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Adipose‐derived mesenchymal stem cells (ASC) hold great promise in the treatment of many disorders including musculoskeletal system, cardiovascular and/or endocrine diseases. However, the cytophysiological condition of cells, used for engraftment seems to be fundamental factor that might determine the effectiveness of clinical therapy. In this study we investigated growth kinetics, senescence, accumulation of oxidative stress factors, mitochondrial biogenesis, autophagy and osteogenic differentiation potential of ASC isolated from horses suffered from equine metabolic syndrome (EMS). We demonstrated that EMS condition impairs multipotency/pluripotency in ASCs causes accumulation of reactive oxygen species and mitochondria deterioration. We found that, cytochrome c is released from mitochondria to the cytoplasm suggesting activation of intrinsic apoptotic pathway in those cells. Moreover, we observed up‐regulation of p21 and decreased ratio of Bcl‐2/BAX. Deteriorations in mitochondria structure caused alternations in osteogenic differentiation of ASCEMS resulting in their decreased proliferation rate and reduced expression of osteogenic markers BMP‐2 and collagen type I. During osteogenic differentiation of ASCEMS, we observed autophagic turnover as probably, an alternative way to generate adenosine triphosphate and amino acids required to increased protein synthesis during differentiation. Downregulation of PGC1α, PARKIN and PDK4 in differentiated ASCEMS confirmed impairments in mitochondrial biogenesis and function. Hence, application of ASCEMS into endocrinological or ortophedical practice requires further investigation and analysis in the context of safeness of their application.

Human adipose tissue is a great source of adult mesenchymal stem cells (MSCs) which are recognized from their ability to self‐renew and differentiation into multiple lineages. MSCs have promised a vast therapeutic potential in treatment many diseases including tissue injury and immune disorders. However, their regenerative potential profoundly depends on patients’ age. Age‐related deterioration of MSC is associated with cellular senescence mainly caused by increased DNA methylation status, accumulation of oxidative stress factors and mitochondria dysfunction. We found that DNA methyltransferase (DNMT) inhibitor i.e. 5‐Azacytidine (5‐AZA) reversed the aged phenotype of MSCs. Proliferation rate of cells cultured with 5‐AZA was increased while the accumulation of oxidative stress factors and DNA methylation status were decreased. Simultaneously the mRNA levels of TET proteins involved in demethylation process were elevated in those cells. Moreover, cells treated with 5‐AZA displayed reduced reactive oxygen species (ROS) accumulation, ameliorated superoxide dismutase activity and increased BCL‐2/BAX ratio in comparison to control group. Our results indicates that, treating MSCs with 5‐AZA can be justified therapeutic intervention, that can slow‐down and even reverse aged‐ related degenerative changes in those cells.

The influences of NSAIDs (Nonsteroidal Anti-inflammatory Drugs) – non-selective metamizole and selectively-acting tolfenamic acid were estimated on morphology, ultrastructure, and cytophysiological activity of canine (Ca) and equine (Eq) adipose-derived mesenchymal stem cells (ASCs). The lowest concentration of metamizole (0.01 mg/mL) stimulated the viability and cytophysiological activity of Ca ASCs and did not affect cell morphology. Stimulated cells possessed a proper, fibroblastic shape, with large, eccentrically located nuclei. Similar effects to those observed in Ca ASCs were found in Eq cells treated with both drugs. Cells cultivated with the intermediate (0.1 mg/mL) doses of NSAIDs displayed proper cell morphology, whereas cells cultivated in intermediate dose (0.1 mg/mL) became more flattened. The highest concentrations (1 mg/mL) of both drugs resulted in a cytotoxic effect in Ca and Eq ASCs. Based on these results, we conclude that stimulation of Ca and Eq ASCs with metamizole as well as Eq ASCs with tolfenamic acid can lead to positive effects only when the lowest drug concentrations are applied. This study indicates a different cellular response of canine and equine ASCs treated with metamizole and tolfenamic acid. The obtained data might be potentially useful in the study of functionalized veterinary biomaterials.

