The effect of various concentrations of iodine potassium iodide on the antimicrobial properties of mineral trioxide aggregate – a pilot study

Abstract – Background: Mineral trioxide aggregate (MTA) is a multi‐purpose dental material with various uses in dentistry. Iodine potassium iodide (IKI) is the most commonly used iodine compound in endodontics. We aimed to assess the antimicrobial activity of tooth‐colored ProRoot MTA combined with IKI. Materials and methods: The antimicrobial activity of IKI was assessed at three concentrations (1%, 2%, and 4%) as the mixing agents combined with MTA against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. For each microorganism, three plates were inoculated with 100 μl of a microbial suspension (McFarland 0.5). Four wells were prepared in each plate. MTA (70 mg) was mixed with any of the three concentrations of IKI (25 μl) or sterile distilled water (25 μl) and placed in each well. The plates were incubated for 24 h at 37°C. Zones of inhibition (ZOI) were measured in millimeters by a blinded observer. Data were analyzed using analysis of variance and the Dunnett t‐test. Results: All MTA mixtures with water or IKI solutions showed inhibitory zones. The mean ZOI of each MTA/IKI mixture was not significantly different from MTA/water mixture (P > 0.05). MTA/1% IKI had smaller ZOI than MTA/water against E. coli, E. faecalis, and C. albicans. MTA/2% IKI showed larger ZOI only against P. aeruginosa. MTA/4% IKI showed larger ZOI against P. aeruginosa and E. coli (P < 0.05). Conclusions: Substitution of IKI solutions (1%, 2%, and 4%) for water did not significantly increase the antimicrobial activity of MTA.     

Antimicrobial action of four herbal plants over mixed-species biofilms of Candida albicans with four different microorganisms

This study aimed to evaluate the antimicrobial effect of four herbal plants glycolic extracts over mixed-species biofilm composed of Candida albicans (C. albicans) and another pathogenic bacterium as alternative therapy to be investigated. Four plants extract of Pfaffia paniculata roots; Hamamelis virginiana leaf, Stryphnodendron barbatiman tree bark and Gymnema sylvestre stem and leaves were tested over multi-species biofilm of C. albicans (ATCC 18804) and Streptococcus mutans (ATCC 35688), Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 4083) or Pseudomonas aeruginosa (ATCC 15442) for 5 min and 24 h and colony forming units per millilitre was calculated. The data were analysed using Kruskal–Wallis with Dunn's test (p ≤ 0.05). All tested extracts showed antimicrobial action over the mixed-species biofilms after 24 h. Some extracts eliminated totally the biofilms. The glycolic extract of P. paniculata, H. virginiana, S. barbatiman and G. sylvestre are effective over mixed-species biofilms and may be indicated as endodontic irrigant or intracanal medication.     

Characterization of L‐cysteine desulfhydrase from Prevotella intermedia

Introduction: Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an l ‐cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product. Methods: The lcs gene homologue cloned into pGEX6p‐1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from l ‐cysteine. Site‐directed mutagenesis was used to convert an amino acid of the Lcs molecule. Results: The purified lcs gene product catalysed the degradation of l ‐cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is l ‐cysteine desulfhydrase. The enzyme required pyridoxal 5′‐phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl₂. The K_m and V_max of the enzyme were 0.7 mm and 4.2 μmol/min/mg, respectively. Replacement of Tyr‐59, Tyr‐118, Asp‐198, and Lys‐233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin. Conclusion: These results show the enzymatic properties of l ‐cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from l ‐cysteine, is closely associated with the pathogenesis of P. intermedia.     

Evaluation of cytotoxicity,antimicrobial activity and physicochemical properties of a calcium aluminate-based endodontic material

A calcium aluminate-based endodontic material, EndoBinder, has been developed in order to reduce MTA negative characteristics, preserving its biological properties and clinical applications.

Objectives

The aim of this study was to evaluate the cytotoxicity, antimicrobial activity, pH, solubility and water sorption of EndoBinder and to compare them with those of white MTA (WMTA).

Material and Methods

Cytotoxicity was assessed through a multiparametric analysis employing 3T3 cells. Antimicrobial activity against Enterococcus faecalis (ATCC 29212), Staphylococcus aureus. (ATCC 25923) and Candida albicans (ATCC 10556) was determined by the agar diffusion method. pH was measured at periods of 3, 24, 72 and 168 hours. Solubility and water sorption evaluation were performed following ISO requirements. Data were statistically analyzed by ANOVA and Tukey`s test with a significance level of 5%.

