Patent Application: US-93283107-A

Abstract:
vapor plasma deposition of titanium metal onto a substrate forms a structured surface that exhibits enhanced cell attachment properties . initially deposited round nanoparticulate surface structures develop tentacles with a spine or thorn - like appearance upon continued deposition under special conditions . the density and size of the formed spinulose particles can be controlled by timing the deposition intervals . a significant increase in osteoblast , fibroblast and endothelial cell attachment is observed on ti spinulose surfaces compared to attachment on nanoparticulate surfaces lacking spinulous nanostructure .

Description:
the present invention provides metal nanostructured surfaces by controlling plasma deposition conditions . in a particular embodiment , spinulose titanium nanostructured surfaces are described , which have strong adhesion for several cell types , including osteoblasts , fibroblasts and endothelial cells . most metals are initially deposited as relatively round plasma - deposited particles . titanium also initially deposits as round nanoparticulates but after several cycles forms spine - like or spinulous projections . most other metals continue to deposit as globular structures with increased deposition times . titanium spinules are relatively uniform and can be grown from plasmas deposited in a range of angles with θs and θc ranging from 0 ° to 80 °. most vacuum based methods for preparing thin films or depositing materials on surfaces are physical vapor deposition or chemical vapor deposition methods . these methods differ from electrodeposition wet chemistry methods . the nanostructured surfaces of the present invention are produced by a modified cyclic plasma arc deposition procedure termed nano plasma deposition ( npd ). the apparatus for producing the ion plasmas is shown in fig1 . npd deposited particles are typically round and may be varied in size and distribution by changing power and / or time of deposition . under certain specified deposition conditions , titanium metal particles develop nanosized spike - like protrusions , which are observed as spinules or small thorny spines . the spinules are distinct in character and appearance from the tiny filiform hairs commonly termed “ whiskers ”, which have been reported as crystalline metallurgical phenomena commonly found associated with metal components in electrical equipment and computer machinery and which can lead to system malfunctions and failures . titanium spinules only form under certain deposition conditions and are distinctly different from such whiskers and from the glancing angle structures ( glad ) reported by others . metal cathode targets are disposed in a vacuum chamber as illustrated in fig1 . an inert gas , typically argon , is introduced into the evacuated chamber and deposition commenced . the substrate 2 is generally positioned 8 - 28 in from the target and deposition is conducted intermittently for periods of approximately 1 - 15 min . during the intervals between depositions , there is no plasma discharge and the inert gas flow can be reduced to zero or stopped completely if desired . the intervals between depositions can be varied and are about 5 - 90 min with a typical run of about 3 - 9 cycles . titanium spinulose nanostructured surfaces appear to be relatively uniform , with the spiky projections ranging in lengths of about 0 . 166 - 1 . 119 μm and widths of about 0 . 156 - 0 . 627 μm depending on number of deposition cycles and distance from the target . the number of cycles can be increased ; however with a fewer number of cycles , generally less than 3 , as shown in fig3 , no spinules are observed . increasing cycles above three results in the emergence of spikey protrusions on some of the deposited round particles , while increasing to 9 or more cycles greatly increases the density of spinulose particles . using similar cycling parameters , aluminum was deposited in what appears to be geometric polygonal structures including some cube - like attachments on surfaces of round particles . spinulose surfaces were not observed . copper , cobalt , silver , nitinol , titanium 6 - 4 , 316l stainless steel , and hafnium also form nanostructured films when deposited under the described cycling conditions . these metals differ in structural detail from npd deposited titanium and aluminum and are more similar to each other in having a globular appearance . nickel , in contrast to the other metals has a more random and “ rough ” appearance . titanium spinulose nanostructures are also formed when the angle of deposition , θs , from the target is varied . the spinules are oriented away from the plasma flow and appear to be more prominent around the circumference of the round particles on which they form as the angle is changed . titanium spinulose surfaces are excellent scaffolds for osteoblast and fibroblast cell attachment . as shown in fig4 and fig5 , titanium spinulose surfaces were superior compared to uncoated surfaces and to surfaces lacking the spinulite structural nanofeatures . fig6 indicates that endothelial cells attached better to ti spinulose surfaces than to uncoated surfaces but were less effective than ti nanostructured surfaces lacking the spinulose features . cell adhesion is better on all deposited spinulose ti substrates with the exception of endothelial cells on a ti spinulose coated titanium substrate , see fig1 . the unique aluminum coatings deposited by the described cycling npd deposition method show good cell adhesion for osteoblasts and endothelial cells , fig1 and fig2 , but not for fibroblast adhesion , fig2 . the following examples are provided as illustrations of the invention and are in no way to be considered limiting . human osteoblast cells ( crl - 11372 ) were purchased from american type culture collection ( rockville , md .) as frozen cultures in complete media : 1 : 1 ham &# 39 ; s f12 medium and dulbecco &# 39 ; s modified eagle &# 39 ; s medium without phenol red with 2 . 5 mm l - glutamine , 10 % fbs and 0 . 3 μg / ml g418 . briefly , the vials were thawed , centrifuged and the cells resuspended in complete media before transfer into a culture device and incubated at 34 ° c . in 5 % carbon dioxide . the cells were then subcultured in complete media after treating with trypsin - edta at either 34 ° c . or 39 ° c . doubling time was 36 hr at 33 . 5 ° c . and 96 hr at 38 . 0 ° c . if not used immediately , the cells were stored frozen in complete media with dmso added to each vial . human fibroblast cells ( crl - 1502 ) were purchased from american type culture collection as frozen cultures in complete media containing eagle &# 39 ; s minimal essential medium with earle &# 39 ; s bss and 2 mm l - glutamine ( emem ) modified to contain 1 . 0 mm sodium pyruvate , 0 . 1 m non - essential amino acids , 1 . 5 g / l sodium bicarbonate supplemented with 10 % fbs and 10 u / ml penicillin / streptomycin . human endothelial cells were purchased from vec technologies ( rensselaer , n . y .) as frozen cultures in mcdb - 131 media . cell sample vials were thawed at 37 ° c ., centrifuged and the cell pellet resuspended in complete media before transfer to a culture device and incubated at 34 ° c . in 5 % carbon dioxide . cells were subcultured by rinsing and adding trypsin - edta before culturing in complete media and incubating at 34 ° c . or 39 ° c . if not used immediately , the cells were rinsed and stored in liquid nitrogen after addition of 10 % fbs and dmso to the vials . substrates used were stainless steel , nitinol , cocrmo alloy , silicon , titanium , glass , silicone , poly ( methyl methacrylate ) pmma , polyurethane ( pu ), polytetrafluoroethylene ( ptfe ), polyvinyl chloride ) ( pvc ), polyethylene terephthalate ( pet ), ultra high molecular weight polyethylene ( uhmwpe ), polyethylene terephthalate glucol ( petg ), polyetheretherketone ( peek ) and polypropylene ( pp ). samples were prepared with an approximate surface area of 1 cm 2 . except for the silicon which was delivered clean , all the other substrate materials were ultrasonically cleaned before deposition in detergent ( chemcrest # 275 at 160 ° f . ), rinsed in deionized water and dried in hot air . clean substrates were then placed in the chamber and exposed to nano - plasma deposition ( npd ). the cathode was commercially pure titanium cathode ( grade 2 ). the substrates were mounted in the vacuum chamber at distances from 8 to 28 in from the cathode ( measured from the centre of the cathode ). the angle between the cathode surface normal and a line from the centre of the cathode to the substrate , θc , was varied in the range 0 - 80 °. the angle between the depositing flux and the substrate surface normal , θs , was varied in the range of 0 - 80 °. the angle between the substrate surface normal and a line from the centre of the cathode to the substrate , θc , was varied in the range of 0 - 80 °. the angle between the depositing flux and the substrate surface , θs , was varied in the range of 0 - 80 °. the chamber was pumped to a base pressure of between 1 . 33 mpa and 0 . 080 mpa . the arc current was varied from a 150 a to 300 a with an argon flow of 0 to 300 standard cubic centimeter per minute ( sccm ). the process was run in cycles , with each cycle consisting of plasma discharge intervals ( varied over the range 1 to 15 minutes ) followed by intervals where there was no discharge and no gas flow ( between 5 and 810 minutes ). each process consisted of 3 - 9 cycles . following plasma deposition , the samples were characterized by scanning electron microscopy ( sem ), and cell adhesion tests with osteoblasts , fibroblasts and endothelial cells . sem images were obtained with a tescan mira field emission instrument ( pittsburgh , pa .) equipped with a se detector , at a magnification of 50 . 