To investigate how delayed blood centrifugation affects the composition of the resultant platelet rich fibrin membrane (PRF, a concentrated growth factor preparation) and its biological effects towards gingival fibroblasts. Blood samples were collected from 18 healthy individuals and centrifuged immediately (T-0), or after a 1–6-minute delay (T-1–6, respectively), to generate PRF. Each PRF membrane was weighed. T-0 and T-6 membranes were incubated for 48 h in cell culture medium at 37 °C to create PRF “releasates” (soluble factors released from the PRF). Human gingival fibroblasts were incubated for 48 h with or without the releasates, followed by RNA isolation and real-time polymerase chain reaction to measure expression of select genes associated with granulation tissue formation, angiogenesis and wound contraction. Additional T-0 and T-6 membranes were used for visualization of leucocyte nuclei and platelets by immunostaining. Immediate centrifugation (T-0) resulted in the largest membranes, T-6 membranes being on average 29

Objective Calcified carotid artery atheroma (CCAA) can be identified incidentally on standard dental panoramic radiographs (DPRs). We sought to (1) determine the prevalence of CCAA on DPRs in a general dental population and (2) establish the proportion of patients in whom this would represent a new statin-indicated condition. Methods We identified patients aged ≥30 with DPRs from 2019 to 2021 from the University of British Columbia Dental Clinic. Patient charts were reviewed for use of lipid-lowering therapies (LLT) and existing statin-indicated conditions. DPRs for each patient were evaluated for the presence and characteristics of CCAA. Results Of 921 patients with a DPR and documented medical history, 548 (59.5 %) were diagnostic for evaluation of CCAA. Although 116/548 (21.2 %) of these patients had evidence of CCAA, only 25.9 % (30/116) were already on LLT; another 20.7 % (24/116) of patients with CCAA had a pre-existing statin-indicated condition but were not on LLT. Therefore, in 53.4 % (62/116) of patients with CCAA-positive DPRs, this constituted a new diagnosis of atherosclerosis not yet treated with LLT, representing 6.7 % (62/921) of the clinic population and 11.3 % of individuals with DPRs of diagnostic quality (62/548). Dyslipidemia, hypertension, coronary artery disease, diabetes, atrial fibrillation, stroke/transient ischemic attack, older age, and male sex were all found to be significant predictors of CCAA. Conclusion CCAA is a common finding among patients with DPRs and in over half of cases, the presence of CCAA represents a new diagnosis of atherosclerosis. The high prevalence of new, untreated atherosclerosis in this population indicates an opportunity for risk factor modification and collaboration between dentists and physicians to optimize patient care.

Standard cell cultures are performed in aqueous media with a low macromolecule concentration compared to tissue microenvironment. In macromolecular crowding (MMC) experiments, synthetic polymeric crowders are added into cell culture media to better mimic macromolecule concentrations found in vivo. However, their effect on cultured cells is incompletely understood and appears context-dependent. Here we show using human gingival fibroblasts, a cell type associated with fast and scarless wound healing, that MMC (standard medium supplemented with Ficoll 70/400) potently modulates fibroblast phenotype and extracellular matrix (ECM) composition compared to standard culture media (nMMC) over time. MMC significantly reduced cell numbers, but increased accumulation of collagen I, cellular fibronectin, and tenascin C, while suppressing level of SPARC (Secreted Protein Acidic and Cysteine Rich). Out of the 75 wound healing and ECM related genes studied, MMC significantly modulated expression of 25 genes compared to nMMC condition. MMC also suppressed myofibroblast markers and promoted deposition of basement membrane molecules collagen IV, laminin 1, and expression of LAMB3 (Laminin Subunit Beta 3) gene. In cell-derived matrices produced by a novel decellularization protocol, the altered molecular composition of MMC matrices was replicated. Thus, MMC may improve cell culture models for research and provide novel approaches for regenerative therapy.

