https://hdl.handle.net/10481/29924 2026-04-10T15:39:50Z https://hdl.handle.net/10481/109554 Bioartificial human corneas generated by tissue engineering. A historical and technical review Alaminos Mingorance, Miguel; Crespo Ferrer, Pascual Vicente; García, José Manuel; Sánchez Quevedo, María Del Carmen; Campos Muñoz, Antonio Jesús Different types of bioartificial corneas have been generated by tissue engineering through combining cells, biomaterials, and bioactive molecules. Orthotypical corneal cells can be obtained from corneal biopsies, and include epithelial, stromal, and endothelial cells, whereas heterotypical cells are obtained from alternative cell sources with corneal differentiation potential, such as mesenchymal stem cells. In turn, two main types of biomaterials have been applied to corneal tissue engineering: those generated by the de-cellularization of natural tissues and biomaterials generated de novo using synthetic or natural biomaterials, especially collagen, fibrin, and agarose. Cells and biomaterials are combined with bioactive factors, inducing cell proliferation and differentiation. A review of previous studies revealed that most bioartificial corneas were not able to fulfill the complex requirements required for clinical translation, which include a thorough preclinical characterization, generation of the tissue as an advanced therapy medicinal product, a clinical research phase, and a final authorization by the European Medicines Agency or another competent regulatory agency. Most authorized products correspond to partial corneal substitutes consisting of one cell type associated or not with a scaffold, and only one product consisting of a human bioartificial cornea containing a fibrin-agarose scaffold and two corneal cell lineages (epithelial and stromal cells) called NANOULCOR was evaluated in patients in the context of an advanced therapy medicinal product. These findings confirm the existence of a bottleneck between basic and clinical research and suggest the need to implement novel clinical studies to develop new therapies that can improve the results of current corneal therapies. This research was supported by grants FIS PI23/00335 and ICI21/00010 (NANOULCOR), funded by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities (Plan Estatal de Investigación Científica, Técnica y de Innovación, and Plan de Recuperación, Transformación y Resiliencia). Co-financed by the European Union. https://hdl.handle.net/10481/109550 Generation of a Bioengineered Substitute of the Human Sclero-Corneal Limbus Using a Novel Decellularization Method Ávila-Fernández, Paula; Sánchez Porras, David; Etayo-Escanilla, Miguel; González Gallardo, Carmen; Alaminos Mingorance, Miguel; Chato Astrain, Jesús; Campos Sánchez, Fernando; García García, Óscar Darío Background: Severe dysfunction of the human limbus associated with limbal stem cell deficiency is a therapeutic challenge, especially when a structural alteration of the limbal niche is associated. Methods: We have evaluated seven decellularization protocols applied to 20 human sclero-corneal limbus, based on the use of SDS (protocol P1), SDS + NaCl (P2), SDS + triton X-100 + SDC + enzymatic treatment (P3), SDS + triton X-100 + SDC + enzymatic treatment + trypsin (P4), sulfobetains + DNAse (P5), sulfobetains + SDC + DNAse (P6) and SDC + DNAse (P7). The decellularization efficiency of each protocol, biocompatibility and safety, as well as their capability to support cell attachment and differentiation, were evaluated. Results: Results showed that the use of protocols P1 to P4, based on strong ionic detergents such as SDS, was not efficient for decellularizing the human limbus. Conversely, protocols P5, P6 and P7 removed more than 95% of DNA while preserving 60–100% of the extracellular matrix components. These protocols were biocompatible, as macrophages cultured with decellularized scaffolds were viable and differentiated to the pro-regenerative M2 phenotype (CD163/CD86 ratio > 2) without inducing a significant increase in reactive oxygen species (ROS). Protocols P6 and P7 supported cell attachment, survival and differentiation of corneal epithelial cells and four types of mesenchymal stem cells cultured on the surface of these scaffolds. Cellularized limbi showed positive expression of several limbal cell markers, especially in scaffolds decellularized with protocol P6. Conclusions: These results support the use of protocol P6 for the generation of human limbal substitutes by tissue engineering using decellularized human limbi. Future studies should determine the clinical potential of the regenerative biomaterial generated in patients with structural limbal damage, particularly in patients with chemical burns and aniridia, where conventional stem cell therapies fail. This research was funded by Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities, grants FIS PI23/00335, FIS PI20/00317 and ICI21/00010 (NANOULCOR) and co-funded by the European Union. Supported by grant CSyF PI-0086-2020 from Consejería de Salud y Consumo, Junta de Andalucía, Spain.; Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics17121540/s1 https://hdl.handle.net/10481/109548 Sox9 prevents retinal degeneration and is required for limbal stem cell differentiation in the adult mouse eye Hurtado, Alicia; López-Soriano, V; Lao Pérez, Miguel; Celis-Barroso, MA; Lazúen, P; Chacón-de-Castro, A; Ramírez Casas, Yolanda; Alaminos