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Calcium phosphate cement (CPC) can fill complex-shaped bone defects and set in situ to form a scaffold with intimate adaptation to neighboring bone. The objectives of this study were to determine (1) the effects of fiber length and alginate microbead volume fraction on CPC mechanical properties, and (2) the effect of cell seeding density of human umbilical cord mesenchymal stem cells (hUCMSCs) on their proliferation and osteodifferentiation on CPC. Adding microbeads to CPC degraded the strength. However, increasing the fiber length improved the mechanical properties. Strength and elastic modulus of CPC-microbead-fiber scaffold matched those reported for cancellous bone. When the cell seeding density was increased from 50k to 300k, the cell viability, osteodifferentiation, and bone mineral synthesis also increased. When the seeding density was further increased to 500k, the osteodifferentiation and mineralization decreased. Hence, the 300k seeding density was optimal for CPC-microbead-fiber under the specified conditions. At day 8, alkaline phosphatase (ALP) gene expression of hUCMSCs with seeding density of 300k was threefold the ALP at 150k, and 200-fold the ALP at 50k. At day 14, osteocalcin and runt-related transcription factor 2 with cell seeding density of 300k was fourfold those at 50k. At day 14, mineralization by hUCMSCs at seeding density of 300k was 5-fold the mineralization at 150k, and 25-fold that at 50k. In conclusion, the effect of stem cell seeding density on CPC was determined for the first time. At low cell densities, cell viability and mineralization increased with seeding density. However, a higher seeding density was not necessarily better, and an optimal seeding density on CPC resulted in the best osteodifferentiation and mineralization. The stem cell-seeded CPC-fiber scaffold with excellent osteodifferentiation and mineralization is promising for orthopedic and craniofacial applications.

Effect of Cell Seeding Density on Proliferation and Osteodifferentiation of Umbilical Cord Stem Cells on Calcium Phosphate Cement-Fiber Scaffold