Effects Of Nanosilicate-incorporated Polycaprolactone Composites On Periodontal Regeneration

Background:Periodontitis destroys periodontal support tissue,and might cause tooth loss.With great progress in regenerative medicine,biomimetic scaffold-mediated tissue regeneration is certified as a promising method to achieve tissue defects.Polycaprolactone(PCL)is extensively investigated in the fields of regenerative medicine,but it has a limited effect on manipulating process of tissue regeneration.Laponite(LAP),as synthetic nanosilicate,is recently used to improve structure and performance of biomaterials in tissue regeneration.However,to date,still no systematic study has been undertaken to explore the bioactivities of LAP-containing PCL in bone tissue and periodontal tissue regeneration,and it is unclear how LAP-containing PCL could regulate the regeneration processes.Objective:PCL/LAP composites with different amounts of LAP were fabricated and the effects on biocompatibility and bioactivity of osteoblasts were evaluated.The influences of porous PCL/LAP scaffolds on bone marrow stromal cells(BMSCs)in vitro and calvarial bone regeneration in vivo were systematically explored.The effects of PCL/LAP electrospun membranes on periodontal ligament cells(PDLCs)and periodontal regeneration were evaluated.Methods:1.LAP-embedded PCL composites were fabricated by a solventexchange method and were characterized by FT-IR,XRD,SEM,water contact angle measurements and compressive experiments.2.PCL/LAP composites were co-cultured with rat osteoblasts to explore their effects on attachments,proliferation,cell viability,osteogenic differentiation and extracellular matrix related gene expression.3.Endothelial cells and osteoclasts were treated with condition medium from osteoblasts cultured with PCL/LAP to explore the effects on angiogenesis and osteoclastogenesis.4.Porous PCL/LAP scaffolds were fabricated by using 3D printing technology and co-cultured with BMSCs to evaluate the effects on cell attachments,proliferation,osteogenesis and pro-angiogenesis.5.Rat calvarial bone defects were prepared and porous PCL/LAP scaffolds were implanted into rat calvarial defects to explore the effects on rat calvarial bone repairing and regeneration.6.PCL/LAP electrospun membranes were fabricated to co-culture with PDLCs to evaluate the effects on cell attachments,proliferation,cell viabilities,osteogenic differentiation and immunomodulation in macrophages.7.PCL/LAP electrospun membranes were used as barrier membranes to be applied in guided tissue regeneration(GTR)therapy of rat periodontal defects to explore the effects on rat periodontal defect repairing and regeneration.Results:1.LAP was distributed in the exfoliated structure without aggregates in PCL/LAP,and the mechanical properties improved after the addition of LAP.2.PCL/LAP didn’t alter attachments and viabilities of rat osteoblasts,but it promoted proliferation,osteogenic differentiation(RUNX2,ALP,OC and BMP-2)and extracellular matrix gene(Col-1 α1,ENPP1 and ANXA6)expression,and potentially activated Wnt/β-Catenin signaling.3.Condition medium from osteoblasts co-cultured with PCL/LAP increased migration,tube formation and angiogenesis-related gene expression of endothelial cells,and decreased osteoclast formation and osteoclastogenesis-related gene and protein expression.4.Porous PCL/LAP scaffolds improved proliferation,ALP expression,mineralized nodule formation,osteogenic differentiation gene and protein expression,and angiogenic cytokine secretion of BMSCs.5.Condition medium from 3D-printed porous PCL/LAP scaffolds co-cultured with BMSCs promoted tube formation and angiogenesis-related gene expression of endothelial cells.6.MicroCT examination and histological analysis indicated more vascularized bone formed after 3D-printed porous PCL/LAP scaffolds implanted into rat calvarial defects.7.PCL/LAP electrospun membrane didn’t affect attachment and proliferation of PDLCs,but it could improve proliferation,ALP expression,mineralized nodules formation and osteogenic differentiation gene and protein(RUNX2,ALP and Col-1 α1)expression of PDLCs.8.Condition medium from PDLCs co-cultured with PCL/LAP electrospun membranes inhibited pro-inflammatory cytokine secretion and M1 macrophage polarization,and promoted M2 macrophage polarization.9.Histological analysis indicated PCL/LAP electrospun membranes promoted rat periodontal defect regeneration in GTR therapy.Conclusions:1.A variety of PCL composites with different amounts of LAP nanoparticles could be successfully fabricated.2.PCL/LAP possessed well biocompatibility and bioactivity.It not only improved proliferation and osteogenensis of osteoblasts,but also regulated angiogenesis and osteoclastogenesis by manipulating paracrine of osteoblasts.3.3D-printed porous PCL/LAP scaffolds possessed good biosafety,promoted proliferation,osteogenesis and angiogenesis of BMSCs and accelerated vascularized bone regeneration.4.PCL/LAP electrospun membranes with well biocompatibility could improve proliferation and osteogenesis of PDLCs,regulated inflammatory responses of macrophages by manipulating paracrine of PDLCs,and promoted periodontal regeneration when it acted as a barrier membrane in GTR therapy.