Wound Regeneration Research Articles

Comparative Study of the Effectiveness of Pulsed and Continuous 450 nm Laser Radiation on Tissue for Layered Removal of Pigmented Nevi

Benign pigmented skin lesions, including congenital giant pigmented nevi (CGPN), are pathological formations located in various skin layers and consisting of melanocyte clusters. CGPN represent a serious psychological and medical problem associated with extensive unaesthetic changes in the patient’s skin appearance. These lesions are currently treated by various methods, including laser radiation. However, such methods often render ineffective and lead to unsatisfactory clinical and aesthetic results, with the incidence of complications in the form of scarring and recurrence sometimes reaching 41%. Therefore, the problem of effective and appropriate treatment of this pathology is relevant, deserving further research. In this work, we conduct an experimental biomedical study into the effect of blue laser radiation with a wavelength (λ) of 450 nm on the tissues of laboratory animals in order to determine its prospects in the treatment of pigmented nevi, including CGPN. Blue laser radiation (λ — 450 nm) was generated by an Lasermed 10-03 device (Russian Engineering Club, LLC, Tula, Russia). An experimental biomedical in vitro research was performed on chilled samples of the liver and muscles of mini-pigs and in vivo on the pigmented skin of live laboratory rats. The dynamics of the regenerative wound process in the exposure areas was studied at different periods, i.e., immediately after exposure and following up to 90 days. A morphometric assessment of the exposure areas was carried out in terms of penetration depth and coagulation changes. The results obtained allowed determination of optimal parameters of 450 nm blue laser radiation for a precision removal of various pigmented skin formations. Laser radiation with a wavelength of 450 nm is a promising treatment approach for pigmented skin lesions, including CGPN.

Morphological characteristics of wound healing with aloe extract, hydrogel, and their combination: experimental research

BACKGROUND: Currently, there remains a high incidence of skin damage due to trauma, chronic diseases, burns, so it is important to study the issues of regeneration and treatment of skin wounds. AIM: To conduct a morphological analysis of wound healing using aloe extract, hydrogel, and their combination in the experiment. MATERIALS AND METHODS: The study involved 40 sexually mature guinea pigs, which were divided into a control group and 4 experimental groups (independent wound healing, application of aloe extract, hydrosorb gel, layer-by-layer application of drugs). Histological skin preparations were prepared on days 3, 7, 14, and 28 of the experiment. In each microslide, in 10 fields of view of the microscope at a magnification of ×50, morphometric parameters were measured in micrometers (µm): the thickness of the purulent-necrotic scab, inflammatory (leukocyte) infiltrate, granulation tissue, the length of the newly formed epithelium on days 3, 7, 14; the thickness of the epithelium, its length and the thickness of the connective tissue in the central part of the regenerate on the 28th day. All data were subjected to statistical processing. RESULTS: The medications led to a decrease in the inflammatory reaction, acceleration of the formation of granulation tissue, and partial wound epithelization by day 7 of the experiment; however, more pronounced signs of wound healing were observed with layer-by-layer application of aloe extract and hydrogel. By day 14, regardless of the application method, the use of medications demonstrated signs of enhanced wound healing. Thus, the length of the epithelium increased by 1.2–1.6 times, the depth of granulation tissue increased by 1.0–1.2 times compared to the control group. By the end of the experiment, all animals had complete closure of the wound with scar formation, while in the experimental groups showed signs of skin remodeling with appendages. CONCLUSION: Morphological analysis showed that aloe extract, hydrosorb gel, and their combination accelerated the wound healing processes of full-thickness skin wounds in the experiment.

