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.


Introduction
In recent years, the focus of medical science has shifted from repair to regeneration. Regenerative medicine aims to recover the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies, able to stimulate and coordinate the processes headed to biological restoration. Regenerative medicine stands out as a top research interest area in medical fields, including both human and veterinary medicine.
Regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), whose safety and efficacy are now considered well established for humans [1,2]. In veterinary medicine, the reality is much diversified. Above all, there is enormous variability in terms of anatomy and physiology among the animal species in which regenerative medicine can be applied (mainly horses and dogs), with consequences on stem cells biology, mechanisms of action, and several aspects on their application, such as minor/major inflammation responses after MSCs administration in vivo. Moreover, sometimes, even between breeds, there is a large variability related to the predisposition to

MSC Isolation Procedures
As reported above, MSCs can be isolated either from 'liquid' sources, such as peripheral blood, or from 'solid' sources, such as adipose tissue. In the first case, the isolation process will employ a gradient centrifugation protocol, whereas, for the second one, an enzymatic treatment will be needed. Afterward, cells are seeded in plates for their adhesion, while the non-adhered ones will be wiped out when replacing culture medium with fresh one [7,33].
Depending on the type of cells and technical factors, usually within a week or 10 days, colonies of adherent cells will appear and gradually cover as a layer the culture plate bottom, until they reach the confluence. Cell layers will be then detached by trypsinization and reseeded to permit a continuous proliferation every time confluence is reached; this step is called 'cell sub-cultivation', and each one is referred to as passage number. MSCs should not be expanded more than four passages for clinical applications to maintain their stemness; indeed, further passages lead to senescence, meaning lower proliferation and morphological as well as biological changes. For instance, previous studies have shown how the onset of senescence is related to a telomere length reduction; senescence of equine BM-MSCs occurs faster than adipose and umbilical cord-derived MSCs [34]. These results indicate the adipose tissue and, to a lesser extent, for difficulties in the collection, the umbilical cord, as preferable choices for tissue regeneration.

MSC Culturing Conditions
The medium used for MSC culture is mainly represented by the Basal Medium Eagle (BME), Minimum Essential Media (MEM), and Dulbecco's Modified Eagle's Medium (DMEM); they are different in the content of amino acids and mineral salts, and the concentration of glucose.
Supplementation of the medium is performed to mimic in vivo conditions in order to sustain cell growth. Foetal bovine serum (FBS) or platelet lysate [35,36] are used in the cell culture medium as a source of growth factors [37], which supports attachment and further expansion of MSCs. The use of antibiotics is important in order to prevent bacterial contaminations, and the most commonly used are penicillin and streptomycin.
The pH value of the medium ranges between 7.2 and 7.4, and in order to keep this value constant, cell plates are usually incubated in 5% CO 2 atmosphere, and the medium culture contains NaHCO 3 as a buffer.

Cryopreservation
Cryopreservation is a conservation method which allows storing biological material at very low temperatures (−196 • C using liquid nitrogen or −80 • C using carbon dioxide). Cryopreservation process turns to be useful for the storage of cells for extended periods; indeed, freezing in liquid nitrogen keeps cells alive in a complete quiescence phase for years. By default, it should be considered that cryopreservation alters or compromises the structure and function of cells; in the worst cases, the ice crystals formed during the freezing process can cause cell damage. For this reason, due to the complexity of the whole process (freezing and thawing), a choice of excipients and process parameters, to protect the cell integrity from stresses, is challenging. These excipients are generally named as cryo-protectants and are mainly divided into two categories-membrane-permeable and impermeable. Cryo-protectants with high permeability are, for example, dimethyl sulfoxide (DMSO), ethylene glycol, methanol, propylene glycol, and dimethylacetamide. They tend to be the most cytotoxic. Cryo-protectants less permeable are methylcellulose (MC), polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), polyethylene glycol, and dextran. In association, FBS is used as a source of protein with synergic cryoprotectant effect [38]. A mixture of DMSO and FBS is considered as the standard cryo-protectant in veterinary medicine, even if the oncogenic and xenogeneic properties of DMSO and FBS, respectively, may alter cells and impact in vitro and in vivo behaviour after implantation [8].
