Tissue Engineered Bone Formation Originating From Bone Marrow Stromal Cell Transferred By Nell-1 Gene In Vitro And Vivo

There are often various causes of bone defects in clinics. They are difficult to treat clinically, especially those of large segments of weight-bearing bones. The technique of tissue engineering, especially the technique of gene-enhanced tissue engineering has brought about a hopeful resolution for the problem. Bone marrow stromal cell is now the most value seed cells of bone tissue engineering, and the target cells of gene-enhanced tissue engineering bone. Nell-1 is a new gene has been cloned recently, who has significantly capability of osteogenesis. The Nell-1 protein could be a new growth factor used in bone tissue engineering. There is no report about BMSc transfected by Nell-1 gene used in mandible rebuilt by the method of tissue engineering now. In this rearch, human Nell-1-expressing replication-deficient retroviral vector (PLNCX2-Nell-1) was reconstructed, Then the ability of PLNCX2-Nell-1 virus granules and the BMSc transferred by Nell-1 gene composited with Fibers Glue (FG) biomaterials to enhance segmental bone defect healing of the Beagles mandible were evaluated. In the present study, human Nell-1-expressing replication-deficient retroviral vector (PLNCX2-Nell-1) was reconstructed using clone technique and recombined DNA technique. Then it was transferred into incasing cells PT67 by liposome-mediated method. The clones of the cells transferred were selected with G418.Targeted cells (BMSc) were infected with the virus granules which secreted from PT67 cells and also selected by G418. The mRNA and protein of Nell-1 gene in transferred cells were determined by RT-PCR, hybridization in situ and immunohistochemistry. Rhodamine phalloidin-DAPI stain and Jet impingement were performanced to detect cell spreading area and cell adhesion strength .The proliferativity of the transferred cell were assayed by methabenzthiazuron (MTT) method. Syntheses of collagen were assessed with 3H-proline incoporation test. Alkaline phosphatase (ALP) , osteocalcin (OC) and laminin(LN)were also detected using enzyme kinetics and radio-immunity methods. The in vivo gene therapy approach was applied in Beagles. Segmental 2.0cm×1.5 cm×0.5 cm defects were created surgically in the Beagles mandible. The FG was used alone , in conjunction with PLNCX2- Nell-1 retroviral granules, PLNCX2 retroviral granules to repair the defects. Besides the defects were filled with 4×106 Nell-1-producing BMSc created through retroviral gene transfer or BMSc uninfected in combination with FG materials . The defect-repairing capability for each of the treatment modalities were assessed by gross observation ,radiographically, histological and immunohistochemistry analysis.We found that:The extracted and purified PLNCX2- Nell-1 contained the correct nucleotide sequence for the full length of Nell-1 cDNA fragment. The recombinant PLNCX2-Nell-1 retroviral vector was constructed successfully. Cells transferred by PLNCX2-Nell-1 expressed abundant human Nell-1 mRNA and protein in the cytoplasm. Positive findings were not found in those cells that were not transferred . Cell spreading and cell adhesion was significantly increased. Cell proliferation was not affected. But collagen ,ALP activity ,OC and LN production in transferred cells increased significantly. All defects repaired by PLNCX2-Nell-1 retroviral granules had healed radiographically at 16 weeks postoperatively compared with no defects in the other groups. Histological analysis of the specimens revealed that defects that had received PLNCX2-Nell-1 retroviral granules were filled with coarse trabecular bone whereas in those that had received PLNCX2, FG materials alone were fibrous tissue.The use of BMSc transferred in conjunction with FG materials exhibited the strongest defect-repairing power. Gross observation,radiographical and histomorphometric analysis revealed a significantly greater total area of bone formation and increased amount and quality of the new bone in the defects than in those treated with the BMSc uninfected.In conclusions:The PLNCX2-Nell-1 was reconstructed successfully. Nell-1 can be transferred and stably expressed in the cultured Beagles bone marrow stem cells. Proliferation and cell cycle of the transferred cell were not affected .Nell-1 gene transfer can be used to induce differentiation of BMSc into osteoblast-like cells. Dirct,local retroviral delivery of Nell-1 led to the healing of segmental bone defect in Beagles that otherwise would not do so. BMSc transferred or not are effective in repairing segmental radius defects . But Nell-1-producing BMSc created by means of retroviral gene transfer is most preferred. So we established the feasibility of ex vivo gene transfer with the use of autologous shot-term cultured BMSc. It may be used to increase the osteogenic capability of BMSc. These dates encourage the future development of genetic approaches to enhancing bone healing in bone tissue engineering.