Antimicrobial efficacy of DJK-5 peptide in combination with EDTA against biofilms in dentinal tubules: Primary irrigation, recovery and re-irrigation.

AIM: To investigate the dynamic recovery of biofilms within dentinal tubules after primary irrigation with different protocols, and to evaluate the efficacy of various re-irrigation protocols on recovered biofilm, considering factors such as smear layer, nutrient conditions, and primary irrigants. METHODOLOGY: A total of 416 mono or multi-species biofilms samples were prepared from human teeth and incubated for 3 weeks. After inducing a smear layer on half of the samples, all specimens were irrigated with one of the following irrigant sequences: (1) 6% NaOCl +17% EDTA, (2) 6% NaOCl +8.5% EDTA, (3) 6% NaOCl and (8.5% EDTA +10 µg/mL DJK-5 antimicrobial peptide), or (4) sterile water. Thirty-two samples were used to assess immediate effect, whilst the rest were re-incubated to assess biofilms recovery. Nutrient conditions were defined based on whether culture media were changed (nutrient-rich) or not (nutrient-poor) during re-incubation. After 16 weeks, recovered biofilms underwent re-irrigation using four additional protocols, with or without DJK-5 peptide, based on primary irrigants. Confocal laser scanning microscopy was employed to evaluate immediate irrigant effects, biofilms recovery intervals (1, 3, 5, 8, 12, and 16 weeks after primary irrigation), and re-irrigation effects at the 16-week. Statistical analysis included one-way anova and two-way mixed anova tests. RESULTS: The DJK-5 peptide irrigation protocols demonstrated the highest killing rates during primary irrigation and resulted in a longer biofilms recovery time of 16 weeks compared to non-peptide protocols (p < .001). Both primary irrigation type and smear layer presence significantly influenced biofilms recovery (p < .001). In the absence of smear layer, re-irrigation efficacy didn't significantly differ from primary irrigation, regardless of primary irrigation type or nutrient conditions. However, with a smear layer present, re-irrigation led to significantly higher proportion of dead bacteria compared to primary irrigation (p < .05). Inclusion of the DJK-5 peptide into the re-irrigation protocol displayed superior killing rate compared to other protocols (p < .001). CONCLUSIONS: Biofilms exhibited susceptibility to both peptide and non-peptide protocols during re-irrigation, irrespective of nutrient conditions or primary irrigation protocols. The DJK-5 peptide irrigation protocols consistently displayed superior effectiveness compared to non-peptide protocols.

Granzyme B promotes matrix metalloproteinase-1 (MMP-1) release from gingival fibroblasts in a PAR1- and Erk1/2-dependent manner: A novel role in periodontal inflammation.

OBJECTIVE: To gain insights into how proteases signal to connective tissues cells in the periodontium. BACKGROUND: The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. METHODS: Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB]GCF ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB-/- ) mice. RESULTS: The [GzmB]GCF in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB-/- mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically. CONCLUSION: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.

Spray-dried microparticles of encapsulated gefitinib for slow-release localized treatment of periodontal disease.

Periodontal disease (PD) can be prevented by local or systemic application of epidermal growth factor receptor inhibitors (EGFRIs) that stabilize αvß6 integrin levels in the periodontal tissue, leading to an increase in the expression of anti-inflammatory cytokines, such as transforming growth factor-ß1. 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 αvß6 integrin levels in oral epithelial cells, while the respective control microparticles showed no effect.

Macromolecular crowding regulates matrix composition and gene expression in human gingival fibroblast cultures.

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.

Platelet factor 4 (CXCL4/PF4) upregulates matrix metalloproteinase-2 (MMP-2) in gingival fibroblasts.

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.

Gingival epithelial cell-derived microvesicles activate mineralization in gingival fibroblasts.

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.

Altered composition of the oral microbiome in integrin beta 6-deficient mouse.

In periodontal disease (PD), bacterial biofilms suppress ß6 integrin expression transforming growth factor-ß1 signaling, resulting in gingival inflammation and bone loss. ß6 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. ß6 integrin-deficient mice develop periodontal inflammation that may relate to dysbiosis in the microbiome that further promotes the disease process.

Decontamination of rough implant surfaces colonized by multispecies oral biofilm by application of leukocyte- and platelet-rich fibrin.

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.

Leucocyte- and platelet-rich fibrin regulates expression of genes related to early wound healing in human gingival fibroblasts.

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.

Epidermal growth factor receptor signaling suppresses αvß6 integrin and promotes periodontal inflammation and bone loss.

In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvß6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-ß1 (TGF-ß1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress ß6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-ß1 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-ß1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the ß6 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.

Inflammasome and cytokine expression profiling in experimental periodontitis in the integrin ß6 null mouse.

