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Infection is a major concern in surgery involving grafting and should be considered thoroughly when designing biomaterials. There is considerable renewed interest in silver nanoparticles (AgNPs) owing to their ability to potentiate antibacterial properties against multiple bacterial strains. This study aimed to develop two antibacterial bone regenerative scaffolds by integrating AgNPs in bovine bone particles (BBX) (Product 1), and a light cross-linked hydrogel GelMA (Product 2). The constructs were characterized using scanning electron microscopy. Metabolic activity of osteoblasts and osteoclasts on the constructs was investigated using PrestoBlue™. Disk diffusion assay was conducted to test the antibacterial properties. The regenerative capacity of the optimized AgNP functionalized BBX and GelMA were tested in a rabbit cranial 6 mm defect model. The presence of AgNPs appears to enhance proliferation of osteoblasts compared to AgNP free controls in vitro. We established that AgNPs can be used at a 100 µg dose that inhibits bacteria, with minimal adverse effects on the bone cells. Our rabbit model revealed that both the BBX and GelMA hydrogels loaded AgNPs were biocompatible with no signs of necrosis or inflammatory response. Grafts functionalized with AgNPs can provide antibacterial protection and simultaneously act as a scaffold for attachment of bone cells.
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The physicochemical properties of grafting materials affect the quality of the osteointegration, resorption rate, and the new bone (NB) formation. This study assessed the physicochemical properties and integration of a low temperature deproteinized bovine bone xenograft (BBX), referred to as optimized MoaBone® (OMB). This novel BBX was physiochemically characterized both pre and post chemical bleaching and sterilization by gamma irradiation. OMB was compared to two commercial BBX; Bio-Oss® (BO) and MoaBone® (MB) using a rabbit cranial model. Residual graft and NB were quantified using histology and micro-computed tomography. Results showed that chemical treatment and gamma irradiation had limited effect on the surface texture. A significant decrease in the collagen content was detected post chemical treatment and in the carbonate content post gamma irradiation. There was no evidence of inflammatory infiltrate, necrosis, or connective tissue encapsulation, and a significant increase of NB in all grafted sites as compared to untreated defects could be observed. However, there was no statistically significant difference between the grafted sites. We conclude that chemical treatment and terminal sterilization strongly impact the final graft's properties. OMB graft showed equivalence with BO for in vivo bone formation and potentially results in lower levels of graft retention.
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Transplante Ósseo , Crânio , Animais , Coelhos , Bovinos , Crânio/patologia , Crânio/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Xenoenxertos , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Temperatura Baixa , Microtomografia por Raio-X , Raios gama , MineraisRESUMO
There is a need to develop bifunctional scaffolds that provide antibacterial protection while encouraging host cell attachment/proliferation. This study evaluates HyStem®-C, and photo-cross-linked GelMA hydrogels for encapsulation and stabilisation of silver nanoparticles (AgNPs). We studied the behaviour of AgNPs and matrix interactions within both hydrogel systems. The cell viability of encapsulated human gingival fibroblasts (HGFs) was determined by Prestoblue® assay and live/dead staining. The release of AgNPs was monitored by inductively coupled plasma-mass spectroscopy. The antibacterial properties of the GelMA-AgNP constructs were determined using disc diffusion. Even distribution of AgNPs in GelMA induced a significant decrease in cell viability (p < 0.0001), whereas AgNP aggregates did not induce cytotoxicity in HyStem®-C. AgNPs doses ≥ 0.5 µg/mL in GelMA were significantly toxic to the HGFs (p < 0.0001). The release of AgNPs from GelMA after 48 h was 20% w/w for 0.1 µg/mL and 51% for 100 µg/mL of AgNPs. At ≥5 µg/mL, a significant intra-construct bactericidal effect was observed. The disc diffusion assay shows that GelMA-incorporated AgNPs were found to be effective against both Escherichia coli and Staphylococcus aureus at 50 and 100 µg/mL, respectively. Visible photo-cross-linked GelMA stably incorporated AgNPs to provide an antimicrobial regenerative construct for oral applications.
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BACKGROUND: Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored. OBJECTIVES: Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models. METHODS: Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models. RESULTS: Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins. CONCLUSIONS: These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema.
