Patent Application: US-201013510906-A

Abstract:
heparan sulphates isolated from male and female murine liver , their characterisation and use in pharmaceutical compositions , methods of treatment and in stem cell culture media suitable for conditions associated with bone repair .

Description:
the details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention , by way of example . it will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details . the following experimental example describes the comparisons of structural and functional properties of mouse gender specific organ heparan sulfate . we describe analyses of hs chains that were purified simultaneously from the gender specific liver mouse tissues , namely male liver heparan sulfate ( mml hs ) and female liver heparan sulfate ( fml hs ) and showed that major gross and fine structural differences exist between male and female liver hs chains . these findings provide further insight into the potential of two differentially sulfated species of hs chains purified from mouse liver tissues to modulate the hmsc proliferation and differentiation . finally we analyzed the gene expression profile of hmsc during osteogenic differentiation . our result showed that a set of osteogenic genes are upregulated during hmsc differentiated into osteoblasts . these results demonstrated that differentially sulfated mml hs and fml hs modulate hmsc proliferation and differentiation . we have applied molecular sieving , enzymatic cleavage and strong anion - exchange hplc methods for structural and composition analyses of intact heparan sulfate chains . the results demonstrated that hs chains purified from gender specific liver tissues are significantly different by a number of parameters . size determination showed male and female liver intact hs chain length are ˜ 100 kda and ˜ 22 kda and comprise 30 - 40 or 40 - 50 and 6 - 8 disaccharide repeats , respectively . this result suggests that the intra - gender specific hs chains show variability across organs . enzymatic depolymerization and disaccharide composition analyses demonstrated significant differences in domain organization and fine structure between gender specific liver hs . n - unsubstituted glucosamine ( δhexaglcnh 3 + , δhexa - glcnh 3 + ( 6s ), δhexa ( 2s )- glcnh 3 + , and n - acetyl glucosamine ( δhexa - glcnac ) are the predominant disaccharide in male mouse liver hs . however , n - sulfated glucosamine ( δhexaglcnso 3 ) is the predominant disaccharide in female mouse liver hs . we showed that structurally different male and female liver hs exerts a different effect on human mesenchymal cells ( hmsc ) proliferation and osteogenic differentiation . finally , the present study demonstrated for the first time that gender specific organ hs chains are distinguishable at the gross and fine structural level , and proved the potential usefulness of gender specific liver hs in modulating hmsc proliferation , osteogenic differentiation and subsequent matrix mineralization . heparin lyase i , ii , and iii from flavobacterium heparium , heparin oligosaccharide ( dp4 - dp26 ), 12 heparin disaccharide standards and de - o - sulfated and de - n - sulfated heparin oligosaccharides were from iduron , uk . chondroitin abc lyase , neuraminidase , pronase ; actinase e , chloroform and methanol were obtained from sigma - aldrich ( st . louis , usa ). hiprep 16 / 10 deae columns , hiprep 26 / 10 desalting columns , superdex 75 10 / 300 gl column , superdex 200 10 / 300 gl column and high molecular weight ( hmw ) and low molecular weight ( lmw ) gel filtration calibration kits were from ge healthcare , sweden . bio - gel p2 was from bio - rad laboratories , hercules , calif . human mesenchymal stem cells ( hmscs ) were from cambrex , usa . all other reagents used were analytical grade . cba male and female mice of 12 - 24 weeks old were used in this study . animals were killed by cervical dislocation , whole liver lobes were loosened from surrounding tissue , and the tissue was cut at the insertion of blood vessels into hepatic hilus to gain all the tissue possible . harvested livers were rinsed with pbs and immediately frozen and kept in − 20 ° c . until processing . liver tissues were cut into small pieces (& lt ; 1 mm 3 ) and homogenized using a homogenizer . fat was removed by washing the homogenized tissues with chloroform / methanol mixtures ( 2 : 1 , 1 : 1 , 1 : 2 ( v / v )). the defatted materials were freeze - dried under vacuum and stored at − 20 ° c . until further use . the dried , defatted tissues were each suspended in 0 . 05 . m tris acetate buffer ( ph 8 . 0 ) and digested for 48 h by actinase e 10 mg / g ( sigma ) at 50 ° c . the proteolyzed homogenates were placed in a boiling water bath for 30 min to deactivate the protease and then centrifuged ( 2500 × g ) for 30 min at room temperature . the recovered supernatant was passed through a hiprep deae column and total glycosaminoglycans ( tgags ) were eluted with high salt . the eluted samples were desalted using a hiprep desalting column and freeze - dried . the samples were quantified for the uronic acid content by carbazole method ( 20 ). the tgags were treated with neuraminidase and chondroitin lyase abc at 37 ° c . for 24 h to remove sialic acids , chondroitin and dermaton sulfate . after chondroitinase digestion , the reaction was terminated by heating in a boiling water bath for 15 min and the digested sample were diluted 1 : 10 