The impact of anodization modification on titanium interaction with human osteoblasts and fibroblasts (in vitro study)

Kamonmard Moungthong; Daneeya Na Nan; Pravej Serichetaphongse; Wareeratn Chengprapakorn

doi:10.1371/journal.pone.0336639

. 2025 Nov 19;20(11):e0336639. doi: 10.1371/journal.pone.0336639

The impact of anodization modification on titanium interaction with human osteoblasts and fibroblasts (in vitro study)

Kamonmard Moungthong ¹, Daneeya Na Nan ², Pravej Serichetaphongse ^3,⁴, Wareeratn Chengprapakorn ^5,^*

Editor: Tapash Ranjan Rautray⁶

PMCID: PMC12629452 PMID: 41259378

Abstract

Title

The impact of new anodization modification on titanium interaction with human osteoblasts and fibroblasts (in vitro)

Purpose

To compare the behavior of osteoblast and fibroblast cells, as well as the surface characteristics, of modified anodized titanium surfaces at different voltages with sandblasted and acid-etched titanium surfaces.

Methods

Twenty titanium discs were fabricated and subjected to 4 types of surface treatments: machined surface, sandblasted and acid-etched surface (SLA), modified anodized A surface at 30V DC and modified anodized B surface at 70V DC. Surface roughness and contact angle were measured. Osteoblast cells were seeded onto titanium discs and evaluated for cell proliferation, Alkaline phosphatase (ALP) activity, Alizarin red staining, and cell morphology with SEM imaging. Immunofluorescent staining was performed to assessed cell adhesion of both osteoblast and fibroblast cells. Statistic analysis was conducted using ANOVA with Tukey post hoc multiple comparisons.

Results

The topography and osteoblast cell spreading on both modified anodized titanium surfaces differed significantly from machined and SLA surfaces (p < 0.05). However, there was no significant difference in osteoblast cell spreading between modified anodized surfaces A and B. In addition, anodized surfaces A and B showed significantly higher ALP activity and Alizarin red staining than both SLA and machined surfaces (p < 0.05). For human gingival fibroblast cells, the modified anodized surfaces exhibited significantly higher cell adherence (p < 0.05), followed by the machined surface, with SLA surfaces showing the least adherence.

Conclusions

The modified anodized surfaces showed superior biocompatibility, enhancing attachment of both osteoblast and fibroblast cells, whereas the SLA surfaces exhibited the least fibroblast cell adherence.

Introduction

Dental implants are widely recognized as a reliable, long-term solution for tooth replacement, with their success largely dependent on effective osseointegration and soft tissue integration [1–4]. Over recent years, significant advancements in implant surface modification have emerged, aiming to accelerate osseointegration for quicker loading times and to reduce the risk of peri-implantitis by inhibiting biofilm development and attachment [5–7]. Common techniques for creating microtopography on implant surfaces include sandblasting, acid etching, plasma spraying, grit blasting, hydroxyapatite coating, and anodization [8,9]. Research indicates that an optimal implant surface roughness, typically ranging between 1–1.5 µm, promotes the osteoblast cell response, thereby enhances osseointegration [5,10–12]. Despite these advancements, the ongoing challenge remains to identify or develop a surface treatment method that maximizes osteoconductive properties while simultaneously preventing bacterial invasion. Anodization is one such surface modification technique showing promise, as it supports both osseointegration and soft tissue integration through favorable cellular responses [13,14]. However, the impact of anodization on soft tissue behavior, particularly at varying voltages, demands further exploration. Analyzing osteoblast and fibroblast cell adhesion on titanium surface anodized at different voltages can provide valuable insights for selecting the most appropriate implants for specific clinical conditions, ultimately guiding dental implant choices more effectively.

Anodization involves electrochemically treating the implant surface, often following a sandblast large grit and acids etch (SLA) treatment, to create an oxide layer [15]. This oxide layer acts as a corrosion barrier with high surface energy [16]. The thickness of the oxide layer is controlled by the anodizing voltage, which also affects the surface color due to wavelength reflection [17–19]. Recent studies have demonstrated that anodization effectively enhances osteoblast adhesion and proliferation, alkaline phosphatase activity, and calcium deposits, thereby improving osseointegration [13,14,20]. For instance, Traini et al. indicated that anodized implant surface with a titanium oxide (TiO₂) coating exhibit lower surface tension and a hydrophilic surface, which fosters improved blood clot formation [21]. This hydrophilic quality is crucial as it facilitates osteoblast cell attachment to the implant surface, enhancing the osseointegration process necessary for immediate implant loading and long-term success [22].

Maintaining peri-implant soft tissue health is critical for the long-term success of dental implants, as peri-implant infections are the primary cause of implant failure, leading to bone loss [23,24]. Hemidesmosome attachment plays a vital role in limiting bacterial infiltration, thus reducing the risk of infection [25–28]. Anodization enhances both osseointegration and soft tissue attachment through favorable responses from osteoblasts and fibroblasts. In vitro studies have demonstrated better fibroblast cell adherence and reduced biofilm formation on anodized surfaces [29,30]. While SLA treatments improve cellular adhesion, they also inadvertently enhance bacterial adherence, which can result in increased inflammation and bone loss compared to polished surfaces [31–33]. In contrast, anodized surfaces exhibit significantly decreased bacterial adherence, presenting a promising alternative for implant surfaces [30,34,35]. Advancements in anodized implant surfaces have shown improved interactions with osteoblasts and gingival fibroblasts. However, there is a lack of comprehensive studies examining cellular responses across different implant surfaces at varying anodization voltages. Our objective is to investigate the effects of different anodization voltages on titanium surfaces, alongside machined and SLA surfaces, focusing on osteoblast and fibroblast cell responses, surface characteristics, and hydrophilicity.

Materials and methods

The study size was calculated from the pilot study. Twenty type IV titanium discs, each measuring 9 mm in diameter and 3 mm in thickness, were utilized according to ISO 5832–2:2018 standards [36]. The discs were divided into four groups, with each group receiving different surface treatment. The control group was machined titanium surface. The first surface modification is sandblasted with large grit and acid etch (SLA). The titanium discs were sandblasted with aluminum oxide (Al₂O₃), followed by acid etching using a mixture of 60% sulfuric acids and 30% hydrochloric acid. Two distinct anodization processes were applied to the surfaces following sandblasting and acid etching. The first one were anodized in a 5% sulfuric solution using a voltage range of 20-30V DC at 0.1 A to get anodized A (blue) surface. The second one were anodized in a 5% sulfuric solution using a voltage range of 60-70V DC at 0.1 A to get anodized B (yellow) surface. After surfaces modifications were completed, all samples underwent a sterilization process by cleansing with alcohol, deionized water, and phosphate buffer saline in preparation for subsequent analysis.

Surface characterization

The surface roughness of the titanium discs was measured using a surface profilometer (Alicona, infinitefocus, Austria) at x10 magnification. Three different locations on each sample were scanned and measured to obtain representative data. The surface morphology of the titanium discs were assessed using scanning electron microscopy (SEM)(JSM 5410LV, JEOL, Japan) at x5000 magnification. The three-dimensional profile images of different titanium surfaces were measured using a surface profilometer (Alicona, infinitefocus, Austria) at x50 magnification.

The surface wettability was determined using the sessile drop method by measuring the water contact angle [37]. The 10 μL sessile droplet of deionized water was deposited vertically on the surface using a micropipette, ensuring no physical contact with the surface. The contact angle was measured 5 times for each samples to obtain consistent data. The results were reported as mean ± standard deviation.

Behavior of osteoblasts and fibroblasts on titanium discs

Human osteoblast cells were obtained from bone fragments during dental implant procedures. Written informed consent was obtained from volunteers aged 20−50 years, with approval from the ethics board of Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2022−098). Bone fracments were harvested between 16 December 2022 and 29 May 2023 and cultured in high glucose-Dulbecco modified eagle medium (DMEM Gibco BRL, Ca, USA) containing 10% fetal bovine serum, 2 mM of L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml, Gibco) and amphotericin B (5 mg/ml). Induction of differentiation to osteoblasts was promoted by culturing with 10 nM dexamethasone, 25 µg/ml L-ascorbic acid, and 10 mM β-glycerophosphate. Cells from the third to the sixth passages were use for experiment after characterization.

Human gingival fibroblast cells were obtained from the molar teeth of 3 patients, aged between 20–30 years, extracted for orthodontic reasons at the Department of Surgery, the Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2024–011) between 6 May 2024 and 20 August 2024. Gingival fibroblasts were scraped from the middle third of the root and cultured for 3–4 weeks in DMEM containing 10% fetal bovine serum, 2 mM of L-glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml) (Gibco) and amphotericin B (5 mg/ml). Cells from the third to the ﬁfth passages were used in the study.

All tissues used to isolate osteoblasts and fibroblasts were obtained from healthy patients. The patients had no history of systemic disease, smoking, or medication that could affect bone or soft tissue healing. Cells were seeded on polystyrene surface and all titanium discs in 48 well plates at a density of 2 × 10⁴ cells/surface. The cultures were incubated for 24 hours at 37 °C. The proliferation rate of osteoblasts and fibroblasts was evaluated using the MTT (methylthiazolytetrazolium) assay. Measurements were taken at 24 hours, 3 days, and 7 days post-seeding. At each time point, the culture media was measured at an absorbance of 570 nm using a microplate reader (Elx800; Biotek, Winooski, VT).

Immunofluorescent staining

Human osteoblast cells were seeded at 2 × 10⁴ cells per well in 24-well plates for 1 hour and 3 hours. Human gingival fibroblast cells were also seeded at the same density and observed after 3 hours. Cells were rinsed twice with 0.1M PBS, fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X 100 for 10 minutes. Cells were incubated overnight with primary antibody vinculin (1:100; Abcam, Grand Island, NY) to stain focal adhesion points. Rhodamine-conjugated phalloidin (1:200; Molecular Probes-Life Technologies, Grand Island, NY) was used to stain actin filaments. Nuclei were stained with 300 nM 4^´,6-diamidino-2-phenylindole, dihydrochloride (DAPI) for 20 min. A glass coverslip was mounted on the titanium surface containing cells using an antifade kit (Vectashield, Vector Laboratories, Burlingame, CA). Fluorescent images were analyzed using a fluorescent microscope (Axiovert 40CFL, Carl Zeiss, Gottingen, Germany). Cell adhesion was quantified by counting the number of cells in 3 fields of view per sample, across three discs for each titanium surface.

