Electrostatic layer-by-layer assembly for fabricating morphology-controlled hydroxyapatite/zirconia composite with enhanced osteogenic performance

Abstract

Background

Yttria-stabilized zirconia (YSZ) represents a promising alternative to titanium for dental and orthopedic implants owing to its mechanical strength and esthetics. However, its bioinertness limits osteoconductivity. Although hydroxyapatite (HAp) coatings can enhance osteointegration, uniform deposition of morphology-controlled HAp on YSZ remains challenging to achieve, limiting implant optimization.

Objectives

This study was aimed at the uniform deposition of morphology-controlled HAp on YSZ using electrostatic layer-by-layer (LBL) assembly to improve biological performance.

Methods

Plate-like HAp was hydrothermally synthesized using dodecanedioic acid and surface-modified with poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate). YSZ discs were similarly charge-modified to enable electrostatic adsorption of HAp. Samples were evaluated using scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and wettability tests. Osteogenic responses were assessed using MC3T3-E1 cells through alkaline phosphatase (ALP) activity and Alizarin Red S staining.

Results

Negatively charged HAp was uniformly deposited on positively charged YSZ, forming a homogeneous coating. Characterization confirmed successful HAp deposition and improved hydrophilicity. HAp/YSZ composites enhanced osteogenic differentiation, exhibiting higher ALP activity and greater calcium deposition than YSZ.

Conclusion

LBL-mediated deposition of morphology-controlled HAp enables uniform coating and enhanced osteogenic activity on YSZ, facilitating advances in next-generation bioactive zirconia implants.

Keywords

Yttria-stabilized zirconia implant morphology-controlled hydroxyapatite layer-by-layer assembly osteoconductivity

Introduction

Zirconia (yttria-stabilized zirconia, YSZ) is widely used in the dental field for applications such as crowns and abutments. YSZ incorporates yttria, and its phase stability, transparency, and mechanical properties are governed by the yttria content. For example, 3 mol% YSZ, which is commonly used today, exhibits high fracture toughness. Increasing yttria content improves transparency but diminishes both toughness and strength. A notable drawback of conventional YSZ is low-temperature degradation (LTD), which arises from spontaneous phase transformation. To address this issue, YSZ containing 1.5 mol% yttria has been developed to enhance fracture toughness by reducing the yttria content and to mitigate the risk of LTD through microstructural control.¹

Recently, YSZ implants have gained attention as potential alternatives to titanium and titanium-alloy fixtures owing to their superior esthetics and biosafety. Although YSZ exhibits excellent mechanical strength, biocompatibility, and esthetic properties, its osteoconductivity remains low.² To address this, researchers have explored surface modifications of YSZ, including coatings of peptides, collagen, bioactive glass, β-tricalcium phosphate, and hydroxyapatite (HAp) to enhance osseointegration.^3–7

HAp is an inorganic compound with a chemical composition similar to that of human bone and teeth and is widely used as a bioactive material in artificial bones, cements, and dental root implants. Research has shown that coating titanium implants with micro-sized, morphology-controlled HAp enhances bone regeneration.⁸ Two key approaches are available for applying HAp coatings: (1) direct deposition of HAp onto the substrate, including sol–gel-derived HAp coatings on YSZ^9,10 and hydrothermal deposition of HAp¹¹; and (2) post-synthesis coating of pre-formed HAp. In post-coating methods, pre-synthesized HAp is applied onto YSZ surfaces using techniques such as laser treatment¹² or chemical bonding.¹³ This approach offers superior control over HAp morphology and crystal orientation, enabling the preparation of needle-like, plate-like, or spherical structures. In general, the morphology and size of HAp significantly influence cellular behavior.¹⁴ However, nanoparticle aggregation hinders uniform coating, establishing a reliable technique to achieve uniform coating of morphology-controlled micro-sized HAp on YSZ remains a major challenge.