Regarding cell transportation, many studies have shown which are the shipping criteria that should be considered-for instance, supplementing media, transport temperature, and other variables, such as the type of container for the shipping. The latter did not show meaningful evidence; in detail, the use of plastic rather than glass containers did not show differences in terms of cell viability after 24 h at room temperature (20)(21)(22) • C) at several MSC concentrations [39]. Shipping temperature is up to cell storage conditions: frozen cells should be transported in dry ice around −80 • C, inside cryovials often wrapped with precooled material and placed in a leak-proof container to prevent direct contact of samples; otherwise, they can be transported in liquid nitrogen to maintain −196 • C temperature. In the literature, it is reported that shipping frozen equine BM-MSCs is the most appropriate method to maintain about 80% of cell viability [40] up to six months [41]. Fresh cells, instead, have been valued differently in several studies: transport was evaluated at different temperature (4 • C, 37 • C, and room temperature 20-22 • C) in PBS or DMEM, either alone [42] or supplemented with FBS or with horse serum [9]. As a result, the majority agreed that MSC transport should be performed at 4 • C, which seems to be the most practical one. According to this, shipping fresh equine MSCs in isotonic saline solution at 4 • C within 24 h is considered ideal for immediate administration, although, in these conditions, MSC viability decreases up to 70% [39,40,43]. Only one study considered room temperature as superior in the case of short-duration transport [43].
The consciousness of differences between the employment of fresh and frozen MSCs is now ongoing. As mentioned before, each step of cryopreservation can affect MSC viability. Concerning this, two reports have demonstrated that while frozen canine AD-MSCs and BM-MSCs had a lower proliferation capacity, lower telomerase activity, and a loss of cells expansion, fresh cells maintained their capacities exploring the same parameters. Similarly, feline cryopreserved AD-MSCs showed lower CD9 and CD105 expression compared to fresh cells [44]. On the other hand, several other studies led to different results. In detail, Martinello et al. proved that, after cryopreservation, equine peripheral blood MSCs conserved their morphology, telomerase activity, proliferation rate, and CD expression pattern [45]. Overall, the use of fresh and cryopreserved MSCs is controversial, and particular care on the cell viability and passage level must be taken before their administration.

Quality Controls
Another important aspect that should be considered when isolating MSCs is their characterization, performing quality, and quantity controls on the starting material and the final product. In such a way, it can ensure the quality, reproducibility, functionality, potency, efficacy, and safety of the clinical product. Required quality controls regard the morphology, immunophenotype, differentiative potential, viability, and sterility testing [46].

Routes for MSC Administration
Although MSCs delivery routes are different, typically, clinical treatment requires the administration of cells using needles. Cells activity and survival could be affected not only by needles type and diameter but, notably, also by following the aspiration and re-injection of MSCs suspension, probably due to the negative pressure during aspiration [47]. Since the passage of MSCs through a needle may affect cell viability, the appropriate catheter aspects should be taken into considerations. Usually, for joint pathologies, for instance, osteoarthritis, MSCs suspension is directly injected intra-articularly. In tendon core lesions, intra-lesional direct administration of MSCs is the preferred route, while administration within the tendon sheath or by regional perfusion with the use of intravenous catheter is used in the case of multi-focal lesions or when the damaged site is difficult to access [48]. Intra-arterial delivery could be effective, but this route is discouraged because of the risk of thrombosis [49]. In the case of focal cartilage lesions, MSCs could be encapsulated into a scaffold and be placed directly into the damage region under arthroscopic guidance [48].

