Cytotoxicity and Epidermal Barrier Function Evaluation of Common Antiseptics for Clinical Use in an Artiﬁcial Autologous Skin Model

Journal of Clinical Medicine

Article Cytotoxicity and Epidermal Barrier Function Evaluation of Common Antiseptics for Clinical Use in an Artiﬁcial Autologous Skin Model

María I. Quiñones-Vico 1,2,3,4 , Ana Fernández-González 1,2,3,* , Elena Pérez-Castejón 1,2,3, Trinidad Montero-Vílchez 2,5 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] (M.I.Q.-V.); [email protected] (E.P.-C.); [email protected] (S.A.-S.) 2 Biosanitary Institute of Granada (ibs. GRANADA), 18014 Granada, Spain; [email protected] 3 Andalusian Network of Design and Translation of Advanced Therapies, 41092 Sevilla, Spain 4 Dermatology Department, School of Medicine, University of Granada, 18014 Granada, Spain 5 Dermatology Department, Virgen de las Nieves University Hospital, 18014 Granada, Spain * Correspondence: [email protected]

Abstract: Bioengineered artificial skin substitutes (BASS) are the main treatment used in addition to autografts when skin injuries involve a large body surface area. Antiseptic/antibiotic treatment is necessary to prevent infections in the BASS implant area. This study aims to evaluate the effect of antiseptics and antibiotics on cell viability, structural integrity, and epidermal barrier function in BASS based on hyaluronic acid during a 28 day follow-up period. Keratinocytes (KTs) and dermal fibroblasts (DFs) were isolated from skin samples and used to establish BASS. The following Citation: Quiñones-Vico, M.I.; antibiotic/antiseptic treatment was applied every 48 h: colistin (1%), chlorhexidine digluconate Fernández-González, A.; (1%), sodium chloride (0.02%), and polyhexanide (0.1%). Cell viability (LIVE/DEAD® assay), Pérez-Castejón, E.; Montero-Vílchez, structural integrity (histological evaluation), and epidermal barrier function (trans-epidermal water T.; Arias-Santiago, S. Cytotoxicity and ® Epidermal Barrier Function loss, (TEWL), Tewameter ) were also evaluated. Cell viability percentage of BASS treated with Evaluation of Common Antiseptics chlorhexidine digluconate was significantly lower (p ≤ 0.001) than the other antiseptics at day 28. for Clinical Use in an Artificial Compared to other treatments, chlorhexidine digluconate and polyhexanide significantly affected the Autologous Skin Model. J. Clin. Med. epithelium. No significant differences were found regarding epidermal barrier. These results may be 2021, 10, 642. https://doi.org/ useful for treatment protocols after implantation of BASS in patients and evaluating them in clinical 10.3390/jcm10040642 practice. BASS represent a suitable model to test in vitro the impact of different treatments of other skin wounds. Academic Editor: Abbas Shafiee Received: 12 January 2021 Keywords: antiseptic/antibiotic testing; bioengineered artificial skin substitute; cell viability; epider- Accepted: 2 February 2021 mal barrier function; regenerative medicine; wound healing Published: 8 February 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. The skin is the largest and one of the most complex organs in the human body, representing 15% of total adult body weight [1,2]. Healthy skin is crucial to maintaining physiological homeostasis as it constitutes a protective barrier against external physical, chemical, and biological agents [3]. The epidermis is the outermost layer of the skin, consisting of a renewable epithe- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. lium whose main function is to prevent the entrance of foreign substances into the body This article is an open access article while allowing water exchange through the skin. The primary cell type in this layer is distributed under the terms and keratinocytes, which are constantly replaced by basal layer stem cells [2]. The dermis is the conditions of the Creative Commons thickest layer located below the epidermis. It is a connective tissue formed mainly by extra- Attribution (CC BY) license (https:// cellular matrix and fibroblasts, which secrete collagen and elastin, providing mechanical creativecommons.org/licenses/by/ strength, flexibility, and elasticity. Beneath this layer the hypodermis is found, an adipose 4.0/). tissue that supplies insulation, cushioning, and an energy storage area [1].

J. Clin. Med. 2021, 10, 642. https://doi.org/10.3390/jcm10040642 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 642 2 of 14

