Abstract
Summary Statement
This 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.
Introduction
Mesenchymal 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 environment they encounter, which leads to our interest in MSC immune-modulation in healing ligaments.
Methods
Forty-four rats underwent bilateral MCL transection. Days 5 and 14 healing were examined comparing two cell doses (1×106 MSCs or 4×106 MSCs). At the time of surgery, fluorescently-labeled rat MSCs (passage 8–10) were injected into the right MCL, while the left MCL served as a control for normal healing. MCLs were collected at the different time points and processed with immunohistochemistry (n=12). Type 1 macrophages (M1) and type 2 macrophages (M2) were quantified spatially within the healing ligaments. Twelve rats with MSC injections underwent mechanical testing. A multiplex cytokine reader measured 10 different cytokines in the healing ligaments at days 5 and 14.
Results
MSCs were detected solely in the healing region and healing region edges at Days 5 and 14 in both dose groups using fluorescence microscopy. At day 5, the higher dose of cells produced significant M2 changes throughout the ligament. There were more M2′s (p=.05) in the distal and proximal healing regions of the normal healing ligament compared to the MSC injection group. There were significant changes in both the low dose and high dose groups at day 14. Fewer M1′s were found in the ends (p=.01) and throughout the MCL (p=.04) in the low dose group. M2′s were decreased in the ends (p=.04), but only in the ligaments that received the higher dose of MSCs. Cytokine analysis showed a greater amount of pro-inflammatory cytokines in the high dose MSC group at Day 5 (IL-1β, IL-2, and Interferon-Y) compared to controls, along with increased IL-12 at Day 14. The low dose MSC injection group demonstrated increased strength with an average failure load of 26.4N compared to 20.9N in the control group (p=.03). Low dose ligaments also exhibited increased stiffness with an average of 12.2 N/mm compared to 10.0 N/mm (p=.01) in control ligaments.
Discussion
MSCs improved healing when applied at an appropriate dose as shown by improved mechanical properties at day 14. Interestingly, the smaller dose of 1 million cells proved more successful than the larger dose of 4 million cells. MSCs also affected the cytokine profile and macrophage phenotype at both healing time points, but not always as expected with regard to inflammatory cells and cytokines.
