| [Background]Stem cells transplantation has been accepted as an effective strategy for repairing and regenerating injured tissues and organs.Autologous and heterologous stem cells transplantation have achieved multiple tissues regeneration in bone,skin,cornea and teeth.The most important issue that hinders regeneration mediated by stem cells is how to regenerate tissue structure and recover physiological function,which is depended on excellent regenerative potent of stem cells participation of host neurovascular bundle and influence of overall health condition.Hence,to elucidate the mutual communication between implanted stem cells and host will be significant for promoting clinical transformation of stem cells.Teeth are important organs in craniomaxillofacial system,which consist of hard enamel,dentin and cementum externally and internal dental pulp tissue.Dental pulp takes a significant role in regulating tooth development,repairing and sensation.Trauma and caries are common risks for dental pulp necrosis,which impedes tooth development and eruption,causes severe pain and threatens human healthy seriously.Currently,the dental pulp regeneration has been achieved in many large animals and humans in clinal trials by transplanting exogenous stem cells or recruiting endogenous stem cells from host.Previous study indicated that the implanted stem cells could not only differentiate into odontoblasts and endothelium but also induce regrowth of neurovascular bundle by paracrine,which provided nutrient for implanted cells and recovered sensory function of regenerated dental pulp.However,the clinical translation of stem cells mediated dental pulp regeneration are hindered by so many factors such as anatomic structure of root canal,source of implanted cells and the regenerative potent of host.Hence,it possesses crucial theoretical significance and practiced value in clinical translation of stem cells mediated dental pulp regeneration by deciphering the integration between implanted stem cells and host component.Mesenchymal stem cells（MSCs）are somatic stem cells with self-renew ability,multi-differentiation potent and low immunogenicity,which are wildly applied in craniofacial tissues regeneration for easy availability in dental pulp,periodontium,alveolar bone and gingiva.The mechanisms of MSCs during tissue regeneration have always been a key problem in the field of stem cells mediated therapy.Recently,extracellular vesicles（EVs）secreted from MSCs are verified to play an important role in therapy and regeneration.Exosomes are small vesicles with bilayer structure,the diameter of which are 30-130 nm.Nucleic acids,lipids,proteins and other bioactive molecules are encapsulated in exosomes,which are important for communication between cells and tissues and regulation in homeostasis and repair.Supplying self-secreted exosomes has promoted dental pulp regeneration obviously,however,considering the nature of EVs in cellular communication,whether there exist exosomal communication between implanted MSCs and host during regeneration still need further exploration.Dental pulp is innervated by numerous sensory nerves derived from trigeminal ganglions（TG）,which is crucial for maintaining stemness and homeostasis and regulating the cellular behavior of surrounding Schwann cells（SCs）,immune cells and stem cells by paracrine.It has been verified that inferior alveolar nerve transection could break dental pulp homeostasis,affect tooth development and formation of dentin and enamel.The recovery of sensory is one of the most important assessment criteria for dental pulp regeneration.Numerous studies declared that MSCs regulated peripheral nerves regeneration by secreting exosomes or neurotrophic factors.Meanwhile,neurons in centra and peripheral nerve system also communicate with surrounding glia cells by exosomes.Hence,how the sensory nerves innervate regenerated dental pulp and whether regrew sensory nerves regulate fate decision of implanted stem cells are still unclear.In conclusion,to investigate the communication between implanted stem cells and host TG,elucidate the fate of MSCs and regulative mechanisms of sensory nerves will have crucial theoretical values for clinical translation of dental pulp regeneration.[Aim]This study aims to decipher the differentiative fate of implanted stem cells,elucidate the important role of host sensory nerves and explore the crucial mechanisms of exosomes mediated mutual communication between host sensory and implanted stem cells by applying SHED（Stem cells from human exfoliated deciduous teeth）mediated heterologous chimeric dental pulp regeneration in rats and minipigs,which provides theoretical foundation for clinical therapeutic strategy of stem cells implantation mediated dental pulp regeneration.