3D-printed biosurfactant-chitosan antibacterial coating for the prevention of silicone-based associated infections.

Infections associated with the surfaces of medical devices represent a critical problem due to biofilm formation and the growing resistance towards antibacterial drugs. This is particularly relevant in commonly used invasive devices such as silicone-based ones where a demand for alternative antibiofilm surfaces is increasing. In this work, an antimicrobial chitosan-biosurfactant hydrogel mesh was produced by 3D-printing. The 3D structure was designed to coat polydimethylsiloxane-based medical devices for infection prevention. Additionally, the porous 3D structure allows the incorporation of customized bioactive components. For this purpose, two biosurfactants (surfactin and sophorolipids) were biosynthesized and tested for their antimicrobial activity. In addition, the printing of surfactant-chitosan-based coatings was optimized, and the resulting 3D structures were characterized (i.e., wettability, FTIR-ATR, antimicrobial activity, and biocompatibility). Compared with surfactin, the results showed a better yield and higher antibacterial activity against Gram-positive bacteria for sophorolipids (SLs). Thus, SLs were used to produce chitosan-based 3D-printed coatings. Overall, the SLs-impregnated coatings showed the best antibacterial activity against Staphylococcus aureus planktonic bacteria (61 % of growth inhibition) and antibiofilm activity (2 log units reduction) when compared to control. Furthermore, concerning biocompatibility, the coatings were cytocompatible towards human dermal fibroblasts. Finally, the coating presented a mesh suitable to be filled with a model bioactive compound (i.e., hyaluronic acid), paving the way to be used for customized therapeutics.

Novel Organic Solvent Nanofiltration Approaches for Microbial Biosurfactants Downstream Processing.

Glycolipid biosurfactants are the most prominent group of microbial biosurfactants, comprising rhamnolipids, sophorolipids and mannosylerythritol lipids (MELs). Usually, large amounts of hydrophobic substrates (e.g., vegetable oils) are used to achieve high titers (~200 g/L) of a crude product of low purity at values limited to 50-60%, contaminated with unconsumed triacylglycerol and residual free fatty acids and monoacylglycerides. The methods reported for the removal of these contaminants use a mixture of organic solvents, compromising solvent recyclability and increasing final process costs. This study reports, for the first time, an innovative downstream method for MELs, in which 90% of the triacylglycerols are separated from the crude MEL mixture in a first stage and the other lipid derivatives (free fatty acids, mono- and diacylglycerols) are removed by organic solvent nanofiltration (OSN). Three commercially available membranes (GMT-oNF-2, PuraMEm-600 and DuramMem-500) and several homemade membranes, casted from 22, 24 or 26% (w/v) polybenzimidazole (PBI) solutions, were assessed for crude MELs purification by diafiltration. A final purity of 87-90% in the MELs was obtained by filtering two diavolumes of methanol or ethyl acetate solutions through a PBI 26% membrane, resulting in MELs losses of 14.7 ± 6.1% and 15.3 ± 2.2%, respectively. Higher biosurfactant purities can be archived using the PBI 26% membrane at higher DV, but at the cost of higher product losses. Namely, in MeOH, the use of 6 DV leads to losses of 32% for MELs and 18% for sophorolipids. To obtain MELs at reagent grade with purities equal or higher than 97%, a two-sequential cascade filtration approach was implemented using the commercial membrane, GMT-oNF. In such a process, MELs with 98% purity was obtained at the cost of 11.6% MELs losses. Finally, decoloration, important in some applications, was successfully assessed using activated carbon. Overall, this study reports a unique solution for microbial biosurfactants production with minimal product losses, enabling solvent recycling and potentially reducing costs.

Improving Chitosan Hydrogels Printability: A Comprehensive Study on Printing Scaffolds for Customized Drug Delivery.

Chitosan is an interesting polymer to produce hydrogels suitable for the 3D printing of customized drug delivery systems. This study aimed at the achievement of chitosan-based scaffolds suitable for the incorporation of active components in the matrix or loaded into the pores. Several scaffolds were printed using different chitosan-based hydrogels. To understand which parameters would have a greater impact on printability, an optimization study was conducted. The scaffolds with the highest printability were obtained with a chitosan hydrogel at 2.5 wt%, a flow speed of 0.15 mm/s and a layer height of 0.41 mm. To improve the chitosan hydrogel printability, starch was added, and a design of experiments with three factors and two responses was carried out to find out the optimal starch supplementation. It was possible to conclude that the addition of starch (13 wt%) to the chitosan hydrogel improved the structural characteristics of the chitosan-based scaffolds. These scaffolds showed potential to be tested in the future as drug-delivery systems.

