Injury response of geriatric mouse patellar tendons

Abstract

Injury adversely impacts the structure and mechanical properties of a tendon, thus causing pain and disability. Previously, we demonstrated that patellar tendons in mature (P150) and aged (P300) mice do not recover original functionality, even 6 weeks after injury, and that uninjured geriatric tendons (P570) are functionally inferior to uninjured mature tendons. In this study, we hypothesized that the repair response in injured geriatric mice would be further compromised, thus undermining patellar tendon function post-injury. Patellar tendons from wild-type mice were injured at 540 days. At 3 and 6 weeks post-surgery, structural, mechanical, and biochemical analyses were performed and compared to uninjured controls. Mechanical properties of geriatric tendons failed to improve after injury. When compared to mature and aged tendons post-injury, it was determined that at no age was there a suitable repair response. In previous studies, we were able to associate the absence of SLRPs with phenotypic changes both early and late in repair. Here we found that SLRPs were significantly decreased after injury, thus offering a possible explanation for why geriatric tendons were unable to mount an adequate repair response. Thus, we conclude that regardless of age after maturity, tendon healing ultimately results in a substandard outcome. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1256-1263, 2016.

Keywords: aging; injury; small leucine rich repeat proteoglycans; tendon.

Fig. 1. Gene Expression of SLRPs in Geratric Tendons after Injury

(A) Expression of Bgn trended downward at 3 weeks and 6 weeks post-injury, relative to an uninjured state, with a significant decrease seen between 3 and 6 weeks post-injury. Expression of Dcn (B) and Fmod (C) significantly decreased post-injury when comparing uninjured to 3 weeks and 6 weeks, though a slight but significant rebound was seen between 3 and 6 weeks for both SLRPs. (D) There was no change in Lum expression across injury state. Box and whisker plots give minimum and maximum range, median (vertical bar), and first and third quartiles for each group. Significance was determined by Mann-Whitney Wilcoxon Test, two-tailed with levels of significance described as a combination of a (uninjured vs. 3 weeks), b (uninjured vs. 6 weeks), or c (3 weeks vs. 6 weeks), and * (p < 0.05) and # (0.05 < p < 0.10).

Fig. 2. Biomechanical Properties of Geriatric Tendons after Injury

Dynamic modulus (A) and viscoelasticity, tangent of the phase angle (B), were examined between uninjured and injured states in geriatric patellar tendons. In performing biomechanical analyses, cross-sectional area of the tendons were also measured (C). (A) At strains of 4% and 8%, significant decreases in dynamic modulus (|E*|) were noted when comparing uninjured P570 tendons to tendons 3 weeks post-injury; in contrast, significant increases in dynamic modulus (|E*|) were noted when comparing geriatric tendons at 3 weeks and 6 weeks post-injury. Dynamic modulus did not differ between uninjured tendons and those 6 weeks post-injury. (B) At strains of 4% and 8%, significant increases in viscoelasticity (tanδ) were noted when comparing uninjured P570 tendons to tendons 3 weeks post-injury and significant decreases were noted when comparing geriatric tendons at 3 weeks and 6 weeks post-injury; however, there were no differences in viscoelasticity between uninjured tendons and those 6 weeks post-injury. (C) Cross-sectional areas of geriatric tendons increased significantly 3 weeks post-injury, but by 6 weeks post-injury mean cross-sectional area is only slightly increased, relative to uninjured geriatric tendon. Measurements depicted are means ± standard deviations. Significance is described as a combination of a (uninjured vs. 3 weeks), b (uninjured vs. 6 weeks), or c (3 weeks vs. 6 weeks), and * (p < 0.05/2) and # (0.05/2 < p <0.10/2).

Fig. 3. Comparative Analysis of Biomechanical Properties Across Age and Injury State

Dynamic modulus (A-C), viscoelasticity (D-F), and cross-sectional areas (G-I) were compared for mature (P150), aged (P300), and geriatric (P570) tendons at the uninjured, 3 weeks post-injury, and 6 weeks post-injury states. Strains are reported and 4% and 8% at 1 Hz frequency. (A) At 4% strain level, dynamic modulus |E*| of the patellar tendon decreased significantly between P150 to P300 and again between P300 to P570. (D) Viscoelasticity (tanδ) increased significantly between P150 to P300 and again between P300 to P570. Results were similar for 8% strains (A, D). At 3 weeks post-injury, |E*| is comparatively greater and tanδ is relatively lower in aged patellar tendons, relative to mature and geriatric tendons (B, E). However, relative to the uninjured states, respectively, |E*| is comparatively lower and tanδ is higher at all ages. By 6 weeks post-injury, all healing patellar tendons regardless of age ultimately demonstrate no difference for either |E*| (C) or tanδ (F). Mean cross-sectional area of uninjured patellar tendons is greater for P300 and P570 mice, relative to P150 mice (G); however, at 3 weeks post-injury mean cross-sectional areas are increased relative to uninjured tendons yet nearly equivalent across the age groups (H). At 6 weeks post-injury, mean cross-sectional area of patellar tendons is slightly greater for P300, relative to P150 (I). Measurements depicted are means ± standard deviations. Significance is described as a combination of a (150 days vs. 300 days), b (150 days vs. 570 days), or c (300 days vs. 570 days), and * (p < 0.05/2) and # (0.05/2 < p <0.10/2).

Fig. 4. Geriatric Tendon Fibril Structure Post-Injury

TEM analysis of fibril diameter distribution (n=4–6 per injury state) for uninjured (A), 3 weeks-post injury (B), and 6 week post-injury geriatric (P570) patellar tendons (C). After injury of geriatric tendons, increased numbers of small diameter fibrils are notable at 3 weeks with even greater numbers of smaller diameter fibrils still persisting at 6 weeks post-injury.