Mingorance, Miguel; Collinson, JM; Burgos Poyatos, Miguel; Jiménez Medina, Rafael; Carmona López, Francisco David; Barrionuevo Jiménez, Francisco Javier Sox9 is a transcription factor with multiple roles during development and in adult organ homeostasis. In the adult eye, Sox9 expression persists in several cell types, including the retinal pigmented epithelium cells and the Müller glial (MG) cells, as well as in the limbal and corneal basal epithelia. To uncover the role of Sox9 in these cell types, we induced the deletion of the gene in adult mice. We found that, after Sox9 ablation, mutant mice undergo a severe process of retinal degeneration characterized by the loss of MG cells and complete depletion of the photoreceptors layer. Moreover, by combining single-cell RNA sequencing and Sox9 lineage tracing, we found that Sox9 is expressed in a basal limbal stem cell population with the ability to form two types of long-lived cell clones involved in stem cell maintenance and homeostasis. Mosaic analysis of Sox9 positive and negative cells confirmed that the gene is essential for limbal stem cell differentiation. Our results show that Sox9 is required for the maintenance of retinal integrity and for limbal stem cell differentiation in the adult mouse eye. https://hdl.handle.net/10481/108702 Extracellular matrix properties of porcine reproductive scaffolds depend on storage temperature Martínez Lopez, Cristina; Scaringi, Maria; García García, Óscar Darío; Chato Astrain, Jesús; Martínez Cáceres, Carlos Manuel; Izquierdo Rico, Mª José; García Vázquez, Francisco Alberto Decellularized extracellular matrix (dECM) scaffolds show great promise in the field of reproductive medicine for the treatment of reproductive tract pathologies, the engineering of artificial organs to restore fertility, or the improvement of assisted reproduction techniques. Although protocols for decellularizing oviducts and uteri to obtain dECM scaffolds are well established, the optimal storage conditions required to maintain the structural and biochemical integrity of these scaffolds remain poorly defined. In this study, we evaluated the effects of one-month storage at three different temperatures (4 °C, −20 °C, and −80 °C) on the stability of dECM scaffolds from porcine oviducts and uteri. Although storage did not alter the macroscopic structure, microscopic analyses showed that storage at −80 °C resulted in a reduction in glycosaminoglycans in the oviduct, while storage at 4 °C altered the histological pattern of uterine scaffolds. Proteomic analysis revealed that freezing conditions (−20 °C and −80 °C) better preserved matrix proteins associated with key functions, such as regeneration and cell adhesion. Frozen storage was also associated with greater biomechanical strength, reduced bacterial growth, and improved biocompatibility of the scaffolds. Together, these results demonstrate that storage conditions directly influence the quality of dECM scaffolds, highlighting that freezing temperatures are more suitable for short-term storage. https://hdl.handle.net/10481/107331 Improving the regenerative microenvironment during tendon healing by using nanostructured fibrin/agarose-based hydrogels in a rat achilles tendon injury model González-Quevedo, David; Díaz-Ramos, Miriam; Chato-Astrain, Jesús; Sánchez-Porras, David; Tamimi, Iskandar; Campos, Antonio; Campos, Fernando; Carriel, Víctor Achilles tendon injuries are a frequent problem in orthopaedic surgery due to their limited healing capacity and the controversy surrounding surgical treatment. In recent years, tissue engineering research has focused on the development of biomaterials to improve this healing process. The aim of this study was to analyze the effect of tendon augmentation with a nanostructured fibrin-agarose hydrogel (NFAH) or genipin cross-linked nanostructured fibrin-agarose hydrogel (GP-NFAH), on the healing process of the Achilles tendon in rats. Methods NFAH, GP-NFAH, and MatriDerm (control) scaffolds were generated (five in each group). A biomechanical and cell-biomaterial- interaction characterization of these biomaterials was then performed: Live/Dead Cell Viability Assay, water-soluble tetrazolium salt-1 (WST-1) assay, and DNA-released after 48 hours. Additionally, a complete section of the left Achilles tendon was made in 24 Wistar rats. Animals were separated into four treatment groups (six in each group): direct repair (Control), tendon repair with MatriDerm, or NFAH, or GP-NFAH. Animals were euthanized for further histological analyses after four or eight weeks post-surgery. The Achilles tendons were harvested and a histopathological analysis was performed. Results Tensile test revealed that NFAH and GP-NFAH had significantly higher overall biomechanical properties compared with MatriDerm. Moreover, biological studies confirmed a high cell viability in all biomaterials, especially in NFAH. In addition, in vivo evaluation of repaired tendons using biomaterials (NFAH, GP-NFAH, and MatriDerm) resulted in better organization of the collagen fibres and cell alignment without clinical complications than direct repair, with a better histological score in GP-NFAH. Conclusion In this animal model we demonstrated that NFAH and GP-NFAH had the potential to improve tendon healing following a surgical repair. However, future studies are needed to determine the clinical usefulness of these engineered strategies.