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Chemical characterisation and fabrication ofchitosanndashsilica hybrid scaff olds with3-glycidoxypropyl trimethoxysilanedagger
Louise S Connella Frederik Romerb Marta Suarezcd Esther M Vallianta Ziyu Zhanga
Peter D Leee Mark E Smithbf John V Hannab and Julian R Jonesa
Chitosan has been explored as a potential component of biomaterials and scaffolds for many tissue
engineering applications Hybrid materials where organic and inorganic networks interpenetrate at the
molecular level have been a particular focus of interest using 3-glycidoxypropyl trimethoxysilane
(GPTMS) as a covalent crosslinker between the networks in a solndashgel process GPTMS contains both an
epoxide ring that can undergo a ring opening reaction with the primary amine of chitosan and a
trimethoxysilane group that can co-condense with silica precursors to form a silica network While manyresearchers have exploited this ring-opening reaction it is not yet fully understood and thus the 1047297nal
product is still a matter of some dispute Here a detailed study of the reaction of GPTMS with chitosan
under different pH conditions was carried out using a combination of solution state and solid state MAS
NMR techniques The reaction of GPTMS with chitosan at the primary amine to form a secondary amine
was con1047297rmed and the rate was found to increase at lower pH However a side-reaction was identi 1047297ed
between GPTMS and water producing a diol species The relative amounts of diol and chitosanndashGPTMS
species were 80 and 20 respectively and this ratio did not vary with pH The functionalisation pH had
an effect on the mechanical properties of 65 wt organic monoliths where the properties of the organic
component became more dominant Scaffolds were fabricated by freeze drying and had pore diameters
in excess of 140 mm and tailorable by altering freezing temperature which were suitable for tissue
engineering applications In both monoliths and scaffolds increasing the organic content disrupted the
inorganic network leading to an increase in silica dissolution in SBF However the dissolution of silica
and chitosan was congruent up to 4 weeks in SBF illustrating the true hybrid nature resulting from
covalent bonding between the networks
Introduction
As the worlds aging population increases the number of indi-
viduals aff ected by bone and cartilage disorders is also
increasing1 To treat these debilitating conditions and address
the scarcity of natural gra material it is necessary for synthetic
materials to be developed that mimic the physical properties of
natural tissue while also stimulating regeneration and remod-
elling by the body2ndash4 Bioactive glass scaff olds have many of the
properties required for tissue engineering scaff olds but they
are too brittle to be used in cyclically loaded applications5 A
so er tougher material is required67 Solndashgel derived hybrid
materials where interpenetrating networks (IPNs) of organic
polymers and inorganic components are covalently bonded8
have shown promise as biodegradable tissue regeneration
scaff olds Natural polymers such as chitosan9ndash14 poly-glutamic
acid15ndash18 and gelatin1920 are covalently bonded to a silica
network via gra ing a silane containing coupling agent to
functional groups along the polymer These hybrid materials
have been formed into scaff olds by various methods including
foaming20 freeze-drying1119 and electrospinning21 The poten-
tial benets of hybrids over conventional composites is that
mechanical properties and dissolution rates can be varied by
a Department of Materials Imperial College London South Kensington Campus SW7
2AZ UK E-mail j ulianrjonesimperialacuk Tel +44 (0) 2075946749b Department of Physics University of Warwick Gibbet Hill Rd Coventry CV4 7AL UK c Fundaci on ITMA Parque Technol ogico de Asturias 33428 Llanera Asturias Spaind Department of Nanostructured Material Centro de Investigaci on en Nanomateriales y
Nanotecnolog ıa Principado de Asturias Parque Tecnol ogico de Asturias 33428
Llanera SpaineSchool of Materials The University of Manchester Oxford Rd M13 9PL UK f The Vice-Chancellors O ffice University House Lancaster University Lancaster LA1
4YW UK
dagger Electronic supplementary information (ESI) available 1H NMR of chitosan and
GPTMS in D2ODCl at pH 4 over time 1Hndash
13C HSQC spectra of fully hydrolysed
GPTMS a er 72 h in D2ODCl at pH 2 29Si MAS NMR spectra of functionalised
chitosan at pH 2 and 4 with table of quantication and thermogravimetric
analysis of 50 wt organic chitosanndashsilica scaff olds a er 0 72 168 and 672 h
in SBF See DOI 101039c3tb21507e
Cite this J Mater Chem B 2014 2668
Received 25th October 2013Accepted 2nd December 2013
DOI 101039c3tb21507e
wwwrscorgMaterialsB
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short time-points indicating that the hydrolysis of the silane
groups was rapid However at 5 min in solution at pH 4 and 6
there were multiple peaks around d1H 053 ppm due to
incomplete hydrolysis of the silane groups (ESI Fig S1dagger)
At pH 2 the hydrolysis was so rapid that no evidence of
partial hydrolysis was observed at 5 min This is in agreement
with Gabrielli et al who observed a pH dependence of the rate of
silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2
protons of the epoxide ring at d1
H 281 ppm and d1
H 261 ppmreduced in intensity over time however this occurred at a much
slower rate than silane hydrolysis New peaks were observed in
the d1H 390ndash330 ppm region although there was considerable
overlap in the 1H NMR spectra making it hard to distinguish
the peaks Using a combination of 13C and HSQC (showing 1H
and 13C coupling through one bond) experiments allowed the
diff erent species to be identied This was conrmed by
repeating the HSQC experiment for GPTMS alone in D2ODCl
a er 72 h at pH 2 where the epoxide ring was fully opened (ESI
Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3
Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C
5939 ppm) and (d1
H 341 ppm d13
C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by
water in solution43 At longer time points but at all pH values
other signals were observed at (d1H 357 ppm d13C 5096 ppm)
and (d1H 357 ppm d13C 5096 ppm) which were attributed to
the reaction of epoxide ring with the primary amine of chitosan
(ndashNH2) to form a secondary amine No other reactions were
