mesenchymal stem cells ( mscs ) have been receiving considerable attention in bone 
 regeneration , both for tissue engineering or cellular therapy .
these cells are capable 
 of self - renewal or differentiation into multi - lineage cells , including osteoblasts and 
 condroblasts , under the appropriate conditions .
mscs have been isolated from different 
 tissues , such as bone marrow , dental pulp , fat , and gingiva .
adipose - derived stem cells ( ascs ) 
 represent one interesting cellular option for clinical applications since these cells 
 are available in large amounts , are easily accessible and isolated , and they demonstrate 
 a fast attachment and proliferation in culture .  
bone morphogenetic proteins ( bmps ) belong to the transforming growth factor  
 ( tgf - b ) superfamily and were identified by their ability to induce bone formation at 
 ectopic sites .
in particular , bmp-2 , 
 bmp-4 , and bmp-7 have been associated with bone development .
bmp-2 has been used for treatment of bone defects in 
 orthopedic , spine , and maxillofacial surgeries .
interestingly , it was 
 observed during the fracture repair that an increase of bmp-4 and bmp-7 occurs in 
 response to the presence of bmp-2 , 
 and it was suggested that bmp-4 and bmp-7 might be able to substitute each other during 
 bone healing , as has been shown in other tissues where these bmps are 
 co - expressed .
therefore , the 
 co - expression of a panel of bmps exert strong synergy , meaning that combination of 
 certain bmps may be more efficacious than single bmp to stimulate osteogenic 
 differentiation , and different bmps may act at different nodal points of 
 osteogenesis . besides the exogenous bmps , it is important to consider 
 the possibility and the effects of biologically active endogenous bmps expressed by 
 human mscs .
( 2009 ) 
 observed that bone marrow - derived stem cells promoted osteogenesis by endogenous bmp-2 , 
 bmp-4 , and bmp-6 .  
classical signaling pathway for bmps begins with the binding of bmp to a dimeric complex 
 of transmembrane serine - threonine kinase receptors , the type i and type ii receptors . 

type i receptors include bmp receptors ( bmprs ) -ia , -ib , and activin receptor type 1 
 ( actr1 ) , while type ii receptors include bmpr - ii , actr2 , and -2b , which are 
 constitutively active kinases .
these bmprs subtypes have different bmp affinities and 
 form receptor complexes prior to or after bmp binding , which is thought to regulate bmp 
 signaling ( smad - dependent versus smad - independent ) .
heteromeric receptor complexes 
 comprising type i and ii receptors lead to ligand - induced phosphorylation of type i 
 receptors . following the activation of this receptor , receptor kinases phosphorylate the 
 transcription factors smad1 , 5 or 8 that subsequently form heteromeric complexes with 
 smad4 and activate the expression of target genes in the nucleus .
reports have shown that 
 mitogen - activated protein kinase ( mapk ) and phosphatidylinositol 3-kinase ( pi-3k ) activity are 
 critical switches for osteogenic differentiation , indicating that other pathways , other 
 than the smad pathway , regulate the mesenchymal stem cells differentiation after bmp 
 exposure .  
the most usual supplementation for in vitro osteogenic differentiation 
 includes ascorbate , -glycerophosphate , and dexamethasone .
differently , some reports have 
 demonstrated that in vitro and in vivo treatments of 
 ascs with bmp-2 had no consistent effect on osteogenic differentiation .
therefore , this study evaluated the effect of a recombinant bmp-2 as 
 well as the endogenous bmp-4 and bmp-7 in the osteogenic differentiation of 
 adipose - derived stem cells ( ascs ) in medium supplemented with ascorbate and 
 -glycerophosphate .
antibiotics / antifungic agentes were purchased from cultilab ( so paulo , sp , 
 brazil ) .
ascorbate , -glycerophosphate , bmp-2 ( recombinant human bmp-2 ) , 
 eosin , paraformaldehyde , -nitrophenol phosphate ( npp ) , and silver 
 nitrate were obtained from sigma - adrich ( st . louis , mo , usa ) . -modified 
 eagle 's minimal essential medium ( mem modified ) and dulbecco 's modified 
 eagle 's medium ( dmem ) were from nutricell ( so paulo , sp , brazil ) . fetal 
 bovine serum , superscript iii , and trizol were 
 purchased from invitrogen ( carlsbad , ca , usa ) .
kit calcium assay was from bioassay 
 systems ( hayward , ca , usa ) .
antibodies anti -bmp4 ( mab1049 ) , -bmp7 ( mab4350 ) , 
 - actin ( 04 - 1116 ) , -smad1 ( 05 - 1459 ) , -phosphorylated smad1/5/8 ( ab3848 ) , 
 -smad4 ( 04 - 1033 ) , anti - rabbit , anti - goat and anti - mouse peroxidase - conjugated 
 antibodies , and immobilon - p membranes were obtained from millipore ( danvers , ma , 
 usa ) .
enhanced chemiluminescence pierce ecl was from thermo scientific ( rockford , il , 
 usa ) .
step one detection system and sybr green 
 pcr master mix were purchased from applied biosystems ( carlsbad , ca , usa ) .
568/10 for human osteoblasts ) and with the 
 understanding and written consent of the voluntaries , human lipoaspirate tissues from 
 three healthy patients ( average age 21 ) , with normal body mass indexes , non - smokers , 
 and not taking any medication , were processed to isolate the ascs , as described by 
 zuk , et al .
the ascs were 
 sub - cultured and used at passage 3 , according to the following groups : asc+om : 
 osteogenic medium ( om ) ( dmem supplemented with 250 m ascorbate and 10 mm 
 -glycerophosphate ) ; ascs+om+bmp2 : om with 50 ng / ml bmp-2 .
mc3t3-e1 
 pre - osteoblasts subclone 4 ( american type culture collection , manassas , va , usa , 
 2593 ) were used as the positive control for the determination of the 
 alkaline phosphatase activity , the evaluation of matrix mineralization , and for the 
 western blotting assays .
mc3t3-e1 cells were cultured according to the 
 recommendations in mem modified with 50 g / ml ascorbate and 10 mm 
 -glycerophosphate . 

