2.1

Sample preparation

Commercially pure grade 2 turned titanium discs (20 mm diameter and 1 mm thickness) as a culture substrate, turned titanium mini-screws (0.5 mm diameter and 5.5 mm length) and grade 1 titanium film (10 mm ² and 0.1 mm thickness) were purchased (Nishimura Co., Ltd., Fukui, Japan). The titanium samples were washed under ultrasonication with a series of ethanol and Milli-Q water after acetone cleaning. The acid-etched surface was prepared by immersion of the turned surface discs in 67% (w/w) sulfuric acid solution at 120 °C for 75 s . Two types of nanotopographical titanium surfaces were prepared by two AH-treatment protocols. In previously reported protocol , the cleaned turned discs were boiled in 5 mol/L sodium hydroxide solution at 60 °C for 24 h. After being washed in Milli-Q water and air-dried, the discs were heated in a furnace with an increase in temperature at a rate of 5 °C/min and sintered at 600 °C for 1 h. After sintering, the discs were naturally cooled. In the other protocol (our modified protocol), the discs were boiled in 10 mol/L sodium hydroxide solution at 90 °C for 24 h, followed by the same subsequent process as previously described. All prepared samples were rinsed by ultrasonication in distilled water for 10 min, and stored in ambient conditions, in the dark, for 4 weeks prior to use. All of the discs and films for culture experimentation were placed on 12-well culture-grade polystyrene plates.

2.2

Surface characterization

The surface topography of the sample discs was evaluated by observation via scanning electron microscopy (SEM; XL30, Philips, Eindhoven, Netherlands) and the surface roughness measurements were evaluated using a 3D-measuring laser microscope (LEXT OLS4000, Olympus, Tokyo, Japan) with a cut-off value of 8 μm and a measurement length of 120 μm. The number and density of nanospikes on both types of the nanotopographic surfaces were analyzed via SEM surface images using an image analyzer (ImageJ, National Institutes of Health, Bethesda, MD, USA). The thickness, porous structure and chemical composition of both types of AH-treated surfaces were evaluated by image analysis of SEM images and elemental analysis using an electron probe microanalyzer (EPMA, JXA-8200, JEOL Ltd., Tokyo, Japan) on cross-sectional specimens of AH-treated titanium films prepared by cutting the film with stainless steel scissors. Three areas were measured per sample, and the data were averaged. The set of measurements was performed in three independent samples.

2.3

Oral fibroblastic cell culture

Fibroblasts were obtained from the palatal tissue of 12-week-old Sprague-Dawley rats by reference to the methodology in previous article . Briefly, the palatal gingiva was washed with 1% PBS and incubated in 0.1 units/ml collagenase/0.8 units/ml dispase solution for 60 min, followed by separating the connective tissue from the epithelial layer. Then, the connective tissue was dissected into small pieces (<1 mm ² ) and digested in 0.25% collagenase for 12 h. The liberated cells were collected and plated in 100 mm plastic tissue culture dishes with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotic–antimycotic solution, containing 100 U ml/L penicillin G sodium, 100 lg ml/L streptomycin sulfate and 250 ng ml/L amphotericin B in a humidified atmosphere of 95% air and 5% CO ₂ at 37 °C. The cells were passaged for a second time when 80% confluency was achieved. After the second passage, the cells were detached using 0.25% Trypsin-1 mM EDTA-4Na and seeded directly onto the titanium discs or films with turned, 5 or 10 M AH-treated or acid-etched surfaces at 3.0 × 10 ⁴ cells/cm ² . The medium was renewed every 3 days. This study protocol was approved by the Animal Research Committee of Tokyo Dental College (Protocol No. 232604).

