Cellular Integration and Vascularisation Promoted by a Resorbable, Particulate-Leached, Cross-Linked Poly(ε-caprolactone) Scaffold

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Cellular Integration and VascularisationPromoted by a Resorbable, Particulate-Leached,Cross-Linked Poly(e-caprolactone) Scaffold

Simon C. Baker, Geraldine Rohman, Jennifer Hinley, Jens Stahlschmidt,Neil R. Cameron, Jennifer Southgate*

Flexible, strong scaffolds were created by crosslinking PCL with 1,6-hexamethylenediisocya-nate, using paraffin beads as a porogen. Particulate leaching generated homogeneous scaffoldswith interconnected spherical pores of 5–200mm. Subcutaneous implantation in rats for3 months resulted in minimal scaffold resorption and anon-inflammatory regenerative host response, withcomplete infiltration by alternatively-activated CD68þ

macrophages. In addition, scaffolds were populatedextensively along microfractures by a stromal matrix,which was highly vascularised and contained a subset ofstromal cells that expressed the anti-inflammatoryCD163 antigen. Such microfractures may be an import-ant physical feature for directing stromal integration andvascularisation events.

Introduction

Tailoring the properties of synthetic polymers to produce

scaffolds that meet the physical requirements for soft tissue

regeneration is a particular challenge. The mechanical

properties of hydroxy-terminated polyester oligomers may

be improved by crosslinking with multifunctional isocya-

nates to produce polymers held together by urethane

S. C. Baker, J. Hinley, J. SouthgateJack Birch Unit of Molecular Carcinogenesis, Department ofBiology, University of York, York YO10 5DD, UKFax: þ44 190 432 8704; E-mail: [email protected]. Rohman, N. R. CameronBiophysical Sciences Institute, and Department of Chemistry,Durham University, Durham DH1 3LE, UKJ. StahlschmidtDepartment of Histopathology, Leeds Institute of MolecularMedicine, St James’s University Hospital, Leeds LS9 7TF, UK

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linkages. For example, a ‘soft’, amorphous PCL diol, a ‘hard’

crystalline poly(hydroxybutyric acid) diol and a diisocya-

nate crosslinker have been combined to produce a polymer

scaffold using a freeze/immersion/precipitation process.[1]

Another report described a thermally-induced phase separa-

tion process, where the diisocyanate used was assimilated as

the ‘hard’ segment and the poly(ethylene glycol) block

present in the PCL diol oligomer formed the ‘soft’ segment.[2]

However, both of these methods led to scaffolds with poorly-

defined porosity. Guelcher and coworkers reported prepara-

tion of injectable crosslinked polyurethane foams produced

by gas blowing using a diisocyanate, PCL triol oligomers and

a fatty acid pore opener; however pores were large (100–

1000mm) and showed both limited reproducibility and

interconnectivity.[3] Scaffold pore size can have profound

effects on cell behaviour and in studies of osteogenic cells on

gelatin scaffolds, attachment was decreased when voids

were increased from�95 to 150mm.[4] Thus, there remains a

need to develop crosslinked polyester scaffolds with a

library.com DOI: 10.1002/mabi.201000415

Cellular Integration and Vascularisation Promoted by a Resorbable . . .

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reproducible, well-defined, interconnected porosity in the

10–100mm range.

The particulate templating approach is ideally suited to

producing materials of well-defined porosity and involves

curing the polymer around a particulate slurry, which is

then removed by leaching. This approach has been

described previously for non-crosslinked materials.[5–7] In

this study, scaffolds were prepared by reaction of the

hydroxyl groups of PCL triol oligomers with 1,6-hexa-

methylenediisocyanate (HMDI) as a crosslinker, in the

presence of paraffin beads as a particulate porogen, which

were subsequently removed by solvent leaching. We show

that this approach allows preparation of scaffolds with

controlled mechanical properties, well-defined porosity

and definable pore diameter.

