Patent Application: US-200913000130-A

Abstract:
isolated active compound of a naturally occurring fraction of enamel matrix derivatives , having at least one of each two n - terminal polypeptide fragments of amelogenin , which are at least 95 % identical to an amino acid sequence as shown in seq . id . no : 1 and / or 2 . the invention relates to the use of said isolated active compound of a fraction and / or of the fraction itself , and / or the at least one of each two polypeptide fragments for use as a medicament and / or for the manufacture of a pharmaceutical composition for a variety of different medical indications such as inducing and / or promoting cementogenesis , bone growth and / or binding between parts of living mineralised tissue , for bonding of a piece of living mineralised tissue to a bonding site on a piece of other living tissue , for endorsing binding between hard tissues , for inducing regeneration of dentin , and / or for filling a mineralized wound cavity and / or tissue defect following from a procedure and / or trauma .

Description:
the separation and purification of the different fractions from complete emd has been performed in serial hplc column processes : 1 ) size exclusion hplc ( tkssw 2 , 000 - 600 × 30 ) in acetonitrile ( acn )/ 0 . 9 % nacl 2 ) reverse phase hplc ( ymc - c8 , 250 × 20 mm ), in a linear gradient of 30 % acn / 0 . 9 % nacl to 60 % can / 0 . 9 % nacl , after this step , with a reverse phase hplc column ( like in step 1 )), the components a ( including the two distinct components a1 and a2 , both of which are & gt ; 20 kd and recognized by anti - amelogenin antibodies ( conventional antibodies against emd , biora ab , se ), b , b1 , b2 , b3 , fraction c , c3 , c4 , d , d2 were showing as distinct peaks and could be separated by fractionating . 2 ) a to separate 4 distinct subfractions ( cp1 , cp2 , cp3 , cp4 ) from the fraction c fraction , a reverse phase hplc column was used ( ace - c4 with a linear acn - acidic gradient ) ( advanced chromatography technology , usa ) 3 ) the last step was intended to remove acn from the samples by using a hi - trap desalting column ( hr 16 / 20 ) ( ammersham biosciences , se ). to determine concentrations of the fractions , a size exclusion hplc column was utilized ( same as in step 1 )) with pbs ). commercially available primary human osteoblasts from both femur and tibia of different donors ( nhost cell system ; cambrex , walkersville , md ., usa ) were grown in osteoblast growth media ( ogm , cambrex ). cultured osteoblasts were exposed to hydrocortisone hemisuccinate ( 200 nm ) and h - glycerophosphate ( 10 mm ) ( cambrex ) in ambient medium to facilitate mineralization . the phenotype of cells was characterized based on the expression levels of alkaline phosphatase ( alp ), collagen type 1 , osteocalcin , and cd44 ( late differentiation marker ), and formation of mineralization nodules . human periodontal ligament cells ( pdl , pooled from 3 donors , biowhittaker , passage 6 ), were treated as described elsewhere ( gestrelius , s ., andersson , c ., lidstrom , d ., hammarstrom , l . & amp ; somerman , m . in vitro studies on periodontal ligament cells and enamel matrix derivative . 3 clin periodontol 24 , 685 - 92 ( 1997 )). the osteosarcoma cell line saos - 2 ( attc htb - 85 ) was obtained from american type culture collection ( rockville , md .). saos - 2 cells were grown in mccoy &# 39 ; s 5a medium ( pm ) supplemented with 10 % fcs and 1 % penicillin - streptomycin solution . the mouse osteoblastic cell line mc3t3 - e1 was obtained from deutsche sammlung von mikroorganismen and zellkulturen ( dsmz no acc 210 ; braunschweig , germany ) and maintained in a - mem ( paa , linz , austria ) containing 20 mm hepes , 10 % fcs ( paa ), and 1 % penicillin - streptomycin solution . the monoclonal osteosarcoma cell line , ohs , was a kind gift from dr . bruland ( the norwegian radium hospital , oslo , norway ). the cells were cultured in rpmi 1640 ( paa ) with 10 % fcs ( paa ), 50 iu / ml penicillin , and 50 g / ml streptomycin . colony - forming - unit fibroblasts ( cfu - f ), human mesenchymal stem cells which were separated and characterized by flow analysis cytometry ( facs ) were obtained from the bone marrow of two voluntary donors ( radium hospital , oslo ) and treated in the same way as the osteoblasts ( mentioned above ). 16 % sds tricine gels , loaded 0 . 