human 
 osteoblast explant cultures were 
 obtained from three patients through biopsies of cortical - porous bone tissue from the 
 mandible , using dental implant drills .
culture media were supplemented with 10% ( v / v ) fetal bovine serum and 1% 
 ( v / v ) antibiotic/ antifungic .  
mc3t3-e1 and ascs ( 9.4x10 cells / well in 24-well plates ) were cultivated 
 for 7 , 14 , 21 , and 28 days .
trizol was used for total protein 
 extraction , and the concentrations were determined according to the lowry 's 
 method .
alp activity was 
 determined by releasing r - nitrophenol ( np ) from r - nitrophenol phosphate 
 ( npp ) . concisely , 0.5 ml of diethanolamine buffer ( 1.02 m , ph 9.8 , 0.6 mm 
 magnesium chloride ) and 0.5 ml of npp were incubated for 2 min at 
 37c .
afterward , 0.1 ml of the extracted protein was added and incubated at 
 37c . following 30 min , 1 ml of 3 n
absorbances were measured ( infinite m200 , tecan , salzburg , grdig , austria ) at 
 405 nm , and the alp activity was calculated from a np standard curve and all 
 values were normalized against the total protein concentration .  
mc3t3-e1 and ascs ( 2x10 cells / well on a 96-well plates ) were cultivated 
 for 21 days .
cells were fixed for 1 h with 3% ( v / v ) aqueous paraformaldehyde and 
 stained with 1% ( w / v ) silver nitrate under light exposure for 1h , and counterstained 
 with eosin .  
mc3t3-e1 and ascs ( 2x10 cells / well on 96-well plates ) were cultivated for 
 5 , 12 , 23 , and 32 days , for the calcium quantification at the extracellular matrix . 

cells were harvested in 200 l of 0.1 n hcl at 4c for 4 h and 
 centrifuged at 10,000 x g for 5 min .
mc3t3-e1 and ascs ( 9.4x10 cells / well on 24 well - plates ) were cultivated 
 for 7 , 14 , 21 , and 28 days .
for the western blotting experiments , 5 g of denatured protein 
 were loaded onto an sds / polyacrylamide gel .
proteins were transferred to immobilon - p 
 membranes , which were incubated overnight with anti -smad1 ( 1:500 ) , -phosphorylated 
 smad1/5/8 ( 1:500 ) , -smad4 ( 1:1000 ) , -bmp4 ( 1:500 ) , -bmp7 ( 2 g / ml ) , or 
 - actin ( 1:2000 ) antibodies .
after incubation with each corresponding 
 secondary antibody , the enhanced chemiluminescence pierce ecl substrate was used for 
 detection , according to the manufacturer 's protocol .  
human osteoblasts and ascs ( 9.4x10 cells / well ) were cultured for 1 , 7 , 
 14 , 21 , and 28 days on 24-well plates .
total rna was isolated with 
 trizol reagent , and cdna transcriptions were performed using 
 oligo ( dt ) primers and superscript iii .
step one 
 detection system and sybr green pcr master mix were used for the 
 qpcr .
the comparative threshold cycle ( ct ) method was used in order to quantify 
 changes in the smad1 , bmpr-2 , osteonectin , and osteocalcin gene expression between 
 the control ( human osteoblasts ) and treated ascs .
the fold difference between the 
 control and treatments was calculated according to 2-ct , where ct=(ct control ) - 
 ( ct treatment ) and ct=(ct target gene ) - ( ct internal control , ribosomal 18 s ) . 

