Prelude to corneal tissue engineering – Gaining control of collagen organization

Abstract

By most standard engineering practice principles, it is premature to credibly discuss the “engineering” of a human cornea. A professional design engineer would assert that we still do not know what a cornea is (and correctly so), therefore we cannot possibly build one. The proof resides in the fact that there are no clinically viable corneas based on classical tissue engineering methods available. This is possibly because tissue engineering in the classical sense (seeding a degradable scaffolding with a population synthetically active cells) does not produce conditions which support the generation of organized tissue. Alternative approaches to the problem are in their infancy and include the methods which attempt to recapitulate development or to produce corneal stromal analogs de novo which require minimal remodeling. Nonetheless, tissue engineering efforts, which have been focused on producing the fundamental functional component of a cornea (organized alternating arrays of collagen or “lamellae”) may have already provided valuable new insights and tools relevant to development, growth, remodeling and pathologies associated with connective tissue in general. This is because engineers ask a fundamentally different question (How can that be done?) than do biological scientists (How is that done?). The difference in inquiry has prompted us to closely examine (and to mimic) development as well as investigate collagen physicochemical behavior so that we may exert control over organization both in cell-culture (in vitro) and on the benchtop (de novo). Our initial results indicate that reproducing corneal stroma-like local and long-range organization of collagen may be simpler than we anticipated while controlling spacing and fibril morphology remains difficult, but perhaps not impossible in the (reasonably) near term.

Polarization microscopy for characterizing fiber orientation of ocular tissues

“…However, the accuracy, repeatability and robustness of the technique when applied to ocular tissues remains to be determined. Tendon and ligaments are generally similar to collagenous ocular tissues in terms of extracellular matrix composition [26], but there are differences in their structural hierarchies [9] and overall organization [4,[27][28][29]. Therefore, it cannot be taken for granted that PLM will apply to ocular tissues as it does to other tissues, and the performance of the technique must be assessed.…”

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

“…27 Precisely how the exquisite two-dimensional plus organization of the corneal stroma arises during development is not well understood. 28 Two competing theories address the deposition of organized collagen. The first suggests that collagen is directly placed (via fibripositors) by the invading neural crest-derived mesenchymal cells, 29 while the second suggests that collagen fibrils condense from concentrated solutions of collagen monomer.…”

“…30 Determining the method by which fibroblastic cells produce organized collagen arrays is not only an important basic science question, but can also inform efforts to engineer load-bearing tissue in general. 28 In a recent investigation by our laboratory, dedifferentiated human stromal keratocytes stratify and secrete a corneallike organization of type I=V collagen lamellae in vitro in a scaffold-free tissue engineering system. 31 To our knowledge, this is one of the first culture system capable of reproducing alternating arrays of collagen with organization similar to the corneal stroma.…”

Lateral Growth Limitation of Corneal Fibrils and Their Lamellar Stacking Depend on Covalent Collagen Cross-linking by Transglutaminase-2 and Lysyl Oxidases, Respectively

“…Keratocytes in culture can deposit a matrix, in which primordia of corneal lamellae are established (6,7), and which is reminiscent of the primary or early secondary corneal stroma (2). However, the role of the macromolecular components remains to be identified.…”