5 μg fraction c and 5 μg emd per lane . western blot of antibody raised against antigen code epo42543 in rabbit sk3184 ( antigen peptide ═ sygyepmgwlh , corresponding to c - terminal of trap ), 1st ab diluted 1 : 500 , 2nd ab ( anti rabbit biotin conjugate , sigma ) diluted 1 : 1 , 000 , avidin alkaline phosphatase conjugate diluted 1 : 20 , 000 ( sigma ). traditional endpoint techniques were used as described elsewhere ( reseland , j . e . et al . leptin is expressed in and secreted from primary cultures of human osteoblasts and promotes bone mineralization . 3 bone miner res 16 , 1426 - 33 ( 2001 )). cells were cultured after treatment with 5 ug / ml peptides or fraction c ( 50 ug / ml emd respectively ) for 24 h , 4d and 7d and messenger rna was extracted using magnetic beads ( dynal a s . oslo , norway ) and cdna was synthesized from cell lysates according to the manufacturer &# 39 ; s instructions ( iscript one - step rt - pcr with sybr green , biorad ). real time pcr has been performed according to the manufacturer &# 39 ; s protocol ( icycler , biorad ). each reaction has been run in duplicate , and results represent the mean values of at least 2 independent cell donors . primary osteoblasts were cultured in t75 culture flasks and cultured after treatment for 24 h , 72 h or 7d respectively and lysed in 7 ml trizol solution ( gibco , usa ). trizol lysates were kept at − 70 ° c . until rna isolation according to the manufacturer &# 39 ; s protocols and further processing ( dep . of medical biochemistry , oslo university , norway ). double stranded cdna and biotin labeled crna probes were made from 5 μg total rna using the superscript choice system ( invitrogen ) and the enzo bioarray respectively . procedures were according to recommendations from affymetrix . this crna was hybridized to hu - 133a chips ( affymetrix ) containing cdna oligonucleotides representing more than 22 , 000 transcripts followed by washing and staining on the genechips fluidics station 450 ( affymetrix ) according to manufacturers instructions . the chips were scanned on the affymetrix genearray ® 2500 scanner . the quality of the rna and probe was controlled by an affymetrix based test measuring the ratio between 5 ′ and 3 ′ mrnas for α - actin and gapdh and found to be highly satisfactory . the datasets were processed by the affymetrix mas5 . 0 software , and signal values representing the expression level of each transcript were generated . each procedure has been done in twice in parallel and the resulting values indicate the mean of at least two donor &# 39 ; s cells in duplicate experiments . study to investigate the efficacy of frcation c on mandibular bone formation . 3 minipigs ( 2 pigs 12 - 18 months , 1 adult pig & gt ; 18 months ) surgery : flap of the lower jaw q3 and q4 , drilled cavities with a diameter of 10 mm treatments : 5 defects treated with a collagen sponge impregnated with fraction c 3 defects treated with fraction c in peg - thiol mixed with peg - acrylate in cekol buffer 2 defects as a negative control with collagen sponges with pbs 1 defect with sham surgery demineralisation : in 0 . 5 m edta ph 8 . 0 ( in situ prep ) for 8 - 12 weeks . changing buffer 2 times a week . histology : after about 12 weeks sectioning and staining for in situ hybridisation and histology . peg in combination with fraction c in 12 / 2005 . 2 . 3 mm diameter defects in the mandibles , 100 ug fraction c / ml gel , 4 animals , 2 defects per animal ( each side one defect ), two time points for healing : 1 and 2 weeks , evaluation by histology group 1 week : 1 ) peg / fraction c vs . peg /- 2 ) peg / fraction c vs . collagen / fraction c group 2 weeks : 3 ) peg / fraction c vs . peg /- 4 ) peg / fraction c vs . collagen /- cells were seeded in 24 wells plates at a density of 20 , 000 cells / well in 1 ml medium ( mdm medium + 10 % fcs ). for each experimental group n = 3 . as controls served cells grown without the addition of peptides . the separate peptides were added immediately after cell seeding at a final concentration of 10 μg / ml medium . after 4 , 8 and 24 hours respectively the media were aspirated and the wells rinsed with 2 × 1 ml pbs and the cell layers were trypsinized with 0 . 