Hydroxyapatite scaffolds infiltrated with thermally crosslinked polycaprolactone fumarate and polycaprolactone itaconate

Hydroxyapatite scaffolds infiltrated with thermally crosslinkedpolycaprolactone fumarate and polycaprolactone itaconate

Shahriar Sharifi,1,2 Yousef Shafieyan,3 Hamid Mirzadeh,4 Shadab Bagheri-Khoulenjani,2

Sayed Mahmood Rabiee,5 Mohammad Imani,1 Mohammad Atai,6

Mohammad Ali Shokrgozar,3 Ali Hatampoor4

1Department of Novel Drug Delivery Systems, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran, Iran2Deparment of Biomedical Engineering, Amirkabir University of Technology, Hafez Ave, P.O. Box 15875-4413, Tehran, Iran3National Cell Bank of Iran, Pasteur Institute of Iran, P.O. Box 1316943551, Tehran, Iran4Deparment of Polymer Engineering, Amirkabir University of Technology, Hafez Ave, P.O. Box 15875-4413, Tehran, Iran5Department of Mechanical Engineering, Babol (Noshirvani) University, Babol, Iran6Department of Polymer Science, Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran, Iran

Received 25 November 2010; accepted 14 February 2011

Published online 27 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33108

Abstract: In this work, two unsaturated derivatives of polycap-

rolactone (PCL), polycaprolactone fumarate (PCLF), and poly-

caprolactone itaconate (PCLI), have been synthesized and used

as an infiltrating polymer to improve the mechanical properties

of brittle hydroxyapatite (HA) scaffolds. PCLF and PCLI were

first synthesized through polyesterification of the low molecu-

lar weight PCL diols with fumaryl chloride and itaconyl chloride

respectively, and then characterized by Fourier transform infra-

red spectroscopy, nuclear magnetic resonance spectroscopy,

gel permeation chromatography, and differential scanning cal-

orimetry analysis. HA scaffolds were sintered using a foam rep-

lication technique, with porosity of about 60%. Polymer-HA

composites were obtained by infiltrating the HA scaffolds with

PCLF and PCLI solution (12.5 and 30 w/v in dichloromethane)

followed by thermal crosslinking. The polymer infiltrated HA

scaffolds were characterized by scanning electron microscopy,

porosimetry, and gravimetrical analysis. The polyesterification

reaction of PCL diols with fumarate chloride was more efficient

than itaconyl chloride and dependent upon the molecular

weight of the initial PCL precursor; the resultant PCLF demon-

strated a degree of substitution of 1.2, 4.2, and 2.7 times higher

than PCLIs. Polymer infiltration improved the compressive

strength of the HA scaffolds, and based upon the type of mac-

romer (PCLF or PCLI) and also their concentration in infiltrating

solution (12.5 or 30 w/v %) compressive strength increased

about 14–328%. In all studied samples, the reinforcement effect

of PCLF infiltration was higher than PCLI. The macromers and

their corresponding infiltrated HA scaffolds did not show any

significant cytotoxicity toward human primary osteogenic sar-

coma cell (G92 cell lines), in vitro. VC 2011 Wiley Periodicals, Inc.

J Biomed Mater Res Part A: 98A: 257–267, 2011.

Key Words: hydroxyapatite, unsaturated polycaprolactone,

infiltration, crosslinking

INTRODUCTION

Given that the inorganic component of the bone is com-prised of calcium phosphates, bioceramics such as hydroxy-apatite (HA), and tricalcium phosphate (TCP) have beenwidely used for bone regeneration applications. Hydroxyap-atite [HA: Ca10(PO4)6(OH)2] has been extensively used overthe past few decades as a biomaterial, because it possessesthe same chemical composition (Ca/P ¼ 1.67) as bone min-erals with favorable features like bioactivity and biocompati-bility.1 Nowadays, HA is being used successfully as a bonefiller, coating for orthopedic implants, filler of inorganic/polymer composites, and substrate for the column chroma-tography of protein and cell culture carriers.2 Nevertheless,the use of ceramic-based materials has been limited due toa lack of proper mechanical properties compared to human

bones’ characteristics. For example, both dense and porousHA suffers from insufficient fracture toughness with respectto cortical bones (KIC ¼ 0.5�1 vs. 2�6 MPa m1/2), whichhinders its use as a monolithic phase in artificial-bone mate-rials.3 To improve the mechanical properties and increasethe toughness of said material, composite approaches mayprove to be a promising solution.4,5 The composite strategycan be carried out in two ways: (1) a biocompatible poly-mer can be reinforced with particulate HA and (2) a bio-compatible polymer can be infiltrated into a ceramic matrixto obtain composites with a continuous ceramic skeleton.3,5–7