2.4

Cellular morphology

On day 1, the culture of gingival fibroblasts on discs with turned, 5 or 10 M AH-treated or acid-etched surfaces, were fixed in 10% neutral buffered formalin for 30 min. The cells were treated with 0.1% Triton X for 5 min, and then stained with 2% rhodamine phalloidin (Actin filament red color, Molecular Probes, OR, USA) in PBS and the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI; Nuclei blue color, Vector, CA, USA) in a mounting agent. Cell morphology and cytoskeletal arrangement were observed under a fluorescent photomicroscope (Axiophoto2, Carl Zeiss Co., Ltd., Jena, Germany). Cell morphometry was analyzed using ImageJ software.

2.5

Cellular attachment and proliferation

The number of cells in the cultures from days 1 and 7 on discs with turned, 5 or 10 M AH-treated or acid-etched surfaces was evaluated by tetrazolium salt-based colorimetry (WST-1, Roche Diagnostics, Tokyo, Japan). Each culture was incubated at 37 °C for 4 h in 1 mL fresh culture media containing 100 μL of WST-1 reagent. The amount of formazan product produced by viable cells was measured by colorimetry at 420 nm using a microplate reader. The proliferative activity of cells was measured by bromodeoxyuridine (BrdU) incorporation during DNA synthesis. On day 4 of surface culture, a 100 mM BrdU solution (100 ml) (Roche Diagnostics, Tokyo, Japan) was added to the culture wells and incubated for 10 h. After denaturing DNA, the cultures were incubated with anti-BrdU antibody conjugated with peroxidase for 90 min and exposed to tetramethylbenzidine for color development. Absorbance was measured using an enzyme-linked immunosorbent assay reader at 370 nm.

2.6

Gene expression analysis

Gene expression was analyzed on days 7 and 14 using reverse-transcriptase polymerase chain reaction (PCR). The total RNA in these cultures was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and a purification column (RNeasy, Qiagen, Valencia, CA, USA). Following DNAse I treatment, reverse transcription of 0.5 mg total RNA was performed using MMLV reverse transcriptase (Clontech, Carlsbad, CA, USA) in the presence of oligo(dT) primer (Clontech). PCR was performed using Taq DNA polymerase (EX Taq; Takara Bio, Madison, WI, USA) to detect collagen I and III mRNA using primer designs and the PCR condition established previously . The forward and backward primers were designed: collagen I: 5′-GGCAACAGTCGATTCACC-3′ and 5′-AGGGCCAATGTCCATTCC-3′, collagen III: 5′-CCTGGACTCAGGGTATC-3′ and 5′-TGCAGGGCCTGGACTACC-3′, respectively. The annealing temperature and the amplification cycles were set at 58 °C and 28 cycles for collagen I and 60 °C and 25 cycles for collagen III. The PCR products were visualized on 1.5% agarose gel by ethidium bromide staining. Band intensity was detected under UV light and normalized with reference to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The gene expression analyses were performed three to five times and representative data sets presented after confirming consistency.

2.7

Collagen production

To quantify collagen production, on day 21, Sirius red staining-based colorimetric assay was employed for cultures on discs. Cultures from different groups were washed with pre-warmed PBS at 37 °C for 1 min and fixed with Bouin’s fluid for 1 h at room temperature. The cultures were washed with ddH ₂ O and treated with 0.2% aqueous phosphomolybdic acid for 1 min. Then, the cultures were washed again with ddH ₂ O and stained with Sirius red dye (C.I. No. 35780, Pfaltz and Bauer, Stamford, CT, USA) dissolved in saturated aqueous picric acid (pH 2.0) at a concentration of 100 mg/100 ml for 90 min with mild shaking. The cultures were washed with 0.01 N hydrochloric acid for 2 min to remove all nonbound dye. Afterwards, 600 μl of 0.1 N sodium hydroxide was added to dissolve the staining using a microplate shaker for 30 min at room temperature. The optical density (OD) of the solution was then measured using a spectrophotometer at 550 nm against 0.1 N sodium hydroxide as a blank. The OD value on each culture was subtracted by the value on the corresponding disk without cells.

