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N. Saeidi, J. W. Ruberti; Molecular Crowding: Towards Explaining the Genesis of Matrix Organization With Application to Corneal Tissue Engineering. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6203.
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© ARVO (1962-2015); The Authors (2016-present)
The extracellular matrices (ECMs) of load-bearing tissues comprise highly-organized arrays of collagen fibrils. Despite their importance, the processes which direct collagen fibril organization are still not well-understood. In the intracellular matrix (ICM) the principal structural biomolecules (e.g. actin and microtubules), undergo a rapid cycles of assembly/disassembly to/from organized filaments. It has been shown that macromolecular crowding is responsible for assembly and organization of the ICM. We hypothesize that molecular crowding may also play a critical role in the genesis of organized ECM. The goal of the present study is to investigate the effect of molecular crowding on the self-assembly and organization of type I collagen monomers, the main structural element of corneal ECM.
Uni or bimolecular crowding conditions (UC or BC) were produced either by extensively concentrating type I atelocollagen molecules (UC - up to 400 mg/ml) or by exposing low concentration collagen monomer (2.7 mg/ml) to concentrations of Polyethylene Glycol (PEG: BC) up to 40% by weight. Collagen fibrillogenesis was initiated by neutralizing the solution and increasing the temperature to 37C. The nano and micro scale organization of the fibrils was investigated by optical and electron microscopy.
Microscopy revealed that UC collagen self-assembled into highly-aligned, alternating lamellae mimetic of native cornea. The fibrillar organization in each lamella was long range and the orientation of the collagen fibrils in the lamellae changed through the construct thickness. Collagen molecules under BC conditions also self-assembled into highly-organized fibrillar arrays. The fibrils mainly appeared in isolated bundles with the number of isolated individual fibrils decreasing at higher concentrations the PEG. Under both BC and UC conditions, the local organization of fibrils improved with increased crowding.
This investigation shows that both UC and BC of collagen result in highly-organized fibrillar bundles which are mimetic of in vivo tissue structure. During development, the ECM is highly-crowded with ECM macromolecules (collagen, elastin, fibronectin, laminin, and hyaluronic acid). This simple in vitro investigation suggests that simple crowding could play a significant role in the formation of highly-anisotropic collagenous matrices.
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