2 ml of trypsin / edta solution to count attached cells by the use of a cellcounter , chemometec a / s . cells were seeded in 48 wells plates at a density of 5 , 000 cells / well / ml medium . for each experimental group n = 3 . as controls served cells grown without addition of peptides . the separate peptides were added immediately after cell seeding at a final concentration of 10 μg / ml medium . after ( 4 ) or 5 days ( wells were checked not to be confluent since the proliferation phase otherwise could be passed ) cells were detached and counted as for the cell attachment assay . cells were seeded in 48 wells plates at a density of 5000 cells / well / ml medium . for each experimental group n = 3 . as controls served medium , cells with medium and peptides in medium without cells ( in case of background values ). the peptides were added immediately after cell seeding at a final concentration of 10 μg / ml medium . after 5 days ( as for the cell proliferation study ) the medium from each well was transferred to eppendorf tubes , centrifugated at 1 , 000 rpm for 5 min . supernatants were kept at − 20 c until the assays were performed . cell layers were washed with 2 × 1 ml pbs . after addition of 0 . 5 ml of milliqwater to the different cell layers bended pipette tips were used to scrape of the cells . cell suspensions were transferred to eppendorf tubes and lysed by the use of an ultrasonic water bath for 10 min and centrifugated at 1000 rpm for 5 min . the cell lysates were kept at − 20 c until the assays were performed . changes of medium ( with peptides included as for the initial seeding of cells ) were made after 3 days . for determining proteins in culture supernatants the following kit assays were used : fig1 . rt - pcr of cultured osteoblasts ( one donor , nho - 3 ). a1 stimulates expression of the following gene products to a greater extend than a2 , most prominently after 7 days : osteocalcin and leptin . values represent the relative concentrations of each protein relative to α - tubulin and are shown as the means of the single results from duplicate experiments . fig2 . rt - pcr of two osteosarcoma cell cultures ( saos - 2 and hos ) stimulated with fractions a1 and a2 : expression of alp and oc increased , especially by treatment with a1 . no effect was observed on the expression of cbfa - 1 and cd44 with a1 , whereas expression of cd44 was slightly reduced after treatment with a2 . n = 3 , values represent the relative concentrations of each protein relative to α - tubulin and are shown as the mixed means from the two cell lines results with ± sd . a1 and a2 have different effects on cultured osteosarcoma cells a1 stimulates bone formation by up - regulation of alp and osteocalcin a2 reduced the expression of cd44 , an osteocyte markerprotein analysis by elisa with elisa , an increase of il - 6 secretion , with a1 more potent than a2 , has been observed ( data not shown ). the increase of il - 6 has to be taken carefully into considerations , since il - 6 is a potent pro - inflammatory factor , which can strongly influence effects of other factors . to further characterize the fraction c from emd , sds page with subsequent western blotting was performed . fraction c showed mainly as a band at around 5 kda ( fig3 ). to compare the effects of pth peptides and emd on primary osteoblasts , cells of two donors were treated synchronously and expression patterns were analysed by affymetrix gene chip analysis . interestingly , 24 h after stimulation , expression of 254 genes were more than 2 - fold changed in the same direction with both emd and pth from non - treated controls . moreover , no genes were found to be oppositely regulated as much as 2 - fold by emd and pth . statistical analysis confirmed a positive correlation between expression changes after stimulation with pth and emd ( correlation coefficient = 0 . 805 ; p & lt ; 0 . 001 ). a subset of these gene expression changes were verified by real time rt - pcr confirming the results obtained with affymetrix analysis ( see table 3 / fig1 ). to compare effects of fraction c in comparison to emd , primary osteoblasts from 1 donor ( nho - 3 ) were stimulated with the 5 kda fraction fraction c and results compared with cells treated with emd . log 2 ( stimulated / control ) values were considered as ≠ 0 only when ≧| 0 . 