2.8

Analysis of deposition method of ECM on surface

For evaluation of surface morphology of ECM, on day 21, the cultures on discs with turned, 5 or 10 M AH-treated, and acid-etched surfaces underwent SEM observation after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%). To determine inclusion of deposited ECM into the nanofeatured surface, day 21 cultures on titanium films with 5 or 10 M AH-treated surfaces were cut with stainless steel scissors after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%). SEM observation with a bird’s eye view and EPMA-based elemental determination on cross-sectional specimens of the film with the superficial culture layer was then undertaken.

2.9

Collagen detachment assay

For evaluation of the mechanical, enzymatic and chemical strengths of deposited collagen on each surface under overloading or inflammatory conditions, day 21 cultures on discs were subjected to ultrasonication or exposure to collagenase or hydrogen peroxide. The culture was then washed twice with PBS. Subsequently, the cultures in PBS were put on ultrasonic equipment (UT105, Sharp, Tokyo, Japan) at 100 W and 35 kHz for 1 min to simulate an overloading condition. To simulate an inflammatory condition, the remaining day 21 cultures were incubated at 37 °C in PBS containing 0.1 U/ml collagenase (Roche Diagnostics) for 1 h or 0.3 mmol/L hydrogen peroxide for 3 h, respectively. After detachment treatment, the cultures were stained with Sirius red and absorbance was measured according to the protocol described above. The OD value on each culture was subtracted by the value on the corresponding disk without cells. The percentage of remaining collagen was calculated as [(Sirius red absorbance of the culture after detachment treatment/the absorbance of the corresponding duplicated day 21 culture without detachment treatment) × 100] (%). In addition, following detachment treatment, some of the day 21 cultures underwent SEM observation after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%) in order to evaluate the morphology of the mode of detachment.

For all culture experiments with quantitative assay except for cellular morphology, three independent cultures were evaluated in each group ( n = 3) using different batch of the cells and there were at least three replicates in each experiment. To assess cellular morphometry, six single cells with typical morphological features were randomly selected from three different points on the titanium surface. The mean value of the seven single cells was set as a representative value for each sample. Each experiment was repeated five times with different discs and cell batches in each group ( n = 5).

2.10

Animal surgery and histological observation

Sixteen-week-old male Japanese white rabbits weighing 3.0 − 3.5 kg ( Fig. 5 A) were used in this study. The rabbits were anesthetized by intramuscular administration of 2.5% thiopental sodium (Ravonal, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan) prior to operation. Following local anesthetic administration of 2% lidocaine with 1:80,000 adrenaline (Xylocaine, Dentsply Sankin, Tokyo, Japan) into the palatal gingiva, the palatal gingival tissue was punched out at a distance of 2.0 mm from the central incisors and the underlying hard palatal plate was drilled for implantation holes using a round bur and stainless steel hand reamer. Titanium mini-screws ( Fig. 5 B) with turned ( Fig. 5 C–E) or 10 M AH-treated surfaces ( Fig. 5 F–H) were bilaterally placed into the hard palatal plate close to the upper central incisors, with the coronal portion of the implant threading running flush with the gingival tissue surface, and the gingival connective tissue contacting the submerged implant surface within 0.5 mm of the most coronal portion of implant threading ( Fig. 5 I). The animals were kept in separate cages for 8 weeks in order to ensure complete healing of peri-implant gingival tissue . The experimental protocols were approved by the Animal Research Committee of Tokyo Dental College (Protocol No. 252602).

2.11

Histological and SEM observation of connective tissue attachment on implants

After 8 weeks of healing, the animals were sacrificed with an intravascular overdose of 2.5% thiopental sodium (Ravonal, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan) and the maxillae, including the central incisors and the titanium implants with periodontal and peri-implant tissue, were harvested. The specimens were fixed in 10% buffered formalin. The specimens were dehydrated in an ascending series of alcohol rinses and embedded in polyester resin (Rigolac 2004; Showa Highpolymer Co., Ltd., Tokyo, Japan) without decalcification. Embedded specimens were cut perpendicular to the long axis of the implants and teeth at a centerline and ground to a thickness of 100 μm with a cutting and grinding system (Maruto Instrument Co., Ltd., Tokyo, Japan). Two sections per specimen were prepared. Each section had a thickness of 30 μm, and was stained with a Villanueva–Goldner stain and observed under light microscopy.

