Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and...

Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca²⁺-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO₄) on MSC migration. In addition, to evaluate the influence of CaSO₄ on MSC differentiation and the potential molecular mechanisms involved.

A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO₄ concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO₄ effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO₄ treatment was also evaluated by qPCR.

CaSO₄ increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO₄ significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO₄-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO₄ and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO₄ effects on MSC migration.

Specific CaSO₄ concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO₄ regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO₄ scaffolds could have potential applications for bone regeneration.