Remodeling of murine intrasynovial tendon adhesions following injury: MMP and neotendon gene expression

Abstract

Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real-time RT-PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp-9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp-2 and Mmp-14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf-5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.

Figure 1

Representative histology sections of normal (A) and repair FDL tendon at days 3 (B), 7 (C), 10 (D), 14 (E), 21 (F), 28 (G), and 35 (H) post-repair (4×). Sections were stained with Alcian Blue/Hematoxylin and OrangeG. Of note is the fibroblastic granulation tissue (*) that fills in the repair site between tendon ends (marked as T) (C–F) and is progressively remodeled with increasingly organized collagen fibers (F–H). Sutures are marked with arrows. Scale bars represent 25 µm.

Figure 2

Gene expression of (A) Mmp-9, (B) Scx, (C) Mmp-2, (D) Mmp-14, (E) Gdf-5, and (F) Smad8 in FDL tendon repair tissue over time up to 35 days post-repair. Total RNA was extracted and pooled from five tendon repairs and processed for real-time RT-PCR. Gene expression was standardized with the internal β-actin control and then normalized by the level of expression in day 3 FDL tendon repairs. Data presented as the mean fold induction (over day 3 repairs) ± SD. *p < 0.05 versus day 3 tendon repair.

Figure 3

In situ hybridization of Scx transcripts (Red) in normal tendon (A) and repair FDL tendons at days 3 (B), 7 (C), 10 (D), 14 (E), and 21 (F) (4×). Area of the tendon is outlined, and tendon tissue is marked as (T); granulation tissue is marked as (*). Scale bars represent 25 µm.

Figure 4

MTP joint flexion ROM at the maximum applied load of 19 g of FDL tendon repairs at multiple time points post-repair (mean ± SD). Asterisk indicates significant difference versus day 0 operated tendons (Bonferroni’s multiple comparison, p < 0.05).

Figure 5

Maximum tensile force of the FDL tendon repairs over time up to 63 days post-op, compared to day 0 repairs (mean ± SD). Label (a) indicates significant differences versus day 0; while label (b) indicates significant differences versus day 42. The dotted line represents the Max Tensile Force of un-operated tendon.