Boosting Titanium Surfaces with Positive Charges: Newly Developed Cationic Coating Combines Anticorrosive and Bactericidal Properties for Implant Application.

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.

Immune response characterization of primary gingival fibroblasts from Grade C periodontitis patients.

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.

Osteogenic Commitment of Human Periodontal Ligament Cells Is Predetermined by Methylation, Chromatin Accessibility and Expression of Key Transcription Factors.

Periodontal ligament stem cells (PDLCs) can be used as a valuable source in cell therapies to regenerate bone tissue. However, the potential therapeutic outcomes are unpredictable due to PDLCs' heterogeneity regarding the capacity for osteoblast differentiation and mineral nodules production. Here, we identify epigenetic (DNA (hydroxy)methylation), chromatin (ATAC-seq) and transcriptional (RNA-seq) differences between PDLCs presenting with low (l) and high (h) osteogenic potential. The primary cell populations were investigated at basal state (cultured in DMEM) and after 10 days of osteogenic stimulation (OM). At a basal state, the expression of transcription factors (TFs) and the presence of gene regulatory regions related to osteogenesis were detected in h-PDLCs in contrast to neuronal differentiation prevalent in l-PDLCs. These differences were also observed under stimulated conditions, with genes and biological processes associated with osteoblast phenotype activated more in h-PDLCs. Importantly, even after the induction, l-PDLCs showed hypermethylation and low expression of genes related to bone development. Furthermore, the analysis of TFs motifs combined with TFs expression suggested the relevance of SP1, SP7 and DLX4 regulation in h-PDLCs, while motifs for SIX and OLIG2 TFs were uniquely enriched in l-PDLCs. Additional analysis including a second l-PDLC population indicated that the high expression of OCT4, SIX3 and PPARG TFs could be predictive of low osteogenic commitment. In summary, several biological processes related to osteoblast commitment were activated in h-PDLCs from the onset, while l-PDLCs showed delay in the activation of the osteoblastic program, restricted by the persistent methylation of gene related to bone development. These processes are pre-determined by distinguishable epigenetic and transcriptional patterns, the recognition of which could help in selection of PDLCs with pre-osteoblastic phenotype.

IL10 promoter rs6667202 polymorphism is functional in health but not in grade c periodontitis patients: A pilot study.

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.

Cementocyte alterations associated with experimentally induced cellular cementum apposition in Hyp mice.

BACKGROUND: Cellular cementum, a mineralized tissue covering apical tooth roots, grows by apposition to maintain the tooth in its occlusal position. We hypothesized that resident cementocytes would show morphological changes in response to cementum apposition, possibly implicating a role in cementum biology. METHODS: Mandibular first molars were induced to super-erupt (EIA) by extraction of maxillary molars, promoting rapid new cementum formation. Tissue and cell responses were analyzed at 6 and/or 21 days post-procedure (dpp). RESULTS: High-resolution micro-computed tomography (micro-CT) and confocal laser scanning microscopy showed increased cellular cementum by 21 dpp. Transmission electron microscopy (TEM) revealed that cementocytes under EIA were 50% larger than control cells, supported by larger pore sizes detected by micro-CT. Cementocytes under EIA displayed ultrastructural changes consistent with increased activity, including increased cytoplasm and nuclear size. We applied EIA to Hyp mutant mice, where cementocytes have perilacunar hypomineralization defects, to test cell and tissue responses in an altered mechanoresponsive milieu. Hyp and WT molars displayed similar super-eruption, with Hyp molars exhibiting 28% increased cellular cementum area versus 22% in WT mice at 21 dpp. Compared to control, Hyp cementocytes featured well-defined, disperse euchromatin and a thick layer of peripherally condensed heterochromatin in nuclei, indicating cellular activity. Immunohistochemistry (IHC) for cementum markers revealed intense dentin matrix protein-1 expression and abnormal osteopontin deposition in Hyp mice. Both WT and Hyp cementocytes expressed gap junction protein, connexin 43. CONCLUSION: This study provides new insights into the EIA model and cementocyte activity in association with new cementum formation.

Subgingival endotoxin and lipoteichoic acid modulate cytokine production in diabetic subjects: A Case-control Study.

