Histological, histochemical, and immunohistochemical characterization of NANOULCOR nanostructured fibrin‑agarose human cornea substitutes generated by tissue engineering

Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 https://doi.org/10.1186/s12916‑024‑03759‑4 RESEARCH Open Access © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. BMC Medicine Histological, histochemical, andimmunohistochemical characterization ofNANOULCOR nanostructured fibrin‑agarose human cornea substitutes generated bytissue engineering Olimpia Ortiz-Arrabal1,2, Cristina Blanco-Elices1,2, Carmen González-Gallardo1,2,3, David Sánchez-Porras1,2, Miguel Etayo-Escanilla1,2, Paula Ávila Fernández1,2, Jesús Chato-Astrain1,2, Óscar-Darío García-García1,2*, Ingrid Garzón1,2* and Miguel Alaminos1,2 Abstract Background Human artificial corneas (HAC) generated by tissue engineering recently demonstrated clinical usefulness in the management of complex corneal diseases. However, the biological mechanisms associated to their regenerative potential need to be elucidated. Methods In the present work, we generated HAC using nanostructured fibrin-agarose biomaterials with cultured corneal epithelial and stromal cells, and we compared the structure and histochemical and immunohistochemical profiles of HAC with control native corneas (CTR-C) and limbus (CTR-L) to determine the level of biomimicry of the HAC with these two native organs. Results HAC tissues consisted of a stratified epithelium and a cellular stromal substitute. The interface between stroma and epithelium was similar to that of CTR-C, without the finger-shaped palisades of Vogt found in CTR-L, and contained a poorly developed basement membrane as determined by PAS histochemistry. Analysis of the stromal layer revealed that HAC contained significantly lower amounts of extracellular matrix components (collagen, proteoglycans, decorin, keratocan, and lumican) than CTR-C and CTR-L, with all samples being devoid of elastic and reticular fibers. At the epithelial level, HAC were strongly positive for several cytokeratins, although KRT5 was lower in HAC as compared to CTR-C and CTR-L. The expression of crystallin lambda was lower in HAC than in control tissues, whereas crystallin alpha-a was similar in HAC and CTR-C. No differences were found among HAC and controls for the cell–cell junction proteins CX43 and TJP1. When specific markers were analyzed, we found that HAC expression profile of KRT3, KRT19, KRT15, and ΔNp63 was more similar to CTR-L than to CTR-C. *Correspondence: Óscar-Darío García-García [email protected] Ingrid Garzón [email protected] Full list of author information is available at the end of the article Page 2 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 Conclusions These results suggest that HAC generated in the laboratory could be structurally and functionally more biomimetic to the structure found at the corneal limbus than to the central cornea, and open the door to the use of these artificial tissues in patients with limbal deficiency. Keywords Cornea, Tissue engineering, Limbal stem cells, Histology, Advanced therapies Background Numerous conditions, including trauma, infections, congenital malformations, degeneration, and other diseases, may affect the transparency of the human cornea and cause blindness [1]. Most of these diseases can be treated by cornea transplantation or keratoplasty. However, this technique is dependent on the availability of donors and is subjected to several limitations that make necessary the search for alternative treatments [2, 3]. In this milieu, tissue engineering techniques allow the development of human artificial corneas (HAC) able to reproduce the structure and physiology of the native cornea [4]. HAC have been generated to the date using different scaffolds, including, among others, the human amniotic membrane [5], decellularized tissues [6, 7], and different hydrogels such as type-I collagen, fibrin, or chitosan [7–9]. To generate a corneal epithelial layer on the surface of most HAC, researchers typically make use of limbal epithelial stem cells (LESCs) isolated from the scleral limbus and expanded in culture [10]. The reason for this is that mature epithelial cells in the central cornea are thought