| Background and objectivePeriodontitis,rheumatoid arthritis,tumors and trauma can lead to bone destruction and defects,and chronic inflammation has been the most common type of disease leading to bone destruction.The stimulation of chronic periodontitis affects the osteo-differentiation ability of periodontal ligament stem cells(PDLSCs)and lead to abnormal bone tissue repair and regeneration,probably because the microenvironment of stem cells is altered due to the stimulation of inflammation,which leads to the change of the biological properties of stem cells.How to effectively promote bone tissue repair and regeneration has been one of the hot spots in medical research.With the development of gene engineering,stem cell gene therapy has provided a new therapeutic direction for repairing bone tissue destruction and defects caused by various reasons,and has become a hot research spot for regeneration and repair of bone tissue defects.PDLSCs have become one of the seed cells for bone defect repair and regeneration because of easy availability and strong self-renewal and multi-differentiation potential.Clinically common,in the chronic inflammatory microenvironment of periodontitis,alveolar bone is destroyed and the osteo-differentiation capacity of PDLSCs is significantly inhibited.So the limited intrinsic regenerative capacity after bone destruction remains an important medical issue.In recent years,various regulatory effects of carbon monoxide releasing molecules(CORMs)have been reported.CORMs is a class of carbonyl compounds that has many functions,such as improving vascular function,protecting the heart,anti-inflammation,anti-apoptosis and so on.It carries and controllably releases carbon monoxide(CO).CORM-3(carbon monoxide releasing molecular-3)is the third generation product of CORMs,which can release CO after being dissolved in water.Compared with other generations,CORM-3 has lower toxicity and more optimized use conditions.How does CORMs affect osteogenesis on hPDLSCs?So far there have been no public reports.In the previous phase,our research group found that CORM-3 could improve the expression of heme oxygenase-1(HO-1)of hPDLSCs.Some scholars also found the high expression of HO-1 caused by CORMs in rat brain astrocytes and other cells.With the related research on the physiological efficacy of CORMs,recent research on the action mechanism of CORMs has gradually involved microRNAs(miRNAs).The discovery of miRNAs and relevant research have brought the differentiation and regulation of stem cells into the post-transcriptional era.MicroRNAs(miRNA)are non-coding small ribonucleic acids with a length of about 19-24 nucleotides that participate in important processes in numerous life activities negatively regulate their target genes.researches reveals that miRNAs make a big difference on the regulation of the proliferation and differentiation of stem cells.miRNAs have been much more involved in a series of important processes in life by binding to target mRNA and directly and specifically knock down protein expression,precisely regulate the signal networks of the cells with a direct control of the network proteins.MiRNA-20b promotes the osteogenic differentiation of BMSCs by targeting the inhibition of PPARy and other activities that activate the BMPs/Runx2 signaling pathway.the Dicer enzyme of the mice was knocked out in vivo,the miRNA effect disappeared,resulting in dysfunction of limb development.Therefore,in this study,we first use hPDLSCs as an experimental model to study the influence of CORM-3 on the osteogenesis of hPDLSCs,and the skull impairment of nude mice is an in vivo research model to investigate the influence of systemic application of CORM-3 on the osteogenesis of hPDLSCs in skull defect area.Also high-throughput miRNAs sequencing of hPDLSCs stimulated by CORM-3 in osteogenesis induction was made.The results showed that under osteogenic induction,CORM-3 stimulation could cause differential expression of miRNAs of hPDLSCs,leading to a up or down of some miRNAs.we further examine the mechanism of CORM-3 on osteogenesis of hPDLSCs at miRNA level.This reseach offer a theory base for improving differentiation ability of seed cells during tissue-regeneration and help optimize miRNA-mediated therapy for stem cellassociated tissue regeneration,offering new direction for the treatmentt of bone destruction or bone trauma.Materials and Methods1.Isolation,culture and identification of hPDLSCsYoung permanent teeth aged 11-18 years were freshly extracted for orthodontic treatment,the PDLSCs were scraped off and incubated through tissue block technique under sterile conditions,and verified