The Layer-by-Layer (LBL) assembly method is a well-established technique for fabricating composite materials.¹⁵ This approach leverages electrostatic interactions to alternately assemble positively and negatively charged components. For instance, Tan et al.¹⁶ demonstrated that modifying the surface charges of SiO₂ and Al₂O₃ using poly(diallyldimethylammonium chloride) (PDDA) as a polycation and poly(sodium 4-styrenesulfonate) (PSS) as a polyanion enabled the fabrication of SiO₂–Al₂O₃ composites with uniformly distributed non-agglomerated Al₂O₃.

Considering these aspects, this study formulated two hypotheses: (i) uniform coating cannot be achieved through the surface charge control process, and (ii) HAp-coated composites do not enhance calcification by the osteoblast cell. Following the method of Horiuchi et al.,¹⁷ morphology-controlled HAp was synthesized using dodecanedioic acid (DDDA) as a template. The synthesized HAp was then deposited onto YSZ containing 1.5 mol% yttria using the LBL method to fabricate HAp/YSZ composites. These composites were subjected to material characterization and in vitro evaluations.

Materials and methods

Preparation of YSZ discs

First, 1.5 mol% yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) powder (Zgaia1.5YB-HT; TOSOH, Yamaguchi, Japan) was uniaxially pressed at 20 MPa for 1 min and sintered at 1350 °C for 2 h in air to produce sintered discs (denoted as 1.5Y) with a diameter of 11 mm and thickness of 1 mm. For comparison, 3.0 mol% Y-TZP powder (TZ-3YSB-E; TOSOH) was prepared in the same manner and sintered at 1500 °C for 2 h to obtain discs (3Y). Untreated 1.5Y and 3Y discs were sandblasted with 50 μm alumina (Al₂O₃; Akiyama Sangyo Co., Osaka, Japan) and etched with 40% hydrofluoric acid (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) for 25 min at room temperature (∼25 °C). These samples were referred to as 1.5Y-SLA and 3Y-SLA, respectively.

Synthesis of HAp

DDDA (6 mmol; FUJIFILM Wako) was dissolved in 36 g ethanol (FUJIFILM Wako). A 60 mL aliquot of 0.1 M CaCl₂ (FUJIFILM Wako) was added dropwise, and the pH was adjusted to 6 using NaOH (FUJIFILM Wako). After stirring at room temperature for 30 min, 30 mL of 0.2 M NaH₂PO₄·2H₂O (Kanto Chemical, Tokyo, Japan) was added dropwise, and NaOH was again added to maintain pH 6. The suspension was subjected to hydrothermal treatment at 150 °C for 18 h (heating rate of 5 °C min⁻¹), followed by washing twice with ethanol and once with deionized water.

Characterization of HAp

The synthesized HAp was freeze-dried. Particle morphology was examined using scanning electron microscopy (SEM; JSM-7900F, JEOL, Tokyo, Japan) and transmission electron microscopy (TEM; JEM-1400Flash, JEOL) at an accelerating voltage of 80 kV. Functional groups were analyzed using Fourier-transform infrared (FT-IR) spectroscopy (FT/IR-4100, JASCO, Tokyo, Japan) with FT-IR resolution: 4 cm⁻¹, number of scans: 25. Crystal structure and orientation were determined using X-ray diffraction analysis (XRD; MiniFlex 600, Rigaku, Tokyo, Japan) with scan speed: 4° min⁻¹, step width: 0.02°.

Coating of hydroxyapatite on zirconia via LBL assembly

A schematic of the coating process is shown in Figure 1. For LBL assembly, PDDA (Sigma-Aldrich, MO, USA) served as the polycation, and PSS (Sigma-Aldrich) was used as the polyanion. Each materials was diluted to 1.0 wt% in 0.5 M NaCl (FUJIFILM Wako) and adjusted to pH 8.5 with NaOH.

Figure 1.

Schematic of HAp/YSZ composite formation via the layer-by-layer method.

To inhibit the aggregation of HAp in solution, it was first hydrophilized with 0.1 wt% sodium deoxycholate (SDC; Sigma-Aldrich). LBL modification was performed sequentially with PDDA and PSS, washing HAp with 0.5 M NaCl between each step. Modified HAp was stored in aqueous solution (pH 8.5) at 0.01 g mL⁻¹.

