Controlled release of canine MSC-derived extracellular vesicles by cationized gelatin hydrogels.

Introduction: Canine mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising form of regenerative therapy. Therapeutic application of EVs remains difficult due to the short half-life of EVs in vivo and their rapid clearance from the body. We have developed cationized gelatin hydrogels that prolong the retention of EVs to overcome this problem. Methods: Canine MSCs were isolated from bone marrow. MSC-derived EVs were isolated from the culture supernatant by ultracentrifugation. Gelatin was mixed with ethylene diamine anhydrate to cationized. Distinct cross-linked cationized gelatin hydrogels were created by thermal dehydration. Hydrogels were implanted into the back subcutis of mice in order to evaluate the degradation profiles. Hydrogels with collagenase were incubated at 37 °C in vitro to quantize the release of EVs from hydrogels. Lipopolysaccharide (LPS)-stimulated BV-2 cells were used to evaluate the immunomodulatory effect of EVs after release from the hydrogels. Results: The cationized gelatin hydrogels suppressed EV release in PBS. More than 60% of immobilized EVs are not released from the hydrogels. The cationized hydrogels released EVs in a sustainable manner and prolonged the retention time of EVs depending on the intensity of cross-linking after degradation by collagenase. The expression of IL-1ß in LPS-stimulated BV-2 cells was lower in EVs released from the hydrogels than in controls. Conclusions: Our results indicate that the controlled release of EVs can be achieved by cationized gelatin hydrogels. The released EVs experimentally confirmed to be effective in reducing proinflammatory response. The cationized gelatin hydrogels appear to be useful biomaterials for releasing canine MSC-derived EVs for regenerative therapy.

Extracellular Vesicles Derived From Canine Mesenchymal Stromal Cells in Serum Free Culture Medium Have Anti-inflammatory Effect on Microglial Cells.

Mesenchymal stem/stromal cells (MSCs) have been used as cell sources for treating dogs with naturally-occurring diseases. Extracellular vesicles (EVs) derived from MSCs are now recognized as pivotal to modulating the immune response and supporting tissue repair. Manufacture of MSC-EVs for clinical application mandates removal of the xeno-proteins, including fetal bovine serum. The objective of this study was to examine whether canine MSCs survived and secreted EVs in serum-free medium (SFM) conditions and to assess the immunomodulatory effect of EVs in vitro. Canine MSCs were found to survive and secrete EVs under SFM conditions. The surface markers of MSCs in the SFM were similar to MSCs in complete culture medium. Canine MSC-EVs had a diameter of ~300 nm and were positive for EV markers. MSC-derived EVs from the serum-free condition reduced the levels of IL-1ß by BV-2 cells in response to LPS stimulation. These results warrant further studies of the use of SFM for producing EVs derived from canine MSCs.

Impaired bone quality characterized by apatite orientation under stress shielding following fixing of a fracture of the radius with a 3D printed Ti-6Al-4V custom-made bone plate in dogs.

Custom-made implants have recently gained attention in veterinary medicine because of their ability to properly fit animal bones having a wide variety of shapes and sizes. The effect of custom-made implants on bone soundness and the regeneration process is not yet clear. We fabricated a 3D printed Ti-6Al-4V custom-made bone plate that fits the shape of the dog radius, and placed it into the radius where an osteotomy had been made. The preferential orientation of the apatite c-axis contributes to the mechanical integrity of the bone and is a reliable measure of bone quality. We determined this parameter as well as the bone shape and bone mineral density (BMD). The bone portion which lies parallel to the bone plate exhibited bone resorption, decreased BMD, and significant degradation of apatite orientation, relative to the portion outside the plate, at 7 months after the operation. This demonstrates the presence of stress shielding in which applied stress is not transmitted to bone due to the insertion of a stiff bone plate. This reduced stress condition clearly influences the bone regeneration process. The apatite orientation in the regenerated site remained different even after 7 months of regeneration, indicating insufficient mechanical function in the regenerated portion. This is the first study in which the apatite orientation and BMD of the radius were evaluated under conditions of stress shielding in dogs. Our results suggest that assessment of bone repair by radiography can indicate the degree of restoration of BMD, but not the apatite orientation.

Canine mesenchymal stromal cell-conditioned medium promotes survival and neurite outgrowth of neural stem cells.

We examined the paracrine action of canine mesenchymal stromal cells (MSCs) derived from bone marrow on the survival and differentiation of neural stem cells (NSCs) in vitro. MSCs were collected from the proximal end of the diaphysis of femur of healthy beagle dogs. The 70-80% confluent MSCs were re-fed with serum-free DMEM. The MSCs were incubated for 48 hr and the supernatant was collected as the conditioned medium (MSC-CM). The survival rate of NSCs in MSC-CM was significantly greater than in the medium without MSC-CM. The percentage of differentiated neurons and neurite length in MSC-CM was also significantly higher than in the medium without MSC-CM. These results suggested that canine MSC-CM promotes stem cell survival and neural differentiation of NSCs.