ER-mitochondria association negatively affects wound healing by regulating NLRP3 activation.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common causative agent of acute bacterial skin and skin-structure infections (ABSSSI), one of the major challenges to the health system worldwide. Although the use of antibiotics as the first line of intervention for MRSA-infected wounds is recommended, important side effects could occur, including cytotoxicity or immune dysregulation, thus affecting the repair process. Here, we show that the oxazolidinone antibiotic linezolid (LZD) impairs wound healing by aberrantly increasing interleukin 1 ß (IL-1ß) production in keratinocytes. Mechanistically, LZD triggers a reactive oxygen species (ROS)-independent mitochondrial damage that culminates in increased tethering between the endoplasmic reticulum (ER) and mitochondria, which in turn activates the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex by promoting its assembly to the mitochondrial surface. Downregulation of ER-mitochondria contact formation is sufficient to inhibit the LZD-driven NLRP3 inflammasome activation and IL-1ß production, restoring wound closure. These results identify the ER-mitochondria association as a key factor for NLRP3 activation and reveal a new mechanism in the regulation of the wound healing process that might be clinically relevant.

Early Biofilm Formation on the Drain Tip after Total Knee Arthroplasty Is Not Associated with Prosthetic Joint Infection: A Pilot Prospective Case Series Study of a Single Center.

BACKGROUND: Periprosthetic joint infection (PJI) is a devastating complication of arthroplasties that could occur during the surgery. The purpose of this study was to analyze the biofilm formation through microbiological culture tests and scanning electron microscopy (SEM) on the tip of surgical drainage removed 24 h after arthroplasty surgery. METHODS: A total of 50 consecutive patients were included in the present prospective observational study. Drains were removed under total aseptic conditions twenty-four hours after surgery. The drain tip was cut in three equal parts of approximately 2-3 cm in length and sent for culture, culture after sonication, and SEM analysis. The degree of biofilm formation was determined using a SEM semi-quantitative scale. RESULTS: From the microbiological analysis, the cultures of four samples were positive. The semi-quantitative SEM analysis showed that no patient had grade 4 of biofilm formation. A total of 8 patients (16%) had grade 3, and 14 patients (28%) had grade 2. Grade 1, scattered cocci with immature biofilm, was contemplated in 16 patients (32%). Finally, 12 patients (24%) had grade 0 with a total absence of bacteria. During the follow-up (up to 36 months), no patient showed short- or long-term infectious complications. CONCLUSIONS: Most of the patients who underwent primary total knee arthroplasty (TKA) showed biofilm formation on the tip of surgical drain 24 h after surgery even though none showed a mature biofilm formation (grade 4). Furthermore, 8% of patients were characterized by a positivity of culture analysis. However, none of the patients included in the study showed signs of PJI up to 3 years of follow-up.

Features of Vat-Photopolymerized Masters for Microfluidic Device Manufacturing.

The growing interest in advancing microfluidic devices for manipulating fluids within micrometer-scale channels has prompted a shift in manufacturing practices, moving from single-component production to medium-size batches. This transition arises due to the impracticality of lab-scale manufacturing methods in accommodating the increased demand. This experimental study focuses on the design of master benchmarks 1-5, taking into consideration critical parameters such as rib width, height, and the relative width-to-height ratio. Notably, benchmarks 4 and 5 featured ribs that were strategically connected to the inlet, outlet, and reaction chamber of the master, enhancing their utility for subsequent replica production. Vat photopolymerization was employed for the fabrication of benchmarks 1-5, while replicas of benchmarks 4 and 5 were generated through polydimethylsiloxane casting. Dimensional investigations of the ribs and channels in both the master benchmarks and replicas were conducted using an optical technique validated through readability analysis based on the Michelson global contrast index. The primary goal was to evaluate the potential applicability of vat photopolymerization technology for efficiently producing microfluidic devices through a streamlined production process. Results indicate that the combination of vat photopolymerization followed by replication is well suited for achieving a minimum rib size of 25 µm in width and an aspect ratio of 1:12 for the master benchmark.

