Murine supraspinatus tendon injury model to identify the cellular origins of rotator cuff healing

Abstract

Purpose of this study To elucidate the origin of cell populations that contribute to rotator cuff healing, we developed a mouse surgical model where a full-thickness, central detachment is created in the supraspinatus. Materials and Methods Three different inducible Cre transgenic mice with Ai9-tdTomato reporter expression (PRG4-9, αSMA-9, and AGC-9) were used to label different cell populations in the shoulder. The defect was created surgically in the supraspinatus. The mice were injected with tamoxifen at surgery to label the cells and sacrificed at 1, 2, and 5 weeks post-operatively. Frozen sections were fluorescently imaged then stained with Toluidine Blue and re-imaged. Results Three notable changes were apparent post-operatively. 1) A long thin layer of tissue formed on the bursal side overlying the supraspinatus tendon. 2) The tendon proximal to the defect initially became hypercellular and disorganized. 3) The distal stump at the insertion underwent minimal remodeling. In the uninjured shoulder, tdTomato expression was seen in the tendon midsubstance and paratenon cell on the bursal side in PRG4-9, in paratenon, blood vessels, and periosteum of acromion in SMA-9, and in articular cartilage, unmineralized fibrocartilage of supraspinatus enthesis, and acromioclavicular joint in AGC-9 mice. In the injured PRG4-9 and SMA-9 mice, the healing tissues contained an abundant number of tdTomato+ cells, while minimal contribution of tdTomato+ cells was seen in AGC-9 mice. Conclusions The study supports the importance of the bursal side of the tendon to rotator cuff healing and PRG4 and αSMA may be markers for these progenitor cells.

Amplifying Bone Marrow Progenitors Expressing α‐Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon‐to‐Bone Repair

“…As αSMA is induced in response to injury in a variety of mesenchymal tissues including tendon, periosteum, and bone marrow (Supplementary Fig. S3), we studied two additional injection groups defined as T 0 (injections on 0, 3, and 6 days post‐surgery) and T 7 (injections on 7, 10, and 13 days post‐surgery).…”

“…This mouse model was previously shown to target an amplifying osteoprogenitor population within the growing metaphysis . In addition, it labels quiescent adult progenitors within periosteum, epitenon, skeletal muscle, and dental pulp that activate in response to injury. Unlike growing bone, fewer quiescent mesenchymal progenitors within the adult bone marrow express αSMA compared with the periosteum, therefore there is minimal contribution of these cells to intramembranous bone formation within the medullary cavity following injury .…”

“…In addition to the transient expression by a subset of quiescent and amplifying progenitors in mesenchymal tissues, αSMA is also a common marker of myofibroblasts . The convenience of a unique molecular marker for these cells have led to a common misconception that mesenchymal cells that expresses αSMA must be myofibroblasts .…”

“…This model labels a resident multipotent progenitor within the bone marrow that gives rise to new bone and adipocytes during growth and development. It also labels quiescent progenitor populations in the periosteum, paratenon, skeletal muscle, and dental pulp that activate in response to injury. In addition, αSMA is highly expressed by these cell populations during the early proliferative phase of the repair response.…”

Biologics and stem cell‐based therapies for rotator cuff repair

“…Although the majority of rodent rotator cuff studies have been carried out in rat, the mouse shoulder anatomy was recently shown to be comparable to rat and human. The very small size of the mouse rotator cuff precluded its use in earlier studies; however, novel microsurgical techniques have now been applied to successfully create partial and full detachment injuries with surgical repair, thereby opening new avenues for mechanistic research . Despite the numerous advantages of rat models, limitations include scar‐mediated healing (human tears do not undergo spontaneous healing), quadrupedal gait, and small scale relative to human .…”