The Research Of Biocompatibility Of Biomaterial Hydroxyapatie In Bone Regeneration

Comminuted fracture often leads to bone defect,so it is difficult to be cured. autogenous bone transplantation can result in donor area bone defect.One kind of donor of bone transplantation,tissue engineering artificial bone,which has wide source and good adaptivity,is becoming the focus of tissue engineering study. Isolation,identification and purification of seed cells is one of the tissue engineering important element.There are many documents about this point,but in the course of fracture healing and bone regeneration,does any more primitive osteoprogenitor cells(undifferentiated cells) or osteoblast-like cells generate and exist? What are their capability of bone formation? How to improve the method of isolation,identification and purification? There is few documents about this point.The rabbit radial bone comminuted fracture model was established to research the histological morphous changes in the course of bone regeneration after bone injury and isolate osteoblast-like cells for providing the tissue engineering research ideal seed cells.Biomaterial supporting frame is another important element of the tissue engineering.Inorganic nonmetals,hydroxyapatite(HA),is widely used in the construction of artificial bone because it is the nature inorganic substance exists in the bone tissue,the main component of bone salts and near to bone formation microenvironment in vivo.Its molecular formula is Ca₁₀(PO₄)₆(OH)₂.In recently years,there are many documents about construction of artificial bone with HA and osteoblast,but most of them are the study of performance of complex in macro level. There are few documents about the toxic action and side effect of HA on osteoblast growth and related detection method.There are also few documents about the molecular biology mechanisms of interaction of osteoblast and HA in micro level. Osteoblasts were cultured with HA to research the effect of HA on osteoblast growth and explore the related potential mechanisms.HA disk without any modification doesn't favor osteoblast growth on it.Some treatment agents are administrated to HA to improve biocompatibility.Among them. collagen typeâ… is the nature organic component of bone and advantageous in the adhesion and growth of osteoblast.The interaction of HA.Collagen typeâ… , prostaglandin E1 and dexamethasone and their influence on osteoblast gene expression about adhesion,proliferation,signal transduction,differentiation and apoptosis were tested in the experiment to provide design of biomaterial supporting frame experiment reference.【Objectives】To provide ideal seed cells and comminuted fracture bone impairment model:To provide the tissue engineering biomaterial research a new convenient and feasible biocompatibility detection method;Investigate the influence of HA,collagen typeâ… .prostaglandin E1 and dexamethasone on osteoblast related genes expression.【Methods】1.Made the model of rabbit bilateral radial bone comminuted fracture bone impairment model.After 14d,operated again to take out the fracture healing tissue of the bone injure.One piece was used to make bone tissue slices and HE staining of broken ends of fractured bone to observe the the histological morphous changes in the course of bone regeneration after bone injury;Another was used to isolate,purify and identify osteoblast-like cells.2.Mouse MC3T3 cells were cultured with biomaterial HA disks.PI-Hoechst33342 double fluorescence dyeing was used to observe the apoptosis of osteoblast.RT-PCR method was used to detected the gene expression of OPN(osteopontin) and Id2 (Inhibitor of differentiation/DNA binding-2) at different culture stage and explored the the molecular biology mechanisms of osteoblast apoptosis.3.Modified HA with administration of Collagen typeâ… ,prostaglandin E1(10ng/ml) and Dexamethasone(10^-7M).Mouse MC3T3 cells were cultured with these agents and tested the gene expression difference of OPN,Id2,ALP(alkaline phosphatase), MAPK(mitogen-activated protein kinase) and AIF(apoptosis inducing factor) in different groups to illuminate the influence of these agents on related genes expression about osteoblast adhesion,proliferation,signal transduction, differentiation and apoptosis.【Results】1.14d after comminuted fracture,bone callus which contained osteoid and cartilaginous tissue formed in the fracture region.Primary osteoblast-like cells were isolated from the bone callus,purified and be identified by morphous observation and diazonium salt-staining to detect AKP expression and proved to be osteoblast-like cells.2.The rate of apoptosis of mouse MC3T3 cells cultured on HA disks was significantly higher than the rate of the control groups.Id2 gene expressed obviously in control group on 5d(early stage) and did not express on 15d(late stage).In HA group,there is no Id2 gene expression on 5d,and there was thinner Id2 expression on 15d.OPN expressed obviously in two groups and two culture duration.There was no significant difference between two groups and two moments.3.Collagen typeâ… enabled MC3T3 cells adhere well and not easy to be eluted.HA cooperated with Collagen typeâ… to improve the gene expression of OPN.HA promoted the gene expression of ALP,MAPK and AIF.But PGE1 could decrease the promotion of AIF gene expression by HA.Collagen typeâ… promoted Id2,OPN.ALP and MAPK gene expression.PGE1 decreased the expression of OPN.ALP and MAPK and increased the expression of Id2.Dex inhibited OPN and MAPK gene expression and promoted Id2 and had no significant effect on ALP and AIF expression.【Conclusions】1.Rabbit primary osteoblast-like cells were successfully isolated,purified and identified from bone callus after bone injure and provided a good model for the research of effect of primary cells in the course of bone regeneration.2.HA disk without any modification could promote MC3T3 cells apoptosis and needs modify to improve its biocompatibility.A new method of detection in situ of biocompatibility of opaque biomaterial which could not be observed directly under light microscope was found.HA influenced Id2 gene expression,in the early stage of cell growth,HA inhibited cells proliferation by decreasing Id2 expression:in the late stage of cell growth,HA induced cells apoptosis by increasing Id2 expression.3.HA provided a good bone formation micro-environment for osteoblast,make cells grow well,appears good characteristic of signal transduction and bone formation and promote osteoblast apoptosis,which plays a certain role in bone remodeling. Collagen typeâ… is good extracellular matrix,which can promote osteoblast adhesion, growth and gene expression.10ng/ml PGE1 inhibited gene expression of OPN,ALP and MAPK;10^-7M Dex decreased the OPN and MAPK gene expression and promoted Id2 and had no significant effect on ALP and AIF expression.These indicate that the two agents at the concentration were not fit for the bone formation and need further research to find optimal concentration interacted with HA.