| Research BackgroundWith the rapid development of stem cell biology,which has become one of the important foundations of tooth/bone tissue engineering research,the theory and method of tissue engineering in the treatment of bone defects is one of the most active research fields in recent years.In the early stage of embryonal development,the neural crest derived cells first migrate to the first gill arch and then form the cranial neural crest derived ectodermal mesenchymal stem cells.Therefore,the maxillary ectodermal mesenchymal stem cells are progenitor cells for the development of maxillofacial hard tissue,including the maxilla and maxilla.P75 neurotrophin receptor?p75NTR?,a single membrane-spanning protein in the TNF receptor family,is a low-affinity receptor capable of binding all neurotrophins.Previous studies have shown that p75NTR plays critical roles in the morphogenesis and development of many embryonic and adult tissues.Its important role in the development of the nervous system has been fully described,but the potential regulatory role in morphogenesis and development of other tissues especially teeth and bones has been rarely reported.In this study,p75NTR-/-mice obtained from Jackson Lab were used to investigated whether p75NTR regulates the alveolar bone development and osteogenic differentiation of murine EMSCs,and further explored the underlying mechanisms associated with the PI3K/Akt/?-catenin pathway.Material and methods:1?Comparison of mandibular bone mass and osteogenic differentiation between p75NTR-/-mice and wild-type?WT?miceFemale p75NTR-/+mice and male p75NTR-/+mice during reproductive period were fed in cages in the animal houses.At 10 days old,2mm tail of newborn mice was cut to extract DNA,and genotype identification was performed to identify p75NTR-/-and WT mice.Mice were conventionally fed to one month old and anesthetized by intraperitoneal injection of 2%pentobarbital sodium?50mg/kg?solution.The mandible was separated and fixed in 4%paraformaldehyde before to be analysed by micro-ct?viva CT 40,Scanco Medical AG?.In order to determine whether the mandibular bone mass of the rapidly growing developing mice was affected by knockouting p75NTR,the 3D image reconstruction and data analysis were conducted using MicroView software 2.2.The same method was used to obtain the mandible of mice at the age of four months to determine whether the mandibular bone mass of mice at the stable growth period was affected by the knockout of p75NTR.RNA and proteins of mandible bone were obtained by liquid nitrogen extraction.PCR and western blot were used to detect whether there were differences in the expression of mineralization-related genes Runx2 and Col1,and to further determine whether there were differences in the mandibular bone differentiation ability of the two genotypes of mice.Take blood method respectively to extract one months and four months old male mice blood samples,then using chemical test serological detection to detect the blood calcium and blood phosphorus,parathyroid hormone and calcitonin to determine whether the differences in bone mass and osteogenetic differentiation ability were related to phosphorus and calcium homeostasis.2?Extraction and identification of E12.5d WT and p75NTR-/-EMSCsp75NTR-/+female mice and p75NTR-/+male mice in the reproductive period were fed in cages and kept in the animal house conventionally.The noon of the day when the vaginal plug of mice were detected was recorded as pregnancy 0.5d.The mice were anesthetized with2%pentobarbital sodium?50mg/kg?and sacrificed by cervical dissection 12.5 days after conception.Abdominal surgery was performed to get the embryos under aseptic conditions.The embryos were washed with sterile PBS and the jaw process tissues of each embryo were cut separately.Chopped organization as far as possible to make it is about 0.1 mm*0.1 mm*0.1 mm of tissue block and 1%collagenase digestion for 20 min.Digestion was stopped by DMEM-F12 that containing 10%fetal bovine serum,1%of penicillin streptomycin.The suspension was then centrifuged at 800 rpm for 5 minutes.The cell pellet was resuspended in DMEM-F12 that containing 10%fetal bovine serum,1%of penicillin streptomycin.The