| BackgroundPulpitis and periodontitis are two of the most common diseases in dental clinics.Pulpitis usually leads to inreversible necrosis of the dental pulp tissue,it could progress into periapical diseases and even cause loss of the teeth.Periodontitis usually leads to chronic progressive destruction of the periodontal tissue,which can further result in teeth loss thus impair the functionality of the oral system.Regeneration of the dental pulp and periodontal tissue shed new light on the treatment of pulpitis and periodontitis.The crux of oral tissue regeneration lies in regeneration of the impaired local tissue thus regain the functionality by using the combination of seed cells,scaffold and growth factors,either all or part of them.Dental pulp stem cells(DPSCs)and periodontal ligament stem cells(PDLSCs)are two of the most popular seed cells for tissue regeneration.The odontogenic differentiation of DPSCs and the osteogenic differentiation of PDLSCs are crucial for dental pulp and periodontal ligament repairmen and regeneration.Since the discoveries of DPSCs and PDLSCs,great strides have been achieved in the area of the regulatory mechanism of differentiation of these two types of cells.However,lots of problems remain to be better constrained and tons of questions to be clarified.We first reported the key regulatory effect of integrin α5(ITGA5)on the odontogenic differentiation of DPSCs.By utilizing an ITGA5-RNAi lentivirus vector,the proliferation and migration capacities of DPSCs were significantly impaired while the mineralization capacity of DPSCs was significantly enhanced.Certain genes relevant to DPSCs odontogenesis were up-regulated,i.e.,Dentin sialophosphoprotein(DSPP),Dentine matrix acidic phosphoprotein 1(DMP1),Alkaline phosphatase(ALP),Secreted protein acidic and cysteine rich(SPARC/Osteonectin,ON),Osteocalcin(OCN),and Bone sialoprotein(BSP).These results suggested that ITGA5 negatively regulates the odontogenic differentiation process of DPSCs though the exact mechanism was still unclear.Other research has shown that ITGA5 positively regulates the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMMSCs).Priming ITGA5 promoted the osteogenic differentiation of BMMSCs in vitro as well as the osteogenesis of BMMSCs in vivo.And yet the involved mechanism has not been fully clarified.To the best of our knowledge,the effect of ITGA5 on the osteogenic differentiation of PDLSCs has not been reported.PDLSCs,DPSCs and BMMSCs share similarities and differences whether from origins or differentiation characters.Thus the effect of ITGA5 on the osteogenic differentiation of PDLSCs seems interesting to explore.Originally,PDLSCs and DPSCs are closer to each other due to the fact that they are both dental mesenchymal stem cells.So it would be fascinating to elucidate and compare the mechanisms of both of the ITGA5-mediated DPSCs odontogenic differentiation and PDLSCs osteogenic differentiation.In this research project,we aimed to find out the involved mechanisms of ITGA5-mediated DPSCs odontogenic differentiation and PDLSCs osteogenic differentiation by the Isobaric tags for relative and absolute quantitation(iTRAQ)proteomic technique and subsequent bioinformatic analysis.Chapter Ⅰ The isolation,culture and identification of human DPSCs and PDLSCsObjectives:To isolate,culture and identify DPSCs and PDLSCs in vitro.Methods:To culture human dental pulp cells and periodontal ligament cells by enzymic digestion of human pulp and periodontal ligament tissue explants.Then the isolated primary cells were screened and purified by a limiting dilution method.The isolated DPSCs and PDLSCs were identified by clone forming assay,cell surface markers detection,and multilineage differentiation assay.Results:Most of the tissue and suspended cells could adhere to the cell culture plate.After 5～10 days,some cells crept from the edge of the tissue explants.These cells exhibited a spindly fibroblast-like shape and they surrounded the tissue explants by a radical or vortex pattern with a great translucency under reserve microscope.The DPSCs and PDLSCs isolated by a limiting dilution method were short-spindle-like in shape.Flat plate monoclonal formation assay showed that the DPSCs and PDLSCs clone rates were both between 30%and 40%.Cell surface markers were detected by flow cytometry and the results showed that both DPSCs and PDLSCs expressed mesenchymal stem cell markers CD29,CD90,CD73,CD44 and CD105,but did not express hematopoietic cell markers CD34,CD45,CD31 and HLA-DR.The osteogenic induction of DPSCs and PDLSCs showed red and brown calcified nodules in different sizes.The adipogenic induction of DPSCs and PDLSCs showed orange fat lipids in the cytoplasm.The chondrogenic induction of DPSCs and PDLSCs showed blue-stained glycosaminoglycan outside the cells.Conclusions:The isolated cells in the present chapter were of