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14 pages, 2562 KiB

Open AccessArticle

Evaluation of Cell Migration and Cytokines Expression Changes under the Radiofrequency Electromagnetic Field on Wound Healing In Vitro Model

by Erica Costantini, Lisa Aielli, Federica Serra, Lorenzo De Dominicis, Katia Falasca, Pamela Di Giovanni and Marcella Reale

Int. J. Mol. Sci. 2022, 23(4), 2205; https://doi.org/10.3390/ijms23042205 - 17 Feb 2022

Cited by 11 | Viewed by 5455

Abstract

Wound healing (WH) proceeds through four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Impaired WH may be the consequence of the alteration of one of these phases and represents a significant health and economic burden to millions of individuals. Thus, new therapeutic strategies [...] Read more.

Wound healing (WH) proceeds through four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Impaired WH may be the consequence of the alteration of one of these phases and represents a significant health and economic burden to millions of individuals. Thus, new therapeutic strategies are the topics of intense research worldwide. Although radiofrequency electromagnetic field (RF-EMF) has many medical applications in rehabilitation, pain associated with musculoskeletal disorders, and degenerative joint disorders, its impact on WH is not fully understood. The process of WH begins just after injury and continues during the inflammatory and proliferative phases. A thorough understanding of the mechanisms by which RF-EMF can improve WH is required before it can be used as a non-invasive, inexpensive, and easily self-applicable therapeutic strategy. Thus, the aim of this study is to explore the therapeutic potential of different exposure setups of RF-EMF to drive faster healing, evaluating the keratinocytes migration, cytokines, and matrix metalloproteinases (MMPs) expression. The results showed that RF-EMF treatment promotes keratinocytes’ migration and regulates the expression of genes involved in healing, such as MMPs, tissue inhibitors of metalloproteinases, and pro/anti-inflammatory cytokines, to improve WH. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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21 pages, 16884 KiB

Open AccessArticle

Single Dose of N-Acetylcysteine in Local Anesthesia Increases Expression of HIF1α, MAPK1, TGFβ1 and Growth Factors in Rat Wound Healing

by Wiktor Paskal, Michał Kopka, Albert Stachura, Adriana M. Paskal, Piotr Pietruski, Kacper Pełka, Alan E. Woessner, Kyle P. Quinn, Ryszard Galus, Jarosław Wejman and Paweł Włodarski

Int. J. Mol. Sci. 2021, 22(16), 8659; https://doi.org/10.3390/ijms22168659 - 12 Aug 2021

Cited by 2 | Viewed by 3057

Abstract

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague–Dawley rats, intradermal injections were made. Lidocaine–epinephrin [...] Read more.

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague–Dawley rats, intradermal injections were made. Lidocaine–epinephrin solutions were supplemented with 0.015%, 0.03% or 0.045% solutions of NAC, or nothing (control group). Scars were harvested on the 3rd, 7th, 14th and 60th day post-surgery. We performed immunohistochemical staining in order to visualize macrophages (anti-CD68), neutrophils (anti-MPO) and newly formed blood vessels (anti-CD31). Additionally, RT-qPCR was used to measure the relative expression of 88 genes involved in the wound healing process. On the 14th day, the number of cells stained with anti-CD68 and anti-CD31 antibodies was significantly larger in the tissues treated with 0.03% NAC compared with the control. Among the selected genes, 52 were upregulated and six were downregulated at different time points. Interestingly, NAC exerted a significant effect on the expression of 45 genes 60 days after its administration. In summation, a 0.03% NAC addition to the pre-incisional anesthetic solution improves neovasculature and increases the macrophages’ concentration at the wound site on the 14th day, as well as altering the expression of numerous genes that are responsible for the regenerative processes. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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20 pages, 4823 KiB

Open AccessArticle

Overexpression of Flii during Murine Embryonic Development Increases Symmetrical Division of Epidermal Progenitor Cells

by Gink N. Yang, Parinaz Ahangar, Xanthe L. Strudwick, Zlatko Kopecki and Allison J. Cowin

Int. J. Mol. Sci. 2021, 22(15), 8235; https://doi.org/10.3390/ijms22158235 - 30 Jul 2021

Cited by 6 | Viewed by 2009

Abstract

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of [...] Read more.

