Improving osteogenic properties of zirconia ceramic via glow discharge plasma-enhanced deposition of amine organic compound
Dental implants have been extensively employed in dental implantology and orthopaedic surgery for various purposes, including facilitating fracture healing, addressing skeletal anomalies, and replacing damaged teeth and joints. Although titanium is commonly utilized as the primary material for manufacturing implants, it has some disadvantages, such as dissemination of its particles into the oral environment and aesthetic concern. Therefore, zirconia was introduced to solve this problem and is currently regarded as the second most commonly utilized material after titanium. Yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) is a promising dental implant material undergoing extensive research. This zirconium oxide exhibits enhanced mechanical characteristics, including a flexural strength of 900 MPa, fracture toughness up to 10 MPa√m, high bending strength, and resistance to corrosion and chemical stability. Moreover, several studies have illustrated that zirconia can reduce bacterial adhesion and biofilm accumulation, thereby lowering the risk of inflammatory reactions. Despite these encouraging results, long-term outcomes are still lacking, and challenges related to zirconia osseointegration need to be addressed. Current research on surface treatments for zirconia implants mainly includes sandblasting and acid etching, laser treatment, powder injection molded (PIM), and sintering. , However, these treatment techniques can negatively impact zirconia's mechanical strength. For instance, residual stress from sandblasting may weaken the material, and heat treatment can reduce its biaxial flexural strength. To address these issues, researchers have endeavored to alter the surface of the material without compromising its physical properties. In our study, we present a low-temperature glow discharge plasma (GDP) treatment combined with amine organic compounds to obtain a bioactive ceramic implant material that enhances interaction with bone cells. GDP treatment is widely used for sterilizing and modifying surface characteristics of biomaterials by employing low atmospheric pressure and low thermal plasma to form functional proteins. This procedure facilitated the surface modification of biodegradable polymers while preserving their inherent characteristics. Combining GDP treatment with allylamine significantly enhances the hydrophilicity of biomaterials, hence effectively maximizing their biocompatibility. Allylamine improves bioactivity by serving as an amine substrate that facilitates the attachment of biologically active proteins to the surfaces. , Prior research has shown that the existence of polar groups on the surface, particularly those abundant in nitrogen, promotes cellular adhesion. Additionally, the plasma polymerized layer is biocompatible, integrates well with surrounding tissue cells, promotes antibacterial properties, and reduces chronic inflammation. Nevertheless, the utilization of bioactive amine organic compounds on zirconia ceramic biomaterials have limited research, further investigations is necessary. Therefore, this study was to examine the unique characteristics and reactivity of allylamine when applied to the surface of zirconia ceramics via GDP treatment, and its interaction with adjacent osteoblast cells. 

