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Biomaterials 32 (2011) 1517e1525

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Biomaterials

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The effect of elastin on chondrocyte adhesion and proliferation onpoly (3-caprolactone)/elastin composites

Nasim Annabi a, Ali Fathi a, Suzanne M. Mithieux b, Penny Martens c, Anthony S. Weiss b,Fariba Dehghani a,*a School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, NSW 2006, Australiab School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, AustraliacGraduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia

a r t i c l e i n f o

Article history:Received 28 September 2010Accepted 12 October 2010Available online 27 November 2010

Keywords:Composite scaffoldPoly (3-caprolactone)ElastinChondrocyte

* Corresponding author. Tel.: þ61 2 9351 4794; faxE-mail address: fdehghani@usyd.edu.au (F. Dehgh

0142-9612/$ e see front matter Crown Copyright � 2doi:10.1016/j.biomaterials.2010.10.024

a b s t r a c t

The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferationwithin the structure of poly (3-caprolactone) (PCL)/elastin composites. The homogenous 3D structurecompositeswere constructed by using high pressure CO2 in two stages. Porous PCL structureswith averagepore sizes of 540 � 21 mm and a high degree of interconnectivity were produced using gas foaming/saltleaching. The PCL scaffolds were then impregnatedwith elastin and cross-linkedwith glutaraldehyde (GA)under high pressure CO2. The effects of elastin and cross-linker concentrations on the characteristics ofcomposites were investigated. Increasing the elastin concentration from 25 mg/ml to 100 mg/ml elevatedthe amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysisshowed that elastin was homogeneously distributed throughout the 3D structure of all composites. Theweight gain of composites increased 2-fold from 15.8 � 0.3 to 38.3 � 0.7 (w/w) % by increasing the elastinconcentration from 25 mg/ml to 50 mg/ml and approached a plateau above this concentration. The pres-ence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the wateruptake ratio of PCLwas enhanced 100-fold from 0.030� 0.005 g liquid/g polymer to 11.80� 0.01 g liquid/gpolymer, when the elastin solution concentration was 50 mg/ml. These composites exhibited lowercompressivemodulus andenergy loss compared topure PCL scaffolds due to their higherwater content andelasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyteadhesion and proliferationwithin the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair.

Crown Copyright � 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Reconstruction and regeneration of organ function often requirethree dimensional (3D) porous scaffolds as a template. Porosity andpore interconnectivity within the scaffold are critical for celladhesion, proliferation, and the diffusion of nutrients and oxygenthroughout the 3D constructs [1,2]. The scaffold should also provideappropriate mechanical properties to support the regeneration ofdamaged tissue [3]. Synthetic biodegradable polymers such as poly(3-caprolactone) (PCL) have superior mechanical propertiescompared to natural polymers and can be easily processed [4,5].However, their applications in tissue engineering are limitedbecause their intrinsic hydrophobicity and absence of cell-recog-nition sites hinder cellular penetration, adhesion, and growth intothe porous structures [6,7]. Synthetic polymers are combined with

: þ61 2 9351 2854.ani).

010 Published by Elsevier Ltd. All

natural polymers such as chitosan [6,7], collagen [8], and elastin [9]to overcome these drawbacks.

Elastin-based biomaterials have great potential for use ashydrogel scaffolds for the regeneration of damaged cartilage [10].Recently, it has been demonstrated that elastin networks playmechanical and biological roles in cartilage repair [11]. In theuppermost zone of articular cartilage, where few cells are present,elastinfibers andmicrofibrils formadense 3Dextracellular networkwith thickness varying from 10 mm to 200 mm [11]. This abundantelastin-rich foundation means that elastin-containing scaffolds canbe useful for the construction of a new generation of cartilagesubstitutes. The results of in vitro studies show that chondrocytes[10,12] and progenitor cells [13] encapsulated in elastin-like poly-peptides (ELPs) undergo cartilagematrix synthesis. In addition, ELPssequences are native tomusculoskeletal tissues, and elicit no knownantigenic response from the host, when implanted subcutaneously[14,15]. Although ELPs provide a physical environment for chon-drocyte differentiation and cartilage matrix synthesis, a major

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N. Annabi et al. / Biomaterials 32 (2011) 1517e15251518

limitation of elastin-based biomaterials, like other natural hydro-gels, is their inability to support the significant loads experienced bycartilaginous tissue in vivo [12,16]. Efforts have been made toincrease themechanical strength of elastin-basedhydrogel scaffoldsthrough enzymatic or chemical cross-linking: a dynamic shearmodulus of less than 5 kPa was achieved using a transglutaminase(Ttg) [12] cross-linker, and compressive stiffness up to 50 kPa wasobtained using b[tris(hydroxymethyl)phosphino]propionic acid(THPP) [17] as a cross-linking agent. However, the compressivestiffness of elastin-based hydrogel scaffolds is still lower than thetarget value for articular cartilage (w500e1000kPa) [18]. Elastin hasrecently been combined with synthetic polymers to produce hybridelectrospun biomaterials with improved mechanical properties forvascular applications where the tensile modulus is greater than400 kPa [19,20].

