(805f) Physiological Programming of Human Embryonic Stem Cells in Osteomimetic Scaffolds

Current methods of treating critical size bone defects (CSDs) include autografts and allografts. Although both approaches have proven clinically successful, major limitations exist including donor-site morbidity, risk of disease transmission and immune-rejection. Tissue engineering provides a promising alternative to circumvent these limitations through the use of autologous cells, three dimensional (3D) scaffolds, and growth factors. The success of tissue engineering strategies, however, relies on the ability of scaffolds to program the desired behavior of transplanted cells. In this sprit, the goal of this study was to develop a scaffold with native bone extracellular matrix (ECM) components to direct bone formation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts were seeded on PLGA sintered-microsphere scaffolds to deposit bone ECM. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. Next, hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and in-vitro bone formation was analyzed at specific time points. Differentiation of hESCs on osteomimetic ECM coated PLGA scaffolds was compared to control groups in which hESCs were differentiated on two dimensional (2D) tissue culture plates, and PLGA scaffolds coated with a non-specific ECM protein cocktail (matrigel). Osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies monitored for 21 days showed a constant increase in the number for hESCs seeded on both groups of PLGA scaffolds and ECM coated PLGA scaffolds. Scanning electron microscopy (SEM) images confirmed cell attachment at day 1 as round hESCs colonies which progressed to differentiated cells exhibiting osteoblast-like morphology by day 28. In differentiation studies, calcium deposition by hESCs at was significantly higher on the osteomimetic ECM coated scaffolds compared to the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of the bone marker protein osteocalcin in hESCs seeded on ECM coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on ECM coated osteomimetic PLGA compared to control groups. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation.