Cristina Cunha Villar

Regeneration of Periodontal Tissues: Guided Tissue Regeneration

The concept that only fibroblasts from the periodontal ligament or undifferentiated mesenchymal cells have the potential to re-create the original periodontal attachment has been long recognized. Based on this concept, guided tissue regeneration has been applied with variable success to regenerate periodontal defects. Quantitative analysis of clinical outcomes after guided tissue regeneration suggests that this therapy is a successful and predictable procedure to treat narrow intrabony defects and class II mandibular furcations, but offers limited benefits in the treatment of other types of periodontal defects.

Immune defence mechanisms and immunoenhancement strategies in oropharyngeal candidiasis

The prevalence of oropharyngeal candidiasis continues to be high, mainly because of an increasing population of immunocompromised patients. Traditional treatment of oropharyngeal candidiasis has relied on the use of antimicrobial drugs. However, unsatisfactory results with drug monotherapy and the emergence of resistant strains have prompted investigations into the potential use of adjunctive immunoenhancing therapies for the treatment of these infections. Here we review the host-recognition systems of Candida albicans, the immune and inflammatory response to infection, and antifungal effector mechanisms. The potential of immune modulation as a therapeutic strategy in oropharyngeal candidiasis is also discussed.

Expression of UME6, a Key Regulator of Candida albicans Hyphal Development, Enhances Biofilm Formation via Hgc1- and Sun41-Dependent Mechanisms

ABSTRACT Biofilm formation is associated with the ability of Candida albicans, the major human fungal pathogen, to resist antifungal therapies and grow on tissues, catheters, and medical devices. In order to better understand the relationship between C. albicans morphology and biofilm formation, we examined biofilms generated in response to expression of UME6, a key filament-specific transcriptional regulator. As UME6 levels rise, C. albicans cells are known to transition from yeast to hyphae, and we also observed a corresponding increase in the level of biofilm formation in vitro. In addition to forming a biofilm, we observed that a C. albicans strain expressing constitutive high levels of UME6 promoted tissue invasion in a reconstituted human three-dimensional model of oropharyngeal candidiasis. Confocal microscopy indicated that both the top and bottom layers of the biofilm generated upon high-level constitutive UME6 expression consist primarily of hyphal cells. UME6-driven biofilm formation was reduced upon deletion of Hgc1, a cyclin-related protein important for hyphal development, as well as Sun41, a putative cell wall glycosidase. Constitutive high-level UME6 expression was also able to completely bypass both the filamentation and biofilm defects of a strain deleted for Efg1, a key transcriptional regulator of these processes. Finally, we show that both Sun41 and Efg1 affect the ability of UME6 to induce certain filament-specific transcripts. Overall, these findings indicate a strong correlation between increased C. albicans hyphal growth and enhanced biofilm formation and also suggest functional relationships between UME6 and other regulators of biofilm development.

Angiogenic activity of an enamel matrix derivative (EMD) and EMD-derived proteins: An experimental study in mice

OBJECTIVES To determine whether all or only certain proteins in an enamel matrix derivative (EMD) are angiogenic. MATERIALS AND METHODS The angiogenic effect was analysed using an in vivo angiogenesis assay. Silicon tubes were filled with or without potential and known angiogenic-modulating factors: (i) an EMD parent, (ii) nine pools of EMD proteins, (iii) fibroblast growth factor/vascular endothelial growth factor and (iv) amelogenin. Silicon tubes were implanted subcutaneously in mice. Dextran-fluorescein isothiocyanate (FITC) was injected via the tail vein, mice were euthanized and tubes were retrieved. Neovascularization was determined by measuring the amount of dextran-FITC within the tubes. RESULTS The greatest angiogenic potential of the EMD parent was at a weight of 125 ng, resulting in a 4.3-fold increase compared with the negative control. Five pools of EMD proteins showed a stronger angiogenic activity than the EMD parent. Pool 5 showed the greatest angiogenic activity, when compared with the negative control (8.1-fold increase) and with 125 ng of the EMD parent (4.2-fold increase). Amelogenin demonstrated a significantly higher angiogenic activity than the negative control (increase up to 4.0-fold) and the EMD parent (increase up to 1.6-fold). CONCLUSIONS EMD parent, recombinant porcine amelogenin and certain pools of EMD proteins induced significant angiogenesis compared with the controls using a standardized in vivo assay.