to be terminally differentiated and unable to proliferate and differentiate exvivo, whereas LESCs have high proliferation potential and can generate large amounts of epithelial cells in culture. In fact, it is well known that LESCs are the stem cells in charge of maintaining corneal epithelial turnover and integrity, and central cornea epithelial cells derive from LESCs [11]. However, the microenvironment of LESCs and mature corneal epithelial cells is very different, and both cell types are known to be structurally and physiologically different [12]. One of the HAC models showing potential clinical usefulness is NANOULCOR, a nanostructured fibrin-agarose human anterior lamellar cornea consisting of human corneal cells and nanostructured fibrin-agarose biomaterials. This HAC showed promising preclinical results in laboratory animals [10, 13] and good biocompatibility and functionality in patients with severe corneal damage enrolled in a preliminary advanced therapies clinical trial [14]. In the present work, we evaluated a fibrin-agarose HAC model using an array of histological, histochemical, and immunohistochemical methods and compared the results with the human native scleral limbus and central cornea in order to determine the level of biomimicry of the HAC with these two native organs. This study could contribute to improve the clinical treatment of patients with severe corneal defects. Methods Cell isolation andculture Primary cultures of human cornea stromal keratocytes and LESCs were obtained from samples of human limbal scleral rings as previously reported [13, 15]. Donor corneas were preserved at 31 °C until 30 days after donation, using tissue culture media at the corneal bank of Andalusia, following the protocols established by this bank, and limbal rings were provided after keratoplasty was carried out. Average age of the donors used in the present work was 51.5 ± 23years. Stromal keratocytes were isolated by enzymatic digestion using a 2mg/mL solution of Clostridium histolyticum type-I collagenase (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 6 h and then cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotics solution (both from Merck, Burlington, MA, USA). LESCs were cultured in an epithelial medium consisting of a mixture of Ham-F12 (150mL), DMEM (300 mL), and FBS (50 mL), supplemented with 1% antibiotic-antimycotics, adenine (24 µg/mL), insulin (5 µg/mL), triiodothyronine (1.3 ng/mL), hydrocortisone (0.4µg/mL), and epidermal growth factor (EGF) (10ng/mL) (all from Merck). In all cases, cells were cultured at 37°C in a humidified incubator with 5.0% CO2 using standard cell culture conditions. The culture medium was changed every 2–3days, and cells were dissociated with 0.25% trypsin–EDTA (Merck) when 70% confluence was reached. Cells corresponding to the 3rd–4th cell passages were used to prevent cell senescence derived from long-term culturing. This research was performed in accordance with guidelines and regulations of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and vision research. This project was approved by the local Human Research and Ethics Committee of the province of Granada (PEIBA) (numbers 1915-N-20 and 2224-N-20). Page 3 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 Generation ofhuman artificial corneal substitutes (HAC) HAC were generated using fibrin-agarose hydrogels with a final concentration of agarose of 0.1%, as previously reported [15, 16]. In brief, a biological substitute of the corneal stroma was first generated by mixing 3.8mL of human plasma with 250 µL of a melted 2% solution of type-VII agarose in phosphate buffer saline (PBS) and 375 µL of DMEM containing 100,000 human keratocytes. To prevent gel fibrinolysis, the mixture was then supplemented with 75 µL of a 100mg/mL solution of tranexamic acid (Amchafibrin™, Fides Ecopharma, Valencia, Spain), and 500 µL of 1% calcium chloride (Merck) was added at the final step to trigger the fibrin polymerization reaction. This mixture was rapidly aliquoted in Transwell cell culture inserts with 0.4 µm porous membranes (Sarstedt, Nümbrecht, Germany) and allowed to jellify at 37°C for at least 6h. Then, 500,000 cultured LESCs were added on top of the stromal substitutes to