Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue-engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue-engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG-RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6-month recovery, the grafted and contralateral shoulders were removed, imaged using X-ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC-RCs reached 31% of the native tendon tangent modulus, which was a modest, non-significant, 11% increase over that of the suture-only repairs. However, the histological analysis showed the regeneration of a native-like enthesis in the ETG-RC-repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289-299, 2018.

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Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle resulting in damage that overwhelms the body's capacity for self‐repair, leading to functional impairment. Thus, the need for technologies that promote regeneration of skeletal muscle fibers to integrate with remaining muscle architecture is imperative. Our lab has developed scaffoldless, tissue‐engineered skeletal muscle units (SMUs) for VML treatment in sheep. Additionally, in order to ensure proper innervation of the SMU myofibers, we developed an engineered neural conduit (ENC) to bridge the SMU and the damaged nerve. This study aims to develop a fabrication method for SMUs and ENCs that restore function following an acute VML injury in a more clinically relevant, load‐bearing model in sheep. Ovine bone marrow stromal cells (BMSCs) were harvested and used to fabricate ENCs. Before 3‐D formation, silicone tubing was pinned in the tissue culture dish to allow the delaminating monolayer to roll around the tubing, creating a lumen (Fig 1A.). For SMU fabrication, semimembranosus muscle was harvested from female lambs and the cell isolation mixture was plated onto tissue culture plastic. When elongating myotubes began to form a network, the plates were shifted to differentiation media until spontaneous delamination of the monolayer occurred. The monolayers were then pinned into 3‐D cylindrical constructs and 2–3 single constructs were placed side‐by‐side and allowed to fuse (Fig 1C.). 2–3 fused SMUs were then sutured together, just prior to implantation (Fig. 1B). A subset of SMUs, fabricated on 60mm dishes, were used to measure contractile and structural properties. All animal procedures were conducted in accordance with The Guide for Care and Use of Laboratory Animals. The biomechanical properties of the SMUs indicated that on average the isometric tetanic force was 657 ± 667 μN. Structural maturation of the constructs was evaluated histologically with H&E, myosin heavy chain, and laminin. Images of developing monolayers were taken 10 days after initial plating and showed a highly aligned and dense myotube network without fibroblast overgrowth. Immunohistochemistry for myosin heavy chain (MF‐20) and laminin showed that the construct is largely composed of aligned muscle. Picrosirius red staining revealed that the ENC's are mostly composed of collagen. We evaluated the development, structure, and function of our SMUs and ENCs throughout the fabrication process. We were successfully able to fabricate and implant 60 SMUs, all 13cm long and 5–10mm in diameter. The biomechanical data showed that we were able to consistently fabricate constructs that met our release criteria for force production. It is important to note that improper alignment of the fibers along the longitudinal axis may have reduced potential force production of the constructs and account for the variability in force data. Histology revealed that the overall structure of the constructs is linear and that the core is not necrotic, indicated by the presence of positively stained‐DAPI...

Anterior cruciate ligament (ACL) tears are a major orthopedic challenge. Based on an incidence of 200,000 ACL repairs per year in the United States, the associated cost is in excess of $4 billion annually. Current ACL reconstruction commonly involves the use of allogenic or autogenic tendon grafts. Outcomes for ACL reconstruction with these techniques are limited by graft availability, donor site morbidity, disease transmission, and incomplete integration of native tissue within the surgically created bone tunnel. Thus, there is a demand for alternative tissue sources for ACL reconstruction. Using human derived mesenchymal stem cells (hMSC) as a starting material, we have successfully fabricated scaffold‐less ligament grafts that form their own extracellular matrix and have used these grafts for ACL reconstruction in a sheep model. To better understand the mechanism of graft development during our fabrication process, hMSCs were cultured and harvested at: Day 0 (initial plating), Day 4 (growth phase), Day 8 (differentiation phase), and Day 12 (3‐D construct) for qPCR analysis of bone and ligament markers. At day 15, 3‐D constructs were tested for biomechanics and histology (Fig. 1). The biomechanics data indicated two significantly different cohorts (n>3 per donor per time point) that could be explained by age (young vs. old). In the young cohort (23–25y), the mean tangent modulus of the engineered ligament was significantly stiffer at 19.0 ± 3.2 kPa compared to the old cohort (33–43 y) at 8.7 ± 1.01 kPa. Average gene expression of ligament markers relative to GAPDH were analyzed via qPCR at defined time points during fabrication. We used a combination of gene targets (Collagen 1, Collagen 3, Tenascin C, and Scleraxis) to confirm commitment of the MSCs toward a ligament lineage and to determine any age‐related differences in gene expression. Compared to undifferentiated hMSCs (Day 0), the young cohort showed significantly increased expression of ligament markers Col‐1 & Ten‐C by Day 4, and Col‐3 by Day 8. The old cohort showed an upward trend in the expression of Col‐1 and TenC, but did not reach significance at any time point, compared to age‐matched and undifferentiated hMSCs. Lastly, both the old and young cohort peaked in expression levels of Scleraxis at day 4, followed by a decline (Fig. 2). Additionally, we assessed the presence of collagen in the fully formed constructs with Picrosirius red staining. The images confirm that collagen was present in the constructs at 72 hours following monolayer roll up and there appears to be, qualitatively, less collagen presence in the construct cross sections fabricated from the cells or the 43 y.o donor compared to the constructs fabricated using cells from the 22 y.o donor. In addition, there is an increase in CSA surface area of dense collagen bands in the constructs derived from the cells of young donors compared to the constructs from the older cohort. Based on these data, we conclude that the use of younger donor cells will yield a stiffer and more robust ligame...

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