Autologous vs Allogeneic MSCs
There are mainly two approaches for stem cell therapy in regenerative medicine-autologous or allogeneic cell transplantation. An autologous transplant uses the patient's stem cells, while allogeneic transplant uses stem cells from a donor. Allogeneic versus autologous MSCs is a hot topic in veterinary regenerative medicine, debating whether there is a difference in terms of safety and efficacy. The use of allogeneic MSCs is subjected to specific national regulations, which are important to consider for trials and clinical applications. However, the use of allogeneic MSCs would represent an important advantage offering availability of banked cells, previously characterised for their safety and biological features, as a proven differentiation capacity. This approach would reduce the typical variability of autologous cellular products, allowing greater homogeneity in treatments and, presumably, results. Moreover, by this approach, it is possible to have a ready-to-use product for appropriate timing of therapeutic applications, without the need to wait for the autologous cell culture expansion.
Using the patient's cells to treat an injury or disease is believed to be safer, but on the other hand, one of the major challenges of this approach is due to timing. Indeed, the timing to expand the right amount of cells for implantation is highly dependent on the harvested tissue, as well as on their reproduction capacity, and thus by age, gender and disease type [50].
Some trials have been conducted on different animal models to compare allogeneic and autologous MSCs. In one of them, self and non-self placenta-derived MSCs were injected into contralateral joints of 16 healthy horses. A comparison was made in terms of lameness evaluation and synovial fluid analysis from 0 h up to 72 h post-injection. The injection of allogeneic MSCs did not provoke a systemic response, while local response such as swelling was minimal, and inflammatory response was not significantly different between the two treatments. Thus, this pre-clinical work is an important step in the development of equine allogeneic stem cell therapies [51]. Furthermore, Shah et al. reported the outcome of the treatment conducted on 203 dogs suffering from osteoarthritis and other joint defects with allogeneic stem cells derived from adipose tissue. Dogs of various breeds and different ages were enrolled in the study. Most of the patients received an intra-articular therapy, while 68 patients were treated intravenously. The large majority of younger animals (90% of < nine-year-old dog) and a large percentage of older patients treated with the allogeneic adult stem cells improved symptoms and demonstrated better quality of life. Only a single patient had a worsening of the symptoms. The large number of dogs enrolled in the study, and the administration routes of the cells, point out the safety of the allogeneic treatment, although the follow-up was limited to 10 weeks [52]. The result of this report strengthens similar findings, where allogeneic MSCs have been able to aid the body's regeneration abilities without causing an immune response or other adverse effects [52,53]. Another proof of allogeneic MSCs safety has been reported by Brandão et al., who analysed autologous and allogeneic AD-MSCs in order to evaluate the inflammatory response in healthy equine tendon [54]. The outcome in the two groups has been compared to a control group where only PBS was administered. After injection, all the groups presented mild pain sensitivity on the second day, which can be explained as a consequence of the healthy tissue response to an injury application. There were no significant differences among the groups in the physical, morphological, thermography, and ultrasonography analyses. Moreover, even the lameness analysis presented similar behaviour between the two cell-treated groups. Interestingly, the authors analysed tissue and cellular response to MSCs administration, concluding that both allogeneic and autologous AD-MSCs did not induce a significant inflammatory response, although a higher number of T lymphocytes have been observed in the group treated with allogeneic cells. Based on their result, the authors suggest that allogeneic MSCs did not have adverse effects in comparison to the autologous cells, thus reinforcing the hypotheses that allogeneic banked cells could be a safe and effective approach to regenerative therapies. According to this, a similar conclusion came from a different study conducted on allogeneic BM-MSCs transplantation in equine tendons. The analysis showed no significant differences compared to the autologous cells [53]. In this latter study, small lesions were created in the equine superficial digital flexor tendon, followed by injection with autologous, allogeneic, or bone marrow supernatant alone, respectively. Post-mortem examinations revealed there was no cell-mediated immune response to the host for both autologous and allogeneic MSC treatment.