Experimental Part

Preparation of Paraffin Beads

Paraffin beads were prepared as described elsewhere.[6] Briefly,

10 g of melted paraffin (melting point: 58–62 8C, ASTM D87, Sigma)

was poured into a 62 8C, 350 mL, aqueous poly(vinyl alcohol) (PVA)

solution (0.35% w/v, 88% hydrolysed PVA, Mw ¼22 000 g �mol�1;

Acros) and stirred for 1 h at 62 8C. The suspension was poured

immediately into 1 L of ice water to solidify the paraffin

micro-drops. Micro-spheres (5–120mm) were selected using a sieve

tower, washed with distilled water and dried at ambient

temperature.

Preparation of Porous Scaffolds

Scaffolds were prepared by a particulate-leaching method under

aseptic conditions. In a typical synthesis, 0.4 g of paraffin beads was

added to a polytetrafluoroethylene (PTFE) mould, compacted and

transferred to a 37 8C oven for 30 min to sinter. 0.1 g PCL triol

oligomer (Mn ¼ 900 g �mol�1, Sigma) was dissolved in 0.3 mL of

dichloromethane (Fisher) before addition of 30mL HMDI (Sigma)

and 10mL dibutyltin dilaurate (Sigma) under a nitrogen atmo-

sphere. The paraffin sphere assembly was cooled to ambient

temperature and the polymer mixture added dropwise. After the

solution had infiltrated completely, pressure was applied for 24 h to

help remove trapped air.[6] Scaffolds were air-dried in the mould for

5 h then removed and the paraffin was extracted by immersing the

scaffold in hexane/dichloromethane (50 vol.-%) for 48 h at ambient

temperature, changing the solvent every 12 h. Next, the porous

scaffold was immersed for 3 h in 100% hexane and air-dried. The

scaffold soluble fraction was determined from the mass percentage

of extractable material. Scaffold structure was varied by changing

the mass of the paraffin sphere content.

Scanning Electron Microscopy (SEM) and Image

Analysis

Fractured scaffold segments were sputter-coated with 7 nm of

chromium. At least five SEM images (Hitachi S-5200) were used to

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Macromol. Biosci. 201

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plot pore frequency versus diameter as analysed using Image J

software (NIH image). Void diameters were corrected statistically

for the underestimation of size that occurs using this method of

measurement. This was achieved by evaluating the average of the

ratio R/r, where R is the actual equatorial void diameter and r is the

diameter value measured from the micrograph. The statistical

factor was calculated from

1, 11, 6

H & Co

h2 ¼ R2 � r2 (1)

The probability that the sectioning takes place at any distance (h)

from the centre is the same for all values of h, so the average

probability value of h is R/2. Replacing this value in Equation (1)

gives

R

r¼ 2

3

� �1=2

(2)

Multiplication of the observed value of the void diameter by this

factor generated a more accurate pore diameter value.

Mercury Intrusion Porosimetry (MIP)

MIP was performed using a Micromeritics AutoPore III 9420.[8]

Intrusion and extrusion mercury contact angles of 1308 were used

and penetrometers had a stem volume of 1.836 mL and a bulb

volume of 5 mL. The intrusion volume always comprised between

45 and 80% of the stem volume. Intrusion pressures for the

polyester scaffolds never exceeded 40 psi.

Density and Porosity Measurements

The porosity of scaffolds was calculated on the basis of their

apparent density r and bulk density r0 according to

Porosity ð%Þ ¼ 1� r

r0

� �� 100 (3)

r values were derived from the dimensions and weights of

scaffolds. Non-porous PCL-XL polymer and paraffin porogen had

r0 values of 1.011 and 0.900 g � cm�3, respectively. Triplicate

measurements were carried out and used to derive mean values.

