Repurposing sarecycline for osteoinductive therapies: an in vitro and ex vivo assessment.

INTRODUCTION: Tetracyclines (TCs) embrace a class of broad-spectrum antibiotics with unrelated effects at sub-antimicrobial levels, including an effective anti-inflammatory activity and stimulation of osteogenesis, allowing their repurposing for different clinical applications. Recently, sarecycline (SA)-a new-generation molecule with a narrower antimicrobial spectrum-was clinically approved due to its anti-inflammatory profile and reduced adverse effects verified with prolonged use. Notwithstanding, little is known about its osteogenic potential, previously verified for early generation TCs. MATERIALS AND METHODS: Accordingly, the present study is focused on the assessment of the response of human bone marrow-derived mesenchymal stromal cells (hBMSCs) to a concentration range of SA, addressing the metabolic activity, morphology and osteoblastic differentiation capability, further detailing the modulation of Wnt, Hedgehog, and Notch signaling pathways. In addition, an ex vivo organotypic bone development system was established in the presence of SA and characterized by microtomographic and histochemical analysis. RESULTS: hBMSCs cultured with SA presented a significantly increased metabolic activity compared to control, with an indistinguishable cell morphology. Moreover, RUNX2 expression was upregulated 2.5-fold, and ALP expression was increased around sevenfold in the presence of SA. Further, GLI2 expression was significantly upregulated, while HEY1 and HNF1A were downregulated, substantiating Hedgehog and Notch signaling pathways' modulation. The ex vivo model developed in the presence of SA presented a significantly enhanced collagen deposition, extended migration areas of osteogenesis, and an increased bone mineral content, substantiating an increased osteogenic development. CONCLUSION: Summarizing, SA is a promising candidate for drug repurposing within therapies envisaging the enhancement of bone healing/regeneration.

Microgap and bacterial microleakage during the osseointegration period: An in vitro assessment of the cover screw and healing abutment in a platform-switched implant system.

STATEMENT OF PROBLEM: Microgap and bacterial microleakage at the implant-prosthetic abutment interface are recognized concerns for implant-supported restorations, leading to inflammation of the peri-implant tissues, with deleterious consequences for crestal bone levels. However, little is known regarding the interface established between the implant and the healing abutment or cover screw placed for the osseointegration phase. PURPOSE: The purpose of this in vitro study was to characterize the implant-cover screw and implant-healing abutment interfaces of a platform-switched implant system to determine the microgap and bacterial microleakage of the system and evaluate the biological response and functionality of an interface sealing agent. MATERIAL AND METHODS: The interfacial microgaps of the implant-healing abutment and implant-cover screw interfaces were characterized by scanning electron microscopy (n=10), and bacterial microleakage was evaluated after colonization with Enterococcus faecalis in a 30-day follow-up (n=10). The sealing efficacy and irritation potential of a silicone-based sealer were determined by using the hen's egg test on chorioallantoic membrane assay. The 2-sample t test was performed to compare means between groups, and data presented with the Kaplan-Meier method were compared statistically by using the log-rank test (α=.05). RESULTS: The interfacial microgap was less than 2.5 µm for both systems. Bacterial microleakage was noted in approximately 50% of the specimens, particularly at early time points, at both the healing abutment and cover screw interfaces. The silicone-based sealer prevented bacterial leakage in the experimental setting. CONCLUSIONS: The implant-healing abutment and implant-cover screw interfaces of the tested system, despite the low microgap, allowed for bacterial microleakage after internal colonization. The use of a nonirritating silicone-based sealing agent effectively sealed the system.

The Osteogenic Assessment of Mineral Trioxide Aggregate-based Endodontic Sealers in an Organotypic Ex Vivo Bone Development Model.

