Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
To study the effect of the repair of rabbit articular cartilage defects by the composite of chondrogenic induction of autologous MSCs and autologous “two-phase” bone matrix gelatin (BMG). Methods Twentyfour healthy adult New Zealand rabbits weighing 2 to 3 kg were divided into group A, B and C with 8 in each. Autologous MSCsderived from group A were cultured in vitro and observed under inverted phase contrast microscope when enough cells through trypsinization transferring in vitro were obtained. Then the growth curves of 1, 3 and 5 passage culture of MSCs were drawn. The 3rd passage MSCs were induced into chondrogenic differentiation by adding TGF-β1 (10 ng/mL), IGF-1 (10 ng/mL) and vitamin C (50 ng/mL) in vitro. At 8 days after induction, the features of chondrocytes were observed under inverted phase contrast microscope, and immunohistochemical staining and Mallory staining were made. Getting out part of the il ium of group A and B, according to the method of Urist, the “two-phase” BMG was acquired. Chondrogenic induction of autologous MSCs was inoculated into the corresponding BMG to set up a composite of cell-carrier, and then it was observed through scanning electric microscope after 3 days of culture. The model of articular cartilage defects of rabbits was made: in group A, autologous cell-carriers were implanted; in group B, there only existed autologous BMG; in group C, there was nothing. At 8, 12 weeks after operation, the gross, HE staining and immunohistochemical staining were made, and grading scales were evaluated according to Wakitani histological grading method. Results Features of MSCs were as follows: the shape of primary cells was shotspindled and of passage cells was long. As to the growth curves of 1, 3 and 5 passage culture of MSCs, passage cells grew slowly for 3 days after being passaged and went into log-growth during the 3rd and the 7th days and into plateau later, but the 3rd passage cells grew best. Observation of MSCs after chondrogenic induction was performed: the shape of cells was ell iptical and the effect of induction was verified by the positive results of collagen type II, S-100 and Mallory staining. Under scanning electricmicroscope, the structure of BMG was good and cells were observed growing in it well. As far as repair of articular cartilage defects are concerned at 8, 12 weeks after transplantation, the defects in group A were repaired by the hyl ine-l ike tissue and the structures of the cartilage surface and normal cartilage were in integrity, and immunohistochemical staining of collagen type II was positive, while those in group B and C were repaired by the fibrous-l ike tissues and the surfaces were irregular. In Wakitanni histological score, at 8 weeks after operation, group A was (3.50 ± 1.51) points, group B was (10.00 ± 1.41) points and group C was (12.00 ± 0.93) points; at 12 weeks, group A was (1.13 ± 0.99) points, group B was (8.38±1.30) points, and group C was (10.13 ± 1.64) points. At different time points, group A was significantly better than group B and C, showing significant differences (P lt; 0.05). Conclusion Induced autologous MSCs and the composite with autologous “two-phase” BMG have the function to repair articular cartilage defects, and they are better than autologous BMG transplanted only or nothing transplanted.