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.

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.

Low Mg content on Ti-Nb-Sn alloy when in contact with eBMMSCs promotes improvement of its biological functions.

Magnesium is a metal used in the composition of titanium alloys and imparts porosity. Due to its osteoconductive, biocompatible and biodegradable characteristics, its application in the development of biomedical materials has become attractive. This study aimed to evaluate the influence of magnesium present in porous Ti-Nb-Sn alloys, which have a low elastic modulus in adhesive, osteogenic properties and the amount of reactive intracellular oxygen species released in mesenchymal stem cells derived from bone marrow equine bone (eBMMSCs). Mechanical properties of the alloy, such as hardness, compressive strength and elastic modulus, were analyzed, as well as surface morphological characteristics through scanning electron microscopy. The evaluation of magnesium ion release was performed by atomic force spectroscopy. The biological characteristics of the alloy, when in contact with the alloy surface and with the culture medium conditioned with the alloy, were studied by SEM and optical microscopy. Confirmation of osteogenic differentiation by alizarin red and detection of ROS using a Muse® Oxidative Stress Kit based on dihydroetide (DHE). The alloy showed an elastic modulus close to cortical bone values. The hardness was close to commercial Ti grade 2, and the compressive strength was greater than the value of cortical bone. The eBMMSCs adhered to the surface of the alloy during the experimental time. Osteogenic differentiation was observed with the treatment of eBMMMSCs with conditioned medium. The eBMMSCs treated with conditioned medium decreased ROS production, indicating a possible antioxidant defense potential of magnesium release.

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.

Evaluation of alginate hydrogel encapsulated mesenchymal stem cell migration in horses.

Osteoarthritis is an incapacitating disease characterized by pain and a progressive decrease in joint mobility. The implantation of mesenchymal stem cells (MSCs) has shown promising results for its treatment. The challenge remains to keep the cells longer at the site of action, increasing their therapeutic potential. The aim of this study was to evaluate the effectiveness of the Qtracker® 655 nanocrystal marking on allogeneic synovial membrane (SM) MSCs, encapsulated in alginate hydrogel, evaluating the migration of these cells. The 10 radiocarpal joints were submitted to arthroscopic surgery (D0), divided into two groups. The chondral defect was treated according to the group: GA free-labelled MSCSM and GB labelled MSCSM microcapsules. Seven days after lesion induction and implantation of labelled cells, biopsies of the lesion site were performed in two animals, and fragments of SM and joint capsule also collected, which were frozen and later processed for fluorescence microscopy. The synovial fluid of the three animals was analyzed by flow cytometry three times - 3, 7 and 21 days after application. The cellular marking with the nanocrystals allowed the visualization of the cells in cartilage, synovial membrane, synovial fluid and articular capsule, but with a predilection for the synovial membrane and the lesion site was scarce. The labelled MSCSM in microcapsules were scarce in the synovial fluid and could be related to the small quantity of MSCs leaving the pores of the microcapsules, also favorable results, as the cells release paracrine effects acting for a long period until the cellular differentiation.

Correction to: Culture of mesenchymal stem cells derived from equine synovial membrane in alginate hydrogel microcapsules.

The original article [1] contained a minor error regarding the mean diameter of the alginate microcapsules described in relation to Fig. 4 in the Results section. The microcapsules had an actual mean diameter of 3000 µm instead of 1000 µm as mistakenly mentioned in the original article.

Allogeneic mesenchymal stem cell transplantation in healthy equine superficial digital flexor tendon: A study of the local inflammatory response.

The superficial digital flexor tendon (SDFT) is a structure frequently affected by injuries in high-performance athletic horses, and there are limited therapeutic options. Regenerative medicine has evolved significantly in treating different illnesses. However, understanding the cellular behaviour during mesenchymal stem cell (MSC) transplantation in healthy tissues is not fully known yet. To address the inflammatory response induced by allogeneic MSC transplantation, this study evaluated the local inflammatory response after the application of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) in the equine tendon compared to an autologous transplant and the control group. Eighteen thoracic limbs (TL) in nine animals were divided into three groups and subjected to the application of AT-MSCs in the healthy tendon. In the allogeneic group (Gallog), the animals received an allogeneic AT-MSC application in the TL. The autologous group (Gauto) received an application of autologous cells in the TL, and in the control group (Gcont), phosphate-buffered saline (PBS) was applied. There were no significant differences among the evaluated groups in the physical, morphological, thermography, and ultrasonography analyses. A higher number of CD3-positive lymphocytes was observed in the Gauto group compared to the control (P < 0.05). Additionally, we did not observe different expressions of CD172 and microvascular density among the groups. The allogeneic transplantation of AT-MSCs did not result in an adverse or inflammatory reaction that compromised the use of these cells in this experiment. Their behaviour was similar to that of autologous transplantation.

