Preliminary Experimental Study On Immunity With Transplantation Of Tissue Engineered Bone(Ted) From Allogeneic Osteblasts(Differentiate From Bmscs,Bone Marrow Stromal Cells) Repair Of Rabbit's Radius Bone Defectd

Objective:To investigate immunity of allogeneic tissue engineered bone with osteoblas-ts (differentiate from BMSCs) immunization to allogeneic T lymphocytes in vitro and the immunomodulatory with transplantation of tissue engineered bone from such cells as seed cells and hydroxyapatite-tricalcium phosphate to repair bone defects, in order to provide the experimental basis for allogeneic osteoblas-ts construct tissue engineered bone repair of bone defects.Methods:1,Bone marrow mesenchymal stem cells(BMSCs) to obtain and identificationTo obtain the Chinese rabbit bone marrow mesenchymal stem cells (BMS-Cs) by density gradient centrifugation and wall sticking, conduct primary culture and sub-culturing, purifying; observe cell morphology, detection of surface mark objects of expression, with MTT method determination of the 3rd generation cell proliferation situation, draw out the growth curve, and the fat induced by oil red O staining.2, Bone marrow mesenchymal stem cells(BMSCs) osteogenic research Taking growth well cells of three generations with osteogenic induction medium for induction and cell morphology was observed and cultured 2days, 5days,8days,lldays,14days respectively.The cells were collected for determination of alkaline phosphatase (ALP) in cells. Then the expression of type I collagen was detected by immunocytochemistry method on 7 days and osteocalcin was detected on 14 days;calcification nod staining by alizarin red staining method on 21 days; cell morphology of cell induction of osteoblast and matrix secretion was observed with scanning electron microscopy.3,Allogeneic osteoblasts regulate T cell subsets in the mixed lymphocyte reaction system (MLR) in vitroTo obtain T lymphocyte cells of the New Zealand White rabbits in peripher-al venous blood by density gradient centrifugation and nylon wool column met-hod,,purified, cultured then to detect its concentration. The mixed lymphocyte reaction system was found with Chinese rabbit osteoblasts (differentiate from MSCs)as stimulate cells, New Zealand rabbit T cells as reactive cells. MTT assay T cell proliferation and flow cytometry assay T-cell apoptosis.4, Allogeneic tissue-engineered bone constructTaking osteoblasts of the Chinese rabbit's as seeds was co-cultured with the HA-TCP scaffolds. Sampling cells was determined by the adhesion rate at 4,8, 12,24 hours respectively after co-culturing. Observing osteoblast cells growth, proliferation and matrix secretion on surface of the HA-TCP scaffolds with electron microscopy when co-cultured for three weeks, drawing the line scanning of cell and expression of type I collagen by RT-PCR detection.5,Preliminary experimental study on allogeneic tissue engineered bone transplanted to repair bone defects in immune(1),Bone tissue engineering (Group I), simple HA-TCP (groupⅡ), autogeno-us bone (III group) were randomly implanted in the rabbit radius bone defect, routine feeding to observe the implant surface changes;(2),To observe the changes of the radius bone defect with X-ray examination at 6 weeks after postoperative, the general situation of bone healing in bone defect and ultrastructural changes with transmission electron microscopy(3),Spleen index were measured by weighted spleen at 4 days,10 days, six weeks after postoperative, the EDU cell staining of spleenic cell proliferation; spleen HE staining to the pathologic changes, to observe an ultrastructural changes of T lymphocytes with transmission electron microscopy; ELISA, detection of spleen cells of IFN-y, IL-10 expression level; RT-PCR detection of expression of IFN-ymRNA and IL-lOmRNA in spleen cells.Results:1,BMSCs were uniform, long spindle by method as density gradient cen-tri-fugation and wall sticking, conduct primary culture and sub-culturing, purifyin-g; cell morphology close to the fusion state can show vortex-like growth; the cell proliferation detection curve shows the cell proliferation of self-update ability; the marker on surface antigen of BMSCs nearly was CD44, CD 105 and only about 2% was CD34 and