Untreated 1.5Y discs were modified by LBL in the sequence PDDA–PSS–PDDA, with intermediate washing steps identical to those for HAp. The YSZ discs were then immersed in the HAp suspension to achieve a coating density of 2 × 10⁻³ g cm⁻². After 1 h of immersion, the discs were rinsed with pH 8.5 aqueous solution to remove unbound particles.

Two experimental groups were prepared: (i) HAp terminated with PDDA applied to YSZ terminated with PSS (pHAp/nYSZ) and (ii) HAp terminated with PSS applied to YSZ terminated with PDDA (nHAp/pYSZ). A control group was prepared, consisting of untreated YSZ immersed in HAp suspension (pH 8.5) for 1 h (HAp/YSZ). All samples were heat-treated at 500 °C for 1 h (5 °C min⁻¹) to remove residual polymer. Because the sample names used in this study are similar and may be confusing, the sample name and definitions are summarized in Table 1.

Table 1.

Definition of each sample.

Sample	Meaning
3Y-SLA	SLA-treated 3Y
1.5Y-SLA	SLA-treated 1.5Y
1.5Y	Untreated 1.5Y
HAp/YSZ	HAp and Untreated 1.5Y composite
pHAp/nYSZ	Positively charged HAp and negatively charged YSZ composite
nHAp/pYSZ	Negatively charged HAp and positively charged YSZ composite

Surface characterization of HAp/YSZ composite

The zeta potential was first measured for both 1.5Y and HAp at each step of the surface-modification process. Specifically, for 1.5Y, LBL modification was performed using the sequence PDDA–PSS–PDDA. The zeta potentials of each step (1.5Y, 1st PDDA, 1st PSS, and 2nd PDDA) were measured using a SurPASS 3 electrokinetic analyzer (Anton Paar, Austria). For HAp, SDC pretreatment was followed by sequential surface modification with PDDA–PSS–PDDA–PSS. Each intermediate sample (HAp, SDC-treated HAp, 1st PDDA, 1st PSS, 2nd PDDA, and 2nd PSS) was dispersed in an aqueous solution at pH 8.5, and zeta potentials were measured using a Litesizer DLS 701 (Anton Paar, Austria).

Thermogravimetric analysis (TGA; Thermo Plus Evo 2, TG-DTA-8122, Rigaku Corporation, Tokyo, Japan; heating rate of 5 °C min⁻¹) was performed to examine the weight changes of 1.5Y and HAp/YSZ composites upon heating.

Surface morphology and elemental composition were characterized by SEM and energy-dispersive X-ray spectroscopy (EDS; JSM-7900F/JED-2300, JEOL Ltd, Tokyo, Japan) after heat treatment at 500 °C. Further surface chemical analyses were conducted using attenuated total reflectance (ATR)-FT-IR, XRD, and X-ray photoelectron spectroscopy (XPS; JPS-9010MC, JEOL Ltd, Tokyo, Japan).

Wettability was evaluated by measuring the water contact angle (n = 5), and surface roughness was analyzed using a laser microscope (OLS4100, Olympus, Tokyo, Japan; n = 3). The adhesion strength between YSZ and HAp was assessed by tensile testing in accordance with ISO 13779-4 (n = 5).¹⁸ Stainless-steel rods were bonded to the HAp-coated surface using a luting agent (Super-Bond Bulk-mix, Sun Medical Co., Ltd, Japan) with a bonding area having a diameter of 3 mm. Tensile loading was applied using a universal testing machine (Autograph AGS-H, Shimadzu, Kyoto, Japan) at a crosshead speed of 2.0 mm min⁻¹. The bond strength (σ) was calculated as

σ = F / A

where F represents the maximum load (N) and A is the cross-sectional area of the bonded interface (mm²). The fracture surfaces of the YSZ and stainless-steel specimens were examined by SEM and EDS.