Injuries affecting the skin can disrupt its functions, so it is essential to quickly heal skin wounds [2]. However, there are many situations when the human body cannot correctly heal itself without medical intervention. In case of deep injuries and severe burns, wound healing is slow and incomplete, leaving the body open to infection and poor thermal and fluid regulation, leading to chronic wounds. Currently, the gold standard treatment for these injuries is to use autologous skin grafts to prevent pathogen entry and maintain skin homeostasis. Nevertheless, due to the limit of available native skin, autologous skin grafts can become challenging when these injuries cover a large body surface area [4,5]. That is the reason why skin substitutes were designed to be used in addition to or as a replacement for autologous skin grafts [6]. The most important functions of these substitutes are the prevention of wound infection, reducing pain, promoting wound healing, and the replacement of normal skin to restore its function [3,5,7]. To generate a bioengineered artificial skin substitute (BASS), it is necessary to develop a dermal stroma substitute with human fibroblasts immersed and later, seed the keratinocytes in the upper layer [8,9]. Regarding the BASS dermal stroma, the use of scaffolds is necessary to mimic the extracellular matrix (ECM) in which fibroblasts are immersed. In addition to anchoring cells, the ECM components influence cell survival, proliferation, function [10], rapidly control homeostasis, and modulate the wound environment to maintain an optimal hydration level [11]. Hyaluronic acid (HA) is the most abundant glycosaminoglycan (GAG) in the skin and, along with collagen, one of the most widely used ECM components in wound dressings. HA stimulates cellular proliferation, cellular migration, and angiogenesis, depending on its size [10,12]. Furthermore, it stimulates early inflammation, which is critical for initiating wound healing, but also reduces long-term inflammation. When compared to autografts, BASS based on fibrin-HA showed similar homeostasis parameters and restored epidermal barrier function in a wound mouse model, promoting the formation of a histological architecture very similar to normal skin [13]. Once the skin substitute is ready and grafted into the patient, treatment with antiseptics and antibiotics is crucial to prevent any infection, which is the main risk in these patients [4]. In patients with more than 40% total body surface area (TBSA), 75% of deaths are due to sepsis [14]. The most common microorganisms that cause invasive infections in these burns are Pseudomonas aeruginosa and Acinetobacter baumanii, followed by Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus [15]. Although there is a wide range of protocols [16,17], there is no gold standard antiseptic treatment in burn care. Antiseptics and antibiotics applied during wound care may affect the viability of skin cells. Few studies have analyzed the impact of antiseptics in cultured fibroblast or keratinocytes [18,19], but no analysis of cytotoxicity and epidermal barrier function have been performed in bioengineered artificial skin substitutes. The main objective of this study was to develop a three-dimensional skin model based on hyaluronic acid scaffold to evaluate in vitro how different treatments (antibiotics and antiseptics), used in clinic, affect cell viability, epithelium integrity, and barrier function. This analysis will provide useful information to select an appropriate antiseptic during the burn care process, but also to treat other skin wounds such as ulcers.

2. Materials and Methods 2.1. Cell Isolation and Culture Fibroblasts and keratinocytes were isolated from skin samples (9 cm2) from reconstructive surgery donors with the patient’s informed consent in compliance with the requirements for donation of human cells and tissues (Royal Decree-Law 9/2014, of July 4). J. Clin. Med. 2021 , 10 , x FOR PEER REVIEW 3 of 14

J. Clin. Med. 2021flow, 10, 642cabin, they were maintained in wash solution for 30 min. Subsequently, scalpel and 3 of 14 forceps were used to separate dermis from epidermis. Hypodermis was discarded. The dermis and epidermis were mechanically processed. The dermis was incubated for 18–24 h in a 2 mg/mLSkin samplessolution were of type transported I collage innase Dulbecco’s (Gibco, phosphate-buffered Thermo Fisher Scientific, saline (DPBS) from California, USA)the and operating neutralized room with to the specific laboratory medi forum the forbeginning dermal of fibroblasts their processing. culture In a laminar (DFM). The epidermisflow cabin, incubated they were with maintained TrypLE Select in wash 10× solution(Gibco, Thermo for 30 min. Fisher Subsequently, Scientific, scalpel and California, USA)forceps for 10 werecycles used of 15 to separatemin per dermiscycle and from neutralized epidermis. Hypodermiswith specific was medium discarded. for the keratinocyte cultureThe dermis (KTM) and [13]. epidermis Cell suspe werensions mechanically were centrifuged processed. at The 300× dermis g for was 10 incubated min. Türk (Sigmafor Aldrich, 18–24 h St in Louis, a 2 mg/mL USA) solution and trypan of type blue I collagenase (Sigma Aldrich, (Gibco, St Thermo Louis, USA) Fisher Scientific, solutions were usedCalifornia, for counting USA) andand neutralizedcell viability with determination, specific medium respectively. for dermal fibroblasts culture (DFM). The epidermis incubated with TrypLE Select 10× (Gibco, Thermo Fisher Scientific, Fibroblasts were seeded at a density of 100,000–140,000 cells/cm 2 (37 °C, 5% CO 2) at California, USA) for 10 cycles of 15 min per cycle and neutralized with specific medium initial processing and at 5000–7000 cells/cm 2 after passing. The human dermal fibroblasts for the keratinocyte culture (KTM) [13]. Cell suspensions were centrifuged at 300× g for used as the feeder10 layer min. for Türk keratinocytes (Sigma Aldrich, culture St Louis, were USA)sub-lethally and trypan irradiated blue (Sigma (50 Gy). Aldrich, The St Louis, equipment used USA)was a solutions BIOBEAM were 8000 used gamma for counting irradiation and cell unit viability with a determination, source of Cs-137 respectively. of 2 2 ◦ 2000 Ci. Dermal irradiatedFibroblasts fibroblasts were seeded were seeded at a density at 10,000 of 100,000–140,000 cells/cm . Keratinocytes cells/cm (37 wereC, 5% CO2) at seeded at 20,000–40,000initial processing cells/cm and2 in atthe 5000–7000 flasks over cells/cm an irradiated2 after passing. feeder. The Cultures human dermal were fibroblasts monitored usingused a Leica as the DM1000 feeder layermicroscope. for keratinocytes culture were sub-lethally irradiated (50 Gy). The equipment used was a BIOBEAM 8000 gamma irradiation unit with a source of Cs-137 of 2.2. Bioengineered2000 Artificial Ci. Dermal Skin Substitute irradiated (BASS fibroblasts) Manufacturing were seeded at 10,000 cells/cm2. Keratinocytes were seeded at 20,000–40,000 cells/cm2 in the flasks over an irradiated feeder. Cultures were The BASS consists of a two-layer cultured scaffold. The upper layer is an epithelium monitored using a Leica DM1000 microscope. of keratinocytes and the lower layer is a fibrin-hyaluronic acid matrix containing fibroblasts. The lower2.2. layer Bioengineered was developed Artificial by Skin mixing Substitute 330(BASS) µL of Manufacturinghuman plasma, 62.4 µL of DFM medium containingThe BASS fibroblasts, consists of 13.4 a two-layer µL of cultured water (B scaffold. Braun The Medical, upperlayer Barcelona, is an epithelium of Spain), 50 µL of keratinocyteshyaluronic acid and (Hyalone, the lower layer Fidia is Ph a fibrin-hyaluronicarma, Abano Terme, acid matrix Italy), containing 24 µL of fibroblasts. calcium chlorideThe (B Braun lower Medical, layer was Barcelona, developed Spain by mixing 10 mg/mL) 330 µ ,andL of 20.2 human µL of plasma, tranexamic 62.4 µ L of DFM acid (Amchafibrin,medium Rottapham, containing Spain). fibroblasts, The volumes 13.4 µ Ldescribed of water above (B Braun correspond Medical, Barcelona,to one Spain), 500 µL scaffold. 50AfterµL of24 hyaluronich, keratinocytes acid (Hyalone, were see Fidiaded to Pharma, constitute Abano the Terme,upper Italy),layer of 24 theµL of calcium BASS and the platechloride was returned (B Braun to Medical, the incubator Barcelona, for Spain,7 days. 10 Acellular mg/mL), substitutes and 20.2 µL without of tranexamic acid cells were fabricated(Amchafibrin, as negative Rottapham, control. Spain). Figure The 1 summarizes volumes described the BASS above manufacturing correspond to one 500 µL process. scaffold. After 24 h, keratinocytes were seeded to constitute the upper layer of the BASS and the plate was returned to the incubator for 7 days. Acellular substitutes without cells were fabricated as negative control. Figure1 summarizes the BASS manufacturing process.