00k and 10 . 00k times at 10 . 00 kv . experiments were carried out with a range of different conditions , substrates and tests . results are shown in tables 1 - 3 . table 1 lists the properties of spinulose titanium deposited on silicon or stainless steel substrates . all examples were run with an argon gas flow of 100 sccm , deposited for 5 min with a 90 min interval between depositions through 9 cycles . the substrates were positioned 13 in from the cathode . θc was 0 ° for all substrates . table 2 shows the effect of alterations in rest interval , distance from cathode , number of cycles and θc for cyclic depositions of titanium on silicon and stainless steel substrates . for these examples , the arc current was 200 a , the argon gas flow 100 sccm and θs was 0 ° for all substrates 316l stainless steel substrates ( 1 . 6 cm 2 × 0 . 1 cm , mirror polished ( mcmaster - carr , elmhurst , ill .) were cleaned in an ultrasonic bath , rinsed with deionized water and dried in air . the substrates were placed in the vacuum chamber on a floating holder approximately 13 in from the cathode surface at an angle of θc = 0 ° with a θs of 0 °. prior to deposition , the chamber was pumped to a base pressure of at least 0 . 088 mpa . the deposition was carried out in 5 minute intervals with 90 min intervals of no arc current and no gas flow . this deposition - pause cycle was repeated 9 times . a 300 a arc discharge was generated in a background of 160 mpa of argon on a pure titanium ( grade 2 ) cathode ( 20 in × 6 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a pronounced spinulose morphology ( see fig2 ) with the fraction of surface area covered by spinulose features estimated to be at least 85 %. a second batch of coated substrates was prepared as above except that deposition was conducted continuously for 45 min . the deposited titanium surface exhibited a rounded surface morphology quite distinct from the spinulose coatings observed using interval deposition , and is referred to here as “ round ” coating ( see fig3 ). human osteoblast , human skin fibroblast and human umbilical artery endothelial cell four hr adhesion tests were carried out on 316l stainless steel coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° and compared with cell adhesion on respective substrates coated with titanium round coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° and with uncoated substrates . the substrates were placed in wells using sterilized tweezers and exposed to uv light for one hour . the substrates were then rinsed with 2 . 0 ml of room temperature ( 1 × pbs ). the desired amount of room temperature complete media ( supplemented with fbs and antibiotic ) was added to each well . the cells were seeded onto the substrates at 2500 cells / cm 2 and incubated at 34 ° c ., 5 % co 2 for four hours . following incubation , the media and non - adherent cells were removed . the substrates were then rinsed with room temperature 1 × pbs and fixed with 4 % paraformaldehyde . the nuclei of adherent cells were fluorescently stained with hoescht stain and counted using a fluorescent microscope . fig4 and fig5 compare the results of the human osteoblast and human skin fibroblast four hr cell adhesion tests on 316l stainless steel coated with a titanium spinulose coating from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° ( 1 ); no coating ( 2 ); and a titanium round coating from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° ( 3 ), respectively . there is an increase in the number of osteoblasts and the number of fibroblasts attached to the spinulose coated substrate compared to the uncoated and the round coated substrates . fig6 compares the results of the human umbilical artery endothelial four hr cell adhesion test on 316l stainless steel coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° ( 1 ); no coating ( 2 ); and a titanium round coating from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° ( 3 ). the spinulous coated substrates show growth inhibition of endothelial cells compared to growth on the round coated substrates while simultaneously demonstrating an increase in adhesion compared to the uncoated substrates . titanium was deposited by nanoplasma deposition as in example 2 on several different substrates . the following materials were used as substrates : ptfe , pet , petg , peek , pmma , pvc , pu , uhmwpe , pp and silicone . ptfe , pet ( 1 cm 2 × 0 . 5 cm ) substrates ( mcmaster - carr ), silicone ( 1 . 3 cm 2 × 0 . 3 cm ) substrates ( mcmaster - carr ), pmma , pvc , pu and uhmwpe ( 1 . 2 cm outer diameter × 1 . 