Zirconia ceramic samples preparation The zirconia ceramic samples (Coho Technology, Taipei, Taiwan) underwent a process utilizing GDP in the presence of argon at 85 Watts (W) for 30 min, and then exposed to the amine compound in a GDP reactor (AST Products Inc., Billerica, MA, USA) as shown in . Three distinct circumstances were applied: 50W, 75W, or 85W, using 13.56 MHz and 100 millitorrs for another 30 min. These conditions are referred to as A50, A75, and A85. Surface morphology and composition analysis The scanning electron microscope (SEM; SU-3500; Hitachi Ltd., Kyoto, Japan) was used to observe surface morphology at 60× and 1,000× magnification, with an accelerating voltage of 20 kV in vacuum mode. To identify the elemental composition of the zirconia surface, elemental energy-dispersive spectroscopy (EDS; SU-3500; Hitachi Ltd.) and X-ray photoelectron spectroscopy (XPS; PHI Quentera II, Ulvac-PHI Inc, Kanagawa, Japan) analysis were used. In EDS, the interaction of the electron beam with the sample generates characteristic X-rays, producing a spectrum that reveals the surface's elemental composition. In XPS, the samples' surfaces were irradiated with monochromatic X-rays from an aluminum (Al Kα) source. The X-rays cause photoelectrons to be emitted from the surface atoms, which were then collected and analyzed by the electron energy analyzer. Functional group analysis Fourier-transform infrared spectroscopy (FTIR; Nicolet iS5 instrument, Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the functional groups of test samples in this study. The prepared samples were placed in contact with the attenuated total reflectance (ATR) crystal. Infrared (IR) light was directed into the ATR crystal, where it underwent multiple internal reflections, creating an evanescent wave that interacts with the surface molecules. This allows for the absorption of wavelengths ranging from 3650 to 650 cm−1, which characterizes the different chemical bonds. Surface hydrophilicity and roughness analysis A 4 μL droplet of ultra-pure water (Millipore-Q system, Merck Millipore, Burlington, MA, USA) was dispensed onto the zirconia disk's surface, and the droplet profile was captured by a contact angle meter (GBX Digidrop, GBX Scientific LTD, Dublin, Ireland) to measure surface hydrophilicity. The contact angle was calculated from multiple measurements taken at three different locations to ensure accuracy. Surface roughness measurements were conducted using an atomic force microscope (AFM) (Nanoview 1000, FSM Precision Inc, Suzhou, China). Samples were prepared by depositing graphene nanosheets onto mica surfaces using a drop-casting method and analyzed in tapping mode. Cell viability and cell adhesion analysis MG-63 human osteoblast-like cells were acquired from the Cell Cultures of the Bioresource Collection and Research Centre (Hsinchu, Taiwan) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Merck KGaA Inc, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, San Jose, CA, USA) under humidified condition with 5% CO 2 at 37 °C. Cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium Bromide (MTT) assay kit (Roche Applied Science, Mannheim, Germany) on 1, 3, 5, and 7 days. MTT working solution was added, followed by incubation for 2 h. The formazan crystals were then dissolved with DMSO, and absorbance was measured at 570 nm using a microplate reader (SpectraMax iD3, Molecular Devices LLC, San Jose, CA, USA). The cell adhesion was observed by immunofluorescent assay. Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA. Cells were then stained with Alexa Fluor 488-phalloidin (Invitrogen, Waltham, MA, USA) and DAPI (Sigma–Aldrich, St. Louis, MO, USA), and visualized using a confocal laser scanning microscope (Leica Microsystem, Wetzlar, Germany). Cell differentiation and mineralization analysis In alkaline phosphatase (ALP) assay (BioVision, Exton, PA, USA), cells were cultured for 1, 3, 5, and 7 days. 500 μL of cell lysates were mixed with assay buffer and incubated with p-nitrophenyl phosphate (pNPP) substrate at 37 °C for 2 h, and absorbance was measured at 405 nm. Mineralization activity was analyzed at 7, 14, and 21 days. Cells were fixed with 70% ethanol for 1 h, washed and stained with Alizarin Red S solution (Sigma–Aldrich) for 10 min. The dye was extracted with 10% acetic acid and measured at 540 nm. Osteoblast related gene expression analysis The gene expression of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox 5 (DLX5), transcription factor SP7 (SP7), osteoprotegerin (OPG) and receptor activator of nuclear factor κ B (RANK) was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) following the protocol described in the previous study. In brief, 1 μL of Trizol (Thermo Fisher) was used to extract RNA and mixed with chloroform. The mixture was centrifuged at 4 °C for 30 min at 15,000 rpm after isopropanol was added. After the mRNA was converted cDNA, quantitative PCR was performed by using Fast SYBR™ Green Master Mix (Thermo Fisher) and analyzed with LightCycler Instrument (Roche Molecular Systems, Pleasanton, CA, USA). The list of primer sequences is provided in . Statistical analysis The data from the experimental analyses were described using means and standard deviations. Data were compared using two-way analysis of variance (ANOVA), Tukey multiple comparison test, and correlation. Simple linear regression was used for multiple comparisons among different groups. Statistical analyses were performed using Prism (v10; GraphPad Software Inc., Boston, MA, USA). Values of P < 0.05 were considered statistically significant. 