Development of a chlorhexidine delivery system based on dental reline acrylic resins.

The high recurrence rate of common denture stomatitis after antifungal treatment is still concerning. This condition is caused by low patient compliance and incomplete local elimination of the main etiological factor - Candida albicans, often associated with other microorganisms, such as Streptococcus species. Impregnating denture materials with antimicrobials for local delivery is a strategy that can overcome the side effects and improve the efficacy of conventional treatments (topical and/or systemic). In this work, we describe the development of three hard autopolymerizing reline acrylic resins (Kooliner, Ufi Gel Hard, and Probase Cold) loaded with different percentages of chlorhexidine (CHX). The novel formulations were characterized based on their antimicrobial activity, mechanical, morphological and surface properties, in-vitro drug release profiles, and cytotoxicity. The addition of CHX in all resins did not change their chemical and mechanical structure. Among all the tested formulations, Probase Cold loaded with 5 wt% CHX showed the most promising results in terms of antimicrobial activity and lack of serious detrimental mechanical, morphological, surface, and biological properties.

Novel Features of Cellulose-Based Films as Sustainable Alternatives for Food Packaging.

Packaging plays an important role in food quality and safety, especially regarding waste and spoilage reduction. The main drawback is that the packaging industry is among the ones that is highly dependent on plastic usage. New alternatives to conventional plastic packaging such as biopolymers-based type are mandatory. Examples are cellulose films and its derivatives. These are among the most used options in the food packaging due to their unique characteristics, such as biocompatibility, environmental sustainability, low price, mechanical properties, and biodegradability. Emerging concepts such as active and intelligent packaging provides new solutions for an extending shelf-life, and it fights some limitations of cellulose films and improves the properties of the packaging. This article reviews the available cellulose polymers and derivatives that are used as sustainable alternatives for food packaging regarding their properties, characteristics, and functionalization towards active properties enhancement. In this way, several types of films that are prepared with cellulose and their derivatives, incorporating antimicrobial and antioxidant compounds, are herein described, and discussed.

Assuring the Biofunctionalization of Silicone Covalently Bonded to Rhamnolipids: Antibiofilm Activity and Biocompatibility.

Silicone-based medical devices composed of polydimethylsiloxane (PDMS) are widely used all over the human body (e.g., urinary stents and catheters, central venous catheters stents) with extreme clinical success. Nevertheless, their abiotic surfaces, being prone to microorganism colonization, are often involved in infection occurrence. Improving PDMS antimicrobial properties by surface functionalization with biosurfactants to prevent related infections has been the goal of different works, but studies that mimic the clinical use of these novel surfaces are missing. This work aims at the biofunctional assessment of PDMS functionalized with rhamnolipids (RLs), using translational tests that more closely mimic the clinical microenvironment. Rhamnolipids were covalently bonded to PDMS, and the obtained surfaces were characterized by contact angle modification assessment, ATR-FTIR analysis and atomic force microscopy imaging. Moreover, a parallel flow chamber was used to assess the Staphylococcus aureus antibiofilm activity of the obtained surfaces under dynamic conditions, and an in vitro characterization with human dermal fibroblast cells in both direct and indirect characterization assays, along with an in vivo subcutaneous implantation assay in the translational rabbit model, was performed. A 1.2 log reduction in S. aureus biofilm was observed after 24 h under flow dynamic conditions. Additionally, functionalized PDMS lessened cell adhesion upon direct contact, while supporting a cytocompatible profile, within an indirect assay. The adequacy of the biological response was further validated upon in vivo subcutaneous tissue implantation. An important step was taken towards biofunctional assessment of RLs-functionalized PDMS, reinforcing their suitability for medical device usage and infection prevention.

Drug Delivery from PCL/Chitosan Multilayer Coatings for Metallic Implants.

