Histological assessment of nanostructured fibrin-agarose skin substitutes grafted in burnt patients. A time-course study

RESEARCH ARTICLE Histological assessment of nanostructured fibrin-agarose skin substitutes grafted in burnt patients. A time-course study Miguel Angel Martin-Piedra 1,2 | Gloria Carmona 3,4 | Fernando Campos 1,2 | Víctor Carriel 1,2 | Ana Fernández-González 2,3,5 | Antonio Campos 1,2 | Natividad Cuende 3 | Ingrid Garz on 1,2 | Purificaci on Gacto 6 | Miguel Alaminos 1,2 1 Tissue Engineering Group, Department of Histology, University of Granada, Spain 2 Instituto de Investigaci on Biosanitaria ibs.GRANADA, Granada, Spain 3 Andalusian Network for the Design and Translation of Advanced Therapies (former Andalusian Initiative for Advanced Therapies) - Fundaci on Andaluza Progreso y Salud, Junta de Andalucía, Seville, Spain; Andalusian Transplant Coordination, Servicio Andaluz de Salud, Seville, Spain 4 Doctoral program in Biomedicine, University of Granada, Granada, Spain 5 Unidad de Producci on Celular e Ingeniería Tisular, Hospital Universitario Virgen de las Nieves, Granada, Spain 6 Burn Unit, University Hospital Virgen del Rocío, Seville, Spain Correspondence Ingrid Garz on, Department of Histology. Faculty of Medicine. University of Granada. Avenida de la Investigaci on 11, E18016, Granada, Spain. Email: [email protected] Purificaci on Gacto, Department of Plastic and Reconstructive Surgery, Virgen del Rocío University Hospital. Av. Manuel Siurot, S/N, E41013, Sevilla, Spain. Email: [email protected]om Ana Fernández-González, Unidad de Producci on Celular e Ingeniería Tisular, Hospital Universitario Virgen de las Nieves, Av. de las Fuerzas Armadas, 2, E18014, Granada, Spain. Email: ana.fernandez.gonzalez@ juntadeandalucia.es Funding information Consejería de Salud y Familias, Junta de Andalucía, Grant/Award Numbers: PE- 0395-2019, PI-0458-2016; Consejería de Transformaci on Econ omica, Industria, Conocimiento y Universidades, Grant/Award Number: B-CTS-450-UGR20; Instituto de Salud Carlos III, Grant/Award Number: AC17/00013 Abstract A previously developed fibrin-agarose skin model—UGRSKIN—showed promising clinical results in severely burnt patients. To determine the histological parameters associated to the biocompatibility and therapeutic effects of this model, we carried out a comprehensive structural and ultrastructural study of UGRSKIN grafted in severely burnt patients after 3 months of follow-up. The grafted epidermis was analogue to native human skin from day 30th onward, revealing well-structured strata with well-differentiated keratinocytes expressing CK5, CK8, CK10, claudin, plakoglobin, filaggrin, and involucrin in a similar way to controls, suggesting that the epidermis was able to mature and differentiate very early. Melanocytes and Langerhans cells were found from day 30th onward, together with a basement membrane, abundant hemidesmosomes and lack of rete ridges. At the dermal layer, we found an interface between the grafted skin and the host tissue at day 30th, which tended to disappear with time. The grafted superficial dermis showed a progressive increase in properlyoriented collagen fibers, elastic fibers and proteoglycans, including decorin, similarly to control dermis at day 60-90th of in vivo follow-up. Blood vessels determined by CD31 and SMA expression were more abundant in grafted skin than controls, whereas lymphatic vessels were more abundant at day 90th. These results contribute to shed light on the histological parameters associated to biocompatibility and therapeutic effect of the UGRSKIN model grafted in patients and demonstrate that the Miguel Angel Martin-Piedra and Gloria Carmona are contributed equally to this study. Received: 20 January 2023 Revised: 10 June 2023 Accepted: 14 June 2023 DOI: 10.1002/btm2.10572 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers. Bioeng Transl Med. 2023;e10572. wileyonlinelibrary.com/journal/btm2 1of19 https://doi.org/10.1002/btm2.10572 bioengineered skin grafted in patients is able to mature and differentiate very early at the epithelial level and after 60–90 days at the dermal level. KEYWORDS artificial skin, burnt patients, histology, tissue engineering 1|INTRODUCTION Large burns are very relevant public health problems causing massive tissue destruction and dramatic pathophysiologic effects on the burnt patient. 1 As an intact healthy skin is necessary to protect the internal tissues, a proper skin barrier is essential for patient survival. 2 However, treatment of these patients is challenging, since the severe and deep skin damage typically destroys the skin stem cells, and the damaged tissue is not able to regenerate by itself. 3 Development of human artificial skin substitutes by tissue engineering offers a promising regenerative treatment for burnt patients. 4,5 In general, these skin substitutes consist of a scaffold biomaterial and skin cells. Although several skin substitutes have been developed, and promising clinical results were demonstrated for some of them, 6,7 an ideal substitute has not been described to the date. In this regard, we developed a bioartificial model of human skin called UGRSKIN or NANOGSKIN, which combines highly biocompatible fibrin-agarose biomaterials containing human dermal fibroblasts with human skin keratinocytes able to form a stratified epithelial layer on top. This skin model was characterized ex vivo and in vivo in laboratory animals using several histological, histochemical, immunohistochemical, and other analysis methods able to demonstrate that the model was safe and efficient. 