sample preparation : sixty commercially pure titanium ( cp - ti , astm b - 265 ) discs ( 2 mm thickness and 12 . 7 mm diameter ) were polished with 320 , 400 , and 600 grit papers using a grinding machine ( buehler , phoenix beta ), ultrasonically cleaned in baths of doubly distilled ( ddw ), acetone , ddw , ethanol , and ddw ( 10 min in each bath ), and then air - dried . two step apatite coating : the discs were immersed in a calcifying solution prepared by mixing 200 mm caco 3 + 100 mm nah 2 po 4 . h 2 o and dissolving the mixed reagents with 2 . 9 % h 3 po 4 solution . the ph of the solution was 2 . 67 at room temperature . the discs were placed on the bottom of a sealed container and kept in oven at 75 ° c . for 24 h . 10 discs were placed in each container with 100 ml of calcifying solution . the discs were then removed from the solution , rinsed three times in distilled water , and air - dried . after removing the discs , the ph of the calcifying solution was 2 . 81 at room temperature . in the second step , to convert the ca — p phase ( monetite ) formed from the first step to apatite , the discs were immersed in the 0 . 2m naoh solution at 75 ° c . for 24 h . these parameters were chosen based on results of several pilot studies . the discs were then removed from the naoh solution , rinsed three times in ddw , and the air - dried . scanning electron microscopy ( sem ) and electron dispersive spectroscopy ( eds ): the shape , size , and coverage of ca — p crystals formed from acidic solution and then their transformation to apatite crystals were analyzed using sem ( jeol - 5400 ) operating at 20 kv . an evax microanalysis system ( eds ) coupled to sem was used to determine the chemical elements of the coating materials in each step of coating procedure . after removing the coating materials using tensile or scratch tester , eds analysis was also carried out on the titanium surface or on the residual coating on substrate . the specimens were coated with gold - palladium prior to sem observation and carbon for eds analysis . one way - anova and tukey statistical tests were used to compare the percentage and ratios of different chemical elements in different areas of the coating . x - ray diffraction ( xrd ): x - ray diffraction ( philips x &# 39 ; pert ) using grazing incident angle of 2 ° was performed to determine the structural and chemical modification on the ti surface after each step of the coating procedure . xrd was carried out using cuk α radiation operating at 45 kv and 40 ma . tensile adhesion test : the tensile adhesion between the coating and the ti substrate was measured using quad - romulus iii system ( quad group inc ., pull down breaking point ). the 3 . 5 mm diameter studs that were pre - coated with a thin layer of epoxy by manufacturer ( quad group inc ) were mounted perpendicularly on each of the coated surface of the 10 ti discs using spring mounting clips . the mounting clip allowed the stud to be perpendicular to the coating surface under a constant pressure during the curing procedure of epoxy . the discs were then placed in an oven and the epoxy was cured at 150 ° c . for 1 h . the loading rate of the tensile test was at 2 n / sec and the tensile strength was measured in mpa . the above method is well - validated in industrial quality control laboratories for measuring the adhesion strength of thin films and complies with mil . std 883 standard test methods . the pre - coated epoxy layer on the stud was thin preventing the penetration of resin to substrate and applies a homogenous resin thickness on the coating . after removing the apatite coating , the samples were analyzed under sem and eds to ensure that the epoxy did not penetrated to the ti substrate . astm c633 , the standard procedure for measuring the adhesion of ha - plasma - sprayed coating , could not be used in this study because the astm procedure can be only applied in coating thickness greater than 0 . 38 mm . scratching test : the scratch test is a common technique used to evaluate the adhesion of a thin coating on the substrate . the adhesion of the coatings obtained in our study was assessed using a csem revetest scratch tester fitted with a rockwell c 200 μm - radius diamond stylus . the point of adhesion failure of the coating from the substrate was detected by an increase in the acoustic signal from the sample . this load is called the critical load lc or failure load measured in n and corresponds to the bonding strength between the coating and substrate . the scratches were generated on the samples by constantly increasing the load at the rate of 100 n / min while the sample was displaced at the constant speed of 10 mm / min . the critical load of coating was measured 10 times . sem analysis showed a uniform coating covering on the entire ti surface after incubation in acidic calcifying solution ( fig1 b compared to 1 a ). the ca — p coating from acidic solution consisted of large and rectangular crystals ( fig2 a and 2b ). xrd identified these crystals as dicalcium phosphate anhydrous ( dcpa ) or monetite , cahpo 4 , the highest peak located at 2θ = 26 . 