Anodization and Its Role in Peri-Implant Tissue Adhesion: A Novel 3D Bioprinting Approach

Abstract

Background: Soft tissue stability around dental implant abutments is critical for maintaining a functional peri-implant seal. Yellow anodization is used to improve the aesthetic and surface characteristics of titanium abutments, yet its epithelial effects under more physiologically relevant 3D conditions remain insufficiently explored. Objective: To develop a 3D bioprinted in vitro peri-implant mucosa model and to compare epithelial cell responses on yellow anodized versus turned titanium abutment surfaces. Methods: Commercial Grade 5 (Ti6Al4V) titanium abutments were anodized and compared with turned controls. A collagen-based 3D bioprinted “collar-like” construct incorporating YD-38 epithelial cells was fabricated using a custom holder system to simulate peri-implant mucosal contact. Samples were cultured for 14 and 21 days. Cell distribution and morphology were assessed by optical microscopy and HE staining, while cytoskeletal organization was evaluated by TRITC-phalloidin/Hoechst staining and confocal microscopy. Quantitative fluorescence analysis was performed at 21 days. Results: Both surfaces supported epithelial coverage in the 3D environment. Anodized specimens showed more pronounced actin cytoskeletal organization and the presence of actin-rich, filamentous cellular extensions compared with turned controls. Quantitative image analysis demonstrated significantly higher TRITC-phalloidin signal intensity at 21 days on anodized samples (p < 0.001). Conclusions: Within the limitations of a 3D epithelial in vitro model using YD-38 cells, yellow anodization was associated with enhanced epithelial cytoskeletal organization compared with turned titanium. The presented 3D bioprinted platform may serve as a practical in vitro tool for screening abutment surface modifications relevant to peri-implant soft tissue integration.

1. Introduction

Dental implants—commonly manufactured from titanium or zirconia—play a central role in contemporary restorative dentistry. Titanium is widely used for its mechanical strength and excellent biocompatibility, while zirconia offers superior aesthetics, especially in the anterior region [1,2].

A typical implant system includes two primary components: the implant fixture, which undergoes osseointegration with the alveolar bone, and the abutment, which connects the fixture to the final prosthesis [3]. Osseointegration enables direct structural and functional contact between the bone and implant surface.

In contrast to natural teeth—supported by the periodontium, including gingiva, periodontal ligament (PDL), cementum, and alveolar bone—peri-implant tissues lack a PDL and its multidirectional collagen fiber orientation. This anatomical difference leads to reduced soft-tissue attachment and more direct force transmission to the bone, making implants more susceptible to microbial penetration and inflammatory conditions such as peri-implant mucositis and peri-implantitis [4].

The integrity of soft tissue attachment around implants plays a crucial role in minimizing peri-implant disease risk, forming a biological seal that is vital for healing. Fibroblasts and epithelial cells contribute to the formation and quality of this seal [5,6]. Abutments, which serve as the interface between the implant and prosthetic restoration, are typically made from titanium or ceramics. Titanium, while mechanically advantageous, may lead to peri-implant mucosal discoloration, with anodization offering an aesthetic improvement by reducing these issues [7]. Titanium-zirconium (TiZr) alloys have been shown to enhance fibroblast activity and biointegration, suggesting they may improve soft tissue integration and reduce implant failure rates [8].

Peri-implantitis, affecting around 22% of dental implants [9], underscores the need for preventive strategies, such as stable osseointegration and bacterial barriers. Surface modifications, like electrochemical treatments and coatings, have demonstrated antibacterial properties, although their long-term clinical efficacy remains uncertain [10]. Titanium anodization, an electrochemical process that forms a microporous oxide film, improves surface properties by removing contaminants, enhancing processability, and making it more cost-efficient for biomedical applications [11,12].

This study explored a 3D bioprinted in vitro approach combining titanium abutments with epithelial cells to better model peri-implant soft tissue interactions and to evaluate the effect of yellow anodization on epithelial organization. This approach aimed to stabilize the biological barrier around the implant, potentially enhancing long-term implant success.

