Ascorbic acid influences on extracellular matrix and structure of rabbit bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The effect of extracellular matrix on stem cells is the focus of tissue engineering. However, there are few reports about the synthesis and secretion of extracellular matrix as well as its effects on cells. OBJECTIVE: To isolate, culture and identify rabbit bone marrow mesenchymal stem cells (BMSCs), and to explore the changes of extracellular matrix and whole structure under the intervention of ascorbic acid. METHODS: Rabbit BMSCs were isolated by differential adherent method of the bone marrow, and the expression of CD44, CD45 and CD31 was identified by flow cytometry. The BMSCs were cultured in the culture medium containing 20 mg/L ascorbic acid. Then the cell morphology, gross structure, ultrastructure, and histological changes of BMSCs were observed. The expression of extracellular matrix related genes was detected by RT-PCR. RESULTS AND CONCLUSION: Over 95% passage 2 BMSCs could express CD44, but the expression levels of CD45 and CD31 were extremely low. Intervention with ascorbic acid enhanced the proliferation of BMMSCs with unclear cell boundaries. A cell-sheet structure formed at 10-14 days after intervention. Hematoxylin-eosin staining results showed a layered cell arrangement, and Masson staining findings showed a large amount of extracellular matrix composition. Abundant endoplasmic reticula and vesicle-like structure were observed under the transmission electron microscope. RT-PCR findings showed that ascorbic acid significantly increased the expression of fibronectin mRNA in the BMSCs (P < 0.05), but slightly increased the mRNA expression of collagen type I. All these findings indicate that ascorbic acid not only increases the proliferation and transformation of rabbit BMSCs, but also promotes the synthesis and secretion of extracellular matrix, which has great potential in tissue engineering applications.