Impact of Smad3 loss of function on scarring and adhesion formation during tendon healing

Abstract

Studies were performed evaluating the role of Smad3, a transcription factor mediating canonical TGF-β signaling, on scarring and adhesion formation using an established flexor digitorum longus (FDL) tendon repair model. In unoperated animals the metatarsophalangeal (MTP) range of motion (ROM) was similar in Smad3(-/-) and wild-type (WT) mice while the basal tensile strength of Smad3(-/-) tendons was significantly (39%) lower than in WT controls. At 14 and 21 days following repair Smad3(-/-) MTP ROM reached approximately 50% of the basal level and was twice that observed in WT tendon repairs, consistent with reduced adhesion formation. Smad3(-/-) and WT maximal tensile repair strength on post-operative day 14 was similar. However, Smad3(-/-) tendon repairs maximal tensile strength on day 21 was 42% lower than observed in matched WT mice, mimicking the relative decrease in strength observed in Smad3(-/-) FDL tendons under basal conditions. Histology showed reduced "healing callus" in Smad3(-/-) tendons while quantitative PCR, in situ hybridization, and immunohistochemistry showed decreased col3a1 and col1a1 and increased MMP9 gene and protein expression in repaired Smad3(-/-) tendons. Thus, Smad3(-/-) mice have reduced collagen and increased MMP9 gene and protein expression and decreased scarring following tendon FDL tendon repair.

Figure 1

Unoperated control tendons and 14 or 21 day post-repair tendons from WT and Smad3^−/− were evaluated for (A) metatarsalphalangeal (MTP) joint flexion range of motion (ROM) at the maximum applied load of 19 grams, (B) Gliding Coefficient, (C) Maximum tensile force, and (D) tensile stiffeness. Results are shown as mean ± SD. *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between WT and Smad3^−/−.

Figure 2

Representative histologic sections of WT and Smad3^−/− unoperated flexor digitorum longus (FDL) tendon and FDL repair tendons on post-operative days 7, 14, and 21. Tendon ends (T) and sutures (↑) are indicated. Sections were stained with alcian blue hematoxylin/orange G eosin and representative images are shown at 100x magnification.

Figure 3

Gene expression of (A) col3a1, (B) col1a1, and (C) MMP9 in WT and Smad3^−/− FDL tendon repair tissue on post-operative days 3, 7, 10 and 14. Total RNA was extracted and from five pooled FDL repair tendons and processed for real-time RT-PCR. Expression was standardized with the internal beta-actin control. Data is presented as the mean fold induction (over WT post-operative day 3 repairs) ± SD. *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between WT and Smad3^−/−.

Figure 4

Gene expression of (A) BMP2, (B) BMP4, (C) BMP6, (D) BMP7 and (E) Smad2 in WT and Smad3^−/− FDL tendon repair tissue on post-operative days 3, 7, 10 and 14. Total RNA was extracted and from five pooled FDL repair tendons and processed for real-time RT-PCR. Expression was standardized with the internal beta-actin control. Data is presented as the mean fold induction (over WT post-operative day 3 repairs) ± SD. *, **, and *** indicate significant differences of p<0.05, p<0.01, and p<0.001, respectively, between WT and Smad3^−/−.

Figure 5

In-situ hybridization of (A) col3a1, (B) col1a1, and (C) MMP9 in FDL repair tendons on post-operative days 3, 7, 10, and 14. Area of the tendon is outlined and tendon ends (T) are indicated. Representative photographs taken at 50x original magnification are shown.

Figure 6

Immunohistochemistry of (A) Type III collagen, (B) Type I collagen, and (C) MMP9 in FDL repair tendons on post-operative days 3, 7, 10, and 14. Tendon ends (T), granulation tissue (G) and sutures (↑) are indicated in representative photomicrographs taken at 200x original magnification.