Tissue engineered cartilage on collagen and PHBV matrices

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Biomaterials 26 (2005) 5187–5197

www.elsevier.com/locate/biomaterials

Tissue engineered cartilage on collagen and PHBV matrices

Gamze Torun Kosea,�, Feza Korkusuzb, Aykut Ozkulc, Yasemin Soysald,Taner Ozdemire, Cemil Yildize, Vasif Hasircif

aDepartment of Genetics and Bioengineering, Yeditepe University, 34755 Istanbul, TurkeybMiddle East Technical University, Medical Center, 06531 Ankara, TurkeycAnkara University, School of Veterinary Medicine, 06063 Ankara, Turkey

dGulhane Military Medical Academy, Department of Genetics, 06018 Ankara, TurkeyeGulhane Military Medical Academy, Department of Orthopedics, 06018 Ankara, Turkey

fMiddle East Technical University, Department of Biological Sciences, Biotechnology Research Unit, 06531 Ankara, Turkey

Received 26 July 2004; accepted 4 January 2005

Abstract

Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium

phosphate (CaP–Gelfixs), and matrices of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) were produced to create a

cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction

spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated

from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was

measured by Western Blot analysis. The foams were seeded with 1� 106 chondrocytes and histological examinations were carried

out to assess cell–matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its

integrity for 21 days, while CaP–Gelfixs was deformed and degraded within 15 days.

Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5mm in diameter and 4mm in depth)

at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte

seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body

reaction. In CaP–Gelfixs matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their

phenotype in both matrices. PHBV had better healing response than CaP–Gelfixs.

Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage

defects.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Cartilage; Tissue engineering; Collagen; PHBV; Biomaterials

1. Introduction

Joint cartilage has a limited capacity of regenerationand artificial matrices for cartilage repair are currenttopics of research [1]. In mature articular cartilage thechondrocyte is a resting cell and functions in a well-regulated balanced system between the cell and matrix [2].

e front matter r 2005 Elsevier Ltd. All rights reserved.

omaterials.2005.01.037

ing author. Tel.: +902165 780617;

80400.

ess: [email protected] (G.T. Kose).

Therefore repair of chondral defects involve a recruitmentof chondrogenic cells to the injury site. Different cellsources may prove effective in creating the desirable tissuehealing response. Chondrocytes or their progenitors havealready been in use for this reason. In osteochondraldefects the marrow progenitor cells can migrate to the siteand begin a reparative response, but this generally leadsto the formation of less durable fibrocartilage rather thanhyaline cartilage. Otherwise isolated and cultured auto-logous chondrocytes may initiate the healing the defectswith entirely hyaline cartilage [3].

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Cartilage tissue engineering techniques involvingscaffolds made from biodegradable and biocompatiblematerials hold great promise for the treatment ofcartilage defects. There are many different kinds ofmaterials such as collagen [4], poly(3-hydroxybutyricacid-co-3-hydroxyvaleric acid) (PHBV) [5], PLA [6],PGA [7], etc. used in this field but still it is not clearwhich matrix is the most suitable.

PLGA has a high degradation rate leading to localacidity. PLLA, on the other hand, has a very lowdegradation rate due to its high crystallinity leavingbehind crystalline particulates even after several yearsleading to incomplete healing [8]. Both materials aresynthetic in origin. Instead, PHBV is of natural origin. Itis biodegradable, biocompatible, and non-toxic [9–11].Its degradation rate is between those of PLLA andPLGA. Therefore, it does not lead to local acidity andalso, since it has lower crystallinity than PLLA, it doesnot leave behind crystalline remnants. Thus, PHBV isbeing increasingly preferred over biodegradable poly-mers of polylactide origin.

In the literature, there are a number of studies abouttissue responses to PHBV materials. Kose et al.observed the formation of bone in in vitro conditionsby culturing rat marrow stromal osteoblasts in biode-gradable, macroporous PHBV matrices over a period of60 days [12,13]. Both visual and biochemical assaysshowed that cultured osteoblasts retained their pheno-type throughout the duration of the experiment.

Malm et al. used the patches of PHB to closeexperimentally induced atrial septal defects in calves[14]. At the end of 12 months no polymer material wasfound. Although, small particles of polymer withpersisting foreign body reaction were observed bypolarized light microscopy, PHB appears to be suitablematerial for human applications.