mesenchymal origin because they expressed mesenchymal stem cell markers but not hematopoietic cell markers.Along with the tissue sources,flat plate monoclonal formation assay and multilineage differentiation assay,the cells isolated in the present chapter were DPSCs and PDLSCs.Chapter Ⅱ The construction of the ITGA5-target lentivirus vectors and the synthesis and detection of ITGA5 priming cyclic peptideObjective:To find effective ways that could interfere with the expression level of ITGA5 in DPSCs and PDLSCs.Methods:To construct ITGA5-inhibited/overexpressed lentivirus vectors and to transfect these vectors with PDLSCs.The validation of the expression levels of ITGA5 in PDLSCs was performed by quantitative real time-polymerase chain reaction(qRT-PCR)and Western blot analysis.To construct DPSCs ITGA5-geneknockout cell line by a clustered regularly interspaced short palindromic repeats/Cas9 endonuclease(CRISPR/Cas9)technique-based double vector system.Total cell DNA was extracted.The ITGA5 knockout efficiency in DPSCs was tested by the surveyor assay.The ITGA5 priming cyclic peptide GA-CRRETAWAC-GA was synthesized.The effects of the synthetic peptide at different concentrations on the ITGA5 expression levels in DPSCs and PDLSCs were tested by qRT-PCR and Western blot analysis.Results:The ITGA5-inhibited lentivirus vector and the ITGA5-overexpressed lentivirus vector could significantly suppress and increase the ITGA5 expression level in PDLSCs,respectively.Three sgRNAs were constructed by a CRISPR/Cas9 technique-based double vector system.After they were transfected with DPSCs,results of the surveyor assay showed that all of them were off-target.The effects of the synthetic peptide at different concentrations on the ITGA5 expression levels in DPSCs and PDLSCs were different.The ITGA5 expression levels in DPSCs decreased first and then increased as the concentration of the peptide went higher.When the concentration was 100 nM,the ITGA5 expression inhibited effect of this peptide reached its maximum.While the ITGA5 expression levels in PDLSCs increased as the concentration of the peptide went higher.When the concentration was 100 nM,the ITGA5 expression promoted effect of this peptide almost reached its maximum.Conclusions:The ITGA5-inhibited lentivirus vector and the ITGA5-overexpressed lentivirus vector could effectively suppress and increase the ITGA5 expression level in PDLSCs,respectively.The experiment of constructing DPSCs ITGA5-geneknockout cell line by a CRISPR/Cas9 technique-based double vector system failed.The most efficient concentration of the ITGA5 priming cyclic peptide on DPSCs and PDLSCs were both 100 nM.Chapter Ⅲ The effects of ITGA5 on the proliferation and migration capacities of human DPSCs and PDLSCsObjectives:To explore the effects of ITGA5 on the proliferation and migration capacities of human DPSCs and PDLSCs.Methods:To test the effect of ITGA5 on the proliferation capacity of DPSCs,DPSCs at the 3rd passage logarithmic phase were under Cell Counting Kit 8(CCK8)proliferation assay and cell cycle analysis by grouping with ITGA5 priming peptide and control.And the transwell migration assay was to test the effect of ITGA5 on the migration capacity of DPSCs.For PDLSCs,experiments were all grouped by ITGA5 priming peptide/control,ITGA5-inhibited lentivirus/control,ITGA5-promoted lentivirus/control.The effects of ITGA5 on the proliferation capacity and the migration capacity of PDLSCs were tested by CCK8 proliferation assay and cell cycle analysis,and the transwell migration assay,respectively.Results:For DPSCs,after treated with the ITGA5 priming cyclic peptide GA-CRRETAWAC-GA,CCK8 proliferation assay and cell cycle analysis showed that the number of interphase cell significantly decreased in the peptide group than in the control group.Transwell migration assay showed that the number of transmembraned cell significantly decreased in the peptide group than in the control group.For PDLSCs,CCK8 proliferation assay and cell cycle analysis showed that the number of interphase cell significantly decreased in the ITGA5-inhibited lentivirus group than in the control group,while significantly increased in the ITGA5-promoted lentivirus group and the peptide group than in the control group.Transwell migration showed that the number of transmembraned cell significantly decreased in the ITGA5-inhibited lentivirus group than in the control group,while significantly increased in the ITGA5-promoted lentivirus group and the peptide group than in the control group.Conclusions:The ITGA5 priming cyclic peptide GA-CRRETAWAC-GA could significantly inhibit the proliferation capacity as well as the migration capacity of DPSCs.The ITGA5-inhibited lentivirus vector could significantly inhibit the proliferation capacity as well as the migration capacity of PDLSCs.Both the ITGA5-promoted lentivirus vector and the ITGA5 