Figure 4: Expression of differentiation markers is increased with alternative seeding techniques and a mineralized template. Alkaline phosphatase (A) and Osteocalcin (B) expression increased significantly in cells seeded by micromass over filtration (ALP: p(day2)<0.001, p(day 8)<0.001; OCN: p(day 8)<0.001, p(day 16) =0.0211) and static seeding ALP: p(day2, day 8)<0.001; OCN: p(day 8, day 16)<0.001). Cells seeded in a mineralized template and calcium rich environment also expressed higher levels of ALP (C) and OCN (D) over cells seeded in PLGA. Cells seeded in mineralized templates expressed significantly more ALP and OCN in all seeding techniques over cells seeded in PLGAonly (ALP: p(day2)0.031, p(day 8)<0.001 p(day 16) <0.001; p(day2)<0.001 OCN: p(day 8)=0.033, p(day 16) < 0.001). Although cells seeded with increased extracellular calcium did not exhibit a significant difference in ALP relative to those seeded in PLGA, it did exhibit significant differences in expression in OCN expression for filtered and static seeded cells (p=0.032, p=0.042 respectively).