For surface analysis of the transmucosal portion of the implants, the remaining animals were sacrificed after 8 weeks of healing using the same method as described above, and the implants were removed by inverse rotation at 20 N/cm ² with a torque device. After removal, the implant specimens were fixed with 2.5% glutaraldehyde and dehydrated in a graded series of ethanol (50–100%). The morphology of the residual tissue at the transmucosal portion of the implant was observed under a SEM.

Eight rabbits were employed in this animal experiment. Two different types of implants placed in each rabbits. Four samples were used for each qualitative SEM and light microscopic observation of histological sections, respectively.

2.12

Nanomechanical analyses of AH-treated surface

The hardness and elastic moduli of the nanofeatured surfaces were measured using a nanoindenter (ENT-2100; Elionix Inc., Tokyo, Japan) with a Berkovich tip. The nanotopographic titanium discs were mounted on the indenter stage and pressed with the maximum load of 0.05 mN at a loading rate of 0.1 mN/min. Subsequently, the sample was unloaded at the same rate after a 30 s pause. The indentation sites were selected under an optical microscope by a blinded technician. Indentation hardness and Young’s modulus were calculated based on the established equations by ISO standards. Indentation hardness was calculated as the maximum indentation force divided by the projected area of contact computed from the load–displacement curve and the area-to-depth function. Young’s modulus was calculated from the unloading segment of the load–displacement curve with an assumed Poisson ratio of 0.3.

Resistance of the nanotopographic surfaces to delamination force was evaluated by a nanoscratch test. The nanotopographic titanium discs were placed on the motorized sliding stage of the nanoscratch tester (CPX2441, Anton Paar Japan K.K., Tokyo, Japan). Scratches were generated with a spherical diamond stylus (tip radius 10 μm) at 500 mN/min at a progressive loading rate. The scratching speed was 5 mm/min. The evolution of the penetration depth, frictional force, and the friction coefficient (frictional force divided by normal force) was plotted against scratch length and force. The scratch path was subsequently observed by a light microscope equipped with the nanoscratch tester, and the point of delamination was determined. The critical load (delaminating force of the surface), defined as the minimal frictional force required for recognizable failure of the surface ( i.e. , exposure of the titanium substrate under microscopic observation), was measured. To validate the determined delamination points, surfaces and cross-sections of the selected scratches were examined by SEM and EPMA.

The coefficient of friction and frictional resistance of AH-treated surfaces were evaluated by a nanofriction and wear test. The nanotopographic titanium discs were placed on a nanotribometer (S/N:01-105, Anton Paar Japan K.K.). Friction tracks were generated with the stainless steel ball of the tribometer by continuous lap sliding with a load of 10 mN, a lapping speed of 5 mm/s and a radius of rotation of 1 mm until the ball reached 400 laps. The profile of the coefficient of friction was plotted against the coefficient of friction and laps. The friction track was subsequently observed by a light microscope equipped with the nanotribometer, and the cross-sectional profile of the friction track was analyzed with a laser microscope (LEXT OLS4000, Olympus, Tokyo, Japan). The cross-sectional area of the wear track, defined as the worn area under the surface, was measured. Four equally spaced points across the track were measured per specimen, and the data were averaged.

For these nanomechanical analyses, three areas were measured per specimen, and the data were averaged. The set of measurements was performed in three independent specimens.

2.13

Statistical analysis

An analysis of variance was used to assess differences among multiple experimental groups, and when appropriate, Bonferroni multiple comparison testing was used. Student’s t -test was used to examine differences between two groups, with a p value <0.05 considered statistically significant.