8–10 In fact, our preclinical studies demonstrated that the biochemical properties and histological structure of the UGRSKIN skin model were similar to the native human skin at both, the epidermal and the dermal layers. Ex vivo, UGRSKIN showed to be biomechanically comparable to the native human skin, with adequate properties as determined by compressive and shear tests. 11 Bioartificial tissues based on fibrin-agarose biomaterials displayed an elastic behavior and its modulus of Young was comparable to native tissues, 11 and our previous time-course analysis carried out for 4 weeks demonstrated that these properties remained stable upon the time in culture. 9 In addition, the optical characterization UGRSKIN model showed that the epidermal layer was able to mature and differentiate being able to absorb most of the incoming UV light, as it is the case of the native skin. 10 When grafted in vivo on immune-deficient animal models, UGRSKIN demonstrated to be highly biocompatible and able to contribute to skin regeneration. 8 One of our main findings was that the bioartificial skin substitute was able to remodel its structure and composition in a time-dependent manner, with a progressive process of extracellular matrix (ECM) remodeling and biosynthesis of crucial fibrillar and non-fibrillar components along with cell differentiation and maturation. 8,9,12 These positive results allowed us to fulfill the requirements of the Spanish Medicines Agency (AEMPS) for advanced therapy medicinal products (ATMP), thus allowing us to transfer this skin substitute to the clinical setting as compassionate use. 13 Preliminary results of the clinical use of the UGRSKIN model in 12 severely burnt patients showed a 75% survival rate. Of the 12 patients, 9 showed a significant improvement and recovery, whereas three patients died mostly due to infections and systemic complications. 13 Although results were encouraging, a comprehensive structural analysis of the histological and biochemical processes involved in tissue regeneration using the UGRSKIN model in humans is still in need. This analysis could provide valuable information regarding tissue biointegration and biofunctionality, and would contribute to a better characterization of the therapeutic effects of this type of ATMP according to the requirements established by the European Medicines Agency. 14 In the present work, we have carried out a comprehensive structural and ultrastructural study of the UGRSKIN model grafted as ATMP in severely burnt patients after 1, 2, and 3 months of follow-up in order to assess the biocompatibility of this model in humans and to determine the histological factors associated to the therapeutic effect of this bioartificial skin. 2|MATERIALS AND METHODS 2.1 |Patients samples The UGRSKIN model of bioartificial human skin was generated using autologous dermal fibroblasts, epidermal keratinocytes and fibrinagarose biomaterials as previously described. 8,13,15 Cultured cells were first obtained from human skin biopsies obtained from patients treated at the burn unit of the University Hospital Virgen del Rocio in Seville (8 male and 4 female patients) and processed at the Cell Production and Tissue Engineering Unit of University Hospital Virgen de las Nieves in Granada, in a GMP facility of the Andalusian Network for the design and translation of Advanced Therapies (And&tAT). Dermal fibroblasts and keratinocytes were isolated using enzymatic digestion and cultured in specific culture media. 8,15 Once primary cell cultures were stablished, we generated a dermal substitute by combining cultured human fibroblasts with fibrin-agarose hydrogels. Human plasma obtained from healthy blood donors was used as a source of fibrin. For 1 mL of dermal substitute, we mixed 760 μLof human plasma with 75 μL of DMEM containing 10,000 cultured dermal fibroblasts and 15 μL of tranexamic acid (Amchafibrin, Fides- Ecofarma, Valencia, Spain). A total of 50 μL of a 2% melted solution of type VII agarose were added, followed by 100 μLof1%CaCl 2 2of19 MARTIN-PIEDRA ET AL. 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (Merck, Darmstadt, Germany), and the mixture was immediately mixedandaliquotedincultureplates.After24hinanincubatorat 37C, 500,000 cultured keratinocytes were seeded on top of the stromal substitute. Finally, the UGRSKIN model was subjected to plastic compression nanostructuration and delivered to the burn unit as an ATMP for autologous use. All this biofabrication procedure as well as the clinical use in each patient were authorized by the Spanish Medicines Agency (AEMPS). Twelve severely burnt patients treatedwiththeUGRSKINmodelwereincludedinthisstudy(n=12). The clinical characteristics of these patients were previously described. 13 After 30, 60, and 90 days of follow-up, a full-thickness biopsy was obtained from the grafted area of each patient using a skin biopsy punch. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Province of Granada (Comité  Etico de Investigaci on, CEIM/CEI protocol number S1900527), and all skin donors provided informed consentfortheirparticipationinthe study. All patients were treated with the approval of the Spanish Medicines Agency (AEMPS) using the hospital exemption and compassionate use for advanced therapy medicinal products (ATMP), as previously described. 