identied suggesting that the only covalent coupling reaction
occurring between chitosan and GPTMS occurred at the primary
amine
The use of quantitative HSQC experiments showed that the
extent of epoxide opening a er 24 h decreased as pH increased
9 68 and 98 mol epoxide ring remained at pH 2 4 and 6
respectively (Fig 4a) This supports the observations of Gabrielli
et al that the opening of the epoxide ring of GPTMS in water is
acid catalysed and hence slightly acidic conditions are required
for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent
nucleophilic attack In contrast with the prediction of Gabrielli
et al altering the pH did not aff ect the relative numbers of diol
and secondary amine species formed the percentage of primary
amines that formed secondary amines remained constant at
around 20 (Fig 4b)
Analysis of 15N MAS NMR of chitosan dissolved at pH 4
quenched in liquid nitrogen and freeze dried showed clearly
that in pure chitosan there were two signals due to acetylated
and deacetylated forms of the chitosan monomer (Fig 5a) A er
24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm
split into two indicating a third nitrogen species is present (Fig 5b)
This is unequivocal evidence that there was a reaction
between chitosan and GPTMS at the primary amine It also
shows that the nucleophilic addition between the amine and
Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions
Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
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8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
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short time-points indicating that the hydrolysis of the silane
groups was rapid However at 5 min in solution at pH 4 and 6
there were multiple peaks around d1H 053 ppm due to
incomplete hydrolysis of the silane groups (ESI Fig S1dagger)
At pH 2 the hydrolysis was so rapid that no evidence of
partial hydrolysis was observed at 5 min This is in agreement
with Gabrielli et al who observed a pH dependence of the rate of
silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2
protons of the epoxide ring at d1
H 281 ppm and d1
H 261 ppmreduced in intensity over time however this occurred at a much
slower rate than silane hydrolysis New peaks were observed in
the d1H 390ndash330 ppm region although there was considerable
overlap in the 1H NMR spectra making it hard to distinguish
the peaks Using a combination of 13C and HSQC (showing 1H
and 13C coupling through one bond) experiments allowed the
diff erent species to be identied This was conrmed by
repeating the HSQC experiment for GPTMS alone in D2ODCl
a er 72 h at pH 2 where the epoxide ring was fully opened (ESI
Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3
Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C
5939 ppm) and (d1
H 341 ppm d13
C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by
water in solution43 At longer time points but at all pH values
other signals were observed at (d1H 357 ppm d13C 5096 ppm)
and (d1H 357 ppm d13C 5096 ppm) which were attributed to
the reaction of epoxide ring with the primary amine of chitosan
(ndashNH2) to form a secondary amine No other reactions were
identied suggesting that the only covalent coupling reaction
occurring between chitosan and GPTMS occurred at the primary
amine
The use of quantitative HSQC experiments showed that the
extent of epoxide opening a er 24 h decreased as pH increased
9 68 and 98 mol epoxide ring remained at pH 2 4 and 6
respectively (Fig 4a) This supports the observations of Gabrielli
et al that the opening of the epoxide ring of GPTMS in water is
acid catalysed and hence slightly acidic conditions are required
for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent
nucleophilic attack In contrast with the prediction of Gabrielli
et al altering the pH did not aff ect the relative numbers of diol
and secondary amine species formed the percentage of primary
amines that formed secondary amines remained constant at
around 20 (Fig 4b)
Analysis of 15N MAS NMR of chitosan dissolved at pH 4
quenched in liquid nitrogen and freeze dried showed clearly
that in pure chitosan there were two signals due to acetylated
and deacetylated forms of the chitosan monomer (Fig 5a) A er
24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm
split into two indicating a third nitrogen species is present (Fig 5b)
This is unequivocal evidence that there was a reaction
between chitosan and GPTMS at the primary amine It also
shows that the nucleophilic addition between the amine and
Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions
Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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Journal of Materials Chemistry B Paper
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
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8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 513
short time-points indicating that the hydrolysis of the silane
groups was rapid However at 5 min in solution at pH 4 and 6
there were multiple peaks around d1H 053 ppm due to
incomplete hydrolysis of the silane groups (ESI Fig S1dagger)
At pH 2 the hydrolysis was so rapid that no evidence of
partial hydrolysis was observed at 5 min This is in agreement
with Gabrielli et al who observed a pH dependence of the rate of
silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2
protons of the epoxide ring at d1
H 281 ppm and d1
H 261 ppmreduced in intensity over time however this occurred at a much
slower rate than silane hydrolysis New peaks were observed in
the d1H 390ndash330 ppm region although there was considerable
overlap in the 1H NMR spectra making it hard to distinguish
the peaks Using a combination of 13C and HSQC (showing 1H
and 13C coupling through one bond) experiments allowed the
diff erent species to be identied This was conrmed by
repeating the HSQC experiment for GPTMS alone in D2ODCl
a er 72 h at pH 2 where the epoxide ring was fully opened (ESI
Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3
Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C
5939 ppm) and (d1
H 341 ppm d13
C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by
water in solution43 At longer time points but at all pH values
other signals were observed at (d1H 357 ppm d13C 5096 ppm)
and (d1H 357 ppm d13C 5096 ppm) which were attributed to
