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Titanium (Ti) is an ideal material for dental implants due to its excellent properties. However, corrosion and mechanical wear lead to Ti ions and particles release, triggering inflammatory responses and bone resorption. To overcome these challenges, surface modification techniques are used, including micro-arc oxidation (MAO). MAO creates adherent, porous coatings on Ti implants with diverse chemical compositions. In this context, zirconia element stands out in its wear and corrosion properties associated with low friction and chemical stability. Therefore, we investigated the impact of adding zirconium oxide (ZrO2) to Ti surfaces through MAO, aiming for improved electrochemical and mechanical properties. Additionally, the antimicrobial and modulatory potentials, cytocompatibility, and proteomic profile of surfaces were investigated. Ti discs were divided into four groups: machined - control (cpTi), treated by MAO with 0.04 M KOH - control (KOH), and two experimental groups incorporating ZrO2 at concentrations of 0.04 M and 0.08 M, composing the KOH@Zr4 and KOH@Zr8 groups. KOH@Zr8 showed higher surface porosity and roughness, even distribution of zirconia, formation of crystalline phases like ZrTiO4, and hydrophilicity. ZrO2 groups showed better mechanical performance including higher hardness values, lower wear area and mass loss, and higher friction coefficient under tribological conditions. The formation of a more compact oxide layer was observed, which favors the electrochemical stability of ZrO2 surfaces. Besides not inducing greater biofilm formation, ZrO2 surfaces reduced the load of pathogenic bacteria evidenced by the DNA-DNA checkerboard analysis. ZrO2 surfaces were cytocompatible with pre-osteoblastic cells. The saliva proteomic profile, evaluated by liquid chromatography coupled with tandem mass spectrometry, was slightly changed by zirconia, with more proteins adsorbed. KOH@Zr8 group notably absorbed proteins crucial for implant biological responses, like albumin and fibronectin. Incorporating ZrO2 improved the mechanical and electrochemical behavior of Ti surfaces, as well as modulated biofilm composition and provided suitable biological responses.

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OBJECTIVE: Periodontal regeneration poses challenges due to the periodontium's complexity, relying on mesenchymal cells from the periodontal ligament (hPDLSCs) to regenerate hard tissues like bone and cementum. While some hPDLSCs have high regeneration potential (HOP-hPDLSCs), most are low potential (LOP-hPDLSCs). This study analyzed hPDLSCs from a single donor to minimize inter-individual variability and focus on key differences in differentiation potentials. DESIGN: This study used RNA-seq, genomic databases, and bioinformatics tools to explore signaling pathways (SPs), biological processes (BPs), and molecular functions (MFs) guiding HOP cells to mineralized matrix production. It also investigated limitations of LOP cells and strategies for enhancing their osteo/cementogenesis. RESULTS: In basal conditions, HOP exhibited a multifunctional gene network with higher expression of genes related to osteo/cementogenesis, cell differentiation, immune modulation, stress response, and hormonal regulation. In contrast, LOP focused on steroid hormone biosynthesis and nucleic acid maintenance. During osteo/cementogenic induction, HOP showed strong modulation of genes related to angiogenesis, cell division, mesenchymal differentiation, and extracellular matrix production. LOP demonstrated neural synaptic-related processes and preserved cellular cytoskeleton integrity. CCKR map signaling and G-protein receptor bindings gained significance during osteo/cementogenesis in HOP-hPDLSCs. Both HOP and LOP shared common BPs related to gastrointestinal and reproductive system development. CONCLUSION: The osteo/cementogenic differentiation of HOP cells may be regulated by CCKR signaling, G-protein bindings, and specific hormonal regulation. LOP cells seem committed to neural mechanisms. This study sheds light on hPDLSCs' complex characteristics, offering a deeper understanding of their differentiation potential for future periodontal regeneration research and therapies.