Results

EndoBinder and WMTA were non-cytotoxic in all tested periods and with the different cell viability parameters. There was no statistical differences between both materials (P>.05). All tested materials were inhibitory by direct contact against all microbial strains tested. EndoBinder and WMTA presented alkaline pH in all tested times with higher values of pH for WMTA (P<.05). Both materials showed values complying with the solubility minimum requirements. However, EndoBinder showed lower solubility than WMTA (P<.05). No statistical differences were observed regarding water sorption (P>.05).

Conclusion

Under these experimental conditions, we concluded that the calcium aluminate-based endodontic material EndoBinder demonstrated suitable biological and physicochemical properties, so it can be suggested as a material of choice in root resorption, perforations and root-end filling.     

Development of a novel resin with antimicrobial properties for dental application

The adhesion of biofilm on dental prostheses is a prerequisite for the occurrence of oral diseases.

Objective

To assess the antimicrobial activity and the mechanical properties of an acrylic resin embedded with nanostructured silver vanadate (β-AgVO₃).

Material and Methods

The minimum inhibitory concentration (MIC) of β-AgVO₃ was studied in relation to the species Staphylococcus aureus ATCC 25923, Streptococcus mutans ATCC 25175, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231. The halo zone of inhibition method was performed in triplicate to determine the inhibitory effect of the modified self-curing acrylic resin Dencor Lay - Clássico^®. The surface hardness and compressive strength were examined. The specimens were prepared according to the percentage of β-AgVO₃ (0%-control, 0.5%, 1%, 2.5%, 5%, and 10%), with a sample size of 9x2 mm for surface hardness and antimicrobial activity tests, and 8x4 mm for the compression test. The values of the microbiologic analysis were compared and evaluated using the Kruskal-Wallis test (α=0.05); the mechanical analysis used the Shapiro-Wilk''s tests, Levene''s test, ANOVA (one-way), and Tukey''s test (α=0.05).

Results

The addition of 10% β-AgVO₃ promoted antimicrobial activity against all strains. The antimicrobial effect was observed at a minimum concentration of 1% for P. aeruginosa, 2.5% for S. aureus, 5% for C. albicans, and 10% for S. mutans. Surface hardness and compressive strength increased significantly with the addition of 0.5% β-AgVO₃ (p<0.05). Higher rates of the nanomaterial did not alter the mechanical properties of the resin in comparison with the control group (p>0.05).

Conclusions

The incorporation of β-AgVO₃ has the potential to promote antimicrobial activity in the acrylic resin. At reduced rates, it improves the mechanical properties, and, at higher rates, it does not promote changes in the control.     

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net‐like fibers comprising DNA and antimicrobial components such as histones, LL‐37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram‐negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA‐ and nucD‐encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg²⁺ and Ca²⁺ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.     

Bioactivity evaluation of calcium silicate‐based endodontic materials used for apexification

This study aims to compare the bioactivity of Biodentine, ProRoot MTA and NeoMTA Plus with regard to their element uptake (Ca, Si and Ca/P) by root canal dentine in a simulated apex (n = 30 each) and evaluate the correlation between the dentine fracture resistance (n = 30 each) and interfacial layer thickness. Specimens immersed in a corrected simulated body solution (c‐SBF) for 1, 30 and 90 days were used. In all test materials, the Ca and Si concentrations in the root dentine were found to be significantly higher, whereas the Ca/P and Si concentrations increased over time (P < 0.05). The dentine fracture resistance showed a difference at only day 30. The dentine fracture resistance of Biodentine and ProRoot MTA was positively correlated with the Si and Ca/P values, and the mean interfacial layer thickness of all specimens. A high biomineralisation capacity of ProRoot MTA and Biodentine, and their positive effects on the dentine fracture resistance during the first 30 days suggest that they may present more advantages than NeoMTA Plus in apexification treatment.     