the reaction of epoxide ring with the primary amine of chitosan
(ndashNH2) to form a secondary amine No other reactions were
identied suggesting that the only covalent coupling reaction
occurring between chitosan and GPTMS occurred at the primary
amine
The use of quantitative HSQC experiments showed that the
extent of epoxide opening a er 24 h decreased as pH increased
9 68 and 98 mol epoxide ring remained at pH 2 4 and 6
respectively (Fig 4a) This supports the observations of Gabrielli
et al that the opening of the epoxide ring of GPTMS in water is
acid catalysed and hence slightly acidic conditions are required
for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent
nucleophilic attack In contrast with the prediction of Gabrielli
et al altering the pH did not aff ect the relative numbers of diol
and secondary amine species formed the percentage of primary
amines that formed secondary amines remained constant at
around 20 (Fig 4b)
Analysis of 15N MAS NMR of chitosan dissolved at pH 4
quenched in liquid nitrogen and freeze dried showed clearly
that in pure chitosan there were two signals due to acetylated
and deacetylated forms of the chitosan monomer (Fig 5a) A er
24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm
split into two indicating a third nitrogen species is present (Fig 5b)
This is unequivocal evidence that there was a reaction
between chitosan and GPTMS at the primary amine It also
shows that the nucleophilic addition between the amine and
Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions
Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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Journal of Materials Chemistry B Paper
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
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8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
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8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 513
short time-points indicating that the hydrolysis of the silane
groups was rapid However at 5 min in solution at pH 4 and 6
there were multiple peaks around d1H 053 ppm due to
incomplete hydrolysis of the silane groups (ESI Fig S1dagger)
At pH 2 the hydrolysis was so rapid that no evidence of
partial hydrolysis was observed at 5 min This is in agreement
with Gabrielli et al who observed a pH dependence of the rate of
silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2
protons of the epoxide ring at d1
H 281 ppm and d1
H 261 ppmreduced in intensity over time however this occurred at a much
slower rate than silane hydrolysis New peaks were observed in
the d1H 390ndash330 ppm region although there was considerable
overlap in the 1H NMR spectra making it hard to distinguish
the peaks Using a combination of 13C and HSQC (showing 1H
and 13C coupling through one bond) experiments allowed the
diff erent species to be identied This was conrmed by
repeating the HSQC experiment for GPTMS alone in D2ODCl
a er 72 h at pH 2 where the epoxide ring was fully opened (ESI
Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3
Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C
5939 ppm) and (d1
H 341 ppm d13
C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by
water in solution43 At longer time points but at all pH values
other signals were observed at (d1H 357 ppm d13C 5096 ppm)
and (d1H 357 ppm d13C 5096 ppm) which were attributed to
the reaction of epoxide ring with the primary amine of chitosan
(ndashNH2) to form a secondary amine No other reactions were
identied suggesting that the only covalent coupling reaction
occurring between chitosan and GPTMS occurred at the primary
amine
The use of quantitative HSQC experiments showed that the
extent of epoxide opening a er 24 h decreased as pH increased
9 68 and 98 mol epoxide ring remained at pH 2 4 and 6
respectively (Fig 4a) This supports the observations of Gabrielli
et al that the opening of the epoxide ring of GPTMS in water is
acid catalysed and hence slightly acidic conditions are required
for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent
nucleophilic attack In contrast with the prediction of Gabrielli
et al altering the pH did not aff ect the relative numbers of diol
and secondary amine species formed the percentage of primary
amines that formed secondary amines remained constant at
around 20 (Fig 4b)
Analysis of 15N MAS NMR of chitosan dissolved at pH 4
quenched in liquid nitrogen and freeze dried showed clearly
that in pure chitosan there were two signals due to acetylated
and deacetylated forms of the chitosan monomer (Fig 5a) A er
24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm
split into two indicating a third nitrogen species is present (Fig 5b)
This is unequivocal evidence that there was a reaction
between chitosan and GPTMS at the primary amine It also
shows that the nucleophilic addition between the amine and
Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions
Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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Journal of Materials Chemistry B Paper
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
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short time-points indicating that the hydrolysis of the silane
groups was rapid However at 5 min in solution at pH 4 and 6
there were multiple peaks around d1H 053 ppm due to
incomplete hydrolysis of the silane groups (ESI Fig S1dagger)
At pH 2 the hydrolysis was so rapid that no evidence of
partial hydrolysis was observed at 5 min This is in agreement
with Gabrielli et al who observed a pH dependence of the rate of
silane hydrolysis in GPTMS43 Peaks attributed to f1 and f2
protons of the epoxide ring at d1
H 281 ppm and d1
H 261 ppmreduced in intensity over time however this occurred at a much
slower rate than silane hydrolysis New peaks were observed in
the d1H 390ndash330 ppm region although there was considerable
overlap in the 1H NMR spectra making it hard to distinguish
the peaks Using a combination of 13C and HSQC (showing 1H
and 13C coupling through one bond) experiments allowed the
diff erent species to be identied This was conrmed by
repeating the HSQC experiment for GPTMS alone in D2ODCl
a er 72 h at pH 2 where the epoxide ring was fully opened (ESI
Fig S2dagger) A fully assigned HSQC spectrum is shown in Fig 3
Peaks at (d1H 373 ppm d13C 6339 ppm) (d1H 348 ppm d13C
5939 ppm) and (d1
