Comparison of Phenotypic Characterization between "Alginate Bead" and "Pellet" Culture Systems as Chondrogenic Differentiation Models for Human Mesenchymal Stem Cells

Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.

Effect of Culture Condition on Chondrocyte Viability Isolated from Articular Cartilage / 대한진단검사의학회지

BACKGROUND: Recent advance in tissue engineering in the biomedical field shed light on the replacement or regeneration of various organs with synthetic substitutes. Currently emerging cartilage tissue engineering therapies involve artificial cartilage fabricated from three dimensional cultures using appro-priate scaffolds. It is mandatory to expand or proliferate the chondrocytes in vitro to prepare the artificial cartilage. The purpose of this study was to find out the most favorable culture conditions for chon-drocyte viability in vitro. METHODS: Articulr chondrocytes or cartilage explants were isolated from the patellofemoral groove of adult pigs. And then we standardized the size and thickness of the cartilage explants as well as preparing alginate-chondrocyte beads for three-dimensional cultures. The cartilage explants, including 10% fetal bovine serum for 10 days, 36 days and passage 6. Cellualr viability was measured by methylthiazol tetrazolium (MTT) assay on monolayer, alginate bead and cartilage explant. SPSS 11.5 was used for data anaylsis. RESULTS: Chondrocytes cultured on monolayers in vitro showed no significant difference in cellular viability until passage 6 following isolation from the patellofemoral groove of adult pigs (P>0.05, n=4). Chondrocyte viability was markedly increased by day 16 both in the monolayer (148%) and three dimensional cultures (245%), and then slightly decreased 126% and 200%, respectively, at day 36. Three dimensional cultures using alginate bead were more favorable for chodrocyte viability than monolayer culture in chondrocyte primary culture (P=0.003, n=6). Chondrocyte viability in the algi-nate bead was increased 300% during 36 days' incubation period (P=0.001, n=3). Cellular viability in the cartilage explant culture was decreased after day 4 in both MTT score (P=0.022, n=10) and MTT OD (P=0.039, n=10). CONCLUSIONS: Three dimensional cultures using alginate bead were the most favorable for chon-drocyte viability in chondrocyte primary cultures.

The Effects of Transglutaminase on Perichondrocyte in Alginate Culture / 대한정형외과연구학회지

PURPOSE: To investigate the effects of transglutaminase in the environment of extracellular matrix on perichondrocyte in alginate culture. MATERIALS AND METHODS: Perichondrocyte cells were isolated from articular cartilage of New Zealand white rabbits by enzymatic digestion and maintained in monolayer culture. After 7 days, the cells were trypsinized and cultured in an alginate bead system. Four groups of the alginate beads were prepared as follow: containing 1 mg/ml of transglutaminase, 10 microgram/ml of fibronectin, mixture of 1mg/ml of TGase and 10 microgram/ml of fibronectin and only perichondrocytes as a control group. Cell proliferation was measure by [Methyl-3H] Thymidine uptake, and proteoglycan synthesis was measure by [35S] Sulfate uptake. The gene expression of integrin-alpha5, integrin-beta1 and type II collagen was analyzed by reverse transcription-polymerase chain reaction. Safranin-O staining was utilized for histological assessment of proteoglycan in extracellular matrix. One-way ANOVA was used to analyze the results statistically. RESULTS: Mixture of transglutaminase and fibronectin exhibited high synthesis rates of proteoglycan and active cell proliferation compared with other groups. The gene expression of type II collagen did not show significant difference between groups. The gene expression of integrin-alpha5 was down-regulated in all groups with time. The gene expression of integrin-beta1 was not down-regulated with time only in mixture of transglutaminase and fibronectin. Histological staining of the secretions by Safranin-O staining was in agreement with the data of proteoglycan synthesis, and Safranin-O staining showed that more cell-to-cell aggregates is developed in the mixture of transglutaminase and fibronectin. CONCLUSION: Mixture of transglutaminase and fibronectin can stimulate chondrocyte proliferation and proteoglycan synthesis, and integrin seems to modulate such interactions.