Patent Application: US-27623003-A

Abstract:
the present invention provides a medical implant material comprising mammalian transglutaminase and a polymer , wherein the transglutaminase is provided in the absence of free divalent metal ions and wherein the polymer is associated with the transglutaminase binding protein . preferably , the transglutaminase is a tissue transglutaminase , which is coated on , impregnated into or covalently linked to the polymer . the polymer may be naturally occuring or synthetic , and may be biodegradable or non - biodegradable . the medical implant material may further comprise a reinforcing agent and / or one or more additional polymers . the invention further provides the use of a mammalian transglutaminase in a method for improving the biocompatibility of a medical implant material , the method comprising the steps of providing a medical implant material comprising a polymer associated with a binding protein for binding the transglutaminase , and treating said material with a mammalian transglutaminase .

Description:
poly ( ε - caprolactone )( pcl )( pcl650 ; solvay interox ) was purchased in pellet form and pressed in a square cavity at a temperature above melting point , in order to form a 4 mm thick meet . pcl discs ( 6 mm in diameter ) were then stamped from the polymer sheet using a cork borer and sterilised under uv light for 1 hour each side prior to use . human osteoblast ( hob ) cells were isolated from explants of trabecular bone dissected from femoral heads following orthopaedic surgery ( as described by disilvio , 1995 ). all cells were cultured in vitro using dulbecco &# 39 ; s modified eagles medium ( dmem ). this was supplemented with 10 % foetal calf serum , 1 % non - essential amino acids , 150 μg / ml ascorbic acid ( bdh , poole , u . k . ), 2 mm l - glutarine , 0 . 02m [ 4 -( 2 - hydroxyethyl )- 1 - piperazineethanesulfonic acid ], ( hepes ), 1 % penicillin / streptomycin ( all obtainable from gibco , brl , paisley , u . k .). the cells were incubated at 37 ° c . in 5 % co 2 , 95 % air . visualisation of human osteoblasts cells on pcl over 4 days using the toluidine blue stain and transmission electron microscopy ( t . e . m ) hob cells were grown on the pcl in 10 % serum containing dmem to initially assess the interaction of cells with this particular polymer surface . the toluidine blue stain allowed the attachment , spreading and proliferation of the cells - on the pcl to be observed . t . e . m . allowed the ultra structure of the cells to be observed on the biomaterial . both these techniques would indicate whether the polymer is biocompatible with this particular cell type . pcl discs were placed in a 96 well plate ( 3 replicates per sample ) and human osteoblast cells ( hobs ) were seeded onto the biomaterial in 10 % serum containing dmem at a density of 1 . 7 × 10 5 / cm 2 . tissue culture plastic ( tcp ) was used as a positive control surface . the plate was then incubated at 37 ° c . in 5 % co 2 . at days 1 and 4 after cell seeding the medium was removed and the cells were washed in sterile phosphate buffered saline ( pbs ). the cells were fixed in 4 % paraformaldehyde and 2 % sucrose for five minutes at room temperatue and then washed twice in pbs . the cells were stained with 1 % toluidine blue diluted in sterile pbs for five minutes at room temperature . the samples were then washed several times in pbs and viewed using light microscopy ( olympus sz - pt ). pcl discs were placed in a 48 - well plate ( 3 replicates per time point ) and hob cells were seeded onto the biomaterial in 10 % serum containing dmem at a density of 1 . 7 × 10 5 / cm 2 . thermanox was used as a positive control spice . the plate was then incubated at 37 ° c . in 5 % co 2 . at days 1 , 2 , 4 and 8 days after cell seeding , the medium was removed and the cells were washed in sterile pbs . the samples were fixed overnight in 1 . 5 % glutaraldehyde in 0 . 1 m sodium cacodylate buffer ( ph 7 , 4 ) then washed in the same buffer and secondary fixed using 1 % osmium tetroxide in millonig &# 39 ; s buffer for 1 hour . the samples were washed in buffer followed by a dehydration step through a series of alcohols , 50 %, 70 %, 90 %, 96 % and 100 % with two 10 - minute changes for each alcohol and 30 minutes in 100 % alcohol . araldite cy212 resin was added to give a ratio 3 : 1 with alcohol , fixed for 1 hour and then transferred to a 1 : 1 resin : alcohol mixture and left overnight and then placed in pure resin for 2 hours under vacuum inltration . the pure resin was changed twice ( 2 hours each change ) and then made into blocks . the polymers were then embedded and cured at 45 ° c . in an oven for 48 hours ( this temperature was used to prevent the pcl from melting ). ultra thin sections were then cut using a reichert ultracut e ultramicrotome , collected on copper grids and stained with 3 % uranylacetate in 50 % methanol for 6 minutes and then reynold &# 39 ; s lead citrate for 6 minutes . the samples were examined using a philips em410 transmission electron microscope at 80 kv . guinea - pig liver tissue transglutaminase ( i . e . type ii transglutaminase , ttg ) obtainable from sigma was immobilised onto pcl using three methods as shown below . each method uses the idea of immobilising the ttg to the pcl via fibronectin because fibronectin has a ttg binding site . the ttg does not have to be active to bind to fibronectin ( i . e . calcium ions are not required ). ethylenediaminetetraacetic acid ( edta ) was added to the ttg to keep the enzyme in its inactive form and prevent the enzyme from cross - linking the fibronectin or itself during the immobilisation . the ttg could be added to the pcl in drop form because the surface of the pcl was hydrophobic . a quantity of 60 μl was enough to cover the whole surface , forming a meniscus on the surface . a solution of 15 μg / ml bovine plasma fibronectin ( gibco ) and 10 μg / ml tissue transglutaminase ( ttg ) in 0 . 1m edta ( ph 7 . 4 . ), was added to the sterile pcl discs ( 60 μl per disc ). this was then left overnight at 4 ° c . after incubation , the solution was removed and the pcl was washed three times with 0 . 1m tris - hcl ( ph 7 . 4 . ), 5 minutes each wash . the discs were then transferred to the tissue culture plates . a 60 μl aliquot of 30 μg / ml bovine plasma fibronectin in sterile distilled water was added to each sterile pcl disc and left overnight in the tissue culture hood to evaporate . once the water had evaporated , a 60 μl aliquot of 20 μg / ml tissue transglutaminase in 5 mm edta ( ph 7 . 4 .) was added to the pcl . this was left to evaporate overnight in the tissue culture hood and then the discs were washed three times in 0 . 1m tris - hcl ( ph 7 . 4 . ), 5 minutes each wash . the discs were then transferred to the tissue culture plates . a 60 μl aliquot of 30 μg / ml bovine plasma fibronectin in sterile distilled water was added to each sterile pcl disc and left overnight in the tissue culture hood to evaporate . once the water had evaporated , a 60 μl aliquot of 20 μg / ml tissue transglutaminase in 5 mm edta ( ph 7 . 4 .) was added to each pcl disc . this was then left for one hour at room temperature . the ttg solution was removed and then the discs were washed three times in 0 . 1m tris - hcl ( ph 7 . 4 . ), 5 minutes each wash and transferred to the tissue culture plates . pcl discs were coated with different concentrations of fibronectin ( 0 to 50 μg / ml ) by the evaporation technique described above . the fibronectin was detected using the fibronectin elisa assay ( gaudry , 1998 ). the polymers were initially washed in 3 × 100 μl of pbs and then blocked with 100 μl of 3 % w / v bovine serum albumin ( bsa ) in pbs . the plate was incubated for 1 hour at room temperature and then washed with 3 × 100 μl of pbs before the addition of 100 μl of the primary antibody ( polyclonal rabbit anti - human plasma fibronectin , diluted 1 : 5000 in blocking buffer ). the plate was incubated for 2 hours at room temperature , then washed in 3 × 100 μl of pbs before the addition of 100 μl of the secondary antibody ( hrp conjugated goat anti - rabbit , diluted 1 : 5000 in blocking buffer ). the plate was incubated for 1 hour at room temperature and then rinsed in 3 × 100 μ 1 of before the addition of 100 μl of 0 . 1m sodium acetate . the hrp was detected using 100 μl of the developer ( 20 mls naoac , 150 μl tmb and 10 μl h 2 o 2 ) and the reaction stopped with 50 μl of 2 . 5m h 2 so 4 . the results were then read in a colourimeter ( titertek multiskan mcc / 340mk2 ) at a wavelength of 450 nm . the elisa used to detect ttg was a modification of that developed by achyuthan et al ., ( 1995 ). this relies upon the ability of ttg to bind specifically to immobilised plasma fibronectin . pcl discs were coated with 60 μl of 30 μg / ml fibronectin in sterile distilled water and then different concentrations of ttg ( 0 - 50 μg / ml ) using the two different methods as described above . the discs were washed in 3 × 100 μl of pbs and then blocked with 100 μl of 3 % w / v bsa in pbs for 1 hour at room temperature . the pcl discs were rinsed with 3 × 100 μl of pbs and bound ttg was detected using 100 μl of anti - monoclonal antibody cub7402 ( diluted 1 : 1000 in blocking buffer ). the primary antibody was left on the polymers for 2 hours at room temperature and then washed in 3 × 100 μl of pbs prior to the addition of 100 μl of the secondary antibody ( hrp - conjugated goat - anti - mouse igg ( sigma ) diluted 1 : 5000 in blocking buffer ). this was left for 1 hour at room temperature and then the hrp detected ( as described above ). to determine if ttg is in its active form when immobilised on the pcl surface to determine if fibronectin and ttg are present on the surface of pcl , the biotin - cadaverine assay was used , ( jones et al ., 1997 ). this method also allows the activity of ttg on the surface of the biomaterial to be quantified . four tissue culture plastic ( tcp ) wells and 4 pcl discs were coated with fibronectin as described above ( 3 replicates per sample were used ). for one of the fibronectin coated pcl samples , ttg was immobilised onto the pcl surface using either of the three different methods also described above . after washing the fibronectin / ttg coated pcl in 3 × 100 μl of 0 . 