Alkaline phosphatase (ALP) activity

Osteogenically differentiated human osteoblast cells were seeded on all titanium discs in 48-well plates at the density of 3x10⁴ cells per well. ALP activity was measured at two time points: after 3 and after 7 days. The cells were incubated with 300 μL of BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) substrate solution (Sigma Life Science, USA) for 30 minutes at 37°C. For quantification, the stained cells were rinsed with 200 µl of 2M KOH to stop the reaction, followed by 300 µl of DMSO to dissolve the intracellular NBT-formazan crystal [38]. The absorbance was measured using a microplate reader at 620 nm (Elx800; Biotek, Winooski, VT) [39].

Alizarin red S staining

Assessment of mineralization was conducted at two time points: after 14 days and after 21 days. Cells were fixed with cold methanol for 10 minutes. Fixed cells were stained with 1% Alizarin Red S solution (Sigma Life Science, USA) for 5 minutes at room temperature to detect calcium deposits. The quantification was performed by dissolved stained sample in 10% of cetylpyridinium chloride and measured for absorbance at 570 nm.

Statistical analysis

Statistical analysis was performed using Prism (GraphPad, La Jolla, CA, USA). Shapiro-Wilk’s test was used to assess the normality of the data and Levene’s test was used to determine the homogeneity of variance. Analysis of Variance (ANOVA) with Tukey post hoc multiple comparisons were conducted to determine the significance of the results. The data presented for all groups are expressed as standard deviation (SD). Significance levels were categorized as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Result

Surface characteristic analysis

The surface roughness (Ra) and root mean square surface roughness (Sa) values for different titanium surface treatments are presented in the Table 1. Statistical analysis indicated that the machined surface (control) had the lowest Ra (0.33 µm) and Sa (0.38 µm) values, which were significantly different from the treated surfaces. Sandblast and acid-etches surface exhibited increased roughness compared to the machined surface, contributing to a more textured appearance. Anodized surfaces (A and B) showed comparable roughness values to each other, with increased roughness compared to the machined surface. However, anodized A displayed slightly higher mean Ra (1.19 µm) and Sa (1.31 µm) values compared to anodized B (1.15 and 1.29 µm) (Fig 1).

Table 1. Surface roughness parameter measurement in Arithmetric Mean Roughness (Ra) and Root mean Square Roughness (Sa), different small letters (alphabet) indicated a significant difference between material types.

Type of surface modification	Ra (mean SD)	Sa (mean SD)
Machined surface (Control)	0.33 µm (0.01)^a	0.38 µm (0.01)^a
Sandblasted and acid-etched	1.08 µm (0.01)^b	1.22 µm (0.03)^b
Anodized A	1.19 µm (0.14)^b	1.31 µm (0.24)^b
Anodized B	1.15 µm (0.06)^b	1.29 µm (0.24)^b

Open in a new tab

The SEM images (Fig 2) revealed that machined surface had relatively uniform, smooth texture characterized by distinct machining lines. SLA surface showed particularly irregular and textured surface morphology. Both anodized surfaces (A and B) exhibited similar rough and pore-filled textures. However, the anodized A surface had deeper valleys, which appeared as pink region in Fig 2.

The surface wettability of different titanium surface treatments was assessed through contact angle measurements, as depicted in Fig 3. Results demonstrated statistically significant differeces in contact angles among all surface treatments (p < 0.05). The machined surface showed the highest contact angle of 65.9 degrees, indicating the lowest wettability. The SLA-treated surface exhibited a moderately lower contact angle at 57.7 degrees in comparison. The anodized A surface had a notably lower contact angle of 20.28 degrees, while the anodized B surface recorded the lowest contact angle of 15.08 degrees, reflecting the highest level of wettability among the samples.

Behavior of osteoblastic and fibroblast cells on titanium surface analysis

Cell cytotoxicity and proliferation

It is observed that both the anodized A and B surfaces showed an increasing proliferation rate when compared to other titanium surfaces (Fig 4). On day 1, the proliferation of the osteoblast cells on the anodized A titanium surface was statistically significantly higher than on all other titanium surfaces (p < 0.05). By day 3, the proliferation rate of both anodized A and B surfaces were statistically significantly higher than those of the machine surface and the sandblasted and acid-etched surface (p < 0.05). However, no statistically significant difference was found between the anodized A and the anodized B groups (p < 0.05). By day 7, there were no significant differences in cell proliferation among test groups, as initial advantages of the anodized A surface diminished overtime. Across time point, significant proliferation changes were found in machined surface titanium between every tested interval (day1 and 3, day 3 and 7, day 1 and 7) The SLA surface exhibited significant differences in proliferation between days 3 and 7 and days 1 and 7 only. Anodized A demonstrated significant changes solely between days 1 and 7, while Anodized B showed significant differences proliferation across all intervals tested.

Immunofluorescent staining

At the 1-hour mark (Fig 5), both anodized A and anodized B surfaces recruited a higher number of osteoblast cells compared to machined and SLA surfaces. Osteoblasts on these anodized surfaces displayed larger sizes, and the focal adhesion molecules (indicated by green fluorescence) were more abundant than those on machined and SLA surfaces. Co-localization of actin and vinculin at the cell borders, appearing orange, was the most prominent on anodized B surface. By the 3-hour time point, osteoblast cells had extended their cytoskeletons and transitioned from a round morphology to polygonal shapes. The highest number of cells were found on anodized A and B surfaces, followed by the SLA and machined surfaces. Co-localization of actin and vinculin was again most abundant on the anodized B surface.

From Fig 6, a statistically significant difference in the number of gingival fibroblast cells was observed between the anodized titanium A, B, and the machined surfaces compared to the SLA surface. The SLA surfaces contained the lowest number of fibroblast cells. Both the polystyrene coverslip and machined surface demonstrated increased cytoskeletal structure and fibroblast cell sizes. Although the anodized A and B surfaces had smaller cell sizes compared to the machined surface, the cytoskeletal distribution and polygonal form were still visible, in contrast to the circular shape and lack of cytoskeletal distribution observed on SLA surfaces.

Alkaline phosphatase

On day 3, there was no statistically significant difference in the alkaline phosphatase levels observed among various surfaces (Fig 7), although the anodized B surface exhibited the highest level among those tested. By day 7, alkaline phosphatase levels on both anodized A and B surfaces were statistically significantly higher than those measured on the machined surface (control). There was no statistically significant difference in alkaline phosphatase levels between the anodized A, anodized B, and the sandblasted/acid-etched surfaces. When comparing ALP activity over time, statistically significant differences were found at both days 3 and 7 for all titanium surface types.

Alizarin red

At day 14, alizarin red staining did not differ significantly between groups, though the anodized B showed a slightly increase (Fig 7). By day 21, anodized B exhibited the highest alizarin red levels, indicating greater mineralization compared to other surfaces. Alizarin red levels on both anodized A and B surfaces were statistically significantly higher than those on the machined surface (control). Additionally, the anodized B surface showed a statistically significantly higher alizarin red level compared to SLA surfaces but there was no significant difference between anodized A and B.

In time-dependent comparison, Alizarin Red staining demonstrated statistically significant differences at both days 14 and 21 among all examined titanium surface categories.

Discussion

Grade IV titanium was chosen for its higher purity, resulting in fewer allergic reactions and better corrosion resistance than Grade V due to the absence of aluminum and vanadium, which supports long-term biocompatibility [40,41]. Previous implant studies have also used Grade IV, making it suitable choice for this experiment [13,42,43]. The anodization process used for anodized A and B surfaces followed sandblasting and acid etching, resulting in a significant increase in surface roughness. This modification created a more pronounced valley and peak topography compared to SLA surfaces, as demonstrated in Fig 2. The roughness of the anodized A and B surfaces, ranging from 1.12 to 1.19 µm, aligns with previous findings that highlight this range as optimal for promoting osteoblast responses [12]. Research indicates that higher anodizing voltages produce rougher surfaces, characterized by numerous and larger micropores, increased crystallinity and a thicker oxide layer. These features collectively enhance osseointegration, as they provide a more suitable environment for bone cell attachment and growth [44–46]. Additionally, the anodized surfaces exhibited the lowest contact angle among different surface treatments tested, indicating superior wettability. The decreased contact angle is directly associated with increased hydrophilicity. Enhanced wettability facilitates better protein adsorption and cell adhesion, thereby promoting cell proliferation and differentiation [47].

This study employed human osteoblast and gingival fibroblast cells to closely replicate realistic cell activities. Previous studies consistently indicate a superior cell response on anodized titanium surfaces compared to machined surfaces [46,48,49]. However, the optimal voltage for promoting osteoblast adhesion, proliferation, and mineralization remains a point of contention in the literature. Some studies have reported that higher voltages yield better cell responses [50,51], whereas others have found no significant differences across various voltages [52,53]. Ercan et al. noted a peak in osteoblast density at 15 volts on day 5 [51]. In this study, we selected lower voltages (20–30 volts) for anodized A and higher voltages (60–70 volts) for anodized B to assess cell proliferation at day 1, 3, 7. Two anodization voltage ranges (20–30 V and 60–70 V) were selected to represent the typical lower and upper limits for forming titanium nanotubular. These ranges, commonly used in research and compatible with available equipment, have also been employed in commercial implant systems (Nobel Biocare and MegaGen) aligning with reported cellular responses [51,54]. The results demonstrated that both anodized A and B surfaces exhibited higher cell growth and cytocompatibility for osteogenesis compared to other titanium surfaces over a 7-day period. These findings align with several studies that have reported enhanced proliferation rates on anodized surfaces compared to machined surface [51,52]. At day 7, cells demonstrated greater adhesion and more extensive spreading on the titanium surface compared to days 1 and 3. Such enhanced attachment and morphology of osteoblasts are favorable for improving implant stability during the healing phase. This finding further supports the clinical concept of immediate loading implants, where rapid and stable cell adhesion to the implant surface is essential for early osseointegration. However, there was no significant difference in osteoblast cells response between the two anodized surfaces. No significant differences in cell adhesion, proliferation, or differentiation were observed between the two anodization voltages for most assays, except for the MTT assay on day 1. This may be explained by a plateau effect, where nanotube morphology and surface chemistry within the tested range were sufficiently similar to elicit comparable cellular responses. It appears that osteoblasts and fibroblasts respond primarily to the presence of nanotubular structures rather than subtle variations in tube diameter. These findings suggest that the general nanotubular architecture is more critical for biological outcomes than precise anodization voltage.

