hiPSCs-Calcium Phosphate Cement Scaffolds Containing Bioactive Molecules for Bone Regeneration

Objectives: The objectives of this study were to: (1) Develop a novel injectable calcium phosphate cement (CPC) containing SB431542; and (2) investigate osteogenic differentiation of hiPSC-derived mesenchymal stem cells (hiPSC-MSCs) on SB431542-CPC for the first time.
Methods: Four hiPSC-MSC-seeded groups were tested: CPC control; CPC with SB431542 inside CPC (CPCSM); CPC with SB431542 in osteogenic medium (CPC+SMM); and CPC with SB431542 in CPC and in medium (CPCSM+SMM). The release profile of SB431542 was assessed by measuring the absorbance at 308 nm. Cell viability was assayed via live/dead staining and a cell counting kit (CCK-8). Osteodifferentiation was measured by RT-PCR (alkaline phosphatase (ALP), Runx2, Collagen I (COL1) and osteocalcin (OC) gene expression), ALP activity and mineral staining.
Results: SB431542 exhibited sustained release from chitosan-CPC scaffolds, reaching a peak of (90±5)% at 21 days. As measured by the CCK-8 assay, SB431542 in CPC increased the proliferation of hiPSC-MSCs (4.36±0.76), compared to CPC control (2.77±0.55) (p<0.05). hiPSC-MSCs differentiated into the osteogenic lineage and synthesized bone minerals. hiPSC-MSCs on CPCSM+SMM had the highest osteogenic expressions (fold change) at 14 days compared to CPC control for alkaline phosphatase [(11.64±0.99) to (2.12±0.42)], osteocalcin [(6.66±0.43) to (2.38±0.21)], collagen[(6.78±0.86) to (1.41±0.32)], and Runx2 [(8.01±0.64) to (2.28±0.30)] (p<0.05). Bone mineral synthesis (fold change) by hiPSC-MSCs with SB431542 was greater than that without SB431542 (p<0.05). At 21d, CPCSM+SMM showed significantly more bone mineral (56.88±3.60) than CPC control (19.28±4.81) (p<0.05).
Conclusions: hiPSC-MSCs with SB431542 showed excellent osteogenic differentiation. CPCSM+SMM had the highest differentiation and mineralization, with no significant difference between CPCSM and CPC+SMM. CPC was a suitable scaffold to deliver hiPSC-MSCs and SB431542 to promote bone regeneration.