Biomechanical, Histologic, and Molecular Evaluation of Tendon Healing in a New Murine Model of Rotator Cuff Repair

Abstract

Purpose: To develop a clinically relevant, robust murine model of rotator cuff tendon repair to examine cellular and molecular mechanisms of healing.

Methods: Sixty C57BL/6 male mice underwent rotator cuff transection and repair using microsurgical techniques. A modified Kessler suturing technique was used prior to tendon detachment. Sutures were passed through 2 intersecting bone tunnels that were made at the tendon attachment site. Mice were sacrificed at 2 and 4 weeks with subsequent biomechanical, histologic, micro-CT, and gene expression evaluations.

Results: Failure forces in the 2- and 4-week groups were 36% and 75% of the intact tendon, respectively. Histologic evaluation revealed complete reattachment of the tendon with no observable gap. Healing occurred by formation of fibrovascular tissue at the tendon-bone interface, similar to larger animal models. Molecular analysis revealed gene expression consistent with gradual healing of the reattached tendon over a period of 4 weeks. Comparisons were made using 1-way analysis of variance.

Conclusions: This model is distinguished by use of microsurgical suturing techniques, which provides a robust, reproducible, and economic animal model to study various aspects of rotator cuff pathology.

Clinical relevance: Improvement of clinical outcomes of rotator cuff pathology requires in-depth understanding of the underlying cellular and molecular mechanisms of healing. This study presents a robust murine model of supraspinatus repair to serve as a standard research tool for basic and translational investigations into signaling pathways, gene expression, and the effect of biologic augmentation approaches.

Fig 1.

Animal allocation.

Fig 2.

Surgical technique of mouse supraspinatus tendon (SST) detachment and repair. (A) An 8-mm incision is made on the lateral aspect of the shoulder. (B) The deltoid muscle is identified with the overlying vasculature. (C and D) The acromioclavicular joint is elevated and the deltoid muscle is minimally dissected to expose the rotator cuff tendons. (E) Using an angled micro Adson forceps, space is developed under the SST. The Adson forceps is kept under the tendon to provide the space required for suturing. (F) A double-needled 6/0 Prolene is used to fashion a modified Kessler suture starting at the midsubstance of the tendon with one needle and completing the configuration with the other needle. (G) With mild traction applied to the sutures, the tendon is then detached from the humerus with a No. 15 blade. (H) Two crossing bone tunnels are drilled using a 30-G needle. The first tunnel is drilled from the anterior extent of the footprint in a posteroinferior direction, and the second tunnel is drilled from the posterior extent of the footprint in an anteroinferior direction. These tunnels exit the humerus around the surgical neck. (I) Needles are then passed through their corresponding tunnels and the sutures are tightened. (SST, supraspinatus tendon.)

Fig 3.

Setup for biomechanical testing in the materials testing system (MTS). The immobilized humerus and the insertion site of the supraspinatus tendon (arrow) are visualized at the center of this image. The load cell is to the right. The computerized image display is off-screen.

Fig 4.

Coronal sections of mouse shoulder specimens at 2 weeks (A and B) and 4 weeks (C and D) following supraspinatus tendon repair, revealed by H&E (A and C) and Safranin O (B and D) histologic stains. The black arrow points to the healing supraspinatus tendon enthesis. Black arrowheads refer to the location of the intersecting transosseous sutures used to achieve tenodesis. Safranin O staining suggests the presence of proteoglycan at the insertion site (B; inset). Magnification is as shown.

Fig 5.

(A) Volume of interest was defined as a hemicylinder incorporating half of the suture tunnel to avoid cortical bone as much as possible. (B) 3D reconstruction of the humeral head in a mouse from 2-week group, with the posterior tunnel in vertical position. Arrows show the position of the bone tunnels.

Fig 6.

Gene expression analysis of enthesis tissue following rotator cuff repair surgery versus native tendon control for 7 genes of interest. Gene expression is reported relative to GAPDH reference. (*P < .05; **P < .01).