Elsevier

Thermochimica Acta

Volume 365, Issues 1–2, 29 December 2000, Pages 119-128
Thermochimica Acta

Changes in collagen structure: drying, dehydrothermal treatment and relation to long term deterioration

https://doi.org/10.1016/S0040-6031(00)00619-5Get rights and content

Abstract

Collagen is the major component of most connective tissues in animals. The structure of the helix and axial molecular packing within fibrils is well documented. Less is known about the structural alterations that occur on drying and the compounded changes that occur on dehydrothermal treatment. The structural properties of collagen and its supramolecular architecture are of importance in these states, since many of the industrial applications of collagen-based materials involve dried collagen or dehydrothermally treated collagen.

The effects of drying and thermal treatment of collagen can be observed by X-ray diffraction, the changes in the diffraction pattern relate to changes in the axial packing of collagen molecules as dehydration occurs. The meridional diffraction series becomes truncated indicating induced structural disorder, the spreading of diffraction features indicate that the molecular orientation is altered for some of the collagen chains or portions of collagen chains within a fibril.

Dehydrothermal treatment of parchment collagen for up to 24 h reduces the axial periodicity of the collagen fibril from 64.5 to 60.0 nm. Analysis of the X-ray diffraction data shows the possible alterations in molecular packing that may explain the structural changes.

Introduction

Collagen is an extracellular matrix protein that has an essential role in maintaining the architecture of multicellular organisms as well as having important industrial uses as leather, sutures, implants and prostheses [1]. It is also important as gelatin in the photographic, cosmetic and food industries [2]. The importance of understanding the relationship between collagen structures and thermal treatment is therefore essential in understanding the modulation of collagen molecular properties. The use of thermal treatment may also have a role in determining the relevance of the degradation sustained by thermal denaturation as a means of accelerated ageing of samples such as parchment and leather [3]. This is in order to compare the effects of degradation induced in a number of days to oxidative damage sustained over a number of centuries.

Collagen molecules are characterized by the sequential Gly-XY triplet structure that is a prerequisite for triple helix formation. Although at least 20 collagen types have been characterized [4], type I collagen is the predominant type being found as the main component of skin, tendon, aorta and bone. The type I collagen triplex is a heteropolymer consisting of two α1 chains and an α2 chain of over 1000 residues in length. The structure of the triple helix has a rope-like nature, where each collagen chain adopts a left-handed helical conformation and the three strands intertwine with a right-handed superhelical twist.

The mechanical integrity of collagen molecules in tissues is often further enhanced by the association of collagen molecules in a specific fibrillar form. The axial packing of collagen molecules in fibrils is well established, since it can be easily visualized by electron microscopy. The axial molecular packing can be described as a step function repeating at 64 nm intervals comprising of gap and overlap regions [5]. In this, each collagen molecule (of approximately 300 nm length) is staggered relative to its nearest neighbour by 67 nm in the native state or ∼64 nm in the dry state. This leads to a fibrillar form where each collagen molecule has a strong molecular connection with neighbouring collagen molecules and an applied force can be transmitted through a fibril to each collagen molecule. A review of the relationship between the collagen molecule, fibrils and mechanical strength is given by Parry [6].

X-ray diffraction of tissues such as tendon gives rise to a complex fibre diagram corresponding to a number of structural features. The diffraction process gives information that is reciprocally related to distances between features in real space. For example, the repeating helix structure of the polyproline II type helix contains a strong periodic function of approximately 0.29 nm corresponding to the axial rise between amino acids; this corresponds to a large diffraction angle and can only be viewed using short sample to detector distances. The axial molecular packing of collagen corresponds to a 67 nm staggering of molecules and results in a sharp series of X-ray peaks nearly parallel to the fibre axis. The first order of this diffraction has a very low diffraction angle and correspondingly can only be detected using long camera lengths. Since the axial packing of collagen is highly regular, the diffraction produces a series of peaks that correspond to the harmonic series based on a 67 nm periodicity at reciprocal spacings of 167,267,367,467 nm, etc. Therefore, although the first order of diffraction may not be observable in a particular camera geometry, the position of a particular order of the diffraction pattern can be identified and the fundamental periodicity determined. This approach is more pertinent than measuring the position of the first order of diffraction since the error in measurement is reduced.

