Rotator cuff tears (RCTs) are common tendon injuries that can cause chronic pain and severe functional disability. Massive RCTs do not heal spontaneously and, in many cases, result in poor clinical outcomes. Specifically, muscle atrophy and fatty inﬁltration correlate with poor outcomes after surgical repair.1 Fatty infiltration of the rotator cuff is a common phenomenon that can lead to permanent structural alterations within the tendon. It has been suggested that changes in muscle fiber orientation (the pennation angle) can cause mesenchymal stem cells to migrate to the interface between muscle fibers and the region of fatty infiltration of the muscle.2 Understanding the factors involved in muscle degeneration and atrophy, and in fatty inﬁltration, may lead to treatments that improve outcomes for patients with massive RCTs. One proposed treatment involves placing continuous mechanical traction on the ends of the torn tendon.2 Findings from this research have indicated that acute tears that become chronic tears are typified by inelasticity and poor function of the muscle–tendon unit. It is therefore important to develop a method that speeds tendon healing without causing the muscle fiber atrophy and pennation angle changes that lead to fatty atrophy, which appears to be an irreversible structural change.
On the basis of the theory that adding mesenchymal cells may improve tendon healing, investigators have studied use of transcription factors (eg, scleraxis) specific to tendogenesis in the embryonal stage.3,4 Nevertheless, certain transcription factors are associated with formation of fibrocartilage in higher concentrations.4 Moreover, decalcified bone matrix increases cartilage formation when added to the tendon repair site.5 Cartilage formation, however, is associated with poorer functional results.6 Thus, there is a need for a method that facilitates faster tendon healing with higher quality tissue formation and less muscle atrophy.
Chitosan, a linear polysaccharide, is associated with scarless healing of soft tissues and prevention of adhesion formation both intraperitoneally and during tendon healing after surgery.7,8 Chitosan tends to precipitate in physiologic pH, thereby mitigating its potency. Fortunately, a chitosan solution that does not precipitate in physiologic conditions was recently developed.9 The solution’s lack of precipitation, coupled with its in situ gelling, allows it to adhere to the repair site long enough to take effect. These characteristics could allow for intimate contact between gel and tendon, facilitating guided-tissue regeneration and preventing adhesion of the rotator cuff to surrounding tissue. By contrast, other biological agents (eg, platelet-rich plasma) are administered as fluid rather than gel and are therefore more susceptible to diffusing from the repair site, mitigating their effects. Thus, chitosan gel is fairly unique among agents.
In the study reported here, we histologically investigated whether a chitosan gel would help improve healing of rotator cuff tendon (acute supraspinatus) tears in a rat model.
Materials and Methods
Supraspinatus Surgical Model
Forty Wistar rats, each weighing between 300 and 400 g, were used in this study. All procedures were approved by the Institutional Animal Care and Use Committee at Rabin Medical Center in Petah Tikva, Israel. The rats were anesthetized with ketamine 90 mg/kg and xylazine 10 mg/kg, both administered intramuscularly, and anesthesia was prolonged as needed with 2% isoflurane, administered by nose cone. The skin was incised 5 cm along the upper back following the midline of the spine. The resulting skin flaps were retracted and the scapula exposed. Careful blunt dissection allowed visualization of the rotator cuff and the trans-scapular arch. A full-thickness incision of the supraspinatus tendon was then made 2 mm distal to the arch. This procedure was performed on both shoulders. For the right supraspinatus tendon, a bioabsorbable chitosan–hydrochloric acid solution (>70% de-acetylated chitosan, molecular weight of 600 kDa; Heppe Medical Chitosan GmbH, Halle, Germany) was sterilely applied to the ends of the tendon (total volume, 0.5 mL) and automatically gelled in situ by heating to about 37°C (rat’s internal body temperature). The tendon ends were subsequently approximated with a single 4-0 Prolene suture (Ethicon, Somerville, New Jersey). The left shoulder (tendon repaired with suture only) served as a control.
The rats were housed for a maximum of 12 weeks after surgery. They were sacrificed (in groups of 5 each) 2 hours, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 12 weeks after surgery. After each rat was sacrificed, both shoulder girdles were harvested, and the sutures were removed from the supraspinatus tendons.
After routine fixation with 4% formalin for 48 hours and decalcification with 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks, the specimens were sectioned with a microtome blade. Care was taken to ensure the plane of the microtome blade was parallel with the longitudinal plane of the supraspinatus muscle and tendon to allow for evaluation of pennation angle. Hematoxylin-eosin staining and Masson trichrome staining were subsequently performed.