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Titanium (Ti) is widely utilized as an implant material; nonetheless, its integration with bone tissue faces limitations due to a patient's comorbidities. To address this challenge, we employed a strategic approach involving the growth of thin films by spin-coating and surface functionalization with etidronate (ETI), alendronate (ALE), and risedronate (RIS). Our methodology involved coating of Ti cp IV disks with thin films of TiO2, hydroxyapatite (HA), and their combinations (1:1 and 1:2 v/v), followed by surface functionalization with ETI, ALE, and RIS. Bisphosphonate-doped films were evaluated in terms of surface morphology and physical-chemical properties by techniques such as electron microscopy, confocal microscopy, and x-ray photoelectron spectroscopy. The antibacterial potential of bisphosphonates alone or functionalized onto the Ti surface was tested against Staphylococcus aureus biofilms. Primary human bone mesenchymal stem cells were used to determine in vitro cell metabolism and mineralization. Although RIS alone did not demonstrate any antibacterial effect as verified by minimum inhibitory concentration assay, when Ti surfaces were functionalized with RIS, partial inhibition of Staphylococcus aureus growth was noted, probably because of the physical-chemical surface properties. Furthermore, samples comprising TiO2/HA (1:1 and 1:2 v/v) showcased an enhancement in the metabolism of nondifferentiated cells and can potentially enhance the differentiation of osteoblastic precursors. All samples demonstrated cell viability higher than 80%. Addition of hydroxyapatite and presence of bisphosphonates increase the metabolic activity and the mineralization of human bone mesenchymal cells. While these findings hold promise, it is necessary to conduct further studies to evaluate the system's performance in vivo and ensure its long-term safety. This research marks a significant stride toward optimizing the efficacy of titanium implants through tailored surface modifications.

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AIM: The aim of the study was to evaluate several mechanical and chemical decontamination methods associated with a newly introduced biofilm matrix disruption strategy for biofilm cleaning and preservation of implant surface features. MATERIALS AND METHODS: Titanium (Ti) discs were obtained by additive manufacturing. Polymicrobial biofilm-covered Ti disc surfaces were decontaminated with mechanical [Ti curette, Teflon curette, Ti brush, water-air jet device, and Er:YAG laser] or chemical [iodopovidone (PVPI) 0.2% to disrupt the extracellular matrix, along with amoxicillin; minocycline; tetracycline; H2 O2 3%; chlorhexidine 0.2%; NaOCl 0.95%; hydrocarbon-oxo-borate-based antiseptic] protocols. The optimal in vitro mechanical/chemical protocol was then tested in combination using an in vivo biofilm model with intra-oral devices. RESULTS: Er:YAG laser treatment displayed optimum surface cleaning by biofilm removal with minimal deleterious damage to the surface, smaller Ti release, good corrosion stability, and improved fibroblast readhesion. NaOCl 0.95% was the most promising agent to reduce in vitro and in vivo biofilms and was even more effective when associated with PVPI 0.2% as a pre-treatment to disrupt the biofilm matrix. The combination of Er:YAG laser followed by PVPI 0.2% plus NaOCl 0.95% promoted efficient decontamination of rough Ti surfaces by disrupting the biofilm matrix and killing remnants of in vivo biofilms formed in the mouth (the only protocol to lead to ~99% biofilm eradication). CONCLUSION: Er:YAG laser + PVPI 0.2% + NaOCl 0.95% can be a reliable decontamination protocol for Ti surfaces, eliminating microbial biofilms without damaging the implant surface.

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Abutment components (i.e., fixtures associated with oral implants) are essentially made of titanium (Ti), which is continuously exposed to the hash oral environment, resulting in scratching. Thus, such components need to be protected, and surface treatments are viable methods for overcoming long-term damage. Diamond-like carbon (DLC), an excellent protective material, is an alternative surface-treatment material for Ti abutments. Here, we demonstrate that a silicon interlayer for DLC film growth and the pulsed-direct current plasma-enhanced chemical vapor deposition (DC-PECVD) method enables the deposition of an enhanced protective DLC film. As a result, the DLC film demonstrated a smooth topography with a compact surface. Furthermore, the DLC film enhanced the mechanical (load-displacement, hardness, and elastic modulus) and tribological properties of Ti as well as increased its corrosion resistance (16-fold), which surpassed that of a bare Ti substrate. The biofilm formed (Streptococcus sanguinis) after 24 h exhibited an equal bacterial load (∼7 Log colony-forming units) for both the groups (Ti and DLC). In addition, the DLC film exhibited good cytocompatibility, owing to its noncytotoxicity toward human gingival fibroblast cells. Therefore, DLC deposition via DC-PECVD can be considered to be a promising protective and cytocompatible alternative for developing implant abutments with enhanced mechanical, tribological, and electrochemical properties.

