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Reduction Of Tendon Fibrosis Using Galectin-3 Inhibitors
Amanda F. Spielman, BS, Michelle Griffin, MD, PhD, Ashley Titan, MD, Heather E. desJardins-Park, BS, Nicholas Guardino, BS, Jennifer Parker, BS, Derrick Wan, MD, Michael Longaker, MD.
Stanford University, Stanford, CA, USA.

PURPOSE: Healing after tendon injuries results in formation of a fibrotic scar, resulting in reduced tendon strength and frequent reinjury. Galectins have shown to be involved in the inflammation and fibrosis of muscle and tendon injuries. Understanding the role of galectins in tendon fibrosis is limited due to the lack of reproducible murine models that evaluate tendon fibrosis. Our study demonstrates a novel Achilles tendon model that can assess tendon fibrosis. We further identify an antifibrotic agent capable of overcoming tendon fibrosis.
METHODS: Adult C57Bl/6 mice (68-week-old) (n = 6) underwent a 1cm skin incision to expose the Achilles tendon. The Achilles tendon then underwent a 50% rupture followed by abrasion of the ruptured tendon with sandpaper. At 14 days post injury tendons were evaluated by histology to confirm tendon fibrosis. Following the confirmation that the murine model can reliably induce tendon fibrosis, adult C57Bl/6 mice (68-week-old) (n = 6) were randomized to three conditions (Fig.1A) 1. Tendon injury (Control group), 2. Tendon injury with simultaneous treatment with a galectin-3 inhibitor (Treatment group), and 3. Skin incision with no tendon injury (Sham group). At post-operative day 14 (POD14), tendons were analyzed for histological and flow cytometry analysis (FACS). Tendon sections underwent immunofluorescent staining for fibrosis (e.g., collagen type I/col-I, collagen type 3/col-3, and alpha smooth muscle actin/a-SMA), vascularization (CD31 staining), inflammation (F4/80 antigen), and were imaged on a confocal microscope.
RESULTS: Histological analysis by H&E and picrosirius red staining confirmed tendon thickening and collagen deposition in the control group compared to the sham group (*p<0.05), validating the reproducibility of the murine model to induce tendon fibrosis. Immunohistochemistry further confirmed greater levels of col-I, col-III, and α-SMA protein expression in the control group compared to the sham group (P <0.05) (Fig.1B). RT-qPCR further confirmed greater gene expression of col- I, col-III, and α-SMA in the control compared to the sham group (*p<0.05) (Fig.1B). Fibroblasts surrounding the fibrotic tendon illustrated high levels of Galectin-1, 2, and 3 protein expression by immunohistochemistry, with galectin-3 being significantly greater in the Control group compared to the Sham group (*p<0.05). Galectin-3 inhibitor treatment decreased the collagen deposition and scarring observed in the Control group (*p<0.05). The Treatment group also demonstrated reduced tendon fibrosis with decreased col- I, col-III, and a-SMA gene and protein expression compared to the Control group (*p<0.05). Flow cytometry analysis further showed reduced numbers of profibrotic fibroblasts (CD26+) in the Treatment compared to the Control group (*p<0.05). Mice receiving the Galectin-3 inhibitor also demonstrated improved vascularization as shown by CD31 staining and a decreased inflammatory infiltrate evidenced by F4/80 macrophage staining (*p<0.05) following tendon injury.
CONCLUSION: Our study provides a novel tendon fibrosis model that is reproducible and cost effective. Targeting galectins using Galectin-3 inhibitors may be an effective method to reduce the tendon fibrosis following injury.


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