Effect Of Cbfa1 On Differentiation Of Dental Follicle Cells Into Osteoblasts/Cementoblasts

Background and ObjectivesPeriodontitis is an inflammatory disease affecting supportive tissues of the teeth, leading to progressive destruction of connective tissue attachment and alveolar bone. One of the most important goals of periodontal treatment is to restore the architecture and function of the periodontium by regenerating the multiple tissues in the periodontium,i.e.the formation of new alveolar bone,cementum,and periodontal ligament.The process of tissue repair and regeneration is similar to that of tissue development,therefore,it is important for periodontal regeneration to explore the mechanism of periodontal tissues development.It is known that periodotium originate from dental follicle cells(DFCs).Beyond their critical role during tooth eruption, DFCs are also thought to contain progenitors of cementoblasts,periodontal ligement fibroblasts and osteoblasts,and have the ability to form cementum,periodontal ligament and alveolar bone during later stage of tooth development.Taken together,it is meaningful to identify the specific factors and/or mechanisms regulating development and differentiation of DFCs,that will provide important information as to which moleculars and cells are required for regeneration of periodontal tissues.The exact mechanisms responsible for initiating DFCs differentiation are not defined and there are many cellular events and factors involved in this complicated progress.Among these factors,transcription factor core binding factorα1(Cbfal) is paid more attention as the master gene of osteoblast differentiation and bone formation.Cbfal is also crucial for dental development.Cbfal deficient mice exhibit an arrest of molar tooth development at the early cap stage.Mutations of Cbfal gene cause cleidocranial dysplasia(CCD),an autosomal dominant disorder in human and mice characterized by defective bone formation.In addition,CCD also shows dental defects including multiple supernumerary teeth,delayed eruption of permanent dentition and so on.The dental abnormalities suggest that Cbfal plays an important role in formation of the dentition.Furthermore,Cbfal expresses in both dental papilla and dental sac,even throughout the dental development,including formation of the periodontium.Cementoblasts,cementocytes,periodontal fibroblast,osteoblasts,and osteocytes all express Cbfal.These data suggest a potential involvement of Cbfal in the differentiation of lining cells in the periodontal space and the development of periodontal tissues.Recently,a large number of in vitro studies imply that forced expression of Cbfal induces osteoblast-specific gene expression in nonosteoblastic cells,including typeⅠcollagen(ColⅠ),alkaline phosphatase(ALP),osteopontin(OPN),bone sialoprotein (BSP) and the skeletal-specific osteocalcin(OC) gene,and disruption of Cbfal by antisense oligonucleotides in osteoblast cultures inhibites expression of osteoblast differentiation markers and formation of mineralized nodules.Furthermore,Cbfal protein binding site is found in the promoter regions of all the major extracellular matrix proteins of mineralizing tissues.All these studies have indicated that Cbfal plays important role in osteobalst/cementoblast by regulating the related genes. However,the regulation of Cbfal on DFCs differentiation is still unclear.Bone morphogenic protein-2(BMP-2) is the most thoroughly researched member of the transforming growth factor-βsuperfamily for the promotion of periodontal regeneration.This molecule has demonstrated potent effects in stimulating osteogenesis and cementogenesis.However,the short half-lives,diffusional limitations,unintended targeting of neighboring non-osseous tissues,and the requirement for cell surface receptors and cofactors may limit the efficiency of BMP-2.At present time,BMP-2 needs to be further explored for its potential use in reconstructive periodontal therapy. So,the aim of this study was to transfect Cbfal into DFCs by retroviral system,to determine the effect of Cbfal on osteoblast/cementoblast related genes expression, and to compare the biological characteristics of Cbfal or BMP-2 overexpressing DFCs,so as to offer some information for periodontal regeneration.Materials and Methods:1.Expression of Cbfal in DFCsMandibles including the first molars were removed from 5～7-day-old BALB/c mice and 5μm sections were prepared.Immunohistochemical and in situ hybridization techniques were adopted to determine the tissue distribution and cellular localization of Cbfal protein and mRNA in dental follicle tissue of BALB/c mice.Then,developing mandibular first molar germs from 5～7-day-old BALB/c mouse were isolated under stereomicroscope.Primary DFCs were obtained by the methods of trypsin digestion and tissue culture,subsequently,DFCs were cultured and subcultured.Total RNA and protein were separately extracted from DFCs culturing in vitro.RT-PCR and Western blot were used to detect the expression of Cbfal mRNA and protein in DFCs.2.Effect of overexpression of Cbfal on differentiation of DFCs into osteoblasts/cementoblastsCbfal recombinant retroviral plasmid vector pLEGFP-IRES-Cbfal was constructed and transfected into the retroviral packaging cells PT67 by LipofectamineTM 2000 to produce Cbfal retrovirus.Stably virus-producing cell line was obtained by G418 selection.The medium from packaging cells comprising virus was added to infect DFCs and chosen by G418 to gain the stably overexpressing Cbfal cells.Real-time RT-PCR,Western blot and mineralization assay were adopted to determine the effect of overexpression of Cbfal on the osteoblast/cementoblast related genes expression and the mineral ability in DFCs.Meanwhile,the enhanced Cbfal—pLEGFP-IRES-Cbfal(E) which deleting of the VWRPY motif was constructed.DFCs overexpressing Cbfal(E) was also chosen by G418 and the expression of osteoblast/cementoblast related genes and the formation of mineral nodules were observed in the Cbfal(E) overexpressing cells. 