Collagen on the other hand is a biological macro-molecule abundant in cartilage tissue and as a result ithas been tested as a carrier material in tissue engineeringapplications. It, however, has a significant degradationrate which lead to loss of mechanical properties beforehealing is complete.

Caplan et al. reported that when mesenchymal stemcells were seeded into collagen gels and implanted inosteochondral defects in rabbits, embryogenesiswas recapitulated and both bone and hyaline cartilagewere formed, although mechanical properties of theregenerated tissues were significantly less than normaland there was some evidence of degeneration after 24weeks [15].

People have tried to overcome this problem byapplying various crosslinking methods and also treat-ments like growth of in situ mineral crystals. In apreliminary study, Yaylaoglu et al. produced a highlyporous HAp (hydroxyapatite)/collagen construct beforeseeding with chondrocytes. The treatment increased the

stability of the foam in the medium and cell proliferationwas observed in the form of ECM deposition [16].

As can be seen from the literature presented aboveboth collagen and PHBV matrices can be used indesigning available structures and surfaces, which helpsguide chondrocyte organization and growth meanwhilemaintaining diffusion of nutrients to the transplantedcells.

In this study, porous calcium phosphate loadedcollagen (CaP–Gelfixs) and PHBV matrices weredesigned to serve as cell carriers for the constructionof tissue engineered cartilage. Surface chemistry andtopography of PHBV carriers were modified usingoxygen plasma treatment. The surface morphologiesand characteristics of both matrices were studied. Invitro cell proliferation was observed by histologicalevaluations. In vivo applicability of this system wasalso investigated by macroscopical and histologicalmethods.

2. Materials and methods

2.1. Preparation of PHBV and collagen foams

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) con-taining 8% by mole of 3-hydroxyvalerate was obtainedfrom Aldrich (Chem. Co., Milwaukee, MI, USA). Inorder to obtain a highly porous matrix with uniformpore sizes, sieved sucrose crystals (300–500 mm, 50 g)were placed in a glass Petri dish. A PHBV solution (6%,w/w) was prepared in chloroform:dichloromethane (1:2,v/v) and poured onto the sucrose crystals until they weresubmerged. They were then air-dried and the resultantfoams were dialyzed against distilled water to remove allthe sucrose, resulting in porous PHBV matrices. Theywere frozen at �70 1C and lyophilized in a freeze dryer(Labconco Freeze Dry, Model 78680, Missouri, USA).The PHBV matrices were then treated with oxygen RF-plasma (Advanced Plasma System, Inc., USA) as disksof 2mm diameter and 1mm thick and sterilized byexposure to gamma radiation (25 kGy).

Calcium phosphate formation on Gelfixs (AbdiIbrahim, Turkey, lyophilized collagen foam (50� 50�50mm3)) was done according to Yaylaoglu et al. [16].Gelfixs was soaked overnight in a phosphoroussolution in Tris buffer (100mM, pH 7.4, 50mM Tris,1% NaN3). The foam was removed, immersed overnightin a calcium solution in Tris buffer (100mM, pH 7.4,50mM Tris, 1% NaN3) and washed with distilled waterfor 2 h. This calcium phosphate formation procedurewas then repeated. The foam was frozen at �70 1C andlyophilized in a freeze dryer. It was then cut into disks of2mm diameter and 1mm thick and sterilized byexposure to gamma radiation (25 kGy).

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2.2. Characterization of PHBV and collagen foams

2.2.1. Scanning electron microscopy (SEM)

Surfaces of the PHBV (6%, w/w) and Gelfixs foamswere all coated with gold and SEM was carried out in aJEOL (Japan, Model JSM 6400) to observe the surfacecharacteristics, pore sizes and the pore distribution ofthe matrix materials.

2.2.2. SEM coupled with energy dispersive spectra

(EDS)

The ED spectrum and the ratio of calcium tophosphate in the CaP–Gelfixs matrix was determinedby the EDS capability of the SEM (NORAN (USA),series 2) after coating the samples with gold undervacuum.