priming cyclic peptide could enhance the proliferation capacity and the migration capacity of PDLSCs.Chapter Ⅳ The effect and involved mechanism of ITGA5 on the odontogenic differentiation of DPSCsObjective:To explore the effect and involved mechanism of ITGA5 on the odontogenic differentiation of DPSCs.Methods:DPSCs were grouped by control group(CONT),osteogenic group(Osteogenic medium,OM),osteogenic+ITGA5 priming peptide(OM+SCP),ITGA5 priming peptide(Synthetic cyclic peptide,SCP),osteogenic+ITGA5 priming peptide+U0126 group(OM+SCP+U0126),osteogenic+ITGA5 priming peptide+LY294002 group(OM+SCP+LY294002).The mineralization capacity of each group was tested by alkaline phosphatase(ALP)activity staining and Alizarin red staining.The expression levels of certain proteins,i.e.,ITGA5,DSPP,phosphorylated focal adhension kinase(pFAK),phosphorylated protein kinase B(pAKT),phosphorylated Extracellular signal-regulated kinases(pERK)and ERK,in each group were tested by Western blot.We utilized iTRAQ technique for sequencing and identifying the differentially expressed proteins between ITGA5-inhibited lentivirus transfected group and control lentivirus transfected group of DPSCs.Subsequent bioinformatic analysis was performed to screen the potential involved pathways and mechanisms.And the results of proteomics were validated by Western blot analysis.Additionally,the effect of the ITGA5 on the odontogenic differentiation of DPSCs in vivo was tested by using a DPSCs plus hydroxyapatite/β-Tricalcium phosphate(HA/β-TCP)granules nude mice subcutaneous transplantation model.DPSCs were grouped by ITGA5-inhibited lentivirus transfected group and control lentivirus transfected group.After 8 weeks,the implants were fixed and demineralized,hematoxylin&eosin(H&E)staining,Wright-Giemsa staining and immunohistochemistry staining were performed in order to observe and compare the odontogenic differentiation capacity of both groups.Results:When compared to CONT group,the ALP activity staining was stronger in the OM group,and much stronger in the OM+SCP group.The staining in the SCP group was similar to that in the CONT group.The staining in both the OM+SCP+U0126 group and the OM+SCP+LY294002 group were significantly lighter.The trends of the Alizarin red staining were consistent with that of the ALP activity staining.Western blot showed that when compared to CONT group,the expression level of ITGA5 was significantly decreased while the expression levels of DSPP,pFAK,pAKT,pERK were significantly increased and the expression level of ERK was unchanged in the OM+SCP group.Either U0126 or LY294002 was added,the expression level of ITGA5 was significantly increased while the expression levels of DSPP,pFAK,pAKT,pERK were significantly decreased and the expression level of ERK remained unchanged.From all the six total cell protein samples(3 from the experimental group and 3 from the control group),13,339 peptides were identified and there were 3,898 proteins that expressed in both the ITGA5-inhibited lentivirus transfected group and control lentivirus transfected group.A total of 67 significantly differentially expressed proteins were identified,55 of which were up-regulated in the ITGA5-inhibited group while 12 were down-regulated.Taken together with the bioinformatic analysis by GeneOntology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment,it suggested that extracellular matrix(ECM)and ECM-receptor activity pathway were involved in the process of ITGA5-mediated DPSCs odontogenic differentiation.Certain components of ECM,such as Secreted protein acidic and cysteine rich(SPARC),Lumican(LUM),Vitronectin(VTN),Prolargin(PRELP),Decorin(DCN),Collagen type Ⅵ alpha 1 chain(COL6A1),Collagen type Ⅵ alpha 2 chain(COL6A2),Collagen type ⅫⅤalpha 1 chain(COL14A1)and Collagen type Ⅴ alpha 1 chain(COL5A1)were up-regulated in the ITGA5-inhibited group than that in the control group.The validation using OM group and OM+SCP group by Western blot analysis showed that the trends of these above proteins were consistent with that in the proteomic analysis.The nude mice subcutaneous transplantation experiment showed that when compared with the control group,there were enhanced osteogenesis and increased fibrous structure tissue,as well as more obvious atypical pulp-dentin complex in the ITGA5-inhibited group.Immunohistochemistry showed abundant DSPP,COL6A2,SPARC and VTN expression in ITGA5-inhibited group than in control group.Conclusions:The ITGA5 priming cyclic peptide GA-CRRETAWAC-GA could promote the odontogenic differentiation of DPSCs under osteogenic induction.Inhibiting the expression level of ITGA5 of DPSCs not only lead to the phosphorylation of certain crucial molecules in the FAK,phosphatidylinositide 3-kinases/protein kinase B(PI3K/AKT),mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinases 1 and 2(MEK1/2/ERK1/2)pathways,but also lead