Study On Periodontal Regeneration Using An Injectable And Thermosensitive Hydrogel With Controlled-Release Of FK506

Background: Periodontitis is a chronic inflammatory disease caused by the interaction between microorganisms and the host's immune system.It causes continuous and irreversible damage to the supporting structures of the teeth(gum,periodontal ligament,alveolar bone,and cementum),and is the leading cause of tooth loss.Moreover,studies have shown that periodontitis is closely related to systemic diseases.As a periodontitis pathogen,Porphyromonas gingivalis(Pg)can cause changes in the intestinal flora,leading to increased permeability of the intestinal epithelium,and trigger systemic inflammation leading to the occurrence of endotoxemia.Increased risk of atherosclerosis,adverse pregnancy outcomes(premature,low birth weight,etc.),rheumatoid arthritis,aspiration pneumonia,and cancer.However,oral antibiotics have many shortcomings in treating periodontitis,such as insufficient antibiotic concentration in periodontal pockets,rapid decline in antibiotic concentrations in plasma to sub-therapeutic levels,and long-term sub-therapeutic doses can easily cause microbial resistance.Therefore,the local drug delivery system has become a current research hotspot.It provides an effective concentration of drugs to periodontal tissues during treatment,which greatly improves the therapeutic effect and reduces the occurrence of side effects.This research uses chitosan-?-sodium glycerol phosphate-gelatin injectable temperature-sensitive hydrogel,which has lots of advantages such as simple clinical operation,small wound area required for injection,and fluidity to fill irregular tissue defect cavity in a liquid state.As one of the main components,chitosan can be used as a drug carrier to control drug release,prolong drug efficacy,reduce toxic and side effects,improve the stability of hydrophobic drugs,and change the route of administration.FK506,also known as tacrolimus,is an antibiotic extracted from the genus Streptomyces and a powerful immunosuppressant commonly used in clinical practice.In recent years,many studies have found that FK506 has anti-inflammatory,and promotes bone formation and repair of bone defects.Since anti-inflammatory and promoting bone formation are the key targets to promote the complete recovery of periodontal tissue,these studies suggest that FK506 has a good application prospect in the treatment of periodontitis.Objective: In this study,we select the appropriate concentration of FK506,and detect the effect of the appropriate concentration of FK506 on the osteogenic differentiation of cells.At the same time,chitosan-?-sodium glycerophosphate-gelatin injectable temperaturesensitive hydrogel was prepared,and FK506,which has anti-inflammatory and osteogenic differentiation effects through local sustained release,was studied in the rat periodontitis model,to provide a theoretical basis for the clinical application of FK506 in the oral cavity and develop new formulations for the treatment of periodontitis.Methods: Experiment 1: Isolate and culture dental pulp cells from human healthy pulp tissue.Investigate the effect of FK506 on cell proliferation by the method of Cell Counting Kit-8 assay.Quantitative Polymerase Chain Reaction was used to detect the expression of osteogenesis related genes,such as RUNX2,ALP,COL-?,OPN,and OCN.Alizarin red staining and CPC quantitative analysis were performed to see the effect of FK506 on biomineralization.Experiment 2: Develop the FK506 loaded hydrogel based on CS,?-GP and GEL.The physicochemical and biological properties were characterized by scanning electron microscope(SEM).Besides,MTT assay was used to detect cell toxicity.Experiment 3: The periodontitis model was established in the first molars of Wistar rats by ligature wire.Then inject hydrogel,FK506 and FK506 hydrogel into the periodontal pocket around the first molar.Micro-CT was used to detect the volume of bone loss,and the CEJ-ABC distance.H&E staining was used to evaluate the inflammatory situation and periodontium regeneration,and immunohistochemical staining was used to detect the expression of COX-2 and MMP-9.The toxicity of CS/?-GP/GEL hydrogel in vivo was also studied by H&E staining of important organs.Results: RT-PCR results showed that FK506 significantly increased the expression of Runx2,ALP,COL-I,OCN,and OPN during the osteogenic differentiation of hDPSCs.Alizarin red staining results indicated that FK506 effectively promoted the mineralization of the hDPSCs extracellular matrix.Scanning electron microscope images showed that the pore size of the hydrogel is uniform.The MTT assay results showed that FK506 contained in hydrogel had no significant inhibitory effect on the proliferation of hDPSCs cells.Micro-CT analysis results showed that FK506 contained in hydrogel can reduce alveolar bone damage caused by periodontitis.Histological analysis showed that there was almost no inflammatory cell infiltration or bone loss in the hydrogel-loaded FK506 treatment group,and a small amount of new bone tissue was seen between the root bifurcation area.Besides,immunohistochemical results showed that the number of COX-2 or MMP-9 positive cells in the FK506 group or hydrogel + FK506 group were significantly decreased.Conclusion: Injectable temperature-sensitive hydrogel sustained-release FK506 can be used for antiinflammatory,periodontal tissue regeneration and it has great potential in the clinical treatment of periodontitis.