generate an epithelial layer [16]. HAC were kept in culture for 4weeks, using the air–liquid culture technique from the third week to promote epithelial stratification and differentiation [15]. Finally, plastic compression nanostructuration was performed to improve the biomechanical properties of HAC, as previously reported [13, 16, 17]. The approximate thickness of the final product was 500µm, as previously published [18], and its optical properties were described elsewhere [18–20]. In general, HAC showed approximately 80% of the light transmittance of the control native corneas [13]. Histological analyses HAC and control human native corneas (CTR-C) and scleral limbi (CTR-L) tissues were fixed for 48h in 4% formaldehyde and embedded in paraffin following standard histology laboratory protocols. Four micrometerthick histological sections were obtained, placed on glass slides, deparaffinized with xylene, cleared in ethanol, and rehydrated in water. In order to analyze the histological structure of each sample, tissue sections were stained with hematoxylin–eosin (HE). In brief, sections were incubated for 3 min in hematoxylin (PanReac AppliChem, Barcelona, Spain), rinsed for 5min in tap water, and stained with eosin (PanReac AppliChem) for 1min. Samples were then washed in distilled water, dehydrated in alcohol series, and coverslipped. Histological images were obtained using a Pannoramic® DESK II DW scanner (3D Histotech, Budapest, Hungary). Analysis ofbasement membrane andextracellular matrix (ECM) components byhistochemistry Deparaffinized tissue sections were subjected to several histochemical methods following previously published protocols [17, 21]. First, basement membrane glycosaminoglycans were stained using the periodic acid-Schiff (PAS) method. In brief, tissue sections were incubated in an oxidant 0.5% periodic acid solution for 5min, incubated in Schiff reagent for 15min, and slightly counterstained with Harris hematoxylin for 20s. Then, relevant components of the extracellular matrix (ECM) were identified using specific histochemical techniques. Elastic fibers were stained with the method of Verhoeff (VER), consisting in a 10min incubation in Verhoeff staining solution, followed by a brief differentiation in 2% ferric chloride. Reticular fibers were identified using the metal reduction protocol of Gomori (RET). In this case, sections were incubated in 1% potassium permanganate, followed by 2% sodium metabisulfite solution and sensibilization with 2% iron alum, incubation in ammoniacal silver and 20% formaldehyde. Differentiation was then performed with 2% gold chloride and 2% thiosulfate. To identify mature collagen fibers in the tissue ECM, we used the picrosirius red histochemical method (PSR), by incubating tissue sections for 30min in sirius red F3B reagent, followed by Harris hematoxylin counterstaining for 5min. ECM proteoglycans were assessed using the alcian blue histochemical method (AB). For this, sections were incubated for 30min in AB solution, washed, and slightly counterstained with nuclear fast red solution for 1min. All histochemical reagents were purchased from PanReac AppliChem. Analysis ofextracellular matrix (ECM) components andepithelial cell markers byimmunohistochemistry andimmunofluorescence Specific components of the tissue ECM and markers of epithelial cells were identified in each sample by immunohistochemistry and immunofluorescence with specific primary antibodies. For cornea ECM components, fibrillar type-I collagen (COL-I) was analyzed, along with decorin (DCN), keratocan (KER), and lumican (LUM). At the epithelial level, we first analyzed globally the presence of cytokeratins using two cytokeratin cocktails (pancytokeratin (PCK) and AE1/AE3). Then, we evaluated the presence of cytokeratin 5 (KRT5), crystallin λ (CRYλ), crystallin alpha-a (CRYαa), connexin 43 (CX43), and tight junction protein 1 (TJP1). As specific markers of mature corneal epithelial cells found at the central cornea, we analyzed the presence of cytokeratin 3 (KRT3), whereas cytokeratin 19 (KRT19) and cytokeratin 15 (KRT15), the limbal isoform of the protein p63 (ΔNp63), were analyzed as specific markers of LESCs residing at the limbal area. All these analyses were carried out using