to deposition of ECM and amplified ECM-receptor activity,thus promote the odontogenic differentiation of DPSCs under osteogenic induction.Chapter Ⅴ The effect and involved mechanism of ITGA5 on the osteogenic differentiation of PDLSCsObjective:To explore the effect and involved mechanism of ITGA5 on the osteogenic differentiation of PDLSCs.Methods:PDLSCs were grouped by control group(CONT),osteogenic group(OM),osteogenic+ITGA5-inhibited lentivirus transfected group(OM+ITGA5i-LV),osteogenic+ITGA5-promoted lentivirus transfected group(OM+ITGA5-LV),osteogenic+ITGA5 priming peptide(OM+SCP),ITGA5 priming peptide(SCP),osteogenic+ITGA5 priming peptide+U0126 group(OM+SCP+U0126),osteogenic+ITGA5 priming peptide+LY294002 group(OM+SCP+LY294002).The mineralization capacity of each group was tested by ALP activity staining and Alizarin red staining.The expression levels of certain proteins,i.e.,ITGA5,COL1A1,ON,Runt-related transcription factor 2(RUNX2),pFAK,pAKT,pERK and ERK,in each group were tested by Western blot.We utilized the iTRAQ technique for sequencing and identifying the differentially expressed proteins between ITGA5-promoted lentivirus transfected group and control lentivirus transfected group of PDLSCs.Subsequent bioinformatic analysis was performed to screen the potential involved pathways and mechanisms.And the results of proteomics were validated by Western blot analysis.Additionally,the effect of the ITGA5 on the osteogenic differentiation of PDLSCs in vivo was tested by using a PDLSCs plus HA/β-TCP granules nude mice subcutaneous transplantation model.PDLSCs were grouped by ITGA5-promoted lentivirus transfected group and control lentivirus transfected group.After 8 weeks,the implants were fixed and demineralized,H&E staining,Wright-Giemsa staining and immunohistochemistry staining were performed in order to observe and compare the osteogenic differentiation capacity of both groups.Results:When compared to CONT group,the ALP activity staining was stronger in the OM group,and much stronger in the OM+SCP group.When compared to OM group,the OM+ITGA5i-LV group showed lighter staining while the OM+ITGA5-LV group stained significantly stronger.The staining in the SCP group and the OM+ITGA5-LV group were similar to that in the CONT group.The staining in both the OM+SCP+U0126 group and the OM+SCP+LY294002 group were significantly lighter.The trends of the Alizarin red staining were consistent with that of the ALP activity staining.Western blot showed that when compared to CONT group,the expression levels of ITGA5,RUNX2,ON,pFAK,pAKT and pERK were significantly increased while the expression level of ERK was unchanged in the OM+SCP group.Either U0126 or LY294002 was added,the expression levels of ITGA5,RUNX2,ON,pFAK,pAKT and pERK were significantly decreased while the expression level of ERK remained unchanged.From all the six total cell protein samples(3 from the experimental group and 3 from the control group),22,451 peptides were identified and there were 3,868 proteins that expressed in both the ITGA5-promoted lentivirus transfected group and control lentivirus transfected group A total of 48 significantly differentially expressed proteins were identified,37 of which were up-regulated in the ITGA5-promoted group while 11 were down-regulated.Taken together with the bioinformatic analysis by GO enrichment and KEGG enrichment,it suggested that cytoskeleton and cell cycle changes were involved in the process of ITGA5-mediated PDLSCs osteogenic differentiation.Certain components of cytoskeleton,such as Keratin,type Ⅱ cytoskeletal 6B(KRT6B)and Keratin,type Ⅰ cytoskeletal 16(KRT16)were up-regulated while Peripherin(PRPH)and Desmin(DES)were down-regulated in the ITGA5-promoted group than that in the control group.The validation using OM group and OM+SCP group by Western blot analysis showed that the trends of these above proteins were consistent with that in the proteomic analysis.The nude mice subcutaneous transplantation experiment showed that when compared with the control group,there were enhanced osteogenesis and increased periodontal ligament-like fibrous structure tissue in the ITGA5-promoted group.Immunohistochemistry showed abundant COL1(Collagen type I)and ON expression in ITGA5-promoted group than in control group.Conclusions:Both the ITGA5-overexpressed lentivirus vector and the ITGA5 priming cyclic peptide GA-CRRETAWAC-GA could promote the osteogenic differentiation of PDLSCs under osteogenic induction.The ITGA5-inhibited lentivirus vector has the opposite effect.Priming the expression level of ITGA5 of PDLSCs not only lead to the phosphorylation of certain crucial molecules in the FAK,PI3K/AKT,MEK1/2/ERK1/2 pathways,but also lead to cytoskeleton and cell cycle changes,thus promote the osteogenic differentiation of PDLSCs under osteogenic induction. |
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