Cell culture

MC3T3-E1 subclone 4 cells (CRL-2593, American Type Culture Collection, USA) were cultured on each sample in complete growth medium containing α-MEM (alpha modification of minimum essential medium) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in a humidified atmosphere containing 5% CO₂.

Cell morphology

Early cell adhesion was assessed by fluorescence microscopy (n = 2). Cells (2.5 × 10⁴ cells mL⁻¹) were seeded onto each sample, incubated for 6 or 24 h, fixed with 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako), and permeabilized with 0.2% Triton X-100 (Kishida Chemical, Osaka, Japan). Cells were stained with rhodamine phalloidin (R415; Thermo Fisher, IL, USA) and 4′,6-diamidino-2-phenylindole (DAPI; VectaShield Plus, Vector Laboratories, CA, USA) and observed using a fluorescence microscope (IX71; Olympus, Tokyo, Japan).

Cell proliferation

Cells (5 × 10⁴ cells/mL) were cultured for 1, 3, or 7 d. Cell proliferation was quantified by measuring absorbance at 450 nm using the Cell Counting Kit-8 (CCK-8; FUJIFILM Wako; n = 6).

Cell differentiation

Early-stage cell differentiation was assessed using an alkaline phosphatase (ALP) assay kit (n = 6). MC3T3-E1 cells were seeded at a density of 2 × 10⁴ cells mL⁻¹. To evaluate the direct effect of the scaffold on osteoblast cell differentiation, the cells were continuously cultured in complete growth medium.¹⁹ After 14 d, the cells were lysed with 0.2% (v/v) Triton X-100. ALP activity was quantified by measuring absorbance at 405 nm using an ALP assay kit (LABALP-M1, FUJIFILM Wako). The total protein content of each sample was determined using a Micro BCA protein assay (Thermo Fisher Scientific, IL, USA), and ALP activity was calculated as the amount of p-nitrophenol generated per total protein content.

Late-stage differentiation was evaluated through Alizarin Red S (ARS) staining (n = 6). Cells were seeded at 2 × 10⁴ cells mL⁻¹ and cultured for 28 d. After fixing with 4% paraformaldehyde in phosphate-buffered saline for 10 min, the cells were washed with deionized water and stained with ARS (FUJIFILM Wako) to visualize calcium deposition. Stained samples were imaged, and the bound dye was extracted using 10% (v/v) acetic acid (Sigma-Aldrich, MO, USA). Calcium deposition was quantified by measuring the absorbance of the extracted dye at 405 nm.

Statistical analysis

Data were analyzed using a statistical software program (IBM SPSS Statistics for Macintosh. V25; IBM Corp., Armonk, USA). For multiple comparisons, one-way analysis of variance was performed at each time point. When homogeneity of variance was confirmed by Levene's test, Tukey's honest significance difference (HSD) test was applied; otherwise, Dunnett's T3 test was used. For comparisons between two groups, an independent t-test was conducted. A p-value of less than 0.05 was considered statistically significant. The quantitative evaluations performed in this study, including the experiments, sample sizes and statistical analyses are concisely summarized in Table 2.

Table 2.

Summary of material characterization (A) and in vitro experiments (B).

(A)
Evaluation	Wettability	Surface roughness	Adhesion strength
Method	Contact angle	Laser mictoscope (Sa)	Tensile test
Sample size (n)	5	3	5
Control group	3Y-SLA	3Y-SLA	–
Statistical analysis	Dunnet's T3 Test	Dunnet's T3 test	Independent t-test
(B)
Evaluation	Cell proliferation	Cell differentiation (early)	Cell differentiation (late)
Method	CCK-8	ALP assay kit	ARS staining
Method	CCK-8	Micro BCA protein assay	ARS staining
Sample size (n)	6	6	6
Control group	3Y-SLA	3Y-SLA	3Y-SLA
Statistical analysis	Tukey's HSD test (3, 7d)	Tukey's HSD test	Dunnet's T3 test
Statistical analysis	Dunnet's T3 test (1d)	Tukey's HSD test	Dunnet's T3 test

Results

Characterization of HAp

SEM and TEM images of the synthesized powder are shown in Figures 2(A) and (B), respectively. The obtained powder exhibited a plate-like morphology with longitudinal dimensions of 0.5–3 μm.