Implant-related infections, mainly caused by Staphylococcus aureus, are a major health concern. Treatment is challenging due to multi-resistant strains and the ability of S. aureus to adhere and form biofilms on bone and implant surfaces. The present work involved the preparation and evaluation of a novel dual polymeric film coating on stainless steel. Chitosan and polycaprolactone (PCL) multilayers, loaded with poly(methyl methacrylate) (PMMA) microspheres encapsulating vancomycin or daptomycin, produced by the dip-coating technique, allowed local antibiotic-controlled delivery for the treatment of implant-related infections. Enhanced adhesion of the film to the metal substrate surface was achieved by mechanical abrasion of its surface. Studies have shown that for both drugs the release occurs by diffusion, but the release profile depends on the type of drug (daptomycin or vancomycin), the pH of the solution, and whether the drug is freestanding (directly incorporated into the films) or encapsulated in PMMA microspheres. Daptomycin freestanding films reached 90% release after 1 day at pH 7.4 and 4 days at pH 5.5. In comparison, films with daptomycin encapsulated microspheres reached 90% release after 2 h at pH 5.5 and 2 days at pH 7.4. Vancomycin encapsulated and freestanding films showed a similar behavior reaching 90% release after 20 h of release at pH 5.5 and 2 and 3 days, respectively, at pH 7.4. Furthermore, daptomycin-loaded films showed activity (assessed by agar diffusion assays) against sensitive (ATCC 25923) and clinically isolated (MRSA) S. aureus strains.

Bonding antimicrobial rhamnolipids onto medical grade PDMS: A strategy to overcome multispecies vascular catheter-related infections.

In clinic there is a demand to solve the drawback of medical devices multispecies related infections. Consequently, different biomaterial surfaces, such as vascular catheters, urgently need improvement regarding their antifouling/antimicrobial properties. In this work, we covalently functionalized medical grade polydimethylsiloxane (PDMS) with antimicrobial rhamnolipids to investigate the biomaterial surface activity towards mono and dual species biofilms. Preparation of surfaces with "piranha" oxidation, followed by APTES bonding and carbodiimide reaction with rhamnolipids effectively bonded these compounds to PDMS surface as confirmed by FTIR-ATR and XPS analysis. Generated surfaces were active towards S. aureus biofilm formation showing a 4.2 log reduction while with S. epidermidis and C. albicans biofilms a reduction of 1.2 and 1.0 log reduction, respectively, was observed. Regarding dual-species testing the higher biofilm log reduction observed was 1.9. Additionally, biocompatibility was assessed by cytocompatibility towards human fibroblastic cells, low platelet activation and absence of vascular irritation. Our work not only sheds light on using covalently bonded rhamnolipids towards dual species biofilms but also highlights the biocompatibility of the obtained PDMS surfaces.

Using plasma-mediated covalent functionalization of rhamnolipids on polydimethylsiloxane towards the antimicrobial improvement of catheter surfaces.

Controlling bacterial biofilm formation on silicone-based bloodstream catheters is of great concern to prevent related-infections. In this study, rhamnolipids (RLs), glycolipid biosurfactants, specifically a RLs mixture and the purified di-RL (RhaRhaC10:0C10:0) were covalently bonded to silicone with the intention of reaching long-lasting antibiofilm surfaces. RLs mixture and di-RL were identified by an UHPLC-MS method that also allowed the confirmation of compound isolation by automated flash chromatography. Silicone surfaces underwent air-plasma treatment, inducing reactive oxygen radicals able to promote the RLs grafting that was confirmed by contact angle, FTIR-ATR and AFM measurements. The antibiofilm activity towards different Gram positive strains was evaluated by colony forming units (CFU) count and confocal laser microscopy. In addition, protein adsorption and biocompatibility were also investigated. RLs were successfully grafted onto silicone and RLs mixture and RhaRhaC10C10:0 functionalized specimens reduced the biofilm formation over 2.3 log units against methicillin sensitive Staphylococcus aureus. Additionally, a decrease of 1 log unit was observed against methicillin resistant S. aureus and S. epidermidis. Functionalized samples showed cytocompatibility towards human dermal fibroblasts, hemocompatibility and no vascular irritation potential. The results mentioned above revealed a synergy between the antimicrobial and the anti-adhesive properties of RLs, making these compounds good candidates for the improvement of the medical devices antibiofilm properties.

