Tendon mineralization is progressive and associated with deterioration of tendon biomechanical properties, and requires BMP-Smad signaling in the mouse Achilles tendon injury model

Abstract

Ectopic tendon mineralization can develop following tendon rupture or trauma surgery. The pathogenesis of ectopic tendon mineralization and its clinical impact have not been fully elucidated yet. In this study, we utilized a mouse Achilles tendon injury model to determine whether ectopic tendon mineralization alters the biomechanical properties of the tendon and whether BMP signaling is involved in this condition. A complete transverse incision was made at the midpoint of the right Achilles tendon in 8-week-old CD1 mice and the gap was left open. Ectopic cartilaginous mass formation was found in the injured tendon by 4weeks post-surgery and ectopic mineralization was detected at 8 to 10weeks post-surgery. Ectopic mineralization grew over time and volume of the mineralized materials of 25-weeks samples was about 2.5 fold bigger than that of 10-weeks samples, indicating that injury-induced ectopic tendon mineralization is progressive. In vitro mechanical testing showed that max force, max stress and mid-substance modulus in the 25-weeks samples were significantly lower than the 10-weeks samples. We observed substantial increases in expression of bone morphogenetic protein family genes in injured tendons 1week post-surgery. Immunohistochemical analysis showed that phosphorylation of both Smad1 and Smad3 was highly increased in injured tendons as early as 1week post-injury and remained high in ectopic chondrogenic lesions 4-weeks post-injury. Treatment with the BMP receptor kinase inhibitor (LDN193189) significantly inhibited injury-induced tendon mineralization. These findings indicate that injury-induced ectopic tendon mineralization is progressive, involves BMP signaling and associated with deterioration of tendon biomechanical properties.

Keywords: Achilles tendon; BMP; Biomechanics; Ectopic mineralization; Injury; Smad.

Figure 1

Tendon mineralization is progressive in mouse Achilles transection model. A and B, The µCT image of injured tendons 10 weeks (A) and 25 weeks (B) post-injury. Arrows indicate mineralized materials. C, Quantification of the volume of mineralized materials (n=10/group). *, P<0.05. Scale bar, 2 mm.

Figure 2

Biomechanical properties, cross sectional area and collagen fiber alignment of uninjured and injured Achilles tendons. Stiffness (A), max force (B), max stress (C), modulus (D), cross sectional area (E) and circular variance of collagen alignment (F) of uninjured tendons (Control) or injured tendons10 weeks and 25 weeks post-injury were measured (n=10/group).

Figure 3

Mineralization and biomechanical properties of the contralateral tendons to the injured Achilles tendons. µCT image (A and B), circular variance of collagen alignment (C), stiffness (D), maximum force (E), maximum stress (F) and modulus (G) of the contralateral tendons to the injured site 10 weeks and 25 weeks post-injury (n=10/group). Scale bar, 2 mm.

Figure 4

Distribution of phospho-Smad1 and -Smad3 in injured Achilles tendons. Images of hematoxylin-eosin (HE, A–G) staining and immunostaining with phospho-Smad1 (p-Smad1, H-N) and -Smad3 (p-Smad3, O–U) of the uninjured (Control, A,B,H,I,O,P) and injured Achilles tendons 2 weeks (2 weeks, C,D,J,K,Q,R) and 4 weeks (4 weeks, E–G, L–N, S–U) post-injury. Images of the mid-substanceO-T; 12.5 mm for G, N and U. G, N and U are magnified images of the boxed areas of F, M and T, respectively.

Figure 5

Inhibition of mineralization by LDN193189 in injured Achilles tendons. LDN193189 (3 mg/kg) or vehicle was daily administrated by intraperitoneal injections from 1 to 3 weeks post-injury (n=10/group). The Achilles tendons were harvested 12 weeks post-injury and subjected to µCT analysis (A) to determine the volume of mineralized materials (B). The sections of tendons were stained with alcian blue and eosin staining (C). The body weight was measured at 2, 4 and 8 weeks post-injury (D) and the tibial length was measured by the X-ray analysis at 12 weeks post-injury (E). *, p<0.05. Scale bars, 2 mm for A; 50 µm for C.