3 cm length ) cylindrical shaped substrates from mcmaster - carr ) were cleaned in an ultrasonic bath , rinsed in deionized water and dried in air . the substrates were placed in the chamber on a floating holder approximately 13 in from the cathode surface at an angle of θc = 0 ° with a θs of 0 °. prior to deposition the chamber was pumped to a base pressure of at least 0 . 907 mpa . the deposition was carried out in 5 minute intervals , with 90 minute pauses in between of no arc current and no gas flow . this deposition - pause cycle was repeated 9 times . a 200 a arc discharge was generated in a background of 440 mpa of argon on a pure titanium ( grade 2 ) cathode ( 20 in by 6 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a pronounced spinulose morphology ( see fig1 ) with a fraction of surface area covered by spinulose features estimated to be at least 85 %. human osteoblast , human skin fibroblast and human umbilical artery endothelial cell four hr adhesion tests were carried out on uhmwpe , ptfe , pvc , pet , silicone , pu and pmma substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° and compared with cell adhesion on the respective uncoated substrates . substrates were placed in wells using sterilized tweezers and exposed to uv light for one hour . each substrate was then rinsed with 2 . 0 ml of room temperature ( 1 × pbs ). the desired amount of room temperature complete media ( supplemented with fbs and antibiotic ) was added to each well . the cells were seeded onto the substrates at 2500 cells / cm 2 and incubated at 34 ° c ., 5 % co 2 for four hours . following incubation , the media and non - adherent cells were removed . the substrates were then rinsed with room temperature ( 1 × pbs ) and fixed with 4 % paraformaldehyde . the nuclei of adherent cells were fluorescently stained with hoescht stain and counted using a fluorescent microscope . fig8 , 9 and 10 compare the results of the human osteoblast , human skin fibroblast and human umbilical artery endothelial cell four hour cell adhesion tests , respectively . fig8 shows an increase in the number of human osteoblast cells that adhered to the surface of the uhmwpe , ptfe , pvc , pet , silicone , pu and pmma substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° over that of the respective uncoated substrates . fig9 shows an increase in the number of human skin fibroblast cells that adhered to the surface of uhmwpe , ptfe , pvc , pet , silicone , pu and pmma substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° over that of the respective uncoated substrates . fig1 shows an increase in the number of human umbilical artery endothelial cells that adhered to the surface of uhmwpe , ptfe , pvc , pet , silicone , pu and pmma substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° over that of the respective uncoated substrates . titanium was deposited by nanoplasma deposition as in example 2 on several different substrates . the following materials were used as substrates : silicon , glass , anodized titanium , titanium , cocrmo , and nitinol . silicon ( 1 cm 2 by 0 . 04 cm ), single crystal ( 100 ) silicon wafers from ( encompass distribution services , llc ), glass ( 18 mm micro cover glass circles ( vwr ) and anodized titanium ( 1 . 25 cm 2 by 0 . 2 cm ), wire cut substrates ( alfa aesar ) were cleaned by blowing compressed air over the surface of the substrates before placing in chamber . titanium ( 1 . 6 cm 2 by 0 . 6 cm , shear cut substrates from mcmaster - carr ), cocrmo ( 1 . 8 cm ( outer diameter )× 1 . 3 cm ( height ), cylindrical shaped , machine cut stubs ( voss metals company , inc . ), nitinol ( 1 . 2 cm ( outer diameter ) by 0 . 6 cm ( length ), cylindrical shaped , machine cut stubs from nitinol devices and components ) were cleaned in an ultrasonic bath , rinsed in deionized water and dried in air . the substrates were placed in the chamber on a floating holder 13 in from the cathode surface at an angle of θc = 0 ° with a θs of 0 °. prior to deposition , the chamber was pumped to a base pressure of at least 0 . 288 mpa . the deposition was carried out in 5 minute intervals , with 90 minute intervals between deposition and no arc current and no gas flow . the deposition - pause cycle was repeated 9 times . a 200 a arc discharge was generated in a background of 160 mpa of argon on a pure titanium ( grade 2 ) cathode ( 20 in × 6 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a pronounced spinulose morphology ( see fig7 ) with a fraction of surface area covered by spinulose features estimated to be at least 85 %. human osteoblast , human skin fibroblast and human umbilical artery endothelial four hr cell adhesion tests were carried out on spinulose coated titanium , cocrmo and nitinol substrates and compared with cell adhesion on the respective uncoated substrates . substrates were placed in wells using sterilized tweezers and exposed to uv light for one hour . each substrate was then rinsed with 2 . 