Latest Trends in Sustainable Polymeric Food Packaging Films.

Food packaging is the best way to protect food while it moves along the entire supply chain to the consumer. However, conventional food packaging poses some problems related to food wastage and excessive plastic production. Considering this, the aim of this work was to examine recent findings related to bio-based alternative food packaging films by means of conventional methodologies and additive manufacturing technologies, such as 3D printing (3D-P), with potential to replace conventional petroleum-based food packaging. Based on the findings, progress in the development of bio-based packaging films, biopolymer-based feedstocks for 3D-P, and innovative food packaging materials produced by this technology was identified. However, the lack of studies suggests that 3D-P has not been well-explored in this field. Nonetheless, it is probable that in the future this technology will be more widely employed in the food packaging field, which could lead to a reduction in plastic production as well as safer food consumption.

Fighting S. aureus catheter-related infections with sophorolipids: Electing an antiadhesive strategy or a release one?

Staphylococcus aureus medical devices related-infections, such as blood stream catheter are of major concern. Their prevention is compulsory and strategies, not prone to the development of resistance, to prevent S. aureus biofilms on catheter surfaces (e.g. silicone) are needed. In this work two different approaches using sophorolipids were studied to prevent S. aureus biofilm formation on medical grade silicone: i) an antiadhesive strategy through covalent bond of sophorolipids to the surface; ii) and a release strategy using isolated most active sophorolipids. Sophorolipids produced by Starmerella bombicola, were characterized by UHPLC-MS and RMN, purified by automatic flash chromatography and tested for their antimicrobial activity towards S. aureus. Highest antimicrobial activity was observed for C18:0 and C18:1 diacetylated lactonic sophorolipids showing a MIC of 50 µg mL-1. Surface modification with acidic or lactonic sophorolipids when evaluating the anti-adhesive or release strategy, respectively, was confirmed by contact angle, FTIR-ATR and AFM analysis. When using a mixture of acidic sophorolipids covalently bonded to silicone surface as antiadhesive strategy cytocompatible surfaces were obtained and a reduction of 90 % on biofilm formation was observed. Nevertheless, if a release strategy is adopted with purified lactonic sophorolipids a higher effect is achieved. Most promising compound was C18:1 diacateylated lactonic sophorolipid that showed no cellular viability reduction when a concentration of 1.5 mg mL-1 was selected and a reduction on biofilm around 5 log units. Results reinforce the applicability of these antimicrobial biosurfactants on preventing biofilms and disclose that their antimicrobial effect is imperative when comparing to their antiadhesive properties.

Seeking faster, alternative methods for glycolipid biosurfactant characterization and purification.

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

Exploring the potential of chitosan-based particles as delivery-carriers for promising antimicrobial glycolipid biosurfactants.

Driven by the need to find alternatives to control Staphylococcus aureus infections, this work describes the development of chitosan-based particulate systems as carriers for antimicrobial glycolipids. By using a simple ionic gelation method stable nanoparticles were obtained showing an encapsulation efficiency of 41.1 ± 8.8 % and 74.2 ± 1.3 % and an average size of 210.0 ± 15.7 nm and 329.6 ± 8.0 nm for sophorolipids and rhamnolipids chitosan-nanoparticles, respectively. Glycolipids incorporation and particle size was correspondingly corroborated by FTIR-ATR and TEM analysis. Rhamnolipids chitosan nanoparticles (RLs-CSp) presented the highest antimicrobial effect towards S. aureus (ATCC 25923) exhibiting a minimal inhibitory concentration of 130 µg/mL and a biofilm inhibition ability of 99 %. Additionally, RLs-CSp did not interfere with human dermal fibroblasts (AG22719) viability and proliferation under the tested conditions. The results revealed that the RLs-CSp were able to inhibit bacterial growth showing adequate cytocompatibility and might become, after additional studies, a valuable approach to prevent S. aureus related infections.

3D-printed platform multi-loaded with bioactive, magnetic nanoparticles and an antibiotic for re-growing bone tissue.

