The Role Of 1, 25-dihydroxyvitamin D₃ As A Cellular Growth Factor In Bone Tissue Engineering

Objective To find out a simple and convenient method in osteoblasts culture from bone marrow and in endothelial cell from umbilical vein in vitro. Methods The bone marrow was obtained by aspiration from healthy adult volunteers, mononuclear cells were then isolated by centrifugation in Ficoll density gradient. Plating was performed in 25ml culture flasks in RPMI-1640 medium and 100ml/L fatal bovine serum. The cells were incubated at 37℃ and relative humidity and in 5% CO₂ environment. After the culture was incubated 10¹2 days, the cells were passaged. The second passaged cells were plated in mineral condition medium containing dexamethasone, beta-sodium gylcerophosphate and ascorbic acid, the mineralized node was stained by Alizarin- Red. Human endothelial cells freshly obtained from human umbilical cords by collagenase digestion of interior of the umbilical vein were cultured using RPMI-1640 supplemented with 20% fetal calf serum. Results Both human marrow stromal osteoblast (hMSO) and human umbilical vein endothelial cells(hUVEC) presented typical morphological and biological characteristics. Mineralized node was observed after hMSO were cultured 14 days in mineralized condition. The immunohistochemical staining of hUVEC showed the cells containing â…§ related antigen. Conclusions hMSO and hUVEC are hopeful excellent seeding-cells for bone tissue engineering.Part 2.Effect of 1,25-dihydroxyvitamin D3 on co-culture of human mahUowstromal osteoblast and human umbilical vein endothelial cell Abstract Objective To evaluate the effect of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on co-culture of human marrow stromal osteoblast(MSO) and umbilical vein endothelial cells(UVEC). Methods Induced human mesenchymal stem cells(MSC) to MSO. Co-culture MSO and UVEC with same number of total cell in 24-well tissue culture plate, with same number of MSO in 96-well tissue culture plate. Evaluate cell activity on the 5th day after 1,25-(OH)2D3 applied, alkaline phosphatase(ALP) activity and osteocalein(OCN) production on the 3rd,6th,12th day after 1,25-(OH)2D3 applied. Results Appling of 1,25-(OH)2D3 has the same effect on MSC comparing co-culture of MSC and VEC, they all made the cell activity , ALP, OCN increased. When 1,25-(OH)2D3 applied to co-culture of MSC and VEC, all the examining indexes increased farther more. Conclusions 1,25-(OH)2D3 can accelerate the differentiation of MSC and when it applied to co-culture of MSO and VEC, the osteogenic capability of MSO is increased farther. Part 3.Compound tissue engineering bone with 1,25-dihydroxyvitamin D3 Abstract: Objective To compound tissue engineering bone with human marrow stromal osteoblast(MSO) , umbilical vein endothelial cells(UVEC) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] oncoral-derived hydroxyapatite (CHA). Study the biocompatibility and cellfunction of of MSO. Methods Prepare CHA with 1,25-(OH)2D3,culture MSO and UVEC on CHA for 3 days.Observe them under scanning electron microscopy and invertedmicroscopy. Evaluate the alkaline phosphatase(ALP) activity andosteocalein(OCN).Results Both MSO and UVEC grow well in vitro, regardless of thepresence of CHA, with biological and morphological characteristicssimilar to those of their normal status. CHA showed no adverse effectson them. The 1,25-(OH)2D3 on CHA increased the ALP and OCN levelof MSO just as the condition without CHA. Conclusion CHA is anoptimal scaffold material for bone tissue engineering, which maypotentially find clinical application for bone defect repair.Part 4.Experimental study on the artificial bone composite of biocoral, 1,25-dihydroxyvitamin D3, human marrow stromal osteoblast and umbilical vein endothelial cellsAbstract Objective To study the ectopic osteogenisis of artificial bone composite of coral-derived hydroxyapatite (CHA), 1,25-dihydroxyvitamin D3, human marrow stromal osteoblast(MSO) and umbilical vein endothelial cells(UVEC). Methods Combined biocoral with 1,25-dihydroxyvitamin D3, human marrow stromal osteoblast and umbilical vein endothelial cells. Implanted the composite under the skin of mice, and Observe them under scanning electron microscopy , microscopy and laser scanning confocal microscope after 4 and 8 weeks. Results The contents of ossification were group A>group B>group C. The chondro-cyte differentiation and matrix formation were observed in local sites after 4 weeks, lamellar bone with bone marrow were formed after 8 weeks. Conclusion The CHA/l,25-dihydroxyvitamin D3/ MSO and UVEC composite possesses a superior osteoinduction and will be a new type of bone substitute to be used in orthopedic surgery.