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 investigate and compare the osteogenic potential of three kinds of calcium phosphate ceramic as carriers for recombinant human bone morphogenetic protein-2(rhBMP-2) in vivo.Methods BCPceramics (HA,TCP,HA/TCP) impregnated with rhBMP-2 (experimental groups) and without rhBMP-2(control groups) were implanted into 6 muscles pockets on the dorsum of 3month-old Wistar rabbits. The rabbits were sacrificed 2, 4 and 8 weeks after implantation and bone induction was estimated by alkaline phosphatase(ALP) activity measurement. The implants were also examined histologically and histomorphometrically by HE staining and computerized graphical analysis. Results The ALPactivity of implants withrhBMP-2 was higher than that of control groups(P＜0.05), but there was no difference between 2 and 4 weeks in experimental groups. In all experimental groups,theimplants exhibited that new bone formation increased with the lapse of time. The amount of new bone formation is more in -HA/rhBMP-2 group than in the other two group in the 2nd and 4th weeks, but there was no difference between them (P＞0.05).In the 8th week, the amount of bone formation was most in HA/TCP with -rhBMP-2, and was more than that in the 2nd and 4th weeks. Whereas in control groups, there was only fibrous connective tissue. Conclusion HA/TCP- is a good carriers of rhBMP-2 and can be used as bone substitutes clinically.
Objective To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
ObjectiveTo compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition. MethodsBMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α(HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days. ResultsThe BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P < 0.05). At each time point, compared with group A, the ALP expression, HIF-1αprotein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P < 0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P < 0.05); compared with group C, the HIF-1αprotein relative expression was significantly up-regulated in group B (P < 0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C. ConclusionHypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.
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.
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 study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of thefemoral head (ANFH). Methods Twenty-four male adult New Zealand rabbits were made the ischemic hind l imb model and divided into 4 groups (n=6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAVhVEGF165- internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind l imb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the prol iferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n=6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineral ization. Results No symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P lt; 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P lt; 0.05). However, there was no significant difference between group C and group D (P gt; 0.05). At 8 weeks after injection, orthotopic ossification and mineral ization were evidently detected in group A and group C, and group A was ber than group C. No obvious evidence of orthotopic ossification and mineral ization were observed in group B and group D. Conclusion rAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.
Objective To observe the changes in the peripheral blood T lymphocyte subsets and the histomorphology of the transplanted tissues in the rabbits in the early stage after transplantation of the tissue engineered boneconstituted by the biologically-derived scaffold and to confirm the feasibility of the biologicallyderived materials as a scaffold in the bone tissue engineering. Methods Forty-eight healthy New Zealand rabbits (weight, 2.0-2.5 kg) with a 1-cm defect were equally and randomly divided into 4 groups: Groups A-D. The partial demineralized freeze-dried bone (PDFDB), the tissue engineered bone constructed by the osteoblasts derived from the lactant rabbit periosteum as a seeding cell, the xenogeneic cancellous bone undergoing the antigen self-digestion, partial demineralization and freeze-driedprocess as a scaffold, and the fresh xenogeneic allografting bone were respectively transplanted into the segmental defects of the rabbit radii in Groups A-D.To examine the effects of the 4 different materials, the flow cytometry was used to observe the changes in the T lymphocyte subsets in the rabbit peripheral blood at 1, 2, and 4 weeks after the operations and to examine the osteogenesis achieved by the 4 materials, the histological observations were also performed at 2, 4, 8, and 12 weeks after the operations. Results Two weeks after the tissue engineered bone transplantation in Group B, the osteoblasts and chondroblasts were found in the apertures of the scaffold, the new bone formation could be observed, the osteoclasts could be seen in the peripheral zone, and some of the netlike frameworks were destroyed and absorbed. Four weeks after the operation, the histological observation revealed that the osteocartilagionous callus turned into a woven bone. The peripheral blood T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1-2 weeks after the operations and in Groups A and B than before the operations and in the other groups (.Plt;0.05)；4 weeks after the operations the T lymphocyte subset of CD4+ was only slightly greater in number than before the operations, but with no statistically significant difference (Pgt;0.05). In Group C, the increase of the T lymphocyte subsets of CD4+ and CD8+ was not significant after the operation (Pgt;0.05). The T lymphocyte subsets of CD4+ and CD8+ were significantly greater in number 1, 2 and 4 weeks after the operations and in Group D than before the operation and in the other groups (Plt;0.05). Conclusion The tissue engineered bone constructed by the partial demineralized freezedried bone as a scaffold does not cause a serious immunologic rejection in the early stage after the transplantation and does not affect its good ability to repair the bone defect. The biologicallyderived bone canbe used as a scaffold in the 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.
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.