The initial reaction at the tissue-implant interface is cell adhesion. Cell expansion and adherence were detected through the interaction between vinculin (green fluorescence) and actin (red fluorescence), which appeared as an orange color in the immunofluorescent images (Fig 5). The force generated by the expanded actin stress fibers, resulting from the partial coupling of vinculin and talin to actin binding, is known as the “actomyosin force machinery” [55]. Vinculin plays an important role in regulate cell adhesion by connecting with the actin cytoskeleton and stabilizing focal adhesions. Differentiating osteoblasts are often characterized by prominent stress fiber bundles along their borders [56,57]. These findings suggest that vinculin is a key indicator of cellular response and interaction with titanium surfaces over time. Our results correspond with those of previous studies [50,58], showing that the bright orange and green fluorescence (indicating vinculin) on anodized A and B surfaces signifies enhanced cell adherence, extension, and differentiation. Immunofluorescent images demonstrated a superior cell response on the anodized titanium surfaces, which was consistent with SEM observations indicating improved osteoblast cell adhesion on anodized surfaces over time (1–3 hours). The hydrophilic nature of the anodized surfaces (characterized by low contact angles) facilitated better osteoblast proliferation, differentiation, and adhesion as evidenced by the presence of filopodia protrusions and microvilli extension.

Normally, fibroblast cells showed good adherence to the machine surface compared to the rough surface (SLA surface) [59,60]. However, a lot of research suggests a microrough surface to promote gingival fibroblast cell attachment [28,61,62]. In this study, the anodized A and B surfaces demonstrated superior cell attachment and spreading compared to the SLA surfaces. Notably, the anodized A surface exhibited the highest fibroblast cell attachment, followed by anodized B, machine surfaces, and the least cell attachment was SLA surfaces respectively. The modified anodized titanium surface (sandblasted before anodization) employed in this work showed results consistent with those of Xu et al., indicating that anodized titanium surfaces improve fibroblast cell responsiveness [63]. Furthermore, several studies have confirmed that fibroblast cells attach more effectively to anodized surfaces in comparison to the control surfaces [29,53,64,65]. Gulati et al. also showed that fibroblast cells exhibit longer filopodia spread and improved cell alignment on the anodized surfaces treated at higher voltages. Additionally, these studies noted and increase in integrinβ1 expression, a receptor that mediates cellular binding onto the implant surface [53]. Figs 4 and 5 of this study further corroborate these findings through extensive cell spreading and organized filopodia structures observed via immunofluorescent imaging. The enhanced fibroblast cell response on the anodized surfaces, illustrated by the growing filopodia, aligns with findings from prior studies. Specifically, vinculin-actin interaction observed in fibroblast cells appeared as orange color in Fig 5, which was most abundant at the borders of the anodized A surface, indicating the highest fibroblast cell adhesion. Vinculin, a crucial component of focal adhesions and cell mechanosensory mechanisms, plays a key role in mediating integrin functions that influence cell motility and behavior [66]. This detailed cellular interaction is critical for the formation of a strong extracellular matrix, thereby enhancing tissue integration and healing. The anodized titanium surface facilitates the gingival fibroblasts adhesion, deposition, and proliferation, which forms a robust bond potentially capable of preventing microbial invasion [6,53,67]. It is noteworthy that human gingival fibroblast response, as observed through immunofluorescence, is superior on the anodized surface compared to the SLA surface, a finding that aligns with the results of Lima et al [29]. The reason is the anodized surfaces had porosities, the TiO₂ oxide layer, and the highest wettability, which is favorable for fibroblast deposition, adhesion, and proliferation [29,68].

ALP activity was used to assess initial osteoblast differentiation, while alizarin red staining was employed to evaluate mineralized nodule formation on titanium surfaces. In this study, ALP activity and alizarin red staining were highest on anodized B surfaces, likely influenced by the applied voltage and the thickness of the oxide layer. The hydrophilicity of the anodized surfaces and the presence of the Ti-OH groups on the surface layer likely promoted this enhanced response [47]. When Ca²⁺ ions combine with OH⁻ groups, the surface gains a positive charge and reacts with phosphate groups to form amorphous calcium phosphate [69]. This observation is supported by Erdogan et al, who found that anodization at the higher voltages resulted in the best alizarin red measurements at 2 weeks, although no significant differences were noted between different anodized voltages [52]. Consequently, increasing anodization voltage appears to enhance oxide layer thickness, improving surface wettability, promoting cell proliferation, spreading, ALP activation, and calcium deposition. In this experiment, although anodized B surfaces exhibited lower contact angles compared to anodized A surfaces, no statistically significant differences were found between ALP activity and alizarin red staining of the anodized A and B surfaces (p < 0.05). Fig 3, 6 and 7 indicate that there is no notable difference in bone formation between machined titanium and SLA surfaces. Typically, fibrin from the blood clots helps osteoblasts adhere under normal physiological conditions. However, because our experiment did not included fibrin, osteoblast attachment to the SLA surface was reduced. On the other hand, the anodized surface’s higher hydrophilicity enhanced cell adhesion, as demonstrated by the immunofluorescent images leading to increase bone formation in line with previous studies [21].

Anodized titanium surfaces show enhanced biocompatibility due to a combination of physicochemical and mechanical properties which enhance surface wettability and promote cell spreading and cell proliferation. From the data present in this study, human osteoblast cells and fibroblast cells respond better on anodized surfaces than on SLA and machined. However, the mechanism of human osteoblast cell and fibroblast cell behavior on the anodized titanium at the molecular level needs further investigation.

Conclusion

The study found that modified anodized titanium surfaces substantially improve osteoblast and fibroblast attachment, proliferation, and differentiation when compared to machined or SLA surfaces. Enhanced surface roughness, porosity and hydrophilicity were identified as key contributors to superior cell responses and stronger tissue integration. These attributes render modified anodized surfaces particularly suitable for applications requiring robust bone and soft tissue integration, such as dental implants. It remains challenging to distinguish the individual effects of surface roughness and hydrophilicity due to the substrates employed; thus, the enhanced cellular response should be regards as a consequence of combined surface features. Notably, high-voltage anodization promotes osteoblast adhesion, whereas low-voltage anodization is more conducive to fibroblast adhesion. Future studies is warrented to optimizie anodization parameters for further improvement in cell responses and to investigate the broader clinical implications of these findings.

Supporting information

S1 Data. Cck.

(XLSX)

pone.0336639.s001.xlsx^{(12.4KB, xlsx)}

S2 Data. Data all manuscript.

(XLSX)