Scaffold structural properties of highporosity and interconnectedpores enhance cell proliferation by allowing nutrient and oxygendiffusion [1]. Such characteristics are particularly important foravascular cartilage tissue replacement constructs. Gas foamingtechnique has been used to create porosity in hydrophobic polymerssuch as poly(lactic acid) [2] and hydrophilic polymers such as elastin[22,23]. The advantages of using high pressure CO2 for theconstruction of porous scaffolds include: decreasing process time,creating homogenous 3D structure, and eliminating the use oforganic solvents [6,8,21]. In this study, dense gas CO2 was used tofabricate porous 3D structures of PCL/elastin composites. The effectsof elastin and cross-linker concentrations on the characteristics offabricated composites were investigated. In vitro cell studies wereconducted to demonstrate the potential of using PCL/elastincomposites for chondrocyte growth and proliferation.

2. Materials and methods

2.1. Materials

a-elastin extracted from bovine ligament was obtained from Elastin Products Co.(Missouri USA). PCL (MW ¼ 80 kDa, Tm ¼ 60 �C, Tg ¼ �60 �C), glutaraldehyde (GA),and sodium chloride (NaCl) were purchased from Sigma (Australia). Food gradecarbon dioxide (99.99% purity) was supplied by BOC. Primary ovine articular carti-lage chondrocytes were generously provided by C.B. Little andM.M. Smith, RaymondPurves Bone and Joint Research Labs, Kolling Institute of Medical Research, Instituteof Bone & Joint Research, University of Sydney. Cells were maintained in Dulbecco’sModified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12 Ham) supple-mented with 10% (v/v) fetal bovine serum (FBS), penicillin and streptomycin. Alltissue culture reagents were obtained from Sigma. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) reagent waspurchased from Promega (Australia).

2.2. Construction of PCL/elastin composite scaffold

2.2.1. Formation of porous PCL scaffoldGas foaming/salt leaching processing was used to make porous 3D PCL scaffolds.

A homogenous mixture of PCL and NaCl particles was first prepared by melt mixing;the mixture was placed in a mould, gas-foamed using carbon dioxide (CO2) asa blowing agent, and soaked in water to leach out salt particles. PCL scaffolds withaverage pore sizes of 540 � 21 mm were produced using CO2 at 65 bar, 70 �C, pro-cessing time of 1 h, depressurization rate of 15 bar/min, and addition of 30 wt% saltparticles (500e700 mm).

2.2.2. Composite PCL/elastin scaffoldsHigh pressure CO2 was used to impregnate elastin into the 3D structure of PCL

scaffold. A PCL scaffold was placed in a Teflon mold inside a high pressure vessel; anelastin solution containing GA was then injected into mold. After the vessel wassealed and allowed to reach thermal equilibrium at 37 �C, the system was pressur-ized with CO2 to 60 bar, isolated and maintained at these conditions for 1 h. Thesystemwas depressurized at 15 bar/min and the sample was collected. The resultingconstruct was washed repeatedly in PBS (10 mM phosphate, 150 mM NaCl; pH 7.4),and then placed in 100 mM Tris in PBS for 1 h to quench the cross-linking reaction.After Tris treatment, the composite scaffold was washed twice and stored in PBS forfurther analysis.

The effects of a-elastin and GA concentrations on the characteristics of thecomposites were assessed. Various concentrations of a-elastin solution including 10,

25, 50, 75, 100 mg/ml and two different concentrations of GA (0.25 and 0.5% (v/v))were used. The amount of elastin embedded in the 3D PCL scaffolds was determinedby gravimetric analysis using the following equation

Weight gain ¼�WComposite �WPCL

�

WPCL� 100

2.3. Scanning electron microscopy (SEM)

SEM images were acquired using a Philips XL30 scanning electron microscope(15 kV) and used to determine the pore characteristics of the composites and toexamine cellular infiltration and adhesion. Lyophilized scaffolds were mounted onaluminiumstubsusing conductive carbonpaint, thengold coatedprior to SEManalysis.