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii^+/−, wild type: WT, transgenic Flii overexpressing: Flii^Tg^/Tg) using Western blot and immunohistochemistry. Flii^+/− embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. Flii^Tg^/Tg led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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18 pages, 7873 KiB

Open AccessArticle

N-Acetylcysteine Added to Local Anesthesia Reduces Scar Area and Width in Early Wound Healing—An Animal Model Study

by Wiktor Paskal, Adriana M. Paskal, Piotr Pietruski, Albert Stachura, Kacper Pełka, Alan E. Woessner, Kyle P. Quinn, Michał Kopka, Ryszard Galus, Jarosław Wejman and Paweł Włodarski

Int. J. Mol. Sci. 2021, 22(14), 7549; https://doi.org/10.3390/ijms22147549 - 14 Jul 2021

Cited by 1 | Viewed by 2791

Abstract

The aim of the study was to evaluate if a pre-incisional N-acetylcysteine (NAC) treatment altered the process of wound healing in a rat model. The dorsal skin of 24 Sprague-Dawley rats was incised in six locations. Before the incisions were made, skin [...] Read more.

The aim of the study was to evaluate if a pre-incisional N-acetylcysteine (NAC) treatment altered the process of wound healing in a rat model. The dorsal skin of 24 Sprague-Dawley rats was incised in six locations. Before the incisions were made, skin was injected either with lidocaine and epinephrine (one side) or with these agents supplemented with 0.015%, 0.03%, or 0.045% NAC (contralaterally). Photographic documentation of the wound healing process was made at 11 time points. Rats were sacrificed 3, 7, 14, or 60 days after incision to excise scars for histological analysis. They included: Abramov scale scoring, histomorphometry analysis, and collagen fiber arrangement assessment. Skin pretreated with 0.03% NAC produced the shortest scars at all analyzed time points, though this result was statistically insignificant. At this NAC concentration the scars had smaller areas on the third day and were narrower on the day 4 compared with all the other groups (p < 0.05). On day 7, at the same concentration of NAC, the scars had a higher superficial concentration index (p = 0.03) and larger dermal proliferation area (p = 0.04). NAC addition to pre-incisional anesthetic solution decreased wound size and width at an early stage of scar formation at all concentrations; however, with optimal results at 0.03% concentration. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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17 pages, 3292 KiB

Open AccessArticle

Calpastatin-Mediated Inhibition of Calpain Ameliorates Skin Scar Formation after Burn Injury

by Cheong Hoon Seo, Hui Song Cui and June-Bum Kim

Int. J. Mol. Sci. 2021, 22(11), 5771; https://doi.org/10.3390/ijms22115771 - 28 May 2021

Cited by 4 | Viewed by 2161

Abstract

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the [...] Read more.

Hypertrophic scars, the most common complication of burn injuries, are characterized by excessive deposition of fibroblast-derived extracellular matrix proteins. Calpain, a calcium-dependent protease, is involved in the fibroblast proliferation and extracellular matrix production observed in certain fibrotic diseases. However, its role in the formation of post-burn hypertrophic skin scars remains largely unknown. Here, calpain expression and activity were assessed in skin fibroblasts obtained directly from patients with third-degree burns, who consequently developed post-burn hypertrophic scars. Furthermore, the antifibrotic effect of calpastatin, an endogenous calpain inhibitor, was evaluated in human fibroblasts and a murine burn model. The activity, mRNA levels, and protein levels of calpain were markedly higher in fibroblasts from the burn wounds of patients than in normal cells. Selective calpain inhibition by calpastatin markedly reduced not only the proliferation of burn-wound fibroblasts but also the mRNA and protein expression of calpain, transforming growth factor-beta 1, α-smooth muscle actin, type I and type III collagens, fibronectin, and vimentin in burn-wound fibroblasts. The anti-scarring effects of calpastatin were validated using a murine burn model by molecular, histological, and visual analyses. This study demonstrates the pathological role of calpain and the antifibrotic effect of calpastatin via calpain inhibition in post-burn hypertrophic scar formation. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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16 pages, 1741 KiB

Open AccessArticle

Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations

by Madelene Å Andersson, Lone Bruhn Madsen, Artur Schmidtchen and Manoj Puthia

Int. J. Mol. Sci. 2021, 22(9), 5045; https://doi.org/10.3390/ijms22095045 - 10 May 2021

Cited by 25 | Viewed by 5920

Abstract

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and reproducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present [...] Read more.