primer 
 reverse 5'-3 ' gccctgttactgccattattgtt ; smad1 mrna expression : primer forward 
 gggactgcctcatgtcatttact .
the results of the alp activity and calcium quantification in all groups 
 were compared through non - parametric one - way analysis of variance ( anova ) followed by 
 the student newman - keuls ( snk ) post hoc test .
results of mrna 
 relative expression in all groups were compared through the student 's t 
 test .
statistical analyses were performed comparing all treatments for each 
 time period separately using the graphpad prism 4 software ( graph pad software inc , 
 san diego , ca , usa ) at a 95% confidence interval .
antibiotics / antifungic agentes were purchased from cultilab ( so paulo , sp , 
 brazil ) .
ascorbate , -glycerophosphate , bmp-2 ( recombinant human bmp-2 ) , 
 eosin , paraformaldehyde , -nitrophenol phosphate ( npp ) , and silver 
 nitrate were obtained from sigma - adrich ( st . louis , mo , usa ) . -modified 
 eagle 's minimal essential medium ( mem modified ) and dulbecco 's modified 
 eagle 's medium ( dmem ) were from nutricell ( so paulo , sp , brazil ) . fetal 
 bovine serum , superscript iii , and trizol were 
 purchased from invitrogen ( carlsbad , ca , usa ) .
kit calcium assay was from bioassay 
 systems ( hayward , ca , usa ) .
antibodies anti -bmp4 ( mab1049 ) , -bmp7 ( mab4350 ) , 
 - actin ( 04 - 1116 ) , -smad1 ( 05 - 1459 ) , -phosphorylated smad1/5/8 ( ab3848 ) , 
 -smad4 ( 04 - 1033 ) , anti - rabbit , anti - goat and anti - mouse peroxidase - conjugated 
 antibodies , and immobilon - p membranes were obtained from millipore ( danvers , ma , 
 usa ) .
enhanced chemiluminescence pierce ecl was from thermo scientific ( rockford , il , 
 usa ) .
step one detection system and sybr green 
 pcr master mix were purchased from applied biosystems ( carlsbad , ca , usa ) .
568/10 for human osteoblasts ) and with the 
 understanding and written consent of the voluntaries , human lipoaspirate tissues from 
 three healthy patients ( average age 21 ) , with normal body mass indexes , non - smokers , 
 and not taking any medication , were processed to isolate the ascs , as described by 
 zuk , et al .
the ascs were 
 sub - cultured and used at passage 3 , according to the following groups : asc+om : 
 osteogenic medium ( om ) ( dmem supplemented with 250 m ascorbate and 10 mm 
 -glycerophosphate ) ; ascs+om+bmp2 : om with 50 ng / ml bmp-2 .
mc3t3-e1 
 pre - osteoblasts subclone 4 ( american type culture collection , manassas , va , usa , 
 2593 ) were used as the positive control for the determination of the 
 alkaline phosphatase activity , the evaluation of matrix mineralization , and for the 
 western blotting assays .
mc3t3-e1 cells were cultured according to the 
 recommendations in mem modified with 50 g / ml ascorbate and 10 mm 
 -glycerophosphate . 