On the Biomechanical Performances of Duplex Stainless Steel Graded Scaffolds Produced by Laser Powder Bed Fusion for Tissue Engineering Applications.

This experimental study aims to extend the know-how on biomechanical performances of duplex stainless steel (DSS) for tissue engineering applications to a graded lattice geometry scaffold based on the F53 DSS (UNS S32750 according to ASTM A182) produced by laser powder bed fusion (LPBF). The same dense-out graded geometry based on rhombic dodecahedral elementary unit cells investigated in previous work on 316L stainless steel (SS) was adopted here for the manufacturing of the F53 DSS scaffold (SF53). Microstructural characterization and mechanical and biological tests were carried out on the SF53 scaffold, using the in vitro behavior of the 316L stainless steel scaffold (S316L) as a control. Results show that microstructure developed as a consequence of different volume energy density (VED) values is mainly responsible for the different mechanical behaviors of SF53 and S316L, both fabricated using the same LPBF manufacturing system. Specifically, the ultimate compressive strength (σUC) and elastic moduli (E) of SF53 are three times and seven times higher than S316L, respectively. Moreover, preliminary biological tests evidenced better cell viability in SF53 than in S316L already after seven days of culture, suggesting SF53 with dense-out graded geometry as a viable alternative to 316L SS for bone tissue engineering applications.

Oleuropein-Rich Gellan Gum/Alginate Films as Innovative Treatments against Photo-Induced Skin Aging.

Olea europaea L. leaf extracts (OLEs) represent highly value-added agro-industrial byproducts, being promising sources of significant antioxidant compounds, such as their main component, oleuropein. In this work, hydrogel films based on low-acyl gellan gum (GG) blended with sodium alginate (NaALG) were loaded with OLE and crosslinked with tartaric acid (TA). The films' ability to act as an antioxidant and photoprotectant against UVA-induced photoaging, thanks to their capability to convey oleuropein to the skin, were examined with the aim of a potential application as facial masks. Biological in vitro performances of the proposed materials were tested on normal human dermal fibroblasts (NhDFs), both under normal conditions and after aging-induced UVA treatment. Overall, our results clearly show the intriguing properties of the proposed hydrogels as effective and fully naturally formulated anti-photoaging smart materials for potential use as facial masks.

Improved biomechanical behavior of 316L graded scaffolds for bone tissue regeneration produced by laser powder bed fusion.

Graded lattice scaffolds based on rhombic dodecahedral (RD) elementary unit cell geometry were manufactured in 316L stainless steel (SS) by laser powder bed fusion (LPBF). Two different strategies based on varying strut thickness layer-by-layer in the building direction were adopted to obtain the graded scaffolds: a) decreasing strut size from core to edge to produce the dense-in (DI) structure and b) increasing strut size in the same direction to produce the dense-out (DO) structure. Both graded structures (DI and DO) were constructed with specular symmetry with respect to the central horizontal axis. Structural, mechanical, and biological characterizations were carried out to evaluate feasibility of designing appropriate biomechanical performances of graded scaffolds in the perspective of bone tissue regeneration. Results showed that mechanical behavior is governed by graded geometry, while printing parameters influence structural properties of the material such as density, textures, and crystallographic phases. The predominant failure mechanism in graded structures initiates in correspondence of thinner struts, due to high stress concentrations on strut junctions. Biological tests evidenced better proliferation of cells in the DO graded scaffold, which in turn exhibits mechanical properties close to cortical bone. The combined control of grading strategy, printing parameters and elementary unit cell geometry can enable implementing scaffolds with improved biomechanical performances for bone tissue regeneration.

A cytokine-induced spheroid-based in vitro model for studying osteoarthritis pathogenesis.