The zirconia ceramic samples (Coho Technology, Taipei, Taiwan) underwent a process utilizing GDP in the presence of argon at 85 Watts (W) for 30 min, and then exposed to the amine compound in a GDP reactor (AST Products Inc., Billerica, MA, USA) as shown in . Three distinct circumstances were applied: 50W, 75W, or 85W, using 13.56 MHz and 100 millitorrs for another 30 min. These conditions are referred to as A50, A75, and A85. 

The scanning electron microscope (SEM; SU-3500; Hitachi Ltd., Kyoto, Japan) was used to observe surface morphology at 60× and 1,000× magnification, with an accelerating voltage of 20 kV in vacuum mode. To identify the elemental composition of the zirconia surface, elemental energy-dispersive spectroscopy (EDS; SU-3500; Hitachi Ltd.) and X-ray photoelectron spectroscopy (XPS; PHI Quentera II, Ulvac-PHI Inc, Kanagawa, Japan) analysis were used. In EDS, the interaction of the electron beam with the sample generates characteristic X-rays, producing a spectrum that reveals the surface's elemental composition. In XPS, the samples' surfaces were irradiated with monochromatic X-rays from an aluminum (Al Kα) source. The X-rays cause photoelectrons to be emitted from the surface atoms, which were then collected and analyzed by the electron energy analyzer. 

Fourier-transform infrared spectroscopy (FTIR; Nicolet iS5 instrument, Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the functional groups of test samples in this study. The prepared samples were placed in contact with the attenuated total reflectance (ATR) crystal. Infrared (IR) light was directed into the ATR crystal, where it underwent multiple internal reflections, creating an evanescent wave that interacts with the surface molecules. This allows for the absorption of wavelengths ranging from 3650 to 650 cm−1, which characterizes the different chemical bonds. 

A 4 μL droplet of ultra-pure water (Millipore-Q system, Merck Millipore, Burlington, MA, USA) was dispensed onto the zirconia disk's surface, and the droplet profile was captured by a contact angle meter (GBX Digidrop, GBX Scientific LTD, Dublin, Ireland) to measure surface hydrophilicity. The contact angle was calculated from multiple measurements taken at three different locations to ensure accuracy. Surface roughness measurements were conducted using an atomic force microscope (AFM) (Nanoview 1000, FSM Precision Inc, Suzhou, China). Samples were prepared by depositing graphene nanosheets onto mica surfaces using a drop-casting method and analyzed in tapping mode. 

MG-63 human osteoblast-like cells were acquired from the Cell Cultures of the Bioresource Collection and Research Centre (Hsinchu, Taiwan) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Merck KGaA Inc, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, San Jose, CA, USA) under humidified condition with 5% CO 2 at 37 °C. Cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium Bromide (MTT) assay kit (Roche Applied Science, Mannheim, Germany) on 1, 3, 5, and 7 days. MTT working solution was added, followed by incubation for 2 h. The formazan crystals were then dissolved with DMSO, and absorbance was measured at 570 nm using a microplate reader (SpectraMax iD3, Molecular Devices LLC, San Jose, CA, USA). The cell adhesion was observed by immunofluorescent assay. Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA. Cells were then stained with Alexa Fluor 488-phalloidin (Invitrogen, Waltham, MA, USA) and DAPI (Sigma–Aldrich, St. Louis, MO, USA), and visualized using a confocal laser scanning microscope (Leica Microsystem, Wetzlar, Germany). 

In alkaline phosphatase (ALP) assay (BioVision, Exton, PA, USA), cells were cultured for 1, 3, 5, and 7 days. 500 μL of cell lysates were mixed with assay buffer and incubated with p-nitrophenyl phosphate (pNPP) substrate at 37 °C for 2 h, and absorbance was measured at 405 nm. Mineralization activity was analyzed at 7, 14, and 21 days. Cells were fixed with 70% ethanol for 1 h, washed and stained with Alizarin Red S solution (Sigma–Aldrich) for 10 min. The dye was extracted with 10% acetic acid and measured at 540 nm. 

