Latest Published paper in Cytokine Biology

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Purpose/Aim of Study: Tendon healing involves a complex, coordinated series of events that, despite new therapies to improve healing, results in scar. The “nude” T-lymphocyte deficient model is a genetic strain exhibiting a loss of function in the Foxn1 gene. Nude mice cannot generate mature T-lymphocytes, are unable to mount many types of adaptive immune responses, and were initially used as skin injury healing models. Their ability to heal external ear injuries, similar to the regenerating MRL mouse model, has since been described. We therefore hypothesized the Foxn1-/- nude T-lymphocyte deficient mouse strain would improve tendon healing.

Materials and Methods: Foxn1-/- T-lymphocyte deficient mice were subjected to Achilles tendon injury. Tissue was collected 7 and 14 days after injury and used to examine mechanical, cellular, compositional, and organizational effects on healing.

Results: T-lymphocyte deficient healing tendons exhibited improved stiffness, smaller wound size, decreased M1/M2 macrophage ratio, increased type III collagen, and an earlier but transient up regulation of endothelial cells compared to genetic controls.

Conclusion: The mechanisms involved in improved tendon healing of the T-lymphocyte deficient mouse may include a number of factors such as the ablation of Foxn1. However, the deficiency in T-lymphocytes and change in M1/M2 ratio may also impact healing by serving a regulating, protective and/or accelerating role. Overall, the clinical implication of modulating the immune cells to stimulate healing are profound and suggests the possibility to manipulate certain subsets of T-lymphocytes and/or macrophages at specific healing stages to significantly enhance musculoskeletal healing.

IHC and histology were performed to identify cellular and ECM changes within the healing tendon on day 7 and 14 injured Achilles tendons. Longitudinal cryosections (approximately 6 sections/slide) were cut at a 5 μm thickness, mounted on Colorfrost Plus microscope slides and maintained at -70 ˚ C. IHC was performed on frozen sections. Cryosections were fixed in acetone, exposed to 3% hydrogen peroxide to eliminate endogenous peroxidase activity, blocked with Rodent Block M (RBM961L, Biocare Medical, Pacheco, CA) and incubated with rabbit or rat primary antibodies. Primary rat monoclonal antibodies (all 1:100 from BioRad, Hercules, CA) specific to mouse F4/80 (MCA497GA), CD206 (MCA2235GA), CD31 (MCA2388GA) were used to detect total macrophages, M2 macrophages, and endothelial cells, respectively. Rabbit polyclonal antibodies were used for type I collagen (1:800, ab34710, Abcam- Serotec, Raleigh, NC) and type III collagen (1:150, ab7778, Abcam- Serotec). Lastly, rabbit monoclonal CCR7 was used to identify M1 macrophages (1:1200, ab32527 Abcam-Serotec). After primary antibody incubation, samples were exposed to rabbit (RMR622H, Biocare Medical, Pacheco, CA) or rat (BRR4016H, Biocare Medical, Pacheco, CA) Horseradish Peroxidase (HRP) Polymer. The bound antibody complex was visualized using diaminobenzidine (DAB, NB314SBD, Innovex Biosciences). Stained sections were dehydrated, cleared, cover-slipped and viewed using light microscopy. After IHC staining, micrographs were collected using a camera-assisted microscope (Nikon Eclipse microscope, model E6000 with an Olympus camera, model DP79). On average, 4.7 (± 0.20) were captured and counted per animal. Images captured for measurement of total macrophages, M1 and M2 macrophages, endothelial cells, type I collagen and type III collagen were quantified via Image J (National Institutes of Health, Bethesda, MD). Measurements were collected 1) within the granulation tissue and 2) within the entire section. More specifically, total protein within the tendon was collected by first outlining the entire tendon using the polygon selection tool in Image J. Protein within the granulation tissue was also measured by outlining, measuring, and enumerating protein specifically within the granulation tissue. Protein was quantified as density/mm2. Tendon cryosections were also Hematoxylin and Eosin (H&E) stained to observe histological morphology of the healing tendon.

 

Regards,
Veronica Thompson
Riya Parker
Associate Editor
Journal of Cytokine Biology