Since about 60% of the bone is composed of inorganicphase and making a composite with a high amount of HA isnot easily achievable due to agglomeration and lack ofproper mixing, the second approach is advantageous for

Correspondence to: S. Sharifi, Biomedical Engineering Department, University Medical Center Groningen, A.Deusinglaan 1, Building 3215, FB40,

9713 AV Groningen, The Netherlands; e-mail: [email protected] (or) Y. Shafieyan, Chemical Engineering Department, McGill University,

3610 University Street, Montreal, Quebec, Canada; e-mail: [email protected]

VC 2011 WILEY PERIODICALS, INC. 257

composite preparation. Furthermore, it provides availabilityfor surface modification to induce more bioactivity.5,8

To date, several studies have been conducted to enhance thefracture toughness of the porous ceramic scaffolds using syn-thetic infiltrating polymers such as poly(glycolic acid), poly(L-lactic acid), poly(D,L-lactic-co-glycolic acid),9,10 polycaprolactone(PCL),4,11–14 dilactic-polylactic acid,15 and natural polymerssuch as nylon67 and collagen.16 Among the aforementionedpolymers, PCL is considered to be a more attractive option dueto its low cost, sustained biodegradability, and availability attuned molecular weights.17–20 Furthermore, PCL’s physiochemi-cal properties can be tuned easily through chemical modifica-tion or copolymerization with other monomers.21–23

In this work, to render PCL into crosslinkable biomate-rial and also improve the mechanical properties of PCLthrough crosslinking reactions and network formation, PCLwas copolymerized with itaconyl chloride and fumarylchloride to form polycaprolactone itaconate (PCLI) andpolycaprolactone fumarate (PCLF), respectively. While thenanoparticulate and microparticulate composites ofhydroxyapatite with unsaturated PCL have previously beenreported,24,25 the infiltration of HA scaffolds with unsatu-rated PCL has not yet been investigated.

The first objective of this study was to synthesize and char-acterize unsaturated derivatives of PCL, which could be used asan infiltrating agent for improving the mechanical properties ofHA scaffolds. Since PCLF was previously synthesized and char-acterized,26–28 this study mainly focuses on synthesis and char-acterization of PCLI macromers, which is reported here for thefirst time. The synthesis of PCLI macromers was confirmed bythe Fourier-transform infrared (FTIR), nuclear magnetic reso-nance (NMR), and differential scanning calorimetry (DSC). Addi-tionally, the molecular weight and its distribution were deter-mined by gel permeation chromatography. Next, theeffectiveness of PCLI and PCLF coatings on mechanical proper-ties enhancement of HA scaffold, prepared by the sacrificial tem-plate method, was investigated. To achieve a good interfacial

adhesion between the organic polymer and inorganic HA scaf-fold in a composite, HA scaffolds were pre-coated with a silanecoupling agent c-methacryloxypropyltrimethoxy, [CH2¼¼C(CH3)C(O)O(CH2)3ASi(OCH3)3] aiming a good bonding betweentwo phases. The cytotoxicity of the macromers and their corre-sponding composites were also evaluated by MTTassay.

MATERIALS AND METHODS

MaterialsPCL diol (Mn of 530, 1250, 2000 g mol�1), itaconyl chloride(IC), fumaryl chloride (FC), propylene oxide (PO), benzoyl per-oxide (BPO), diammonium hydrogen phosphate ((NH4)2PO4),calcium nitrate (Ca(NO3)2), N-Vinylpyrrolidone (NVP), penicil-lin, 3-(Methacryloyloxy)propyl]trimethoxysilane (A174), andstreptomycin were purchased from Aldrich Chemical Corp.Methylene chloride (DCM) was obtained from Merck (Ger-many). Prior to reaction, a certain amount of PCL diol wasdried overnight under a vacuum at 50�C. Prior to use, DCM(Merck) was dried over the calcium hydride. Itaconyl chlorideand fumaryl chloride were purified by distillation at 90 and160�C, respectively. All other chemical reagents and solventswere analytical grade and were used as received.

Synthesis of PCLI and PCLFScheme 1 illustrates the sequential reaction steps for thesynthesis of PCLI and PCLF. To synthesis PCLI, PCL diol, ita-conyl chloride, and PO were reacted in 1:0.99:2 molar ratios.In a typical synthesis, 10 g of PCL diol 530 was dissolved in100 mL dried DCM and then 2.18 g PO was added to this so-lution. IC (3.11 g) was dissolved in 25 mL of anhydrous DCMand was then added drop-wise to the stirred solution undernitrogen atmosphere, using a reflux condenser at the ambienttemperature. After the addition of itaconyl chloride solutionto PCL diol solution, the mixture was stirred at the ambienttemperature for an additional 24 h. Upon completion of thisreaction, the solution was washed several times with waterto eliminate residual PO and chlorinated PO. After solvent

SCHEME 1. Schematic of PCLI and PCLF synthesis.