Conventional Versus Regenerative Methods for Wound Healing: A Comparative Experimental Study on a Sheep Model

Background and Objectives: Wound healing is a complex process involving cellular, anatomical, and functional repair, often hindered in chronic wounds associated with diseases like diabetes and vascular disorders. This study investigated the efficacy of conventional and regenerative wound healing approaches in a sheep surgical wound model. Materials and Methods: Six female Bergamasca sheep underwent five full-thickness skin lesions treated with various methods: sterile gauze (control), chlorhexidine, sodium hypochlorite, micronized dermis system application, and dermal matrix. Wound healing progression was monitored over 42 days through wound dimension measurements, exudate analysis, and histopathological evaluations. Results: The results indicated that all wounds healed completely by day 42, with significant reductions in wound size and exudate over time. Notably, Micronized dermis system application and dermal matrix treatments showed a faster evolution in exudate characteristics and improved collagen reorganization compared to other treatments. Histological analysis revealed earlier neovascularization and better reconstitution of hair follicles in these groups. Despite the lack of significant differences in healing time, both regenerative approaches enhanced wound healing phases, contributing to exudate control, angiogenesis promotion, and reduced scar formation. Conclusions: The findings suggest that while micronized dermis system application and dermal matrix do not accelerate acute wound healing compared to conventional methods, they offer potential benefits in managing exudate and improving tissue regeneration, warranting further investigation in chronic wound scenarios.

Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

Mammalian wounds leave visible scars, and there are no methods for complete regeneration. However, mouse fetuses regenerate their skin, including epidermal and dermal structures, up to embryonic day (E)13. This regeneration pattern requires the formation of actin cables in the wound margin epithelium; however, the molecular mechanisms are not fully understood. Rac1 alters actin in cells and is involved in the formation of filopodia. We investigated whether actin remodeling and skin regeneration patterns can be reproduced through the regulation of Rac1 signaling. Rac1 expression was downregulated in E13 wounds and upregulated after E15 when scars remained. NSC23766, a Rac1-specific inhibitor, altered actin dynamics at the cell margin from filopodia formation to cable formation and inhibited the migration of mouse epidermal keratinocyte, PAM212, by Rac1 signaling suppression. NSC23766 suppressed Rac1 activity and completely regenerated the fetal mouse wounds, even at E14, by changing actin dynamics. Knocked-out Rac1 transgenic mice experienced delayed epithelialization of wounds with suppressed epidermal migration in adults; however, in fetuses, complete wound regeneration via Rac1 signal suppression was observed. Therefore, Rac1 suppression in the wound epidermis can achieve regenerative wound healing in fetuses and may be a potential candidate for healing scars.

Application of Fetal Membranes and Natural Materials for Wound and Tissue Repair

The human fetal membrane is a globally accepted biological biomaterial for wound and tissue repair and regeneration in numerous fields, including dermatology, ophthalmology, and more recently orthopedics, maxillofacial and oral surgery, and nerve regeneration. Both cells and matrix components of amnion and chorion are beneficial, releasing a diverse range of growth factors, cytokines, peptides, and soluble extracellular matrix components. Beside fetal membranes, numerous natural materials have also been reported to promote wound healing. The biological properties of these materials may potentiate the pro-healing action of fetal membranes. Comparison of such materials with fetal membranes has been scant, and their combined use with fetal membranes has been underexplored. This review presents an up-to-date overview of (i) clinical applications of human fetal membranes in wound healing and tissue regeneration; (ii) studies comparing human fetal membranes with natural materials for promoting wound healing; and (iii) the literature on the combined use of fetal membranes and natural pro-healing materials.

In Vitro Assessment of Injectable Bone Marrow Aspirate Concentrates Compared to Injectable Platelet-Rich Fibrin.

Injectable platelet-rich fibrin (iPRF), a liquid form of PRF that is prepared from peripheral blood without anticoagulants, promotes tissue wound healing and regeneration. The present study focused on iPRF-like bone marrow aspirate concentrate (iBMAC) prepared without anticoagulant, and the regenerative potential of iPRF and iBMAC was compared in vitro. iPRF and iBMAC were prepared from the same New Zealand white rabbits. The cytocompatibility and regenerative potential of each concentrate were evaluated using primary rabbit gingival fibroblasts and osteoblasts. Both gingival fibroblasts and osteoblasts treated with each concentrate exhibited excellent cell viability. Interestingly, compared to cells treated with iPRF, cells treated with iBMAC demonstrated significantly greater migration potential. Furthermore, higher mRNA levels of transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and collagen I (COL1) were observed in gingival fibroblasts treated with iBMAC than in those treated with iPRF. Compared with osteoblasts treated with iPRF, osteoblasts treated with iBMAC exhibited greater differentiation potential, as indicated by increased osteocalcin (OCN) expression and mineralization capability. The results of the in vitro study suggest that, compared with iPRF, iBMAC may promote wound healing and bone regeneration more effectively. However, further preclinical and clinical studies are needed to confirm the regenerative potential of iBMAC in the body.