H 341 ppm d13
C 5939 ppm) were attributedto the formation of a diol when epoxide rings are opened by
water in solution43 At longer time points but at all pH values
other signals were observed at (d1H 357 ppm d13C 5096 ppm)
and (d1H 357 ppm d13C 5096 ppm) which were attributed to
the reaction of epoxide ring with the primary amine of chitosan
(ndashNH2) to form a secondary amine No other reactions were
identied suggesting that the only covalent coupling reaction
occurring between chitosan and GPTMS occurred at the primary
amine
The use of quantitative HSQC experiments showed that the
extent of epoxide opening a er 24 h decreased as pH increased
9 68 and 98 mol epoxide ring remained at pH 2 4 and 6
respectively (Fig 4a) This supports the observations of Gabrielli
et al that the opening of the epoxide ring of GPTMS in water is
acid catalysed and hence slightly acidic conditions are required
for the reaction with nucleophilic species Gabrielli et al alsopostulated that too much formation of diol would prevent
nucleophilic attack In contrast with the prediction of Gabrielli
et al altering the pH did not aff ect the relative numbers of diol
and secondary amine species formed the percentage of primary
amines that formed secondary amines remained constant at
around 20 (Fig 4b)
Analysis of 15N MAS NMR of chitosan dissolved at pH 4
quenched in liquid nitrogen and freeze dried showed clearly
that in pure chitosan there were two signals due to acetylated
and deacetylated forms of the chitosan monomer (Fig 5a) A er
24 h reaction with GPTMS at pH 4 the signal at d15N 350 ppm
split into two indicating a third nitrogen species is present (Fig 5b)
This is unequivocal evidence that there was a reaction
between chitosan and GPTMS at the primary amine It also
shows that the nucleophilic addition between the amine and
Fig 3 Fully assigned quantitative HSQC NMR spectrum of chitosanfunctionalised with GPTMS for 24 h at pH 4 with corresponding 1H and13C 1D spectra showing the potential products and side reactions
Fig 4 The quantitative HSQC NMR experiments were used tocalculate (a) mol of unopened epoxide secondary amine productand diol side-product and (b) relative amounts of secondary amineproduct and diol product of the reacted epoxide at pH 2 4 and 6 for24 h
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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Journal of Materials Chemistry B Paper
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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Journal of Materials Chemistry B Paper
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
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the epoxide ring is the only covalent bonding which occurs
between the amine and GPTMS However it should be noted
that this does not rule out the possibility that hydrogen bonding
may occur between amine amide or hydroxyl species or that all
of the epoxide groups will react
FTIR spectra of the chitosan functionalised with GPTMS for
24 h at pH 2 and pH 4 (Fig 6a) are very similar to the pure
chitosan FTIR spectrum Minor diff erences arise at 1507 cm1
where the secondary amide peak reduced in intensity at pH 4This is potentially due to hydrogen bonding of the amine group
in chitosan which is more prominent at pH 4 because fewer of
the amine groups were converted to secondary amines There is
no evidence of the epoxide ring remaining at either pH 2 or pH 4
as the bands for CndashOndashC stretching of GPTMS would be expected
at 909 cm1 and 846 cm131 This is potentially due to the small
amount of GPTMS used relative to the amount of chitosan and
diol formation which reduces the relative amount of epoxide
ring further Mahony et al showed in a silicagelatin system that
the bands corresponding to unopened epoxide ring could not
be distinguished until a molar ratio of GPTMS to gelatin of 1500
was used (at pH 5)
20
Structural characterisation of hybrid monoliths
The chemical structure of the hybrids was characterised in
order to determine the eff ect of pH and organic content on the
monoliths FTIR spectra of hybrid monoliths (Fig 6b) fabri-
cated by combining hydrolysed TEOS with the chitosanndashGPTMS
solution at pH 4 or pH 2 to give a composition of 65 wt
organic show a strong SindashOndashSi stretching band that appeared at
1020 cm1 The band at 934 cm1 was attributed to non-
bridging SindashOH bonds and appears moreintenseat pH 2 than at
pH 4 indicating a more condensed network at pH 4 The
primary and secondary amide bands of chitosan were retainedat 1600 cm1 and 1500 cm1 In a similar fashion to the func-
tionalised chitosan at pH 4 the intensity of the secondary
amine reduced whereas little change was observed at pH 2
Again this may be attributed to more prominent hydrogen
bonding at pH 429Si MAS NMR can be used to quantify the connectivity of a
silica network The nomenclature Qn is used to describe silica
species where the silicon is bonded by n bridging oxygens and 4
n non-bridging oxygens whereas Tn is used to describe a
silicon atom bonded to a carbon (as in GPTMS) with n bridging
oxygens with 3 n non-bridging oxygens 29Si MAS NMR spectra
showed that the hybrid monoliths had a partially condensedsilica network comprising of distinct Tn and Qn species which
correspond to CndashSi(OndashSi)n(OH)3n and Si(OndashSi)n(OH)4n
respectively44
Peak tting of the one pulse MAS 29Si NMR spectra allowed
quantication of each of the silicon species present in 65 wt
organic hybrids (spectra shown in Fig 7 and calculated
percentage abundance of silicon species in Table 1) In agree-
ment with the FTIR results the hybrids synthesized at pH 4
were more highly condensed than at pH 2 as indicated by the
higher numbers of Q4 and T3 species present In fact at pH 4
there were no Q2 species present whereas there were 50 04
Fig 5 15N MAS NMR of (a) pure chitosan and (b) chitosan reacted withGPTMS at pH 4 for 24 h
Fig 6 (a) FTIR spectra of pure chitosan and chitosan functionalisedwith GPTMS at pH 2 and 4 (b) FTIR spectra of pure chitosan andchitosanndashsilica hybrid monoliths with 65 wt organic where thefunctionalisation step was carried out at pH 2 and 4
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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Journal of Materials Chemistry B Paper
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
