|Year : 2022 | Volume
| Issue : 3 | Page : 217-222
An In vitro evaluation of effect of implant abutment on human gingival epithelial keratinocytes
Paulami Bagchi1, Ahmed Ali Alfawzan2, Swaroopkumar M Magar3, Ritu Priya4, Anuraj Singh Kochhar5, Sneh Agrawal6, Faris Jaser AlMutairi7
1 Department of Prosthodontics Including Crown Bridge and Implantology, DY Patil Dental School, Lohegaon, Pune, Maharastra, India
2 Department of Preventive Dentistry, College of Dentistry in Ar Rass, Qassim University, Al-Qassim Region, 52571, Kingdom of Saudi Arabia
3 Department of Prosthodontics, Saraswati Dhanwantari Dental College and Research Institute, Perbhani, Maharashtra, India
4 Department of Conservative Dentistry and Endodontics, Sardar Patel Post Graduate Dental and Medical Sciences, Lucknow, Utter Pradesh, India
5 Department of Orthodontics, Former Consultant Max Hospital, Gugaon, India
6 Department of Periodontology, Bharati Vidyapeeth Deemed to be Dental College and Hospital, Navi Mumbai, Maharastra, India
7 Department of Maxillofacial Surgery and Diagnostic Sciences, College of Dentistry, Qassim University, Al Qassim Region, Kingdom of Saudi Arabia
|Date of Submission||19-Dec-2020|
|Date of Decision||04-Feb-2021|
|Date of Acceptance||02-Jun-2021|
|Date of Web Publication||26-Sep-2022|
Swaroopkumar M Magar
Department of Prosthodontics, Saraswati Dhanwantari Dental College and Research Institute, Perbhani, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Abutment surfaces are being designed to promote gingival soft tissue attachment and integration. This confirms implant survival for long term by forming a seal around the prosthetics. Objectives: This study was done to compare the biocompatibility of three implant abutments: titanium uncoated, Ti-nitride coated, and modified polyetheretherketone (PEEK) with human gingival keratinocytes. Materials and Methods: The titanium-uncoated, titanium-nitride-coated, and modified PEEK discs (13 mm × 3 mm) were fabricated and compared with uncoated polyester cell culture discs, which were used as controls. These three implant abutments were evaluated for biocompatibility with respect to human gingival keratinocytes for viability, morphology, proliferation, and migration by scanning electron microscopy imaging and scratch wound healing assays. Measurements of roughness show changes between the investigated surfaces. Results: Keratinocytes cultured on all examined surfaces indicated adhesion and attachment. An assay of cell viability showed no substantial variances among the groups. The modified PEEK surface showed greater cell proliferation and migration among the three abutment materials. Conclusion: All three abutment material surface types showed similar epithelial biological responses. However, modified PEEK material showed the highest biocompatibility.
| Abstract in French|| |
Contexte: Les surfaces des piliers sont conçues pour favoriser la fixation et l'intégration des tissus mous gingivaux. Cela confirme l'implantation survie à long terme en formant un joint autour des prothèses. Objectifs : Cette étude a été réalisée pour comparer la biocompatibilité de trois Piliers Implantaires : titane non revêtu, revêtu de nitrure de titane et polyétheréthercétone modifié (PEEK) avec des kératinocytes gingivaux humains. Matériaux et Méthodes: les disques PEEK non revêtus de titane, revêtus de nitrure de titane et modifiés (13 mm × 3 mm) ont été fabriqués et comparés à des disques de culture cellulaire en polyester non revêtus, qui ont été utilisés comme témoins. Ces trois piliers implantaires ont été évalués pour biocompatibilité vis-à-vis des kératinocytes gingivaux humains pour la viabilité, la morphologie, la prolifération et la migration par balayage électronique l'imagerie microscopique et les tests de cicatrisation des plaies. Les mesures de rugosité montrent des changements entre les surfaces étudiées. Résultats: Les kératinocytes cultivés sur toutes les surfaces examinées ont indiqué une adhérence et une fixation. Un test de viabilité cellulaire n'a montré aucune écarts entre les groupes. La surface PEEK modifiée a montré une plus grande prolifération et migration cellulaire parmi les trois matériaux de pilier. Conclusion: Les trois types de surfaces de matériaux de pilier ont montré des réponses biologiques épithéliales similaires. Cependant, le matériau PEEK modifié a montré la biocompatibilité la plus élevée.
