A comprehensive study of adult stromal cells derived from mesenchymal tissues and their application in tendon regeneration

This research project focused on study of adult stromal cells derived from equine and canine mesenchymal tissue and the application of these cell sources in musculoskeletal injuries, particularly in tendon repair. Adult MSCs gained general attention in both human and veterinary medicine, however despite the advances in this field, much studies are needed in order to better understand MSCs behavior. With this goal the following research project focused on: - A comprehensive characterization and study in vitro of mesenchymal stromal cells derived from peripheral blood of horses (ePB-MSCs) and from adipose tissue of dog (cA-MSCs). Moreover the possibility to cryopreserve these cells in the long-term period and the delivery methods of both ePB-MSCs and cA-MSCs in the short-term period was verified. - The use of MSCs from peripheral blood and from adipose tissue in the tendon regeneration process was investigated in two different work: the use of adipose tissue derived MSCs in the re-cellularization of a human biocompatible scaffold, and the use of MSCs derived from ovine peripheral blood injected in experimental tendon lesions of the deep digital flexor tendon (DDFT) in a ovine model. The full characterization of both ePB-MSCs and cA-MSCs was achieved using FACS sorting in order to identify the Cluster of Differentiation (CD) expression on the surface of these cells, by the study of population doubling time (PDT), the analysis of telomerase enzyme presence by Real Time PCR, and the alkaline phosphatase positivity. The differentiative potential of both ePB-MSCs and cA-MSCs was assessed by the in vitro induction of these cells into osteo, muscular and adipogenic lineages and verified by expression of tissue specific gene, like PPAR-y, Desmin, and Osteopontin (SPP-1). The same characterization and differentiative potential were then verified on ePB-MSCs and cA-MSCs after 1 year. Results obtained from this study furnished novel information on adult MSCs and confirmed the possibility to cryopreserve these cells in the long-term period for their potential applications in clinical field. The study on delivery of both ePB-MSCs and cA-MSCs in the short-term period, performed by exposing cells to different media, time and temperature, lead to the conclusion that cells could be delivered in PBS at room temperature no longer than 9-12 hrs; cells were also monitored for CD expression, for apoptotic resistance and s-galactosidase activity during different time intervals. The second challenge of this research project was on study of regeneration capability of adult MSCs derived from peripheral blood and from adipose tissue in tendon pathologies. A first work was performed using MSCs derived from human lipoaspirate in order to recellularize a human tendon scaffold to be used as insert in full-thickness lesions of flexor hand tendons. A previous re-cellularization was successfully obtained using Tris solution and enzymatic digestion, and the absence of native genonic DNA were assessed by standard PCR. Moreover a good protocol for re-cellularization was obtained, using a collagen matrix gel, which helped a good penetration of stromal cells inside the biocompatible scaffold. Secondly a comparation study among the effect of PB-MSCs, platelet rich plasma (PRP) or combination of both on tendon healing was performed using ovine model. Experimental lesions were made using collagenase 1A injection in the left deep digital flexor tendon (DDFT) of 18 Bergamasca sheep, using the right DDFT as internal control (lesioned, not treated). Sheep were further divided in two groups, the first euthanized after 1 month and the second after 4 months, and tendon were then analyzed by histological and immunohistochemical assays. Results obtained after 4 months showed a significant difference between the treated tendon of all group respect to the relative internal control (lesioned, not treated); notably after 4 months the group that received the treatment with MSCs alone or in combination with PRP, showed significant improvement of the healing process respect to PRP treated group, becoming similar to the normal healthy tendons.