Sensory Nerve Regulates Lineage Commitment Of Mesenchymal Stromal Cells By Tuning Sympathetic Tones

Part? Sensory Nerves Regulate Mesenchymal Stromal Cells DifferentiationObjective:Study on whether sensory nerves can regulate mesenchymal stromal cells(MSCs)differentiation,whether balance between osteogenesis and adipogenesis change when sensory nerves knocks out.Method:The cre-loxp system was introduced to generate conditional knock out mice Advilin-TrkA^-/-which knock out TrkA receptor on peripheral sensory nerves inhibit sensory nerves growth in this part.Bone mass and bone marrow fat composition was measured by?CT and osmium tetroxide(OsO4).Osteoblasts and adipocytes were stained by osteocalcin(OCN)and perilipin by immunofluorescence,trilineage differentiation experiment was performed by crystal violet,Alize Red and Oil red staining after MSCs cultured in vitro.Osteogenic progenitor cells(OPCs)and adipogenic progenitor cells(APCs)was counted and sorted by flow cytometry.Result:Bone volume/tissue volume(BV/TV)and trabecular thickness(Tb.Th)significantly decreased in 3-month-old TrkAAvil^-/-mice relative to their WT littermates(P<0.05).Fat was significantly increased in the bone marrow of 3-month-old TrkAAvil^-/-mice compared with the WT controls,as evidenced by a markedly higher number of adipocytes and fat droplets in decalcified femurs stained with OsO4(P<0.05).Immunostaining(IF)of OCN and perilipin showed a significant increase in the number of adipocytes and decrease in the number of osteoblasts in 3-month-old TrkAAvil^-/-mice relative to their WT littermates(P<0.05).Colony-forming-unit fibroblast(CFU-F),adipocyte(CFU-AD),and osteoblast(CFU-OB)assays with bone marrow cells showed a significant increase in adipogenesis and decreases in osteogenesis and CFU-F in TrkAAvil^-/-mice relative to WT littermates(P<0.05).Flow cytometry analysis showed that APCs significantly increased,and OPCs decreased.Importantly,the population of MSCs also decreased in TrkAAvil^-/-mice(P<0.05).Conclusion:Bone mass significantly decrease in the conditional genetic sensory nerves knock out mice,accompanied by adipocytes composition in bone marrow.Osteoblasts decreased and adipocytes increased in the sensory nerves knock out mice by IF,and OPCs also showed same trend by flow cytometry.Conditional knock out sensory nerves showed lower bone mass and much higher bone marrow fat tissue,which indicated that sensory nerves could participated regulation of MSCs differentiation and proliferation.Part ? PGE2 Derived from Osteoblasts Regulate Mesenchymal Stromal Cells Differentiation Through Sensory Nerves EP4 ReceptorObjective:Study on whether prostaglandin E2(PGE2)can regulate mesenchymal stromal cells(MSCs)differentiation through sensory nerve,search the primary cell secrete PGE2,raise the potential mechanism that sensory nerves regulate MSCs.Method:Introduced the conditional genetic knock out mice Avil-EP4^-/-which knock out EP4 receptor on the peripheral sensory nerves,and conditional knock out cyclooxygenase 2(COX2)from osteoblast mice OCN-COX2^-/-.Bone mass and bone marrow fat composition was measured by ?CT and OsO4 staining.Osteoblasts and adipocytes were stained by OCN and perilipin by immunofluorescence,trilineage differentiation experiment was performed by crystal violet,Alize Red and Oil red staining after MSCs cultured in vitro.OPCs and APCs was counted and sorted by flow cytometry.Inject PGE2 degradation enzyme inhibitor SW033291 on the transgenic mice mentioned above,detect the change of bone mass,osteoblasts and adipocytes by ?CT and OsO4 staining and IF.Result:Bone mass significantly decreased in the Avil-EP4^-/-and OCN-COX2^-/-relative to the wild type mice(P<0.05),accompanied by fat tissue composition in bone marrow(P<0.05).Injection of SW033291 significantly increased the number of osteoblasts and decreased the number of adipocytes in WT mice,and these effects were absent in EP4Avil-/and OCN-COX2^-/-mice,indicating that sensory nerves regulates MSCs through EP4 signaling.Moreover,MSCs isolated from EP4Avil-/-and OCN-COX2^-/-mice showed an increase of CFU-AD,and a decrease in CFU-OB and CFU-F capability compared with SSCs from their WT littermates(P<0.05).The frequency of APCs increased,whereas the frequencies of OPCs and MSCs decreased in EP4Avil-/-and OCN-COX2^-/-mice relative to WT littermates(P<0.05).Prop injection can rescue phenotype of increased fat tissue composition and decreased bone mass in EP4Avil^-/-and OCN-COX2^-/-mice.Importantly,propranolol promoted osteogenesis and inhibited adipogenesis(P<0.05).Conclusion:PGE2 derived from osteoblasts target EP4 receptor on sensory nerves regulate MSCs proliferation and differentiation.Part ? PGE2-Sensory Nerves-Sympathetic Nerve Axis Regulate LepR+MSCs differentiation and Mechanism in Bone Fracture Repair Objective:Study on the specific population of MSCs that response PGE2 sensory nerves signal,analyze the mechanism in bone fracture healing process.Method:Introduce transgenic lineage tracing mice LepR-cre;YFP as inject Capsaicin on the this mice to generate sensory nerves denervation model,observe bone mass and bone marrow area fat composition by ?CT and OsO4 staining after capsaicin injection.Adrenergic ?2 receptor inhibitor propranolol(Prop)injected on EP4Avil^-/-and OCN-COX2^-/-mice to test sympathetic nerves involved with PGE2 sensory js axis.LepR and adipocytes were stained by GFP and perilipin by immunofluorescence to do the in vivo fate mapping experiment to test whether increased adipocytes original from LepR+MSCs after capsaicin injection.We established femur bone fracture model on the mice LepR-cre;YFP injected with Capsaicin,SW033291 and Vehicle.After lmonth the ?CT,SO/FG,Masson staining and IF(OCN&perilipin)was introduce in this part to observe the bone fracture repair and microenvironment change.Result:?CT analysis showed that BV/TV and Tb.Th were significantly reduced in the capsaicin-injection group compared with vehicle group(P<0.05).The marrow fat was significantly increased in the bone marrow of capsaicin-injected mice relative to vehicle controls as evidenced by markedly more adipocytes and fat droplets in decalcified femurs stained with OsO4(P<0.05).Adipogenesis was increased and osteogenesis was decreased after capsaicin injection relative to the control group(P<0.05).In vivo fate mapping assay showed that YFP+adipocytes were significantly increased after sensory denervation.Propranolol,not SW033291,could lower the number of YFP+adipocytes,which were induced by sensory denervation.Injection of capsaicin reduced BV relative to the vehicle group in ?CT analysis,and bone formation was significantly lower in the capsaicin group,as shown by SO/FG staining.Masson staining showed that bone callus volume was also significantly decreased with injection of capsaicin.OCN+osteoblasts were less detectable,whereas adipocytes increased significantly with injection of capsaicin(P<0.05).Importantly,the increased adipocytes were primarily YFP+,indicating they were derived from LepR+cells.Conclusion:LepR+MSCs is the major population of MSC that response PGE2-sensory nerves-sympathetic nerves axis.Importantly,this mechanism participated in bone fracture repair process.