Dania.docx J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 17 The Effect of Pores in Dual Nano Hydroxyapatite Coating on Thermally Oxidized Commercial Pure Titanium: Mechanical and Histological Evaluation Dania Fawzi Mahmood, B.D.S.(1) Salah A. Mohammed, B.D.S., M.Sc. (2) ABSTRACT Background:In this study,TiO2 layer was thermally grown as a diffusion barrier on CP Ti substrate prior to electrophoretic deposition of HA coatings, to improve the coating’s compatibility also macro and micro pores in nano Hydroxyapatite dual coatings were created and their effect on the bond strength between the bone and implant was evaluated. Materials and methods: Electrophoretic Deposition technique (EPD) was used to obtain coatings for each one of four types of Hydroxyapatite(HA)on CP Ti screws (micro HA, nano HA, dual nano HA with micro pores, dual nano HA with macro pores) where carbon particles used as fugitive material to be removed by thermal treatment to create porosity.For examination of the changes occurred on the substrate, SEM, SPM and XRD used, coatings characterized by XRD, SEM and interfacial shear strength measurements. Results:The results mentioned the formation of rutilenano TiO2 with, SEM showed that the size of pores in HA coatings corresponded to the size of carbon particles. Statistical analysis of the removal torque tests showed highest means of the single nano HA coating at 2 and 4 weeks implantation intervals. Histological analysisrevealed a faster reaction of bone and higher osteoblasts activity towards thermally oxidized CP Ti implants coated with single nano HA coating. Conclusion:Carbon particles as a fugitive material within nano HA coat produced porosity.Presence of pores 1µ in nano HA coats did not achieve highest removal torque values nor highest osteoblasts activity in 2 and 4 weeks implantation intervals. Keywords:Titanium, Thermal oxidization, Nano Hydroxyapatite, Coatings, porosity. (J Bagh Coll Dentistry 2016; 28(1):17-25). INTRODUCTION One of the major issues with hydroxyapatite coating on a metallic substrate is certain level of porosity in the hydroxyapatite films can be achieved using electrophoretic deposition but with limits where no macro pores are evident, only small pores up to 1 μm observed in the coating. Similar surface morphology of the nanohydroxyapatite(1,2)and Bioglass® coatings(3) formed by electrophoretic deposition followed by sintering was reported.But the necessity of porosity in bone regeneration has been shown by many studies(4,5). Pores are necessary for bone tissue formation because they allow migration and proliferation of osteobalsts and mesenchymal cells, as well as vascularization(6). In addition, a porous structure improves mechanical interlocking between the implant biomaterial and the surrounding natural bone, providing greater mechanical stability at this critical interface(7). Thermal treatment is one of the many techniques described(8)to produce porosity in scaffolds used for bone regeneration by extruding fugitive materials from thermoplastic ceramics to produce a 3- directional macroporous structure. The production of porous tubes was performed by means of electrophoretic deposition for use in biomedical application. (1)Master student, Department of Prosthetic Dentistry, College of Dentistry, University of Baghdad. (2)Assistant Professor, Department of Prosthetic Dentistry, College of Dentistry, University of Baghdad. By means of the hydrothermal process, the mixing of carbon nanotubes and hydroxyapatite was obtained. The hydroxyapatite porous tubes processed with electrophoretic deposition (DC 60V) had a thickness of 310 µ after sintering (1200°C, for 60 min). After the sintering process, porous crack free hydroxyapatite micro tubes were obtained. The experiments conducted reveal that the hydroxyapatite-multi wall carbon nano tubes combination enables the production of complex- shaped ceramics using the electrophoretic deposition process(9). MATERIALS AND METHODS In vitro Commercial available CP Ti grade 2 substrate, micro and nano HA powders were used. 1. Substrate Thermal Oxidization: Mirror polished Cp Ti substrates were ultrasonicated in water bath for 30 min, followed by acetone wash(10) then were heated at 650⁰C for 8h in oven (11). 2. CarbonPowder Preparation:Graphite blocks were ground by ball milling and sieved into two groups according to particles size range; 1~50µ micro and 60~125µ macro particles. 