suspension containing cells was then cultured at 37°C in a 5%CO2-humidifed incubator.Subsequently,the tail and limb tissues of the corresponding fetal mice were cut for DNA extraction and genotype identification.Then,two kinds of E12.5d EMSCs were detected and identified from cell morphology,cell cloning,cell proliferation,cell surface markers and cell apoptosis by flow cytometry.3?Comparison of osteogenic differentiation between E12.5d WT and p75NTR-/-EMSCsE12.5d WT and p75NTR-/-EMSCs were cultured in DMEM/F12 containing 10%FBS and 1%antibiotics.Third passage WT and p75NTR-/-EMSCs were used for the experiments.The osteogenic differentiation medium??-MEM supplemented with 10%FBS,1%antibiotics,10 mM/L?-glycerol phosphate,100 n M dexamethasone,and 50 mg/ml ascorbic acid?was replaced every 3 days.On days 7 and 21,the cells were stained using an ALP staining kit or alizarin red separately.On days 7,14 and 21 cells were extracted RNA and proteins to detect the expression of related genes by RealTime-PCR and western blot.4?Detection of EMSC osteogenic differentiation regulated by the PI3K/Akt/?-catenin pathwayp75NTR-/-EMSCs and WT EMSCs were induced for 7 days and then total RNA were exerted for RNA-sequencing.GO analysis were applied to identify the functional categories of differentially expressed mRNAs.KEGG analysis were applied and to detect the different expressed genes which showed that PI3K/Akt signaling pathway was significantly down regulated in p75NTR-/-EMSCs group.To further evaluate whether the PI3K/Akt pathway is involved in regulating EMSC osteogenic differentiation,the wild-type EMSCs were treated with the PI3K inhibitor LY294002 and PI3K agonist 740Y-P during osteogenic induction.On days 7,the cells were stained using an ALP staining kit.Furthermore,induced cells were extracted RNA and proteins to detect the expression of related genes by RealTime-PCR and western blot.5?Detection of the relationship between p75NTR and PI3K/Akt/?-catenin pathway during the osteogenic differentiation of EMSCsp75NTR was overexpressed or knockdown in wild-type EMSCs by lentivirus transfection separately.Then the LY294002 was added to p75NTR overexpressed group while 740Y-P was added to p75NTR knockdown group to regulate the PI3K/Akt pathway,divided into 6 research groups:p75NTR-overexpressed?pLJM1-p75NTR?EMSCs and negative control EMSCs?p LJM?,p75NTR-knockdown?shp75NTR?EMSCs and negative control EMSCs?shNC?,p75NTR-overexpressed EMSCs that treated with the PI3K inhibitor LY294002?p LJM1-p75NTR+LY294002?and p75NTR-kncokdown EMSCs that treated with the PI3K agonist 740Y-P?shp75NTR+740Y-P?.On days 7,total RNA and proteins of six groups were extracted to detected the related genes and protein expression levels.After osteogenic cultured for 7 and 21 days,the ALP staining and alizarin red staining was exerted separately.6?Detection of the pro-osteogenic effects of NGF in EMSCs regulated by deletion of the extracellular domain of p75NTR(p75NTRECD)We treated WT and p75NTR-/-EMSCs with 100 ng/m L exogenous NGF to investigate whether the NGF could influence the potential of osteogenic differentiation in murine EMSCs,divided into 4 research groups:WT EMSCs group?WT EMSCs+NGF group?p75NTR-/-EMSCs group?p75NTR-/-EMSCs+NGF group.On days 7,total RNA and proteins of six groups were extracted to detected the related genes and protein expression levels.And the ALP staining was also exerted on days 7.Results:1??1?Micro-CT analysis showed that bone mass in the 1-month-old and 4-month-old male p75NTR-/-mice was strikingly reduced,with significantly decreased bone volume?BV?,BV/total volume?TV?,trabecular number?Tb.N?and trabecular thickness?Tb.Th?comparing to the bone mass in the wild-type control mice.Furthermore,the knockout mice showed increased trabecular separation?Tb.Sp?.?2?We further detected the expression of Runx2 and Col1 in 4-month-old mice by extracting total mandibular bone RNA and proteins,finding that both Runx2 and Col1 levels were lower in p75NTR-/-mice.?3?We examined serum calcium,phosphorus,PTH,and CT levels in the two kinds of mice and no significant differences in the levels of these parameters were detected.2??1?Both WT and p75NTR-/-EMSCs exhibited a