258 SHARIFI ET AL. HA SCAFFOLDS INFILTRATED WITH THERMALLY CROSSLINKED PCLF AND PCLI

removal through rotoevaporation, the synthesized PCLI wasobtained. The polymer was then dried in a vacuum oven at30�C for 12 h and then stored at �20�C until being used.The same procedure was applied to synthesize PCLI fromPCL diol 1250 and 2000 g mol�1. PCLFs were also synthe-sized according to the method reported by Sharifi et al.26 Inthis study, the number listed after PCLI or PCLF indicates themolecular weight of its precursor PCL diol.

HA scaffold preparationHA scaffolds were prepared using hydroxyapatite powdervia the polyurethane foam reticulate method. HA powderwas first synthesized through a chemical precipitation pro-cess via the reaction of nitrate calcium, hydrogen phosphateammonium, and ammonia, as has been described previ-ously.29 In the next step, a slurry mixture, which was laterused for coating the scarifying template, was made accord-ing to the recipe listed in Table I. To this end, all ingredientswere milled for an hour using a planetary ball mill. Follow-ing the slurry preparation, polyurethane foam templateswere replicated with the slurry mixture by a repeated dip-ping-and-drying process. The samples were then dried inthe air and the coated substrates were then heated at a rateof 0.5�C/min up to 600�C and held for an hour at this tem-perature. Then, samples were rapidly heated to 1200�C witha heating rate of 10�C/min and held for 3 h at thistemperature before being cooled in the furnace. Samplesdemonstrating a good consistency were obtained throughthis process and used for further experiments.

Functionalization of scaffold’s surfaceThe silanization of the HA scaffolds was done according tothe literature.30 In brief, 2 g of MPS was dissolved into 50 g

aqueous solution with ethanol concentration of 30 wt %.The pH value was adjusted to 3.5 using acetic acid to cata-lyze the hydrolysis of the methoxy groups of silane. HA scaf-folds were then immersed in the resultant liquid under mildstirring conditions and then heated to 50�C until the solventvaporized. The silane was finally condensed by heat treat-ment at 120�C for 1 h. The samples were then washed sev-eral times with deionized water/ethanol to removeunbounded silane.

Infiltrating of HA scaffold with crosslinked PCLIand PCLFSurface functionalized HA scaffolds were used to fabricatethe composite scaffolds. At first, infiltrating solutions wereprepared at two different concentrations of PCLI and PCLF(12.5 and 30 w/v %) by dissolving a certain amount ofPCLI and PCLF in DCM. Then, BPO (2 wt % of correspond-ing PCLI or PCLF) and NVP (5 wt % corresponding PCLI orPCLF) were added to this solution as an initiator and reac-tive diluents, respectively. To infiltrate the HA scaffolds, thesamples were immersed in 50 mL of infiltration solution atambient temperature and left for 24 h. Then, the wet blockswere transferred to a vacuum oven (0.3 atm) and were keptthere for 20 min to evaporate the solvent. The sampleswere cured for 2 h at 90�C to induce the curing reactionand then 2 h at 120�C in a forced air convection oven tocomplete the crosslinking reaction. The compositions of for-mulations are presented in Table II. The first number in thesample code indicates the molecular weight of PCL diolused for synthesis of PCLI or PCLF and the second numberstands for the concentration of macromere in the infiltrationsolution.

Characterization

Characterization of unsaturated copolymersThe chemical structure and formation of macromers wereconfirmed using FTIR and NMR spectroscopy. FTIR spectra(4000–400 cm�1) were acquired using a Bruker, Equinox55 spectrophotometer. A thin film of macromer was caston a KBr disk, and the spectrum was collected with 32scans and a resolution of 4 cm�1. 1HNMR spectra were col-lected in the ambient temperatures using a NMR apparatus

TABLE I. Composition of Slurry Used for HA Scaffolds

Preparation

Component Content (wt %)

Powder of bioceramic 45%Distilled water 43%Magnesia 4%Binder 5%Antifoaming agent 3%

TABLE II. Formulations of Infiltration Solutions

Sample Code

Concentration of Macromers

in Infiltrating Solution

(W/V%)

Concentration of

NVP in Infiltrating Solution

(W/V%)

BPO Concentration in

Infiltrating Solution

(W/V%)

HA-PCLI530-12.5 12.5 0.65 0.25HA-PCLI530-30 30 1.57 0.61HA-PCLI1250-30 30 1.57 0.61HA-PCLI2000-30 30 1.57 0.61HA-PCLF530-12.5 12.5 0.65 0.25HA-PCLF530-30 30 1.57 0.61HA-PCLF1250-30 30 1.57 0.61HA-PCLF2000-30 30 1.57 0.61HA – – –

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | AUG 2011 VOL 98A, ISSUE 2 259

(Bruker, 400 MHz, Germany). CDCl3 was used as the sol-vent and chemical shifts were given in ppm from the signalof tetramethylsilane (TMS). The pulse angle and thenumber of scans were 90� and 32, respectively. 13CNMRspectra were collected by the same apparatus and usingthe same solvent. These tests were conducted in ambienttemperature.

