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 investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. Methods TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro, and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Results Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. Conclusion TBC have good biocompatibility and can be used to repair bone defect in clinic.
ObjectiveTo study the preservation effect of true bone ceramics (TBC) prepared by high-temperature calcination of bovine bone on alveolar ridge of canine extraction socket.MethodsSix healthy Beagle dogs (aged 1.5-2 years) were selected to extract the second and fourth premolars of both mandibles and the second premolars of the maxilla. The left extraction socket was implanted with TBC as the experimental group, and the right side was implanted with the calcined bovine bone (CBB) as the control group, to observe the alveolar ridge preservation effect. Three dogs were euthanized after general observation at 1 and 6 months after operation respectively. After separating the maxilla and mandible, cone beam CT (CBCT) was performed to measure the average gray value of the graft site and the adjacent reference area (the area between the roots of the adjacent third premolar) and calculate the gray scale ratio between the bone graft site and the reference area. Histological observation was made on the bone graft site to evaluate the new bone formation.ResultsGeneral observation showed that the wounds of both groups were basically healed at 2 weeks after operation, and the bone graft materials were not exposed. The wounds healed well at 1 and 6 months after operation without swelling. The results of CBCT showed that the residual material was found in both groups at 1 month after operation, and no significant residual material was found in both groups at 6 months after operation, and the alveolar ridge height of the bone graft area was not significantly reduced. There was no significant difference in the bone mineral density between the experimental group and the control group. The gray scale ratios of the experimental group at 1 month and 6 months after operation were 0.97±0.14 and 0.93±0.06, respectively, and were 0.99±0.16 and 0.94±0.05 in control group, showing no significant difference between the two groups (t=−1.030, P=0.333; t=−0.770, P=0.466). HE staining observation showed that a large number of bone graft materials did not degrade and new bone formed around the grafts in both groups at 1 month after operation; the bone graft materials were absorbed and a large number of new bones were formed in both groups at 6 months after operation.ConclusionTBC can maintain bone mineral density and have good osteoconductivity in the alveolar ridge site preservation experiment of dogs, and can be used for alveolar ridge site preservation.