| Objective To detect the expression of hBMP-7 in rabbit bone marrow stem cell (BMSc) transferred by retroviral vector mediated hBMP-7 gene. Proliferation and differentiation of BMSc transferred by hBMP-7 gene or not were also elucidated at the same time. Then the ability of PLNCX2-hBMP7 virus granules and the BMSc transferred by hBMP-7 gene composited with PLLA(poly-L-lactide) biomaterials to enhance segmental bone defect healing of the rabbit radius were evaluated.Methods Human BMP7-expressing replication-deficient retroviral vector (PLNCX2-hBMP7) 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 hBMP-7 gene in transferred cells were determined by RT-PCR, hybridization in situ and immunohistochemistry. The proliferativity of the transferred cell were assayed by methabenzthiazuron (MTT) method and flow cytometer. 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 rabbits. Segmental 1.5 cm defects were created surgically in the radius of New Zealand white rabbits. The Poly -L-lactide (PLLA) was used alone , in conjunction with PLNCX2-hBMP7 retroviral granules, PLNCX2 retroviral granules to repair the defects. Besides the defects were filled with 4X 106 BMP7-producing BMSc created through retroviral gene transfer or BMSc uninfected in combination with PLLA materials . The defect-repairing capability for each of the treatment modalities were assessed by gross observation ,radiographically, histologicaland immunohistochemistry analysis.Results The extracted and purified PLNCX2-hBMP7 contained the correct nucleotide sequence for the full length of hBMP-7 cDNA fragment. The recombinant PLNCX2-hBMP7 retroviral vector was constructed successfully. Cells transferred by PLNCX2-hBMP7 expressed abundant human BMP7 mRNA and protein in the cytoplasm. Positive findings were not found in those cells that were not transferred .Cell proliferation was not affected. But col 1 age , ALP activity ,OC and LN production in transferred cells increased significantly. All defects repaired by PLNCX2-hBMP7 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-hBMP7 retroviral granules were filled with coarse trabecular bone whereas in those that had received PLNCX2, PLLA materials alone were fibrous tissue.The use of BMSc transferred in conjunction with PLLA 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.Conclusions The PLNCX2-hBMP7 was reconstructed successfully. Human BMP-7 can be transferred and stably expressed in the cultured rabbit bone marrow stem cells. Proliferation and cell cycle of the transferred cell were not affected . BMP7 gene transfer can be used to induce differentiation of BMSc into osteoblast-like cells. Dirct, local retroviral delivery of hBMP7 led to the healing of segmental bone defect in rabbits that otherwise would not do so. BMSc transferred or not are effective in repairing segmental radius defects . But BMP-7-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 a...
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