Figure 1. Schematic representation of the bioengineered artiﬁcial skin substitutes (BASS) manufacturing process.Created with BioRender.com. Figure 1. Schematic representation of the bioengineered artificial skin substitutes (BASS) manufacturing process. Created with BioRender.com .

2.3. Antiseptic/Antibiotic Test

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A total of 24-well cell culture plates (Thermo Fisher Scientific, CA, USA) of the BASS were2.3. Antiseptic/Antibiotic treated with three Test different antiseptics and one antibiotic for 28 days. Controls were left withoutA total oftreatment. 24-well cell Viability culture platesassay (Thermoand histolo Fishergy were Scientiﬁc, performed CA, USA) at ofdays the 7, BASS 14, 21, and 28.were The treated antibiotic with threeused differentwas colistin antiseptics 1% (G.E.S., and one Ge antibioticnéricos Españoles for 28 days. Laboratorio Controls were S.A., Ma- left without treatment. Viability assay and histology were performed at days 7, 14, 21, drid, Spain) and the antiseptics used were chlorhexidine digluconate 1% (HiBiSCRUB ®, and 28. The antibiotic used was colistin 1% (G.E.S., Genéricos Españoles Laboratorio S.A., MolnlyckeMadrid, Spain) Health and Care the antiseptics AB, Madrid, used Spain), were chlorhexidine sodium ch digluconateloride 0.02% 1% (Sonoma (HiBiSCRUB Pharmaceu-®, ticals,Molnlycke CA, USA), Health and Care polyhexanide AB, Madrid, Spain), 0.1% sodium(B Braun chloride Medical, 0.02% Barcelona, (Sonoma Pharmaceuti-Spain). Two differ- entcals, protocols CA, USA), of and antiseptic polyhexanide application 0.1% (B were Braun tested Medical,, long Barcelona, (24 h) Spain).and short Two (4 different min) applica- tionprotocols every of 48 antiseptic h, to replicate application their were clinical tested, use. long Therefore, (24 h) and treatment short (4 min) duration application was 24 h for colistinevery 48 and h, to sodium replicate chloride their clinical and use. 4 min Therefore, for chlorhex treatmentidine duration digluconate was 24 hand for colistinpolyhexanide accordingand sodium to chloride previous and toxicity 4 min for studies chlorhexidine [19]. The digluconate antiseptic/antibiotic and polyhexanide test accordingwas performed threeto previous times toxicity( n = 3). studiesFigure [219 represents]. The antiseptic/antibiotic the antibiotic/antisepti test wasc performed test. three times (n = 3). Figure2 represents the antibiotic/antiseptic test.

Figure 2. Schematic representation of the antibiotic/antiseptic test. Created with BioRender.com. Figure 2. Schematic representation of the antibiotic/antiseptic test. Created with BioRender.com . 2.4. Viability Assay 2.4. ViabilityThe primary Assay outcome of this study was to determine cell viability after the different treatments described above. Viability assay at days 7, 14, 21, and 28 was performed using LIVE/DEADThe primary® Cell outcome Viability of Assay this study (Thermo was Fisher to determine Scientific, cell CA, viability USA). This after method the different treatmentsallows colorimetric described discrimination above. Viability between assay living at and days dead 7, 14, cells. 21, After and adding28 was the performed staining using LIVE/DEADsolution, the® plate Cell wasViability incubated Assay at room(Thermo temperature Fisher Scientific, and darkness CA,for USA). 30 min. This Then, method al- lowsstaining colorimetric solution was discrimination removed, scaffolds between were washedliving and with dead Dulbecco’s cells. After phosphate-buffered adding the staining solution,saline (DPBS, the plate Sigma was Aldrich, incubated St. Louis, at room MO, USA) temperatu and placedre and in slidesdarkness for fluorescence for 30 min. Then, stainingmeasurement solution at 405 was nm removed, using a Leica scaffolds DM2000 were microscope. washed with The obtainedDulbecco’s images phosphate-buff- were eredanalyzed saline by (DPBS, the scientific Sigma image Aldrich, analysis St. programLouis, MO, Image US J.A) and placed in slides for fluorescence measurement at 405 nm using a Leica DM2000 microscope. The obtained images were analyzed by the scientific image analysis program Image J.