Given the lack of in vitro models faithfully reproducing the osteoarthritis (OA) disease on-set, this work aimed at manufacturing a reliable and predictive in vitro cytokine-based Articular Cartilage (AC) model to study OA progression. Cell spheroids of primary human fetal chondrocytes (FCs) and h-TERT mesenchymal stem cells differentiated chondrocytes (Y201-C) were analysed in terms of growth kinetics, cells proliferation and apoptosis over 10 days of culture, in healthy condition or in presence of cytokines (interleukin-1ß, -6 and TNF-α). Then, the spheroids were assembled into chondrospheres using a bottom-up strategy, to obtain an in vitro cytokines-induced OA model. The resulting chondrospheres were evaluated for gene expression and anabolic ECM proteins. Compared to the healthy environment, the simulated OA environment induced chondrocyte hyperproliferation and apoptotic pathway, decreased expression of anabolic ECM proteins, and diminished biosynthetic activity, resembling features of early-stage OA. These characteristics were observed for both Y201-C and HC at high and low concentrations of cytokines. Both HC and Y201-C demonstrated the suitability for the manufacturing of a scaffold-free in vitro OA model to facilitate studies into OA pathogenesis and therapeutic strategies. Our approach provides a faithful reproduction of early-stage osteoarthritis, demonstrating the ability of obtaining different disease severity by tuning the concentration of OA-related cytokines. Given the advantages in easy access and more reproducible performance, Y201-C may represent a more favourable source of chondrocytes for establishing more standardized protocols to obtain OA models.

Influence of Trabecular Geometry on Scaffold Mechanical Behavior and MG-63 Cell Viability.

In a scaffold-based approach for bone tissue regeneration, the control over morphometry allows for balancing scaffold biomechanical performances. In this experimental work, trabecular geometry was obtained by a generative design process, and scaffolds were manufactured by vat photopolymerization with 60% (P60), 70% (P70) and 80% (P80) total porosity. The mechanical and biological performances of the produced scaffolds were investigated, and the results were correlated with morphometric parameters, aiming to investigate the influence of trabecular geometry on the elastic modulus, the ultimate compressive strength of scaffolds and MG-63 human osteosarcoma cell viability. The results showed that P60 trabecular geometry allows for matching the mechanical requirements of human mandibular trabecular bone. From the statistical analysis, a general trend can be inferred, suggesting strut thickness, the degree of anisotropy, connectivity density and specific surface as the main morphometric parameters influencing the biomechanical behavior of trabecular scaffolds, in the perspective of tissue engineering applications.

Bacillus subtilis Modulated the Expression of Osteogenic Markers in a Human Osteoblast Cell Line.

Several in vivo trials have previously demonstrated the beneficial effects of the administration of various probiotic forms on bone health. In this study, we explored the potency of two probiotics, Bacillus subtilis and Lactococcus lactis, alone or in combination with vitamin D (VD), to modulate the transcription of genes involved in the ossification process in a human osteoblast cell line. Genes that mark the "osteoblast proliferation phase", such as RUNX2, TGFB1, and ALPL, "extracellular matrix (ECM) maturation", such as SPP1 and SPARC, as well as "ECM mineralization", such as BGN, BGLAP, and DCN, were all highly expressed in osteoblasts treated with B. subtilis extract. The observed increase in the transcription of the ALPL mRNA was further in agreement with its protein levels as observed by Western blot and immunofluorescence. Therefore, this higher transcription and translation of alkaline phosphatase in osteoblasts treated with the B. subtilis extract, indicated its substantial osteogenic impact on human osteoblasts. Although both the probiotic extracts showed no osteogenic synergy with VD, treatment with B. subtilis alone could increase the ECM mineralization, outperforming the effects of L. lactis and even VD. Furthermore, these results supported the validity of employing probiotic extracts rather than live cells to investigate the effects of probiotics in the in vitro systems.

An In Vitro Engineered Osteochondral Model as Tool to Study Osteoarthritis Environment.

