Osteoblasts were cultured and isolated from a piece of tibial pettiosteum of four New-Zealandrabbits. After subeultured,these cells Were incubatd in vitro with tritiated thvmidine for 36 hoursand then these labeled cells were implanted in the subeutaneous layer of the defects of the auriclarcartilage and the radial bone, After 2 weeks and 4 weeks respectively, these rabbits were killed andthe spoimens were obtained from the site where the cells had been transplanted. The transformation of these cells was observed by autoradiographic method. The results indicated that nearly all of the cultured cells were labeled. After 2 weeks, it was observed that many labeled osteoblasts were in different stages of differentiation, some were beried by extracellular matrix and resembled osteocyte, thers were differentiated into chondrocyte-like cell. In addition, some labeled osteoblasts were congregated in the form of multinucleated osteoclast. After 4 weeks , in the subcutaneous layer the labeled osteoblasts were changed to osteoid tissue and in the defect of the auricular crtilage these cells transformed into chondritic tissue; moreover, those labeled osteoblsts which had been implanted into the radial defect had differentiated into typical bone tissue. The results of this research indicated that the osteoblasts isolated from the periosteum if reimplanted to the same donor might be possible to repair the bone and cartilage defects.
Objective To evaluate the bone regenerative potential of reconbinant human bone morphogenetic protein 2(rhBMP-2) / collagen on adult rat calvarial bone. Methods A tight subperiosteal pocket was produced under both sides ofthe temporal muscle in rats. rhBMP-2 / collagen was implanted in one side and collagen alone was implanted in the other side as control. The rats were sacrificed 2, 4 and 8 weeks after operation. The specimen was harvested and examined histologically. For morphometric analysis, the thickness of the temporal bone of both sides was measured and compared. Results The rhBMP-2 / collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage production. The ossification process in the rhBMP-2 / collagen occurred directly through bone formation, similar to intramembranous ossification. Conclusion rhBMP-2 / collagen is an effective material as a biological onlay implant.
ObjectiveTo review the development and applications of hypoxia-inducible factor 1α (HIF-1α) in the strategy of tissue engineered angiogenesis and osteogenesis.
MethodThe literature about HIF-1α in tissue engineering technology was reviewed, analyzed, and summarized.
ResultsHIF-1α plays a key role in angiogenic-osteogenic coupling, and as an upstream regulator, HIF-1α can regulate the expressions of its target genes related with angiogenesis and osteogenesis. In addition, HIF-1α not only can control and improve the angiogenesis, but also has important significance in proliferation and differentiation of seed cells, especially stem cells, which is the foundation for bone healing.
ConclusionsWith the development of tissue engineering technology, the problems in the applications of HIF-1α, such as the effective dose of targeting controlled-release, pro-inflammatory effect, and carcinogenicity, will be explored and solved in the future, so it can be used better in clinical.
Objective To evaluate the osteogenesis of bi phasic ceramic-l ike biologic bone (BCBB) with tissue engineering in repairing segmental bone defects. Methods BMSCs isolated from the femoral and tibial marrow of 2-weekold Japanese white rabbit were cultured to passage 3. Then 20 μL of the cell suspension at a concentration of 1 × 107 cells/mLwere seeded into 15 mm × 15 mm × 5 mm BCBB block; the construction of tissue engineered BCBB was completed after 8 days of compound culture. Forty-eight adult Japanese white rabbits were randomly divided into groups A, B, C and D, then BCBBs cultured with BMSCs in vitro for 8 days (group A) and only BCBBs without BMSCs (group B) were respectively implanted into the radius segmental bone defects of rabbits, autogenous il iac bone graft (group C) and empty defect (group D) were used as controls. The specimens were examined after 4, 8, 12 and 24 weeks, the osteogenesis was evaluated through X-ray radiograph and histology examination. Results X-ray examination: the border between the material and host’s bone was clear after 4 weeks, and blurred after 8 weeks in group A and group B; the density of some part of the edge of the material was similar to that of radius and there was high density imaging in the materials of group A after 12 weeks; there was much high density imaging in the materials of group B after 12 weeks. The medullary cavity of bone was formed and l ittle high density imaging in the materials of group A after 24 weeks. Some high density imaging still existed in the materials of group B after 24 weeks. The X-ray evaluated scores showed that the scores of group A was higher than that of group B, and there was significant difference between group A and group B after 12 and 24 weeks (P lt; 0.05). Histological examination: there was new bone formation in the materials and also new bone grew adhesively on the surface of BCBB in group A. While in group B only new bone grew and attached to the surface of BCBB. BCBB degraded more with the time and more new bone formed. The histological evaluation showed that the bone forming area in group A was more than that in group B, and there was significant difference between group A and group B (P lt; 0.05). Conclusion The osteogenesis of BCBB with tissue engineering was superior to only BCBB, BCBB could be used as a scaffold of bone tissue engineering.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
ObjectiveTo explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice.
