Objective To investigate the behavior of rat calvarial osteoblasts cultured on chitosan-gelatin/hydroxyapatite (CSGel/HA) composite scaffolds. Methods The rat calvarial osteoblasts (the 3rd passage) were seeded at a density of 1.01×106 cells/ml onto the CS-Gel/HA composite scaffolds having porosity 85.20%, 90.40% and 95.80%. Cell number was counted after cultured for 3 days,1 week, 2 weeks and 3 weeks. Cell proliferation, bone-like tissue formation, and mineralization were separately detected by HE, von Kossa histological stainingtechniques. Results The CS-Gel/HA composite scaffolds supported the attachmentof seeded rat calvarial osteoblasts. Cells proliferated faster in scaffold withhigher porosity 90.40% and 95.80% than scaffold with lower porosity 85.20%. The osteoblasts/scaffold constructs were feasible for mineral deposition, and bonelike tissue formation in 3 weeks. Conclusion This study suggests the feasibility of using CS-Gel/HA composite scaffolds for 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.
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.
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 sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique. METHODS: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case. RESULTS: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed. CONCLUSION: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
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 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 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.
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 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.