fibroblast-like morphology.?2?No significant difference of the colony formation rate,cell proliferation capacity and cell apoptosis were found between WT and p75NTR-/-EMSCs.?3?The mesenchymal stem cell surface markers CD14,CD90,CD146 and CD166 were all expressed in both WT and p75NTR-/-EMSCs,while the haematopoietic marker CD45 was not expressed.Furthermore,the p75NTR expression rate in wild-type E12.5d EMSCs was 23.75%.3??1?The protein and mRNA levels of Runx2 and Col1 in p75NTR-/-EMSCs were much lower than those in WT EMSCs following 7,14 and 21 days of osteogenic induction.?2?On days 7,less ALP staining was observed in p75NTR-/-EMSCs than that in WT EMSCs.?3?Alizarin red staining showed that p75NTR-/-EMSCs exhibited fewer and lighter mineralized nodules following 21 days of osteogenic induction.4??1?RNA-seq analysis showed that the differentially expressed genes between two kinds of EMSCs were highly involved in the PI3K/Akt signalling pathway.?2?The inhibitor LY294002 markedly suppressed PI3K and Akt phosphorylation without influencing total PI3K and Akt expression,and impaired Runx2 and Col1 expression in EMSCs.Furthermore,the ALP staining intensity was decreased in the LY294002-treated group.In contrast,740Y-P significantly activated PI3K and Akt phosphorylation without influencing total PI3K and Akt expression in EMSCs during osteogenic induction.Higher protein and mRNA levels of Runx2 and Col1 were detected in 740Y-P-treated EMSCs.Meanwhile,the ALP staining intensity was also increased in the 740Y-P group compared to the control group.Moreover,when PI3K/Akt pathway was regulated with agonist or inhibitor in EMSCs during osteogenic differentiation,both?-catenin and active?-catenin showed the same up or down regulated trend.5??1?We successfully forced overexpression of p75NTR in p75NTR EMSCs group,and knocked down p75NTR in shp75NTR EMSCs group.?2?The protein expression levels of Runx2?Col1?p-PI3K?p-Akt??-catenin and active?-catenin were markedly up-regulated in p75NTR-overexpressed EMSCs during osteogenic differentiation.?3?The protein expression levels of Runx2?Col1?p-PI3K?p-Akt??-catenin and active?-catenin were markedly down-regulated in p75NTR knockdown EMSCs during osteogenic differentiation.?4?LY294002 attenuated the increased expression levels of Runx2 and Col1 caused by p75NTR overexpression.?5?740Y-P markedly increased expression levels of Runx2 and Col1 in p75NTR knock-down EMSCs during osteogenic differentiation.?6?The ALP and alizarin red staining were significantly increased/decreased consisting to the western blot and RT-PCR results.6??1?The mineralization related genes Runx2,Col1 and the PI3K/AKT/?-catenin related genes p-PI3K,p-Akt,?-catenin and active?-catenin expression was upregulated by NGF in WT EMSCs.The NGF treatment did not obviously enhance Runx2,Col1,p-PI3K,p-Akt,?-catenin and active?-catenin in the p75NTR-/-group.?2?Both ALP and alizarin red staining depth of the NGF-treated WT group was the darker than the untreated group,while the NGF treatment did not obviously enhance the staining depth of the p75NTR-/-group.Conclusion:1?p75NTR might participate in the regulation of mouse alveolar bone.2?Both WT and p75NTR-/-EMSCs were identified as mesenchymal stem cells.Additionally,no significant difference of the colony formation rate,cell proliferation capacity and cell apoptosis were found between WT and p75NTR-/-EMSCs.3?p75NTR could positively regulate osteogenic differentiation of EMSCs via enhancing PI3K/Akt/?-catenin pathway.4?p75NTRECD play an important role in NGF regulated osteogenic differentiation of EMSCs.In conclusion,our experiments demonstrated that the deletion of p75NTR reduced alveolar bone mass and may attenuated the alveolar bone development in mice.p75NTR positively regulated osteogenic differentiation of EMSCs via enhancing the PI3K/Akt/?-catenin pathway.Moreover,p75NTRECD seemed to play an important role in NGF promoted osteogenic differentiation in murine EMSCs.Our findings may enrich the understanding of maxillofacial development,and support the use of p75NTR and PI3K/Akt/?-catenin pathway as potential targets for management of alveolar bone engineering. |
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