2.1

Sample preparation

Commercially pure grade 2 turned titanium discs (20 mm diameter and 1 mm thickness) as a culture substrate, turned titanium mini-screws (0.5 mm diameter and 5.5 mm length) and grade 1 titanium film (10 mm ² and 0.1 mm thickness) were purchased (Nishimura Co., Ltd., Fukui, Japan). The titanium samples were washed under ultrasonication with a series of ethanol and Milli-Q water after acetone cleaning. The acid-etched surface was prepared by immersion of the turned surface discs in 67% (w/w) sulfuric acid solution at 120 °C for 75 s . Two types of nanotopographical titanium surfaces were prepared by two AH-treatment protocols. In previously reported protocol , the cleaned turned discs were boiled in 5 mol/L sodium hydroxide solution at 60 °C for 24 h. After being washed in Milli-Q water and air-dried, the discs were heated in a furnace with an increase in temperature at a rate of 5 °C/min and sintered at 600 °C for 1 h. After sintering, the discs were naturally cooled. In the other protocol (our modified protocol), the discs were boiled in 10 mol/L sodium hydroxide solution at 90 °C for 24 h, followed by the same subsequent process as previously described. All prepared samples were rinsed by ultrasonication in distilled water for 10 min, and stored in ambient conditions, in the dark, for 4 weeks prior to use. All of the discs and films for culture experimentation were placed on 12-well culture-grade polystyrene plates.

2.2

Surface characterization

The surface topography of the sample discs was evaluated by observation via scanning electron microscopy (SEM; XL30, Philips, Eindhoven, Netherlands) and the surface roughness measurements were evaluated using a 3D-measuring laser microscope (LEXT OLS4000, Olympus, Tokyo, Japan) with a cut-off value of 8 μm and a measurement length of 120 μm. The number and density of nanospikes on both types of the nanotopographic surfaces were analyzed via SEM surface images using an image analyzer (ImageJ, National Institutes of Health, Bethesda, MD, USA). The thickness, porous structure and chemical composition of both types of AH-treated surfaces were evaluated by image analysis of SEM images and elemental analysis using an electron probe microanalyzer (EPMA, JXA-8200, JEOL Ltd., Tokyo, Japan) on cross-sectional specimens of AH-treated titanium films prepared by cutting the film with stainless steel scissors. Three areas were measured per sample, and the data were averaged. The set of measurements was performed in three independent samples.

2.3

Oral fibroblastic cell culture

Fibroblasts were obtained from the palatal tissue of 12-week-old Sprague-Dawley rats by reference to the methodology in previous article . Briefly, the palatal gingiva was washed with 1% PBS and incubated in 0.1 units/ml collagenase/0.8 units/ml dispase solution for 60 min, followed by separating the connective tissue from the epithelial layer. Then, the connective tissue was dissected into small pieces (<1 mm ² ) and digested in 0.25% collagenase for 12 h. The liberated cells were collected and plated in 100 mm plastic tissue culture dishes with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotic–antimycotic solution, containing 100 U ml/L penicillin G sodium, 100 lg ml/L streptomycin sulfate and 250 ng ml/L amphotericin B in a humidified atmosphere of 95% air and 5% CO ₂ at 37 °C. The cells were passaged for a second time when 80% confluency was achieved. After the second passage, the cells were detached using 0.25% Trypsin-1 mM EDTA-4Na and seeded directly onto the titanium discs or films with turned, 5 or 10 M AH-treated or acid-etched surfaces at 3.0 × 10 ⁴ cells/cm ² . The medium was renewed every 3 days. This study protocol was approved by the Animal Research Committee of Tokyo Dental College (Protocol No. 232604).

