Cell-free multi-layered collagen-based scaffolds demonstrate layer specific regeneration of functional osteochondral tissue in caprine joints.

Developing repair strategies for osteochondral tissue presents complex challenges due to its interfacial nature and complex zonal structure, consisting of subchondral bone, intermediate calcified cartilage and the superficial cartilage regions. In this study, the long term ability of a multi-layered biomimetic collagen-based scaffold to repair osteochondral defects is investigated in a large animal model: namely critical sized lateral trochlear ridge (TR) and medial femoral condyle (MC) defects in the caprine stifle joint. The study thus presents the first data in a clinically applicable large animal model. Scaffold fixation and early integration was demonstrated at 2 weeks post implantation. Macroscopic analysis demonstrated improved healing in the multi-layered scaffold group compared to empty defects and a market approved synthetic polymer osteochondral scaffold groups at 6 and 12 months post implantation. Radiological analysis demonstrated superior subchondral bone formation in both defect sites in the multi-layered scaffold group as early as 3 months, with complete regeneration of subchondral bone by 12 months. Histological analysis confirmed the formation of well-structured subchondral trabecular bone and hyaline-like cartilage tissue in the multi-layered scaffold group by 12 months with restoration of the anatomical tidemark. Demonstration of improved healing following treatment with this natural polymer scaffold, through the recruitment of host cells with no requirement for pre-culture, shows the potential of this device for the treatment of patients presenting with osteochondal lesions

Exploring stable-based behaviour and behaviour switching for the detection of bilateral pain in equines

Efficient and sensitive animal pain detection approaches are increasingly studied with the goal of improving animal welfare and monitoring the efficacy of treatment and rehabilitation. The aim of this study was to determine the potential of various behaviours as sensitive indicators of subtle inflammation states in equines. The long-term goal of this research is to understand how to objectively and remotely classify behaviours that are associated with inflammation using wearable inertial sensor technologies. This study represents a proof-of concept investigation to ascertain what behavioural indices might be important in long-term monitoring of mild bilateral inflammation and recovery with a view to translating the approach to a technology-enabled remote monitoring paradigm. Bilateral synovitis of the intercarpal joints was induced in seven equines using lipopoly saccharide (0.25 ng) at time zero. The horses were confined to stables and monitored intermittently over seven days by stable-fixed video cameras. White blood cell count, carpal circumference and food availability were recorded across the study. An ethogram was created to manually annotate behaviours from video footage following lameness induction at seven different timepoints across a 1-week period. Behaviour data were processed extracting the duration, frequency and variability of behaviours. One-way repeated measures ANOVA revealed a significant time effect for white blood cell count and behaviour switching. There were no significant changes in carpal circumferences and heart rate measures over the sampling period. Food availability appears to be an important contextual factor that should be considered in pain-related behavioural studies. We conclude that behaviour variability may be a promising indicator of subtle bilateral inflammation which should be further explored in larger controlled trials and different pain presentations. Future work will seek to optimise grouping of behaviours associated with inflammation that can be detected using wearable technologies for future remote monitoring protocols

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

The Feasibility of Equine Field-Based Postural Sway Analysis Using a Single Inertial Sensor

(1) Background: Postural sway is frequently used to quantify human postural control, balance, injury, and neurological deficits. However, there is considerably less research investigating the value of the metric in horses. Much of the existing equine postural sway research uses force or pressure plates to examine the centre of pressure, inferring change at the centre of mass (COM). This study looks at the inverse, using an inertial measurement unit (IMU) on the withers to investigate change at the COM, exploring the potential of postural sway evaluation in the applied domain. (2) Methods: The lipopolysaccharide model was used to induce transient bilateral lameness in seven equines. Horses were monitored intermittently by a withers fixed IMU over seven days. (3) Results: There was a significant effect of time on total protein, carpal circumference, and white blood cell count in the horses, indicating the presence of, and recovery from, inflammation. There was a greater amplitude of displacement in the craniocaudal (CC) versus the mediolateral (ML) direction. A significant difference was observed in the amplitude of displacement in the ML direction between 4–12 h and 168 h. (4) Conclusions: The significant reduction in ML displacement during the acute inflammation period alongside greater overall CC displacement may be a compensatory behaviour for bilateral lameness

The Feasibility of Equine Field-Based Postural Sway Analysis Using a Single Inertial Sensor

(1) Background: Postural sway is frequently used to quantify human postural control, balance, injury, and neurological deficits. However, there is considerably less research investigating the value of the metric in horses. Much of the existing equine postural sway research uses force or pressure plates to examine the centre of pressure, inferring change at the centre of mass (COM). This study looks at the inverse, using an inertial measurement unit (IMU) on the withers to investigate change at the COM, exploring the potential of postural sway evaluation in the applied domain. (2) Methods: The lipopolysaccharide model was used to induce transient bilateral lameness in seven equines. Horses were monitored intermittently by a withers fixed IMU over seven days. (3) Results: There was a significant effect of time on total protein, carpal circumference, and white blood cell count in the horses, indicating the presence of, and recovery from, inflammation. There was a greater amplitude of displacement in the craniocaudal (CC) versus the mediolateral (ML) direction. A significant difference was observed in the amplitude of displacement in the ML direction between 4–12 h and 168 h. (4) Conclusions: The significant reduction in ML displacement during the acute inflammation period alongside greater overall CC displacement may be a compensatory behaviour for bilateral lameness

Repair of large osteochondritis dissecans lesions using a multi-layered collagenbased osteochondral graft substitute in an equine athlete.

Osteochondral lesions resulting from osteochondritis dissecans are problematic to treat and present a significant challenge for clinicians. The aims of this study were to investigate the use of a scaffold-assisted microfracture approach, employing a novel, multilayered, collagen-based, osteochondral graft substitute in the treatment of severe osteochondritis dissecans of both lateral femoral trochlear ridges in an equine athlete, and to assess the potential of this novel scaffold to enhance repair of the osteochondral unit. A 15 month-old female filly presented with large osteochondritis dissecans lesions involving both femoral lateral trochlear ridges. After routine arthroscopic debridement and microfracture of the subchondral bone, multilayered osteochondral defect repair scaffolds were implanted into the fragmentation beds in both left and right femoropatellar joints via mini-arthrotomies. Exploratory arthroscopy 5 months postimplantation revealed smooth cartilaginous repair tissue, contiguous with the adjacent cartilage, covering the defect. At 22-month follow up, the filly had no signs of lameness and was exercising at her intended level. Radiographically, although still slightly flattened, the femoral trochlear ridges were smooth, with no evidence of osteoarthritis. Ultrasonographically, the defects were filled with bone and covered with an overlying cartilaginous layer, with the trochlear ridge contour almost entirely restored. This report demonstrates the effective clinical use of this novel, multilayered, osteochondral defect repair scaffold in the treatment of osteochondritis dissecans of an equine athlete. The successful repair achieved here using this novel scaffold in an equine patient with large bilateral lesions shows the potential for clinical translation in the treatment of human patients presenting with osteochondral defect

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.European CommissionScience Foundation IrelandWellcome Trust Institutional Strategic Support Fund Clinician Stimulus Scheme (CK)SFI Synthesis and Solid State Pharmaceutical Centr