The GPC experiments were performed using a GPCinstrument (Agilant 1100) consisting of 10 mL PLGEl col-umn (104 A�, 103 A� , 102 A�, 500–400 kDa). Polystyrene ofa known molecular weight was used as a calibration stand-ard and reagent grade THF was used as a mobile phaseeluting at a flow rate of 1.0 mL/min. A 100 lL sample of0.1 mg/mL solution in THF, which was filtered through a0.2 lm filter prior to use, was injected for all measure-ments. The melting point (Tm), the glass transition tempera-ture (Tg), and the crystallinity of the macromers were eval-uated using a TA instrument with a 920 differentialscanning calorimeter under a nitrogen gas flow rate of 50mL/min and a heating rate of 10�C/min via heating from�100 to 100�C. First, the specimens were heated from �80to 100�C at a heating rate of 10�C/min and then quenchedrapidly to �100�C, and after 2 min, a second scan wasrecorded. The glass transition temperature, Tg, was taken asa midpoint of the heat capacity change. The melting temper-ature (Tm) and the heat of fusion (Hm) were determinedfrom the maximum endothermic peaks position and theintegration of the endothermic area. The crystallinity of themacromers was measured using similar methods, as previ-ously described in this article.31

Characterization of HA scaffoldsThe morphology of the HA scaffolds and polymer infiltratedHA scaffolds were evaluated using scanning electron micros-copy (SEM; Vega II XMU instrument Tescan, Czech Repub-lic). Scaffolds were cooled with liquid nitrogen, carefullysectioned with a razor, mounted on the aluminum stubsusing carbon tape, and then sputter coated with gold tobecome ready for SEM observation.

FTIR spectroscopy was performed to determine the sur-face chemistry. For the FTIR sample preparation, silanizedHA scaffolds, and nonsilanized HA scaffolds were pulverizedin mortar. The resultant fine powder was mixed with KBrand pressed. Then, FTIR spectra were collected usingBruker, Equinox 55 spectrophotometer. The porosity ofthese samples was determined using the Archimedes tech-nique. Samples were first saturated with water, weighed inair (W1), then submerged in water again, and finallyweighed once again (W2). The porosity was calculated usingthe following formula:

Porosityð%Þ ¼ 100� ðW2 �W1

qwÞ=V (1)

where qw is the density of water and V is the theoreticalvolume of HA scaffolds (10 � 10 � 10 mm3). The porosityof the sintered bodies was calculated as the average valueof six determinations.

Mechanical propertiesMechanical behavior was examined using a Zwick/ Roell2005 apparatus with a crosshead speed of 0.01 mm/s. Thesample with cubic geometry (10 � 10 � 10 mm3) was usedfor the test. The compressive strengths were determinedfrom mechanical test recordings. The tests were done intriplicate.

Statistical analysisStatistical comparisons were made via analysis of variances(ANOVA). The student t test was utilized to compare thedata between groups. In all statistical evaluations, p < 0.05was considered statistically significant.

In vitro cytotoxicity assayThe cytotoxicity of samples was evaluated using the dime-thylthiazol diphenyl tetrazolium bromide (MTT) assay. Inbrief, G-292 cultured cells (human primary osteogenic sar-coma cell line, National Cell Bank of Iran (NCBI), PasteurInstitute of Iran were harvested with a 0.25% tripsin-EDTAsolution (Sigma) in a phosphate-buffered saline (PBS; pH7.4) and re-suspended in the culture medium (RPMI-1640supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 mg/mL streptomycin). The cells wereseeded at a density of 1 � 104 cells/well in the 96-wellmicrotiter plates. These plates were incubated overnight at37�C in a humidified atmosphere of 5% CO2 in air. The mac-romers (PCLI and PCLF), silane coated HA scaffolds, andtheir corresponding composites (HA scaffolds infiltratedwith PCLI and PCLF) were sterilized with 70% ethanol andadded to RPMI/1640 media at a concentration of 0.2 g/mLaccording to the ISO 10993 standard. Afterwards, the mix-ture was incubated for 72 h. Subsequently, the extractionmedia was diluted with fresh RPMI-1640 containing 10%FBS in 50 v/v percent, which was then added to G-292cells. The negative controls were comprised of six G-292cultured wells with no sample [tissue culture polystyrene(TPS)] whereas latex was used as a positive control. After24 h of incubation, the extraction media were removed, thecells washed with PBS, and MTT solution (0.5 mg/mL in therespective medium) was added to each well in an amountequivalent to 10% of the culture medium. The plates wereincubated at 37�C, 95% relative humidity, and 5% CO2 for 5h. The MTT solution was then removed and an appropriateamount of DMSO/2-propanol 1:1(v/v) solution was addedto dissolve the formazone crystals. The solution in each wellwas mixed and optical densities (OD) were measured at570 nm [multiwell microplate reader (ICN, Switzerland)].The cell viability was normalized to that of cells cultured inthe culture media without any extracts.