Figure 2.

Characterization of morphology-controlled hydroxyapatite. (A) SEM and (B) TEM images of the synthesized powder (×20k and ×10k, respectively). (C) FT-IR spectra and (D) XRD patterns of the synthesized powder.

The FT-IR spectra and XRD patterns of the synthesized powder are shown in Figures 2(C) and (D), respectively. In the FT-IR spectra, characteristic absorption peaks of HAp were observed at 1100–960cm⁻¹ and 600–560 cm⁻¹, corresponding to ${PO}_{4}^{3 –}$ stretching and bending vibrations, respectively. Additionally, an OH⁻ stretching peak was observed at 3570 cm⁻¹.¹⁷ The XRD pattern of the synthesized powder exhibited diffraction peaks consistent with those of standard HAp (ICDD No. 01-074-0565), with a particularly strong reflection observed at the (300) plane compared with the reference pattern.

Characterization of HAp/YSZ composites

Zeta potential measurements for HAp and 1.5Y at each modification step are presented in Figures 3(A) and (B), respectively. For HAp, the initial zeta potential at pH 8.5 was −9.81 mV, which increased to −3.42 mV after SDC treatment. Subsequent LBL treatments alternately shifted the surface potential to positive and negative values after PDDA and PSS adsorption, respectively. Notably, the magnitude of charge reversal was greater after PSS adsorption than after PDDA, for both the first and second layers. For 1.5Y, the initial zeta potential was −16.79 mV. After sequential LBL modification, alternating charge reversal was observed, confirming effective surface functionalization with PDDA (positive) and PSS (negative) layers.

Figure 3.

Zeta potential of (A) HAp and (B) YSZ. (C) TGA curves of 1.5Y and HAp/YSZ composites. (D)SEM images (×300 and ×2k) and EDS mapping images (×2k) of 1.5Y and HAp/YSZ composites.

TGA results for 1.5Y (without SLA treatment) and HAp/YSZ composites are presented in Figure 3(C). The weight of all samples increased gradually with increasing temperature. The extent of weight gain followed the order 1.5Y > HAp/YSZ > pHAp/nYSZ ≈ nHAp/pYSZ. Above 200 °C, the weight-temperature profile of the HAp/YSZ group became nearly parallel to that of 1.5Y. Above 500 °C, both experimental groups showed trends similar to those of the other samples.

Representative SEM and EDS images of 1.5Y (without SLA treatment) and the HAp/YSZ composites are shown in Figure 3(D). Particle adsorption onto the YSZ surface was observed in all samples except 1.5Y. In contrast, particle aggregation was evident in the HAp/YSZ group. In the experimental groups (pHAp/nYSZ and nHAp/pYSZ), HAp particles were distributed over the entire YSZ surface, with nHAp/pYSZ exhibiting the most uniform coating. EDS mapping confirmed localized Ca aggregation in pHAp/nYSZ, whereas Ca was homogenously distributed in the case of nHAp/pYSZ.

FT-IR spectra and XRD patterns of the HAp/YSZ composites are shown in Figures 4(A) and (B), respectively. In the FT-IR spectra, strong ${PO}_{4}^{3 –}$ peaks appeared at 1100–960 cm⁻¹ and 600–560 cm⁻¹ in the experimental groups. The XRD profiles of 1.5Y and the HAp/YSZ composites were similar to those of YSZ. However, when magnified at 30° ≤ 2θ ≤ 38°, a characteristic HAp peak was detected in the experimental groups. XPS spectra (Figure 4C) revealed Na 1s (∼1080 eV), Ca(LMM) (∼970 eV), and P 2p (∼140 eV) signals in the HAp/YSZ, pHAp/nYSZ, and nHAp/pYSZ groups, which were absent in 1.5Y. Compared with the HAp/YSZ, both experimental groups showed reduced Zr 3s/3p/3d peak intensities and increased Ca 2s/2p signals.