13 2.2 |Histological, histochemical, and immunohistochemical analyses Five types of samples were analyzed in the present work: ex vivo samples (UGRSKIN model of skin before clinical application), biopsies of patients treated with the UGRSKIN model after 30, 60, and 90 days of follow-up and normal skin control samples. In each case, histological analysis was carried out using transmission electron microscopy (TEM) and light microscopy analysis. For TEM, samples were fixed in 2.5% buffered glutaraldehyde, postfixed in 1% osmium tetroxide, and dehydrated in graded acetone series. Then samples were embedded in Spurr's resin and stained with aqueous uranylacetate and lead citrate. Ultrathin sections were examined using a EM902 microscope (Carl Zeiss Medi-tec, Oberkochen, Germany). For light microscopy, samples fixed in 10% buffered formalin, embedded in paraffin, and sectioned in 5-μm-thick sections, which were stained with hematoxylin and eosin and histologically analyzed using a Nikon Eclipse 90i light microscope. To analyze specific components of the human skin dermis ECM, the following histochemical methods were performed: to identify mature collagen fibers, the picrosirius red method was used and samples were observed using a light microscope. To assess the presence of mature, properly oriented collagen bundles, the same samples were analyzed using polarized light microscopy. Elastic fibers were stained by Verhoeff histochemistry, whereas proteoglycans were stained with alcian blue. In addition, Schiff periodic acid staining (PAS) methods were used to identity the basement membranes glycosaminoglycans. All these methods were used using routine protocols described elsewhere. 16–18 Other relevant components of the human epidermis and dermis were identified using specific immunohistochemical procedures, including epithelial cytokeratins (CK5, CK8, and CK10), cell-cell junctions markers (plakoglobin and claudin), epithelial differentiation proteins (filaggrin and involucrin), specific epithelial cells (melanocytes and Langerhans cells), components of the ECM (type-V collagen and decorin), blood vessels (positive for CD31 and SMA) and lymphatic vessels (positive for D240), as well as proliferating cells (positive for Ki67). In brief, tissue sections were deparaffinized and rehydrated. Heat-induced epitope retrieval was performed using citrate buffer pH 6.0 at 95C and samples were prehybridized with normal horse serum (Vector Laboratories, Burlingame, CA) for 1 h and incubated with primary antibodies overnight (see Supplementary Table S1 for references and concentrations). After rinsing,sampleswerehybridizedwithperoxidase-conjugatedsec- ondary antibodies for 1 h at room temperature and revealed with diaminobenzidine-DAB-(Vector Laboratories). Finally, tissue sections were counterstained with Harris hematoxylin for 5 s, rinsed, dehydrated, and mounted. For claudin and plakoglobin samples were analyzed by immunofluorescence. In both cases, tissue sections were processed as described above, but secondary antibodies conjugated with Cy3 or FITC (Sigma-Aldrich) were used at 1:500 dilution, and samples were counterstained with DAPI mounting medium (Vector Laboratories, Burlingame, CA, United States). 2.3 |Quantitative analyses All tissue sections subjected to histochemical and immunohistochemical analysis were analyzed using a Nikon Eclipse 90i light microscope (Nikon Corp., Tokyo, Japan). In each case, images were obtained from each sample subjected to the same technique using exactly the same parameters and configuration (exposure, intensity, background, white balance, etc.). Then, images were analyzed to quantify the intensity signal using a quantitative approach method as previously reported for the human skin. 18 On the one hand, the number of blood and lymphatic vessels found per mm 2 of superficial or profound dermis was independently quantified by three independent histologists, and averages were calculated. In the case of Ki67, the number of cells showing positive signal was quantified per mm of the basal layer of the epidermis and expressed as number of cells per mm of epithelium (CPM), since proliferating cells reside at the basal stratum of the epidermis. The same method was used to determine the number of cells showing positive staining signal for melanocytes and Langerhans cell markers. Furthermore, the results of the histochemical and immunohistochemical analyses were quantified using an automatic method based on the ImageJ software (National Institutes of Health, USA). For picrosirius red with non-polarized light, alcian blue, and PAS histochemistry, and for the immunohistochemical expression of epidermal markers (cytokeratins, cell-cell junctions, and differentiation markers), type-V collagen and decorin, average sample intensity was calculated MARTIN-PIEDRA ET AL.3of19 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License by randomly selecting 10 points in each type of sample (n=10), as previously described. 19,20 For polarized light picrosirius red and Verhoeff, we calculated the area of the dermis that was occupied by the stained fibers by using the area fraction option of the software. In the case of the dermal markers, the superficial and the profound dermis were analyzed separately. 2.4 |Statistical analysis For each sample and for each analysis method, results were expressed as averages and standard errors of the mean, and as medians and quartile ranges (corresponding to the first and third quartiles). Results of the quantitative analyses were compared with control native human skin and with ex vivo samples. First, each distribution was evaluated using the Shapiro–Wilk test to determine the normality of each distribution. Results of this analysis showed that the distributions were not normal and, therefore, did not fulfill the criteria for parametric analysis. Therefore, comparison of the results obtained for each type of sample were compared with controls and with ex vivo samples using the Mann–Whitney Utest, which is designed for the comparison of two non-parametric distributions. These analyses were performed using the Real Statistics software and pvalues below 0.05 were considered statistically significant for the doubletailed tests. 