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Dermatite Atópica , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Queratinócitos/metabolismo , Epiderme/metabolismo , Inflamação , Diferenciação Celular , Infecções Estafilocócicas/patologiaRESUMO
BACKGROUND: Surgical safety checklist (SSC) use benefits veterinary patients, but endorsement and implementation are essential for these benefits to be observed. METHODS: A cross-sectional survey assessed UK veterinary professionals' attitudes towards and usage of SSCs and identified factors associated with poorer attitude or failure to use SSCs. RESULTS: Of 513 respondents, 70% used SSCs. Of these, 87.1% used SSCs for every surgical procedure, 19.1% adapted SSCs for different procedures and 61.1% had a standard operating procedure detailing how to use SSCs. Attitudes towards SSC use were favourable, with increased positive attitude associated with employing at least one registered veterinary nurse with a post-qualifying qualification (p < 0.001), current SSC use (p < 0.001), undertaking self-directed reading (p = 0.033) or completing SSC-relevant post-qualification continuing professional development (p = 0.005). Factors associated with veterinary practices not using SSCs included Practice Standards Scheme (PSS) non-membership (odds ratio [OR] 2.0, 1.1-3.4), no RCVS hospital status (OR 1.9, 1.1-3.5) or being a mixed first-opinion veterinary practice (OR 2.4, 1.2-5.0). LIMITATIONS: Study limitations include sampling methodology and non-validated attitudinal scale usage. CONCLUSION: Most respondents used SSCs. Familiarity, education and the RCVS PSS were associated with improved uptake and attitudes, but mixed practice was associated with reduced usage.
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Lista de Checagem , Hospitais , Animais , Lista de Checagem/métodos , Estudos Transversais , Atitude do Pessoal de Saúde , Reino Unido , Segurança do PacienteRESUMO
Ideal bone grafting scaffolds are osteoinductive, osteoconductive, and encourage osteogenesis through the remodeling processes of bone resorption, new bone formation, and successful integration or replacement; however, achieving this trifecta remains challenging. Production methods of bone grafts, such as thermal processing, can have significant effects on the degree of cell-surface interactions via wide-scale changes in the material properties. Here, we investigated the effects of small incremental changes at low thermal processing temperatures on the degree of osteoclast and osteoblast attachment, proliferation, and differentiation. Bovine bone scaffolds were prepared at 100, 130, 160, 190, and 220 °C and compared with a commercial control, Bio-Oss®. Osteoclast attachment and activity were significantly higher on lower temperature processed bone and were not present ≥190 °C. The highest osteoblast proliferation and differentiation were obtained from treatments at 130 and 160 °C. Similarly, qRT2-PCR assays highlighted osteoblasts attached to bone processed at 130 and 160 °C as demonstrating the highest osteogenic gene expression. This study demonstrated the significant effects of small-scale processing changes on bone graft materials in vitro, which may translate to a tailored approach of cellular response in vivo.
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Bovine bone grafts (BBX) require protein removal as part of the manufacturing process to reduce antigenicity and, in consequence, to be safely used in humans. Deproteinisation may have direct effects on the characteristics of the bone material and on in vivo material performance. This research aimed to comprehensively study the physicochemical and mechanical properties of BBX processed at low deproteinisation processing temperatures. Cubes of bovine bone (8 mm3) were treated with temperatures between 100 °C and 220 °C at 30 °C intervals and with pressures ranging from 1.01 to 24.58 Bar. The samples were characterised topographically and mechanically using scanning electron microscopy (SEM), atomic force microscopy (AFM), and uniaxial bending tests. The organic content and the chemical composition were determined using thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR). X-ray diffraction (XRD) and FTIR were also used to quantitatively determine the specimen crystallinity. Increasing temperature/pressure was associated with decreasing protein levels and compressive strength and increasing surface irregularities and crystallinity. The findings suggest that low-temperature processed bone is likely to exhibit a rapid in vivo degradation rate. The deproteinisation temperature can be adjusted to tailor the graft properties for specific applications.