triplicates (n = 3 for each type of tissue). On the one hand, COL-I, DCN, KER, LUM, PCK, AE1/AE3, KRT3, KRT5, KRT15, KRT19, CRYλ, and Page 4 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 ΔNp63 markers were detected by immunohistochemistry. Briefly, tissue sections were dewaxed and rehydrated, and then antigen retrieval was carried out at 95 °C. Endogenous peroxidase was blocked with 3% of H2O2 in PBS, and nonspecific antibody binding sites were blocked with 1 × casein and normal horse serum (both, from Vector Laboratories, Burlingame, CA). Then, samples were incubated with primary antibodies overnight at 4°C in a humid chamber following the technical details summarized in Additional file1: TableS1. After washing with PBS, samples were incubated with readyto-use secondary antibodies labeled with peroxidase for 1h at room temperature (RT), and antibody binding was detected using a diaminobenzidine substrate kit (DAB) (both, from Vector Laboratories). Finally, samples were briefly counterstained with Harris hematoxylin. On the other hand, CRYαa, CX43, and TJP1 were analyzed by immunofluorescence. As described above for immunohistochemistry, tissue sections were dewaxed and rehydrated, and then antigen retrieval was performed at 95°C. Unspecific sites were blocked with 1 × casein and normal horse serum (both from Vector Laboratories) and samples were incubated with specific primary antibodies overnight at 4°C in a humid chamber as detailed in Additional file1: TableS1. Then, secondary antibodies conjugated with a fluorescent pigment (FITC or Cy3) were applied for 1h at room temperature, and samples were counterstained with mounting medium with DAPI (Vector Laboratories). Images were obtained and analyzed using a Nikon Eclipse 90i fluorescence microscope. Quantification andstatistical analysis Results obtained for the histochemical and immunohistochemical analysis of ECM components were quantified with the ImageJ software (National Institutes of Health, Bethesda, MD, USA), as previously described [21]. First, the staining intensity was calculated in each histological image for each analysis method giving a positive signal, by randomly selecting 15 points per sample type, and asking the software to calculate the signal staining intensity (INT). Then, we evaluated the percentage of area that was occupied by positively stained structures, by randomly selecting 15 square areas of 50 × 50µm per sample type, and the area fraction (AF) was automatically assessed by the program. Results corresponding to the immunohistochemistry and immunofluorescence analysis of epithelial cell markers were semiquantitatively analyzed, as previously suggested [13, 16, 22]. In this case, the staining signal was categorized in each sample as negative ( −), slightly positive (+ / −), positive ( +), very positive (+ +), or strongly positive (+ + +). These analyses were carried out by three independent expert histologists in order to reduce potential biases. A workflow describing the quantification process is shown in Fig.1. Quantitative results were compared among the three groups of study (CTR-C, CTR-L, and HAC). First, we Fig. 1 Analysis of the histochemical staining signal in the different samples analyzed in the present work. Samples were fixed, embedded in paraffin, sectioned, and stained with each method, and the staining intensity was automatically quantified to determine both the signal intensity (INT) ant the area fraction (AF) Page 5 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 analyzed if each variable differed from a normal distribution using the Shapiro–Wilk test. As this test showed that most distributions were not normal, pairwise comparisons between two specific groups of study were carried out using the non-parametric test of Mann–Whitney. Statistical p values below 0.05 were considered statistically significant for the two-tailed test. Statistical testing was carried out using the Real Statistics Resource Pack software (Release 7.2) available at https:// www. realstati stics. com/ (Purdue University, West Lafayette, IN, USA). Results Histological analysis As shown in Fig.2, the histological analysis of CTR-C using HE showed a stratified epithelium consisting of around 4–5 cell strata, in