3|RESULTS 3.1 |Characterization of the epidermal layer of UGRSKIN grafted in patients 3.1.1 | Histological characterization of the epidermis using light and electron microscopy Analysis of the epidermal layer of UGRSKIN grafted in patients for 30, 60, and 90 days and stained with HE revealed that this layer was highly differentiated and properly integrated at all study times. As shown in Figure 1, the grafted skin epidermis consisted of four cell strata that were analogue to control native human skin (basal, spinosum, granulosum and corneum strata). However, the skin was devoid of the typical epithelial rete ridges and dermal papillae found in control skin. At the ultrastructural level (Figure 2), analysis of samples using TEM confirmed that the epidermal layer was highly differentiated at all study times. First, we found that the superficial stratum of all samples consisted of several layers of terminally differentiated keratinocytes (skin corneocytes). Then, we found that the granulosum stratum was composed by epithelial keratinocytes containing abundant keratohyalin electron-dense granules. At the spinosum stratum, cells showed numerous intercellular junctions, especially, desmosomes. Finally, the basal stratum consisted of elongated keratinocytes whose shape was compatible with the basal cells of the human epithelium. Interestingly, isolated Langerhans cells and melanocytes were detected within the epidermal layer. No differences were found among samples corresponding to 30, 60, and 90 days of follow-up. 3.1.2 | Immunohistochemical and immunofluorescence characterization of the skin epidermis Analysis of epithelial markers at the epidermal layer was carried out by immunofluorescence and immunohistochemistry. First, we assessed the expression of three epithelial cytokeratins (Figure 3and Tables 1and 2). For CK8, our results revealed that all samples were negative, and no differences with the control native skin were detected. For CK5, we found that that the lowest expression corresponded to the UGRSKIN samples kept in culture, with differences with control native skin being statistically significant. However, differences between samples grafted in patients and controls were nonsignificant. When CK10 was analyzed, we found very low staining signal in samples kept ex vivo, whereas controls and in vivo samples showed very strong signal. Differences with controls were statistically significant for samples kept ex vivo and 30-days in vivo skin tissues (Figure 3and Tables 1and 2). Then, we evaluated expression of two types of cell-cell junctions proteins playing a key role in maintaining the epithelial barrier function in each type of sample. As shown in Figure 4and Tables 1and 2, we found strong positive immunofluorescence expression of claudin and plakoglobin proteins in grafted skin from day 30 onward, with values comparable to control native skin, although expression was very low in ex vivo samples, which were significantly lower than control skin. In addition, we found that the bioartificial human skin keratinocytes expressed filaggrin and involucrin, two proteins related to terminal differentiation of the human skin epidermis. As shown in Figure 4and Tables 1and 2, suprabasal strata showed strong positive immunohistochemical signal for these two proteins, as it was the case of control skin. In contrast, expression was slight or negative in ex vivo samples. Differences with controls were significant for ex vivo samples and for 30-days in vivo skin in the case of involucrin. Furthermore, we quantified the number of melanocytes and Langerhans cells in the epithelium of UGRSKIN after identifying each cell type by immunohistochemistry (Figure 5and Tables 1and 2). In this regard, we found that control skin had an average and standard error of 28.6 ± 1.4 melanocytes per mm of epithelium (CPM), whereas ex vivo samples were devoid of these cells (0.0 ± 0.0 CPM) and skin grafted in patients showed a progressively increasing number of melanocytes (0.2 ± 0.1 CPM at day 30, 17.8 ± 0.7 at day 60 and 14.4 ± 1.4 at day 90 of follow-up). A very similar situation was found for Langerhans cells, with controls showing 61.2 ± 1.7 CPM of epithelium and samples grafted in patients showing 5.8 ± 0.2, 19 ± 1.1 and 23.2 ± 1.0 CPM after 30, 60, and 90 days, respectively, whereas ex vivo 4of19 MARTIN-PIEDRA ET AL. 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License samples were negative (0.0 ± 0.0 CPM). Differences with control native skin were statistically significant for both the melanocytes and Langerhans cells number (p< 0.05) (Table 2). Finally, analysis of proliferating cells showing positive Ki67 signal (Figure 5and Tables 1and 2) revealed the presence of 60.0 ± 2.6 proliferating CPM of epithelium of native control skin. However, the FIGURE 1 Analysis of UGRSKIN grafted in patients and control native skin using HE staining. The top panel shows a low magnification image of the epidermis, dermis and hypodermis layers of the skin (full-thickness), whereas each layer is shown at higher magnification in the lower panels. EPI, epidermis; HD, hypodermis; PD, profound dermis; SD, superficial dermis. The interface between the superficial and profound dermis is labeled with arrows. Scale bars: 100 μm. MARTIN-PIEDRA ET AL.5of19 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License number of proliferating cells was significantly lower (p< 0.05) in ex vivo samples (44.2 ± 2.6 CPM) and in skin grafted in patients for 30 days (44.6 ± 2.2 CPM), although no statistical differences with controls were found at 60 and 90 days (51.4 ± 0.8 and 64.6 ± 1.5 CPM, respectively). 