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OBJECTIVES: Investigate the effect of aging on the wear behavior of glazed vs polished monolithic zirconia and to establish if glazing provides protection against low temperature degradation. METHODS: 40 1-mm-diameter spheres made from four differently treated monolithic zirconia (VITA YZ® HT); polished, polished-aged, glazed and glazed-aged (n = 10), were tested in a wear testing machine (UFW200) against bovine enamel in artificial saliva as per the following settings (ISO20808:2016): ball-on-disc configuration, 5 N vertical load, 0.1 m/s sliding speed, 400 m sliding distance and 37 °C temperature. Vertical substance loss (mm) wear of zirconia and enamel specimens was measured. Data were statistically analyzed with Kruskal-Wallis one-way ANOVA test (α > 0.05). RESULTS: Glazed-aged zirconia specimens resulted in the greatest amount of enamel wear (0.823 mm ± 0.157) followed by glazed (0.729 mm ± 0.289), polished-aged (0.377 mm ± 0.201) then polished (0.247 mm ± 0.125). In the groups with the same surface finish, aging showed no statistical difference in wear (P > 0.008). Glazing resulted in a higher enamel wear compared with polishing that was statistically significant (P < 0.008) except when the polished specimens were aged and the glazed specimens were not aged. SIGNIFICANCE: Aging increases abrasiveness of monolithic zirconia regardless of the type of surface finish. The effect of aging is "latent" and only revealed under mechanical loading during wear simulation which increases surface roughness and wear by adversely affecting zirconia's mechanical properties, making it less capable to maintain its initial surface smoothness. The glaze layer may protect zirconia from LTD, however, it is susceptible to aging which further increases its abrasiveness.
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Polimento Dentário , Porcelana Dentária , Animais , Bovinos , Teste de Materiais , Propriedades de Superfície , ZircônioRESUMO
Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.
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Fatores Reguladores de Interferon/metabolismo , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Epiderme/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fatores Reguladores de Interferon/genética , Transdução de Sinais , Transcrição Gênica , Fator de Necrose Tumoral alfa/genéticaRESUMO
Manuka oil, an essential oil derived from the Leptospermum scoparium, has been traditionally used for wound care and as a topical antibacterial, antifungal, and anti-inflammatory. However, the essential oil is not well retained at mucosal sites, such as the oral cavity, where the benefits of the aforementioned properties could be utilized toward the treatment of persistent biofilms. Within this study, L. scoparium essential oil was incorporated into a semisolid emulsion for improved delivery. The safety profile of L. scoparium essential oil on human gingival fibroblasts was determined via cell viability, cytotoxicity, and caspase activation. The minimal bactericidal concentration of L. scoparium essential oil was determined, and the emulsion's antibiofilm effects visualized using confocal laser scanning microscopy. L. scoparium essential oil demonstrated a lower IC50 (0.02% at 48 h) when compared to the clinical control chlorhexidine (0.002% at 48 h) and displayed lower cumulative cytotoxicity. Higher concentrations of L. scoparium essential oil (≥ 0.1%) at 6 h resulted in higher caspase 3/7 activation, suggesting an apoptotic pathway of cell death. A minimal bactericidal concentration of 0.1% w/w was observed for 6 oral bacteria and 0.01% w/v for Porphyromonas gingivalis. Textural and rheometric analysis indicated increased stability of emulsion with a 1â:â3 ratio of L. scoparium essential oil: Oryza sativa carrier oil. The optimized 5% w/w L. scoparium essential oil emulsion showed increased bactericidal penetrative effects on Streptococci gordonii biofilms compared to oil alone and to chlorhexidine controls. This study has demonstrated the safety, formulation, and antimicrobial activity of L. scoparium essential oil emulsion for potential antibacterial applications at mucosal sites.
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Leptospermum , Óleos Voláteis , Antibacterianos/farmacologia , Biofilmes , Emulsões , Óleos Voláteis/farmacologiaRESUMO
The synthesis and specific surface functionalization of antimicrobial silver nanoparticles (AgNPs) and their incorporation into an alginate hydrogel is described. Divalent cation-mediated ionic crosslinking was used to disperse the AgNPs throughout the gel, made possible by -COO- cross-linking sites provided by the surface-enhanced nanoparticles, inspired by the classic egg-box model crosslinking of calcium alginate. An AgNP concentration, 10-20 µg g-1 increased hygrogel elasticity, viscosity, and shear resistance by 45, 30, and 31% respectively. Cryo-TEM revealed evenly distributed AgNP assemblies of discrete AgNPs throughout the gel matrices. FTIR-ATR indicated AgNPs were involved in alginate carboxylate-Ca2+-COO-AgNP crossbridging, which was not achieved through mixing of AgNPs into preformed gels. Live/dead fluorometric assays determined a minimal bactericidal concentration of 25 µg g-1 Ag for 6 microorganisms. Anti-biofilm assays showed species-dependent cell death of 44 -61%, with limited silver ion release of 0.41% and 1.1% after 7 days for Gram positive and negative bacteria, respectively.