pone.0336639.s002.xlsx^{(33.6KB, xlsx)}

Acknowledgments

This study was supported by the Prosthodontic Department, Faculty of Dentistry, Chulalongkorn University. Center of Excellence for Dental Stem Cell Biology, Chulalongkorn University. Oral Biology Research Center, Chulalongkorn University. Dental Material R&D Center, Chulalongkorn University. We thank Dr. Prasit Pavasant for technical assistance.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Pandey C, Rokaya D, Bhattarai BP. Contemporary Concepts in Osseointegration of Dental Implants: A Review. Biomed Res Int. 2022;2022:6170452. doi: 10.1155/2022/6170452 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Buser D, Mericske-Stern R, Bernard JP, Behneke A, Behneke N, Hirt HP, et al. Long-term evaluation of non-submerged ITI implants. Part 1: 8-year life table analysis of a prospective multi-center study with 2359 implants. Clin Oral Implants Res. 1997;8(3):161–72. doi: 10.1034/j.1600-0501.1997.080302.x [DOI] [PubMed] [Google Scholar]
3.Javed F, Ahmed HB, Crespi R, Romanos GE. Role of primary stability for successful osseointegration of dental implants: Factors of influence and evaluation. Interv Med Appl Sci. 2013;5(4):162–7. doi: 10.1556/IMAS.5.2013.4.3 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Abrahamsson I, Berglundh T, Linder E, Lang NP, Lindhe J. Early bone formation adjacent to rough and turned endosseous implant surfaces. An experimental study in the dog. Clin Oral Implants Res. 2004;15(4):381–92. doi: 10.1111/j.1600-0501.2004.01082.x [DOI] [PubMed] [Google Scholar]
5.Rosa MB, Albrektsson T, Francischone CE, Schwartz Filho HO, Wennerberg A. The influence of surface treatment on the implant roughness pattern. J Appl Oral Sci. 2012;20(5):550–5. doi: 10.1590/s1678-77572012000500010 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Smeets R, Stadlinger B, Schwarz F, Beck-Broichsitter B, Jung O, Precht C, et al. Impact of Dental Implant Surface Modifications on Osseointegration. Biomed Res Int. 2016;2016:6285620. doi: 10.1155/2016/6285620 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Tarnow DP, Emtiaz S, Classi A. Immediate loading of threaded implants at stage 1 surgery in edentulous arches: ten consecutive case reports with 1- to 5-year data. Int J Oral Maxillofac Implants. 1997;12(3):319–24. [PubMed] [Google Scholar]
8.Wennerberg A, Albrektsson T. On implant surfaces: a review of current knowledge and opinions. Int J Oral Maxillofac Implants. 2010;25(1):63–74. [PubMed] [Google Scholar]
9.Le Guéhennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater. 2007;23(7):844–54. doi: 10.1016/j.dental.2006.06.025 [DOI] [PubMed] [Google Scholar]
10.Andrukhov O, Huber R, Shi B, Berner S, Rausch-Fan X, Moritz A, et al. Proliferation, behavior, and differentiation of osteoblasts on surfaces of different microroughness. Dent Mater. 2016;32(11):1374–84. doi: 10.1016/j.dental.2016.08.217 [DOI] [PubMed] [Google Scholar]
11.Feller L, Jadwat Y, Khammissa RAG, Meyerov R, Schechter I, Lemmer J. Cellular responses evoked by different surface characteristics of intraosseous titanium implants. Biomed Res Int. 2015;2015:171945. doi: 10.1155/2015/171945 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Wennerberg A, Albrektsson T. Suggested guidelines for the topographic evaluation of implant surfaces. Int J Oral Maxillofac Implants. 2000;15(3):331–44. [PubMed] [Google Scholar]
13.Zhu X, Chen J, Scheideler L, Reichl R, Geis-Gerstorfer J. Effects of topography and composition of titanium surface oxides on osteoblast responses. Biomaterials. 2004;25(18):4087–103. doi: 10.1016/j.biomaterials.2003.11.011 [DOI] [PubMed] [Google Scholar]
14.Yang Y, Oh N, Liu Y, Chen W, Oh S, Appleford M, et al. Enhancing osseointegration using surface-modified titanium implants. JOM. 2006;58(7):71–6. doi: 10.1007/s11837-006-0146-1 [DOI] [Google Scholar]
15.Sul Y-T, Byon E, Wennerberg A. Surface characteristics of electrochemically oxidized implants and acid-etched implants: surface chemistry, morphology, pore configurations, oxide thickness, crystal structure, and roughness. Int J Oral Maxillofac Implants. 2008;23(4):631–40. [PubMed] [Google Scholar]
16.Karambakhsh A, Afshar A, Ghahramani S, Malekinejad P. Pure Commercial Titanium Color Anodizing and Corrosion Resistance. J of Materi Eng and Perform. 2011;20(9):1690–6. doi: 10.1007/s11665-011-9860-0 [DOI] [Google Scholar]
17.Karambakhsh A, Afshar A, Malekinejad P. Corrosion Resistance and Color Properties of Anodized Ti-6Al-4V. J of Materi Eng and Perform. 2010;21(1):121–7. doi: 10.1007/s11665-010-9791-1 [DOI] [Google Scholar]
18.Wadhwani C, Brindis M, Kattadiyil MT, O’Brien R, Chung K-H. Colorizing titanium-6aluminum-4vanadium alloy using electrochemical anodization: Developing a color chart. J Prosthet Dent. 2018;119(1):26–8. doi: 10.1016/j.prosdent.2017.02.010 [DOI] [PubMed] [Google Scholar]
19.Charrière R, Lacaille G, Pedeferri MP, Faucheu J, Delafosse D. Characterization of the gonioapparent character of colored anodized titanium surfaces. Color Research & Application. 2014;40(5):483–90. doi: 10.1002/col.21911 [DOI] [Google Scholar]
20.Sharma A, McQuillan AJ, Sharma LA, Waddell JN, Shibata Y, Duncan WJ. Spark anodization of titanium-zirconium alloy: surface characterization and bioactivity assessment. J Mater Sci Mater Med. 2015;26(8):221. doi: 10.1007/s10856-015-5555-7 [DOI] [PubMed] [Google Scholar]
21.Traini T, Murmura G, Sinjari B, Perfetti G, Scarano A, D’Arcangelo C, et al. The Surface Anodization of Titanium Dental Implants Improves Blood Clot Formation Followed by Osseointegration. Coatings. 2018;8(7):252. doi: 10.3390/coatings8070252 [DOI] [Google Scholar]
22.Wennerberg A, Jimbo R, Stübinger S, Obrecht M, Dard M, Berner S. Nanostructures and hydrophilicity influence osseointegration: a biomechanical study in the rabbit tibia. Clin Oral Implants Res. 2014;25(9):1041–50. doi: 10.1111/clr.12213 [DOI] [PubMed] [Google Scholar]
23.Zitzmann NU, Berglundh T. Definition and prevalence of peri-implant diseases. J Clin Periodontol. 2008;35(8 Suppl):286–91. doi: 10.1111/j.1600-051X.2008.01274.x [DOI] [PubMed] [Google Scholar]
24.John V, Shin D, Marlow A, Hamada Y. Peri-Implant Bone Loss and Peri-Implantitis: A Report of Three Cases and Review of the Literature. Case Rep Dent. 2016;2016:2491714. doi: 10.1155/2016/2491714 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Corvino E, Pesce P, Mura R, Marcano E, Canullo L. Influence of Modified Titanium Abutment Surface on Peri-implant Soft Tissue Behavior: A Systematic Review of In Vitro Studies. Int J Oral Maxillofac Implants. 2020;35(3):503–19. doi: 10.11607/jomi.8110 [DOI] [PubMed] [Google Scholar]
26.Kim J-J, Lee J-H, Kim JC, Lee J-B, Yeo I-SL. Biological Responses to the Transitional Area of Dental Implants: Material- and Structure-Dependent Responses of Peri-Implant Tissue to Abutments. Materials (Basel). 2019;13(1):72. doi: 10.3390/ma13010072 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Schupbach P, Glauser R. The defense architecture of the human periimplant mucosa: a histological study. J Prosthet Dent. 2007;97(6 Suppl):S15-25. doi: 10.1016/S0022-3913(07)60004-3 [DOI] [PubMed] [Google Scholar]
28.Canullo L, Annunziata M, Pesce P, Tommasato G, Nastri L, Guida L. Influence of abutment material and modifications on peri-implant soft-tissue attachment: A systematic review and meta-analysis of histological animal studies. J Prosthet Dent. 2021;125(3):426–36. doi: 10.1016/j.prosdent.2020.01.025 [DOI] [PubMed] [Google Scholar]
29.Lima JHCD, Robbs PCM, Tude EMO, De Aza PN, Costa EMD, Scarano A, et al. Fibroblasts and osteoblasts behavior after contact with different titanium surfaces used as implant abutment: An in vitro experimental study. Heliyon. 2024;10(3):e25038. doi: 10.1016/j.heliyon.2024.e25038 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Genova T, Chinigò G, Munaron L, Rivolo P, Luganini A, Gribaudo G, et al. Bacterial and Cellular Response to Yellow-Shaded Surface Modifications for Dental Implant Abutments. Biomolecules. 2022;12(11):1718. doi: 10.3390/biom12111718 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Schmidlin PR, Müller P, Attin T, Wieland M, Hofer D, Guggenheim B. Polyspecies biofilm formation on implant surfaces with different surface characteristics. J Appl Oral Sci. 2013;21(1):48–55. doi: 10.1590/1678-7757201302312 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Crawford RJ, Webb HK, Truong VK, Hasan J, Ivanova EP. Surface topographical factors influencing bacterial attachment. Adv Colloid Interface Sci. 2012;179–182:142–9. doi: 10.1016/j.cis.2012.06.015 [DOI] [PubMed] [Google Scholar]
33.Berglundh T, Gotfredsen K, Zitzmann NU, Lang NP, Lindhe J. Spontaneous progression of ligature induced peri-implantitis at implants with different surface roughness: an experimental study in dogs. Clin Oral Implants Res. 2007;18(5):655–61. doi: 10.1111/j.1600-0501.2007.01397.x [DOI] [PubMed] [Google Scholar]
34.Del Curto B, Brunella MF, Giordano C, Pedeferri MP, Valtulina V, Visai L, et al. Decreased bacterial adhesion to surface-treated titanium. Int J Artif Organs. 2005;28(7):718–30. doi: 10.1177/039139880502800711 [DOI] [PubMed] [Google Scholar]
35.Caous JS, Lövenklev M, Fäldt J, Langton M. Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH. BMC Oral Health. 2013;13:4. doi: 10.1186/1472-6831-13-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Standardization IOf. ISO 5832-2:2018(en) Implants for surgery — Metallic materials — Part 2: Unalloyed titanium. 2018.
37.Schuster JM, Schvezov CE, Rosenberger MR. Influence of Experimental Variables on the Measure of Contact Angle in Metals Using the Sessile Drop Method. Procedia Materials Science. 2015;8:742–51. doi: 10.1016/j.mspro.2015.04.131 [DOI] [Google Scholar]
38.Choi HS, Kim JW, Cha Y-N, Kim C. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem. 2006;27(1):31–44. doi: 10.1080/15321810500403722 [DOI] [PubMed] [Google Scholar]
39.Rook GA, Steele J, Umar S, Dockrell HM. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods. 1985;82(1):161–7. doi: 10.1016/0022-1759(85)90235-2 [DOI] [PubMed] [Google Scholar]
40.Vora HD, Shanker Rajamure R, Dahotre SN, Ho Y-H, Banerjee R, Dahotre NB. Integrated experimental and theoretical approach for corrosion and wear evaluation of laser surface nitrided, Ti-6Al-4V biomaterial in physiological solution. J Mech Behav Biomed Mater. 2014;37:153–64. doi: 10.1016/j.jmbbm.2014.05.017 [DOI] [PubMed] [Google Scholar]
41.Koike M, Lockwood PE, Wataha JC, Okabe T. Initial cytotoxicity of novel titanium alloys. J Biomed Mater Res B Appl Biomater. 2007;83(2):327–31. doi: 10.1002/jbm.b.30799 [DOI] [PubMed] [Google Scholar]
42.Cordeiro JM, Barão VAR. Is there scientific evidence favoring the substitution of commercially pure titanium with titanium alloys for the manufacture of dental implants?. Mater Sci Eng C Mater Biol Appl. 2017;71:1201–15. doi: 10.1016/j.msec.2016.10.025 [DOI] [PubMed] [Google Scholar]
43.Jain S, Williamson RS, Janorkar AV, Griggs JA, Roach MD. Osteoblast response to nanostructured and phosphorus-enhanced titanium anodization surfaces. J Biomater Appl. 2019;34(3):419–30. doi: 10.1177/0885328219852741 [DOI] [PubMed] [Google Scholar]
44.Zhu X, Kim KH, Jeong Y. Anodic oxide films containing Ca and P of titanium biomaterial. Biomaterials. 2001;22(16):2199–206. doi: 10.1016/s0142-9612(00)00394-x [DOI] [PubMed] [Google Scholar]
45.Sul Y-T, Johansson CB, Jeong Y, Wennerberg A, Albrektsson T. Resonance frequency and removal torque analysis of implants with turned and anodized surface oxides. Clin Oral Implants Res. 2002;13(3):252–9. doi: 10.1034/j.1600-0501.2002.130304.x [DOI] [PubMed] [Google Scholar]
46.Kim M-H, Park K, Choi K-H, Kim S-H, Kim SE, Jeong C-M, et al. Cell adhesion and in vivo osseointegration of sandblasted/acid etched/anodized dental implants. Int J Mol Sci. 2015;16(5):10324–36. doi: 10.3390/ijms160510324 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, et al. High surface energy enhances cell response to titanium substrate microstructure. J Biomed Mater Res A. 2005;74(1):49–58. doi: 10.1002/jbm.a.30320 [DOI] [PubMed] [Google Scholar]
48.De Angelis E, Ravanetti F, Cacchioli A, Corradi A, Giordano C, Candiani G, et al. Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium treated by a novel multiphase anodic spark deposition process. J Biomed Mater Res B Appl Biomater. 2009;88(1):280–9. doi: 10.1002/jbm.b.31179 [DOI] [PubMed] [Google Scholar]
49.Sista S, Nouri A, Li Y, Wen C, Hodgson PD, Pande G. Cell biological responses of osteoblasts on anodized nanotubular surface of a titanium-zirconium alloy. J Biomed Mater Res A. 2013;101(12):3416–30. doi: 10.1002/jbm.a.34638 [DOI] [PubMed] [Google Scholar]
50.Voltrova B, Hybasek V, Blahnova V, Sepitka J, Lukasova V, Vocetkova K, et al. Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface. RSC Adv. 2019;9(20):11341–55. doi: 10.1039/c9ra00761j [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Ercan B, Webster TJ. Greater osteoblast proliferation on anodized nanotubular titanium upon electrical stimulation. Int J Nanomedicine. 2008;3(4):477–85. doi: 10.2147/ijn.s3780 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Erdogan YK, Ercan B. Anodized Nanostructured 316L Stainless Steel Enhances Osteoblast Functions and Exhibits Anti-Fouling Properties. ACS Biomater Sci Eng. 2023;9(2):693–704. doi: 10.1021/acsbiomaterials.2c01072 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Gulati K, Moon H-J, Kumar PTS, Han P, Ivanovski S. Anodized anisotropic titanium surfaces for enhanced guidance of gingival fibroblasts. Mater Sci Eng C Mater Biol Appl. 2020;112:110860. doi: 10.1016/j.msec.2020.110860 [DOI] [PubMed] [Google Scholar]
54.Michalska-Domańska M, Nyga P, Czerwiński M. Ethanol-based electrolyte for nanotubular anodic TiO2 formation. Corrosion Science. 2018;134:99–102. doi: 10.1016/j.corsci.2018.02.012 [DOI] [Google Scholar]
55.Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM. Differential transmission of actin motion within focal adhesions. Science. 2007;315(5808):111–5. doi: 10.1126/science.1135085 [DOI] [PubMed] [Google Scholar]
56.Rodríguez JP, González M, Ríos S, Cambiazo V. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem. 2004;93(4):721–31. doi: 10.1002/jcb.20234 [DOI] [PubMed] [Google Scholar]
57.Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci U S A. 2010;107(11):4872–7. doi: 10.1073/pnas.0903269107 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Das K, Bose S, Bandyopadhyay A. Surface modifications and cell-materials interactions with anodized Ti. Acta Biomater. 2007;3(4):573–85. doi: 10.1016/j.actbio.2006.12.003 [DOI] [PubMed] [Google Scholar]
59.Atsuta I, Ayukawa Y, Furuhashi A, Ogino Y, Moriyama Y, Tsukiyama Y, et al. In vivo and in vitro studies of epithelial cell behavior around titanium implants with machined and rough surfaces. Clin Implant Dent Relat Res. 2014;16(5):772–81. doi: 10.1111/cid.12043 [DOI] [PubMed] [Google Scholar]
60.An N, Rausch-fan X, Wieland M, Matejka M, Andrukhov O, Schedle A. Initial attachment, subsequent cell proliferation/viability and gene expression of epithelial cells related to attachment and wound healing in response to different titanium surfaces. Dent Mater. 2012;28(12):1207–14. doi: 10.1016/j.dental.2012.08.007 [DOI] [PubMed] [Google Scholar]
61.Teng F-Y, Ko C-L, Kuo H-N, Hu J-J, Lin J-H, Lou C-W, et al. A comparison of epithelial cells, fibroblasts, and osteoblasts in dental implant titanium topographies. Bioinorg Chem Appl. 2012;2012:687291. doi: 10.1155/2012/687291 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Grössner-Schreiber B, Herzog M, Hedderich J, Dück A, Hannig M, Griepentrog M. Focal adhesion contact formation by fibroblasts cultured on surface-modified dental implants: an in vitro study. Clin Oral Implants Res. 2006;17(6):736–45. doi: 10.1111/j.1600-0501.2006.01277.x [DOI] [PubMed] [Google Scholar]
63.Xu R, Hu X, Yu X, Wan S, Wu F, Ouyang J, et al. Micro-/nano-topography of selective laser melting titanium enhances adhesion and proliferation and regulates adhesion-related gene expressions of human gingival fibroblasts and human gingival epithelial cells. Int J Nanomedicine. 2018;13:5045–57. doi: 10.2147/IJN.S166661 [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Ma Q, Wang W, Chu PK, Mei S, Ji K, Jin L, et al. Concentration- and time-dependent response of human gingival fibroblasts to fibroblast growth factor 2 immobilized on titanium dental implants. Int J Nanomedicine. 2012;7:1965–76. doi: 10.2147/IJN.S29538 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Ferrà-Cañellas MDM, Llopis-Grimalt MA, Monjo M, Ramis JM. Tuning Nanopore Diameter of Titanium Surfaces to Improve Human Gingival Fibroblast Response. Int J Mol Sci. 2018;19(10):2881. doi: 10.3390/ijms19102881 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta. 2004;1692(2–3):103–19. doi: 10.1016/j.bbamcr.2004.04.007 [DOI] [PubMed] [Google Scholar]
67.Ivanoff C-J, Widmark G, Johansson C, Wennerberg A. Histologic evaluation of bone response to oxidized and turned titanium micro-implants in human jawbone. Int J Oral Maxillofac Implants. 2003;18(3):341–8. [PubMed] [Google Scholar]
68.Miura S, Takebe J. Biological behavior of fibroblast-like cells cultured on anodized-hydrothermally treated titanium with a nanotopographic surface structure. J Prosthodont Res. 2012;56(3):178–86. doi: 10.1016/j.jpor.2011.11.004 [DOI] [PubMed] [Google Scholar]
69.Brunette DM, Tengvall P, Textor M, Thomsen P. Titanium in medicine: material science, surface science, engineering, biological responses and medical applications. Springer. 2001. [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0336639.r001

