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.
Objective To study the method of inducing human marrow mesenchymal stem cells (MSCs) into osteoblasts directionally and to identify osteogenesis characteristics. Methods MSCs were isolated from adult marrow using density gradient separation method and were cultured in conditioned medium containing Dex 10 -8 mol/L，β-GP 10 mmol/L，and AA 50 μg/ml. The MSCs attachment formed soon and passage 3 cells were chosen to check osteogenesis characteristics, including alkaline phosphatase assay with modified calcium-cobalt staining method, type Ⅰ collagen assay with immunohistochemistry, osteopontin and osteonectin assay with in situ hybridization and calcium nodes assay with Von Kossa staining. Results Passage 3 MSCs had typical appearance of osteoblasts and could be passaged continuously till passage 10. The rate of ALP expression was 85%. The expressions of collagen type Ⅰ, osteopontin and osteonectin were positive and calcium nodeswere seen by Von Kossa staining. Conclusion We have successfully induced human MSCs into osteoblasts; the induced cells have typical osteogenesis characteristics.
ObjectiveTo review the research progress of induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) transfected by double-gene. MethodsThe recent literature concerning the comparative research of induced osteogenesis of BMSCs transfected by double-gene was extensively reviewed. The characteristics of BMSCs, the advantage and effect of synergistic inductive osteogenesis, the application prospect and problems of BMSCs transfected by double-gene were summarized. ResultsThe effect of induced osteogenesis concerning BMSCs transfected by double-gene is far superior to single gene transfection and the activity of osteoblast is also significantly increased. The research used in bone tissue engineering experiment also obtain good effect. ConclusionInduced osteogenesis of BMSCs transfected by double-gene is able to make up for the lack of a single gene transfection and has great development prospects in the orthopaedic field.
ObjectiveTo investigate the osteogenic differentiation potential and the biological features of synovium-derived mesenchymal stem cells (SMSCs) in vitro and to observe the osteogenic capability of the composite scaffolds constructed with SMSCs and hydroxylapatite/chitosan/poly L-latic acid (HA/CS/PLLA) in vivo. MethodSMSCs were separated and cultured with adherent method and enzymatic digestion method. Specific phenotypes of SMSCs were detected by flow cytometry after purification. Then, SMSCs were identified by oil red O staining, alkaline phosphatase (ALP) staining, and alizarin red staining after adipogenic and osteogenic induction, respectively. In vitro experiments:the expressions of osteogenic related genes[osteocalcin (OCN), collagen type I, ALP, and Runx-2] were detected by real-time fluorescent quantitative PCR at 1, 7, 14, 21, and 28 days after osteogenic induction; ALP activities were also determined by ELISA at 1, 3, 5, 7, 9, and 11 days after osteogenic induction; meanwhile, extracellular matrix calcium mineralization was detected by alizarin red S method at 7, 14, 21, and 28 days after osteogenic induction; the normal SMSCs were harvested as control group. In vivo experiments:Twenty-four Sprague Dawley (SD) rats were randomly divided into experimental group (n=12) and control group (n=12) . The 3rd passage SMSCs were seeded on HA/CS/PLLA to construct composite scaffolds, after adhesion for 72 hours in vitro, the composite scaffolds were implanted into the right thigh muscle of 12 SD rats as experimental group; HA/CS/PLLA was implanted into the right thigh muscle of the other 12 SD rats as control group. At 4 and 8 weeks after implantation, the scaffolds were harvested for X-ray film and histological examination to observe ectopic bone formation. ResultsThe positive rates of CD147, CD90, CD105, and CD44 were more than 95%, while the positive rates of CD117, CD34, CD14, and CD45 were less than 10%. Oil red O staining demonstrated red lipid droplets in the cytoplasm, and alizarin red staining showed flaky red calcifications, and cytoplasm was dyed brown by the ALP staining. The mRNA expressions of collagen type I, ALP, and Runx-2 were significantly increased at 7 days after osteogenic induction, and OCN mRNA expression was significantly increased at 14 days after osteogenic induction; ALP activity was significantly higher at 5, 7, 9, 11 days after osteogenic induction in the SMSC-induced group than control group and reached a maximum at 7 days (P<0.05) . Calcium mineralization was significantly enhanced at 14 days after osteogenic induction, and gradually increased with time (P<0.05) ; moreover, it was significantly higher in the SMSC-induced group than control group (P<0.05) . X-ray and histological examination demonstrated that the new bone tissues formed in 2 groups, but bone formation content of the experimental group was significantly more than that of the control group at 4 and 8 weeks after implantation (P<0.05) . ConclusionsSMSCs can be induced into osteoblasts both in vitro and in vivo, so SMSCs might be a promising seed cells for bone tissue engineering.