Gene Therapy with Tetracycline-Regulated Human Recombinant COLIA1 cDNA Direct Adenoviral Delivery Enhances Fracture Healing in Osteoporotic Rats.

A number of previous studies have indicated that the genetic variation at the collage type I alpha 1 (COLIA1) gene locus influences susceptibility to osteoporosis. However, seldom have studies reported the effect of gene delivery using an adenovirus vector carrying human recombinant COLIA1 cDNA on stimulating osteogenic activity of osteoblasts and enhancing fracture healing of ovariectomized rats. The current study was performed to demonstrate whether direct gene delivery using an adenovirus vector carrying human recombinant COLIA1 cDNA could stimulate osteogenic activity of osteoblast in vitro and enhance fracture healing of ovariectomized rats in vivo. In vitro, the tet-on system regulated COLIA1 gene adenovirus was constructed and transfected to osteoblasts. COLIA1 mRNA and collagen type I levels were assessed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay to determine whether adenovirus transfected successfully. Osteogenic activity of the osteoblasts was assessed by alkaline phosphatase activity, immunohistochemical staining, immunofluorescent staining, mineralized matrix formation, and extracellular calcium levels. In vivo, adenovirus-delivered COLIA1 gene was injected into the fracture site of the tibia in an ovariectomized rat model of osteoporosis, and bone callus condition was assessed to determine whether the COLIA1 gene could accelerate osteoporotic fracture healing. In vitro, the results showed that COLIA1 gene adenovirus transfection could increase osteoblast COLIA1 gene expression and collagen type I protein synthesis, increase alkaline phosphatase activity, and stimulate calcium nodules formation, which exhibited a direct osteogenic effect on the osteoblasts. In vivo, local injection of COLIA1 gene adenovirus increased collagen type I expression, restored bone mineral density, and accelerated fracture healing in ovariectomized rats, without increasing serum collagen type I and liver COLIA1 mRNA levels. This study suggests direct gene delivery using an adenovirus carrying human COLIA1 cDNA can stimulate the osteogenic activity of osteoblasts in vitro and enhance bone fracture healing in vivo. The tet-on system is an ideal gene regulatory system for effective and safe regulation of the therapeutic gene.