Influence of ageing on self‐etch adhesives: one‐step vs. two‐step systems

The aim of this study was to evaluate microtensile bond strength (μTBS) to dentine, interfacial nanoleakage expression, and stability after ageing, of two‐step vs. one‐step self‐etch adhesives. Human molars were cut to expose middle/deep dentine, assigned to groups (n = 15), and treated with the following bonding systems: (i) Optibond XTR (a two‐step self‐etch adhesive; Kerr), (ii) Clearfil SE Bond (a two‐step self‐etch adhesive; Kuraray), (iii) Adper Easy Bond (a one‐step self‐etch adhesive; 3M ESPE), and (iv) Bond Force (a one‐step self‐etch adhesive; Tokuyama). Specimens were processed for μTBS testing after 24 h, 6 months, or 1 yr of storage in artificial saliva at 37°C. Nanoleakage expression was examined in similarly processed additional specimens. At baseline the μTBS results ranked in the following order: Adper Easy Bond = Optibond XTR ≥Clearfil SE = Bond Force, and interfacial nanoleakage analysis showed Clearfil SE Bond = Adper Easy Bond = Optibond XTR> Bond Force. After 1 yr of storage, Optibond XTR, Clearfil SE Bond, and Adper Easy Bond showed higher μTBS and lower interfacial nanoleakage expression compared with Bond Force. In conclusion, immediate bond strength, nanoleakage expression, and stability over time were not related to the number of steps of the bonding systems, but to their chemical formulations.     

Efficacy and retrievability of root canal filling using calcium silicate‐based and epoxy resin‐based root canal sealers with matched obturation techniques

This study aimed to compare the efficiency of root canal filling procedures and the retrievability of the filling material with various sealers. Forty‐three patients assigned to endodontic treatment with (i) continuous wave of condensation technique (CTW) with AH‐plus (ii) single‐cone technique (SCT) with EndoSeal MTA. The spent time, voids entrapping and postoperative symptoms were evaluated. To evaluate the retrievability, mandibular premolar (n = 60) were divided into four groups: AH‐plus/CTW, EndoSeal MTA/SCT, MTA Fillapex/SCT and EndoSequence BC Sealer/SCT. The time required removing the filled materials and remnant score were examined. EndoSeal MTA/SCT showed significantly shorter time of filling procedure. The number of void did not show significant differences between two techniques. No patients showed clinical signs during the follow‐up periods. There were no significant differences between group AH‐plus and EndoSeal MTA for remnant score. A certain calcium silicate‐based sealer with SCT may give similar clinical efficiencies as much as continuous‐wave technique using AH‐plus sealer.     

The effect of bleaching agents on the compressive strength of calcium silicate‐based materials

The present study aimed to evaluate the effect of sodium perborate on the compressive strength of calcium silicate‐based materials. ProRoot wMTA, MTA Plus, NeoMTA Plus and Biodentine discs with 5 mm thickness and 4 mm diameter were prepared. Thirty discs from each material were used for compressive strength testing and divided into two groups: control and bleaching (n = 15). The sodium perborate was mixed with 30% hydrogen peroxide in a creamy consistency and placed on the surface of the specimens. Specimens were tested in an Instron machine, and compressive strength values were recorded and compared. The data were analysed using one‐way anova and post hoc Tukey tests. Compressive strength of all tested materials significantly decreased after bleaching (P < 0.05). SEM examination revealed deterioration on materials’ surfaces after bleaching. Application of sodium perborate and hydrogen peroxide reduced the compressive strength of ProRoot wMTA, MTA Plus, NeoMTA Plus and Biodentine.     

Indirect caries‐preventive effect of silver diamine fluoride on adjacent dental substrate: A single‐section demineralization study

This study assessed the indirect effect of 38% silver diamine fluoride (SDF) on demineralization of adjacent untreated sound and pre‐demineralized enamel and dentine using a single‐section model for digital transverse microradiography (TMR‐D). Forty‐eight bovine dentine single sections were demineralized, stratified (n = 12) according to integrated mineral loss (ΔZ), and treated with SDF or deionized water (DIW). Each “treated dentine” section was attached between untreated sound and pre‐demineralized enamel or dentine and then subjected to demineralization. ΔZ and lesion depths (LD) of all specimens at baseline, 24 and 48 h demineralization, and after treatment of “treated dentine” were quantified using TMR‐D. Fluoride in the demineralization solution of SDF clusters was determined using an ion‐selective electrode. ΔZ and LD of sound and ΔZ of pre‐demineralized enamel adjacent to SDF‐treated dentine did not increase over time. All untreated dentine demineralized significantly; however, ΔZ of sound dentine adjacent to SDF‐treated specimen was still significantly lower than control. SDF‐treated dentine remineralized and released fluoride even after 48 h. Consistent with clinical findings, when applied only to demineralized teeth in this chemical model, 38% SDF completely inhibited demineralization in adjacent untreated sound enamel. Demineralization prevention was observed to a lesser extent in adjacent pre‐demineralized enamel but not in dentine.     