human 
 osteoblast explant cultures were 
 obtained from three patients through biopsies of cortical - porous bone tissue from the 
 mandible , using dental implant drills .
culture media were supplemented with 10% ( v / v ) fetal bovine serum and 1% 
 ( v / v ) antibiotic/ antifungic .
mc3t3-e1 and ascs ( 9.4x10 cells / well in 24-well plates ) were cultivated 
 for 7 , 14 , 21 , and 28 days .
trizol was used for total protein 
 extraction , and the concentrations were determined according to the lowry 's 
 method .
alp activity was 
 determined by releasing r - nitrophenol ( np ) from r - nitrophenol phosphate 
 ( npp ) .
concisely , 0.5 ml of diethanolamine buffer ( 1.02 m , ph 9.8 , 0.6 mm 
 magnesium chloride ) and 0.5 ml of npp were incubated for 2 min at 
 37c . afterward , 0.1 ml of the extracted protein was added and incubated at 
 37c .
following 30 min , 1 ml of 3 n naoh was used to stop the reaction . 

absorbances were measured ( infinite m200 , tecan , salzburg , grdig , austria ) at 
 405 nm , and the alp activity was calculated from a np standard curve and all 
 values were normalized against the total protein concentration .
mc3t3-e1 and ascs ( 2x10 cells / well on a 96-well plates ) were cultivated 
 for 21 days .
cells were fixed for 1 h with 3% ( v / v ) aqueous paraformaldehyde and 
 stained with 1% ( w / v ) silver nitrate under light exposure for 1h , and counterstained 
 with eosin .  
mc3t3-e1 and ascs ( 2x10 cells / well on 96-well plates ) were cultivated for 
 5 , 12 , 23 , and 32 days , for the calcium quantification at the extracellular matrix . 

cells were harvested in 200 l of 0.1 n hcl at 4c for 4 h and 
 centrifuged at 10,000 x g for 5 min .
mc3t3-e1 and ascs ( 9.4x10 cells / well on 24 well - plates ) were cultivated 
 for 7 , 14 , 21 , and 28 days . following cells lysis ,
the protein quantification was 
 performed . for the western blotting experiments , 5 g of denatured protein 

proteins were transferred to immobilon - p 
 membranes , which were incubated overnight with anti -smad1 ( 1:500 ) , -phosphorylated 
 smad1/5/8 ( 1:500 ) , -smad4 ( 1:1000 ) , -bmp4 ( 1:500 ) , -bmp7 ( 2 g / ml ) , or 
 - actin ( 1:2000 ) antibodies .
after incubation with each corresponding 
 secondary antibody , the enhanced chemiluminescence pierce ecl substrate was used for 
 detection , according to the manufacturer 's protocol .
human osteoblasts and ascs ( 9.4x10 cells / well ) were cultured for 1 , 7 , 
 14 , 21 , and 28 days on 24-well plates .
total rna was isolated with 
 trizol reagent , and cdna transcriptions were performed using 
 oligo ( dt ) primers and superscript iii .
step one 
 detection system and sybr green pcr master mix were used for the 
 qpcr .
the comparative threshold cycle ( ct ) method was used in order to quantify 
 changes in the smad1 , bmpr-2 , osteonectin , and osteocalcin gene expression between 
 the control ( human osteoblasts ) and treated ascs . the fold difference between the 
 control and treatments
was calculated according to 2-ct , where ct=(ct control ) - 
 ( ct treatment ) and ct=(ct target gene ) - ( ct internal control , ribosomal 18 s ) . 