2.5. Histological Analysis One of the secondary outcomes of this study was to analyze the BASS histological structure. For that, BASS were collected at days 7, 14, 21, and 28, fixed in 4% phosphate- buffered formalin (Merck, Damstadt, Germany), embedded in paraffin, and cut into 4 µm

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J. Clin. Med. 2021 , 10 , x FOR PEER REVIEW 5 of 14 2.5. Histological Analysis One of the secondary outcomes of this study was to analyze the BASS histological structure. For that, BASS were collected at days 7, 14, 21, and 28, fixed in 4% phosphate- sections.buffered The formalin sections (Merck, were stained Damstadt, with Germany), hematoxyli embeddedn and eosin in paraffin,(Merck, Damstadt, and cut into Ger- 4 µm many)sections. to reveal The the sections histological were stained structure. with hematoxylin and eosin (Merck, Damstadt, Ger- many) to reveal the histological structure. 2.6. Epidermal Barrier Function Evaluation 2.6.Another Epidermal secondary Barrier Function outcome Evaluation of this research was to assess the BASS functionality throughAnother the barrier secondary function outcome evaluation. of this In order research to evaluate was to assess the barrier the BASS function functionality of the BASS,through trans-epidermal the barrier functionwater loss evaluation. (TEWL) was In measure order tod evaluate after 14 thedays barrier of treatment function using of the a TewameterBASS, trans-epidermal® TM 300 (Courage water loss+ Khazaka (TEWL) Electronic, was measured Köln, after German 14 daysy) in of 6-well treatment cell cul- using turea Tewameter plates (24 ® mmTM diameter, 300 (Courage Thermo + Khazaka Fisher Scientif Electronic,ic, CA, Köln, USA) Germany) of the BASS.in 6-well The cell Tewameterculture plates® probe (24 indirectly mm diameter, measures Thermo the gradient Fisher Scientific,of the wate CA,r evaporation USA) of the density BASS. of The ® theTewameter skin throughprobe two pairs indirectly of sensors measures (temperature the gradient and relative of the humidity) water evaporation inside a hollow density cylinder.of the The skin physical through basis two pairsfor measurement of sensors (temperature is the law of diffusion and relative discovered humidity) by Adolf inside a Fickhollow in 1855: cylinder. dm/dt = The −D*A*dp/dx physical basis, where for A measurement is the surface is (m the2), lawm, the of diffusionwater transported discovered 2 (g),by t, the Adolf time Fick (h), in D,1855: the diffusion dm/dt = constant−D*A*dp/dx, (= 0.0877where g/m(h A ismmHg)), the surface p is (mthe), vapor m, the pres- water suretransported of the atmosphere (g), t, the (mm time Hg), (h), D, and the x diffusionis the distan constantce from (= the 0.0877 surface g/m(h of mmHg)),the skin to p the is the measurementvapor pressure point of (m). the atmosphere (mm Hg), and x is the distance from the surface of the skinBefore to the taking measurement the measurement, point (m). dehydration by controlled pressure of the BASS using a glassBefore disc taking of 85 g the as measurement,shot for 2 min dehydrationwas necessary by to controlled improve their pressure biomechanical of the BASS propertiesusing a glass[13]. The disc dehydrated of 85 g as shot BASS for was 2 min placed was necessaryin the Franz to cell improve and 0.5 their g of biomechanical mupirocin 20 propertiesmg/g (excipients; [13]. The macrogol dehydrated 400 BASSand polyethylengl was placed inycol the 3350, Franz ISDIN, cell and Barcelona, 0.5 g of mupirocin Spain) was20 applied. mg/g (excipients; The probe was macrogol used to 400 take and the polyethylenglycol measurements (Figure 3350, ISDIN, 3) and Barcelona,MPA software Spain) was applied. The probe was used to take the measurements (Figure3) and MPA software (Multi Probe Adapter, Courage + Khazaka Electronic, Köln, Germany) to analyze the re- (Multi Probe Adapter, Courage + Khazaka Electronic, Köln, Germany) to analyze the sulting data. Mupirocin is a topical antibiotic used to treat superficial skin infections such resulting data. Mupirocin is a topical antibiotic used to treat superficial skin infections such as impetigo, folliculitis, and furunculosis, as well as other skin diseases, and is widely as impetigo, folliculitis, and furunculosis, as well as other skin diseases, and is widely used used during the wound healing process in patients with severe burns or skin ulcers. Fig- during the wound healing process in patients with severe burns or skin ulcers. Figure3 ure 3 summarizes the epidermal barrier function evaluation. summarizes the epidermal barrier function evaluation.

Figure 3. Schematic representation of the trans-epidermal water loss (TEWL) measurement after the antibi- Figure 3. Schematic representation of the trans-epidermal water loss (TEWL) measurement after the antibiotic/antiseptic otic/antiseptic treatments. treatments.