Tissue integration of collagen‐based matrices: an experimental study in mice

OBJECTIVES To test whether or not tissue integration, biodegradation, and new blood vessel formation in two collagen-based matrices depend on the level of chemical cross-linking. MATERIAL AND METHODS Two collagen matrices with high (CM1) and low (CM2) levels of chemical cross-linking were randomly implanted in two pouches in 14 athymic nude mice. Three and 6 weeks later, the animals were euthanized. Histologic and histomorphometric measurements were performed on paraffin-embedded sections. RESULTS Both collagen matrices integrated well into the surrounding soft tissues. The level of cross-linking and duration of implantation had an effect on the formation of new blood vessels. More blood vessels (n = in absolute numbers) were found in outer compartments compared to the central compartments of the matrices, reaching 5.6 (CM2) vs. 4.3 (CM1) at 3 weeks, and 5.3 (CM2) vs. 7.3 (CM1) at 6 weeks. Similarly, connective tissue formation increased for both matrices between 3 and 6 weeks, whereas the amount of remaining collagen network gradually decreased over time being more pronounced for CM1 (-50%) compared to CM2 (-15%). CONCLUSIONS The degree of cross-linking was negatively correlated for all outcome measures resulting in improved tissue integration, superior matrix stability and enhanced angiogenic patterns for the less cross-linked collagen matrix (CM2) in this experimental study in mice.

Trafficking of Candida albicans through oral epithelial endocytic compartments.

Oral epithelial cells are the first cells that interact with C. albicans during the establishment of oropharyngeal candidiasis. Following initial adhesion, C. albicans invades oral epithelial cells by inducing its own endocytosis and gains access to epithelial vacuolar compartments. Epithelial endocytic pathways are key innate immune mechanisms in host defense. We examined the trafficking of C. albicans through oral epithelial endocytic compartments. We present evidence that C. albicans is internalized by oral epithelial cells through actin-dependent clathrin-mediated endocytosis and is taken into vacuolar compartments immediately following its internalization. C. albicans-containing endosomes transiently acquired early endosomal marker EEA1, but showed marked defects in acquisition of late endosomal marker LAMP1 and lysosomal marker cathepsin D. Defective endolysosomal maturation may partially explain the inability of oral epithelial cells to kill C. albicans.

Anticandidal Activity and Biocompatibility of a Rechargeable Antifungal Denture Material

OBJECTIVES Candida-associated denture stomatitis is a recurrent and debilitating oral mucosal disease. Development of anticandidal denture materials represents a promising strategy to manage this condition. We have previously shown that miconazole incorporated in methacrylic acid (MAA) copolymerized diurethane dimethacrylate (UDMA) denture materials has long-term anticandidal activity. In this study, we examined the ability of culture medium conditioned with drug-free- or miconazole-MAA-UDMA discs to prevent Candida infection in an in vitro oral epithelial cell/Candida albicans coculture system. MATERIALS AND METHODS Candida albicans (C. albicans)-induced OKF6/TERT-2 cell damage was quantified by the release of lactate dehydrogenase from epithelial cells, cytokine production was quantified using protein cytokine arrays, and the expression of C. albicans genes was measured by RT-qPCR. RESULTS Candida albicans had limited growth with altered expression levels of secreted aspartyl proteinase-2 and -5 in culture medium conditioned by miconazole-MAA-UDMA discs. Significantly, the ability of C. albicans to induce oral epithelial cell damage and trigger epithelial proinflammatory cytokine production was also inhibited by miconazole disc conditioned media. CONCLUSION Miconazole released from MAA-UDMA denture materials effectively prevents the development of candidal infection in an in vitro oral epithelial system. Further characterization of this drug-rechargeable denture material is warranted.

Matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases, and inflammation in cyclosporine A-induced gingival enlargement: a pilot in vitro study using a three-dimensional model of the human oral mucosa.

