Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.     

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self-repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)-2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long-term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real-time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP-2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP-2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP-2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT.     

Viability,proliferation and phenotype maintenance in cryopreserved human iliac apophyseal chondrocytes

Cryopreservation preserves cells at low temperature and creates a reserve for future use while executing the clinical translation. Unlike articular chondrocyte, cryopreservation protocol and its outcome are not described in iliac apophyseal chondrocytes, a potential source of chondrocytes in cartilage engineering. This study for the first time describes the cryopreservation of human iliac apophyseal chondrocytes. Four cartilage samples were procured from iliac crests of children undergoing hip surgery after consent. The total chondrocyte yield was divided into two groups. First group was grown as monolayer while second group was cryopreserved following the slow cooling method in the medium containing 10 % Dimethyl sulfoxide for 3 months. Group two cells were also grown as a monolayer following thawing. Viability, time to confluence, population doubling time and phenotype maintenance were compared for both the groups. Viability was 65.75 % after 3 months of cryopreservation at ?196 °C, as compared to 94.19 % for fresh chondrocytes (p = 0.001). Fresh and cryopreserved cells reached confluence on 10th and 15th day of culture respectively. Population doubling time was significantly more in fresh than cryopreserved chondrocytes on 10th (p = 0.0006) and 15th day (p = 0.0002) in culture. Both fresh and cryopreserved cells maintain their chondrocyte phenotype as assessed by immunocytochemistry. Relative gene expression by real time polymerase chain reaction showed similar upregulation of mRNA of Collagen 2, SOX 9, Aggrecan and Collagen 1 in cryopreserved chondrocyte as compared to fresh chondrocyte. Iliac apophyseal chondrocytes cryopreserved for 3 months maintained the phenotype successfully 2 weeks after thawing in culture. The viability and proliferation rates after thawing were adequate for a clinical translation of these cells.     

Xenogeneic transplantation of articular chondrocytes into full-thickness articular cartilage defects in minipigs: fate of cells and the role of macrophages

Xenogeneic or allogeneic chondrocytes hold great potential to build up new cartilage in vivo. However, immune rejection is a major concern for the utility of universal donor-derived cells. In order to verify the reported immune privilege of chondrocytes in vivo, the aim of this study was to assess engraftment of human articular chondrocytes (HAC) in minipig knee cartilage defects and their contribution to cartilage regeneration. HAC were transplanted matrix-assisted within two hydrogels into full-thickness cartilage defects of minipigs or implanted ectopically into immune deficient mice to assess redifferentiation capacity. At 2 and 4 weeks after surgery, cell-persistence and host cell invasion were monitored by species-specific in situ hybridization and RT-PCR. Early tissue regeneration was evaluated by histomorphometry and a modified O’Driscoll score. HAC capable of successful in vivo chondrogenic redifferentiation persisted at ectopic sites for 4 weeks in both carrier materials. Early defect regeneration involved extensive host cell invasion and a decline of HAC to less than 5 % of initial cell numbers in 6/12 defects within 2 weeks. Few clusters of persisting HAC within collagen type II-rich tissue were surrounded by porcine macrophages. Four weeks after cell transplantation, most of the defects contained well-integrated cell-rich tissue free of human cells with no apparent difference between hydrogel carriers. In summary, HAC failed to engraft in porcine articular cartilage defects despite their ability for successful in vivo redifferentiation. The co-localization of macrophages to hydrogel-implanted HAC suggests active graft rejection without evidence for an immune-privileged status of xenogeneic chondrocytes in a large animal joint.     

Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study

To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide^®), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O’Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.     

Repair of Ear Cartilage Defects with Allogenic Bone Marrow Mesenchymal Stem Cells in Rabbits

The study aims to investigate the feasibility of repairing cartilaginous defects with chondrocytes induced from allogenic bone marrow mesenchymal stem cells (BMMSC) in rabbits’ ear. BMMSCs were isolated and purified from New Zealand rabbits, in vitro amplified, and cultured in chondrocyte induction medium in order to acquire chondrocytes. After 3 weeks of induction, their phenotypes were confirmed as chondrocytes, then they were implanted onto novel polymeric scaffolds made from Poly (dl-lactide-co-glycolide) (PLGA) embedded with chitosan nonwoven cloth. The experimental group was transplanted with tissue engineering cartilaginous grafts composed of chondrogenetic BMMSC/scaffolds; the scaffold group was treated with scaffolds without cells, while in the control group, nothing was implanted. Specimens were taken at 6, 12, and 18 weeks after implantation, and the healing condition was observed by hematoxylin-eosin staining and toluidine blue staining. The right and left ears with cartilage defects of eighteen rabbits were randomly divided into three groups. In the experimental group, after 18 weeks of transplantation, the gross observation indicated that the cartilaginous defects were completely repaired by chondrocytes with smooth surface and similar color with the surrounding tissue. Hematoxylin-eosin staining and toluidine blue staining suggested that the defective area was filled with mature cartilage cells with obvious lacunae but without obvious boundaries with the normal cartilage tissue, and that the new cartilage cells were evenly distributed with homogeneously dyed cytoplasm and smaller in size. The chondrocyte induced from allogenic BMMSC can be used to repair cartilage defects in rabbit’s ear.     

A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage

This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2–1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2–1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.     

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo . When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo , the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo . These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo , however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.     