primer 
 reverse 5'-3 ' gccctgttactgccattattgtt ; smad1 mrna expression : primer forward 
 gggactgcctcatgtcatttact .
primer reverse 
 aactcgtcacagtccggattg ; osteonectin mrna expression : primer forward 
 cgggtgaagaagatccatgag . primer reverse ctgccagtgtacagggaagatg .
the results of the alp activity and calcium quantification in all groups 
 were compared through non - parametric one - way analysis of variance ( anova ) followed by 
 the student newman - keuls ( snk ) post hoc test .
results of mrna 
 relative expression in all groups were compared through the student 's t 
 test .
statistical analyses were performed comparing all treatments for each 
 time period separately using the graphpad prism 4 software ( graph pad software inc , 
 san diego , ca , usa ) at a 95% confidence interval .
ascs+om and asc+om+bmp2 demonstrated similar alp activities , and mc3t3-e1 cells 
 ( positive control ) showed the highest activity ( r=0.0004 ) at day 7 .
ascs+om+bmp2 
 up - regulated the alp activity compared to acs+om and mc3t3-e1 at day 14 
 ( =0.00379 ) . at day 21 , ascs+om+bmp2 and mc3t3-e1 demonstrated the highest 
 activities ( r=0.0087 ) .
there was no statistically significant difference among the 
 groups at day 28 ( >0.05 ) ( figure 1 ) .  
different letters refer to statistical significant differences ( anova / snk , 
 <0.05 ) von kossa staining demonstrated that all groups presented phosphate at the 
 extracellular matrix at day 21 ( figure 2a - c ) .
adipose - derived 
 stem cells ( ascs ) with osteogenic medium ( om ) .
bar=500 m ; b ) ascs with om 
 supplemented with bmp-2 ; c ) mc3t3-e1 cells ( positive control ) ; d ) calcium 
 quantification at days 12 , 23 , and 32 .
different letters refer to statistical 
 significant differences ( anova / snk , <0.05 ) at day 5 , calcium deposition was not observed at the extracellular matrix . at day 12 , 
 mc3t3-e1 demonstrated the highest calcium amount ( r<0.0001 ) , ascs+om and 
 ascs+om+bmp2 showed similar results .
there was no statistically significant 
 difference among the groups ( r>0.05 ) at days 23 and 32 ( figure 2d ) .  
ascs+om+bmp2 expressed more smad1 when compared to mc3t3-e1 and ascs+om at day 7 . at 
 days 14 and 21 , all groups demonstrated similar smad1 expressions .
ascs+om 
 demonstrated the lowest expression at day 28 ( figure 
 3 ) .  
protein expression evaluated by 
 western blotting , at days 7 , 14 , 21 , and 28 only mc3t3-e1 expressed phosphorylated smad1/5/8 at day 7 .
all groups demonstrated 
 similar expression of these proteins at day 14 . at day 21 , ascs+om+bmp2 expressed 
 more than ascs+om , while mc3t3-e1 stopped these expressions .
all groups had no 
 expression of these phosphorylated proteins at day 28 ( figure 3 ) .  
ascs+om showed the highest smad4 expression at day 7 . at day 14 , mc3t3-e1 
 demonstrated the highest expression followed by ascs+om+bmp2 and then by ascs+om . 

mc3t3-e1 demonstrated the highest level of smad4 at day 21 , followed by ascs+om and 
 then by ascs+om+bmp2 . at day 28
, mc3t3-e1 showed the highest expression , while 
 ascs+om and ascs+om+bmp2 demonstrated similar levels of smad4 ( figure 3 ) .  
ascs+om 
 demonstrated the highest bmp-4 expression followed by ascs+om+bmp2 and then mc3t3-e1 
 at day 21 .
figure 3 did not show differences 
 between ascs+om and ascs+om+bmp2 groups at day 28 , while mc3t3-e1 demonstrated a weak 
 expression .  
bmp-7 expressions were smaller than the other groups at day 28 
 ( figure 3 ) .  
figure 4a shows that bmpr - ii relative mrna 
 expressions were similar for ascs+om and ascs+om+bmp2 ( >0.05 ) at days 1 , 
 7 , 14 , and 21 . at day 28
a ) bmpr - ii mrna expression ; b ) smad1 mrna expression ; 
 c ) osteocalcin mrna relative expression ; d ) osteonectin mrna expression . 

different letters refer to statistical significant differences ( t test , 
 <0.05 ) ascs+om+bmp2 showed the highest levels of smad1 relative mrna expression at days 1 
 ( <0.0001 ) , 7 ( =0.0002 ) , and 21 ( =0.0008 ) .
ascs+om 
 demonstrated the highest expressions at days 14 ( =0.0014 ) and 28 
 ( =0.0003 ) ( figure 4b ) .  
osteocalcin and osteonectin relative mrna expressions were similar for all groups for 
 all time periods ( >0.05 ) ( figure 4c 
 and 4d ) .
ascs+om and asc+om+bmp2 demonstrated similar alp activities , and mc3t3-e1 cells 
 ( positive control ) showed the highest activity ( r=0.0004 ) at day 7 .
ascs+om+bmp2 
 up - regulated the alp activity compared to acs+om and mc3t3-e1 at day 14 
 ( =0.00379 ) . at day 21 , ascs+om+bmp2 and mc3t3-e1 demonstrated the highest 
 activities ( r=0.0087 ) .
there was no statistically significant difference among the 
 groups at day 28 ( >0.05 ) ( figure 1 ) .  
von kossa staining demonstrated that all groups presented phosphate at the 
 extracellular matrix at day 21 ( figure 2a - c ) .
adipose - derived 
 stem cells ( ascs ) with osteogenic medium ( om ) .
bar=500 m ; b ) ascs with om 
 supplemented with bmp-2 ; c ) mc3t3-e1 cells ( positive control ) ; d ) calcium 
 quantification at days 12 , 23 , and 32 .
different letters refer to statistical 
 significant differences ( anova / snk , <0.05 ) at day 5 , calcium deposition was not observed at the extracellular matrix . at day 12 , 
 mc3t3-e1 demonstrated the highest calcium amount ( r<0.0001 ) , ascs+om and 
 ascs+om+bmp2 showed similar results .
there was no statistically significant 
 difference among the groups ( r>0.05 ) at days 23 and 32 ( figure 2d ) .
ascs+om+bmp2 expressed more smad1 when compared to mc3t3-e1 and ascs+om at day 7 . at 
 days 14 and 21 , all groups demonstrated similar smad1 expressions .
ascs+om 
 demonstrated the lowest expression at day 28 ( figure 
 3 ) .  
protein expression evaluated by 
 western blotting , at days 7 , 14 , 21 , and 28 only mc3t3-e1 expressed phosphorylated smad1/5/8 at day 7 .
all groups demonstrated 
 similar expression of these proteins at day 14 . at day 21 , ascs+om+bmp2 expressed 
 more than ascs+om , while mc3t3-e1 stopped these expressions .
all groups had no 
 expression of these phosphorylated proteins at day 28 ( figure 3 ) .  
ascs+om showed the highest smad4 expression at day 7 . at day 14 , mc3t3-e1 
 demonstrated the highest expression followed by ascs+om+bmp2 and then by ascs+om . 