Titanium, despite its mechanical benefits, may cause peri-implant mucosal discoloration due to its gray color and rarely allergic reactions. One of the simplest surface modifications is the anodization, which improves its aesthetics by reducing visibility [7]. Anodization is an electrochemical method often used in patients with thin gingival biotype. This process alters the gray color of the titanium abutment to a pink or yellow color [13], which is not so visible through the thin gingiva. Some studies examined the interactions between anodized surfaces and bone tissue, but the investigated yellow anodized surface was not tested in respect of peri-implant soft tissue integration [12,14,15,16,17]. In our previous work, we characterized turned and yellow anodized Grade 5 (Ti6Al4V) titanium disks using SEM/AFM and surface chemistry analyses. Yellow anodization produced a granular surface topography compared with the circular grooves of turned samples and increased nanoscale roughness. The anodized oxide layer showed a shift towards higher Ti(IV) content and improved hydrophilicity. These previously reported findings provided the rationale to test whether such surface features could also enhance epithelial organization in a 3D peri-implant-like in vitro setting [18].

Previous in vitro and in vivo studies have shown that anodized or nanostructured titanium surfaces can modulate gingival fibroblast behavior and collagen fiber orientation at the peri-implant interface [19]. More recent work has reported enhanced attachment and proliferation of oral keratinocytes and mesenchymal stromal cells on modified or anodized abutment surfaces compared with turned controls, suggesting a broader soft-tissue benefit of such surface treatments [20,21]. Other approaches have focused on periodontal ligament stem cell–based or hydroxyapatite/β-TCP composite systems to engineer ligament-like attachments around implants, further highlighting the potential of bioactive interfaces to improve soft-tissue integration [22]. However, these models primarily emphasize fibroblastic or PDL-related responses, and a standardized 3D in vitro system dedicated to epithelial organization around modified abutment surfaces is still lacking; the present study aims to address this gap.

While fibroblasts and epithelial cells both contribute to peri-implant soft tissue sealing, the present study focuses on epithelial responses in a 3D collagen-based model.

The aims of this study were to develop a 3D bioprinted in vitro peri-implant mucosa model using epithelial cells and to compare epithelial attachment and cytoskeletal organization on yellow anodized versus turned titanium abutment surfaces.

The null hypothesis was that yellow anodization would not alter epithelial coverage or cytoskeletal organization compared with turned titanium abutments in the 3D in vitro model.

2. Materials and Methods

2.1. Sample Preparation

Commercially available titanium (Ti) abutments (Grade 5, Ti6Al4V alloy; Cortex Dental Implants Industries Ltd., Shlomi, Israel) were selected for this study. The specimens were designed in accordance with the geometry of standard gingival formers, which served as control samples. Two distinct titanium (Ti) surfaces were compared in the study: a turned control group and an anodized test group.

The anodization procedure [18] followed the protocol established by Wieland Edelmetalle GmbH (Pforzheim, Germany). Briefly, titanium samples were first immersed in an acid pickling solution for 30 s to remove surface contaminants, rinsed with tap water, and subsequently anodized at 52 V and 62 Ah for 180 s using a titanium coloring electrolyte (Wieland Edelmetalle GmbH, Pforzheim, Germany). The process was powered by a regulated current source (Power Station PE 1028, Plating Electronic GmbH, Sexau, Germany). Following anodization, the samples were immersed in denatured alcohol for 25 s, rinsed, air-dried with compressed air, and subjected to ultrasonic cleaning in distilled water (two 60 s cycles) before final drying for 20 min. The resulting yellow anodized layer exhibited a uniform microporous morphology and enhanced hydrophilicity compared to turned controls, as confirmed by previous characterization studies [18].