These results seem to be promising and lead to the conclusion that allogeneic rather than autologous MSCs could be used for regenerative medicine purposes in veterinary medicine. Hence, it would be important to implement studies related to the immunomodulation of MSCs in order to understand cell response better and reinforce, accordingly, therapeutic applications. In particular, immunoreactivity tests performed both in vitro and in vivo using allogeneic MSCs are important to guarantee safe and effective therapy. Moreover, further in-depth studies still need to be performed to understand the real behaviour of the stem cells at the site of application and their crosstalk with resident cells [55].
Finally, most of the clinical applications to investigate MSCs behaviour are conducted on experimentally damaged tissue models. Some authors, indeed, suggest that to evaluate MSCs action mechanisms and attitude better, it would be necessary to apply cells in healthy tissues [53]. The injection of allogeneic MSCs in the considered area would allow the assessment of the possible local inflammatory reaction. Thus, this could reveal the interaction of any other aspects, proving that alterations are caused by cell transplantation [56]. Working on healthy tissues is also important to predict the side effects of allogeneic MSC transplantation in animals. All the considered studies regarding autologous vs allogeneic MSCs are summarised in Table 1. All groups presented mild pain sensitivity, there were no significant differences among the groups in the physical, morphological, thermography, and ultrasonography analyses.
Also, the lameness analysis presented similar behaviour between the two cell-treated groups. Both allogeneic and autologous AD-MSCs did not induce a significant inflammatory response, although a higher number of T lymphocytes have been found in the allogeneic treatment.
1 × 10 7 cells for each application resuspended in PBS.

MSCs in the Veterinary Field: Disease Targets
Musculoskeletal disorders (MSDs) represent common pathologies in veterinary clinical practice. MSDs affect the osteoarticular apparatus, including muscles, bones, and joints. They are associated with painful symptoms that can be both acute and chronic. These disorders often result from overuse injuries, muscle fatigue, inflammation of the tendon structure, or intervertebral disk degeneration of the vertebral column. Examples of MSDs include osteoarthritis (OA), tendon ligament injury (TLI), and intervertebral disk degeneration (IVDD) [57].
Up to now, treatment options for MSDs include systemic or intra-articular administration of anti-inflammatory drugs, hyaluronic acid (HA), cells-based products including platelet-rich plasma (PRP), and autologous/ allogeneic cells implantation [57,58]. Since MSDs have a high prevalence, regenerative therapies, including the use of MSCs, have been brought to the attention of veterinary practitioners as an alternative to the more traditional treatments [54,59,60]. Below, the employment of MSCs to treat each specific MSD is reported.

Osteoarthritis
OA is the most common form of arthritis related to the progressive degeneration of articular cartilage and subchondral bone, leading to severely debilitating conditions. It is a chronic and irreversible condition involving the cycle of inflammation and tissue degradation [61].
In recent years, MSCs have been proposed as a therapy for the treatment of OA in both dogs and horses. The currently available evidence of the MSCs effectiveness and safety profile is confirmed in the clinical trials reported in Table 2. In all cases, whether it is a dog or a horse, veterinarian practitioners directly administered MSCs into the joint by intra-articular injection [40,62].
Regarding dogs' treatments, the most common approach is to use adipose tissue as cells source, favoured by the possibility of minimal-invasive collection. In detail, as reported in a review, different clinical trials on the use of AD-MSCs have been investigated comparing the AD-MSCs alone or in combination either with intra-articular autologous PRP or HA as a chondroprotective agent [63]. Considering the common outcomes of the studies reported in Table 2, the most important element is the safety and efficacy of the therapy, expressed in term of the absence of any side effects (including local and systemic inflammation), reduction of lameness, improvement of joint functionality, and pain reduction. As a general trend, better endpoints were noticed in dogs treated with AD-MSCs associated with plasma rich in growth factor (PRGF-Endoret) [64] than AD-MSC alone [65]. It has been indeed demonstrated that in vitro PRP releases growth factors, including Transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), epidermal (EGF), and insulin-like growth factor (IGF) that affect cartilage regeneration [66]. Another study demonstrated the superiority of AD-MSCs alone over PRP at the six-month follow-up, although outcomes were not described beyond this period [67]. OA in dogs has also been treated by injection of AD-MSC suspended in saline solution, comparing the outcome to dogs treated using saline solution only, as a control group. This experiment was reproduced afterwards by the same group of research administering a higher amount of cells and with a longer follow-up. In fact, since the pain grade of the affected joint was severe, the success rate after the injection was not significantly high in the first study; however, clear evidence of the efficacy of MSCs therapy was observed in the second work [68,69].