INTRODUCTION: Mineral trioxide aggregate (MTA)-based sealers are endodontic materials with widespread success in distinct clinical applications, potentially embracing direct contact with the bone tissue. Bone response to these materials has been traditionally addressed in vitro. Nonetheless, translational data are limited by the absence of native cell-to-cell and cell-to-matrix interactions that hinder the representativeness of the analysis. Ex vivo organotypic systems, relying on the culture of explanted biological tissues, preserve the cell/tissue composition, reproducing the spatial and organizational in situ complexity. This study was grounded on an innovative research approach, relying on the assessment of an ex vivo organotypic bone tissue culture system to address the osteogenic response to 3 distinct MTA-based sealers. METHODS: Embryonic chick femurs were isolated and grown ex vivo for 11 days in the presence of MTA Plus (Avalon Biomed Inc, Bradenton, FL), ProRoot MTA (Dentsply Tulsa Dental, Hohnson City, Germany), Biodentine (Septodont, Saint Maurdes Fosses, France), or AH Plus (Dentsply Sirona, Konstanz, Germany); the latter was used as a control material. Femurs were characterized by histologic, histochemical, and histomorphometric analysis. Gene expression assessment of relevant osteogenic markers was conducted by quantitative polymerase chain reaction. RESULTS: All MTA-based sealers presented an enhanced osteogenic performance compared with AH Plus. Histochemical and histomorphometric analyses support the increased activation of the osteogenic program by MTA-based sealers, with enhanced collagenous matrix deposition and tissue mineralization. Gene expression analysis supported the enhanced activation of the osteogenic program. Comparatively, ProRoot MTA induced the highest osteogenic functionality on the characterized femurs. CONCLUSIONS: MTA-based sealers enhanced the osteogenic activity within the assayed organotypic bone model, which was found to be a sensitive system for the assessment of osteogenic modulation mediated by endodontic sealers.

Nano-hydroxyapatite in oral care cosmetics: characterization and cytotoxicity assessment.

Nano-hydroxyapatite has been used as an oral care ingredient, being incorporated in several products for the treatment of dental hypersensitivity and enamel remineralisation. Despite its promising results, regulatory and safety concerns have been discussed and questioned by the European Scientific Committee on Consumer Safety (SCCS) regarding the usage of hydroxyapatite nanoparticles in oral care products. In this work, a commercially available nano-hydroxyapatite was characterized and its cytocompatibility towards human gingival fibroblasts was evaluated, as well as its irritation potential using the in vitro HET-CAM assay. All the conditions chosen in this study tried to simulate the tooth brushing procedure and the hydroxyapatite nanoparticles levels normally incorporated in oral care products. The commercial hydroxyapatite nanoparticles used in this study exhibited a rod-like morphology and the expected chemical and phase composition. The set of in vitro cytotoxicity parameters accessed showed that these nanoparticles are highly cytocompatible towards human gingival fibroblasts. Additionally, these nanoparticles did not possess any irritation potential on HET-CAM assay. This study clarifies the issues raised by SCCS and it concludes that this specific nano-hydroxyapatite is cytocompatible, as these nanoparticles did not alter the normal behaviour of the cells. Therefore, they are safe to be used in oral care products.

Engineering a multifunctional 3D-printed PLA-collagen-minocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration.

3D-printing and additive manufacturing can be powerful techniques to design customized structures and produce synthetic bone grafts with multifunctional effects suitable for bone repair. In our work we aimed the development of novel multifunctionalized 3D printed poly(lactic acid) (PLA) scaffolds with bioinspired surface coatings able to reduce bacterial biofilm formation while favoring human bone marrow-derived mesenchymal stem cells (hMSCs) activity. For that purpose, 3D printing was used to prepare PLA scaffolds that were further multifunctionalized with collagen (Col), minocycline (MH) and bioinspired citrate- hydroxyapatite nanoparticles (cHA). PLA-Col-MH-cHA scaffolds provide a closer structural support approximation to native bone architecture with uniform macroporous, adequate wettability and an excellent compressive strength. The addition of MH resulted in an adequate antibiotic release profile that by being compatible with local drug delivery therapy was translated into antibacterial activities against Staphylococcus aureus, a main pathogen associated to bone-related infections. Subsequently, the hMSCs response to these scaffolds revealed that the incorporation of cHA significantly stimulated the adhesion, proliferation and osteogenesis-related gene expression (RUNX2, OCN and OPN) of hMSCs. Furthermore, the association of a bioinspired material (cHA) with the antibiotic MH resulted in a combined effect of an enhanced osteogenic activity. These findings, together with the antibiofilm activity depicted strengthen the appropriateness of this 3D-printed PLA-Col-MH-cHA scaffold for future use in bone repair. By targeting bone repair while mitigating the typical infections associated to bone implants, our 3D scaffolds deliver an integrated strategy with the combined effects further envisaging an increase in the success rate of bone-implanted devices.

