OBJECTIVE: To investigate the influence of basic fibroblast growth factor (bFGF) on adhesion characteristics of osteoblasts, aimed at the important problem in bone tissue engineering of how to promote the adherence of osteoblasts to extracellular matrix materials. METHODS: 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml bFGF were used to induce bone marrow stromal-derived osteoblasts of rabbit for 24 hours before incubation, and the common culture medium as the control. The attached cells were calculated with stereology method at 0.5 hour, 1st hour, 2nd hour, 4th hour, 8th hour after seeding. RESULTS: The number of attached cells was significant higher in the experimental group when induced by 10 ng/ml bFGF than that in the control group (P lt; 0.01); the number did not increase with the increase of bFGF concentration and there was no significant difference between the experimental group induced by 100 ng/ml bFGF and control group, and the number was even obviously lower in the experimental group when induced by 200 ng/ml than the control group (P lt; 0.01). CONCLUSION: bFGF can influence the adhesion characteristics of osteoblasts, 10 ng/ml bFGF can promote the adherence of osteoblasts to matrix materials, but 200 ng/ml bFGF may inhibit cell adhesion.
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 establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
Objective To review the research progress of osteoblastextracellular matrix(ECM) and its application in bone tissue engineering. Methods The recentrelated literatures were extensively reviewed. Results The ECM was complex in its components. The configuration of cell and cell’s adhesion, migration, proliferation, and differentiation were subject to the ECM. The bioactivity of the tissue engineering products was revealed by ECM, which predicted the product’s efficiency in clinic application. Conclusion ECM has the potential to become the effective index in evaluating tissue engineered products.
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 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 study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signal ing factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH’s function in bone metabolism. Methods Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkal ine phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to α-MEM supplemented with 1%FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently ithcold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative eal-time PCR. Results Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Al izarin red staining showed the formation of red mineral ized nodules in the special mineral ization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P lt; 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P lt; 0.05); the expression of β-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P lt; 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1- 34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P lt; 0.05). Conclusion In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.
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.