The Study Of Neural Crest-tendon Derived Stem Cells And-peripheral Nerves Cooperatively Involve In Tendon Regeneration

Tendon is a type of dense connective tissue connecting muscle to bone.The tendon is susceptible to injury from excess mechanical strain experienced during sports or other daily activities.However,limited knowledge of tendon natural repair hinder the new therapeutic developments.The repair of injured tendon depends on synthesis of excetral cellular matrix(ECM)by cells and the condition of local microenvironment.When tendon is injured,tenocytes synthesize a large amount of ECM(Collagen)to form tissues.However,mature tenocytes have low cell proliferation rate,this feature compromises tendon remodeling.Therefore,a specific tendon progenitor/stem cells would differentitate into functional tenocytes involving in the regeneration.Until now,the big divergence of tendon progenitor/stem cells in origin,distribution,and immune-phenotype has not yet reached to the agreement.In the previous study,we found,during the process of tendon repair,there is a type of neural crest characteristic stem cells involve in the tissue remodeling.The activation of these cells are of significance for promoting the tendon regeneration.One of the key point of this study is to investigate how those neural crest origin-tendon derived stem cells(NCO-TDSCs)could be activated,differentiating into functional tenocytes,then accelerating tendon regeneration.Besides,another important key point focus on where those cells reside in the body,and how they migrate into the wound site when tendon repair.As is known,all the cells of peripheral nerves in the paratenon area are developed from neural crest(NC)origin.Thus,it is worthy to explore whether NCO-TDSCs come from the ingrowth peripheral nerves when tendon injury.Based on the above issues,many studies are carried out.The main experiments and resutls are as follows:(1)Platelet-rich plasma(PRP)activates tendon derived stem cells to promote regeneration of Achilles tendon ruptureNCO-TDSCs exhibit clonogenic and multi-differentiation abilities were isolated from Achilles tendon via enzyme digestion.NCO-TDSCs express many stem cell biomarkers: nucleostemin(NS),Oct-4,SSEA-4,?-SMA,CD29,CD44,CD90,Sox10,Snail,and P75.In the in vitro study,PRGF(activated PRP)significantly enhanced cell DNA synthesis,improved viability and promoted proliferation,while facilitating cell migration and the recruitment of TDSCs.In addition,TDSCs were mixed with collagen and PRP to form collagen-TDSC constructs(CTC)and PRP-collagen-TDSC constructs(PCTCs).After 3 weeks of culture in vitro,we found that most of the encapsulated TDSCs in the CTCs and PCTCs were still alive,while cells in the PCTCs showed a more aligned arrangement compared to the CTCs.In addition,the micro-structure of PCTC showed more obvious fiber-like tissues and formed a cyclic microvascular structure.The tenocyte-related genes types I and III collagen,Tenascin-C and Scleraxis of TDSCs in the PCTCs and CTCs were upregulated with time,and PCTCs showed more significance than CTCs.After in vivo transplantation,the CTCs and PCTCs showed stimulatory effects on Achilles tendon healing.Moreover,the PCTCs improved the macroscopic appearance,histological morphology and biomechanical strength of ruptured Achilles tendon better than CTC.(2)Peripheral nerves assist angiogenesis in the injured tendonSox10-Cre/ROSA26-Flox-RFP were adopt to trace peripheral nervous cell lineage.Both in the patellar tendon window defect and Achilles tendon rupture models,RFP labeled nerve cells surround neovessel.For the patellar tendon window defect,1 week post-injury,ingrowth peripheral nerves sprout into the wound site,and involve in the unmature vessel formation.In the severer Achille tendon rupture injury,based on the immunofluorescent staining identification,peripheral nerves rapidly sprout into the midpotion of injured site,some nerve tissues secrete Neuropeptide Y1 while the related neovessels express Neuropeptide Y1 receptor.These results show a relation between ingrowth nerves and angiogenesis in injured tendon.(3)Neural crest origin cells migrate from nerves and participate in Achille tendon remodelingSox10-Cre/ROSA26-Flox-RFP tansgenic mice were used for tracing possible neural crest origin cells involving in remodeling of Achille tendon rupture.Within 4 weeks following Achilles tendon rupture,many RFP positive cells migrate from ingrowth peripheral nerves into wound site,these RFP positive cells express neuron biomarker: Tuj1,Schwann cell biomarker: S100?,NCO-TDSCs unspecific markers: P75 and Sox10,fibroblast biomarkers: FAP and Vimentin.In vivo results demonstrate the NC-derived cells migrate from nerves are a mixed cell group.The RFP positive cells were isolated from Achille tendon of 2 weeks post-injury,the RFP cells account for about 27% of the total isolated cells.Similar as in vivo immunofluorescent testing,the RFP positive cells include different type of cells which express Tuj1,S100?,P75,FSP1,FAP and Sox10 respectively.In addition,part of the RFP positive cells possess multi-differentiation potency.These results confirm that the RFP positive cells consist of neuron,Schwann cells,NCO-TDSCs,fibroblast.All in all,this study demonstrate:(1)PRP can activate NCO-TDSCs to improve the quality of Achilles tendon rupture healing in the early stages.(2)NC derived peripheral nerves sprout into the wound site involving in angiogenesis.(3)NCO-TDSCs partially come from the ingrowth peripheral nerves in the injured tendon.In addition,a group of cells derived from neural crest origin migrated from the ingrowth nerve,exhibit fibroblast characteristics,and may participate in tendon regeneration.