Development of Metabolically Active and Native-Like Novel Osteoligament Tissue through Pre-Developed Osteochondral and Ligamentous Tissue Coupling

Biological fixation or combination of different tissue grafts poses significant clinical challenges. The clinical success in grafting soft and hard tissues is limited by the lack of functional design to establish successful graft interface integration. The proposed plan is based on the hierarchical development of single tissue constructs to biphasic scaffolds. It is a three step fabrication process starting from osteochondral tissue development, through ligament tissue fabrication and finally combining both to form osteoligament multi-phase construct. Among the three-step process, the primary step is the fabrication of heat sintered poly(lactide-co-glycolide) (PLGA)/hydroxyapatite (HAP) microsphere-gelatin/chondroitin-6-sulfate /hyaluronic acid cryogel, and heat sintered PLGA-heat sintered PLGA/HAP osteochondral scaffold. The bone part will be made with gradient HAP concentration in which lower concentrations will be at the sintered PLGA microparticles or cryogel-based cartilage side, which is separated by a PLGA barrier film. Scaffolds will be analyzed for its physico-chemical properties through standard protocols. The cellular response at each part of the scaffold will be evaluated by co-culture of rabbit osteo-induced bone marrow mesenchymal stem cells and chondrocytes. In vivo experiments will include subcutaneous implantation in nude mice and repair of rabbit knee osteochondral defects. Second part of the study will focus on the development of tissue engineering ligament scaffold, in which self-twisting-yarn fabrication method will be implemented using custom-made setup. Poly(caprolactone) (PCL) nanofibers, PCL microfibers, and platelet-rich plasma (PRP)-loaded PCL nanofibers will be electrospun to a custom-made umbrella-shaped multi-stranded needle collector to form twisted yarn-type scaffolds resembling ligament structure. Each single yarn/cord will be braid or wired with another one to form ligament scaffolds with suitable thickness. Culturing anterior cruciate ligament (ACL) fibroblasts or ligament-derived stem cells in PCL/PRP yarn-type scaffolds is expected to accelerate the ligament regeneration process much better than conventional techniques. Other than scaffold characterization and in vitro cell response, both nude mice and rabbits models will be used for in evaluation of ligament regeneration. The third part of the proposal comes with a tie-locking scaffold design through the successful combination of cell-seeded in vitro cultured osteochondral and ligamentous scaffolds. Cylindrical shaped graded HAP osteochondral scaffolds with custom-generated orifice at the terminals will be combined with the in vitro regenerated ligament scaffolds, by tying the latter through the orifice in the former. The design of the interface will be in such a way that it could exactly resemble the bone-ACL interface as in the native tissue. Rabbit knee joint implantation of the osteochondral construct will be performed to evaluate the formation of the four distinct but continuous regions: ligament, non-calcified fibrocartilage, calcified fibrocartilage and bone. Apart from physico-chemical and gross view analysis, histological and immunohistochemical characterizations will prove the potential of the biphasic osteoligament tissue regeneration. Considering the statistics of current global tissue demands and availability, the proposed multiphase tissue model is believed to be able to regenerate hetero-tissue junctions suitable for joint tissue.

Project ID:PC10507-0236
External Project ID:MOST105-2314-B182-009