Influence of in vitro differentiation status on the in vivo bone regeneration of cell/chitosan microspheres using a rat cranial defect model

Abstract

The aim of this study was to investigate the influence of the in vitro osteogenic differentiation status on the in vivo bone regeneration of cell/chitosan microspheres qualitatively and quantitatively. To this end, rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were seeded onto apatite-coated chitosan microspheres. The constructs were osteogenically differentiated for 0, 7, 14, and 21 days followed by calvarial defect implantation in vivo for up to 8 weeks. In vitro studies showed that BMSCs in the constructs proliferated from day 0 to day 7. The activity and gene expression of alkaline phosphatise increased from day 0 to day 14 and then decreased. The gene expression of collagen type I and osteocalcin peaked at day 21. In vivo, constructs retrieved from day 0 group were filled with fibrous tissues and capillaries, but no bone formation was observed. Constructs retrieved from day 7 and day 21 groups showed progressive bone formation, whereas those retrieved from day 14 group had the highest percentage of bone formation. These data suggested that to generate a substantial amount of bone in vivo, not only the in vitro osteogenic differentiation was necessary, but also the period of pre-differentiation was important for the cell-scaffold constructs. The period of pre-differentiation for 14 days was found to be the most suitable for chitosan microspheres.

Keywords:

• Bone regeneration
• osteogenic differentiation
• mesenchymal stromal cells
• apatite-coated chitosan microspheres

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant 81570956, 31600757, and 81870744; the Natural Science Foundation of Hunan Province under Grant 2016JJ3168; the Health and Family Planning Commission Project of Hunan Province under B2019193. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.