[Methods]1.Stem cells from human exfoliated deciduous teeth collaborating with host cells to regenerate chimeric dental pulp heteroulogously.The deciduous teeth were collected in clinical from patients aged 6-12 for SHED primary culture;The model of dental pulp regeneration was performed in incisors of minipigs and maxillary first molars of rats individually.The morphology of dental root canal between SHED implantation and normal teeth was assessed by micro-CT（Micro-computed tomography）;the histological structure of dental pulp was compared with HE（Hematoxylin-Eosin）staining and immunofluorescence staining,including odontoblast layer and neurovascular bundle.The fate decision of implanted SHED were analyzed with single cell RNA sequencing（sc RNA-seq）and immunofluorescence staining was used to detect main differentiated cell types of implanted cells.2.The role of implanted stem cells-derived exosomes in inducing the regrowth of host trigeminal nerve axons during dental pulp regeneration.The function of implanted stem cells derived exosomes were verified by dental pulp regeneration in minipigs.The effect of inhibiting stem cells derived exosomes secretion on dental pulp regeneration was observed by implanting GW4869 pretreated SHED.SHED derived exosomes（S-Exo）were harvested by ultracentrifugation.Di R labeled S-Exo were injected into the root canal of maxillary first molars of rats.Live imaging is used to track the in vivo fate of S-Exo during the process of pulp regeneration.PKH26 labeled S-Exo were injected into the root canal and immunofluorescence staining were used to observe their cellular location in TG.CD63-m Cherry overexpressing lentivirus were used to label SHED and implanted into the pulp cavity of the first molar in rats to further verify the above results.Rat trigeminal ganglion neurons（TGN）were primary cultured in vitro to verify the promoting effect of S-Exo on neural axon regeneration.3.Detecting the effect of host derived T-Exo on SHED differentiation into Schwann cells（SCs）.The TG secreted exosomes were labeled in vivo by AAV9-hsyn-m Cherry-CD63 injection,which was used to tracing TG derived exosomes（T-Exo）during dental pulp regeneration.T-Exo were harvested and identified for comparing the endocytic preference between SHED and TGN.Using an in vivo rat model of dental pulp regeneration,the biological effects of S-Exo and T-Exo on dental nerves regeneration and Schwann cells（SCs）differentiation during pulp regeneration were elucidated by inhibiting exosome release from SHED and TGN with GW4869.Additionally,direct and indirect co-culture models of SHED and TGN were established in vitro,and the biological effects of their respective exosomes on dental pulp regeneration were further clarified by inhibiting exosome release from both cell types.4.The role of sequential exosomal communication between implanted stem cells and host trigeminal ganglions in of dental pulp nerves regeneration.The axonal regeneration and SCs differentiation were analyzed at different time point with 2,4,6 and8 weeks after SHED and TGN cocultured and subcutaneous implanted in immunocompromised mice.The exosomal internalization of TGN were inhibited by Dynasore and secretion were decreased by sh Rab27a lentivirus transfection individually,which were cocultured with sh RAB27A-SHED to observe axonal regeneration and SCs differentiation.Coculturing of TGN and sh RAB27A-SHED in Transwell under identical treatment conditions to clarify their paracrine regulation of dental nerves regeneration and SCs differentiation.The morphology and expression of SCs related markers were detect in SHED after being stimulated by S-Exo,T-Exo and exosomes derived from TGN after internalizing S-Exo（ST-Exo）,of which were also directly co-cultured with TGN and implanted subcutaneously in immunocompromised mice to verify the role in inducing SCs differentiation.The protein contents in T-Exo and ST-Exo were analyzed by proteomic analysis,which were further validated by Western blot.Finally,the different expressed proteins co-located with CD63 were verified in rat TG with dental pulp regeneration.5.The detection of functional molecular and mechanism in SHED differentiating into SC mediated by ST-Exo.Lentivirus transfection was used to downregulate the expression of TUFM and GFM2 in ST-Exo to verify the most important molecular in inducing SC differentiation.The biological function of TUFM in inducing SCs differentiation was evidenced by detecting morphology of SHED,expression of SCs related markers as well as directly co-culture and immunocompromised mice implantation.To investigate the mechanisms of ST-Exo-induced SCs differentiation,the levels of oxidative phosphorylation（OXPHOS）and mitochondrial autophagy in SHED were measured after the addition of ST-Exo.By knocking down the expression of TUFM in SHED using lentivirus and supplementing with ST-Exo and Rapamycin,it was further validated that ST-Exo regulated OXPHOS and mitochondrial autophagy by delivering TUFM to induce the differentiation of SHED into SCs.