5 |. platelet - derived growth factor receptor , alpha polypeptide ( pdgfra ): cell proliferation /// platelet - derived growth factor , alpha - receptor activity /// atp binding /// transferase activity ; integral to plasma membrane fibronectin 1 ( fn1 ): cell motility /// cell adhesion /// signal transduction ; cell adhesion molecule activity extracellular matrix /// extracellular space /// soluble fraction /// inflammatory_response_pathway genes that are down - regulated by fraction c , but not regulated by emd wnt1 inducible signaling pathway protein 1 : regulation of cell growth /// cell adhesion /// signal transduction /// cell - cell signaling /// cell growth and / or maintenance integrin , alpha v ( vitronectin receptor , alpha polypeptide , antigen cd51 ): cell - matrix adhesion /// integrin - mediated signaling pathway genes that are up - regulated by emd but not regulated by fraction c mmp - 14 ( matrix metalloproteinase 14 ) ( membrane - inserted ): proteolysis and peptidolysis actin , alpha 2 , smooth muscle , aorta ( acta2 ): muscle development ; motor activity /// structural constituent of cytoskeleton /// structural constituent of muscle ; striated muscle thin filament /// actin filament angiopoletin 1 : angiogenesis /// signal transduction aggrecan 1 ( chondroitin sulfate proteoglycan 1 ): cell adhesion /// heterophilic cell adhesion vinculin : cell adhesion dentin sialophosphoprotein ( dspp ): extracellular matrix lamin a / c thrombospondin 1 : cell motility /// cell adhesion /// development /// neurogenesis /// blood coagulation follistatin ( fst ): development /// negative regulation of follicle - stimulating hormone secretion activin inhibitor activity extracellular tgf - beta signaling pathway wingless - type mmtv integration site family , member 5a ( wnt5a ): signal transduction /// frizzled - 2 signaling pathway /// cell - cell signaling /// development /// embryogenesis and morphogenesis /// soluble fraction ; wnt_signaling genes that are down - regulated by emd but not regulated by fraction c cd97 antigen : cell motility /// inflammatory response /// immune response /// cell adhesion /// cell surface receptor linked signal transduction /// g - protein coupled receptor protein signaling pathway /// cell - cell signaling transforming growth factor , beta receptor ii ( 70 / 80 kda ) ( tgfbr2 ): protein amino acid phosphory - dation /// transmembrane receptor protein serine / threonine kinase signaling pathway /// tgf - beta ligand binding to type ii receptor /// positive regulation of cell proliferation fgf - 2 ( fibroblast growth factor 2 ( basic )): regulation of cell cycle /// activation of mapk /// angiogenesis /// chemotaxis /// signal transduction /// ras protein signal transduction /// cell - cell signaling /// histogenesis and organogenesis /// neurogenesis /// muscle development /// cell proliferation cd14 antigen : phagocytosis /// apoptosis /// inflammatory response /// immune response /// cell surface receptor linked signal transduction leptin receptor : energy reserve metabolism /// cell surface receptor linked signal transduction /// development dead ( asp - glu - ala - asp ) box polypeptide 24 c ) conventional and real time rt - pcr and protein expression by human osteoblasts table 4a ) conventional and real time rt - pcr analysis of human tibia and femur osteoblasts treated with single fractions separated from emd . the values represent the mean values from two donors in duplicate . values marked with stars show results from real time rt - pcr , not marked values have been generated by conventional rt - pcr . 4b ) expression of proteins into the culture medium after stimulation with single fractions separated from emd . the values represent the mean values from two donors in duplicate . d ) elisa analysis of factors secreted by human osteoblasts ( nho ) or mesenchymal stem cells ( cfu - f ) moreover , preliminary experiments with cfu - f cells stimulated with fraction c and emd showed for both agents a tendency of reducing expression levels of the pro - inflammatory cytokines il - 1β , tnf - α , and il - 8 ( assayed with elisa , data not shown ). some of different fractions from emd exert effects on different cells in vitro . the focus on fraction c and b3 is based on these experiments showing the most relevant modifications in the expression of genes and proteins related to osteogenesis . first animal experimental results are with fraction c in combination with collagen as a carrier matrix . the use of collagen is controversial , due to its potential to induce inflammation when applied in vivo , but on the other hand , it is the currently best - characterized carrier for this kind of applications . first results ( n = 1 ) from affymetrix gene array