Effects Of Human Interleukin-10 Gene Therapy On Bone Metabolism And Implant Osseointegration In Ovariectomized Rats

Objective This study aimed to explore the effects of hIL-10 gene therapy on bone metabolism and implant osseointegration in ovariectomized rats by delivering the plasmids (EX-A0007-M03) carried with human interleukin-10 (IL-10) gene into rats via hydrodynamic tail vein injection. Thus it provided evidence for hIL-10 gene therapy on bone metabolism diseases and improvement of osseointegration.Methods 1. After amplification and identification of the plasmids (EX-A0007-M03) carried with human interleukin-10 (IL-10) gene, the plasmids were transfected into 293T and RAW264.7 cells with liposome. Fluorescence microscopy and ELISA were used to detect the expression of target gene.Osteoclast formation and bone absorption experiments were carried out to identify the biological function of hIL-10. 2. Using hIL-10 as a reporter gene, this study transfered the plasmids into rats by hydrodynamic tail vein injection, and observed the effects of different injection conditions on the hIL-10 expression in vivo, which were verificated through the frozen section, ELISA and immunohistochemistry. 3. The ovaries were completely excised bilaterally in 3 months old female SD rats and factors like the body weight, uterine pathology, serum ALP, Ca, P concentration, dual energy X ray, pathology and biomechanics of bone tissue were used to validate that the model of postmenopausal osteoporosis had been successfully established. 4. In the basis of the successful establishment of postmenopausal osteoporosis, the rats were transferred with hIL-10 plasmids by hydrodynamic tail vein injection weekly. In 2, 4, 8 weeks after injection, the animals were sacrificed, with body weight, serum ALP, Ca, P, TRAP, IL-1βconcentration, dual energy X ray, bone pathology and biomechanical testing to detect the effects of hIL-10 gene therapy on bone metabolism. 5. After the successful establishment of postmenopausal osteoporosis, the titanium implants were placed in the tibia and the rats were transferred with hIL-10 plasmids by hydrodynamic tail vein injection weekly. In 2, 4, 8 weeks after injection, the animals were sacrificed, with the effects of hIL-10 gene therapy on the osseointegraton of implants detected by methods like X-ray, micro-CT, bone-grinding slice techniques.Results 1. EX-A0007-M03 was confirmed to carry the foreign gene of hIL-10 by gel electrophoresis and DNA sequencing. EX-A0007-M03 was transiently transfected into 293T and RAW264.7 cells successfully by liposome, with some cells emitting green fluorescence. ELISA detection confirmed that the target gene was expressed; the hIL-10 protein had the biological function to inhibit osteoclast formation and resorption. 2. It was demonstrated from the frozen section, ELISA and immunohistochemistry that hIL-10 were highly expressed in rats after hydrodynamic tail vein injection. 3. In 12 weeks after ovariectomy, the rats developed the features of high turnover osteoporosis with the increased body weight and the serum ALP concentration, atrophy of the uterus, significant loss and diminished mechanical properties of bone. 4. The increase of TNF-αand TRAP 5b induced by OVX could be significantly inhibited after hIL-10 gene therapy (P<0.05), but the hIL-10 gene therapy had no effect on bone metabolism in osteoporosis or normal rats. 5.The hIL-10 gene therapy had no effect on osseointegration in osteoporosis or normal rats (P>0.05).Conclusion 1. hIL-10 plasmids have the biological function to inhibit osteoclast formation and bone resorption after transfection. 2. It is a simple, safe and effective approach to express hIL-10 gene in rats. 3. Female SD rats ovariectomized at the age of 3 months could be an ideal animal model for postmenopausal osteoporosis 12 weeks later. 4. hIL-10 gene therapy had no significant effect on bone metabolism and osseointegration in osteoporosis or normal rats. For a better understanding of IL-10 on metabolic bone disease, further studies are required.