The Role of Mesenchymal Stem Cell Secretome (Extracellular Microvesicles and Exosomes) in Animals' Musculoskeletal and Neurologic-Related Disorders.

The advances in regenerative medicine are very important for the development of medicine and the discovery of stem cells has shown a greater capacity to raise the level of therapeutic quality while their use becomes more accessible, especially in their mesenchymal form. In veterinary medicine, it is not different. The use of those cells, as well as recent advances related to the use of their extracellular vesicles, demonstrates a great opportunity to enhance therapeutic methods and ensure more life quality for patients, which can be in clinical or surgical treatments. Knowing the advances in these modalities and the growing clinical and surgery research and demands for innovations in orthopedic and neurology medicines, this paper aimed to review the literature about the methodologies of use and applications such as the pathways of action and the advances that were postulated for microvesicles and exosomes derived from mesenchymal stem cells in veterinary medicine, especially for musculoskeletal disorders and related injuries.

In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album.

INTRODUCTION: The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. OBJECTIVE: This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). METHODS: MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. RESULTS: At the concentration of 10 µL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 µL/mL presented absolute percentages of initial apoptosis twice as high as at 10 µL/mL, which was similar to the control (untreated group). CONCLUSION: The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

Induced resistance to ifosfamide in osteosarcoma cells suggests a more aggressive tumor profile.

Aims: Osteosarcoma (OS) is the most common primary malignant bone sarcoma among children and adolescents. Treatment is based on neo-adjuvant and adjuvant chemotherapy, using the standard drugs cisplatin, methotrexate, doxorubicin, and ifosfamide (IFO). Due to the high capacity of tumor resistance, the current work aimed to analyze genes related to cycle control and cell differentiation in OS cells sensitive to and with induced resistance to IFO. This was to assess whether the differentiated expression of these genes may affect resistance to the drug IFO used in OS treatment, and thus establish possible biomarkers of disease progression. Materials and methods: In this work, the treatment-sensitive OS U2OS lineage was used, and the same lineage was submitted to the process of induction of IFO resistance. These cells were evaluated by MTT, migration and proliferation assays and submitted to gene expression analysis. Key findings: The results demonstrate that after induction of resistance to IFO, resistant U2OS cells show a more aggressive tumor behavior, with greater capacity for cell migration, proliferation, and invasion compared to sensitive cells. Gene analysis indicates that resistance-induced cells have differentiated expression of the genes EPB41L3, GADD45A, IER3, OXCT1, UBE2L6, UBE2A ALPL, and EFNB2. Our results suggest new perspectives on possible resistance biomarkers, especially the genes EFNB2 and EPB41L3, given that these genes have rarely been studied their expression linked to osteosarcoma. They show how the resistance induction model can be useful for studies on tumor cell behavior.

Canine atopic dermatitis attenuated by mesenchymal stem cells.

OBJECTIVE: To evaluate the use of mesenchymal stem cells (MSCs) in the attenuation of canine atopic dermatitis (AD). MATERIALS AND METHODS: Sixteen dogs were selected and divided into three groups, mild, moderate, and severe, according to the Canine Atopic Dermatitis Extent and Severity Index (CADESI-4). They were evaluated for 82 days. The protocol recommended in this experiment was to inject 2 × 106/kg bodyweight of MSC's in all groups by the intravenous route with intervals of applications of 21 days. The degree of pruritus was evaluated by examining the visual analog scale, the CADESI-4, the histopathology of the skin, hematological and biochemical parameters, the pyogenic effect of MSCs, and the thickness of the epidermis. RESULTS: There was a significant difference in the reduction of epidermal thickness in the moderate and severe groups. Hematological, biochemical, and body temperature parameters remained within normal limits for the species with no side effects. CONCLUSION: MSCs attenuated the clinical signs of AD.

Intraovarian injection of mesenchymal stem cells improves oocyte yield and in vitro embryo production in a bovine model of fertility loss.