RESULTS AND DISCUSSION

Synthesis of infiltrating polymersAs PCLF has been synthesized, fully characterized, anddiscussed previously,26,27,31 this section focuses on relayingthe results and discussion related to PCLI. Whenever it isrelevant, references to previous studies regarding to PCLFcharacteristics are incorporated into the discussion.


The FTIR spectra of PCLI and their corresponding diolare presented in Figure 1. The peak at 3448 cm�1 wasrelated to the terminal hydroxyl group of PCL. Adsorptionat 2940 and 2865 cm�1, can be attributed to CAH stretch-ing. The peak at 1734 cm�1 was caused by C¼¼O stretchingof the ester functional groups. The peak at 1106 cm�1 isresulted from asymmetrical CAOAC stretching. The newpeak in 1630 cm�1 in PCLI spectrum can be attributed toC¼¼C bonds, which indicates the introduction of an itaconategroup in the PCL backbone. The intensity of the terminalhydroxyl peak was also lower in synthesized copolymer,compared to their parent, which indicated a higher molecu-lar weight of the copolymers compared to their diols.

The introduction of itaconate in the PCL structure wasconfirmed by 1HNMR and 13CNMR spectrophotometry.Figure 2(a) illustrates the 1HNMR of the PCLI and its corre-sponding PCL diol. A dual chemical shift, with peak positionat 3.2 ppm, and two triplets, with peak positions at 5.6 and6.5 ppm, in the 1HNMR spectrum of the PCLI were attrib-uted to the protons of the methyl group of an unsaturateditaconyl group. The dual chemical shift, with peak positionat 3.2 ppm, was also attributed to the protons of the methylattached to a carbon situated next to a C¼¼C group.

The 13CNMR spectra of the PCLI and its initial diol areshown in Figure 2(b). In the spectrum of the PCLI, fivechemical shifts, with peak positions at 37, 119, 134, 167,and 174 ppm, were assigned to a parent PCL diol. The peakat 169 and 171 ppm were due to a carboxyl unit of the newester, which resulted from the esterification of a PCL diolwith itaconyl chloride. Two peaks, at 130 and 139 ppm,were assigned to the carbons of an unsaturated pendinggroup. The peak at 37 ppm was attributable to the carbonof a methylene group attached to an unsaturated itaconatependent group. The data obtained by the NMR spectroscopyalso was in accordance with the FTIR data, thus confirmingthe presence of an itaconyl group along the structure ofthe PCL.

The synthesis of PCLI was also confirmed by GPC. Mn,Mw, and PDI of parents PCL diols as well as their synthe-sized PCLI are listed in Table III. Due to the usage of poly-styrene standards, the measured molecular weights of thePCL diols were significantly higher than their nominal val-ues. As shown in Table III, the molecular weights of thePCLIs are 4.12, 2.04, and 2.03 times greater than the weightof their corresponding precursor diols. The molecularweight increment, were interpreted as an index for the reac-tion efficiency. The increase in the molecular weight of thePCLI synthesized using PCL diol 530 was significantlyhigher than other samples which is probably due to thesmaller size of the polymer chain and the higher chain mo-bility. For comparison, the molecular weights of correspond-ing PCLFs are also presented in Table III. It is worthwhile tonote that the PCLIs had a significant lower molecular weightcompared to the PCLFs that were synthesized using similarPCL diol parents. This was probably attributable to thelower reactivity of the itaconyl chloride compared to thefumaryl chloride.

The thermal properties of the PCLI macromers and theircorresponding diols are given in Table IV. According to theDSC results, for all synthesized copolymers, the Tg of thepolymer increased after copolymerization. The increase inTg was more significant for the polymer made from PCL diol530 (21%) compared to the PCL synthesized from a highermolecular weight PCL diol (10 and 6% for the PCLI synthe-sized from PCL 1250 and PCL 2000, respectively). Theincrease in Tg can be attributed to an increase in molecularweight according to the Fox-Flory equation. Furthermore,the melting point of the polymers decreased 9, 10, and 9%comparing to their corresponding diol after copoly-merization (Table IV) which was likely due to the decreasein crystallinity of the PCLI. The decrease in Tm was likelydue to the decrease in crystallinity of the PCLI. The additionof itaconyl groups to the PCL chains may have inhibited thecrystallization of the PCL; consequently, the crystallinity

FIGURE 1. FTIR spectra of synthesized PCLI. (a) PCL diol 1250. (b) PCLI 1250. [Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


decreased. In general, the introduction of an itaconate groupinto the backbone of the PCL had the same effect as havingintroduced a fumarate group, except for the fact that ita-conyl chloride was less reactive compared to fumaryl chlo-ride toward the polycaprolactone diol and that the resultingmacromer demonstrated a lesser degree of unsaturationcompared to the PCLF.