3. EPD:Thermally oxidized CP Ti substrate was used for both electrodes. J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 18 Single Layer Micro HA: The distance between the electrodes was10 mm., suspension preparedfroml00g micro HA in 1 liter Ethanol (12) andin order to determine the most suitable time and voltage that would give the best HA film 3 voltages were used (30, 40 and 60) V and for each voltages 3 times were used (2, 3 and 5) minutes finally sintering was carried out at 800 C under argon for 2 hours(13). Single Layer Nano HA: The distance between the electrodes was 5 mm., suspension preparedof 0.5g nanoHA in 100 ml Ethanolin order to determine the most suitable time and voltage that would give the best HA film 3 voltages used (40, 50 and 60), V were used for each voltages 3 times (4, 5 and 6) minutes finally sintering was carried out at 900 C under argon for 2 hours. Dual Layer Nano HA: For the basal layer, the distance between the electrodes was 5mm, suspension was preparedfrom0.5g nanoHA in 100 ml Ethanol the deposition voltage and time were40V/ 6 minutes finally sintering was carried out at 900 C under argon for 2 hours (1). For the superficial layer,the distance between the electrodes was 5mm., suspension was preparedfrom0.5g nanoHA in 100 ml Ethanol with 1, 1.5, 2 (14), 10 and 20 wt. percentage micro or macro carbon particlesaccording to the desired pores sizes, the deposition voltage and time were40V/6 minutesfinally sintering was carried out at 700 C under air for 2 hours(15). 4.Characterization Both TiO2 and HA coatings phase analysis was done by X-ray Diffractometer (SHIMADZU, XRD- 6000, Japan), microstructural analysis was done by optical microscope (Nikon Eclipse ME 600L/441002,Japan with digital camera type DXM 1200 F) and (Vega Easy Probe Scanning Electron Microscope, Spain), thickness measurement was done by microprocess coating thickness gauge (ERICHSEN GMBH & CKG, D-5870 HEMER SUNDWIG, W-GERMANY). For TiO2 topography analysis scanning probe microscope (Angestrom Advanced Inc., SPM-AA3000) was used. Shear strength measurements for the HA coatings were done according to ASTM F1044 –11by using automatic extensometer (Instron 1122, England with load cell of 500 N). In vivo 1. Implant Preparation: Eighty screws machined from cp Ti grade 2,10mm rod by using Lathe machine, then they were thermally oxidized and divided into four groups according to the coatings type; micro HA, nano HA, nano HA with micro pores and nano HA with macro pores coated screws finally the coated screws were sterilized with gamma irradiation dose of 2.5-3.0 mega rad using gamma cells 220 with Co60source. 2. Implantation and Removal Torque and Histological Tests: 16 rabbits were divided into 2 groups for each healing interval (2 and 4 weeks) each one consist of 8 animals, one of them were sacrificed for histological study, while the other 7 were sacrificed for mechanical test by torque removal test. Four implants implanted in the tibiae of each rabbit, in the right tibia implant coated with nano HA implanted medially and implant coated with micro, HA implanted laterally in the same tibia, while in the left tibia of the same rabbit implant coated with nano with micro pores, HA implanted medially and implant coated with nano with macro pores implanted laterally in the same left tibia. RESULTS Phase Identification: XRD patterns of unoxidizedCp Ti substratesand oxidized substrates were obtained. Upon peak matching with the relevant ICDD files, before oxidation the peaks corresponded to α–Ti and after thermal oxidation of the substrates the peaks corresponded to formation of rutile phase of TiO2 as shown in figure 1. XRD pattern of EPD deposited and sintered HA coatings on substrates shows there isn’t any unstablephase formed during sintering like TTCP, TCP or calcium oxide,crystallite size in the grains of nano HA coatings was estimated according to sherrer equation (d = B ג / ß cos ), For the single layer of nano HA, the particle size or diameter ranges from 23.1 ~ 138.6 nm and for the dual layer nano HA the particle size or diameter ranges from 20 ~ 198 nm.The results supported the formation of HA coating on oxidized CPTi substrate and absence of any other reaction products.No peaks for Titania (TiO2) are detected in the XRD spectrum, this shows that the sintered HAP coating completely cover the entire substrate as shown in figure 2. J BagJ Bagh College Dentistry Restorative Dentistry Unoxidized CP Ti grade 2 Microstructural Optical microscope observations f substrate optical microscope formation of scales after thermal treatment 3.SEM showed the formation