Cell-seeded scaffolds were fixed with 2% (v/v) GA in 0.1 M Na-cacodylate bufferwith 0.1 M sucrose for 1 h at 37 �C. Samples underwent post-fixationwith 1% osmiumin 0.1 M Na-cacodylate for 1 h and were then dehydrated in ethanol solutions at 70%,80%, 90% and 3 times 100% for 10 min each. For drying, the samples were immersedfor 3 min in 100% hexamethyldisilazane then transferred to a desiccator for 25 min toavoid water contamination. Finally they were mounted on stubs and sputter coatedwith 10 nm gold.

2.4. Fourier transform infrared (FTIR) spectroscopy

FTIR analysis was used to qualitatively characterize the functional groups ofelastin and PCL, and to confirm the 3D penetration of elastin into the PCL scaffolds.FTIR spectrawere collected at the resolution of 2 cm�1 and signal average of 32 scansin each interferogram over the range of 1900e1400 cm�1 using a Varian 660 IR FTIRspectrometer. Composite scaffolds with thickness of 3 mm were used for FTIRanalysis. The depth of elastin penetration into the PCL scaffolds was evaluated byperforming FTIR analysis on the top surface and two layers cut from within thecomposites (approximately 1 mm and 2 mm below the surface).

2.5. Water uptake properties

The water uptake ratio of the PCL/a-elastin composite scaffolds was evaluated at37 �C in PBS. The scaffolds were lyophilized prior to use and were weighed dry. Thesamples were then soaked in 10 ml PBS for 24 h. The excess liquid was removed fromthe wet samples and the water uptake ratio was calculated based on a ratio of theincrease inmass to that of thedry sample. The reporteddata at each conditionwas theaverage measurement for at least three scaffolds.

2.6. Mechanical characterization: Compressive properties

Uniaxial compression tests were performed in an unconfined state using anInstron (Model 5543) with a 500 N load cell according to the testing proceduredescribed previously [22,23]. Compression tests were performed on PCL scaffold,PCL/a-elastin composite, and pure a-elastin hydrogel. Prior to mechanical testing,the samples were soaked for 2 h in PBS. The thickness (3 � 0.1 mm) and diameter(12.5 � 0.7 mm) of each sample was then measured using digital callipers. Thecompressive properties of the samples were tested in the hydrated state, in PBS, atroom temperature. The samples were subjected to a loading and unloading cycleand the compression (mm) and load (N) were collected at a cross speed of 30 mm/sand 40% final strain level. The compressive modulus was obtained as the tangentslope of the stress-strain curve between 10% and 20% strain level. In addition, for allsamples, the energy loss based on the compression cycle was computed. Threespecimens were tested for each sample type (PCL scaffold, PCL/a-elastin composite,and pure a-elastin hydrogel). The composite PCL/a-elastin was prepared by sub-jecting a PCL scaffold soaked in an aqueous solution containing 50 mg/ml elastin and0.25% (v/v) GA to high pressure CO2 as described above.

2.7. In vitro cell culture

The ability of cells to grow into the 3D structure of the PCL/a-elastin compositesand pure PCL scaffolds was assessed using MTS and SEM analysis. Scaffolds madeusing 50 mg/ml elastin and 0.25% (v/v) GAwere transferred into a 48-well plate andwashed twice with ethanol to sterilize the materials. The scaffolds were thenwashed at least twice with culture media to remove any residual ethanol andequilibrated in culture media (DMEM/F-12 Ham, 10% FBS, pen-strep) at 37 �Covernight. The cells were then seeded onto the scaffolds at 1.6 � 105 cells/well.Unseeded hydrogels located in adjacent wells were used for comparison. The scaf-folds were kept in a CO2 incubator for 7 days at 37 �C, after which they were fixed toassess cell penetration and growth using SEM analysis.

2.7.1. Proliferation/cytotoxicity assayMTS analysis was performed at time intervals of 1, 4, and 7 days to determine

cellular proliferation and viability within the scaffolds. Following culturing for theappropriate time the cell-seeded scaffolds were moved to a new 48-wellplatewhere250-ml fresh medium and 50-ml Cell Titer 96 Aqueous One reagent were then addedinto eachwell. The scaffolds were kept in a CO2 incubator at 37 �C for 1 h then read at

Fig. 1. SEM images of composite scaffolds made with high pressure CO2 using (a,b) 25 mg/ml, (c,d) 50 mg/ml, (e,f) 75 mg/ml, and (g,h) 100 mg/ml. Arrowheads in the images showcross-linked elastin. 0.25% (v/v) GA was used.

