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Acute and chronic wounds present a significant healthcare challenge, requiring innovative solutions for effective treatment. The exploitation of natural by-products with advanced cell regeneration potential and plant-based materials, which possess bioactive properties, is an innovative topic in wound management. This study investigates the potential of donkey gelatin and keratin for blending with natural bioactive extracts such as sumac, curcumin, and oak acorn to fabricate antioxidant and antimicrobial nanofibers with accelerated wound healing processes. The fabricated nanofibers possess good in vitro biocompatibility, except for the sumac-based donkey nanofibers, where cell viability significantly dropped to 56.25% (p < 0.05 compared to non-treated cells). The nanofiber dimensions showed structural similarities to human extracellular matrix components, providing an ideal microenvironment for tissue regeneration. The donkey nanofiber-based sumac and curcumin extracts presented a higher dissolution in the first 10 min (74% and 72%). Curcumin extract showed similar antimicrobial and antifungal performances to rivanol, while acorn and sumac extracts demonstrated similar values to each other. In vitro tests performed on murine fibroblast cells demonstrated high migration rates of 89% and 85% after 24 h in the case of acorn and curcumin nanofibers, respectively, underscoring the potential of these nanofibers as versatile platforms for advanced wound care applications.

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The present study aimed to conduct a comparative investigation of the biological properties of phenolic and polysaccharide extracts obtained using an ultrasound-assisted technique from Aloe vera gel and their effects on each stage of the wound healing process in in vitro experimental models. HPLC analysis showed that the phenolic extract contained aloin, ferulic, and caffeic acid, as well as quercetin dihydrate, as major compounds. Capillary zone electrophoresis indicated the prevalence of mannose and glucose in the polysaccharide extract. Cell culture testing revealed the anti-inflammatory properties of the phenolic extract at a concentration of 0.25 mg/mL through significant inhibition of pro-inflammatory cytokines—up to 28% TNF-α and 11% IL-8 secretion—in inflamed THP-1-derived macrophages, while a pro-inflammatory effect was observed at 0.5 mg/mL. The phenolic extract induced 18% stimulation of L929 fibroblast proliferation at a concentration of 0.5 mg/mL, enhanced the cell migration rate by 20%, and increased collagen type I synthesis by 18%. Moreover, the phenolic extract exhibited superior antioxidant properties by scavenging free DPPH (IC50 of 2.50 mg/mL) and ABTS (16.47 mM TE/g) radicals, and 46% inhibition of intracellular reactive oxygen species (ROS) production was achieved. The polysaccharide extract demonstrated a greater increase in collagen synthesis up to 25%, as well as antibacterial activity against Staphylococcus aureus with a bacteriostatic effect at 25 mg/mL and a bactericidal one at 50 mg/mL. All these findings indicate that the phenolic extract might be more beneficial in formulations intended for the initial phases of wound healing, such as inflammation and proliferation, while the polysaccharide extract could be more suitable for use during the remodeling stage. Moreover, they might be combined with other biomaterials, acting as efficient dressings with anti-inflammatory, antioxidant, and antibacterial properties for rapid recovery of chronic wounds. © 2024 by the authors.

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The complex composition of honey plays a crucial role in wound healing, exhibiting varying effects at different stages of the healing process. This study investigated seven honey varieties sourced from different regions of Romania using in vitro experimental models developed in macrophage-like, fibroblast, and keratinocyte cell lines to explore the mechanisms by which honey promoted the healing process. This study assessed the impact of honey on inflammatory cytokine production in macrophage-like cells, cell proliferation and collagen synthesis in fibroblasts, and cell proliferation and migration in keratinocytes. Additionally, correlation analysis was conducted to examine the relationship between honey composition and its biological properties. Honey varieties presented both anti- and pro-inflammatory effects. Moreover, they displayed dose-dependent pro-proliferative effects, stimulating collagen synthesis and cell migration, thereby enhancing the re-epithelialization process. The Pearson coefficient analysis indicated a strong positive correlation between biological activities and phenolic content. Additionally, there was a medium positive correlation with the ascorbic acid content and a medium negative correlation with the glucose content in the different honey varieties. Romanian honey varieties rich in phenolics showed potential in modulating inflammation, proliferation, collagen synthesis, and cell migration, suggesting their suitability for further evaluation and development of innovative dressings for skin tissue regeneration.