The gene expression of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox 5 (DLX5), transcription factor SP7 (SP7), osteoprotegerin (OPG) and receptor activator of nuclear factor κ B (RANK) was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) following the protocol described in the previous study. In brief, 1 μL of Trizol (Thermo Fisher) was used to extract RNA and mixed with chloroform. The mixture was centrifuged at 4 °C for 30 min at 15,000 rpm after isopropanol was added. After the mRNA was converted cDNA, quantitative PCR was performed by using Fast SYBR™ Green Master Mix (Thermo Fisher) and analyzed with LightCycler Instrument (Roche Molecular Systems, Pleasanton, CA, USA). The list of primer sequences is provided in . 

The data from the experimental analyses were described using means and standard deviations. Data were compared using two-way analysis of variance (ANOVA), Tukey multiple comparison test, and correlation. Simple linear regression was used for multiple comparisons among different groups. Statistical analyses were performed using Prism (v10; GraphPad Software Inc., Boston, MA, USA). Values of P < 0.05 were considered statistically significant. 

Examination of surface morphology The SEM images revealed that control zirconia disk exhibited a smoother surface compared to other surface-treated disks ( A). Significantly, the allylamine-treated samples exhibited irregular bulbous patterns on their surface, which were particularly pronounced at 1000 x magnification. When the GDP allylamine wattage increased from A50 to A85, the rounded bulbous patterns and surface deposits became more noticeable. Analysis of surface composition EDS results showed that control zirconia had the lowest carbon (C) and nitrogen (N) content while having the highest oxygen (O) concentration ( B). Allylamine-treated samples exhibited increased levels of C and N as the GDP wattage elevated. XPS analysis showed that the C, N, and O content of the control zirconia disk were 57.74% (±17.8), 1.81% (±0.51), and 29.5% (±13.7), respectively. The C and N contents of all allylamine-treated groups were higher compared to the control zirconia disk ( A). FTIR functional groups analysis The samples treated with allylamine exhibited the presence of aromatic C–H, C–O stretching, N–H stretching, C ˆ C, and C–H bonds ( C). The data indicated that with increasing concentration of allylamine, the visibility of these functional groups became more pronounced, and the intensity of absorption bands increased compared to the untreated zirconia sample. Measurement of surface hydrophilicity and roughness The water droplets contact angle on the surface of the untreated zirconia disk is 72.03° (±3.61°) while 31.80° (±2.81°) for A50, 40.30° (±0.96°) for A75, and 53.53° (±1.78°) for A85 ( A and B). There was a statistically significant difference between the control and allylamine-treated groups. The control zirconia disk had an average roughness (Ra) value of 0.76703 ± 0.1810 μm. After allylamine treatment, Ra vale elevated to 1.0090 ± 0.5007 μm (A50), 1.7691 ± 0.6627 μm (A75), and 2.0774 ± 0.6615 μm (A85). Significant differences were found only for A85 group when compared with control and A50 group. The 3D surface roughness illustration is shown in E and F. Cell viability and cell adhesion analysis On day 3 and day 5, the A50 group exhibited the highest MG-63 cell viability, reaching 183% and 193%, respectively, with all allylamine-treated groups showing significantly higher viability than the control. By day 7, the A50 group maintained the highest viability at 270%, followed by A75 at 195% and A85 at 183% ( A). The cell adhesion analysis showed that MG-63 cells began to grow and firmly adhere to the zirconia surface within 12 h, and demonstrate the osteoblast-like robust adhesion to the zirconia surface after 24 h. Cell differentiation and mineralization analysis The ALP enzyme activity increased by the 7th day ( ). The A50 group exhibited the highest enzyme expression level among all groups, with a value of 0.6203 U/L. The A85 group recorded 0.5548 U/L on day 7, while the A75 group had the second-highest value of 0.5617 U/L. Significant differences were observed between the control and allylamine-treated groups from day 3 to day 7. Qualitative and quantitative evaluations of calcium (Ca) deposition in the extracellular matrix showed that the A50 and A75 groups had the highest Ca content at day 14 and day 21, Furthermore, there was a statistically significant difference between A50 groups and the control disk on day 21 ( ). Osteogenic gene expression analysis The expression levels of osteogenic gene markers demonstrated a gradual increase over the cultivation period ( ). On day 14 and 21, the A50 exhibited the highest expression of all markers, except for OPG, which was highest in the A85 group. Additionally, the SP7 and DLX5 markers increased initially until day 7 but began to decline by day 14. On day 21, ALP levels showed a statistically significant difference between the A50 and A85 groups, while OC levels were significantly different between the A50 group and both A75 and A85 groups. 

