j. Soc. Cosmet. Chem., 82, 163-169 (May/June 1987) Crosslinking in coilaDen (rat tail tendon) D. G. KAPLAN, A. B. FURTEK, and J. J. AKLONIS, Department of Chemistry, University of Southern California, Los Angeles, CA 90089 Received October 9, 1986. Synopsis Mechanical creep experiments have been carried out on swollen denatured tail tendons from rats of various ages. Even though covalent crosslinks may be present in these tendons, our results show that there are not enough effective crosslinks to result in a network structure capable of exhibiting equilibrium elastic be- havior in mechanical experiments. Tendons with additional synthetically incorporated crosslinks clearly show such equilibrium behavior. This being the situation, it is doubtful that the natural crosslinks in collagenous materials are an important influence on mechanical strength of tendon as has been claimed. INTRODUCTION Crosslinking can cause substantial changes in the physical and chemical properties of polymeric materials. The aging of collagen, a high molecular weight polymer, and subsequent changes in its properties, have frequently been ascribed to variations in the extent of crosslinking (1-8). The high tensile strength of collagenous tissues has also been associated with the presence of these covalent crosslinks (9-11). There have been numerous chemical methods used to study crosslinking in collagen (12-19), and such work has resulted in the identification of several chemical species which bind collagen chains together. However, not all high-strength polymeric materials contain covalent crosslinks. For example, semi-crystalline polymers are known which have mechanical strengths supe- rior to corresponding amorphous polymers even when uncrosslinked. Examples of such materials are polyethylene, polypropylene, and Mylar. In such materials, even moderate crosslinking results in negligible changes in physical properties below the crystal melting temperature. Kevlar, one of the strongest synthetic polymers known, is a crys- talline polymer in which no covalent crosslinks are present. Partial crystallinity is found in naturally occurring polymers also. Since the crystalline phase generally has a higher density than the amorphous phase, a partially crystalline structure will contain density and refractive index fluctuations. The white opalescent character of some collagenous tissues results from the scattering of visible light by the partially crystalline structure of native collagen. Thus, the semicrystalline nature of collagen alone could be the major contributor to its high mechanical strength. Despite the precision of various chemical methods which might be used to quantify the level of crosslinking in collagen, a chemical assessment of the importance of such cross- 163

164 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS links in influencing the mechanical properties of the material could be misleading. This possibility arises since not all crosslinks need be effective in influencing the mechanical properties of a polymer. For example, if crosslinks were found at moderately high con- centration in localized areas of the collagen matrix while they were essentially absent in other areas, the material would likely behave as though it were uncrosslinked in me- chanical experiments while appearing to be crosslinked from a chemical point of view. Thus, the topological positioning of crosslinks as well as their overall concentration are important factors in determining the contributions which crosslinks make to mechan- ical properties of a collagenous material. We have studied methods based on rubber elasticity theory (20) which may be used to assess the number of effective crosslinks in collagenous materials. This theory states that for an isotropic elastic material, the elongation of a stressed sample can be related to the degree of crosslinking. Since rat tail tendon is neither isotropic nor elastic due to its partially crystalline character, we destroyed the crystallinity by denaturation (21) and plasticized (22) the denatured tendon to make it elastic at room temperature. Rubber elasticity theory is based on thermodynamic arguments, and therefore all analyzable data must be time-independent. This paper discusses some surprising results we found when trying to use rubber elasticity theory to characterize rat tail tendon collagen. EXPERIMENTAL Samples of almost pure collagen were obtained from the tails of male white Wistar rats (Mission Labs, Rosemead, CA). The rats ranged in ages from 2 months to over 2 years. Upon removal from the distal end, the samples, which were approximately 10 cm long and . 15 mm in diameter, were washed in excess distilled water and allowed to air dry for 3 hours. They were stored under dry nitrogen and calcium sulfate desiccant at 4øC until use. Storage times did not exceed 3 months. Since our experimental plan involved using rubber elasticity theory, the samples had to behave in a way describable by this equation of state. As mentioned above, natural rat tail tendon, being highly crystalline, is not consistent with this model thus the crys- talline nature of the tendon was eliminated by denaturation. Prior to denaturation, samples selected for uniformity of dimensions and straightness were soaked for 1 hour in distilled water. Samples were then denatured at 70øC for 30 seconds, also in distilled water. This particular set of denaturation conditions was chosen since complete denaturation was necessary and yet the possible effects of hydrol- ysis and excessive swelling by water had to be avoided. In preliminary experiments (21) we observed that these were the mildest conditions which resulted in a totally amor- phous sample as evidenced by visual and X-ray examinations. These samples were then dried overnight in a desiccator. The glass transition temperature of pure collagen is in excess of 200øC, and rather than attempt mechanical experiments above this high temperature where the material would be elastic and presumably obey rubber elasticity theory, it was decided to depress the glass transition temperature through plasticization (22). This is a well-known technique in which the bulk polymer is diluted or swollen with a low molecular weight solvent. The denatured tendons were swollen in glycerol for 24 hours at 60øC. In previous work (23), glycerol proved to be a liquid capable of swelling tendons sufficiently to obtain an