Assessment of Coagulase-Negative Staphylococci and Lactic Acid Bacteria Isolated from Portuguese Dry Fermented Sausages as Potential Starters Based on Their Biogenic Amine Profile.

The aim of this study was to evaluate the decarboxylase activity of coagulase-negative staphylococci (CNS) and lactic acid bacteria (LAB) involved in meat products fermentation, in order to characterize and select the strains most suitable to be used as safe starter cultures. Isolates were obtained from traditional Portuguese dry fermented meat sausages, identified by PCR and characterized according to their technological properties. Lactobacilli and enterococci were assessed for their bacteriocinogenic potential. Biogenic amines (BA) were screened by culture method and analyzed by RP-HPLC/UV. The screening method, compared with chromatographic analysis, was not reliable for CNS and LAB strains selection. Tyramine decarboxylase activity was present in CNS strains, with a slight production of amines. No other hazardous BA were produced. Among lactobacilli, moderate production of tyramine was related only to Lactobacillus curvatus, with some strains producing putrescine or 2-phenylethylamine. Enterococci were high and moderate producers of tyramine and 2-phenylethylamine, respectively. Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus carnosus, independent of their genetic and technological profiles and BA production, were adequate for use in meat products, according to the data. Lactobacillus plantarum and Lactobacillus sakei strains could also be selected for starters. PRACTICAL APPLICATION: The selection of coagulase-negative staphylococci and lactic acid bacteria (LAB) isolates were based on their production of biogenic amines in order to avoid this potential hazard production in meat products. The most suitable isolates could be used as safe starter cultures in meat products industry. The staphylococci and LAB selected will achieve particular organoleptic characteristics in meat products and bioprotection from pathogens.

In vivo assessment of a new multifunctional coating architecture for improved Mg alloy biocompatibility.

Magnesium alloys are regarded as potential biodegradable load-bearing biomaterials for orthopedic applications due to their physico-chemical and biomechanical properties. However, their clinical applicability is restricted by their high degradation rate, which limits the physiological reconstruction of the neighbouring tissues. In this work, a multifunctional coating architecture was developed on an AZ31 alloy by conjoining an anodization process with the deposition of a polymeric-based layer consisting of polyether imine reinforced with hydroxyapatite nanoparticles, aiming at improved control of the corrosion activity and biological performance of the Mg substrate. Anodization and coating protocols were evaluated either independently or combined for corrosion resistance and biological behaviour, i.e. the irritation potential and angiogenic capability within a chicken chorioallantoic membrane assay, and bone tissue response following tibia implantation within a rabbit model. Electrochemical impedance spectroscopy (EIS) analysis showed that coated Mg constructs, particularly anodized plus coated with AZ31, exhibited excellent stability compared to the anodized alloy and, particularly, to the bare AZ31. Microtomographic evaluation of the implanted samples correlated with these degradation results. Mg constructs displayed a non-irritating behaviour, and were associated with high levels of vascular ingrowth. Bone ingrowth neighbouring the implanted constructs was observed for all samples, with coated and anodized plus coated samples presenting the highest bone formation. Gene expression analysis suggested that the enhanced bone tissue formation was associated with the boost in osteogenic activity through Runx2 upregulation, following the activation of PGC-1α/ERRα signaling. Overall, the developed multifunctional coatings appear to be a promising strategy to obtain safe and bioactive biodegradable Mg-based implants with potential applications within bone tissue.