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 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 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 evaluate the osteogenesis of three bio-bone derived materials in repairing segmental bone defects. Methods Sixty Japanese rabbits were made 10 mm radius segmental defects and divided into 5 groups(groups A, B, C ,D and E,n=12). Composite fully deproteinised bone(CFDB, group A), partially deproteinised bone(PDPB, group B), partially decalcified bone(PDCB, group C), autogenous iliac bone graft(group D) and no implant(group E) were implanted into the radius segmental bone defects of rabbits. The specimens were examined after 4, 8, 12 and 24 weeks; the osteogenesis was evaluated through X-ray radiograph and undecalcified solid tissue histological examination.Results The border between the material and host’s bone was distinct after 4 weeks and blurred after 8 weeks; the density of partial edge of the material was similar to that of radii after 12 weeks. The medullary cavity of bone reopened in group B; the density of most defect area was similar to that of the host bone and there was a few high density shadow in group C; the density of most defect area was higher than that of host bone in group A after 24 weeks. There was no significant difference in radiograph scoring between groups A, B and C after 4 weeks and 8 weeks(P＞0.05); the scores of group B and C were higher than that of group A after 12 weeks(P＜0.05); and the scores were arranged as follow: group Dgt;group Bgt; group Cgt;group A after24 weeks(P＜0.05). Bone callusgrew toward defect area and new bone adhered to the material after 4 weeks and 8 weeks; more new bone formed, and the materials were absorbed and degraded with time. The quantity of bone formation was more in group D than in group B andin group B than in group C and in group C than in group A after 24 weeks(P＜0.05).Conclusion PDPB had good osteogenesis in repairing the segmental bone defect, PDCB was inferior to it, both PDPB and PDCB are fit to repair segmental bone defect. Both of them were inferior to autogenous bone.
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 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.
ObjectiveTo investigate the regulation of human bone marrow mesenchymal stem cells (hBMSCs) osteogenic and adipogenic differentiations mediated by Wnt10b adenoviral vector in vitro. MethodsThe hBMSCs from ilial bone tissue in adults at passage 4 were infected by Wnt10b gene expression adenoviral vector (group A), Wnt10b-shRNA adenoviral vector (group B), and empty vector (group C), and non-transfected hBMSCs served as the blank control group. Then the cells were cultured separately in the circumstance of osteogenic induction, adipogenic induction, and non-induction. The alkaline phosphatase (ALP) staining, alizarin red staining, and oil red O staining were used to detect the osteogenic and adipogenic differentiations; real-time fluorescent quantitative PCR and Western blot were used to analyze the expressions of osteoblast and adipocyte genes and proteins. ResultsThe results of ALP staining were positive after osteogenic induction, group A showed strong staining, and group B showed the weakest staining. The results of alizarin red staining showed that there were a lot of patchy confluent brown mineralized nodules in group A; a few punctate brown mineralized nodules were seen in group B; and many punctuate brown mineralized nodules were found in groups C and D. The results of oil red O staining showed strong staining in groups B, C, and D after adipogenic induction, especially in group B; scattered or small clustered staining was observed in group A. The expressions of osteoblast genes and proteins were significantly higher in group A than groups B, C, and D, and in groups C and D than group B by real-time fluorescent quantitative PCR and Western blot test; however, the expressions of adipocyte genes and proteins showed a contrary tendency. ConclusionThe high level expression of Wnt10b can enhance osteogenic differentiation of hBMSCs, and the low level expression of Wnt10b can increase adipogenic differentiation of hBMSCs.
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 study the bone formation and osteogenesis after transplantation of human periosteal mesenchymal stem cells(PMSC). METHODS Suspension of PMSC which obtained from cell culture of periosteal segments in vitro were injected into the backs of nude mice subcutaneously, and the fracture site of neck of femur in old person. RESULTS Subdermal nodules were observed by naked eyes after 11 days of transplantation. 4 weeks later, their anatomic diameter reached 2-7 mm(averaged 3.6 mm). It was proved that the subdermal nodules were trabecular ball trapped with fibrous tissue. The nodules were investigated by human special apoB gene with PCR, and the test of anti-human-tissue precipitin reaction(AHTPR). The results of PCR and AHTPR were positive reaction. There were no subdermal nodules formed in the sites of injection of frozen-melted PMSC or culture medium. The new callus in the sites of fracture were tested by PCR test, and two kinds of apoB gene products were detected. CONCLUSION The results indicated that the implanted PMSC could form new bone directly in nude mice, and the cells of donor and recipient all could form new bone.
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.