N. Annabi et al. / Biomaterials 32 (2011) 1517e1525 1519

490 nm using a microplate reader (Bio Rad 680). Values obtained for unseededcomposites were used as background.

3. Results and discussion

The objective of this study was to create PCL/elastin compositeswith enhanced mechanical properties and pore characteristics suit-able for load-bearing tissueengineering applications suchas cartilagereplacement. In this study, PCL scaffolds were impregnated withvarying concentrations of elastin and cross-linked under high pres-sure CO2. The effects of elastin and GA concentrations on the char-acteristics of composites made under high pressure were

investigated. Initial experiments demonstrated that PCL/elastincomposites were not formedwhen elastin concentrationswas below25mg/ml. Therefore, elastin concentrations in the range of25e100 mg/ml and twodifferent cross-linker concentrations, 0.5 and0.25% (v/v) GA, were used to assess the effect of elastin and GAconcentrations on the properties of hybrid scaffolds.

3.1. Pore structure of composite scaffolds

Large pores in the scaffold can allow effective nutrient supply,gas diffusion and metabolic waste removal, but can also lead tolow cell attachment and intracellular signaling, while small pores

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have opposite effects whereby cell attachment is promoted butthere is poor nutrient and gas delivery [24]. Consequently, theconstruction of scaffolds containing both macropores and micro-pores may provide the essential physical support for cellulargrowth.

SEM images in Fig. 1 show the effect of elastin concentrations onthepore characteristics of composites.AconstantGAconcentrationof0.25% (v/v)was used to investigate the effect of elastin concentration.In all the composites a microporous structure of cross-linked elastinwas formed within the macropores of PCL. The use of increasingelastin solution concentrations led to the embedding of a denserelastin structure within the PCL macropores. Open PCL pores thatcontained a coating of elastin matrix were observed on both the topsurface (Fig.1a) andcross-section (Fig.1b)of composite scaffoldwhenthe elastin solution concentration was 25mg/ml. At 50 mg/mla substantial porous elastin matrix was deposited within the PCLmacropores (Fig. 1c and d). When the elastin solution concentrationwas further increased to 75mg/ml and 100 mg/ml, the top surfaceimages indicated a covering of elastin (Fig. 1e and g). The cross-section images showed that the PCLmacroporeswere almost entirelyfilled with a porous elastin matrix (Fig. 1f and h).

3.1.1. The effect of GA concentrationThe effect of GAconcentrations onporemorphologyof composite

PCL/elastin made at high pressure CO2 is shown in Fig. 2. Increasingthe GA concentration from 0.25% (v/v) to 0.5% (v/v) did not affect thestructures of pores in the cross sections of the samples; however, ithad an impact on the pores on the top surfaces of the composites. Asshown in Fig. 2c and d, the top surfaces of both samples, producedusing 25 mg/ml and 50 mg/ml elastin solutions, were covered witha layer of cross-linked elastin when a higher concentration of GA

Fig. 2. SEM images from the top surfaces of composite scaffolds made with high pressure COand 50 mg/ml in b, d.

(0.5% (v/v) was used. However, open pores were observed on the topsurfaces of compositeswhenusing aGA concentration of 0.25% (v/v),as shown in Fig. 2a and b. Increasing the concentration of GAenhanced the rate anddegree of cross-linkingof elastin; this resultedin the formation of densely packed cross-linked layers of elastin onthe top surfaces that might inhibit cellular growth into the 3Dstructures. Consequently, in this study, 0.25% (v/v) was used tomakecomposite scaffolds.

3.2. FTIR analysis on composites

FTIR analysis was used to compare elastin penetration withinthe 3D structures of composites made with different concentra-tions of elastin. As shown in Fig. 3, FTIR spectra of elastin hydrogelshowed two main peaks at 1535 cm�1, and 1655 cm�1 corre-sponding to the amide II and amide I bands, respectively. Similarpeaks were observed for bovine elastin and k-elastin [25], humanelastin [26], bovine tropoelastin [27], a-elastin [28,29], two elastin-like poly(pentapeptides) [30], and synthetic elastin hydrogels [31].The FTIR spectra of PCL showed a main peak at 1725 cm�1 attrib-uted to C]O stretching; this peak was also observed in previousstudies for electrospun PCL samples [32]. The presence of peaksassigned to PCL and elastin on the top surfaces and 1 mm and 2 mminto the hybrid scaffoldsmade using elastin solution concentrationsof 25 mg/ml and 50 mg/ml confirmed the presence of elastin in alllayers of the composites (Fig. 3a and b). FTIR analysis on hybridscaffolds produced when using higher concentrations of elastin (i.e.75 mg/ml and 100 mg/ml) demonstrated elastin penetration intothe PCL scaffold but the absence of peaks assigned to PCL on the topsurfaces of these composites (Fig. 3c and d). This confirmed theresults obtained from SEM analysis, indicating that the top surfaces