Mots-clés: Biocompatibilité, implant, kératinocytes, prothèses, microscopie électronique à balayage
Keywords: Biocompatibility, implant, keratinocytes, prosthetics, scanning electron microscopy
|How to cite this article:|
Bagchi P, Alfawzan AA, Magar SM, Priya R, Kochhar AS, Agrawal S, AlMutairi FJ. An In vitro evaluation of effect of implant abutment on human gingival epithelial keratinocytes. Ann Afr Med 2022;21:217-22
|How to cite this URL:|
Bagchi P, Alfawzan AA, Magar SM, Priya R, Kochhar AS, Agrawal S, AlMutairi FJ. An In vitro evaluation of effect of implant abutment on human gingival epithelial keratinocytes. Ann Afr Med [serial online] 2022 [cited 2023 Mar 21];21:217-22. Available from: https://www.annalsafrmed.org/text.asp?2022/21/3/217/356822
| Introduction|| |
In last few years, the paradigm of prosthetic dentistry has been changed drastically. Dental implants are one of the most opted modalities for teeth replacement.
The surrounding oral flora and its interaction with the dental implants play an important role in the success or failure of implants. The long-term success of dental implants not only requires a sustained osseointegration but also the integrity of a biological seal formed by the peri-mucosal tissue surrounding the implants.
Microorganisms are colonized as soon as implant surfaces are exposed to the human oral cavity., The initial bacterial adhesion on implants is the first and essential step in the geneses of complex peri-implant biofilms, which, in turn, may result in peri-implantitis and loss of the supporting bone. Approximately one-third of patients with dental implants and one-fifth of all dental implants experienced peri-implantitis. Studies have demonstrated that surface properties of dental implants have a great impact on early bacterial adhesion.,
A variety of bacterial species can penetrate the transmucosal tissue surrounding the dental implant, whereby these tissues should ideally act as a barrier between the oral environment/bacterial infection and peri-implant bone. Improved marginal seal between implant and abutment is significant to avoid the bacterial microleakage that may inhibit peri-implant tissue health. Therefore, several researches concentrated on human gingival epithelial keratinocytes (HGEK) as important cells associated with soft tissue interaction, i.e. implant attachment and lining.,,,
Titanium is known for its excellent biocompatibility and outstanding mechanical properties. Localized inflammatory responses of the bone and periodontal tissues observed in peri-implantitis have also been attributed to titanium degradation and release of metallic ions due to wear, fatigue, and corrosion at the implant–abutment surface connection.,,
Another well-known type of implant abutment is made of a high-performance thermoplastic polymer called polyetheretherketone (PEEK), which is in the meanwhile considered as an alternative biomaterial to metallic implant, as PEEK neither releases ions, forms by-products, nor corrodes or degrades. It can be considered as a biologically satisfactory material for implant abutments although there is a lack of literature concerning the influence of modified PEEK abutments on oral gingival keratinocytes cell proliferation and wound healing response in comparison to titanium abutments.
Since the mid-1980s, titanium nitride (TiN) coatings have been in use for dental applications. The purpose of the titanium coating is to deliver better abrasion/wear resistance, surface hardness, lower friction, and corrosion resistance, as well as better useful collaboration with neighboring biological and material substrates.
Surface roughness and wettability are observed as the greatest important surface factors persuading microbial addition on implants. Increased surface roughness on implant surfaces correlates with faster and firmer integration into the surrounding bone.
The abutment surface adjustment and micro- and macroretention may increase the crowns' retentive strength of cement. The implant abutment profile may have implications on the establishment of surrounding epithelial attachment leading to necessary initial healing and physiologic gingival contour.
Therefore, the aim of the present in vitro study was to characterize the cell response of HGEK cultured on the abutment surface of three different abutment materials.
| Materials and Methods|| |
This in vitro study was done in the department of prosthodontics and oral implantology after obtaining approval from the institutional ethics committee. Informed consent was obtained from all participants.
