Rabbit Articular Chondrocytes Research Articles

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.

Role of fibroblast growth factor 8 (FGF8) in animal models of osteoarthritis

IntroductionFibroblast growth factor 8 (FGF8) is isolated as an androgen-induced growth factor, and has recently been shown to contribute to limb morphogenesis. The aim of the present study was to clarify the role of FGF8 in animal models of osteoarthritis (OA).MethodsThe expression of FGF8 in the partial meniscectomy model of OA in the rabbit knee was examined by immunohistochemistry. The effect of intraperitoneal administration of anti-FGF8 antibody was tested in a model of OA that employed injection of monoiodoacetic acid or FGF8 into the knee joint of rats. The effect of FGF8 was also tested using cultured chondrocytes. Rabbit articular chondrocytes were treated with FGF8 for 48 hours, and the production of matrix metalloproteinase and the degradation of sulfated glycosaminoglycan in the extracellular matrix (ECM) were measured.ResultsThe expression of FGF8 in hyperplastic synovial cells and fibroblasts was induced in the meniscectomized OA model, whereas little or no expression was detected in normal synovium. Injection of FGF8 into rat knee joints induced the degradation of the ECM, which was suppressed by anti-FGF8 antibody. In the monoiodoacetic acid-induced arthritis model, anti-FGF8 antibody reduced ECM release into the synovial cavity. In cultured chondrocytes, FGF8 induced the release of matrix metalloproteinase 3 and prostaglandin E2, and caused degradation of the ECM. The combination of FGF8 and IL-1α accelerated the degradation of the ECM. Anti-FGF8 antibody suppressed the effects of FGF8 on the cells.ConclusionFGF8 is produced by injured synovium and enhances the production of protease and prostaglandin E2 from inflamed synoviocytes. Degradation of the ECM is enhanced by FGF8. FGF8 may therefore participate in the degradation of cartilage and exacerbation of osteoarthritis.

The roles of tyrosine kinase and phosphatase in the activation process of the volume sensitive Cl− current (ICl,vol) in rabbit articular chondrocytes

The roles of tyrosine kinase and phosphatase in the activation process of the volume sensitive Cl− current (ICl,vol) in rabbit articular chondrocytes

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells–fibrin suspension and polymerized by dropping thrombin–sodium chloride (CaCl2) solution. A PLGA–cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold–cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.

Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation

Baculovirus is an emerging gene delivery vector, thanks to a number of unique advantages. Herein, we genetically modified the rabbit articular chondrocytes with a recombinant baculovirus (Bac-CB) encoding bone morphogenetic protein-2 (BMP-2), which conferred high level BMP-2 expression and triggered the re-differentiation of dedifferentiated third passage (P3) chondrocytes in the monolayer culture. The transduced and mock-transduced P3 cells were seeded into porous scaffolds and cultured in either the dishes or the rotating-shaft bioreactor (RSB), a novel bioreactor imparting a dynamic, two-phase culture environment. Neither mock-transduced constructs in the RSB culture nor the Bac-CB-transduced constructs in the static culture grew into uniform cartilaginous tissues. Only the Bac-CB-transduced constructs cultured in the RSB for 3 weeks resulted in cartilaginous tissues with hyaline appearance, uniform cell distribution, cartilage-specific gene expression and considerably enhanced cartilage-specific extracellular matrix deposition, as determined by histological staining, reverse transcription-PCR analyses and biochemical assays. This is the first study demonstrating that combination of baculovirus-mediated growth factor expression and RSB culture synergistically enhanced in vitro creation of cartilaginous tissues from dedifferentiated chondrocytes. Since baculovirus transduction is generally considered safe, this approach represents a viable alternative to stimulate the formation of engineered cartilage in a more cost-effective way than the growth factor supplementation.

17 -Oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors

To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease adamts-5 expression in interleukin 1β-treated chondrocytes

Objective:To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesised sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1β.Methods:Primary cultured rabbit articular chondrocytes...

