Basic Science Research into New Treatments for Shoulder Joint Disease ­ My Experience at the University of

June 2018. I worked in the Human Soft Tissue Research Laboratory, which is part of the Department of Orthopedic Surgery at the University of Connecticut. I performed basic research to develop new treatments for shoulder joint disease with Prof. Augustus D. Mazzocca, who has done extensive work in biological and biomechanical orthopedics, especially for shoulder disease. I was fortunate to be able to work alongside seven highly motivated German orthopedic surgeons from the Department of Orthopaedic Sports Medicine, Technical University of Munich. During my 2 years at the University of Connecticut, I performed biological, biomechanical, and clinical studies of shoulder surgery and observed the medical and cultural differences between Japan, the USA, and Germany. In this review, I summarize the biological and biomechanical studies that I performed at the University

Before I began studying in the USA, I was involved in several basic science and clinical studies on rotator cuff disease in Japan. First, we completed an in vivo study of transgenic mice (antioxidant enzyme Sod1-deficient mice) to investigate the mechanism of rotator cuff degeneration, which is one of the reasons for rotator cuff tear. 1) This study found that intracellular oxidative stress induces degeneration of the supraspinatus enthesis, resulting in rotator cuff tears. 1) Second, we analyzed the effect of antioxidant treatment on oxidative stress in the rotator cuff in Sod1-deficient mice, and showed that antioxidant treatment (vitamin C administration) attenuated histologic changes in the supraspinatus entheses induced by Sod1 deficiency. 2) Third, we performed a clinical study that used semi-quantitative imaging with MRI T2 mapping to analyze the healing process after rotator cuff repair, and showed that the T2 value of the repaired tendon was highest 3 months postoperatively and was decreased at 6 and 12 months postoperatively, suggesting that the healing process after rotator cuff repair may continue for at least 12 months postoperatively. These studies on the mechanism of rotator cuff degeneration, antioxidant treatment for rotator cuff degeneration, and the healing process of the repaired rotator cuff stoked my interest in regeneration of the rotator cuff. Therefore, I contacted Prof. Mazzocca to perform basic science research on the regeneration of the rotator cuff in his laboratory through the International Committee of the Japan Shoulder Society. In 2016, 2 years after my first contact with Prof. Mazzocca, I went to the USA and started performing basic science research on shoulder disease.
For 2 years, I performed biological, biomechanical, and clinical studies of orthopedic surgery, especially for shoulder disease. 3)-19) I was fortunate to be able to work alongside seven highly motivated German orthopedic surgeons from the Department of Orthopaedic Sports Medicine, Technical University of Munich (Figure-1). The 2 years that I spent doing research in the USA enabled me to understand the medical and cultural differences between Japan, the USA, and Germany. In this review, I summarize the biological and biomechanical studies that I performed at the University of Connecticut.

Background
Rotator cuff tears are the most common tendon injury in orthopedic patients, and are associated with shoulder pain and dysfunction. Arthroscopic rotator cuff repair (ARCR) is a well-established procedure. However, despite advances in ARCR, recurrent rotator cuff tears remain a major challenge, with re-tears occurring in up to 94% of cases. 20)-23) Hypovascularity and a poor healing environment have been hypothesized to facilitate tear initiation and prevent tendon healing after repair. To reduce re-tear rates, biological augmentation has garnered interest as a means of improving the healing potential of the repaired tendon. Tendon healing has been supported using the application of different growth factors, platelet concentrates, mesenchymal stem cells (MSCs), and scaffolds. However, there is currently no gold standard for the biological augmentation of rotator cuff repair.
Study #1: Examining the potency of subacromial bursal cells as potential augmentation for rotator cuff healing: An in vitro study 12) The purpose of study #1 was to compare the potency of MSCs (proliferation and differentiation capacity) derived from the subacromial bursa versus a bone marrow aspirate (BMA) in patients undergoing ARCR. BMA is a major source of MSCs and one of the most widely used biological augmentations in orthopedic surgery. Subacromial bursa and BMA were harvested arthroscopically from 13 patients undergoing arthroscopic primary rotator cuff repair. Bone marrow was aspirated from the proximal humerus and concentrated using an automated system (Angel System; Arthrex). Subacromial bursa was collected from two sites (over the rotator cuff tendon and muscle) and digested with collagenase to isolate a single cellular fraction. Proliferation, number of colony-forming units, differentiation potential, and gene expression were compared between the cells derived from each specimen. The cells derived from the subacromial bursa by collagenase digestion had significantly better proliferation and differentiation ability compared with cells derived from BMA by automated concentration (Figure-2).
Study #2: Comparison of preparation techniques for isolating subacromial bursa-derived cells as potential augmentation for rotator cuff repair 14) Enzymatic (collagenase) digestion of bursal tissue is a substantial barrier to clinical application, as there is concern that any remaining active collagenase may further damage the injured tissues. In addition, enzymatic protocols require 2 to 3 hours of processing time, making them infeasible in a clinical scenario. The aim of study #2 was to identify an effective, nonenzymatic method of maximizing the yield of subacromial bursa-derived nucleated cells for augmenting rotator cuff repair. Subacromial bursa (minimum 0.2 g) was collected prospectively from over the supraspinatus in seven patients with at least one full-thickness tendon tear who were undergoing arthroscopic primary rotator cuff repair. Samples were analyzed prospectively after being processed using four different methods: (1) mechanical digestion with scissors (chopping), (2) collagenase digestion, (3) mechanical digestion with a tissue homogenizer, and (4) whole tissue with minimal manipulation (Figure-3). Tissue processed using each of the four methods was plated and cultured in low oxygen tension and placed in a humidified incubator for 7 days. Following incubation, cellularity was assessed with a nucleated cell count using a Coulter Counter. Flow cytometry was performed on the non-enzymatic method that demonstrated the greatest cell count to confirm the presence of MSCs. Mechanical isolation of subacromial bursa-derived cells using mechanical digestion (chopping technique) demonstrated a similar nucleated cell count compared with cells obtained using collagenase digestion. Flow cytometry confirmed the presence of MSCs in the subacromial bursa. Cells that were derived from chopping and cultured for flow cytometry revealed the presence of MSC-specific surface markers (CD90, CD105, CD73) and multiple differentiation potentials (osteogenesis, chondrogenesis, and adipogenesis).