Epithelial αvß6 integrin participates in immune surveillance in many organs, including the gastrointestinal track. Expression of αvß6 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 ß6 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 Il1b was also significantly increased in both groups. However, the expression of Il18 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, αvß6 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.

Integrin αvß6: Structure, function and role in health and disease.

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 αvß6 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-ß1 (TGF-ß1) to modulate innate immune surveillance in lungs and skin and along the gastrointestinal tract, and to maintain epithelial stem cell quiescence. The loss of αvß6 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 αvß6 integrin expression and aberrant TGF-ß1 activation and function are associated with organ fibrosis and cancer. Therefore, αvß6 integrin serves as an attractive target for cancer imaging and for fibrosis and cancer therapy.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts.

Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

Connexin 43 Hemichannels Regulate the Expression of Wound Healing-Associated Genes in Human Gingival Fibroblasts.

Connexin 43 (Cx43) is the most ubiquitous connexin in various cells, and presents as hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We have recently shown that Cx43 abundance was strongly reduced in fibroblasts of human gingival wounds, and blocking Cx43 function in cultured human gingival fibroblasts (GFBLs) strongly regulated the expression of wound healing-related genes. However, it is not known whether these responses involved Cx43 HCs or GJs. Here we show that Cx43 assembled into distinct GJ and HC plaques in GFBLs both in vivo and in vitro. Specific blockage of Cx43 HC function by TAT-Gap19, a Cx43 mimetic peptide, significantly upregulated the expression of several MMPs, TGF-ß signaling molecules, Tenascin-C, and VEGF-A, while pro-fibrotic molecules, including several extracellular matrix proteins and myofibroblast and cell contractility-related molecules, were significantly downregulated. These changes were linked with TAT-Gap19-induced suppression of ATP signaling and activation of the ERK1/2 signaling pathway. Collectively, our data suggest that reduced Cx43 HC function could promote fast and scarless gingival wound healing. Thus, selective suppression of Cx43 HCs may provide a novel target to modulate wound healing.

Suppression of αvß6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells.

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 αvß6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-ß1, 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 αvß6 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 ß6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1ß and -6. Furthermore, GECs with ß6 integrin siRNA knockdown showed increased interleukin-1ß expression, indicating that αvß6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed ß6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvß6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts.

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-ß signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts.

Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial-stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing-associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P<0.05) out of 80 genes investigated, including matrix metalloproteinase-1 and -3, interleukin-6 and -8, and genes associated with transforming growth factor-ß signaling. Similar changes were observed at the protein level. MVs from normal epidermal keratinocytes showed similar response to HaCaT cells. KC-MVs activated ERK1/2, JNK, Smad, and p38 signaling pathways in fibroblasts with ERK1/2 signaling having the most prominent role in the MV-induced gene expression changes. KC-MVs stimulated fibroblast migration and induced fibroblast-mediated endothelial tube formation but did not affect collagen gel contraction by fibroblasts. The results demonstrate that keratinocyte microvesicles have a strong and a specific regulatory effect on fibroblasts that may modulate several aspects of wound healing.

Expression and function of connexin 43 in human gingival wound healing and fibroblasts.

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-ß1, 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/ß and TGF-ß 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/ß and TGF-ß signaling pathways, and may promote fast and scarless wound healing in human gingiva.

Integrins in Wound Healing.

Significance: Regulation of cell adhesions during tissue repair is fundamentally important for cell migration, proliferation, and protein production. All cells interact with extracellular matrix proteins with cell surface integrin receptors that convey signals from the environment into the nucleus, regulating gene expression and cell behavior. Integrins also interact with a variety of other proteins, such as growth factors, their receptors, and proteolytic enzymes. Re-epithelialization and granulation tissue formation are crucially dependent on the temporospatial function of multiple integrins. This review explains how integrins function in wound repair. Recent Advances: Certain integrins can activate latent transforming growth factor beta-1 (TGF-ß1) that modulates wound inflammation and granulation tissue formation. Dysregulation of TGF-ß1 function is associated with scarring and fibrotic disorders. Therefore, these integrins represent targets for therapeutic intervention in fibrosis. Critical Issues: Integrins have multifaceted functions and extensive crosstalk with other cell surface receptors and molecules. Moreover, in aberrant healing, integrins may assume different functions, further increasing the complexity of their functionality. Discovering and understanding the role that integrins play in wound healing provides an opportunity to identify the mechanisms for medical conditions, such as excessive scarring, chronic wounds, and even cancer. Future Directions: Integrin functions in acute and chronic wounds should be further addressed in models better mimicking human wounds. Application of any products in acute or chronic wounds will potentially alter integrin functions that need to be carefully considered in the design.

Formation of cartilage and synovial tissue by human gingival stem cells.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.