Aging does not alter tendon mechanical properties during homeostasis, but does impair flexor tendon healing

Abstract

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study, we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3 and 27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3 to 15 months, and 3 to 27 months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31-month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22-month old mice, relative to 4-month old mice. No changes in cell proliferation were observed between young and aged, however, a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2716-2724, 2017.

Keywords: aging; biomechanics; tendon; tendon healing; tenocytes.

Figure 1. Male and female uninjured tendons from multiple sites display few alterations in mechanical properties with age

FCU, FDU and tail fascicles were mechanically tested at 3, 6, 15 and 27 months (22-27 months for male FCU) to identify age-related changes in toe stiffness, linear stiffness, transition extension and maximum load. Apart from an increased FCU toe stiffness at 3 months (p=0.027 versus 15 months and p=0.012 versus 27 months) and an increased linear stiffness at 3 months (p=0.0013 versus 9 months, p=0.012 versus 15 months and p=0.011 versus 27 months) in female mice, no age-associated changes in mechanical properties were found. (*) indicates p<0.05.

Figure 2. FDL tendons exhibit microstructural changes in composition without a change in cellular proliferation with age

Yellow arrows indicate an observable collagen fiber re-alignment (perpendicular ‘score’ marks in the tendon) in the aged tendon seen with Alcian Blue/ Hematoxylin/ Orange G staining in 1D, and a change in composition with picrosirius red staining in 1E. No proliferating cells are seen with PCNA staining within the tendon body in young or aged FDL tendons (1C, F).

Figure 3. Aged tendons display reduced mechanical properties following injury

At 14 days post-surgery, max load, stiffness, and yield load were significantly decreased in the aged FDL tendon (3A, B, C) (p=0.036, p=0.048, and p = 0.044 respectively). The tested values for energy to max were not significantly different between young and aged tendon repairs, despite a slight observable decrease in the aged tendon (3D) (p=0.1).

Figure 4. Aged tendons exhibit decreased granulation tissue following injury

At D14 after surgical transection and repair, aged FDL noticeably deposit a reduced amount of granualation tissue surrounding the site of injury following ABHOG staining (dotted light blue line and arrows). Tendons are outlined in black and marked with a “T”.

Figure 5. Aged tendons heal with thinner, more disorganized collagen fibers bridging the ends

Picrosirius red staining reveals robust collagen deposition between tendon ends in the aged mice, as compared to the thinner fibers seen bridging the aged tendons (white arrows). Native tendon is outlined in red and marked with a “T”.

Figure 6. Comparable levels of proliferating cells are seen in the granulation tissue in young vs. aged FDL repairs

PCNA staining done at D14 following surgy on the FDL in young and aged mice does not indicate an appreciable difference in cellular proliferation surrounding the injury site at this time point. Granulation tissue is denoted by the pink dotted line, and native tendons with blue and marked by a “T”.