3.Comparison of osteoblast/cementoblast related genes expression in DFCs overexpressing Cbfal or BMP-2Cbfal and BMP-2 recombinant retroviral plasmid vectors-pLEGFP-IRES-Cbfal and pLEGFP-IRES-BMP-2 were seperately transfected into the packaging cells PT67 to produce retrovirus containing Cbfal or BMP-2.The medium from packaging cells comprising virus was added to infect DFCs and chosen by G418 to gain the cells stably overexpressing Cbfal or BMP-2.Real-time RT-PCR,Westem blot and mineralization assay were adopted to investigate and compare the effect of overexpression of Cbfal and BMP2 on the osteoblast/cementoblast related genes expression and the biomineralization.Results1.Expression of Cbfal in DFCsDeveloping mandibular first molar germs from 5～7-day-old BALB/c mouse were at later bell stage,the epithelial root sheath had already innerly folded,but the root still could not be seen.Dental follicle tissue was a loose fibrous connective tissue surrounding the developing dental germ.There was Cbfal mRNA but not Cbfal protein expressing in DFCs by histochemical observation.For the DFCs culturing in vitro,Cbfal mRNA was positive by RT-PCR,while Cbfal protein was not detected by Western blot.2.Overexpression of Cbfal enhanced differentiation of DFCs into osteoblasts/cementoblastsCbfal recombinant retroviral plasmid vector pLEGFP-IRES-Cbfal was identified correct by restriction endonuclease and the sequence was consistent with that in GeneBank.Cbfal retrovirus packaged by PT67 was added to infect of DFCs and antibiotic selection 2 weeks to obtain stably overexpressing Cbfal cells.The green fluorescence could be observed under converse microscope.Real-time RT-PCR analysis confirmed that the transfected cells were overexpressing(5-to 10-fold) Cbfal gene comparing with that in the control cells(P＜0.01).The presence of Cbfal protein in cell extracts was detected by Western blot with Cbfal antibody.Positive result was seen only in Cbfal retrovirus transduced cells.DFCs stably overexpressing Cbfal was successfully constructed by retroviral system.Real-time RT-PCR analysis demonstrated that all osteoblast/cementoblast related genes(ColⅠ,ALP,OPN,BSP,OC,CAP and CP23) were expressing in DFCs.In DFCs overexpressing Cbfal the expression of BSP,OC,OPN,ColⅠ,CAP and CP23 were higher than those in DFCs and DFCs infected by retrovirus empty vector (P＜0.01),while the expression of ALP was inhibited(P＜0.01).Cbfal(E) increased more expression ofOC,OPN,ColⅠand CP23 than Cbfal did(P＜0.01),while Cbfal increased more expression of CAP than Cbfal(E)(P＜0.01).For BSP and ALP,the effect of Cbfal and Cbfal(E) did not seem diffenent(P＞0.05).Mineralization assay showed that mineral nodule was observed in both DFCs and DFCs infected by virus,and the nodule was increasing with the time of culture.The mineral nodule formed in DFCs overexressing Cbfal was significantly more when compared with that in wild DFCs(P＜0.01).Furthermore,in the DFCs overexpressing Cbfal(E) the formation of nodules was the most(P＜0.05).3.Comparison of osteoblast/cementoblast related genes expression in DFCs overexpressing Cbfal or BMP-2BMP-2 recombinant retroviral plasmid vector pLEGFP-IRES-BMP-2 was identified correct by restriction endonuclease and the sequence was consistent with that in GeneBank.Cbfal and BMP-2 retrovirus produced by PT67 were separately added to infect DFCs and chosen by G418 to obtain stably overexpressing Cbfal or BMP-2 cells.The green fluorescence could be observed in infected cells under converse microscope.Real-time RT-PCR analysis confirmed that the transfected cells were overexpressing aim gene.Western blot also showed that the aim gene protein was expressing in the Cbfal,BMP-2 retrovirus transduced cells.In DFCs overexpressing Cbfal the expression of OPN,ColⅠ,BSP and CAP were more than those in DFCs overexpressing BMP-2(P＜0.01),while BMP-2 enhenced more expression of CP23 than Cbfal did(P＜0.01).Both in DFCs overexpressing Cbfal or BMP-2,the expression of ALP was decreased(P＜0.01),but the expression of OC was not affectd(P＞0.05).In mineralization assay,the mineral nodule formed in DFCs overexressing Cbfal was significantly more than that in DFCs overexpressing BMP-2(P＜0.01),while overexpression of BMP-2 seemed to have no effect on mineralization(P＞0.05).Conclusions:1.DFCs stably overexpressing Cbfal,BMP-2 are successfully obtained by retrovirus infection.2.Cbfal may induce the differentiation of DFCs into ostoblast/cementoblast phenotype by increasing the expression of osteoblast/cementoblast-related genes.3.By retroviral gene transfer approach,overexpression of Cbfal enhences more expression of osteoblast/cementoblast-related genes than overexpression of BMP-2 in DFCs,which suggests that Cbfal may be more important than BMP-2 in periodontal development and regeneration.