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Along with poor implant-bone integration, peri-implant diseases are the major causes of implant failure. Although such diseases are primarily triggered by biofilm accumulation, a complex inflammatory process in response to corrosive-related metallic ions/debris has also been recognized as a risk factor. In this regard, by boosting the titanium (Ti) surface with silane-based positive charges, cationic coatings have gained increasing attention due to their ability to kill pathogens and may be favorable for corrosion resistance. Nevertheless, the development of a cationic coating that combines such properties in addition to having a favorable topography for implant osseointegration is lacking. Because introducing hydroxyl (-OH) groups to Ti is essential to increase chemical bonds with silane, Ti pretreatment is of utmost importance to achieve such polarization. In this study, plasma electrolytic oxidation (PEO) was investigated as a new route to pretreat Ti with OH groups while providing favorable properties for implant application compared with traditional hydrothermal treatment (HT). To produce bactericidal and corrosion-resistant cationic coatings, after pretreatment with PEO or HT (Step 1), surface silanization was subsequently performed via immersion-based functionalization with 3-aminopropyltriethoxysilane (APTES) (Step 2). In the end, five groups were assessed: untreated Ti (Ti), HT, PEO, HT+APTES, and PEO+APTES. PEO created a porous surface with increased roughness and better mechanical and tribological properties compared with HT and Ti. The introduction of -OH groups by HT and PEO was confirmed by Fourier transform infrared spectroscopy and the increase in wettability producing superhydrophilic surfaces. After silanization, the surfaces were polarized to hydrophobic ones, and an increase in the amine functional group was observed by X-ray photoelectron spectroscopy, demonstrating a considerable amount of positive ions. Such protonation may explain the enhanced corrosion resistance and dead bacteria (Streptococcus aureus and Escherichia coli) found for PEO+APTES. All groups presented noncytotoxic properties with similar blood plasma protein adsorption capacity vs the Ti control. Our findings provide new insights into developing next-generation cationic coatings by suggesting that a tailorable porous and oxide coating produced by PEO has promise in designing enhanced cationic surfaces targeting biomedical and dental implant applications.

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BACKGROUND: Grade C, Stage 3-4 Periodontitis (Perio4C) is a rapidly destructive disease caused by an unequilibrated immune response that starts after the primary contact of the periodontopathogens with the gingival tissue. However, it is still unclear how this imbalanced response initiates and what is the role of the connective tissue cells in the progression of this disease. Thus, this study aims to assess the local immune response of Perio4C patients through the exposure of primary gingival fibroblast cells (GFs) with Aggregatibacter actinomycetemcomitans protein extract (AaPE) and the quantification of the inflammatory cytokines interleukin (IL)-4, IL-17, tumor necrosis factor (TNF)-α, IL-1ß, interferon (IFN)-γ, and IL-10 super-family members (IL-10, IL-19, and IL-24) secreted by them. METHODS: Gingival biopsies from nine periodontally health (PH) and eight Perio4C patients were harvested, and the primary culture of GFs was obtained. The cells were exposed to AaPE (5 and 20 µg/ml) and 12-myristate 13-phorbol acetate and ionomycin - calcium salt (PMA). The supernatant was collected after 1.5 and 3 h, and a cytokine panel was evaluated. RESULTS: Clustering analysis indicated dissimilar and stimuli-dependent cytokine production between Perio4C and PH subjects. Perio4C GFs presented lower production of IL-4, TNF-α, IFN-γ, IL-17, IL-10, IL-24, and IL-19, while IL-1ß levels were similar to the PH group, leading to a disruption in the pro-/anti-inflammatory cytokine ratio (p < 0.05). IL-1ß and IL-10 super-family were the most discriminative representants for PH and Perio4C, respectively. CONCLUSION: GFs from individuals with Perio4C tended to hypo-respond to stimulation with AaPE, producing lower concentrations of some pro- and anti-inflammatory molecules, trending to develop a pro-inflammatory extracellular environment.

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Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 µM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

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BACKGROUND AND OBJECTIVE: Previous studies have demonstrated an association between the IL10 promoter rs6667202 (C > A) single-nucleotide polymorphism (SNP) and grade C, stage 3 or 4 periodontitis (Perio4C) in the Brazilian population, where the altered A allele was detected more frequently in these patients. However, no functional analysis of this variation has yet been performed. Thus, the objective of this preliminary study was to evaluate the functionality of rs6667202 in gingival fibroblasts (GFs) of individuals with Perio4C and with periodontal health (PH) stimulated with Aggregatibacter actinomycetencomitans protein extract (AaPE). METHODS: Patients with PH and Perio4C were segregated according to their genotype (AA, AC, or CC), and a biopsy was performed to establish the culture of the GFs. After GFs exposure to AaPE at 5 µg/ml for 1.5 h, RNA was extracted to analyze IL10 expression by qPCR. Aliquots of the cell's supernatant were subjected to immunoenzymatic analysis (MAGpix) to detect interleukin-10 (IL-10). RESULTS: In PH, the genotypes AA and AC are related to less expression of IL10 (p = 0.027 and p < 0.0001) and less production of IL-10 (p = 0.002 and p = 0.001), when compared to CC. In Perio4C, there was no statistical difference between the genotypes (p > 0.05), although a lower IL-10 expression and release compared with PH CC was seen (p = 0.033 and p < 0.001). CONCLUSION: The rs6667202 SNP is functional in PH, as it decreases the expression and production of IL-10. In Perio4C, other factors may be masking its action by altering the IL-10's response.

