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María I Quiñones-Vico,1– 4,* Ana Ubago-Rodríguez,1– 3,* Ana Fernández-González,1– 3 Raquel Sanabria-de la Torre,2,5 Álvaro Sierra-Sánchez,1– 3,5 Trinidad Montero-Vilchez,2,6 Manuel Sánchez-Díaz,2,6 José L Arias,2,7,8 Salvador Arias-Santiago1– 4,6 1Cell Production and Tissue Engineering Unit, Virgen de las Nieves University Hospital, Granada, 18014, Spain; 2Instituto de Investigación Biosanitaria Granada ibs.GRANADA, Granada, 18014, Spain; 3Andalusian Network of Design and Translation of Advanced Therapies, Sevilla, 41092, Spain; 4Medicine Department, School of Medicine, University of Granada, Granada, 18016, Spain; 5Department of Biochemistry and Molecular Biology IIi and Immunology, School of Medicine, University of Granada, Granada, 18016, Spain; 6Dermatology Department, Virgen de las Nieves University Hospital, Granada, 18014, Spain; 7Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Granada, Granada, 18071, Spain; 8Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, 18100, Spain*These authors contributed equally to this workCorrespondence: Ana Fernández-González, Email ana.fernandez.gonzalez@juntadeandalucia.esAbstract: Pseudomonas aeruginosa (P. aeruginosa) is a common nosocomial pathogen that can cause severe infections in critically ill patients. Due to its resistance to multiple drugs, it is challenging to treat, which can result in serious illness and death. Conventional treatments for infected wounds often involve the topical or systemic application of antibiotics, which can lead to systemic toxicity and the development of drug resistance. The combination of wound dressings that promote wound healing with nanoparticles (NPs) represents a revolutionary strategy for optimizing the safety and efficacy of antibiotics. This review assesses a systematic search to identify the latest approaches where the evaluation of wound dressings loaded with antibiotic NPs is conducted. The properties of NPs, the features of wound dressings, the antimicrobial activity and biocompatibility of the different strategies are analyzed. The results indicate that most research in this field is focused on dressings loaded with silver NPs (57.1%) or other inorganic materials (22.4%). Wound dressings loaded with polymeric NPs and carbon-based NPs represent 14.3% and 6.1% of the evaluated studies, respectively. Nevertheless, there are no clinical trials that have evaluated the efficacy of NPs-loaded wound dressings in patients. Further research is required to ensure the safety of these treatments and to translate the findings from the bench to the bedside. Keywords: hydrogel, skin infection, wound dressing, P. aeruginosa, nanoparticles

Background: There is not an ideal biomaterial for tissue‐engineered skin substitutes (TESSs), and most of the studies or existing therapies use xenogeneic origin natural biomaterials or biosynthetic scaffolds. Objective: To analyse clinical, histological integration and homeostasis parameters of a human TESS manufactured with fibrin–hyaluronic acid biomaterial (HA‐Skin), grafted in immunodeficient mice for 8 weeks, and compared with the gold standard treatment (Autograft), a human TESS manufactured with fibrin–agarose biomaterial (AG‐Skin) and secondary wound healing dressings. Methods: Human TESSs and autografts were implanted into BALB/c mice after surgical excision. Secondary wound healing approach was achieved with biosynthetic collagen wound dressing (Biobrane®) and fibrin–hyaluronic acid or fibrin–agarose biomaterial without cells (Total N = 44). Clinical integration and homeostasis parameters were evaluated every two weeks for two months. Histological and immunohistochemical analyses were performed four and eight weeks after grafting. Results: HA‐Skin, AG‐Skin and Autograft groups showed a proper clinical integration and epithelization eight weeks later. Scar evaluation revealed better results for Autograft and HA‐Skin. Homeostasis analysis indicated similar values of transepidermal water loss and elasticity between HA‐Skin (6.42 ± 0.75 g/h/m2, 0.42 ± 0.08 AU), Autograft (6.91 ± 1.28 g/h/m2, 0.40 ± 0.08 AU) and healthy mouse skin (6.40 ± 0.43 g/h/m2, 0.35 ± 0.03 AU). Histological results showed that human TESSs and autografts presented better skin structuration and higher expression of cytokeratins. Conclusions: This study suggests that human TESS based on fibrin–hyaluronic acid biomaterial could be suitable for clinical application in the treatment of several dermatological pathologies (wound healing). [ABSTRACT FROM AUTHOR]