The sample size was estimated with a confidence level of 95% that the real value was within ± 5% of the measured/surveyed value. It was done with formula; where z is the z score, ε is the margin of error, N is population size, and p̂ is the population proportion. Hence, the obtained sample size was 74 and it was rounded off to 80 total sample discs. These total 80 discs were divided with 20 each material of three test abutment and one control group and evaluated for biocompatibility. The titanium uncoated, TiN-coated, and modified PEEK discs (13 mm × 3 mm) were used as provided by manufacturer (Zimmer Biomet 3i, Palm Beach Gardens, Florida, USA) and compared with uncoated polyester cell culture discs, which were used as controls. The images of the different materials disc surfaces were obtained by using scanning electron microscopy (SEM, Shimadzu SS-550, Japan).
Surface disc images were recorded and SEM specifications were used under vacuum with 2 mm distance and with 5 kV secondary electron detector. Surface roughness was measured by perthometer (Ra in μm), values were determined using a 0.6 mm cutoff value and 4 mm as the length measurement. Roughness measurements were made at different points in two distinct discs for each material.
Using a contact angle meter, surface wettability of each surface type was inspected. One microliter drop of deionized water was measured in two different discs per material at three distinct points and after 3s of water droplets application. Image-Pro Plus version 6.0 (Media Cybernetics Inc., Bethesda, MD, USA) was used to photograph the contact angle with water. A contact angle >90° considered hydrophobic and closer to 0° was considered hydrophilic. Measurement of each material wettability was a result of average values obtained from the three discs per material.
For the cell culture, gingiva, a source of tissue was taken from the retromolar pad area of 60 patients undergoing oral surgeries. Gingiva was sectioned into minor pieces (2–3 mm2) and fibroblasts were secluded by sequential trypsin and collagenase digestion, after exclusion of epithelial sheet dispase. The HGEK were then cultured with complete Dulbecco's Modified Eagle Medium (cDMEM), consisting of DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine (EuroClone), and 1% penicillin/streptomycin (EuroClone).
Then, the cells were harvested with a trypsin treatment. HGEK were then detached from the culture plates and seeded onto the modified PEEK, TiN-coated, and uncoated Ti discs at a density of 2 × 104 cells/disk in 12-well plastic tissue culture plates. Then, 50 μL of cDMEM comprising the cell suspension was placed directly onto each disc's surface to permit adhesion of cell. The discs were then incubated for 2 h at 37°C and 5% CO2 before adding 1 mL of culture medium. The cells were cultured in cDMEM at 37°C with 5% CO2 for up to 21 days, replacing the medium every 7 days.
Cell attachment and morphology
Surfaces were analyzed using SEM to quantify cell attachment. Using SEM, cells on the discs were evaluated and images were documented at magnification of 10x and 100x.
Cell viability and proliferation
To examine the cell viability, HGEK cells were seeded on the different disc materials and tested with a tetrazolium bromide colorimetric assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT). This was done according to the method proposed by Denizot and Lang, with minor modifications. The culture medium was harvested, then the cells were incubated for 3 h at 37°C in 1 mL of 0.5 mg/mL MTT solution prepared in phosphate-buffered saline solution. After eliminating the MTT preparation with a pipette, 0.5 mL of 10% dimethyl sulfoxide in isopropanol was mixed for 30 min at 37°C. For each sample, optical density values at 570 nm were recorded in duplicate on 200 μL aliquots deposited in 27-well plates using a multilabel plate reader.