Apoptosis induced by NO via phosphorylation of p38 MAPK that stimulates NF‐κB, p53 and caspase‐3 activation in rabbit articular chondrocytes

Nitric oxide (NO), reported as an important inducer of apoptosis, plays a considerable role in the pathogenetic mechanisms of articular diseases. This research aimed at investigating the role of p38 MAPK signal transduction pathway on apoptosis induced by NO in rabbit articular chondrocytes. In the present study, NO was produced by a novel NO donor NOC-18. Rabbit articular chondrocytes were cultured as monolayer, and the first passage cells were used for the experiments. We detected apoptosis induced by NO using Annexin V-FITC/PI flow cytometry and TUNEL assay. Measurement of caspase-3 has reflected its activity level. Western blotting was performed to show the protein expressions of p38, NF-kappaB, p53 and caspase-3. Furthermore, we examined the inhibitory effects in the NO pathway with p38-specific inhibitor SB203580. Treatment with NOC-18 caused accelerated apoptosis in a concentration dependent manner. This acceleration was able to be reduced when added to SB203580. Besides, the inhibitor could significantly decrease NO-induced p38, NF-kappaB, p53 and caspase-3 protein expressions, as well as caspase-3 intracellular activity (P<0.05). These results suggest that p38 MAPK signal transduction pathway is critical to NO-induced chondrocyte apoptosis, and p38 plays a role by way of stimulating NF-kappaB, p53 and caspase-3 activation.

Poster 245: In Vitro Model of Cartilage Degradation With Chondrocytes in a Three-Dimensional Culture System–Time-Dependent Involvement of MMP-3 and TIMP-1, -2 and -3

A trial to evaluate the protective role of TIMP-1, −2 and −3 against IL-1β-induced cartilage degradation using rabbit articular chondrocytes in a three-dimensional culture system. Chondrocytes were cultured in alginate beads with IL-1 β or TGF-β alone or both. The glycosaminoglycan(GAG) concentration in the culture medium was determined by use of the DMMB assay; and the levels of TIMP-1, −2 and proMMP-3 were measured with their respective sandwich EIAs. Sections of the beads were prepared and stained with Toluidine blue or anti-TIMP-1 −2, −3 antibodies. The number of chondrocytes negative for pericellular proteoglycan staining [PG(-)chondrocyte], the number of TIMP-positive chondrocytes [TIMP (+)chondrocyte] and positive staining of TIMP-3 in the extracellular matrix was counted. RT-PCR was performed to evaluate the gene expressions of TIMP-1, −2, −3 and MMP-3. The number of TIMP-1(+)chondrocytes, TIMP-1 concentration in the culture media and TIMP-1-gene expression were all increased maximally as early as 6 hours after IL-1β stimulation, and were then gradually decreased. The number of positive staining of TIMP-3 in the cell was increased after 12 hours. GAG and proMMP-3 concentrations in the culture media were increased gradually with time. The numbers of both TIMP-3(+)chondrocytes and positive staining of TIMP-3 in the extracellular matrix were significantly increased in TGF-β group after 12 hours pretreatment compared with those in IL-1β group. ProMMP-3 concentration in the culture media was significantly decreased compared with IL-1β group through all the time examined. TIMP-1 was suggested to play a protective role for the early stage of cartilage degradation induced by IL-1β. TIMP-3, however, seems to follow TIMP-1 in its protective role. TGF-β augmented protective effects of TIMPs.

In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects

To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell–collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur ( n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM±3.5%) in rabbit articular chondrocytes, 74.7% (SEM±1.8%) in human articular chondrocytes and 52.7% (SEM±5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM±8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular β-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.

Human Amniotic Membrane as a Delivery Matrix for Articular Cartilage Repair

The purpose of this study is to evaluate the feasibility of human amniotic membrane (HAM) as a chondrocyte carrier by assessing cell proliferation and maintenance of phenotype in vitro and cartilage regeneration in vivo. Intact HAM was treated with 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA) for 15 min and the epithelial cells removed to make a denuded HAM. Rabbit articular chondrocytes were then seeded on three different HAM substrates: the epithelial side of intact HAM (IHE), basement side of denuded HAM (DHB), and stromal side of denuded HAM (DHS). These cell-substrate specimens were cultured for up to 4 weeks, and cell proliferation rate and phenotypic stability were examined at weeks 1 and 4. While chondrocytes grew in monolayer fashion on the surface of IHE and DHB substrates, the cells seeded in DHS penetrated and spread into the whole thickness of the stromal layer. The proliferating activity of chondrocytes in DHB was continuously up-regulated. A similar proliferating activity was observed in DHS in the first week, which remained stable for up to 4 weeks. The expression of type II collagen gradually increased with time in the DHS group, while it gradually decreased in the DHB group or was not detected at all in the IHE group. These results suggested that denuded HAM was able to support chondrocyte proliferation and maintenance of phenotype in vitro, seemingly more favorable when DHS was used. Based on this data, the DHS with chondrocytes was used to cover rabbit osteochondral defect with the stromal side facing in. The defect area was successfully regenerated with hyaline cartilage in the Safranin-O stain and International Cartilage Repair Society (ICRS) scoring after 8 weeks of implantation. In conclusion, our findings suggest that denuded HAM could be one of the ideal cell carrier matrices for cartilage regeneration.