[Results]1.SHED successfully regenerated functional dental pulp in vivo by heterologous implantation.The implantation of SHED into the pulp cavity of miniature pigs achieved pulp nerve regeneration including the formation of odontoblast layer,blood vessels,and SCs-enwrapped pulp nerves.As the implantation time increases,the density of nerve fibers and the number of SCs around the nerves in the regenerated pulp also gradually increased.sc RNA-seq analysis of the fate of implanted stem cells revealed that they differentiated into endothelial cells,odontoblasts,SCs,etc.GFP-labeled SHED were also used to track implanted cells and found that implanted SHED differentiated into vascular endothelial cells,odontoblasts,and SCs.Human mitochondria（h-mit）were used to label implanted SHED,and co-localization with the SC marker GFAP was observed,but not with the neuronal markerβIII Tubulin（βIII Tub）.Immunofluorescence staining confirmed that regenerated nerve fibers expressed the sensory nerve markers CGRP,TRPV1,and TRPM8,indicating their origin from the host sensory nerves.2.Exosomes derived from implanted stem cells were delivered into host TG and promoted TGN axon growth.Compared to the control group,the effect of GW4869pretreatment on SHED-mediated in-situ pulp regeneration in miniature pigs was significantly inhibited.The arrangement of the odontoblast layer was sparse and disordered and the number of blood vessels,nerves,and SCs was decreased.PKH26 and Di R dyes were used to label S-Exo and injected into the pulp cavity of the first molar of rats,which could delivered into the neuronal cell bodies of the host TG.The implantation of CD63-m Cherry SHED further confirmed that S-Exo could enter TGN during pulp regeneration.Adding S-Exo to rat TGN cultured in vitro significantly promoted nerve axon growth,but adding S-Exo to SHED did not change the expression level of SCs related markers.3.T-Exo were delivered to the regenerated dental pulp and induced SCs differentiation of implanted SHED.During dental pulp regeneration,rat T-Exo labeled in vivo with AAV9-hsyn-m Cherry-CD63 were delivered to the pulp cavity where SHED were implanted.In vitro endocytosis comparison experiments have shown that T-Exo were preferentially engulfed by SHED,while S-Exo were preferentially engulfed by TGN.In rat in-situ pulp regeneration experiments,inhibiting the secretion of T-Exo leaded to a decrease in SCs differentiation,while inhibiting the secretion of S-Exo inhibited the growth of pulp nerves into the pulp cavity.In in vitro directly co-culture model,inhibiting the secretion of T-Exo with GW4869 or sh Rab27a mainly leaded to a decrease in SCs differentiation,while inhibiting the secretion of S-Exo mainly leaded to a decrease in nerve axon density.4.Exosomes secreted from TGN after internalizing S-Exo（ST-Exo）possessed enhanced ability in inducing SC differentiation.In vitro co-culture models of SHED and TGN at different time points indicate that the speed of nerve axon growth is faster than SCs differentiation.The co-culture results showed that after adding S-Exo to co-culture system,the density of nerve axons increased significantly,and the number of co-cultured SHED differentiating into SCs also increased significantly.When Dynasore inhibited the endocytosis of S-Exo by TGN,axonal growth was inhibited and SCs differentiation decreased significantly.However,when sh Rab27a inhibited the exosomes secretion of TGN,even when stimulated with S-Exo,the differentiation of surrounding SHED into SCs did not increase significantly,indicating that the ability of TGN stimulated by S-Exo to induce SCs differentiation was significantly enhanced,and the fate of SCs differentiation was regulated through exosomes.Further experiments showed that compared to S-Exo and T-Exo,ST-Exo could significantly induce the differentiation of SHED into SCs.Proteomics results indicated that compared to T-Exo,ST-Exo could carry more mitochondrial-derived proteins such as TUFM and GFM2.5.ST-Exo delivered TUFM regulated SC differentiation by activating OXPHOS and mitophagy.When the expression levels of TUFM and GFM2 in ST-Exo were knocked down respectively,it was found that only when TUFM knockdown of ST-Exosh TUFM decreased the ability to induce SCs differentiation significantly,and it was unable to increase the OXPHOS level of SHED.To further clarify the specific mechanisms,knocking down the expression of TUFM in SHED with sh TUFM lentivirus transfection also inhibited SCs differentiation,while supplementation with ST-Exo or Rapamycin restored SCs differentiation.Western blot and immunofluorescence staining showed that TUFM mainly activated OXPHOS by regulating mitochondrial autophagy,promoting the differentiation of SHED into SCs.[Conclusions]1.SHED regenerated functional dental pulp in vivo heterologously;2.SHED promoted axonal regeneration and decided dental pulp regeneration by secreting exosomes.3.Mutual communication between implanted SHED and host TG by exosomes mediated dental nerves regeneration.4.Compared to S-Exo and T-Exo,ST-Exo possessed enhanced ability in inducing SCs differentiation.5.TUFM in ST-Exo was the significant molecular in regulating SCs differentiation.6.TUFM in ST-Exo regulated SHED differentiating into SCs by activating OXPHOS and mitophagy.This study demonstrated the therapeutic role of human stem cells in heterologous chimeric dental pulp regeneration,analyzed the significant biological nature in regeneration mediated by mutual exosomes communication between implanted stem cells and host TG,and confirmed the regulation and mechanisms of ST-Exo in inducing SC differentiation,which provided important theoretical foundation for stem cell based clinical translation strategies for dental pulp regeneration. |
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