The Effect Of Kangfuxin On Accelerating The Healing Of Tooth Extraction Socket Through CCL2/Angiogenesis

Background: Promoting extraction socket healing after toothextraction is critical for occlusal reconstruction.Kangfuxin(KFX)has therapeutic potential for wound healing,but its role in accelerating the healing of toothextraction socket is still unclear.Here,we aimed to explore the mechanism of KFX in accelerating the toothextraction socket healing by promoting angiogenesis.Methods: The bilateral first molar of C57BL/6 were extracted,and mice were divided into normal saline(NS)groups and KFX groups,respectively.Maxillary were examined by scanning electron microscope(SEM),micro-computed tomography(micro-CT),and histology and histomorphometric analysis.Mouse bone marrow mesenchymal stem cells(m BMSCs),human periodontal ligament stem cells(hPDLSCs)and human dental pulp stem cells(hDPSCs)were treated with0.0008 mg/ml KFX under osteogenic induction.Differentially expressed genes in m BMSCs was screened out based on RNA-sequencing(RNA-seq),and examined in various stem cells.Alkaline phosphatase staining was used to examine the osteogenic differentiation in stem cells.Immunofluorescence staining was used to detect the angiogenesis in the toothextraction site of mice.Function-based experiments were performed in vitro,including wound healing,transwell migration and tube formation assay.After transfection of CCL2 specific small interfering RNA(si CCL2),the alteration of the migration and angiogenesis in the human umbilical vein endothelial cells(HUVECs)treated withKFX-treated-conditioned medium(KFX-CM)were observed.Results: The micro-CT and hematoxylin and eosin(H&E)staining showed that bone mass was increased in KFX groups;SEM and Picrosirius red staining revealed that the number of collagens,especially type I collagen,was formed after KFX treatment;and Goldner trichrome staining verified the increased mineralization in KFX groups.To reveal the mechanism in osteogenesis,RNA-seq of BMSCs filtered 25 DEGs,with21 upregulate and 4 downregulate.Based on the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis,chemokines-related genes were enriched,and Ccl2 increased approximately 3 time after KFX treatment,while the expression of Runx2,Col1α2 had no significant difference.However,the expression of CCL2,RUNX2 and COL1A2 bothin m RNA and protein level showed significant increase in hPDLSCs and hDPSCs.Notably,the CM of hPDLSCs and hDPSCs,whichtreated withKFX,could promote migration and angiogenesis in HUVECs,because of upregulating the expression of CCL2 protein.Ccl2 knockdown abolishes CM-induced endothelial cell migration and angiogenesis.Immunofluorescence staining identified that the blood vessels and type H vessels were increased in KFX groups in vivo.Conclusion:1.KFX can promote the healing of the mouse toothextraction socket.2.KFX can promote angiogenesis and accelerate the healing of toothextraction wound by upregulating the expression of CCL2.