Preliminary Research Of Specialat-rich Sequence-binding Protein 2 In Acting Synergistically On BMP9-induced Osteo/odontoblastic Differentiation Of Mouse Incisor Mesenchymal Stem Cells

Background:Bone and tooth defect or loss seriously affect facial features and masticatory function and is a complicated problem in oral clinical treatment.With the development of tissue regeneration engineering recently years,it provide a probability in the treatment of bone and tooth defect or loss.Located in the mouse incisor apical mesenchyme,mouse incisor mesenchymal stem cells(MSCs)not only maintain self-renewal but also possess osteo/odontogenic differentiation potential under inducing by bio-factors so that could ensure incisor continuously growth.Mouse incisor MSCs provide excellent seed cells to the research of tissue regeneration engineering.Exploring key regulators of osteo/odontogenic differentiation in mouse incisor MSCs is benefic to solve the clinical problem of bone and tooth tissue defect or loss.Special AT-rich sequence-binding protein 2(SATB2)is a multifunctional regulator involved in transcription.SATB2 deletion or mutation would lead to craniomaxillofacial dysplasia and delay tooth crown forming and root development abnormalities.As one of the most potent factor in BMPs to induce differentiation of stem cells,bone morphogenetic protein 9(BMP9),also called growth differentiation factor 2,has been reported to play a pivotal role in tooth development and odontogenic stem cell(like mouse incisor MSCs)differentiation.It was reported that the symptoms of SATB2 deletion or mutation were similar to bone morphogenetic protein(BMP)loss-of-function phenotypes.In the researches of osteogenic differentiation in stem cells,both SATB2 and BMP9 could promote the osteotogenic differentiation capability in BMSCs.However,researches about the role of SATB2 in oste/odontogenic differentiation of odontogenic stem cells and whether SATB2 acted synergistically with the potent osteogenic factor BMP9 in inducing osteo/odontogenic differentiation are rare.Objective: The purposes of this study were to investigate the effects of SATB2 in osteo/odontogenic differentiation of mouse incisor MSCs and whether SATB2 could synergize with BMP9 in regulating osteo/odontogenic differentiation of mouse incisor MSCs.Methods: Immunofluorescence staining,RT-q PCR analysis and Western Blot were used to detect the expression of SATB2 and BMP9 in C57BL6 mouse(4 week-old)incisor apical tissue.Mouse incisor MSCs were isolated and identified.Adenovirus-mediated Satb2 overexpression and silence in mouse incisor MSCs were validated by RT-q PCR analysis and Western Blot.CCK-8 and crystal violet assay were used to assess the proliferation capability of mouse incisor MSCs.RT-q PCR was used to detect the expression of MSCs markers(Cd90 and Cd29)and osteo/odontoblastic transcription factors m RNA in mouse incisor MSCs.Alkaline phosphatases(ALP)staining,activity assay,Alizarin red S mineralization staining and quantitative analysis were used to evaluate the alkaline phosphatases forming and calcium depositing in the mouse incisor MSCs.Then Gel MA hydrogel with different concentration was synthesized.The proliferation and viability of mouse incisor MSCs in Gel MA hydrogel were detected by CCK-8 assays and live/dead staining.The effect of SATB2 on BMP9-induced alkaline phosphatases forming and calcium deposition in Gel MA hydrogel were analyzed by ALP staining and ARS staining.Critically sized defects were made in rat calvaria and implanted Gel MA hydrogel with mouse incisor MSCs which infected with adenovirus.To evaluate new bone formation,Micro-CT images were acquired at 10 weeks and were then analyzed to measure volume(bone volume fraction,BV/TV)and quantity(number of trabeculae,Tb.N).H&E and Masson Trichrome staining were also carried out to detect trabecular bone mature and mineralized and bone matrices formation.Results: 1.Immunofluorescence staining indicated that SATB2 was found to be expressed in 4 week-old mouse incisor mesenchyme area and co-expressed with Bmp9 in pre-odontoblasts/odontoblasts.RT-q PCR analysis and Western blot revealed that Satb2 and Bmp9 m RNA and protein were expressing in the incisor apical tissue.2.Mouse incisor MSCs were isolated and cultured.Immunofluorescence staining indicated that mouse incisor MSCs were successfully obtained and these cells could be used in our next research.RT-q PCR and Western blot analyses showed that SATB2 was effectively overexpressed and silenced in mouse incisor MSCs.3.Mouse incisor MSCs which infected with Ad Satb2 exhibited more excellent proliferation,increased the expression of mesenchymal stem cell markers(Cd90 and Cd29),osteoblast transcription factors(Runx2,Opn and Ocn)and odontoblastic differentiation markers(Dspp and Dmp1)and enhanced ALP staining,activity and calcium deposition.However,silencing SATB2 suppressed all.4.Mouse incisor MSCs were induced by Ad Bmp9 after infecting with Ad Satb2 or Adsi Satb2.ALP and ARS staining and quantitative analysis showed that SATB2 enhanced BMP9-induced mouse incisor MSCs forming alkaline phosphatases and depositing calcium while si SATB2 suppressed BMP9-induced capability.RT-q PCR indicated that Runx2 and Opn were upregulated in the process which Satb2 acted synergistically role in Bmp9-induced osteo/odontogenic differentiation.5.In 20%(w/v)Gel MA hydrogel,mouse incisor MSCs not only exhibited high proliferation and viability but also showed differentiation capability.These results indicated that 20%(w/v)Gel MA hydrogel could be an excellent scaffolding material and be used for tissue regeneration engineering.The defects in rat calvarial bone were filled with Gel MA hydrogel which embedded with mouse incisor MSCs that infected with Ad Satb2,Adsi Satb2,Ad Bmp9,Ad Satb2+Ad Bmp9 or Adsi Satb2+Ad Bmp9.Quantitative analysis of the Micro-CT images indicated that the volume(bone volume fraction,BV/TV)and quantity(number of trabeculae,Tb.N)of new bony tissue were increased after SATB2 synergized BMP9-induced mouse incisor MSCs.H&E staining and Masson trichrome staining results revealed that trabecular bone formation,maturity and mineralization were significantly increased by combined transfection with Ad Satb2 and Ad Bmp9 in mouse incisor MSCs.si SATB2 significantly suppressed trabecular bone formation,maturity and mineralization so that depressed BMP9-induced mouse incisor MSCs healing calvarial bone in rats.Conclusion: SATB2 promotes proliferation,stemness and osteo/odontogenic differentiation of mouse incisor MSCs.SATB2 acts synergistically role with the potent osteogenic factor BMP9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs.Therefore,SATB2 can cooperate with BMP9 as a new efficacious bio-factor for osteogenic regeneration and tooth engineering.