Inhibiting dipeptidyl peptidase 4 positive fibroblasts using zinc sulfide cellulose nanofiber scaffolds to achieve scarless healing

Wound regeneration with integral function and cutaneous appendages remains challenging in wound dressing applications. Cellulose nanofibers (CNF) exhibit remarkable characteristics in wound dressing applications; however, their utility in the wound healing process is limited by insufficient scar inhibition and regenerative healing. Herein, inspired by fibroblast heterogeneity mediating wound healing and skin regeneration, we developed a CNF scaffold designed to block Dipeptidyl Peptidase 4 positive (DPP4+) fibroblasts for regenerative healing. CNF encapsulated sitagliptin (SITA) and zinc sulfide nanoparticles (NZnS), namely CNF@SITA@NZnS, to fabricate a novel biomaterial for scar reduction and regenerative healing. The scaffold promoted scarless healing and hair follicle regeneration in rats. In vivo experiments, the wounds in the scaffold showed less skin fibrosis, a better collagen ratio and more new hair follicles. In vitro experiments showed that the scaffold material promoted scarless healing, possibly by inhibiting the secretion of extracellular matrix and fibroblast-to-myofibroblast conversion. The promotion of hair follicle regeneration by the scaffold material may be due to promotion of the migration and proliferation of human hair follicle papilla cells. RNA sequencing is performed to explore the underlying mechanisms, which can activate ECM-receptor interaction pathway in favor of the wound healing process. The inhibiting effect of CNF@SITA@NZnS scaffold on DPP4+ fibroblasts can be a potential target to reduce scarring and promote skin regeneration.

Translation Prospects of a Novel ECM‐Silk Fibroin/Alginate 3D‐Printed Scaffold for Treatment of Full‐Thickness Skin Wounds: An In Vitro and In Vivo Study

ABSTRACTThe development of bioinks incorporating extracellular matrix (ECM) has attracted significant interest for creating three‐dimensional (3D)‐printed structures that simulate natural skin, aiming to facilitate profound wound healing. In our study, we utilized the potential of human placenta, renowned for its abundant structural proteins and growth factors essential for wound recovery, as a basis for an ECM‐based bioink. Different concentrations (1.5%, 3% and 5% w/v) of decellularized/solubilized placental ECM were integrated into silk fibroin/alginate to generate a printable bioink. The biocompatibility of the printed hydrogels was studied in vitro. Our refined ECM‐based bioink at a 5% w/v concentration was administered to full‐thickness wounds in a mouse model. The ECM‐based frameworks, due to their distinct structure, created a non‐cytotoxic environment conducive to in vitro cell adhesion, infiltration, and proliferation. Crucially, they did not provoke an adverse immune reaction in the host. Implanting the 3D‐printed ECM scaffold into deep wounds resulted in increased formation of granulation tissue, angiogenesis, and re‐epithelialization compared to scaffolds lacking ECM and untreated wounds. Our findings decisively demonstrate that the 5% ECM 3D scaffold promotes regeneration of deep wounds in vivo, creating a skin substitute with cellular organization closely resembling normal skin. This advancement sets the stage for future clinical exploration and holds tremendous promise for advancing wound healing therapies.

Optimization of an Affordable and Efficient Skin Allograft Composite with Excellent Biomechanical and Biological Properties Suitable for the Regeneration of Deep Skin Wounds: A Preclinical Study.