which basal cells displayed a typical columnar morphology, whereas apical cells were spindle-shaped, flattened, squamous cells. Analysis of CTR-L revealed the typical finger-shaped palisades of Vogt in which LESCs project towards the subjacent corneal stroma containing blood vessels. In addition, the histological analysis of HAC tissues revealed the presence of an epithelial layer with 5–6 cell strata, with cells displaying an elongated morphology showing very few differences among cell strata. When the stromal layer was analyzed, we found that both control tissues (CTR-C and CTR-L) consisted of a dense network of collagen fibers organized in thin lamellae containing disperse stromal keratocytes. In turn, HAC showed a randomly organized extracellular matrix of fibrin-agarose with abundant scattered keratocytes. No specialized structures, such as the Vogt palisades or blood vessels were found in HAC. Analysis ofthebasement membrane using PAS histochemistry When the different samples were stained with the PAS histochemical method, we found a PAS-positive signal at the interface between the epithelial and the stromal tissues of both the CTR-C and CTR-L, revealing the presence of a well-differentiated basement membrane at this level. When HAC tissues were analyzed, we found a positive staining of the epithelial cells, suggesting the presence of glycoproteins at this level, with a slight positive signal at the interface between the epithelial and the stromal layers, suggesting that HAC were devoid of a wellstructured, mature basement membrane (Fig.2). Identification ofcorneal stroma ECM components byhistochemistry andimmunohistochemistry On the one hand, we analyzed the presence of relevant fibrillar components of the corneal stroma ECM in control tissues and bioengineered corneas (Fig.3). First, we found a negative staining signal for VHF and RET in CTR-C, CTR-L, and HAC, suggesting that elastic and reticular fibers were not present in these three tissues. Then, the analysis of collagen fibers was positive in all samples and was therefore quantified (Table1). Quantification showed that of the results of the PSR histochemistry showed very strong staining signal intensity and area fraction (AF) in CTR-C, and very low signal in HAC, Fig. 2 Analysis of control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC) using hematoxylin–eosin (HE) and PAS histochemistry. Three samples were analyzed per type of tissue (n = 3). Images are shown at different magnifications for each method. Black arrows highlight illustrative PAS-positive areas in each sample. Scale bars: 50 μm Page 6 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 Fig. 3 Histochemical and immunohistochemical analysis of ECM components of the corneal stroma of control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC). Three samples were analyzed per type of tissue (n = 3). VHF, Verhoeff histochemistry for elastic fibers; RET, Gomori’s reticulin histochemistry for the identification of reticular fibers; PSR, picrosirius red histochemical method for collagen fibers; COL-I, immunohistochemistry for type-I collagen; AB, alcian blue histochemistry for proteoglycans; DCN, immunohistochemistry for decorin; KER, immunohistochemistry for keratocan; LUM, immunohistochemistry for lumican. Top images correspond to histological microphotographs, whereas the signal quantification is represented, for those markers showing positive signal, in the lower panel. Scale bars: 50 μm Table 1 Quantitative expression analysis of the ECM components showing a positive staining signal using histochemical and immunohistochemical methods in control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC). INT, signal intensity for each analysis method; AF, area fraction (percentage of tissue area occupied by positive signal for each analysis method). Columns to the left show averages and standard deviations, whereas statistical p values for the pairwise comparisons are shown to the right. PSR, picrosirius red histochemical method for collagen fibers; COL-I, immunohistochemistry for type-I collagen; AB, alcian blue histochemistry for proteoglycans; DCN, immunohistochemistry for decorin; KER, immunohistochemistry for keratocan; LUM, immunohistochemistry