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This study aimed to evaluate the hypoglycemic potential, antioxidant activity and prebiotic activity of a hydroalcoholic extract of blueberry pomace (BP), an aqueous extract of chia seeds (CS) and a novel combination of BP-CS extracts (BCM) for further use as ingredient of functional food. Spectrometric and HPLC analyses were used to characterize the total phenolic and flavonoid content and composition of BP, while CS was analyzed for total carbohydrate content. Data showed that the BCM mixture exerted an inhibition of alpha-amylase activity, which was 1.36 times higher than that of BP and 1.25 higher than CS extract. The mixture also showed better scavenging activity of free DPPH radicals than individual extracts, and had an IC50 value of 603.12 mu g/mL. In vitro testing indicated that both serum- and colon-reaching products of simulated intestinal digestion of BCM presented the capacity to protect Caco-2 intestinal cells against oxidative stress by inhibition of reactive oxygen species production. In addition, the colon-reaching product of BCM digestion had the capacity to significantly (p < 0.05) stimulate the growth of Lactobacillus rhamnosus and Lactobacillus acidophilus, revealing a prebiotic potential. All these results indicated that improved biological activity of the novel combination of BP and CS extracts could be due to the synergistic action of constituents. The combination is recommended for further testing and the development of novel functional food for controlling type 2 diabetes and gastrointestinal conditions.

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The present research aims to describe a new methodology to obtain biocompatible hydrogels based on Aloe vera used for wound healing applications. The properties of two hydrogels (differing in Aloe vera concentration, AV5 and AV10) prepared by an all-green synthesis method from raw, natural, renewable and bioavailable materials such as salicylic acid, allantoin and xanthan gum were investigated. The morphology of the Aloe vera based hydrogel biomaterials was studied by SEM analysis. The rheological properties of the hydrogels, as well as their cell viability, biocompatibility and cytotoxicity, were determined. The antibacterial activity of Aloe vera based hydrogels was evaluated both on Gram-positive, Staphylococcus aureus and on Gram-negative, Pseudomonas aeruginosa strains. The obtained novel green Aloe vera based hydrogels showed good antibacterial properties. In vitro scratch assay demonstrated the capacity of both AV5 and AV10 hydrogels to accelerate cell proliferation and migration and induce closure of a wounded area. A corroboration of all morphological, rheological, cytocompatibility and cell viability results indicates that this Aloe vera based hydrogel may be suitable for wound healing applications.

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Lonicera caerulaea L. and Aronia melanocarpa (Michx.) Elliot fruits are frequently used for their health benefits as they are rich in bioactive compounds. They are recognized as a source of natural and valuable phytonutrients, which makes them a superfood. L. caerulea presents antioxidant activity three to five times higher than other berries which are more commonly consumed, such as blackberries or strawberries. In addition, their ascorbic acid level is the highest among fruits. The species A. melanocarpa is considered one of the richest known sources of antioxidants, surpassing currants, cranberries, blueberries, elderberries, and gooseberries, and contains one of the highest amounts of sorbitol. The non-edible leaves of genus Aronia became more extensively analyzed as a byproduct or waste material due to their high polyphenol, flavonoid, and phenolic acid content, along with a small amount of anthocyanins, which are used as ingredients in nutraceuticals, herbal teas, bio-cosmetics, cosmeceuticals, food and by the pharmaceutical industry. These plants are a rich source of vitamins, tocopherols, folic acid, and carotenoids. However, they remain outside of mainstream fruit consumption, being well known only to a small audience. This review aims to shed light on L. caerulaea and A. melanocarpa and their bioactive compounds as healthy superfoods with antioxidant, anti-inflammatory, antitumor, antimicrobial, and anti-diabetic effects, and hepato-, cardio-, and neuro-protective potential. In this view, we hope to promote their cultivation and processing, increase their commercial availability, and also highlight the ability of these species to be used as potential nutraceutical sources, helpful for human health.

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Diabetes is a condition correlated with a high number of diagnosed chronic wounds as a result of a complex pathophysiological mechanism. Diabetic chronic wounds are characterized by disorganized and longer stages, compared to normal wound healing. Natural polymer hydrogels can act as good wound dressings due to their versatile physicochemical properties, represented mainly by high water content and good biocompatibility. Natural bioactive hydrogels are polymers loaded with bioactive compounds providing antibacterial and antioxidant properties, modulation of inflammation and adherence to wounded tissue, compared to traditional dressings, which enables promising future applications for diabetic wound healing. Natural bioactive compounds, such as polyphenols, polysaccharides and proteins have great advantages in promoting chronic wound healing in diabetes due to their antioxidant, anti-inflammatory, antimicrobial, anti-allergic and wound healing properties. The present paper aims to review the wound healing mechanisms underlining the main issues of chronic wounds and those specifically occurring in diabetes. Also, the review highlights the recent state of the art related to the effect of hydrogels enriched with natural bioactive compounds developed as biocompatible functional materials for improving diabetic-related chronic wound healing and providing novel therapeutic strategies that could prevent limb amputation and increase the quality of life in diabetic patients.