5 ° ( fig3 b ). a preferred orientation along the ( 111 ) lattice planes of the monetite structure was observed from the xrd pattern ( fig3 b ). the ratio of the intensity of ( 111 ) plane to that of ( 020 ) in the diffraction pattern of the coating was about 2 . 5 , compared to a ratio of 1 . 5 observed in the diffraction pattern of monetite powder ( jcpds # 71 - 1759 ). higher intensity ratio in the coating compared to that in powder diffraction pattern indicates preferred orientation along in the monetite coating ( 111 ). table 1 shows the levels ( percentage ) of different chemical elements identified in the monetite coating using eds . the ca / p atomic weight ratio from eds was 1 . 16 . sodium ( na ) was not detected in the monetite coating . in the second step , the discs coated with monetite obtained from the first step were immersed in naoh solution at 75 ° c . for 24 h . xrd analysis showed that monetite coating was transformed to apatite , with the highest peak ( 211 ) at 2θ = 31 . 8 ° ( fig3 d ). the xrd pattern of apatite coating ( fig3 c ) showed ( 002 )/( 300 ) lattice planes intensity ratio of 1 . 5 compared to a ratio of 0 . 67 for the apatite powder ( jcpds # 09 - 0432 ), indicating preferred orientation along the ( 002 ) planes of the apatite crystals in the coating . the apatite crystals were agglomerated following the outline of the initially formed large monetite crystals ( fig2 c and 2d ). sem of the cross section of the coating showed porosity and a thickness of approximately 30 μm ( fig2 e ). table 1 summarizes the atomic percentage of ca , p , and na obtained using eds analysis in different areas of the coating . the ca / p ratio of the apatite coating was higher than that of the monetite coating ( 1 . 79 vs . 1 . 16 ). a low percentage of na was detected in the apatite coating ( table 1 ), probably due to the partial incorporation of na + ions into apatite structure ( na - for - ca substitution ) during the transformation of the monetite to apatite in naoh solution . fig4 a shows the scratch test data plotted for four different measurements . scratches made on each coating were reproducible in the failure mode . the failure load ( critical load ) at which the coating starts to be removed and the ti surface to become visible was measured at the point of the first increase in acoustic signal from the ti surface ( arrows in fig4 a and fig4 b ). the average of failure load of the coating was 13 . 1 ± 1 . 3n . fig4 b and 4c show the scratch track on apatite coating . no fracture or chipping was observed at the border or inside the scratch track and the coating materials were squashed along of the track . just before the point of the removal of the coating from the substrate , small lateral tensile cracks were observed inside of the scratch track ( fig4 c ). the mean tensile strength in different areas of the coating was 5 . 2 ± 2 . 1 mpa . no trace of epoxy was detected on substrate after coating removal demonstrating the absence of penetration of the resin to the substrate . sem and eds analysis showed — evidence of the presence of — coating residues on the areas from which the coating was removed during the tensile and scratch tests ( fig5 a , 5b and table 1 ). even after the coating appeared visually to have been removed , eds demonstrated the presence of calcium and phosphorous elements on the ti surface . the numbers on fig5 a and 5b correspond to the areas on which the eds analysis was performed . after the tensile test , the ca / p ratio of the coating residues in different areas was significantly lower ( p & lt ; 0 . 05 ) than that of coating surface ( table 1 ). decreasing ca / p ratio was also observed in the coating towards the ti substrate in the scratch track of the scratching test ( table 1 ). considering a na - for - ca substitution in the apatite , ( ca + na )/ p ratio would give a better estimation of the calcium phosphate phase of the coating . although the percent of na increased in the coating residues , the ( ca + na )/ p ratio was still lower in the coating residues close to the substrate compared to that away from the substrate and closer to the surface ( table 1 ). eds analysis of the ti surface ( after removing the apatite coating ) demonstrated the presence of ca — p compounds as well as a higher percentage of na —. the higher percentage of na on titanium surface could be due to the formation of na titanate during the monetite - to - apatite transformation in naoh solution at 75 ° c . the foregoing results show that homogenous and adherent ca — p coating ( monetite ) formed on titanium discs during the immersion of the substrates in the acidic calcifying solution . the low ph of the calcifying solution has several effects on the