Figure 4.

Characterization of HAp/YSZ composite. (A) ATR-FT-IR spectra, (B) XRD patterns and (C) XPS spectra of 1.5Y and HAp/YSZ composites.

Water contact angle measurements are shown in Figure 5(A). Mean angles for 3Y-SLA, 1.5Y, 1.5Y-SLA, HAp/YSZ, pHAp/nYSZ, and nHAp/pYSZ were 12°, 43°, 15°, 35°, 5°, and 15°, respectively. Contact angles for 3Y-SLA, 1.5Y-SLA, and the experimental groups were significantly lower than those for untreated 1.5Y and the HAp/YSZ. Surface roughness measurements (Figure 5B) showed that 3Y-SLA, 1.5Y-SLA, and pHAp/nYSZ had significantly higher roughness compared with 1.5Y, HAp/YSZ, and nHAp/pYSZ.

Figure 5.

(A) Water contact angles of YSZ and HAp/YSZ composites. (B) Surface roughness of YSZ and HAp/YSZ composites. Different letters indicate significant differences (Dunnett T3 test, p < 0.05). (C) Adhesive strength between YSZ and HAp in the HAp/YSZ composites (independent-samples t-test, p < 0.05). Corresponding SEM images and Ca EDS mapping images of fracture surfaces (×5k).

Tensile test results for the experimental groups are shown in Figure 5(C). The adhesive strengths of pHAp/nYSZ and nHAp/pYSZ were 10.39 and 10.08 MPa, respectively, with no significant difference between the two groups. Calcium signals were detected on both the YSZ and stainless-steel sides of the fractured specimens, confirming that failure occurred within the HAp coating layer in both cases.

Behavior of MC3T3-E1 cells on HAp/YSZ composites

Fluorescence images acquired at 6 and 24 h after seeding are shown in Figure 6(A). Actin filaments, stained with rhodamine phalloidin appear red; and nuclei, stained with DAPI, appear blue. At 6 h, cells adhered to all sample surfaces. By 24 h, cells, especially those in the experimental groups, exhibited pronounced elongation and well-developed filopodia.

Figure 6.

(A) Fluorescence images of MC3T3-E1 cells on 3Y-SLA, 1.5Y-SLA, and HAp/YSZ composites at 6 and 24 h (×20k). (B) Cell proliferation measured by absorbance at 1, 3, and 7 d. Dunnett T3 test was used for day 1, and Tukey's HSD test for days 3 and 7 (p < 0.05). (C) ALP activity after 14 d. (D) Quantitative calcium deposition and (E) representative Alizarin Red S staining images after 28 d. ALP activity was analyzed using Tukey's HSD test; calcium deposition was analyzed using Dunnett T3 test.

Cell proliferation at 1, 3, and 7 d post-seeding is shown in Figure 6(B). Significant differences were observed between 3Y-SLA and pHAp/nYSZ at day 1 and between 1.5Y-SLA and both pHAp/nYSZ and nHAp/pYSZ at day 3. No significant differences were detected at day 7.

Early osteogenic differentiation, assessed by ALP activity, is illustrated in Figure 6(C). ALP activity was significantly higher in the experimental groups compared with the control groups, with no significant difference between pHAp/nYSZ and nHAp/pYSZ.

Late differentiation, evaluated by ARS staining, is shown in Figures 6(D) and (E). The experimental groups exhibited stronger staining than the control groups, with nHAp/pYSZ showing the most uniform and extensive mineralization. Quantitative analysis revealed significantly higher optical density for nHAp/pYSZ compared with all other groups.

Discussion

We developed HAp/YSZ composite materials by uniformly coating morphology-controlled HAp onto YSZ using an LBL technique based on electrostatic surface-charge control. To the best of our knowledge, this work represents the first successful demonstration of uniform HAp coating on YSZ via electrostatic adsorption. Furthermore, the resulting HAp/YSZ composites exhibited a high calcification capacity in vitro. Based on these findings, the null hypotheses presented in the preceding section were rejected.

