Cytotoxicity and Wound Closure Evaluation in Skin Cell Lines after Treatment with Common Antiseptics for Clinical Use

Citation: Ortega-Llamas, L.; Quiñones-Vico, M.I.; García-Valdivia, M.; Fernández-González, A.; Ubago-Rodríguez, A.; Sanabria-de la Torre, R.; Arias-Santiago, S. Cytotoxicity and Wound Closure Evaluation in Skin Cell Lines after Treatment with Common Antiseptics for Clinical Use. Cells 2022,11, 1395. https://doi.org/10.3390/ cells11091395 Academic Editor: Pei-Hui Lin Received: 12 March 2022 Accepted: 18 April 2022 Published: 20 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Cytotoxicity and Wound Closure Evaluation in Skin Cell Lines after Treatment with Common Antiseptics for Clinical Use Laura Ortega-Llamas 1,2,3,†, María I. Quiñones-Vico 1,2,3,4,† , Marta García-Valdivia 1,2,3, Ana Fernández-González 1,2,3,* , Ana Ubago-Rodríguez 1,2,3,4 , Raquel Sanabria-de la Torre 1,2,3 and Salvador Arias-Santiago 1,2,3,4,5 1 Cell Production and Tissue Engineering Unit, Virgen de las Nieves University Hospital, 18014 Granada, Spain; [email protected] (L.O.-L.); [email protected] (M.I.Q.-V.); [email protected] (M.G.-V.); [email protected] (A.U.-R.); [email protected] (R.S.-d.l.T.); [email protected] (S.A.-S.) 2Biosanitary Institute of Granada (ibs.GRANADA), 18014 Granada, Spain 3Andalusian Network of Design and Translation of Advanced Therapies, 41092 Seville, Spain 4Dermatology Department, School of Medicine, University of Granada, 18014 Granada, Spain 5Dermatology Department, Virgen de las Nieves University Hospital, 18014 Granada, Spain *Correspondence: [email protected] † These authors contributed equally to this work. Abstract: In recent years, new therapies, such as skin cell lines injections, have emerged to promote re-epithelialization of damaged areas such as chronic ulcers or to treat patients with severe burns. Antiseptics are commonly used during wound clinical management to avoid serious infections, but they may delay the healing process due to their apparent cytotoxicity to skin cells. The cytotoxicity of ethanol, chlorhexidine digluconate, sodium hypochlorite, povidone iodine and polyhexanide was evaluated in this in vitro study on human fibroblasts and keratinocytes. Treatments were applied to each cell type culture every 48 h for 14 days. To determine the cytotoxic of antiseptics, cell viability (Live/Dead ® ) and cell proliferation (AlamarBlue ™ ) assays were performed on cell monolayers. Cell migration capacity was evaluated with a wound closure assay. Results showed how chlorhexidine digluconate and ethanol significantly reduced the viability of keratinocytes and inhibited cell migration. Povidone iodine followed by chlorhexidine digluconate significantly reduced fibroblast cell viability. Povidone iodine also inhibited cell migration. Sodium hypochlorite was the least detrimental to both cell types. If epithelial integrity is affected, the wound healing process may be altered, so the information gathered in this study may be useful in selecting the least aggressive antiseptic after treatment with new emerging therapies. Keywords: antiseptics; cell migration; cytotoxicity; fibroblasts; keratinocytes; wound healing; wound regeneration 1. Introduction The skin is the first physical protective barrier in the human body against water, microorganisms, mechanical trauma, chemical substances and damage caused by ultraviolet light. It is composed of three layers: the epidermis, dermis and hypodermis [1,2]. In the epidermis, there are four types of cells that form a stratified epithelium. Keratinocytes are the most frequent, and, other less abundant cell types are found among them: melanocytes, Langerhans cells and Merkel cells. The dermis is the layer beneath the epidermis. It consists mainly of connective tissue, fibroblasts and collagen fibers. The hypodermis is the deepest layer, located below the dermis and mainly composed of adipocytes [1,2]. During the regeneration of damaged skin, processes such as cell proliferation, cell migration, secretion of growth factors and specific cytokines, among others, are present. An alteration in any of these events can delay the wound healing process and lead to a chronic wound, which may be the focus of significant bacterial colonization [ 3 ]. Burns or Cells 2022,11, 1395. https://doi.org/10.3390/cells11091395 https://www.mdpi.com/journal/cells Cells 2022,11, 1395 2 of 18 hard-to-heal ulcers promote bacterial colonization and biofilm