2.4

Cellular morphology

On day 1, the culture of gingival fibroblasts on discs with turned, 5 or 10 M AH-treated or acid-etched surfaces, were fixed in 10% neutral buffered formalin for 30 min. The cells were treated with 0.1% Triton X for 5 min, and then stained with 2% rhodamine phalloidin (Actin filament red color, Molecular Probes, OR, USA) in PBS and the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI; Nuclei blue color, Vector, CA, USA) in a mounting agent. Cell morphology and cytoskeletal arrangement were observed under a fluorescent photomicroscope (Axiophoto2, Carl Zeiss Co., Ltd., Jena, Germany). Cell morphometry was analyzed using ImageJ software.

2.5

Cellular attachment and proliferation

The number of cells in the cultures from days 1 and 7 on discs with turned, 5 or 10 M AH-treated or acid-etched surfaces was evaluated by tetrazolium salt-based colorimetry (WST-1, Roche Diagnostics, Tokyo, Japan). Each culture was incubated at 37 °C for 4 h in 1 mL fresh culture media containing 100 μL of WST-1 reagent. The amount of formazan product produced by viable cells was measured by colorimetry at 420 nm using a microplate reader. The proliferative activity of cells was measured by bromodeoxyuridine (BrdU) incorporation during DNA synthesis. On day 4 of surface culture, a 100 mM BrdU solution (100 ml) (Roche Diagnostics, Tokyo, Japan) was added to the culture wells and incubated for 10 h. After denaturing DNA, the cultures were incubated with anti-BrdU antibody conjugated with peroxidase for 90 min and exposed to tetramethylbenzidine for color development. Absorbance was measured using an enzyme-linked immunosorbent assay reader at 370 nm.

2.6

Gene expression analysis

Gene expression was analyzed on days 7 and 14 using reverse-transcriptase polymerase chain reaction (PCR). The total RNA in these cultures was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and a purification column (RNeasy, Qiagen, Valencia, CA, USA). Following DNAse I treatment, reverse transcription of 0.5 mg total RNA was performed using MMLV reverse transcriptase (Clontech, Carlsbad, CA, USA) in the presence of oligo(dT) primer (Clontech). PCR was performed using Taq DNA polymerase (EX Taq; Takara Bio, Madison, WI, USA) to detect collagen I and III mRNA using primer designs and the PCR condition established previously . The forward and backward primers were designed: collagen I: 5′-GGCAACAGTCGATTCACC-3′ and 5′-AGGGCCAATGTCCATTCC-3′, collagen III: 5′-CCTGGACTCAGGGTATC-3′ and 5′-TGCAGGGCCTGGACTACC-3′, respectively. The annealing temperature and the amplification cycles were set at 58 °C and 28 cycles for collagen I and 60 °C and 25 cycles for collagen III. The PCR products were visualized on 1.5% agarose gel by ethidium bromide staining. Band intensity was detected under UV light and normalized with reference to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The gene expression analyses were performed three to five times and representative data sets presented after confirming consistency.

2.7

Collagen production

To quantify collagen production, on day 21, Sirius red staining-based colorimetric assay was employed for cultures on discs. Cultures from different groups were washed with pre-warmed PBS at 37 °C for 1 min and fixed with Bouin’s fluid for 1 h at room temperature. The cultures were washed with ddH ₂ O and treated with 0.2% aqueous phosphomolybdic acid for 1 min. Then, the cultures were washed again with ddH ₂ O and stained with Sirius red dye (C.I. No. 35780, Pfaltz and Bauer, Stamford, CT, USA) dissolved in saturated aqueous picric acid (pH 2.0) at a concentration of 100 mg/100 ml for 90 min with mild shaking. The cultures were washed with 0.01 N hydrochloric acid for 2 min to remove all nonbound dye. Afterwards, 600 μl of 0.1 N sodium hydroxide was added to dissolve the staining using a microplate shaker for 30 min at room temperature. The optical density (OD) of the solution was then measured using a spectrophotometer at 550 nm against 0.1 N sodium hydroxide as a blank. The OD value on each culture was subtracted by the value on the corresponding disk without cells.