The porosity of scaffolds with open-cell structures was

estimated using Equation (4) where rp, rs and fw represent the

density of polymer, density of porogen and the paraffin weight

fraction, respectively,[7]

Theoretical porosity ð%Þ ¼ fw=rs

fw=rs þ ð1�fwÞ=rp

(4)

Dynamic Mechanical Analysis (DMA)

The storage and loss moduli were measured for each scaffold type at

37 8C using a dynamic mechanical analyser (DMA Q800 V7.3 Build

119). Round discs of �1 mm thickness and 10 mm diameter were

used in all experiments. Triplicate samples were mounted in a

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S. C. Baker, G. Rohman, J. Hinley, J. Stahlschmidt, N. R. Cameron, J. Southgate

compression clamp and 20 min after the temperature reached a

steady state, a fixed oscillation frequency of 1 Hz was applied and

analysed four times per scaffold.

Differential Scanning Calorimetry

DSC analyses were carried out with a Pyris 1 calorimeter under a

nitrogen atmosphere. Samples were scanned twice from �90 to

þ150 8C. The heating rate was 10 8C �min�1 and the second run was

recorded after quenching. The glass transition temperature (Tg) was

determined at the mid-point from the second scan.

Attenuated Total Reflection Fourier-Transform

Infrared (ATR-FTIR) Spectroscopy

ATR-FTIR spectra of scaffold samples were generated using a

Thermo Nicolet Nexus FTIR spectrometer controlled by OMNIC

software Version 5.1. The specimen was mounted on a SMART

OMNIC sampler connected to the FTIR Nexus and spectra were

obtained by accumulating 32 scans in the range 600–4 000 cm�1

with a resolution of 4 cm�1.

In vitro Degradation Study

Polyester scaffolds were sterilised in 70 vol.-% ethanol solution for

3 h, before extensive washing in sterile distilled water and

transfer to cell culture medium (Dulbecco’s modified Eagle’s

medium, DMEM) without antibiotics. The scaffold sections were

immersed in 10 mL medium and incubated at 37 8C in a humidified

atmosphere of 5% CO2 in air; pH was monitored periodically. After

7 d, the samples were washed extensively in distilled water and

air-dried to a constant mass. Any mass loss was determined

by calculation from the initial mass of the scaffolds and their

residual mass after drying. Additionally, scaffolds were analysed

by ATR-FTIR and SEM.

Cell Culture

Finite stromal cell lines were established from surgical samples of

human urinary tract and maintained as described in detail

elsewhere.[9] The growth medium consisted of DMEM containing

5 vol.-% fetal bovine serum. Cell cultures were maintained at 37 8Cin a 90% humidified atmosphere of 10% CO2 in air. Cells were

passaged at confluence using a standard trypsin and versene

protocol. Scaffolds of 1 mm diameter and 10 mm thicknesses

were sterilised in 70 vol.-% ethanol for 1 h with five washes in

sterile phosphate-buffered saline (PBS) and equilibration in growth

medium before use in cell culture. For cell seeding, excess liquid was

removed from scaffolds using a sterile filter paper wick and

scaffolds were suspended on a stainless steel ring (8 mm internal

diameter) to prevent capillary action drawing the cell suspension

through the scaffolds and onto the substrate below. 20mL of cell

suspension containing 105 cells was pipetted onto the upper

surface and left for 2 h to allow cell attachment, before flooding

with medium. Seeded scaffolds were maintained submerged in

growth medium in 6-well plates (bacterial grade plastic), for up to 7



d, with the medium replenished every 2–3 d. Six replicate scaffolds

were analysed at each time point.

Modified MTT Assay

A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay was performed as described in detail

elsewhere.[10] MTT (2 mL, 0.5 mg �mL�1) was added to each well

of a 24-well plate containing six replicate scaffold cultures and left

to incubate for 4 h at 37 8C. Scaffolds were air-dried thoroughly on

filter paper and placed in glass vials. 800mL of dimethyl sulfoxide

was added to the scaffolds and vortexed to dissolve the formazan

crystals. Absorbance was measured in a glass cuvette at 570 nm

using a Jasco V560 spectrophotometer. Scaffolds maintained in

culture without cells and analysed as above were used for

background subtraction.