BACKGROUND It has been suggested that cyclosporine A (CsA) induces gingival enlargement by promoting an increase in the gingival extracellular matrix (ECM). Nonetheless, the variable occurrence of CsA-induced gingival enlargement in patients receiving this medication indicates a multifactorial pathogenesis. Clinical observations suggest that local inflammation is associated with the development and severity of CsA-induced gingival enlargement. Therefore, the purpose of this study is to investigate the effects of CsA and inflammation on the production of ECM homeostatic mediators. METHODS The effects of CsA and inflammation (as assessed using interleukin [IL]-1β) on the secretion of mediators involved in ECM homeostasis were determined using fibroblast monolayers and three-dimensional (3D) models of the human oral mucosa. Fibroblast monolayers and 3D cultures were treated with CsA alone or in combination with IL-1β for up to 72 hours, and the secretion of matrix metalloproteinases (MMPs) 1, 2, 3, 8, 9, 10, and 13 and tissue inhibitors of MMPs (TIMPs) 1, 2, and 4 into the culture medium was assessed using enzyme-linked immunoassay-based antibody arrays. RESULTS Fibroblast monolayers responded to CsA with no changes in the secretion of ECM mediators. Conversely, 3D cultures responded to CsA treatment with a reduction in MMP-10 secretion. IL-1β alone triggered higher secretory levels of MMPs in both fibroblast monolayers (MMP-3 and MMP-10) and 3D cultures (MMP-9 and MMP-10). Importantly, fibroblast monolayers and 3D cultures treated with a combination of IL-1β and CsA showed a decrease in the MMP-1/TIMP-1 ratio. CONCLUSIONS These data support the hypothesis that inflammation may alter the pathogenesis of CsA-induced gingival enlargement by promoting a synergistic decrease in the MMP-1/TIMP-1 ratio.

Effectiveness of Intraoral Chlorhexidine Protocols in the Prevention of Ventilator-Associated Pneumonia: Meta-Analysis and Systematic Review

BACKGROUND: Ventilator-associated pneumonia (VAP) is common in critical patients and related with increased morbidity and mortality. We conducted a systematic review and meta-analysis, with intention-to-treat analysis, of randomized controlled clinical trials that assessed the effectiveness of different intraoral chlorhexidine protocols for the prevention of VAP. METHODS: Search strategies were developed for the MEDLINE, EMBASE, and LILACS databases. MeSH terms were combined with Boolean operators and used to search the databases. Eligible studies were randomized controlled trials of mechanically ventilated subjects receiving oral care with chlorhexidine or standard oral care protocols consisting of or associated with the use of a placebo or no chemicals. Pooled estimates of the relative risk and corresponding 95% CIs were calculated with random effects models, and heterogeneity was assessed with the Cochran Q statistic and I2. RESULTS: The 13 included studies provided data on 1,640 subjects that were randomly allocated to chlorhexidine (n = 834) or control (n = 806) treatments. A preliminary analysis revealed that oral application of chlorhexidine fails to promote a significant reduction in VAP incidence (relative risk 0.80, 95% CI 0.59–1.07, I2 = 45%). However, subgroup analyses showed that chlorhexidine prevents VAP development when used at 2% concentration (relative risk 0.53, 95% CI 0.31–0.91, I2 = 0%) or 4 times/d (relative risk 0.56, 95% CI 0.38–0.81, I2 = 0%). CONCLUSIONS: We found that oral care with chlorhexidine is effective in reducing VAP incidence in the adult population if administered at 2% concentration or 4 times/d.

Efficacy of stem cells on the healing of peri-implant defects: systematic review of preclinical studies

This systematic review considers the evidence from animal studies evaluating the effectiveness of mesenchymal stem cells (MSC) in the treatment of intraoral peri‐implant defects. MEDLINE, EMBASE, and LILACS databases were searched for quantitative preclinical controlled animal model studies that evaluated the effect of MSC on bone healing at intraoral peri‐implant bone defects. The primary outcome was the amount of (re‐)osseointegration reported as bone‐to‐implant contact in the defect area. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses statement guidelines. Ten studies met the inclusion criteria. Only one study induced peri‐implant inflammation to produce peri‐implant bone defects. In all others, defects were surgically created at implant installation. Differences in defect morphology were identified among the studies. Both xenogenous and autogenous MSC were used to treat peri‐implant defects. These included bone marrow‐derived MSC, periodontal ligament‐derived MSC, umbilical cord MSC, bone marrow‐derived mononuclear cells, and peripheral blood mononuclear cells. Meta‐analysis was not possible because of heterogeneities in study designs. Nonetheless, in most studies, local MSC implantation was not associated with adverse effects and had a positive effect on bone healing around peri‐implant defects. Combination of MSC with membranes and bioactive factors appears to provide improved treatment outcomes. In large animal models, intraoral use of MSC may provide beneficial effects on bone healing within peri‐implant defects. The various degrees of success of MSC in peri‐implant bone healing are likely to be related to the use of cells from various populations, tissues, and donor species. However, human safety and efficacy must be demonstrated before its clinical use can be considered.