Abstract: The translation of current tissue engineering approaches to clinical application is somehow limited by the use of scaffolding materials. Recently a number of in vitro scaffold-free three-dimensional culture techniques have been developed. These techniques realize the assembly of tissue-like structures including but not limited to spheroids, blood vessels and cartilage. In particular, cells can now self-assemble to form planar tissue-like structures at the interface of an aqueous-two-phase system (ATPS). The unique advantage of this technique is that without a solid substrate, planar tissue-like structures can now be assembled rapidly with very simple procedures. This technique can potentially be very useful for tissue engineering in eye because of its ability to direct cells to form monolayer. In this talk, we will introduce what ATPS is and its current applications in biomedical research. We will then present an approach to assemble cell sheets in ATPS using both primary cells isolated from porcine eyes and other cell lines. The physiological relevance of these eye-related cell sheets as well as their potentials in ophthalmic research and applications will be discussed.
Background: The purpose of this infrastructure is to provide to the Network researchers a database and diverse related tools for the anatomical and functional analysis of the normal, pathological and surgical cornea.
Methods: This database is composed of normal and pathological individuals, totaling more than 36,000 patients. It includes anatomical and functional imaging data, physiological optics data, psychometric and clinical data (medical history, surgical parameters, acuteness, etc.). Various corneal topography tools were added, giving the database a unique character: tools for analyzing individual maps, average map tools for the study and comparison of populations, 3D modeling and visualization tools, statistical tools, etc. There are also screening tools for detecting various corneal conditions (LASIK, PRK, RK, keratoconus) and for secure data exchange between colleagues.
Results: Several studies were made in recent years thanks to this common infrastructure. For example, this database has provided important information regarding the evolution of the 3D shape of the normal cornea with age and ametropia and has confirmed the mirror symmetry of corneas for the right and the left eyes (enantiomorphism). The different stages of Fuchs’ dystrophy were also characterized to provide essential knowledge for surgery of the posterior layer of the cornea. Our database also allowed studying the anatomy of the wounds and the shape of the cornea before and after a transfixing transplant or an endothelial transplant (DSAEK and DSEK). The data on the characterization of experimentally transplanted corneas with corneal equivalents generated by tissue engineering and the recent addition of clinical data on the replacement of a diseased cornea with a synthetic corneal equivalent (keratoprosthesis) also resulted in several publications. More recently, the database has allowed to develop innovative algorithms to determine the optimal shape of an implant according to the clinical parameters of the recipient. On the other hand, we also demonstrated that the 3D shape of the cornea can be used as a biometric characteristic (such as fingerprints) for identification of individuals for various applications ranging from forensics to secure border crossings. Consequently, a new multimodal database (cornea + iris + eventually retina) was created for the purpose of biometric identifications. This database provides a unique set of anatomical and functional tools for the analysis of the cornea. It is characterized by the scientific quality and large quantity of accumulated information on the cornea and the high-level tools to exploit its content.
Conclusions: The common infrastructure is easily accessible to all VHRN members on request. The database will also be accessible online in 2018 (see http://cvl.concordia.ca for more information).
Background: Because of its superficial anatomical localization, the cornea is particularly vulnerable to abrasive forces and various traumas, which can lead to significant visual impairments. Upon injury of the corneal epithelium, there are important changes that occur in the composition of the underlying extracellular matrix (ECM). Those changes are perceived by the integrins that recognize the ECM components as their ligand and activate different intracellular signalling pathways, ultimately leading to reepithelialisation and reorganization of the injured epithelium, both of which are necessary in order to restore the visual properties of the cornea. The goal of this study was to analyse the impact of the pharmacological inhibition of specific signal transduction mediators of integrin-dependant signalling pathways on corneal wound healing using both monolayers of hCECs and tissue-engineered human corneas (hTECs) as in vitro models.