1m tris - hcl ( ph , 7 . 4 . ), 100 μl of homogenising buffer consisting of 0 . 25 mm sucrose , 5 mm tris - hcl ( ph 7 . 4 .) and 2 mm edta ( h 7 . 4 .) was added and incubated for different time periods up to 15 hours at 37 ° c . this was then tested for ttg activity . the other three fibronectin coated pcl discs were used for controls , which consisted of adding ttg in directly homogenising buffer containing either calcium ( positive control ) or edta ( negative control ). the other negative control was homogenising buffer only . the four tcp wells coated with fibronectin were to enable ttg activity to be analysed in the homogenising buffer taken from the fibronectin / ttg coated pcl surface and for three controls which are the same as mentioned above . the pcl or tcp were initially blocked with 100 μl of 3 % bsa in 0 . 1m tis - hcl ( ph 7 . 4 .) at room temperature for 1 hour and then washed with 3 × 100 . μl of 0 . 1m tris - hcl . for the positive controls , 100 μl of the following solution was added to the fibronectin coated tcp and pcl : 20 μg / ml guinea pig ttg , 5 mm cacl 2 , 3 . 85 mm dtt and 0 . 4 % biotin - cadaverine in homogenising buffer . for the negative controls 100 μl of the following solution was added to the fibronectin coated tcp and pcl : 20 μg / m guinea pig ttg , 5 mm edta , 3 . 85 mm dtt and 0 . 4 % biotin - cadaverine in homogenising buffer . to rule out any biomaterial interaction with the assay , another control was used which consisted of adding the following solution to fibronectin coated pcl and tcp : 5 mm cacl 2 , 3 . 85 mm dtt and 0 . 4 % biotin - cadaverine in homogeising buffer . this was also added to the fibronectin and ttg coated pcl . to the 100 μl of homogenising buffer that had been incubated with the immobilised fibronectin / ttg surface , 10 mm cacl 2 , 3 . 85 mm dtt and 0 . 4 % bc was added . the samples were then left for 2 hours at 37 ° c . after this time 100 μl of 5 mm edta in 0 . 1m tris - hcl was added to the wells , left for 10 minutes and then washed in 2 × 100 μl of 0 . 1m tris - hcl . 100 μl of extravidin peroxidase in 3 % bsa was added ( 1 : 5000 dilution ) and left at 37 ° c . for 1 h . the wells were washed with 3 × 100 μl of 0 . 1m tris - hcl and 100 μl of 0 . 1m naoac ( ph 6 . 0 ). 100 μl of the developer was added ( 20 mls naoac , 150 μl tmb and 10 μl h 2 o 2 ) and the reaction stopped with 50 μl of 2 . 5m h 2 so 4 . the results were then read in a colourimeter at a wavelength of 450 nm . to determine the effect tissue transglutaminase has on the spreading of human osteoblast cells on poly ( ε - caprolactone ) initially sterile pcl discs were coated with fibronectin and ttg as described above . controls consisted of pcl , pcl coated with fibronectin and tcp . the hob cells were harvested using trypsin , followed by blocking in serum containing medium and then resuspended twice in serum free medium . cells were seeded onto the samples at 3 . 4 × 10 5 cells / cm 2 in serum free medium . cells were also seeded onto tcp in serum continuing medium ( positive control surface ). all samples were done in triplicate . at 1 , 2 , 4 and 6 hours , the samples were washed twice with pbs and fixed in 1 . 5 % paraformaldehyde in sodium cocodylate buffer for 30 minutes at room temperature . the samples were dehydrated in a graded series of ethanol ( 60 - 100 %) and then left to evaporate in hexomethyldisiazane ( hmds ) overnight . the samples were then gold coated and viewed using a philips 501b scanning electron microscope . at 30 minutes , 1 hour and 3 hours the samples were washed in 2 × 200 μl of pbs and fixed in 200 μl of 1 . 5 % paraformaldehyde in 0 . 1m sodium cocodylate buffer ( ph 7 . 4 .) for 30 minutes at room temperature . the cells were then washed in 2 × 200 μl of sterile double distilled water and viewed using a philips xl 30 environmental scanning electron microscope equipped with a field emission gun ( feg - esem ) in wet mode . the degree of cell spreading was scored as type i - iii ( sinha et al ., 1994 ). to determine the effect tissue transglutaminase has on the differentiation of human osteoblast cells on poly ( ε - caprolactone ) ( i ) determination of the minimum amount of foetal calf serum in dmem required to stimulate human osteoblast cell proliferation on pcl initially , the minimum amount of foetal calf serum ( fcs ) in the medium required to stimulate hob cell proliferation was determined . the cells were harvested using trypsin , followed by blocking in serum containing medium and then resuspended twice in serum free medium . the cells were then seeded into a 96 tissue culture plate ( falcon ) in either , 100 μl of 0 %, 2 %, 4 %, 6 % or 10 % fcs at a density of 1 . 