Effect of acid-etching procedure on selected physical properties of mineral trioxide aggregate

Aim To evaluate the effect of acid‐etch procedures on the compressive strength and surface microhardness of tooth‐coloured mineral trioxide aggregate (MTA). Methodology White ProRoot MTA (Dentsply Tulsa Dental) was mixed and packed into cylindrical tubes of 4 mm in diameter and 6 mm in height. Three groups, each of 15 specimens were subjected to an acid‐etch procedure either 4, 24 or 96 h after mixing. The compressive strength was measured and compared with unetched control groups. Differences between groups were analysed using the Kruskall–Wallis test. A further batch of cylindrical specimens of 6 mm in diameter and 12 mm in height were prepared for testing surface microhardness. Three groups of 15 specimens were subjected to the acid‐etch procedure at either 4, 24 or 96 h following mixing. Data were subjected to one‐way anova . Changes in the surface microstructure before and after the acid‐etch procedures were analysed using a scanning electron microscope (SEM). Results There was a general trend for the compressive strength and surface microhardness of specimens to increase with time. In terms of compressive strength, the increase was significant between 4 h and the other time periods for both experimental and control groups (P < 0.0001); however, there was no significant difference between 24 and 96 h. The increase in surface microhardness was significant between 4, 24 and 96 h (P < 0.0001). In addition, there was a significant difference between experimental and control groups at all time periods (P < 0.0001). SEM examination revealed morphological differences between the intact and the etched MTA surfaces. Conclusions Acid‐etch procedures affected the compressive strength and surface microhardness of ProRoot MTA. This indicates that it may be better to postpone restorative procedures for at least 96 h after mixing MTA. Etching created surface changes that might have the potential to enhance bonding of resinous materials.     

Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model

The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth‐associated biofilm. This in vitro study aimed to investigate changes in biofilm composition and structure, during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva‐containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species‐specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms.     

Antifungal Activity of Endosequence Root Repair Material and Mineral Trioxide Aggregate

Introduction

The purpose of this study was to investigate the antifungal activity of Endosequence Root Repair Material (ERRM; Brasseler USA, Savannah, GA) as compared with mineral trioxide aggregate (MTA) using Candida albicans.

Methods

All materials were packed into sterilized intravenous tubing to obtain standardized samples and allowed to set for 3 or 24 hours and then exposed to a suspension of C. albicans for incubations of 24 or 48 hours. To analyze the mechanisms of the material's antifungal activity, additional samples of each test material were prepared in the same manner and allowed to set for 24 hours; these were then incubated in a culture medium for 24 hours. The pH of each conditioned media was measured before transferring to wells containing C. albicans. The development of biofilm was analyzed after 24 and 48 hours with 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-([phenyl amino] carbonyl)-2H-tetrazolium hydroxide reduction assay.

Results

Materials in both experimental groups significantly limited biofilm formation at each interval (ie, 24 and 48 hours). After incubating for a 24-hour period in the presence of C. albicans, ERRM in both experimental groups showed a reduction in biofilm formation that was statistically significant in comparison with MTA. However, when set for 24 hours and incubated for 48 hours, gray MTA and white MTA showed a more substantial reduction in biofilm formation than comparable samples of ERRM. Cultured media conditioned with test materials showed statistically significant antifungal biofilm activity after 48 hours.

Conclusions

All materials tested have comparable antifungal biofilm activity. It appeared that changing the environment, such as the pH, contributed to this activity.     

Protease‐activated receptor 2 mediates interleukin‐8 and intercellular adhesion molecule‐1 expression in response to Aggregatibacter actinomycetemcomitans

Introduction:  We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. Methods:  Human gingival cells (Ca9‐22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease‐activated receptor 2 (PAR‐2). Results:  The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract‐induced expression of interleukin‐8 (IL‐8) and intercellular adhesion molecule‐1 (ICAM‐1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression was significantly decreased in PAR‐2/siRNA‐transfected cells. Conclusions:  A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression in gingival epithelial cells is mediated by PAR‐2.     

