Phytoestrogen (Daidzein) Promotes Chondrogenic Phenotype of Human Chondrocytes in 2D and 3D Culture Systems

Clinical investigations have shown a significant relationship between osteoarthritis (OA) and estrogens levels in menopausalwomen. Therefore, treatment with exogenous estrogens has been shownto decrease the risk ofOA.However, the effect estrogen has not been clearly demonstrated in the chondrocytes using phytoestrogens, which lack the specific side-effects of estrogens, may provide an alternative therapy. This study was designed to examine the possible effects of phytoestrogen (daidzein) on human chondrocyte phenotype and extracellular matrix formation. Phytoestrogens which lack the specific side-effects of estrogens may provide beneficial effect without causing hormone based side effect. Human chondrocytes cells were cultured in 2D (flask) and 3D (PCL-CA scaffold) systems. Daidzein cytotoxic effect was determined by MTT assay. Chondrocyte cellular content of glycosaminoglycans (GAGs), total collagen and chondrogenic gene expression were determined in both culture systems after treatment with daidzein.Daidzein showedtime-dependent and dose-independent effects on chondrocyte bioactivity.Thecompound at low doses showed significant (p0.05). The expression levels of Fibronectin, Laminin and Integrin b1were significantly increased especially in3Dculture system. This studywas illustrated the potential positive effects of daidzein onmaintenance of human chondrocyte phenotype and extracellular matrix formation suggesting an attractive and viable alternative therapy for OA.

The Effect of Placenta Extract on Proliferation and Differentiation of Human Chondrocytes

PURPOSE: The isolated human chondrocytes for cartilage reconstruction and transplantation presents a major problem as these cells would change biologically in vitro. For more effective applications of these cells in the clinical field, it is necessary to get a large amount of cells in a short period without affecting their function and phenotype. METHODS: This study reports the effects of placenta extract on chondrocytes in vitro. We initiated this study on the basis of the hypothesis that placenta extract can influence both the proliferation of chondrocytes and their biologic functions(for example, to express cell specific gene or to produce their own extracellular matrix). Chondrocytes in monolayer culture with or without placenta extract were collected and analyzed by MTT assay, ECM assay, and RT-PCR. RESULTS: Placenta extract stimulated the proliferation of chondrocytes in monolayer culture. The phenotype of chondrocytes was well maintained during the expansion in monolayers. Chondrocytes expanded in the presence of placenta extract produced ECM, glycosaminoglycan, abundantly. Compared to chondrocyte expanded in culture medium only, chondrocytes expanded with placenta extract demonstrated higher COL2A1 expression that was biochemically comparable to primary chondrocytes. This study provides an evidence that placenta extract is helpful to expand chondrocytes during tissue cultivation, to maintain their differentiated phenotype and to promote their function. CONCLUSION: These results suggest that placenta extract during cultivation play an important role in controlling cell behaviors. Furthermore, these results provide a biologic basis for cartilage tissue engineering.

The Effects of bFGF, VEGF and Micromass Culture on Proliferation and Differentiation of Human Chondrocytes

The acquisition of human chondrocytes for transplantation and cartilage coverage presents a major problem as these cells dedifferentiate rapidly during expansion in monolayer culture. Dedifferentiated chondrocytes change their shapes, metabolic states, and programs of matrix biosynthesis. We initiated this study on the basis of the hypothesis that bFGF, VEGF, and micromass culture can influence both the proliferation and their ability to express COL2A1 gene as a chondrogenic marker and Cbfa1 gene as an osteogenic marker. Chondrocytes in monolayer and micromass culture with or without bFGF and VEGF in vitro were collected and analyzed. In results, bFGF stimulated the proliferation of chondrocytes in monolayer culture. VEGF also stimulated the proliferation, but was less effective. The phenotype of chondrocytes was gradually changed in monolayer culture. Chondrocytes expanded in the presence of bFGF became dedifferentiated. However, dedifferentiated chondrocytes fully maintained their potential for redifferentiation in response to environmental changes. After transferring in micromass culture, chondrocytes which expanded with bFGF demonstrated high COL2A1 expression that was biochemically comparable to primary chondrocytes. Chondrocytes which expanded with VEGF demonstrated high Cbfa1 expression in both monolayer and micromass culture with passage times. This study provides that bFGF is needed to expand chondrocytes during tissue cultivation and additional three-dimensional environment is needed to maintain their differentiated phenotype. VEGF initiates the osteogenic potential during the chondrocyte expansion especially in micromass culture.