indicate that emd seems to be generally more regulation - active for osteoblasts than fraction c . this was confirmed with elisa analysis of primary osteoblasts for soluble rank and il - 6 , which were both upregulated after exposure to emd , whereas fraction c ( table 5 ) did not induce such a change in these cells . however , real time rt - pcr showed a bone - specific upregulation of cbfa - 1 in primary human osteoblasts and of osteocalcin in mesenchymal stem cells , whereas emd did not show this effect ( table 4 ). this indicates a less broad , but rather more osteogenic effect of fraction c compared to emd . additionally , the pro - inflammatory potential of fraction c seems to be differentiated compared to that of emd . 2 . an in vitro study to evaluate the effect of fractions of emd and synthetic peptides on periodontal ligament ( pdl ) cells to study the effect of synthetic peptides based on fractions from emd ( seq id no : 1 , 2 , 3 , or 4 ) on cell proliferation , the activity of ap , and the expression of osteocalcin ( oc ) and tgf - beta . synthesized peptides , 1 mg / ml stock in h2o . before addition to the cells the peptide solution will be sterile filtrated , 0 . 22 μm and concentration measured by uv absorption a280 nm . different concentrations will be prepared by diluting stock solution in sterile 0 . 1 % acetic acid . peptide solutions will be added to the cells at concentrations of 1 / 5 / 20 / 100 μg / ml . emd ( positive control will be applied to the cells at a final conc . of 100 μg / ml ( positive control ). cells will be treated with the corresponding volume ( to the application of emd ) of 0 . 1 % acetic acid as negative controls . cells will be seeded in 24 wells plates at a density of 5 , 000 cells / well / ml medium in the presence of the stimulating factors for 1 , 2 or 5 days in parallel plates . for the group stimulated 5d , the medium will be changed ( including stimulating factors ) after 2d . ap activity ( early response ) will be determined in cell lysates according to standard protocols . culture supernatants will be collected and frozen at − 20 ° c . until determining concentrations of tgf - beta ( early response ) and osteocalcin ( late response ). 3 . 5 kda component of enamel matrix derivative possesses osteogenic properties analysis of emd by high performance liquid chromatography revealed the presence of three main components : ( 1 ) a 20 kda protein [ fraction a ], ( 2 ) two proteins of 12 and 9 kda , and ( 3 ) a 5 kda peptide [ fraction c ]. two of these components ( fraction a and c ) have been purified and characterized . the fraction a protein corresponds to the full length amelogenin protein and the fraction c is the n - terminal part of this protein ( 3 ). the aim of the present study was to examine the effect of these two emd components on osteoblasts . methods : confluent cultures of mg63 human osteoblast - like cells and normal human osteoblasts were treated with or without emd , recombinant human amelogenin ( rhamel ), fraction a ( 0 . 01 - 100 μg / ml ) or fraction c ( 0 . 1 - 250 μg / ml ) for 24 hours . effects on dna content and alkaline phosphatase specific activity ( alp ), and osteocalcin ( ocn ), osteoprotegerin ( opg ), vascular endothelial growth factor a ( vegf - a ) and fibroblast growth factor - 2 ( fgf - 2 ) levels in the conditioned media were determined . results : fraction c reduced dna content of mg63 cells in a dose - dependent manner and increased osteoblast differentiation markers like alkaline phosphatase and osteocalcin with peak increases at 10 mg / ml . the peptide also increased local factors like opg , vegf and fgf in a dose - dependent manner . the effects of the fraction c were similar to those of the fraction a , emd , and rhamel . moreover , normal human osteoblasts responded in a similar manner to mg63 cells . see fig4 . conclusions : these results indicate that the fraction c component of emdogain possesses osteogenic activities and that the osteogenic effects of amelogenin may be due the n - terminal region of the protein . 4 . 