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.
Objective To review the progress, methods and obstacles in the differentiation of embryonic stem cells(ESCs) into osteoblasts in vitro. Methods The recent literature concerned with the differentiation of ESCs into the osteoblasts was extensively reviewed and briefly summarized. Results ESCs was a good tool for derivation of obsteoblasts.Conclusion The study on the induction of ESCsinto the osteogenic lineage provides a model for analyzing the molecular processes of osteoblasts development in vivo and establishes the foundation for the use of ESCs in skeletal tissue repair.
Objective To study the effect of various storage methods on cellular compatibility of bio-derived bone. Methods Freeze dried biomaterials had been stored in two different preservation solutions for three months, while the biomaterials stored for the same time were observed as control group. The experiment was divided into groups A, B, C and D according to different storage methods (group A: with materials stored in preservation solution 1; group B:with materials stored in preservation solution 2; group C:with freeze-dried materials; and group D: simple osteoblasts). Osteoblasts at 2×106/ml had been cocultured with materials for 1, 3, 5, and 7 days.The cell-material complexwas observed under phase microscope and electronic scanning microscope to evaluate the adhesion and growth of osteoblasts; the cell viability and alkaline phosphatase(ALP) activity were measured,and the cell cycle wasanalysed by flow cytometer.WTHZ〗Results Osteoblasts adhered to materials preserved by different methods,differentiated and proliferated in the hole of materials. The difference of cell viability was not significant between three groups on day 1 andday 3. The cell viability of osteoblasts adhered to three materials was Agt;Cgt;B group on day 5 and day 7 (Plt;0.01，Plt;0.05). The ALP activity of osteoblasts adhered to three materials was Agt;Cgt;B group on day 7(Plt;0.01).The cell cycle of differentgroups did not change significantly,the abnormal cells were not seen. Conclusion The choice of proper preservation solution can optimize the cellular compatibility of bio-derived bone.
Objective To investigate the effect of 1,25(OH)2VD3 on differentiation of embryonic stem cells (ESCs) into osteoblasts. Methods Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies (EBs) were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums: group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C (VC, 50 μg/mL) and β-glycerophosphate (β-GP, 50 mmol/L); group C, inwhich EBs medium was the same as that of group B and 5 × 104 osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC (50 μg/mL), β-GP (50 mmol/L) and 1,25(OH)2VD(4 × 10-9 mol/L), and 5 × 104 osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days. The RQ-PCR was performed to measure the mRNA expressions of osteocalcin (OCN). Al izarin red S staining was performed to count the bone nodules. Results The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outl ine and different sizes were evident by microscope. Al izarin red S staining testified the number of bone noudles in groups A, B, C and D was 20 ± 8, 18 ± 5, 31 ± 1 and 50 ± 1, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 ± 1.17, 20.29 ± 1.03, 18.84 ± 4.07 and 32.15 ± 5.23, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). Conclusion The combined action of 1,25(OH)2VD(4 × 10-9 mol/L), VC, and β-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.
Objective To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
OBJECTIVE To investigate the feasibility of freeze-dried demineralized bone matrix (FDBM) as scaffold material in bone tissue engineering. METHODS Osteoblasts which were isolated from cranial periosteum of New Zealand rabbits were cultured as the seeding cells, then the cells were cocultured with heterogenous FDBM in vitro. The cell-material complex was observed under phase microscope, light microscope and electronic scanning microscope in order to evaluate the interaction between cells and FDBM. RESULTS Eight hours after coculture, the osteoblasts adhered to FDBM scaffolds. Seven days later, the osteoblasts differentiated and proliferated in FDBM network. Extracellular matrix was secreted and calcium nodes were formed among osteoblasts. CONCLUSION FDBM is a good scaffold material for the bone tissue engineering.
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 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 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 review the research progress of osteoblasts in the hematopoietic microenvironment of bone marrow and regulatory pathways and mechanisms. Methods The advances in the osteoblasts as crucial components for hematopoietic microenvironment in bone marrow, regulation to osteoblasts and hematopoietic stem cells(HSCs), and correlative singal pathways and mechanisms were introduced based on the recent related literature. Results Evidence indicates that osteoblasts are crucial components of the hematopoietic microenvironments in adult bone marrow. The osteoblasts maintainthe quiescence of primitive HSCs by the signaling receptorsligands, secreted cell factors and celladhesion molecules and by regulating other cells in the niche. The quiescent primitive HSCs persist stem cell characteristic which has unlimited selfrenewal and multipotent differentiation potential. Conclusion The further understanding of the relationship between osteoblasts and hematopoietic microenvironment should lead to development of new strategies directed toward clinical therapeutics of HSCs transplantation.