formation. These infections can cause sepsis with fatal consequences for the patient. Therefore, antimicrobial treatments with antibiotics or antiseptics are often required for successful wound healing [ 4 , 5 ]. The correct proliferation of fibroblasts and keratinocytes during the skin regeneration process is essential. Moreover, these cells are mainly involved in the inflammatory response, one of the most crucial phases for the epithelial regeneration process [3]. In order to prevent and treat localized skin infections in clinic, the use of antiseptics has been encouraged due to the emergence of bacterial resistance to antibiotics. Antiseptics are clinically applied topically on the skin to prevent infection due to their ability to reduce microorganisms [ 6 ]. Applying antiseptics at low concentrations is recommended since, according to previous studies, high concentrations can damage the treated tissue and affect the cell viability and migration capacity of fibroblasts and keratinocytes, which can delay the healing process [ 6 , 7 ]. Despite their clinical use for wound healing, antiseptics and antibiotics can affect the viability of skin cells, but there are few studies analyzing the impact of antiseptics on fibroblasts and keratinocytes cultured in vitro [8,9]. The main objective of this study was to evaluate the cytotoxicity of common antiseptics for clinical use on fibroblasts and keratinocytes involved in epithelial regeneration in concentrations ranging from the concentration used in clinical practice to a 1% dilution of the stock solution. In addition, it provides information about the effect of these antiseptics on two essential aspects of the wound healing process: cell migration and proliferation. This research provides useful evidence for selecting an adequate antiseptic treatment during wound management where cell suspension or BASS are used as advanced therapy. 2. Materials and Methods 2.1. Cell Isolation and Culture Human fibroblasts (HFs) were isolated from skin samples (9 cm 2 ) from plastic, dermatological or urological surgery with the prior consent of the patients in compliance with the requirements for human cell and tissue donation (Royal Decree-Law 9/2014, of 4 July) [ 9 ]. The dermis and epidermis were separated by mechanical processing. The dermis was incubated for 18–24 h in a 2 mg/mL solution of type I collagenase (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA). After the incubation time, the dermis was neutralized with a specific medium for dermal fibroblasts (DFM). Cell suspensions were centrifuged at 1000 rpm for 10 min. Türk (Sigma Aldrich, St. Louis, MO, USA) and Trypan Blue (Sigma Aldrich, St. Louis, MO, USA) solutions were used for cell counting and determining viability after initial processing. Fibroblasts were seeded at a density of 100,000–140,000 cells/cm2at initial processing and at 5000–7000 cells/cm2after passing. The immortalized human keratinocyte cell line HaCaT was used as a model to study of keratinocyte cytotoxicity [ 10 ]. HaCaT cells were seeded at a density of 10,000 cells/cm 2 . 2.2. Cell Viability Assays with Antiseptics HFs and HaCaT cells were seeded in 24-well plates (Thermo Fisher Scientific, Carlsbad, CA, USA) at a density of 10,000 cells/cm 2 . Cell culture monolayers in 24-well plates were treated with five different antiseptics which were applied for three minutes every 48 h for 14 days. For this purpose, the medium was removed before each treatment. Subsequently, 500 µ L of the antiseptic solution was applied to each well. After three minutes, the wells were washed with Dulbecco’s phosphate-buffered saline solution (DPBS, Sigma Aldrich, St. Louis, MO, USA) and finally the medium was added to the treated cell wells and incubated at 37 ◦C, 5% CO2until the next treatments. The antiseptics used were: 70% ethanol (Betamadrileño SL, Madrid, Spain), 2% chlorhexidine digluconate (HiBiSCRUB ® , Molnlycke Health Care AB, Madrid, Spain), 0.02% sodium hypochlorite (Microdacyn, Sonoma Pharmaceuticals, CA, USA), povidone iodine 100 mg/mL (LAINCO, SA, Barcelona, Spain) and 0.1% polyhexanide (Prontosan, B Braun Medical, Barcelona, Spain), all approved for clinical use. Cells 2022,11, 1395 3 of 18 A range of concentrations of the stock solution of each antiseptic was used: 1%, 5%, 10%, 50% and 100% (concentration used in the clinic). All solutions were diluted in DPBS. A preliminary study was used to determine the concentration which provided