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whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
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Journal of Materials Chemistry B Paper
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quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
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present at pH 2 Calculation of the degree of condensation ( Dc)
gave values of 927 and 902 for pH 4 and 2 respectively The
more condensed network is due to the fact that at pH lt22 the
transition state of condensation is stabilised by the ethoxy and
methoxy groups of TEOS and GPTMS The partially hydrolysed
silica precursor condenses faster leading to chains of silica
network with a large number of non-bridging oxygens Theopposite is true at pH gt22 where fully hydrolysed precursors
condense fastest leading to highly condensed silica networks
with fewer non-bridging species45 Repeating 29Si MAS NMR for
the functionalised chitosan shows only Tn species as expected
as there was no TEOS present (ESI Fig S3dagger) However it was
observed that within 5 min condensation had occurred
between the GPTMS molecules so that at pH 2 up to 60 of the
GPTMS was present in a T3 form (ESI Table S1dagger) This would
render the molecule unable to condense further when TEOS is
introduced potentially leading to two distinct silica networks
that do not interpenetrate The signicance of this is unknown
and further investigation is required to establish the degree of
interaction between the two networks
SEM images of the fracture surfaces of the monoliths fabri-cated with 35 and 65 wt organic at pH 4 and pH 2 all show that
no macroscale phase separation occurred during hybrid
synthesis at any composition (Fig 8) Agglomerated particle
morphologies typical of that formed by the solndashgel process46
were observed This is due to silica nanoparticles that agglom-
erate and fuse to form a mesoporous silica gel46 The apparent
particle diameters were similar for samples made at pH 2 and
pH 4 (compare Fig 8a with b and 8c with d) but larger particles
are observed as organic content increased The particle size of
the 35 wt organic hybrids was more typical for solndashgel silica
microstructures so the larger particle size is likely due to chi-
tosan polymer coating the surface of the silica particles
Mechanical and dissolution properties of monoliths
From compression tests hybrid monoliths containing 35 wt
organic exhibited brittle behaviour with a strain at fracture of 4
to 8 Increasing the chitosan content reduced the brittle
character as shown by the deformation prior to fracture for 65
wt organic monoliths whereas 35 wt organic monoliths
failed catastrophically (Fig 9) The increase in chitosan content
also increased the strain at fracture to around 48 This had the
eff ect of reducing the compressive modulus of the monoliths
Table 1 Percentage abundance of silicon species present in 65 wtorganic hybrids functionalised at pH 4 and 2
pH Q4 Q3 Q2 T 3 T 2 Dc
4 642 08 225 07 NA 82 06 52 09 9272 600 05 251 04 50 04 69 07 30 04 902
Fig 7 29Si MAS NMR spectra of 65 wt organic hybrids synthesized at(a) pH 4 and (b) pH 2 showing the peak 1047297tting used to calculate theabundance of each silicon species
Fig 8 Fracture surfaces of hybrid monoliths imaged by SEM with (aand b) 35 wt organic and (c and d) 65 wt organic contents andfunctionalised at (a and c) pH 4 and (b and d) pH 2 Aggregated particlemorphologies typical of solndashgel silica glasses are observed moleculeunable to condense further when TEOS is introduced potentiallyleading to two distinct silica networks that do not interpenetrate Thesigni1047297cance of this is unknown and further investigation is required toestablish the degree of interaction between the two networks
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Journal of Materials Chemistry B Paper
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8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
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freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
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Journal of Materials Chemistry B Paper
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irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 677
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1113
whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
678 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 813
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 913
freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
676 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1013
irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 677
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1113
whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
678 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 913
freeze-dried Chitosan has been chosen for scaff old synthesis by
freeze-drying as the polymer forms sheets between the ice
crystals as the sol is forced out of the solidifying pure water
where ultimately the ice crystals form the interconnected pore
structure of the scaff olds4950
Hybrid scaff old morphology
Investigation of the morphology of the scaff
olds by SEM(Fig 11) showed that reducing freezing temperature reduced
the pore diameters This can be attributed to the higher degree
of supercooling that occurs at lower freezing temperatures
hence increasing the nucleation rate of ice crystals Although
more ice crystals form the lower temperatures means that the
growth of the crystals is slower resulting in many small ice
crystals and hence smaller pores in the nal scaff old The pores
were elongated and angular with a certain degree of direction-
ality as the gels tended to freeze from the outside-in with a
protrusion forming in the centre where the ice forced the gel as
it expanded during freezing
Pore interconnectivity and interconnect size is o en more
important that pore size Mercury porosimetry uses a model toobtain the diameters of pores that constrict the mercury intru-
sion as a function of pressure Analysis of the modal pore
interconnect diameters by mercury porosimetry conrmed that
the interconnect diameter reduced as the freezing temperature
reduced The scaff olds frozen at 20 C had modal pore
diameters of 178 47 mm and 156 7 mm 80 C were 150
39 mm and 140 15 mm and those quenched in liquid nitrogen
