The Use Of TLR2 Modified BMSCs For Enhanced Bone Regeneration In The Inflammatory Micro-environment

Objects: The repair of periodontal bone tissue defects in patients with periodontitis is one of the major challenges for dentists.Stem cell-based bone regeneration has been considered as a promising strategy to restore the lost periodontal bone tissue.However,the local inflammatory environment of periodontal tissue in patients,who suffered periodontal disease,affects stem cell-based periodontal bone regeneration.In this study,we tested the effect of TLR2 on BMSCs mediated alveolar bone regeneration by establishing a TLR2 gene-modified canine BMSCs using a lentivirus.Peptidoglycan(PGN),a polymer consisting of sugars and amino acids form the cell wall of most bacteria,was used to activate the TLR2 signaling pathway in canine BMSCs.Lastly,the TLR2 modified BMSCs were delivered by collagen gel and then injected into canine tooth extraction sockets,and new bone regeneration was detected after two weeks.Materials and methods: 1.Canine bone marrow stem cells(BMSCs)were isolated and cultured.Stem cell markers on the surface of BMSCs were identified by flow cytometry.The osteogenic,chondrogenic and lipogenic differentiation abilities of BMSCs were identified,too.2.In vitro,Lv-TLR2 and Lv-GFP were constructed and transfected into BMSCs.TLR2 gene expression of BMSCs were detected by immunofluorescence staining and Western blot.3.PGN at a final concentration of 10?g/m L was used to activate TLR2 signaling pathway in TLR2-BMSCs.The expression of osteogenic and angiogenic related markers was evaluated with RT-PCR and Western blot.The expression of osteocalcin(OCN)was evaluated using immunofluorescence.ALP staining was also performed.4.The cell/collagen gel complex was injected into athymic nude mice subcutaneously.The implants of each group were harvested two weeks after transplantation and sections were observed using a fluorescence microscope.5.An animal model of periodontitis in canine premolars was established.After extraction of the affected tooth,BMSCs/ collagen gel complex was injected into the tooth extraction socket.Sequential fluorescence labeling analysis was used to evaluate the effect of bone regeneration in inflammatory environment.Results: 1.Canine BMSCs were successfully isolated from bone marrow.Flow cytometry analysis showed that these cells negatively expressed hematopoietic markers and positively stained for stem cell markers.Canine BMSCs can be differentiated into adipocytes,osteoblasts,and chondrocytes sucessfully.2.Both immunofluorescence staining and Western blot confirmed that BMSCs could stably express TLR2 target protein after Lv-TLR2 transfection.3.Under the stimulation of PGN,RT-PCR showed that the expression of osteogenic and angiogenic related genes including Runx2,ALP,OCN and VEGF was significantly up-regulated in BMSC-TLR2 group compared with other three groups.Western blot showed that the expression of Hif-1?,VEGF and BMP2 was significantly up-regulated in TLR2-modified BMSCs.4.Two weeks after BMSCs/ collagen gel material constructs implanted subcutaneously to nude mice,new bone formation was found.Lv-TLR2 gene transfection greatly enhanced new bone formation.5.BMSCs transfected with TLR2 gene were injected into the tooth extraction socket of canine periodontitis model.Micro-CT,hard tissue sections and sequential fluorescence labeling analysis all showed that TLR2 gene modified BMSCs could form new bone more quickly and can achieve better repair effect.6.Conclusions: PGN can promote BMSCs mediated-vascularized bone regeneration through TLR2 receptor.This finding may contribute to stem cell-based bone tissue regeneration in an inflammatory microenvironment,especially in periodontal bone regeneration.