Indirect Immunofluorescence Microscopy

Scaffolds were fixed in zinc salts (0.1 M Tris, 0.05% w/v calcium

acetate, 0.5% w/v zinc acetate and 0.05% w/v zinc chloride at

pH¼ 7.4) for 1 h, dehydrated through several changes of ethanol

and embedded in polyester wax at 37 8C (VWR, UK). 8mm sections

were floated onto Superfrost slides (VWR) and dewaxed in ethanol.

Fluorescein isothiocyanate (FITC-) tagged phalloidin was applied to

sections for 1 h. Sections were washed four times in TBS containing

0.25% Tween 20, with 0.1 mg �mL�1 Hoechst 33258 (Sigma) added

to the penultimate wash to visualise nuclei. To prevent photo-

bleaching, slides were mounted in 0.1% w/v p-phenylenediami-

nodihydrochloride (Sigma) in 90% glycerol adjusted to pH¼ 8.0 and

examined by epifluorescence and bright-field illumination on an

Olympus BX60 microscope. Images were acquired digitally and

combined using Image Pro Plus software (Media Cybernetics,

Wokingham, UK).

In vivo Studies and Immunohistological Analysis

Scaffolds of �1 mm in thickness and 10 mm in diameter were

sterilised in 70 vol.-% ethanol for 1 h, before extensive washing

in multiple changes of sterile PBS. Work was approved by the

University of Durham Ethical Review Committee and was carried

out in a designated establishment using standard operating

procedures by trained personnel under a project licence issued

by the UK Home Office. Sterile scaffolds were implanted into the

ventral subcutaneous pocket between the skin and underlying

muscle of the body wall of Piebald-Viral-Glaxo (PVG) rats. Animals

were sacrificed after 3 months, at which time scaffolds were excised

by dissection to maintain the surrounding tissue intact and fixed in

zinc salts. Fixed scaffolds were dehydrated through several changes

of ethanol and embedded in polyester wax (VWR) at 37 8C. Samples

were sectioned at 5mm, floated onto Superfrost slides (VWR) and

dewaxed.

For immunoperoxidase labelling, endogenous avidin and biotin

binding sites were blocked (Vector), sections were labelled with

primary antibodies (Table 1) and following washing, bound antibody

was immunodetected using an Animal Research Kit according to

manufacturer’s instructions (DAKO, Ely). The peroxidase reaction

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Table 1. Details of primary antibodies used to label proteinsexpressed by cells within scaffolds.

Antigen Antibody Host Supplier

CD2 OX-34 Mouse AbD Serotec

CD3 CT-CD3 Mouse Caltag

CD68 ED1 Mouse AbD Serotec

CD163 ED2 Mouse AbD Serotec

Collagen Type I COL1 Mouse Sigma

Fibronectin L9 Rabbit Made in house

Smooth Muscle Actin 1A4 Mouse Sigma

Vimentin V9 Mouse DAKOFigure 1. Normalised ATR-FTIR spectra of PCL-XL scaffoldsobtained by the particulate-leaching method and prepared with75 wt.-% of paraffin beads (diameter 5–120mm). (a) Scaffoldbefore paraffin extraction; (b) Porous scaffold after paraffinextraction; (c) Porous scaffold after incubation for 7 d in 10 mLof growth medium at 37 8C in a humidified atmosphere of 5% CO2in air. Scaffolds were pre-sterilised for 3 h in 70 vol.-% ethanolsolution. The arrow indicates a band characteristic of paraffinwax.


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was developed using diaminobenzidine (Sigma) substrate and

sections were counterstained in haematoxylin and mounted in

Faramount (DAKO). Relevant negative and positive controls were

included in all experiments as specificity and method controls.

