Effect of Extracorporeal Shock Wave Therapy on Equine Umbilical Cord Blood Mesenchymal Stromal Cells in vitro

Presented by: Ramés Salcedo-Jiménez

 

Authors: Ramés Salcedo-Jiménez1, Judith Koenig1, Olivia Lee2, Tom Gibson1, Pavneesh Madan2, Thomas Koch2

Affiliations: 1Department of Clinical Studies, University of Guelph, Ontario, Canada 2Department of Biomedical Sciences, University of Guelph, Ontario, Canada

Introduction: Extracorporeal shock wave therapy (ESWT) has been shown to induce different biological effects on a variety of cells, including regulation and stimulation of their function and metabolism. ESWT can promote different biological responses such as proliferation, migration, and regenerations of cells. Recent studies have shown that mesenchymal stromal cells (MSCs) secrete factors that enhance regeneration of tissues, stimulate proliferation and differentiation of cells and decrease inflammatory and immune-reactions. Clinically, the combination of these two therapies has been used as treatment for tendon and ligament lesions in horses; however, there are no scientific evidences supporting this combination of therapies in vivo.

Hypothesis / Objectives: The objectives of this study were to evaluate if ESWT affects equine umbilical cord blood mesenchymal stromal cells’ (CB-MSCs) proliferation, metabolic activity, migration, and differentiation, and if those cells would maintain their immunomodulatory properties in comparison to untreated cells in vitro.

Materials and Methods: Three cryovials of equine CB-MSCs at passage 3 were thawed, and the cells were treated at a confluence of 90% in T-25 cell culture flasks. The in vitro shock wave treatment was performed using an electrohydraulic shock wave generator attached to a water bath using the following parameters: energy flux density 0.1 mJ/mm2, frequency of 3Hz, and 300 impulses. All experiments were performed as triplicates. Proliferation, viability, migration and immunomodulatory properties of the cells were evaluated. CB-MSCs were induced to evaluate their trilineage differentiation potential.

Results: All the equine CB-MSC showed an elongated, spindle-shape fibroblast-like morphology. No difference in proliferation was observed after treatment. ESWT treated cells showed a significant difference (p = 0.0002) in their metabolic activity as evaluated by the Alamar blue assay. Positive adipogenic, osteogenic, and chondrogenic induction for all cell cultures was confirmed. For adipogenesis (p = 0.0002) and osteogenesis (p < .0001) ESWT treated cells showed significant higher potential for differentiation as evaluated by the amount of intracellular lipids and alkaline phosphatase activity respectively. ESWT treated cells maintained their immunomodulatory properties as evaluated by the lymphocyte proliferation assay.

Conclusions: In the present study we demonstrated that equine CB-MSCs are sensitive to ESWT. The proliferation and immunomodulatory properties were not adversely influenced while the metabolic activity was increased. Equine CB-MSCs maintained their multilineage differentiation potential after treatment and increased their potency towards adipogenic and osteogenic lineage, which is in accordance to previous studies. ESWT has an effect on equine CB-MSCs and combining these two therapies might have a synergism when utilized for treatment of musculoskeletal injuries in horses. Further studies are warranted to determine if these effects are observed in vivo and with different types of MSCs.

Acknowledgements, Funding, and Conflicts of Interest: Funding: Equine Guelph.