5 kda component of enamel matrix derivative induces osteoblast differentiation emd ( enamel matrix derivative ) is a protein complex enriched in amelogenins which correspond to the bioactive part of straumann emdogain ®. recent published analysis of emd by high performance liquid chromatography revealed the presence of its three main components : ( 1 ) 20 kda , ( 2 ) [ 12 + 9 ] kda , ( 3 ) 5 kda . two of these components ( 20 kda , 5 kda ) have been purified : the first one ( 20 kda ) is suspected to correspond to the full length amelogenin protein and the second one ( 5 kda ) to the n - terminal part of this protein . these two components have been tested in cell cultures comparatively to a recombinant human amelogenin ( rhamel ) over - expressed in escherichia coli and to the emd complex . mg63 osteoblast - like cells , originally isolated from a human osteosarcoma , were obtained from the american type culture collection ( rockville , md .). these cells are well characterized and exhibit numerous osteoblastic traits , including increased alkaline phosphatase activity and osteocalcin synthesis in response to 1α , 25 ( oh ) 2d3 . moreover , observations using mg63 cells have been confirmed using normal human osteoblasts , normal mouse calvarial osteoblasts , fetal rat calvarial cells and other osteoblast cell lines , and the results correlate with clinical performance in animals and humans . mg63 cells were cultured in dulbecco &# 39 ; s modified eagle medium ( dmem ) containing 10 % fetal bovine serum ( fbs ) and 1 % penicillin and streptomycin at 37 ° c . in an atmosphere of 5 % co2 and 100 % humidity . cells were seeded at 15 , 000 cells / well , media were changed every 48 h . at confluence the different concentration of the material was added to the culture for 24 we used two determinants of osteoblast differentiation in day - 7 cultures : alkaline phosphatase specific activity [ orthophosphoric monoester phosphohydrolase , alkaline ; e . c . 3 . 1 . 3 . 1 ] of cell lysates , and osteocalcin content of the conditioned media . cells . alkaline phosphatase is an early marker of differentiation and reaches its highest levels as mineralization is initiated . osteocalcin is a late marker of differentiation and increases as mineral is deposited . lysates were prepared using isolated cells collected by centrifugation after counting . enzyme activity was assayed by measuring the release of para - nitrophenol from para - nitrophenylphosphate at ph 10 . 2 and results were normalized to protein content of the cell lysates . the levels of osteocalcin in the conditioned media were measured using a commercially available radioimmunoassay kit ( human osteocalcin ria kit , biomedical technologies , stoughton , mass .) and normalized to dna content . the conditioned media from the day - 7 cultures were also assayed for growth factors and cytokines . osteoprotegerin was measured using enzyme - linked immunosorbent assay ( elisa ) kit ( dy805 osteoprotegerin duoset , r & amp ; d systems , minneapolis , minn .). vegf was assesseds using an elisa kit ( rnd systems ). briefly , 100 ul of conditioned media was added to precoated plates and incubated for two hours . a specific detection antibody was added to the plate and incubated for an additional two hours . the absorbance of the samples was read using a microplate reader and the results analyzed using a standard curve . dna measurment - cells were sonicated in 0 . 5 % trition - x 100 and dna was measured using quant - it ™ pico green ® kit ( invitrogen ), which measures double - stranded dna . the amount of dna was measured using a fluorescence microplate reader using a dna standard from 0 . 2 to 200 ng of dna . 5 . identification of the active compound of fraction c ( 5 kda ; c1rp or c2rp ) identification of the active compound of fraction c ( 5 kda ; c1rp or c2rp ). we have tested synthetic peptides of amelogenin ( exon1 , 3 and 5 ) of various lengths . fraction c , having a molecular weight of 5250 . 4 g / mol was used at a final concentration of 5 μg / ml ( 0 . 9 μmol / l ). the peptides were used in a molar concentration similar to fraction c . wbra001 enhanced osteocalcin and cd44 expression and secretion similar to fraction c , the smaller peptides had no effect on the bone markers in osteoblasts . 