Fig. 2: Collagen damage is evident in results from biochemical, fracture toughness, and mechanical tests.

Based on the trypsin-hydroxyproline assay, two general categories of collagen damage were defined. The two groups were undamaged collagen (UC), including samples from RT, 100 °C, and 130 °C, and damaged collagen (DC), including the 160 °C and 190 °C samples. All bar-chart data are represented by mean ± standard deviation. a The trypsin-hydroxyproline assay (N = 8–11/group), which measures the percent of damaged collagen, shows an increase in collagen damage with increases in heat-treatment temperature. Very little collagen damage was present in RT, 100 °C, and 130 °C groups. The 160 °C and 190 °C groups possessed much more damage than the lower-temperature groups. A Tukey multiple comparison tests showed that all groups were significantly different from one another. b The crack-resistance curves (R-curves) from the in situ SRμCT toughness test (N = 2/group) shows a marked decrease in toughness from the UC groups to the DC groups. c Flexural strength tests (N = 6–8/group) were performed to obtain ultimate strain, work to fracture, and Young’s modulus. The ultimate strain was reduced from the UC groups to the DC groups. A Tukey test revealed that all temperatures were significantly different from each other except the RT and the 130 °C. d Work-to-fracture was greatly reduced from the UC groups to the DC groups. Tukey test showed that all groups were significantly different from each other except the RT and 100 °C, and the 130 °C and 160 °C. e Young’s modulus is largely unaffected by heat treatment; there was no significant difference between the UC and DC groups. A Tukey test revealed that the groups that were significantly different from each other include the RT and 100 °C, and the RT and 130 °C.