Effect of type Ⅰ collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells

摘要

Objective: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type Ⅰ collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). Methods: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type Ⅰ collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type Ⅰ collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [~3H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin ( OCN), alkaline phosphatase ( ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction ( RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). Results: Type Ⅰ collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm +-141cpm vs. 1797 cpm +- 118 cpm, P =0.017; 8 h, 2311 cpm +- 113 cpm vs. 1891 cpm +- 103 cpm, P = 0.01). The cells which cultured on PLGA coated with type Ⅰ collagen showed significantly higher cell proliferation than controls on the 7th day (1021 cpm +- 159 cpm vs. 451 cpm +- 67 cpm, P = 0.002), the 14th day (1472 cpm +- 82 cpm vs. 583 cpm +- 67 cpm, P < 0.001) and 21th day (1728 cpm +- 78 cpm vs. 632 cpm +- 55 cpm, P < 0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type Ⅰ collagen on the 21th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type Ⅰ collagen while cylindric or round cells in controls. Conclusions: Type Ⅰ collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.