A Translational Model for Repeated Episodes of Joint Inflammation: Welfare, Clinical and Synovial Fluid Biomarker Assessment.

This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.

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

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.

Treatment effects of intra-articular triamcinolone acetonide in an equine model of recurrent joint inflammation.

BACKGROUND: Intra-articular triamcinolone acetonide is a widely used treatment for joint inflammation despite limited scientific evidence of its efficacy. OBJECTIVES: To investigate if intra-articular triamcinolone acetonide has sustained anti-inflammatory effects using an equine model of repeated joint inflammation. STUDY DESIGN: Randomised controlled experimental study. METHOD: For three consecutive cycles 2 weeks apart, inflammation was induced in both middle carpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After the first LPS injection only, treatment with 12 mg triamcinolone acetonide (TA) followed in one randomly assigned joint, while the contralateral joint was treated with sterile saline (control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded and synovial fluid samples were analysed for various biomarkers (total protein, WBCC; PGE2 ; CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed timepoints (post injection hours 0, 8, 24, 72 and 168). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were tested using a linear mixed model for repeated measures with horse as a random effect, and time and treatment as fixed effects. RESULTS: The TA treated joints showed significantly higher peak synovial GAG concentrations (Difference in means 283.1875 µg/mL, 95% CI 179.8, 386.6, P < 0.000), and PGE2 levels (Difference in means 77.8025 pg/mL, 95% CI 21.2, 134.4, P < 0.007) after the first inflammation induction. Significantly lower TP levels were seen with TA treatment after the second induction (Difference in means -7.5 g/L, 95% CI -14.8, -0.20, P < 0.04) . Significantly lower WBCC levels were noted with TA treatment after the first (Difference in means -23.7125 × 109  cells/L, 95% CI -46.7, -0.7, P < 0.04) and second (Difference in means -35.95 × 109  cells/L, 95% CI -59.0, -12.9, P < 0.002) inflammation inductions. Significantly lower general MMP activity was also seen with TA treatment after the second inflammation inductions (Difference in means -51.65 RFU/s, 95% CI -92.4, -10.9, P < 0.01). MAIN LIMITATIONS: This experimental study cannot fully reflect natural joint disease. CONCLUSIONS: In this model, intra-articular TA seems to have some anti-inflammatory activity (demonstrated by reductions in TP, WBCC and general MMP activity) up to 2 weeks post treatment but not at 4 weeks. This anti-inflammatory effect appeared to outlast a shorter-lived, potentially detrimental effect illustrated by increased synovial GAG and PGE2 levels after the first induction.

Intra-articular delivery of a nanocomplex comprising salmon calcitonin, hyaluronic acid, and chitosan using an equine model of joint inflammation.

Polyelectrolyte nanoparticle constructs (NPs) comprising salmon calcitonin (sCT), chitosan (CS), and hyaluronic acid (HA) were previously established as having anti-inflammatory potential when injected via the intra-articular (i.a.) route to a mouse model. We attempted to translate the formulation to a large animal model, the lipopolysaccharide (LPS)-stimulated equine model of joint inflammation. The aim was to manufacture under aseptic conditions to produce sterile pyrogen-free NPs, to confirm physicochemical characteristics, and to test toxicity and efficacy in a pilot study. NP dispersions were successfully formulated using pharmaceutical-grade source materials and were aseptically manufactured under GMP-simulated conditions in a grade A modular aseptic processing workstation. The NP formulation had no detectable pathogen or endotoxin contamination. NPs were then tested versus a lactated Ringer's solution control following single i.a. injections to the radiocarpal joints of two groups of four horses pre-treated with LPS, followed by arthrocentesis at set intervals over 1 week. There was no evidence of treatment-related toxicity over the period. While there were no differences between clinical read-outs of the NP and the control, two synovial fluid-derived biomarkers associated with cartilage turnover revealed a beneficial effect of NPs. In conclusion, NPs comprising well-known materials were manufactured for an equine i.a.-injectable pilot study and yielded no NP-attributable toxicity. Evidence of NP-associated benefit at the level of secondary endpoints was detected as a result of decreases in synovial fluid inflammatory biomarkers.