To control the morphology of HAp, samples were synthesized following the procedure reported by Horiuchi et al.,¹⁷ resulting in plate-like crystals with longitudinal dimensions of 0.5–3 μm. Lebre et al.¹⁴ reported that small needle-shaped HAp particles can induce inflammatory responses. In contrast, the HAp synthesized in this study exhibited a plate-like morphology, confirming that morphological control was achieved. Suzuki et al.²⁰ demonstrated that plate-like HAp with preferential exposure of the a-plane enhances ion exchange owing to the high density of exposed calcium atoms, thereby promoting early-stage osteogenic differentiation. Thus, the plate-like HAp obtained in the present study is expected to contribute to improved biocompatibility.²¹

In composite materials fabricated using the LBL method, molecules bearing opposite charges (positive and negative) are known to be uniformly adsorbed.^16,22 Consistent with this, as shown in Figure 3(D), HAp adsorption was observed across the entire surface in the LBL-treated groups, with a particularly homogeneous dispersion in the nHAp/pYSZ samples. In contrast, HAp aggregation was observed in the HAp/YSZ and pHAp/nYSZ samples. In the LBL process, molecular aggregation tends to occur when the absolute zeta potential is below ±40 mV.^23,24 Previous reports indicate that HAp typically exhibits zeta potential less than ±40 mV,^25,26 suggesting that aggregation is prone to occur in the HAp/YSZ group. Indeed, as shown in Figure 3(A), untreated HAp exhibited a low zeta potential of −9.81 mV, which likely promoted aggregation. Tan et al.^16,27 reported that prior hydrophilization with SDC can improve surface charge for molecules with zeta potentials below ±40 mV. In the present study, the zeta potential after PDDA treatment remained below +10 mV for both the first and second PDDA layers, indicating insufficient positive charge modification. In contrast, after PSS treatment, the absolute zeta potential exceeded 50 mV, reflecting sufficient negative charge modification. These results suggest that HAp aggregation in the pHAp/nYSZ samples was attributable to inadequate charge modification, whereas uniform HAp adsorption in nHAp/pYSZ samples was achieved through effective LBL assembly. Negatively charged surfaces have been reported to optimize osteoblast adhesion and proliferation, whereas positively charged surfaces, although promising for antibacterial applications and immunomodulation, are often associated with undesirable pro-inflammatory responses.²⁸ Although the final surface charge of the composite materials obtained in this study was not directly determined by zeta potential analysis, the untreated HAp exhibited a negative zeta potential. Therefore, it is likely that the surface potential of the nHAp/pYSZ composite was also negative. This surface characteristic suggests that the developed nHAp/pYSZ composite may contribute to enhanced osteoconductive performance.

In contrast, increased surface roughness is generally considered to have a positive effect on osteoblast proliferation and differentiation. However, in the present study, nHAp/pYSZ significantly enhanced mineralization despite exhibiting a smoother surface than 3Y-SLA and 1.5Y-SLA. These results suggest that the chemical composition and crystal structure of HAp exert a more dominant influence on osteogenic behavior than the zirconia surface itself. Furthermore, compared with pHAp/nYSZ, the uniform coating achieved in nHAp/pYSZ resulted in superior osteoconductive performance.

Because SDC is a surfactant commonly used as a cytolytic agent and is known to exhibit cytotoxicity,²⁹ heat treatment was applied to remove it before initiating cell experiments. As shown in Figure 3(C), TGA results indicated that all samples exhibited weight gains with increasing temperature, with two inflection points observed at approximately 200 °C and 500 °C. Below 200 °C, the experimental groups gained weight more slowly than 1.5Y. Between 200 °C and 500 °C, the HAp/YSZ and 1.5Y groups showed parallel weight-gain trends, whereas the weight gain of the experimental groups was more gradual. Above 500 °C, the trends of the experimental groups were nearly identical to 1.5Y and HAp/YSZ groups. These results suggest that adsorbed moisture was removed by 200 °C, and residual SDC and polymers were eliminated by 500 °C. Accordingly, all coated samples were heat-treated at 500 °C prior to biological evaluation.