Deep skin wounds require grafting with a skin substitute for treatment. Despite many attempts in the development of an affordable and efficient skin substitute, the repair of deep skin wounds still remains challenging. In the current study, we present a 3D sponge composite made from human placenta (a disposable organ) and sodium alginate with exceptional properties for skin tissue engineering applications. Toward this goal, different proportions of alginate (Alg) and decellularized placenta scaffold (DPS) were composited and freeze-dried to generate a 3D sponge with the desired biomechanical and biological features. Comprehensive in vitro, in ovo, and in vivo characterizations were performed to assess the morphology, physical structure, mechanical behaviors, angiogenic potential, and wound healing properties of the composites. Through these analyses, the scaffold with optimal proportions of Alg (50%) and DPS (50%) was found to have superior properties. The optimized scaffold (Alg50/DPS50) was applied to the full-thickness wounds created in rats. Our data revealed that the addition of DPS to the Alg solution caused a significant improvement in the mechanical characteristics of the scaffold. Remarkably, the fabricated composite scaffold exhibited mechanical properties similar to those of native skin tissue. When implanted into the full-thickness wounds, the Alg50/DPS50 composite scaffold promoted angiogenesis, re-epithelialization, and granulation tissue formation, as compared to the group without a scaffold. Overall, our findings underscore the potential value of this hybrid scaffold for enhancing skin wound healing and suggest an Alg50/DPS50 composite for clinical investigations.

Programmable Artificial Skins Accomplish Antiscar Healing with Multiple Appendage Regeneration.

Functional appendage regeneration is essential for skin rehabilitation, but it has always failed by current existing healing approaches, owing to their inefficacy in preventing disfiguring scars. In this study, a novel regeneration-directing artificial skin (RDAS) system is presented, which is based on the rational design of multi-layered hydrogels that closely mimic natural skin matrices. By leveraging the programmability and architectural rigidity of DNA components, without the need for exogenous cell transplantation, such RDAS effectively minimizes tissue fibrosis by accurately guiding the regenerative process in wound fibroblasts, enabling rapid scarless wound repair, restoration of dermal function, and successful in situ regeneration of multiple appendages, such as hair follicles (HFs), sebaceous glands (SGs), and sweat glands (SwGs). Therefore, the RDAS offers a cell-free antiscarring therapeutic strategy for regenerative wound healing, resulting in improved outcomes. This innovative approach holds great potential for future clinical applications and burn rehabilitation.

Programmed BRD9 Degradation and Hedgehog Signaling Activation via Silk-Based Core-Shell Microneedles Promote Diabetic Wound Healing.

Wound healing impairment in diabetes mellitus is associated with an excessive inflammatory response and defective regeneration capability with suppressed Hedgehog (Hh) signaling. The bromodomain protein BRD9, a subunit of the non-canonical BAF chromatin-remodeling complex, is critical for macrophage inflammatory response. However, whether the epigenetic drug BRD9 degrader can attenuate the sustained inflammatory state of wounds in diabetes remains unclear. Without a bona fide immune microenvironment, Hh signaling activation fails to effectively rescue the suppressed proliferative ability of dermal fibroblasts and the vascularization of endothelial cells. Therefore, a silk-based core-shell microneedle (MN) patch is proposed to dynamically modulate the wound immune microenvironment and the regeneration process. Specifically, the BRD9 degrader released from the shell of the MNs mitigated the excessive inflammatory response in the early phase. Subsequently, the positively charged Hh signaling agonist is released from the negatively charged core of the silk fibroin nanofibers and promotes the phase transition from inflammation to regeneration, including re-epithelialization, collagen deposition, and angiogenesis. These findings suggest that the programmed silk-based core-shell MN patch is an ideal therapeutic strategy for effective skin regeneration in diabetic wounds.

Engineered Multifunctional BioHJzyme via Tuning D‐Band Center for Postoperative Infected Wound Regeneration of Tumor Resection

