(761g) Injectable Bone Regeneration Therapy: Encapsulation of Mesenchymal Stem Cells and Osteoconductive Ceramic

Current repair and regeneration therapies for major bone loss or trauma are inadequate.  Improved therapies would fill defects left by bone fractures, as well as regenerate the bone by stimulating deposition of a mineralized matrix.  Ideally, this new therapy could incorporate mesenchymal stem cells (MSCs) with a resorbable, osteoconductive ceramic in an injectable formulation that would facilitate filling and repair of irregularly shaped bone defects. In this study, the encapsulation of MSCs with osteoconductive hydroxyappatite (HAP) microcarriers was investigated as a potential bone regeneration therapy. The encapsulation method, termed “complex coacervation,” involved formation of an insoluble polyelectrolyte complex between the anionic glycosaminoglycan chondroitin 4-sulfate (CSA), and the cationic polysaccharide chitosan. In short, a CSA solution with suspended MSCs and HAP microcarriers was extruded as 400 micron diameter droplets and collected into stirred chitosan, thus forming an insoluble complex capsule around the CSA-MSC-HAP droplet. After washes with PBS, capsules were transferred to culture. Osteogenic differentiation was induced in the encapsulated MSC using a standard cocktail. Results were compared to dish-cultured and encapsulated MSC without HAP microcarriers as controls. The MSC formed aggregates within the microcapsules and also adhered to the capsule walls and the HAP microcarriers. After 18 days in osteogenic culture, MSCs encapsulated with HAP microcarriers exhibited extensive deposition of mineralized matrix, compared to control capsules without induced osteogenesis. Mineralization was visualized via fluorescence of tetracycline incorporated into the newly synthesized matrix. Mineral deposition was evident within cell layers, around HAP microcarriers, and within the CSA-chitosan capsule wall. Calcein AM/ethidium homodimer staining indicated that MSC encapsulated with HAP microcarriers retained viability through at least 30 days of culture. Results to date indicate that CSA-chitosan microcapsules containing HAP microcarriers support osteogenic differentiation and rapid mineralization of cultured MSCs, and may be suitable as an injectable, bone-regenerating therapeutic. These microcapsule cultures are being further analyzed to quantify MSC metabolism, matrix deposition, and cell-matrix organization in both osteogenic and expansion culture media. Additional analyses for alkaline phosphatase activity and bone-specific proteins will be used to confirm differentiation to an osteogenic lineage. Mechanical properties of the cultured capsules will also be evaluated by uniaxial compression.