166 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2.160 '-' 2.150 w 2.140 LLI • 2.13o 2.1 1 2 5 4 5 6 7 8 TIME (Days) Figure 1. Creep behavior of a denatured tendon from a 26-month-old rat presented on a linear time scale. 2,160 2,150 u- ,,, 2,140 2.130 2.1200 ,1 .3 .4 .5 .6 .7 LOG TIME (Days) Figure 2. Data from Figure 1 plotted on a logarithmic time scale.

CROSSLINKING IN COLLAGEN 167 To further test the conclusion that the samples were, in fact, very lightly or non-uni- formly crosslinked, it was decided to induce additional crosslinks into the network synthetically to see if equilibrium behavior might be observed under these circum- stances. Treating tendons directly after denaturation with a 0.1% parabenzoquinone in water at room temperature for various amounts of time ranging from 5 minutes to several hours has been reported to incorporate crosslinking (24). The samples prepared in this way had considerably differing crosslink densities, depending on crosslinking times (21). After crosslinking, the samples were washed and then dried overnight in a desiccator prior to swelling. All experiments were carried out as described previously. In Figure 3, the creep behavior of a tendon taken from a 3-month-old rat and cross- linked for 60 minutes in a 0.1% parabenzoquinone solution is plotted. Here once again, we have plotted the behavior appropriately as log time versus length. It is clear that this sample with synthetically induced crosslinks attains an equilibrium length quickly. These experiments were repeated for tendons taken from rats ages 6, 12, and 18 months. In each of these samples, it was found that the non-crosslinked samples did not reach an equilibrium extension length during an experimental time frame of more than 8 days at 40øC, whereas all the synthetically crosslinked tendons tested reached equilibrium extension lengths within 60 minutes. CONCLUSIONS Numerous theories (1-8) have been proposed relating aging and the disease process in connective tissue to crosslinking. Similarly, failure to synthesize viable crosslinks also 1.750 -- 1,740- 1.730-- -,8 -.6 -,4 -,2 0 ,2 ,4 ,6 LOG TIME (Hours) Figure 3. Creep behavior of a denatured tendon from a 3-month-old rat plotted on a logarithmic time scale synthetic crosslinks have been added by treatment with a 0.1% parabenzoquinone solution for 1.0 hr.