Composite System of PLCL Scaffold and Heparin-Based Hydrogel for Regeneration of Partial-Thickness Cartilage Defects

Delivering isolated chondrocytes with matrix is a promising approach to promote the cartilage repair. The present study attempted to combine the advantages of porous scaffold and hydrogel in delivering chondrocytes to partial-thickness cartilage defects. An electrospun, gelatin-incorporated PLCL scaffold mechanically similar to natural cartilage was fabricated, and chondrocytes were seeded using an injectable heparin-based hydrogel for efficient cell seeding. The scaffold/hydrogel composite showed more enhanced expression of chondrogenic genes and production of GAGs than those prepared without hydrogel. In addition, significant cartilage formation showing good integration with surrounding, similar to natural cartilage, was observed by scaffold/hydrogel composite system in partial-thickness defects of rabbit knees while no regeneration was observed in control defects. Although no exogenous chondrogenic factors were added, it was evident that the scaffold/hydrogel composite system was highly effective and better than the scaffold alone system without hydrogel for cartilage regeneration both in vitro and in vivo.     

Src kinase inhibition promotes the chondrocyte phenotype

Regulated differentiation of chondrocytes is essential for both normal skeletal development and maintenance of articular cartilage. The intracellular pathways that control these events are incompletely understood, and our ability to modulate the chondrocyte phenotype in vivo or in vitro is therefore limited. Here we examine the role played by one prominent group of intracellular signalling proteins, the Src family kinases, in regulating the chondrocyte phenotype. We show that the Src family kinase Lyn exhibits a dynamic expression pattern in the chondrogenic cell line ATDC5 and in a mixed population of embryonic mouse chondrocytes in high-density monolayer culture. Inhibition of Src kinase activity using the pharmacological compound PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine) strongly reduced the number of primary mouse chondrocytes. In parallel, PP2 treatment increased the expression of both early markers (such as Sox9, collagen type II, aggrecan and xylosyltransferases) and late markers (collagen type X, Indian hedgehog and p57) markers of chondrocyte differentiation. Interestingly, PP2 repressed the expression of the Src family members Lyn, Frk and Hck. It also reversed morphological de-differentiation of chondrocytes in monolayer culture and induced rounding of chondrocytes, and reduced stress fibre formation and focal adhesion kinase phosphorylation. We conclude that the Src kinase inhibitor PP2 promotes chondrogenic gene expression and morphology in monolayer culture. Strategies to block Src activity might therefore be useful both in tissue engineering of cartilage and in the maintenance of the chondrocyte phenotype in diseases such as osteoarthritis.     

The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell’s chondrogenic induction.     

Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints

Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

Novel effects of CCN3 that may direct the differentiation of chondrocytes

Identification and characterization of local molecules directing the differentiation of chondrocytes to either transient or permanent cartilage are major issues in cartilage biology. Here, we found CCN family protein 3 (CCN3) was abundantly produced in rat developing epiphyseal cartilage. Evaluations in vitro showed that CCN3 repressed epiphyseal chondrocyte proliferation, while promoting matrix production in multiple assays performed. Furthermore, CCN3 enhanced the articular chondrocytic phenotype; whereas it repressed the one representing endochondral ossification. Additionally, the phenotype of growth plate chondrocytes and chondrogenic progenitors also appeared to be affected by CCN3 in a similar manner. These findings suggest a significant role of CCN3 in inducing chondrocytes to articular ones during joint formation.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Chondrogenic differentiation potential of osteoarthritic chondrocytes and their possible use in matrix-associated autologous chondrocyte transplantation

Introduction  

Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA.     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

Development and phenotypic characterization of a high density in vitro model of auricular chondrocytes with applications in reconstructive plastic surgery

Cultivation of phenotypically stable auricular chondrocytes will have applications in autologous chondrocyte transplantation and reconstructive surgery of cartilage. Chondrocytes grown in monolayer culture rapidly dedifferentiate assuming a fibroblast-like morphology and lose their cartilage-specific pattern of gene expression. Three-dimensional high-density culture models mimic more closely the in vivo conditions of cartilage. Therefore, this study was undertaken to test whether the high-density cultures might serve as a suitable model system to acquire phenotypically and functionally differentiated auricular chondrocytes from porcine cartilage. Freshly isolated porcine auricular chondrocytes were cultured for 7 passages in monolayer culture. From each passage (passage 0 and 1-7) cells were introduced to high-density cultures and examined by transmission electron microscopy. Western blotting was used to analyse the expression of cartilage-specific markers, such as collagen type II and cartilage specific proteoglycan, fibronectin, cell adhesion and signal transduction receptor beta1-integrin, matrix metalloproteinases (MMP-9, MMP-13), cyclo-oxygenase (COX)-2 and the apoptosis commitment marker, activated caspase-3. When dedifferentiated auricular chondrocytes from monolayer passages 0-4 were cultured in high-density culture, they recovered their chondrocytic phenotype and formed cartilage nodules surrounded by fibroblast-like cells and synthesised collagen type II, proteoglycans, fibronectin and beta1-integrins. However, chondrocytes from monolayer passages 5-7 did not redifferentiate to chondrocytes even when transferred to high-density culture, and did not synthesize a chondrocyte-specific extracellular matrix. Instead, they produced increasing amounts of MMP-9, MMP-13, COX-2, activated caspase-3 and underwent apoptosis. Three-dimensional high-density cultures may therefore be used to obtain sufficient quantities of fully differentiated auricular chondrocytes for autologous chondrocyte transplantation and reconstructive plastic surgery.