were 21 12 mm and 23 20 mm for 50 wt and 65 wt organic
respectively (Fig 12)
A guide for a suitable interconnect diameter for bone tissue
engineering scaff olds is 100 mm51 At 20 C and 80 C the
interconnect diameters were well above 100 mm Quenching in
liquid nitrogen caused a signicant decrease in pore intercon-
nect diameter The interconnect diameters of 65 wt organic
and 50 wt organic scaff olds were similar at each freezing
temperature however the total porosity of the scaff olds varied with composition (967 02 and 975 02 for 50 wt and
65 wt organic respectively Table 3) This is due to the water
content of the gels prior to freeze-drying The scaff olds with
higher organic content contained relatively more chitosan
solution (17 mg mL1) and so also contain more water When
the water is frozen and removed during freeze-drying the ulti-
mate result is to increase the porosity of the scaff olds
mCT images of the 65 wt organic scaff olds frozen at 20 C
and 80 C shown in Fig 13 illustrate the angular and
Fig 12 Modal pore interconnect diameters calculated from inter-connect diameters determined by mercury porosimetry
Table 3 Percentage porosity of scaffolds with organic content andfreezing temperature
Organic content (wt) Freezing temp (C) Porosity ()
65 20 975 0480 975 01196 975 02
50 20 969 0280 967 02196 964 01
(Mean SD n frac14 10)
Fig 13 X-ray microtomography (mCT) of 65 wt organic scaffoldfrozen at (a) 20 C and (b) 80 C illustrating the elongated andirregular pore morphology typical of freeze-drying
Fig 11 Images of the morphology of 65 wt organic and 50 wtorganic hybrid scaffolds formed by freeze drying at differenttemperatures by SEM The decreasing pore size as the freezingtemperature reduced can be observed clearly
676 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1013
irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 677
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1113
whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
678 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1013
irregular pore morphologies that are characteristic of scaff olds
fabricated via freeze-drying Applying 3D image analysis tech-
niques the modal pore diameter of the 20 C 65 wt organic
scaff old was 313 mm and the modal interconnect diameter was
189 mm which is in good agreement with the mercury poros-
imetry data The images also showed that the scaff olds were well
interconnected important for tissue ingrowth and vasculariza-
tion The mean tortuosity of the scaff olds another property
which may be important for successful regeneration of tissue was measured by mercury porosimetry as 193 023 165
024 and 137 031 for 20 C 80 C and 196 C scaff olds
respectively This is within the range reported for cancellous
bone by Pakula et al of 11 to 2852
Mechanical behaviour of the chitosanndashsilica hybrid scaff olds
The mechanical properties of the scaff olds were investigated
under compression and the data is presented in Table 4
A slight increase in the compressive modulus was observed
at 50 wt organic compared with 65 wt organic however due
to the highly porous nature of the scaff olds there was a large
degree of scatter within the data and the diff erence was not
statistically signicant The strain at failure did not vary with
freezing temperature although a small increase in compressive
modulus and compressive strengths was observed for samples
quenched in liquid nitrogen At 875 699 and 1430 kPa for20C 80 C and liquid nitrogen 50 wt organic hybrids
respectively and 808 620 and 1030 kPa for20 C80 C and
liquid nitrogen 65 wt organic hybrid scaff olds respectively
the compressive strengths are far too low for load sharing
applications for bone regeneration as originally intended This
is due to the very high porosities of the scaff olds The freezedrying method does not give control of percentage porosity
Given the promising mechanical properties of the monolith
samples if the porosity were reduced then the compressive
strengths may be increased making them more suitable for
bone regeneration scaff olds Alternatively these scaff olds may
be used in non-load sharing applications such as cartilage
regeneration These scaff olds may be particularly attractive for
cartilage regeneration due to the elongated pore morphologies
and since chitosan has a similar structure to anionic glycos-
aminoglycans found in articular cartilage53
Dissolution behaviour of hybrid scaff olds
The silicon release in SBF as measured in triplicate by ICP-OES
(Fig 10b) was very rapid for both the 65 wt and 50 wt
organic scaff olds The fastest rate of silicon release was up to 8
h with the silicon concentration in solution plateauing at
around 80 g L1 and 90 g L1 for 50 and 65 wt organic
respectively a er 24 h As with the monolith hybrid samples
greater silicon release was observed for higher organic content
hybrids due to disruption of the silica network by the organic
component Phosphorus and calcium ion concentrations did
not vary over the timescale of the experiment (data not pre-
sented) and so it can be concluded that no apatite formed on
the sample surfaces as expected
FTIR analysis of the remaining solids a er 4 weeks in SBF
(Fig 14) showed that the amide I and II bands were retained
although there was a signicant reduction in the intensity of the
amide II band This indicates that there was still chitosan
remaining in the hybrid a er the dissolution study conrmed
by thermogravimetric analysis (TGA ESI Fig S4dagger) The weight
loss by TGA between 200 C and 600 C of the 50 wt organic
scaff old prior to immersion in SBF due to combustion of theorganic component was 38 wt A er 72 h immersion this
increased to 40 wt and then remained constant at 1 w and 4 w
This suggests that there is rapid silica dissolution within the
rst 72 h as also indicated by the ICP-OES dissolution proles
Table 4 Table Mechanical properties of freeze-dried hybrid scaffolds
Organiccontent (wt)
Freezing temp (C)
Compressmodulus (MPa)
Failurestress (kPa)
Strain at failure ()
65 20 085 032 808 289 119 3980 073 029 620 176 116 48196 137 064 1030 452 87 32
50 20 106 050 875 419 119 6480 091 040 699 213 78 27196 108 014 1430 713 145 75
(Mean SD n frac14 10)
Fig 14 FTIR of hybrid scaffolds before and after 4 w immersion in SBFof (a) 65wt organic and (b) 50 wt organic scaffolds Thepresence ofamide I and II bands indicates chitosan remains in the scaffolds afterimmersion