Osteoarthritis (OA) is a joint degenerative pathology characterized by mechanical and inflammatory damages affecting synovium, articular cartilage (AC), and subchondral bone (SB). Several in vitro, in vivo, and ex vivo models are developed to study OA, but to date the identification of specific pharmacological targets seems to be hindered by the lack of models with predictive capabilities. This study reports the development of a biomimetic in vitro model of AC and SB interface. Gellan gum methacrylated and chondroitin sulfate/dopamine hydrogels are used for the AC portion, whereas polylactic acid functionalized with gelatin and nanohydroxyapatite for the SB. The physiological behavior of immortalized stem cells (Y201s) and Y201s differentiated in chondrocytes (Y201-Cs), respectively, for the SB and AC, is demonstrated over 21 days of culture in vitro in healthy and pathological conditions, whilst modeling the onset of cytokines-induced OA. The key metrics are: lower glycosaminoglycans production and increased calcification given by a higher Collagen X content, in the AC deep layer; higher expression of pro-angiogenic factor (vegf) and decreased expression of osteogenic markers (coll1, spp1, runx2) in the SB. This novel approach provides a new tool for studying the development and progression of OA.

Altered type I collagen networking in osteoporotic human femoral head revealed by histomorphometric and Fourier transform infrared imaging correlated analyses.

Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell-matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein-2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.

Tetraspanin CD9 Expression Predicts Sentinel Node Status in Patients with Cutaneous Melanoma.

The tetraspanin CD9 is considered a metastasis suppressor in many cancers, however its role is highly debated. Currently, little is known about CD9 prognostic value in cutaneous melanoma. Our aim was to analyse CD9 expression in melanocytic nevi and primary cutaneous melanomas through immunohistochemistry and immunofluorescence approaches to determine its correlation with invasiveness and metastatic potential. CD9 displayed homogeneous staining in all melanocytic nevi. In contrast, it showed a complete loss of reactivity in all thin melanomas. Interestingly, CD9 was re-expressed in 46% of intermediate and thick melanomas in small tumor clusters predominantly located at sites of invasion near or inside the blood or lymphatic vessels. The most notable finding is that all CD9 stained melanomas presented sentinel node positivity. Additionally, a direct association between CD9 expression and presence of distant metastasis was reported. Finally, we confirm that CD9 expression is consistent with an early protective role against tumorigenesis, however, our data endorse in melanoma a specific function of CD9 in vascular dissemination during late tumor progression. The presence of CD9 hotspots could be essential for melanoma cell invasion in lymphatic and endothelial vessels. CD9 could be a valid prognostic factor for lymph node metastasis risk.

3D Printed Scaffold Based on Type I Collagen/PLGA_TGF-ß1 Nanoparticles Mimicking the Growth Factor Footprint of Human Bone Tissue.

In bone regenerative strategies, the controlled release of growth factors is one of the main aspects for successful tissue regeneration. Recent trends in the drug delivery field increased the interest in the development of biodegradable systems able to protect and transport active agents. In the present study, we designed degradable poly(lactic-co-glycolic)acid (PLGA) nanocarriers suitable for the release of Transforming Growth Factor-beta 1 (TGF-ß1), a key molecule in the management of bone cells behaviour. Spherical TGF-ß1-containing PLGA (PLGA_TGF-ß1) nanoparticles (ca.250 nm) exhibiting high encapsulation efficiency (ca.64%) were successfully synthesized. The TGF-ß1 nanocarriers were subsequently combined with type I collagen for the fabrication of nanostructured 3D printed scaffolds able to mimic the TGF-ß1 presence in the human bone extracellular matrix (ECM). The homogeneous hybrid formulation underwent a comprehensive rheological characterisation in view of 3D printing. The 3D printed collagen-based scaffolds (10 mm × 10 mm × 1 mm) successfully mimicked the TGF-ß1 presence in human bone ECM as assessed by immunohistochemical TGF-ß1 staining, covering ca.3.4% of the whole scaffold area. Moreover, the collagenous matrix was able to reduce the initial burst release observed in the first 24 h from about 38% for the PLGA_TGF-ß1 alone to 14.5%, proving that the nanocarriers incorporation into collagen allows achieving sustained release kinetics.

Study of the Adhesion of the Human Gut Microbiota on Electrospun Structures.