2 using (a, b) 0.25% (v/v) GA, and (c, d) 0.5% (v/v) GA. 25 mg/ml elastin was used in a, c

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Fig. 3. FTIR spectra for pure a-elastin hydrogel, PCL, and composite PCL/a-elastin scaffolds produced under high pressure CO2 using (a) 25 mg/ml, (b) 50 mg/ml, (c) 75 mg/ml, and(d) 100 mg/ml elastin solution. 0.25% (v/v) GA was used. Layer 1: top surface, Layer 2: 1 mm depth, Layer 3: 2 mm depth).

N. Annabi et al. / Biomaterials 32 (2011) 1517e1525 1521

N. Annabi et al. / Biomaterials 32 (2011) 1517e15251522

of composites produced at higher concentrations of elastin werecovered with a cross-linked elastin layer.

3.3. Water uptake properties and weight gain

The weight gain and water uptake ratio of the composites areshown in Fig. 4. The mass gain of the composites was enhanced 2-fold from 15.8� 0.3 (w/w) % to 38.3 � 0.7 (w/w) % when the elastinsolution concentration was increased from 25 mg/ml to 50 mg/ml,and approached a saturation level, when the concentration excee-ded 50 mg/ml. Elastin was completely diffused within the 3Dstructures of PCL, when using concentrations below 50 mg/ml;however, at higher concentrations of elastin the excess amount ofcross-linked elastin on the top surface of PCL detached during thewashing stepwithPBS. Consequently, no significantweight gainwasobtained at higher concentrations of elastin. Mei et al. also reportedthat the weight gain of composite PCL/chitosanwas enhanced from6.6 to 8.4 (w/w) % as the concentrations of chitosan solutions wereincreased from 0.5 (w/w) % to 2 (w/w) % [6].

Water uptake ratio of a scaffold is an important factor, whichaffects oxygen and nutrient transfer within the scaffolds. The wateruptake ratioof compositeswasmeasured inPBSat37 �C.As shown inFig. 4b, the water uptake ratio of samples increased from 3.5 � 0.2 gliquid/g polymer to 6.7 � 0.7 g liquid/g polymer, and 10.2 � 0.1 gliquid/g polymer when elastin solution concentrationwas increasedfrom 25 mg/ml to 50 mg/ml, and 75 mg/ml respectively. Furtherincrease of elastin solution concentration to 100 mg/ml had nosignificant impact on water uptake properties. The presence ofelastin within the 3D PCL scaffolds imparted hydrophilicity to thehybrid scaffold and improved its water uptake properties more than100-fold compared to pure PCL scaffolds.

3.4. Mechanical characterization

3.4.1. Compressive propertiesThe compressive stress-strain curves of PCL scaffold, PCL/elastin

composites, and pure elastin hydrogels are shown in Fig. 5. As shownin Table 1, the compression modulus of composites was1.30 � 0.07 MPa, 1000-fold higher than pure elastin hydrogels(1.13�0.2 kPa) and lower than PCL scaffolds (1.53�0.18 MPa) i.e. thePCL scaffolds were stiffer than that of either the composites or pureelastin hydrogels. The presence of elastin within the PCL matriximparted hydrophilicity to the scaffold and increased its wateruptake content. In general, samples with greater water uptake havea lower compressive modulus [23,33,34]. Therefore, the lowercompressive modulus of the composites is probably due to their

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higher water uptake as compared to PCL scaffolds. Wan et al. alsoreported that the compressive modulus of PCL scaffolds made bysolvent casting/salt leaching processing was decreased from23.2�1.13 MPa to 5.8� 0.31 MPawhen itwasblendedwith chitosan[7]. In this study PCL scaffolds with average pore size of188.4 � 8.4 mm was used to form composite PCL/chitosan [7];consequently, the smaller pore size led to a higher compressivemodulus compared to the PCL scaffolds constructed in this studywhich contained macropores of 540 � 21 mm. Generally, the poresizes of scaffold have a substantial effect on the mechanical proper-ties,with the stiffness of the scaffold decreasing as porosity increases[2]. The PCL/elastin composites exhibited higher compressivemodulus compared to rTE/a-elastin composite hydrogels producedat high pressure CO2 (4.9e5.8 kPa) [22]. The compressivemodulus ofcomposites in this study comfortably exceeded the target value forarticular cartilage of 500e1000 kPa [18].

