Summary StatementThis study explores the therapeutic use of MSCs to enhance ligament healing from an immuno-modulatory perspective. We report improved healing with MSC treatment, but inconsistent effects on inflammatory markers.IntroductionMesenchymal stem cell (MSC) use continues to hold untapped potential as a therapeutic agent because: 1) MSCs have the ability to differentiate into several different connective tissues such as cartilage, bone, muscle and fat (1–3), and 2) MSCs can modulate immune and inflammatory responses that affect healing (4, 5). This paradigm shift from differentiation to immune modulation is being studied for different applications (6). Several studies suggest MSCs decrease inflammation by reducing pro-inflammatory cytokines and changing the macrophage phenotype from M1 (classically-activated) to M2 (alternatively-activated) (7–10). However, their immune-modulatory effects within a healing ligament remain unexplored. MSCs can behave differently depending on the tissue and healing ...

A normal healing response after ligament and tendon rupture results in scar formation and an inferior tissue that fails to emulate its original structure, composition, and function. More regenerative healing (closer to the original) can be obtained through early suppression of inflammatory cells and associated cytokines. Examination of the immune mediated response of mesenchymal stem/stromal cells (MSCs) during healing indicates that MSCs reprogram macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Based on these studies our objective was to treat ligament and tendon injuries with MSCs in order to modulate their inflammatory response. Our initial studies using allogeneic cells demonstrated an in vivo dose dependency of MSCs on ligament healing. Medial collateral ligaments (MCLs) treated with 1 × 106 (low dose) MSCs exhibited less inflammation and a reduced number of M1 macrophages compared to ligaments treated with 4 × 106 (high dose) MSCs. Strength of ligament was also improved with the low dose treatment. We then examined the in vivo effects of MSCs that had been preconditioned to be more anti-inflammatory. Treatment with these preconditioned MSCs was compared with normally processed (unconditioned) MSCs using the rat Achilles tendon and MCL healing models. Pre-conditioned MSCs significantly reduced inflammation by increasing the M2 macrophages and decreasing the M1 macrophages. Most importantly, treatment with pre-conditioned MSCs improved tissue strength to levels comparable to intact tissue. Overall, pre-conditioned MSC-treatment out-performed unconditioned MSCs to improve ligament and tendon healing by stimulating a more robust, paracrine-mediated immunosuppressive response.

Stem cells have shown promise as a therapeutic intervention by enhancing skeletal muscle regeneration following muscle injury. The purpose of this study was to determine the effect of mouse mesenchymal stem cells (MSCs) on muscle function following a single stretch injury in the calf muscle of C57BL/67 mice. A custom isokinetic device was used to induce a single stretch injury. An intramuscular injection of MSCs or saline was administered three days post-injury. Mechanical testing to measure peak isometric joint torque in vivo was done immediately and at seven or 14 days post-injury. Susceptibility to reinjury was assessed in the soleus muscle using an in situ repeated eccentric contraction (ECC) protocol. In vivo isometric torque of the plantar flexors dropped immediately following stretch injury by 50%. Treatment with MSCs attenuated the torque deficit at seven days, while there were no differences in torque deficit between groups at 14 days. In situ ECC testing of the soleus showed a significant specific force drop following injury, with the MSC group demonstrating a protective effect at seven and 14 days. These results demonstrate transient improvement in isometric torque and reduced susceptibility to reinjury following single stretch injury with intramuscular injection of MSCs.