MethodsMC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I).
ResultsThe results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P < 0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P < 0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P < 0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P < 0.05).
ConclusionThe appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.
Objective
To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro.
Methods
The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days.
Results
After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D.
Conclusion
BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.
Objective To evaluate the osteogenic potential of human bone marrow mesenchymal stem cells (MSCs) transferred with human bone morphogenetic protein 2(BMP 2) gene by adenovirus. Methods The MSCs were isolated from human bone marrow and cultured in vitro. They were divided into 3 groups: Adv hBMP 2 transduced group; Adv βgal transduced group; untransduced group. Western immunoblot analysis, alkaline phosphatase(ALP) staining, Von Kossa staining, and a quantitative ALP activity assay were performed. Nine unde mice received injection into a thigh muscle to test the osteoinductivity of the three types of cells. Results In the Adv-hBMP-2 transprotein; most MSCs were stained positively for ALP activity 9 day after transduction; the MSCs reached the peak of ALP activity 12 day after transduction; the calcified nodes formed 21 days after transduction. The ectopic bones formed in the thigh muscles of the nude mice. Little bone formation was observed in the other groups 4 weeks after cell injection. Conclusion Adenovirus mediated hBMP-2 gene transfection can induce osteogenesis of human bone marrow mesenchymal stem cells.
OBJECTIVE To observe the osteogenesis of percutaneous autogenous bone marrow grafting in cicatricial bone defect, to seek a good method for treating fracture nonunion. METHODS Eighteen rabbits were adopted in this study. 1 cm bone defect model was made in each side of radius, 6 weeks later, 2 ml autogenous bone marrow was injected in the right radial bone defect as experimental group, 2 ml autogenous peripheral blood in the left side as control group. X-ray features, histologic changes, Ca and P content in the site of bone defect were studied in various times. Also 15 patients were treated clinically for the nonunion fracture, the average time from nonunion to bone marrow grafting was 13 months. RESULTS In experimental group, the increasing new bone tissue were observed in X-ray and histologic examination. While in control group, no osteogenesis was observed. Ca and P content of experimental group was higher than that of control group. For the 15 patients, 13 cases healed in 5-9 months, 2 cases failed. CONCLUSION Percutaneous autogenous bone marrow grafting is capable of osteogenesis in the cicatricial bone defects. It can be used in nonunion cases which are not fit for operation of bone grafting because of poor condition of the skin.
Objective To establish a method of isolating and culturing adult human bloodderived mesenchymal stem cells(MSCs) and to investigate their osteogenic potential in vitro. Methods Thirty peripheral blood sampleswere collected from 30adult volunteers(15 ml per person).Adult human MSCs derived from peripheral blood were isolated from the lymphocyte separation fluid fraction of mononuclear cells, cultured in α-Modified Eagle’s Medium with low glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype of MSCs was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage grew in the presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L β-glycerophosphate,50 μmol/L vitamin C, and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, the expression level of collagen typeI, osteocalcin and osteonectin were determined. And the calcium tubercle formation would be examined after the continual one-month culture of the fifth passage. Results MSCs exsited in the pheripheral blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27±0.22/106 mononuclear cells. The MSCs expressed CD44,CD54,CD105,and CD166，but did not CD14, CD34, CD45,and CD31.Under the function of osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression levels of collagen type Ⅰ, osteocalcin and osteonectin. And the calcium tubercle formation was examined throughtetracycline fluorescence labeling method. Conclusion The isolation and cultureconditions established for adult human MSCs may select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived MSCs possess osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.