considerable cell viability and showed the most significant difference between different treatments. Finally, the 1% concentration of the stock antiseptic solution (0.7% ethanol, 0.02% chlorhexidine digluconate, 0.0002% sodium hypochlorite, 1 mg/mL povidone iodine and 0.001% polyhexanide) was tested three times and statistical analyses were performed with this concentration. Cell viability and proliferation were determined at days 3, 7, 10 and 14 using two different protocols: Live/Dead ® Cell Viability Assay (Thermo Fisher Scientific, Carlsbad, CA, USA) and AlamarBlue assay (Invitrogen ™ alamarBlue ™ HS Cell Viability Reagent, Thermo Fisher Scientific, Waltham, MA, USA). 2.2.1. Live/Dead®Cell Viability Assay The Live/Dead ® cell viability assay is a colorimetric assay which consists of preparing a staining solution that combines two fluorescent reagents, calcein AM (green fluorescence, Ex/Em 494/517 nm) and ethidium homodimer-1 (red fluorescence, Ex/Em 517/617 nm). It allows live cells to be differentiated from dead cells by staining them green and red, respectively. The Live/Dead ® staining solution was applied on days 3, 7, 10 and 14 of treatment. Then, it was incubated in the dark for 30 min at room temperature. After the incubation time, the solution was removed, the plates were washed with DPBS, and fluorescence was measured at 405 nm using a Leica DM2000 microscope (Leica, Wetzlar, Germany). The images obtained were analyzed using ImageJ v1.47 software (National Institutes of Health, Bethesda, MD, USA) to determine the percentage of cell viability. 2.2.2. AlamarBlue Cell Proliferation Assay AlamarBlue ™ HS Cell Viability Reagent is a ready-to-use resazurin-based solution which allows proliferation to be quantified from the reducing capacity of living cells [11]. On days 3, 7, 10 and 14 of treatment, alamarBlue reagent was added to each well (10% vol/vol), incubated in the dark at 37 ◦ C, 5% CO 2 for four hours and fluorescence was measured at 560/590 nm using a 96-well plate [ 11 ]. Cell proliferation was measured by the degree of reduction in the reagent and the concentration of live cells (cells/cm 2 ) after each treatment, by establishing a standard curve. Measurements at 1% concentration of stock solutions of the antiseptics were tested three times. 2.3. Wound Closure Assay A wound closure assay, also known as a Scratch Test, was performed to determine the impact of antiseptics on cell migration and wound closure. HFs and HaCaT cells were seeded in 12-well plates (Thermo Fisher Scientific, Carlsbad, CA, USA) at a density of 10,000 cells/cm 2 . When the cells were confluent and adherent to the plate, the DFM was removed and each well was scratched with a sterile 10 µ L pipette tip to simulate wound formation [ 12 ]. Cells were then treated for three minutes with the different antiseptics mentioned above, at a concentration of 1% of the clinically used stock solution. Control cells were left untreated. Cell debris and traces of the antiseptic solution were removed by washing with DPBS [ 12 ]. Then, the cells were incubated and images were taken of the scraped area of each well until the control was completely closed. The images were analyzed with ImageJ software. Equations (1) and (2) were used to calculate the percentage of wound closure and cell migration rate ( µ m/h), respectively [ 13 ]. The percentage of wound closure at hour 0 was considered 0% and the percentage of reduction of the scratched area was calculated at 6, 12 and 24 h for HFs and at 12, 24, 36 Cells 2022,11, 1395 4 of 18 and 48 h for HaCaT cells. Wound closure was monitored until the control was completely closed in each cell line [12,13]. Wound closure (%)=At=0−At At=0×100 (1) Cell migration rate µm h=Wi−Wf t(2) where “ At=0 ” is the area of the initial wound just after scratching, “ At ” is the wound area after “n” hours of initial scratching, “W i ” is the average of the initial wound width in µ m, “W f ” is the average of the final wound width in µ m and “t” is the time of the assay (in hours) until the control wound closed [12]. 2.4. Statistical Analysis Statistical analysis of the data was carried out using the program GraphPad Prism (GraphPad Prism 8.0 Software, Inc., La Jolla, CA, USA). The data obtained are expressed as the mean ± the standard error of the mean (SEM). For the analysis, a factorial analysis of variance (ANOVA) was applied to determine the effect of each factor present. Once the ANOVA test was applied, a post hoc analysis was performed with the Tukey’s test for all factors to determine the degree of significance when comparing the factor classes. p ≤ 0.05 was considered statistically significant. 