OBJECTIVES:Peri-implantitis (PI) is caused by bacteria in the peri-implant space but the consensus on microbial profile is still lacking. Current microbial sampling of PI lesions has largely focused on analyzing bacterial species that have been shed from the implant surface and captured in the pocket fluid. The purpose of the present study was to investigate the morphotypes of bacteria in biofilm covering the implant threads and explore whether certain morphotypes were associated with PI.METHODS:Fourteen failed implants were removed and instantly processed for scanning electron microscope analysis. The implants were imaged at three equally divided sub-crestal levels of the exposed area. Bacterial morphotypes were identified and quantified by three examiners. Mobility and years in function were correlated to the presence of different morphotypes.RESULTS:The implants demonstrated the presence of variable bacterial morphotypes that did not correlate to disease progression in our study. Some implants were dominated by filaments and others showed the presence of combinations of cocci/rods or spirilles/spirochetes. In general, all implants showed variable morphologic biofilm composition. However, individual implants tended to have similar composition throughout the entire implant. Rods and filaments were dominant morphotypes throughout the surfaces and cocci showed increased presence toward the apical third. There were some differences in the biofilm morphology with mobility and time in function.CONCLUSIONS:The profiles of bacterial biofilm morphotypes in failing implants with similar clinical presentations were highly variable. While there were significant differences between implants, similar morphotypes in individual implants were often found throughout the entire surface.

Periodontal disease (PD) can be prevented by local or systemic application of epidermal growth factor receptor inhibitors (EGFRIs) that stabilize αvβ6 integrin levels in the periodontal tissue, leading to an increase in the expression of anti-inflammatory cytokines, such as transforming growth factor-β1. Systemic EGFRIs have side effects and, therefore, local treatment of PD applied into the periodontal pockets would be preferrable. Thus, we have developed slow-release three-layered microparticles of gefitinib, a commercially available EGFRI. A combination of different polymers [cellulose acetate butyrate (CAB), Poly (D, L-lactide-co-glycolide) (PLGA) and ethyl cellulose (EC)] and sugars [D-mannose, D-mannitol and D-(+)-trehalose dihydrate] were used for the encapsulation. The optimal formulation was composed of CAB, EC, PLGA, mannose and gefitinib (0.59, 0.24, 0.09, 1, and 0.005 mg/ml, respectively; labeled CEP-gef), and created microparticles of 5.7 ± 2.3 µm in diameter, encapsulation efficiency of 99.98%, and a release rate of more than 300 h. A suspension of this microparticle formulation blocked EGFR phosphorylation and restored αvβ6 integrin levels in oral epithelial cells, while the respective control microparticles showed no effect.

In periodontal disease (PD), bacterial biofilms suppress β6 integrin expression transforming growth factor-β1 signaling, resulting in gingival inflammation and bone loss. β6 integrin-null (Itgb6-/- ) mice develop spontaneous PD. The aim of this study was to unravel potential differences in oral microbiome in wild-type (WT) and Itgb6-/- FVB mice. Mouse oral microbiome was analyzed from 3- and 6-month-old WT and Itgb6-/- . The periodontal inflammation and spontaneous bone loss were present in 3-month-old and advanced in 6-month-old Itgb6-/- mice. The observed amplicon sequence variants (ASVs) of alpha diversity showed close similarity in 3-month-old and 6-month-old Itgb6-/- mice. Chao1 and ACE methods revealed that the microbiome in Itgb6-/- mice showed less diversity compared to the WT. UniFrac Principal Coordinate analyses (PCoA) showed a clear spatial segregation and clustering between Itgb6-/- and WT mice in general, and between 3-month- and 6-month-old WT mice. Weighted PCoA showed the tight clustering and distinct separation of individual mouse samples within Itgb6-/- and WT. The most abundant microbial classes varied between the sample groups. However, the genus Aggregatibacter significantly increased in the 6-month-old Itgb6-/- mice. β6 integrin-deficient mice develop periodontal inflammation that may relate to dysbiosis in the microbiome that further promotes the disease process.

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX , BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Abstract Objectives Decontamination of biofilm‐colonized rough implant surfaces remains challenging. We investigated the effect of airflow with glycine powder (AFG) on decontamination of mature oral multispecies biofilm from a sandblasted and acid etched (SLA) titanium surface. Materials and Methods Subgingival dental plaque was cultured on SLA disks anaerobically for 21 days. AFG with various settings and distances was applied directly on the disks with or without previous rinse of 0.9% NaCl. The specimens were then analyzed through scanning electron microscope and remaining bacteria on the implant surface were quantified and statistically compared. Results Mature oral biofilm with cocci and rods as major morphotypes, as well as spiral‐ and filamentous‐shaped organisms, was formed on the untreated disks. Saline rinsing removed the thick biofilm layer but left numerous of coccoid bacteria in rough surface pits. AFG effectively removed most of the bacteria from the pits. Both 25% and 50% power settings were equally effective at 3‐mm distance. With 50% power, AFG successfully removed bacteria at both 3‐ and 6‐mm distance. When AFG was applied on native biofilm without prior rinsing with saline, it effectively removed the biofilm including bacteria in the pits. Conclusion Application of AFG appears effective in removing bacteria from rough implant surfaces.