as the origin of cells.The stem cell characteristics of hPDLSCs were verified by adipogenic/osteogenic induction.Flow cytometry is used to identify osteogenic related factors.2.Influence of CORM-3 on the vitality of hPDLSCshPDLSCs were incubated in a-MEM including 10%FBS and CORM-3(0,100,200,400 and 800 ?M),24 h later,CCK-8 medium(10 ?L)is added into the indicated well.After 1 day,the vitality of the cell in indicated well was assayed three times through CCK-8 method.3.Effects of CORM-3 on osteogenesis of hPDLSCs400 ?M CORM-3 was selected for following study.HPDLSCs were cultured in four groups:Osteogenic group,hPDLSCs are cultivated with osteogenesis medium at 37°;CORM-3+Osteogenic group,firstly,cells are treated with CORM-3 medium at room temperature for 1 day,followed by complete replacement with osteogenesis medium.Degassed CORM-3+Osteogenic group,firstly,cells are treated with degassed CORM-3 for 24 h,followed by complete replacement with osteogenesis medium;cells in control group,cells were cultivated in a-MEM including 10%FBS.The medium of every group was refreshed every 3 days.The mRNA/protein expression of Runx2/ALP/OPN(encoding gene SPP1,NM000582.3),and ALP/mineralization activity,were examined at day 3,7,and 14.4.The influence of CORM-3 on the repair of cranial trauma of nude mouseThere is a complete randomized group division of nude mice,CORM3+hPDLSC group:defects are laid with collagen membranes seeded with hPDLSCs in the presence of CORM-3 intraperitoneal injection.Normal saline+hPDLSC group:defects are laid with collagen membranes seeded with hPDLSCs in the presence of normal saline intraperitoneal injection;CORM-3 group:defects are laid with collagen membranes with a CORM-3 solution injection intraperitoneally;normal saline group(control group):defects is laid with collagen membranes with a intraperitoneal injection of normal saline.A full-thickness cranial bone defect model with an outer diameter of 4 mm was created in each of the bilateral regions of the skull of nude mice,and the cranial material is draw out for HE?MASSON dye at 1 month after operation and Microscopic CT scan at 7 weeks after operation to measure new bone mass of every group.Image J,Evaluation V6.5-3,GraphPad Prism.6,software(CT Analyser;Bruker)are used to measure and analyze new bone.5.The screening?verification of differently expressed microRNAs of hPDLSCs in osteogenesis with the application of CORM-3Cells from the "CORM-3+osteogenesis induction 24 h group" and "osteogenesis induction 24 h group" are extracted and sent to NovoGen sequencing company for miRNA high-throughput sequencing.The differently expressed miRNAs are screened out and analyzed by database and for qRT-PCR validation.6.The effect of hsa-miRNA-195-5p on the osteogenesis of hPDLSCs with the application of CORM-3By over-expression and inhibition of hsa-miR-195-5p by cell transfection,alizarin red staining,qRT-PCR and Western blot(WB)were used to detect the difference of osteogenesis of transfected cells.The hPDLSCs are set to 7 groups:osteogenic group:hPDLSCs are incubated in osteogenesis solution;CORM-3+osteogenic group:hPDLSCs are incubated in CORM-3 osteogenesis medium;mimics group:hsa-miR-195-5p mimics is transfected into hPDLSCs for 12 h and replaced with osteogenic induction medium containing 400 ?M CORM-3;mimics NC group:hPDLSCs are transfected with hsa-miR-195-5p mimics NC for 12 h and changed with osteogenic medium containing CORM-3;control group:hPDLSCs are cultivated in ?-MEM including CORM-3;inhibitor group:hsa-miR-195-5p inhibitor is transfected into hPDLSCs for 12h and change with CORM-3 osteogenic medium;inhibitor NC group:inhibitor NC was transfected into hPDLSCs for 12 h and change with osteogenic induction medium containing CORM-3.qRT-PCR?WB were used to test Runx2,OPN mRNA and protein in each group at day 3 and day 7;Alizarin red dye is used to detect the mineralization of each group at 14 days.semi-quantitative analysis was performed.7.Target gene prediction and validation of hsa-miRNA-195-5pAccording to target gene prediction databases(Targetscan 5.0 and miRanda),and in combination with bioinformatics analysis to predict the target gene of hsa-miR195-5p,BMPR1A,a target gene related to osteogenesis,was selected for subsequent study.Secondly,overexpress or inhibite hsa-miR-195-5p by a transfection method,detect the expression level of intracellular BMPR1A protein,and analyze gray value by Image J software;Also,BMPR1A is verified by double luciferase report experiment.8.The effect of BMPR1A on hPDLSCs in the osteogenesis with the application of CORM-3BMPR1A mRNA?protein expression during osteogenesisof hPDLSCs with the application of CORM-3 are tested by qRT-PCR and WB method and statistically analyzed.Intracellular BMPR1A was overexpressed or inhibited by transient transfection into two parts:BMPR1A overexpression and silencing.The hPDLSCs are set to 6 groups,CORM-3+osteogenic group,cells are incubated in osteogenenesis solution containing CORM-3;BMPR1A overexpression group:cells are transfected with BMPR1A overexpression vector for lday and refreshed with osteogenesis medium including 400?M CORM-3;BMPR1A NC group:cells were transfected with BMPR1A vector(empty vector)for 1 day and refreshed with osteogenic induction medium including CORM-3;control group:cells are cultivated in ?