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and reproducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, P. aeruginosa and S. aureus. We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains successfully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of antibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indicating that the demonstrated method mirrors typical in vivo infections. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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18 pages, 2358 KiB

Open AccessArticle

Exosomes Secreted from Amniotic Membrane Contribute to Its Anti-Fibrotic Activity

by Yong Mao, Vimal Jacob, Amit Singal, Shunyao Lei, Min Sung Park, Mariana R.N. Lima, Chaoyang Li, Sandeep Dhall, Malathi Sathyamoorthy and Joachim Kohn

Int. J. Mol. Sci. 2021, 22(4), 2055; https://doi.org/10.3390/ijms22042055 - 19 Feb 2021

Cited by 10 | Viewed by 2437

Abstract

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by [...] Read more.

Amniotic membranes (AM) have anti-fibrotic activity. Exosomes (nano-sized vesicles) function as conduits for intercellular transfer and contain all the necessary components to induce the resolution of fibrosis. In this study, we tested the hypothesis that the anti-fibrotic activity of AM is mediated by exosomes. AM-derived exosomes or amniotic stromal cell-derived exosomes were isolated and characterized. Anti-fibrotic activity of exosomes was evaluated using human hepatic stellate cells (LX-2), an in vitro model of fibrosis. Exosomes isolated from AM tissue-conditioned media had an average size of 75 nm. Exosomes significantly inhibited the proliferation of TGFβ1-activated LX-2 but had no effect on the proliferation of non-activated LX-2 cells. Exosomes also reduced the migration of LX-2 in a scratch wound assay. Furthermore, exosomes reduced the gene expression of pro-fibrotic markers such as COL1A1, ACTA, and TGFβ1 in LX-2 cells. Interestingly, exosomes isolated from AM tissue under hypoxic conditions seemed to show a stronger anti-fibrotic activity than exosomes isolated from tissue under normoxic conditions. Exosomes released by in vitro cultured AM stromal cells were smaller in size compared with tissue exosomes and also showed anti-fibrotic activity on LX-2 cells. In conclusion, AM-tissue-released exosomes contribute to the anti-fibrotic activity of AM. This is the first report of isolation, characterization, and functional evaluation of exosomes derived from amniotic tissues with the direct comparison between tissue-derived exosomes and cultured cell-derived exosomes. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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20 pages, 2894 KiB

Open AccessArticle

Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes

by Magdalena Misiura, Weronika Baszanowska, Ilona Ościłowska, Jerzy Pałka and Wojciech Miltyk

Int. J. Mol. Sci. 2020, 21(23), 9243; https://doi.org/10.3390/ijms21239243 - 03 Dec 2020

Cited by 21 | Viewed by 2766

Abstract

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. [...] Read more.

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor β₁ (TGF-β₁), and β₁-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of β₁-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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Review

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16 pages, 1909 KiB

Open AccessReview

The Ambivalent Role of Skin Microbiota and Adrenaline in Wound Healing and the Interplay between Them

by Arif Luqman and Friedrich Götz

Int. J. Mol. Sci. 2021, 22(9), 4996; https://doi.org/10.3390/ijms22094996 - 08 May 2021

Cited by 10 | Viewed by 4646

Abstract

After skin injury, wound healing sets into motion a dynamic process to repair and replace devitalized tissues. The healing process can be divided into four overlapping phases: hemostasis, inflammation, proliferation, and maturation. Skin microbiota has been reported to participate in orchestrating the wound [...] Read more.

After skin injury, wound healing sets into motion a dynamic process to repair and replace devitalized tissues. The healing process can be divided into four overlapping phases: hemostasis, inflammation, proliferation, and maturation. Skin microbiota has been reported to participate in orchestrating the wound healing both in negative and positive ways. Many studies reported that skin microbiota can impose negative and positive effects on the wound. Recent findings have shown that many bacterial species on human skin are able to convert aromatic amino acids into so-called trace amines (TAs) and convert corresponding precursors into dopamine and serotonin, which are all released into the environment. As a stress reaction, wounded epithelial cells release the hormone adrenaline (epinephrine), which activates the β2-adrenergic receptor (β2-AR), impairing the migration ability of keratinocytes and thus re-epithelization. This is where TAs come into play, as they act as antagonists of β2-AR and thus attenuate the effects of adrenaline. The result is that not only TAs but also TA-producing skin bacteria accelerate wound healing. Adrenergic receptors (ARs) play a key role in many physiological and disease-related processes and are expressed in numerous cell types. In this review, we describe the role of ARs in relation to wound healing in keratinocytes, immune cells, fibroblasts, and blood vessels and the possible role of the skin microbiota in wound healing. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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14 pages, 1480 KiB