3. Results 3.1. High Concentrations of Antiseptics Cause a Total Reduction of Cell Viability in Skin Cell Lines All treatments caused high toxicity in both HaCaT cells and HFs when the concentrations used clinically were applied (70% ethanol, 2% chlorhexidine digluconate, 0.02% sodium hypochlorite, povidone iodine 100 mg/mL and 0.1% polyhexanide). At day 3, treatments with these concentrations caused 100% cell death compared to the untreated control. When the concentration of the stock solutions for the antiseptics tested was reduced to 50%, 10% or 5%, the impact on cell viability remained highly noticeable in all treatments. A significant reduction in cell viability was observed when comparing each treatment with the untreated control. However, after treatment with sodium hypochlorite, a smaller reduction in cell viability was observed. Sodium hypochlorite was found to be the least toxic antiseptic on skin cell lines at the concentrations tested (Figures S1–S10). 3.2. Common Antiseptics for Clinical Use Diluted to 1% Affect Cell Viability in Skin Cell Lines 3.2.1. Chlorhexidine Digluconate and Ethanol Affected the Viability of HaCaT Cells More than the Other Treatments The concentration range tested was reduced to 1% of the stock solutions. At this concentration, treatments with chlorhexidine digluconate and ethanol in HaCaT cells significantly reduced the percentage of living cells compared to the other treatments, turning out to be the most toxic antiseptics for this cell line. However, polyhexanide and sodium hypochlorite reflected similar cell viability to the untreated control (Figure 1and Table S1). Significant differences were observed between the different antiseptics and days of treatment (Figure 2). At day 3, 7 and 10, significant differences were observed in cell viability after treatment with chlorhexidine digluconate compared to ethanol, sodium hypochlorite, polyhexanide and the control (Figure 2a–c). At day 14, a significant reduction in cell viability was observed after treatment with chlorhexidine digluconate and ethanol compared to sodium hypochlorite, polyhexanide, povidone iodine and the control (Figure 2d). Cells 2022,11, 1395 5 of 18 Figure 1. LIVE/DEAD ® images of HaCaT cells after each treatment at 1% of stock solution and the control at days 3, 7, 10 and 14. ( a – d ) HaCaT cells after ethanol (0.7%) treatment at days 3, 7, 10 and 14, respectively. ( e – h ) HaCaT cells after chlorhexidine digluconate (0.02%) treatment at day 3, 7, 10 and 14, respectively. ( i – l ) HaCaT cells after sodium hypochlorite (0.0002%) treatment at days 3, 7, 10 and 14, respectively. ( m – p ) HaCaT cells after povidone iodine (1 mg/mL) treatment at days 3, 7, 10 and 14, respectively. ( q – t ) HaCaT cells after polyhexanide (0.001%) treatment on days 3, 7, 10 and 14, respectively. ( u – x ) Control (without treatment) at days 3, 7, 10 and 14, respectively. Dead cells are represented in red and live cells in green. n= 3. Magnification 10×. 3.2.2. Povidone Iodine and Chlorhexidine Digluconate Reduced HFs Cell Viability Compared to the Other Treatments In HFs at 1% concentration of antiseptics in the stock solution, chlorhexidine digluconate, ethanol and povidone iodine significantly reduced the percentage of live cells compared to the other treatments. Povidone iodine and chlorhexidine digluconate were the antiseptics that caused the greatest impact. However, treatment with sodium hypochlorite reflected a cell viability similar to the untreated control (Figure 3and Table S2). At day 3, cell viability was significantly reduced after treatment with chlorhexidine digluconate and povidone iodine, compared to ethanol, sodium hypochlorite, polyhexanide and the control (Figure 4a). At day 7, a significant decrease in cell viability was seen after treatment with povidone iodine compared to the other treatments (Figure 4b). At days 7, 10 and 14, a significant reduction in cell viability was observed after treatment with chlorhexidine digluconate compared to ethanol, sodium hypochlorite, polyhexanide and the control (Figure 4b,d). In addition, after treatment with povidone iodine, a significant reduction in cell viability was observed at days 10 and 14 compared to ethanol, sodium hypochlorite, polyhexanide and the control (Figure 4c,d). Cells 2022,11, 1395 6 of 18 Cells 2022, 11, x FOR PEER REVIEW 6 of 19 Figure 2. Statistical analysis of cell viability in HaCaT cells after antiseptic treatment on days 3, 7, 10 and 14. Bar graph of cell viability percentage in HaCaT cells at (a) day 3, (b) day 7, (c) day 10, and (d) day 14 of treatment. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. n = 3. p values ≤ 0.05 were considered statistically significant. Figure 2. Statistical analysis of cell viability in HaCaT cells after antiseptic treatment on days 3, 7, 10 and 14. Bar graph of cell viability percentage in HaCaT cells at ( a ) day 3, ( b ) day 7, ( c ) day 10, and ( d ) day 14 of treatment. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. n= 3. pvalues ≤ 0.05 were considered statistically significant. Cells 2022,11, 1395 7 of 18 Figure 3. LIVE/DEAD ® images of HFs after each treatment with 1% antiseptic stock solution and the control at days 3, 7, 10 and 14. ( a – d ) HFs after ethanol (0.7%) treatment at days 3, 7, 10 and 14, respectively. ( e – h ) HFs after chlorhexidine digluconate (0.02%) treatment at days 3, 7, 10 and 14, respectively. ( i – l ) HFs after sodium hypochlorite (0.0002%) treatment at days 3, 7, 10 and 14, respectively. ( m – p ) HFs after povidone iodine (1 mg/mL) treatment at days 3, 7, 10 and 14, respectively. ( q – t ) HFs after polyhexanide (0.001%) treatment at days 3, 7, 10 and 14, respectively. ( u – x ). Control at days 3, 7, 10 and 14, respectively. Dead cells are represented in red and live cells in green. n=3. Magnification 10×. 3.3. Common Antiseptics for Clinical Use Reduce Cell Growth and Proliferation in Skin Cell Lines 3.3.1. Chlorhexidine Digluconate and Povidone Iodine Significantly Affect HaCaT Cells Growth Compared to the Other Treatments In HaCaT cells, no significant differences in the number of live cells/cm 2 were observed compared to the control and between the different treatments at day 3. On day 7, a significant reduction in cell density was observed after treatment with chlorhexidine digluconate compared to ethanol, sodium hypochlorite, polyhexanide and the control, and after treatment with povidone iodine compared to the control. On day 10, a significant decrease in cell density was observed after chlorhexidine digluconate and povidone iodine treatments with respect to ethanol, sodium hypochlorite, polyhexanide and the control. At day 14, treatment with ethanol produced a significant reduction in cell density compared to the control. Likewise, on day 14, a significant reduction in cell density was observed after treatment with chlorhexidine digluconate compared to treatments with sodium hypochlorite, polyhexanide and the control. Finally, on day 14, treatment with povidone iodine caused a significant reduction in cell density compared to treatment with polyhexanide and the control (Figure 5a,b). Cells 2022,11, 1395 8 of 18 Cells 2022, 11, x FOR PEER REVIEW 8 of 19 Figure 4. Statistical analysis of cell viability in HFs after antiseptic treatment on days 3, 7, 10 and 14. Bar graph of cell viability percentage in HFs at (a) day 3, (b) day 7, (c) day 10, and (d) day 14 of treatment. ** p ≤ 0.01; **** p ≤ 0.0001. n = 3. p values ≤ 0.05 were considered statistically significant. Figure 4. Statistical analysis of cell viability in HFs after antiseptic treatment on days 3, 7, 10 and 14. Bar graph of cell viability percentage in HFs at ( a ) day 3, ( b ) day 7, ( c ) day 10, and ( d ) day 14 of treatment. ** p ≤ 0.01; **** p ≤ 0.0001. n= 3. pvalues ≤ 0.05 were considered statistically significant. Cells 2022,11, 1395 9 of 18 Cells 2022, 11, x FOR PEER REVIEW 9 of 19 3.3. Common Antiseptics for Clinical Use Reduce Cell Growth and Proliferation in Skin Cell Lines 3.3.1. Chlorhexidine Digluconate and Povidone Iodine Significantly Affect HaCaT Cells Growth Compared to the Other Treatments In HaCaT cells, no significant differences in the number of live cells/cm2 were observed compared to the control and between the different treatments at day 3. On day 7, a significant reduction in cell density was observed after treatment with chlorhexidine digluconate compared to ethanol, sodium hypochlorite, polyhexanide and the control, and after treatment with povidone iodine compared to the control. On day 10, a significant decrease in cell density was observed after chlorhexidine digluconate and povidone iodine treatments with respect to ethanol, sodium