highly porous su after thermal oxidization, Microscope Thermally HA coating after sintering the optical microscope observations showed that th uniform coating layer of formed at 40V/2 minutes while for the nano HA coatings nano HA the number of the carbon particles after 1 hour sintering under air proceeded to complete absence after 2 hour h College Dentistry Restorative Dentistry Figure 1: Unoxidized CP Ti grade 2 Microstructural Analysis: ptical microscope observations f substrates before and after thermal oxidization optical microscope, formation of uniform layer of rainbow colored oxide es after thermal treatment showed the formation highly porous su after thermal oxidization, Figure 3: Optical Figure 4:Scanning Microscope for the hermally Grown TiO HA coating after sintering the optical microscope observations showed that th uniform coating layer of formed at 40V/2 minutes while for the nano HA was formed at 40V/6 minutes. While for nano HA with carbon particles the number of the carbon particles after 1 hour sintering under air proceeded to complete absence after 2 hour, as shown in h College Dentistry Restorative Dentistry Figure 1:XRD Patterns Unoxidized CP Ti grade 2 Substrates. nalysis: ptical microscope observations f before and after thermal oxidization observation showed uniform layer of rainbow colored oxide es after thermal treatment as shown in showed the formation highly porous su after thermal oxidization,figure 4. OpticalMicrograph Scanning Electron the TiO2Barrier. rown TiO2. HA coating after sintering the optical microscope observations showed that th uniform coating layer of the micro HA coatings was formed at 40V/2 minutes while for the nano HA was formed at 40V/6 minutes. While for with carbon particles showed decrease in the number of the carbon particles after 1 hour sintering under air proceeded to complete absence as shown in figure 5. h College Dentistry Vol. 28 atterns of Oxidized ubstrates. ptical microscope observations for Cp Ti before and after thermal oxidization observation showed that uniform layer of rainbow colored oxide as shown in f showed the formation highly porous su icrograph lectron arrier. View for HA coating after sintering the optical microscope observations showed that the most the micro HA coatings was formed at 40V/2 minutes while for the nano HA was formed at 40V/6 minutes. While for showed decrease in the number of the carbon particles after 1 hour sintering under air proceeded to complete absence 8(1), March 19 xidizedandFigure 2: or Cp Ti before and after thermal oxidization that the uniform layer of rainbow colored oxide figure showed the formation highly porous surface for HA coating after sintering the optical e most the micro HA coatings was formed at 40V/2 minutes while for the nano HA was formed at 40V/6 minutes. While for showed decrease in the number of the carbon particles after 1 hour sintering under air proceeded to complete absence (A) Before S M Nano HA, Carbon 1% Total Powder Weight nano HA absence of macro pores, the nano HA coatings contained pores up to 1µ only F with 40 Voltz and 2 Minute Electrophoretic Deposition, Showing the Surface M March 2016 Figure 2:XRD (A) Before S Air (C) After Sintering in A Magnification. Hours, at 50&100X Figure 5:Optical Microscopic Image for Nano HA, Carbon 1% Total Powder Weight Micro Particles C SEM for the micro nano HA coating, absence of macro pores, the nano HA coatings contained pores up to 1µ only Figure 6: SEM for Micro HA Coat A with 40 Voltz and 2 Minute Electrophoretic Deposition, Showing the Surface M at (100 and 168 XRD Patterns (A) Before Sintering,(B) (C) After Sintering in A agnification.for One Hour, at 100X for 2 ours, at 50&100XM Optical Microscopic Image for Nano HA, Carbon 1% Total Powder Weight Micro Particles C SEM for the micro HA coating, f coating,figure 7 after sintering showed the absence of macro pores, the nano HA coatings contained pores up to 1µ only igure 6: SEM for Micro HA Coat A with 40 Voltz and 2 Minute Electrophoretic Deposition, Showing the Surface M at (100 and 168) X SEM M The effect of HA. (B) After Sintering in (C) After Sintering in Airat 100X for One Hour, at 100X for 2 Magnification. Optical Microscopic Image for Nano HA, Carbon 1% Total Powder Weight Micro Particles Coating. HA coating, f after sintering showed the absence of macro pores, the nano HA coatings contained pores up to 1µ only as shown in igure 6: SEM for Micro HA Coat A with 40 Voltz and 2 Minute Electrophoretic Deposition, Showing the Surface