5 kda amelogenins , isolated from emd batch 3113 was identified to contain both ; c1rp or c2rp ( 1 - 43 and 1 - 45 trap ), whereas the fraction c isolated from emd batch 9121 contains not only c1rp and c2r ( 1 - 43 and 1 - 45 trap ), but 2 further , unidentified minor peaks . the effect of the different isoforms on expression of bone markers was tested on osteoblasts . the effect on leptin , il - 6 and opg secretion was similar for the mix of ; c1rp and c2rp ( 3113 ) and fraction c . the mix ( 3113 ) induced an acute (& lt ; 24 h ) release of osteocalcin to the culture medium ( figure below ). mrna expression was however increased by fraction c after & gt ; 3 days of incubation . the isolated fractions of c1rp and c2rp induced a higher ldh activity in the culture medium than fraction c and the mix of peptides ( 3113 ). this slightly more toxic effect on the cells made it difficult to see any difference in effect between these two purified fractions . conclusion ; the mix of c1rp and c2rp ( 3113 ) was more potent than fraction c alone in stimulating early osteoblast differentiation . at later stages the fraction c is a more potent inducer of osteocalcin expression . in the present context , both , bone and cementum formation , are included as mineralized tissues . the extracellular matrix ( ecm ) consists mainly of type i collagen . osteogenic cells synthesize alkaline phosphatase ( alp ) playing a key role in mineralization ( incorporation of calcium into the ecm ). therefore , alp is used as an early marker for osteogenic differentiation in vitro . alp increases within the first days and decreases when mineralization takes place ( detection of osteocalcin ). minimum essential medium eagle ( mem )+ 10 % fetal calf serum + 1 % penicilin - streptomycin (× 100 )+ 1 % non essential amino acid solution (× 100 )+ 1 % l - glutamine ( 200 mm ) seeding density mg63 cells in passage 4 were seeded with a density of 10 , 000 cells / cm2 on 96 wells culture plates . sampling was performed after 24 hours and 7 days in vitro . to determine the proliferation capacity , we used a colorimetric immunoassay based on the measurement of bromodeoxyuridine , brdu incorporation during dna synthesis . 10 μl of brdu labelling solution ( roche , penzberg , germany ) were added to cell culture and cells were incubated for 2 hours at 37 ° c . the labeling and quantification was carried out as described in the instruction manual . absorbance was measured with versamax microplate reader at 450 nm with a reference wavelength of 690 nm . results are reported as optical density ( od ). furthermore , the proliferation reagent wst - 1 test was performed to assess cell proliferation and viability . wst - 1 solution was added in a final solution of 1 : 10 to the monolayer culture and cells were incubated at 37 ° c . for a further 1 hour . the absorbance of the supernatants was measured spectrophotometrically using versamax micro plate reader ( molecular devices , california , usa ) at 420 nm - 480 nm with a reference wavelength of 600 nm . results are reported as optical density ( od ). for the determination of alp activity the enzyme activity of the supernatant was assayed spectrophotometrically at 405 nm as the release of p - nitrophenol ( sigma , st . louis , mo ., usa ) from p - nitrophenyl - phosphate over time . mg63 cells were lysed with pbs and 0 . 05 % triton - x100 on day 1 and 7 after treatment . the alp was expressed as pm / minute / mg of protein . ( s : synthesized ; rp : reverse phase ; c1 : first peak of the chromatogram ; c2 : second peak of the chromatogram ) we have performed 1 experiment with n = 5 . sampling on day 1 ( 1 day in vitro ) and on day 7 ( 7 days in vitro ) the results indicate that within the first 24 hours after treatment proliferation capacity was initiated , demonstrated by the increased od values due to brdu incorporation . after 7 days proliferative activity of mg63 cells is decreased with confluence and matrix maturation . fraction c treatment caused an increase of 10 % in proliferative activity after 24 hours . a maximum of 24 % increase was detected in cells supplemented with the combination of the synthesized c1rp and c2rp . with regard to the high proliferation capacity after 24 hours of treatment , mg63 cells demonstrate an increased mitochondrial activity after 7 days . the study showed a stimulation of cell viability and proliferation by all components compared to the negative control ( untreated monolayer cultures ). synthesized peptides increase the proliferation of mg63 by 45 %, whereby the phosphorylation seems not to enhance the effect . it can be concluded that there is a clear effect of fraction c components ( c1rp , c2rp ) on cell proliferation and viability after 7 days in culture , the combination of c1rp + c2rp does increase the viability . alkaline phosphatase is an early marker for osteogenic maturation in vitro . therefore discussion should