7 × 10 5 cells / cm 2 . the negative control used . consisted of medium without cells , and the positive control consisted of cells in 10 % serum containing medium . the cells were incubated at 37 ° c ., 5 % co 2 for either 1 , 2 , 4 , 6 or 8 days . at each of these time points the medium was removed from the wells and the cells washed in 2 × 100 μl of sterile pbs . 100 μl of sterile double distilled water was added to each well and the cells lysed by the freeze thaw method , which involved placing the samples at − 80 ° c . for 20 minutes and then at 37 ° c . for 15 minutes . this was repeated three times . the dna content of each sample was determined using the dna hoechst assay ( rago et al ., 1990 ) ( see below ). the above experiment was repeated using pcl instead of tcp , however 7 % and 10 % fcs in the medium was used and the cells were incubated for either 1 or 3 days . the negative control consisted of medium without cells . the positive control consisted of cells in 10 % serum containing medium on tcp . this assay allows cellular dna content to be wed using the fluorochrome ; bisbenzimidazole ( hoechst 33258 ; sigma ). it works by a shift in the emission wavelength of hoechst 33258 upon binding of cellular dna . this results in a linear relationship between fluorescence and dna content over a broad range of dna . this reaction has been shown to be highly specific , and other cellular contents such as rna , protein and carbohydrates do not cause significant fluorescence . hoechst 33258 was dissolved in sterile deionised water to a final concentration of 1 mg / ml and then diluted 1 in 50 with tne buffer ( ph 7 . 4 .). tne buffer consisted of 10mm tris ( bdh ), 2 mm sodium chloride ( sigma ) and 1 mm diaminoethanetetra - acetic acid ( edta ) ( fisons ). it has previously been shown that crude cellular extracts assayed in the presence of high salt concentrations ( as in the tne buffer ) yield higher fluorescence due to dissociation of dna and chromatin with improved exposure of dna binding sites . 100 μl of the diluted hoechst 33258 was then added to 100 μl of the cell lysates along with a range of positive dna calf thymus standards ( range 0 - 20 μg / ml ) ( obtainable from sigma ). the fluorescence was then read at 360 nm ( excitation filter ) and 460 nm ( emission filter ) using a cytofluor ™ fluorescent plate reader ( millipore ). ( iii ) the differentiation of human osteoblast cells on the fibronectin / tissue transglutaminase coated pcl using the alkaline phosphatase assay the sterile pcl discs were coated with fibronectin and ttg as described in section 2 . 4 . 3 . controls consisted of pcl , pcl coated with fn and also tcp . the hob cells were harvested using trypsin , followed by blocking in serum containing medium and then resuspended twice in serum free medium . cells were seeded onto the polymers and tcp at 1 . 7 × 10 5 cells / cm 2 in 10 % serum containing medium . the cells were incubated for 2 days at 37 ° c ., 5 % co 2 . after this dime period , the medium was removed , 100 μl of pbs added and then 100 μl of sterile distilled water added . the cells were lysed using the freeze thaw method , whereby the cells were frozen at − 80 ° c . for 20 minutes and then thawed for 15 minutes at 37 ° c . ( this was repeated three times ). the samples were then diluted 1 in 2 with sterile double distilled water . the dna hoechst assay ( see above ) and the alkaline phosphatase assay were then performed on the cell lysates ( see below ). alkaline phosphatase ( alp ) is a marker of early bone cell differentiation . this photometric assay allows alp activity to be quantified . the principle of the assay is the conversion of 4 - nitrophenylphosphate ( substrate ) and water to phosphate and 4 - nitrophenolate in the presence of active alp . twenty - five ml of buffer ( ph 9 . 8 ) ( containing 1 . 0 mol / l diethanolamine , 0 . 5 mmol / l mgcl 2 and 0 . 225 mol / l nacl ) was added to 0 . 35 g of the substrate ( consisting of 255 μmol 4 - nitrophenylphosphate ). 