Polymeric platforms obtained by three-dimensional (3D) printing are becoming increasingly important as multifunctional therapeutic systems for bone treatment applications. In particularly, researchers aim to control bacterial biofilm on these 3D-platforms and enhance re-growing bone tissue, at the same time. This study aimed to fabricate a 3D-printed polylactic acid platform loaded with hydroxyapatite (HA), iron oxide nanoparticles (IONPs) and an antibiotic (minocycline) with tuneable properties and multistimuli response. IONPs were produced by a facile chemical co-precipitation method showing an average diameter between 11 and 15 nm and a superparamagnetic behaviour which was preserved when loaded into the 3D-platforms. The presence of two types of nanoparticles (IONPs and HA) modify the nanomorphological/nanotopographical feature of the 3D-platforms justifying their adequate bioactivity profile and in vitro cellular effects on immortalized and primary osteoblasts, including cytocompatibility and increased osteogenesis-related gene expression (RUNX2, BGLAP and SPP1). Disk diffusion assays and SEM analysis confirmed the effect of the 3D-platforms loaded with minocycline against Staphylococcus aureus. Altogether results showed that fabricated 3D-platforms combined the exact therapeutic antibiofilm dose of the antibiotic against S. aureus, with the enhanced osteogenic stimulation of the HA and IONPs nanoparticles which is a disruptive approach for bone targeting applications.

A Glance at Antimicrobial Strategies to Prevent Catheter-Associated Medical Infections.

Urinary and intravascular catheters are two of the most used invasive medical devices; however, microbial colonization of catheter surfaces is responsible for most healthcare-associated infections (HAIs). Several antimicrobial-coated catheters are available, but recurrent antibiotic therapy can decrease their potential activity against resistant bacterial strains. The aim of this Review is to question the actual effectiveness of currently used (coated) catheters and describe the progress and promise of alternative antimicrobial coatings. Different strategies have been reviewed with the common goal of preventing biofilm formation on catheters, including release-based approaches using antibiotics, antiseptics, nitric oxide, 5-fluorouracil, and silver as well as contact-killing approaches employing quaternary ammonium compounds, chitosan, antimicrobial peptides, and enzymes. All of these strategies have given proof of antimicrobial efficacy by modifying the physiology of pathogens or disrupting their structural integrity. The aim for synergistic approaches using multitarget processes and the combination of both antifouling and bactericidal properties holds potential for the near future. Despite intensive research in biofilm preventive strategies, laboratorial studies still present some limitations since experimental conditions usually are not the same and also differ from biological conditions encountered when the catheter is inserted in the human body. Consequently, in most cases, the efficacy data obtained from in vitro studies is not properly reflected in the clinical setting. Thus, further well-designed clinical trials and additional cytotoxicity studies are needed to prove the efficacy and safety of the developed antimicrobial strategies in the prevention of biofilm formation at catheter surfaces.

A scope at antifouling strategies to prevent catheter-associated infections.

The use of invasive medical devices is becoming more common nowadays, with catheters representing one of the most used medical devices. However, there is a risk of infection associated with the use of these devices, since they are made of materials that are prone to bacterial adhesion with biofilm formation, often requiring catheter removal as the only therapeutic option. Catheter-related urinary tract infections (CAUTIs) and central line-associated bloodstream infections (CLABSIs) are among the most common causes of healthcare-associated infections (HAIs) worldwide while endotracheal intubation is responsible for ventilator-associated pneumonia (VAP). Therefore, to avoid the use of biocides due to the potential risk of bacterial resistance development, antifouling strategies aiming at the prevention of bacterial adherence and colonization of catheter surfaces represent important alternative measures. This review is focused on the main strategies that are able to modify the physical or chemical properties of biomaterials, leading to the creation of antiadhesive surfaces. The most promising approaches include coating the surfaces with hydrophilic polymers, such as poly(ethylene glycol) (PEG), poly(acrylamide) and poly(acrylates), betaine-based zwitterionic polymers and amphiphilic polymers or the use of bulk-modified poly(urethanes). Natural polysaccharides and its modifications with heparin, have also been used to improve hemocompatibility. Recently developed bioinspired techniques yielding very promising results in the prevention of bacterial adhesion and colonization of surfaces include slippery liquid-infused porous surfaces (SLIPS) based on the superhydrophilic rim of the pitcher plant and the Sharklet topography inspired by the shark skin, which are potential candidates as surface-modifying approaches for biomedical devices. Concerning the potential application of most of these strategies in catheters, more in vivo studies and clinical trials are needed to assure their efficacy and safety for possible future use.

