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 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.
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 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.
ObjectiveTo study the effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. MethodsCalvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL (0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups). The absorbance (A) values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration (experimental group) for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. ResultsThe cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture (P<0.05). The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups (P<0.05) except 0.01 and 0.05 mg/mL groups (P>0.05). The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. ConclusionTitanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.
ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.
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 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 the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.