HA scaffolds structures and surface functionalizationThe macrostructure of the sintered HA scaffold was con-trolled by the porous structure of the polymer substrate.Porosity is one of the most important factors affecting themorphological properties of biomaterial’s scaffolds in boneregeneration processes. After sintering, the ceramicresembled the polymer matrix texture, giving rise to a struc-ture characterized by several macropores, whose size(>100–200 lm) could assure osteoconduction after implan-tation [Fig. 3(a)].

To increase the bondage strength of the HA surface andthe polymeric coating, the surface of the porous HA scaf-folds were functionalized using a silane coupling agent(MPS). In Figure 3(b), the FTIR spectra of the HA scaffolds

are compared before and after silanization. Two new peaks,positioned at 1650 and 2800 cm�1, were attributed to acarbonyl group (C¼¼O) and the CH3 groups of MPS, respec-tively. The peak at 1640 cm�1 was also due to the C¼¼Cbonds of the MPS.

Crosslinking of the infiltrating polymersThe uncured PCLFs or PCLIs macromers were paste-likeweak solids and could not improve the mechanical proper-ties of the HA scaffolds in this state. To be applicable as anefficient, tough coating, and reduce the brittleness of HAscaffolds, the infiltrating polymer in the HA scaffolds werecrosslinked. Although PCLF and PCLI are self-crosslinkablepolymers, to facilitate a crosslinking reaction and toimprove crosslinking efficacy, a small amount of the NVPmonomer was added. As NVP is a small molecule, it pos-sessed more mobility than the PCLI or PCLF chains andbridged two adjacent unsaturated itaconyl or fumarategroups of the PCLI or the PCLF respectively. The unsatu-rated bond of the MPS could also be coupled with NVP, aswell as an unsaturated group of the PCLI or PCLF, thus lead-ing to an improved adhesion between coating layer and

FIGURE 2. 1HNMR and 13CNMR spectra PCLI and its precursor diol. (a) 1HNMR of PCL diol530 and PCLI530. (b) 13CNMR of PCL diol530 and

PCLI530.


substrate. The gel content of all composites was determinedby a swelling study and was above 70%, thus indicating theefficiency of said crosslinking reaction.

Mechanical propertiesTable V demonstrates the compressive stress of the samplesalong with the porosity and the weight increment after infil-tration. In all samples, it was found that the compressive

strength of the infiltrated scaffolds were higher than thenoninfiltrated scaffolds. Since the composite strengthdepends on macro porosity, its disruption and pore size,polymer infiltration into microporous areas and coatingonto the struts of the HA scaffolds could have reduced thesize of any possible defect of the HA and resulted in aceramic-polymer composite struts which had increasedcompressive strengths compared to noncoated one. In all

TABLE IV. Tm, Tg, and the Crystallinity Synthesized PCLI,

PCLF, and Their Precursor Diols

Sample Tm (�C) Tg (�C) Crystallinity (%)

PCL diol 530 35.3 �78.5 35PCL diol 1250 56 �68 48PCL diol 2000 63 �64 56PCLI 530 32 �62 31PCLI 1250 50 �61 40PCLI 2000 57 �60 49PCLF 530 30 �55 30PCLF 1250 44 �50 36PCLF 2000 56 �40 44

FIGURE 2. Continued

TABLE III. Molecular Weights and Their Distribution of

Synthesized PCLI, PCLF, and Their Precursor Diols

Sample Mn (g mol�1) Mw (g mol�1) PDI

PCL diol 530 970 1570 1.62PCL diol 1250 2450 3750 1.53PCL diol 2000 4030 6480 1.6PCLI 530 4000 5500 1.3PCLI 1250 5000 7500 1.5PCLI 2000 8200 15,000 1.8PCLF 530 6000 9300 1.55PCLF 1250 21,400 30,000 1.39PCLF 2000 22,000 37,700 1.71

ORIGINAL ARTICLE


studied samples, the macroporosity of the samplesdepended on the macromer type and the thickness of theinfiltrating solution reduced between 3 and 26% after infil-tration. In samples infiltrated using 12.5 w/v % infiltrationsolutions (HA-PCLI530-12.5 and HA-PCLF530-12.5), no sig-nificant changes in the macroporosity of scaffolds wereseen. However, these samples demonstrated slight weightincrement after infiltration, thus indicating that the micro-spores and some defects in the ceramic struts were infil-trated with the polymer, which led to an increase in the

compressive strength of the coated scaffolds. The morphol-ogy of the coating was also observed with SEM for the HAscaffold coated with PCLF530 using 12.5 w/v % coatingsolution, which indicated that the ceramic part was success-fully infiltrated with the polymer (Fig. 4).