Although the adhesion of bacteria on surfaces is a widely studied process, to date, most of the works focus on a single species of microorganisms and are aimed at evaluating the antimicrobial properties of biomaterials. Here, we describe how a complex microbial community, i.e., the human gut microbiota, adheres to a surface to form stable biofilms. Two electrospun structures made of natural, i.e., gelatin, and synthetic, i.e., polycaprolactone, polymers were used to study their ability to both promote the adhesion of the human gut microbiota and support microbial growth in vitro. Due to the different wettabilities of the two surfaces, a mucin coating was also added to the structures to decouple the effect of bulk and surface properties on microbial adhesion. The developed biofilm was quantified and monitored using live/dead imaging and scanning electron microscopy. The results indicated that the electrospun gelatin structure without the mucin coating was the optimal choice for developing a 3D in vitro model of the human gut microbiota.

The multifaced role of HtrA1 in the development of joint and skeletal disorders.

HtrA1 (High temperature requirement A1) family proteins include four members, widely conserved from prokaryotes to eukaryotes, named HtrA1, HtrA2, HtrA3 and HtrA4. HtrA1 is a serine protease involved in a variety of biological functions regulating many signaling pathways degrading specific components and playing key roles in many human diseases such as neurodegenerative disorders, pregnancy complications and cancer. Due to its role in the breakdown of many ExtraCellular Matrix (ECM) components of articular cartilage such as fibronectin, decorin and aggrecan, HtrA1 encouraged many researches on studying its role in several skeletal diseases (SDs). These studies were further inspired by the fact that HtrA1 is able to regulate the signaling of one of the most important cytokines involved in SDs, the TGFß-1. This review aims to summarize the data currently available on the role of HtrA1 in skeletal diseases such as Osteoporosis, Rheumatoid Arthritis, Osteoarthritis and Intervertebral Disc Degeneration (IDD). The use of HtrA1 as a marker of frailty in geriatric medicine would represent a powerful tool for identifying older individuals at risk of developing skeletal disorders, evaluating an appropriate intervention to improve quality care in these people avoiding or improving age-related SDs in the elderly population.

Oxidative stress in retinal pigment epithelium impairs stem cells: a vicious cycle in age-related macular degeneration.

Aging, chronic oxidative stress, and inflammation are major pathogenic factors in the development and progression of age-related macular degeneration (AMD) with the loss of retinal pigment epithelium (RPE). The human RPE contains a subpopulation of progenitors (i.e., RPE stem cells-RPESCs) whose role in the RPE homeostasis is under investigation. We evaluated the paracrine effects of mature RPE cells exposed to oxidative stress (H2O2) on RPESCs behavior through co-cultural, morphofunctional, and bioinformatic approaches. RPESCs showed a decline in proliferation, an increase of the senescence-associated ß-galactosidase activity, the acquisition of a senescent-like secretory phenotype (SASP), and the reduction of their stemness and differentiation competencies. IL-6 and Superoxide Dismutase 2 (SOD2) seem to be key molecules in RPESCs response to oxidative stress. Our results get insight into stress-induced senescent-associated molecular mechanisms implicated in AMD pathogenesis. The presence of chronic oxidative stress in the microenvironment reduces the RPESCs abilities, inducing and/or maintaining a pro-inflammatory retinal milieu that in turn could affect AMD onset and progression.

Metabolic Comorbidities in Pediatric Atopic Dermatitis: A Narrative Review.

Atopic dermatitis (AD) is an itchy dermatitis with multifactorial aetiology, chronic-recurrent course, and typical distribution of lesions according to the age, affecting the 10-20% of pediatric population. Patients with AD, including children, suffer from many metabolic comorbidities, including metabolic syndrome, being overweight, obesity, dyslipidaemia, and arterial hypertension, all of which had a prevalence that was demonstrated to be higher than in healthy patients. The association between AD and metabolic comorbidities is multifactorial and involves the deregulation of immune system. In fact, hypertrophic adipose tissue produces soluble adipokines involved in inflammation and immunity, which stimulate the production of pro-inflammatory cytokines, responsible for a chronic low-grade inflammatory state and a higher predisposition to hypersensitivity reactions. Especially in pediatric population with AD, these metabolic disorders are usually underestimated and are associated with long term sequelae and an increased risk of a cardiovascular event, which may also occur later in adult age. Therefore, metabolic comorbidities should be carefully evaluated and early treated in children with AD, to minimize the long-term risk of cardiovascular events.