hypochlorite, polyhexanide and the control. At day 14, treatment with ethanol produced a significant reduction in cell density compared to the control. Likewise, on day 14, a significant reduction in cell density was observed after treatment with chlorhexidine digluconate compared to treatments with sodium hypochlorite, polyhexanide and the control. Finally, on day 14, treatment with povidone iodine caused a significant reduction in cell density compared to treatment with polyhexanide and the control (Figure 5a,b). Figure 5. Graphical representation of cell density in HaCaT cells ( a , b ) and HFs ( c , d ) after each treatment and the control. ( a ) Line graph of cell density in HaCaT cells after each treatment and the control at days 3, 7, 10 and 14. ( b ) Bar graph with statistical analysis of cell density in HaCaT cells after antiseptic treatment and the control at days 3, 7, 10 and 14. ( c ) Line graph of cell density in HFs after each treatment and the control at days 3, 7, 10 and 14. ( d ) Bar graph with statistical analysis of cell density in HFs after antiseptic treatment and the control at days 3, 7, 10 and 14. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. n= 3. pvalues ≤ 0.05 were considered statistically significant. 3.3.2. Chlorhexidine Digluconate and Povidone Iodine and Ethanol Had Greater Impact on HF Proliferation Compared to the Other Treatments In HFs, significant differences were observed in the number of live cells/cm 2 according to the treatment applied and compared to the control. At day 3, cell density was significantly reduced after chlorhexidine digluconate and povidone iodine treatments compared to ethanol, sodium hypochlorite, polyhexanide and the control. At day 7, treatment with ethanol significantly reduced cell density compared to sodium hypochlorite and the control. Furthermore, at day 7, treatment with chlorhexidine digluconate and povidone iodine resulted in significantly lower cell density than cells treated with sodium hypochlorite and the control. Finally, at day 7, treatment with polyhexanide significantly reduced cell density compared to the control and sodium hypochlorite. On days 10 and 14, after treatment with ethanol, chlorhexidine digluconate, povidone iodine and polyhexanide, a great impact Cells 2022,11, 1395 16 of 18 Funding: The work of María I. Quiñones Vico is supported by a predoctoral fellowship (BOE 22/10/2019) from the Ministry of Science, Innovation and Universities of Spain. This study is part of her doctoral research in the Biomedicine’s program of University of Granada. This research has received competitive funding in the call for grants for the financing of Research, Development and Innovation in Biomedicine and Health Sciences in Andalusia, for the year 2019 (PIGE-0242-2019) and from the Carlos III Health Institute (PI17/02083). Institutional Review Board Statement: The study was approved by the Provincial Ethics Committee of Granada (Spain). Informed Consent Statement: Informed consent was obtained from all surgery donors in compliance with the requirements for donation of human cells and tissues (Royal Decree-Law 9/2014, of 4 July). Data Availability Statement: Data is contained within the article. Acknowledgments: We gratefully acknowledge financial support from the Ministry of Health and Families of the Andalusian Regional Government (PIGE-0242-2019) and from the Carlos III Health Institute (PI17/02083). The work of María I. Quiñones Vico was supported by a predoctoral fellowship (BOE22/10/2019) from the Ministry of Science, Innovation and Universities of Spain. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations BASS Bioengineered Autologous Skin Substitute CHLO Chlorhexidine DFM Dermal Fibroblast Medium DPBS Dulbecco’s phosphate-buffered saline HFs Human Fibroblasts hMSCs Human Mesenchymal Stem Cells hAMSCs Human Amniotic Membrane derived MSCs hPMSCs Human Placenta derived MSCs hUC-MSCs Human Umbilical Cord derived MSCs ROS Reactive Oxygen Species SDS Sodium Dodecyl Sulfate SEM Standard Error of the Mean References 1. Jia, T.; Qiao, W.; Yao, Q.; Wu, W.; Kaku, K. Treatment with docosahexaenoic acid improves epidermal keratinocyte differentiation and ameliorates inflammation in human keratinocytes and reconstructed human epidermis models. Molecules 2019 ,24, 3156. [CrossRef] [PubMed] 2. Arda, O.; Göksügür, N. Basic histological structure and functions of facial skin. Clin. 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