Influence of inflammation on the expression of microRNA-140 in extracellular vesicles from 2D and 3D culture models of synovial-membrane-derived stem cells.

Background: In osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an in vitro inflammatory environment. Methods: Four experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1ß and TNFα. The culture media samples were collected at 24 h, 72 h, and 120 h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level. Results: Encapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72 h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72 h and in the 2D-OA group at 120 h (p < 0.001). However, the 3D-OA culture showed higher expression of the mRNA Adamts5 in the EVs at 120 h. Conclusion: The responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.

Biocompatibility of hydrogel derived from equine tendon extracellular matrix in horses subcutaneous tissue.

Tendinopathies account for a substantial proportion of musculoskeletal injuries. To improve treatment outcomes for partial and total tendon ruptures, new therapies are under investigation. These include the application of mesenchymal stem cells (MSCs) and biocompatible scaffolds derived from the Extracellular Matrix (ECM). Synthetic polymer hydrogels have not demonstrated results as promising as those achieved with ECM hydrogels sourced from the original tissue. This study aimed to evaluate the biocompatibility of a hydrogel formulated from equine tendon ECM. Six horses were administered three subcutaneous doses of the hydrogel, with a saline solution serving as a control. Biopsies were conducted on days 7, 14, and 56 post-application to gauge the hydrogel's impact. Throughout the experiment, the horse's physical condition remained stable. Thermographic analyses revealed a temperature increase in the treated groups compared to the control group within the initial 12 h. The von Frey test, used to measure the mechanical nociceptive threshold, also showed significant differences between the treated group and the control group at 6 h, 21 days, and 28 days. Histopathological analyses identified an inflammatory response on day 7, which was absent on days 14 and 56. Transmission electron microscopy indicated a decrease in inflammatory cellularity, while immunohistochemistry staining suggested an increased presence of inflammatory factors on day 14. In summary, the hydrogel is easily injectable, triggers a temporary local inflammatory response, and integrates into the adjacent tissue from day 14 onwards.

Flunixin Meglumine Is Superior to Meloxicam for Providing Analgesia after Surgical Castration in 2-Month-Old Goats.

Farm animals are exposed to various painful procedures during their productive lives, making it necessary to implement anesthetic and analgesic protocols. However, there are few studies evaluating the effectiveness of these drugs. Our objective was to compare the analgesic effects of two nonsteroidal anti-inflammatory drugs (NSAIDs): meloxicam (MEL) and flunixin meglumine (FLU), in goat kids subjected to surgical castration under local anesthesia. Anglo-Nubian goat kids (60 days old) were allocated into two groups: MEL (n = 9), and FLU (n = 8), each administered 5 min before starting castration. All had been previously subjected to local anesthesia with lidocaine, injected bilaterally into the testes, plus subcutaneous in the scrotal raphe. Pain sensitivity was evaluated using the von Frey monofilaments test. Reactions were recorded before castration (M0), immediately after castration (M1), and once-daily for three consecutive days post-castration (M2, M3, and M4, respectively). Pain assessments were conducted in three body regions: at four points of the scrotum (dorsal and ventral; left and right lateral; R1); medial region of the pelvic limb, gracilis muscle (R2); and hypogastric region of the abdomen (R3). MEL goats had considerably greater pain reaction in R1 and R2 over time, mainly in M2; therefore, FLU was a more effective analgesic than MEL, resulting in less pain reaction.

Production of Cytotoxic Antibodies After Intra-Articular Injection of Allogeneic Synovial Membrane Mesenchymal Stem Cells With and Without LPS Administration.

Allogeneic mesenchymal stem cells (MSC) are widely used in clinical routine due to the shorter expansion time and reliability of its quality. However, some recipients can produce alloantibodies that recognize MSCs and activate the immune system, resulting in cell death. Although antibody production was already described after MSC injection, no previous studies described the immune response after intra-articular MSC injection in acute synovitis. This study aimed to evaluate the influence of inflammation on immune response after single and repeated intra-articular injections of synovial membrane MSC (SMMSC). Horses were divided in three groups: control group (AUTO) received autologous synovial membrane MSCs; whereas group two (ALLO) received allogeneic SMMSCs and group three (ALLO LPS) was submitted to acute experimental synovitis 8 h before SMMSCs injection. The procedure was repeated for all groups for 28 days. Physical and lameness evaluations and synovial fluid analysis were performed. Sera from all animals were obtained before and every 7 days after each injection up to 4 weeks, to perform microcytotoxicity assays incubating donor SMMSCs with recipients' sera. The first injection caused a mild and transient synovitis in all groups, becoming more evident and longer in ALLO and ALLO LPS groups after the second injection. Microcytotoxicity assays revealed significant antibody production as soon as 7 days after SMMSC injection in ALLO and ALLO LPS groups, and cytotoxicity scores of both groups showed no differences at any time point, being equally different from AUTO group. Although inflammation is capable of inducing MHC expression in MSCs, which enhances immune recognition, cytotoxicity scores were equally high in ALLO and ALLO LPS groups, making it difficult to determine the potentiation effect of inflammation on antibody production. Our findings suggest that inflammation does not display a pivotal role in immune recognition on first allogeneic MSC injection. In a translational way, since specific antibodies were produced against MSCs, patients that need more than one MSC injection may benefit from a first allogeneic injection followed by subsequent autologous injections.

Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties.

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.

In Vitro Biological Performance of Alginate Hydrogel Capsules for Stem Cell Delivery.

Encapsulation of biological components in hydrogels is a well described method for controlled drug delivery of proteins, tissue engineering and intestinal colonization with beneficial bacteria. Given the potential of tissue engineering in clinical practice, this study aimed to evaluate the feasibility of encapsulation of adipose tissue-derived mesenchymal stem cells (MSCs) of mules in sodium alginate. We evaluated capsule morphology and cell viability, immunophenotype and release after encapsulation. Circular and irregular pores were observed on the hydrogel surface, in which MSCs were present and alive. Capsules demonstrated good capacity of absorption of liquid and cell viability was consistently high through the time points, indicating proper nutrient diffusion. Flow cytometry showed stability of stem cell surface markers, whereas immunohistochemistry revealed the expression of CD44 and absence of MHC-II through 7 days of culture. Stem cell encapsulation in sodium alginate hydrogel is a feasible technique that does not compromise cell viability and preserves their undifferentiated status, becoming a relevant option to further studies of tridimensional culture systems and in vivo bioactive agents delivery.

Physical and biological characterizations of TiNbSn/(Mg) system produced by powder metallurgy for use as prostheses material.

Titanium scaffolds with non-toxic ß stabilizing elements (Nb and Sn), Ti-34Nb-6Sn (TNS), and with magnesium as spacer (TNS/M), were processed by powder metallurgy, and sintered at 800 °C. The X-ray diffraction (XRD) pattern showed that materials are biphasic alloys, presenting 45 to 42% (wt %) in hcp (α-phase) and the rest is bcc (ß-phase), and the presence of a slight peak relating to TiO2 in both materials. Pores of approximately 50 µm for TNS and 300 µm to TNS/M were observed in the micrographic analysis by scanning electron microscopy (SEM). The wettability was higher for TNS/M compared to TNS. The elastic modulus was higher for TNS compared to TNS/M. Stem cells derived from equine bone marrow (BMMSCs) were used for in vitro assays. The morphologic and adhesion evaluation after 72 h, carried out by direct contact assay with the materials showed that the BMMSCs were anchored and adhered to the porous scaffolds, in the way the cytoplasmic extension was observed. The cellular migration, using the "wound healing" method, was significant for the groups treated with conditioned medium with materials in 24 h. Osteogenic differentiation of BMMSCs, assessed by calcium deposition and staining with Alizarin Red, was greater in the conditioned medium with TNS/M in 10 days of culture. Since the biological effects was good and the elastic modulus decreased in the system with magnesium is a promising new content titanium alloy for biomedical application.

Accuracy of differences in blood and peritoneal glucose to differentiate between septic and non-septic peritonitis in horses.

Difference in blood and peritoneal glucose (DBPG) is used in clinical practice to support a diagnosis of septic peritonitis in horses. It is inexpensive, easy and rapid to perform. The aim of this study was to evaluate the accuracy of the DBPG to differentiate between septic and non-septic peritonitis in horses. Blood and peritoneal fluids were harvested from suspected animals. Plasma and peritoneal glucose levels, total nucleated cell count, direct microscopic and microbiological examinations of the peritoneal fluid were evaluated. Using DBPG levels, the animals were classified into two groups: difference ≥ 50 mg/dL (positive test) and difference < 50 mg/dL (negative test). Positive microbiological examination and/or presence of bacteria in direct microscopic examination was used as a gold standard to detect septic peritonitis. The accuracy parameters analysed were: sensitivity, specificity, and positive/negative predictive values, for which the results were respectively: 0.23, 0.91, 0.60 and 0.67. Due to poor accuracy, other cut-off margins and peritoneal glucose concentrations were evaluated. The test was considered most accurate when the DBPG was zero with sensitivity, specificity, and positive/negative predictive values of 0.85, 0.82, 0.73, 0.90 respectively. Peritoneal glucose concentrations alone were not a reliable feature to detect peritonitis. DBPG ≥50 mg/dL, widely used for the diagnosis of septic peritonitis, does not have a good accuracy and the DBPG = 0 has a better accuracy for detecting the disease.