2.2. Cell Culture

Gingival squamous cell carcinoma cell line YD-38 was procured from the European Collection of Authenticated Cell Cultures (ECACC). Cells were cryopreserved in liquid nitrogen. Thawing was achieved by rapidly immersing the frozen ampoule in a 37 °C water bath for 1–2 min. The contents of the ampoule were centrifuged at 1000 rpm for 5 min in RPMI (Roswell Park Memorial Institute 1640 medium, Biosera, Cholet, France) supplemented with 2 mM L-glutamine (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) 10% Fetal Bovine Serum (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillin-streptomycin solution (Biosera, Cholet, France) to remove the cryoprotectant agent DMSO (dimethyl sulfoxide, Sigma-Aldrich, Co., St. Louis, MO, USA). Harvested cells were pipetted into a 25 cm² flask containing 5 mL of medium. Following a 3-day incubation period, the culture reached confluence, and the cells were passaged into four new flasks at least three times before investigations.

From the confluent cultures spent media were aspirated and flasks were rinsed three times with PBS (Dulbecco’s Phosphate-Buffered Saline, Biosera, Cholet, France), and the cells were detached from the bottom of the flask by a 5 min trypsinization with Trypsin-EDTA 1× solution (Biosera, Cholet, France), and were centrifuged at 1000 RPM for 5 min. The centrifugation was followed by resuspending the cells in fresh media and harvested cells were divided into 2–4 equal parts at passages. Cultures were grown under standard conditions at 37 °C under a humidified atmosphere containing 5% CO₂.

2.3. Design and Fabrication of the 3D Bioprinted System

To support cell integration, custom 3D-printed plastic holders were designed with a tapered, perforated structure at the 3D Printing and Visualisation Centre, University of Pécs (Pécs, Hungary). These holders securely fit the abutments, and because of the holes, allow cell passage, optimizing tissue interaction (Figure 1). YD-38 cells were incorporated into the collagen bioink prior to printing to generate a cell-laden collar-like construct. The “doughnut-shape” ensured sufficient contact area and is responsible for the fixation of the cell-containing matrix. The final profile was 3D-printed using PLA (Polylactic Acid, Herz GmbH, Neuwied, Germany) threads for quality control.

A 3D printer (BioX Cellink, San Diego, CA, USA) was used to print the cell-containing matrix (Figure 2A) around laboratory analogs with a gingival former inside the holder in a biosafety cabinet (22G nozzle, 30 kPa pressure, 3 mm/s speed, pre-cooled syringes, cartridge, nozzles and Luer lock adaptors and printhead). The matrix was pH Neutral Collagen Bioink (Lifeink^® 200, Collagen Bioink, Advanced Biomatrix Inc., Carlsbad, CA, USA). The construct was then filled with RPMI medium (Figure 2B) to support cell viability and incubated for 2 and 3 weeks at 37 °C under a humidified atmosphere containing 5% CO₂, changing the media every 2–3 days.

2.4. Optical Microscopy and Histological Evaluation

After 14 and 21 days of incubation, control and anodized cell–matrix samples were embedded in Cryomatrix (Thermo Fisher Scientific, Waltham, MA, USA) after the removal of the titanium specimens and stored at −80 °C. The samples were sectioned (cell-collagen matrix) with Leica CM1860 UV cryostat and stained with Hematoxylin and Eosin (HE) (Sigma-Aldrich, Co., St. Louis, MO, USA) for histological analysis [23]. Samples were washed with PBS, (Biosera, Cholet, France), fixed with 4% paraformaldehyde (Affymetrix Inc, Santa Clara, CA, USA), and incubated with staining solutions. Visualization was conducted at 40× magnification using EVOS XL Core microscope (Thermo Fisher Scientific, Waltham, MA, USA) [24].

2.5. Fluorescence Microscopy and Cytoskeletal Analysis

Fluorescence staining was also performed, utilizing TRITC-phalloidin Sigma-Aldrich, Co., St. Louis, MO, USA for the actin cytoskeleton and Hoechst dye (Sigma-Aldrich, Co., St. Louis, MO, USA) for nuclei. For fluorescence imaging YD38 cells cultured in 3D were fixed with 4% paraformaldehyde at room temperature for 20 min. The samples were incubated in 0.2% Triton X-100 (AppliChem GmbH, Darmstadt, Germany) solution for 5 min at room temperature, followed by two washes with PBS. TRITC-phalloidin (1:400 dilution with PBS) staining was performed for 60 min in the dark. After two washes with PBS Hoechst (1:10,000 dilution in PBS) staining was performed for 5 min in the dark, followed by two washing steps. The samples were stored in 4 °C in the dark until imaging.

