Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering

Supported Ministry of Science and Innovation (Instituto de Salud Carlos III), grants FIS PI20/0317 and ICI21/00010 (NANOULCOR). Supported by grant CSyF PI-0086-2020 from Consejería de Salud y Familias, Junta de Andalucía, Spain and grant B-CTS-504-UGR20 (Programa Operativo FEDER Andalucía 2014-2020, University of Granada and Consejería de Transformación Económica, Industria, Conocimiento y Universidades). Cofinanced by the European Regional Development Fund (ERDF) through the “Una manera de hacer Europa” program.The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1124995/ full#supplementary-material SUPPLEMENTARY FIGURE S1 Immunofluorescence analysis of the OAC and HAC tissues kept ex vivo and grafted in vivo and control native human corneas (H) and rabbit corneas (R) using an anti-human mitochondria primary antibody. Scale bar: 50 μm (applicable to all images).Purpose: We carried out a histological characterization analysis of the stromal layer of human heterotypic cornea substitutes generated with extra-corneal cells to determine their putative usefulness in tissue engineering. Methods: Human bioartificial corneas were generated using nanostructured fibrin-agarose biomaterials with corneal stromal cells immersed within. To generate heterotypical corneas, umbilical cord Wharton’s jelly stem cells (HWJSC) were cultured on the surface of the stromal substitutes to obtain an epithelial-like layer. These bioartificial corneas were compared with control native human corneas and with orthotypical corneas generated with human corneal epithelial cells on top of the stromal substitute. Both the corneal stroma and the basement membrane were analyzed using histological, histochemical and immunohistochemical methods in samples kept in culture and grafted in vivo for 12 months in the rabbit cornea. Results: Our results showed that the stroma of the bioartificial corneas kept ex vivo showed very low levels of fibrillar and non-fibrillar components of the tissue extracellular matrix. However, in vivo implantation resulted in a significant increase of the contents of collagen, proteoglycans, decorin, keratocan and lumican in the corneal stroma, showing higher levels of maturation and spatial organization of these components. Heterotypical corneas grafted in vivo for 12 months showed significantly higher contents of collagen fibers, proteoglycans and keratocan. When the basement membrane was analyzed, we found that all corneas grafted in vivo showed intense PAS signal and higher contents of nidogen-1, although the levels found in human native corneas was not reached, and a rudimentary basement membrane was observed using transmission electron microscopy. At the epithelial level, HWJSC used to generate an epithelial-like layer in ex vivo corneas were mostly negative for p63, whereas orthotypical corneas and heterotypical corneas grafted in vivo were positive. Conclusion: These results support the possibility of generating bioengineered artificial corneas using non-corneal HWJSC. Although heterotypical corneas were not completely biomimetic to the native human corneas, especially ex vivo, in vivo grafted corneas demonstrated to be highly biocompatible, and the animal cornea became properly differentiated at the stroma and basement membrane compartments. These findings open the door to the future clinical use of these bioartificial corneas.Ministry of Science and Innovation (Instituto de Salud Carlos III) FIS PI20/0317 ICI21/00010Junta de Andalucia PI-0086-2020University of Granada and Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades) B-CTS-504-UGR20European Commissio

Analysis of cell proliferation of 9 TMJF cell passages by microarray.

<p>Average expression of the proliferation-related genes PCNA (in blue) and MKI67 (in red) are shown for each cell passage.</p

Intracellular ionic concentrations of potassium, sodium, chlorine and K/Na ratio of 9 consecutive cell passages of TMJF cells.

<p>Statistically significant differences between two consecutive cell passages are labeled with asterisks. All values are expressed as millimoles of each element per kilogram of cell dry weight and are shown as mean ± standard deviation.</p

Statistical <i>p</i> values for the comparisons of the cell viability levels as determined by trypan blue dye exclusion test, and LIVE/DEAD™ Cell viability assay and the intracellular ionic concentration of calcium, chlorine, potassium, magnesium, sodium, phosphorous and sulfur in 9 consecutive cell passages of TMJF.

<p>Pairwise comparisons between two consecutive cell passages were performed by using the Wilcoxon non-parametric test. The global significant differences among the 9 cell passages were analyzed by the W test of Kendall. Statistically significant <i>p</i> values are shown with asterisks (*).</p

Cell viability index of 9 consecutive cell passages (P1 to P9) of TMJF obtained by normalization of trypan blue dye test, LIVE/DEAD™ Cell viability assay and EPXMA (K/Na ratio) values.

<p>Cell viability index of 9 consecutive cell passages (P1 to P9) of TMJF obtained by normalization of trypan blue dye test, LIVE/DEAD™ Cell viability assay and EPXMA (K/Na ratio) values.</p

Analysis of cell viability and ionic content of 9 consecutive cell passages (P1 to P9) of TMJF.

<p>The percentage of live cells in each cell passage as determined by Trypan Blue dye exclusion test and the combined LIVE/DEAD™ Assay are shown in the first two rows. In the following rows, the intracellular ionic concentrations of calcium, chlorine, potassium, magnesium, sodium, phosphorous, sulfur and K/Na ratio are shown. In all cases, both the mean values and standard deviation are shown for each cell passage and technique. Ionic concentrations are expressed in millimoles of each element per kilogram of cell dry weight.</p

Expression of ECM components along 9 consecutive cell passages of TMJF.

<p>Each gene has been classified as <b>ECM-F</b> (ECM- fibrillar component), <b>ECM-GAGS</b> (ECM-glycosaminoglycans), <b>ECM-MGPS</b> (ECM- multiadhesive gluproteins), <b>ECM-OG</b> (other genes with a function in ECM), <b>ECM-PGS</b> (ECM-proteoglycans). The correlation between gene expression and cell passaging as determined by the Pearson (r) correlation test is shown in the last column. All genes with a positive correlation with cell passaging (r >0.700) are shown with asterisks (*)<b>.</b> Genes with a negative correlation with cell passaging (r <−0.700) are shown with double-asterisks (**).</p

Photographic images of control and decellularized SI samples using a patterned surface to estimate transparency.

<p>Images correspond to control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).</p

Scanning electron microscopy (SEM) images of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>Scale bar represents 50 µm.</p

Average results of the evaluation of decellularization efficiency, semi-quantitative analysis of tissue structure and quantitative analysis of fibrillar and non-fibrillar ECM components in control non-decellularized intestine and in all decellularization global groups.

<p>Nuclear cell removal and the staining signal for picrosirius, Gomori reticulin, PAS and Alcian blue correspond to percentages in reference of control non-decellularized intestine samples and are expressed as means plus/minus standard deviations. Tissue DNA quantification is shown as nanograms of DNA per milligram of dry weight of tissue. HE results are shown as median values and quartiles Q1–Q3 and correspond to the following classification: (0) highly organized tissue, (1) low levels of disorganization, (2) intermediate levels of disorganization, (3) high tissue disorganization. The analysis of tissue structure by SEM (interfibrillar spaces and collagen fibers) is shown in a scale ranging between (−) (small and regular interfibrillar spaces and highly disorganized fibers and disrupted three-dimensional structure) and (+++) (very large and irregular interfibrillar spaces and highly organized fibers and adequate three-dimensional structure) as defined in the methods section.</p