An adhesion strength of at least 15 MPa is generally required⁹; however, in the present study, all experimental groups exhibited relatively low adhesion strengths of approximately 10 MPa between HAp and YSZ, which is below this criterion. Fracture occurred within the coating layer in all cases (Figure 5C). In a previous study,⁹ when HAp was deposited onto zirconia using the sol–gel method, the adhesion strength exceeded 40 MPa, indicating substantially stronger bonding. Kubicki et al.³⁰ reported that adhesion strength may result from the crystal growth of HAp and closure of micropores within the coating. Compared with direct-deposition methods, the post-synthesis coating approach used in this study tends to form a multilayered structure with micropores, which may weaken interfacial bonding strength between the coating layers. Therefore, further studies are required to achieve a monolayer HAp coating with improved adhesion strength.

As shown in Figure 5(A), both the experimental groups and SLA-treated YSZ surfaces became superhydrophilic after processing. Superhydrophilicity has been reported to enhance early cell adhesion,³¹ consistent with the pronounced filopodia observed in the experimental groups after 24 h (Figure 6A). However, superhydrophilicity does not necessarily promote long-term proliferation or differentiation.^32,33 In agreement with this, we found no significant differences in cell proliferation among the groups at day 7. In contrast, cell differentiation assays revealed higher ALP activity in the experimental groups at day 14, and notably, nHAp/pYSZ exhibited the greatest calcium deposition at day 28 compared with the SLA group. In addition, the differentiation assay was performed without using osteogenic differentiation medium in this study. Wang et al.¹⁹ who conducted differentiation experiments using both normal and osteogenic differentiation medium, reported that the coating groups exhibited high differentiation even in normal medium. The authors¹⁹ suggested that such coatings may be advantageous for the repair of bone-related tissues even under conditions with no or limited concentrations of osteogenic inducers. The results of the present study indicate that HAp not only promotes osteogenic differentiation but may also possess similar performance in the absence of inducers.

Several limitations of this study should be acknowledged. First, although the zeta potentials of HAp and YSZ were individually measured, the zeta potential of the fabricated composite materials was not directly evaluated. Second, osteogenic differentiation was assessed by quantitative analyses of alkaline phosphatase activity and calcium deposition; however, gene expression analysis by quantitative real-time PCR (qRT-PCR) was not performed. Therefore, data on osteogenic differentiation markers at the transcriptional level were not obtained, and further studies are required to address these limitations.

Conclusions

Morphology-controlled HAp was successfully synthesized, and an HAp/YSZ composites were fabricated using the LBL method. The synthesized HAp exhibited a plate-like morphology with a well-controlled structure. Moreover, negatively charge-modified HAp was uniformly deposited onto positively charge-modified YSZ surfaces, demonstrating the effectiveness of the LBL method for achieving homogeneous coatings. In vitro experiments demonstrated enhanced cellular differentiation on the composite surface, suggesting that morphology-controlled HAp may contribute to improved osteoconductive performance of YSZ-based implants.

Footnotes

ORCID iD

Kosuke Nozaki

Ethical considerations

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Author contributions

Satsuki Tanaka: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft.

Kosuke Nozaki: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft.

Kimihiro Yamashita: Methodology, Writing – review & editing.

Yuki Nakano: Investigation, Writing – review & editing.

Kazuaki Hashimoto: Investigation, Writing – review & editing.

Shiho Otake: Methodology, Writing – review & editing.

Noriyuki Wakabayashi: Project administration, Writing – review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The following financial support was received for the research, authorship, and/or publication of this article: Ministry of Education, Culture, Sports, Science and Technology of Japan (23K09269); Kazuchika Okura Memorial Foundation's research grant; Grant-in-Aid for the Cooperative Research Project of Creation of Life Innovation Materials for Interdisciplinary and International Researcher Development from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); and Cooperative Research Project of the Research Center for Biomedical Engineering.

Declaration of conflicting interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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