Open AccessReview

Systematic Review: Adipose-Derived Mesenchymal Stem Cells, Platelet-Rich Plasma and Biomaterials as New Regenerative Strategies in Chronic Skin Wounds and Soft Tissue Defects

by Pietro Gentile and Simone Garcovich

Int. J. Mol. Sci. 2021, 22(4), 1538; https://doi.org/10.3390/ijms22041538 - 03 Feb 2021

Cited by 81 | Viewed by 5296

Abstract

The number of clinical trials evaluating adipose-derived mesenchymal stem cells (AD-MSCs), platelet-rich plasma (PRP), and biomaterials efficacy in regenerative plastic surgery has exponentially increased during the last ten years. AD-MSCs are easily accessible from various fat depots and show intrinsic plasticity in giving [...] Read more.

The number of clinical trials evaluating adipose-derived mesenchymal stem cells (AD-MSCs), platelet-rich plasma (PRP), and biomaterials efficacy in regenerative plastic surgery has exponentially increased during the last ten years. AD-MSCs are easily accessible from various fat depots and show intrinsic plasticity in giving rise to cell types involved in wound healing and angiogenesis. AD-MSCs have been used in the treatment of soft tissue defects and chronic wounds, employed in conjunction with a fat grafting technique or with dermal substitute scaffolds and platelet-rich plasma. In this systematic review, an overview of the current knowledge on this topic has been provided, based on existing studies and the authors’ experience. A multistep search of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus database, and Cochrane databases has been performed to identify papers on AD-MSCs, PRP, and biomaterials used in soft tissue defects and chronic wounds. Of the 2136 articles initially identified, 422 articles focusing on regenerative strategies in wound healing were selected and, consequently, only 278 articles apparently related to AD-MSC, PRP, and biomaterials were initially assessed for eligibility. Of these, 85 articles were excluded as pre-clinical, experimental, and in vitro studies. For the above-mentioned reasons, 193 articles were selected; of this amount, 121 letters, expert opinions, commentary, and editorials were removed. The remaining 72 articles, strictly regarding the use of AD-MSCs, PRP, and biomaterials in chronic skin wounds and soft tissue defects, were analyzed. The studies included had to match predetermined criteria according to the patients, intervention, comparator, outcomes, and study design (PICOS) approach. The information analyzed highlights the safety and efficacy of AD-MSCs, PRP, and biomaterials on soft tissue defects and chronic wounds, without major side effects. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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15 pages, 1254 KiB

Open AccessReview

Mesenchymal Stem Cell Secretome as an Emerging Cell-Free Alternative for Improving Wound Repair

by Parinaz Ahangar, Stuart J. Mills and Allison J. Cowin

Int. J. Mol. Sci. 2020, 21(19), 7038; https://doi.org/10.3390/ijms21197038 - 24 Sep 2020

Cited by 99 | Viewed by 12679

Abstract

The use of mesenchymal stem cells (MSC) for the treatment of cutaneous wounds is currently of enormous interest. However, the broad translation of cell therapies into clinical use is hampered by their efficacy, safety, manufacturing and cost. MSCs release a broad repertoire of [...] Read more.

The use of mesenchymal stem cells (MSC) for the treatment of cutaneous wounds is currently of enormous interest. However, the broad translation of cell therapies into clinical use is hampered by their efficacy, safety, manufacturing and cost. MSCs release a broad repertoire of trophic factors and immunomodulatory cytokines, referred to as the MSC secretome, that has considerable potential for the treatment of cutaneous wounds as a cell-free therapy. In this review, we outline the current status of MSCs as a treatment for cutaneous wounds and introduce the potential of the MSC secretome as a cell-free alternative for wound repair. We discuss the challenges and provide insights and perspectives for the future development of the MSC secretome as well as identify its potential clinical translation into a therapeutic treatment. Full article

(This article belongs to the Special Issue New Innovations in Wound Healing and Repair 2.0)

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