2.7. Statistical Analysis

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2.7. Statistical Analysis The obtained data are presented as the mean ± standard error of the mean (SEM). For the data analysis, the statistical program R was used (R Development Core Team (2011). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). A generalized linear model was applied for development of the statistical analysis of the data and normal distribution of the residues was verified. On the linear model, an analysis of variance (ANOVA) factorial was performed, to evaluate the effect of each factor present. Once ANOVA was performed, a post hoc analysis was performed with Tukey’s test for all factors to evaluate the degree of significance when comparing the factor classes. Values of p ≤ 0.05 were considered statistically significant.

3. Results 3.1. Chlorhexidine Digluconate Affects Cell Viability to a Greater Extent Compared to the Other Treatments Chlorhexidine digluconate greatly reduced cell viability compared to the other treatments. This was reflected in the number of dead cells (Figure4) as well as in the viability percentage (Table1, Figure5). No significant differences among the days of the evaluation period for each treatment were found (Figure6). However, the analysis revealed significant differences among the different treatments in each day of the evaluation (Figure7). Specifically, on day 7 of treatment (Figure7a), there was a significant reduction in the cell viability after chlorhexidine digluconate compared to colistin (p ≤ 0.001) and sodium chloride (p ≤ 0.001) treatments, and the control (non-treated BASS (p ≤ 0.001); and after polyhexanide compared to colistin (p = 0.0272), sodium chloride (p = 0.0282) treatments, and the control (p = 0.0210). At day 14 (Figure7b), there was only significant differences between chlorhexidine digluconate treatment compared to sodium chloride treatment (p = 0.0472) and the control (p = 0.0179). At day 21 (Figure7c), cell viability was significantly lower after chlorhexidine digluconate treatment compared to colistin (p = 00836), sodium chloride (p = 0.00497), and polyhexanide (p = 0.01415) treatments, and the control (p = 0.00127). Lastly, at day 28 of the evaluation period (Figure7d), there was a significant difference between chlorhexidine digluconate treatment and the control (p = 0.0246). Therefore, compared to the control, chlorhexidine digluconate followed by polyhexanide more affected cell viability than the other treatments after the antiseptic/antibiotic test.

Table 1. Cell viability percentage media for each treatment and control at days 7, 14, 21, and 28; n = 3.

Treatment Day 7 Day 14 Day 21 Day 28 Colistin 89.35% 75.03% 77.7% 47.34% Chlorhexidine digluconate 33.16% 41.86% 21.81% 9.69% Sodium chloride 89.15% 82.18% 82.08% 62.34% Polyhexanide 56.66% 53.99% 73.37% 59.41% Control 90.83% 89.76% 94.47% 94.96% J. Clin. Med. 2021 , 10 , x FOR PEER REVIEW 6 of 14

The obtained data are presented as the mean ± standard error of the mean (SEM). For the data analysis, the statistical program R was used (R Development Core Team (2011). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). A generalized linear model was applied for development of the statistical analysis of the data and normal distribution of the residues was verified. On the linear model, an analysis of variance (ANOVA) factorial was performed, to evaluate the effect of each factor present. Once ANOVA was performed, a post hoc analysis was performed with Tukey’s test for all factors to evaluate the degree of significance when comparing the factor classes. Values of p ≤ 0.05 were considered statistically significant.

3. Results 3.1. Chlorhexidine Digluconate Affects Cell Viability to a Greater Extent Compared to the Other Treatments J. Clin. Med. 2021, 10, 642 Chlorhexidine digluconate greatly reduced cell viability compared to the other treat- 7 of 14 ments. This was reflected in the number of dead cells (Figure 4) as well as in the viability percentage (Table 1, Figure 5).

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Table 1. Cell viability percentage media for each treatment and control at days 7, 14, 21, and 28; n = 3.

Treatment Day 7 Day 14 Day 21 Day 28 Figure 4. LIVE/DEAD® images of BASS after each treatment and control at days 7, 14, 21, and 28. (a–d) BASS after colistin Figure 4. LIVE/DEAD® images of BASS after each treatment and control at days 7, 14, 21, and 28. (a–d) BASS after colistin (1%) treatment at dayColistin 7, 14, 21, and 28 respectivel 89.35%y. (e– h) BASS after 75.03% chlorhexidine 77.7%digluconate (1%) 47.34% treatme nt at day 7, (1%) treatment14, 21, andChlorhexidine at 28, day respectively. 7, 14, 21, digluconate and( i–l) 28BASS respectively. after sodium 33.16% (e –chlorideh) BASS (0.02%) after 41.86% chlorhexidine treatment at d 21.81%ay digluconate 7, 14, 21, and (1%) 28, 9.69 treatmentrespectively.% at ( daym– 7, 14, 21, andp) 28, BASS respectively. after polyhexanideSodium (i–l) BASSchloride (0.01%) after treatment sodium chloride at 89.15% days 7, (0.02%) 14, 21 and treatment 82.18% 28 respectively. at day 7,82.08% ( q 14,–t) 21, Control and 28,at days respectively. 62.34% 7, 14, 21, and (m–p 28) BASS after polyhexaniderespectively. Live (0.01%)Polyhexanide cells treatmentare represented at days in green 7, 14, 56.66% a 21nd anddead 28 cells, respectively. in 53.99% red. n = (3.q –Magnificationt) Control 73.37% at 10 days×. 7, 14, 59.41% 21, and 28 respectively. Live cells are representedControl in green and dead cells, 90.83% in red. n = 3. Magniﬁcation 89.76% 10× 94.47%. 94.96%

100 Colistin Day 7 Chlo. Digluconate Day 7 Sodium Chloride Day 7 80 Polyhexanide Day 7 Control Day 7 60 Colistin Day 14 Chlo. Digluconate Day 14 Sodium Chloride Day 14 40 Polyhexanide Day 14

Cell (%) Cell Viability Control Day 14 Colistin Day 21 20 Chlo. Digluconate Day 21 Sodium Chloride Day 21 Polyhexanide Day 21 0 Control Day 21 Colistin Day 28 Chlo. Digluconate Day 28 Sodium Chloride Day 28 Polyhexanide Day 28 Control Day 28

FigureFigure 5. Bar 5 .graph Bar graph of cell of cell viability viability percentage percentage media media forfor eacheach treatment treatment and and control control at days at days 7, 14, 7, 21, 14, and 21, 28. and n 28.= 3. n = 3.