To examine the impact of MSCs in the treatment of equine model of OA, several clinical studies have been performed. A recent review compares intra-articular injection of MSCs in both naturally occurring and induced equine OA; results have been variable, which may be caused by the changing environment, follow-up, MSC dosage and source, as well as inter-observer differences in subjective outcome parameters [70]. Considering bone marrow as MSCs source, autologous cells have been dispersed in HA, and their effect has been compared to the HA alone used as a control [71]. Horses had defects arthroscopically created on both stifle joints and received an intra-articular injection; after 12 months, horses were euthanised. Results showed no significant improvement, but evaluation post-mortem performing histologic and immunohistochemical analyses confirmed a significant increase in joint repair. Naturally occurring OA treated with BM-MSCs dissolved in HA have also been investigated, post-surgery (arthroscopy), supporting the amelioration of horse condition after cells injection even after 24 months [72]. When treating naturally occurring OA, there is more variation because duration and severity of the disease vary, and it is difficult to judge whether or not the treatment with MSCs results efficacious and in what extent [73]. Such conditions often lead to a lack of objective conclusions due to, for example, of variation in joints treated and lack of control groups [41]. Comparison between cell treatments and elective drugs has been explored; in detail, the injection of autologous AD-MSC versus steroid drugs (betamethasone) was evaluated up to 180 days [74]. At the end of the period, no inflammatory response was observed in any groups, and improvements were noticeable in AD-MSC treated horses but not in the one cured with betamethasone and group control. Recently, Broeckx et al. [75] proposed a somehow alternative approach to the therapy of equine degenerative joint disease. Their randomised, double-blinded, and placebo-controlled clinical trial enrolled 75 horses affected by fetlock joint osteoarthritis. The treated group of fifty horses received a single intraarticular injection of blood-derived, allogeneic chondrogenic-induced MSCs. Cells were resuspended in allogeneic plasma. The authors report better scores for treated animals regarding lameness, flexion test, joint effusion both at short (3-18 weeks) and long-term (one year) follow up. The novelty of this study lies in the use of blood-derived MSCs, to replace the most widely used adipose-tissue or bone marrow-derived cells and their combination to allogeneic plasma to improve cell viability, replication, and chondrogenic differentiation. All the MSCs were prepared from a single donor and pre-differentiated at P9; no adverse effects are reported following the application of allogeneic MSCs and plasma.

Tendon Ligament Injury
Tendon ligament injury (TLI) affects a large part of the equine population ranging from acute traumatic ruptures to chronic overuse and degenerative tendinopathies [76]. The outcomes of conventional therapies are quite often unsatisfactory due to the poor regeneration capacities of tendon tissue in equine species [77,78]. In fact, apart from a primary inflammatory reaction following the injury, spontaneous healing is characterised by fibroplasia and can be referred to as a repair rather than a regeneration process [79]. The repairing system leads to the formation of cellular scar tissue with low extracellular matrix organization, in which stiffness is increased, but elasticity is decreased compared to the original tendon tissue [55,[79][80][81]. Indeed, the primary outcomes investigated to evaluate tendon regeneration are the stiffness, modulus of elasticity, histological score, DNA content, vascularity, and compositional parameters, which could be considered indicators of regeneration, when compared to levels observed in the normal tendon.