ABSTRACT In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvβ6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress β6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-β1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-β1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the β6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD. This article has an associated First Person interview with the first author of the paper.

BACKGROUND:Decontamination of biofilm-infected rough implant surfaces is challenging. Platelet rich blood products have been shown to have anti-microbial properties against periodontal pathogens. Our aim was to investigate the effect of a potential biological implant surface disinfectant, leukocyte- and platelet-rich fibrin (L-PRF), on a mature oral multispecies biofilm on a rough titanium surface. METHODS:Sandblasted, large grit, acid-etched (SLA) titanium disks were inoculated with subgingival dental plaque and cultured anaerobically for 21 days. The L-PRF membranes were collected from 12 donors in three trials (four donors in each trial). The disks were rinsed with 0.9% NaCl and exposed to the cell-rich portion of the L-PRF membranes for 48 hours followed by scanning electron microscope (SEM) analysis immediately or after rinsing with 0.9% NaCl prior to fixation. The presence of platelet factor-4 in the rinse samples was analyzed by Western blotting. Remaining bacteria were quantified from SEM images of the implant surfaces and their numbers statistically compared. RESULTS:The L-PRF-treated samples without rinsing displayed numerous cells with multiple pseudopodia in immediate contact with bacteria that appeared perforated and increased in size. The cells were identified as platelets based on morphological criteria and by positive reaction for platelet factor-4 by Western blotting. After post-treatment rinsing, the L-PRF-treated disks displayed a significant reduction in bacterial counts (in average 92% reduction). CONCLUSION:Application of L-PRF significantly reduced bacterial counts on contaminated SLA titanium surface, most likely through anti-microbial action by platelets.

BACKGROUND:Leucocyte- and platelet-rich fibrin (L-PRF) is a blood-derived biomaterial rich in leucocytes and platelets embedded in a high-density fibrin network that can be compressed into a membrane and used in surgical applications to stimulate tissue regeneration and wound healing, especially in oral cavity. This study aimed to determine the combined effects of the growth factors and cells present in L-PRF on fibroblasts that directly face the L-PRF membranes placed during surgical procedures.METHODS:The effect of L-PRF from six donors on the expression of 84 key wound healing genes in normal human gingival fibroblasts was tested by RT-qPCR.RESULTS:L-PRF significantly regulated the expression of 33 fibroblast genes (39%), including interleukins, myofibroblast-, extracellular matrix- and angiogenesis-associated genes, and matrix metalloproteinase-1 and -3. L-PRF regulated fibroblast gene expression both time- and dose-dependently, and the effects were mediated by mitogen-activated protein kinases ERK1/2, JNK and p38. L-PRF also stimulated fibroblast wound closure and promoted the ability of fibroblasts to induce endothelial tube formation. L-PRF-induced gene expression changes in fibroblast were similar to those observed in early human and pig wounds.CONCLUSIONS:This study provides new insights into the biological mechanism by which L-PRF regulates key gingival fibroblast functions important in wound healing.

Pyogenic granuloma (PG) is typically a localised and vascular granulation tissue growth that occurs on both the oral cavity and the skin. PG is the most common gingival tumour, although it can also be found on the lips, tongue and buccal mucosa (Neville and Day 2002). PG commonly exists as a soft tissue vascular lesion with interdental attachment that rarely exceeds over 2.5 cm in size (Laine 2002). The surface of PG may be ulcerated causing spontaneous bleeding, but the lesion is painless unless it is damaged by the opposing teeth during mastication. In addition to inflammation induced by biofilm and physical trauma, the development of PG may also be influenced by the hormonal factors, because of the high incidence of oral PG in pregnant women (Neville and Day 2002). A rare case of oral PG with an unusual large size which developed in the anterior palatal region of a pregnant woman is reported here.

