these interactions stimulate a subpopulation of mesenchymal cells to differentiate into odontoblasts , which , in turn , form the primary dentin . but , after dentinal damage these processes may be reactivated , which is subsequently associated with the formation of tertiary dentin including reactive and reparative dentin . in response to minor injuries , the primary odontoblast survive and reactionary tertiary dentin is formed ; meanwhile , reparative dentinogenesis occurs after more intense injuries that lead to odontoblast death . these all demonstrate that postnatal dental pulp contains cells with stem cell properties . they are highly proliferative , clonogenic , and capable of regenerating a tissue ; the properties that clearly define them as stem cells . stem cells have several unique properties include the ability of self - renewal , proliferation , differentiation and they can give rise to specialized cell types . stem cells are categorized as embryonic and adult . in comparison to other types of adult stem cells used in tissue engineering , mesenchymal stem cells are more promising for therapeutic purposes . the most common source of mesenchymal stem cells is bone marrow ; however , it has limitations such as the small number of cells obtained by biopsy and painful process . currently , several types of adult stem cells have been isolated from teeth , namely dental pulp stem cells ( dpscs ) , stem cells from human exfoliated deciduous teeth ( sheds ) , periodontal ligament stem cells ( pdlscs ) , stem cells from apical papilla ( scaps ) , and dental follicle progenitor stem cells ( dfpcs ) . sheds are a population of highly proliferative cells which can differentiate into osteogenic and odontogenic cells , adipocytes , and neural cells . they have several distinct advantages over dental pulp stem cells among which are increased cell population doublings , higher proliferation rate , ease of access , lack of tissue destruction at donor site , reduction or elimination of pain and discomfort , and the possibility of obtaining cells from young patients . these cells can be regarded as the possible sources of stem cells for future tissue engineering applications . another field of research following the isolation of stem cells from the desired source is to find the appropriate materials to add to the cell culture medium so that the most efficient material for the proliferation and differentiation of stem cells is introduced . vitamin d3 metabolites such as 1 , 25-dihydroxyvitamin d3 are the key factors in regulation of bone metabolism . studies have shown that 1 , 25-dihydroxyvitamin d3 increases the alp activity and osteogenic differentiation of normal permanent dental pulp as well as dental follicle stem cells . considering the differences between these cells and deciduous stem cells , the current study was designed to investigate the effect of 1,25-dihydroxyvitamin d3 on osteogenic differentiation ( alkaline phosphatase activity and alizarin red staining ) of stem cells of exfoliated deciduous teeth . fifteen sound exfoliated human deciduous teeth were collected from 10 healthy children aged 6 to 11 years old ( 4 girls and 6 boys ) . prior to extraction , the patients received oral health education , professional 
 prophylaxis was done for each patient , and they used 0.2% chlorhexidine mouthwash for one minute . it was immersed in a digestive solution containing 100 u / ml penicillin , 100 mg / ml streptomycin , and 500 mg / ml claritromycin in 4 ml 0.1 m phosphate - buffered saline ( pbs ) 
 with the addition of 3 mg / ml type i collagenase and 4 mg / ml dispase , for 1 hour at 37c . the solution was filtered through 70m falcon strainers ( falcon ; fisher , usa ) . after filtration , the cells were immersed in -mem ( minimum essential medium ) ( gibco ; germany ) , added with 10% fetal bovine serum ( fbs ) ( gibco ; germany ) , 100m 2p - ascorbic acid , 
 2 mm l - glutamine , 100 u / ml penicillin , and 100 g / ml streptomycin . the flasks were incubated at 37c in 
 a 5% co2 , and the medium was changed twice a week . after the third passage , the cells were isolated from the culture plate by using 25% trypsin - edta and were cultured for 24 hours . when the cell density reached 80% , they were used for osteogenic differentiation . 
  as the control group , the cells were cultured in osteogenic cell culture medium containing -mem ( gibco ; germany ) , 10% fbs , 50 g / ml l - ascorbic acid 2-phosphate , 50 mm -glycerophosphate , and 0.1 m dexamethasone ( sigma ) . as the case group , the cells were cultured in osteogenic culture medium supplemented with 100 nm1 and 25(oh)2d3 . after 21 days of culture , alkaline phosphatase ( alp ) activity was analyzed with a commercially available p - nitrophenylphosphate tablet set ( pars azmoon ; iran ) . likewise , the mineralization was analyzed by alizarin red staining ( ecm 815 ; millipore , usa ) . for alizarin red staining , the cell cultures were rinsed twice with fbs and fixed with 10% formaldehyde for 15 minutes at room temperature . then , the cultures were stained with alizarin red solution for 20 minutes at room temperature . the total mineralized tissues were counted according to the manufacturer 's instructions of alizarin red . quantitative analysis of alizarin red staining was performed by determining the od405 ( optical density ) values of a set of known alizarin red concentrations and comparing these values with the obtained values ( figure 1 ) . for the analysis of normal distribution of data , non - parametric kolmogorov - smirnov test was used . after the third passage , the cells were isolated from the culture plate by using 25% trypsin - edta and were cultured for 24 hours . when the cell density reached 80% , they were used for osteogenic differentiation . 