Effects of growth factors on heparin‐carrying polystyrene‐coated atelocollagen scaffold for articular cartilage tissue engineering

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

In Vivo Cartilage Tissue Engineering Using a Cell‐Derived Extracellular Matrix Scaffold

We have observed in our previous study that a cell-derived extracellular matrix (ECM) scaffold could assure the growth of a cartilage tissue construct in vitro. The purpose of the present study was to evaluate the feasibility of a chondrocyte-seeded cell-derived ECM scaffold by implanting it in vivo in nude mouse. A porous cell-derived ECM scaffold was prepared with a freeze-drying protocol using porcine chondrocytes. Rabbit articular chondrocytes were seeded onto the scaffold and cultured for 2 days in vitro, and then implanted into the nude mouse subcutaneously. They were retrieved at 1, 2, and 3 weeks postimplantation. Under macroscopic analysis, the cartilage-like tissue formation matured with time and developed a smooth, white surface. Contrary to the control (in which no cells were seeded), the size of the neocartilage tissue increased slightly by the third week and remained more stable. Total glycosaminoglycan (GAG) content and the GAG/DNA ratio increased significantly with time in the chemical analysis. The histology exhibited a sustained accumulation of newly synthesized sulfated proteoglycans. Immunohistochemistry, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) clearly identified type II collagen at all time points. Compressive strength of in vivo neocartilage increased from 0.45 +/- 0.06 MPa at 1 week to 1.18 +/- 0.17 MPa at 3 weeks. In conclusion, this study demonstrated that the cell-derived ECM scaffold could provide chondrocytes with favorable in vivo environment to produce a hyaline-like cartilage tissue.

Paclitaxel regulates cell cycle, dedifferentiation and COX‐2 expression in primary culture

Paclitaxel is one of the chemotherapeutic agents frequently used in the treatment of various cancers. Paclitaxel works by promoting assembly of microtubules and stabilizing them, prventing depolymerzation. This inability to depolymerize microtubules prevents cellular replication. In this study, we used rabbit articular chondrocytes as models to determine whether paclitaxel-induced G2/M arrest might be associated with cell cycle related proteins. In chondrocytes during paclitaxel treatment, normally flat cells became rounded and in shape. Flow cytometry analysis revealed that paclitaxel accumulate cells in the G2/M phase of the cell cycle. To further cell cycle delay is mediated by mitotic cell cycle related proteins that include Cyclin B, Cdc2, Rb. Taken together, paclitaxel induced cell cycle delay. Paclitaxel-mediated mitotic arrest is associated with the modulation of cell cycle related proteins expression. Also we next investigated the effects of paclitaxel on dedifferentiation and cyclooxygenase-2 (COX-2) expression in rabbit articular chondrocytes. Immunohistochemistry and immunofluorescene study revealed that paclitaxel caused a induced dedifferentiation and suppressed COX-2 expression. Furthermore, paclitaxel enhanced prostaglandin E2 synthesis dependent of time course but independent of concentration. Thus, our data suggest that paclitaxel contributes to the expression of COX-2 and dedifferentiation in articular chondrocytes.

ERK-1/-2 and p38 Kinase Oppositely Regulate 15-deoxy-Δ12,14-prostaglandinJ2-Induced PPAR-γ Activation That Mediates Dedifferentiation But Not Cyclooxygenase-2 Expression in Articular Chondrocytes

Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-γ, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-γ activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-γ. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-γ activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-γ activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-γ-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-γ-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-γ and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.

Rabbit Articular Chondrocytes Seeded on Collagen-Chitosan-GAG Scaffold for Cartilage Tissue Engineering In Vivo

In this study, we prepared a tri-copolymer porous matrices by natural polymer, collagen (Col), Chitosan (Chi) and Chondroitin (CS). Rabbit articular chondrocytes were isolated from the shoulder articular joints of a rabbit, seeded in Col-Chi-CS scaffold, and implanted subcutaneously in the dorsum of athymic nude mice to tissue engineer articular cartilage in vivo. In vitro studies show that Chondrocytes adhered to the scaffold, where they proliferated and secreted extracellular matrices with time, filling the space within the scaffold. The results of hematoxylin and eosin staining scanning electron microscopy revealed that most of the chondrocytes maintained their typically rounded morphology. After 28 days of culture within Col-Chi-CS scaffold in vitro, the results of histological staining showed forming of cartilage-specific morphological appearance and structural characteristics such as lacunae. Subcutaneous implantation studies in nude mice demonstrated that a homogeneous cartilaginous tissue, which was similar to those of natural cartilage, formed when chondrocytes were seeded in Col-Chi-CS matrix after implant 12 weeks. The tri-copolymer matrix could therefore have potential applications as a three-dimensional scaffold for cartilage tissue engineering.