mc3t3-e1 demonstrated the highest level of smad4 at day 21 , followed by ascs+om and 
 then by ascs+om+bmp2 . at day 28
, mc3t3-e1 showed the highest expression , while 
 ascs+om and ascs+om+bmp2 demonstrated similar levels of smad4 ( figure 3 ) .  
ascs+om 
 demonstrated the highest bmp-4 expression followed by ascs+om+bmp2 and then mc3t3-e1 
 at day 21 .
figure 3 did not show differences 
 between ascs+om and ascs+om+bmp2 groups at day 28 , while mc3t3-e1 demonstrated a weak 
 expression .  
ascs+om bmp-7 expressions were smaller than the other groups at day 28 
 ( figure 3 ) .
figure 4a shows that bmpr - ii relative mrna 
 expressions were similar for ascs+om and ascs+om+bmp2 ( >0.05 ) at days 1 , 
 7 , 14 , and 21 . at day 28
a ) bmpr - ii mrna expression ; b ) smad1 mrna expression ; 
 c ) osteocalcin mrna relative expression ; d ) osteonectin mrna expression . 

different letters refer to statistical significant differences ( t test , 
 <0.05 ) ascs+om+bmp2 showed the highest levels of smad1 relative mrna expression at days 1 
 ( <0.0001 ) , 7 ( =0.0002 ) , and 21 ( =0.0008 ) .
ascs+om 
 demonstrated the highest expressions at days 14 ( =0.0014 ) and 28 
 ( =0.0003 ) ( figure 4b ) .  
osteocalcin and osteonectin relative mrna expressions were similar for all groups for 
 all time periods ( >0.05 ) ( figure 4c 
 and 4d ) .
bmp-2 is considered a potent local factor that promotes bone formation and has been used as an osteogenic 
 supplementation .
this report evaluated the effects of bmp-2 associated with 
 ascorbate and -glycerophosphate to induce osteogenic differentiation of human 
 ascs during the entire in vitro osteogenic process ( day 1 to 32 ) , and 
 the effects of endogenous bmp-4 and bmp-7 in this process .
herein , efforts were made to 
 study osteogenic markers , such as alp activity , smads , bmpr - ii , osteonectin , 
 osteocalcin , and calcium deposition , as discussed below .  
an enzymatic hydrolysis activity of alp is necessary to initiate biological 
 mineralization by hydrolyzing -glycerophosphate , which serves as an additional 
 source of phosphate ions .
some 
 proteins are involved in the extracellular matrix deposition and mineralization during 
 bone formation , such as osteonectin and osteocalcin .
osteonectin is an intermediate 
 phase glycoprotein that binds calcium and type i collagen with a high affinity . 

