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N. Saeidi, K. N. Portale, J. W. Ruberti; Direct Optical and Quick Freeze Deep Etch Imaging of Self-Assembled, Cholesterically Dense Collagen Gels. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1865.
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© ARVO (1962-2015); The Authors (2016-present)
Self-assembled collagen gels derived from dilute solutions of collagen (3.0 mg/ml) are typically weak and highly disordered. Such gels are a poor starting point from which to begin the engineering of a corneal stroma. Cholesteric packing of collagen monomers prior to assembly could increase the strength of the resulting matrix and possibly induce fibril organization. However, little is known about gross morphology of cholesterically assembled collagen matrices or about the nanoscale structure of collagen fibrils assembled under cholesteric conditions.
Cold, acid-soluble bovine collagen monomers (3.0 mg/mL, Inamed Biomaterials, Fremont, CA) in PBS were dialyzed against 40% solution of 20k MW Polyethylene Glycol (PEG) using 3.5 KDa dialysis tubing for 24 hr to reach desired concentration (ranging from 20 to 30 mg/mL). While collagen was kept at 4°C, it was then neutralized using 1:8 ratio of PBS to collagen and the pH of the solution was adjusted to be in the range of 6.8 to 7.4 using 0.1M NaOH. 1 mL of the final solution was injected between two clean coverslips which were 250 microns apart. The system was then kept at 4°C for 30 minutes to allow the cholesteric solution to "re-order". The monomer solution was then exposed to 37°C for 45 min to initiate collagen fibrillogenesis. Differential Interference Contrast imaging (DIC - 60x objective; 1.4 NA) was performed on self-assembled collagen to study large scale organization of fibrils. The ultrastructure of the fibrils was investigated using Quick-Freeze, Deep-Etch (QFDE) electron microscopy.
Direct optical imaging (DIC) of the assembled collagen revealed an extremely dense matrix comprising individual fibrils which filled the space between the cover slips. The fibrils were often aligned over long distances albeit far (25-30 microns) from the glass surfaces. This indicates that the surface energy of the cover slips may influence the organization of fibrils. QFDE micrographs showed a dense array of fibrils of ~100 nm diameter. Many of the fibrils exhibited the typical D-periodic banding. Often, when banded fibrils were adjacent and aligned, the period of the banding was in phase.
Cholesteric packing of collagen monomers does induce organization over long distances (hundreds of microns) in self-assembled fibrillar collagen gels. Such a mechanism may be used to construct strong, organized scaffolds for corneal stromal tissue engineering. The "in-phase" banding on adjacent collagen fibrils suggests that the organized packing of highly concentrated monomers can be reflected in the fibrils following assembly.
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