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Background aims The aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-β1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions. Methods Non-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted. Results Administration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-β1 secretion in irradiated human chondrocytes was also demonstrated. Conclusions Four studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.

Bone morphogenetic protein 2 (BMP2) was selected as a transgene to regenerate osteoporotic bone defects after several BMPs were tested using a bone formation study in nude mice. Human chondrocytes were transduced with a BMP2-containing retroviral vector, and single clones were selected. The cells were characterized over numerous passages for growth and BMP2 expression. The single clones were irradiated and tested for viability. BMP2 expression lasted for 3 weeks before dying off completely after approximately 1 month. Irradiated and non-irradiated transduced chondrocytes successfully healed fractures in osteoporotic rats induced by ovariectomy. The osteoinducing effect of irradiated cells was better than that of their non-irradiated counterparts or a chondrocytes-only control. This study showed that delivering BMP2 from the transduced and irradiated chondrocytes could be an effective and safe method of repairing osteoporotic bone fractures.

The purpose of this study was to investigate the efficacy of cartilage regeneration when using a mixture of transforming growth factor-beta1 (TGF-beta1)-producing human chondrocytes (hChon-TGF-beta1) and primary human chondrocytes (hChon) ("mixed cells"), compared with either hChon-TGF-beta1 or hChon cells alone. Specifically, mixed cells or hChon cells were first injected intradermally into the backs of immune-deficient nude mice to test the feasibility of cartilage formation in vivo. Both the mixed cells and the hChon-TGF-beta1 cells alone induced cartilage formation in nude mice, whereas hChon cells alone did not. To further test the efficacy of the cells in generating cartilage, an artificially induced partial thickness defect of the femoral condyle of a rabbit knee joint was loaded with hChon-TGF-beta1 cells with or without mixing additional untransduced hChon cells, and hyaline cartilage regeneration was observed at 4 or 6 weeks. The efficiency of complete filling of the defect and the quality of tissue generated after implanting were evaluated on the basis of a histological grading system modified from O'Driscoll et al. (J. Bone Joint Surg. 70A, 595, 1988). Significantly, mixed cells (14.2 +/- 0.9) produced significantly better results than hChon-TGF-beta1 (9.0 +/- 1.7) or hChon (8.0 +/- 1.8) cells alone. Histological and immunohistochemical staining of the newly repaired tissues produced after treatment with either mixed cells or hChon-TGF-beta1 cells alone showed hyaline cartilage- like characteristics. These results suggest that the implantation of mixed cells may be a clinically efficient method of regenerating hyaline articular cartilage.

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.

Bone fractures such as pelvic fracture or sternal fracture could be a serious, life-threatening event requiring emergency medical care and lengthy rehabilitation. We have selected BMP-2 as a possible answer for the treatment of severe bone fractures. BMP-2 is known as the powerful inducers of osteogenic differentiation. Due to the short half-life of BMPs in vivo, continuous production of BMPs by gene therapy could be one of the good methods for the successful regeneration of a bone defect. We have tested ex-vivo approach for the regeneration of critical sized bone defect. We investigated whether xenogeneic BMP-2 producing cells had the ability to regenerate cortical bone at the diaphysis of a long bone in a rabbit without using a scaffold. We have constructed BMP-2 producing NIH3T3 and human foreskin fibroblasts. The expression levels of BMP2 were confirmed by ELISA. Osteogenic activities were determined by alkaline phaosphatase assay. Radiographic analysis was performed at 1, 2, 3, 4, 5 and 6 weeks after injection of the cells. Radiological union was observed beginning at 3 weeks after injection and complete thickness cortical bone was observed at 6 weeks after injection. The tibia was harvested at 6 weeks and histological examination was performed. The regenerated bone was almost identical to the normal cortical bone. Fibrous tissue was formed at the end of normal bone without the evidence of bone formation in the control.Immunohistochemical and histological staining was used to analyze the regenerated tissue. At one week after injection, inflammatory cells were observed. At four weeks after injection, cortical was similar to normal bone. At 6 weeks after injection, the structure of regenerated bone tissue was almost identical to that of the normal bone tissue.Osteocalcin expression was observed from regenerated bone osteocytes. Our results suggest that bone regeneration with BMP-2 could have induced the osteoblastic transformation of the pre-osteogenic cells which were recruited into the defect area. These results show that ex vivo gene therapy using BMP-2 producing fibroblasts could be an effective tool for the promoting of bone formation.