Adjuvant intraperitoneal ceftriaxone in the treatment of septic peritonitis in horses.

BACKGROUND: Intraperitoneal administration of ceftriaxone maintains therapeutic abdominal concentrations for 24 hours in healthy horses. Therefore, it is a possible treatment for septic peritonitis. The aim of this study was to evaluate the efficacy of ceftriaxone as an adjuvant treatment in horses with septic peritonitis. METHODS: Twenty-six horses with clinical signs, sonography and/or laboratory findings of septic peritonitis were included. Peritoneal fluid was collected for microbiological culture and in vitro microbial sensitivity profile assessment. Daily intraperitoneal administration of ceftriaxone (25 mg/kg) was initiated with supportive and systemic antimicrobial treatment. The animals were divided into three groups: group 1-gastrointestinal tract injuries and abdominal surgery (excluding perforations/ruptures); group 2-not related to changes in the gastrointestinal tract; group 3-secondary to intestinal rupture and/or faeces contamination. RESULTS: The mean success rate of the treatment was 77 per cent (20/26 animals), with success rates of 84.6 per cent in group 1; 87.5 per cent, group 2; and 40 per cent, group 3. CONCLUSIONS: This is the first study to report adjuvant intraperitoneal treatment ceftriaxone for septic peritonitis in horses and indicates that this treatment can successfully treat septic peritonitis in horses.

Manejo e transporte de equinos fraturados / Management and transportation of fractured equine patients / Manejo y transporte de equinos fracturados

O atendimento emergencial e o transporte de equinos fraturados a centros de referência capacitados constituem procedimentos fundamentais para o sucesso de sua recuperação. Após a execução das medidas de estabilização do paciente, manejo das feridas e, se possível, avaliação radiográfica, realiza-se a imobilização externa da fratura de acordo com o local do membro onde esta se encontra, para melhor neutralização das forças atuantes. A imobilização de fraturas do esqueleto apendicular consiste basicamente no emprego de uma bandagem de Robert-Jones, sobreposta por uma ou mais talas posicionada(s) no(s) aspecto(s) dorsal, lateral, medial ou caudal/palmar/plantar. As fraturas cranianas e mandibulares podem ocasionar distúrbios neurológicos e oculares, temporários ou permanentes, e frequentemente acometem a cavidade nasal e os seios paranasais, gerando comprometimento respiratório. O transporte dos pacientes fraturados deve ser efetuado da maneira mais segura possível, com a finalidade de evitar maiores complicações do quadro do paciente. Deste modo, obtêm-se maior possibilidade de recuperação e melhor prognóstico para estes animais.(AU)

The emergency care and transportation of fractured equine patients to trained veterinary hospitals are fundamental procedures for the success of its recovery. After the stabilization procedures, wound management, and, if possible, radiographic evaluation on the patient, external immobilization of the fracture is performed depending on the place where the fracture is located, in order to better neutralize the acting forces. The immobilization of appendicular skeleton fractures consists on basically using Robert-Jones? bandage, overlapped with one or more splints positioned on the dorsal, lateral, medial or caudal/palmar/plantar aspect. Skull fractures may cause neurological and ocular disorders, both temporary or permanent, and frequently affect nasal cavity and paranasal sinuses, leading to respiratory problems. The transportation of fractured patients must be carried out in the safest way as possible, in order to avoid further complication to the patient?s condition. Thus, greater recovery possibility and better prognosis for these animals are obtained.(AU)

El atendimiento de emergencia y el transporte de equinos fracturados a centros de referencia capacitados, constituyen procedimientos fundamentales para el éxito de su recuperación. Tras la ejecución de medidas de estabilización del paciente, manejo de las heridas y, si posible, evaluación radiográfica, se realiza la inmovilización externa de la fractura de acuerdo con el lugar del miembro donde ésta se encuentre, para mejor neutralización de las fuerzas actuantes. La inmovilización de fracturas del esqueleto apendicular consiste en utilizar un vendaje de Robert-Jones, sobrepuesto por una o más férulas posicionadas de forma dorsal, lateral, medial o caudal/palmar/plantar. Las fracturas craneanas y mandibulares pueden causar disturbios neurológicos y oculares, temporarios o permanentes, y frecuentemente acometen la cavidad nasal y los senos paranasales, generando comprometimiento respiratorio. El transporte de los pacientes fracturados debe ser efectuado de la manera más segura posible, con el fin de evitar mayores complicaciones en el cuadro del paciente. De este modo se obtiene mayor posibilidad de recuperación y mejor pronostico para estos animales.(AU)