Morphology SEM Magnification. The effect of After Sintering in at 100X for One Hour, at 100X for 2 agnification. Optical Microscopic Image for Nano HA, Carbon 1% Total Powder Weight HA coating, figure 6 and after sintering showed the absence of macro pores, the nano HA coatings as shown in figure 8. igure 6: SEM for Micro HA Coat Achieved with 40 Voltz and 2 Minute Electrophoretic orphology agnification. of J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 20 Figure 7: SEM for Nano HA Coat Achieved with 40 Voltz and 6 Minute Electrophoretic Deposition after Sintering, Showing the Surface Morphology at SEM Magnification 100 and 500 X. Figure 8: SEM Shows the Surface Morphology of Nano HA after Sintering, Showing the Typical Pore Size in the Nano HA Coat, SEM Magnification 14.76Kx. SEM for the nano HA with carbon before sintering showed the inclusion of the carbon particles within the nano layer as shown in figure 9, and after sintering showed that for the nano HA coatings with micro and macro carbon particles , the formation of few number of pores corresponded in size of the carbon particle size at 1, 1.5 and 2 % from the total powder weight concentrations, for the nano HA with micro carbon particles the number of pores increased when 10% concentration was used, up to critical level of 20% concentration were large devoid areas of the superficial layer detected as shown in figure 10, while for the nano HA with macro carbon particles when 10% concentration was used, formation of nano HA whiskers were detected, and when 20% concentration was used, pores up to 85µ were detectedfigure 11. Figure 9: SEM Image of the Dual Nano HA Coat with 10% Micro Carbon, before Sintering, Shows the Sistribution of the Sarbon Micro Particles within Nano HA (arrows). (A) (B) Figure 10:SEM of Dual Nano HA Coat /micro Carbon Particles with 2 Concentrations:(A) 10 % and (B) 20 % from the Total Powder Weight, after Sintering at 700ºC for 2hrs in Air Atmosphere. (A) (B) Figure 11:SEM of Dual Nano HA Coat/Macro Carbon Particles with 2 Concentrations:(A) 10 % from the Total Powder Weight at 25 Kx SEM Magnification, and (B) 20 % from the Total Powder Weight at 500X SEM Magnification, after Sintering at 700ºC for 2hrs in Air Atmosphere. Thickness Measurements: Formation of 1.6µ thick TiO2 detected, while for the micro and nano HA coatings, the thickness increased with increasing both voltage and time, while for the dual nano layer when the voltage and time fixed the thickness was constant (68µ). SPM measurementsshowed the formation of oxide with nano topography as shown in figure 12. Figure 12: SPM Shows the TiO2 Barrier has Nano Surface Roughness. J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 21 Shear Strength Measurements: The highest magnitude for the dual nano HA, followed by single layer nano HA and the least magnitude was for micro HA coatings, as shown in table 1. Table 1: Values for the Mean Failure Load (Newton) with Failing Stresses or Adhesion Strength (MPa) for each Type of Coating Type of coat Mean failure load /N Adhesion strength / MPa Micro HA 20 0.4 Nano HA 57 1.14 Nano HA with micro pore 100 2 Nano HA with macro pore 78 1.6 Removal Torque Test: Removal torque test revealed that the highest torque mean values were for the screws coated with nano HA at 2 and 4implantationintervals, and statistical analysis done by ANOVA at p=0.05 showed highly significant difference between removaltorque meanvalues for screws coated with four different coatings at eachimplantation intervalsof 2 and 4 weeks, while there was a significant difference only for the dual coat with pores 50 µ between 2 and 4 weeks implantation intervals. Histological Evaluation: Historical evaluation revealed that there was a faster reaction of bone and higher osteoblasts activity towards thermally oxidized CP Ti implants coated with nano HA compared to thermally oxidized CP Ti implants coated with micro HA at 2 and 4 weeks implantation intervals,as shown in figure 13 and figure 14. Early formation of Haversian system observed after 2 weeks of implantation at bone implant interface in screws coated with nano HA with macro pores,as shown in figure 15. Figure 13: Optical Microscopesshow Newly Formed Bone Trabeculaearound the Four Different Types of HA Coatings at 2 Weeks Implantation Intervals. Figure 14: Optical Microscopesshow Newly Formed Bone Trabeculaearound the Fourdifferent Types of HA Coatings at 4 Weeks Implantation Intervals. Figure 15: Optical Microscope Shows Early Formation of HaversianSystem at Bone