A Pilot Research Of Wnt Signal Pathway Regulated Osteogenic Differentiation Of Jawbone Marrow Mesenchymal Stem Cells From Ectodermal Dysplasia Patient

[Background]The ectodermal dysplasia(ED)is not a single disorder,but a series of related syndromes which was caused by 77 different identified genetic mutations and can be passed to next generation.The appearance of ED in oral cavity are mainly about teeth malformations,hypodontia or anodontia,decreased secretion of gland and atrophy of jaw bone.The treatment of hypodontia or anodontia is removable denture or implant-fixed denture.However,this therapy protocpol is obstructed by the atrophy jaw bone which reduce the retention of removable denture and increase the difficulty of implant.Thus,it was crucial to explore the mechanism of ED and develop some effective therapy procedure.Moreover,if the problem of atrophy jaw bone can be resolved,the implant procedure can be an acceptable way to reconstruct the occlusion of patients.As above,we hope to regenerate the bone mass of alveolar bone by the application of tissue engineering method with mesenchymal stem cells.Mesenchymal stem cell(MSC)is a group of multipotent cell from mesoderm and ectoderm in the early embryo,with potency to differentiate into osteocytes,adipocytes,hepatocytes,and so on.MSCs take an important role in the regeneration of defect organs andtissues,including the bone tissues.Due to a part of MSCs came from the ectoderm in the early embryo and regulate the bone formation,we chose MSCs to investigate the mechanism of ED and the bone mass atrophy of ED patients.Identified gene mutations of ED were mainly the TP63 gene and EDA/EDAR/EDARADD gene.Some signal pathways were closely related with ED.Canonical Wnt/β-Catenin pathway regulated the cells proliferation and differentiation during the early embryo state,suggesting a strong relationship with MSCs.In addition,Wnt/β-Catenin pathway can interact with TP63 and EDA/EDAR/EDARADD,regulating the progress of ED.In here we researched the Wnt/β-Catenin pathway in cell proliferation and differentiation of ED patients’MSCs,hoping for exploration the mechanism of ED and find some therapy method.Still,we look forward to augmenting the atrophy jaw bone of ED patients to get a better prognosis of implant surgery.[Objective]To explore the mechanism of Wnt/β-Catenin pathway induced MSCs ontogenesis differentiation in ED patients,looking forward to augmenting the atrophy jaw bone of ED patients,[Method]First,we isolated MSCs from the jaw bone marrow of ED patients and normal patients,and cultured in vitro.To determine the specific surface marker of MSC,we use flow cytometry.The CCK-8 cell proliferation assay was performed to compare the growth speed of MSCs between the ED group and normal group while the colony-formation assay to determine the cell survival rate of these two group.After we identified the cell characteristics,we were going to compare the osteogenesis ability under the Wnt/β-Catenin pathway regulation between two groups.We use CHIR-99021,an activator of Wnt/β-Catenin pathway while XAV-939,an inhibitor,to treat the two groups of MSCs under an osteogenesis environment about 2-4 weeks.2 weeks after,we detected the RUNX2 protein expression through the western blot assay and the RUNX2 transcription by PCR.We used alizarin red s to stained the mineralization after 4 weeks,and measured the amount and size of the calcification nodes which represent the ability of osteogenesis.[Results]Both two groups of MSCs were successfully isolated and passaged,with favorable stability.The flow cytometry results showed that the CD73 and CD90 markers were high expressed in both groups,while the CD34 and CD45 were low expressed,indicating that both two groups of cells held the specific marker of MSCs,and they were not hemopoietic stem cells.CCK-8 proliferation assay suggested that the growth speed of normal groups was obviously higher than ED groups,which may be a reason that the bone mass of ED patients was insufficient.According to the colony-formation assay,both two groups of cells can form cell colonies.Under the Wnt/β-Catenin pathway regulation,for normal group,the numbers and size of calcification nodes were increased with CHIR-99021 processed,especially with the high concentration of CHIR-99021,while the XAV-939 decreased the osteogenesis of MSCs in normal group.In contrast,the CHIR-99021 inhibited the osteogenesis in ED group and XAV-939 could enhanced this effect.The inhibition and enhancement seems to be concentration dependent.[Conclusion]The MSCs between Ectodermal Dysplasia group and control group showed no obvious differences in the ability to form colonies,the expression of specific marker and osteogenesis.However,the cell proliferation of Ectodermal Dysplasia group appeared more slowly than the control group,which may be a candidate factor in jawbone atrophy.The activator CHIR-99021 and Inhibitor XAV-939 toward Wnt signal pathway showed a different effect in regulating the osteogenesis process.Under the induced of XAV-939,the osteogenesis ability of JBMMSCs from Ectodermal dysplasia patient was increased.