-MEM mediun(10%FBS).siRNA group:BMPR1A siRNA transfected cells are cultivated for 1 day and replaced with osteogenesis medium containing CORM-3;siRNA NC group:siRNA NC transfected cells were incubated for 1 day and replaced with osteogenesis medium containing CORM-3;qRT-PCR and WB method were used to detect ALP and Runx2 mRNA and protein in each group at day 3 and day 7 respectively.Calcification of each group is observed by alizarin red dye at day 14 and analysis is performed.9.Rescue assay of overexpressed target gene BMPR1AHsa-miR-195-5p mimics are cotransfected with BMPR1A overexpression vector/empty vector by cell transfection.The calcification of each group is examined by alizarin red dye at 2 weeks,and analyzed statistically.Results1.Isolation,culture and identification of hPDLSCshPDLSCs are identified by cell morphology and characteristic antigens on cell surface.Primary cultured hPDLSCs exhibite a specific morphology of radial pattern.After 21 days induction,osteogenic?lipogenic differentiation are confirmed by the mineralized nodules mass and fat granule.Monoclonal cell masses show the clusters of cells grow from a cell.The analysis from Flow cytometric reveal that hPDLSCs is low or negatively expressed CD45(0.16%)and CD34(1.39%),while highly positively expressing CD44(99.9%),CD90(99.9%)and CD105(99.7%)(FigurelD)consistent with the phenotypic profile of hPDLSCs.2.The influence of CORM-3 on the vitality of hPDLSCThe CCK-8 test shows that 200 and 400 ?M CORM-3 improve the proliferation of hPDLSCs,however,400 ?M CORM-3 showed a more significant promotion than 200?M CORM-3(P<0.05).In comparison with the control group,800 ?M CORM-3 treatment causes a great decrease of cell vitality(P<0.05).According to the above,400 ?M is selected as the experimental concentration of CORM-3 for the subsequent study.3.Effects of CORM-3 on osteogenesis of hPDLSCsIn the CORM-3+osteogenic group,mRNAs and proteins levels of OPN(SSP1),Runx2,and ALP were higher than in the other groups at day 3,7 and 14(P<0.001).Cells in degassed CORM-3+osteogenic group dose not show great difference in mRNA/protein expression of ALP?Runx2 and OPN(SSP1))with comparition to the osteogenic group.On day 3?7 and 14,ALP vitality was higher in CORM3+osteogenic group than that of the other groups significantly(P<0.001).In addition,at each time point,there was no significant difference in ALP activity between the degassed CORM-3+osteogenic and osteogenic groups.AR staining and semiquantitative analysis showed that on day 14 and 21,the degree of cell mineralization was obviously higher in CORM-3+osteogenic group than the rest of the groups(P<0.001).Calcium deposition of the osteogenic group is much more than that in the control group(P<0.05).Mineralization of the degassed CORM-3+osteogenic group is not significantly different from that of osteogenic group.4.The influence of CORM-3 on the repair of cranial defects in nude mouseHE staining and Masson staining showed that the CORM-3+hPDLSC group found a larger area of new bone in the defect area,and the ratio of new bone is greatly higher than the rest of the other groups(P<0.05).The percentage of new bone in CORM-3 and normal saline+hPDLSC group was greatly higher than that of control group(P<0.05).Only some cells in control group scatter in the skull wound.Micro-CT analysis showed that the new bone in the CORM-3+hPDLSC group had larger areas of high density in the skull defect area,the bone volume fraction(BV/TV)in CORM-3+hPDLSC group is clearly larger than that in the other groups.The Tb.N and Tb.Th in the CORM-3+hPDLSC group were the highest of the four groups(P<0.05),whereas Tb.Sp is clearly lower than the rest groups(P<0.05).Tb.N?Tb.Th and Tb.Sp is no statistically different between the CORM-3 group and the normal saline+hPDLSC group.5.The screening and verification of differentially expressed miRNAs of hPDLSCs in osteogenesis with the application of CORM-3(1)Cluster analysis of miRNAs from the results appears that 8 miRNAs are elevated and 2 miRNAs are decreased,and the changes of 10 miRNAs are statistically significant.