Implant Interface in Screws Coated with Nano HA with Macro Pores after 2 Weeks of Implantation Intervals. J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 22 DISCUSSION Thermally Grown TiO2 Barrier In the current work, a transition layer of thermally grown titanium oxide is intentionally grown between cpTi and EPD grown HA coating to prevent HA decomposition as a result OF ionic transport from metallic substrate to HA(1).The presence of a TiO2 layer also introduces ceramic– ceramic (TiO2–HA) interface in place of metal– ceramic (cpTi /HA) interface, hence reducing the thermal expansion coefficient mismatch between the coating and substrate (16). The Surface Morphology of Thermally Oxidized Sample Both optical microscopic and SEM image clearly reveals the presence of oxide smooth scales throughout the surface without spallation. Obviously, the duration of oxidation is considerably high to achieve a good surface coverage with the oxide scale and this agrees with other findings (11). Pores are also evident throughout the surface,this microstructural inhomogenities might be generated during the growth of oxide islands and their orientation on the surface, where thermal oxidation would lead to an increase in surface roughness of the oxide film due to the differential oxidation rate of individual grains of the polycrystalline Alloy. Structural Characteristics of Untreated and Thermally OxidizedSamples The XRD pattern of the untreated CP Ti sample is entirely comprised of hexagonal α-phase, while XRD pattern of thermally oxidized sample exhibit the presence of dominant rutile phase peaks, this disagrees with Kumar et al (11).Weak intensity α-Ti peaks are also present and this agrees withKumar et al(11),where α-Ti peaks are evident in thermally oxidized CP Ti sample having a thin oxide layer and this may be due to that Cu–Kα radiation could penetrate to a depth of 10~20 μm, much larger than the thickness of the oxide film where the average thickness of the oxide layer is 1.6µ.From the data obtained from the XRD and according to Scherer equation, the grain size of TiO2 has nano measurements suggesting the presence ofnano surface roughness supported by the measurement provided from the scanning probe microscope, this may be due to the effect of thermal oxidation conditions like temperature and time. Oxide Film Thickness The oxide film average thickness is 1.6µ, measured by microprocess coating thickness gauge, and this disagree withJain et al and Kumar et al(10,11),who reported the formation of thicker oxide layer may be due to different thermal oxidation conditions.The surface morphology, oxide grains size and the evolution of surface topography of the oxide film may suggest the mechanism of the growth mode for the oxide barrier which involves the formation of a thin oxide scale followed by its agglomeration and growth, to completely cover the surface, versus oxide film formation mode according to which the nucleation of oxide takes place throughout the surface when it immediately comes in contact with oxygen (11). HA coatings Morphology of HA Coatings In thepresent study cracks observed across the single layer nano and micro HA which is probably due to thermal expansion mismatch of HA and CP Ti substrate. The thermal expansion coefficient of titanium substrate is much lower than that of HA, so large thermal contraction mismatch would arise and tend to induce the formation of cracks when cooled from the elevated temperatures of sintering; besides, a significant firing shrinkage during sintering will lead to the formation of cracks in coatings as well this agrees with Wang et al (17). Cracks increase in size with increase of the thickness of coating layers, whether the increase of the thickness was due to increase in time or in applied voltage this agrees with the findings of Meng et al(13). Cracks were clearly seen in the top coating layers in the dual coat approach and it appeared that there is no distinct border between the first and the second coating layers, this agrees with Wei et al(1)and may suggest that a strong seamless bonding formed between these two coating layers.The porosity in the single layer micro HA differs from that in nano HA, since porosity is particle size dependent where larger than pores present in micro HA sample compared with nano HA sample, no macro pores are evident, only small pores up to 1μm observed in the coating, this agrees with other findings(2,3). Carbon particles with particle size (1~125) µ, was successfully electrophoretically