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Osteochondral structure reconstruction by tissue engineering, a challenge in regenerative medicine, requires a scaffold that ensures both articular cartilage and subchondral bone remodeling. Functional hydrogels and scaffolds present a strategy for the controlled delivery of signaling molecules (growth factors and therapeutic drugs) and are considered a promising therapeutic approach. Icariin is a pharmacologically-active small molecule of prenylated flavonol glycoside and the main bioactive flavonoid isolated from Epimedium spp. The in vitro and in vivo testing of icariin showed chondrogenic and ostseoinductive effects, comparable to bone morphogenetic proteins, and suggested its use as an alternative to growth factors, representing a low-cost, promising approach for osteochondral regeneration. This paper reviews the complex structure of the osteochondral tissue, underlining the main aspects of osteochondral defects and those specifically occurring in osteoarthritis. The significance of icariin's structure and the extraction methods were emphasized. Studies revealing the valuable chondrogenic and osteogenic effects of icariin for osteochondral restoration were also reviewed. The review highlighted th recent state-of-the-art related to hydrogels and scaffolds enriched with icariin developed as biocompatible materials for osteochondral regeneration strategies.

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The aim of this study was to investigate the melissopalynology, physicochemical characteristics, antioxidant and antibacterial activity of seven honey samples harvested from different geographical regions and climates of Romania. The melissopalynological analysis revealed that monofloral and multifloral samples contained a wide diversity of minor pollen types from Romanian flora. The moisture, pH and free acidity values were within international limit. HPLC analysis indicated high content of fructose and glucose and low content of sucrose. Bioactive compounds including proteins, phenolics, flavonoids and ascorbic acid were present in variable quantities, according to the botanical origin and geographical area. The highest phenolics and ascorbic acid content was in multifloral honeys from Crisana mountain and meadow and the extrafloral honeydew honey. The same honey samples have exerted free radical scavenging and antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The free radical scavenging activity was strongly correlated to phenolics and ascorbic acid content, while the antimicrobial activity was medium correlated only to phenolics content. In conclusion, the selected Romanian honey samples with best antioxidant and antimicrobial activity could be further tested for the development of novel biomedical products.

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Research background. Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Experimental approach. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Results and conclusions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. Novelty and scientific contribution. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena.

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Over the past years, research attention has been focusing more on waste-derived, naturally derived, and renewable materials, in the view of a more sustainable economy. In this work, different topical formulations were obtained from the valorization of marine and agro-industrial by-products and the use of Carbopol 940 as gelling agent. In particular, the combination of extracts obtained from the marine snail, Rapanosa venosa, with Cladophora vagabunda and grape pomace extracts, was investigated for wound healing purposes. Rapana venosa has demonstrated wound healing properties and antioxidant activity. Similarly, grape pomace extracts have been shown to accelerate the healing process. However, their synergic use has not been explored yet. To this aim, four different formulations were produced. Three formulations differed for the presence of a different extract of Rapana venosa: marine collagen, marine gelatin, and collagen hydrolysate, while another formulation used mammalian gelatin as further control. Physico-chemical properties of the extracts as well as of the formulations were analyzed. Furthermore, thermal stability was evaluated by thermogravimetric analysis. Antioxidant capacity and biological behavior, in terms of cytocompatibility, wound healing, and antimicrobial potential, were assessed. The results highlighted for all the formulations (i) a good conservation and thermal stability in time, (ii) a neutralizing activity against free radicals, (iii) and high degree of cytocompatibility and tissue regeneration potential. In particular, collagen, gelatin, and collagen hydrolysate obtained from the Rapana venosa marine snail represent an important, valuable alternative to mammalian products.

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Severe skin injuries, including burns, represent a real concern for the global health-care system and therefore, there is an increased interest in developing wound dressings, in order to stimulate and enhance skin tissue repair. The aim of this study was to design novel hybrid materials, biomimetic to skin extracellular matrix and enriched with silver nanoparticles (nAg), in order to provide both dermal tissue regeneration and antimicrobial activity. Two material variants (variant A and variant B) consisting of type I collagen (COL), chondroitin sulfate (CS) and k-elastin peptides (EL) enriched with positively-charged nAg, were conditioned as membranes. UV exposure ensured both sterilisation and cross-linking of the materials. Physico-chemical characterization of the hybrid biomaterials showed values of density and swelling degree higher than those of COL membrane, while the process of in vitro degradation followed a similar pattern. Infrared spectroscopy and x-ray diffraction indicated alterations of the characteristic structural features and crystallinity of COL after blending with CS and EL and nAg embedding. Scanning electron microscopy observations revealed different surface morphologies of the hybrid membranes, according to their composition. In vitro studies on L929 fibroblasts and HaCaT keratinocytes showed that both hybrid membranes exhibited good cytocompatibility and promoted higher cell proliferation compared to COL sample, as evaluated by MTT and Live/Dead assays. The presence of actin filaments highlighted by fluorescent labelling confirmed the fibroblast and keratinocyte adhesion onto the surface of hybrid membranes. Most importantly, both materials showed an increased wound healing ability in an in vitro scratch assay model, stimulating cell migration at 24 h post-seeding. In addition, good antimicrobial activity was recorded, especially against Gram-positive bacterial strain. Altogether, our findings recommend COL-CS-EL-nAg hybrid membranes as good candidates for wound healing acceleration and bioengineering of skin tissue.