The next step was the evaluation of cell migration and motility by a scratch wound assay. HGEK were seeded onto Ti-uncoated, TiN-coated, and PEEK disc surfaces at a concentration of 2 × 104 cells/well in 27-well plates and cultivated under serum starvation and 10 μg/mL mitomycin C to block cell proliferation. The serum starvation was employed 16 h before the in vitro making of the “wound” scratch to avoid cell proliferation during cell migration. No alteration in cell counting and cell number was achieved at the start and end of the experiment. Then, each disc was artificially wounded by creating a scratch with a plastic pipette 10 μL tip on the cell monolayer. Cells were treated with 0.5 μM fluorescent dye Hoechst 33,342 for 30 min to create nuclei labeling. The dye solution was removed and cells were washed with fresh medium and cultured for additional 12 h and 24 h. Images of scratch wounds were captured using a fluorescence inverted microscope at 0, 12, and 24 h after wounding and wound width measurements were subtracted from wound width at time zero to obtain the net wound closure. Additional experiments were conducted for images recorded only at 0, 12, and 24 h. The average width of the scratches at diverse intervals was assessed after cell migration and distance between wound edges was measured. The wound closure areas were calculated after 24 h with ImageJ (Software 1.48q, Rayne Rasband, National Institutes of Health, USA). To avoid scratch width variation, “relative wound closure” area (RWC) was calculated (RWC [%] = wound closure area [pixel] ×100 [%]/× [pixel]). Experiments were repetitive at least three times.
The data/observations were tabulated and subjected to Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp) for analysis. The paired t-test was applied for comparison between the individual groups. Statistically significant differences in the Ra surface values were determined by ANOVA. P < 0.05 was measured as statistically important.
| Results|| |
A statistically insignificant difference was observed between the materials at 24 h (P = 0.1256) in cell viability analysis [Figure 1]. The cell viability was observed to be increased after 48 h of cell culture, though statistically nonsignificant difference was observed between the groups (P > 0.05). HGEK cell orientation on Ti-uncoated and TiN groups was parallel to disc microgroove directions after 14 days, whereas the cells present on PEEK discs were in random orientation and direction [Figure 2].
|Figure 2: Scanning electron microscopy images obtained from human gingival epithelial keratinocytes grown on the disks|
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Cell proliferation and migration
It was observed that PEEK materials stimulate the proliferation of HGEK cells significantly after 7 days of growth in all three experimental groups as compared to control. Similarly, Ti uncoated and TiN coated also showed the same effect, although statistically nonsignificant differences were observed between the groups [P > 0.04, [Figure 3]]. Regarding the cell migration, scratch distances and width closure were obtained by software comparison between images from 7 days up to 21 days. It was also observed that after 7 days, the HGEK cells migrated and covered almost 50% of the wound area for all abutment discs, whereas the TiN showed lower migration and uncoated Ti showed least as compared to PEEK material.
|Figure 3: Cell proliferation over disc material compared with control disc|
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Initial wound edges marked initial cell migration and were used to identify the decrease in wound width throughout the experiment. A statistically significant difference in migration distance was found for PEEK discs when compared to the control, while no difference in motility was found on Ti-uncoated and TiN-coated discs [* P < 0.01, [Figure 4]]. After 21 days, no significant change regarding cell migration was observed between the different materials.
In terms of the proliferation rate of fibroblasts seeded onto the disks at different times by means of the MTT test, the biocompatibility of the Ti disks was tested. The HGEK were able to adhere to the disks and proliferate. The cell proliferation increased over the course of time up to 14 days in culture. After 21 days from seeding, cell proliferation was found to be nearly the same level as that seen at 14 days, thus indicating that cell culture reached confluence. No substantial variances appeared among the types of disk surface. The cell colonization of the samples is apparent from the SEM images. After 14 days from seeding, HGFs were seen to be well spread and attached to the surface of the samples, creating an uninterrupted monolayer. Then, the cells reached confluence and stopped proliferating, at 21 days of culture.
Abutment material surface characterization
The mean surface roughness values Ra (mean ± standard deviation) of the uncoated Ti, TiN, and PEEK discs accounted for 0.069 ± 0.005, 0.081 ± 0.004, and 0.725 ± 0.011 μm, respectively [Table 1]. The Ra values of PEEK and uncoated Ti were found to be significantly lower than the TiN-coated specimens (P < 0.02 for both the comparisons). Histologically, the unevenness on the abutment discs encompassed parallel longitudinal grooves and ridges. The grooves were spaced on uncoated Ti, TiN, and PEEK which were approximately 1.22 ± 1.10, 3.14 ± 1.12, and 1.54 ± 1.40 μm apart, respectively, as can be seen in SEM micrographs. A statistically significant difference (P < 0.05) in the distances between grooves was found at a microlevels and submicron levels. However, under ×100k magnification, nanostructures were visible on PEEK surfaces. The wettability (median water contact angles) was found to be altered of each specimen between the materials. TiN coated showed significantly higher contact angles (96.2°) than the hydrophilic uncoated Ti and PEEK surface [71.2° and 91.3°, P < 0.02, [Table 1]]. Surface roughness of the discs was not altered after presentation of wettability in any of the parameters qualified.