times with water . the diluted samples were passed through the hiprep deae column and hs were eluted with high salt . the eluted samples were desalted using a hiprep desalting column and the resulting hs samples were freeze - dried . the samples were quantified for uronic acid content by carbazole method ( 20 ). hs samples were run on a superdex 75 ( 10 / 300 ) column for sizing of the gag chains . the superdex 75 column separated homogenous populations of hs chains were further analyzed on a superdex 200 column to confirm their sizes . protein hmw and lmw protein standards ( ge healthcare ) and even numbered heparin oligosaccharides derived from heparin ( highly sulfated ) were used to calibrate the sizing column . the purified hs samples were treated with heparinase i , heparinase ii or heparinase iii . dried samples ( 100 μg ) were dissolved in 100 mm sodium acetate / 0 . 2 m calcium acetate ; ph 7 . 0 and incubated with 10 mu / ml of heparinase i , or heparinase ii or heparinase iii in the same buffer at 37 ° c . for 16 hand then a second aliquot of enzyme added and incubated for a further 6 h . the heparinase i or heparinase ii or heparinase iii digested hs samples were analyzed on biogel p - 10 column ( 1 × 120 cm ) equilibrated with 0 . 25 m nh 4 hco 3 . even numbered oligosaccharides derived from heparin were used as standards to calibrate the column . each sample was sequentially digested for a recovery of disaccharides for sax - hplc analysis ; for this the samples were digested at 37 ° c . as follows : heparinase i for 3 h , heparinase iii for 2 h , heparinase ii for 16 h , and finally an aliquot of each lyases for 6 h . samples were run on a bio - gel p - 2 column ( 1 × 120 cm ) equilibrated with 0 . 25 m nh 4 hco 3 to recover the disaccharides . the disaccharide products were freeze - dried prior to disaccharide analysis . the constituent of lyase - derived disaccharides were resolved by sax - hplc on a propac pa1 analytical column ( 4 × 250 mm ) linked to a dionex ics - 3000 hplc system . after equilibration in the mobile phase ( double distilled water adjusted to ph 3 . 5 with hcl ) at 1 ml / min , samples were injected and disaccharides eluted with a linear gradient of sodium chloride from 0 - 1 m over 60 min in the same mobile phase . the elution was monitored for uv absorbance at 232 nm . disaccharides were identified by comparison with the elution positions of known 12 disaccharide standards from iduron . human mesenchymal stem cells ( hmscs ) ( cambrex , usa ) were plated in maintenance medium consisting of dmem ( 1000 mg / l glucose ), 10 % fetal calf serum ( fcs ), 2 mm l - glutamine and 100 u / ml penicillin - streptomycin sulfate at 37 ° c . in a humidified atmosphere with 5 % co 2 . to assess their proliferation , assays were performed to determine cell number utilizing the guava pca - 96 system ( guava technologies ) as per the manufacturer &# 39 ; s instructions . briefly , cells were seeded at 3 , 000 cells / cm 2 in 48 - well plates and allowed to adhere overnight . the following day the cells were re - fed with the same medium with or without different concentrations of hs ( 12 . 5 , 1 . 25 , 0 . 625 , 0 . 312 and 0 . 156 μg / ml ) and the medium were changed every 3 days . proliferation of cells was assessed by guava viacount on 1 , 3 , 5 and 7 day to determine the total viable cells . cells were washed in pbs , pelleted with 0 . 125 % trypsin , the trypsin neutralized , the pellet resuspended in 400 μl of pbs with the addition of 4 μl of flex reagent , incubated for 10 min and cell number and viability determined utilizing the guava viacount software . the interaction between hs and growth factor was determined using gag binding plates ( iduron ) according to manufacturer &# 39 ; s instructions . briefly , hs was coated on the plate prior to adding growth factors . bound growth factor was detected using biotinylated antibodies ( r & amp ; d system ). human mesenchymal stem cells ( hmscs ) were plated in maintenance medium consisting of dmem ( 1000 mg / l glucose ), 10 % fetal calf serum ( fcs ), 2 mm l - glutamine and 100 u / ml penicillin - streptomycin sulfate at 37 ° c . in a humidified atmosphere with 5 % co 2 . to assess their proliferation , assays were performed to determine cell number utilizing the guava pca - 96 system ( millipore , usa ) as per the manufacturer &# 39 ; s instructions . briefly , cells were seeded at 3 , 000 cells / cm 2 in 48 - well plates and allowed to adhere overnight . the following day the cells were re - fed with the same medium with or without different concentrations of hs ( 12 . 5 , 1 . 25 , 0 . 625 , 0 . 312 and 0 . 156 μg / ml ) and the medium changed every 3 days . proliferation of cells was assessed on days 1 , 3 , 5 and 7 to determine total viable cells . cells were washed in pbs , pelleted with 0 . 125 % trypsin , the trypsin neutralized , the pellet resuspended in 400 μl of pbs with the addition of 4 μl of flex reagent , incubated for 10 min and cell number and viability determined utilizing the guava viacount software . mscs were seeded in designated media at 5 , 000 / cm 2 in six - well plates and cultured until subconfluent . cells were washed and deprived of fcs for 24 h and then treated with hs or fgf - 2 alone , or together with the fgfr1 - blocking drug su5402 ( calbiochem ) at 10 μm in dmso for 60 min . cells were lysed in 300 μl of ice - cold lysis buffer [ 150 mm nacl , 10 mm tris ph 7 . 