Decision Letter 0

Tapash Rautray

27 Aug 2025

Dear Dr. Chengprapakorn,

Please submit your revised manuscript by Oct 11 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Tapash Ranjan Rautray

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include a separate caption for each figure in your manuscript.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Additional Editor Comments:

Dear Authors,

Thank you for submitting your manuscript to PLOS One. After thorough evaluation by Reviewers, it was found that your study addresses an important topic relating to the influence of titanium surface anodization on osteoblast and fibroblast behavior. The reviewers acknowledge the relevance of your work. However, both reviewers have raised substantive concerns that must be addressed before the manuscript can be considered for publication.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The study by the authors addresses a pertinent, yet unaddressed, question related to the effect of different anodizations of the Ti surface on the cellular response. Using an in vitro setup, the authors demonstrate that anodization, irrespective of its associated technique, improves cell viability, adhesion, and the regenerative response of osteoblasts and fibroblasts in comparison to a machined control. This work is of interest and may motivate future in vivo studies to probe the effects of different anodization protocols on osseointegration prior to any translation to patients.

That being said, the study has several concerns that the authors are invited to address.

1. The biological effect of titanium surface anodization in comparison to a machined titanium surface has been extensively studied in vitro and in vivo. In line with what the authors demonstrated, anodization triggers pro-regenerative signalling. Importantly, this results from coupled modulation of the inflammatory response. Inflammatory cells are the first cellular actors to encounter the titanium surface in tissues. However, the authors largely overlook this crucial aspect and instead focus on the regenerative cells.

The reviewer judges that it is critical for the current work to provide complementary experiments employing cells representative of the initial inflammatory phase, such as THP-1 cells. This would substantially aid the mechanistic understanding of the differential responses between the surfaces, beyond the current interpretation based solely on cell adhesion.

Please address by providing additional data.

2. Why did the authors choose Grade IV titanium, which is most frequently utilised in orthopaedics, while Grade V is mostly exclusive to the dental field? Such a choice does not support the translational value of the study. Please explain and consider adding a dedicated section on this point in the Discussion.

3. How do the authors justify the selected anodization voltages (30 and 70 V)? Please clearly explain the reasoning behind this in the Discussion.

4. While the authors showcase the improved regenerative response via ALP and Alizarin Red assays, confirmation at the gene level appears necessary to support the conclusions. Please complement with additional data.

5. Were the tissues used to obtain osteoblasts and fibroblasts all from healthy patients? Please provide further information on the patients from whom tissues were harvested for the fibroblast and osteoblast experiments.

6. Please complement the ALP assay and the Alizarin Red data with associated microscopy images that support the quantitative data.

7. How do the authors interpret the absence of differences in the biological effects between different surface anodization voltages? Please dedicate a section to this in the Discussion.

8. Please clarify whether the variance shown in all graphs is SEM or SD. This information should be incorporated in the Statistics section of the Methods and in each figure caption.

9. Table 1 does not fully and clearly disclose the intergroup statistics for the comparison of the surface characteristics. Instead, by showing a graph (for example, a bar graph), a reader would more easily note these differences. Please address.

10. While the authors describe the statistical comparison between the experimental groups, no time-dependent comparison is provided within each group. This would help highlight the kinetic changes of the different studied parameters. Please complement accordingly.

11. For transparency purposes, please add plots to the bar graphs.

Reviewer #2: The authors tested the effects of anodization on titanium substrates on osteoblast and fibroblast cell behaviors. The surface topography and surface chemistry of titanium discs were modified with sandblasting/acid etching and anodization. Primary human osteoblasts and fibroblasts were cultured and the authors measured cell response.

Specific Comments:

1. The authors state at the end of the Introduction that “Our objective is to investigate the effects of different anodization voltages on titanium surfaces, alongside machined and SLA surfaces, focusing on osteoblast and fibroblast cell responses, surface characteristics, and hydrophilicity” which suggests the manuscript may be focused on investigating anodization engineering conditions. The authors even write “However, the impact of anodization on soft tissue behavior, particularly at varying voltages, demands further exploration” and ”However, there is a lack of comprehensive studies examining cellular responses across different implant surfaces at varying anodization voltages”. Yet only two voltages were tested (20-30 V and 60-70 V). Is this a sufficient range to test to address gaps in the literature? In all the assays performed, the authors found no statistical difference between the two anodization voltages save for the MTT assay on day 1. The authors point out in the Discussion that anodization voltages did have statistically significant effects in some studies, but not here and offer no further discussion.

2. The Conclusion states “The increased surface roughness and porosity, along with enhanced hydrophilicity, contribute to better cell responses and stronger tissue integration”. The effects of surface roughness and hydrophilicity are not clearly demonstrated by the choice of substrates and the presented data. The substrates used in this study are: machined (control), sandblasted and acid etched (SLA), SLA + anodization at 20-30 V, and SLA + anodization at 60-70 V. Anodized surfaces have roughness, so what is the appropriate control surface to test the effects of anodization? Is there a synergistic effect of roughness and hydrophilicity? It is not clear how one can conclude that roughness or hydrophilicity independently have an effect on cellular responses given these choices of substrates.

For the effect of surface roughness, the comparison of the control to SLA is a valid comparison. However, the presented data and statistical analysis do not support the conclusion that roughness has an effect. Figures 3, 6, 7 shows no statistical difference between machined surfaces and SLA, save for cell adhesion for fibroblasts on hour 3.

3. Figure 3 shows results of the MTT assay after 1,3, and 7 days. The error bars are quite consistent across measurements, except on day 7. Any explanation? Was any outlier test performed?