Introduction: To evaluate risk indicators associated with implant failure and relationship between bone levels and soft-tissue health of anodized implants placed in private practice. Material and Methods: Partially or completely edentulous patients who received an anodized implant between 2003 and 2013 were included. Univariate and multivariate analysis was used to identify the relationship between study variables and implant failure. Mean marginal bone level changes (MBL Delta) were assessed using periapical radiographs. Periimplant soft tissue was evaluated using a modified bleeding index (implant mucosal index, IMI). Results: A total of 1087 implants placed in 414 patients were followed for 3.9 +/- 2.7 years. The cumulative implant survival rate after 10 years of function was 97.0%. Shorter (P = 0.0068) and maxillary implants (P = 0.0314) were associated with lower implant survival rate. Mean MBL decreased from -0.16 +/- 0.43 mm at baseline to -0.53 +/- 0.53 mm 8 to 10 years later. Implants with healthier mucosa were associated with less bone loss. Conclusions: Implants with an anodized surface showed a high long-term survival rate in a daily practice. Longer implants and implants placed in the mandible were associated with greater survival. Immediate loading and tapered design did not affect implant survival. Profuse multipoint bleeding and suppuration on recall were associated with greater bone loss.

The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias.

Integrins are cell surface receptors that traditionally mediate cell-to-extracellular matrix and cell-to-cell adhesion. They can, however, also bind a large repertoire of other molecules. Integrin αvβ6 is exclusively expressed in epithelial cells where it can, for example, serve as a fibronectin receptor. However, its hallmark function is to activate transforming growth factor-β1 (TGF-β1) to modulate innate immune surveillance in lungs and skin and along the gastrointestinal tract, and to maintain epithelial stem cell quiescence. The loss of αvβ6 integrin function in mice and humans leads to an altered immune response in lungs and skin, amelogenesis imperfecta, periodontal disease and, in some cases, alopecia. Elevated αvβ6 integrin expression and aberrant TGF-β1 activation and function are associated with organ fibrosis and cancer. Therefore, αvβ6 integrin serves as an attractive target for cancer imaging and for fibrosis and cancer therapy.

Aims: Abundance of connexin 43 (Cx43), a transmembrane protein that forms hemichannels (HCs) and gap junctions (GJs), is dynamically regulated in human gingival fibroblasts (GFBLs) during wound healing. This may be important for fast and scarless gingival wound healing as Cx43 is involved in key cell functions important during this process. Our aim was to uncover the factors that regulate Cx43 expression and abundance in GFBLs. We hypothesized that cytokines and growth factors released during wound healing coordinately regulate Cx43 abundance in GFBLs. Results: TGF-beta 1, -beta 2, -beta 3, PGE2 and IL-1 beta significantly upregulated, while TNF-alpha and IFN-gamma downregulated Cx43 in cultured GFBLs. TGF-beta 1, -beta 2, -beta 3, IL-1 beta and IFN-gamma modulated Cx43 abundance at both mRNA and protein levels, while TNF-alpha and PGE2 regulated only Cx43 protein abundance, suggesting involvement of distinct transcriptional/post-transcriptional and translational/post-translational mechanisms, respectively. TGF-beta 1-induced upregulation of Cx43 was mediated by TGF beta RI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL-1 beta, TNF-alpha and IFN-gamma that inhibited SMAD2/3 phosphorylation. Conclusion: Regulation of Cx43 abundance in GFBLs involves transcriptional/post-transcriptional and translational/post-translational mechanisms that are distinctly modulated by an interplay between TGF-beta isoforms and PGE2, IL-1 beta, TNF-alpha and IFN-gamma.

