Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw

Abstract

This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4-1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed.

Fig 1. Freezing-induced expansion of collagen fibril.

Average confinement of single collagen molecule in the fibril is modeled by considering an equilateral hexagonal unit cell for confinement based on a quasi-hexagonal packing state [44, 45]. Unit cell expands as intrafibrillar fluid freezes. d_m is tropocollagen nominal diameter, d is confinement diameter, d_c is the length of a side of the unit cell.

Fig 2

(A) Representative MTDSC heating thermograms that show endothermic peaks of denaturation for F/T versus UF collagen in hydrogels and molecular solutions. Lines and shaded regions indicate the mean and standard deviation respectively. Denaturation temperature of collagen in (B) molecular solution and (C) hydrogel. ‘*’ indicates significant difference (p < 0.05).

Fig 3. Effects of F/T conditions on post-thaw denaturation temperature.

‘*’ indicates significant difference (p < 0.05).

Fig 4. Recovery of collagen post-thaw thermal stability by use of cryoprotectant, DMSO.

Fig 5. Freezing-induced morphological changes in collagen hydrogels at network and fibril levels.

(A) Representative SEM images. Scale bars are 5 μm and 500 nm for top and bottom panels respectively. (B) Hydrogel fibril diameter distributions.

Fig 6. Prediction of denaturation temperature decrease upon F/T by computational modeling.

(A) The change in denaturation temperature upon hypothetical expansion of a tightly packed fibril (minimum porosity). (B) The amount of freezing-induced fibril expansion and change in denaturation temperature as a function of unfrozen fibril porosity.