Musculoskeletal disorders (MSDs) have an extensive and growing impact representing a major health problem worldwide. Despite years of research, the understanding of these diseases and the control on their progression has not yet been achieved; indeed, different therapeutic approaches are being investigated to prevent the disorders or to promote recovery and/or regeneration of the musculoskeletal system, including muscles, bones, and joints [1
]. MSDs often result from traumatic injuries due, for example, to vigorous physical activity leading to excessive muscle fatigue, damage of joint structures (articular cartilage, subchondral bone, synovial membrane), and inflammation or lesion of the tendon/ligament structure. Structural and genetic factors are also associated with musculoskeletal diseases onset and progression [3
]. MSDs represent a high percentage of orthopedic clinical pathologies in veterinary practice and, mainly, in equine practice. Due to the low healing capacity of tissues involved in joint and tendon disorders, there is a tendency to develop chronic diseases with a significant clinical relevance [4
]. Beyond the traditional treatments (local or systemic nonsteroidal anti-inflammatory drugs, intralesional steroids, correct shoeing, chondroprotectants such as hyaluronic acid), innovative regenerative therapies, including the use of Mesenchymal Stem/Stromal Cells (MSCs), have emerged over the past ten years as promising agents to promote local repair. Especially in the veterinary field, many clinical applications of cell-based therapies have been described, starting from the early 2000s with the first application of MSCs on the equine model [5
]. Despite the considerable amount of data suggesting the safety and efficacy of MSCs in experimental animal models and preclinical studies, cell-based therapies are not yet routinely applied in the clinic [7
]. Over the years, the therapeutic application of MSCs has known a paradigm shift: Nowadays, their engraftment, proliferation, and differentiation properties are not considered key features of their therapeutic abilities. Instead, the main actors of MSCs benefit are now considered several bioactive molecules (including proteins, cytokines, chemokines, growth factors, but also nucleic acid) released by cells and involved in cell-to-cell communication and cross-talk [11
]. Known as secretome, this complex set of secreted factors and vesicles seems to represent a valid alternative as a cell-free therapy, offering several advantages in comparison to cell-based therapy, such as lower immunogenicity, ease of storage and handling, and lower cost ensuring usefulness and feasibility in the clinic [12
]. However, addressing a suitable medicinal product is challenging, and further investigations are needed to shed light on secretome composition, properties, and in vivo behavior. Indeed, while evaluating secretome safety and efficacy for clinical use, many aspects, ranging from production processes to molecular characterization, must be defined.
In this study, an injectable freeze-dried pharmaceutical powder containing equine secretome has been formulated (Lyosecretome); the technological process has been validated in an authorized facility for veterinary clinical-grade medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical powder properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarkers; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms (sterility, bacterial endotoxin, and Mycoplasma); and (v) in vitro potency tests, and efficacy on proliferative induction of selected cell lines. Finally, putative proteins responsible for the biological effects have been identified by a Lyosecretome proteomic investigation based on nano Liquid Chromatography coupled with high-resolution mass spectrometry.
This study aimed to propose a procedure for a scalable production of equine Lyosecretome, define its characteristics, and pave the way for its use in in vivo preclinical studies in animals affected by musculoskeletal disorders. To this aim, secretome production was induced by serum starvation of expanded AD-MSCs, and then the freeze-dried secretome was characterized in terms of qualitative and quantitative parameters. Experimental evidence indicates that MSCs can be isolated from almost every tissue of the body [15
], even if it is still unclear whether cells derived from different sources share the same biological and therapeutical features. However, compared to the Lyosecretome prepared from MSCs isolated from human adipose tissue, we found similar categories, including genetic information processing, immune response, and stress response. Adipose tissue and bone marrow are the preferred sources to isolate and expand MSCs for equine clinical applications [13
]. In our study, AD-MSCs were used for the preparation of Lyosecretome.
