The purpose of this study was to establish an acid-etching procedure for altering the Ca/P ratio of the nanostructured surface of hydroxyapatite (HAP) by using surface chemical and morphological analyses (XPS, XRD, SEM, surface roughness, and wettability) and to evaluate the
Hydroxyapatite [Ca10(PO4)6(OH)2] (HAP) and
Presently, scaffolds fabricated from HAP that exhibit high porosity and pore interconnectivity are used clinically [
Dorozhkin [
These 5 steps show that, throughout the process of HAP dissolution, the composition of the surface changes to Ca3(PO4)2 (tricalcium phosphate, TCP) and CaHPO4 (dicalcium phosphate dehydrate, DCPD). Then, Bertazzo et al. [
Modified schematic diagram representing the phenomena that occur on the surface of hydroxyapatite (Ca10(PO4)6(OH)2: HAP) after implantation, illustrated by Bertazzo et al. [
We put forth the hypothesis that the acid-etching procedure, altering the Ca/P ratio of the HAP surface directly by phosphoric acid, that has been described in this study can cause the bioactive surfaces to mimic the initial phases proposed by Bertazzo et al. [
HAP plates (thickness, 2 mm; width, 10 mm; length, 10 mm) (APP-101; Pentax, Tokyo, Japan) were used in this study. HAP plates were treated with 10%, 20%, 30%, 40%, 50%, or 60% phosphoric acid [H3PO4] (lot no. T1949; Sigma-Aldrich Japan, Tokyo, Japan) solution for 10 minutes at 25°C, followed by rinsing 3 times with ultrapure water (MilliQ water: >18 MΩcm) (HAP—10% PA, HAP—20% PA, HAP—30% PA, HAP—40% PA, HAP—50% PA, and HAP—60% PA).
HAP, HAP—10% PA, HAP—20% PA, HAP—30% PA, HAP—40% PA, HAP—50% PA, and HAP—60% PA plates were mounted individually onto stubs with insulating tape. The surfaces of the plates were chemically analyzed using an X-ray photoelectron spectroscopy (XPS) instrument (AXIS-HS; Kratos, Manchester, UK). The measurements were performed
The thin films of the HAP and HAP—30% PA plates were analyzed using an XRD instrument (Ultima IV; Rigaku, Osaka, Japan). Samples were scanned with Cu-K
A HAP—30% PA plate that was stored under dry conditions at 25°C for 12 hours (HAP—30% PA—12 h) was added as a new sample. The surface characteristics of HAP, HAP—30% PA, and HAP—30% PA—12 h were evaluated by the following methods.
Samples were analyzed using an XPS instrument at incidence angles of 90, 75, 60, 45, 30, and 15 degrees. The measurements were obtained under the above-mentioned conditions. The data were analyzed using one-way ANOVA and Tukey’s test for multiple comparisons (
An SEM (VE-8800; Keyence, Osaka, Japan) was used to observe the surface topography of samples. The surface roughness (Ra) of samples was determined using a confocal laser scanning microscope (VK-8500; Keyence, Osaka, Japan). The Ra value (
Osteoblast-like cells (MC3T3-E1 derivative cell lines of mouse calvaria (RIKEN BioResource Center, Tsukuba, Japan)) were cultured in Dulbecco’s Modified Eagle Medium (D-MEM) (lot no. RNBB4045; Sigma-Aldrich Japan, Tokyo, Japan) containing 10% fetal bovine serum (lot nos. F0926, 027K03911; Sigma-Aldrich Japan, Tokyo, Japan) and 1% penicillin-streptomycin (lot nos. P0781, 060M0811; Sigma-Aldrich Japan, Tokyo, Japan) at 37°C in a humidified atmosphere of 5% CO2. The medium was changed twice a week.
MC3T3-E1 cells (1 × 104) were seeded onto HAP, HAP—30% PA, and HAP—30% PA—12 h plates sterilized by ultraviolet (UV) irradiation for 4 hours. The cells were incubated at 37°C and 5% CO2. Initial cell adhesion was evaluated at 0.5 and 24 hours and cell proliferation at 1, 4, and 7 days. Cells were seeded on tissue culture plates and incubated as controls over each incubation period.