Influence of synovial Joint Fluid of Rheumatic and Osteoarthritic on Chondrocyte in vitro / 대한정형외과연구학회지

PURPOSE: Earlier work suggested that two cytokines inhibit synthesis of type II collagen and of aggrecans by chondrocytes and they depress chondrocyte proliferation, but there was little report how the chondrocyte is modulated by culture conditions such as the joint fluids of the rheumatoid arthritis and that of the osteoarthritis. The purpose of this investigation was to determine whether RA(rheumatic arthritis) or OA(Osteoarthritis) joint fluid influence proliferation and differentiation in cultured human articular chondrocytes. MATERIALS AND METHODS: Human chondrocytes were cultured in a standard media (DMEM and 10% FBS), RA and OA joint fluid were added to media at the concentration of 20, 40 and 60% respectively for 1, 3 and 6days. 3H-thymidine and 3H-uridine uptake of cultured chondrocytes were measured as indicators of cell proliferation. Synthesis of human collagen type I, II was estimated by the RT-PCR procedures. RESULTS: 3H-thymidine uptake of the chondrocyte cultured in RA SF(synovial fluid) medium at 2 and 4 days; its uptake in the group treated by RA SF 20%, 40%, 60% increased more significantly than that in control group (P0.05). 3H-uridine uptake of the chondrocyte cultured in RA SF medium at 2 and 4 days; its uptake of the group treated by RA SF 20%, 40%, 60% increased more significantly than that of control group (P0.05). Human type I collagen mRNA expressions of the chondrocyte markedly increased in RA and OA SF mixed groups. Human type II collagen mRNA expressions of the chondrocyte were reduced in RA and OA SF mixed groups, especially RA SF 60% mixed groups. CONCLUSION: RA and OA SF increased the proliferation of the articular chondrocyte, but its decreased the differentiation of the chondrocyte. RA and OA SF may change the phenotype of the articular chondrocyte and this phenomenon was more outstanding in RA SF.

In Vitro Effects of Threapeutic Low Energy Ultrasound on Cultured Humon Articular Chondrocyte / 대한정형외과학회잡지

Ultrasound has been applied therapeutically to accelerate connective tissue healing and healing of long bone fracture. We performed this study to evaluate the effect of therapeutic low energy ultrasound on cultured human articular chondrocyte. Chondrocytes were isolated from articular cartilage of knee joint of young adult during notchy plasty of ACL reconstructive procedure using collagenase and cultured in DMEM solution. Therapeutic ultrasounds ( Intensity ; 1W/cm2, 0.75W/cm2, 0.25W/cm2. Frequency ; 1 MHz) were applied at the cultured human articular cartilage for 0, 30, 60, 90 seconds, repectively. 3H-Thymidine uptake, 3H-Uridine uptake and production of collagen Type I, Type II were evaluated on 3 days, and 5 days of culture. Decreased cell proliferation, decreased thymidine and uridine uptake, decreased production of human collagen Type I and complete loss of production of human collagen Type II were showed after high dose ultrasound application( 1W/cm2, 0.75W/cm2, 1MHz). After low dose ultrasound application( 0.25W/cm2, 1MHz), there were increased cell proliferation and increased production of human collagen Type II and production of Type I collagen was not changed after 3 days and 5 days. In summary, this study showed that decreased cell proliferation and collagen synthesis were observed with application of high dose ultrasound, whereas, increased cell proliferation and human collagen Type II synthesis were observed with application of low dose ultrasound.