An Experimental Study On Heterotopic Transplantation Of Bone Marrow Mesenchymal-derived Stem Cells Transfected With Dentin Matrix Protein 1

Objective: The loss of tooth, an important organ of oromaxillo-facial region, not only affect people's beauty of facial, but also the function of chewing and pronunctiton. Now, current treatment for tooth loss are fixed partial denture , removable partial denture or dental implants. For the past few years, the develop of tissue engineering provide a new strategy for tooth loss and reseachers give more attention on tooth tissue engineering. Bone marrow marrow-derived mesenchymal sem cells (BM-MSCs) is a promising seed cells for many tissue engineering , including tooth engineering. In our study, we transfected porcine BM-MSCs with dentin matrix protein 1 (DMP1) gene which is related to tooth development. Then the transgenic cells were transplated ectopicly in the thigh muscle of autologous or allogenic individuals. Two month after transplantation, animals were sacrificed and specimens were examined histologically and immunologically to investigate the survival of the transplanted cells and their biological beheavies, whether there were secreted matrix or calcification happening. Our experiment provided an evidence of the election of tooth tissue engineerin seed cells. Methods:1 Isolation and cultivation of porcine BM-MSCsBone marrow aspirates were obtained from two minipigs which named P-â… ,P-â…¡seperately. Briefly, BM-MSCs were isolated by their adherence to plastic. They were cultured in DMEM supplemented with 10% FBS at 37℃with 5%CO₂ in culture flasks. The non-adherent cells were washed out gradually by changing the medium. The cells from two animals were namedâ… -BM-MSCs,â…¡-BM-MSCs seperately。2 Identification of BM-MSCs phenotypeThe surface antigens including CD34,CD38,CD45,HLA-DR,CD90,CD44,CD147 were detected by flow cytometry. (It was done at tumor research institute of the forth hospital of hebei medical university)3 DMP1 gene transfectionThe third passage cells were elected to be transfected. Before transfection cells were plated at 1×10⁵ into 6-cell culture dishes. Liposome-mediated transfection were performed on cells at 60% confluence. After 48h, the cells were diluted 1:10, cultured in the medium with G418 at the cocentration of 500μg/ml for 14 days. The medium was changed every 4 days. Then transfected cells namedâ… -BM-MSCs- DMP1,â…¡-BM-MSCs- DMP1 were harvested for further use.4 Immunohistochemistry staining of transgenic cellsFirst, cells were cultured on coverslips in the 6-cell culture dishes, then the expression of DMP1 and dentin sialoprotein (DSP) in cells ofâ… -BM-MSCs- DMP1,â…¡-BM-MSCs- DMP1 was examined by immunocytochemical SP staining. The antibody titre was 1:200. The untransfected cells were used as negetive controls.5 Ectopic transplantation of transgenic cellsBefore transplantation, cells ofâ… -BM-MSCs- DMP1 andâ…¡-BM-MSCs- DMP1 were suspended in PBS at a density of 1×10⁷/ml. Then the cell suspension were injected in the wall thigh muscle of proximal part lower limbs as follows (4 sites per limb and 200μl per site):â… -BM-MSCs- DMP1 in the left lower limb of P-â… ,â…¡-BM-MSCs- DMP1 in the right lower limb of P-â…¡(autologous team);â…¡-BM-MSCs- DMP1 in the right lower limb of P-â… ,â… -BM-MSCs- DMP1 in the left lower limb of P-â…¡(allogenous team).6 Histological and immunohistochemical investigation of specimenAnimals were sacrificed two month later and the graft samples were harvested, followed by fixation in 4% paraformaldehyde. Then samples were embeded in paraffin, cut at 4μm, and stained in hematoxylin and eosin (H&E) and immunohistochemistry. The fate of the cells were investigated to see whether there were dentin-like tissue formation and delected the expression of DSP in the transplanted cells.Result:1 BM-MSCs were isolated and cultured successfully from porcine, the result of flow cytometry indicated they were from mesenchymal tissue.2 BM-MSCs were transfected with DMP1 gene by liposome-mediated method successfully. The result of immunohistological staining showed that DMP1,DSP were positive in transgenic cells.3 The HE staining results of autologous transplantation team showed that transplanted cells secreted matrix and mineralized. We can also investigated the formation of dentin-like organization. The expression of DSP of some tranplanted cells in the dentin-like tissue were positive.4 Evident inflammatory reaction were investigated in the allogenous team by HE staining. The tissue in the central region was necrosised and the surroundings were connective tissue in which some transplanted cells can be seen. These cells had no activity of matrix secretion, but showed positive in DSP immunohistochemical staining.Conclution:1 BM-MSCs were easily isolated and cultured ex vivo and can be tranfected with DMP1 gene.2 The result of ectopic transplantation indicated that autologous BM-MSCs- DMP1 may be a promising seed cells for tooth tissue engineering while the use of allogenous BM-MSCs- DMP1 should be further discussed because of evident inflammation.