4 , 2 mm edta , 1 % triton x - 100 , 0 . 1 % sodium dodecyl sulfate ( sds ), 0 . 5 % igepal ] supplemented with 0 . 2 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride and 30 μl of protease inhibitor cocktail ( sigma ). the lysate was incubated on ice for 10 min , passed through a 21 - gauge needle and then centrifuged at 10 , 000 g for 10 min at 4 ° c . to remove cellular debris . protein content was determined using a protein assay kit ( bio - rad ) following the manufacturer &# 39 ; s instructions . protein ( 20 μg ) was mixed with an equal volume of 2 × laemmli buffer , boiled for 5 min , and then separated by sds polyacrylamide gel electrophoresis on 8 % gels . the protein was then transblotted onto nitrocellulose membranes ( amersham biosciences ) using a trans - blot ® sd cell semi - dry transfer apparatus ( bio - rad ) for 15 min at 20 v . the membranes were blocked in 5 % nonfat milk in tris - buffered saline tween - 20 ( tbst ) ( 1 . 5m nacl , 1m tris , ph 7 . 4 , 1 % tween ) for 1 h and incubated overnight at 4 ° c . with rabbit anti - actin , rabbit anti - erk1 / 2 , monoclonal anti - diphosphorylated erk1 / 2 ( sigma ). the membranes were washed with tbst and incubated for 1 h with anti - rabbit igg - or anti - mouse igg - hrp - conjugated secondary antibodies ( southern biotech , birmingham , ala .). following washes , the membranes were covered with supersignal ® west pico chemiluminescent substrate ( pierce , rockford , ill .) for 5 min then exposed on film . effect of male and female liver hs on hmsc osteogenic differentiation to confirm the effect of osteogenic potential of male and female mouse liver hs , mineralization assays were performed on triplicate cultures of human mesenchymal stem cells ( hmscs ) ( cambrex , usa ). the cells were seeded in triplicate at a density of 3000 cells / cm 2 in 12 well plates in osteogenic medium ( maintenance medium supplemented with 10 nm dexamethasone , 25 μg / ml l - ascorbic acid - 2 - phosphate and 10 mm b - glycerophosphate ) and let them adhere for 24 h . the following day the cells were re - fed with the same medium with or without two different concentrations of hs ( 1 μg / ml or 300 ng / ml ) and grown for 3 weeks and the medium were changed every 3 days . to stain for the accumulation of calcium within the matrix , cell monolayers were washed with pbs × 3 and incubated with 4 % paraformaldehyde ( pfa ) for 10 min at room temperature ( rt ). the monolayers were then washed with double - distilled water ( ddh 2 o )× 3 , incubated with 1 % alizarin red stain for 10 min , washed again with ddh 2 o × 3 and air dried . images were taken using an olympus bx51 microscope , dp70 camera , and dpcontroler software v1 . 1 . 1 . 65 . for phosphate nodule staining , cell monolayers were then washed with pbs × 3 and incubated with 4 % pfa for 10 min at rt . cell monolayers were then washed with ddh 2 o × 3 and incubated for 30 min in 1 % silver nitrate under uv light . the monolayers were then washed with ddh 2 o × 3 , incubated with 5 % sodium thiosulfate for 2 min and washed again with ddh 2 o × 3 and air dried . images were taken using an olympus bx51 microscope , dp70 camera , and dpcontroler software v1 . 1 . 1 . 65 . bioquant image analysis ® software ( bioquant image analysis corporation , tn , usa ) was used to quantify the average density of alizarin red s and von kossa stained wells . briefly , cultures were performed in triplicate 21 day cultures . mml hs and fml hs treated cultures for each concentration were performed on the same plate . to ensure the same light intensity and exposure within the samples , images of the plates were taken using an epson perfection 1670 scanner . using the image analysis software , the digital images were loaded into the field of view and a consistent region of interest ( roi ) was placed over each well . the average density of the wells was recorded for each sample and is reported in units of grayscale . in these measurements , a darker stain correlates with minimal light transmission giving a higher density value . the data is presented as this value subtracted from the value for white light , giving apparent stain signal intensity . human mesenchymal cells were seeded in triplicate at a density of 3000 cells / cm 2 in 12 well plates in osteogenic medium ( maintenance medium supplemented with 10 nm dexamethasone , 25 μg / ml l - ascorbic acid - 2 - phosphate and 10 mm b - glycerophosphate ) and let them adhere for 24 h . the following day the cells were re - fed with the same medium with or without two different concentrations of hs ( 1 μg / ml or 300 ng / ml ) and grown for 3 weeks and the medium were changed every 3 days . total rna was extracted at day 7 , 14 and 21 days using a nucleospin ® rna ii kit according to the manufacturer &# 39 ; s instructions ( macherey - nagel , easton , pa .). first strand complementary dna ( cdna ) synthesis was carried out on total rna using superscript iii reverse transcriptase according to the manufacturer &# 39 ; s instructions ( invitrogen , carlsbad , calif .). real time pcr primers and probe sets ( described in table 1 ) for quantitative real - time pcr were designed using primer express ( ver . 1 . 