Objectives: The bone tissue engineering primarily focuses on three-dimensional co-culture systems, which physical and biological properties resemble the cell matrix of actual tissues. The complex dialogue between bone-forming and endothelial cells (ECs) in a tissue-engineered construct will directly regulate angiogenesis and bone regeneration. The purpose of this study was to investigate whether co-culture between osteogenic and angiogenic cells derived by bone mesenchymal stem cells (MSCs) could affect cell activities and new bone formation.

Materials and methods: Mesenchymal stem cells were dually induced to differentiate into osteogenic cells (OMSCs) and ECs; both cell types were co-cultured at different ratios to investigate their effects and underlying mechanisms through ELISA, RT-qPCR and MTT assays. The selected cell mixture was transplanted onto a nano-hydroxyapatite/polyurethane (n-HA/PU) scaffold to form a cell-scaffold construct that was implanted in the rat femoral condyles. Histology and micro-CT were examined for further verification.

Results: ELISA and gene expression studies revealed that co-cultured OMSCs/ECs (0.5/1.5) significantly elevated the transcription levels of osteogenic genes such as ALP, Col-I and OCN, as well as transcription factors Msx2, Runx2 and Osterix; it also upregulated angiogenic factors of vascular endothelial growth factor (VEGF) and CD31 when compared with cells cultured alone or in other ratios. The optimized OMSCs/ECs group had more abundant calcium phosphate crystal deposition, further facilitated their bone formation in vivo.

Conclusions: The OMSCs/ECs-scaffold constructs at an optimal cell ratio (0.5/1.5) achieved enhanced osteogenic and angiogenic factor expression and biomineralization, which resulted in more effective bone formation.