Alterações morfológicas na capsula do casco em potros Crioulos do nascimento ao desmame / Morphometric changes in the hoof capsule of Criollo foals from birth to weaning

South America has numerous Criollo horse breeding farms; however, information on foal hoof growth is still limited and identifying the ideal periods to apply corrective trimming is a frequent concern for horse owners. In the present study, a morphometric analysis of hoof growth was performed on 46 Criollo foals from birth to weaning (0-8 months). Results showed that hoof growth rate was higher in the first four months followed by a decrease until the eighth month. Average growth rate of the hoof was 0.21cm per month, whereas that of the heel was 0.14cm per month. However, no significant differences were observed between medial and lateral heel length or between limbs. Coronary band perimeter and solar width showed an average increase of 0.97cm and 0.46cm per month, respectively, and were significantly correlated (r=0.99, P≤0.01). The characteristic most positively correlated to biometric variables was foal age, followed by solar width, toe length, and coronary band perimeter. In conclusion, hoof length increase in Criollo foals was more intensive during the first four months of life.

A América do Sul possui um grande número de criatórios da raça Crioula, no entanto, há uma carência de informações sobre o desenvolvimento natural dos cascos dos potros, tornando a identificação de períodos ideais para o casqueamento corretivo uma dúvida frequente entre os criadores. Portanto, o objetivo deste estudo foi realizar a biometria natural (do nascimento aos 8 meses) no casco de 46 potros da raça Crioula. Os resultados indicaram uma taxa de crescimento mais rápido nos primeiros 4 meses, com subsequente desaceleração até o desmame. O crescimento médio do casco foi em média 0,21cm mo-1, enquanto o comprimento do talão foi de 0,14cm mo-1. Não foram observadas diferenças significativas no equilíbrio médio/lateral do casco ou entre os membros durante o período experimental. O perímetro da banda coronária e a largura solar do casco apresentaram um crescimento médio de 0,97 e 0,46cm mo-1, respectivamente, e foram altamente correlacionados (r=0,99, P≤0,01). A idade dos potros foi a característica mais correlacionada positivamente, seguida da largura solar, do comprimento do casco e do perímetro da banda coronária. Nós concluímos que o crescimento do casco em potros da raça Crioula foi mais intenso durante os quatro primeiros meses de vida.

Alterações morfológicas na capsula do casco em potros Crioulos do nascimento ao desmame / Morphometric changes in the hoof capsule of Criollo foals from birth to weaning

South America has numerous Criollo horse breeding farms; however, information on foal hoof growth is still limited and identifying the ideal periods to apply corrective trimming is a frequent concern for horse owners. In the present study, a morphometric analysis of hoof growth was performed on 46 Criollo foals from birth to weaning (0-8 months). Results showed that hoof growth rate was higher in the first four months followed by a decrease until the eighth month. Average growth rate of the hoof was 0.21cm per month, whereas that of the heel was 0.14cm per month. However, no significant differences were observed between medial and lateral heel length or between limbs. Coronary band perimeter and solar width showed an average increase of 0.97cm and 0.46cm per month, respectively, and were significantly correlated (r=0.99, P≤0.01). The characteristic most positively correlated to biometric variables was foal age, followed by solar width, toe length, and coronary band perimeter. In conclusion, hoof length increase in Criollo foals was more intensive during the first four months of life.(AU)

A América do Sul possui um grande número de criatórios da raça Crioula, no entanto, há uma carência de informações sobre o desenvolvimento natural dos cascos dos potros, tornando a identificação de períodos ideais para o casqueamento corretivo uma dúvida frequente entre os criadores. Portanto, o objetivo deste estudo foi realizar a biometria natural (do nascimento aos 8 meses) no casco de 46 potros da raça Crioula. Os resultados indicaram uma taxa de crescimento mais rápido nos primeiros 4 meses, com subsequente desaceleração até o desmame. O crescimento médio do casco foi em média 0,21cm mo-1, enquanto o comprimento do talão foi de 0,14cm mo-1. Não foram observadas diferenças significativas no equilíbrio médio/lateral do casco ou entre os membros durante o período experimental. O perímetro da banda coronária e a largura solar do casco apresentaram um crescimento médio de 0,97 e 0,46cm mo-1, respectivamente, e foram altamente correlacionados (r=0,99, P≤0,01). A idade dos potros foi a característica mais correlacionada positivamente, seguida da largura solar, do comprimento do casco e do perímetro da banda coronária. Nós concluímos que o crescimento do casco em potros da raça Crioula foi mais intenso durante os quatro primeiros meses de vida.(AU)