The Phylogenetically Conserved Molluscan Chitinase-like Protein 1 (Cg-Clp1), Homologue of Human HC-gp39, Stimulates Proliferation and Regulates Synthesis of Extracellular Matrix Components of Mammalian Chondrocytes

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Encapsulation of Chondrocytes in Photopolymerizable Styrenated Gelatin for Cartilage Tissue Engineering

We have developed a photopolymerizable styrenated gelatin that can cross-link through polymerization induced by irradiation with visible light. The purpose of this study was to investigate the feasibility of using photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation. As visible light activates camphorquinone added as a photoinitiator, free radicals induce the polymerization of the gelatin macromer; the styrenated gelatin then becomes cross-linked. Rabbit articular chondrocytes were cultured in styrenated gelatin hydrogels and also in collagen gels as a control. After being cultured in the gels, the cells were collected from both gels and counted. Reverse transcriptase-polymerase chain reaction, histological examination, and quantification of the synthesized glycosaminoglycan were performed. On average, 26% of the embedded cells were collected from the gelatin hydrogel immediately after the crosslinking reaction. The surviving chondrocytes expressed the mRNA of type II collagen and aggrecan core protein and produced a cartilaginous matrix throughout the gelatin after 3 weeks. A slightly insufficient accumulation of the matrix was found in the internal region of the gelatin hydrogels, suggesting that less permeability for nutrients due to the high concentration and closely packed structure resulted in less cell viability. Although some limitations became evident, these results indicate that it may be possible to use photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation.

Biological characterization of rabbit's articular chondrocytes by confluent culture in vitro

To obtain large amount of differentiated chondrocytes in vitro, examine and compare the biological characterization of rabbits' articular chondrocyte cultured in different density in vitvo. From November 2001 to June 2004, articulate tissues were obtained from the joints of the adult rabbits. Chondrocytes were isolated from the cartilage tissue with type II collagenase digestion and cultured in DMEM/F-12 supplemented with 20% fetal bovine serum (FBS). The chondrocytes were cultured with low density of monolayer culture and high density of confluent culture respectively. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. When chondrocytes cultured in monolayer and in low density, it proliferated rapidly during the three generations, but with the same time, dedifferentiation was also rapid. After the third passage, most of the passage cells lost the phenotype, and the proliferation also stagnated. While chondrocytes cultured in high density, dedifferentiation slowed down. And even the phenotypes of the dedifferentiated chondrocyte which were cultured in low density could reduced partly by followed high density culture. Culture chondrocytes by high density in vitro can effectively maintain the differentiated phenotype of chondrocyte. It also keeps the proliferation character as monolayer culture. The dedifferentiated chondrocyte caused by many passages could redifferentiate partly. So it is indicated that confluent culture of original or expanded chondrocytes in high density is a better culture methods than culture in low density.

In vitro gene transfection into rabbit articular chondrocytes mediated by recombinant adeno-associated virus vector

To investigate the in vitro efficiency of recombinant adeno-associated virus (rAAV2) vector-mediated gene transfection into the rabbit articular chondrocytes. Articular chondrocytes were isolated from New Zealand rabbits, cultured, and transfected with rAAV2 containing enhanced green fluorescent protein (eGFP) gene at different values multiplicity of infection (MOI). Successful transfection of chondrocytes was confirmed by detection of recombinant enhanced green fluorescent protein using inverted fluorescence microscopy. The transfection efficiency was determined using the fluorescence-activated cell sorter. A maximum level of 4.76% was set as the background autofluorescence in live, uninfected chondrocytes. The percentage of eGFP-positive chondrocytes increased in a vector dose-dependent manner. When the MOI value was <1 x 10(5) v.g./cell the transfection rate was positively linearly correlated with the dose. However, the MOI > 10(5) v.g./cell did not markedly increase the transfection rate. The greatest population of eGFP-expressing cells was 97.7% and the highest mean fluorescence intensity was 17.4 7 days after transfection. EGFP gene expression remained high until 56 days after transfection. The in vitro efficiency of rAAV2 vector-mediated eGFP gene transfection into rabbit articular chondrocytes was high. The eGFP expression can be maintained for a rather long time. EGFP gene is an ideal tracer for chondrocytes transduction.