Sirtuin 6 (SIRT6) is a NAD-dependent deacetylase involved in lifespan regulation. To evaluate the effect of SIRT6 on osteogenesis, rat bone marrow mesenchymal stem cells (rBMSCs) with enhanced or reduced SIRT6 function were developed. We observed that SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin, while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor-jB (NF-jB) transcriptional activity and upregulated the expression of acetyl-NF-jB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF-jB inhibitor BAY 11-7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of collagen/chitosan/hydroxyapatite scaffold could significantly boost new bone formation in rat cranial critical-sized defects, as determined by microcomputed tomography and histological examination. These data confirm that SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF-jB signaling.
In this paper we developed two types of chitosan-based microspheres with and without biomimetic apatite coatings and compared their potential as injectable scaffolds for bone regeneration. The microspheres were obtained by emulsion cross-linking (E0) and coacervate precipitation (C0), respectively. They were then biomimetically coated with apatite to become E1 and C1 microspheres. The physicochemical properties and biocompatibility of the microspheres were characterized. Both E0 and C0 microspheres presented favorable ranges of diameter, density and Rockwell hardness. However, there were differences in the degree of cross-linking, shape, morphology, degradation rate, swelling rate, pH value after PBS immersion and the biocompatibility between E0 and C0. The apatite coating was successfully prepared for both C0 and E0, which enhanced the attachment, proliferation and differentiation of MC3T3-E1 cells. In conclusion, our results suggest the feasibility of using chitosan microspheres as a potential injectable scaffold. Both the preparation method and the biomimetic apatite coating contribute to their biological properties.
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