3.2 |Characterization of the human skin basement membrane by histochemistry and electron microscopy Histochemical staining of UGRSKIN using the PAS method showed that all skin biopsies obtained from patients grafted with this skin model showed positive staining at the basement membrane, and the staining intensity was similar to control native human skin, whereas ex vivo samples showed significantly lower PAS staining intensity (Figure 5and Tables 1and 2). In addition, we confirmed the presence of a well-differentiated basement membrane at the ultrastructural level using TEM (Figure 2). In this regard, we found that skin grafted in patients for 30, 60, and 90 days showed a well-defined basement membrane at the dermo-epidermal junction, which was associated to numerous hemidesmosomes in basal keratinocytes (Figure 2). 3.3 |Characterization of the dermal layer of UGRSKIN grafted in patients 3.3.1 | Histological characterization of the dermal layer using light and electron microscopy When the dermis of the grafted skin was evaluated using HE staining (Figure 1), we found that this layer consisted of a dense fibrillar structure in which abundant stromal cells and blood vessels were immersed. As expected, control native skin showed a superficial papillary dermis with numerous cells and capillaries, and a profound reticular dermis rich in collagen fibers. However, the dermo-epidermal junction of UGRSKIN grafted in patients was flat, and a clear distinction between papillary and reticular dermis was not found. Interestingly, all in vivo samples showed an interface separating a superficial FIGURE 2 TEM analysis of UGRSKIN grafted in patients. The left panel corresponds to the analysis of the epidermal layer, whereas the basement layer and dermis are shown at the right. (a–c) corneum stratum of UGRSKIN after 30, 60, and 90 days of follow-up; (d–f) granulosum stratum; (g, h, i) spinosum stratum; (j–l) basal stratum; (m–o) basement membrane; (p–r): fibers of the dermal layer; (s–x) cells at the dermal layer. BV, blood vessel; CO, collagen fibers; EL, elastin fibers; FB, fibroblasts; LC, Langerhans cell; MC, melanocyte cell. White arrowheads: keratohyaline granules; black arrows: desmosome cell-cell junctions; yellow arrowheads: basement membrane; red arrows: hemidesmosomes. 6of19 MARTIN-PIEDRA ET AL. 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License dermis from a profound dermis, especially in the case of the biopsies corresponding to 30 days of follow-up, which probably corresponds to the limit between the grafted bioartificial skin and the remaining native connective tissue of the patient. This interface tended to progressively disappear with time and was almost absent in 90-days biopsies. Analysis of the superficial dermis of in vivo samples showed abundant cells and vessels, as it was the case of the controls. However, the profound dermis was richer in collagen fibers and contained less stromal cells, especially in biopsies taken after 90 days of followup, whilst 30-days biopsies showed numerous cells immersed in the dermal extracellular matrix. No differences were found between the native skin and the in vivo samples for the hypodermal layer of the human skin (Figure 1). Ultrastructural evaluation of the dermis of grafted UGRSKIN showed that the dermis was well-differentiated in all in vivo samples (Figure 2). All samples contained an abundant extracellular matrix with capillary blood vessels and numerous collagen and elastic fibers. Dermal fibroblasts containing abundant organelles such as ribosomes and endoplasmic reticulum were found, suggesting that these cells were metabolically active at all study times. No differences were found among the different in vivo samples at the ultrastructural level. 3.3.2 | Histochemical and immunohistochemical characterization of the dermal layer Fibrillar and non-fibrillar components of the human skin dermis extracellular matrix were assessed by histochemistry and immunohistochemistry. First, the presence of collagen fibers was assessed by picrosirius red histochemistry (Figure 6and Tables 2and 3). Results showed that the signal intensity of UGRSKIN samples was significantly lower in ex vivo samples as compared to control skin. For the superficial dermis, intensity of biopsies at 30 and 60 days was significantly lower than controls (p< 0.05), but samples at 90 days were similar to control skin. Interestingly, differences with control skin were nonsignificant for the profound dermis. Then, we wanted to assess the presence of mature and properly oriented collagen bundles using polarized light microscopy (Figure 6and Tables 2and 3). For the superficial dermis, we found that the area occupied by mature collagen was significantly higher in control skin as compared to ex vivo samples and biopsies at days 30 and 60. For the profound dermis, biopsies taken after 30 days had significantly less area occupied by mature collagen than controls, whereas in vivo samples at 60 and 90 days were similar to native skin. Then, we analyzed the presence of type-V collagen by immunohistochemistry (Figure 7and Tables 2and 3). Strikingly, expression of this fibrillar component of the extracellular matrix at the superficial layer of the dermis was slight in native skin, and expression was significantly higher in in vivo samples at 30, 60, and 90 days, with ex vivo samples showing significantly lower signal for this protein. When the profound dermis was considered, we found that native skin and biopsies corresponding to 30 days showed similar expression of type-V collagen than control skin, whilst samples at 60 and 90 days were significantly higher. Furthermore, Verhoeff histochemistry