2.2.3. Porosity analysis

Average pore size and the pore distribution of thePHBV and Gelfixs foams were determined using theScion Image Analysis Program (National Institutes ofHealth, USA) which is a public domain image proces-sing and analysis program for Macintosh.

2.2.4. Void volume calculations

Volume of pores inside the polymers was determinedto find out the volume available for cell loading. Porevolume fraction inside the matrices was determined tofind out the volume available for cell loading. Pre-weighed foam disks (7mm diameter and 1.9mmthickness) were immersed in water, and repeated cyclesof vacuum and aeration was applied to achievepenetration of water into the matrix. After equilibration,wet weights of the foams were recorded. Foamthicknesses were measured using a micrometer (ErsteQualitat, Germany) and from these dimensions samplevolumes were determined. Total volume of the poreswas calculated from the weight of loaded water. Theirratio gave the pore volume fraction.

2.3. In vitro chondrocyte– biomaterial interaction studies

2.3.1. Cell culture

Articular cartilage slices were obtained from proximalhumerus of 22 week—old male adult local rabbits(3250 g) under anesthesia (IM injection of xylazine(5mg/kg) and ketamine HCl (100mg/kg). They werewashed with sterile physiological saline and incubatedwith collagenase Type II (1mg/mL; Sigma, St. Louis,USA) at 37 1C overnight. After incubation, cells werecentrifuged at 1200 rpm for 10min and cultured on T-25flasks in RPMI 1640 with 20% FCS, 100 units/mLpenicillin and streptomycin. The cultures were followedfor two weeks and media changed twice a week. Theviability of cartilage cells grown in culture medium weredetermined by Trypan blue staining.

2.3.2. Characterization of chondrocytes

Assessment of Type II collagen in cell culture wasused as the exact determination method of chondrocyticphenotype. Characterization of chondrocytes and qua-litative measurement of Type II collagen was performedon the cell pellet with Western blot analysis.

2D chondrocyte culture of sixth passage was scrapedinto its own medium and centrifuged at 1200 rpm for10min at 4 1C. The cell pellet was then washed twiceusing ice-cold PBS with protease inhibitors (1mMEDTA and 1 mM PMSF) without phenol red. Theresulting pellet was weighed and resuspended in thesame PBS. Following two freeze-thaw cycles at �80 1C,40 mL cell suspension was boiled in reducing buffercontaining Tris base, SDS, glycerol, glycine and b-mercaptoethanol for 5min. Proteins were separated in5% and 10% SDS–Polyacrylamide gel and one set of thesamples was transferred to polyvinylidine difluoridemembrane. Other set was stained with Coomassie Blue.The membrane was blocked in TBS containing 3% non-fat milk powder, 1% Horse Serum and 0.05% Tween 20overnight at room temperature. After excessive washingin TBS-T, the blot was incubated with primary antibody(Anti-collagen II Mab, Clon 2B1.5, MS-235-P, Neo-Markers, CA, USA) for 1 h at 37 1C. After a series ofwashing, it was incubated with the secondary antibody(anti-mouse IgG labelled with Biotin, AmershamBuchler GmbH, Germany) for 1 h at 37 1C. Themembrane was washed again and incubated withstreptavidin–biotin complex labelled with HRPO. Bandswere visualized using 4-chloronaphthol and H2O2 (3%).

2.3.3. Seeding of chondrocytes on polymeric foams

Chondrocytes obtained from shoulder joint of rabbitswere reproduced in monolayer cultures and thencollected from flasks with trypsinization. Trypan blueviability test was employed to estimate the number ofliving chondrocytes. 1� 106 viable cells were seeded onPHBV and Gelfixs matrices in 24 well plates. Both cell-seeded and control matrices were incubated in vitro for7 and 15 days. At the end of each time period, matriceswere removed from the medium and put into formalde-hyde buffer (10%).

2.3.4. Histological evaluation for in vitro studies

Histological staining of PHBV and Gelfixs foamswas carried out for visualization of cells and demonstra-tion of tissue formation. For light microscopy on plasticembedded semi-thin sections, polymer-cell samples werefixed in 10% formaldehyde. They were then processed ina graded series of ethanol and placed in flat molds toembed in resin (Araldite CY212, Agar, Germany). Theywere cut into 3–5 mm thick semi-thin sections andstained with Hematoxylin–Eosin (HE) and Papanico-loau (PAP). Stained semi-thin sections were examined

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by at least two investigators for the cellular compositionand new tissue formation in the pores of the implant.

