An Experimental Study On Biological Features And Osteogenic Potential Of Bone Marrow Mesenchymal Stem Cells Cultured In Vitro

Objectives: To establish a reproducible system for the culture and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) in vitro. To investigate the self-renewing capacity, the osteogenic potential, cell surface molecules, and apoptosis of purified mesenchymal stem cells (MSCs) during extensive sub-cultivation and osteogenic differentiation. To characterize effect of dexamethasone on the MSCs cultured in vitro. To determine in vivo osteogenic potential in size-critical bone defect of culture-expanded rabbit autologous MSCs induced by osteogenic supplement (OS).Methods: 1. MSCs-enriched low density fraction was isolated from total marrow cells centrifuged with a 1.073g/mL Percoll. The MSCs were cultured in DMEM-LG (control medium) at 37℃ in a humidified atmosphere containing 95% air and 5% CO₂. The MSCs were observed daily and cell cycle were analyzed. 2. For osteogenic induction, the MSCs were cultured with OS medium containing Dexamethasone (Dex), sodium β-glycerophosphate (β-GP), and ascorbic acid (AsA). The MSCs induced by OS were examined by Alkaline Phosphatase (ALP), Von Kossa staining, collagen-1 (COL-1) and osteocalcin (OC) immunohistochemical staining. ALP enzyme activity of the cell layer was also measured. 3. The cell surface molecules on MSCs treated with OS for the indicated days and MSCs not induced at indicated passage was investigated by flowcytometry. 4. The apoptosis ratios of MSCs at indicated passage were measured by flowcytometry, their self-renewing capacity was also determined. 5. Human MSCs were treated with Dex at indicated concentration. Effect of Dex on cell proliferation, ALP expression, and apoptosis ratios of human MSCs was investigated. 6. Animal experiment: Rabbit MSCs purified by Percoll liquid were culture-expanded with control medium in vitro. When the number of cells arrived at 10⁸, the MSCs were treated with OS for 1 week. The MSCs induced by OS were collected and percutaneously injected into radius bone defects of the indicated rabbit which the MSCs derived from, respectively.Autologous bone marrow was implanted in the contralateral limb as controls. Repair of bone defects was valuated at various time points by radiography, histology, quantitative histomorphometry, and biomechanics.Results: 1. The human MSCs attached to the surface of plates, exhibiting a fibroblast-like spindle shape. In vitro culture, MSCs proliferated quickly to form colonies, showing no contact inhibition. Treatment of hMSCs with OS resulted in a morphological transformation of the cells from long spindle-like into cuboidal-like shapes. There were more mitochondria, rough endoplasmic reticulum and lysosomes in the osteo-induced MSCs than the control group. The cell cycle analysis showed that only 24.6% of MSCs were in proliferation phases(S+G2+M). Human MSCs in vitro culture exhibited a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. The results of ALP, Von Kossa staining, COL-1 and OC immunohistochemical staining of osteo-induced MSCs were positive, while those of the control group were negative. 2. These expanded attached MSCs were positive for CD29, CD44, CD71, CD90, CD105, CD166. In contrast, the cells were negative for CD 14, CD34, CD38, CD45. The positive rates of CD71, CD90, and CD 166 decreased when the MSCs treated with OS, while showed no statistically significant difference among passages 1, 4, 7, 10. A significant increase in ALP activity in OS group was observed with maximal activity occurring on day 12, reaching 4 to 5-fold of the control. This increase in ALP activity was consistent for induced group regardless of whether they were extensively subcultured. 3. With increasing passage number, the hMSCs growth rates were slower and the apoptosis ratios increased. At passage 7 and beyond, not including passage 4, the difference in comparison to passage 1 cultures is statistically significant. 4. Dex inhibited the MSCs proliferation while enhanced the ALP activity at high concentration (10" mol/L and above), P>0.01. The difference at low concentration (10"9 mol/L) is not statistically significant. Dex increased the apoptosis of MSCs even at a concentration of 10'9 mol/L. The apoptosis ratios of MSCs by Dex is dose-dependent. 5. At both 4th week and 8th week, the osteo-induced MSCs induced more bony callus than control group respectively, while no or little bone formation was found in blank group. The healing ratios of induced, control group, and blank group were 12/12, 10/12, 0/2 respectively at 12th week. Histomorphometrical analysis showed that trabecular bone volume (TB V) and mineral apposition rate (MAR) of OS group were much more than those of control group, PO.01. Biomechanical strength of osteo-induced group were superior...