ABSTRACT
Gene therapy is a promising clinical tool that is no longer limited as a method to supplement genetic deficits, but rather is considered reliable for delivering proteins to specific tissues or cells. Recombinant adeno-associated virus (rAAV) vector is one of the most potent gene transfer vehicles. Many biomaterials have been used in reconstructive surgery, but their biological inactivity has limited their use. To overcome shortcomings of available bone-related biomaterials, we investigated the combination of rAAV with biomaterials. Taking advantage of the method of lyophilizing rAAV onto biomaterials, we showed that an rAAV coating successfully induced beta-galactosidase protein expression by rat fibroblasts on hydroxyapatite, beta-tricalcium phosphate, and titanium alloy in vitro. beta-Galactosidase expression was detected for 8 weeks after implantation of rAAV-coated hydroxyapatite into rat back muscles in vivo. A coating of bone morphogenetic protein-2-expressing rAAV induced significant de novo bone formation on hydroxyapatite in rat back muscles. Our study demonstrates that the combination of lyophilized rAAV and biomaterials presents a promising strategy for bone regenerative medicine.
Subject(s)
Adenoviridae/genetics , Bone Morphogenetic Protein 2/genetics , Bone Regeneration/physiology , Gene Transfer Techniques , Genetic Therapy/methods , Animals , Calcium Phosphates , Cells, Cultured , Coated Materials, Biocompatible , Durapatite , Fibroblasts/cytology , Fibroblasts/physiology , Freeze Drying , Plasmids/genetics , Rats , Rats, Wistar , Titanium , Transduction, Genetic/methods , beta-Galactosidase/geneticsABSTRACT
Porous beta-tricalcium phosphate (TCP) has been known to have osteoinductive potential in ectopic site implantation in canine models without the use of osteoinductive substances or cell transplantation. Prostaglandin E2 receptors, particularly EP4, are known to play an important role in osteogenesis. EP4 agonists have been demonstrated to have positive effects on bone remodeling and bone morphogenic protein-induced ectopic bone formation in rodent models. We examined the efficiency of porousbeta-TCP-induced osteoinduction in beagles by the graded-release EP4 agonist, which was injected at their buttocks intramuscularly. Newly formed bone was observed on and after 3 weeks in the EP4 agonist-injected, while on and after 6 weeks in control groups, respectively. After that the bone resorption and the beta-TCP degradation was accelerated in EP4 agonist-injected group. Tartarate-resistant acid phosphatase-positive cells appeared in each group before bone formation, and the number of these cells reduced gradually; however, more multinucleated and larger cells appeared, particularly in the injected group. Injection of an EP4 agonist was proved to accelerate the osteoinduction and degradation of beta-TCP in canine model. Histological analysis revealed that the EP4 agonist stimulated osteoclastogenesis before bone formation.