0 ; applied biosystems ) and synthesized by proligo ( proligo llc , boulder , colo .). quantitative pcr ( qpcr ) was then performed to assess the relative expression of the target genes in hmscs . this was carried out on an abi prism 7000 sequence detection system ( applied biosystems , warrington , uk ) using 600 nmol / l forward and reverse primers , 250 nmol / l probe , and taqman pcr master mix ( abi applied biosystems ). reactions were run using the thermal profile ; with an initial 10 min activation step at 95 ° c . followed by 45 cycles of 95 ° c . for 20 sec ; 55 ° c . for 10 sec , 60 ° c . for 30 sec and 72 ° c . for 40 sec . biological triplicates were measured in triplicates and data were normalized to 18s ribosomal rna expression in each sample . purification and sizing of intact gender specific mouse liver hs chains the male and female mouse hs - gags chains were isolated using deae anion - exchange chromatography . subsequent separation on superdex 75 demonstrated that the male liver hs chains eluted a major high molecular weight and a smaller small molecular weight peak ( fig1 a ). in contrast to male , female liver hs chains eluted a major small molecular weight and small high molecular weight peak ( fig1 b ). thus in each case , the original hs gag chain preparation was composed of two distinct populations of heparan sulfate chains . we further separated the high molecular weight peak of mml hs and small molecular weight peak of fml hs . we re - ran the separated mml high and fml low molecular weight hs peaks on superdex 75 column to check the homogeneity . the superdex 75 separated homogenous population of mml hs and fml hs chains were sodium borohydrate treated to release the linker oligosaccharide from the peptide backbone and fractionated on superdex 200 columns to determine the size of the hs chains . separation on superdex 200 demonstrated that the mml hs eluted with a kav of 0 . 31 and the fml hs eluted with a kav of 0 . 54 ( fig1 c and d ). the relative molecular weights of male and female liver hs chains were determined to be 100 , 000 and 22 , 000 da , respectively . in addition to this the superdex 200 column was calibrated with the available even numbered heparin oligosaccharides from iduron , ranging from dp2 to dp26 . the fml hs chains have the same . kav of dp12 but the mml hs chains are above dp26 . from these data we concluded that the intact fml hs chain composed of approximately 6 - 8 disaccharides repeats and mml hs chain is composed of approximately 30 - 40 or 40 - 50 disaccharides repeats . the calculated values showed significant variation in chain lengths between mml hs and fml hs . heparinase treated male and female liver hs were applied to a bio - gel p10 column ( 1 × 120 cm ) to access the distribution and ratios of the resulting oligosaccharides . representative patterns of the male and female liver hs after heparinase i digestion are shown in fig2 a and b . digestion by heparinase i was more extensive in fml hs than mml hs . the male liver hs depolymerization profile showed approximately 10 % linkages susceptible to heparinase i . there was a corresponding majority of resistant regions of larger size to be excluded from the bio - gel p10 column ( fig2 a ). however , the female liver hs depolymerization profile showed 60 % linkages are susceptible to heparinase i to yield di - and tetrasaccharides . a correspondingly smaller percentage of resistant regions of larger oligosaccharides are still eluted in the void volume ( fig2 b ). the splitting of the disaccharide peak is due to almost complete resolution of the disaccharides into di - and tri - sulfated . this reflects the small clusters of the heparinase i susceptible sites in the hs chains . the heparinase ii digested mml hs profile was the same as heparinase i digestion ( fig2 c ). on the other hand fml hs completely digested into di and tetrasaccharides and almost no material remained in the void volume ( fig2 d ). heparinase iii digested approximately 80 % of the mml hs chains , the resulting digested fragments were mainly di - and tetrasaccharides and a smaller portion still eluted in the void volume ( fig2 e ). the fml hs chains completely digested into di -, tetra -, and hexasaccharides and almost no material remained in the void volume ( fig2 f ). this approach demonstrated a significant gross structural difference between mml hs and fml hs . the fine structural differences in the disaccharide content of the male and female liver hs chains were analyzed after exhaustive digestion with a mixture of heparinases i , ii and iii and separation on bio - gel p2 column ( 1 × 120 cm ), followed by strong anion - exchange hplc . the resulting disaccharide peaks were identified by reference to well characterized disaccharide standards from iduron . the sax - hplc separated mml hs and fml hs disaccharide profile was shown in fig3 a and b . the area under each peak was used to obtain the disaccharide composition of each sample . comparisons of the male and female liver hs showed that , n - unsubstituted glcnh 3 + ( δhexua - glcnh 3 + , δhexua - glcnh 3 + ( 6s ), δhexua ( 2s )- glcnh 3 + ) and n - acetylated ( δhexua - glcnac ) are the predominant disaccharide in the mml hs . however , n - sulfated ( δhexua - glcnso 3 ) is the predominant disaccharide in the fml hs ( table 2 ). the sax - hplc data showed a great distinction between the two heparan sulfates in that the mml hs total sulfation was 66 % with 18 % n - sulfation , 26 % 6 - o - sulfation and 22 % 2 - o - sulfation and the fml hs total sulfation was 85 % with 62 % n - sulfation , 12 % 6 - o - sulfation and 11 % 2 - o - sulfation ( fig4 a ). mml hs and fml hs was composed of 57 % and 30 % n - unsubstituted disaccharides , respectively . this is higher than the 1 . 