The resilience of natural elastin allows for reversible defor-mation without loss of energy [35]. Generally, the energy loss forthe composite scaffolds was lower than PCL scaffolds, demon-strating a decrease in hysteresis for the composites. As indicated inTable 1, the composite scaffold exhibited lower energy loss at48.1 � 5.1% compared to PCL scaffolds (62.2 � 4.1%). This was dueto the presence of elastin within the structure of PCL, whichimparted elasticity to the constructs and reduced the energy loss.The lowest energy loss of 22.1 �1.3% was observed for pure elastinhydrogels which compares with the 22.7% energy loss of nativecartilage [36].

3.5. In vitro cell proliferation using composite PCL/elastin scaffolds

PCL/elastin composites using 50 mg/ml elastinwere assessed fortheir cell interactive capabilities. The high mechanical properties ofthis composite (compressive modulus of 1.3 � 0.07 MPa) and thepresence of open pores on both the top surface and throughout thecross-section were expected to provide an appropriate physicalsupport for chondrocyte penetration and growth within the 3Dconstructs. The growth and proliferation of sheep articular cartilagechondrocytes in hybrid scaffolds were examined by MTS and SEManalysis to demonstrate the feasibility of using 3D composites forload-bearing tissue engineering applications including cartilagereplacement. MTS analysis on PCL scaffolds and PCL/elastincomposites is shown in Fig. 6. As indicated in Fig. 6a, cell numbersincreased over time for the PCL scaffolds and PCL/elastin compos-ites. While these results suggest that cellular proliferation washigher on the PCL scaffolds compared to the composites, anexamination of the scaffolds themselves indicated this was unlikely

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Fig. 5. Unconfined compressive behavior of (a) PCL scaffold, (b) pure elastin hydrogel, and (c) composite PCL/elastin. 50 mg/ml, and 100 mg/ml elastin solutions were used toconstruct composites, and pure elastin hydrogels, respectively.

N. Annabi et al. / Biomaterials 32 (2011) 1517e1525 1523

to be the case. Following the MTS reaction, the composite scaffoldshad turned an intense purple color while the PCL scaffolds wereless intense. The intense color of the composite is likely due toa substantial adsorption of the formazan product to the elastinwithin the composite scaffolds. As such a quantitative analysis ofcell proliferation within the scaffolds was not possible but colorcomparisons yielded a more robust appreciation of proliferationthan the supernatant. Measurement of scaffold color intensitiesusing Image J software is shown in Fig. 6b.

SEM images of chondrocytes cultured on PCL scaffolds and PCL/elastin composites are shown in Fig. 7. The cells attached andproliferated on the top surface (Fig. 7c) and into the 3D structure(Fig. 7def) of PCL/elastin composites due to the presence of cross-linked elastin within the large pores of PCL scaffold. However, iso-lated single colonies were observed on the top surface (Fig. 7a) andalso cross-section (Fig. 7b) of PCL scaffolds. Thiswas likely due to thelack of cell-recognition signals and hydrophobic surface propertiesof PCL that impeded cell adhesion under these conditions. Incontrast, the presence of elastin within the pores of PCL scaffoldspromoted cellular attachment and growth. The results of in vitrostudies demonstrated that the porous PCL/elastin composite is

Table 1Compressive modulus and energy loss of elastin hydrogels, PCL/elastin compositesand PCL scaffolds.

Construct Compressive modulus(MPa)

Energy loss(%)

Elastin 0.001 � 0.0002 22.1 � 1.3PCL/Elastin 1.31 � 0.07 48.1 � 5.1PCL 1.53 � 0.18 62.2 � 4.1

Fig. 6. MTS analysis on PCL scaffolds and PCL/elastin composites. (a) Cell viabilitymeasured by MTS assay at 1, 4, and 7 post-seeding, (b) color intensities of PCL andcomposite scaffolds measured using Image J software.

Fig. 7. Images of cells cultured on (a, b) PCL scaffold, and (cef) PCL/elastin composites. Top surfaces are shown in a and c, cross sections in b and def, arrowheads in the images showrepresentative cells 50 mg/ml elastin solution was used to form composite scaffolds.

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a suitable biomaterial for chondrocyte proliferation and growthunder these conditions.