ABSTRACTTendon healing is a complex coordinated series of events resulting in protracted recovery, limited regeneration, and scar formation. Mesenchymal stem cell (MSC) therapy has shown promise as a new technology to enhance soft tissue and bone healing. A challenge with MSC therapy involves the ability to consistently control the inflammatory response and subsequent healing. Previous studies suggest that preconditioning MSCs with inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1β may accelerate cutaneous wound closure. The objective of this study was to therefore elucidate these effects in tendon. That is, the in vivo healing effects of TNF‐α primed MSCs were studied using a rat Achilles segmental defect model. Rat Achilles tendons were subjected to a unilateral 3 mm segmental defect and repaired with either a PLG scaffold alone, MSC‐seeded PLG scaffold, or TNF‐α‐primed MSC‐seeded PLG scaffold. Achilles tendons were analyzed at 2 and 4 weeks post‐injury. In vivo, MSCs, regardless of priming, increased IL‐10 production and reduced the inflammatory factor, IL‐1α. Primed MSCs reduced IL‐12 production and the number of M1 macrophages, as well as increased the percent of M2 macrophages, and synthesis of the anti‐inflammatory factor IL‐4. Primed MSC treatment also increased the concentration of type I procollagen in the healing tissue and increased failure stress of the tendon 4 weeks post‐injury. Taken together delivery of TNF‐α primed MSCs via 3D PLG scaffold modulated macrophage polarization and cytokine production to further accentuate the more regenerative MSC‐induced healing response. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:269–280, 2017.

Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 h using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1 μg/ml. Rat MCLs were surgically transected and administered 1 × 10 6 cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing.

Objectives: Although anterior cruciate ligament reconstruction is one of the most common procedures in the field of sports medicine, less than 50% can return to previous activity level partially due to poor biological incorporation of tendon to bone. The aim of the study is to investigate the biological and mechanical effects of Mesenchymal Stem Cell (MSC) and Bone Marrow (BM) treatment for early tendon graft healing in a bone tunnel. Methods: 60 Wistar Albino rats were enrolled in the study and divided in 3 groups equal in number. In all animals a bilateral 1.2 mm bone tunnel was created in proximal tibia. 15 mm Digital Flexor tendons were harvested from each lower extremity and passed from each tunnel. Medial ends of the graft was secured to bone using a custom made endobutton while lateral portion was sutured to periosteum. 100μl whole Bone marrow was aspirated from group 1 animals and injected into the tunnel after tendon transfer while 106 MSCs in 50μl HBSS was injected in the tunnel in group 2. Group 3 was taken as control. Tendon to bone healing was evaluated via immunohistochemistry and mechanical testing at 2 and 6 weeks. Results: At 2 weeks the tendon to bone interface tissue was found to be stronger in the BM treatment group whereas there was no difference in stiffness between group 1 and 2. Interface tissue width was found to be significantly decreased in the BM group compared to control and MSC treatment groups (p=0.0012, p=0.0001). At 2 weeks the number of M1 macrophages at the interface tissue was found to be significantly low in the BM group while M2 macrophages were highest in the MSC group. Cellularity was observed significantly high in the MSC group while tissue density was lowest in the BM group due to accelerated graft degradation at 2 weeks. Conclusion: Although both Bone Marrow and Mesenchymal Stem Cell treatments enhance tendon to bone healing by accelerating the remodeling process, Bone Marrow treatment reveals a more anti-inflammatory healing with improved mechanical properties.

Mesenchymal stem cells (MSCs) have potential therapeutic applications for musculoskeletal injuries due to their ability to differentiate into several tissue cell types and modulate immune and inflammatory responses. These immune-modulatory properties were examined in vivo during early stage rat medial collateral ligament healing. Two different cell doses (low dose 1 × 106 or high dose 4 × 106 MSCs) were administered at the time of injury and compared with normal ligament healing at days 5 and 14 post-injury. At both times, the high dose MSC group demonstrated a significant decrease in M2 macrophages compared to controls. At day 14, fewer M1 macrophages were detected in the low dose group compared to the high dose group. These results, along with significant changes in procollagen I, proliferating cells, and endothelialization suggest that MSCs can alter the cellular response during healing in a dose-dependent manner. The higher dose ligaments also had increased expression of several pro-inflammatory cytokines at day 5 (IL-1β, IFNγ, IL-2) and increased expression of IL-12 at day 14. Mechanical testing at day 14 revealed increased failure strength and stiffness in low dose ligaments compared to controls. Based on these improved mechanical properties, MSCs enhanced functional healing when applied at a lower dose. Different doses of MSCs uniquely affected the cellular response and cytokine expression in healing ligaments. Interestingly, the lower dose of cells proved to be most effective in improving functional properties.