for lumican. Statistically significant differences are highlighted with asterisks (*) CTR-C CTR-L HAC CTR-C vs. CTR-L CTR-C vs. HAC CTR-L vs. HAC PSR INT 122.93 ± 26.12 69.53 ± 33.08 20.00 ± 0.00 0.0001* < 0.0001* < 0.0001* AF 88.19 ± 5.25 33.76 ± 14.36 1.79 ± 0.90 < 0.0001* < 0.0001* < 0.0001* COL-I INT 48.07 ± 24.94 25.80 ± 23.99 8.20 ± 4.55 0.0164* < 0.0001* 0.0367* AF 21.39 ± 4.27 11.05 ± 6.68 2.51 ± 2.85 0.0001* < 0.0001* < 0.0001* AB INT 54.13 ± 11.23 45.67 ± 27.99 20.60 ± 10.89 0.4363 < 0.0001* 0.0128* AF 67.06 ± 6.41 42.03 ± 7.63 13.50 ± 4.59 < 0.0001* < 0.0001* < 0.0001* DCN INT 74.47 ± 26.26 92.27 ± 52.63 24.87 ± 16.92 0.4124 < 0.0001* 0.0001* AF 63.80 ± 3.86 47.85 ± 11.25 2.49 ± 2.94 0.0002* < 0.0001* < 0.0001* KER INT 122.87 ± 26.16 74.13 ± 17.08 22.40 ± 12.16 < 0.0001* < 0.0001* < 0.0001* AF 99.08 ± 0.60 54.70 ± 11.59 2.86 ± 4.80 < 0.0001* < 0.0001* < 0.0001* LUM INT 95.33 ± 19.51 107.27 ± 15.67 46.2 ± 15.63 0.0675 < 0.0001* < 0.0001* AF 92.24 ± 3.36 83.68 ± 11.46 10.27 ± 2.51 0.0453* < 0.0001* < 0.0001* Page 7 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 with CTR-L showing intermediate values. Differences were statistically significant for all pairwise comparisons. When type-I collagen was identified by immunohistochemistry, we also found that the highest intensity and AF values corresponded to CTR-C, followed by CTR-L, and the lowest results were found in HAC. Differences were statistically significant. On the other hand, several non-fibrillar components of the corneal stroma ECM were identified by histochemistry and immunohistochemistry. As shown in Fig.3 and Table 1, the analysis of tissue proteoglycans using AB revealed intense staining signal in CTR-C and CTR-L, with non-significant differences between both tissues for the staining intensity, although the area fraction was significantly higher in CTR-C. Both the CTR-C and CTR-L showed significantly higher intensity and AF than HAC. Then, we identified three specific ECM proteins using immunohistochemistry. For DCN, the highest intensity corresponded to CTR-C and CTR-L, with non-significant differences between both types of samples, and the highest intensity was found in HAC, with statistically significant differences with CTR-C and CTR-L. However, we found that the highest AF was found in DCN, followed by CTR-L and the lowest, in HAC, with differences among samples being statistically significant. For KER, our results revealed that the highest intensity and AF corresponded to CTR-C, with significant differences with CTR-L and HAC, and the lowest intensity and AF were found in HAC, with significant differences with CTR-C and CTR-L. Finally, our analysis showed that LUM intensity was very high in CTR-C and CTR-L, with non-significant differences between both types of samples, and the lowest intensity was found in HAC that was significantly lower than CTR-C and CTR-L. However, differences were statistically significant for the area fraction, with the highest values found in CTR-C, and the lowest, in HAC. Analysis ofepithelial cell markers Epithelial characterization of the different samples analyzed in this work (Fig.4 and Table2) was first carried out by analyzing the presence of several non-specific cytokeratins by immunohistochemistry. In this milieu, the use of two pancytokeratin cocktails (PCK and AE1/ AE3) confirmed the presence of global human cytokeratins in all samples, with very positive PCK signal in CTR-C and CTR-L and strongly positive signal in HAC, and strongly positive AE1/AE3 signal for all sample types. For KRT5, we found a strongly positive signal in CTR-C and CTR-L and a very positive signal in HAC. Then, we evaluated the presence of two corneal crystallins playing an important role in maintaining corneal transparency. For CRYλ, results were strongly positive for both control Fig. 4 Evaluation of epithelial cell markers in control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC) using immunohistochemistry and immunofluorescence. Three samples were analyzed per type of tissue (n = 3). PCK, human cytokeratin cocktail pancytokeratin; AE1/AE3, human cytokeratin cocktail AE1/AE3; KRT5, cytokeratin 5; CRYλ, crystallin λ; CRYαa, crystallin alpha a; CX43, connexin 43; TJP1, tight junction protein 