BACKGROUND: Periodontal disease and diabetes mellitus (DM) are highly prevalent and interrelated diseases, resulting in altered host response microbiota. Thus, this study aimed to evaluate the impact of DM on local levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and their relationship with cytokines and matrix metalloproteinases' (MMPs) profile. METHODS: This case-control study included diabetic (n = 15) and non-diabetic (n = 15) subjects presenting Stage 3-4, Grade C, Periodontitis. Gingival crevicular fluid (GCF) was collected, and LPS and LTA levels were analyzed by enzyme-linked immunosorbent assay (ELISA), while IFN-γ, IL-10, IL-17, IL-1ß, IL-4, MMP-2, and MMP-9 were measured by LUMINEX/MAGpix. Mann-Whitney and Spearman's correlation tests were used to compared and to correlate variables (p < 0.05). RESULTS: Higher levels of LTA, LPS, IL-10, IL-1ß, and MMP-2 (p < 0.05) and lower levels of IL-17 were found in the DM group (p < 0.05). Non-diabetic subjects presented higher LPS, IFN-γ, IL-17, and MMP-2 levels and lower IL-10 concentration (p < 0.05). No significant correlation was seen between LPS and cytokine profile in non-diabetic. Local levels of LTA were positively correlated with IL-17 and MMP-2 and negatively with IL-10. CONCLUSION: LTA and LPS drove the inflammatory profile through the modulation of cytokines and MMPs in a different manner in DM and non-diabetic subjects.

Hydroxyapatite/ß-tricalcium phosphate and enamel matrix derivative for treatment of proximal class II furcation defects: a randomized clinical trial.

OBJECTIVE: To clinically evaluate proximal furcations treated with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) isolated or combined with enamel matrix derivative (EMD). MATERIAL AND METHODS: Thirty patients, presenting at least one proximal class II furcation defect, probing pocket depth (PPD) ≥5 mm and bleeding on probing, were included. The defects were assigned to the HA/ß-TCP group (n = 15); open-flap debridement (OFD) + HA/ß-TCP filling, or, HA/ß-TCP-EMD group (n = 15); OFD + HA/ß-TCP + EMD filling. Plaque (PI) and gingival index (GI), PPD, relative gingival margin position (RGMP), vertical and horizontal attachment level (RVAL and RHAL), vertical and horizontal bone level (RVBL and RHBL), and furcation diagnosis were evaluated at baseline and at 6 months. RESULTS: Both groups presented improvements after therapies (p < 0.05); however, no inter-group differences could be seen in any single parameter (p > 0.05). At 6 months, the gains in rVCAL in the HA/ß-TCP and HA/ß-TCP-EMD groups were 1.47 ± 0.99 and 2.10 ± 0.87 mm, while the RHCAL gains were 1.47 ± 1.46 and 1.57 ± 1.58 mm (p > 0.05). The RVBL and RHBL gains for the HA/ß-TCP and HA/ß-TCP-EMD group were 1.47 ± 1.13 and 1.70 ± 1.26 mm, and 1.90 ± 1.11 and 1.70 ± 1.37 mm respectively (p > 0.05). The HA/ß-TCP-EMD group showed seven closed furcations versus four in the HA/ß-TCP group (p > 0.05). CONCLUSION: Both treatments lead to improvements in all clinical variables studied in the present trial. However, the closure of proximal class II furcation defects is still unpredictable.

Periosteum-derived cells as an alternative to bone marrow cells for bone tissue engineering around dental implants. A histomorphometric study in beagle dogs.

BACKGROUND: The aim of this study is to investigate the potential use of periosteum-derived cells (PCs) for tissue engineering in peri-implant defects. METHODS: Bone marrow cells (BMCs) and PCs were harvested from seven adult beagle dogs, cultured in vitro, and phenotypically characterized with regard to their osteogenic properties. The animals were then subjected to teeth extraction, and 3 months later, two implant sites were drilled, bone dehiscences created, and dental implants placed. Dehiscences were randomly assigned to one of two groups: PCs (PCs + carrier) and BMCs (BMCs + carrier). After 3 months, the animals were sacrificed and the implants with adjacent hard tissues were processed for undecalcified sections. Bone-to-implant contact, bone fill within the limits of implant threads, and new bone area in a zone lateral to the implant were histometrically obtained. RESULTS: In vitro, phenotypic characterization demonstrated that both cell populations presented osteogenic potential, as identified by the mineral nodule formation and the expression of bone markers. Histometrically, an intergroup analysis showed that both cell-treated defects had similar bone fill within the limits of implant threads and bone-to-implant contact (P >0.05), and although a trend toward higher new bone area values was found for the PC group, there was no significant difference between the experimental groups (P >0.05). CONCLUSIONS: Periosteal and bone marrow cells presented a similar potential for bone reconstruction. As such, periosteum may be considered as an alternative source of osteogenic cells in implant dentistry.