OBJECTIVE: To isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI). METHODS: BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation. RESULTS: Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days. CONCLUSION: Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
There is a great hope to treat long bone defects with bioactive artificial bone constructed by osteoblasts and biomaterials, in which the key point is to provide an optimum environment for the normal function of osteoblasts. The cellular sociological characteristics of osteoblasts were summarized and it was suggested that the ideal bioactive artificial bone should be composed of inorganic and organic materials together with cellular components such as osteoblasts and vascular endothelial cells, and combined with control release of growth factors, following its implantation it could be vascularized very soon and merged with the host bone by bony consolidation.
Objective To investigate the effect of WO-1 on the proliferation and differentiation of human embryonic osteoblasts (HEO) and to provide research methods of bone tissue engineering. Methods HEO were isolated from periosteum and calvaria and then cultrued in vitro. The doseeffect relationship between WO-1 concentration and biological effect of HEO was evaluated by growth curve and 3 H-TdR count. The effect of WO-1 on cell activity and proliferation was investigated by cloning efficiency,cell cycle analysis was determined by flow cytometer and morphological was examined through transmission electron microscope. Moreover, the effect of WO-1 on osteoblastic function was evaluated at protein and mRNA levels by ALP activity, 3 H-proline incorporation, osteocalcin secretion (RIA) and mRNA expression of type I collagen and osteocalcin (RT-PCR). Results The proliferation of HEO was inhibited in high concentration of WO-1,while it was promoted in low concentration of WO-1. The optimal dose was 8 μg/ml, and there was dose-effect relationship in the certain range of WO-1 concentration (0.25 μg/ml to 8 μg/ml). In 8 μg/ml of WO-1, the cloning efficiency and cloning volume of HEO were inereased, population doubling time was decreased.All indexes of ostoblastic function including ALP activity, type I collagen synthesis and osteocalcin secertion were inereased, the more sufficed cell organs were observed under transmission electron microscope than control group(P＜0.05). Conclusion WO-1 can promote the cell activity and proliferation of HEO cultured in vitro inlow concentration, enhance the synthesis of extracellular mamix, such as type Icollagen and osteocalcin, and accelerate the mineralization of osteoid. WO-1 can be used as a stimulant of proliferation and differentiation of HEO in the research of bone tissue engineering, which provide the theoretical basis in clinical application.
OBJECTIVE: To evaluate the cellular compatibility of three natural xenogeneic bone derived biomaterials. METHODS: Three types of natural xenogeneic bone derived biomaterials were made with physical and chemical treatment, composite fully deproteinized bone(CFDB), partially deproteinized bone(PDPB) and partially decalcified bone(PDCB). Three types biomaterials were cocultured with human embryonic periosteal osteoblasts. The cell growth, attachment, cell cycle, alkaline phosphatase activity were detected to evaluate the cellular compatibility to biomaterials. RESULTS: Osteoblasts attached on all three biomaterials and grew well, the effect of three biomaterials on cell proliferation was PDCB gt; PDPB gt; CFDB. The cell cycle was not obviously affected by three biomaterials. The effect of three biomaterials on alkaline phosphatase activity of osteoblasts was PDCB gt; PDPB gt; CFDB. CONCLUSION: CFDB,PDPB,PDCB have good cellular compatibility without cytotoxic and tumorigenicity, CFDB is the best. The three biomaterials can be used as scaffold materials of bone tissue engineering.
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) gene transfer on the biological characteristics of osteoblasts. Methods The expression of TGF-β1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-β1 activity in the supernatant (minklung epithelium cell growth -inhibition test). The effects of gene transfer andsupernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3 H-TdR and MTT. Results The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-β1 gene could express TGF-β1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1∶1, 1∶2, 1∶4, could inhibit growth of Mv-1-Lu evidently and the ratios ofinhibition were 16.3%, 22.7%, 28.2% respectively. TGF-β1 gene transfer hadno effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALPactivity of osteoblasts. Conclusion TGF-β1 gene transfer promotes the expression of TGF-β1 and the biological characteristics of trasfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
Objective To investigate the possibility of ectomesenchymal stem cell of human embryo facial process in differentiating into osteoblasts.Methods Ectomesenchymal stem cells of human embryo facial process were isolated and cultured in mineralized promoting solution containing 10 mmol/L β-glycerophosphate, 100 μg/ml ascorbic acid and 10 nmol/L dexamethasone supplemented with 15% FBS. The morphological change was observed by phase contrast microscopy. The characteristics of cells was identified by immunohistochemistry assay. Alkaline phosphatase activity was tested and the form of mineralized nodules was tested with Von Kossa staining. The expression of osteocalcin was identified by RT-PCR.Results There were significant changes in the shape of the cells after 3 days cultured in mineralized promoting solution. The cells became larger and the shape changed from fibroblast-like to multilateral. The result for anticollogen typeⅠstaining was positive. The alkaline phosphatase activity increased. Mineralized nodules were formed aftercultured 25 days by Von Kossa staining. RT-PCR assay showed induced cells expressed osteocalcin.Conclusion Ectomesenchymal stem cells of humanembryo facial process can be induced to differentiate into osteoblasts by mineralized promoting solution.
OBJECTIVE: To study the expression of type I collagen and its receptor system-integrin alpha 2 beta 1 in different passages of osteoblasts. METHODS: The expression of type I collagen and integrin alpha 2 beta 1 in the primary, sixth and fifteenth passage of osteoblasts were detected by S-P immunohistological staining technique, and their mRNA expression by quantity RT-PCR technique. RESULTS: Type I collagen and integrin alpha 2 beta 1 were expressed in different passages of osteoblasts and there was no significant difference among three passages by immunohistological technique. Their mRNA expression was gradually decreased with subculture. CONCLUSION: Type I collagen promotes the adhesion and phenotype expression of osteoblasts through its receptor-integrin alpha 2 beta 1. The reductive expression of type I collagen-receptor system will decline the phenotype of osteoblasts.
Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.