Ultrafiltration was chosen for the easy scalability to prepare large batches because it presents several advantages compared to other techniques commonly used to isolate MSC-secretome, including the affordable costs and the shorter time requested by the entire process [16
]. Furthermore, this technique grants the choice between filtration modules with different molecular weight cut-offs (MWCO), allowing the preferred retention based on the desired yield in small vesicles (as, for example, extracellular vesicles) or the whole secretome. Other conventional methods commonly used for isolating EVs, such as ultracentrifugation, present several limitations regarding the possible damage of EVs membranes, due to the high g-force required by the centrifugation steps [17
]. In the current study, we selected a relatively low MWCO able to retain the whole secretome to ensure a large-spectrum therapeutic potential to the preparation, even though several studies support the hypothesis that the EVs fraction alone might be sufficient to promote healing of the injured tissues [18
]. A biotechnological product such as secretome can be thermolabile, and thus it cannot be dried applying thermal methods, responsible for possible modification and/or degradation of bioactive components (e.g., protein). For this reason, to reach a water-free product, the lyophilization process was considered. Lyophilization guarantees the maintenance of sterile conditions when changing from liquid to a powder state; moreover, the final product is easy to store and requires a short reconstitution time of the dried product; long-term stability is also provided [19
]. Unfortunately, this technique has several limitations: Costs, limited capacity, long processing time (it lasts approximately 4 days), and difficulties in the “scale up” from laboratory to industrial production. Thus, its use is justified when the product’s value is sufficiently high, such as producing biotechnological drugs, vaccines, vitamins, antibiotics, liposomes, and oncological products. The freeze-drying method creates freezing and drying stresses that can alter the stability of the biological product; hence, before proceeding, the addiction of a cryoprotectant is needed. The cryoprotectant guarantees the protection of both protein and vesicles lipidic layer from the damages of ice crystals formed during the freezing step and inhibits vesicle size alteration by aggregation. In this regard, mannitol is the most suitable cryoprotectant due to its important stabilizing effects on freeze-dried pharmaceutical products [19
]. Mannitol properties have been widely discussed and investigated [20
]. Its role is to ensure long-term stability, easy reconstitution, and maintenance of biological features and activities. However, the use of mannitol still remain controversial and many other excipients, such as trehalose, can be adopted instead [22
]. In the current work, mannitol was able to avoid vesicles/particles aggregation as revealed by NTA analysis; moreover, it prevented vesicle structure modification in terms of morphology as shown by SEM analysis. This latter aspect was also ensured by applying ultrafiltration process for the secretome isolation, rather than ultracentrifugation, which can possibly break-up vesicles membrane due to the high shear forces.
FTIR spectra revealed similar bands for each of the three batches, where the presence of lipids and protein content was confirmed. Overall, all samples’ spectra were overlapping, thus ensuring the reproducibility of the batch production process. DSC has been employed to demonstrate the purity, polymorphic form, melting point, and thermal behavior of EV lipid bilayers, which can be affected in terms of stability under adverse conditions [23
]. The lyophilization process was conducted successfully for all batches, as confirmed by TGA analysis, with a very low water residual and good thermal stability.
Regarding the analysis performed by the ExoView, the method is based on a direct-biomarker association that captures particles into a chip. The technique allows discriminating whether a protein is expressed on the surface of the exosome or in the exosome by fluorescence analysis of the captured particles. Since a specific chip for analysis of equine secretome is not available, the microarray specific to human proteins was used. Although not validated for equine species, the chip is based on highly conserved proteins, and a cross-reaction is plausible. We observed differences between the analysis of the three batches. The number of exosomes presenting a double correlation anti-CD-81/CD-9 human antibody was higher than the other correlations; moreover, CD-9 binding is probably the most affine to horse secretome’s surface protein due to a higher protein match.
The anti-elastase activity tested on equine Lyosecretome provides the rationale for using secretome in regenerative medicine [24
]. High protease expression is a feature commonly found in several musculoskeletal disorders contributing to their onset and evolution. In this context, protease inhibitors work, contributing to restoring extracellular matrix homeostasis. Our results showed a dose-dependent trend for each batch. Significant variability was observed between the different preparation, probably caused by an intrinsic biological variability between the cell populations used for their preparation.
In vitro metabolic activity assays are the gold standard for studying cell viability and proliferation. In the present work, MTT assay was performed on four different cell types, i.e., AD-MSCs, SF-MSCs, tenocytes, and chondrocytes, considered potential targets for the use of Lyosecretome in musculoskeletal disorders. No cytotoxic effect was observed after Lyosecretome treatment in any of the four different cell lines, independently of the used concentration. Lyosecretome was able to maintain cell replication when compared to serum-free medium. A dose-dependent effect was observed for each cell line, and statistically significant differences were observed as a function of the cell type. Cell metabolic activity ranged from 30% to 75% compared to medium supplemented with FBS, with the higher stimulus observed at a Lyosecretome concentration of 200 mg/mL. Interestingly, at this concentration, tenocytes, chondrocyte, and SF-MSCs were more responsive than AD-MSCs, reaching a plateau at 200 mg/mL.
