Treatment Effects of Intra-Articular Allogenic Mesenchymal Stem Cell Secretome in an Equine Model of Joint Inflammation

Abstract

Background: Allogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation.

Objectives: To investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation.

Study design: Randomized positively and negatively controlled experimental study.

Method: In phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology.

Results: In phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs.

Main limitations: This study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control.

Conclusions: In this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment.

Keywords: equine model; joint inflammation; lipopolysaccharide (LPS); mesenchymal stem cells; secretome.

Figure 1

Overview of study design. The preparation steps were carried out in advance of the experimental period. Bone marrow derived mesenchymal stem cells (MSCs) were harvested from donor horses in Colorado State University (CSU) Veterinary Teaching Hospital and cultured, frozen and stored according to their standard protocols. Later, MSCs were transported, still frozen, to Erasmus MC in Rotterdam, where they were thawed and cultured and then used to prepare Mesenchymal stem cell secretome (MSC-secretome) treatments. Cells from the same cell lines as used for the MSC preparation were also transported to Dublin, where the final preparatory steps and viability assessment were performed immediately prior to their use in Phase 2 of the experiment. The experimental period represents 3 weeks in total. PIH (Post Induction Hour) indicates time in hours after induction of inflammation with intra-articular injection of 0.25 ng of lipopolysaccharide (LPS) in each radiocarpal joint (RCJ) of 8 horses. At PIH₁2, one randomly selected RCJ of each horse was injected with intra-articular mesenchymal stem cell secretome and the contralateral joint was injected with medium (negative control). Following PIH₁168, horses had a washout period (7 days) during which they were on pasture rest. At PIH₂2, the RCJ that had previously been treated with intra-articular mesenchymal stem cell secretome was again treated with intra-articular mesenchymal stem cell secretome and the contralateral joint was injected with mesenchymal stem cells (positive control).

Figure 2

Phase 1 synovial white blood cell counts, total protein, and joint circumference. (A) Synovial White Blood Cell Count, (B) Synovial Fluid Total Protein, and (C) Joint Circumference over time following induction of inflammation with intra-articular injection of 0.25 ng of LPS in the left and right radiocarpal joints of horses at PIH₁ 0 (n = 8 horses). Joints were treated with either intra-articular mesenchymal stem cell (MSC) secretome or medium (negative control) at PIH₁2. Boxes depict median and interquartile ranges; whiskers denote minimum and maximum values. For the Synovial White Blood Cell Count, the box and whiskers for the first timepoint are not visible on the graph as the values for these were very low with each measurement recorded being < 1 × 10⁹ cells/L. *p < 0.05, indicating time points where there are significant treatment effects.

Figure 3

Phase 1 synovial fluid glycosaminoglycan, prostaglandin F2α and tumor necrosis factor α. (A) Glycosaminoglycan, (B) Prostaglandin F2α, and (C) Tumor Necrosis Factor α concentrations in synovial fluid over time following induction of inflammation with intra-articular injection of 0.25 ng of LPS in the left and right radiocarpal joints of horses at PIH₁ 0 (n = 8 horses). Joints were treated with either intra-articular mesenchymal stem cell (MSC)-secretome or medium (negative control) at PIH₁2. Boxes depict median and interquartile ranges; whiskers denote minimum and maximum values. ***p < 0.001, indicating timepoints where there are significant treatment effects.

Figure 4

Phase 2 synovial white blood cell counts, total protein and joint circumference. (A) Synovial White Blood Cell Count, (B) Synovial Fluid Total Protein, and (C) Joint Circumference over time following induction of inflammation with intra-articular injection of 0.25 ng of LPS in the left and right radiocarpal joints of horses at PIH₂ 0 (n = 8 horses). Joints were treated with either intra-articular mesenchymal stem cell (MSC)-secretome of mesenchymal stem cells (MSCs) (positive control) at PIH₂2. Boxes depict median and interquartile ranges; whiskers denote minimum and maximum values. For the Synovial White Blood Cell Count the box and whiskers for the first timepoint are not visible on the graph as the values for these were very low with each measurement recorded is < 1 × 10⁹ cells/L.

Figure 5

Phase 2 synovial fluid glycosaminoglycan, prostaglandin f2α and tumor necrosis factor α. (A) Glycosaminoglycan, (B) Prostaglandin F2α, and (C) Tumor Necrosis Factor α concentrations in synovial fluid over time following induction of inflammation with intra-articular injection of 0.25 ng of LPS in the left and right radiocarpal joints of horses at PIH₂ 0 (n = 8 horses). Joints were treated with either intra-articular mesenchymal stem cell (MSC)-secretome of mesenchymal stem cells (MSCs) (positive control) at PIH₂2. Boxes depict median and interquartile ranges; whiskers denote minimum and maximum values.