4. Conclusions

PCL scaffold properties were substantially enhanced for tissueengineering applications by the integration of elastin into thematrix. The concentration of elastin was optimized to achievemechanical properties and pore characteristics suitable for carti-lage tissue engineering. Elastin was uniformly distributed andcross-linked within PCL macropores under high pressure CO2.Increasing elastin concentrations enhanced water uptake ratio andweight gain of composites. While the mechanical properties ofcomposites were lower than pure PCL scaffolds, they remainedwithin the range suitable for cartilage repair. The PCL/elastin scaf-folds exhibited lower energy loss compared to PCL scaffolds, con-firming an increase in elastic properties which is attributed toelastin within the constructs. The composites supported chon-drocytes proliferation and growth within the 3D structures. ThePCL/elastin scaffolds may be contemplated as suitable biomaterialsfor tissue engineering applications such as cartilage repair.

Acknowledgements

The authors thank Christopher Little and Margaret Smith forproviding ovine chondrocytes and acknowledge financial supportfrom the Australian Research Council and the National Health andMedical Research Council.

Appendix

Figures with essential colour discrimination. Figs. 1 and 7 in thisarticle may be difficult in interpret in black and white. The fullcolour images can be found in the online version, at doi:10.1016/j.biomaterials.2010.10.024.

References

[1] Ma PX. Biomimetic materials for tissue engineering. Adv Drug Deliv Rev2008;60:184e98.

[2] Annabi N, Nichol JW, Zhong X, Ji C, Koshy S, Khademhosseini A, et al.Controlling the porosity and microarchitecture of hydrogels for tissue engi-neering. Tissue Eng Part B Rev 2010;16:371e83.

[3] Langer RS, Vacanti JP. Tissue engineering. Science 1993;260:920e6.

N. Annabi et al. / Biomaterials 32 (2011) 1517e1525 1525

[4] Sarasam A, Madihally SV. Characterization of chitosan-polycaprolactoneblends for tissue engineering applications. Biomaterials 2005;26:5500e8.

[5] Averous L, Moro L, Dole P, Fringant C. Properties of thermoplastic blends:starch-polycaprolactone. Polymer 2000;41:4157e67.

[6] Mei N, Chen G, Zhou P, Chen X, Shao Z-Z, Pan L-F, et al. Biocompatibility ofpoly(3-caprolactone) scaffold modified by chitosan-the fibroblasts prolifera-tion in vitro. J Biomater Appl 2005;19:323e39.

[7] Wan Y, Wu H, Cao X, Dalai S. Compressive mechanical properties andbiodegradability of porous poly(caprolactone)/chitosan scaffolds. PolymDegrad Stab 2008;93:1736e41.

[8] Chen G, Ushida T, Tateishi T. A biodegradable hybrid sponge nested withcollagen microsponges. J Biomed Mater Res 2000;51:273e9.

[9] Almine JF, Bax DV, Mithieux SM, Nivison-Smith L, Rnjak J, Waterhouse A, et al.Elastin-based materials. Chem Soc Rev 2010;39:3371e9.

[10] Betre H, Setton LA, Meyer DE, Chilkoti A. Characterization of a geneticallyengineered elastin-like polypeptide for cartilaginous tissue repair. Bio-macromolecules 2002;3:910e6.

[11] Yu J, Urban JPG. The elastic network of articular cartilage: an immunohisto-chemical study of elastin fibres and microfibrils. J Anat 2010;216:533e41.

[12] McHale MK, Setton LA, Chilkoti A. Synthesis and in vitro evaluation of enzy-matically cross-linked elastin-like polypeptide gels for cartilaginous tissuerepair. Tissue Eng 2005;11:1768e79.

[13] Betre H, Ong SR, Guilak F, Chilkoti A, Fermor B, Setton LA. Chondrocyticdifferentiation of human adipose-derived adult stem cells in elastin-likepolypeptide. Biomaterials 2006;27:91e9.

[14] Urry DW, Parker TM, Reid MC, Gowda DC. Biocompatibility of the bioelasticmaterials, poly(GVGVP) and its gamma-irradiation cross-linked matrix:summary of generic biological test results. J Bioact Compat Polym 1991;6:263e82.

[15] Sandberg LB, Soskel NT, Leslie JG. Elastin structure, biosynthesis, and relationto disease states. N Engl J Med 1981;304:566e79.

[16] Nettles DL, Kitaoka K, Hanson NA, Flahiff CM, Mata BA, Hsu EW, et al. In situcrosslinking elastin-like polypeptide gels for application to articular cartilagerepair in a goat osteochondral defect model. Tissue Eng 2008;14:1133e40.

