Research on Hydrogen Peroxide Induced Degradation of Human Articular Chondrocyte Extracellular Matrix in Vitro / 中国运动医学杂志

Objective To explore the influence of elevating the oxygen pressure on articular chondrocytes in vitro.Method A hydrogen peroxide induced human articular chondrocyte damage model was established.Then the articular chondrocyte viability was detected using the CCK-8 kit.Collagen Ⅱ(COL Ⅱ),The expression levels of aggrecan (ACAN),matrix metalloproteinase 13 (MMP13) and adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were detected using the realtime PCR and Western blotting.Result The viability of articular chondrocytes improved at 12 h but decreased at 24 h after the stimulation of hydrogen peroxide.Twenty-four hours later,the average expression level of COL Ⅱ and ACAN decreased(P＜0.05),while that of MMP13 and ADAMTS5 elevated(P＞0.05).Conclusion Hydrogen peroxide induced elevation of the extracellular oxygen pressure can influence the synthesis and degradation of the articular chondrocyte extracellular matrix.

The expression of type Ⅱ collagen in articular chondrocyte of osteoarthritis / 复旦学报（医学版）

Objective To investigate the expression of type Ⅱ collagen in the articular chondrocyte of osteoarthritis (OA) patients and normal human. Methods The samples of articular cartilage were obtained from the patients undergoing total joint replacement, including 8 primary OA patients, 8 secondary OA patients and 9 normal subjects. Type Ⅱ collagen expression in chondrocyte was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The expressin of type Ⅱ collagen mRNA in normal OA group was higher than that in primary OA group and secondary OA group with a statistical difference (P=0.014), while there was no statistical difference between primary OA group and secondary OA group(P=0.716). Conclusions The reduction of type Ⅱ collagen expression leads to the change of collagen directly and possibly plays an important role in OA, which is the common pathway of the occurrence of both the primary and secondary OA.

Cross-talk between Integrin and SMAD Signal Pathway in Articular Chondrocyte / 대한정형외과연구학회지

PURPOSE: Identifying the signal cross-talk between integrin signaling cascade and TGF-beta 1 signaling cascade in articular chondrocytes. MATERIALS AND METHODS: To analyze integrin or TGF-beta 1 mediated signaling pathways from extracellular stimuli, type II collagen was coated on the cell culture plate and TGF-beta 1 was added to cell culture media. Chondrocytes were cultured in the conditioned media with each or both stimuli. Altered activation of signaling proteins detected with western blot technique. RESULTS: More rapid attachment of cells was observed in the type II collagen coated group than non-coated group. The phosphorylated SMAD 2 and 3 were expressed in the type II collagen coated group and synergistically up-regulated phosphorylation in the co-treated group. The phosphorylated FAK at tyrosine 925 was activated by TGF-beta 1 treatment and synergistically up-regulated by both stimuli. But there was no meaningfully changed phosphorylation of extracellular signal regulated protein kinase (ERK) 1/2 and p38, as known downstream molecules of FAK cascade. CONCLUSION: This result means that SMAD 2, SMAD 3 and tyrosine 925 of FAK are involved in this signal cross-talking in articular chondrocytes.

Mitochondrial DNA deletion mutations in articular chondrocytes of cartilage affected by osteoarthritis / 中南大学学报(医学版)

Objective To detect the changes of mitochondrion DNA (mtDNA) sequence in articular chondrocytes of cartilage affected by osteoarthritis and to clarified the pathogenetic mechanism of osteoarthritis. Methods We analyzed the mtDNA 4 977 bp deletion mutations of articular chondrocytes in 10 patients with osteoarthritis and 3 normal cartilages using the gap-PCR amplification method. We designed a two round PCR detection method, in which total DNA was isolated from articular chondrocytes as the template of the first round PCR reaction and products from the first round were the template in the second round reaction. Results The results of the first rounds of PCR reaction showed the mtDNA 524 bp amplified products in the osteoarthritis group and in the corresponding peripheral blood samples were not detected, but the 533 bp products were detected. However, the results of the second round reaction revealed that the 524 bp zones were detected in 2 of the 10 osteoarthritis patients and the corresponding peripheral blood samples were not detected. The 533 bp products were detected in all specimens. The mtDNA 524 bp amplified products in all the normal articular chondrocytes and the corresponding peripheral white blood cells contrast were not detected in both rounds PCR. Conclusion This was the first study to evaluate the mtDNA 4 799 bp large fragment deletion mutational accumulation between nt8 470～nt13 447 of articular chondrocytes in osteoarthritic cartilage. Osteoarthritis may be related to mtDNA mutation of articular chondrocytes.

Effects of IGF (Insulin-like growth factor) on sub-populated articular chondrocytes by Percoll density gradient / 대한정형외과연구학회지