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OBJECTIVES: Assessing the evidence and comparing the levels of cytokines in gingival crevicular fluid (GCF) of periodontal healthy sites of smokers and nonsmokers. MATERIALS AND METHODS: Seven databases were surveyed for observational studies up to April 8, 2021. Studies comparing cytokine levels on GCF in periodontally healthy sites of smokers vs. nonsmokers were included in the study. The risk of bias was evaluated using NIH (2014) tool. For meta-analyses, levels in GCF were analyzed, followed by evidence certainty assessment using the GRADE approach. RESULTS: Eighteen studies were included for qualitative evaluation, and eight were included in meta-analysis. Qualitatively, despite high heterogeneity and risk of bias observed among the studies, most of them presented no significant difference in the gingival crevicular cytokine fluid levels between groups. Regarding meta-analyses, interleukin-8 (IL-8) and superoxide dismutase (SOD) levels in GCF were analyzed. The significant difference was observed only in SOD levels, where heavy smokers had lower levels compared to nonsmokers (MD - 30.06 [- 40.17, - 19.96], p = 0.07, 95%CI), as well as light smokers had lower levels compared to nonsmokers (MD - 15.22 [- 16.05, - 14.39], p < 0.00001, 95%CI). CONCLUSION: No distinct GCF cytokine profiles were detected for smokers and non-smokers. However, despite the limitations observed in the included studies, lower levels of SOD were identified in smokers. CLINICAL RELEVANCE: Indicating a distinct GCF profile of cytokines in periodontal healthy smokers may help to understand the mechanism whereby smoking may affect the host response.

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Dental adhesives hydrolyze in the mouth. This study investigated the water sorption (SOR), solubility (SOL) and cytotoxicity (CYTO) of experimental adhesives containing nitrogen-doped titanium dioxide nanoparticles (N_TiO2). Specimens (n = 15/group [SOR, SOL]; n = 10/group [CYTO]) of unaltered Clearfil SE Protect (CSP), OptiBond Solo Plus (OSP), Adper Scotchbond (ASB) and experimental adhesives (OSP + 25% or 30% of N_TiO2) were fabricated, desiccated (37 °C) and tested for SOR and SOL according to ISO Specification 4049 (2009). CYTO specimens were UV-sterilized (8 J/cm2) and monomer extracted in growth medium (1, 3 or 7 days). Human pulp cells were isolated and seeded (0.5 × 104) for MTT assay. SOR and SOL data was analyzed using GLM and SNK (α = 0.05) and CYTO data was analyzed with Kruskal-Wallis and SNK tests (α = 0.05). SOR and SOL values ranged from 25.80 µg/mm3 (30% N_TiO2) to 28.01 µg/mm3 (OSP) and 23.88 µg/mm3 (30% N_TiO2) to 25.39 µg/mm3 (25% N_TiO2). CYTO results indicated that pulp cells exposed to experimental materials displayed comparable viabilities (p > 0.05) to those of OSP. Experimental materials displayed comparable SOR, SOL and CYTO values (p > 0.05) when compared to unaltered materials. N_TiO2 incorporation have not adversely impacted SOR, SOL and CYTO properties of unaltered adhesives.

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BACKGROUND: Promoting the directional attachment of gingiva to the dental implant leads to the formation of tight connective tissue which acts as a seal against the penetration of oral bacteria. Such a directional growth is mostly governed by the surface texture. MATERIAL AND METHODS: In this study, three different methods, mechanical structuring, chemical etching and laser treatment, have been explored for their applicability in promoting cellular attachment and alignment of human primary gingival fibroblasts (HGFIBs). RESULTS: The effectiveness of mechanical structuring was shown as a simple and a cost-effective method to create patterns to align HGIFIBs. CONCLUSION: Combining mechanical structuring with chemical etching enhanced both cellular attachment and the cellular alignment.

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