Study On The Construction Of Tissue Engineering Bone By Heterogenous Bone Acellular Extracellular Matrix Which Was Compounded By The Osteoblast And The Vascular Endothelial Cell

The culture of seed cell is an essential element in tissue engineering. Osteoblast is the seed cell in tissue engineering, but the base for the repairing of bone defect in tissue engineering is still its vascularization. Co - culture of the osteoblast and the vascular endothelial cell can not only promote the proliferative activity of the osteoblast but also can accelerate the revascularization of the tissue engineering bone. Thus it can help to increase the ability of tissue engineering bone in the repairing of bone defect. With the rapid development of the society, the traffic and mechanization are improved. The complicated and severe fracture caused by the high - speed and high - capacity trauma will result in bone defect and nonunion of the fracture. The bone source and the site for supplying bone were limited in autografting, meanwhile it is difficult in the modeling of self - tissue, therefore its clinical application is limited. There is certain im-munologic rejection in the isotype heterogenous bone grafting and the tissue engineering bone provides a new way for solving this problem. In this experiment, the osteoblast and the vascular endothelial cell were compounded by using the a-cellular rib of pig. Then the tissue engineering bone was constructed and implanted into the animal. Observe and study the biological compatibility, try to find out the pathological process of in vivo ossification, bone defect repairing and revascularizarion for the heterogenous bone grafting. Thus it will provide the theological base for the clinical application of tissue engineering bone.Method1. The culture of the seed cell in tissue engineering and the study of cellular compatibility1.1 Cell cultureCulture the osteoblast (A group) and vascular endothelial cell( B group) of the rabbit, and co - culture the osteoblast and vascular endothelial cell ( C group).1.2The index of observation1.2.1 Observation under the inverted phase contrast microscope Observation of the morphology and the growth of the cells in the 3 groupsunder the inverted phase contrast microscope1.2.2 Immunocytochemical stainImmunocytochemical stain of type I collagen to identify the osteoblast. Immunocytochemical stain of the vascular factor Vffl to identify the vascular endothelial cell.1.2.3 Histological observationThe sections were made by the co - cultured cells. Three days later, observe the mixed growth of the two kinds of cells after HE stain and Masson tri-chrome stain.1.2.4 Test the activity of alkaline phosphatase( ALP)Test the activity of ALP by using the method of paranitrobenzene phosphate. Observe whether the vascular endothelial would affect the ALP activity produced by the osteoblast.1.2.5 Test the cellular activity by the method of methythiazolyl tetrazolium bromide( MTT)Analyse the growth and proliferation of the cells in the 3 groups.2. Study the effect of heterogenous bone acellular extracellular matrix (AECM) on the biological compatibility between the osteoblasts and the vascular endothelial cells2.1 Prepare framework material for the heterogenous bone acellular extra-cellular matrixThe fresh rib of the pig was defatted, deantigened and decellular by hydrogen peroxide solution, chloroform/methanol and Triton X - 100, then it was made into the framework material of the heterogenous bone acellular extracellular matrix and was observed under the scanning electron microscope.2. 2 Compound of the cell with the heterogenous acellular extracellular matrixThe 3 groups of rabbit cells were compounded with the acellular rib of the pig individually. Observe and study the biological compatibility, antigenicity and the biological toxicity of the heterogenous acellular extracellular matrix.2.3 The indexes of observation2. 3.1 Observation under the inverted phase contrast microscope Observe the adhesion of the cells with the material , the distribution of thecells in the pores of the material, and the growth, differentiation and proliferation of the cell.2.3.2 His...