Tendon repair is compromised in a high fat diet-induced mouse model of obesity and type 2 diabetes

Abstract

Introduction: The obesity epidemic has resulted in a large increase in type 2 diabetes (T2D). While some secondary complications of T2D are well recognized and their cellular and molecular mechanisms are defined, the impact of T2D on the musculoskeletal system is less understood. Clinical evidence suggests that tendon strength and repair are compromised. Here, a mouse model of obesity and T2D recapitulates the deleterious effects of this condition on tendon repair.

Methods: Male C57BL/6J mice at 5 weeks of age were placed on a high fat (HF)(60% kcal) or low fat (10% kcal) diet for 12 weeks. The flexor digitorum longus (FDL) tendon was then injured by puncturing it with a beveled needle. Progression of FDL tendon healing was assessed through biomechanical and histological analysis at 0, 7, 14 and 28 days post-injury.

Results: HF-fed mice displayed increased body weight and elevated fasting glucose levels, both consistent with T2D. No differences in biomechanical properties of the uninjured FDL tendon were observed after 12 weeks on HF versus lean diets, but decreased maximum force in uninjured tendons from HF-fed mice was observed at 24 weeks. Following puncture injury, tendons from HF-fed mice displayed impaired biomechanical properties at day 28 post injury. In support of defective repair in the HF-fed mice, histological examination of the injury site showed a smaller area of repair and lower cell content in the repair area of HF-fed mice. Insulin receptors were expressed in most cells at the injury site regardless of diet.

Discussion: The HF-diet mouse model of obesity and T2D reproduces the impaired tendon healing that is observed in this patient population. The exact mechanism is unknown, but we hypothesize that a cellular defect, perhaps involving insulin resistance, leads to decreased proliferation or recruitment to the injury site, and ultimately contributes to defective tendon healing.

Figure 1. Murine “Stab Injury” model.

A 23-gauge needle (outlined by blue dotted line) approaches the surface of an exposed and elevated FDL tendon (A) prior to creating the stab injury defect (depicted by black oval). Picrosirius red stained longitudinal sections of (B) injured (blue arrow) and (C) uninjured, sham operated FDL tendons are visualized under polarized light (5× magnification). Note that the tissue section in (B) is oriented through the center of the stab injury. Maximum force (D), work to maximum force (E) and stiffness (F) were measured in isolated FDL tendons immediately following a puncture injury produced by various needle gauge sizes. Results are represented as mean ± SEM (n = 7–9). *** p<0.001, ** p<0.01 and * p<0.05.

Figure 2. Long-term effects of high fat diet on biomechanical properties of FDL tendon in the absence of injury.

Five-week-old C57BL/6 male mice were placed on HF (60% kcal from fat) or lean control diets (10% kcal from fat) for 12 weeks. After 12 weeks on diet, half of the HF-fed mice were switched to the lean diet and continued on this diet for an additional 12 weeks along with comparable numbers of mice continuously fed the lean diet. Body weight (A) and fasting glucose levels (B) were determined in all groups at the 12 and 24 week time points. Maximum force (C,F), work to maximum force (D,G) and stiffness (E,H) were measured in FDL tendons harvested at 12 weeks (C,D,E) and 24 weeks (F,G,H) from each of the diet groups. Results are represented as mean ± SEM (n = 7 at 12 weeks and n = 5–6 at 24 weeks). *** p<0.001 and * p<0.05.

Figure 3. Effect of diet on biomechanical properties of the FDL tendon during injury repair.

Five-week-old C57BL/6 male mice were placed on HF (60% kcal from fat) or lean control diets (10% kcal from fat) for 12 weeks. Following stab injury, mice were maintained on their respective diets until sacrifice. FDL tendons were harvested at the time points indicated and biomechanical properties determined. Maximum force (A, D), work to maximum force (B, E) and stiffness (C, F) were measured in injured (A,B,C) and contralateral control tendons (D,E,F). Values for injured tendons were normalized to the respective contralateral tendons at 7,14 and 28 days. Results are represented as mean ± SEM (n = 6–8). Body weight (H) and fasting blood glucose (I) were measured at the time of injury and again at tissue harvesting. Results are represented as mean ± SEM (n = 9–10). *** p<0.001, ** p<0.01 and * p<0.05.

Figure 4. Effect of high fat diet on tissue repair, cellular recruitment and collagen organization/fiber alignment during the FDL tendon repair process.

Mice were placed on HF or lean control diets for 12 weeks. Following stab injury, mice were maintained on their respective diets until sacrifice. FDL tendons were harvested at 7 day intervals for 28 days and processed for histologic analysis. Longitudinal sections of injured FDL tendons at day 14 from mice on lean (A) or HF (B) diets were stained with hematoxylin-eosin (5× magnification). Area of repair tissue (increased cellularity) was quantitated as a fraction of total tendon area (C). The number of cells per unit area of repair tissue was determined (D). Longitudinal sections of uninjured (E,F) and injured FDL (G–H) tendons at day 28 were stained with picrosirius red and visualized under polarized light (20× magnification). Brighter color intensities indicate organized and aligned collagen fibers while zones of disorganized or absent collagen appear dark. Results are represented as mean ± SEM (n = 2–4). * p<0.05.

Figure 5. Insulin receptors are expressed by cells involved in the FDL tendon repair process.

Longitudinal sections of FDL tendons from lean (A) and HF (B) diet-fed mice at day 28 post-injury were stained for insulin receptors (20× magnification). Strong positive staining (brown) was present in the majority of cells in the repair zone regardless of dietary treatment.