Results

Preparation of Scaffolds

Paraffin beads (5–120mm diameter and 53–91 wt.-% weight

fraction) were used to produce PCL-XL scaffolds; the

quantity of soluble porogen extracted with hexane/

dichloromethane is reported in Table 2. Taking into account

the soluble fraction from non-porous PCL-XL (sample A in

Table 2), the extractable material wt.-% was very close to

the initial paraffin bead weight fraction in every case,

indicating efficient paraffin extraction. This was supported

by the loss of the characteristic 720 cm�1 band associated

with paraffin in the ATR-FTIR spectrum (Figure 1), and the

absence of a 60 8C paraffin melting peak in the DSC.

After extraction, all scaffolds exhibited glass transition

temperatures of �30 8C with no melting temperature,

Table 2. Characterisation of scaffolds with increasing paraffin weigh

Scaffold fw

(paraffin)

Solution

fractiona)

App

dens

wt.-% wt.-% g�c

A (non-porous) 0 5.2 1.0

B 53 58.1 0.4

C 75 78.5 0.2

D 83 87.8 0.1

E 91 94.9 0.1

a)Solution fraction was calculated as the mass percentage of extracta

(%)¼ (1�r/r0)�100 with r0 ¼1.011g � cm�1 for non-porous PCL-XL n

compression clamping, ND¼not determined.




whilst the softening point of the PCL triol precursor was

30 8C.

Characterisation of Scaffolds

SEM showed spherical pores (Figure 2) whose walls became

thinner and interconnectivity increased as the paraffin

bead weight fraction was raised from 53 to 75 wt.-%. From

75 to 91 wt.-%, the distribution shifted towards larger pore

size and smaller interconnecting holes; the SEM analysis

was confirmed by the MIP data (Figure 3). The average pore

size was smaller than the mean paraffin bead diameter, but

the distributions showed a higher variance (5–200mm,

Figure 3).

The theoretical porosity was strongly determined by the

paraffin weight fraction [Equation (4)] and was close to

t fraction following porogen extraction.

arent

ity rb)Porosityc) Storage

modulusd)Loss

modulusd)

m–3 % MPa MPa

11 0 1.25� 0.10 0.025� 0.01

34 57.1 ND ND

46 75.7 0.41� 0.02 0.042� 0.01

76 82.6 0.40� 0.02 0.071� 0.01

10 89.1 0.35� 0.02 0.074� 0.01

bles after extraction; b)r¼mporous material/Vporous material;c)Porosity

etworks (scaffold A); d)Moduli at 37 8C determined by DMA using

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Figure 2. Scanning electron microscopy micrographs of cross-sections of porous PCL-XL scaffolds obtained by the particulate-leachingmethod and prepared with various weight fractions (ww) of paraffin beads (diameter 5–120mm).

Figure 3. Paraffin bead size distribution and pore size distributions of porous PCL-XL scaffolds obtained by the particulate-leaching methodand prepared with various weight fractions (ww) of paraffin beads. The pore size distributions were determined by image analysis of SEMmicrographs and by mercury intrusion porosimetry. Data for 53 wt.-% porogen scaffold were not recorded because this material wasdeemed to have too low a degree of interconnection to be useful.

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Figure 4. Porosity of porous particulate-leached PCL-XL scaffoldsobtained by the particulate-leaching method versus weight frac-tion of paraffin porogen (ww). Theoretical porosity line wascalculated from Equation (4) and experimental data weremeasured with the mass and volume method according toEquation (3).


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linear throughout the tested range. Experimental scaffold

porosity values closely matched the theoretical (Figure 4),

ranging from 57.1 to 89.1% (Table 2), except at the lowest

porogen levels (53 wt.-%), where the MIP values were

Figure 5. The in vitro biological performance of particulate-leached PCstained scaffold showing overall cell attachment, predominantly to thVery little cell migration into the bulk of the material was observed. (B)stromal cells on the PCL-XL scaffolds. (C and D) Magnified images of ceand Hoechst 33258 show cells make little contact with the scaffold




slightly higher than the theoretical value (Figure 4). This

was probably due to additional pores formed by greater

evaporative phase separation at high polymer content.

