Effects of collagen digestion have been defined up to the fibril level. be used as a simple and reliable model of mechanically altered tissue samples. mechanical testing apparatus. Slack length of the muscle strips was defined as the length at which the muscle strips experienced 5 mN of tensile force. After determining their slack lengths, the samples were submitted to cyclic loading. Initially, samples were preloaded up to 120 % of their slack length. Once preloaded, samples were cycled at a rate of 1 1 mm/s and an amplitude of 20% of their slack buy 93-35-6 length (Figure 2). Samples were submitted to 10 pre-conditioning load cycles. Afterwards, samples were allowed to rest for five minutes prior to the actual cyclic testing. During each subsequent test, samples were submitted to 5 loading cycles. Figure 2 The cyclic loading process. The force versus time plot (top), shows the five loading cycles. A sample profile of the displacement of the tendon versus time is seen in the bottom figure. Samples were preloaded to 120% slack length (Ls) and cycled at amplitude … All samples were submitted to 4 progressive rounds of cyclic testing with 5 minute breaks between each round. Each round contained 5 cyclic loading/unloading tests with the displacement amplitude and displacement rate the same as the preconditioning cycles. During the five minute break, 4 muscle samples were soaked in solutions containing 0.25%, 0.125%, 0.0625%, and 0% type II clostridium histolyticum, respectively, derived bacterial collagenase (GIBCO, Invitrogen Corporation), in Rees-Simpson solution. As tensile strain is known to greatly reduce skeletal muscles susceptibility to enzymatic digestion, samples were left in a relaxed state during digestion periods14. For each sample, passive stiffness was determined from the slope of the linear portion of the fifth hysteresis buy 93-35-6 curve generated by each cyclic test (Figure 3). The linear portion was defined as the region between the peak force and 25% of the peak force. Slope of the linear region was calculated by means of a linear regression. The samples of the same collagenase concentration were averaged. To normalize the stiffness, each data set was multiplied by the scaling factor necessary to make its maximum data point equal to 1. Figure 3 An example of the force versus displacement curve, as obtained during mechanical testing. Stiffness was calculated using the hysteresis curve corresponding to the fifth cycle of each round of cyclic tests. Stiffness was calculated as the slope in the … One-way-ANOVA test was performed on stiffness values of the control specimens and the specimens digested with HMOX1 various collagenase concentrations at the end of 15 minutes. Tukeys post-hoc buy 93-35-6 test was performed to identify the pair(s) with statistically significant difference. In addition, at each collagenase concentration, linear regression was performed between the normalized stiffness and the digestion time. Histology As a means to verify collagen changes, light microscopy was used to determine the histological effects of the collagen digestion process. Following the mechanical measurement, each sample was fixed in 10% formalin. Once sufficiently bound, samples were imbedded in paraffin. Five 4m thick longitudinal slices were taken from each muscle strip. Slices were fixed to slides and stained using a standard Masson Tri-chrome technique. Slides were imaged at 40x, 100x, and 400x magnification. Collagen networks were identified as the blue stained structures surrounding the red stained muscle fibers. Degree of collagen degradation was assessed on a qualitative basis by the same observer. Results Mechanical testing Regression analysis revealed strong linear relationship (r2=0.97 for 0.0625% concentration; r2=0.98 for 0.125% concentration; r2=0.99 for 0.25% concentration) between normalized stiffness and digestion time at all concentrations (Figure 4). Samples digested in all the three collagenase concentrations exhibited statistically significant reduction in normalized stiffness at the end of the tests (p<0.01) when compared to the control samples. In addition, the difference in normalized stiffness reduction between samples treated with 0.0625% collagenase and those treated with 0.125% collagenase was statistically insignificant (p=0.5). The differences.