osteocalcin is produced exclusively in bone , dentin , and cementum and it represents 
 approximately 20% of non - collagenous proteins .
finally , a functional assay of terminally differentiated 
 osteoblasts is the extracellular matrix mineralization , which can be evaluated by the 
 calcium quantification .  
in general , we observed that the supplementation with bmp-2 did not increase the levels 
 of osteogenic markers in the initial , intermediate , or final phases of human ascs 
 differentiation .
our results demonstrated that these cells expressed endogenous bmp-4 
 and bmp-7 , and we were encouraged to suggest that these endogenous bmps were 
 biologically active due to the observed induction of alp activity , upregulation of 
 osteogenic genes ( osteocalcin and osteonectin ) , and increase of calcium deposition in 
 cells treated without bmp-2 .
( 2009 ) observed that human bone marrow - derived 
 mesenchymal cells were able to express endogenous bmp-4 , but not bmp-7 .
herein , the peak of alp activity , which indicates the initial phase of extracellular 
 matrix mineralization , occurred at day 7 . at that stage , ascs treated in the presence or 
 absence of bmp-2 demonstrated similar results , suggesting that bmp-2 supplementation did 
 not improve the initial phase of osteogenic differentiation , in accordance with song , et 
 al .
( 2007 ) ( human bone 
 marrow - derived stem cells ) and differing from wang , et al .
it is important 
 to appreciate that explanations made with bmps in human cells can be extrapolated to the 
 human system with more precision .  
the presence of bmp-2 in medium supplementation did not up - regulate the bmpr - ii mrna 
 expression , similarly to that found by wang , et al . (
we also observed a similar expression of osteocalcin and 
 osteonectin mrna relative expression for ascs treated with or without bmp-2 .
( 2011 ) demonstrated that 
 induction of ascs with om containing bmp-2 for 7 days significantly augmented expression 
 of osteopontin and osteocalcin , and decreased alp and osteonectin gene expressions . 