deposited as a composite with nano HA on CP Ti substrate, smaller percentages of the carbon particles from the total powder weight didn’t give distinct pores, while increasing the percentage of carbon particles weight from the total powder weight in the deposition process increased the number of the pores in the coatings after sintering this may be due to increased inclusion of carbon particle.The size of the pores in the coatings seems to correspond roughly to the size of the carbon particles used, they are smaller, may be due to thermal contraction after cooling from sintering, except for 20% J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 23 concentration of carbon with particle size (1~50)µ from the total powder weight, carbon particles deposited in large accumulations leaving no clear morphology pores after they had been removed by sintering, only large areas devoid from the second layer. SEM image of dual nano HA coat electrophoretically deposited with carbon particles sizes ranges from 60 µ to 125 µ with 10 % concentration from the total powder weight after sintering, it shows the formation of HA nano whiskers; they are rod like particles with high aspect ratio (length to diameter), they possea great influence on osseointegration, and improving mechanical properties, as well as the nano HA whiskers resemble both the size of the nano apatite and the anisotropy in the natural bone(18). HA Coatings Micro Thickness From the results,electrophoretic deposition indicated that the coating thicknessincreases with deposition time under a constant voltage condition and this agreed with the findings ofZhitomirsky(19).AlsoEPD indicated that the coating thicknessincreases with applied voltage under a constant deposition time, and this agreed with the findings of Meng et al(13). Although the time and voltage were the same for the first and second layer of the dual coat but the thickness differs between them, where the first layer is thicker, and this may be due to the voltage drop across the first deposited and sintered layer.Also the result shows that the carbon particles size didn’t have any effect on the thickness of the dual coat where after sintering the thickness of the both nano HA with micro and macro pores did have same thickness. Structural Characteristics of HA Coatings The XRD patterns for the electrophoretically deposited micro HA coat, nano HA single layer coat and for nano HA dual layer coat showed pure phase of HA after sintering and there is no presence of any decomposition phase, like TTCP, αTCp or CaO upon comparison with ICDL files, this disagrees withRuys et al(16). No decomposed phases are reported may be due to the presence of TiO2 barrier that prevent that act as diffusion barrier between the substrate and the coat, as well as for the dual coat might be protected from the metal substrate ion migration and subsequent protection from decomposition, is that the first layer of the coat acts as a diffusion barrier, and application of the first coating layer also created a large diffusion distance to be traversed by the diffusing ions. While the peaks of the micro HA coat after sintering are broad as an index of poor crystallization,the peaks of the nano HA coat both single and dual layers are sharp with low background as an index for high crystallinity and this agrees with Wong et al(17). Crystallite size in the grains of nano HA coatings after sintering was estimated by using Scherrer’s formula, for the single layer nano HA the crystal size or diameter ranges from 23.1 ~ 138.6 nm and for the dual layer nano HA the crystal size or diameter ranges from 20 ~ 198 nm and this agrees with other findings(10,20).The three XRD patterns of micro HA, nano single layer and nano dual layer coatings did not include peaks for the substrate (α-Ti), and this may be due to that the sintered HAP coating completely cover the entire substrate. This disagrees with Wong et aland Jain et al(10,20).The three XRD patterns of micro HA, nano single layer and nano dual layer coatings possess high crystallinity as indicated by sharp peaks, this agrees with Wong et al(20), also the peaks corresponding to nano HA are much broader than those of micro HA sample which is due to its smaller grain size. Shear Testing of HA Coatings To access the reliability of HA coatings when used in vivo; that is the adhesive strengthor the interfacial shear strengthof the screw coatings upon tightening in the bone during implantation process, the ASTM F1044 was performed for the micro , nano , nano with micro pores and nano with macro pores HA coatings. In thepresent study, the fracture always happened at the interface of HA coating and Ti substrate, for all types of coatings implying that the test was viable and acceptableand this agrees withChen et al(21)and disagrees withWei et al(1). The value of adhesive strength was least for micro HA then nano HA,nano with macro pores and the highest value was for nano HA with micro pores. in general the adhesive strength for the nano was higher than that for the micro HA, this may be due to that the nano particles provide larger interfacial area with the substrate, this agrees with Sun et al(22)also the larger pores in micro HA sample compared with nano HA sample,and the larger the pore the more stress concentration and weaker mechanical properties. The larger adhesive strength for the dual layer nano HA as compared with single layer may be due to that when the second coating layer was coated on top of the primary layer, it filled in the cracks in the primary layer, thereby creating coating integrity(1).Although they didn’t found correlation between the observed degree of cracking and adhesive strength of nano HA dual coatings.Coat integrity may also explains the reason why nano HA J Bagh College Dentistry Vol. 28(1), March 2016 The effect of Restorative Dentistry 24 coat with micro pores has larger adhesive strength than the coat with macro pore. Mechanical Test In the present study, it has been shown that a difference between different time periods was presentwere the minimum torque value was seen in 2 weeks implantation time while the maximum value was observed in the 4 weeks implantation periods for the four groups.This agreed with the study of Johansson and Albrektsson(23)thatdemonstrated an increase in the removal torque with time. It has been suggested that this increase depends on an increasing bone-to- metal contact with time as a result of a progressive bone formation and remodeling around the implant during healing, whichsubstantially improved the mechanical capacity. A comparison between the different implantation periods in this study shows that after 2 weeks of implantation a higher torque value was needed to remove the screws coated with nanoHA, followed by the screws coated with micro HA, then the screws coated with nano HA with macro pores and finally the screws coated with nano HA with micro pores.After 4 weeks of implantation, a higher torque value was needed to remove the screws coated with nano HA, followed by the screws coated with nano HA with macro pores, then the screws coated with nano HA with micro pores and finally the screws coated with micro HA,this may be due to the effect of the surface topography of the HA coat , where removal torque values revealed an increased retention for the chemically modified implants that exhibit specific nano topography, this agrees with Meirelles et al(24). In the present study there was a significant difference in torque value between all 4 groups of implant screws coated with nano ,micro , nano with micro pores and nano with macro pores HA,at two periods of healing interval (2 and 4 weeks).Regarding the torque values with implantation time, the findings of present study showed that there is significant increase in the torque value after 4 weeks of implantation, only for the screws coated with nano HA with macro pores at P 0.05, this may reflects the importance of the HA coat pores with macro dimension on osteobalsts and osseointegration. Histological Test The histological analysis of all groups showed a new bone trabeculae formation, with active osteoblats on borders and osteoblast rim activity is also evident, and pre-osteocytes are trapped in the newly formed matrices.The bone trabeculae are thicker at both implantation intervals for the nano HA coated implants when compared to micro HA coated implants, this is due to nanotopography against micro topography which enhance bone formation. This agrees with Meirelles et al(24). Theimplant coated with nano HA with macro pores in the histological microphotograph showed beginning of Haversian system formation at 2 weeks implantation interval this may be due to that macro pores might provide encouraging environment for the progenitor cells to proliferate and differentiate. REFERENCES 1. Wei M, Ruys AJ, Milthorpe BK, Sorrell CC, Evans JH. Electrophoretic Deposition of Hydroxyapatite Coatings on Metal Substrates: ANanoparticulate Dual-Coating approach. 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