Fixed and stained cells were imaged with a Nikon C2+ confocal laser scanning microscope (Nikon, Tokyo, Japan) system equipped with violet-diode (405 nm), multiline argon (457–517 nm), and solid-state (543, 561 nm and 633 nm) lasers. A large overview image of each sample was taken first using a 10× dry objective using Nis-Elements C imaging software. Next, five 12-bit images were taken at 2048 × 2048 resolution in a cross-layout with a 20× objective. Post-acquisition image processing was performed using Fiji software 20250514-1117.

2.6. Quantitative Image Analysis and Statistics

Statistical analysis was performed with GraphPad Prism software 10.6.1. Three independent abutment samples per group were analyzed. For each sample, five images were acquired, and the mean TRITC-phalloidin signal of these images was calculated to obtain one value per sample. Group comparisons were performed using these sample-level means. Data are presented as mean ± SEM (n = 3 per group), in line with common practice for exploratory in vitro studies. The measurements were performed with Fiji software. As this was an exploratory in vitro study, no a priori power calculation was performed; the sample size (n = 3 per group) was chosen based on previous similar 3D in vitro experiments and technical feasibility.

3. Results

3.1. Optical Microscopic Evaluation of Epithelial Coverage and Morphology

Optical microscopy and (HE) staining confirmed epithelial cell adhesion and coverage on both surfaces after 14 and 21 days of incubation (Figure 3) [24]. At day 14, confluent cell layers were visible on both surface types, indicating initial epithelial establishment within the collagen-based hydrogel matrix.

By day 21, an evident increase in cell density and nuclear number was observed, particularly on the anodized samples. Cells exhibited elongated morphologies with aligned cytoplasmic extensions, suggesting increased cellular organization and higher cell density. In contrast, cells on the turned surfaces appeared less uniformly distributed, with fewer intercellular connections. These findings indicate that anodized titanium was associated with increased cell density and coverage over prolonged culture periods.

3.2. Fluorescence Microscopy and Cytoskeletal Organization

Fluorescence microscopy images show epithelial cells after 14 days of cultivation on both control and anodized substrates. The cytoskeleton is stained red with TRITC-phalloidin, while nuclei are counterstained with Hoechst dye. Filamentous extensions originating from the cells are indicated within the green squares. These filamentous structures were observed exclusively in the anodized group, whereas the non-anodized control group exhibited only intercellular contact formation.

The analysis of the fluorescent microscopic images highlights another positive outcome of the study. The first filaments can be observed in the red-stained cytoskeleton, labeled with TRITC-phalloidin, demonstrating the formation of filamentous, actin-rich cellular extensions (Figure 4). Since, as previously described, a collar-like profile of the cell-containing matrix was created, it can be assumed that these extensions are oriented towards the abutment surface. However, during the removal of the cell–matrix from the abutment, this profile was ruptured.

The turned (control) and the anodized samples were compared based on their fluorescent emission of TRITC-phalloidin dye, staining the cytoskeleton of the epithelial cells. Quantitative analysis was performed with GraphPad Prism and Fiji software. Both groups (3 control samples and 3 anodized samples) were incubated for 21 days. The software quantified the TRITC-phalloidin-positive fluorescence signal on the images using identical acquisition and analysis parameters for all samples. Afterwards the mean values were compared (Figure 5).

4. Discussion

The primary hypothesis was that anodized titanium surfaces would enhance peri-abutment soft-tissue integration by promoting epithelial cell attachment, actin cytoskeletal organization, and early filament formation compared with turned titanium. The findings support this hypothesis: anodized surfaces demonstrated superior cellular adhesion, increased cytoskeletal organization, and exclusive formation of filamentous extensions, which may indicate enhanced cell–surface interaction and cytoskeletal maturation.

