OBJECTIVE: To investigate the effects of three-dimentional culture in bioderived material modified by Pluronic F-127 on the growth and function of rabbit periosteal osteoblast in vitro. METHODS: Bio-derived materials were from fresh pig ribs and were modified by Pluronic F-127. Then rabbit periosteal osteoblasts were cultured in bio-derived materials(group A), in the modified bio-derived materials(group B) and on the plastic surfaces as a control (group C), respectively. During a 7-day period, the status of growth, cell viability and alkaline phosphatase(ALP) activity were measured. RESULTS: Osteoblasts attached, elongated and grew well on the modified bio-derived materials. There were no significant difference in osteogenesis and ALP activity between group A and group B(P gt; 0.05). The osteogenesis and ALP activity in groups A, B were less than those in group C (P lt; 0.01). CONCLUSION: Pluronic F-127 can be used for a carrier for bioactive factors to modify bio-derived material.
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.
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 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 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 analyze MC3T3E1 cell morphology, prol iferation, and osteogenic differentiation in fibrin gel (FG) so as to lay a fundament for use of FG in tissue engneering. Methods MC3T3E1 cells were incubated in three concentrations (20, 10 and 5 mg/mL)of FG as the experimental groups (groups A, B and C) and in the common medium culture as the control group (group D). The cell morphology and distribution in FG were observed by inverted phase contrast microscope and confocal laser scanning microscope at different time. The cell prol iferation was assessed by fluorospectrophotometer. The alkal ine phosphatase (ALP) activity was detected by automatic biochemistry analyses and von Kossa staining was used to analyze calcium salts mineralization. RT-PCR was used to analyze the ALP and bone sialoprotein (BSP)mRNA expression at 14 and 21 days. Results In groups A, B and C, the MC3T3E1 cells had long processes which connected each other and formed network; but fusiform or cube cells were observed in group D at 21 days. The fluorescence intensity was increased gradually with time, was the highest at 14 days and the lowest at 28 days in group D; it was highest in groups A, B and C at 28 days, there were statistically significant differences when compared with group D (P lt; 0.05). The ALP activity was increased gradually with time, and it was the highest at 28 days in group D and at 21 days in groups A and B, there were significant differences (P lt; 0.05), no statistically significant differences compared with group D at other time points (P gt; 0.05). The mineral ization nodus were seen at 21 and 28 days in group A, but no mineral ization nodus was seen in group D at 28 days. The RT-PCR results showed the mRNA expressions of ALP and BSP were enhanced in group A when compared with group D (P lt; 0.05). Conclusion The osteogenic differentiation was most obvious and cell prol iferation was most active after 21 days of incubation in FG.
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.
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 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 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.