Benefits of Polydopamine as Particle/Matrix Interface in Polylactide/PD-BaSO4 Scaffolds.

This work reports the versatility of polydopamine (PD) when applied as a particle coating in a composite of polylactide (PLA). Polydopamine was observed to increase the particle-matrix interface strength and facilitate the adsorption of drugs to the material surface. Here, barium sulfate radiopaque particles were functionalized with polydopamine and integrated into a polylactide matrix, leading to the formulation of a biodegradable and X-ray opaque material with enhanced mechanical properties. Polydopamine functionalized barium sulfate particles also facilitated the adsorption and release of the antibiotic levofloxacin. Analysis of the antibacterial capacity of these composites and the metabolic activity and proliferation of human dermal fibroblasts in vitro demonstrated that these materials are non-cytotoxic and can be 3D printed to formulate complex biocompatible materials for bone fixation devices.

Citrate zinc hydroxyapatite nanorods with enhanced cytocompatibility and osteogenesis for bone regeneration.

The development of biomaterials that mimicking the hydroxyapatite nanoparticles existent in the immature bone tissue is crucial, especially to accelerate the bone remodeling and regeneration. In this work, it was developed for the first time, hydroxyapatite nanoparticles (NPs) incorporating citrate and zinc (cit-Zn-Hap) in their composition towards a one-step hydrothermal procedure. For comparison purposes, hydroxyapatite NPs incorporating only zinc (Zn-Hap) or citrate (cit-Hap), as well as hydroxyapatite without any of these elements (Hap) were synthesised. The physicochemical characterization was carried out reveling that, the presence of zinc on hydroxyapatite (cit-Zn-Hap), reduced the size of nanoparticles, changed the phosphate environment and decreased the surface charge when compared with cit-Hap nanoparticles. The osteogenic potential of cit-Zn-Hap NPs was analysed in human bone marrow-derived stromal cells (BMSCs), in the absence of osteoinductive factors. NPs were internalized by endocytosis appearing trapped in endosomes and lysosomes scattered through the cytoplasm. Exposure to these NPs resulted in a significant induction of ALP activity, extracellular matrix mineralization, and gene expression of early and later osteogenic transcription factors, as well as of osteoblastic markers. The osteoinductive effect might be regulated, at least in part, by the increased signalling through the canonical WNT pathway. Evaluation of the cell behaviour following exposure to Zn-Hap and cit-Hap strongly suggested a synergistic effect of citrate and Zn in cit-Zn-Hap NPs towards the induction of the osteogenic commitment and functionality of BMSCs. These findings will allow the design of new biomimetic hydroxyapatite nanoparticles with great potential for bone regeneration.

Understanding intracellular trafficking and anti-inflammatory effects of minocycline chitosan-nanoparticles in human gingival fibroblasts for periodontal disease treatment.

Periodontal diseases remain a challenge due to a complex interplay of factors involving a chronic inflammatory activation and bacteria internalization in periodontal cells. In this work, chitosan-nanoparticles loaded with minocycline (MH-NPs), a tetracycline with antimicrobial and anti-inflammatory effects, were developed for in situ delivery in the periodontal milieu aiming to improve drug effectiveness. A general cytocompatibility evaluation and a detailed approach to address the cellular uptake process, trafficking pathways and the modulation of relevant inflammatory gene expression was conducted using human gingival fibroblasts. Results show that MH-NPs with an adequate cytocompatible profile can be internalized by distinct endocytic processes (macropinocytosis and clathrin-mediated endocytosis). The ability to modulate autophagy with the delivery within the same endosomal/lysosomal pathway as periodontal pathogens was observed, which increases the intracellular drug effectiveness. Porphyromonas gingivalis LPS-stimulated cultures, grown in the presence of MH-NPs, were found to express significantly reduced levels of inflammation-related markers (IL-1b, TNFα, CXCL-8, NFKB1). These nanoparticles can be potentially used in periodontal disease treatment conjoining the ability of intracellular drug targeting with significant anti-inflammatory effects.

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.