1. Higher magnification inserts at the bottom of the image correspond to the areas highlighted with dotted red squares in the HAC tissues. Scale bars: 50 μm Page 8 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 tissues (CTR-C and CTR-L), whereas HAC showed very positive staining signal. For CRYαa, the signal was strongly positive in CTR-C and HAC and very positive in CTR-L. Finally, the analysis of two relevant cell–cell junction proteins (CX43 and TJP1) revealed a very positive signal in the three types of tissues analyzed here (CTR-C, CTR-L, and HAC). Analysis ofspecific markers ofmature epithelial cells andLESCs When the expression of the corneal epithelium cell marker KRT3 was evaluated in the three types of tissues, we found a strongly positive signal at the epithelial layer of CTR-C, a positive signal at the origin of the limbal niche of CTR-L, with negative signal in the cells corresponding to the Vogt palisades, and a slightly positive signal in the epithelium of HAC tissue substitutes (Fig.5 and Table2). Then, we evaluated several specific markers of LESCs using immunohistochemical and immunofluorescence methods (Fig. 5 and Table 2). Results of this analysis showed that KRT19 was positive in CTR-C and a very positive in CTR-L and HAC. For KRT15, signal was negative in the epithelial cells of the CTR-C and very positive in CTR-L and HAC. Finally, our analysis of the limbal stem cell marker ΔNp63 using immunofluorescence Table 2 Results of the semiquantitative expression analysis of relevant epithelial cell markers in control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC) using immunohistochemistry and immunofluorescence. PCK, human cytokeratin cocktail pancytokeratin; AE1/AE3, human cytokeratin cocktail AE1/ AE3; KRT5, cytokeratin 5; CRYλ, crystallin λ; CRYαa, crystallin alpha a; CX43, connexin 43; TJP1, tight junction protein 1; KRT3, cytokeratin 3; KRT19, cytokeratin 19; KRT15, cytokeratin 15; ΔNp63, limbal isoform of the protein p63. The staining signal was assessed as negative ( −), slightly positive (+ / −), positive ( +), very positive (+ +), or strongly positive (+ + +) CTR-C CTR-L HAC PCK + + + + + + + AE1/AE3 + + + + + + + + + KTR5 + + + + + + + + CRYλ + + + + + + + + CRYαa + + + + + + + + CX43 + + + + + + TJP1 + + + + + + KRT3 + + + + + / − KRT19 + + + + + KRT15 - + + + + ΔNp63 + + + + + Fig. 5 Analysis of specific markers of the epithelial cells of the human cornea and limbus in control human native corneas (CTR-C), control human native scleral limbi (CTR-L), and human artificial corneas (HAC) using immunohistochemistry. Three samples were analyzed per type of tissue (n = 3). KRT3, cytokeratin 3; KRT19, cytokeratin 19; KRT15, cytokeratin 15; ΔNp63, limbal isoform of the protein p63. Higher magnification inserts at the bottom of the image correspond to the areas highlighted with dotted red squares in the HAC tissues. Scale bars: 50 μm Page 9 of 12 Ortiz‑Arrabaletal. BMC Medicine (2024) 22:531 showed positive signal in CTR-C and a very positive signal in CTR-L and HAC. Discussion The recent development of novel advanced therapies medicinal products allowed the regenerative treatment of severe diseases for which a curative therapy has not been described [23]. In the field of ophthalmology, corneal conditions affecting the structure of this organ are very difficult to manage, and novel alternative treatments are in need. In this context, the use of the NANOULCOR advanced therapies medicinal product (ATMP) offered promising results in one of the first clinical trials carried out in Europe with a bioartificial cornea substitute consisting of the two most external layers of the cornea— epithelium and stroma [17]. Although the results of this clinical trial have been positive, evaluation of the molecular mechanisms associated to the clinical efficiency of ATMPs used clinically is necessary for a proper characterization of these products. One of the unsolved questions related to the clinical effectiveness of NANOULCOR is whether this ATMP retain the undifferentiation phenotype of LESCs, or if this product resembles the differentiated status of the human mature central cornea. Although this HAC was generated with cultured LESCs, it has been demonstrated that