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Simple Summary  Recently, mesenchymal stromal cells have been proposed as a valid approach for treating osteoarthritis diseases, mainly due to the secretion of soluble factors and extracellular vesicles known as secretome. In this paper, an injectable freeze-dried pharmaceutical powder containing canine mesenchymal stromal cells-secretome (Lyosecretome) has been formulated, and in vitro potency experiments were conducted. Furthermore, the Lyosecretome was applied in dogs affected by osteoarthritis to assess its safety. Results indicated that intra-articular injection of allogeneic Lyosecretome is safe and does not induce an adverse response, thus paving the way for future studies regarding the efficacy.    Abstract  In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 10 6  cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.

Canine Mesenchymal Cell Lyosecretome Production and Safety Evaluation after Allogenic Intraarticular Injection in Osteoarthritic Dogs