The effect observed following Lyosecretome treatment, in particular elastase inhibition, could be attributable, at least partially, to its protein content. In fact, in addition to proteins involved in stress response, including the Heat Shock Proteins, we found protease/peptidase inhibitors among the protein classes better represented. In particular, the putative relevant role of some of them, such as SERPINs, was also suggested by their presence among the most abundant proteins (SERPIN1F) and proteins defined as hubs (SERPINH1). Moreover, about 10% of the identified proteins were annotated to be involved in the inflammatory response. In addition to SERPINE1, annotated to also be involved in “Response to bacterium”, the presence of proteins with anti-inflammatory activity (IL10RA, LTA4H, MXRA5 [25
], RARRES2 [26
], TEK, ANXA1 [28
]) emerged. Some of them, including MXRA5 (Chemerin), have been previously associated with osteoarthritis, and anti-inflammatory and anti-fibrotic properties have been proposed for this protein suggesting its potential role in chronic inflammation [29
]. In this scenario, MACF1 has also been detected in Lyosecretome; this protein plays a core function in wound healing cell migration [30
], primarily due to its ability to coordinate interaction between actin, microtubule, and cell junctions. Concerning the cytokine profile, several proteins with cytokine/chemokine activity have been observed in the Lyosecretome, i.e., IL-6, IL-7, CSF-1, IL10RA, IL12RB2, CXCL6, GDF7, and Wnt5B. IL-6 and IL-7 are two pro-inflammatory cytokines involved in MSCs immunomodulation [32
]. The presence of IL-6 agrees with a recent paper by Bundgaard et al. that also demonstrated the presence of CXCL6 in equine bone marrow-derived MSCs [34
]. Pro-inflammatory and chondrogenic treatments modulated the expression of both proteins. IL6, acting as a pro-inflammatory mediator, contributes to host defense during infection and tissue injury, but anti-inflammatory properties have also been suggested depending on the presence of other cytokines [35
]. CSF-1 plays a role in inflammatory processes and promotes bone regeneration and mobilization of vascular and osteogenic progenitor cells [36
]. CSF-1 presence has also been demonstrated in equine bone-marrow-derived MSCs secretome [34
]. GDF7 and WNT5B are two secreted signaling proteins whose role has been suggested in tenogenic (GDF7) and osteogenic and chondrogenic (WNT5B) differentiation pathways of MSCs [37
]. IL10RA mediates the immunosuppressive signal of interleukin 10, an anti-inflammatory cytokine, already observed in bone marrow-derived human MSCs secretome [39
Highly expressed proteins belonging to the class I small leucine-rich proteoglycan (SLRP) family, including LUM (lumican), DCN (decorin), and BGN (biglycan), were found among proteins defined as hubs. SLRP family comprises a group of extracellular matrix components distributed in most tissue whose gene expression is modified in OA [40
]. Lumican, one of the most abundant proteins found in the secretome, regulates collagen fibril organization, and its expression is reduced in OA cartilage [40
]. Interestingly, lumican is a major component of the corneal stroma, and its therapeutic use has been proposed for ocular lesions [41
]. Furthermore, lumican is considered a key effector in normal wound repair [42
]. Decorin plays a role in fibril assembly in tension-bearing tissues. It may contribute to stabilizing the cartilage matrix in OA [43
]. Decorin upregulation has been proposed as an attempt to reduce aggrecan fragmentation in post-traumatic OA, and decorin-based therapeutics have been suggested to attenuate OA progression [44
]. Concerning biglycan, this extracellular matrix component contributes to the regulation of chondrogenesis and ECM turnover in OA [45
]. Interestingly, the Lyosecretome prepared from equine adipose tissue-derived MSCs shares with the equine bone-marrow-derived MSCs secretome described by Bundgaard et al. [34
] a number of proteins related to cartilage biology, i.e., LUM, DCN, COMP (cartilage oligomeric matrix protein), SCRG1 (stimulator of chondrogenesis-1), EPYC (Epiphycan), GAS6 (growth arrest specific 6).
Regarding Lyosecretome as a pharmaceutical product, the following acceptance criteria were defined: (i) Fulfilment of specific requirements by the cellular source: Viability over 98%, capable for trilineage differentiation and negative for bacterial/viral contamination. (ii) Protein and lipid content were between 10–13 and 0.6–1 μg per mg of powder, respectively, mean EV size in the range of 100–200 nm, EV concentration in the range of 1–4 × 108
particles when re-suspended in dH2
O at 1 mg/mL, expression of CD-9, CD-63, and CD-81 markers, negative for bacterial/viral contamination, water content less than 2%, fulfilment of the criteria required for freeze-dried (cake aspect) and injectable formulations (osmolarity, pH, absence of visible particles after resuspension in physiological solution). The assessment on the final product has to be performed by characterization techniques that can be validated at pharmaceutical grade and have an acceptable cost to be routinely implemented [14
]. (iii) Functional aspects should be included to demonstrate that the final product, after having undergone all the isolation, purification, and formulation processes, maintains biological activity. In detail, anti-elastase activity was higher than 20% at 20 mg/mL in dH2
O. Finally, due to the limited experience in preparing equine Lyosecretome (these are the first scaled-up batches prepared following ISO 9001:2018 for validation) the source of variability cannot be fully investigated yet. With a more consistent number of batches prepared, it will be possible to adopt specific measures to minimize the batch-to-batch variability.