No significant differences among the days of the evaluation period for each treatment were found (Figure 6). However, the analysis revealed significant differences among the different treatments in each day of the evaluation (Figure 7). Specifically, on day 7 of treatment (Figure 7a), there was a significant reduction in the cell viability after chlorhexidine digluconate compared to colistin ( p ≤ 0.001) and sodium chloride (p ≤ 0.001) treatments, and the control (non-treated BASS (p ≤ 0.001); and after polyhexanide compared to colistin (p = 0.0272), sodium chloride ( p = 0.0282) treatments, and the control (p = 0.0210). At day 14 (Figure 7b), there was only significant differences between chlorhexidine digluconate treatment compared to sodium chloride treatment ( p = 0.0472) and the control (p = 0.0179). At day 21 (Figure 7c), cell viability was significantly lower after chlorhexidine digluconate treatment compared to colistin ( p = 00836), sodium chloride ( p = 0.00497), and polyhexanide ( p = 0.01415) treatments, and the control (p = 0.00127). Lastly, at day 28 of the evaluation period (Figure 7d), there was a significant difference between chlorhexidine digluconate treatment and the control (p = 0.0246). Therefore, compared to the control, chlorhexidine digluconate followed by polyhexanide more affected cell viability than the other treatments after the antiseptic/antibiotic test.

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FigureFigure 6. 6. IndividualIndividual statistical statistical analysis analysis of of antiseptic/antib antiseptic/antibioticiotic viability viability test.test. BoxplotBoxplot ofof cellcell viability viability percentage percentage after after (a )(a col-) colistinistin (1%) (1%) treatment, treatment, (b ()b chlorhexidine) chlorhexidine digluconate digluconate (1%) (1%) treatment, treatment, (c) ( sodiumc) sodium chloride chloride (0.02%) (0.02%) treatment, treatment, (d) ( polyhexanided) polyhexanide(0.1%) (0.1%) treatment, treatment, and (ande) for (e) the for control. the control.n = 3. n N/S:= 3. N/S: no signiﬁcantno significant differences. differences.

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Figure 7. Statistical analysis of the days of treatment in the antiseptic/antibiotic viability (Table1) treatment. Boxplot of Figurecell viability7. Statistical percentage analysis at of (a )the day days 7, (b of) day treatment 14, (c) day in th 21,e antiseptic/antibiotic and (d) day 28 of treatment. viability N/S: (Table no 7 signiﬁcant) treatment. differences; Boxplot of cell* viabilityp ≤ 0.05; percentage ** p ≤ 0.01; ***at (pa≤) day0.001. 7, (nb)= day 3. The 14, values(c) day of 21,p ≤ and0.05 (d were) day considered 28 of treatment. statistically N/S: no signiﬁcant. significant differences; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. n = 3. The values of p ≤ 0.05 were considered statistically significant.

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3.2. Chlorhexidine Digluconate and Polyhexanide AffectAffect the Epithelium Integrity to a Greater Extent Compared to the Other TreatmentsTreatments In addition to the viabilityviability test,test, histologicalhistological analysisanalysis waswas performedperformed toto evaluateevaluate thethe skin integrity. As shown in Figure 88,, thethe BASSBASS treatedtreated withwith chlorhexidinechlorhexidine digluconate,digluconate, followed by those treated with polyhexanide,polyhexanide, more deteriorateddeteriorated compared to the otherother treatments, since very few cells were appreciated inin the epidermis. Regarding the dermis, an important important reduction reduction in in ﬁbroblasts fibroblasts population population was wa seens seen after after these these antiseptics antiseptics compared com- to colistin and sodium chloride treatments. For colistin and sodium chloride, a stratiﬁcation pared to colistin and sodium chloride treatments. For colistin and sodium chloride, a strat- of the epithelium over time was observed (similar to control group), which suggested the ification of the epithelium over time was observed (similar to control group), which sug- integrity preservation by these treatments. gested the integrity preservation by these treatments.