Valuable female cattle are continuously subject to follicular puncture (ovum pick-up - OPU). This technique is commonly used for in-vitro embryo production, but may result in ovarian lesion. Mesenchymal stem cells (MSC) ameliorate the function of injured tissues, but their use to treat ovarian lesions in cattle has not been established. We investigated whether a local injection of MSC would reduce the negative effects of repeated OPU under acute and chronic scenarios in bovines. First, we performed four OPU sessions and injected 2.5 × 106 MSCs immediately after the 4th OPU procedure (n = 5). The treated organs (right ovary) were compared to their saline-treated counterparts (left), and presented superior production of oocytes and embryos in the three following OPU sessions (P < 0.05). Then, cows with progressive fertility loss went through three OPU sessions. Animals received MSC, saline, or MSC + FSH in both ovaries after the first OPU. In the two following OPU sessions, the MSC and MSC + FSH - treated groups failed to present any significant alteration in the number of oocytes and embryos compared to saline-treated animals. Thus, MSC have beneficial effects on the fertility of OPU-lesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

Homoeopathic Viscum album (10-3) is More Cytotoxic to in vitro Culture of Human Breast Cancer Cell Line than to Human Mesenchymal Stem Cells

BackgroundBreast cancer has been considered a public health problem and homeopathic treatments are becoming increasingly recommended due to its ways of action and absence of adverse effects. MCF-7 is an adenocarcinoma of human breast cell line useful as preclinicalmodel to screen therapeutic agents such as ultra-diluted Viscum album, an European plant which extract is commonly used in cancer therapy. AIMS MCF-7 and mesenchymal stem cells (MSC) were used to evaluate the in vitrocytotoxicity of homoeopathic Viscum album 1x10-3(VA3X). Methodscells were cultured for 24 hours in controlled environment (37.5oC and 5% CO2) in 96-well plates. After this time, VA3X was added to the culture medium in concentrations varying from 10 to 100 L/mL.A control group was maintained with culture medium only. Cells were cultivated for 48 hours in these conditions for evaluation of cell viability by MTT assay. ResultsHigher cytotoxicity was observed in MCF-7 when compared to MSC, as the lower concentration of VA3X was capable of inducing tumor cell death and not healthy cell death. The MTT assay results were that 42 L/mL of VA3X reduced MCF-7 cells viability to 50% and 62 L/mL reduced MSC cells to the same percentage, what means that tumor cells are more sensible to VA3X than heathy cells. ConclusionViscum albumpresented higher cytotoxic action on human breast cancer cell line culture than on mesenchymal stem cells. This medicine is extensively used against cancer, and the use of the homoeopathic form of it brings new possibilities as no or fewer adverse effects would be present.(AU)

Hemato-biochemical profile of meat cattle submitted to different types of pre-loading handling and transport times.

Pre-loading handling and conditions of transport are related to welfare, disease risk and product quality of production animals. These steps continue to be one of the major animal management problems in Brazil. This study evaluated the effects of different types of pre-loading handling and road transport times on the haematological and biochemical traits of cattle. Eighteen male cattle were submitted to three travel times (24, 48 and 72 h) in a truck soon after load using different types of pre-loading handling: traditional (rough handling), training (gentle handling) and use of flags to movement cattle. Haematological traits, blood biochemical measures as well as blood and faecal cortisol were analysed in order to assess animal welfare and physiological status. The traditional management showed to be more stressful, also had animals with a greater number of neutrophils and lower numbers of lymphocytes than handling with flags, showing that animals submitted to more stressful situations can have compromised immune system. Serum aspartate aminotransferase concentrations were within the reference levels and when taken together with increased creatine kinase patterns observed indicate muscle damage in traditional management. Decrease in glucose concentrations over time from traditional management to flag management was observed, while fructosamine was increased in traditional management with 72 h of travel. When taken together, all reported factors, immune, enzymatic, energetic and hormonal, indicate that the quality of pre-loading handling and time of transport were determinant for animal welfare, its homeostatic balance and sanitary conditions.