2.8

Analysis of deposition method of ECM on surface

For evaluation of surface morphology of ECM, on day 21, the cultures on discs with turned, 5 or 10 M AH-treated, and acid-etched surfaces underwent SEM observation after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%). To determine inclusion of deposited ECM into the nanofeatured surface, day 21 cultures on titanium films with 5 or 10 M AH-treated surfaces were cut with stainless steel scissors after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%). SEM observation with a bird’s eye view and EPMA-based elemental determination on cross-sectional specimens of the film with the superficial culture layer was then undertaken.

2.9

Collagen detachment assay

For evaluation of the mechanical, enzymatic and chemical strengths of deposited collagen on each surface under overloading or inflammatory conditions, day 21 cultures on discs were subjected to ultrasonication or exposure to collagenase or hydrogen peroxide. The culture was then washed twice with PBS. Subsequently, the cultures in PBS were put on ultrasonic equipment (UT105, Sharp, Tokyo, Japan) at 100 W and 35 kHz for 1 min to simulate an overloading condition. To simulate an inflammatory condition, the remaining day 21 cultures were incubated at 37 °C in PBS containing 0.1 U/ml collagenase (Roche Diagnostics) for 1 h or 0.3 mmol/L hydrogen peroxide for 3 h, respectively. After detachment treatment, the cultures were stained with Sirius red and absorbance was measured according to the protocol described above. The OD value on each culture was subtracted by the value on the corresponding disk without cells. The percentage of remaining collagen was calculated as [(Sirius red absorbance of the culture after detachment treatment/the absorbance of the corresponding duplicated day 21 culture without detachment treatment) × 100] (%). In addition, following detachment treatment, some of the day 21 cultures underwent SEM observation after fixation with 2.5% glutaraldehyde and dehydration in a graded series of ethanol (50–100%) in order to evaluate the morphology of the mode of detachment.

For all culture experiments with quantitative assay except for cellular morphology, three independent cultures were evaluated in each group ( n = 3) using different batch of the cells and there were at least three replicates in each experiment. To assess cellular morphometry, six single cells with typical morphological features were randomly selected from three different points on the titanium surface. The mean value of the seven single cells was set as a representative value for each sample. Each experiment was repeated five times with different discs and cell batches in each group ( n = 5).

2.10

Animal surgery and histological observation

Sixteen-week-old male Japanese white rabbits weighing 3.0 − 3.5 kg ( Fig. 5 A) were used in this study. The rabbits were anesthetized by intramuscular administration of 2.5% thiopental sodium (Ravonal, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan) prior to operation. Following local anesthetic administration of 2% lidocaine with 1:80,000 adrenaline (Xylocaine, Dentsply Sankin, Tokyo, Japan) into the palatal gingiva, the palatal gingival tissue was punched out at a distance of 2.0 mm from the central incisors and the underlying hard palatal plate was drilled for implantation holes using a round bur and stainless steel hand reamer. Titanium mini-screws ( Fig. 5 B) with turned ( Fig. 5 C–E) or 10 M AH-treated surfaces ( Fig. 5 F–H) were bilaterally placed into the hard palatal plate close to the upper central incisors, with the coronal portion of the implant threading running flush with the gingival tissue surface, and the gingival connective tissue contacting the submerged implant surface within 0.5 mm of the most coronal portion of implant threading ( Fig. 5 I). The animals were kept in separate cages for 8 weeks in order to ensure complete healing of peri-implant gingival tissue . The experimental protocols were approved by the Animal Research Committee of Tokyo Dental College (Protocol No. 252602).

2.11

Histological and SEM observation of connective tissue attachment on implants

After 8 weeks of healing, the animals were sacrificed with an intravascular overdose of 2.5% thiopental sodium (Ravonal, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan) and the maxillae, including the central incisors and the titanium implants with periodontal and peri-implant tissue, were harvested. The specimens were fixed in 10% buffered formalin. The specimens were dehydrated in an ascending series of alcohol rinses and embedded in polyester resin (Rigolac 2004; Showa Highpolymer Co., Ltd., Tokyo, Japan) without decalcification. Embedded specimens were cut perpendicular to the long axis of the implants and teeth at a centerline and ground to a thickness of 100 μm with a cutting and grinding system (Maruto Instrument Co., Ltd., Tokyo, Japan). Two sections per specimen were prepared. Each section had a thickness of 30 μm, and was stained with a Villanueva–Goldner stain and observed under light microscopy.