2.4. In vivo chondrocyte– biomaterial interaction studies

2.4.1. Implantation of chondrocyte seeded PHBV and

collagen matrices

Twenty healthy, albino, mature, local rabbits, aged 4months and weighing approximately 3250 g, werepurchased from Institute of Farming Animals (Ankara,Turkey). Standard laboratory food and water wereprovided constantly. All procedures were in fullcompliance with Turkish Law 6343/2, VeterinaryMedicine Deontology Regulation 6.7.26, and with theHelsinki Declaration of Animal Rights.

Anesthesia was induced with an intramuscular injec-tion of ketamine (50mg/kg) and xylazine (10mg/kg). Afull thickness cartilage defect (4.5mm in diameter and4mm in depth) through the articular cartilage and intothe subchondral bone was created on the patellar grooveon both knees using an electric drill. The animals weredivided into two groups. Defects in Group One (n ¼ 8)were treated with Gelfixs and Group Two (n ¼ 8) weretreated with PHBV. Chondrocyte-seeded matrices wereimplanted in right knees. Left knees served as control(matrices without cell). Implants were shaped andplaced into the defect and fixed with a self-tapping(0.4mm in diameter) cancellous screw. The wounds wereclosed using sterile, nonabsorbable suture material. Allrabbits were returned to their cages and allowed to movefreely after surgery; none had abnormal gait or mobilitydifficulty. After 8 and 20 weeks, animals were eutha-nized by diethyl ether inhalation. The transplantedtissues were harvested and stored in 10% neutral-buffered formalin until embedding for histology.

2.4.2. Macroscopical and histological evaluations for in

vivo studies

When the animals were killed, the knees were assessedfor contractures and adhesions. After collection of theknee joints, the distal femur, patella and synovium wereexamined macroscopically. The cartilage defect site andadjacent tissue were evaluated by two blinded observersand scored. Four criteria were evaluated according toNiederauer et al. [20]; (1) Edge integration, (2) Smooth-ness of the cartilage surface, (3) Cartilage surface degreeof filling, and (4) Color of cartilage opacity ortranslucency of the neocartilage. The grading in macro-scopical evaluation was between 0 and 8 where 8 was thebest (normal) cartilage.

For the histological evaluations, cartilage specimenswere fixed in 10% phosphate buffered formalin (pH 7.0)at room temperature. They were then rinsed in bufferand after macroscopic detection, fixed in formic acid(10%) and decalcified in nitric acid (10%) for 5–7 days.They were dehydrated in a graded series of ethanol and

embedded in paraffin. Three–five micrometer thick,longitudinal and transverse sections were prepared witha rotary microtome (Microm, HM 360, Germany) andstained with haematoxylin and eosin. Stained sections (aminimum of ten sections obtained from different levelsof each tissue) were examined by two investigatorsand documented with an Olympus microscope (ModelBH-2, Japan).

The sections of the grafted area were examined for thequality of the repair process. A semi-quantitativegrading scale was used according to Wakitani et al.[17]. Scoring system included; (1) Cell morphology, (2)Matrix staining, (3) Surface regularity, (4) Thickness ofcartilage, (5) Integration of donor to host adjacentcartilage. Normal articular cartilage achieves a totalscore of 12 points.

2.5. Statistical analysis

All measurements were expressed as means 7standard deviations. Data was analyzed using para-metric (one-way and two-way analysis of variance,paired T-test) and non-parametric (Kruskal-Wallis) testto assess statistical significance.

3. Results

3.1. Characterization of the polymeric matrices

3.1.1. SEM

The surface characteristics, average pore sizes and thepore distribution of the untreated and unloaded PHBV,sucrose loaded and oxygen plasma treated PHBV (6%,w/w), Gelfixs and calcium phosphate loaded Gelfixs

(CaP–Gelfixs) foams were observed by SEM.It was observed that untreated and unloaded PHBV

had a more ordered, compact and smooth surface(Fig. 1a). The amount of the pores was not sufficient forproper osteoblast growth. In order to obtain moreporous foams, sucrose crystals (300–500 mm) wereloaded into the structure during foam preparation andthey were later leached out leaving voids behind. Also, amore hydrophilic PHBV structure than that of theuntreated ones was obtained after oxygen RF-plasmatreatment (Fig. 1b).