In recent years, research has focused on regenerative therapies and tissue engineering approaches, with the aim to recover the original function of the damaged tendon. Typically, as the tendon core lesion is clearly visible by ultrasonography, the application of MSCs is simply performed by injection of the cell suspension directly into the damaged tissue [82]. Specifically, some studies have been conducted to evaluate the role of MSCs on equine tendon healing, injecting cells derived from different sources (mainly adipose tissue and bone marrow), autologous or allogeneic, alone or associated with other treatments (PRP, HA) [80,83]. Although most of the reports are not blinded or do not provide sufficient information about controls, the literature describes encouraging outcomes, giving evidence of the benefit and safety of MSC application for tendon regeneration [84] MSCs treatment has been described either in healthy animals, in which experimental lesions were induced surgically or by collagenase gel in the superficial digital flexor tendon [80,85]; or in naturally occurring TLI [84,86,87]. Romero et al. evaluated tissue healing in an experimental tendonitis model after administration of autologous bone marrow and adipose tissue-derived MSCs and platelet-rich plasma (PRP). BM-MSCs and PRP produced similar results, although PRP-treatment resulted in higher expression of COL3A1 and ACAN genes, suggesting lower tendon regeneration. Although all the treatments showed beneficial effects compared to the control group, the authors concluded that BM-MSCs might provide better healing properties [88]. Brandao et al. [45] studied the local inflammatory response of tendon injected with autologous or allogeneic AD-MSCs, concluding that no adverse or inflammatory reaction was observed in horses treated with allogeneic cells. Actually, allogeneic cells have been extensively used also for the treatment of naturally occurring tendon lesions supporting the safety of allogeneic cells administration in the horse [85][86][87]89]. In most clinical studies, outcomes are evaluated comparing results to conventional therapies in term of re-injury rate following the return to activity. Although this approach does not give an accurate indication about the quality of tissue recovery, it provides practical information about the possibility of the animal to return to their previous normal activities. Interestingly, cell treatment resulted in a significantly lower re-injury rate in comparison to conventional therapies. Generally, re-injury rate following traditional treatment such as hyaluronan, beta aminopropionitrile fumarate or polysulfated glycosaminoglycans, ranges between 23-80% [77,90], while the re-injury rate following MSCs medication is reported to be lower. In this regard, Pacini et al. observed that nine out of 11 racehorses could return to competition without any re-injury event, within a follow-up period of two years [91]. Similar results have been observed in a different clinical report, where after MSCs treatment, the majority of the patients returned to their previous activities, avoiding re-injury [84]. Smith et al. conducted a clinical study on 82 racehorses and 24 other sports horses; after rehabilitation follow-up, only 13-36% of the horses were re-injured, depending on their disciplines [92]. Smith et al. [81] compared the mechanical and morphological characteristics of the tendon extracellular matrix in horses affected by spontaneous tendonitis. BM-MSC treated tendons demonstrated improvements in several parameters compared to not treated tendons, providing evidence on the role of cell therapy on tendon healing in naturally occurring tendonitis.
Regarding tendinopathy in canine clinical reports, the scenario is way less documented and investigated than horses; nevertheless, the beneficial effects of MSCs seen in few studies, are quite significant. Although tendonitis has not a high prevalence in dogs, supraspinatus tendinopathy (ST) represents a quite common cause of forelimb lameness. Aetiology is probably related to overuse from chronic repetitive activity. Canapp et al. applied adipose-tissue-derived MSCs in combination with PRP to the treatment of 55 dogs with ST [93]. Based on ultrasonography and objective gait analysis results, the authors suggest that MSCs administration is a promising therapy for ST. More recently, the same group extended the study to the use bone marrow aspirate concentrate (BMAC) combined with PRP, observing positive sonographic results with improvement of tendon size (significant reduction of the affected tendon), fibre pattern and echogenicity, even if only 13.8% dogs treated recovered entirely to a normal fibre pattern at 90 days post-treatment [94]. This could be related to the short follow-up in comparison to the longer time needed for tendon healing.
