Autologous protein solution mitigates the inflammatory cascade in stimulated equine chondrocyte

 

[um_loggedin show_lock=yes]Authors: Kyla F. Ortved, DVM, PhD, DACVS, DACVSMR; Michael E. Dodson, BA, MBS; Alexis L. Gale, BSc; Renata L. Linardi, DVM, PhD

Affiliations:
Department of Clinical Studies, New Bolton Center, University of Pennsylvania

Introduction: Osteoarthritis is a progressive disease estimated to affect 60% of horses. Several intra-articular orthobiologics are available to treat joint damage and prevent OA, including platelet-rich plasma (PRP) and autologous conditioned serum (ACS). PRP efficacy is due to degranulation of α-granules releasing growth factors including PDGF, TGF-β, FGF, VEGF, etc. that modulate the healing response in damaged tissue. ACS is produced through incubation and centrifugation of blood such that interleukin-1 receptor antagonist protein (IL-1Ra) and other anti-inflammatory cytokines are concentrated. Recently, autologous protein solution (Pro-Stride, Owl Manor Veterinary), a product that combines the benefits of PRP with ACS has been developed.

Hypothesis/Objectives: The aim of this study was to evaluate the effect of APS on the inflammatory cascade in chondrocytes cultured in vitro. We hypothesized that stimulated chondrocytes cultured in APS would have a significantly decreased pro-inflammatory and catabolic response compared to ACS, as evaluated by gene expression and protein synthesis.

Materials and Methods: Blood was collected from 6 horses (2-8yrs) for preparation of APS (Pro-Stride) and ACS (IRAP-II). Cartilage was then harvested from the femoral trochlea and chondrocytes cultured in transwell plates. Treatment groups included: 1)Control, 2)APS, and 3)ACS. Two hours prior to stimulation, serum free media, APS or ACS was added to the top of the transwell. Cells were then stimulated with IL-1b+TNF-a for 48 hours following which medium was collected and RNA isolated. MMP3, MMP13, IL-1b, TNF-a, and IL-10 was quantified using a multiplex assay. IL-1Ra concentration was quantified using ELISA. Gene expression was determined for IL-1b, TNF-a, MMP3, and MMP13.

Results:
Stimulation of chondrocytes with IL-1b/TNF-a significantly increased supernatant concentration of MMP3 and MMP13. Pre-treatment of stimulated chondrocytes with Pro-Stride led to significantly decreased production of both MMP3 and MMP13 compared to untreated controls (Fig 1). Pre-treatment with ACS had similar effects on stimulated chondrocytes. IL-1b concentration in culture supernatants was decreased in Pro-Stride treated chondrocytes compared to untreated controls, however, this did not reach statistical significance (Fig 2). TNF-a was not affected by pre-treatment with Pro-Stride or ACS. IL-10 and IL-1Ra was significantly increased in both unstimulated and IL-1b/TNF-a stimulated chondrocytes treated with Pro-Stride compared to untreated and ACS treated chondrocytes (Fig 3). Gene expression data is pending.

Conclusions: Treatment of inflamed chondrocytes with APS led to significant decreases in production of two central degradative enzymes involved in breakdown of the extra-cellular matrix (ECM), MMP3 and MMP13. ECM breakdown leads to decreased compressive and tensile strength of cartilage and further joint degeneration. Elevated concentrations of IL-10 and IL-1Ra, both potent anti-inflammatory cytokines, in Pro-Stride may play a role in mitigating production of MMPs. APS effectively decreases the inflammatory and catabolic cascade in stimulated chondrocytes in vitro. This combinatorial orthobiologic may be an effective intra-articular therapeutic following joint trauma as it can limit post-traumatic inflammation and prevent development of osteoarthritis.

Acknowledgements, Funding, and Conflicts of Interest: Funded by Owl Manor Veterinary.

Figures:

Ortved Pro Stride Figures (PDF)

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