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IntroductionOne of the challenges in treating Clostridioides difficile infection (CDI) is that the bacterium forms biofilms, a critical virulence mechanism known to promote antibiotic resistance and, as a result, consequently, a higher recurrence of the disease. The goal of this study was to compare the ability of three MLST Clade 2 strains to form a biofilm in vitro: ICC-45 (ribotype SLO231/UK[CE]821), a ST41 toxinotype IXb isolated in Brazil; and two epidemic NAP1/027/ST01 strains: NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica and the reference epidemic strain NAP1/027/ST01 (R20291); and ATCC700057, a non-toxigenic strain.MethodsThe ability of strains to form biofilm was evaluated using crystal violet staining. In addition, samples were stained with the Film Tracer biofilm matrix (Invitrogen®) and the biofilm matrix thickness was measured using confocal microscopy. The matrix architecture was determined using Scanning electron microscop. Confocal microscopy was used to detect the presence of toxin A (tcdA) using an anti-Clostridioides difficile TcdA antibody. The expression of virulence genes (tcdA, tcdB, tcdC, cdtB, spo0A, slpA, cwp66 and cwp84) was examined, as well as the effect of antibiotics metronidazole (MTZ) and vancomycin (VAN) on biofilm growth.ResultsAll of the strains tested formed a moderate biofilm with 1.1 <DO570nm>3.5. After 72h, biofilm biomass of the NAP1/027/ST01 epidemic strains (LIBA5756 and R20291) was significantly higher than ICC-45 and ATCC 700057 biofilms, as confirmed by electron and confocal microscopy. At 120h, the LIBA5756 biofilm biomass decreased compared to other strains. The toxigenic strains R20291 or LIBA 5756 had higher expression of genes tcdA, tcdB, tcdC, cdtA, slpA and spo0A than ICC-45, but there were no significant differences in the expression levels of cdtB, cwp66 and cwp84. In epidemic strains, VAN and MTZ inhibited biofilm formation; however, in the ICC-45 strain, MIC concentrations of VAN and MIC and 4MIC of MTZ did not inhibit biofilm formation.ConclusionThe three MLST Clade 2 isolated from different rybotipes, two of which were isolated from Latin America, are competent biofilm-forming bacteria, indicating their ability to induce C. difficile infection recurrence, making treatment difficult.

Objectives We aimed to evaluate the effect of nanohydroxyapatite morphology and its interaction with anionic collagen on osteoblast activity. Materials and Methods Murine osteoblasts were incubated with a commercial collagen scaffold (as a control) or collagen-nanohydroxyapatite scaffolds (Col-HANP) for 24 and 48 hours for viability and proliferation assessments by MTT and Ki67 immunofluorescence, respectively. The hydroxyapatite nanoparticles were synthesized in three different morphologies/sizes (labeled as Col-HANP 0h, as Col-HANP 2h, and as Col-HANP 5h) as a function of the hydrothermal synthetic approach. Osteoblast's activity was investigated by bone alkaline phosphatase activity (ALP) and Von Kossa mineralization assays. For biocompatibility evaluation, the scaffolds were implanted subcutaneously in the dorsum of male Wistar rats for 7 and 15 days. Results The incubation of cells with Col-HANP 5h for 48h resulted in a significant increase in their proliferation and activity. The implantation of Col-HANP 5h in the subcutaneous tissue presented decreased recruitment of inflammatory cells and IL-1β levels on day 7, as well as an increase in collagen synthesis on day 15 compared to collagen and control groups. Conclusions The significant effects on osteoblasts proliferation and activity illustrate the potential application of Col-HANP 5h scaffold as a promising strategy for bone tissue engineering.

Injectable facial fillers are excellent options for treating facial aging, wrinkles, and contour defects. Both polycaprolactone (PCL) and hyaluronic acid (HA) have been used to restore lost tissue volume and improve facial contour. However, the mechanisms involved in the effect of these biomaterials still need to be fully understood. The present work aims to establish an experimental model to investigate cellular and morphological changes in the skin of Wistar rats in response to HA and PCL to understand the mechanisms associated with these effects. The subcutaneous tissue of the back of Wistar rats was used as a reception area for biomaterials, represented by the commercial products Ellansé®, containing polycaprolactone (PCL) and Juvederm Voluma®, containing hyaluronic acid (HA). Animals were euthanized afetr 30 or 60 days, and skin samples were collected from treated and untreated animals (CONTROL) for histological and immunohistochemical evaluation for IBA-1, TGF-β, and FGF. Analysis of type I and type III collagen deposition, neovascularization, and adipose tissue was performed. On histological examination, HA appeared as an amorphous, basophilic material interspersed with connective tissue bundles. The skin fragments with PCL showed intense cell proliferation, with foreign body giant cells and a higher capillary proliferation than the HA group. More vessels were observed in the HA and PCL groups compared to the CONTROL group. A significant increase in fibroblasts and fibrocytes was observed in skin fragments inoculated with HA and PCL, associated with increased FGF expression. The number of fibroblasts was significantly higher in the PCL group than HA. The PCL group showed higher immunostaining for IBA-1 and TGF-β than the CONTROL and HA groups. Collagen deposition was observed in the treated groups, especially type III collagen in the PCL group, when compared to HA. Our morphological results demonstrated stimulation of fibroblastic activity and active-related tissue regeneration, with increased vascular proliferation and expression of markers related to tissue proliferation, mainly associated with the PCL group. We also observed increased adipose tissue, although further studies are needed to confirm these findings.