Tissue Biomarkers Predicting Lymph Node Status in Cutaneous Melanoma.

Cutaneous melanoma is a severe neoplasm that shows early invasiveness of the lymph nodes draining the primary site, with increased risk of distant metastases and recurrence. The tissue biomarker identification could be a new frontier to predict the risk of early lymph node invasiveness, especially in cases considered by current guidelines to be at low risk of lymph node involvement and not requiring evaluation of the sentinel lymph node (SLN). For this reason, we present a narrative review of the literature, seeking to provide an overview of current tissue biomarkers, particularly vascular endothelium growth factors (VEGF), Tetraspanin CD9, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), D2-40, and gene expression profile test (31-GEP). Among these, 31-GEP seems to be able to provide a distinction between low or high risk for positive SLN classes. VEGF receptor-3 and CD9 expression may be independent predictors of positive SLN. Lastly, LYVE-1 and D2-40 allow an easier assessment of lymph vascular invasion, which can be considered a good predictor of SLN status. In conclusion, biomarkers to assess the lymph node status of cutaneous melanoma patients may play an important role in those cases where the clinician is in doubt whether or not to perform SLN biopsy.

Insights into oxidative stress in bone tissue and novel challenges for biomaterials.

The presence of Reactive Oxygen Species (ROS) in bone can influence resident cells behaviour as well as the extra-cellular matrix composition and the tissue architecture. Aging, in addition to excessive overloads, unbalanced diet, smoking, predisposing genetic factors, lead to an increase of ROS and, if it is accompanied with an inappropriate production of scavengers, promotes the generation of oxidative stress that encourages bone catabolism. Furthermore, bone injuries can be triggered by numerous events such as road and sports accidents or tumour resection. Although bone tissue possesses a well-known repair and regeneration capacity, these mechanisms are inefficient in repairing large size defects and bone grafts are often necessary. ROS play a fundamental role in response after the implant introduction and can influence its success. This review provides insights on the mechanisms of oxidative stress generated by an implant in vivo and suitable ways for its modulation. The local delivery of active molecules, such as polyphenols, enhanced bone biomaterial integration evidencing that the management of the oxidative stress is a target for the effectiveness of an implant. Polyphenols have been widely used in medicine for cardiovascular, neurodegenerative, bone disorders and cancer, thanks to their antioxidant and anti-inflammatory properties. In addition, the perspective of new smart biomaterials and molecular medicine for the oxidative stress modulation in a programmable way, by the use of ROS responsive materials or by the targeting of selective molecular pathways involved in ROS generation, will be analysed and discussed critically.

Innovative Eco-Friendly Hydrogel Film for Berberine Delivery in Skin Applications.

Hydrogel formulations (masks or patches, without tissue support) represent the new frontier for customizable skin beauty and health. The employment of these materials is becoming popular in wound dressing, to speed up the healing process while protecting the affected area, as well as to provide a moisturizing reservoir, control the inflammatory process and the onset of bacterial development. Most of these hydrogels are acrylic-based at present, not biodegradable and potentially toxic, due to acrylic monomers residues. In this work, we selected a new class of cellulose-derived and biodegradable hydrogel films to incorporate and convey an active compound for dermatological issues. Films were obtained from a combination of different polysaccharides and clays, and berberine hydrochloride, a polyphenolic molecule showing anti-inflammatory, immunomodulatory, antibacterial and antioxidant properties, was chosen and then embedded in the hydrogel films. These innovative hydrogel-based systems were characterized in terms of water uptake profile, in vitro cytocompatibility and skin permeation kinetics by Franz diffusion cell. Berberine permeation fitted well to Korsmeyer-Peppas kinetic model and achieved a release higher than 100 µg/cm2 within 24 h. The latter study, exploiting a reliable skin model membrane, together with the biological assessment, gained insights into the most promising formulation for future investigations.