Small Extracellular Vesicles Derived From Periodontal Ligament Stem Cells Regulate Pyroptosis Of THP-1 Macrophages And Periodontal Bone Regeneration

Objective:Pyroptosis is a common pathogenesis in inflammatory diseases and may be related to the occurrence and development of periodontitis.Periodontal ligament stem cells(PDLSCs)can achieve tissue regeneration by immunomodulatory effects.However,whether there is an association between periodontitis and pyroptosis and whether pyroptosis can be regulated by PDLSCs paracrine factors are still unclear.The aim of this study is to investigate the relationship between pyroptosis and periodontitis,and to further verify whether the key paracrine factor-small extracellular vesicles(sEVs)of PDLSCs can mediate pyroptosis by regulating the NF-κB/NLRP3/GSDMD pathway,thereby improving the periodontal inflammatory microenvironment and promoting tissue regeneration.Method:1.Determine whether there is an association between clinical periodontal inflammation and pyroptosis: Clinical normal and inflammatory gingival tissues were collected,and paraffin-embedded sections were prepared.hematoxylin-eosin(HE)staining and immunohistochemistry(IHC)staining were used to observe the expression of GSDMD,a key factor related to pyroptosis,and CD68,a macrophage marker in inflammatory tissues.Tissue m RNA was extracted,and quantitative Real-Time PCR(q RT-PCR)was used to further verify the expression of factors related to the classical pyroptosis pathway in inflammatory gingival tissues;2.Extraction and identification of primary PDLSCs and sEVs: Intact isolated teeth(donors aged 18-26 years old with periodontal and general health)obtained from orthodontic or third wisdom tooth extraction were collected clinically,and the periodontal ligament in the middle third of the root was removed.PDLSCs were extracted and cultured by tissue explant method.The stemness of PDLSCs was identified by cell morphology fluorescence staining,colony formation assay,osteogenic/adipogenic induction and flow cytometry.sEVs were extracted from PDLSCs of passage 3-5 by differential centrifugation and identified by transmission electron microscopy(TEM),particle size analysis,and western blot;3.Establishment of THP-1 macrophages pyroptosis model in vitro: THP-1 monocytes were induced to adhere to the wall by phorbol myristate acetate(PMA).The cells were treated with P.gingivalis-LPS at different concentrations(0.1,1,and 10μg/ml)for 11 hours,followed by 5m M ATP for 1 hour.CCK-8 assay was used to detect the cell viability under different induction conditions.The cell membrane rupture was observed by double staining with Hoechst 33342 and PI.The expressions of pyroptosis-related factors induced by P.gingivalis-LPS at different concentrations were detected by q RT-PCR and western blot;4.Regulation of PDLSCs paracrine factors on pyroptosis of THP-1 macrophages:(1)Cell-cell co-culture model: THP-1 cells were seeded in the lower layer of transwell and induced to adhere to the wall,then induced pyroptosis.PDLSCs were seeded in the upper transwell layer,and when the cell confluence reached 80-90%,the PDLSCS were co-cultured with THP-1 macrophages for 24 hours.(2)sEVs-cell co-culture model: THP-1macrophages were co-cultured with different concentrations of sEVs(10μg/ml,50μg/ml)for24 hours and 48 hours after pyroptosis induction.The m RNA and protein were extracted from the cells in the two co-culture methods for subsequent q RT-PCR and western blot analysis;5.Construction of hydroxybutyl chitosan hydrogel(HBCH)containing sEVs derived from PDLSCs(HBCH/P-sEVs)thermosensitive system and its effect on PDLSCs: sEVs(1mg/ml)suspended in PBS were added to the colloidal HBCH,and HBCH/P-sEVs-l(5%v/v)and HBCH/P-sEVs-h(10%v/v)were prepared by alternately mixing HBCH/P-sEVs-l and HBCH/P-sEVs-h by vortex and centrifugation.HBCH/P-sEVs was incubated at 37℃ to form a gel state and then placed in the culture medium for 24 h.After collection and centrifugation,the subsequent conditioned medium was made.PDLSCs were seeded in conditioned medium,and the proliferation of PDLSCs was analyzed by CCK-8.The osteogenic differentiation of PDLSCs was analyzed by western blot and alkaline phosphatase(ALP)staining;6.Verification of pyroptosis regulation and osteogenesis of HBCH/P-sEVs in vivo: An inflammatory bone defect model was established in rats by silk ligation combined with mandibular bone removal,and the bone defect was filled with material.Micro-CT,Masson staining,HE staining,and IHC staining were used to determine the expression of bone formation and pyroptosis-related factors.Results:1.GSDMD,a key factor of pyroptosis,and CD68,a macrophage marker,were highly expressed in inflammatory tissues,and they were co-localized.Several factors related to the classical pyroptosis pathway are highly expressed at the gene level in inflammatory tissues;2.After culture,the morphology of the cells was fibroblast-like,with obvious colony-forming ability.Under induction,the cells could form lipid droplets and calcification nodules.The membrane structure of sEVs was observed under TEM,and the particle size was about 130 nm.The positive markers Alix,TSG101 and CD68 were successfully expressed;3.P.gingivalis-LPS combined with ATP could successfully induce the high expression of the factors involved in the classical pyroptosis-related pathway in THP-1 macrophages in vitro,and the expression of pyroptosis-related factors was concentration-dependent within the concentration range of 0.1-10μg/ml of P.gingivalis-LPS;4.In the cell-cell co-culture mode,the pyroptosis-related factors NF-κB,NLRP3,ASC,caspase-1,GSDMD and IL-1β in the co-culture pyroptosis group were significantly lower than those in the mono-culture pyroptosis group.In the sEVs co-culture mode,the expression of caspase-1 and GSDMD in the co-culture pyroptosis group was significantly lower than that in the mono-culture pyroptosis group;5.Both HBCH and HBCH/P-sEVs could achieve bidirectional reversibility of sol-gel transition under the change of temperature.The phase transition temperatures of HBCH,HBCH/P-sEVs-l and HBCH/P-sEVs-h were 17℃,16.5℃ and 16℃,respectively.Fourier transform spectroscopy showed that the degree of group substitution of HBCH was changed compared with chitosan hydrogel(CSH),while HBCH/P-sEVs and HBCH shared the same characteristics.The release rate of sEVs in HBCH/P-sEVs was 20% per day for the first 7days,reached a plateau after 10 days,and the release rate could reach more than 80% on 14days;6.HBCH/P-sEVs conditioned medium could significantly promote the proliferation and osteogenic differentiation of PDLSCs;7.In vivo,HBCH/P-sEVs significantly promoted alveolar bone regeneration and reduced the infiltration of inflammatory cells and the expression of caspase-1 and GSDMD in the inflammatory microenvironment.Conclusions:1.Macrophage pyroptosis is involved in the development of periodontitis;2.P.gingivalis-LPS combined with ATP can successfully induce classical pyroptosis of THP-1 macrophages in vitro;3.PDLSCs paracrine factors can inhibit THP-1 macrophages pyroptosis by regulating NF-κB/NLRP3/GSDMD pathway in vitro;4.sEVs derived from PDLSCs can be combined with temperator-sensitive HBCH to produce a repair and regeneration system suitable for the clinical treatment of periodontitis.