The advantage of X-ray diffraction is that the intensity terms relating to the structural repeat can be observed in the hydrated state where the fundamental axial periodicity (D) is 67 nm. Up to 140 meridional diffraction peaks have been observed in the hydrated state, showing the high degree of crystallinity along a fibril axis [7]. The use of X-ray diffraction has allowed the molecular packing structure of collagen to be relatively well defined as a microfibrillar structure that contains specific molecular kinks [8].

The conformation of collagen molecular packing in the dehydrated state has been studied to a lesser extent than the fully hydrated tissue. However, it could be said that information from electron microscopy is effectively an examination in the dehydrated state [9]. The main effect of drying has been the observation that the axial repeat decreases from 67 nm to the region of 64 nm. The parameters of the collagen helix were in part determined by the use of X-ray diffraction of stretched dehydrated tendon samples [10]. However, the molecular packing in the dehydrated state is less well resolved. Conventional X-ray diffraction has the advantage that the diffraction data correspond to several thousand fibrils being observed at any one time, changes in the diffraction data therefore are inherently statistically significant in comparison to examining single fibrils.

The dehydrothermal treatment of collagen (typically 120°C dry heat for up to 24 h) marks a progression from the air dried state of fibrillar collagen. In this treatment, more water is driven off from the collagen molecules and also there are changes induced in the character of amino acids of the collagen chain that may result from oxidative damage or crosslinking [11]. Dehydrothermal treatment has been used to examine the changes in the strength and solubility of collagen-based products for the biopharmaceutical industry, and also as a means of developing regimens for accelerated ageing. One application of this is in the examination of the ageing of historic parchment manuscripts, where oxidative damage and molecular cleavage may have been occurring over a number of centuries.

The purpose of this study is to examine the changes in molecular packing that occur on the drying and dehydrothermal treatment of collagen. This allows a better understanding of the changes that may occur relating to the axial molecular packing of collagen on drying and subsequent thermal treatment, and also to assess the suitability of dehydrothermal treatment as a suitable technique for accelerated ageing.

Section snippets

Samples

A variety of samples were examined by X-ray diffraction. Rat tail tendon samples of typical diameter 200 μm were dissected from 3-month-old Wistar rats and maintained in a physiological buffered saline. Samples were air dried (for 24 h as isolated tendons at room temperature), some were used for dehydrothermal treatment. Parchment samples of new and ancient parchments, mostly bookbindings from the Royal Library, Denmark, were a gift from Dr. Larson of the Danish School of Conservation. The unaged

X-ray diffraction and collagen structure

X-ray diffraction can provide information about the molecular packing of collagen. The principal changes of interest here are the effects on the axial molecular packing of the collagen molecules in a fibril. The highly regulated molecular organization along a fibril axis ensures that in the native hydrated state, the molecular packing results in a quasi-crystalline lattice structure [13]. The axial projection of the repeating electron density profile corresponds to a series of sharp reflections

Conclusions

The process of dehydration of collagen and subsequent changes in molecular properties on dehydrothermal treatment are complex, and a number of factors may be involved in the alteration of the D period. The principal alteration in molecular packing from the hydrated state is probably the collapse of the gap/overlap and the partial shearing of unit cell contents within the gap region upon loss of water. Dehydrothermal treatment completes this structural phase change by reducing the molecular

Acknowledgements

We wish to thank Kurt Nielsen of the Danish Technical University, Lyngby and Andrew Miller of the University of Stirling for helpful advice. We also wish to thank staff at the CLRC SRS Daresbury beamlines 2.1 and 7.2 for technical support.

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