4. Figure 4 shows SEM images of cells. The authors describe morphological changes of cells on different substrates. SEM preparation is harsh for cells (fixation, washes, drying) and morphology may not be representative of in vitro, much less in vivo conditions. How useful is SEM imaging for cell morphology characterization? The authors performed fluorescent imaging, which may be a better dataset to perform morphological analysis. Furthermore, morphology such as shape and size can be quantified. The authors should support their discussion of morphological changes with quantitative image analysis.

Minor comments: Scale bars on SEM and fluorescence images are difficult to read, please enlarge. Suggest labeling sample conditions in the figure instead of just in the figure caption to help the reader.

5. The manuscript mentions a pilot study in the Materials and Methods section. What are the details of this pilot study? These details may would be suitable for Supporting Materials.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Nov 19;20(11):e0336639. doi: 10.1371/journal.pone.0336639.r002

Author response to Decision Letter 1

15 Oct 2025

Letter to Reviewer 1

1. The biological effect of titanium surface anodization in comparison to a machined titanium surface has been extensively studied in vitro and in vivo. In line with what the authors demonstrated, anodization triggers pro- regenerative signalling. Importantly, this results from coupled modulation of the inflammatory response. Inflammatory cells are the first cellular actors to encounter the titanium surface in tissues. However, the authors largely overlook this crucial aspect and instead focus on the regenerative cells.

- We sincerely thank the reviewer for this insightful and valuable comment. We fully agree that inflammatory cells represent the first line of interaction with the titanium surface and play a critical role in orchestrating subsequent regenerative processes. We acknowledge that the present study focused primarily on regenerative cells, and that the absence of experiments involving inflammatory cells, such as THP-1 cells, is a limitation.

Unfortunately, as primary human-derived cells were employed in the present work, the limited availability of these cells precluded us from conducting further experiments with inflammatory cell models at this stage. We will certainly consider incorporating inflammatory cell models in our future studies to provide a more comprehensive mechanistic understanding of the biological responses to anodized versus machined titanium surfaces.

- The dedicated section has been added to the Discussion at page 16

3. How do the authors justify the selected anodization voltages (30 and 70 V)? Please clearly explain the reasoning behind this in the Discussion.

- Justification for the selected voltages was added to the Discussion at page 17

Two anodization voltage ranges (20–30 V and 60–70 V) were selected to represent the typical lower and upper limits for forming titanium nanotubular. These ranges, commonly used in research and compatible with available equipment, have also been emploted in commercial implant systems (Nobel Biocare and MegaGen) aligning with reported cellular responses[51, 54].

- We sincerely thank the reviewer for this important question. As primary human-derived cells were employed in the present work, the limited availability of these cells precluded us from conducting further experiments with inflammatory cell models at this stage. However, we sincerely appreciate the suggestion and will take it into consideration for future investigations.

- The dedicated section has been added to the Materials and Methods page 6

We sincerely thank the reviewer for this important question. All tissues used to isolate osteoblasts and fibroblasts were obtained from healthy patients undergoing third molar extraction or implant surgery. The donors had no history of systemic disease, smoking, or medication-usage that could affect bone or soft tissue healing. Ethical approval for the use of these tissues was obtained from the ethics board of Chulalongkorn University, and written informed consent was provided by all participants.

6. Please complement the ALP assay and the Alizarin Red data with associated microscopy images that support the quantitative data.

- We have incorporated the reviewer’s recommendation in providing additional data on titanium surfaces, specifically images from the Alizarin Red staining test.

7. How do the authors interpret the absence of differences in the biological effects between different surface anodization voltages? Please dedicate a section to this in the Discussion.

- The recommended section has been added to the Discussion at page 17

This may be explained by a plateau effect, where nanotube morphology and surface chemistry within the tested range were sufficiently similar to elicit comparable cellular responses. It appears that osteoblasts and fibroblasts respond primarily to the presence of nanotubular structures rather than subtle variations in tube diameter. These findings suggest that the general nanotubular architecture is more critical for biological outcomes than the precise anodization voltage.

8. Please clarify whether the variance shown in all graphs is SEM or SD. This information should be incorporated in the Statistics section of the Methods and in each figure caption.

- The information has been added to the Statistics section at page 8

For clarification, the data presented for all groups are expressed as standard deviation (SD).

- A bar graph has been included to facilitate clearer interpretation and improve the reader’s understanding of the results.

- The additional time-dependent comparison has been added at page 10, 12

We thank the reviewer for the comment. In the revised analysis, our MTT cell proliferation results showed that machined titanium exhibited statistically significant differences between days 1 and 3, days 3 and 7, and days 1 and 7. The SLA surface exhibited significant differences between days 3 and 7, days 1 and 7, with no significant difference between days 1 and 3. Anodized A demonstrated significant differences only between days 1 and 7, with no significance detected between days 1 and 3 or days 3 and 7. In contrast, Anodized B exhibited statistically significant differences across all comparisons (days 1 and 3, days 3 and 7, and days 1 and 7). Furthermore, ALP activity showed statistically significant differences at both days 3 and 7 across all titanium surface types, while Alizarin Red staining demonstrated statistically significant differences at both days 14 and 21 across all titanium surface types.

11. For transparency purposes, please add plots to the bar graphs.

- The recommended bar plot graphs were added to enhance clarity and facilitate easier interpretation of the results.

Letter to Reviewer 2

- Further discussion has been added to page 17

No significant differences in cell adhesion, proliferation, or differentiation were observed between the two anodization voltages for most assays, except for the MTT assay on day 1. This may be explained by a plateau effect, where nanotube morphology and surface chemistry within the tested range were sufficiently similar to elicit comparable cellular responses. It appears that osteoblasts and fibroblasts respond primarily to the presence of nanotubular structures rather than subtle variations in tube diameter. These findings suggest that the general nanotubular architecture is more critical for biological outcomes than precise anodization voltage.

- We thank the reviewer for this important methodological point. We agree that with the current substrate set (machined, SLA, SLA + anodization at 20–30 V, SLA + anodization at 60–70 V) it is not possible to attribute observed biological effects uniquely to either surface roughness or hydrophilicity because the anodization treatment alters both parameters simultaneously. Therefore, the present study can only support the conclusion that the combined surface modifications produced by anodization (increased roughness together with increased hydrophilicity) are associated with improved cell responses and mineralization; it cannot demonstrate independent effects of roughness or hydrophilicity. Complementary characterization and analyses should include AFM/profilometry (quantitative roughness metrics), XPS/EDS (surface chemistry), and statistical tests for interaction (e.g., two-way ANOVA) to determine whether effects are additive or synergistic. Because these additional controls were not included in the current manuscript, we have revised the Conclusion and Discussion to make this limitation explicit and to avoid implying independent causation. We now state that anodization-produced changes in roughness and hydrophilicity together correlate with enhanced cell responses, and we have added the above experimental approaches to the Future Work section as planned studies to disentangle independent and synergistic effects.

- The conclusion was changed at page 17

It remains challenging to distinguish the individual effects of surface roughness and hydrophilicity due to the substrates employed; thus, the enhanced cellular response should be regards as a consequence of combined surface features

- the explanation was added at page 17

Figure 3, 6 and 7 indicate that there is no notable difference in bone formation between machined titanium and SLA surfaces. Typically, fibrin from the blood clots helps osteoblasts adhere under normal physiological conditions. However, because our experiment did not included fibrin, osteoblast attachment to the SLA surface was reduced. On the other hand, the anodized surface’s higher hydrophilicity enhanced cell adhesion, as demonstrated by the immunofluorescent images leading to increase bone formation in line with previous studies [21].

3. Figure 3 shows results of the MTT assay after 1,3, and 7 days. The error bars are quite consistent across measurements, except on day 7. Any explanation? Was any outlier test performed?

- We thank the reviewer for this comment. The increased variability observed, particularly on day 7, is most likely attributable to biological differences in cell growth rates among primary human-derived cells from different donors, rather than to experimental error. All data points were carefully examined, and no outliers were identified.

Minor comments: Scale bars on SEM and fluorescence images are difficult to read, please enlarge. Suggest labeling

sample conditions in the figure instead of just in the figure caption to help the reader.

- SEM imaging was performed as the initial protocol, while fluorescence imaging was subsequently incorporated to evaluate cell adhesion on the titanium surface. We agree that fluorescence images provide a more robust dataset for morphological analysis and have accordingly followed the reviewer’s suggestion to utilize them. The scale bars and sample conditions have been clearly provided in the revised figures according to the reviewer’s recommendation.

5. The manuscript mentions a pilot study in the Materials and Methods section. What are the details of this pilot study? These details may would be suitable for Supporting Materials.

- We thank the reviewer for raising this important point. The pilot study mentioned in the Materials and Methods section was conducted to optimize the experimental conditions prior to the main study. Specifically, it was performed to validate cell seeding density, culture duration, and staining protocols on the titanium discs to ensure reproducibility and adequate cell attachment for subsequent assays. The relevant details have been previously published online in ThaiJo under the title “Characteristics and osteoblast cell responses of the anodized titanium and sandblasted and acid-etched titanium surfaces.”

Attachment

Submitted filename: Letter to Reviewer 51068.docx

pone.0336639.s003.docx^{(35.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0336639.r003

Decision Letter 1

Tapash Rautray

29 Oct 2025

The impact of anodization modification on titanium interaction with human osteoblasts and fibroblasts (in vitro study)

PONE-D-25-30704R1

Dear Dr. Chengprapakorn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tapash Ranjan Rautray

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have now complied with the comments of the Reviewers. So, it can be accepted in present form

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0336639.r004

Acceptance letter

Tapash Rautray

PONE-D-25-30704R1

PLOS ONE

Dear Dr. Chengprapakorn,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tapash Ranjan Rautray

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data. Cck.

(XLSX)

pone.0336639.s001.xlsx^{(12.4KB, xlsx)}

S2 Data. Data all manuscript.

