Development of a Novel In-Vivo Equine Model System to Define the Role of the Tendon Inflammatory Cytokine Environment on the Timing and Efficacy of Mesenchymal Stem Cell Treatment
Presented by: Lauren V. Schnabel
Authors: Lauren V. Schnabel (1,2), Jessica M. Gilbertie (1,2), Alix K. Berglund (1,2), Kristen Messenger (3), Matthew B. Fisher (2,4) Caityn R. Horne (1)
Affiliations: 1) Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University (2) Comparative Medicine Institute, North Carolina State University (3) Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University (4) Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina – Chapel Hill
Introduction: A key question relating to mesenchymal stem cell (MSC) therapy for tendon injuries is the timing of treatment. Although the traditional paradigm is to wait until the acute inflammation has subsided in the tendon before initiating MSC treatment, we propose the opposite: that the peak inflammatory environment, characterized by high levels of pro-inflammatory cytokines, is in fact the most beneficial for MSC transplantation as it will will cause the MSCs to produce pro-healing cytokines and growth factors critical for tendon repair. In order to study this, however, we first need a way to characterize the tendon inflammatory environment.
Hypothesis / Objectives: The objective of this study was to develop an in-vivo equine model system to continuously measure cytokine levels within surgically-induced superficial digital flexor tendon (SDFT) lesions. Our hypothesis was that we could implant commercially available 30kDa ultrafiltration probes (Basi®) within SDFT lesions in order to accomplish this.
Materials and Methods: Surgical lesions were created using a 3.5mm arthroscopic bone-cutter burr in the mid metacarpal region of the SDFT in both forelimbs of a horse under general anesthesia. Lesion creation and ultrafiltration probe placement was guided by and confirmed with ultrasonography (Fig. 1). Following recovery from anesthesia, each probe was connected to a Vacutainer™ device and secured to the limb with a bandage. Vacutainers™ were changed every 24 hours and tendon effluent samples stored until analyzed for concentrations of IL-1α, IL-1β, TNF-α, and IL-6 via ELISA. Following euthanasia, tendon samples were harvested with the probes in place and sectioned for histopathology.
Results: Each probe consistently yielded ~1.5mL of sample fluid per 24 hours for 16 days. The horse remained comfortable and was able to exercise freely (walk and trot) with the system in place. Detectable levels of all cytokines except TNF-α were found, with IL-1β having the highest and most well defined peak in concentration around day 3 (Fig. 2). Histopathology revealed a minimal foreign body response (Fig. 3). We later had probes of larger pore size made for us by Basi® in order to recover cytokines such as TNF-α that are actively secreted as homodimers/trimers, which proved successful in-vitro (Fig. 4).
Conclusions: This proof-of-concept study did prove that ultrafiltration probes can be placed within surgically-induced equine SDFT lesions and yield samples of sufficient volume and quality for analyses, but future studies should be performed with 100kDa probes in order to capture all cytokines of interest. In these future studies we will utilize proteomics instead of ELISAs to examine all potential cytokines and will also perform in vitro assays using tendon effluent as culture media for MSCs in order to determine which cytokine environment (peak inflammatory or post-inflammatory) leads to an enhanced MSC secretome for tendon healing.
Acknowledgements, Funding, and Conflicts of Interest: This work was supported by the Fund for Orthopedic Research in honor of Gus and Equine athletes (F.O.R.G.E; LVS). The authors would like to thank Mrs. Julie Long for her technical assistance and the NCSU Laboratory Animal Resources staff for their help with animal care and handling. The authors declare no conflicts of interest.