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In the present study, we evaluated for the first time the photoprotective effect of fish bone bioactive peptides (FBBP) preparation isolated from silver carp (Hypophthalmichthys molitrix) discarded tissue using in vitro experimental models of skin cells exposed to ultraviolet B (UVB) irradiation and stressing agents. FBBP preparation was obtained by papain treatment of minced bones and centrifugal ultrafiltration, and the molecular weight (MW) distribution was characterized by size exclusion and reversed-phase high performance liquid chromatography (RP-HPLC). In vitro assessment of the effect of FBBP pretreatment in UVB-irradiated L929 fibroblasts and HaCaT keratinocytes revealed their cytoprotective activity. Their capacity to efficiently reduce reactive oxygen species (ROS) production and lipid peroxidation varied in a dose-dependent manner, and it was greater in fibroblasts. A decrease of proinflammatory cytokines secretion, in particular of tumor necrosis factor alpha (TNF-alpha), was found after FBBP pretreatment of THP-1-derived inflamed macrophages. Melanin production and tyrosinase activity investigated in UVB-irradiated Mel-Juso cells were lowered in direct relation to FBBP concentrations. FBBP fractions with high radical scavenging activity were separated by ion exchange chromatography, and two collagenic sequences were identified. All these results offer new scientific data on aquaculture fish bone-derived peptides confirming their ability to control the antioxidant, anti-inflammatory and pigmentation processes developed during UV irradiation of skin cells and recommend their use as valuable natural ingredients of photoprotective cosmeceutical products.

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Acid-soluble, undenatured, type I collagen (BSC) isolated, for the first time, from gilthead bream skin and the novel fabricated 3D porous wound dressing were analyzed for physicochemical and biological properties, in order to offer a safe alternative to commercial bovine collagen (BC) products. SDS-polyacrylamide analysis confirmed the purity of BSC preparation. The hydroxyproline content and temperature of denaturation of BSC were lower than those of BC, in accordance with the structural data recorded by FT-IR spectroscopy. However, certain concentrations of BSC stimulated the cell metabolism of L929 fibroblasts in a higher proportion than BC. The 3D wound dressing presented high porosity and low surface hydrophobicity that could help cell attachment and growth. The rapid biodegradation of BSC wound dressing could explain the improved in vitro cell migration and wound closure rate. In conclusion, the skin of gilthead bream from the Black Sea coast represented a valuable source for the biomedical industry, providing biocompatible, biodegradable collagen and 3D porous wound dressing, as novel material with enhanced wound healing activity.

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This study focuses on combining different bioprocessing tools in order to develop an in-depth engineering approach for enhancing the biological properties of two valuable food by-products, namely fish waste and yellow onion skins, in a single new bioactive formulation. Bone tissue from phytophagous carp (Hypophthalmichthys molitrix) was used to obtain bioactive peptides through papain-assisted hydrolysis. The peptides with molecular weight lower than 3 kDa were characterized through MALDI-ToF/ToF mass spectrometry and bioinformatics tools. As a prerequisite for microencapsulation, the ability of these peptides to bind the flavonoids extracted from yellow onion skins was further tested through fluorescence quenching measurements. The results obtained demonstrate a considerable binding potency with a binding value of 10(6) and also the presence of one single or one class of binding site during the interaction process of flavonoids with peptides, in which the main forces involved are hydrogen bonds and van der Waals interactions. In the freeze-drying microencapsulation process, an efficiency for total flavonoids of 88.68 +/- 2.37% was obtained, considering the total flavonoids and total polyphenols from the powder of 75.72 +/- 2.58 quercetin equivalents/g dry weight (DW) and 97.32 +/- 2.80 gallic acid equivalents/g DW, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test on the L929 cell line cultivated in the presence of different concentrations of microencapsulated samples (0.05-1.5 mg/mL) proved no sign of cytotoxicity, the cell viability being over 80% for all the samples.

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