50 μl of this solution was then added to 50 μl of the cell lysate sample and the absorbance read in a cytofluorimeter ( anthos labtec instruments ) at 450 nm ( measuring filter ) and 620 nm ( reference filter ). human osteoblast cells were grown on the pcl in 10 % serum containing dmem to initially assess the interaction of cells with this particular polymer surface . toluidine blue staining allowed the attachment , spreading and proliferation of the cells on the pcl . to be observed . transmission electron microscopy ( t . e . m .) allowed the ultra structure of the cells to be observed in order to assess their response to the biomaterial surface . this would give an indication as to whether the polymer is biocompatible with this particular cell type . fig1 and 2 show hob cells stained with toluidine blue at days 1 and 4 on pcl respectively . fig3 and 4 show hob cells stained with toluidine blue at days 1 and 4 on tcp respectively . these results clearly indicate that hob cells attach and spread on the pcl in dmem containing 10 % foetal calf serum as compared to the positive control ( tcp ). there is also a definite increase in cell population from day 1 to day 4 on both the tcp and pcl surfaces . the t . e . m . results also indicated that the hob cells proliferate on the pcl surface as shown by multilayer formation over time . the ultra structure of hob cells on pcl was seen to be normal . however at 8 days of culture in dmem containing 10 % foetal calf serum , it was found that very few cells remained on the surface . it was thought that the cells must have been removed during the washing processes . the elisa technique clearly demonstrated the binding of fibronectin to pcl ( see fig5 ) the concentration of bound fibronectin increased proportionally up to 50 μg / ml . the modified elisa technique also demonstrated the binding of ttg to fibronectin coated pcl when ttg was immobilised by either evaporation overnight or by incubation for 1 h at room temperature ( see fig6 and 7 respectively ). fig6 and 7 clearly show that ttg can be immobilised to pcl using both methods . the maximum concentration of ttg that can be immobilised to the fibronectin coated pcl is approximately 30 μg / ml for both immobilisation techniques and the half maximum binding capacity is approximately 15 μg / ml . the activity of tissue transglutaminase , after immobilisation onto the biomaterial surface ( i ) quantitative evaluation of the activity of ttg on pcl when immobilised in a 0 . 1m edta solution together with fibronectin by incubation at 4 ° c . the activity of ttg on the pcl surface was evaluated using the biotin - cadaverine incorporation assay ( see above ). 15 μg / ml of fibronectin and 10 μg / ml ttg were immobilised onto the pcl ice by incubation together in a 0 . 1m edta solution overnight at 4 ° c . the data in fig8 show that ttg is not active on the pcl surface or in the homogenising buffer removed from the same surface . this suggests that either fibronectin was not immobilised onto the pcl using ibis method ( which needs to be confirmed using the elisa technique ) or the fibronectin adsorbed to the pcl was in a configuration unfavourable for ttg cross - linking . the positive control for pcl showed no significant ttg activity either , which again suggests that fibronectin is not present on the surface or is inaccessible for ttg cross - linking due to its unfavourable configuration . another alternative would be that ttg did not bind to the fibronectin and was in an inactive form hence no activity was seen in the homogenising buffer either . in further experiments the fibronectin and ttg concentrations were increased . it was also thought that 0 . 1m edta solution was too high a concentration for ttg binding as edta at this concentration might interfere with the ttg binding site on fibronectin molecule . five mm edta was used in the preceding experiments ( which is commonly used in the biotin cadaverine incorporation assay because it is high enough to inhibit ttg activity and yet low enough to allow the ttg to bind to the fibronectin ). ( ii ) quantitative evaluation of the activity of ttg on fibronectin - coated pcl when immobilised by evaporation in 5 mm edta . the activity of ttg on the pcl surface was evaluated using the biotin - cadaverine incorporation assay ( see above ). the ttg was immobilised onto the pcl by initially coating the surface with 30 μg / ml fibronectin by evaporation . a 5 mm solution of 20 μg / ml ttg was then added to the pcl in the same way ( see above for details ). the data in fig9 show that the evaporation method allows the fibronectin to be immobilised onto the pcl in a configuration that is favourable for ttg cross - linking ( see pcl - positive control , which shows that addition of ttg gives rise to biotin cadaverine incorporation on the fibronectin coated pcl surface . the presence of , fibronectin on the pcl surface when immobilised by this method was also confirmed by the elisa assay ( see fig5 ). fig9 also illustrates that the immobilised ttg is not active on the pcl when it is immobilised by evaporation overnight even though the elisa results show that it is present on the surface ( see fig6 ). there was no significant ttg activity in the homogenising buffer which was removed from the fibronectin / ttg pcl surface , which also confirms that the ttg is present on the surface of the polymer . ( iii ) quantitative evaluation of the activity of ttg on fibronectin - coated pcl when immobilised by incubation in 5 mm edta for one hour at room temperature the activity of tg on the pcl surface was evaluated using the biotin - cadaverine incorporation assay ( see section 2 . 