0 ml of room temperature ( 1 × pbs ). the desired amount of room temperature complete media ( supplemented with fbs and antibiotic ) was added to each well . the cells were seeded onto the substrates at 2500 cells / cm 2 and incubated at 34 ° c ., 5 % co 2 for four hrs . following incubation , the media and non - adherent cells were removed . the substrates were then rinsed with room temperature ( 1 × pbs ) and fixed with 4 % paraformaldehyde . the nuclei of adherent cells were fluorescently stained with hoescht stain and counted using a fluorescent microscope . fig8 , 9 and 10 compare the results of the human osteoblast , human skin fibroblast and human umbilical artery endothelial four hour cell adhesion test , respectively . fig8 shows an increase in the number of human osteoblast cells that adhered to the surface of the spinulose coated titanium , cocrmo and nitinol substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° over that of the respective uncoated substrates . fig9 shows an increase in the number of human skin fibroblast cells that adhered to the surface of cocrmo and nitinol substrates coated with a titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 ° over that of the respective uncoated substrate . titanium substrates coated with titanium spinulose coating formed from a titanium plasma deposited at an angle of θc = 0 ° with a θs of 0 °, however , showed growth inhibition of the human umbilical artery endothelial cells ( see fig1 ) over that of the respective uncoated substrate . silicon ( 1 cm 2 by 0 . 04 cm , single crystal ( 100 ) wafer ( encompass distribution services , llc ) substrates were cleaned by blowing compressed air over the surface of the substrates before placing in chamber . 316l stainless steel substrates ( 160 cm 2 × 0 . 1 cm ), mirror polished ( mcmaster - carr ) were cleaned in an ultrasonic bath , rinsed in deionized water and dried in air . the substrates were placed in the chamber on a floating holder approximately 13 in from the cathode and at a θ c = 0 ° with a θ s ranging from 45 ° to 80 °. prior to deposition the chamber was pumped to a base pressure of at least 0 . 088 mpa . the deposition was carried out in 5 min intervals , with 90 min pauses intervals of no arc current and no gas flow . this deposition - pause cycle was repeated 9 times . a 300 a arc discharge was generated in a background of 160 mpa of argon on a pure titanium ( grade 2 ) cathode ( 20 in × 6 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a pronounced oblique angle spinulose morphology ( see fig1 ) with at least 85 % of the surface covered by oblique angle spinulose features . a second batch of coated substrates was prepared as described above with the exception that the deposition protocol did not include intermittent depositions , such that deposition was conducted continuously for 45 min . these coatings exhibited a rounded surface morphology quite distinct from the spinulose coatings and is referred to here as “ round ” coating ( see fig3 ). human osteoblast , human skin fibroblast and human umbilical artery endothelial four hr cell adhesion tests were carried out on 316l stainless steel substrates coated with oblique angle titanium spinulose coating formed from a titanium plasma deposited at an angle of θs = 45 ° with a θc of 0 ° and compared with cell adhesion on respective substrates coated with titanium spinulose coating formed from a titanium plasma deposited at an angle of θs = 0 ° with a θc of 0 °, titanium round coating formed from a titanium plasma deposited at an angle of θs = 0 ° with a θc of 0 ° and with no coating . substrates were placed in wells using sterilized tweezers and exposed to uv light for one hr . each substrate was then rinsed with 2 . 0 ml of room temperature ( 1 × pbs ). the desired amount of room temperature complete media ( supplemented with fbs and antibiotic ) was added to each well . the cells were seeded onto the substrates at 2500 cells / cm 2 and incubated at 34 ° c ., 5 % co 2 for four hrs . following incubation , the media and non - adherent cells were removed . the substrates were then rinsed with room temperature ( 1 × pbs ) and fixed with 4 % paraformaldehyde . the nuclei of adherent cells were fluorescently stained with hoescht stain and counted using a fluorescent microscope . fig1 , 14 and 15 compare the results of the human osteoblast , human skin fibroblast and human umbilical artery endothelial four hr cell adhesion test on 316l stainless steel coated with an oblique titanium spinulose coating formed from a titanium plasma deposited at an angle of θ s = 45 ° with a θ c of 0 °; a titanium spinulose coating formed from a titanium plasma deposited at an angle of θ s = 0 ° with a θ c of 0 ° ( as seen in fig2 ); uncoated and round coating formed from a titanium plasma deposited