Fluoride And Chloride Containing Bioactive Glasses Enhance Osteogenesis And Angiogenesis In Vitro And In Vivo

Background:Bioactive glasses（BGs）are a type of synthetic bioactive material with good biocompatibility.BGs were attractive synthetic bone substitutes owing to their abilities to degrade,form bone-like mineral and stimulate bone regeneration.Therefore,BGs have good prospects for application in the field of bone defect repair.Previous studies showed that BGs properties can be regulated by adding different elements.Fluorine induces cellular phosphorylation and regulates osteoblast activity.And fluorine plays an important role in bone formation and mineralization.Chlorine is widely found in the human body and has effects such as p H regulation and cell volume stabilization.Our previous studies showed that the incorporation of fluoride into alkali-free bioactive silicate glass promoted osteogenesis to some extent in vitro,while the incorporation of chloride facilitated glass degradation and accelerated the formation of hydroxyapatite.The incorporation of fluoride and chloride promotes the degradation of glass and the formation of apatite.However,whether there is a synergistic effect of incorporating fluoride and chloride on further enhancement of osteogenesis and angiogenesis in vitro and in vivo was unknown.Objectives:Synthesized three halide-containing BGs with fluoride only,or chloride only,or mixed fluoride and chloride,investigated their physicochemical properties and osteogenic and angiogenic effects both in vitro and in vivo.Methods:The melt-quench synthesis route was applied to produce three bioactive glasses with around 6 mol%fluoride（GPF）or chloride（GPCl）or mixed fluoride and chloride（GPFCl）.Evaluation of material structure and biological ability by DSC,FTIR,ICP-OES,ISE.The osteogenic differentiation of h PDLSCs stimulated by BGs was detected by RT-q PCR,Western blot and immunofluorescence experiments.The in vivo osteogenic effect of BGs was investigated by the SD rat calvarial defect model combined with Micro-CT and histological staining.Results:1.The MAS-NMR spectral results show a chemical shift around3 ppm for³¹P in the three glasses.The 19F chemical shift around-95 ppm for GPF and GPFCl.2.The results showed that the addition of both fluoride and chloride in a bioactive silicate glass could combine the structural roles of both,leading to a faster apatite formation than the glass with the presence of fluoride only and a more stable fluorapatite formation than the glass with the presence of chloride only,which formed hydroxyapatite upon immersion.3.The studied BGs were cytocompatable,as suggested by the cytotoxicity evaluation of h PDLSCs cultivated in the extracted BGs-conditioned culture media.4.More importantly,these BGs stimulated osteogenic differentiation of h PDLSCs without adding grow factor as indicated by the fact that BGs-conditioned media up-regulated the expression of BMP-2,OPN and VEGF of h PDLSCs and promoted the formation of bone nodules and collagen in vitro.By comparison,the incorporation of fluoride facilitated the expression of osteogenic-related biomarkers and bone nodule formation preferentially,while the incorporation of chloride induced the expression of angiogenic-related biomarkers and collagen formation.5.The in vivo investigation results demonstrated that the developed halide-containing BGs accelerated the process of bone regeneration,while the glass with mixed fluoride and chloride showed the most significant promotion effect amongst the three BGs.Conclusion:Therefore,our findings revealed that all the BGs studied in this project have good bioactive activity.Fluoride and chloride containing bioactive glasses promotes osteogenic differentiation of h PDLSCs without the addition of an osteogenic induction solution and all promote new bone formation in vivo.GPFCl combines the advantages of both F and Cl to providing the best osteogenic effect.