Finally, a study made a comparison between BMAC and AD-MSC added to PRP for the therapy of cranial cruciate ligament tear in dogs [94]. In this research, the dogs recruited were different in terms of breeds, age, weight, and sex; the outcomes were based on diagnostic stifle arthroscopy, radiographs, orthopaedic examinations, gait evaluation, and functional questionnaire. Results proved cells efficacy, but there are limitations due to a lack of standardization of PRP preparations the uncertain number of administered cells. This could have affected the outcomes of the two treatments (AD-MSC and BM-MSC). While all these results are encouraging, the long-term success of MSC treatment remains to be proven.
Although semitendinosus myopathy is not tendonitis, this fibrotic musculoskeletal disorder of working dogs affects a muscle whose long tendon is part of the Achilles tendon. Gibson et al. recently conducted a study using a single administration of AD-MSCs to treat semitendinosus myopathy in 11 police dogs, comparing follow-up at six months and one year. At six months follow-up, all patients had returned to work, while at long term follow-up, the dogs were still active in their previous activity, showing an improvement in gait [95]. The authors do not report a recurrence of the disease, and most of the dogs worked until retirement, suggesting a long-term efficacy of the therapy. Table 3 reports all the considered studies regarding TLI treated with MSCs. Intralesional administration of 10 × 10 6 AD-MSC in in 1 mL of PC. 1 mL of PBS was used as a control group After 16 weeks improvements were reported for AD-MSC group.
In detail: decrease of the lesion progression and inflammatory reaction, better organization of collagen fibres, an increase of blood flow. No difference in terms of gene expression was found.  After 45 weeks, all treatments had beneficial effects, but in detail, data suggest BM-MSCs might be the better approach for tendon healing.
[88] [ 87] TLI-SDFT Horses (n = 11) Autologous BM-MSC Injections of at least 1 × 10 6 of BM-MSCs were re-suspended in 1.5 mL of autologous serum Patients were back to their sports activities, without having suffered a re-injury [91] TLI-SDFT Horses (n = 12) Autologous BM-MSC suspended in 2 mL of BM supernatant Implantation of 10 × 10 6 BM-MSCs were suspended in 2 mL of citrated BM supernatant. 2 mL of PBS were used as a control group In six months, there were significant benefits in terms of safety and healing tendon process (reduced stiffness, histological showed better organization and reduction in re-injury rate). 2-4 mL of BM-MSCs + PRP or 1-2 mL of AD-MSCs + PRP was injected intra-articularly into the stifle (volume depended on the dog's size) Neither treatment was superior to the other in terms of outcome (90 days). [94]

Intervertebral Disk Degeneration
Intervertebral disk degeneration (IVDD) is a complex multifactorial process considered as the primary cause of lower back pain in humans. Since intervertebral disk degeneration is incurable, all available treatment strategies are mainly focused on pain relief only [97]. Therefore, efforts are dedicated to the development of strategies able to regenerate, or at least repair, and preserve the functioning of the intervertebral disk structure. Among these, intradiscal injection of MSCs has become highly topical in experimental and clinical investigations.