was used to identify elastic fibers in each type of sample (Figure 7and Tables 2and 3). Quantification of the area of dermis occupied by these fibers FIGURE 3 Immunohistochemical analysis of cytokeratins 5, 8, and 10 expression in the epidermal layer of UGRSKIN kept ex vivo, UGRSKIN grafted in patients for 30, 60, and 90 days and control human native skin. Scale bars: 100 μm. Histograms to the right show the average and standard deviation results of the staining signal quantification for each marker expressed as intensity units (I.U.). Significant differences with control native skin are labeled with C, whereas significant differences with samples kept ex vivo are labeled with E. Statistical comparisons were carried out with 10 samples per experimental group. MARTIN-PIEDRA ET AL.7of19 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License showed that ex vivo samples and biopsies obtained at day 30 was significantly lower than control native skin at both the superficial and profound dermis, although no differences were found at day 60 and 90. Once the main fibrillar components of the dermis extracellular matrix was evaluated, we analyzed the presence of proteoglycans in each sample type. On the one hand, alcian blue histochemistry revealed that proteoglycans were present in all samples, with the signal intensity found in native skin being similar to the rest of samples, except for the superficial dermis of biopsies at 30 days, which showed significantly lower alcian blue intensity than controls (Figure 8and Tables 2and 3). On the other hand, the presence of decorin, a proteoglycan playing a crucial role in dermal matrix assembly, was determined by immunohistochemistry. As shown in Figure 8and Tables 2 and 3, we found that decorin expression was significantly lower in ex vivo samples and in the superficial dermis of skin grafted for 30 days as compared to control skin dermis. However, decorin expression was comparable to controls for the superficial dermis of 60 and 90-days biopsies and for the profound dermis of 30, 60, and 90-days biopsies. 3.3.3 | Analysis of blood and lymphatic vessels at the dermal layer In the first place, we analyzed the presence of blood vessels in the dermis of the different samples analyzed in this work using CD31 and SMA immunohistochemistry (Figure 9and Tables 2and 3). Results showed that abundant well-structured blood vessels were found in all samples except for the ex vivo UGRSKIN. Quantification of CD31-positive vessels revealed that blood vessels were mostly present at the superficial dermis (around 3-fold as compared to profound dermis) in control skin and in all patient biopsies. Interestingly, we found that biopsies obtained at day 30, 60, and 90 contained a significantly higher number of CD31-positive structures than control skin in both the superficial and profound dermis. Very similar results were found for SMA-positive blood vessels. In both cases, the number of blood vessels found at the different days of follow-up were very similar, with abundant vessels found from day 30 onward. Regarding lymphatic vessels, our analysis using D240 antibodies revealed that this type of vessel was less abundant in the skin than blood vessels and that the superficial dermis of control skin was more TABLE 1 Histochemical and immunohistochemical analysis of the main epithelial markers and histochemical staining of the basement membrane using PAS in control native skin and UGRSKIN kept ex vivo and grafted in patients for 30, 60, and 90 days. Marker Layer UGRSKIN ex vivo UGRSKIN 30 days UGRSKIN 60 days UGRSKIN 90 days Control CK5 Epidermis 19.2 ± 2.1 30.4 ± 2.8 31.6 ± 1.9 30.1 ± 1.9 38.0 ± 3.2 16.0 [14.3–22.5]*29.0 [25.0–34.0] 30.5 [29.0–31.8] 31.0 [25.0–35.8] 35.5 [32.8–40.3] CK8 Epidermis 31.7 ± 2.9 34.2 ± 1.0 33.7 ± 1.4 33.0 ± 1.0 34.8 ± 2.6 27.5 [25.3–39.5] 34.0 [33.3–35.0] 32.5 [31.0–35.5] 32.5 [32.0–35.3] 35.5 [35.0–39.5] CK10 Epidermis 56.8 ± 7.6 134.1 ± 10.2 150.2 ± 9.1 152.0 ± 8.7 169.1 ± 4.0 51.5 [43.8–57.8]*145.0 [136.0–151.3] 161.5 [132.8–167.5] 158.0 [155.0–160.8] 176.0 [159.0–178.5] Claudin Epidermis 24.6 ± 7.6 98.4 ± 14.3 107.2 ± 11.0 105.6 ± 22.4 119.1 ± 10.4 14.5 [4.0–44.8]*103.5 [62.0–117.3] 102.5 [95.3–114.0] 89.5 [70.0–143.0] 125.5 [93.8–146.5] Plakoglobin Epidermis 50.2 ± 11.6 90.3 ± 10.2 93.2 ± 7.6 98.5 ± 3.8 87.5 ± 4.8 59.0 [21.8–79.3]*96.5 [71.0–116.8] 96.0 [90.3–108.0] 101.0 [94.0–105.8] 83.0 [78.8–87.8] Filaggrin Epidermis 65.3 ± 7.6 175.8 ± 7.4 179.2 ± 14.8 186.4 ± 6.1 170.7 ± 5.4 57.0 [55.5–66.3]*170.5 [161.3–194.8] 189.5 [154.5–211.3] 193.5 [173.5–197.8] 170.5 [161.0–180.8] Involucrin Epidermis 34.1 ± 4.5 66.9 ± 3.6 85.4 ± 5.1 89.0 ± 3.5 96.1 ± 5.1 32.5 [28.3–38.5]*67.0 [57.8–78.0]*83.5 [71.0–96.3] 87.0 [79.5–97.3] 98.0 [92.8–106.0] Melan-A Epidermis 0.0 ± 0.0 0.2 ± 0.1 17.8 ± 0.7 14.4 ± 1.4 28.6 ± 1.4 0.0 [0.0–0.0]*0.0 [0.0–0.0]*18.5 [15.3–19.8]*12.0 [11.0–18.8]*27.5 [25.3–32.8] CD1a Epidermis 0.0 ± 0.0 5.8 ± 0.2 19.0 ± 1.1 23.2 ± 1.0 61.2 ± 1.7 0.0 [0.0–0.0]*6.0 [5.0–6.0]*19.5 [16.5–20.0]*22.5 [21.0–26.5]*61.5 [58.0–65.8] Ki67 Epidermis 44.2 ± 2.6 44.6 ± 2.2 51.4 ± 0.8 64.6 ± 1.5 60.0 ± 2.6 41.5 [38.5–50.8]*42.5 [40.5–48.0]*51.0 [50.0–52.8] 62.5 [61.0–68.8] 60.0 [56.0–64.8] PAS Basement membrane 10.3 ± 4.3 53.4 ± 4.6 60.8 ± 6.0 58.8 ± 12.5 63.7 ± 5.2 3.5 [2.0–11.8]*54.5 [40.8–63.8] 60.0 [48.8–77.0] 48.5 [40.3–52.5] 64.0 [53.0–72.3] Note: Results correspond to the quantitative analysis of staining intensity, except for Melan-A, CD1a and Ki67, corresponding to the number of positive cells found per millimeter of epidermis. Results are shown as average ± standard error of the mean followed by the median and quartile range [first and third quartiles]. *pvalues below 0.05 as compared to control native skin are labeled with asterisks. 