Although both samples coated with PCLF530 andPCLI530 showed higher compressive strength compared tothe neat HA scaffolds, the HA-PCLF530 composites werestronger than the HA-PCLI530 composites. One possible rea-son to explain this result could be the difference in crosslink

FIGURE 3. (a,b) SEM picture of polyurethane template and the resulting HA scaffolds. (c) FTIR spectra of HA scaffolds before and after

silanization.

TABLE V. Properties of Crosslinked PCLI and PCLF Infiltrated HA Scaffolds

Sample

Concentration of

Macromer in Solution

(W/V %)

Porosity

(By Archimedes

%)

Weight Increment

(%)

Ultimate

Compressive Stress

(MPa)

HA-PCLI530-12.5 12.5 56.5 6 3.4 4 6 0.24 8 6 0.5HA-PCLI530-30 30 54 6 2 6 6 0.78 8.7 6 0.4HA-PCLI1250-30 30 53.2 6 3 7 6 0.41 9 6 0.9HA-PCLI2000-30 30 51 6 3 10 6 0.54 10 6 1.05HA-PCLF530-12.5 12.5 57.7 6 2.8 6 6 0.21 12 6 1HA-PCLF530-30 30 52 6 1.2 15 6 0.69 16.22 6 1.5HA-PCLF1250-30 30 47 6 1.2 19 6 0.87 18 6 0.8HA-PCLF2000-30 30 44 6 4.2 25 6 0.94 30 6 1.3HA 0 60 6 2 0 7.1 6 1.6


densities, as the number of unsaturated bonds was higher inPCLF530 than PCLI530. Another reason may be due to thefact that the double bonds of the fumaric acid present in thebackbone structure were more susceptible to self-crosslink-ing or crosslinking with NVP than the double bonds of ita-conic acid present in the backbone structure under the stud-ied condition.32 By increasing the thickness of the infiltratingsolution, the compressive strength increased about 128 and22% in samples infiltrated with PCLF530 and PCLI530,respectively. However, while the relative weight increment ofthe samples increased up to 15% with an increase in the con-centration of the infiltration solution, the porosity of the scaf-folds decreased significantly (13%). In other words, asstrength is known to be influenced strongly by porosity, theimproved compressive strength demonstrated here is due toa combination effect of polymer infiltration in macroporosi-ties and microporosities and polymer coating on struts.33

Increasing the molecular weight of the PCLF and the PCLIalso significantly affected the compressive strength of theinfiltrated structures. As the infiltration solution was pre-pared by weight percentage, the solution for the PCLF2000was thicker than the PCLF1250 because the PCLF2000 had ahigher molecular weight. Consequently, the relative weightincrements for samples infiltrated with the PCLF2000 was31% higher than with the PCLF1250. The same trend wasalso observed for the HA scaffolds infiltrated with PCLI1250and PCLI2000. However, as the molecular weight of the PCLImacromers were less than PCLFs and consequently the vis-cosity of the infiltrating solutions were less, the weight incre-ment was less compared to samples infiltrated with PCLFs.Similar to the above-mentioned; the relatively high compres-sive strength of these samples can be attributed to the syner-gic effect of porosity reduction and strong polymer coating. Infact, as the chains become longer, the molecular entangle-

ments and the intermolecular forces increased as the chainsno longer slipped along one other. The difficulty in untanglingtheir chains makes polymers strong and resilient; thus, theHA scaffolds infiltrated with a higher molecular weight of thePCLFs demonstrated a higher strength compared to the PCLFwith a lower molecular weight. The same behavior was alsoobserved for HA scaffolds infiltrated with PCLI1250 andPCLI2000. As the PCLIs had lower molecular weights com-pared to PCLFs, along with possibly less reactivity of thependent itaconate groups, the networks and the infiltratingcoating were weaker than the PCLFs. Although the PCLFscoatings were stronger than the PCLIs in terms of compres-sive strength, nevertheless the PCLI coatings may be advanta-geous since itaconic acid, which is the degradation product ofthe PCLI and has a PKa of 3.85, which has less acidity powerthan lactic acid (PKa 3.08) and fumaric acid (PKa 3.03); there-fore, they may show much better long-term biocompatibilitycompared to the common polyesters like PLA.