(XLSX)

pone.0336639.s002.xlsx^{(33.6KB, xlsx)}

Attachment

Submitted filename: Letter to Reviewer 51068.docx

pone.0336639.s003.docx^{(35.4KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0336639.ref001] 1.Pandey C, Rokaya D, Bhattarai BP. Contemporary Concepts in Osseointegration of Dental Implants: A Review. Biomed Res Int. 2022;2022:6170452. doi: 10.1155/2022/6170452 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref002] 2.Buser D, Mericske-Stern R, Bernard JP, Behneke A, Behneke N, Hirt HP, et al. Long-term evaluation of non-submerged ITI implants. Part 1: 8-year life table analysis of a prospective multi-center study with 2359 implants. Clin Oral Implants Res. 1997;8(3):161–72. doi: 10.1034/j.1600-0501.1997.080302.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref003] 3.Javed F, Ahmed HB, Crespi R, Romanos GE. Role of primary stability for successful osseointegration of dental implants: Factors of influence and evaluation. Interv Med Appl Sci. 2013;5(4):162–7. doi: 10.1556/IMAS.5.2013.4.3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref004] 4.Abrahamsson I, Berglundh T, Linder E, Lang NP, Lindhe J. Early bone formation adjacent to rough and turned endosseous implant surfaces. An experimental study in the dog. Clin Oral Implants Res. 2004;15(4):381–92. doi: 10.1111/j.1600-0501.2004.01082.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref005] 5.Rosa MB, Albrektsson T, Francischone CE, Schwartz Filho HO, Wennerberg A. The influence of surface treatment on the implant roughness pattern. J Appl Oral Sci. 2012;20(5):550–5. doi: 10.1590/s1678-77572012000500010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref006] 6.Smeets R, Stadlinger B, Schwarz F, Beck-Broichsitter B, Jung O, Precht C, et al. Impact of Dental Implant Surface Modifications on Osseointegration. Biomed Res Int. 2016;2016:6285620. doi: 10.1155/2016/6285620 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref007] 7.Tarnow DP, Emtiaz S, Classi A. Immediate loading of threaded implants at stage 1 surgery in edentulous arches: ten consecutive case reports with 1- to 5-year data. Int J Oral Maxillofac Implants. 1997;12(3):319–24. [PubMed] [Google Scholar]

[pone.0336639.ref008] 8.Wennerberg A, Albrektsson T. On implant surfaces: a review of current knowledge and opinions. Int J Oral Maxillofac Implants. 2010;25(1):63–74. [PubMed] [Google Scholar]

[pone.0336639.ref009] 9.Le Guéhennec L, Soueidan A, Layrolle P, Amouriq Y. Surface treatments of titanium dental implants for rapid osseointegration. Dent Mater. 2007;23(7):844–54. doi: 10.1016/j.dental.2006.06.025 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref010] 10.Andrukhov O, Huber R, Shi B, Berner S, Rausch-Fan X, Moritz A, et al. Proliferation, behavior, and differentiation of osteoblasts on surfaces of different microroughness. Dent Mater. 2016;32(11):1374–84. doi: 10.1016/j.dental.2016.08.217 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref011] 11.Feller L, Jadwat Y, Khammissa RAG, Meyerov R, Schechter I, Lemmer J. Cellular responses evoked by different surface characteristics of intraosseous titanium implants. Biomed Res Int. 2015;2015:171945. doi: 10.1155/2015/171945 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref012] 12.Wennerberg A, Albrektsson T. Suggested guidelines for the topographic evaluation of implant surfaces. Int J Oral Maxillofac Implants. 2000;15(3):331–44. [PubMed] [Google Scholar]

[pone.0336639.ref013] 13.Zhu X, Chen J, Scheideler L, Reichl R, Geis-Gerstorfer J. Effects of topography and composition of titanium surface oxides on osteoblast responses. Biomaterials. 2004;25(18):4087–103. doi: 10.1016/j.biomaterials.2003.11.011 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref014] 14.Yang Y, Oh N, Liu Y, Chen W, Oh S, Appleford M, et al. Enhancing osseointegration using surface-modified titanium implants. JOM. 2006;58(7):71–6. doi: 10.1007/s11837-006-0146-1 [DOI] [Google Scholar]

[pone.0336639.ref015] 15.Sul Y-T, Byon E, Wennerberg A. Surface characteristics of electrochemically oxidized implants and acid-etched implants: surface chemistry, morphology, pore configurations, oxide thickness, crystal structure, and roughness. Int J Oral Maxillofac Implants. 2008;23(4):631–40. [PubMed] [Google Scholar]

[pone.0336639.ref016] 16.Karambakhsh A, Afshar A, Ghahramani S, Malekinejad P. Pure Commercial Titanium Color Anodizing and Corrosion Resistance. J of Materi Eng and Perform. 2011;20(9):1690–6. doi: 10.1007/s11665-011-9860-0 [DOI] [Google Scholar]

[pone.0336639.ref017] 17.Karambakhsh A, Afshar A, Malekinejad P. Corrosion Resistance and Color Properties of Anodized Ti-6Al-4V. J of Materi Eng and Perform. 2010;21(1):121–7. doi: 10.1007/s11665-010-9791-1 [DOI] [Google Scholar]

[pone.0336639.ref018] 18.Wadhwani C, Brindis M, Kattadiyil MT, O’Brien R, Chung K-H. Colorizing titanium-6aluminum-4vanadium alloy using electrochemical anodization: Developing a color chart. J Prosthet Dent. 2018;119(1):26–8. doi: 10.1016/j.prosdent.2017.02.010 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref019] 19.Charrière R, Lacaille G, Pedeferri MP, Faucheu J, Delafosse D. Characterization of the gonioapparent character of colored anodized titanium surfaces. Color Research & Application. 2014;40(5):483–90. doi: 10.1002/col.21911 [DOI] [Google Scholar]

[pone.0336639.ref020] 20.Sharma A, McQuillan AJ, Sharma LA, Waddell JN, Shibata Y, Duncan WJ. Spark anodization of titanium-zirconium alloy: surface characterization and bioactivity assessment. J Mater Sci Mater Med. 2015;26(8):221. doi: 10.1007/s10856-015-5555-7 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref021] 21.Traini T, Murmura G, Sinjari B, Perfetti G, Scarano A, D’Arcangelo C, et al. The Surface Anodization of Titanium Dental Implants Improves Blood Clot Formation Followed by Osseointegration. Coatings. 2018;8(7):252. doi: 10.3390/coatings8070252 [DOI] [Google Scholar]

[pone.0336639.ref022] 22.Wennerberg A, Jimbo R, Stübinger S, Obrecht M, Dard M, Berner S. Nanostructures and hydrophilicity influence osseointegration: a biomechanical study in the rabbit tibia. Clin Oral Implants Res. 2014;25(9):1041–50. doi: 10.1111/clr.12213 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref023] 23.Zitzmann NU, Berglundh T. Definition and prevalence of peri-implant diseases. J Clin Periodontol. 2008;35(8 Suppl):286–91. doi: 10.1111/j.1600-051X.2008.01274.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref024] 24.John V, Shin D, Marlow A, Hamada Y. Peri-Implant Bone Loss and Peri-Implantitis: A Report of Three Cases and Review of the Literature. Case Rep Dent. 2016;2016:2491714. doi: 10.1155/2016/2491714 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref025] 25.Corvino E, Pesce P, Mura R, Marcano E, Canullo L. Influence of Modified Titanium Abutment Surface on Peri-implant Soft Tissue Behavior: A Systematic Review of In Vitro Studies. Int J Oral Maxillofac Implants. 2020;35(3):503–19. doi: 10.11607/jomi.8110 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref026] 26.Kim J-J, Lee J-H, Kim JC, Lee J-B, Yeo I-SL. Biological Responses to the Transitional Area of Dental Implants: Material- and Structure-Dependent Responses of Peri-Implant Tissue to Abutments. Materials (Basel). 2019;13(1):72. doi: 10.3390/ma13010072 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref027] 27.Schupbach P, Glauser R. The defense architecture of the human periimplant mucosa: a histological study. J Prosthet Dent. 2007;97(6 Suppl):S15-25. doi: 10.1016/S0022-3913(07)60004-3 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref028] 28.Canullo L, Annunziata M, Pesce P, Tommasato G, Nastri L, Guida L. Influence of abutment material and modifications on peri-implant soft-tissue attachment: A systematic review and meta-analysis of histological animal studies. J Prosthet Dent. 2021;125(3):426–36. doi: 10.1016/j.prosdent.2020.01.025 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref029] 29.Lima JHCD, Robbs PCM, Tude EMO, De Aza PN, Costa EMD, Scarano A, et al. Fibroblasts and osteoblasts behavior after contact with different titanium surfaces used as implant abutment: An in vitro experimental study. Heliyon. 2024;10(3):e25038. doi: 10.1016/j.heliyon.2024.e25038 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref030] 30.Genova T, Chinigò G, Munaron L, Rivolo P, Luganini A, Gribaudo G, et al. Bacterial and Cellular Response to Yellow-Shaded Surface Modifications for Dental Implant Abutments. Biomolecules. 2022;12(11):1718. doi: 10.3390/biom12111718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref031] 31.Schmidlin PR, Müller P, Attin T, Wieland M, Hofer D, Guggenheim B. Polyspecies biofilm formation on implant surfaces with different surface characteristics. J Appl Oral Sci. 2013;21(1):48–55. doi: 10.1590/1678-7757201302312 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref032] 32.Crawford RJ, Webb HK, Truong VK, Hasan J, Ivanova EP. Surface topographical factors influencing bacterial attachment. Adv Colloid Interface Sci. 2012;179–182:142–9. doi: 10.1016/j.cis.2012.06.015 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref033] 33.Berglundh T, Gotfredsen K, Zitzmann NU, Lang NP, Lindhe J. Spontaneous progression of ligature induced peri-implantitis at implants with different surface roughness: an experimental study in dogs. Clin Oral Implants Res. 2007;18(5):655–61. doi: 10.1111/j.1600-0501.2007.01397.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref034] 34.Del Curto B, Brunella MF, Giordano C, Pedeferri MP, Valtulina V, Visai L, et al. Decreased bacterial adhesion to surface-treated titanium. Int J Artif Organs. 2005;28(7):718–30. doi: 10.1177/039139880502800711 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref035] 35.Caous JS, Lövenklev M, Fäldt J, Langton M. Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH. BMC Oral Health. 2013;13:4. doi: 10.1186/1472-6831-13-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref036] 36.Standardization IOf. ISO 5832-2:2018(en) Implants for surgery — Metallic materials — Part 2: Unalloyed titanium. 2018.