2 to 7 . 5 % previously reported with various porcine , bovine and rat tissues ( 21 , 22 ). non - sulfated , mono - sulfated and di - sulfated n - unsubstituted disaccharides ( ahexuagicnh 3 + , δhexua - gicnh 3 + ( 6s ), δhexua ( 2s )- gicnh 3 + and δhexua ( 2s )- gicnh 3 + ( 6s )) have been detected in both male and female liver hs with different percentages ( fig4 b ). these results demonstrated major structural differences between gender specific liver hs chains at the gross and fine level . gag binding plates revealed that male liver hs binds to all growth factors tested ( fgf2 , bmp2 , pdgf and vegf ; fig4 c ) whereas female live hs selectively bound only fgf2 and bmp2 ( fig4 d ). to assess the possibility of structural differences identified between the male and female liver hs to modulate hmsc proliferation , hmscs were treated with varying concentration of hs and viable cell number was counted over a 7 - day culture period in normal maintenance or differentiation ( osteogenic ) medium . in growth medium , mml hs dose dependently increased the cell number , the highest cell number was obtained with the highest concentration of hs ( 12 . 5 μg / ml ). however , fml hs at lower concentration ( 152 ng / ml ) increased the highest cell number and higher concentrations gave a relative decrease in the cell number ( fig5 a and b ). however , in contrast to the maintenance medium , mml hs at lower concentration ( 312 ng / ml ) in the osteogenic medium increased the highest cell number , and fml hs at a higher concentration ( 12 . 5 μg / ml ) increased the highest cell number ( fig5 c and d ). notably for fml hs , there was an inverse relationship with decreasing doses giving a greater proliferative effect ( fig5 a & amp ; b ). in all cases , liver hs irrespective of gender , stimulated proliferation of hmscs in the presence of 10 % fcs . taken separately , fml - hs was maximally proliferative at 156 ng / ml , whereas mml - hs was maximally proliferative at 12 . 5 μg / ml . notably , despite this dosing effect , both hss produced similar numbers of hmscs over a 7 - day period ( 2 - 2 . 5 × 10 5 ). the gag - binding plates suggested that the liver hs isolates had much greater affinities for fgf2 than pdgfbb , vegf165 or bmp2 , factors known to be physiologically relevant for hmscs ( fig1 ). in order to confirm whether the mechanism involved in triggering the proliferation by the liver hs was via fgf2 , as hs is a co - receptor for the fgf / fgfr complex and as fgfr1 is a high affinity receptor for fgf - 2 binding , we next sought to verify whether the inhibition of this receptor with the chemical inhibitor su5402 could affect liver hs &# 39 ; s intracellular signalling activity . activation of the erk cascade is required for fgf - induced mitogenesis . thus we sought to verify if liver hs , like fgf - 2 , could induce erk activation in low serum conditions . as expected if the hs was involved directly with fgfr1 , both male and female hs were able to stimulate the phosphorylation of erk1 / 2 ( fig5 e , with protein levels quantified in 5f ); the effects for both liver hs species could be blocked with the su5402 . knowing that mml hs maximally binds fgf2 and increased hmsc proliferation , we next examined whether this effect was mediated by fgfr signaling . pulsing with su5402 had an inhibitory effect on the basal growth of hmscs while also preventing hs and fgf2 induced proliferation ( fig5 g ). this result further confirmed that the mechanism involved in triggering the proliferation by the liver hs was via fgf2 / fgfr signaling . we showed that structurally different male and female liver hs exerts a different effect on hmsc proliferation in maintenance and osteogenic medium . next we examined whether these effects could still be observed when the cells were induced to differentiate down the osteoblast lineage . photomicrography showed the osteogenic differentiation of hmsc with or without liver hs in osteogenic media for 21 days ( fig6 a ). our results clearly show that male and female liver hs increases hmsc mineralization in dose dependent manner . mml hs at lower concentration ( 300 ng / ml ) induces high mineralization equal to the higher concentration ( 1 μg / ml ) of fml hs . the effect of liver hs on the osteogenic differentiation of hmsc to mineralize in vitro can be visualized by alizarin red sand von kossa staining . mml hs at 300 ng / ml and 1 μg / ml after 21 days , an extensive mineralization noted . fml hs at 300 ng / ml concentration did not show increased mineralization as compared to control and mml hs . but at 1 μg / ml female liver hs increased mineralization equal to 300 ng / ml mml hs . female liver hs at 300 ng / ml was almost equal to control ( fig6 b ). measurement of calcium - bound alizarin red s dye showed higher calcium accumulation in male hs treated samples than mml hs ( fig6 c ). measurement of mineralized bone nodules by von kossa staining showed higher bone nodules in mml hs treated cells than fml hs ( fig6 c ). we next investigated the effect of liver hs on the expression of mrna transcripts related to osteoblast differentiation using qpcr . the expression levels of alkaline phosphatase ( alp ), an early transcription factor essential for osteoblast differentiation was markedly increased at the 7 day culture period . the mml hs at 300 ng / ml or 1 μg / ml treated cells showed higher