Initial cell adhesion and cell proliferation were analyzed by a Cell Counting Kit-8 assay (Dojindo Laboratory, Kumamoto, Japan), a colorimetric assay that uses a tetrazolium salt, WST-8, to determine viable cell number. After each incubation period, the growth medium in each well was removed by aspiration and replaced with a mixture of WST-8 and D-MEM in a 1 : 10 ratio. After 2 hours of incubation at 37°C and 5% CO2, a 110
MC3T3-E1 cells (10 × 104) were seeded onto HAP, HAP—30% PA, and HAP—30% PA—12 h plates as described previously. Alkaline phosphatase (ALP) activity was evaluated at 7, 14, and 21 days after obtaining a confluent cell monolayer. Real-time polymerase chain reaction (PCR) analysis of ALP was carried out at 14, 21, and 28 days after obtaining a confluent cell monolayer.
ALP activity was determined by the SensoLyte
Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). The total RNA concentration was adjusted to 10 ng/
The XPS-determined binding energies (eV) of Ca 2p, P 2p, and
XPS-determined binding energies (eV) of Ca 2p, P 2p, and
Sample | Binding energy (eV) | At. % | Ca/P | ||||||
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Ca 2p | P 2p |
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C 1s | O 1s | Ca 2p | P 2p | |||
HAP | Mean |
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214.06a |
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1.65 |
SD |
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0.05 |
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0.02 | |
HAP—10% PA | Mean |
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214.07a |
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1.54c |
SD |
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0.05 |
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0.01 | |
HAP—20% PA | Mean |
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214.05a |
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1.51b |
SD |
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0.05 |
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0.02 | |
HAP—30% PA | Mean |
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214.07a |
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1.50b |
SD |
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0.05 |
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0.03 | |
HAP—40% PA | Mean |
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214.06a |
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1.56c,d |
SD |
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0.05 |
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0.02 | |
HAP—50% PA | Mean |
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214.03a |
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1.57c,d |
SD |
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0.05 |
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0.04 | |
HAP—60% PA | Mean |
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214.05a |
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1.58d |
SD |
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0.05 |
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0.02 |
The presence of the same superscript letter for the values indicates that there were no significant differences among the samples (
The XRD spectra of HAP and HAP—30% PA thin films, referenced to the ICDD standard for HAP (no. 01-072-1243), are shown in Figure
X-ray diffraction (XRD) spectra of HAP and HAP—30% PA thin films referenced to the ICDD standard for HAP (no. 01-072-1243). There was no difference between the XRD spectra of HAP and HAP—30% PA, and the chemical composition of their thin films was assumed to be the same.
The XPS-determined binding energies (eV) of Ca 2p, P 2p, and
XPS-determined binding energies (eV) of Ca 2p, P 2p, and
Sample | Angle of incidence (degrees) | Binding energy (eV) | Ca/P | ||
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Ca 2p | P 2p |
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HAP |
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Mean |
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214.08a | 1.65b | |
SD |
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HAP—30% PA |
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Mean | 346.85 | 132.78 | 214.08a | 1.45b | |
SD |
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HAP—30% PA—12 h |
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Mean |
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1.09b | |
SD |
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SEM images for HAP, HAP—30% PA, and HAP—30% PA—12 h are shown in Figure
SEM images of (a) HAP, (b) HAP—30% PA, and (c) HAP—30% PA—12 h.
The surface roughness and wettability for HAP, HAP—30% PA, and HAP—30% PA—12 h are shown in Table
Surface roughness and wettability for HAP, HAP—30% PA, and HAP—30% PA—12 h.
