Exosomes From Hypoxia-Pretreated Periodontal Ligament Stem Cells Promote Periodontal Tissue Regeneration

Background and ObjectivePeriodontitis is one of the most common oral diseases,with a high incidence,and is the leading cause of tooth loss in adults,seriously affecting the quality of life of individuals.It is characterized by the progressive destruction of periodontal supporting tissues.Therefore,the goal of periodontitis treatment is to achieve structural and functional regeneration of periodontal tissue.while stem cell therapy has made some strides in achieving tissue regeneration,a host of problems of transplanted stem cell expansion in vitro,such as replicative aging,differentiation potential diminishing or even futile,and relevant immunogenic response make them encounter great challenges on the path toward clinical translation.Recently,some studies have shown that the regenerative effect of transplanted mesenchymal stem cells(MSCs)is mainly mediated by paracrine functions,in which exosomes(EXs)play a crucial role.The application of EXs not only avoids biosafety issues such as teratogenicity,tumorigenicity,and mutagenicity,but also has low immunogenicity,easier accessibility,and more convenient utilization and functionalization modifications compared with conventional transplanted stem cells.Meanwhile,EXs can simulate various biological behaviors of stem cells to further encourage the regeneration of tissues.Periodontal ligament stem cells(PDLSCs),which are the seed cells for periodontal tissue regeneration,secrete EXs that have been demonstrated to exhibit outstanding tissue regenerative potential in inflammatory periodontal bone defects.Consequently,the clinical application of PDLSC-EXs provides a new strategy to fulfill the regeneration of periodontal tissues.However,there are still some urgent problems in the process of EXs transformation,such as a low acquisition rate,regenerative potential has yet to be improved,and the targeting rate must still be optimized.In terms of relevant mechanisms,microribonucleic acid(miRNA)is recognized as a key molecule mediating the involvement of EXs in tissue repair and regeneration processes.The process by which miRNAs are secreted from cells to EXs and subsequently function is specifically altered by various stimulatory conditions,which affect the biological function and regenerative potential.Previous studies of our group have shown that hypoxia pretreatment significantly enhances PDLSCs-based periodontal tissue regeneration by improving cellular biological functions such as proliferation,migration,and osteogenic differentiation.The results of pre-experiments showed that PDLSCs after hypoxia pretreatment were capable of secreting more EXs.And hypoxia PDLSC-EXs were more easily taken up by PDLSCs.The present study intends to explore the effects of hypoxic pretreatment on the biological functions of PDLSC-EXs and their capability to promote periodontal tissue regeneration based on the above-mentioned previous studies and to preliminarily elucidate the intrinsic mechanism of action.The study results are expected to accelerate the clinical translation process of EXs,effectively improve the efficiency of periodontal tissue regeneration,and provide a new feasible strategy for the treatment of periodontitis.MethodFirstly,the chosen hypoxia condition in our previous work was used to treat PDLSCs,and PDLSC-EXs were extracted and identified.Secondly,the effects of PDLSC-EXs on the biological functions of PDLSCs including proliferation,migration,osteogenic differentiation,and immunomodulation were observed under hypoxia conditions.Then,Gelatin methacryloyl(GelMA)gels were loaded with PDLSC-EXs under two different treatment conditions and transplanted into a rat periodontal defect model to evaluate the effect of periodontal tissue regeneration.The molecular mechanisms were further explored by screening differentially expressed miRNAs in PDLSC-EXs before and after hypoxia pretreatment by high-throughput sequencing and validated by microfluidic and quantitative real-time polymerase chain reaction(qRT-PCR)techniques.ResultsIn terms of particle size,surface protein expression,and concentration,hypoxia-pretreated PDLSC-EXs were superior to normoxia PDLSC-EXs.Co-culture of normoxia and hypoxia pretreated PDLSC-EXs with PDLSCs and macrophages respectively,showed that PDLSC-EXs under hypoxia pretreatment could significantly enhance the biological functions of PDLSCs including proliferation,migration,and osteogenic differentiation.It also could significantly promote the polarization of macrophages to the M2 type.PDLSC-EXs under two different treatment conditions loaded with GelMA gel were transplanted into periodontal defects of rats,and both Micro-CT and H&E staining results showed that the regenerative potential of PDLSC-EXs under hypoxia pretreatment was superior to normoxia pretreatment.Immunofluorescence staining showed that hypoxia PDLSC-EXs significantly promoted more endogenous stem cell homing.It is further confirmed by immunofluorescence staining that PDLSC-EXs under hypoxia pretreatment promoted the polarization of M2-type macrophages,and thus facilitated tissue repair.Analysis of differentially expressed miRNAs in PDLSC-EXs before and after hypoxia pretreatment by high-throughput sequencing revealed that two miRNAs were up-regulated(has-miR-125b-5p,novel-has-miR239-5p)and three miRNAs(has-miR-323a-3p,has-miR-3074-5p,has-miR-409-3p)were down-regulated under hypoxia condition,which was initially verified by microfluidic and qRT-PCR technology.Twenty target genes and eight signaling pathways closely related to cell proliferation,migration,osteogenic differentiation,and immunity were further predicted.ConclusionHypoxia pretreatment can promote PDLSC-EXs secretion,boost the efficacy of PDLSC-EXs for tissue regeneration in defect areas,enhance the recruitment of stem cells in the regenerative region,elevate the biological functions of recruited stem cells involving proliferation and osteogenic differentiation,modulate the local immune response,and thus to promote periodontal tissue regeneration.This project could provide new strategies for periodontal tissue engineering,and establish the theoretical basis for elucidating the regulation of hypoxia stimulation on regenerative microenvironment through EXs.