281 USE OF MESENCHYMAL STEM CELLS COLLECTED FROM BONE MARROW OF ADULT DOGS FOR THE TREATMENT OF NON-UNION BONE FRACTURES

Abstract

The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy since they have the potential to replicate as undifferentiated cells and can be induced to differentiate into bone, fat, cartilage, tendon, muscle, and other tissues. The establishment of a pattern to culture MSC is the first step to start further experiments including MSC differentiation, cell therapy, and autologous transplants. In the present study, three dogs presenting non-union bone fractures, with one to two years of evolution, one in the middle third of the femur and the other two on the distal third of the radius and the ulna, were submitted to stem cell transplantation. After general anesthesia (IV) MSCs were aspirated from the head of the humerus from each dog and centrifuged at 1500 rpm for 10 minutes to eliminate the serum and the fat. The material was resuspended in a 1/1 proportion with DMEM High Glucose (GIBCO, Grand Island, NY, USA) and centrifuged with 7 mL of Ficoll-Paque (density 1.077 g mL^–1; Amersham Biosciences, Sao Paulo, Brazil) at 1500 rpm for 40 min. The middle ring formed was aspirated and washed in DMEM High Glucose. The pellet was resuspended in DMEM High glucose with 20% fetal calf serum, penicillin, amphotericin B, and streptomycin. Primary cultures were established and subcultivated for as many as 4 passages. MSCs were cultured in humidified incubators with 5% CO₂ in air and allowed to adhere for 120 h, followed by media change every 3 to 4 days. When cultures reached more than 90% confluence, adherent cells were detached with 0.05% trypsin-EDTA (GIBCO) and replanted (passage) at a density of 2 × 10⁶ per 175 cm² flask. To confirm the lineage of the MSCs, anti-vimentin immunocytochemistry was performed. After about 15 days of culture the cells were resuspended at a concentration of 2 × 10⁷ cells mL^–1 and prepared for transfer. The animals were subjected to a surgical procedure where the metallic implant (n = 3) and the fibrous tissue present in the non-union fracture site were removed and the fracture was stabilized with the use of steel bone plate and screws. At the end of the fracture stabilization, the cells were transferred directly into the fracture site. Radiographic exams were performed on the post-surgical site immediately after the surgery and monthly until complete bone healing, which was considered satisfactory 5 to 8 months after the surgery in all animals. The clinical results indicate that the therapy with homologous mesenchymal stem cells is a promising and efficient method to treat non-union bone fractures in dogs.

This work was supported by FAPESP (grants 06/54575-0 and 06/56738-4).