The study further aimed to investigate the influence of anodized titanium on peri-implant soft-tissue behavior using a 3D-bioprinted in vitro model that closely recapitulates the biological interface between epithelial cells and abutment materials. The results suggest that electrochemical anodization enhances epithelial responses by promoting greater epithelial coverage and higher cell density, more organized cytoskeletal architecture, and increased filament formation relative to turned control surfaces.

These observations are consistent with previous reports showing that anodization modifies titanium’s surface chemistry and microtopography in ways that favor soft-tissue integration. The anodic oxidation process generates a uniform TiO2 layer with increased roughness and hydrophilicity—surface characteristics known to enhance protein adsorption and integrin-mediated cell adhesion. Importantly, these mechanistic interpretations are supported by our previous surface characterization of turned and yellow anodized Grade 5 (Ti6Al4V) titanium disks, which demonstrated increased nanoscale roughness and hydrophilicity after anodization. Although those measurements were not repeated on the current abutment geometry, the observed epithelial actin organization and actin-rich extensions in the 3D model are consistent with the previously reported surface trends. In the present study, the pronounced actin filament formation on anodized samples suggests more advanced cytoskeletal organization and cell–surface interaction, supporting the establishment of a stronger mechanical interface between cells and the implant surface.

A crucial finding was that filament formation occurred only in the anodized group, while the non-anodized control group exhibited merely intercellular contact formation. This suggests a potential advantage of surface anodization in promoting epithelial cell–surface interaction in this 3D model.

These findings are consistent with previous reports. Prior studies have shown enhanced viability of gingival fibroblasts on electrochemically anodized titanium [19], and have demonstrated improved adhesion of human keratinocytes and mesenchymal stem cells to anodized titanium surfaces [20]. While these studies included fibroblasts and other cell types, the present work focused exclusively on epithelial responses in a 3D collagen-based model.

In comparison to an in vivo study [21], which involved a 21-day incubation of a periodontal ligament stem cell and bone marrow mesenchymal cell mixture, the present study employed a 3D in vitro approach with observation periods of 14 and 21 days. Despite these methodological differences, both studies reported structured tissue organization; however, the present work focused on epithelial cytoskeletal responses in a simplified 3D model.

Although hydroxyapatite-coated systems have shown promising performance in other models, the present study focused on the effect of yellow anodization on epithelial behavior in a 3D in vitro setting [22].

Although this investigation did not involve comparative material testing, the findings contribute to the growing body of evidence supporting the use of anodization to improve cellular adhesion and epithelial organization on titanium surfaces. The study employs a limited sample size and an epithelial carcinoma cell line, which does not fully replicate primary gingival cell behavior. Additionally, the in vitro 3D model cannot entirely reproduce the biomechanical and immunological conditions of the oral environment. Future studies should incorporate primary cells and in vivo assessment. Future investigations could explore combinatory surface modifications—such as hydroxyapatite coatings—in the development of advanced biohybrid abutment systems. Moreover, quantitative analysis was restricted to actin-associated fluorescence intensity at a single time point (21 days), and no extracellular matrix markers or functional assays were included; therefore, the conclusions are limited to epithelial coverage and cytoskeletal organization.

5. Conclusions

This study established a 3D-printed in vitro model that effectively mimics the peri-implant soft-tissue environment and enables assessment of abutment biointegration. Anodized titanium surfaces promoted enhanced cytoskeletal organization and filament formation, suggesting a potentially more favorable epithelial interface that may be relevant for peri-implant soft tissue stability, particularly in thin gingival biotypes. Filament formation occurred exclusively on anodized surfaces.

This research established a 3D bioprinted in vitro model to evaluate epithelial interactions with titanium abutment surfaces. Within the limitations of an epithelial carcinoma cell line and a simplified 3D environment, yellow anodized titanium promoted more organized actin cytoskeletal architecture and the appearance of filamentous, actin-rich cellular extensions compared to turned controls. The model provides a practical in vitro platform for screening abutment surface modifications related to peri-implant soft tissue integration. Future studies should validate these findings using non-malignant primary gingival epithelial cells and more complex co-culture systems.