Human Adipose tissue-Mesenchimal Stromal Cells (hAT-MSCs) are one of the most common cell therapy used in recent clinical trials due to their immunomodulation capacity. One issue that arises in pursuit of this type of application is the method of conservation and maintenance of differentiation potential of hAT-MSCs after undergoing a period of time. Understanding the storage and post-thaw characteristics of these cells is essential for both preclinical and clinical research to ensure a good product. Using the potential benefits of hAT-MSCs to develop tissue engineering strategies and cell-based therapies requires efficient cryopreservation of these cells for extended periods of time without loss of their multipotentiality. To analyse the influence of four cryoprotective agents and long-term storage (19 months) in hAT-MSCs culture. Post-thaw cell viability, stromal cell-surface markers and osteogenic and adipogenic differentiation potential of hAT-MSCs stored for 19 months. After 19 months of cryopreservation, the hAT-MSCs present optimal viabilities (>79%), maintain their typical morphology, the expression of phenotypic markers (CD90+, CD73+, CD 105+, CD44+ and CD11b-,CD45-, HLA-DR-) and the capacity for adipogenic and osteogenic differentiation. This study shows that different cryoprotective agents (CPAs) used and a long-term storage do not affect the multipotentiality of the hAT-MSCs. The best CPA for maintaining the functionality of the hAT-MSCs is 10% Me2SO, 54% DMEM, 36% FBS. [ABSTRACT FROM AUTHOR]

The aim of this study was evaluated the epidermal barrier function in an artificial bioengineered skin substitutes (ABSS) based on hyaluronic acid biomaterial. The study was performed evaluating specific skin ointments at different test period. Dermal fibroblasts (DF) and Keratinocytes (KT) were enzymatically isolated from 9 cm2 skin biopsies. After 4-5 weeks in culture, DF were part of dermal fibrin-hyaluronic acid stroma. KT (obtained using feeder-free method) were seeded on the surface to establish ABSS. Flow cytometry assay was carried out to phenotypically evaluate of keratinocytes: CD49f (integrin α6) and CD90 (fibroblast cell surface marker). Cell viability (LIVE/DEAD® assay), structural integrity (histological evaluation) and epidermal barrier function (transepidermal water loss, TEWL: Tewameter®) were measured after 5, 15 and 30 minutes to apply beclomethasone, mupirocin and vaseline. Acellular ABSS were used as controls. Specific cell culture parameters were evaluated after final recovery of keratinocytes. The cell expansion rate was significantly higher (p≤0.001%) with the CNT-57S media were supplemented with human platelet lysate. CD49f percentage was higher in keratinocytes with CNT-57S (95.2%). The CD90 percentage was lower in keratinocytes expanded with CNT-57S human lysate supplemented (1.60%). A viability assay was carried out in ABSS at first (85.1%) and second week (87.8%). Finally, barrier function evaluation of ABSS was carried out by measuring TEWL in the first and in the second week of maintenance. TEWL values in acellular ABSS were higher than in cellular ABSS, although the difference was not significant. Cells improved ABSS barrier function, thus favouring the reduction of water loss. Regarding the evaluation period, significant differences of TEWL were observed, decreasing the values in the second week. This may be due to the growth of the epithelial layer in cellular ABSS, which contributes to improvement of the barrier function. TEWL measures also decreased over the measurement time, although the differences were not significant. No difference was observed with respect to the type of ointment. In conclusion, keratinocytes culture was optimized using animal component-free and feeder-free method. A novel way to evaluate keratinocytes contribution to epidermal barrier function has been development in vitro. [ABSTRACT FROM AUTHOR]