CROSSLINKING IN COLLAGEN 165 elastomer at room temperature furthermore, it has a low enough vapor pressure to eliminate measurable swelling agent loss under our experimental conditions. After swelling, surface glycerol was removed by resting the sample on a piece of filter paper in a desiccator for 1 day. The actual experiment measured sample creep in which the strain under constant stress was recorded. A 2.5-cm straight portion of sample with the most uniform shape was cut from the total tendon and weighed. This piece would be weighed again after the mechanical experiment to check for glycerol loss. For all the mechanical testing re- ported, no change in weight of the swollen sample was detected. Black ink marks of negligible weight which were used as reference points were then made near each end of the sample, which was held in an adjustable screw clamp and hung inside a dry box in preparation for measurement. The dry box was equipped with an internal heater and blower, and dry nitrogen purged the box. With ordinary insulation installed around the box, temperature stability was + 2øC during the course of the experiment. Although various experimental temperatures were used in our thermoelastic studies (21), the ex- periments reported here were all carried out at 40 ø -+ 2øC. All lengths were measured with a cathetometer (-+ .005 cm) placed outside the dry box. Each sample was allowed to equilibrate at the experimental temperature for 30 minutes before appropriate weights were suspended from it. The weight ranged from 0. 1241 gram to 0.4216 gram. Lengths were recorded during stressing. RESULTS AND DISCUSSION The results of a tensile creep experiment carried out at 40øC on a tendon of a 26- month-old rat prepared according to the procedure described above are shown in Figure 1. Here the sample length between bench marks is plotted versus time after the appli- cation of the load. As might be expected, the sample length increases as a function of time and appears to be approaching an equilibrium value at longer times. It is tempting to extrapolate this behavior to an equilibrium extension and use this extrapolated value for analysis via rubber elasticity theory. However, in the study of physical properties of polymers it is well known that such results are often misleading. Since polymer mole- cules are very long, contributions from short-range as well as long-range molecular motions influence observed behavior. Furthermore, short-range molecular motions occur rapidly while longer-range molecular motions may be much slower. Because polymer molecules are so long, these various time scales of relaxation are grossly dif- ferent. In order to accurately access both long-range and short-range relaxation in the same figure, it is always necessary to plot system response as a function of the logarithm of time if any conclusions concerning approaches to equilibrium behavior are to be drawn from experimental data. In view of this fact, the data in Figure 1 have been replotted as length versus log time in Figure 2. Here it is clear that no approach to equilibrium behavior is observed even on an extended time scale involving creep for several days. Thus, any attempt to analyze such data via an equilibrium theory is un- founded. More importantly, however, these observations indicate that the tendon studied, rather than being extensively crosslinked, was so lightly crosslinked that slow relaxation was observed. This tendon was taken from an old rat in which substantial crosslinking would be expected (1). Similar behavior was observed with tendons from animals ranging in age from 2 months to 26 months.

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.