PURPOSE: Articular chondrocytes have been known to have heterogeneity in articular cartilage. The different responses of chondrocytes to various cytokines and growth factors have been reported. These variations are likely a result of metabolic differences among the cell populations. We used the Percoll density gradient method to separate chondrocytes from articular cartilage into distinct subpopulations. Several growth factors are known to enhance the synthesis of cartilage matrix. In particular, IGF has specific anabolic effects. Addition of IGF to chondrocytes increased the synthesis of proteoglycans and collagen type-II while inhibiting the degradation and release of proteoglycans. MATERIALS AND METHODS: Chondrocytes were isolated from rabbit knee articular cartilage by collagenase digestion. In brief, male rabbits weighing 250g were euthanized by injecting an overdose of Nembutal, and nonfibrillated articular cartilage of the knee was removed by sterile dissection. Isotonic Percoll was mixed with 10x PBS to give a 60% stock solution. This was further diluted with PBS to give Percoll concentrations of 10, 20, 30, 40, 50, and 60%. RT-PCR, western blot analysis, immunocytochemistry, and immunohistochemistry were done for examination of collagen type II and aggrecan as the specific marker of extracellular matrix and proteoglycan synthesis on cultured chondrocytes. RESULTS: The sub-populated cells were proliferated variously. On the other hand, the addition of IGF to the sub-populated cells increased the proliferation in all fractions. Also the expression of collagen type-II and TIMP-2 was increased by IGF treatment. After alginate culture, collagen type-II expression was not significantly different between the IGF treated and the control groups in high density fractions. However, the addition of IGF to chondrocytes increased the expression of collagen type-II in low density fractions. The expression of collagen type-II after IGF addition was decreased in monolayer culture while it was increased in alginate culture. CONCLUSION: The effects of IGF are various among the subpopulated chondrocytes. These results will provide useful information for the separation of articular chondrocytes with an active metabolic activity and extracellular matrix for the investigation of the pathogenesis of articular cartilage.

Development of Bioreactor for Subjecting Cell to Versatile Physical Forces / 대한정형외과연구학회지

PURPOSE: Articular cartilage is under continuous mechanical stresses during daily activity. The mechanical force must also be applied during the culturing process to produce a phenotypically correct tissue. We have developed bioreactor, capable to apply the three main fluid-induced stresses: shear stress, compression, and hydrostatic pressure. The objective of this study was to investigate the effects of bioreactor on chondrocyte proliferation and matrix synthesis in articular chondrocytes and to determine the most appropiate chondrogenesis biomechanical environment. MATERIALS AND METHODS: Articular cartilage was harvested from the rabbit knee. Isolated chondrocytes from articular cartilage were cultured in static culture and bioreactor culture. Bioreactor culture condition was fluid rate of 0.2 cm/sec and shear stress of 0.6x10-3 dyn/cm2 After 3 days, the effects of fluid-induced shear stress were evaluated by measuring the cell proliferation, observation of cell morphology and expression of cartilage specitic ECM using Histology, and Immunocytochemistrical staining. RESULTS: We have developed bioreactor and subjected chondrocytes to fluid-induced shear stress of 0.6x10-3 dyn/cm2 for 3 days. We observed changes in chondrocyte shape, orientation, and nodule formation. In metabolic studies, the application of fluid-induced shear stress to articular chondrocytes resulted in a significant increase in the proliferation of chondrocytes and the synthesis of type II collagen compared to that of in the static culture. CONCLUSION: From these results, it was concluded that the bioreactor which we developed produced appropriate chondrogenesis biomechanical environment.

The Modulation of Integrin Expression by the Extracellular Matrix in Articular Chondrocytes

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.

The effect of proliferation and phenotype by ascorbic acid on human articular chondrocytes culture / 대한정형외과연구학회지

For the successful autologous chondrocyte transplantation, it is important to maximize the number of chondrocyte and maintain its original morphology and phenotypic change of the chondrocyte in the culture. In this study, the effect of ascorbic acid and human serum which are known to promote cell proliferation and collagen synthesis was observed in the culture of human chondrocyte. Media were prepared with the conditions of fetal bovine Serum(FBS) treated group, FBS +ascorbic acid(asc) treated group, human serum(HuS) treated group, and HuS+asc treated group, respectively. Proliferation was measured by cell counting using trypan-blue staining method. We used to determine the degree of expression of aggrecan of mRNA and type II collagen using RT-PCR. Type II collagen in cultured cell and medium was measured by western blot analysis and proteoglycan synthesis by DMB (Dimethylene Blue) assay. Under all conditions, aggrecan on mRNA level was well expressed. On the other hand, expression of type II collagen was reduced on HuS treated group than FBS treated group, and ascorbic acid treated groups showed decreased expression of type II collagen. Western blot analysis showed increased expression of type II collagen on HuS treated group than FBS treated group, and ascorbic acid treated groups showed increased level. HuS+asc treated group showed the most significant effect than the other groups. The increased effects of ascorbic acid on the proliferation and collagen synthesis were more prominent in the culture with human serum. It might be due to the synergic effect with some growth factors which were present in human serum.

Effects of human serum and TGF-beta on proliferation and redifferentiation of human articular chondrocytes / 대한정형외과연구학회지

In monolayer culture, articular chondrocytes are well known to proliferate and dedifferentiate by seum and transforming growth factor-beta(TGF-beta). These dedifferentiated cells regain the ability to express type II collagen in alginate bead culture. In this study, the effects of human serum and TGF-beta on the proliferation and phenotypical change of human chondrocytes were examined in both monolayer and alginate bead culture. Proliferation was measured by 3H-thymidine incorporation and cell counting, chondrocytic phenotype by Western blot analysis of type II collagen expression, and proteoglycan synthesis by dimethylmethylene blue assay. Both human serum and TGF-beta synergistically increased the proliferation of chondrocytes in monolayer culture. Human serum had effect to maintain the type II collagen expression, even with enhanced level, in monolayer culture and showed redifferentiation in alginate culture, similar to fetal bovine serum control. TGF-beta enhanced the production of proteoglycan in monolayer culture. In conclusion, the present study demonstrated that human serum and TGF-beta could be used as potent additives to increase chondrocyte proliferation and maintain its phenotype.