Mesenchymal Stromal Cell Secretome Is Affected by Tissue Source and Donor Age.

Variation in mesenchymal stromal cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs are attributed to secretion of biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome remains largely unknown. Here, we examined the influence of donor age, sex, and tissue source, on the protein profile of the equine MSC secretome. We used dynamic metabolic labeling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically secreted based on the rate of label incorporation into newly synthesized proteins released into the extracellular space. Next, we analyzed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of CTHRC1 and LOX, further validated with orthogonal techniques. Due to the lack of flow cytometry characterization of MSC surface markers, the analysis could not account for the potential effect of cell population heterogeneity. This study provides evidence that tissue source and donor age contribute to differences in the protein composition of MSC secretomes which may influence the effects of MSC therapy.

Temporal extracellular vesicle protein changes following intraarticular treatment with integrin α10ß1-selected mesenchymal stem cells in equine osteoarthritis.

Introduction: Equine osteoarthritis (OA) is a heterogeneous, degenerative disease of the musculoskeletal system with multifactorial causation, characterized by a joint metabolic imbalance. Extracellular vesicles are nanoparticles involved in intracellular communication. Mesenchymal stem cell (MSC) therapy is a form of regenerative medicine that utilizes their properties to repair damaged tissues. Despite its wide use in veterinary practice, the exact mechanism of action of MSCs is not fully understood. The aim of this study was to determine the synovial fluid extracellular vesicle protein cargo following integrin α10ß1-selected mesenchymal stem cell (integrin α10-MSC) treatment in an experimental model of equine osteoarthritis with longitudinal sampling. Methods: Adipose tissue derived, integrin α10-MSCs were injected intraarticularly in six horses 18 days after experimental induction of OA. Synovial fluid samples were collected at day 0, 18, 21, 28, 35, and 70. Synovial fluid was processed and extracellular vesicles were isolated and characterized. Extracellular vesicle cargo was then analyzed using data independent acquisition mass spectrometry proteomics. Results: A total of 442 proteins were identified across all samples, with 48 proteins differentially expressed (FDR ≤ 0.05) between sham-operated control joint without MSC treatment and OA joint treated with MSCs. The most significant pathways following functional enrichment analysis of the differentially abundant protein dataset were serine endopeptidase activity (p = 0.023), complement activation (classical pathway) (p = 0.023), and collagen containing extracellular matrix (p = 0.034). Due to the lack of an OA group without MSC treatment, findings cannot be directly correlated to only MSCs. Discussion: To date this is the first study to quantify the global extracellular vesicle proteome in synovial fluid following MSC treatment of osteoarthritis. Changes in the proteome of the synovial fluid-derived EVs following MSC injection suggest EVs may play a role in mediating the effect of cell therapy through altered joint homeostasis. This is an important step toward understanding the potential therapeutic mechanisms of MSC therapy, ultimately enabling the improvement of therapeutic efficacy.

Gene expression analysis of subchondral bone, cartilage, and synovium in naturally occurring equine palmar/plantar osteochondral disease.

Osteoarthritis (OA) is a disease of the entire joint but the relationship between pathological events in various joint tissues is poorly understood. We examined concurrent changes in bone, cartilage, and synovium in a naturally occurring equine model of joint degeneration. Joints (n = 64) were grossly assessed for palmar/plantar osteochondral disease (POD) in racehorses that required euthanasia for unrelated reasons and assigned a grade of 0 (n = 34), 1 (n = 17), 2 or 3 (n = 13) using a recognized grading scheme. Synovium, cartilage, and subchondral bone were collected for histological and gene expression analysis. Relations between POD grade, cartilage histological score, and gene expression levels were examined using one-way analysis of variance or Kruskal-Wallis test and Spearman's correlation coefficient with corrections for multiple comparisons. Cartilage histological score increased in joints with POD grade 1 (p = 0.002) and 2 or 3 (p < 0.001) compared to 0. At grade 1, expression of COL1A1, COL2A1, and MMP1 increased and BGN decreased in subchondral bone while expression of BGN and ACAN decreased in cartilage. These changes further progressed at grades 2 and 3. POD grades 2 and 3 were associated with decreased expression of osteoclast inhibitor OPG and increased markers of cartilage degeneration (MMP13, COL1A1). Expression of the vascular endothelial growth factor decreased with POD grade and negatively correlated with cartilage histological score. Synovium showed no histological or transcriptomic changes related to pathology grade. Cartilage degeneration in POD is likely to be secondary to remodeling of the subchondral bone. Limited activation of proinflammatory and catabolic genes and moderate synovial pathology suggests distinct molecular phenotype of POD compared with OA.

An Evaluation of Current Preventative Measures Used in Equine Practice to Maintain Distal Forelimb Functionality: A Mini Review.