into fat-induced oil red staining was positive.2,Bone marrow mesenchymal stem cell osteogenic cell morphology gradual-ly becomes long spindle or short spindle, polygonal, and finally into a colony cascading growth. ALP content was increasing higher and there was significant differences (p＜0.05) compared with the control group during induction. Type I collagen was positive at 7 days; osteocalcin was positive at 14 days and calcified nodules was positive at 21 days; cell grow well and secretion of matrix.3,Cells adhesion rate were 44±1.5%,68±1.3%,78±1.0%,84±2.0%, 87±1.8% at 4h,8h,12h,24h,36h respectively.After 36h, cell adhesion rate of each HA-TCP material is more than 85%. After three weeks, osteoblasts were growth strong and good shape on the HA-TCP material surface and extension into the material porosity, matrix secretion well. 4,Animals with postoperative wound in each group healed well and there are inflammatory response in local and systemic. After six weeks, bone junction ofⅠgroup andⅢgroup are almost healed butⅡgroup are not satisfied with the X-ray showing. The bone ofⅢgroup and the radius bone defect is basically to achieve bone healing. Three groups of bone defects in bone healing:Ⅲgroup＞Ⅰgroup＞Ⅱgroup. Transmission electron microscopy revealed osteoblast in three groups of bone defect broke into the collagen fibers and fiber fibers and rich in collagen fibers. There are significant difference among three groups; I group composed of fiber fiber, rich in collagen fibers; andⅡgroup composed of fibers fibers, collagen fibers relatively small; fiber fiber composed of autologous bone, the most abundant collagen fibers.5,Spleen index of the animals in each group after postoperative at four days; ten days, six weeks is a slightly higher in the same group among at earlier and a slightly lower at late, P＞0.05; but there are no difference among three groups, p＞0.05; HE staining showed that spleen morphology was normal at each time points, the splenic nodule was uniform size, spleen red pulp and spleen white pulp was no significant difference; just the blood vessels of the early red pulp is a little expansion, congestion, but white pulp significantly expanded; lymphocytic of splenic nodule was slightly reduce;form of spleen no significant change. Red pulp morphology returned to normal at late, splenic lymphocytes to reduce the earlier. Spleen cell proliferation is a slight higher in the same group among at earlier and a slightly lower at late, P＞0.05; but there are no difference among three groups, p>0.05.Electron microscopy showed that the lymphocyte structure of the autogenous group was normal, the lymphocyte's mitochondria of tissue engineering bone group and pure HA-ACP group are varying, degrees of vacuolization of mitochondrial cristae, disorders, swelling, agglutination; endoplasmic reticulum expanding, widen the gap of endoplasmic reticulu-m;degranulation of endoplasmic reticulum; chromatin were condensation and margination, chromosome was loose, increased the golgi complex and lysosomes of different levels significantly; dilated rough surfaced endoplasmic reticulum and degranulation of mitochondria. The expression of IFN-y, IL-10 and IFN-ymRNA, IL-10mRN in spleen cells at 4days, lOdays, six weeks after postoperative is a slight higher in the same group among at earlier and a slightly lower at late, P＞0.05;there were no significant difference, and no statistical significance among at three groups, P＞0.05.Conclusion:1,BMSCs separation and extraction operation is simple and there is strong growth and proliferation capacity, can be induced to differentiate into osteoblasts, in line with the basic conditions for tissue engineering cell.2,Allogeneic osteoblasts can inhibit the proliferation of T cells in vitro and mainly induced apoptosis of CD4+ T cell subsets masses.3,Tissue engineered bone which is constructed by allogeneic osteoblasts (diff-erentiate from BMSCs) and the HA-TCP materials in vitro is simple and precise and meet the needs of the bone tissue engineering basically.4,Tissue engineered bone which is constructed by allogeneic osteoblasts (diff-erentiate from BMSCs) osteogenic ability is powerful in vivo, immunity is very low, can be used as bone defect repair materials.