Disorders of the locomotive system, especially those occurring due to degenerative changes of the joints, are serious problems in daily veterinary medical practice. Steroid injections are the main way of treating these disorders. However, this approach brings usually only temporary effects of pain relief, and may cause many side effects. Alternative therapies focus on regeneration of damaged tissue using adult mesenchymal stem cells (MSCs). Since 2002, the great plasticity and immunomodulatory properties of MSCs isolated from adipose tissue (AdMSCs) have been used successfully in the treatment of degenerative joint diseases (DJD) of both dogs and horses. Possible simultaneous application of steroid therapy and stem cell transplantation could improve the commonly used clinical procedure. In this paper, the influence of the two steroid drugs (betamethasone and methylprednisolone) on AdMSCs was evaluated on the basis of morphology and proliferation rate. Both steroids positively influenced the viability and proliferation state of cells in a concentration of 0.01 mg/ml and 0.1 mg/ml, respectively. However, the concentration of 1 mg/ml had a cytotoxic effect. Moreover, the lower dosage of steroid drugs used in the experiment did not affect the morphology of cells and significantly increased cellular activity. In conclusion, our data demonstrate the stimulating effect of steroid drugs on cell morphology, proliferation rate and cytophysiological activity. These findings may influence the use of stem cells and steroids in applied regenerative veterinary medical practice in the future.

Equine metabolic syndrome (EMS) is a metabolic disorder characterized by excessive obesity and or/regional adiposity, insulin resistance (IR) and prior or current laminitis. In the course of the EMS, an important role exert systemic inflammation caused by excessive expression of proinflammatory cytokines. The aim of the current investigation was to examine relationships between expression IL-6 and TNF-α in adipose tissue and their concentration in peripheral blood in horses with EMS. On the basis of range of research procedures, horses were divided into two groups: group A (EMS horses, n=8) and group B (healthy horses with overweight, n=8). The concentration of the proinflammatory cytokines in the peripheral blood and their expression in the adipose tissue were determined. The results of the EMS group showed numerous macrophages and lymphocytes infiltration and increased diameters of adipocytes (P

Estudios recientes han revelado propiedades pluripotentes del tejido graso aislado de celulas madre mesenquimales. Estas celulas se utilizan con exito como factor terapeutico para muchos trastornos locomotores, siendo aun mas eficaz que las celulas madre de medula osea. Aisladas y cultivadas, AD-MSC se observaron, fotografiaron y midieron comparar celulas de dos especies diferentes.

In this article we demonstrate the efficiency of autologous transplantations of adipose-derived mesenchymal stem cells for equine bone spavin treatment. Horses qualified to the study were divided into three groups: (i) research - treated with intra-articular injections of autologous stem cells, (ii) comparison treated with steroid drugs and (iii) control - untreated. All animals underwent comprehensive clinical examination before and after treatment. Our research confirms the long-term beneficial influence resulting from stem cell therapy in horse bone spavin treatment, in contrast to routine steroid usage.

A main symptom of equine metabolic syndrome (EMS) in ponies is pathological obesity characterized by abnormal accumulation of fat deposits and inflammation. In this study, we analyzed the expression of two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in subcutaneous adipose tissue and the correlation with serum concentrations in peripheral blood of Welsh ponies. Based on clinical examination findings, the animals were divided into two groups: ponies affected with EMS (n = 8) and obese ponies (n = 8). The adipose tissue was examined using immunohistochemical analysis while concentrations IL-6 and TNF-a were measured using enzyme-linked immunosorbent assays (ELISAs). Additionally, histological characterization of the adipose tissue was performed. The results obtained showed that IL-6 expression in adipose tissue biopsies derived from animals with EMS was enhanced while TNF-α levels of both groups were comparable. Compared to the obese ponies, EMS animals also had significantly elevated levels of serum IL-6 and TNF-α. Histological analysis revealed macrophage infiltration and fibrosis in adipose tissue preparations from the EMS group. These data suggest that IL-6 may play a key role in the course of EMS in Welsh ponies. Our findings also demonstrated that analysis of pro-inflammatory cytokines levels in serum may serve as an additional tool for diagnosing EMS.