Horses are used in a variety of equestrian disciplines predisposing them to musculoskeletal injury or disease including osteoarthritis and tendinopathy. As a result, a number of preventative measures are used within equine medicine and husbandry, ranging from therapeutic shoeing to the use of nutraceuticals. Despite their popularity and routine use evidence base and clinical outcomes are variable, bringing into question the efficacy of these prophylactic measures. In recent years a small number of studies have been performed examining the effect of specific strategies in order to quantify the preventative and protective claims such modalities have on joint and forelimb health. Few have robustly demonstrated a capacity to protect the limb by reducing inflammation, or promoting regenerative pathways. This review focusses on performance horses specifically, and the resounding theme that emerges in current research is the need for longitudinal studies to inform scientific conclusions surrounding single and multi-modal use. Furthermore, there is a requirement to prioritise evidence-based medicine to inform optimal clinical practice.

Revisiting predictive biomarkers of musculoskeletal injury in thoroughbred racehorses: longitudinal study in polish population.

BACKGROUND: High prevalence of musculoskeletal disorders in racehorses and its impact on horse welfare and racing economics call for improved measures of injury diagnosis and prevention. Serum biomarkers of bone and cartilage metabolism have previously shown promise in prediction of musculoskeletal injuries in horses. This study aimed to re-evaluate usability of the predictive serum biomarkers identified in North American Thoroughbred racehorses in a geographically distinct group of Polish Thoroughbreds. RESULTS: Serum concentrations of bone and cartilage biomarkers: osteocalcin, c-terminal telopeptide of type I collagen, total glycosaminoglycans (GAG), chondroitin sulfate epitope and c-propeptide of type II procollagen (CPII) were evaluated in the beginning and the next 3 months of one racing season in a cohort of twenty-six 2-year-old Polish racehorses. Exit criteria were diagnosis of musculoskeletal injury, leading to > 5 days off training (n = 8), or completion of 3 study months with no training interruptions (n = 18). Normalized results and matching archival data from 35 2-year-old North American racehorses was used for logistic regression analysis to identify universal predictors of injury. Mean GAG and CPII levels were lower in injured group comparing to control, which is consistent with previous findings in racehorses. These biomarkers were also identified as predictors of injury in the mixed population model. Population origin had no significant effect on predictive value of evaluated biomarkers (Wald test p = 0.137). Decreased osteocalcin and increased c-terminal telopeptide of type I collagen levels in injured horses comparing to controls were specific for Polish population and signalized disruption in bone turnover homeostasis. CONCLUSIONS: Changes in serum GAG and CPII in racehorses at risk of injury appear to be similar across distinct populations while dynamics of serum bone marker is more population-specific.

Donor age affects proteome composition of tenocyte-derived engineered tendon.

BACKGROUND: The concept of tissue engineering is to deliver to the injury site biological scaffolds carrying functional cells that will enhance healing response. The preferred cell source is autologous in order to reduce immune response in the treated individual. However, in elderly patients age-related changes in synthetic activity of the implanted cells and subsequent alterations in tissue protein content may affect therapeutic outcomes. In this study we investigated the effect of donor age on proteome composition of tenocyte-derived tendon tissue-engineered constructs. RESULTS: Liquid chromatography tandem mass spectrometry was used to assess the proteome of tissue-engineered constructs derived from young and old equine tenocytes. Ageing was associated with altered extracellular matrix composition, especially accumulation of collagens (type I, III and XIV), and lower cytoskeletal turnover. Proteins involved in cell responsiveness to mechanical stimuli and cell-extracellular matrix interaction (calponin 1, palladin, caldesmon 1, cortactin) were affected. CONCLUSIONS: This study demonstrated significant changes in proteome of engineered tendon derived from young and old tenocytes, indicating the impact of donor age on composition of autologous constructs.

The Effect of Different Types of Musculoskeletal Injuries on Blood Concentration of Serum Amyloid A in Thoroughbred Racehorses.

BACKGROUND: Training-induced muscle, skeletal and joint trauma may result in acute phase response reflected by the changes in the blood concentration of serum amyloid A (SAA) in racehorses. It remains yet unclear if such systemic reaction could be triggered by sport injuries and what is the impact of different types of musculoskeletal trauma on SAA concentrations in racehorses. This study aimed to determine changes in the SAA blood concentration in racehorses with different types of injuries of musculoskeletal system. MATERIALS AND METHODS: The study involved 28 racehorses diagnosed after the race with bone fractures (n = 7), dorsal metacarpal disease (n = 11), joint trauma (n = 4) or tendon and muscle trauma (n = 6) and 28 healthy control racehorses. Serum samples were collected twice, between 1 and 4 days of the injury or succesful completion of the race. SAA concentration was measured using the commercial ELISA kit. Differences between mean SAA concentration in respective groups were analyzed using ANOVA and Tukey post-hoc test. RESULTS: Mean SAA concentration within the first 4 days of the injury of muscle and tendon was significantly higher than in bone fractures, dorsal metacarpal disease, joint trauma or in the healthy horses (p<0,001). There were no significant differences between the other groups. CONCLUSIONS: Strain injuries of muscle and tendons can cause a moderate increase in SAA blood concentration in racehorses, reflecting the occurrence of the acute phase response. Similar reaction is not observed in the stress-related bone injuries.