The SEM images revealed that control zirconia disk exhibited a smoother surface compared to other surface-treated disks ( A). Significantly, the allylamine-treated samples exhibited irregular bulbous patterns on their surface, which were particularly pronounced at 1000 x magnification. When the GDP allylamine wattage increased from A50 to A85, the rounded bulbous patterns and surface deposits became more noticeable. 

EDS results showed that control zirconia had the lowest carbon (C) and nitrogen (N) content while having the highest oxygen (O) concentration ( B). Allylamine-treated samples exhibited increased levels of C and N as the GDP wattage elevated. XPS analysis showed that the C, N, and O content of the control zirconia disk were 57.74% (±17.8), 1.81% (±0.51), and 29.5% (±13.7), respectively. The C and N contents of all allylamine-treated groups were higher compared to the control zirconia disk ( A). 

The samples treated with allylamine exhibited the presence of aromatic C–H, C–O stretching, N–H stretching, C ˆ C, and C–H bonds ( C). The data indicated that with increasing concentration of allylamine, the visibility of these functional groups became more pronounced, and the intensity of absorption bands increased compared to the untreated zirconia sample. 

The water droplets contact angle on the surface of the untreated zirconia disk is 72.03° (±3.61°) while 31.80° (±2.81°) for A50, 40.30° (±0.96°) for A75, and 53.53° (±1.78°) for A85 ( A and B). There was a statistically significant difference between the control and allylamine-treated groups. The control zirconia disk had an average roughness (Ra) value of 0.76703 ± 0.1810 μm. After allylamine treatment, Ra vale elevated to 1.0090 ± 0.5007 μm (A50), 1.7691 ± 0.6627 μm (A75), and 2.0774 ± 0.6615 μm (A85). Significant differences were found only for A85 group when compared with control and A50 group. The 3D surface roughness illustration is shown in E and F. 

On day 3 and day 5, the A50 group exhibited the highest MG-63 cell viability, reaching 183% and 193%, respectively, with all allylamine-treated groups showing significantly higher viability than the control. By day 7, the A50 group maintained the highest viability at 270%, followed by A75 at 195% and A85 at 183% ( A). The cell adhesion analysis showed that MG-63 cells began to grow and firmly adhere to the zirconia surface within 12 h, and demonstrate the osteoblast-like robust adhesion to the zirconia surface after 24 h. 

The ALP enzyme activity increased by the 7th day ( ). The A50 group exhibited the highest enzyme expression level among all groups, with a value of 0.6203 U/L. The A85 group recorded 0.5548 U/L on day 7, while the A75 group had the second-highest value of 0.5617 U/L. Significant differences were observed between the control and allylamine-treated groups from day 3 to day 7. Qualitative and quantitative evaluations of calcium (Ca) deposition in the extracellular matrix showed that the A50 and A75 groups had the highest Ca content at day 14 and day 21, Furthermore, there was a statistically significant difference between A50 groups and the control disk on day 21 ( ). 

The expression levels of osteogenic gene markers demonstrated a gradual increase over the cultivation period ( ). On day 14 and 21, the A50 exhibited the highest expression of all markers, except for OPG, which was highest in the A85 group. Additionally, the SP7 and DLX5 markers increased initially until day 7 but began to decline by day 14. On day 21, ALP levels showed a statistically significant difference between the A50 and A85 groups, while OC levels were significantly different between the A50 group and both A75 and A85 groups. 