  as the control group , the cells were cultured in osteogenic cell culture medium containing -mem ( gibco ; germany ) , 10% fbs , 50 g / ml l - ascorbic acid 2-phosphate , 50 mm -glycerophosphate , and 0.1 m dexamethasone ( sigma ) . as the case group , after 21 days of culture , alkaline phosphatase ( alp ) activity was analyzed with a commercially available p - nitrophenylphosphate tablet set ( pars azmoon ; iran ) . likewise , the mineralization was analyzed by alizarin red staining ( ecm 815 ; millipore , usa ) . for alizarin red staining , the cell cultures were rinsed twice with fbs and fixed with 10% formaldehyde for 15 minutes at room temperature . then , the cultures were stained with alizarin red solution for 20 minutes at room temperature . the total mineralized tissues were counted according to the manufacturer 's instructions of alizarin red . quantitative analysis of alizarin red staining was performed by determining the od405 ( optical density ) values of a set of known alizarin red concentrations and comparing these values with the obtained values ( figure 1 ) . for the analysis of normal distribution of data , non - parametric kolmogorov - smirnov test was used . 
  concentration of alizarin red according to the standard optical density . the medium was observed by using an inverted light microscope on the days 1 , 3 , 7 , and in the third passage . the morphology of the cells remained constant during cell passage ( figure 2 ) . dental pulp stem cells of deciduous teeth at day 3 ( 100 magnification ) . the results of kolmogorov - smirnov test indicated the normal distribution of data . 
 compared with the control group , significant increase was observed in alp activity of sheds after being treated with 1,25(oh)2d3 ( p= 0.002 ) ( table 1 ) .   the t - test results of alkaline phosphatase activity quantification of alizarin red staining demonstrated that cells exposed to 1,25(oh)2d3 induced higher mineralized nodules ( p < 0.001 ) ( table 2 , figure 3 ) . the t - test results of alizarin red staining alizarin red staining of mineralized deposits without ( a , b , c ) and with vitamin d metabolite ( d , e , f ) . osteogenic differentiation before staining ( a and d ) ( 40 magnification ) ; after staining at 40x ( b and e ) and after staining at 400x magnification ( c and f ) . the medium was observed by using an inverted light microscope on the days 1 , 3 , 7 , and in the third passage . the morphology of the cells remained constant during cell passage ( figure 2 ) . dental pulp stem cells of deciduous teeth at day 3 ( 100 magnification ) . the results of kolmogorov - smirnov test indicated the normal distribution of data . 
 compared with the control group , significant increase was observed in alp activity of sheds after being treated with 1,25(oh)2d3 ( p= 0.002 ) ( table 1 ) . quantification of alizarin red staining demonstrated that cells exposed to 1,25(oh)2d3 induced higher mineralized nodules ( p < 0.001 ) ( table 2 , figure 3 ) . the t - test results of alizarin red staining alizarin red staining of mineralized deposits without ( a , b , c ) and with vitamin d metabolite ( d , e , f ) . osteogenic differentiation before staining ( a and d ) ( 40 magnification ) ; after staining at 40x ( b and e ) and after staining at 400x magnification ( c and f ) . this study aimed to isolate mesenchymal stem cells from exfoliated deciduous teeth in order to evaluate the effects of 1,25(oh)2d3 on their osteogenic differentiation . several studies have tried to find the appropriate method for regeneration of damaged bone which is an issue of paramount importance in dentistry . regarding the systemic 
 advantages of 1,25 ( oh ) 2d3 in bone remodeling and wound healing , in addition to its considerable role in tooth formation 
 emphasized by in vivo and clinical studies , the present study was designed to investigate the effect of 1,25(oh)2d3 on osteogenic differentiation of shed . deciduous teeth , therefore , 
 may be an ideal resource of stem cells to induce bone regeneration . mesenchymal 
 stem cells have been extensively characterized in vitro by the expression of markers such as stro-1 , cd146 or cd44 . the international society for cellular therapy declares that one of the required criteria to define human mesenchymal stem cells is expression of cd105 , cd73 , and cd90 ; in addition to lack of expression of cd45 , cd34 , cd14 , cd11b , cd79a , cd19 , and hla - dr surface molecules . among the limitations of this study was the impossibility of performing flow cytometric analysis of cell surface due to the limited technical and financial resources . if these cells were end cells , they would not differentiate into other cell lines , so they were certainly considered stem cells . based on previous studies , differentiation of mesenchymal stem cells such as dpscs and sheds into osteoblast - like cells is 
 induced by dexamethasone , ascorbic acid , and -glycerophosphate . these osteogenic 
 supplements are the most commonly used osteogenic inducer for human mesenchymal stem cells in vitro . 1,25(oh)2d3 was found to 
 be more effective than dexamethasone in osteogenic differentiation of adipose - derived stem cells . therefore , 
 despite the different categories of stem cells , the effect of 1,25(oh)2d3 was the same on osteogenic differentiation potential . the in vitro studies on osteoblastic cells revealed that 1,25(oh)2d3 increased the alkaline phosphatase activity . it was reported that adding 1,25(oh)2d3 ( 10 and 100 nm ) increased the alp activity of dental pulp stem cells and dental follicle stem cells . likewise , the current study showed that 1,25(oh)2d3 ( 100 nm ) enhanced the alp activity in sheds . so , the response of stem cells in primary and permanent teeth are the same in this regard . 
  on the other side , bakopoulou et al . detected that the alp activity was significantly reduced in presence of hema ( 0.5 mm and 0.1 mm ) and tegdma ( 0.25 mm , 0.1 mm ) in osteogenic culture differentiation , which was unlike the effect of vitamin d3.khanna-jain et al . reported that dental pulp stem cells and dental follicle stem cells formed mineralized matrix when treated with vitamin d3 metabolites in osteogenic differentiation culture . the alizarin red staining in our study demonstrated that the cells exposed to 1,25(oh)2d3 induced higher mineralized nodules . application of 1,25 ( oh ) 2d3 in tissue engineering is a suggestion which needs more investigations .