AbstractThe catalytic therapy for abundant •OH, 1O2, and •O2− provides an efficient tactic and methodology for rapid tumor/bacteria killing, whereas the limitation is focused on the catalytic efficiency owing to the in‐built energy band of catalytic materials. Thus, d‐band center tuning BioHJzyme is devised, which is composed of zinc tellurate/manganese dioxide anchored by glucose oxidase (GOx) with anti‐tumor and anti‐bacteria properties for postoperative infected wound regeneration of tumor. The GOx depletes glucose to produce H2O2, intercepting the glucose metabolism. The D‐BioHJzyme can catalyze the produced H2O2 to highly lethal •OH with POD‐mimetic activity owing to the tunned d‐band center strategy decreasing the adsorption energy of the intermediate in the catalytical process, while the oxygen originated from H2O2 and Mn2+ can be catalyzed into 1O2. The GPx‐mimetic activity of it can impair the antioxidant system of tumor. In vivo studies show that the BioHJzyme exhibits robust abilities against bacteria and tumors by •OH/1O2 production and promotes the apoptosis owing the Te. Besides, the BioHJzyme can accelerate cutaneous regeneration by M1/M2 regulation and the angiogenesis/collagen deposition. This work enlightens a powerful platform for the remedy of postoperative infected wound regeneration of tumor resection using an engineered BioHJzyme.

In situ photocrosslinkable hydrogel treats radiation-induced skin injury by ROS elimination and inflammation regulation

The clinical management of radiation-induced skin injury (RSI) poses a significant challenge, primarily due to the acute damage caused by an overabundance of reactive oxygen species (ROS) and the ongoing inflammatory microenvironment. Here, we designed a dual-network hydrogel composed of 5 % (w/v) Pluronic F127 diacrylate and 2 % (w/v) hyaluronic acid methacryloyl, termed the FH hydrogel. To confer antioxidant and anti-inflammation properties to the hydrogel, we incorporated PVP-modified Prussian blue nanoparticles (PPBs) and resveratrol (Res) to form PHF@Res hydrogels. PHF@Res hydrogels not only exhibited multiple free radical scavenging activities (DPPH, ABTS), but also displayed multiple enzyme-like activities (POD-, catalase). Meanwhile, PHF@Res-2 hydrogels significantly suppressed intracellular ROS and promoted the migration of fibroblasts in a high-oxidative stress environment. Moreover, in the RSI mouse model, the PHF@Res-2 hydrogel regulated inflammatory factors and collagen deposition, significantly reduced epithelial hyperplasia, promoted limb regeneration and neovascularization, and accelerated wound healing, outperforming the commercial antiradiation formulation, Kangfuxin. The PHF@Res-2 hydrogel dressing shows great potential in accelerating wound healing in RSI, offering tremendous promise for clinical wound management and regeneration.

Fabrication and Properties of Hydrogel Dressings Based on Genipin Crosslinked Chondroitin Sulfate and Chitosan.

Multifunctional hydrogel dressings remain highly sought after for the promotion of skin wound regeneration. In the present study, multifunctional CHS-DA/HACC (CH) hydrogels with an interpenetrated network were constructed using hydroxypropyl trimethyl ammonium chloride modified chitosan (HACC) and dopamine-modified chondroitin sulfate (CHS-DA), using genipin as crosslinker. The synthesis of HACC and CHS-DA was effectively confirmed using Fourier transform infrared (FT-IR) analysis and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The prepared CH hydrogels exhibited a network of interconnected pores within the microstructure. Furthermore, rheological testing demonstrated that CH hydrogels exhibited strong mechanical properties, stability, and injectability. Further characterization investigations showed that the CH hydrogels showed favorable self-healing and self-adhesion properties. It was also shown that increasing HACC concentration ratio was positively correlated with the antibacterial activity of CH hydrogels, as evidenced by their resistance to Escherichia coli and Staphylococcus aureus. Additionally, Cell Counting Kit-8 (CCK-8) tests, fluorescent images, and a cell scratch assay demonstrated that CH hydrogels had good biocompatibility and cell migration ability. The multifunctional interpenetrated network hydrogels were shown to have good antibacterial properties, antioxidant properties, stable storage modulus and loss modulus, injectable properties, self-healing properties, and biocompatibility, highlighting their potential as wound dressings in wound healing applications.

Priming Mesenchymal Stem Cells with Lipopolysaccharide Boosts the Immunomodulatory and Regenerative Activity of Secreted Extracellular Vesicles

Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 × 108 particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies.

Zinc-based Polyoxometalate Nanozyme Functionalized Hydrogels for optimizing the Hyperglycemic-Immune Microenvironment to Promote Diabetic Wound Regeneration.