Background
The optimal surgical procedure for acromioclavicular (AC) joint dislocations is still under debate, with more than 160 techniques described in the literature. High-grade AC dislocation (> type 3 lesion using the Rockwood classification) comprises ruptures of both the AC ligament complex (ACLC) and coracoclavicular (CC) ligament. Until 5 years ago, the procedures for AC joint dislocation focused on CC stabilization. However, persistent posterior instability of the AC joint has been observed after CC stabilization without AC stabilization, and patients with posterior instability have substantially poorer clinical results.

Study #3: Repair of the entire superior acromioclavicular ligament complex best restores posterior translation and rotational stability 9)
The purpose of study #3 was to evaluate the specific regional contributions to posterior translational and rotational stability of the superior half of the ACLC, which the surgeon can easily access and repair or reconstruct. The superior half of the ACLC was divided into three regions. Region A (0°60°) comprised the anterior 1/3 of the superior half of the ACLC, Region B (60°120°) comprised the superior 1/3 of the superior half of the ACLC, and Region C (120°180°) comprised the posterior 1/3 of the superior half of the ACLC. Fifteen fresh-frozen cadaveric shoulders were used. Biomechanical testing was performed to evaluate the resistance force against passive posterior translation (10 mm) and the resistance torque against passive posterior rotation (20°) under the following four conditions: (1) intact (n = 15), (2) with the ACLC dissected (n = 15), (3) specimens randomly divided into three groups by region of suturing (Region A, B, or C; n = 5 per group), (4) after suturing additional regions (Region A + B (0°120°), Region B + C (60° 180°), or Region A + C (0°60°, 120°180°); n = 5 per group). The results revealed that each segment of the superior ACLC makes different contributions to the posterior translational and rotational stability of the AC joint (Figure-4). Based on these findings, surgical techniques restoring the entire superior ACLC are recommended to address both the posterior translational and rotational stability of the AC joint.

Study #4: Reconstruction of the acromioclavicular ligament complex using a dermal allograft: A biomechanical analysis 13)
Based on the results of study #3, we developed a surgical technique to reconstruct the superior half of the ACLC using a dermal allograft (Figure-5: ACLC patch plus an anatomic CC reconstruction (ACCR)). The purpose of study #4 was to analyze the posterior translational and rotational stability of the AC joint following reconstruction of the superior ACLC using a dermal allograft. Six freshfrozen cadaveric shoulders were used. The resistance force against posterior translation (10 mm) and torque against posterior rotation (20°) were measured. Specimens were first tested with an intact ACLC and CC ligaments. The ACLC and CC ligaments were then transected to simulate a Type III/V AC joint dislocation. Each specimen was then tested under three conditions in the following order: (1) ACLC patch reconstruction alone, (2) ACLC patch with an ACCR using a semitendinosus allograft, and (3) transected ACLC with an ACCR only. The ACLC patch plus ACCR technique achieved a posterior translational and rotational stability closest to the native construct.
Study #5: Posterior rotational and translational stability in acromioclavicular ligament complex reconstruction: A comparative biomechanical analysis in cadaveric specimens 10) The purpose of study #5 was to evaluate the posterior translational and rotational stability of an ACLC reconstruction with a dermal allograft (ACLC patch) compared with three internal suture brace augmentation techniques ( Figure-6: ACLC patch, oblique brace, anterior brace, and x-frame brace). A total of 28 cadaveric shoulders were

Conclusion
I performed biological and biomechanical studies to develop new treatments for shoulder joint disease at the University of Connecticut. Based on these studies performed at the University of Connecticut, I started several clinical studies for shoulder disease at Juntendo University. First, for rotator cuff repair, I returned subacromial bursa tissue on repaired rotator cuff to improve tendon healing and started the randomized controlled trial.
Second, for treatment of AC dislocation, I performed the oblique brace (AC) with CC ligaments repair for acute AC dislocation with posterior instability.