[pone.0336639.ref037] 37.Schuster JM, Schvezov CE, Rosenberger MR. Influence of Experimental Variables on the Measure of Contact Angle in Metals Using the Sessile Drop Method. Procedia Materials Science. 2015;8:742–51. doi: 10.1016/j.mspro.2015.04.131 [DOI] [Google Scholar]

[pone.0336639.ref038] 38.Choi HS, Kim JW, Cha Y-N, Kim C. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. J Immunoassay Immunochem. 2006;27(1):31–44. doi: 10.1080/15321810500403722 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref039] 39.Rook GA, Steele J, Umar S, Dockrell HM. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J Immunol Methods. 1985;82(1):161–7. doi: 10.1016/0022-1759(85)90235-2 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref040] 40.Vora HD, Shanker Rajamure R, Dahotre SN, Ho Y-H, Banerjee R, Dahotre NB. Integrated experimental and theoretical approach for corrosion and wear evaluation of laser surface nitrided, Ti-6Al-4V biomaterial in physiological solution. J Mech Behav Biomed Mater. 2014;37:153–64. doi: 10.1016/j.jmbbm.2014.05.017 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref041] 41.Koike M, Lockwood PE, Wataha JC, Okabe T. Initial cytotoxicity of novel titanium alloys. J Biomed Mater Res B Appl Biomater. 2007;83(2):327–31. doi: 10.1002/jbm.b.30799 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref042] 42.Cordeiro JM, Barão VAR. Is there scientific evidence favoring the substitution of commercially pure titanium with titanium alloys for the manufacture of dental implants?. Mater Sci Eng C Mater Biol Appl. 2017;71:1201–15. doi: 10.1016/j.msec.2016.10.025 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref043] 43.Jain S, Williamson RS, Janorkar AV, Griggs JA, Roach MD. Osteoblast response to nanostructured and phosphorus-enhanced titanium anodization surfaces. J Biomater Appl. 2019;34(3):419–30. doi: 10.1177/0885328219852741 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref044] 44.Zhu X, Kim KH, Jeong Y. Anodic oxide films containing Ca and P of titanium biomaterial. Biomaterials. 2001;22(16):2199–206. doi: 10.1016/s0142-9612(00)00394-x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref045] 45.Sul Y-T, Johansson CB, Jeong Y, Wennerberg A, Albrektsson T. Resonance frequency and removal torque analysis of implants with turned and anodized surface oxides. Clin Oral Implants Res. 2002;13(3):252–9. doi: 10.1034/j.1600-0501.2002.130304.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref046] 46.Kim M-H, Park K, Choi K-H, Kim S-H, Kim SE, Jeong C-M, et al. Cell adhesion and in vivo osseointegration of sandblasted/acid etched/anodized dental implants. Int J Mol Sci. 2015;16(5):10324–36. doi: 10.3390/ijms160510324 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref047] 47.Zhao G, Schwartz Z, Wieland M, Rupp F, Geis-Gerstorfer J, Cochran DL, et al. High surface energy enhances cell response to titanium substrate microstructure. J Biomed Mater Res A. 2005;74(1):49–58. doi: 10.1002/jbm.a.30320 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref048] 48.De Angelis E, Ravanetti F, Cacchioli A, Corradi A, Giordano C, Candiani G, et al. Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium treated by a novel multiphase anodic spark deposition process. J Biomed Mater Res B Appl Biomater. 2009;88(1):280–9. doi: 10.1002/jbm.b.31179 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref049] 49.Sista S, Nouri A, Li Y, Wen C, Hodgson PD, Pande G. Cell biological responses of osteoblasts on anodized nanotubular surface of a titanium-zirconium alloy. J Biomed Mater Res A. 2013;101(12):3416–30. doi: 10.1002/jbm.a.34638 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref050] 50.Voltrova B, Hybasek V, Blahnova V, Sepitka J, Lukasova V, Vocetkova K, et al. Different diameters of titanium dioxide nanotubes modulate Saos-2 osteoblast-like cell adhesion and osteogenic differentiation and nanomechanical properties of the surface. RSC Adv. 2019;9(20):11341–55. doi: 10.1039/c9ra00761j [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref051] 51.Ercan B, Webster TJ. Greater osteoblast proliferation on anodized nanotubular titanium upon electrical stimulation. Int J Nanomedicine. 2008;3(4):477–85. doi: 10.2147/ijn.s3780 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref052] 52.Erdogan YK, Ercan B. Anodized Nanostructured 316L Stainless Steel Enhances Osteoblast Functions and Exhibits Anti-Fouling Properties. ACS Biomater Sci Eng. 2023;9(2):693–704. doi: 10.1021/acsbiomaterials.2c01072 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref053] 53.Gulati K, Moon H-J, Kumar PTS, Han P, Ivanovski S. Anodized anisotropic titanium surfaces for enhanced guidance of gingival fibroblasts. Mater Sci Eng C Mater Biol Appl. 2020;112:110860. doi: 10.1016/j.msec.2020.110860 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref054] 54.Michalska-Domańska M, Nyga P, Czerwiński M. Ethanol-based electrolyte for nanotubular anodic TiO2 formation. Corrosion Science. 2018;134:99–102. doi: 10.1016/j.corsci.2018.02.012 [DOI] [Google Scholar]

[pone.0336639.ref055] 55.Hu K, Ji L, Applegate KT, Danuser G, Waterman-Storer CM. Differential transmission of actin motion within focal adhesions. Science. 2007;315(5808):111–5. doi: 10.1126/science.1135085 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref056] 56.Rodríguez JP, González M, Ríos S, Cambiazo V. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem. 2004;93(4):721–31. doi: 10.1002/jcb.20234 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref057] 57.Kilian KA, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci U S A. 2010;107(11):4872–7. doi: 10.1073/pnas.0903269107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref058] 58.Das K, Bose S, Bandyopadhyay A. Surface modifications and cell-materials interactions with anodized Ti. Acta Biomater. 2007;3(4):573–85. doi: 10.1016/j.actbio.2006.12.003 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref059] 59.Atsuta I, Ayukawa Y, Furuhashi A, Ogino Y, Moriyama Y, Tsukiyama Y, et al. In vivo and in vitro studies of epithelial cell behavior around titanium implants with machined and rough surfaces. Clin Implant Dent Relat Res. 2014;16(5):772–81. doi: 10.1111/cid.12043 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref060] 60.An N, Rausch-fan X, Wieland M, Matejka M, Andrukhov O, Schedle A. Initial attachment, subsequent cell proliferation/viability and gene expression of epithelial cells related to attachment and wound healing in response to different titanium surfaces. Dent Mater. 2012;28(12):1207–14. doi: 10.1016/j.dental.2012.08.007 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref061] 61.Teng F-Y, Ko C-L, Kuo H-N, Hu J-J, Lin J-H, Lou C-W, et al. A comparison of epithelial cells, fibroblasts, and osteoblasts in dental implant titanium topographies. Bioinorg Chem Appl. 2012;2012:687291. doi: 10.1155/2012/687291 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref062] 62.Grössner-Schreiber B, Herzog M, Hedderich J, Dück A, Hannig M, Griepentrog M. Focal adhesion contact formation by fibroblasts cultured on surface-modified dental implants: an in vitro study. Clin Oral Implants Res. 2006;17(6):736–45. doi: 10.1111/j.1600-0501.2006.01277.x [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref063] 63.Xu R, Hu X, Yu X, Wan S, Wu F, Ouyang J, et al. Micro-/nano-topography of selective laser melting titanium enhances adhesion and proliferation and regulates adhesion-related gene expressions of human gingival fibroblasts and human gingival epithelial cells. Int J Nanomedicine. 2018;13:5045–57. doi: 10.2147/IJN.S166661 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref064] 64.Ma Q, Wang W, Chu PK, Mei S, Ji K, Jin L, et al. Concentration- and time-dependent response of human gingival fibroblasts to fibroblast growth factor 2 immobilized on titanium dental implants. Int J Nanomedicine. 2012;7:1965–76. doi: 10.2147/IJN.S29538 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref065] 65.Ferrà-Cañellas MDM, Llopis-Grimalt MA, Monjo M, Ramis JM. Tuning Nanopore Diameter of Titanium Surfaces to Improve Human Gingival Fibroblast Response. Int J Mol Sci. 2018;19(10):2881. doi: 10.3390/ijms19102881 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336639.ref066] 66.Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta. 2004;1692(2–3):103–19. doi: 10.1016/j.bbamcr.2004.04.007 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref067] 67.Ivanoff C-J, Widmark G, Johansson C, Wennerberg A. Histologic evaluation of bone response to oxidized and turned titanium micro-implants in human jawbone. Int J Oral Maxillofac Implants. 2003;18(3):341–8. [PubMed] [Google Scholar]

[pone.0336639.ref068] 68.Miura S, Takebe J. Biological behavior of fibroblast-like cells cultured on anodized-hydrothermally treated titanium with a nanotopographic surface structure. J Prosthodont Res. 2012;56(3):178–86. doi: 10.1016/j.jpor.2011.11.004 [DOI] [PubMed] [Google Scholar]

[pone.0336639.ref069] 69.Brunette DM, Tengvall P, Textor M, Thomsen P. Titanium in medicine: material science, surface science, engineering, biological responses and medical applications. Springer. 2001. [Google Scholar]

PERMALINK

The impact of anodization modification on titanium interaction with human osteoblasts and fibroblasts (in vitro study)

Kamonmard Moungthong

Daneeya Na Nan

Pravej Serichetaphongse

Wareeratn Chengprapakorn

Roles

Abstract

Title

Purpose

Methods

Results

Conclusions

Introduction

Materials and methods

Surface characterization

Behavior of osteoblasts and fibroblasts on titanium discs

Immunofluorescent staining

Alkaline phosphatase (ALP) activity

Alizarin red S staining

Statistical analysis

Result

Surface characteristic analysis

Table 1. Surface roughness parameter measurement in Arithmetric Mean Roughness (Ra) and Root mean Square Roughness (Sa), different small letters (alphabet) indicated a significant difference between material types.

Fig 1. The surface roughness (Ra) and root mean square surface roughness (Sa) values of different titanium surface treatments.

Fig 3. The average measurement of the contact angle on various titanium surfaces.

Behavior of osteoblastic and fibroblast cells on titanium surface analysis

Cell cytotoxicity and proliferation

Fig 4. Osteoblast cell viability and proliferation assay after 1, 3, and 7 days.

Immunofluorescent staining

Fig 5. Immunofluorescent staining of osteoblast and gingival fibroblast cell for 1 and 3 hours.

Fig 6. Cell amount of osteoblast and fibroblast cells on each type of titanium surface at 1 hour and 3 hours.

Alkaline phosphatase

Fig 7. Alkaline phosphatase activity after 3, and 7 days (left), Alizarin Red test after 14, and 21 days (right), Signification level at p < 0.05 (*), p < 0.001 (**), p < 0.005 (***), p < 0.0001(****).

Alizarin red

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tapash Rautray

Roles

Author response to Decision Letter 1

Decision Letter 1

Tapash Rautray

Roles

Acceptance letter

Tapash Rautray

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 7. Alkaline phosphatase activity after 3, and 7 days (left), Alizarin Red test after 14, and 21 days (right), Signification level at p < 0.05 (), p < 0.001* (**), p < 0.005 (***), p < 0.0001(****).