coating procedure . the low ph of the solution permitted higher a concentration of the calcium and phosphate ions in the calcifying solution . in a neutral calcifying solution ( ph 7 ), the concentrations of calcium and phosphate ions are limited and at higher concentrations , ca — p crystals start to precipitate in the solution . using a calcifying solution containing a higher concentration of calcium and phosphate results in a better coverage of the deposited ca — p coating . an acidic solution also affects the topography and chemistry of the ti surface . several studies have reported the effects of various acids on topography and chemistry of titanium surface . [ kim w d . effects of acid treatments on in vitro bioactivity of titanium . m . s . thesis , new york university 2002 .] acid etching could improve bone / implant attachment by improving the mechanical interlock between the implant and the host bone . surface topography has also been shown to have an effect on osteoblasts cell morphology , proliferation , and differentiation . [ boyan b d , lohmann c h , dean d d , sylvia v l , cochran d l , schwartz z . mechanisms involved in osteoblast response to implant surface morphology . annu rev mater res 31 : 357 - 371 , 2001 ; poisoned l , reybier k , jaffrezic n , comte v , lagneau c , lissac m , martelet c . relationship between surface properties ( roughness , wettability ) of titanium and titanium alloys and cell behaviour . mat sci eng c - bio s 23 : 551 - 560 , 2003 ] acid etching creates pits and increases the surface roughness and surface area of implant . higher surface area could improve the adsorption of proteins and growth factors on materials that in turn may affect cells behavior . on the other hand , a rougher surface could provide a higher number of nucleation sites for the growing calcium phosphate crystals , thereby improving both coverage and adhesion of the coating due to the mechanical bonding between the coating crystals and the ti surface . both acid and alkali pre - treatment have been shown to increase the thickness of oxide layer on the ti or ti alloy surface . the titanium oxide plays a critical role on the absorption of organic layer ( e . g . proteins ) or mineral ( e . g . calcium phosphate ) on the ti surface . in the presence of water , the titanium oxide layer becomes hydroxylated and forms ti — oh groups . the ti — oh groups on titanium surface are amphoteric , exhibiting both acidic and basic properties . acidic hydroxide binds to two ti atoms and basic hydroxide binds to one ti atom . the following equations reactions show the acidic and basic reactions of ti — oh with water molecule : ti — oh ( acidic hydroxide )+ h 2 o [ ti — o ] − + h 3 o + ( acidic reaction ) ti — oh ( basic hydroxide )+ h 2 o [ ti — oh 2 ] + + oh − ( basic reaction ) acidic hydroxide gives proton and causes a negative charged surface ( acidic reaction ), while basic hydroxide accepts proton and makes a positive charged surface ( basic reaction ). in an acidic solution ( e . g ., calcifying solution ), the higher availability of protons promote more basic reactions occur causing a positively charged surface . on the other hand , in a basic solution , the proton detached from acidic hydroxide ( acidic reaction ) resulting in a negatively charged surface . in our method , immersion of titanium discs in acidic calcifying solution could have resulted in a positive surface charge on the substrate , attracting the phosphate ions and creating the nucleation sites for calcium phosphate ( monetite ) deposition . phosphoric acid used as of one of ingredient of calcifying solution provides phosphate groups ( h 2 po 4 − , hpo 4 2 − , and po 4 3 − ), that could be attracted to the positive charge of [ ti — oh 2 + ] and form — further nucleation sites for ca — p deposition . furthermore , acid treatment could also increase the thickness of tio 2 layer . higher roughness , thicker tio 2 layer , and positively charged surface could all contribute to a higher attachment of the monetite layer formed from acidic calcifying solution . it should be mentioned that the higher concentration of positive charge reduces the calcium concentration on titanium surface and also decreases the substitution of phosphate ions for the basic hydroxide group . as mentioned above , in acidic solution , the equilibrium of basic reaction is shifted to right side resulting in a lower number of basic hydroxide present on the ti surface . depending on the ph , temperature , and composition of the calcifying solution , different types of ca — p compounds can form or transform from one type to another type . for example , under acidic condition ( ph 2 to 5 ) and at low temperature ( 25 ° c . to 60 ° c .) dicalcium phosphate dihydrate ( dcpd ), cahpo 4 . 