Scanning electron micrographs of untreated andcalcium phosphate containing Gelfixs showed that theyboth had porous structures but the untreated oneappeared to have a more delicate structure with a finermatrix (Fig. 2a and b). The structure of the Gelfixs wassignificantly altered upon modification by calciumphosphate deposition. This also substantially decreasedthe degradation rate of the matrix material.

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Fig. 1. Scanning electron micrograph of: (a) untreated PHBV (6%, w/

w) foams (no sucrose leaching to create pores), (b) RF-oxygen plasma

treated (100W, 20min) PHBV (6%, w/w) foam with sucrose-leaching

(300–500mm).Fig. 2. Scanning electron micrograph of: (a) untreated, (b) calcium

phosphate loaded lyophilized collagen (Gelfixs) foam.

G.T. Kose et al. / Biomaterials 26 (2005) 5187–5197 5191

3.1.2. SEM coupled with energy dispersive spectra

The presence and the ratio of calcium phosphate inCaP–Gelfixs matrix was determined by the EDSattachment of the SEM. The presence of the peaks dueto calcium and phosphorus was observed. Calcium andphosphate provided more stability to the collagenstructure and matrices were stiffened. Therefore, CaP–Gelfixs was easy to handle and shape, and applicationto the defect was not difficult like in unloadedcollagen [18].

In the CaP–Gelfixs sample, the ratio of calciumto phosphorus was found to be 2.26. This is muchhigher than the targeted 1.67, a ratio specific forhydroxyapatite in cortical bone but similar to theresults of a calcium phosphate deposition on gelatinmembrane (2.33) [16]. Even though CaP–Gelfixs con-struct does not closely mimic the natural cartilage but itprobably is much more durable than alternativeconstructs.

3.1.3. Porosity analysis

Image analysis was carried out to characterizethe constructs in terms of their porosity. Morespecifically the aim was to calculate the averagepore size and the pore distribution of the matrices.They were examined using the Scion Image AnalysisProgram by NIH. Image analysis results showedthat the porosity (fraction of the surface that isvoid) of the untreated and unloaded PHBV (6%)was 0.428. Its pore size distribution was homogeneous.The porosity of sucrose loaded and oxygenplasma treated PHBV (6%) (100W 20min) was foundas 0.656.

The porosities of untreated Gelfixs, and CaP–Gel-fixs were found as 0.610 and 0.575, respectively.Although the porosity of CaP–Gelfixs is somewhat lessthan that of untreated one, it does not inhibit the growthof chondrocyte inside the matrix.

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3.1.4. Void volume calculations

Volume of pores inside the untreated and unloadedPHBV, sucrose loaded and oxygen plasma treatedPHBV (6%, w/w), Gelfixs and CaP–Gelfixs foamswas determined (Table 1).

Void volume of the sucrose loaded (300–500 mm) andoxygen plasma treated (100W, 20min) PHBV wasfound to be slightly more than that of untreatedand unloaded one, 9.16mL/g dry foam vs. 8.95mL/gdry foam, respectively. The volume of the pores inGelfixs is much more than that of Gelfixs–calciumphosphate foams; 29.96mL/g dry foam vs. 13.22mL/gdry foam, respectively, due to loading of calciumphosphate into the structure of the latter. Void volumestudies revealed that Gelfixs was significantly moreporous than PHBV.

Table 1

Void volumes of PHBV and Gelfixs foams

Type of foams Void volume (mL/g dry foam)

PHBV 6% 8.9571.55

PHBV 6%aOP 9.1671.03

Gelfixs 29.9678.17

CaP–Gelfixs 13.2275.65

aOP: Oxygen plasma treated foam (100W,10min).