Sample | Surface roughness | Wettability | |
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Ra ( |
Contact angle (degrees) | ||
HAP | Mean | 0.25 | 102.10c |
SD | 0.06 | 2.98 | |
HAP—30% PA | Mean | 0.91a | 55.13c |
SD | 0.05 | 0.35 | |
HAP—30% PA—12 h | Mean | 0.96a,b | 13.67c |
SD | 0.04 | 3.54 |
Surface roughness:
Wettability:
The initial cell adhesion of MC3T3-E1 cells to HAP, HAP—30% PA, and HAP—30% PA—12 h at 0.5 and 24 hours is shown in Figure
Initial cell adhesion of MC3T3-E1 osteoblast-like cells to HAP, HAP—30% PA, and HAP—30% PA—12 h at 0.5 and 24 hours. At 0.5 hours, the number of cells adherent to HAP—30% PA—12 h was significantly greater than that for HAP (
The proliferation of MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 1, 4, and 7 days is shown in Figure
Cell proliferation of MC3T3-E1 osteoblast-like cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 1, 4, and 7 days. At 4 and 7 days, cell proliferation on HAP—30% PA and HAP—30% PA—12 h was significantly higher than that on HAP (
The ALP activity of MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 7, 14, and 21 days after obtaining a confluent cell monolayer is shown in Figure
ALP activities of MC3T3-E1 osteoblast-like cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 7, 14, and 21 days after obtaining a confluent cell monolayer. At 14 days, the ALP activities of the cells on HAP—30% PA and HAP—30% PA—12 h were significantly higher than that on HAP (
Relative mRNA levels for ALP in MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 14, 21, and 28 days after obtaining a confluent cell monolayer are shown in Figure
Relative mRNA levels for ALP in MC3T3-E1 osteoblast-like cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 14, 21, and 28 days after obtaining a confluent cell monolayer. At 14 days, there was no mRNA expression on HAP, but the expression on HAP—30% PA—12 h and HAP—30% PA was estimated in sequence. At 21 days, the expression of ALP on HAP was estimated, and the expression on HAP, HAP—30% PA, and HAP—30% PA—12 h was increased at 28 days. There were no statistically significant differences among expressions at any time (
Modification to the HAP surface by using etching with 10%, 20%, 30%, 40%, 50%, and 60% phosphoric acid altered the Ca/P ratio from 1.50 to 1.58. The Ca/P ratio of HAP—30% PA was the smallest and matched the theoretical value of TCP (1.50). However, there were no significant differences among the binding energies of
XRD analysis showed that the chemical composition of the thin films (a level of 100 nm) for HAP and HAP—30% PA was assumed to be the same. This finding does not illustrate that the changed Ca/P ratio leads to alteration of the chemical composition of the surface. Thus, the nanostructured surface of HAP—30% PA has not yet been confirmed as being modified to TCP, although the Ca/P ratio was 1.50.
The Ca/P ratio of HAP—30% PA—12 h was approximated to the Ca/P ratio of DCPD (1.00), but this finding was obtained accidentally. The XPS depth profiling analyses for HAP, HAP—30% PA, and HAP—30% PA—12 h were performed to evaluate the chemical compositions of their nanostructured surfaces. The binding energy of
The reasons for reduction of the Ca/P ratio of the modified surface were (i) Ca defect and (ii) P abundance. P abundance could not be considered because the surface treated with 30% phosphoric acid solution was rinsed with ultrapure water, and the expansion of the break of the grain boundary of HAP was not observed in the SEM image of HAP—30% PA—12 h. The 30% phosphoric acid etching process promotes decalcification of the surface, resulting in the reduction of the Ca/P ratio by Ca defects. As for the reduction of the Ca/P ratio of HAP—30% PA—12 h to 1.00, it is believed that the Ca in the outer layer of the nanostructured surface is supplied to the deep layer for 12 hours to equilibrate the amorphous calcium phosphate.
Reduction in the Ca/P ratio leads to high solubility of the surface. Additionally, surface modification induces the rougher and wetter surface of HAP. Generally, a contact angle of more than 65 degrees indicates a hydrophobic surface, and a contact angle of less than 65 degrees indicates a hydrophilic surface. Since the contact angle of HAP (102.10 degrees) is greater than 65 degrees, the hydrophobic surface becomes more intense with an increase in surface roughness. However, in this study, the contact angle grew smaller with an increase in surface roughness, resulting in a super hydrophilic surface. These findings demonstrate that the surface might have been modified both morphometrically and chemically.
We expected that the
Furthermore, we will investigate the response of osteoclasts to HAP—30% PA and HAP—30% PA—12 h because osteoclasts regulating bone metabolism are coupled with osteoblasts in a direct or indirect manner
In this study, we established the acid-etching procedure to alter the Ca/P ratio of the nanostructured surface of HAP by surface chemical and morphological analyses and evaluated the
For a SensoLyte
This study was supported in part by a Grant-in-Aid for Scientific Research (nos. 21592452 and 24592915) from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (2009–2011 and 2012–2014). The authors would like to thank Editage for providing editorial assistance.