|Table 1: Arithmetic average of surface roughness Ra (means and standard deviations [μm]) and wettability (means and standard deviations [°]) of the three tested abutment materials|
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| Discussion|| |
The long-term success of dental implants largely depends on the surrounding soft tissues, which should provide a protective seal against bacterial colonization to prevent peri-implant disease., An ideal transmucosal implant constituent should not only enable epithelial and connective tissue attachment and its preservation over time but also diminishes bacterial colonization and plaque gathering. Plaque formation is mostly influenced by parameters such as the surface roughness and chemical composition of abutments and the transmucosal portions of implants. Rougher surfaces facilitate bacterial colonization and rapid biofilm maturation, whereas smoother surfaces provide a less suitable substrate for bacteria.
Modified PEEK was used in this study and compared to titanium-uncoated and TiN-coated abutment materials, with regard to their influence on HGEK.
Gould et al. assessed the epithelial cells attachment mechanism to titanium and concluded that epithelial attachment to titanium was similar to the tooth surface; similarly, we found epithelial attachment in our study. Ziebart et al. evaluated the endothelial progenitor cell (EPC) and titanium implant surface interface and observed that modSLA surface encourages an undifferentiated phenotype of EPCs that has the capacity to secrete growth factors in pronounced amounts.
Ramenzoni et al. assessed the consequence of Modified PEEK Implant Abutments on Epithelial Keratinocytes Migration of Human Gingiva and they found that all the tested groups were biocompatible, while modified PEEK surface had better cell proliferation and biocompatibility in contrast to titanium and zirconia, this is similar to our findings.
The microorganism reservoirs found around dental implants and which contribute to implant failure seem to be interchangeable to the ones identified around natural teeth. For this reason, to diminish or elude bacterial adhesion and further surface colonization, it is important to consider the antimicrobial properties of the material substratum. Both surface roughness and wettability hold a direct effect on bacterial adhesion.
All tested three abutment material surface types showed similar epithelial biological responses. However, modified PEEK material showed the highest biocompatibility. It is in accordance to the study conducted by Ma. Thomas and Patrick also observed the same result in their studies. It also showed positive cell response and changes in wettability after surface treatment. It is in accordance with the study done by Schwitalla and Muller and Koutouzis et al. Roffel et al. evaluated the interactions among human oral gingival tissue and titanium abutments by histomorphometric and immunohistochemical examination and concluded that reconstructed human gingiva model showed human epithelial tissue attachment to dental abutments and expression of protein markers in soft tissue attachment and integration. Zhao et al. indicated that nanosized hydroxyapatite PEEK is a biocompative material.
A modified PEEK surface presented enhanced hydrophilicity with a water contact angle of 90.3°, which is due to increased cellular migration and proliferation. A dental abutment surface can positively affect its interaction with the adjacent tissues by improved biomaterial wettability. It greatly improves cell adhesion and migration in the current study which is in accordance with the study conducted by Ramenzoni et al.
Further research is required with different coating on abutments and the consideration of inflammation and bacterial endotoxins over the abutment surfaces. Future cell culture studies should be conducted to determine the effect of inflammatory conditions on the relationship between soft tissues and biomaterial interface.
| Conclusion|| |
PEEK surface was observed to have more biocompatibility and have a positive impact on viability, migration, and proliferation of HGEK as compared to uncoated and coated titanium abutment. TiN coated also showed a greater antibacterial activity. All the surfaces viewed and examined in this present study proved to be safe for use as implant abutments.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]