Figure 8. Hematoxylin and eosin staining of BASS after each t treatmentreatment and control at days 7, 14, 21,21, andand 28.28. ((aa––d)) BASSBASS after colistin (1%) treatment at day 7, 14, 21, and 28, respectively; (e–h) BASS after chlorhexidine digluconate (1%) treat- after colistin (1%) treatment at day 7, 14, 21, and 28, respectively; (e–h) BASS after chlorhexidine digluconate (1%) treatment ment at day 7, 14, 21, and 28, respectively; (i–l) BASS after sodium chloride (0.02%) treatment at day 7, 14, 21, and 28, at day 7, 14, 21, and 28, respectively; (i–l) BASS after sodium chloride (0.02%) treatment at day 7, 14, 21, and 28, respectively; respectively; (m–p) BASS after Polyhexanide (0.01%) treatment at days 7, 14, 21, and 28, respectively; (q–t) Control at days m–p q t (7, 14,) 21 BASS and after28 respectively. Polyhexanide n = (0.01%) 3. Magnification treatment 10 at× days. 7, 14, 21, and 28, respectively; ( – ) Control at days 7, 14, 21 and 28 respectively. n = 3. Magnification 10×. 3.3. Skin Barrier Function Was Not Significantly Affected after the Antibiotic/Antiseptic Test 3.3. Skin Barrier Function Was Not Significantly Affected after the Antibiotic/Antiseptic Test As shown in Figure 9, no significant differences were appreciated after mupirocin applicationAs shown between in Figure the four9, no groups significant used differencesand the control were ( p appreciated > 0.05) (Figure after 9a) mupirocin and there wereapplication no significant between differences the four groups in TEWL used andvalues the thro controlugh ( thep > 0.05)evaluation (Figure time9a) and( p > there 0.05) were no significant differences in TEWL values through the evaluation time (p > 0.05)

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(Figure 99b).b). However,However, TEWLTEWL valuesvalues wereweresigniﬁcantly significantly higher higher ( (pp ≤≤ 0.001) in the acellular control (absence of barrier) regarding the differentdifferent treatmentstreatments andand thethe control.control.

Figure 9. StatisticalStatistical analysis analysis of of TEWL TEWL measurement. measurement. ( a) ( aBoxplot) Boxplot of ofBASS BASS TEWL TEWL values values regarding regarding the thetreatme treatmentnt used. used. ( b) (Boxplotb) Boxplot of BASS of BASS TEWL TEWL values values regarding regarding the theevaluatio evaluationn period. period. *** ***p ≤ p0.001,≤ 0.001, N/S: N/S: no significant no signiﬁcant differences differences..

4. Discussion Discussion InIn this study,study, chlorhexidine chlorhexidine digluconate digluconate seriously seriously affected affected cell cell viability viability and and epithelium epithe- liumintegrity, integrity, while while no significant no significant differences differences were observedwere observed in the in barrier the barrier function function evaluation. evaluation.Bioengineered artificial skin substitutes (BASS) are used in regenerative medicine as an advanceBioengineered therapy artificial for injuries skin covering substitutes large (BASS) body surfaceare used areas, in regenerative especially burns. medicine These as anskin advance substitutes, therapy composed for injuries of both covering a dermis large and body an surface epidermis, areas, should especially be easy burns. to handle, These skinmust substitutes, permanently composed ensure the of skinboth barrier a dermis function, and an and epidermis, should notshould induce be easy a host to immune handle, mustresponse permanently [1,3,7]. ensure the skin barrier function, and should not induce a host immune responseOnce [1,3,7]. the BASS is transplanted to the patient, treatment with antiseptics and antibiotics is crucialOnce tothe prevent BASS is any transplanted infection. However,to the patient, there treat is noment evidence with antiseptics of the impact and of antibi- these oticstreatments is crucial on the to viabilityprevent ofany the infection. cells that However, constitute there the BASS. is no If evidence a treatment of greatlythe impact affects of thesecell viability, treatments this on could the resultviability in an of alterationthe cells that of skin constitute properties, the BASS. highlighting If a treatment the epithelium greatly affectsintegrity cell and viability, its barrier this function,could result which in an is alt essentialeration toof regulateskin properties, temperature, highlighting water lossthe epitheliumand protect integrity from mechanical and its barrier injuries function, and external which agents is essential [18]. Among to regulate all the temperature, antiseptics waterand antibiotics loss and availableprotect from [16, 17mechanical], four of the injuries more and widely external used inagents clinic [18]. were Among selected all to the be antisepticsevaluated inand terms antibiotics of cell viability available and [16,17], barrier four function. of the more widely used in clinic were selectedChlorhexidine to be evaluated is a in powerful terms of antisepticcell viability with and evidence barrier function. of a beneficial role in burn careChlorhexidine [19]. It is a positively is a powerful charged antiseptic bisbiguanide with atevidenc physiologice of a beneficial pH that binds role in to burn the nega- care [19].tively It charged is a positively cell walls charged of bacteria, bisbiguanide leading toat a ph disruptionysiologic ofpH microbial that binds cell to membranes the negatively [20]. chargedSeveral incell vitro wallsstudies of bacteria, have reported leading cytotoxicityto a disruption on cultured of microbial cells, cell whereas membranesin vivo [20].and Severalclinical datain vitro results studies seem have to bereported controversial. cytotoxicityIn vivo on, cultured although cells, it is widelywhereas used in vivo in clinic and clinicaldue to itsdata broad-spectrum results seem to antimicrobial be controversial. activity, In adversevivo, although effects haveit is widely been reported used in inclinic the dueliterature. to its broad-spectrum For instance, chlorhexidine antimicrobial is activity, occasionally adverse associated effects have with been contact reported dermatitis, in the literature.and rarely For with instance, anaphylaxis chlorhexidine and hypersensitivity is occasionally reactions associated such aswith urticaria contact [21 dermatitis,–23]. Skin andallergic rarely reactions with anaphylaxis and burns and are reportedhypersensitivity mainly re onactions extremely such lowas urticaria birth weight [21–23]. infants, Skin allergicbut these reactions adverse and effects burns can are occur reported on adults mainly as wellon extremely [19,24]. Furthermore, low birth weight if it contactsinfants, butthe these ocular adverse surface, effects progressive can occur corneal on adults damage as we canll [19,24]. occur, Furthermore, causing toxic if effectsit contacts on the epithelial layers of the cornea and conjunctiva at low concentrations and leading to irre-

