Novel hiPSC-based tri-culture for pre-vascularization of calcium phosphate scaffold to enhance bone and vessel formation

•A novel tri-culture system was developed to elicit pre-vascularization of calcium phosphate cement (CPC) scaffold.•Tri-culture group favored formation of tubular structures, elevated angiogenic and osteogenic markers, and mineralization.•In vivo study demonstrated that the tri-cultured group exhibited a robust capacity for bone regeneration.•The novel pre-vascularization strategy will likely increase the success of stem cell-based bone regenerative therapy.

Vascularization of tissue-engineered bone is a critical step in maintaining cell viability and advancing cell performance in vivo. In this study, a novel tri-culture system was developed to elicit pre-vascularization of calcium phosphate cement (CPC) scaffold in which human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSMSCs) were seeded together with human umbilical vein endothelial cells (HUVECs) and pericytes. In a two-step methodology design, we first performed osteoinduction of the seeded hiPSMSCs on the CPC scaffold and then incorporated HUVECs and pericytes to the hiPSMSC-colonized CPC scaffold under a favorable culturing condition, with an objective to form a stable and functional capillary-like vascular network that sustained the engineered osseous tissue. The angiogenic and osteogenic effects of various culture strategies were studied and compared in nude rat model with cranial bone defects: (1) CPC scaffold alone (CPC control); (2) Pericyte-seeded CPC (CPC-pericytes); (3) HUVEC-seeded CPC (CPC-HUVECs); (4) hiPSMSC-seeded CPC (CPC-hiPSMSCs); (5) HUVECs co-cultured with hiPSMSCs on CPC (bi-culture group) and (6) HUVECs and pericytes co-cultured with hiPSMSCs on CPC (tri-culture group). After 12 weeks, tri-culture group showed the highest amount of new bone (new bone area fraction of (45.3 ± 2.7) %, p < 0.05) and vessel formation (new blood vessel density of (50.7 ± 3.8) vessels/mm2, p < 0.05) in all groups. Our results demonstrated that the tri-culture strategy was effective in promoting angiogenesis and osteogenesis for bone tissue engineering.