Epithelial alpha v beta 6 integrin participates in immune surveillance in many organs, including the gastrointestinal track. Expression of alpha v beta 6 integrin is reduced in the junctional epithelium of the gingiva in periodontal diseases, and mutations in the ITGB6 gene are associated with these diseases in humans and mice. The aim of this study was to unravel potential differences in the inflammatory responses in the periodontal tissues of FVB wild-type (WT) and beta 6 integrin-null (Itgb6(-/-)) mice, using a ligature-induced periodontitis model and assessing inflammation, bone loss and expression profiles of 34 genes associated with periodontal disease. Using micro-CT and histology, we demonstrated more advanced inflammation and bone loss in the control and ligatured Itgb6(-/-) mice compared to the WT animals. Neutrophil and macrophage marker genes were significantly upregulated by ligation in both WT and Itgb6(-/-) mice while the expression of T-cell and B-cell markers was downregulated, suggesting acute-type of inflammation. Expression of inflammasome NLRP3-related genes Nlpr3 and Illb was also significantly increased in both groups. However, the expression of 1118 was significantly lower in non ligatured Itgb6(-/-) mice than in the WT mice and was further downregulated in both groups by the ligatures. IL 18 mediates many effects of the AIM2 inflammasome, including regulation of the microbiome. Interestingly, expression of Aim2 was significantly lower in both control and ligatured Itgb6(-/-) mice than in WT animals. Overall, ligature-induced periodontitis was associated with increased expression of pro-inflammatory cytokines, chemokines and osteoclastogenic regulatory molecules. Another significant difference between the Itgb6(-/-) and WT mice was that mRNA expression of the anti-inflammatory cytokine IL-10 was increased in ligatured WT mice but reduced in the Itgb6(-/-) mice. In conclusion, alpha v beta 6 integrin in junctional epithelium of the gingiva appears to positively regulate the expression of the AIM2 inflammasome and anti-inflammatory IL-10, thus providing protection against periodontal inflammation.

Bone is a composite material composed of collagen and calcium phosphate (CaP) mineral. The collagen gives bone its flexibility while the inorganic material gives bone its resilience. The CaP in bone is similar in composition and structure to the mineral hydroxyapatite (HA) and is bioactive, osteoinductive and osteoconductive. Therefore synthetic versions of bone apatite (BA) have been developed to address the demand for autologous bone graft substitutes. Synthetic HA (s-HA) are stiff and strong, but brittle. These lack of physical attributes limit the use of synthetic apatites in situations where no physical loading of the apatite occurs. s-HA chemical properties differ from BA and thus change the physical and mechanical properties of the material. Consequently, s-HA is more chemically stable than BA and thus its resorption rate is slower than the rate of bone regeneration. One solution to this problem is to introduce a faster resorbing CaP, such as β-tricalcium phosphate (β-TCP), when synthesizing the material creating a biphasic (s-HA and β-TCP) formulation of calcium phosphate (BCP). The focus of this review is to introduce the major differences between BCP and biological apatites and how material scientists have overcome the inadequacies of the synthetic counterparts. Examples of BCP performance in vitro and in vivo following structural and chemical modifications are provided as well as novel ultrastructural data. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2493-2512, 2018.

BACKGROUNDPeri-implantitis is a complex polymicrobial biofilm-induced inflammatory osteolytic gingival infection that results in orofacial implant failures. To the best knowledge of the authors, there are no preclinical in vivo studies in implant dentistry that have investigated the inflammatory response to known microbial biofilms observed in humans. The aim of this study is to develop a novel peri-implant rat model using an established model of polymicrobial periodontitis.METHODSWistar rats were used for the study of experimental peri-implantitis. One month after extraction of maxillary first molars, a titanium mini-implant was inserted. Two months after implant healing, implants were uncovered, and abutment fixing was done using cyanoacrylate to prevent abutment loosening. Rats were separated into two groups (group A: polymicrobial-infected and group B: sham-infected). One week after healing of abutments, rats were infected with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia for 12 weeks. Bacterial colonization, bone resorption, and implant inflammation were evaluated by polymerase chain reaction (PCR), microcomputed tomography, and histology, respectively.RESULTSThree rats with four implants in the infection group and two rats with three implants in the sham-infection group were analyzed. PCR analysis revealed presence of bacterial genomic DNA, and infection elicited significant immunoglobulin (Ig)G and IgM antibody responses, indicating bacterial colonization/infection around implants. Infection induced an enhanced mean distance from implant platform to the first bone-to-implant contact, extensive peri-implantitis with advanced bone resorption, and extensive inflammation with granulation tissue and polymorphonuclear leukocytes.CONCLUSIONSTo the best knowledge of the authors, this is the first study to develop a novel rat model of polymicrobial peri-implantitis. With modifications to improve implant retention it could offer significant advantages for studies of initiation and progression of peri-implantitis.