**5. Effects on tissue-level (material) properties**

In our previous studies, we used mathematical methods to estimate tissue-level biomaterial properties. Through calculating long bone's cross-sectional moment of inertia, we normalized load-displacement data to stress and strain. Under such conditions, ERT rats' worse structural material properties disappeared. Additionally, exercise and control groups showed no differences in yield stress, yield toughness or elastic modulus (Young's modulus),55 suggesting that endurance training is not harmful for bone material properties. ERT's benefits on the post-yield biomaterial behaviors seemed to be more size-independent and associated with tissue-level (e.g. bone matrix, collagen) changes. Because measuring post-yield mechanical properties using beam bending theory is only valid in the pre-yield regime,75 reporting post-yield stress, strain or toughness is inappropriate. Therefore, we discussed this tissue-level adaptation base on the post-yield parameters measured from load-displacement data. As shown in our two recent studies, either moderate ERT or endurance swimming training benefits bone tissue more in terms of energy absorption capacity,55,77 especially in post-yield energy. Similar results of enhanced post-yield behavior were provided by another ERT animal study, which showed a short-term treadmill running (21 days) enhanced tibia post-yield deformation in mice.78 Moreover, such effects on postyield behavior changes seem to apply not only to endurance training. After a short-term (5 days) freefall landing exercise, Wistar rats revealed an increased post-yield energy absorption in ulnae.79 Such an enhanced absorption capability is more likely due to tissuelevel (e.g. bone matrix, collagen orientation etc) changes rather than structural adaptation. As mentioned in previous studies, tissue-level properties can be divided into the inorganic mineral phase (e.g., hydroxyapatite), which determines tissue stiffness and strength, 80, 81

and the organic bone matrix, which plays a key role in energy absorption, 82, 83 It has been suggested that the networks of collagen, one of the major components of bone matrix, could affect the energy dissipation between the yield point and fracture point in bone tissue.84-86

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Collagen fiber orientation (CFO) has been measured by circularly polarized light microscopes as one parameter to represent the collagen network and to predict post-yield energy of bone tissue.84 Hence, the post-yield behavior revealed by ERT rats' bone tissue could partially stem from a highly organized collagen fiber network. Though the information regarding collagen orientation in the present study is not available in our previous rodent studies,55,77 it has been reported that dogs after one-year of intensive endurance running (40km/day) revealed a higher organization of collagen fibers in bone tissues.87 Such highly organized collagen fibers seemed to be able to compensate for the 10% BMD decrease in running dogs. Thus, a highly organized CFO would be expected in rodent studies. As mentioned above, rats subjected to short-term freefall landing exercise (5 days, 10 or 30 times per day) from a height of 40cm also showed enhanced post-yield energy of ulnae.79 In that study, authors tried to measure the CFO of cross-sectional ulnae. Unfortunately, no difference in CFO between exercise and control groups was found. One major reason for this lack of significant results could be species difference. That is, CFO analysis might not be sensitive enough to detect biomaterial differences in rodents. To date, CFO-related analysis in bone tissue specimens have all been obtained from big mammals,88, 89 which have more mature Haversian's systems and visible osteons. However, in smaller mammals (e.g. young adult rodents), it is not possible to find Haversian's systems or complete osteon.90, 91 Per our observation, the organization of collagen fiber tends to be relatively irregular in rats and, thus, CFO analysis might not be sensitive enough to predict post-yield material properties.

On the other hand, cross-links within collagen networks might be another contributor to changes in tissue post-yield behaviors.92 In an exercise-related study, Kohn and colleagues verified that cortical toughness enhanced by a 21-day ERT could be correlated with the overall maturity of collagen cross-links.93

In addition to individually measure CFO or cross-links, the biomaterial properties (e.g. tissue strength or tissue post-yield behaviors) might benefit from better integration between collagen and its crosslinks. Related measurement methods are awaited and are worthy of further study.

Finally, microdamage is another factor influencing tissue's post-yield behavior. Accumulation of microdamages (or microcracks) would lead to a fragile bone with lower capability in post-yield energy dissipation.84, 94 However, such accumulated microdamages seemed to be more related to aging. Also, as in CFO-related studies, microdamage studies have been more frequently done in big mammals. Whether microdamage measurement can be performed on exercise-related rodent study needs further verification.