Fig. 3. Western blot for

3.2. In vitro chondrocyte– biomaterial interaction

3.2.1. Characterization of chondrocytes

2D chondrocyte cultures were examined for collagentype II expression by Western Blot. After incubationwith anti collagen type II Ab, formation of collagen typeII (96 kDa) band was observed in Fig. 3. This bandindicates the presence of collagen type II that is anindicator of chondrocyte phenotype.

3.2.2. Histological evaluations for in vitro study

In the third week of the monolayer culture, the cellspresented a fibroblast like morphology and some cellcolonies showed round morphology. Macroscopic ex-amination revealed that PHBV (with or withoutchondrocytes) maintained its form for 21 days whileCaP–Gelfixs was deformed and completely degradedwithin 15 days.

In vitro histology revealed that, cells on the PHBVand CaP–Gelfixs were at subconfluent densities. Cellsadhered to both matrices after the first day of theseeding. The percentage of the plated cells on bothmatrices was gradually increased after seeding on thematerials and reached the subconfluent level 14 daysafter the seeding.

Histological sections of the chondrocyte seededmatrix composite with PAP stain revealed that cells

collagen type II.

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were adherent to matrix surfaces and they produced amixochondroid matrix (Fig. 4a and b). Cells showedprominent binucleation, cohesive group formation,

Fig. 4. (a,b) In vitro photomicrographs of the chondrocyte seeded

matrices (a) CaP Gelfix, (b) PHBV) H&E� 400. Cells maintained their

phenotype in both biomaterials.

nuclear polymorphism and contained perinuclear va-cuoles after 21 days in culture.

3.3. In vivo chondrocyte– biomaterial interaction

The introduction of an implant into osteochondraldefects with the appropriate physical structure andchemical composition may improve the reparativeresponse by providing a scaffold that allows theorganization of the reparative cells and their productswithin the defect itself [19].

At the end of 8 weeks and 20 weeks, 4 animals weresacrificed for each group and macroscopical–histologi-cal examinations were performed.

3.3.1. Macroscopical findings

Macroscopical results at 8 and 20 weeks are presentedin Fig. 5a. The score obtained according to Niederaueret al. [20] was a cumulative result of individualparameters. In Fig. 5a, the scores with both materialtypes were better when the samples were seeded withcells. This was true for both time points. Also, the scoresfor PHBV samples were higher than those of theCaP–Gelfix for both time points.

0

1

2

3

4

5

6

7

8

Samples

Mac

rosc

op

y S

core

s

Time(Weeks)

(Weeks)

With Cell With CellWithout Cell Without CellPHBVCa-P Gelfix

n=4n=4n=4n=4n=4 n=4 n=4 n=4

0

2

4

6

8

10

12

Samples

His

tolo

gy

Sco

res

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With Cell With CellWithout Cell Without Cell

Time

n=4 n=4 n=4 n=4n=4 n=4 n=4 n=4

8 20 8 20 8 20 8 20

8 20 8 20 8 20 8 20

(a)

(b)

Fig. 5. Results of repair evaluation through (a) macroscopical and, (b)

histological examination. Macroscopical scoring is between 0 and 8,

with 8 being the best (normal cartilage). Histological scoring is

between 0 and 12, with 12 being the best (normal cartilage). (R: right

knee, L: left knee, I: CaP–collagen (Gelfixs) matrix, II: PHBV matrix).

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Detailed evaluations of macroscopical results areprovided below.

At 8 weeks, the gross examinations of the knee jointsin Group One (CaP–Gelfixs) showed that there wereminimal abrasions on the opposing articulating surfacesand no inflammation of the synovial membrane andother joint tissues was noted. The regenerative tissue didnot fill the entire defect area. The margin of the graftand some rough areas on the surface were visible.Integration into native cartilage was progressing. Thecolor of the neocartilage was opaque.

In Group Two (PHBV), regenerative tissues hadsimilar color with adjacent cartilage at the end of 8weeks. Tissue integration with the native cartilage hadbeen nearly complete and resurfacing was seen. Surfaceof the implant became thicker and translucent. Mildinflammatory reaction and synovitis were seen on theopposite articular surface of the patella and femoralcondyles.

At 20 weeks, in Group One (CaP–Gelfixs), noreactions occurred on the patellar cartilage. The fissureswere observed in the neocartilage. In the cell seededCaP–Gelfixs matrices, complete resurfacing was de-tected and surface of the reparative tissue was smooth.Hardness of the reparative tissue was more than therecipient cartilage in the unseeded CaP–Gelfixs im-planted group.