For surface analysis of the transmucosal portion of the implants, the remaining animals were sacrificed after 8 weeks of healing using the same method as described above, and the implants were removed by inverse rotation at 20 N/cm ² with a torque device. After removal, the implant specimens were fixed with 2.5% glutaraldehyde and dehydrated in a graded series of ethanol (50–100%). The morphology of the residual tissue at the transmucosal portion of the implant was observed under a SEM.

Eight rabbits were employed in this animal experiment. Two different types of implants placed in each rabbits. Four samples were used for each qualitative SEM and light microscopic observation of histological sections, respectively.

2.12

Nanomechanical analyses of AH-treated surface

The hardness and elastic moduli of the nanofeatured surfaces were measured using a nanoindenter (ENT-2100; Elionix Inc., Tokyo, Japan) with a Berkovich tip. The nanotopographic titanium discs were mounted on the indenter stage and pressed with the maximum load of 0.05 mN at a loading rate of 0.1 mN/min. Subsequently, the sample was unloaded at the same rate after a 30 s pause. The indentation sites were selected under an optical microscope by a blinded technician. Indentation hardness and Young’s modulus were calculated based on the established equations by ISO standards. Indentation hardness was calculated as the maximum indentation force divided by the projected area of contact computed from the load–displacement curve and the area-to-depth function. Young’s modulus was calculated from the unloading segment of the load–displacement curve with an assumed Poisson ratio of 0.3.

Resistance of the nanotopographic surfaces to delamination force was evaluated by a nanoscratch test. The nanotopographic titanium discs were placed on the motorized sliding stage of the nanoscratch tester (CPX2441, Anton Paar Japan K.K., Tokyo, Japan). Scratches were generated with a spherical diamond stylus (tip radius 10 μm) at 500 mN/min at a progressive loading rate. The scratching speed was 5 mm/min. The evolution of the penetration depth, frictional force, and the friction coefficient (frictional force divided by normal force) was plotted against scratch length and force. The scratch path was subsequently observed by a light microscope equipped with the nanoscratch tester, and the point of delamination was determined. The critical load (delaminating force of the surface), defined as the minimal frictional force required for recognizable failure of the surface ( i.e. , exposure of the titanium substrate under microscopic observation), was measured. To validate the determined delamination points, surfaces and cross-sections of the selected scratches were examined by SEM and EPMA.

The coefficient of friction and frictional resistance of AH-treated surfaces were evaluated by a nanofriction and wear test. The nanotopographic titanium discs were placed on a nanotribometer (S/N:01-105, Anton Paar Japan K.K.). Friction tracks were generated with the stainless steel ball of the tribometer by continuous lap sliding with a load of 10 mN, a lapping speed of 5 mm/s and a radius of rotation of 1 mm until the ball reached 400 laps. The profile of the coefficient of friction was plotted against the coefficient of friction and laps. The friction track was subsequently observed by a light microscope equipped with the nanotribometer, and the cross-sectional profile of the friction track was analyzed with a laser microscope (LEXT OLS4000, Olympus, Tokyo, Japan). The cross-sectional area of the wear track, defined as the worn area under the surface, was measured. Four equally spaced points across the track were measured per specimen, and the data were averaged.

For these nanomechanical analyses, three areas were measured per specimen, and the data were averaged. The set of measurements was performed in three independent specimens.

2.13

Statistical analysis

An analysis of variance was used to assess differences among multiple experimental groups, and when appropriate, Bonferroni multiple comparison testing was used. Student’s t -test was used to examine differences between two groups, with a p value <0.05 considered statistically significant.