The storage moduli (E0) for non-porous scaffolds were

�1.25 MPa and the loss moduli (E00)�0.025 MPa (Table 2). As

expected, E0 decreased with increasing porosity (0.35–

0.41MPa) whilst E00 increased (0.042–0.047 MPa, Table 2).

In vitro Degradation of Scaffolds

Scaffolds incubated under sterile conditions at 37 8C in

medium without antibiotics for seven d showed the lack of

any measurable loss of mass, even though 37 8C is above

the glass transition for PCL. Equally, there was no pH

change or colour variation in the medium which would have

indicated acid hydrolysis and/or microbiological infection.

The absence of degradation was confirmed by no observed

change in the ATR-FTIR spectrum (Figure 1). Furthermore,

image analysis after the incubation period revealed no

macroscopic pore size variation (data not shown).

In vitro Study

Staining of nuclei with Hoechst 33258 revealed colonies of

several cells thick on the surface of the scaffolds (indicated

with arrows in Figure 5A). MTT analysis indicated that

L-XL scaffolds was assessed over a 7 d time course. (A) Hoechst 33258-e material surface and within microfractures inside the material core.MTT assays illustrate the lack of proliferation by human urinary tract

lls within scaffold microfractures stained with FITC-tagged phalloidinmaterial and do not display any alignment of actin networks.

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stromal cells attached to the scaffolds

but showed little or no proliferation

(Figure 5B). There was also very little

cellular migration into the scaffolds apart

from along occasional fracture lines

within the scaffold (areas boxed in

Figure 5A and expanded in Figure 5C

and 5D). Phalloidin labelling revealed

little organisation of actin micro-

filaments into stress fibres within cells

and no alignment of cytoskeletal net-

works between adjacent cells (Figure 5C

and 5D). Additionally it was observed

that cells growing within the scaffold

showed limited interaction with the

material at small contact points and

preferentially attached to other cells

(Figure 5C and 5D).

Figure 6. Particulate-leached PCL-XL scaffolds were implanted subcutaneously into ratsfor 3 months and subsequently assessed macroscopically (A). van Gieson staininghighlighted small calibre vessels within the paucicellular capsule (arrows, B) andnew tissue structure including vascularisation inside the scaffold (C). White scalebar (top left) represents 1 cm and applies only to ’A’. Black scale bars denote 500mm.

In vivo Study

At sacrifice, no change in the weight of

rats was noted and the skin overlying the

implants was unremarkable, with scaf-

folds showing no gross reduction in size (Figure 6A).

Histologically, scaffolds were found embedded within the

striated muscle of the rat body wall, surrounded by a

paucicellular connective tissue sheath �300mm thick and

containing small calibre vessels, concentrated towards the

implant interface (Figure 6B). The core of the scaffolds was

completely cellularised, but whereas some regions had

retained the pore structure of the biomaterial, in other

regions it had been replaced by a highly vascularised stroma

(Figure 6C) consisting of dispersed cells embedded within a

regenerative matrix of collagen type I and fibronectin

(Figure 7).

CD68 was used to identify macrophages, which

were found in direct apposition to the surface of the

Figure 7. Particulate-leached PCL-XL scaffolds were implanted subcutaneously into ratsfor 3 months and subsequently labelled by immunoperoxidase for collagen type I andfibronectin. Black scale bar (bottom right) denotes 500mm and applies to both images inthe panel.

biomaterial pores (Figure 8). Some

fusion of CD68þ macrophages into

foreign body giant cells following

‘frustrated phagocytosis’ was observed

(Figure 8).

Immunolabelling for smooth muscle

actin (SMA) indicated an absence

of myofibroblasts from the stromal

tissue and highlighted extensive

vascularisation within the stroma-

forming regions of the scaffold;

such vessels were absent from

macrophage-rich regions of the

scaffold (Figure 9). Vimentin was simi-

larly distributed to SMA, and together

identified the formation of blood-filled



vessels within the constructs (Figure 9). There was

no evidence of inflammation, with no lymphocytes

detected (Figure 10).