In Group Two (PHBV), the color of the repair tissuewas whitish and it had a smooth surface at the end of 20weeks. Integration to host cartilage was completed.Complete resurfacing in the grafted area was seen. Mildsynovitis and osteophyte formation were seen. Synovialinflammation was not prominent in the chondrocyteseeded implants. Also color of the neocartilage wassimilar to the nature cartilage and had becometransparent.

3.3.2. Histological evaluation

Histological results at 8 and 20 weeks are presented inFig. 5b. The score is a cumulative result of five differentparameters obtained according to Wakitani et al. [17].In this figure, the scores with both material types werebetter when the samples were seeded with cells exceptPHBV at eight weeks. The scores for CaP–Gelfixsamples were higher than those of the PHBV for bothtime points.

More detailed evaluation of the histological results ispresented below.

At week 8, in Group One (CaP–Gelfixs), surface ofthe defect area was partially smooth. Reparative tissuein the defect demonstrated partially fibrocartilageformation. On the base and the edges of the defect,integration of the material to the host was evident.But bone formation was observed in the grafted area.Matrix staining for hyaline cartilage failed except thenoncalcified top cartilage zone. Hypocellularity and

cluster formation were irregular in the entire reparativetissue.

In osteochondral defect repair, it has been hypothe-sized that the invasion of cells from the subchondralbone may produce fibrous tissue [20]. Chemical proper-ties of the material allow attachment of cells withcontrolled cell-implant interactions to allow bothsynthesis of matrix components and differentiation ofthe progenitor cells [19]. The possible reason of boneinvasion in the CaP–Gelfixs group was osteoblasticdifferentiation of the adherent stem cells from the bonemarrow in to the pores of the CaP–Gelfixs. On thecontrary, bone formation in the chondrocyte seededmatrices was lower than unseeded matrices. This may beexplained as follows; presence of the cells in the scaffoldin the defect area may prevent mesenchymal cells frominvading the graft until the grafted chondrocytes haveproduced hyaline like matrices [21]. In another study,the formation of hyaline-like cartilage was determined inthe unseed matrices [20]. Cells from the adjacentcartilage may have contributed to the repair processand without in vitro seeding of chondrocytes, thesematrices have been facilitated the intrinsic repaircapacity of articular cartilage.

In this study, chondrocyte seeded CaP–Gelfixs

revealed better histological scores. Particularly roundshaped chondrocytes and metachromatic matrix stain-ing was clearly evident on the surface of the neocartilagebut endochondral bone filled up to the level of tidemark,too.

In Group Two (PHBV) hyaline like cartilage on thesurface of the regenerative tissue was observed and thisneocartilage was well integrated to the adjacent cartilageat the end of 8 weeks. Polymer remnants and cavitieswere evident around the periphery of the defect. Slightinflammatory cell infiltration was observed. The centralportion of the reparative tissue and neocartilage wasslightly depressed. The top layer consisted of mostlyhyaline cartilage. Noncalcified top layer had a rim ofchondrogenic cells and cluster formation. Unlike theGroup One (CaP–Gelfixs), bone formation in thereparative tissue was not prominent. Thickness of thecartilage was about half of the adjacent cartilage. In thedefects where unseeded matrices applied, fibrous tissuedevelopment, foreign body reaction and neovasculariza-tion in the areas that were close to host bone wereobserved. Degradation of the PHBV matrices leftcavities in the reparative tissue, but the remnants ofpolymer were observed about 1/3 of the grafted area.

At week 20, in Group One (CaP–Gelfixs), thearticular surface of the implant was partially coveredwith hyaline like cartilage (Fig. 6a). Bone formation atthe base and the side of the defect was more obviousthan PHBV matrices and therefore the composite graftfixed to the host more rigidly than PHBV. Although thescores for integration and surface filling of the defects

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Fig. 6. Histological examinations of: (a) Calcium phosphate loaded collagen (Gelfixs) foam, (b,c) PHBV foam at the end of 20 weeks. Thickness and

cellularity of the neocartilage were clearly superior than adjacent cartilage (b). Hyaline like cartilage was filled the defect (c) (H&E� 50).

