(192d) An Injectable Bone Regeneration Therapy Using Mesenchymal Stem Cells Encapsulated in GAG-Chitosan-Hydroxyappatite Microcapsules

Current repair and regeneration therapies for uniquely shaped functional bones are inadequate or invasive. Specifically, current scaffold treatments fail to regenerate the shape of unique craniofacial bones, and many injectable bone regeneration techniques cannot provide compression resistance until a significant mineralized matrix has been deposited by osteoblasts. An improved therapy would fill the irregularly shaped defects left by complex bone fractures and provide compression resistance until sufficient mineralized matrix deposition has occurred. Ideally, this new therapy could incorporate patient-derived mesenchymal stem cells (MSCs) with a resorbable, osteoconductive ceramic in an injectable formulation that would facilitate filling and repair of irregularly shaped bone defects. This study investigates the encapsulation of MSCs with hydroxyappatite (HAP) granules as a potential bone regeneration therapy. The encapsulation method, termed “complex coacervation,” involves formation of an insoluble polyelectrolyte complex between the anionic glycosaminoglycan chondroitin 4-sulfate (CSA), and the cationic polysaccharide chitosan. Briefly, a CSA solution with suspended MSCs and HAP granules was extruded as 400 micron diameter droplets and collected into rapidly stirred chitosan.  The insoluble polyelectrolyte complex is formed at the droplet-chitosan interface, and encapsulates the MSCs and HAP granules suspended in the CSA solution. After washes with PBS, the resulting capsules were transferred to culture, and osteogeneic induction of the encapsulated MSCs was induced using a standard cocktail. Dish-cultured MSCs and MSCs encapsulated without HAP granules were used as controls. MSCs formed aggregates within the microcapsules and adhered to capsule walls and HAP granules. Results indicated that CSA-chitosan microcapsules containing HAP granules supported MSC proliferation, osteogenic differentiation, and mineralized matrix deposition over a 30 day culture period. New mineralized matrix and viable cell distribution were visualized via fluorescence of tetracycline and Calcein Red-Orange, respectively. These microcapsules are being further analyzed for cell-matrix organization, quantification of calcified matrix, and alkaline phosphatase activity in both osteogenic and expansion culture media. Additional analyses for the bone specific proteins osteocalcin and ostepontin will confirm osteogenesis of encapsulated MSCs. The mechanical properties of microcapsules will be measured using uniaxial compression and correlated with cell-specific parameters and HAP loading level. Current results indicate that CSA-chitosan microcapsules containing HAP granules may be suitable as the foundation of an injectable, bone-regenerating therapeutic.