Root canal filling in primary molars without successors: Mineral trioxide aggregate versus gutta‐percha/AH‐Plus

The aim of this study was to compare the clinical and radiographical success of mineral trioxide aggregate (MTA) and gutta‐percha/AH‐Plus used as a root canal filling material in primary second molars without successors. A total of 16 patients (9 girls, 7 boys) aged 6–13 years (mean: 10.5) were selected and randomly distributed into the treatment groups. Children were recalled for clinical and radiographic examination at 6, 12, 18, 24 and 36 months. Differences in treatment outcomes were analysed using chi‐squared and Fisher's exact tests. Clinically, there was no significant difference in the success rates between the groups at the end of a 3‐year follow‐up period (MTA: 100%; Gutta‐percha/AH‐Plus: 70%) (P > 0.05). However, radiographically, there was a significant difference between the groups (MTA: 80%; gutta‐percha/AH‐Plus: 30%) (P < 0.05). The present study showed that MTA can be recommended for use in root canal treatment of primary molars without successors based on better radiographic success.     

Bond strength between dentine and a novel fast‐setting calcium silicate cement with fluoride

The aim of this study was to evaluate the dentine bond strength of a novel fast‐setting calcium silicate cement (Protooth) versus a calcium hydroxide‐based cement (Dycal), a calcium silicate cement (ProRoot MTA), and a glass ionomer cement (Ketac‐Molar). Mid‐root dentine slices of 1 mm thickness were obtained from human maxillary incisors. After enlarging the lumen of the canal to 1.3 mm, the cavities were randomly filled with test materials. Samples were immersed in physiological‐like solution. The push‐out bond strength was tested on days 1, 28, and 180 (n = 12). Failure types of bonding were determined using a stereomicroscope. We analysed the data using linear regression. Dycal and day 1 were considered as reference for cement type and assessment time, respectively. Protooth, Ketac‐Molar, and ProRoot MTA demonstrated higher push‐out bond strength than Dycal. The push‐out bond strength in the Protooth group increased on day 28 and 180. The bond strength of Ketac‐Molar was significantly reduced on day 28. Dycal showed a significant decrease in bond strength on day 180 compared with that on day 1 and 28. Mixed failure was the dominant failure type. Protooth bonding to dentine was increased with time, in contrast to that of ProRoot MTA, Dycal, and Ketac‐Molar, as a function of time.     

Long‐term evaluation (20 years) of the outcomes of coronally advanced flap in the treatment of single recession‐type defects

1 Background

Coronally advanced flap (CAF) has been considered as one of the most predictable and versatile root coverage procedures. Thus, the aims of this study are two‐fold: 1) to evaluate the long‐term outcomes following CAF in the treatment of gingival recession (GR); and 2) to explore the influence of several tooth/patient‐related factors on the stability of gingival margin at 5, 10, and 20 years after surgery.

2 Methods

Ninety‐four patients with 97 GR (73 Miller`s Class I and 24 Miller's Class III) were treated with CAF in a private practice between 1984 and 1996. Recession depth (RD), probing depth (PD), keratinized tissue (KT) width and patient/tooth‐associated variables were recorded for each GR at baseline, 1 year, 5 years, 10 years, 15 years and 20 years after surgery. Parametric, non‐parametric, and logistic regression statistics were used throughout the study

3 Results

A total of 72 patients with 72 GR were available for analysis at the 20‐year follow‐up examination (final patients’ dropout rate = 23.4%). Statistically significant improvements were found for RD in all evaluations (P < 0.05). Little more than a half (56%) of the sites treated with CAF did not display RD changes between the short‐term (i.e., 1 year) and long‐term (i.e., 20 years) examinations. Overall, mean root coverage (MRC) decreased from 68.59% to 56.11%. The achievement of complete root coverage (CRC) 1 year after treatment was associated to GR not presenting interdental tissue loss (P = 0.001), the root condition (i.e., lack of non‐carious cervical lesion [step] – P < 0.001), an attached KT band ≥ 2 mm (P = 0.019), and baseline RD (P = 0.020). GR recurrence seemed to be influenced by age, RD at 1‐year follow‐up, sites displaying an attached KT < 2 mm and interdental tissue loss.

4 Conclusions

The aging process, the condition of the interdental periodontal tissue, and the presence of an attached KT band < 2 mm seem to be negative factors influencing the stability of the gingival margin during the 20‐year observation period.