Cell culture studiesMTT assay was performed to investigate the biocompatibil-ity of the synthesized macromers and their correspondingcomposites. There was no statistically significant differencebetween the viability of all the cells in the extraction fluidof the PCLF macromers in comparison with the negativecontrol groups at the concentration studied here (p < 0.05).The PCLI macromers also did not show any meaningful tox-icity toward G292 cell lines [Fig. 5(a)].

FIGURE 5. Cell viability of macromers and their corresponding com-

posites. (a) Macromers. (b) Polymer infiltrated HA scaffolds.

FIGURE 4. SEM picture of infiltrated HA scaffolds PCLF530 (12.5 w/v

% in DCM was used for HA scaffolds infiltration).

ORIGINAL ARTICLE


According to the MTT results, PCLF infiltrated macromerswere also nontoxic towards cells. At all molecular weights,however, the HA scaffolds infiltrated with thinner infiltratingsolution (12.5 w/v %) showed slightly higher proliferationrates compared to the scaffolds infiltrated with the thickerPCLF solutions [Fig. 5(b)]. This behavior can be attributed tothe presence of more free HA areas in composites infiltratedwith thinner polymer solution. The effect of HA on increasingthe cell proliferation has been reported by other researchers.For instance, Wang et al.25 showed that by increasing the HAcontent in crosslinked PCLF530/HA nanocomposites, theadsorption of extracellular matrix proteins from the culturemedium increased, and consequently, cell attachment andproliferation increased strongly. Similarly, some studies havedemonstrated that the calcium concentration in an extracellu-lar environment plays an important role in increasing theproliferation and differentiation of osteoblastic cells by mem-brane-mediated ionic transfer.34,35

The HA scaffolds infiltrated with PCLI, however, demon-strated a slightly different cellular response compared to thePCLF-coated HA scaffolds. Although the HA scaffolds thatwere infiltrated with a thinner solution (HA-PCLI530-12.5)showed an increase in cell proliferation rate due to the reasondiscussed above, samples coated with PCLI1250 andPCLI2000, (HA-PCLI1250-30 and HA-PCLI2000-30) exhibiteda lower cytocompatibility compared to their correspondingcomposites. As PCLI itself is biocompatible, this behavior canbe attributed to the trace amounts of low molecular weightoligomers of the NVP, which might have not been involved inthe crosslinking reaction. Having used the as-prepared sam-ples for the cytotoxicity assessment, the trace amount of NVPcould have leached out from the samples and caused thelower biocompatibility response compared to that of theircomposites. Since the molecular weight between crosslinks(Mc) in PCLI530 was less than PCLI1250 and PCLI2000, thenetwork was denser, and consequently, the release of a possi-ble unreacted oligomer was harder compared with PCLI1250and PCLI2000. Hence, the HA scaffolds infiltrated withPCLI1250 and PCLI2000 showed lower cytocompatibilitycompared to those infiltrated with PCLI530. As the degree ofoligomerization and the resultant number of unsaturatedbonds were higher in the PCLFs compared to the PCLIs, thenetworks that were prepared using the PCLF had highercrosslink densities, and consequently, the probable leach outof unreacted oligomers was retarded. Therefore, PCLF compo-sites did not show any significant cytotoxicity. This was incon-sistent with our previous study, in which we showed that in acrosslinked PCLF formulation with 10 wt % NVP, the sol partdid not contain any unreacted NVP.36

CONCLUSION

In this study, porous HA scaffolds were modified through anin situ crosslinking reaction of two unsaturated copolymersof the PCL to form organic networks, which infiltrated intothe porous inorganic structure. The PCL diol with three mo-lecular weights was copolymerized with fumaryl chlorideand itaconyl chloride to yield unsaturated copolymers. Theincorporation of the itaconate and fumarate groups into the

backbones of polymers was confirmed by NMR and FTIRspectroscopy. The esterification reaction of the PCL diolswith the fumarate group was more efficient than itaconategroup as the resulting macromers synthesized from the PCLdiol 530, 1250, and 2000 had a degree of substitution 1.2,4.2, and 2.7 times higher than the PCLIs. The compressivestrength of the modified HA scaffolds were influenced bythe concentration of the coating solution, molecular weightof the infiltrating polymer, and type of the macromer. Ingeneral, the compressive strength of the coated scaffoldswas increased about 14–328%. In all samples studiedherein, the PCLF-coated HA scaffold had a significantlyhigher compressive strength than the PCLI-coated scaffolds.According to in vitro cytotoxicity evaluations, neither themacromers nor the infiltrated HA scaffolds showed anyadverse cytotoxicity.

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ORIGINAL ARTICLE

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Hydroxyapatite scaffolds infiltrated with thermally crosslinked polycaprolactone fumarate and polycaprolactone itaconate