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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.

Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva.

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.

Oral Wound Healing: Cell Biology and Clinical Management brings experts from around the world together to provide an authoritative reference on the processes, principles and clinical management of wound healing in the oral mucosa. Promoting a thorough understanding of current research on the topic, this new resource draws together thinking on the basic biological processes of wound healing in the oral environment, as well as providing more detailed information and discussion on processes such as inflammation, reepithelialization and angiogenesis. Beyond this, the book goes on to examine topics pertinent to the effective clinical management of oral wound healing, bringing together chapters on large dento-facial defects, dental implants, periodontal regeneration, and pulp healing.An essential synthesis of current research and clinical applications, Oral Wound Healing will be an indispensable resource for dental specialists, oral and maxillofacial surgeons as well as researchers in oral medicine and biology.

Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-β signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.

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.

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvβ6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-β1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6 −/− mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvβ6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of β6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1β and -6. Furthermore, GECs with β6 integrin siRNA knockdown showed increased interleukin-1β expression, indicating that αvβ6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed β6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvβ6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

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.

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.

Integrin αvβ6 is an epithelial-specific receptor that binds and activates latent transforming growth factor-β1 (TGF-β1). TGF-β1 has been implicated as an endogenous inducer of hair follicle (HF) regression during hair cycling. We hypothesized that αvβ6 integrin–mediated TGF-β1 signaling regulates hair regeneration and HF involution. In wild-type (WT) mice, the expression of integrin αvβ6 was strongly upregulated in the outer root sheath (ORS) during early hair regeneration, and was specifically enhanced in the HF bulge region. Expression gradually decreased in late anagen and remained restricted to the bulge region in the catagen and telogen stage HFs. The first spontaneous hair cycle was not altered in β6 integrin knockout (β6−/−) mice. However, after depilation, β6−/− mice exhibited retarded HF regression compared with WT controls. β6−/− follicles contained significantly higher numbers of proliferating Ki67-positive keratinocytes than WT follicles at an identical cycle stage. The β6−/− follicles also demonstrated significantly reduced levels of TGF-β1 expression and Smad2 phosphorylation during early anagen and anagen–catagen transition. Our study indicates that αvβ6 integrin has an important inhibitory role in keratinocyte proliferation in both HFs and interfollicular epidermis. Thus, downregulated TGF-β1 signaling in β6−/− mice may affect bulge niche stem cell behavior.