Effects Of Osteogenesis Differentiation Of HJBMSCs By Exosome Of HPDLSCs In Type 2 Diabetes Mellitus With Periodontitis

Objective:Periodontal inflammation develops rapidly in patients suffered from type 2 diabetes mellitus(T2DM)and periodontitis,and it is seriously difficult to control periodontal inflammation.Our previous study showed that the biological properties of human periodontal membrane stem cells(h PDLSCs)be changed due to the influence of related virulence factor of the periodontal microenvironment in T2 DM with periodontitis.It’s unclear whether inflammatory periodontal membrane stem cells affect adjacent jaw bone marrow mesenchymal stem cells(h BMSCs)and exacerbate alveolar bone destruction through paracrine pathway.Therefore,this study intends to extract exosomes of periodontal membrane stem cells derived from patients with T2 DM with periodontitis,and observe its effect on osteogenic differentiation of h JBMSCs.To explore the mechanism of accelerated resorption of alveolar bone in type 2 diabetes patients with periodontitis and provide theoretical basis for the treatment of the disease,we screen differentially expressed exosomal mi RAN that is derived from periodontal membrane stem cells in patients with T2 DM and periodontitis by high-throughput sequencing.Methods:1)The periodontal tissues of healthy persons(H-h PDLSCs),simple periodontitis patients(P-h PDLSCs)and T2 DM with periodontitis(DP-h PDLSCs)were collected.h PDLSCs were cultured by enzyme digestion method,and h JBMSCs were cultured by bone tissue block method.The four kinds of cells were purified and cultured by routine passage.Phenotypes were identified by flow cytometry and the differentiation potential to osteogenic、chondrogenic and adipogenic differentiation was determined.Exosomes(H-EXO,P-EXO,DP-EXO)were extracted,and identified by transmission electron microscopy(TEM)、Nanoparticle Tracking Analysis(NTA)and Western Blot(WB).2)Respectively,H-EXO、P-EXO、DP-EXO were added into the culture medium,and cultured with h JBMSCs for 16 h.The results that exosomes were ingested by h JBMSCs was observed by inverted fluorescence microscope.The exosomes were added into the osteogenic induction medium to induce the osteogenesis of h JBMSCs.Alkaline phosphatase activity was detected after 7 days,mineralized nodules were detected by alizarin red staining 21 days later.The osteogenic related genes and proteins(ALP,Runx2,BMP2,col1)were detected by RT-PCR and WB.3)Mi RNAs of H-EXO、P-EXO and DP-EXO were tested by high-throughput sequencing screened differentially expressed mi RNAs target genes.Target Scan and Mir Base databases predicted target genes,and KEGG and GO enrichment analysis were used to explore the functions and pathways involved in the differentially expressed genes Results:1)H-h PDLSCs,P-h PDLSCs,DP-h PDLSCs and h JBMSCs were successfully cultured.Flow cytometry results showed that the four kinds of cells high expressed surface antigen of mesenchymal stem cell.The differentiative potential of DP-h PDLSCs to osteogenesis,chondrogenesis and adipogenesis decreased.The results of exosomal identification showed that H-EXO,P-EXO and DP-EXO were consistent with the characteristics of exosomes.2)Under inverted fluorescence microscope,it was observed that H-EXO,P-EXO and DP-EXO could be absorbed by h JBMSCs.ALP staining and alizarin red staining showed that H-EXO group could promote osteogenic differentiation of h JBMSCs,P-EXO group had no significant difference with OS group,DP-EXO group inhibited osteogenic differentiation of h JBMSCs.RT-PCR and WB results showed that H-EXO could promote the expression of osteogenesis related genes and proteins in h JBMSCs,P-EXO had no statistical significance with the OS group,while DP-EXO inhibited the expression of osteogenesis related genes and proteins in h JBMSCs(P < 0.05).3)GO enrichment analysis showed that the first three most significant enrichment in biological processes were signal transduction,regulation of transcription,DNA-templated,and the positive regulation of transcription by RNA polymerase Ⅱ.In cell composition,the first five most significant enrichment are membrane,cytoplasm,nucleus,integrate component of membrane,cytosol.In molecular functions,the first three most significant enrichment are respectively: protein binding,mental ion binding,DNA binding.Through KEGG enrichment analysis,the most significant five pathways were found: cancer pathway,PI3K-Akt signaling pathway,Ras signaling pathway,regulation of actin cytoskeleton,and proteoglycans in cancer.In DP-EXO,mi R-23a-3p expresses higher expression of H-EXO and P-EXO..Conclusion: The periodontal microenvironment combined with periodontitis and type 2diabetes mellitus can reduce the multiple differentiation potential of h PDLSCs,and the exosomes secreted by DP-h PDLSCs can reduce the osteogenic differentiation potential of h JBMSCs,thus reducing the ability to self-repair the alveolar bone.The changes of periodontal microenvironment can change the exosomal mi RNA of periodontal ligament stem cells.Mir-23a-3p may be the key mi RNA for DP-EXO to inhibit the osteogenic differentiation of h JBMSCs.However,the mechanism of changing the exosomal mi RNA content of h PDLSCs and the osteogenic differentiation potential of h JBMSCs still needs to be further studied.