Biomimetic calcium-silicate cements support differentiation of human orofacial mesenchymal stem cells

Abstract

Introduction: Human orofacial bone mesenchymal stem cells (OFMSCs) from maxilla and mandible have robust osteogenic regenerative properties on the basis of our previous reports that demonstrate phenotypic and functional differences between jaw and axial bone mesenchymal stem cells in same individuals. Furthermore, a combination of OFMSCs with bioactive calcium-releasing cements can potentially improve OFMSC multilineage differentiation capacity, but biocompatibility of calcium-silicate cements with OFMSCs is still unclear. We tested the hypothesis that material extracts of calcium-releasing calcium-silicate cements support biomimetic microenvironment for survival and differentiation of human OFMSCs.

Methods: Two experimental calcium-silicate cements, (1) calcium-silicate mineral powder (wTC) containing dicalcium and tricalcium-silicate, calcium sulfate, and calcium chloride and (2) wTC doped with alpha-tricalcium phosphate (wTC-αTCP), were designed and prepared. Cement setting times were assessed by Gilmore needles, ability to release calcium and hydroxyl ions was assessed by potentiometric methods, and OFMSC attachment to calcium-silicate discs was assessed. Calcium-silicate material extracts were tested for ability to support OFMSC survival and in vitro/in vivo differentiation.

Results: Fewer OFMSCs attached to calcium-silicate discs relative to tissue culture plastic (P = .001). Extracts of calcium-silicate cements sustained OFMSC survival, maintained steady state levels of vascular cell adhesion molecule-1, alkaline phosphatase, and bone sialoprotein while up-regulating their respective gene transcripts. Adipogenic and in vivo bone regenerative capacities of OFMSCs were also unaffected by calcium-silicate extracts.

Conclusions: Ion-releasing calcium-silicate cements support a biomimetic microenvironment conducive to survival and differentiation of OFMSCs. Combination of OFMSCs and calcium-silicate cement can potentially promote tissue regeneration in periapical bone defects.

Figure 1

Effect of calcium silicate cements on attachment and survival of OFMSCs. Representative images of calcium silicate cements within the PVC mold (A, top panel) and after recovery from the mold (A, lower panel) indicate rigidity of the calcium silicate discs was maintained irrespective of setting parameters. OFMSC attachment to silicate discs was significantly lower (p = 0.001) than cells attached to tissue culture plastic (B). Plastic adherence in α-MEM culture medium (C) was reduced when OFMSCs were introduced to cement extracts (D, E) as demonstrated by floating cells and partial loss of cell-to-cell contact. Metabolically active cells in calcium silicate extracts and α-MEM were similar based on WST-1 survival assay (F) and there were no significant differences between surviving cells in wTC and wTC-αTCP extracts (p = 0.074). [*** = p ≤ 0.001].

Figure 2

Effects of calcium silicate cements on in vitro differentiation of OFMSCs. Representative Western blot bands of control (α-MEM), wTC and wTC-αTCP extract-treated OFMSCs proteins showed immunoreativity to vascular cell adhesion marker-1 (VCAM-1), alkaline phosphatase (ALP), bone sialoprotein (BSP) and α-tubulin [protein loading control] (A). Densitometric analysis of immunoreactive bands (B) showed minimal changes in VCAM-1 and BSP levels relative to control cells but ALP level was slightly more reduced in OFMCS exposed to wTC-αTCP extracts. Real time PCR amplification of specific primers for the same markers demonstrated significant upregulation of their gene transcripts with ALP > VCAM-1 > BSP (C) [PCR data normalized to non-osteogenic controls and TATA binding protein as housekeeping gene]. There were no significant differences between the two cement types.

Figure 3

Effect of calcium silicate cements on adipogenesis and in vivo bone regeneration. Lipid aggregates (black arrow heads) were randomly distributed around majority of α-MEM-cultured (control) OFMSCs (A), while wTC and wTC-αTCP extract-treated cells displayed fewer but structurally larger lipid aggregates (B, C) than control cells. Similarly OFMSCs pre-treated with material extracts regenerated appreciable in vivo bone (black solid arrows) structurally similar to those of control cells (D, E, F); and there were no differences in amount of bone regenerated by extract-treated and α-MEM-treated OFMSCs (G) [* = hydroxyapatite carrier; clear arrow = fibrous tissues].