2h 2 o is the preferred ca — p phase . under the conditions of our method ( ph 2 , 75 ° c . ), monetite or dicalcium phosphate anhydrous ( dcpa ), cahpo 4 , was the stable ca — p phase . monetite converted to apatite crystal in a basic solution , as demonstrated in earlier studies . legeros r z . calcium phosphates in oral biology and medicine . volume 15 . san francisco : karger ; 1991 . 201 p . ; [ legeros r z . the unit - cell dimensions of human enamel apatite : effect of chloride incorporation . arch oral biol 20 : 63 - 71 , 1974 . ; legeros r z , legeros j p , trautz o r , shirra w p . conversion of monetite , cahpo4 to apatites : effect of carbonate on the crystallinity and the morphology of apatite crystallites . adv x - ray anal 14 : 57 - 66 , 1971 ]. the transformation of one type of calcium phosphate to another type is in fact a dissolution / re - precipitation process . [ legeros r z . calcium phosphates in oral biology and medicine . volume 15 . san francisco : karger ; 1991 . 201 p . ; the initial ca — p phase dissolves in acidic or basic solutions and the second phase re - precipitates from the supersaturated solution . the sem micrographs showed that the apatite crystals precipitated following the outline of the initially formed monetite crystals , in a similar manner as that observed for the transformation of octacalcium phosphate , ocp , to apatite . eds analysis showed the presence of na ions in the apatite coating but was not detected in the monetite coating indicating that na - for - ca substitution occurred in the apatite during the transformation of monetite to apatite in the naoh solution . the ca / p molar ratio of the apatite coating was calculated as 1 . 79 . however , when na - for - ca substitution in the apatite was considered , the ( ca + na )/ p molar ratio was calculated as 1 . 94 . the ca / p molar ratio of stoichiometric hydroxyapatite , ca 10 ( po 4 ) 6 ( oh ) 2 , is 1 . 67 , lower than that calculated for the apatite coating in this study . the higher value of the ca / p molar ratio obtained for the coating may be due to the partial substitution of carbonate for phosphate ( carbonate type b ). the ca / p molar ratio for the monetite coating was calculated as 1 . 16 , slightly higher than that of a stoichiometric monetite , cahpo 4 , with a ca / p molar ratio of 1 . 0 . eds analysis obtained from areas from which the apatite coating was removed or scratched off demonstrated the presence of a calcium phosphate phase but with at a lower ( ca + na )/ p or ca / p ratio compared to that of the intact coating surface ( table 1 ). lower ca / p ratio of the coating materials at the coating / ti interface could be due to positive surface charge ( formed because of acidic calcifying solution ), that attracts more phosphate ions and leads to a lower ca / p ratio . compared to the apatite coating surface , the percentage of na was higher on the ti surface or coating residues after removing or scratching off the apatite coating . the higher percentage of na on titanium surface or in the coating layer closer to substrate may be explained by the partial incorporation of na in the apatite structure ( na - for - ca substitution ) during the transformation of monetite to apatite in the naoh solution and by the formation of na titanate layer on titanium surface 29 during the monetite - to - apatite transformation . the higher percentage of na could be in fact due to the higher percentage of na on the titanium substrate and not in the remaining apatite coating . formation of na titanate at the substrate / coating interface could also cause an increase in the coating adhesion . the tensile strength of apatite coating prepared in the foregoing was about 5 . 2 mpa . tensile strength of plasma - sprayed coating has been reported to range from about 5 to 54 mpa . this wide range of tensile strength values reported for plasma - sprayed ha coating may be due to the penetration of the resin to the ti substrate used in the tensile test ( especially with thin or porous coatings ) leading to inaccurate measurements . another source of inaccuracy in measuring tensile strength of the plasma - sprayed ha coating could be the misaligning of the tensile force causing a partially shear force during the measurement of tensile strength . in our method , it was observed that the failure occurred within the coating itself rather than at the coating / titanium interface , indicating low cohesive strength of coating compared to its adhesive strength on titanium surface . even for the samples on which the coating was visually removed , eds revealed the presence of remaining ca — p crystals on titanium surface . the coating adhesion using csem scratch test was measured at about 13 . 1 ± 1 . 3n . this value was much higher than those reported for sputtering and laser deposition coatings which were 38 . 47 mn and 1 . 