8of19 MARTIN-PIEDRA ET AL. 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TABLE 2 Statistical analysis of the quantitative results obtained by histochemistry and immunohistochemistry. Marker Layer Control vs. UGRSKIN ex vivo Control vs. UGRSKIN 30 days Control vs. UGRSKIN 60 days Control vs. UGRSKIN 90 days UGRSKIN ex vivo vs. UGRSKIN 30 days UGRSKIN ex vivo vs. UGRSKIN 60 days UGRSKIN ex vivo vs. UGRSKIN 90 days UGRSKIN 30 days vs. UGRSKIN 60 days UGRSKIN 30 days vs. UGRSKIN 90 days UGRSKIN 60 days vs. UGRSKIN 90 days CK5 Epidermis 0.0002*0.0524 0.0524 0.0753 0.0039*0.0011*0.0021*0.5787 0.7394 0.8534 CK8 Epidermis 0.315 0.1431 0.1431 0.0753 0.1903 0.123 0.1903 0.4813 0.2176 0.9118 CK10 Epidermis <0.0001*0.0007*0.123 0.123 0.0001*<0.0001*<0.0001*0.2176 0.0288*0.6305 Claudin Epidermis <0.0001*0.1431 0.393 0.4359 0.0003*0.0001*0.0015*0.7394 0.8534 0.7394 Plakoglobin Epidermis 0.0288*0.5288 0.2176 0.1051 0.0185*0.0052*0.0005*0.9705 0.8534 0.8534 Filaggrin Epidermis <0.0001*0.6842 0.315 0.063 <0.0001*<0.0001*<0.0001*0.6305 0.3527 0.9705 Involucrin Epidermis <0.0001*0.0005*0.1431 0.123 0.0002*<0.0001*<0.0001*0.0232*0.0007*0.4359 Melan-A Epidermis <0.0001*<0.0001*<0.0001*<0.0001*0.4813 <0.0001*<0.0001*<0.0001*<0.0001*0.1051 CD1a Epidermis <0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*0.0115* Ki67 Epidermis 0.0007*0.0005*0.0147*0.2475 0.6842 0.0753 <0.0001*0.0232*<0.0001*<0.0001* PAS Basement membrane <0.0001*0.1903 0.6305 0.123 <0.0001*<0.0001*0.0001*0.4359 0.5787 0.2475 Picrosirius red Superficial dermis <0.0001*0.0147*0.0039*0.2475 0.0052*0.0005*<0.0001*0.123 0.063 0.1903 Profound dermis <0.0001*0.7394 0.9118 0.393 <0.0001*<0.0001*<0.0001*0.9118 0.2475 0.6842 Polarized picrosirius red Superficial dermis 0.0079*0.0079*0.0079*0.9999 0.0079*0.0079*0.0079*0.0079*0.0079*0.0952 Profound dermis 0.0079*0.0079*0.6905 0.5476 0.0079*0.0079*0.0079*0.0079*0.0079*0.0317* Type-V collagen Superficial dermis <0.0001*0.0355*0.0147*0.0089*<0.0001*<0.0001*<0.0001*0.0185*0.0005*<0.0001* Profound dermis <0.0001*0.7959 0.0005*0.0001*<0.0001*<0.0001*<0.0001*0.0355*0.0039*0.393 Verhoeff Superficial dermis 0.0079*0.0079*0.3095 0.2222 0.1508 0.0079*0.0159*0.0079*0.1508 0.5476 Profound dermis 0.0079*0.0079*0.0079*0.0079*0.0159*0.0079*0.0079*0.0079*0.0079*0.0159* Alcian blue Superficial dermis 0.0232*0.0185*0.063 0.2799 0.0288*0.8534 0.0433*0.0068*0.0892 0.7394 Profound dermis 0.063 0.1431 0.8534 0.9118 0.0115*0.0288*0.0753 0.2475 0.1051 0.8534 Decorin Superficial dermis 0.0089*0.0185*0.9705 0.5787 0.5787 0.0115*0.0007*0.0007*0.3527 0.5288 Profound dermis 0.0005*0.8534 0.5288 0.9118 0.0002*<0.0001*0.0003*0.7394 0.4359 0.5787 CD31 Superficial dermis <0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001* Profound dermis 0.0002*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*0.2799 0.9705 0.2475 SMA Superficial dermis <0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001*<0.0001* Profound dermis 0.0002*<0.0001*<0.0001*0.0001*<0.0001*<0.0001*<0.0001*0.0039*<0.0001*0.0002* D240 Superficial dermis <0.0001*<0.0001*0.8534 <0.0001*0.063 0.0002*<0.0001*0.4359 0.0115*<0.0001* Profound dermis 0.0002*0.5288 0.0892 0.7394 0.063 0.0002*0.0002*0.0433*0.2799 0.1903 Note: Values correspond to statistical pvalues for the comparison of two specific groups using the Mann–Whitney Ustatistical test. *pvalues below 0.05 are labeled with asterisks. MARTIN-PIEDRA ET AL.9of19 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License and the smooth muscle layer of medium and large-size vessels. 49 These results are in accordance with previous reports showing that the use of autologous skin micrografts for the treatment of posttraumatic skin defects was associated to an early expression of CD31 and SMA positive vessels, leading to skin regeneration and avoiding the outcome scar. 33 Although a higher amount of blood vessels was found at the superficial dermis, we found that grafted skin vessels were not organized into upper and lower horizontal plexuses as it is the case of the normal human skin as previously reported. 50 The rapid biointegration process of the artificial skin and the fact that the host tissue was severely injured could be the responsible of this vessel distribution in the patients' dermis. Although future studies should be carried out in this sense, it is likely that vessels could reorganize and distribute in a different manner at longer follow-up times. Regarding lymphatic vessels, we found very few differences between the profound dermis of the grafted skin and the native skin. However, the number of lymphatic vessels found in samples grafted for 30 days was significantly lower than controls at the superficial dermis, and this number tended to increase with time. It has been proposed that lymphatic vessels are key actors controlling tissue microenvironment that play a major role in multiple physiological and pathophysiological processes, such as control of tissue hydration and regulation of immune functions. 51 Therefore, our findings point out that the regenerating skin is also capable of attracting abundant lymphatic vessels from the host tissue in order to ensure a normal physiology of the regenerated skin. Finally, our analysis of the subcutaneous adipose tissue (hypodermis) showed no differences with control skin, and this structure was formed by adipocytes and are subdivided by multiple septae of connective tissue in all samples, at previously reported for the normal FIGURE 9 Analysis of blood and lymphatic vessels in the dermal layer of control skin and UGRSKIN using CD31, SMA, and D240 immunohistochemistry. For each type of sample, images were taken at the superficial and profound dermis. Scale bars: 100 μm. Histograms to the right show the average and standard deviation results of the quantitative analysis of vessels showing positive expression of CD31, SMA, and D240 per mm 2 of superficial and profound dermis Significant differences with control native skin are labeled with C, whereas significant differences with samples kept ex vivo are labeled with E. Statistical comparisons were carried out with 10 samples per experimental group. 