In diabetic wounds, hyperglycemia-induced cytotoxicity and impaired immune microenvironment plasticity directly hinder the wound healing process. Regulation of the hyperglycemic microenvironment and remodeling of the immune microenvironment are crucial. Here, we developed a nanozymatic functionalized regenerative microenvironmental regulator (AHAMA/CS-GOx@Zn-POM) for the effective repair of diabetic wounds. This novel construct integrated an aldehyde and methacrylic anhydride-modified hyaluronic acid hydrogel (AHAMA) and chitosan nanoparticles (CS NPs) encapsulating zinc-based polymetallic oxonate nanozyme (Zn-POM) and glucose oxidase (GOx), facilitating a sustained release of release of both enzymes. The GOx catalyzed glucose to gluconic acid and (H₂O₂), thereby alleviating the effects of the hyperglycemic microenvironment on wound healing. Zn-POM exhibited catalase and superoxide dismutase activities to scavenge reactive oxygen species and H₂O₂, a by-product of glucose degradation. Additionally, Zn-POM induced M1 macrophage reprogramming to the M2 phenotype by inhibiting the MAPK/IL-17 signaling diminishing pro-inflammatory cytokines, and upregulating the expression of anti-inflammatory mediators, thus remodeling the immune microenvironment and enhancing angiogenesis and collagen regeneration within wounds. In a rat diabetic wound model, the application of AHAMA/CS-GOx@Zn-POM enhanced neovascularization and collagen deposition, accelerating the wound healing process. Therefore, the regenerative microenvironment regulator AHAMA/CS-GOx@Zn-POM can achieve the effective conversion of a pathological microenvironment to regenerative microenvironment through integrated control of the hyperglycemic-immune microenvironment, offering a novel strategy for the treatment of diabetic wounds.

De-novo transcriptome of anterior epimorphic regeneration in Perionyx excavatus

Perionyx excavatus, an indigenous earthworm possesses exceptional regeneration capacity. Their anterior regeneration features wound closure, regeneration induction and morphogenesis of damaged organs. This study involved a complete analysis of their transcriptomic dataset, with an emphasis on identifying the genes expressed during regeneration and predicting their implications in the process of regeneration and morphogenesis. Control (first ten segments) and test (5th day blastema) RNA samples of biological replicates were isolated and sequenced on NovaSeq. 6000 using PE150 read length. An average of 98.64% of high-quality data was retained with assembly showing better continuity with the average transcript length with 823 bp and N50 value of 1,858 bp. This is the first report on the comparative transcriptome of P. excavatus during anterior regeneration and this study will shed light on the complexity of annelid regeneration.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

Injectable Oxygen-Carrying Microsphere Hydrogel for Dynamic Regulation of Redox Microenvironment of Wounds.

The delayed healing of infected wounds can be attributed to the increased production of reactive oxygen species (ROS) and consequent damages to vascellum and tissue, resulting in a hypoxic wound environment that further exacerbates inflammation. Current clinical treatments including hyperbaric oxygen therapy and antibiotic treatment fail to provide sustained oxygenation and drug-free resistance to infection. To propose a dynamic oxygen regulation strategy, this study develops a composite hydrogel with ROS-scavenging system and oxygen-releasing microspheres in the wound dressing. The hydrogel itself reduces cellular damage by removing ROS derived from immune cells. Simultaneously, the sustained release of oxygen from microspheres improves cell survival and migration in hypoxic environments, promoting angiogenesis and collagen regeneration. The combination of ROS scavenging and oxygenation enables the wound dressing to achieve drug-free anti-infection through activating immune modulation, inhibiting the secretion of pro-inflammatory cytokines interleukin-6, and promoting tissue regeneration in both acute and infected wounds of rat skins. Thus, the composite hydrogel dressing proposed in this work shows great potential for dynamic redox regulation of infected wounds and accelerates wound healing without drugs.

Proteomic analysis of the human amniotic mesenchymal stromal cell secretome by integrated approaches via filter-aided sample preparation

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SignificanceIn this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.