[17] Lim DW, Nettles DL, Setton LA, Chilkoti A. Rapid cross-linking of elastin-likepolypeptides with (hydroxymethyl)phosphines in aqueous solution. Bio-macromolecules 2007;8:1463e70.

[18] Setton LA, Nettles DL, Lim DW, Guilak F, Chilkoti A. Genetically engineeredelastin-like polypeptides for cartilage tissue regeneration. Osteoarthr Cartil2007;15:B2e5.

[19] Wise SG, ByromMJ,WaterhouseA, BannonPG,NgMK,WeissAS. Amultilayeredsynthetic human elastin/polycaprolactone hybrid vascular graft with tailoredmechanical properties. Acta Biomater; 2010. doi:10.1016/j.actbio.2010.07.022.

[20] Stitzel J, Liu J, Lee SJ, Komura M, Berry J, Soker S, et al. Controlled fabrication ofa biological vascular substitute. Biomaterials 2006;27:1088e94.

[21] Coombes AGA, Verderio E, Shaw B, Li X, Griffin M, Downes S. Biocomposites ofnon-crosslinked natural and synthetic polymers. Biomaterials 2002;23:2113e8.

[22] Annabi N, Mithieux SM, Weiss AS, Dehghani F. Cross-linked open-pore elastichydrogels based on tropoelastin, elastin and high pressure CO2. Biomaterials2010;31:1655e65.

[23] Annabi N, Mithieux SM, Boughton EA, Ruys AJ, Weiss AS, Dehghani F.Synthesis of highly porous crosslinked elastin hydrogels and their interactionwith fibroblasts in vitro. Biomaterials 2009;30:4550e7.

[24] Lien S-M, Ko L-Y, Huang T-J. Effect of pore size on ECM secretion and cellgrowth in gelatin scaffold for articular cartilage tissue engineering. Acta Bio-mater 2009;5:670e9.

[25] Debelle L, Alix AJP, Jacob M-P, Huvenne J-P, Berjot M, Sombret B, et al. Bovineelastin and k-elastin secondary structure determination by optical spectros-copies. J Biol Chem 1995;270:26099e103.

[26] Debelle L, AlixAJP,Wei SM, JacobM-P,Huvenne J-P, BerjotM, et al. The secondarystructure and architecture of human elastin. Eur J Biochem 1998;258:533e9.

[27] Debelle L, Alix AJP. Optical spectroscopic determination of bovine tropoelastinmolecular model. J Mol Struct 1995;348:321e4.

[28] Mammi M, Gotte L, Pezzin G. Evidence for order in the structure of alpha-elastin. Nature 1968;220:371e3.

[29] Dehghani F, Annabi N, Valtchev P, Mithieux SM, Weiss AS, Kazarian SG, et al.Effect of dense gas CO2 on the coacervation of elastin. Biomacromolecules2008;9:1100e5.

[30] Schmidt P, Dybal J, Rodriguez-Cabello JC, Reboto V. Role of water in struc-tural changes of poly(AVGVP) and poly(GVGVP) studied by FTIR and ramanspectroscopy and ab initio calculations. Biomacromolecules 2005;6:697e706.

[31] Mithieux SM, Rasko JEJ, Weiss AS. Synthetic elastin hydrogels derived frommassive elastic assemblies of self-organized human protein monomers.Biomaterials 2004;25:4921e7.

[32] Nirmala R, Nam KT, Park DK, Woo-Il B, Navamathavan R, Kim HY. Structural,thermal, mechanical and bioactivity evaluation of silver-loaded bovine bonehydroxyapatite grafted poly([3]-caprolactone) nanofibers via electrospinning.Surf Coat Technol 2010;205:174e81.

[33] Srokowski EM,WoodhouseKA.Development and characterisationofnovel cross-linked bio-elastomeric materials. J Biomater Sci Polym Ed 2008;19:785e99.

[34] Vieth S, Bellingham CM, Keeley FW, Hodge SM, Rousseau D. Microstructuraland tensile properties of elastin-based polypeptides crosslinked with genipinand pyrroloquinoline quinone. Biopolymers 2007;85:199e206.

[35] Gosline J, Lillie M, Carrington E, Guerette P, Ortlepp C, Savage K. Elasticproteins: biological roles and mechanical properties. Philos Trans R Soc Lond,B, Biol Sci 2002;357:121e32.

[36] Ding M, Dalstra M, Linde F, Hvid I. Mechanical properties of the normalhuman tibial cartilage-bone complex in relation to age. Clin Biomech1998;13:351e8.