Regenerative therapies for equine degenerative joint disease: a preliminary study

Abstract

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.

Figure 1. Representative images of peripheral blood (PB)-derived mesenchymal stem cells (MSCs) in their undifferentiated state (A & C) and chondrogenic induced (B & D) after Hematoxylin (A & B) and Crystal Violet (C & D) stainings.

The typical chondrogenic morphology and lacune formation (black arrow) can be noticed after induction. Scale bars represent 50 µm.

Figure 2. Results of RT-PCR for the gene expression of collagen (Col) type II, aggrecan and cartilage oligomeric matrix protein (COMP) in the native MSCs (Ctrl) and chondrogenic induced MSCs (Ind).

Values are given as the mean of three measurements ± SEM.

Figure 3. Representative images of peripheral blood (PB)-derived mesenchymal stem cells (MSCs) in their undifferentiated state (A & C) and chondrogenic induced (B & D) after Alcian Blue (A & B) and Safranin O (C & D) stainings.

Glycosaminoglycan production (black arrows) can be noticed after induction. Scale bars represent 50 µm.

Figure 4. Immunocytochemistry on adhesive mesenchymal stem cells (MSCs) using Ki67 (A), collagen (Col) type II (B), vimentin (C) and p63 (D).

Native MSCs were negative for p63 and positive for Ki67, Col II and vimentin, whereas chondrogenic induced MSCs were positive for p63, Col II and vimentin with a decreased signal for Ki67 (arrows = negative nuclei). The relevant isotype controls were negative. Scale bar represents 50 µm.

Figure 5. Flow cytometry confirmed a low expression of major histocompatibility complex (MHC) class I and no expression of MHC class II on the native MSCs and chondrogenic induced MSCs.

The light and dark grey histograms represent the relevant isotype control staining and marker antibody staining, respectively with the corresponding percentage of mean positive cells ± SEM.

Figure 6. Clinical evolution scores of the different treatments at different time points in the first study (A).

Values are given as the mean ± SEM. Diagrams represent the clinical evolution scores of 30 horses treated with native mesenchymal stem cells (MSCs) and PRP (Combination 1, n = 15) or chondrogenic induced MSCs and PRP (Combination 2, n = 15) in the second study (B).