7 mn respectively , using 50 μm - radius stylus . [ inagaki m , yokogawa y , kameyama i . bond strength improvement of hydroxyapatite / titanium composite coating by partial nitriding during rf - thermal plasma spraying . surf coat tech 173 : 1 - 8 , 2003 . ozeki k , yuhta t , fukui y , aoki h , nishimura i . a functionally graded titanium / hydroxyapatite film obtained by sputtering . j mater sci - mat med 13 : 253 - 258 , 2002 .] whereas for ha coating prepared using electrochemical deposition , 20n was needed to scraped off the coating from the substrate . [ kuo m c , yen s k . the process of electrochemical deposited hydroxyapatite coatings on biomedical titanium at room temperature . mat sci eng c - bio s 20 : 153 - 160 , 2002 .] it should be noted that the results of scratch test varies as a function of the coating thickness , coating ductility and brittleness , shape and size of stylus , the rate of applying the load , and substrate hardness . in our study the coating was squashed and displaced to both sides of the scratch track , without any chipping , or fracturing . the acoustic signal started to increase ( the point of reading the critical load ) at the point when the coating started to be removed from the substrate and the ti surface became visible ( adhesive failure ). before the failure point of the coating , small lateral cracks were observed inside the track due to coating delamination . the absence of fracturing indicates that the coating materials were squashed without detaching from the substrate . fernandez - pardas et al [ femandez - pradas j m , cleries l , martinez e , sardin g , esteve j , morenza j l . influence of thickness on the properties of hydroxyapatite coatings deposited by krf laser ablation . biomaterials 22 : 2171 - 2175 , 2001 ]. reported that when the coating deform without fracturing , and did not detach from the substrate even when subjected to 18n load . squashing of coating materials without fracturing could be the reason for the big difference between the critical loads measured in our study compared to those reported for the other type of coating . for the coating prepared by pulsed laser deposition technique , arias et al . [ arias j l , mayor m b , pou j , leng y , leon b , perez - amor m . micro - and nano - testing of calcium phosphate coatings produced by pulsed laser deposition . biomaterials 24 : 3403 - 3408 , 2003 ] reported that although the first increase in acoustic signal was read at 3 . 5n ( due to the formation of fractures ), the coating was partially removed and the substrate became visible at 9 . 6n load . the adhesion of the coating obtained from our method was much higher than that of coating prepared in calcifying solution at 37 ° c . and ph 7 . 2 . as mentioned above positive surface charge of titanium and higher surface roughness ( both due to acidic ph of calcifying solution ) could increase the number of nucleation site for monetite deposition . phosphate groups ( h 2 po 4 − , hpo 4 2 − , and po 4 3 − ) provided by phosphoric acid and nah 2 po 4 . h 2 o could bond to [ ti — oh 2 ] + group , create nucleation sites and increase the attachment of the calcium phosphate coating . furthermore , formation of na titanate during the transformation of monetite to apatite in naoh solution , could also contribute to a higher coating / substrate adhesion . the foregoing demonstrates the value of chemical deposition from acidic calcifying solution as an alternative coating technique . the coatings prepared using the present method were porous and composed of crystalline phase of apatite . good coating adhesion was obtained compared to other low temperature coating methods . in our method , using dilute phosphoric acid in calcifying solution made a positive surface charge [ ti — oh 2 ]+ which bonds to the phosphate groups ( h 2 po 4 − , hpo 4 2 − , and po 4 3 − ) and provides more nucleation sites for calcium phosphate deposition . furthermore , acidic calcifying solution could also modify the ti surface topography and increase the mechanical interlock between the substrate and ca — p coating . formation of na titanate during the transformation of monetite crystals to apatite could also favor apatite deposition and adhesion . while the present invention has been set forth in terms of specific embodiments thereof , the instant disclosure is such that numerous variations upon the invention are now enabled to those skilled in the art , which variations yet reside within the scope of the present teaching . accordingly , the invention is to be basically construed and limited only by the scope and spirit of the claims now appended hereto .
an adherent bioactive calcium phosphate coating is formed on a titanium or titanium alloy substrate by immersing the substrate in an acidic calcium phosphate solution to form a non - apatitic calcium phosphate coating on the substrate . in a second step the coated substrate can be converted to a less reactive coating by being immersed into a basic or neutral solution to convert the coating into an apatite . however if a relatively reactive coating is desired , the second step can be dispensed with .