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versible keratitis at higher concentrations [24,25]. Middle ear ototoxicity with sensorineural deafness has been reported [24]. In terms of wound healing, the role of chlorhexidine is controversial as well. It has been reported that the use of antiseptics such us chlorhexidine inhibits wound healing in vivo [26], although other reports hold that the concentration of chlorhexidine, which is cytotoxic in vitro, is not cytotoxic in vivo and does not interfere with the re-epithelialization and healing process of the wound [19,27]. Regarding in vitro cytotoxicity, chlorhexidine has significant adverse effects on dermal fibroblast growth [20] and significantly reduces cell migration and survival of myoblasts and osteoblasts [28]. In our BASS model, chlorhexidine digluconate reduced the cell viability to a large extent in comparison with colistin, sodium chloride. and polyhexanide. Compared to the control, chlorhexidine followed by polyhexanide more affected cell viability than the other antiseptics. The reduction in the cell viability resulted in deterioration of the epithelium integrity and this could alter the barrier function. The trans-epidermal water loss is widely used as a marker for skin barrier function in vivo and in vitro [29–31]. When skin is damaged, its barrier function is impaired, resulting in a higher water loss [32]. Therefore, if the cell viability of the BASS is reduced and its epithelium is deteriorated, this should be traduced into higher water loss and poor absorption of the dermatological agent used to treat wounds. In our study, no significant differences in TEWL between groups were observed. However, the absence of epithelial barrier in the acellular BASS revealed significantly higher TEWL values compared to the different treatments and the control. Although chlorhexidine significantly reduces cell viability, this was not reflected in the TEWL measurement. More in vivo and in vitro studies with longer follow-up periods may be necessary to assess epidermal barrier function differences between groups and to determine when alterations on epithelial barrier are produced. The use of suitable probes for in vitro long follow-up measurements is highly recommended [30,32,33]. Furthermore, for future studies, the antiseptics and antibiotics treatments could be tested on BASS simu- lating skin disease models. This could be achieved by damaging the BASS through different methods [34,35] or by developing them using fibroblasts and keratinocytes from patients with different skin pathologies. To the best of our knowledge, the limitations of this research include the need to improve the barrier function evaluation using suitable probes for in vitro TEWL measurements and to introduce BASS skin disease models that could simulate different types of damaged barriers.

5. Conclusions The role of chlorhexidine in burn care is still controversial since its broad spectrum of action is well-established but effects on wound healing and reepithelization are still contradictory. In this study, chlorhexidine digluconate seriously affected the cell viability and epithelium integrity, while no signiﬁcant differences were observed in the barrier function evaluation. These results could help determine the appropriate treatment after BASS implantation or antiseptic use for wound healing, although further research is necessary to propose new treatment protocols after implantation and evaluating them in clinical practice. Furthermore, our BASS could be a suitable model to study in vitro the impact of different antiseptics/antibiotics for the treatment of other skin wounds such us ulcers or cutaneous detects.

Author Contributions: Conceptualization, M.I.Q.-V. and S.A.-S.; Data curation, A.F.-G.; Formal analysis, A.F.-G.; Funding acquisition, S.A.-S.; Investigation, M.I.Q.-V. and E.P.-C.; Methodology, M.I.Q.-V., A.F.-G., E.P.-C., and T.M.-V.; Project administration, S.A.-S.; Resources, M.I.Q.-V. and A.F.-G.; Software, A.F.-G.; Supervision, A.F.-G. and S.A.-S.; Validation, M.I.Q.-V., A.F.-G., and E.P.-C.; Visualization, M.I.Q.-V. and A.F.-G.; Writing—original draft, M.I.Q.-V.; Writing—review & edit- ing, M.I.Q.-V., A.F.-G., and S.A.-S. All authors have read and agreed to the published version of the manuscript. Funding: The work of María I. Quiñones Vico was supported by a predoctoral fellowship (BOE 22/10/2019) from the Ministry of Science, Innovation and Universities of Spain and by a research J. Clin. Med. 2021, 10, 642 13 of 14

initiation grant of the University of Granada. This study is part of her doctoral research in the Biomedicine’s program of University of Granada. This study was funded by the Carlos III Health Institute of Spain through the project PI13/02576 and PI17/02083 (co-funded by European Regional Development Fund “A way to make Europe”) and Andalusian Regional Government Financial (SAS PI-0458-2016). 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 reconstructive surgery donors in compliance with the requirements for donation of human cells and tissues (Royal Decree- Law 9/2014, of July 4). Data Availability Statement: Data is contained within the article. Acknowledgments: We thank to Purificación Gacto-Sánchez from the Plastic Surgery Department, Burns Unit Manager, of the Virgen del Rocío University Hospital, Seville, Spain, for the knowledge provided about antiseptic application protocols used in clinic. We appreciate help provided by the Radiotherapy Department and Pathology Department of Virgen de las Nieves University Hospital of Granada (Spain). We gratefully acknowledge financial support from the Carlos III Health Institute of Spain (PI13/02576 and PI17/02083) and Andalusian Regional Government Financial (SAS PI- 0458-2016). The work of María I. Quiñones Vico was supported by a predoctoral fellowship (BOE 22/10/2019) from the Ministry of Science, Innovation and Universities of Spain and by a research initiation grant of the University of Granada. This study is part of her doctoral research in the Biomedicine’s program of University of Granada. Conflicts of Interest: The authors declare no conflict of interest.

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