7 for details ). the ttg was immobilised onto the pcl by initially coating the surface with 30 μg / ml fibronectin by evaporation . a 5 mm solution of 20 μg / m ttg was then added to the pcl for 1 hour at room temperature ( see above for details ). fig9 and 10 both show that the evaporation method allows the fibronectin to be immobilised onto the biomaterial surface in a configuration that is favourable for ttg crossing ( see pcl positive control , which shows that addition of ttg gives rise to biotin cadaverine incorporation on the fibronectin coated pcl . fig1 also illustrates that immobilised ttg is present on the biomaterial surface and is active when immobilised for 1 h at room temperature . the elisa technique ( see fig7 ) confirms that the ttg can be immobilised by this method . the homogenising buffer that was removed from the pcl that had previously been coated with fibronectin and ttg , showed no ttg activity when compared to the tcp negative control which confirms that ttg was immobilised to the pcl . hob cells on tissue culture plastic ( tcp ) with and without 10 % senum in the medium and pcl , pcl + fibronectin and pcl + fibronectin + ttg in serum free medium were cultured for 2 or 4 hours . after this , they were fixed and viewed using sa g electron microscopy ( s . e . m .). the results clearly demonstrated that cell spreading occurred before 2 hours on the fibronectin / ttg coated pcl surface . some distortion of the pcl surface occurred during the hydration step during s . e . m . sample preparation . the experiment was therefore repeated using smaller time points and viewed using environmental scanning electron microscopy ( e . s . e . m . ), which doesn &# 39 ; t require a hydration step when preparing the sample . the samples were viewed in wet mode and the results are shown in fig1 - 21 . fig1 - 14 shows the degree of spreading of hobs on various substrates at 30 minutes after cell seeding . the results clearly show that in 30 minutes the cells have attached and spread to the fibronectin / ttg coated pcl surface . some of the cells are already at the late stages of cell spreading . the rate at which these cells spread was quicker than cells seeded on tcp in serum containing medium or on tie fibronectin coated pcl surface . it can be clearly seen that cells on pcl alone are rounded in morphology and show no signs of spreading at this particular time point . after 1 hour incubation on the different surfaces , similar results were obtained ( see fig1 - 18 ). the cells were spreading quicker on pcl coated with fibronectin + ttg than on fibronectin coated pcl or tcp . the cells on the fibronectin + ttg coated pcl , showed a more flattened morphology than seen at 30 minutes . cells on pcl still remained rounded at this time point . fig1 - 21 show the degree of spreading 3 hours after seeding the hob cells in serum free medium . the cells clearly remained rounded in morphology on the pcl . a lot of cells had detached from the biomaterial surface illustrating that hob cells require adhesion proteins to spread on pcl . however , after 3 hours on the pcl + fibronectin and pcl + fibronectin + ttg surfaces the cells were flat in morphology and had formed a monolayer . fig2 and 23 show graphical data to summarise the effects ttg has on hob cell spreading after 30 and 60 minutes ( as viewed using e . s . e . m .). the degree of cell spreading was scored as type i - iii . type i was classed as cells that have attached to the surface but remain rounded in morphology . type ii cells are classed as cells that have attached to the surface and have started to spread . type iii cells are classed as those which have attached and spread out flat on the surface ( late stage of spreading ). it can therefore be concluded from fig1 - 23 that hob cells respond immediately to the ttg on the pcl surface , which subsequently causes earlier cell spreading . this surface is far more preferential to pcl alone or pcl coated with fibronectin . initially the minimum serum content of medium required for hob cells to proliferate on tcp was investigated . the intention was to allow minimal interference of serum proteins with ttg when investigating its role in the differentiation of hob cells when coated on pcl . fig2 clearly shows that hob cells cannot proliferate in serum free medium on tcp . they can proliferate with as little as 2 % serum im the medium . however the rate of proliferation is significantly slower than that of cells in the medium with a serum content of 4 % or more . when the proliferation of hob cells on pcl in varying amounts of serum containing medium was studied , it was found that a much higher serum content was required ( see fig2 ). there was no significant difference in dna concentration between day 1 and day 3 when the cells were cultured in 7 % serum cont medium . however , the dna concentration was significantly higher between day 1 and day 3 for cells cultured in 10 % serum containing medium . these results illustrate that the minimum content of serum in dmem required for hob cell proliferation on pcl , is 10 %. at day 3 , the number of cells on tissue culture plastic was significantly greater than the number of cells on pcl when both are cultured in 10 % serum contating medium . the study of implant surface and biomaterial tissue interface reactions is essential for the continued improvement of implant performance . a review by blitterwijk et al ., ( 1991 ) discusses the importance of the reactions of cells at implant surfaces in determining the biocompatibility of the implant . current research involves making bioactive materials , which will allow the integration of the material with the body . many workers have introduced the concept of combining synthetic polymers with natural polymers to enhance biocompatibility . in this study poly ( ε - caprolactone ) ( pcl ) was chosen and the natural polymer fibronectin was immobilised onto this surface . in a further attempt to stabilise and facilitate compatibility of the cell - biomaterial interface , the enzyme tissue transglutaminase ( ttg ) was also immobilised onto the fibronectin coated pcl surface . this enzyme catalyses the post - translational modification of proteins by forming inter and intramolecular ε ( γ - glutamyl ) lysine cross - links in inter - and intracellular proteins . the bonds that form are stable , covalent and resistant to chemical , enzymatic and physical disruption . the cross - linking of extracellular proteins is thought to enhance cellular responses such as cell attachment , spreading and differentiation at the biomaterial interface . however ttg may act as a receptor adhesive protein without protein crosslinking via its interaction with the b 1 and b 3 integrins ( see gaudry et al ., 1999 , exp . cell . res . 252 , 104 - 113 ; akimov et al ., 2000 , j . cell . biol . 148 , 825 - 838 ). jurgensen et al ., ( 1997 ) showed that ttg could be a new biological ‘ glue ’ for cartilage - cartilage interfaces . tissue tg has 62 % greater adhesive strength than that of tissucol , a commercially available fibrin - glue preparation jones et al ., ( 1997 ) showed that human endothelial cells transfected with anti - sense ttg , showed a decrease in cell adhesion and spreading . the theory of ttg being involved in cell attachment is also supported by the findings of gentile et al ., ( 1992 ) using balb - c 3t3 fibroblasts , borge et al ., ( 1996 ) using chondrocytes and verderio et al ., ( 1998 ) using swiss 3t3 fibroblasts . in the present study , three different methods were used to try and immobilise fibronectin and ttg . all methods were based on the theory that ttg would bind to the immobilised fibronectin on the pcl , because fibronectin has a ttg binding site ( jeong et al ., 1995 ). this binding has been shown to be linked with the first 7 residues at the n terminal domain of ttg . however , reduction and alkylation of fibronectin , destroys its ability to associate with the ttg , suggesting that some features of its tertiary structure are necessary for the binding of the enzyme . the ttg was immobilised onto the fibronectin coated pcl surface in either 0 . 1 m or 5 mm edta to prevent ttg cross - linking occurring before the biotin - cadaverine experiment was performed . it was hoped that this would not affect the binding properties of the enzyme . jeong et al ., ( 1995 ) showed that binding occurs in the absence of ca 2 + and that the alteration in the conformation of the enzyme is not essential for the formation of the fibronectin - ttg complex . the three methods of immobilising ttg to pcl were , a ) adding fibronectin and ttg in solution overnight at 4 ° c ., b ) immobilising fibronectin onto the surface by evaporation and then immobilising ttg onto the surface by evaporation , c ) immobilising fibronectin onto the surface by evaporation and then adding ttg and leaving it for 1 h at room temperature . the elisa techniques demonstrated the presence of fibronectin on pcl when immobilised by evaporation and also demonstrated the presence of ttg on fibronectin coated pcl when immobilised by either evaporation overnight or by incubation for 1 h at room temperature . the activity of ttg on the pcl surface was evaluated using the biotin - cadaverine incorporation assay . it was found that ttg was not active on the surface of pcl when immobilised in solution together with fibronectin . an