In our study, the SEM observation validated the effective grafting of amine compounds onto the zirconia samples. Both EDS and XPS analyses show a progressive increase in carbon and nitrogen elements as the wattage of allylamine increases ( B). The presence of allylamine, with the formula of C 3 H 5 NH 2 , is evident due to the contribution of its carbon and nitrogen atoms. Additionally, FTIR analysis determined the chemical bonds and functional groups present in the allylamine-treated groups, including aromatic C–H, C–O, N–H, and C ˆ C bonds, which are attributed to its C 3 H 5 NH 2 chemical structure. There is a significant correlation between contact angle and the concentration of allylamine ( B). Lower concentrations of allylamine result in lower contact angles and improved hydrophilicity. Increased allylamine concentration leads to a more textured surface. One in vitro research by Gavilán et al., has demonstrated that the roughness of a biomaterial can impact the initial development and metabolism of cells, ultimately enhancing clinical outcomes. Our modifications resulted in a surface Ra value of 1.009 to 2.077 μm, compared to the control ceramic's Ra of 0.7670 μm. A moderate correlation (r = 0.7609) exists between increasing allylamine concentration and surface roughness ( D). Allylamine treatment enhances roughness, which in turn boosts osteoblast activity and bone apposition. Fischer et al. reported a similar Ra of 1.2 μm after sandblasting with alumina and hydrofluoric acid etching. Additionally, a classical paper by Albrektsson and Wennerberg found that a Ra of 1–2 μm promotes optimal osseointegration, which aligns with our roughness data around 2 μm. The MTT assay indirectly assesses cell vitality, growth, and mitochondrial function. On day 7, the A50 group had the highest cell viability, followed by A75 and A85. This result supports Wanqi Yu's study, which found greater MG-63 cell proliferation and adherence on a plasma-enhanced chemical vapor-coated surface. The increased proliferation may be attributed to enhanced cell adhesion and spreading observed at 12 and 24 h in allylamine-treated samples, due to the cell-attractive properties of the amine groups. Kunz et al. also showed that osteoblasts benefit from the positive charges of a plasma-polymerized amine layer, despite reduced spreading ability. Following the initial 12-h cultivation period ( B), MG-63 cells showed greater adhesion and proliferation on allylamine-treated samples. The A50 group exhibited the highest cell growth at both 12 and 24 h, eventually covering the entire surface. Afterwards, phosphatase activity and calcium mineralization, key markers of osteoblast development and bone mineralization were conducted ( , ), and the A50 group had the highest ALP activity and calcium deposits compared to the other groups. Tang et al. similarly found that UV-exposed TiO 2 -modified zirconia promoted cell proliferation, increased ALP activity, and enhanced mineralization. The gene expression markers aligned with cell differentiation and proliferation results, which were evaluated in this study. ALP, an early osteogenic marker, and OC, which is associated with the bone formation process, were both elevated. DLX5 and SP7, early-stage osteoblast differentiation markers, showed upregulation until day 7 before downregulating. Elevated levels of RANK and OPG on day 7 indicate substantial bone remodeling, which is essential for the production of new osteoblast cells and bone. Nishizaki et al. demonstrated that a nanostructured zirconia/alumina composite implant improved ALP activity and osteogenic gene expression. Similarly, Chaves et al. achieved increased osteogenic differentiation of stem cells derived from adipose tissue by polymerizing amines through plasma treatment. However, the study still has some limitations associated with plaque adhesion and soft tissue integration properties. It should be further investigated in future research. The properties of zirconia ceramics were significantly enhanced by combining amine compounds with surface modification using GDP. Both surface hydrophilicity and roughness of the zirconia ceramic were notably improved after surface modification. Most importantly, amine grafting markedly enhanced the material's bioactivity, improving cell viability, differentiation, and osteogenic properties, thereby overcoming zirconia's inherent bio-inertness. The optimal outcomes were observed with the allylamine 50- Watts group. These findings open new avenues for enhancing the performance of zirconia-based implants, potentially leading to better clinical outcomes in dental implants and orthopedic applications. 

The authors have declared that there are no competing interests. 

