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.

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.

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.