G.T. Kose et al. / Biomaterials 26 (2005) 5187–5197 5195

were better than those of eight weeks, hyaline cartilagedevelopment was not significantly increased in time.Mild cluster formation and hypocellularity were alsonoted. Surface erosion and fissure formation were rarelyobserved on the top layer.

In Group Two (PHBV), hyaline like cartilage hadfilled the defect area entirely at the end of 20 weeks.Surface of the reparative tissue was smooth and steadyintegration of the grafted tissue with the adjacentnormal cartilage was observed (Fig. 6b). Thickness of

the reparative tissue was the same as that of the normalcartilage and in some areas it was thicker than adjacentcartilage. Neocartilage demonstrated cluster and colum-nar appearance of chondrogenic cells. Metachromatic,hyaline like matrix staining was also evident. Anotheraspect of the cartilage repair process was not only theformation of hyaline cartilage but also the so-calledneocartilage that protects the surrounding cartilagefrom further deterioration [22]. A slight degree ofinflammatory reaction in the reparative tissue was

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observed and it was less than that of the eighth weekspecimens. Scaffold was nearly absorbed, therefore,remnants of scaffold were rarely observed (Fig. 6c).

Chondrocyte loaded scaffold composites stimulatedrepair of hyaline like cartilage more than the unseededscaffolds. The reparative tissue which was produced bychondrocyte seeded scaffolds had better histologicalscores and marked hyaline like characteristics. At weekeight chondrocytes of the repair tissue showed pre-dominantly cluster formation, whereas at week 20chondrocytes showed both cluster formation andcolumnar appearance. The mean filling ratio of thedefect area with reparative tissue at week eight forGroup One (CaP–Gelfixs) and Two (PHBV) were 81%and 72%, respectively, and at week twenty 96% and91%, respectively. No bone formation was observed atthe articular surface in both groups.

4. Conclusion

The scaffolds (CaP–Gelfixs and PHBV) evaluated inthis study allowed sufficient adhesion and proliferationof chondrocytes under in vitro conditions. Also thesescaffolds supported the differentiated chondrocytephenotype and induced the production of cartilagematrix. In vivo histological scores revealed that chon-drocytes in the matrices remained adherent and activelyparticipated in neocartilage formation in the wholephase of repair process.

In this study, cartilage defects treated with chondro-cyte seeded PHBV showed better healing response thanchondrocyte seeded CaP–Gelfixs matrices. Chondro-cyte seeded matrices have better results becauseadherent cells were not dispersed by joint fluid andremained stable in the defect. Reparative tissues werenot replaced by mesenchymal cells and no degenerationon the normal articular surfaces was seen; in the PHBVgroup, however, a mild synovial inflammation wasobserved.

This study also showed that PHBV matrices per-mitted appropriate gradual degradation and allowedtissue remodelling to take place. PHBV matrices showedthe early cartilage formation resembling normal articu-lar cartilage. In the late phase of the experiment, maturehyaline like cartilage formation was observed inchondrocyte seeded PHBV scaffolds. Thickness of therepaired cartilage was almost the same or in some casestwice as that of the adjacent cartilage.

CaP–Gelfixs showed scant hyaline-like cartilageformation in the early phase of the experiment, but inthe late phase moderate hyaline-like cartilage formationwas observed in chondrocyte seeded CaP–Gelfixs

implants. In CaP–Gelfixs group, fibrocartilage forma-tion and bone invasion was noted in the late phase of theexperiment.

It was observed in this study that PHBV matrices aremore appropriate carriers compared to the CaP–Gelfixs

for in vitro chondrocyte tissue engineering.It thus appears that tissue engineering of cartilage

using collagen matrix containing calcium phosphate(CaP–Gelfixs) and PHBV matrices has a seriouspotential. Studies on the enhancement of the neocarti-lage repair and increasing cell attachment are inprogress.

Acknowledgement

The authors would like to acknowledge the contribu-tions of Assoc. Prof. Dr. Hasan Bilgili of AnkaraUniversity, Faculty of Veterinary Medicine, Depart-ment of Orthopaedic Surgery in the in vivo phase.

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