(2)Up-regulated miRNAs:hsa-miR-132-3p,hsa-miR--212-3p;down-regulated miRNAs:hsa-miR-195-5p,hsa-miR-199-5p(3)hsa-miRNA-132-3p,hsa-miRNA-212-3,hsa-miRNA-195-5p,hsa-miRNA199b-5p are choosed to verified the results by qRT-PCR.(4)hPDLSCs are incubated in CORM-3 osteogenesis medium and osteogenesis medium alone for lday.The hsa-miRNA-212-3p expression is elevated and the expression of hsa-miRNA-195-5p and hsa-miRNA-199b-5p is decreased,and the results are statistically different(P<0.05).There is no statistical difference for miRNA-132-3p,comparing to the control group,which is inconsistent with the sequencing results.The hsa-miR-195-5p,whose expression was down-regulated in osteo-differentiation with CORM-3 application,was selected for further study6.The effects of hsa-miRNA-195-5p on the osteogenesis of hPDLSCs with CORM-3 applicationAfter enhancing the expression of hsa-miR-195-5p,the results of alizarin red dye?WB and qRT-PCR for ALP and Runx2 show the osteogenesis of hPDLSCs was greatly inhibited;conversely,the inhibition of hsa-miRNA-195-5p enhance the osteogenesis.7.Target gene validation of hsa-miR-195-5p(1)Bioinformatics analysis combined with luciferase reporter gene assay identify BMPR1A is the target gene of hsa-miRNA-195-5p.(2)BMPR1A protein expression after overexpression of hsa-miR-195-5p by cell transfection technique for 48 h showed a significant decrease,in contrast,inhibition of hsa-miR-195-5p for 48 h resulted in a significant increase of BMPR1A expression by western blot detection.(P<0.05).8.The effects of BMPR1A in osteogenesis of hPDLSCs with CORM-3 applicationCORM-3 contribute to a big rise in the mRNA and protein expression of BMPR1A during osteogenic differentiation.After elevating BMPR1A by cell transfection method,the mRNA/protein levels of Runx2 and ALP at day 3?7 and 14 days calcification are obviously enhanced(P<0.05);while the opposite result appears after decreasing BMPR1A.9.Rescue assay of overexpressed target gene BMPR1Ahsa-miR-195-5p mimics are cotransfected with BMPR1A overexpression vector,and the mRNA and protein expression levels of Runx2 and ALP rebound after 3 and 7 days,and are close to the CORM-3+osteogenic group;the mineralization of the cotransfection with BMPR1A overexpression vector was also close to that in the CORM-3+osteogenic group after 14 days,the semi quantitative analysis shows that there is no statistical difference(P>0.05),and the calcification capacity is significantly enhanced when hsa-miR-195-5p mimics are cotransfected with BMPR1A overexpression vector compared to with BMPR1A empty vector.Conclusions1.CORM-3(400 ?M)enhances osteogenesis of hPDLSCs via CO release.2.CORM-3 induce hPDLSCs to promote the repair of cranial defects in nude mice.3.The first correlated differently expressed microRNA expression profile of hPDLSCs in osteogenesis with the application of CORM-3 was established.4.Hsa-miRNA-195-5p targets BMPR1A to promote osteogenesis of hPDLSCs with CORM-3 application.In this process,hsa-miR-195-5p expression is down-regulated and negatively regulated the process,BMPR1A protein expression is elevated and BMPR1A positively regulated the process.Cotransfection of BMPR1A overexpression vector with hsa-miR-195-5p mimics was able to rescue the diminished osteogenesis brought about by hsa-miR-195-5p mimics.Innovation and SignificanceIn this research,hPDLSCs are used for an in vitro study model.we report that CORM-3 promote osteogenic differentiation of hPDLSCs via CO release from CORM-3 through the study of the related indicators of osteogenesis.The cranial coloboma of nude mice is used for an in vivo study model,we report that CORM-3 induced hPDLSCs to promote the repair of cranial defects in nude mice through the study of the newly bone formation,which offer new theory basis for the treatment of bone defects and lay a research base for the next study and repair of bone wound.Based on the in vivo and in vitro studies combined with the results of miRNA gene sequencing of hPDLSCs with CORM-3 application in osteogenesis,the differentially expressed genes are identified.According to bioinformatics prediction and experiments,the target genes were identified and verified.we further develop the study of related mechanisms and report that CORM-3 promotes the osteogenic differentiation of hPDLSCs via miR195 5p/BMPRIA.This study offers new ideas for the repair of osteoclasia,and provides a theoretical basis and new molecular targets for the treatment of osteoclasia at the molecular biology level. |
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