organotypic culture systems used during fabrication of NANOULCOR are able to induce partial cell differentiation [13, 15]. In the present work, we first demonstrated that the bioartificial tissues generated by tissue engineering reproduced the histological structure of the central cornea and were devoid of the Vogt palisades found in CTR-L. In fact, HAC consisted of a stromal substitute with a stratified epithelium on top, and the interface between both tissues was flat, in agreement with our previous publications [10, 16, 17], and as known for the normal central cornea [24]. When the basement membrane was analyzed, we confirmed our previous results suggesting that an incipient structure was present in HAC kept in culture, and exposition to the invivo environment is required for the terminal differentiation of this basement membrane [17]. To determine the degree of biomimicry of HAC, and their similarity to control tissues, we then analyzed some relevant components of the human cornea stroma ECM. For the fibrillar components, we found that both the native tissues and the HAC were devoid of reticular and elastic fibers. These findings are not surprising, since the fine and definite structure of the corneal layers is crucial for a proper transparency [25], and the presence of thick non-collagenous fibers, especially the elastic fibers, could impair corneal transparency. It has been demonstrated that the human native cornea has elastic properties [26]. However, the presence of elastic fibers in the human cornea has not been described, and some reports suggest that corneal elasticity may be associated to elastinfree microfibril bundles [27]. Previous studies from our group also showed that HAC are devoid of fibrillar and elastic fibers [16, 17]. However, all tissues analyzed in the present work were rich in collagen fibers, especially the native CTR-C. It is well known that the corneal stroma consists of abundant well-organized of collagen fibrils arranged in the corneal stroma, and this structure is crucial for corneal transparency [25]. In contrast, several authors found that the collagen fibers number, concentration, and organization is significantly reduced in the sclero-corneal limbus [25], as found in our study. Finally, the lowest amount of collagen fibers corresponded to HAC. These results are in agreement with our previous findings demonstrating that cornea substitutes kept in culture show very low collagen contents that cannot be detected using histochemistry, and only very sensitive immunohistochemical methods can identify this fibers in HAC kept exvivo [16]. These previous reports also demonstrated that invivo grafting was able to significantly increase the presence of collagen fibers [17]. For the non-fibrillar components of the ECM, our results showed that HAC contained significantly lower amounts of proteoglycans, decorin, keratocan, and lumican than CTR-C and CTR-L, whereas all these components, except for keratocan, were similar in CTR-C and CTR-L. Again, these results coincide with previous reports suggesting that the bioartificial tissues kept in culture tend to show very low differentiation levels and express low amounts of these non-fibrillar components, whereas HAC implanted invivo in laboratory animals significantly increased these components as a result of the invivo environment and stromal-epidermal interaction [13, 17]. These results imply that the HAC generated in this work significantly differed from CTR-C and CTR-L in terms of ECM composition, suggesting that the stromal layer of these artificial tissues could be very undifferentiated. However, the lack of blood vessels and palisades of Vogt in HAC suggests that bioengineered corneas could morphologically more similar to CTR-C than CTR-L. On the other hand, we analyzed the epithelial layer of HAC and compared their immunohistochemical and immunofluorescence profile with the control tissues. First, we found that HAC were able to express high amounts of non-specific cytokeratins, as it was the case of CTR-C and CTR-L. Keratins are essential elements of the cytoskeleton of human epithelial cells [28], and their presence in HAC confirms that epithelial layer of artificial corneas retains certain differentiation profile. In