Methods: hTECs were produced by the self-assembly approach and wounded with a 8-mm diameter biopsy punch. Total RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The wounded tissues were then incubated with the WNK1 inhibitor WNK463 and wound healing was monitored over a period of 6 days. Control corneas were incubated with the vehicle alone (DMSO). The impact of WNK1 inhibition on hCECs monolayers was determined using a scratch wound assay.
Results: Gene profiling analyses and protein kinases arrays revealed important alterations in the expression and activity of several mediators from the integrin-dependent signalling pathways in response to the ECM changes taking place during corneal wound healing. Among these, WNK1 is considerably activated through phosphorylation during corneal wound healing. The pharmacological inhibition of WNK1 by WNK463 significantly reduced the dynamic of corneal wound closure in our hTECs and hCECs monolayers compared to their respective negative controls.
Conclusions: These results allowed the identification of WNK1 kinase as an important player for a proper healing of the cornea. Also, these results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and they may lead to the identification of new therapeutic targets in the field of corneal wounds.
Background: The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.
Methods: Cells from human choroids were isolated and cultured. Culture purity was assessed using immunostaining (CD31, HMB45, vimentin, keratins 8/18). To engineer the choroid, fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix (ECM) assembly. Endothelial cells, melanocytes or RPE cells were separately seeded on the stromal substitutes. Choroidal substitutes were further characterized by histology, mass spectrometry, immunostaining, and compared to native human choroids.
Results: The technique used to isolate choroidal cells yielded pure cultures of fibroblasts, melanocytes and vascular endothelial cells. The stromal substitutes engineered using the self-assembly approach were composed of collagen (types I, VI, XII and XIV), proteoglycans (decorin, lumican) and other ECM proteins. Protein expression was confirmed using immunostaining. Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.
Conclusions: This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells. This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.
Background: Disruption of the microstructure in corneal stroma can lead to the loss of transparency. The lack of a characterization method for the microstructure prevents such scaffolds to be implemented in tissue transplantation. The non-invasive, three-dimensional (3D) rendering multiphoton microscopy (MPM) poses the potential to solve this problem.
Methods: MPM images and data analyses were performed with three kinds of samples with known and different quality. Isosurfaces (ISOs) were constructed for the evaluation of void volume and collagen distribution.
Results: The differences in the microstructures of these samples were revealed with clear indications and links to their behaviours in rehydration and possible transparency. According to this analysis, the scaffold with the highest void space ratio amongst the three presented the highest successful rates to be thoroughly rehydrated.
Conclusions: Such a method can be developed for assessing the quality of tissue engineered corneas, or donated corneas, and be useful as a powerful research tool in cornea related research.
Abstract: Corneal blindness represents one of the world’s three major causes of blindness, and the fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To address the growing need for corneal transplants two main approaches are being pursued: allogenic and bioengineering cornea. Bioengineering corneas are constructed by naturally generating an extracellular matrix (ECM) component as the scaffold structure with or without corneal cells. It is well established that the scaffold structure directs the fate of cells, therefore, the fabrication of the correct scaffold structure components could produce an ideal corneal substitute, able to mimic the native corneal function. Another key factor in the construction of tissue engineering cornea is seed cells. However, unlike the epithelium and stroma cells, human cornea endothelium cells (HCECs) are notorious for having a limited proliferative capacity in vivo because of the mitotic block at the G1 phase of the cell cycle due to “contact-inhibition”. This review will focus on the main concepts of recent progress towards the scaffold and seed cells, especially endothelial cells for bioengineering cornea, along with future perspectives.
Abstract: Since the 21st century, the development of corneal tissue engineering technology has been developing rapidly. With the progress of biomaterials, cell culture and tissue engineering technology, tissue engineering cornea has gained great development in both basic scientific research and clinical application. In particular, tissue engineered corneal scaffolds are the core components of tissue engineered corneas. It is the focus of current research on tissue engineering cornea to search for scaffolds with good biocompatibility, high safety and good biomechanical properties. In this paper, the recent research progress of tissue engineering corneal materials is reviewed.