explanation for this would be that fibronectin is inaccessible for ttg cross - linking due to its unfavourable configuration or alternatively , the fibronectin did not bind to the pcl at 4 ° c . it was also found that ttg was not active on the fibronectin coated pcl surface when the ttg was immobilised by the evaporation method . an explanation for this would be that the evaporation method renders the ttg inactive as it was shown that under these circumstances ttg is present on the pcl surface ( as demonstrated by the elisa technique ). when the ttg was immobilised by incubation for 1 h at room temperature however , it was found to be active on the surface . tissue transglutaminase is regulated by calcium and nucleotides ( smethurst and griffin , 1996 ). they found that ttg was active at 100 μm calcium and when atp or gtp were absent or in very low concentrations . in the cytoplasm of a resting energy - rich cell , the atp levels may be as high as 8 - 11 mm and gtp levels between 50 and 300 μm , with a proportion of each being bound to cytosolic proteins , giving lower free nucleotide concentrations . in the resting state the free calcium is around 100 - 200 μm . under these conditions their data suggest that ttg activity in the cytosol would be switched off . however , in dmem ( the medium that human osteoblast cells are cultured in ) or in the in vivo situation there is & gt ; 100 μm calcium , which is enough to activate the ttg on the biomaterial surface . when the human osteoblast cells were seeded onto the fibronectin / ttg coated pcl surface , ttg had a profound effect on cell morphology ( as viewed using e . s . e . m .). the results clearly demonstrated that cell spreading in serum free medium occurred 30 minutes after cell seeding unlike the cells on the pcl surface which remained rounded and unlike the cells on the pcl + fibronectin surface which had only just started to spread . the cells on the fibronectin / ttg coated surface spread quicker than cells seeded on tissue culture plastic in 10 % serum containing medium ( positive control ). after 1 hour incubation on the different surfaces , similar results were obtained and 3 hours after cell seeding the human osteoblast cells had formed a monolayer on pcl + fibronectin , pcl + fibronectin + ttg and on tissue culture plastic . the cells on pcl however , were still rounded in morphology and only a few cells were present indicating that some cells had probably detached during the hour . it can therefore be concluded from the e . s . e . m . results , that human osteoblast cells immediately respond to the ttg on the pcl surface , which subsequently causes earlier cell spreading . this surface is far more preferential to pcl alone , pcl coated with just fibronectin and tissue culture plastic . cells initially attach to the biomaterial by physicochemical factors , i . e . charge , surface free energy or the water content of the biomaterial ( schamberger and gardella , 1994 ) and then strongly adhere to ecm proteins , which have been deposited on the biomaterial surface . it is thought that initially the cells attach to the fibronectin coated pcl surface . evidence then suggests that ttg cross - links the fibronectin with other cell surface bound proteins forming a stabilised extracellular matrix on the biomaterial face . cross - linking of the surface bound proteins may also trigger the activation of integrins . the cell may also utilise the ttg as an adhesion protein in association with the β1 integrin in order for it to attach to the biomaterial surface . gaudry et al ., ( 1999 ) has shown that ttg co - localises with the β1 integrin and akimov et al ., 2000 , j . cell . biol . 148 , 825 - 8 has suggested that ttg may mediate the interaction between the b1 and b3 integrins and fibronectin . the differentiation of human osteoblast cells on the ttg coated pcl surface was investigated . it was necessary to first establish the lowest amount of serum required in dmem that will allow osteoblast proliferation . the results showed that cells can proliferate in as little as 2 % serum containi medium on tissue culture plastic , however they require a minimum of 10 % serum containing medium when cultured on pcl . the rate of proliferation is also slower on pcl than on tissue culture plastic . the differentiation of cells on the tcp . pcl , pcl + fn and pcl + fn + ttg surfaces was measured 2 days after cell seeding using the alkaline phosphatase activity assay and was expressed as per μg dna . the preliminary results indicate that ttg , does not have any detrimental effect on the alkaline phosphatase activity / differentiation of hob cells on pcl when compared to pcl coated with fibronectin . however , this needs to be investigated at later time points . kaartinen et al ., ( 1999 ), however believes that the cross - 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