at an angle of θ c = 0 ° with a θ s of 0 ° ( fig3 and 4 ), respectively . fig1 and 14 show an increase in the number of human osteoblast and human skin fibroblast cells on the substrates coated with the oblique spinulose coating compared to the respective uncoated and round coated substrates . fig1 shows that for the human umbilical artery endothelial cells , the oblique titanium spinulose coated substrates inhibited the growth of the cells compared to growth on the respective round coated substrates . for all cell types , the spinulose coating formed from a deposition angle of θ s = 0 ° with a θ c of 0 ° demonstrated an increase in cell adhesion compared to the oblique spinulose coated substrates or to the uncoated substrates . 316l stainless steel substrates ( 160 cm 2 × 0 . 1 cm ), mirror polished ( mcmaster - carr ) were cleaned in an ultrasonic bath , rinsed with deionized water and dried in air . the substrates were placed in the chamber on a floating holder approximately perpendicular to the cathode surface , 8 - 13 in from the cathode with a θ s of 0 ° to 90 ° with a θ c of 0 - 80 °. prior to deposition the chamber was pumped to a base pressure of better than 1 . 45 mpa . the deposition was carried out in 5 minute intervals , with 90 minute pauses in between of no arc current and no gas flow . the deposition - pause cycle was repeated 9 times . a 200 a arc discharge was generated in a background of 160 mpa of argon on a pure titanium ( grade 2 ) cathode ( 20 × 6 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a spinulose morphology ( see fig2 , 17 and 18 ). as θ c approaches 0 °, there is a gradual progression in formation of the spinulose morphology . 316l stainless steel substrates ( 160 cm 2 × 0 . 1 cm ) mirror polished ( mcmaster - carr ) were cleaned in an ultrasonic bath , rinsed with deionized water and dried in hot air . the substrates were placed in the chamber on a floating holder approximately 13 inches away from the cathode at an angle of θ c = 0 ° with a θ s of 0 °. prior to deposition the chamber was pumped to a base pressure of better than 22 . 7 mpa . the deposition was carried out in 5 min intervals with 60 min pauses in between of no arc current and no gas flow . the deposition - pause cycle was repeated 3 times . a 150 a arc discharge was generated in a background on 667 mpa of argon on a pure aluminum cathode ( 4 in × 2 in ). samples were imaged in a feg - sem ( tescan mira ), operated with an accelerating voltage of 10 kv . the images showed a pronounced geometric morphology ( see fig1 ). human osteoblast , human skin fibroblast and human umbilical artery endothelial cell four hr adhesion tests were carried out on 316l stainless steel substrates coated with aluminum geometric coating formed from an aluminum plasma deposited at an angle of θ c = 0 ° with a θ s of 0 ° and compared to the respective uncoated substrates . substrates were placed in wells using sterilized tweezers and exposed to uv light for one hr . each substrate was then rinsed with 2 . 0 ml of room temperature ( 1 × pbs ). the desired amount of room temperature complete media ( supplemented with fbs and antibiotic ) was added to each well . the cells were seeded onto the substrates at 2500 cells / cm 2 and incubated at 34 ° c ., 5 % co 2 for four hrs . following incubation , the media and non - adherent cells were removed . the substrates were then rinsed with room temperature ( 1 × pbs ) and fixed with 4 % paraformaldehyde . the nuclei of adherent cells were fluorescently stained with hoescht stain and counted using a fluorescent microscope . fig1 , 20 and 21 compare the results of the human osteoblast , human skin fibroblast and human umbilical artery endothelial four hour cell adhesion test on 316l stainless steel substrates coated with an aluminum geometric coating formed from aluminum plasma deposited at an angle of θ c = 0 ° with a θ s of 0 ° and uncoated respectively . fig1 and 20 show that there was an increase in the number of osteoblasts and endothelial cells attached to the geometric particle coated substrates over that of the uncoated substrates . fig2 shows that the geometric particle coated 316 stainless steel inhibited growth of fibroblast cells to a greater degree than uncoated substrates suzuki , m ., nagai , k ., kinoshita , s ., nakajima , k ., and kimura , k ., “ vapor phase growth of al whiskers induced by glancing angle deposition at high temperature ” applied physics letters 89 , 133103 ( 2006 ). u . s . pat . application pub . no . 2004 / 0228898 ( ross and guagliano , nov . 18 , 2004 ) barsoum , m . w ., hoffman , e . n ., doherty , r . d ., gupta , s ., and zavaliangos , a . “ driving force and mechanism for spontaneous metal whisker formation ” phys . rev . lett ., v . 93 ( 20 ), 12 nov . 2004 , 206104 ( 1 - 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