Intraarticular injection of relaxin-2 alleviates shoulder arthrofibrosis

Abstract

Arthrofibrosis is a prevalent condition affecting greater than 5% of the general population and leads to a painful decrease in joint range of motion (ROM) and loss of independence due to pathologic accumulation of periarticular scar tissue. Current treatment options are limited in effectiveness and do not address the underlying cause of the condition: accumulation of fibrotic collagenous tissue. Herein, the naturally occurring peptide hormone relaxin-2 is administered for the treatment of adhesive capsulitis (frozen shoulder) and to restore glenohumeral ROM in shoulder arthrofibrosis. Recombinant human relaxin-2 down-regulates type I collagen and α smooth muscle actin production and increases intracellular cAMP concentration in human fibroblast-like synoviocytes, consistent with a mechanism of extracellular matrix degradation and remodeling. Pharmacokinetic profiling of a bolus administration into the glenohumeral joint space reveals the brief systemic and intraarticular (IA) half-lives of relaxin-2: 0.96 h and 0.62 h, respectively. Furthermore, using an established, immobilization murine model of shoulder arthrofibrosis, multiple IA injections of human relaxin-2 significantly improve ROM, returning it to baseline measurements collected before limb immobilization. This is in contrast to single IA (sIA) or multiple i.v. (mIV) injections of relaxin-2 with which the ROM remains constrained. The histological hallmarks of contracture (e.g., fibrotic adhesions and reduced joint space) are absent in the animals treated with multiple IA injections of relaxin-2 compared with the untreated control and the sIA- and mIV-treated animals. As these findings show, local delivery of relaxin-2 is an innovative treatment of shoulder arthrofibrosis.

Keywords: arthrofibrosis; fibrosis; frozen shoulder; relaxin.

Fig. 1.

In vitro biochemical investigation of relaxin-2 in synoviocytes. (A) Western blot (Inset) analysis reveals that collagen I expression decreases to 25% with 100 ng/mL of relaxin-2 in the presence of TGF-β relative to a GAPDH loading control. (B) Relaxin-2 treatment stimulates cAMP production at 100 ng/mL, consistent with the down-regulation in collagen I. (C) Human synoviocytes treated with relaxin show a decrease in myofibroblast differentiation. (I) Untreated synoviocytes (red, membrane; blue, nucleus) show minimal expression of αSMA (green). (II) Synoviocytes treated with 5 ng/mL TGFβ1 show increased myofibroblast differentiation as denoted by increased αSMA expression. (III) Treatment with 100 ng/mL relaxin-2 precludes myofibroblast differentiation in the presence of TGFβ1.

Fig. 2.

Relaxin-2 does not change the healthy architecture of the synovial joint space. Over the course of 24 h, the integrity of the synovial joint space is maintained. (Left) Anti-relaxin-2 staining shows the accumulation of relaxin-2 around the periphery of the synovial joint space without infiltration into the humeral head. After 24 h (Bottom Left), relaxin-2 staining is minimal, indicating that it has been dispersed from the synovial joint. (Middle) The cellular organization and architecture of the relaxin-2 treatment remains healthy. There is no notable increase in cell death. (Right) Safranin-O staining shows that the integrity of the joint is maintained. After 24 h, the margins of GAG and cartilage content remain the same as indicated by the orange surface on the humeral head and the joint capsule.

Fig. 3.

mIA relaxin-2 induces a significant change in total ROM postsurgery. (A) Temporal results of the total ROM are presented as means with a 95% CI. Healthy baseline describes a healthy control, whereas untreated control describes the immobilized control group without any treatment. Day 0 signifies suture removal and the first measurement. Significance is defined at α = 0.05. (B) Temporal results of external ROM. (C) Temporal results of internal ROM. (D) Normalized torque-angle curve of the final measurement; shaded colored regions signify a 95% CI. Negative angles and torques denote external rotation and positive angles denote internal rotation.

Fig. 4.

Coronal histologic sections of the affected humeral head show a reversal of the fibrotic phenotype. Lateral and medial directions correspond to the left and the right of the image, respectively. Colored planes transect the humerus, where the color-coordinated slices were obtained. (A) H&E images are taken at 2.5× magnification. Subsequent H&E images are taken at 10× magnification. (B) Section stained for fibronectin taken at 2.5× magnification. Subsequent fibronectin images are taken at 5× magnification. (I) denotes healthy baseline, (II) denotes untreated control, and (III) denotes mIA relaxin-2–treated group.