expression of alp when compared to control . fml hs at higher concentration ( 1 μg / ml ) upregulated alp expression to become comparable with 300 ng / ml mml hs . in mml hs treated hmsc the expression levels of bone sialoprotein ( bsp2 ), osteopontin ( opn ) and runx2 late transcription factors essential for osteoblast differentiation were upregulated at both concentrations tested over the 21 day culture period compared to controls . on the other hand , the expression of these markers was significantly upregulated at higher concentration 1 μg / ml of fml hs treated hmsc , which is comparable to lower concentration ( 300 ng / ml ) of mml hs treated cells ( fig7 ). the present morphological and genetic results demonstrate that gender specific liver hs differentially modulate osteogenic differentiation of hmsc . hs biosynthesis is a complex multi - step process that occurs in a very specific and sequential manner via membrane - bound enzymes in the endoplasmic reticulum and golgi apparatus . heparan sulfate is a ubiquitous component of the extracellular matrix in a wide variety of animal species and tissues . the fine structure of the chains depends on the regulated expression of multiple biosynthetic enzymes , such as glycosyltransferases , sulphotransferase and an epimerase . structural differences and the presence of n - unsubstituted amino groups were observed in the heparan sulfates from different tissues and species ( 19 , 21 , 22 ). in this study we describe the detailed analysis of the structural and functional properties of the hs chains purified from male and female mouse liver tissue . the results presented suggest the existence of two distinct and separate populations of heparan sulfates . they are structurally different , changes that could be construed to be significant as they could be confirmed at the functional level . glycosaminoglycans are linear , sulphated , negatively charged polysaccharides that have molecular weights of roughly 10 - 100 kda ( 23 ). the sec - hplc data showed that the relative molecular weight of mml hs was 100 kda and intact hs chain size was approximately 30 - 40 or 40 - 50 disaccharide repeats , which is comparable to the hs chain length observed from seven rat tissues ( 21 ). the fml hs had an apparent molecular weight of 22 kda and intact hs chain was approximately 6 - 8 disaccharide repeats . this is consistent with the previous report that , fully sulfated heparin 12 mer ( dp12 ) showed the same molecular mass of 22 kda ( 24 ). we purified hs from different age groups of male and female mouse liver tissues ( data not shown ), and they all gave the result shown in fig1 . this confirmed that hs chain size and structure was gender specific and not dependent on the age of the tissue . glycosaminoglycan degrading enzymes heparinase i , ii and iii are useful analytical tools for investigating the composition and structural sequence of various hs chains and each heparinase has distinct substrate specificities ( 25 ). heparinase i has high specificity for highly sulfated disaccharides and cleaves hs chains with sulfate - rich regions at an n - sulfated glucosamine / sulfated - iduronic acid ( glcns (± 6s )- idoua ( 2s )) ( 26 - 30 ). the size of the fragments generated , compared to the undigested chain gives , therefore , the frequency of these areas of high sulfation . separation of the hs chains from male and female liver hs after digestion with heparinase i gave different profiles . mml hs showed less sensitivity to heparinase i , this reflects that there is no close proximity of heparinase - susceptible sites in the male liver hs which probably exist in relatively small clusters and at the non - reducing end . this is similar to the result obtained with skin hs , the idoua residue essential for heparinise i action is present in lower concentrations ( 6 . 5 - 10 % of total hexua ) ( 31 - 33 ). female liver hs showed high sensitivity to heparinase i , with ˜ 65 % of the linkages cleaved to yield dp2 - dp6 as the major product and 35 % yield longer oligosaccharides . this reflects a closer proximity of heparinase - susceptible sites in fml hs than in mml hs . in contrast to heparinase i , heparinase iii cleaves hs chains in n - acetylated or n - sulfated disaccharides regions ( glcnac / glcns - hexa ) with a preference for glca over idoa . it will tolerate 6 - o sulfation of the amino sugar but is inhibited by 2 - o sulfation of idoa ( 34 , 35 ). heparinase iii also cleaves non - sulfated , n - unsubstituted disaccharide ( glca - glcnh 3 + ) ( 36 ). heparinase iii cleaved 80 % of material from mml hs into di - and tetrasaccharide units . this is similar to the 78 % obtained for endothelial hs ( 37 ) but substantially less than the 63 % disaccharide result obtained with skin fibroblasts ( 31 ). however , female liver hs was completely degraded into di - and tetrasaccharides . this is unique when compared to other previous hs studied . the susceptible sites were highly contiguous , and the resistant fragments alternated to form tetrasaccharides . these results suggest that fml hs is composed of mostly n - acetylated , n - sulfated and n - unsubstituted disaccharide . heparinase ii has broad substrate specificity ( 27 , 35 , 38 ) and particularly it has two distinct active sites , one of which is heparinase i like , whereas the other is heparinase iii like ( 39 ). heparinase ii also has substrate specificity on unsaturated disaccharides that are o - sulfated at c2 of uronic acid or c6 of glcnh 3 + ( 40 ). mml hs showed less sensitivity , while fml hs showed high sensitivity to heparinase ii . this reflects the close proximity of heparinase ii susceptible sites in fml hs . the specificity of individual heparinase analyses of the male and female hs showed both to have a distinctive structure and composition . disaccharide composition analyses showed mml hs is less sulfated than fml hs . this is consistent with the heparinase i digestion profile of mml hs and fml hs ( fig2 a and b ). comparing male and female liver hs it can be seen that δhexua ( 2s )- glcns ( 6s ) and δhexua - glcns are the most likely to be the non - reducing end sulphated disaccharides for both hs . this finding is consistent with the previous report that the non - reducing end of bovine kidney hs is heavily sulfated and especially n - sulfated ( 41 ). mml hs showed ˜ 70 % disaccharides were less sulfated , but heparinase ii digestion produce only ˜ 10 disaccharides , this is because the n - acetylated saccharides ( glcaglcnac ) continuously present in the reducing end are cleaved into disaccharides , but the n - sulfated , n - unsubstituted and 6 - o - n - unsubstituted saccharides present on the n - acetyl / n - sulfated region in the middle of the hs chain . this enzyme cleaves these saccharides , because of the longer chain length with concomitant higher mass it was excluded in the void volume of the biogel p10 column ( fig2 c ). however , in fml hs ˜ 90 % saccharides were less sulfated , so the complete chain was cleaved by heparinse ii to make disaccharides ( fig2 d ). this reflects the close proximity of heparinase ii susceptible sites in the whole fml hs chains . mml hs heparinase iii digestion produced ˜ 80 % di and tetrasaccharides . in mml hs 8 % trisulfated saccharides present on the non - reducing end followed by 12 % 2 - o - n - unsubstituted saccharides , and these saccharides were resistant to heparinase iii , so in biogel p10 separation some longer chains excluded in the void volume ( fig2 e ). however , female liver hs was completely degraded into di - and tetrasaccharides . this showed the n - acetylated saccharides start with the reducing end followed by n - unsubstituted and n - sulfated saccharides and trisulfated - saccharide in the non - reducing end . the susceptible sites were highly contiguous , and the resistant fragments alternated to form tetrasaccharides ( fig2 f ). the structure of male and female mouse liver intact hs chains has been predicted with combined data obtained from chain length , heparinase i , ii and iii digestion , and % composition of differentially sulphated disaccharides . the superdex 200 sizing column profile showed that the intact mml hs and fml hs chains composed of 30 - 40 or 40 - 50 and 6 - 8 disaccharides repeat , respectively . mml hs chains may consist of a substantial proportion of alternating acetylated glucosamine sequence ( glca - glcnac ) in the reducing end ( na domain ) and n - unsubstituted glucosamine are the major proportion of mixed sequences in the middle of the hs chain , which contains δhexaglcnh 3 + , δhexa - glcnh 3 + ( 6s ), δhexa ( 2s )- glcnh 3 + , δhexa ( 2s )- glcnac ( 6s ) and a minor portion of δhexa - glcnso 3 ( na / ns domain ). there are also relatively a minor proportion of about 4 - 5 trisulfated disaccharides ( δhexa ( 2s )- glcns ( 6s )) sequence on the s - domain in the non - reducing end , as illustrated in fig8 . fml hs chain may consist of a major proportion of n - sulfated glucosamine ( δhexa - glcnso 3 ) and also a substantial proportion of n - unsubstituted glucosamine in the middle of the hs chain , which contains δhexa - glcnh 3 + , δhexa - glcnh 3 + ( 6s ), hexa ( 2s )- glcnh 3 + ( na / ns domain ). there are also a minor proportion of δhexa - glcnac present on the reducing end ( n - acetyl domain ) and also a minor proportion of δhexa ( 2s )- glcns ( 6s ) present on the non - reducing end ( n - sulfated domain ) as illustrated in fig8 . the present study demonstrated that the hs chains purified from gender specific liver tissue are , undeniably , almost completely distinguishable at the gross and fine structural level , therefore the question arises as to how these heparan sulfates exhibit their biologically different function . we chose human mesenchymal stem cells ( hmsc ) for biological functional studies , because hmsc are a potential source for cell based therapeutic studies . the use of hmsc in bone tissue engineering applications offers a powerful new tool in the development of clinical strategies for the repair of various bone disorders including osteogenesis imperfecta and osteoporesis . our lab previously showed that heparan sulfate isolated from neuroepithelial cells increases the proliferation and osteogenic differentiation of hmsc ( 12 ). the present study showed that male and female liver hs has been shown to increase proliferation and differentiation of hmsc . we demonstrated that fml hs at lower concentration increased highest cell number in maintenance medium and mml hs at lower concentration increased highest cell number in osteogenic medium . the degree of sulfation was an important determinant of heparan sulfate ability to effect the proliferation and mineralization of hmsc . we hypothesized that fml hs with high n - sulfation was important for hmsc proliferation in normal maintenance medium , mml hs with high n - unsubstituted glcnh 3 + was