Veterinary Regenerative Medicine for Musculoskeletal Disorders: Can Mesenchymal Stem/Stromal Cells and Their Secretome Be the New Frontier?

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials

Three-Dimensional Bioprinted Controlled Release Scaffold Containing Mesenchymal Stem/Stromal Lyosecretome for Bone Regeneration: Sterile Manufacturing and In Vitro Biological Efficacy

Recently, 3D-printed scaffolds for the controlled release of mesenchymal stem cell (MSC) freeze-dried secretome (Lyosecretome) have been proposed to enhance scaffold osteoinduction and osteoconduction; coprinting of poly(ε-caprolactone) (PCL) with alginate hydrogels allows adequate mechanical strength to be combined with the modulable kinetics of the active principle release. This study represents the feasibility study for the sterile production of coprinted scaffolds and the proof of concept for their in vitro biological efficacy. Sterile scaffolds were obtained, and Lyosecretome enhanced their colonization by MSCs, sustaining differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralized matrix detected by alizarin red was significantly higher for the Lyosecretome scaffolds. The amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the Lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with the Lyosecretome scaffolds after 14 and 28 days. Overall, these results prove the role of MSC secretome, coprinted in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC secretome are now available for in vivo pre-clinical tests of bone regeneration

Canine mesenchymal cell lyosecretome production and safety evaluation after allogenic intraarticular injection in osteoarthritic dogs

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 char-acterized 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 activ-ity, 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 intraarticular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 106 cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyalu-ronic 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, func-tional 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

Equine mesenchymal stem/stromal cells freeze-dried secretome (Lyosecretome) for the treatment of musculoskeletal diseases: Production process validation and batch release test for clinical use

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchy-mal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome pro-teomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases

Silk-based Drug Delivery Systems

Silk proteins show excellent biocompatibility, controllable biodegradability and non-immunogenicity, and as such are studied extensively worldwide for biomedical applications. In particular, there is increasing interest in their use for drug delivery systems. This focussed book on silk proteins for drug delivery systems, delves into a key emerging area to outline the concepts and define the field. Covering spider silk and silk worm cocoons, the editors elucidate the extraction, structure and properties of silk sericin and silk fibroin. Showing how these proteins are employed in micro and nano drug delivery systems, their use in pre-clinical and clinical trials, and closing with chapter on sustainability- driven innovation in the pharma industry, this book is ideal for graduates and researchers in biomaterials science and pharmaceutical science

Sistemi microparticellari per la veicolazione di farmaci, cellule e molecole bioattive.

L’impiego di sistemi microparticellari per la veicolazione, il direzionamento e il rilascio controllato di farmaci è ormai consolidato nella terapia farmacologica di molte patologie; nel corso degli anni sono state messe a punto svariate tecnologie sia per la realizzazione di prototipi, sia per la produzione industriale di sistemi sofisticati. E’ stato possibile, pertanto, ottimizzare le potenzialità terapeutiche di farmaci di origine sintetica e biotecnologica. Recentemente tali sistemi sono stati proposti anche per la veicolazione di cellule nel campo delle Terapie Avanzate e nella medicina rigenerativa: le tecnologie farmaceutiche classiche sono state utilizzate per la realizzazione di forme farmaceutiche innovative iniettabili o impiantabili. In questo contesto, vengono descritti i più comuni sistemi microparticellari, ed in particolare microcapsule e microsfere, impiegati per la somministrazione transmucosale e parenterale di farmaci. Vengono trattate, inoltre, alcune problematiche relative alla veicolazione di cellule destinate all’impiego in protocolli clinici, mediante processi validabili e secondo le linee guida europee per la produzione di medicinali per le Terapie Avanzate