CD163 has previously been reported to identify

macrophages of an alternatively-activated (M2) pheno-

type as distinct from the classically-activated (M1)

state.[11–13] Within the surrounding connective tissue,

CD68þ cells were rare, whereas CD163þ cells were

diffuse but unequivocal. By contrast, stromal regions

in the scaffold core contained a dispersed CD163þ

population within the stroma; these cells were

mutually-exclusive from the biomaterial-associated

CD68þ cells, as demonstrated by the labelling of serial

sections (Figure 8).

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Figure 8. Serial 5mm sections were taken of particulate-leached PCL-XL scaffolds3 months post implantation in rats to investigate the potential expression of CD163by CD68þ macrophages. The serial sections were labelled with antibodies against CD68or CD163 and micrographs were taken of identical areas of the serial sections in oneCD68-rich area (top) and a distinct CD163-rich region (bottom). The micrographs showthat none of the CD68þ cells in the CD68-rich area express CD163. Furthermore, in aregion rich in CD163 there was no evidence of CD68 expression in CD163þ cells. Blackscale bar (bottom right) denotes 500mm and applies to all images in the panel.


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Discussion

Regenerative medicine has driven the need for biomaterials

with a range of different properties, including the balance

between structural integrity and biodegradability, and the

ability to promote tissue integration. Here we describe a

novel biomaterial that both retains structural integrity and

shows evidence of harnessing a tissue integrative response.

Hydroxyl group crosslinking of PCL oligomers and

particulate-leaching produced tunable scaffolds with

Figure 9. Particulate-leached PCL-XL scaffolds were implanted subcutaneously into ratsfor 3 months and subsequently labelled by immunoperoxidase for smooth muscle actinand vimentin. Black scale bar (bottom right) denotes 500mm and applies to both imagesin the panel.



� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhe

well-defined pores, wherein the wall

thinness and intervoid connectivity

increased with the paraffin weight frac-

tion. A porogen content of 75 wt.-% was

taken as the minimum threshold for the

production of scaffolds with an appro-

priate degree of interconnection. Our

previous studies have indicated that

biomimicry of mechanical properties

can enhance tissue-specific cell prolifera-

tion[10,14] and the elastic modulus of the

PCL-XL scaffolds described here is a

further step towards generating scaffolds

for soft-tissue applications.

The PCL-XL biomaterial was not found

to be suitable for in vitro tissue engineer-

ing applications; there was little cellular

infiltration and cell attachment was

more than fivefold less efficient than on

emulsion-templated PCL scaffolds, which

were evaluated in parallel but reported

elsewhere.[10] Poor in vitro performance

is commonly considered grounds for

halting development of a new bio-

material; however, in this study parallel

in vitro and in vivo investigations were

performed, with scaffolds that were

implanted subcutaneously revealing

the more promising results.

The invasion of cells into PCL-XL scaffolds appeared to be

facilitated by micro-fractures in the structure, which

probably formed as the PCL was cured during material

synthesis. Thus in vitro, the few stromal cells that were

observed within the scaffolds were all located within such

faults in the material and in vivo, the highly vascularised

stromal matrix appeared to have developed along such

fractures towards the core of the material. Although not

originally designed as a feature, these fracture channels

may indicate an exploitable approach

for directing specific cellularisation and

vascularisation events within a bio-

material core.

The macrophage is considered to be a

major determinant of the host response,

although the precise relationships

between biomaterial properties, modu-

lation of the macrophage-activated

phenotype and outcome of the host/

biomaterial interaction are poorly under-

stood. In this case macrophages had

completely infiltrated and lined the

pores of the biomaterial and showed

some evidence of giant cell formation

through the process of frustrated

im625

Figure 10. Particulate-leached PCL-XL scaffolds were implanted subcutaneously into ratsfor 3 months and subsequently labelled by immunoperoxidase for CD2 and CD3. A lackof CD2þ and CD3þ cells suggests limited recruitment of lymphocytes. Black scale bar(bottom right) denotes 500mm and applies to both images in the panel.