important for hmsc proliferation in osteogenic medium and also mml hs with high n - unsubstituted glcnh 3 + was important for hmsc osteogenic differentiation . we tested the effect of different de - o - sulfated heparin on hmsc growth in normal and osteogenic differentiation medium . our data showed that only de - n - sulfated heparin does not increase hmsc proliferation when compared to 10 % serum in normal growth medium and 2 - de - o - sulfated or 6 - de - o - sulfated heparin has less effect on proliferation ( data not shown ). however , de - n - sulfated , 2 - de - o - sulfated or 6 - de - o - sulfated heparin had no effect on the hmsc proliferation in osteogenic medium ( data not shown ). in addition to this we tested the differentiation ability of both hs on confluent cells , the hs were added to post confluence culture of hmsc , the results showed there is no difference in the alizarin red s and von kossa staining after 7 , 14 and 21 days culture period ( data not shown ). from the above result , we suggested two possibilities to explain the effect of mml hs at lower concentration inducing high osteogenic differentiation of hmsc : ( 1 ) in osteogenic medium lower concentration of mml hs treated hmsc produce higher cell number than fml hs treated cells for 5 day culture period . because of the higher cell number in mml hs treated cells , the initiation of osteoblast phenotype starts earlier than fml hs treated cells , this is correlated with the higher mineralization in mml hs treated cells . yang et al ( 42 ) showed that the degree of hs sulfation was an important determinant for hmsc osteogenic differentiation and mineralization . bovine aortic smooth muscle cells ( basmc ) mineralization was inhibited by highly sulfated heparin , but n - desulfated heparin or less sulfated dermatan sulfate had no inhibition . our data showed that total sulfation of mml hs ( 66 % with only 18 % n - sulfation ) was lesser than the fml hs ( 85 % with 62 % n - sulfation ), the less n - sulfated mml hs at lower concentration 300 ng / ml showed higher osteogenic differentiation and mineralization . these findings indicated that , overall , charge is the significant determinant for hmsc osteogenic differentiation and mineralization . in control experiments , the effect of different de - o - sulfated heparins on hmsc growth in normal and osteogenic differentiation medium was also assessed . only de - n - sulfated heparin failed to improve hmsc proliferation ; 2 - de - o - sulfated or 6 - de - o - sulfated heparin had less effect on proliferation and neither de - n - sulfated , 2 - de - o - sulfated nor 6 - de - o - sulfated heparin had any effect on the hmsc proliferation in osteogenic medium ( data not shown ). lower concentrations of mml hs are presumably able to induce greater levels of osteogenic differentiation in hmscs because cell proliferation was greater over the 5 days , so resulting in faster rates of confluence , which in turn triggered more efficient osteoblast phenotypic development through better matrix mineralization ; yang et al . ( 46 ) have also shown that the degree of hs sulfation is an important determinant of rates hmsc osteogenic differentiation and mineralization . bovine aortic smooth muscle cell mineralization is inhibited by highly sulphated heparin , but n - desulfated heparin or the less sulfated dermatan sulfate has no inhibitory activity . our data however shows that mml hs , with its lesser sulfation levels ( 65 % with only 11 % n - sulfation ) was better at triggering osteogenic differentiation and mineralization than the fml hs ( 85 % with 61 % nsulfation ). these findings indicate that hs structure , and not charge density , is the significant determinant for osteogenic differentiation and mineralization . we showed that gender specific liver hs modulate hmsc proliferation and differentiation . osteogenic differentiation has been genetically confirmed by sequential mrna expression pattern of osteoblast associated marker genes and their apparition during the differentiation of hmsc . our real - time pcr data demonstrated that hs treatment increases osteogenic differentiation related genes alp , bsp2 , opn and runx2 . the role of these genes on osteogenic differentiation of msc has been well defined ( 43 - 45 ). alp is known to be an early marker for osteoprogenitor cells ; its maximum expression was observed on day 7 of the differention period and decreased after day 7 until day 21 during the mineralization phase . bone sialoprotein ( bsp2 ) was described as a late marker of developing osteoblasts appearing with matrix mineralization and was maximally expressed on day 21 for both hs treated samples . these results suggest that liver hs induces upregulation of osteogenesis related genes , which facilitate hmsc to respond to osteogenic signals and subsequently matrix mineralization . finally , morphological and genetic analysis proved the potential usefulness of gender specific liver hs on hmsc proliferation and osteogenic differentiation . the abbreviations used are : gags , glycosaminoglycans ; hs , heparan sulfate ; tgags , total glycosaminoglycans ; mml hs , male mouse liver hepran sulfate ; fml hs , female mouse liver heparan sulfate ; hmsc , human mesenchymal stem cells ; alp , alkaline phosphatase ; bsp2 , bone sioloprotein 2 ; opn , osteopontin , dp , degree of polymerization . 1 . casu , b ., and lindahl , u . 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