Objective To study the biological behavior of osteoblast and vascular endothelial cell culture. Methods The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortox of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C(co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibilitywere observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase(ALP) activity were measured. Results The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells .The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B(P＜0.01), and it was higher in group A than in group B(P＜0.05). In group C, the cellproliferation were increased slowly early, but fast later. Conclusion Thecellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity,and the combined cultured cells have greater proliferation ability.
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 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 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.
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 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 find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissueeng ineering． Methods There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone，10 mmol/L beta-glycero-phosphate，50 μg/ml retin oic acid) on growth of MSCs isolated from the rat femur and the tibia． MSCs were cultured in the DMEM medium (the c ontrol group) and in the osteoinductive culture medium (the inductor group)；fur thermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group)．The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured．The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymase chain reaction (RT-PCR)． Results There were more cells in the osteoblast group than in the control group(31.73±3.31×104 V S. 24.33±3.04×104, Plt;0.05), but there were fewer cells in the inductor gro up(16.23±2.44×104, Plt;0.05). There was no significant difference in th e cell number between the combined treatment group (21.54±2.29×104) and th e control group（Pgt;0.05）．The ALP activity was higher in the combined trea tment group （2.01±0.56 U）than in the control group (1.27±0.43 U), in the inductor group（1.27±0.43 U）, and in the osteoblast group （0.77±0.19 U）.The osteocalcin mRNA was expressed in the three treat ment groups but was not expressed in the control group. The significantly higher leve l of the osteocalcin mRNA was expressed in the inductor group（0.783±0.094）and in the combined treatment group（0.814±0.071）than in the osteoblast group（0.302±0.026） (Plt;0.05). Conclusion The combined use of t he osteoblast and the inductor can induce marrow stromal cells. Their combined u se does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering．
Osteoblasts were cultured and isolated from a piece of tibial pettiosteum of four New-Zealandrabbits. After subeultured,these cells Were incubatd in vitro with tritiated thvmidine for 36 hoursand then these labeled cells were implanted in the subeutaneous layer of the defects of the auriclarcartilage and the radial bone, After 2 weeks and 4 weeks respectively, these rabbits were killed andthe spoimens were obtained from the site where the cells had been transplanted. The transformation of these cells was observed by autoradiographic method. The results indicated that nearly all of the cultured cells were labeled. After 2 weeks, it was observed that many labeled osteoblasts were in different stages of differentiation, some were beried by extracellular matrix and resembled osteocyte, thers were differentiated into chondrocyte-like cell. In addition, some labeled osteoblasts were congregated in the form of multinucleated osteoclast. After 4 weeks , in the subcutaneous layer the labeled osteoblasts were changed to osteoid tissue and in the defect of the auricular crtilage these cells transformed into chondritic tissue; moreover, those labeled osteoblsts which had been implanted into the radial defect had differentiated into typical bone tissue. The results of this research indicated that the osteoblasts isolated from the periosteum if reimplanted to the same donor might be possible to repair the bone and cartilage defects.
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 investigate the influence of different dose levels of hydroxyapatite/tricalcium phosphate (HA/TCP) on the proliferation and alkalinephosphatase (ALP) activity of rabbit osteoblasts. Methods Three different doselevels of HA/TCP (10%, 40%, 70%) were co-cultivated with rabbit osteoblasts respectively. The proliferation and ALP expression capacity of osteoblasts were examined with MTT method and enzyme histochemistry once every 24 hours until 5 days. Three control groups of other materials were treated and examined in the sameway: rabbit osteoblasts as normal control; polyvinylchloride as positive control; titanium alloy as negative control. Results There was remarkable timeeffect relationship in the proliferation of osteoblasts. Ten percent HA/TCP did not affect osteoblasts growth while 40% HA/TCP could slow the cell growth rate down though time-effect relationship still existed. The proliferation of osteoblasts stagnated when co-cultivated with 70% HA/TCP. On the other hand, 10% HA/TCP could cause reversible damage on ALP activity of osteoblasts, whereas when the dose was40%, and the cultivation lasted 6 days the damage was irreversible. Three different dose levels of titanium alloy (10%, 40%, 70%) had no effect on the proliferation or ALP activity of osteoblasts. Conclusion Dosage is an important factor affecting the biocompatibility evaluation of biomaterial. It suggests that dose choosing should be more specified upon each individual biomaterial. It also indicates that ALP may be a good supplementary index of the cell compatibility of material.
