Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

Purpose: Human cornea substitutes generated by tissue engineering currently require limbal stem cells for the generation of orthotypical epithelial cell cultures. We recently reported that bioengineered corneas can be fabricated in vitro from a heterotypical source obtained from Wharton's jelly in the human umbilical cord (HWJSC). Methods: Here, we generated a partial thickness cornea model based on plastic compression nanostructured fibrin-agarose biomaterials with cornea epithelial cells on top, as an orthotypical model (HOC), or with HWJSC, as a heterotypical model (HHC), and determined their potential in vivo usefulness by implantation in an animal model. Results: No major side effects were seen 3 and 12 months after implantation of either bioengineered partial cornea model in rabbit corneas. Clinical results determined by slit lamp and optical coherence tomography were positive after 12 months. Histological and immunohistochemical findings demonstrated that in vitro HOC and HHC had moderate levels of stromal and epithelial cell marker expression, whereas in vivo grafted corneas were more similar to control corneas. Conclusion: These results suggest that both models are potentially useful to treat diseases requiring anterior cornea replacement, and that HHC may be an efficient alternative to the use of HOC which circumvents the need to generate cornea epithelial cell cultures.

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.

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.

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