mainly focus on results of day 1 rather than day 7 after treatment . alp activity is typically reduced after 7 days when mineralization takes place . the data indicate that frac c enhance the alp activity after 1 day of about 125 % compared to the negative control , whereby the single components ( c1 / c2rp ) as well as the combination do not show a significant effect . furthermore , synthesized peptides demonstrate low alp activities after 1 day , independent from their phosphorylation status , similar to the negative control . the combination ( sc1rp - p + sc2rp - p ) of both peptides seems to have a slightly inhibitory effect on alp activity , which has to be discussed concerning the peptide interaction . also the positive control ( emd ) showed a decrease of alp activity versus negative control , which has not been demonstrated by supplementation of 100 μg / ml emd ( not shown ). higher concentrations of emd demonstrate a 1 . 5 fold increase of alp activity ( versus negative control ) after 1 day , and still increasing after 7 days ( not shown ). this has also been demonstrated by boyan et al . in former studies , showing almost no effect on the proliferation but on differentiation . aim : to study attachment of pdl cells on different emd fractions using xtt assay for assessing of cell number solution of emd and fractions are already prepared in bicarbonate buffer for glp49 / 37 . 200 ul solutions are poured in each well ( amount of proteins added to wells is about 5 times lower than in glp49 / 37 ( 200 ul / well instead of 1 ml ) as well area is 5 . 5 times smaller ) incubation overnight at 4 c the next day , the plates are washed 2 times with 200 ul pbs before adding the cells one flask of subconfluent pdl mix cells is washed 2 times in pbs and incubated with trypsin / edta for 2 - 3 min . trypsin is neutralized with medium , cells are spin down and counted . a cell suspension at 50 000 cells / ml is prepared and 200 ul is added in each well ( 10 000 cells / well ) after 4 and 8 - hour incubation , floating cells are removed by two washes with 200 ul pbs wells are refilled with 150 ul normal medium and 75 ul xtt mix is added in each well one extra row containing 150 ul medium and 75 ul xtt mix is added to serve as blank . cells are incubated for 2 hours before reading the plate at 480 nm with wavelength reference at 650 compare to glp59 / 03 pdl mix cells of this experiment attached much quicker to the uncoated plastic . a significant number of cells exhibited spreading in the uncoated condition after 4 - hour incubation ( in glp59 / 03 , cells remained round in the uncoated wells after an incubation up to 8 hours ) see fig1 . the results show a pattern very similar to those obtained in glp49 / 37 . however the differences between the control and the difference fractions or emd are not as high ( only one condition was significantly different of the control ), suggesting that cell number assessment with xtt kit is not very sensitive . another explanation is that the non - specific attachment of pdl cells is higher in 96 - well plate than in 24 - well plate reducing the effect ( see remark ) 1 . kawase , t . et al . enamel matrix derivative ( emdogain ) rapidly stimulates phosphorylation of the map kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells . 3 periodontal res 36 , 367 - 76 ( 2001 ). 2 . lyngstadaas , s . p ., lundberg , e ., ekdahl , h ., andersson , c . & amp ; gestrelius , s . autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative . j clin periodontol 28 , 181 - 8 ( 2001 ). 3 . kawase , t ., okuda , k ., yoshie , h . & amp ; burns , d . m . anti - tgf - beta antibody blocks enamel matrix derivative - induced upregulation of p21waf1 / cipl and prevents its inhibition of human oral epithelial cell proliferation . 3 periodontal res 37 , 255 - 62 ( 2002 ). 4 . haase , h . r . & amp ; bartold , p . m . enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells . 3 periodontol 72 , 341 - 8 ( 2001 ). 5 . van der pauw , m . t ., van den bos , t ., everts , v . & amp ; beertsen , w . enamel matrix - derived protein stimulates attachment of periodontal ligament fibroblasts and enhances alkaline phosphatase activity and transforming growth factor beta1 release of periodontal ligament and gingival fibroblasts . 3 periodontol 71 , 31 - 43 ( 2000 ). 6 . tokiyasu , y ., takata , t ., saygin , e . & amp ; 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