however , when the exposure time to bmp-2 was increased to either 21 or 28 days ( the 
 terminal differentiation stages of ascs ) any changes to the gene expression , 
 attributable to bmp-2 was lost .
( 2006 ) observed similar osteonectin expression in mouse ascs 
 cultures using control medium and osteogenic medium with bmp-2 .
complementary , our 
 results demonstrated that ascs treated with or without exogenous bmp-2 showed similar 
 calcium deposition at the extracellular matrix , as found by song , et al .
conversely , beloti and 
 rosa ( 2005 ) demonstrated that 
 osteoblast differentiation of human bone marrow cells did not occur only in the presence 
 of ascorbate and -glycerophosphate .
( 2010 ) observed that the addition of 
 bmp-2 or bmp-6 to dexamethasone , ascorbic acid , and -glycerophosphate did not 
 enhance osteogenic differentiation of human periodontal ligament cells , since they 
 observed that neither of the bmps induced in vitro mineralization , 
 despite the increase of alp activity .  
bmp-2 supplementation led to the highest levels of smad1 relative mrna expression at 
 days 1 , 7 , and 21 , the highest expression of smad1 at day 7 , and the highest expression 
 of smad4 at day 14 .
( 2009 ) demonstrated that the treatment of osteoblast - like cells with bmp-4 increases 
 smad1/5 phosphorylation .
( 2001 ) also observed that the bmp-4 activation in c2c12 mouse muscle myoblast cell line 
 occurs via the smad pathway .
( 2009 ) demonstrated that 
 osteogenesis by endogenous bmp-4 and bmp-6 was independent of smad activation and 
 dependent on phosphatidylinositol 3-kinase ( pi-3k ) activation in marrow - derived stem 
 cells .
therefore , bmp-4 activation has been related to pathways dependent or independent 
 of smads .
these different findings may be due to the different lineage cell type , 
 species , and culture conditions .
( 2011 ) did not observe any significant changes in smad1/5/8 
 phosphorylation levels upon bmp-2 induction .
additionally , these authors observed that 
 the removal of dexamethasone from the bmp-2 did not significantly change the 
 mineralization capacity of ascs from that measured when samples were induced with bmp-2 
 and om containing dexamethasone .
such a finding would suggest that the lack of effect of 
 bmp-2 was not due to any interference by the presence of dexamethasone .  
despite that the presence of bmp-2 in the osteogenic supplementation led to the highest 
 expression of smads , this protein did not increase the alp activity at the peak of this 
 enzyme , the expression of osteonectin and osteocalcin mrna , or the calcium deposition at 
 the extracellular matrix , an indicator of osteoblast final maturation .
( 2003 ) observed that 
 ascs cultured with bmp-2 demonstrated more alp and matrix calcification than the 
 positive osteoblast control . considering that both treatments ( ascs+om and ascs+om+bmp2 ) 
 led to similar effects in osteogenic differentiation , the effects of exogenous bmp-2 
 could be counterbalanced by the highest expression of endogenous bmp-4 and bmp-7 . 
 herein , probably endogenous bmp-4 and bmp-7 pathways were independent of smad 
 activation , corroborating seib , et al .
there are a number of possible explanations for the reasons 
 that ascs had demonstrated similar osteogenic differentiation in the presence or absence 
 of bmp-2 , including the maturation of osteogenic progenitors during culture , emergence 
 of a dominant cell type or spontaneous differentiation of mscs into the 
 osteoblasts .
although a combination of these factors could be 
 responsible for the results herein , endogenous bmps production could be a critical 
 factor in any of these scenarios .
moreover , the 
 supplementation of ascs with bmp-2 did not increase the level of osteogenic markers in 
 the initial ( alp activity ) , intermediate ( osteonectin and osteocalcin ) , or final 
 ( calcium deposition ) phases suggesting that the exogenous addition of bmp-2 did not 
 improve the in vitro osteogenesis process of human ascs .
the authors would like to thank to cnpq / mct and capes / mec ( brazil ) for their research 
 fellowships .
william f. zambuzzi ( universidade do grande rio ) for gently 
 providing the mc3t3-e1 cells .
the authors would also like to thank msc lucas barcelos 
 oliveira for his proficient editorial assistance .
this work was partially supported 
 by mct / sebrae/ finep ( grant number 23080.039510/2007 - 12 ) .
 bone morphogenetic protein type 2 ( bmp-2 ) is a potent local factor , which promotes 
 bone formation and has been used as an osteogenic supplement for mesenchymal stem 
 cells.objectivesthis study evaluated the effect of a recombinant bmp-2 as well as the endogenous 
 bmp-4 and bmp-7 in the osteogenic differentiation of adipose - derived stem cells 
 ( ascs ) in medium supplemented with ascorbate and -glycerophosphate . 
 material and methodshuman ascs were treated with osteogenic medium in the presence ( ascs+om+bmp-2 ) or 
 absence ( ascs+om ) of bmp-2 . 
 the alkaline phosphatase ( alp ) activity was determined 
 and the extracellular matrix mineralization was evaluated by von kossa staining 
 and calcium quantification . 
 the expressions of bmp-4 , bmp-7 , smad1 , smad4 , and 
 phosphorylated smad1/5/8 were analyzed by western blotting . 
 relative mrna 
 expressions of smad1 , bmp receptor type ii ( bmpr - ii ) , osteonectin , and osteocalcin 
 were evaluated by qpcr . 
 results : ascs+om demonstrated the highest expression of 
 bmp-4 and bmp-7 at days 21 and 7 , respectively , the highest levels of bmpr - ii mrna 
 expression at day 28 , and the highest levels of smad1 mrna at days 14 and 28 . 
 
 ascs+om+bmp-2 demonstrated the highest levels of smad1 mrna expression at days 1 , 
 7 , and 21 , the highest expression of smad1 at day 7 , the highest expression of 
 smad4 at day 14 , the highest alp activity at days 14 and 21 , and expression of 
 phosphorylated smad1/5/8 at day 7 . 
 ascs+om and ascs+om+bmp2 showed similar alp 
 activity at days 7 and 28 , similar osteonectin and osteocalcin mrna expression at 
 all time periods , and similar calcium depositions at all time periods . 
 conclusionswe concluded that human ascs expressed endogenous bmp-4 and bmp-7 . 
 moreover , the 
 supplementation of ascs with bmp-2 did not increase the level of osteogenic 
 markers in the initial ( alp activity ) , intermediate ( osteonectin and osteocalcin ) , 
 or final ( calcium deposition ) phases , suggesting that the exogenous addition of 
 bmp-2 did not improve the in vitro osteogenesis process of human 
 ascs . 
INTRODUCTION
MATERIALS AND METHODS
Reagents
Isolation and culture of human adipose-derived stem cells 
Determination of alkaline phosphatase activity 
Evaluation of matrix mineralization 
Western blotting 
qPCR 
Statistical analysis 
RESULTS
BMP-2 improved the alkaline phosphatase activity 
BMP-2 did not improve the calcium at the extracellular matrix 
Expression of osteoblast-related proteins 
Relative expression of osteoblast-related mRNA 
DISCUSSION
CONCLUSIONS
ACKNOWLEDGEMENTS