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phagocytosis. Generally, such a response is associated with

chronic inflammation leading to eventual isolation of the

implant by encapsulation. However, in this case there was a

complete absence of lymphocyte or other inflammatory

cells, suggesting that the CD68þ macrophages were

alternatively-activated. In addition, the capsule sheath

was sparse and the centre of the implant showed evidence

of tissue remodelling and integration, with major vascu-

larisation and stromal tissue formation.

CD163 is a scavenger haemoglobin/haptoglobin recep-

tor, expression of which is regulated by pro- and anti-

inflammatory mediators, which down- and up-regulate

expression respectively.[15] CD163 stimulation leads to

generation of anti-inflammatory products[16,17] and has

been used as a marker of an alternatively-activated

macrophage phenotype associated with natural biomater-

ials.[11–13] Whilst the expression of CD163 has to-date only

been reported on cells of the monocyte/macrophage

lineage (reviewed[18]), the evidence presented here indi-

cates that broader expression of CD163 may be found in

association with stromal integration of a synthetic scaffold.

In this study, CD68þ macrophages were never CD163þ,

whereas CD163 was expressed by a mutually-exclusive

population of elongated CD68– cells amongst infiltrating

stromal cells within the scaffold and in the surrounding

connective tissues. Given the association of CD163 with

wound-healing and angiogenesis,[19] its expression by cells

in and around the implant may have contributed to a non-

inflammatory regenerative response.

In vitro studies have suggested that in the context of

biomaterials, macrophages may not achieve a canonical

alternatively-activated state, but continue to secrete

chemokines associated with classical-activation and lead-

ing to the conclusion that a unique ‘biomaterial activation

chemokine profile’ may exist.[20] Interpreting the findings

of the present study in the light of the natural biomaterial

literature[11–13] suggests that depending on their precise

chemical/topographical properties, different biomaterials


� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinhe

may elicit a macrophage activation

profile unique to each material.

The novel material described here sup-

ported a non-inflammatory tissue inte-

grative/remodelling response, including

extensive vascularisation when implanted

in rats. If reproduced in man, this could

indicate the use of these scaffolds for a

number of applications. The authors ori-

ginally studied these materials with a view

to their application in urinary tract recon-

struction, but these scaffolds have proper-

ties that could be useful as a patching/

bulking support structure in general/

reconstructive surgery, including hernia

and fistula repair.

Conclusion

The particulate-leached crosslinked PCL foam described has

mechanical properties that make it a candidate for soft tissue

regenerative applications. The evidence from the in vivo

study indicates that the material harnesses a non-inflam-

matory remodelling response in the host that serves to drive

a functional, vascularised tissue regenerative response.

Abbreviations

ATR-FTIR A
ttenuated total reflection Fourier-transform
infrared spectroscopy

DMA D
ynamic mechanical analysis
DMEM D
ulbecco’s modified Eagle’s medium
DSC D
ifferential scanning calorimetry
HMDI 1
,6-hexamethylenediisocyanate
MIP M
ercury intrusion porosimetry
PCL P
oly(e-caprolactone)
PCL-XL C
rosslinked PCL
PTFE P
olytetrafluoroethylene
PVA P
oly(vinyl alcohol)
SEM S
canning electron microscopy
Acknowledgements: This research was funded by the Biotechno-logy and Biological Sciences Research Council (BBS/B/03130). J. S.holds a research chair supported by York Against Cancer. Theauthors would like to thank Professor Colin Jahoda (DurhamUniversity) for supporting the in vivo studies, Professor EileenIngham (University of Leeds) for helpful discussions and clinicalcolleagues for supplying tissues.

Received: October 9, 2010; Revised: December 30, 2010; Publishedonline: February 22, 2011; DOI: 10.1002/mabi.201000415

im www.MaterialsViews.com


www.mbs-journal.de

Keywords: biomaterials; CD163; CD68; macrophages; polyesters

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