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 677
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1113
whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
678 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1113
whereas chitosan dissolution was slower However a er the
rst 72 h the two components are released at the same rate so
that the relative composition remains constant up to 4 w in SBF
Congruent dissolution seen here a er 72 h is one of the
dening features of a successful hybrid material and so this is a
promising result for the long term mechanical and chemical
stability of the chitosanndashsilica hybrid
Although the assessment of biological activity is beyond the
scope of this article similar chitosanndash
GPTMS systems have beenstudied previously in vivo and in vitro10ndash12363754 The good prolif-
eration of osteoblastic MG63 cell cultures on chitosanndashsilica
hybrid membranes and freeze dried scaff olds with varying
GPTMS and TEOS contents showed that the hybrid materials
were biocompatible101137 Compared with pure chitosan scaff olds
and membranes the hybrid materials showed better prolifera-
tion and multilayers of well spread MG63 cells a er 6 days in cell
culture10 however the type of silica species present aff ected the
behaviour of the cells with an increase in TEOS promoting
osteodiff erentiation rather than proliferation as seen in hybrids
with high GPTMS contents but no TEOS37 Scaff olds freeze dried
at
20
C exhibited cell penetration deep inside the materialindicating good interconnectivity and permeability11 In vivo
studies were carried out in adult female Wistar rats to determine
the biocompatibility of chitosanndashGPTMS freeze-dried scaff olds
and membranes54 For each animal three 2 2 cm samples were
implanted into 3 cm long dorsal incisions and were recovered
a er 1 2 4 and 8 weeks From the results of these studies the
authors are condent that the chitosanndashsilica hybrid materials
presented here would be suitable for tissue regeneration appli-
cations particularly the highly porous freeze dried scaff olds
Conclusions
Summary of eff ect of pH on monolith hybrids A combination of solution and solid state NMR techniques
showed a reaction between the epoxide ring of GPTMS and
chitosan at the primary amine Following the reaction at three
diff erent pH values has shown that this reaction was acid
catalyzed with signicantly more epoxide ring opening at pH 2
than at pH 4 or 6 However it was also shown that an unwanted
side reaction occurred between water and the epoxide ring
resulting in diol formation and that this was the dominant
reaction at all pH values Hydrolysis of the methoxysilane
groups of GPTMS was rapid under acidic conditions however
condensation occurred simultaneously so that within 5 min T3
species are present in GPTMS Fabricating monolith hybrids was achieved by introducing the functionalised chitosan into a
sol of hydrolysed TEOS The silica network of the monoliths was
less condensed when chitosan was functionalised at pH 2
compared with those functionalised at pH 4 This had the eff ect
of increasing the rate of silica dissolution in SBF for the pH 2
sample The eff ect of pH on mechanical properties was minimal
at 35 wt organic as the brittle nature of the silica phase
appeared to predominate However at 65 wt organic the
organic phase had a more signicant eff ect on the mechanical
properties as the elongation at failure was increased from 7 to
40 The samples fabricated at pH 2 which had a greater
degree of coupling between the chitosan and GPTMS showed a
slight increase in compressive modulus
Summary of the fabrication and characterisation of hybrid
scaff olds
Chitosanndashsilica hybrid scaff olds were fabricated by combining
the solndashgel process with a freeze-drying step Chitosan was
functionalised using pre-determined optimum pH conditionsand compositions of 50 wt and 65 wt organic Freezing
temperatures had a dramatic eff ect on the modal pore inter-
connect diameter Scaff olds fabricated by quenching in liquid
nitrogen had interconnect diameters of 20ndash23 mm which is too
small for tissue engineering applications Scaff olds frozen
at 20 and 80 C are suitable as they have pore interconnects
well in excess of 100 mm the critical value required for tissue
engineering scaff olds The compressive strengths of the scaf-
folds were too low to be used in load-sharing applications
primarily due to their high porosities of 96ndash97 Reducing the
porosity will increase the compressive strengths of the scaff olds
for alternative applications such as non-load bearing cartilage
regeneration may be more appropriate A 4 weeks dissolution
study in SBF showed that silicon release was rapid within the
rst 24 h but a er this time the chitosan and silica are released
at the same rate so that the relative composition of the hybrid
remains unchanged a er 72 h up to 4 weeks This is an
important result that points towards long term mechanical
stability and chemical activity of the scaff olds
Here for the rst time
A combination of solution and solid state NMR techniques
have been used to probe the functionalisation reaction between
chitosan and GPTMS
It has been shown that covalent bonding occurs between
the primary amine of chitosan and the epoxide of GPTMS toform a secondary amine allowing covalent coupling between
chitosan and a silica network
The extent of reaction at diff erent pH values was quantied
to show that both the reactions of GPTMS with water and with
chitosan are acid catalyzed and that the relative amounts of
product and side-product does not depend on pH
That functionalisation pH was shown to have an impact on
the mechanical properties of hybrids at 65 wt where the
properties of the organic component become more dominant
That high organic content was shown to disrupt the silica
network speeding up the rate of silica dissolution in both
monolith and scaff old hybrids
The interconnect diameters were quantied for freeze-
dried chitosan scaff olds and conrmed that 20 and80 C are
appropriate freezing temperatures for fabricating tissue engi-
neering scaff olds
Chitosan and silicon were shown to be released congru-
ently when immersed in SBF for up to 4 w
Acknowledgements
The authors would like to thank Mr Peter Haycock Department
of Chemistry Imperial College London for carrying out the