16 of 19 MARTIN-PIEDRA ET AL. 23806761, 0, Downloaded from https://aiche.onlinelibrary.wiley.com/doi/10.1002/btm2.10572 by Universidad De Granada, Wiley Online Library on [06/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License human skin. 52 Most likely, these findings correspond to profound layers of the patient skin that were not irreversibly burned. The present study has several limitations. Although UGRSKIN model has demonstrated potential usefulness to regenerate all skin layers, the real regenerative potential and clinical outcomes of patients treated with UGRSKIN should be determined in the future using a larger cohort of patients. In addition, the UGRSKIN model should be improved to generate more biomimetic skin substitutes showing the typical rete ridges, a vascular network and skin appendages to obtain a more biomimetic skin substitute able to reproduce the structure of the native skin. In addition, this skin model is strictly dependent on the availability of skin biopsies obtained from burnt patients, and the time required to generate the UGRSKIN substitute should be shortened in the future. These drawbacks could be surpassed by using alternative cell sources, as previously reported. 15 In conclusion, our comprehensive histological, histochemical and immunohistochemical characterization of the UGRSKIN bioartificial skin model grafted in patients provides several clues explaining the very good biocompatibility and clinical efficiency of this ATMP used in severely burnt patients. In general, we found that the epithelial layer tends to mature and differentiate very rapidly from the first day of the analysis, and this could be one of the main factors contributing to patient survival. Similarly, the dermal layer demonstrated to be highly biocompatible, but required longer periods of time to fully differentiate and integrate at the host tissue. Future studies should be carried out to determine the stability of the regenerated skin after longer periods of time. AUTHOR CONTRIBUTIONS Miguel Angel Martin-Piedra: Data curation (equal); investigation (equal); writing –original draft (equal); writing –review and editing (equal). Gloria Carmona: Data curation (equal); investigation (equal); writing –original draft (equal); writing –review and editing (equal). Fernando Campos: Formal analysis (equal); methodology (equal). Víctor Carriel: Data curation (equal); formal analysis (equal); methodology (equal). Ana Fernández-González: Formal analysis (equal); methodology (equal). Antonio Campos: Conceptualization (equal); funding acquisition (equal); project administration (equal). Natividad Cuende: Project administration (equal); validation (equal). Ingrid Garz on: Conceptualization (equal); methodology (equal); supervision (equal); validation (equal); writing –review and editing (equal). Purificaci on Gacto: Conceptualization (equal); project administration (equal); resources (equal). Miguel Alaminos: Conceptualization (equal); funding acquisition (equal); project administration (equal); supervision (equal); writing –review and editing (equal). ACKNOWLEDGMENTS This study was supported by AC17/00013 (NanoGSkin project) by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities, through AES 2017 within the EuroNanoMed framework, EU and by grant PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain. Additional support was provided through grants B-CTS-450-UGR20 (proyectos de I +D+ienel marco del Programa Operativo FEDER Andalucía 2014-2020, University of Granada and Consejería de Transformaci on Econ omica, Industria, Conocimiento y Universidades) and PI-0458-2016 (Consejería de Salud y Familias, Junta de Andalucía, Spain). This project was cofinanced by the European Regional Development Fund (ERDF) through the “Una manera de hacer Europa”program. Authors are thankful to Fabiola Bermejo-Casares for her valuable technical contribution to this work. This study is part of the Doctoral Thesis of Gloria Carmona. CONFLICT OF INTEREST STATEMENT Authors declare that they do not have any conflict of interest. PEER REVIEW The peer review history for this article is available at https://www. webofscience.com/api/gateway/wos/peer-review/10.1002/btm2. 10572. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Miguel Angel Martin-Piedra https://orcid.org/0000-0002-4639- 3175 Víctor Carriel https://orcid.org/0000-0002-8114-5644 Ingrid Garz on https://orcid.org/0000-0001-5944-0578 Miguel Alaminos https://orcid.org/0000-0003-4876-2672 REFERENCES 1. Burgess M, Valdera F, Varon D, Kankuri E, Nuutila K. The immune and regenerative response to burn injury. Cell. 2022;11(19):3073- 3096. doi:10.3390/cells11193073 2. Karppinen SM, Heljasvaara R, Gullberg D, Tasanen K, Pihlajaniemi T. Toward understanding scarless skin wound healing and pathological scarring. F1000Res. 2019;8:787-797. doi:10.12688/f1000research. 18293.1 3. Amini-Nik S, Dolp R, Eylert G, et al. Stem cells derived from burned skin—the future of burn care. EBioMedicine. 2018;37:509-520. doi:10. 1016/j.ebiom.2018.10.014 4. Yoon D, Cho YS, Joo SY, Seo CH, Cho YS. 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See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License