678 | J Mater Chem B 2014 2 668ndash680 This journal is copy The Royal Society of Chemistry 2014
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1213
quantitative HSQC experiments This research has been funded
by the EPSRC (EPE0570981 EPE0516691 and EPI0208611)
and the Department of Materials Imperial College London
EMV was a Natural Sciences and Engineering Research Council
of Canada (NSERC) Canadian Centennial Scholar MS was
supported by Ficyt under the Argo program JVH and MES
acknowledge support for the solid-state NMR facilities at War-
wick used in this research which were funded by EPSRC and the
University of Warwick NMR was also partially funded throughthe Birmingham Science City projects which were supported by
Advantage West Midlands (AWM) and the European Regional
Development Fund (ERDF) JVH and MES acknowledge EPSRC
support for FR via project EPI0046881
Notes and references
1 R Burge B Dawson-Hughes D H Solomon J B Wong
A King and A Tosteson J Bone Miner Res 2007 22 465ndash
475
2 L L Hench and J M Polak Science 2002 295 1014ndash1017
3 R Langer and D A Tirrell Nature 2004 428 487ndash
4924 J R Jones J Eur Ceram Soc 2009 29 1275ndash1281
5 M M Pereira J R Jones and L L Hench Adv Appl Ceram
2005 104 35ndash42
6 J R Jones Acta Biomater 2013 9 4457ndash4486
7 E M Valliant and J R Jones So Matter 2011 7 5083ndash5095
8 B M Novak Adv Mater 1993 5 422ndash433
9 Y Shirosaki C M Botelho M A Lopes and J D Santos J
Nanosci Nanotechnol 2009 9 3714ndash3719
10 Y Shirosaki K Tsuru S Hayakawa A Osaka M Lopes
J Santos M Costa and M Fernandes Acta Biomater
2009 5 346ndash355
11 Y Shirosaki T Okayama K Tsuru S Hayakawa and
A Osaka Chem Eng J 2008 137 122ndash
12812 Y Shirosaki K Tsuru S Hayakawa A Osaka M A Lopes
J D Santos and M H Fernandes Biomaterials 2005 26
485ndash493
13 M J Simoes A Gartner Y Shirosaki R M Gil da Costa
P P Cortez F Gartner J D Santos M A Lopes
S Geuna A S Varejao and A C Mauricio Acta Med Port
2011 24 43ndash52
14 G Toskas C Cherif R-D Hund E Laourine B Mahltig
A Fahmi C Heinemann and T Hanke Carbohydr Polym
2013 94 713ndash722
15 E M Valliant F Romer D Wang D S McPhail
M E Smith J V Hanna and J R Jones Acta Biomater2013 9 7662ndash7671
16 G Poologasundarampillai C Ionescu O Tsigkou
M Murugesan R G Hill M M Stevens J V Hanna
M E Smith and J R Jones J Mater Chem 2010 20 8952
17 G Poologasundarampillai B Yu O Tsigkou E Valliant
S Yue P D Lee R W Hamilton M M Stevens
T Kasuga and J R Jones So Matter 2012 8 4822ndash4832
18 M-Y Koh C Ohtsuki and T Miyazaki J Biomater Appl
2011 25 581ndash594
19 L Ren K Tsuru S Hayakawa and A Osaka Biomaterials
2002 23 4765ndash4773
20 O Mahony O Tsigkou C Ionescu C Minelli L Ling
R Hanly M E Smith M M Stevens and J R Jones Adv
Funct Mater 2010 20 3835ndash3845
21 C Gao Q Gao Y Li M N Rahaman A Teramoto and
K Abe J Appl Polym Sci 2013 127 2588ndash2599
22 S V Madihally and H W T Matthew Biomaterials 1999 20
1133ndash1142
23 M Rinaudo G Pavlov and J Desbrieres Polymer 1999 40
7029ndash
703224 M Rinaudo G Pavlov and J Desbrieres Int J Polym Anal
Charact 1999 5 267ndash276
25 S Minami M Morimoto Y Okamoto H Saimoto and
Y Shigemasa in Materials Science of Chitin and Chitosan
ed T Uragami and S Tokura Kodansha Ltd Tokyo 2006
ch 7 pp 191ndash217
26 S-H Rhee J-Y Choi and H-M Kim Biomaterials 2002 23
4915ndash4921
27 A Osaka S Hayakawa K Tsuru S Takashima M Kubo and
Y Shirosaki J R Soc Interface 2005 2 335ndash340
28 Y Liu Y Su and J Lai Polymer 2004 45 6831ndash6837
29 A-C Chao J Membr Sci 2008 311 306ndash
31830 J G Varghese R S Karuppannan and M Y Kariduraganavar
J Chem Eng Data 2010 55 2084ndash2092
31 P Innocenzi T Kidchob and T Yoko J Sol-Gel Sci Technol
2005 35 225ndash235
32 S S Rashidova D S Shakarova O N Ruzimuradov
D T Satubaldieva S V Zalyalieva O A Shpigun
V P Varlamov and B D Kabulov J Chromatogr B Anal
Technol Biomed Life Sci 2004 800 49ndash53
33 F Al-Sagheer and S Muslim J Nanomater 2010 2010 1ndash8
34 S Prochazkova K M V arum and K Ostgaard Carbohydr
Polym 1999 38 115ndash122
35 L Gabrielli L S Connell L Russo J Jimenez-Barbero
F Nicotra L Cipolla and J R Jones RSC Adv 2014 41841ndash1848
36 Y Shirosaki K Tsuru H Moribayashi S Hayakawa
Y Nakamura I R Gibson and A Osaka J Ceram Soc
Jpn 2010 118 989ndash992
37 Y Shirosaki K Tsuru S Hayakawa Y Nakamura
I R Gibson and A Osaka in Bioceramics Development and
Applications ed S Kim The Korean Society for
Biomaterials 2009 vol 22 pp 217ndash220
38 S Heikkinen M M Toikka P T Karhunen and
I A Kilpelainen J Am Chem Soc 2003 125 4362ndash4367
39 J R Jones G Poologasundarampillai R C Atwood
D Bernard and P D Lee Biomaterials 2007 28 1404ndash
141340 R C Atwood J R Jones P D Lee and L L Hench Scr
Mater 2004 51 1029ndash1033
41 S Yue P D Lee G Poologasundarampillai and J R Jones
Acta Biomater 2011 7 2637ndash2643
42 T Kokubo and H Takadama Biomaterials 2006 27 2907ndash2915
43 L Gabrielli L Russo A Poveda J R Jones F Nicotra
J Jimenez-Barbero and L Cipolla Chemistry 2013 19
7856ndash7864
44 K J D MacKenzie and M E Smith Multinuclear Solid-State
Nuclear Magnetic Resonance of Inorganic Materials Elsevier
Science 2002
This journal is copy The Royal Society of Chemistry 2014 J Mater Chem B 2014 2 668ndash680 | 679
Paper Journal of Materials Chemistry B
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online
8112019 Connell(2014)_JmatChemB(Chitosan-Si Hybrid Scaffolds)
httpslidepdfcomreaderfullconnell2014jmatchembchitosan-si-hybrid-scaffolds 1313
45 J D Wright and N A J M Sommerdijk Sol ndash gel materials
chemistry and applications Taylor amp Francis Ltd London 2000
46 S Lin C Ionescu K J Pike M E Smith and J R Jones J
Mater Chem 2009 19 1276
47 J Zhong and D C Greenspan J Biomed Mater Res 2000
53 694ndash701
48 K Tsuru C Ohtsuki A Osaka T Iwamoto and
J D Mackenzie J Mater Sci Mater Med 1997 8 157ndash161
49 S Deville E Saiz R K Nalla and A P Tomsia Science 2006311 515ndash518
50 S Deville Adv Eng Mater 2008 10 155ndash169
51 S F Hulbert S J Morrison and J J Klawitter J Biomed
Mater Res 1972 6 347ndash374
52 M Pakula F Padilla P Laugier and M Kaczmarek J Acoust
Soc Am 2008 123 2415ndash2423
53 A Di Martino M Sittinger and M V Risbud Biomaterials
2005 26 5983ndash5990
54 S Amado M J Simoes P A S Armada da Silva A L Lu ıs
Y Shirosaki M A Lopes J D Santos F Fregnan
G Gambarotta S Raimondo M Fornaro A P Veloso A S P Varejao A C Maurıcio and S Geuna Biomaterials
2008 29 4409ndash4419
Journal of Materials Chemistry B Paper
View Article Online