Dogs with IVDD are the only patients where medical and surgical approaches similar to humans are used [63]. For this reason, canine IVDD is considered a valuable and reliable disease model for the investigations of novel and effective healing treatment for human IVDD. In a recent study by Steffen et al. [98], six dogs suffering from naturally occurring degenerative disc disease received autologous bone-marrow-derived MSCs. Although results showed no adverse effects, the authors were not able to demonstrate by MRI any apparent regenerative effect of the treatment. This work was pioneering in naturally occurring IVDD, thus negative result may be caused by biological and biomechanical differences between injury-induced and naturally occurring IVD degeneration. To improve the therapeutic approach, the authors, in a following study [99], introduced collagen microcarriers as a scaffold for MSCs. Twenty dogs affected by spontaneous lumbosacral IVD degeneration confirmed by MRI and clinical signs (lumbosacral back pain) were included in the study. Autologous MSCs were isolated from bone marrow and, before the injection, MSCs were mixed with collagen microcarriers, as a delivery system, with or without TGF-β1 crosslinking. After decompressing surgery, dogs were divided into three groups, which received three different intradiscal injections of 1) intradiscal injection of MSC-microcarriers, 2) MSC-TGF-β1-microcarriers, of 3) microcarriers only. Clinical performance and Pfirrmann grading, assessed through magnetic resonance imaging, were evaluated at 10 months after the injection. In vivo injection was successful in all dogs, and clinical functioning returned to normality. However, post-operative Pfirrmann grade remained unchanged in all dogs, and the undesired side-effect formation of Schmorl's nodes occurred in 45% of the dogs. This side effect was reduced by halving the injection volume. Therefore, clinical improvement was observed in all groups, despite the formation of Schmorl's nodes, but microcarriers and MSCs failed to regenerate the structure of degenerated IVD [99]. A successful work was conducted using fetal allogeneic BM-MSCs on seven dogs in which, by the end of the period of 90 days, all dogs had an improvement in functional movements and were able to take steps; moreover, none of them presented adverse symptoms at any time [100]. Two years later, a randomised control case report was conducted on 34 dogs suffering from IVDD disorder with no deep pain; in this study, surgery was evaluated alone and in combination with transplantation of allogenic AD-MSC into the spinal cord. Neurological progress was noticed, and the success rate for the AD-MSCs group was significantly higher (77.8%) than surgery alone, thus demonstrating the potential therapeutic efficacy of MSCs [101].
Even though the use of MSCs in veterinary regenerative medicine for IVDD treatment seems to be promising, as mentioned above, there are also clinical results that suggest that MSCs are not always capable of repairing the damaged tissue, at least using the therapeutic protocols proposed so far [102]. Table 4 reports all the considered studies regarding IVDD treated with MSCs.  Ten months after treatment: injection was successful in all dogs; thus, they returned to normality. Schmorl's nodes were found as side effects. Six months after treatment: Improvement in the neurological exam and better endpoint with AD-MSCs application rather than surgery only. [101]

Animal Spontaneous Pathologies as Potential Preclinical Models for Human Therapy
Experimental animal models are still widely used to study biological properties and therapeutic potential of MSCs in regenerative medicine. From this point of view, a key point is the choice of the animal species to develop a useful and scientifically relevant model. Resorting to laboratory animal species (mice, rats, rabbits) to establish the safety and efficacy of novel therapeutic strategies leads to the use of induced experimental models very far from the clinical reality of human medicine, making their use uninformative and leading to the sacrifice of a high number of animal lives. This aspect is in contrast with the European Parliament directive (Directive 2010/63/EU) which limits the use of animal models and suggests to the scientific community the use of alternative methods, as a result of the 3Rs principle promoted by Russell and Burch in 1959 [103].
Using spontaneous animal pathologies may be a strategy. Specifically, referring to naturally occurring, spontaneous diseases of domestic animals (primarily dog, cat and horse) allows using model diseases much more similar to human ones from pathogenesis, evolution and biological mechanisms of healing. Thus, companion animal clinical models represent an important contribution to human studies [104]; indeed, similarities concerning symptoms, etiopathology, biomarkers [105] and even genetic [63] can be found with the human counterpart. Also, dogs and cats share with humans the living environment and underlying pathologies (e.g., obesity, diabetes) which often influence the pathological onset, evolution, and healing processes. Finally, their relatively long life expectancies make companion animals a model for long-term studies. Using spontaneous pathologies of these animals for the development of innovative regenerative therapies is, therefore, of extreme interest for human and veterinary medicine, thus avoiding experimental animal models.
Musculoskeletal disorders are an important example of this concept. Cell therapies based on the application of MSCs aim to regenerate damaged tissues exploiting tissue's intrinsic potential for repair. The complexity of the healing processes induced by MSCs makes it necessary to use natural pathologies (and not experimentally induced disease models) to understand the real therapeutic potential of the cells. In dogs and horses, musculoskeletal disorders have a high prevalence and are considered quite similar to those developed in humans. Innovative cell therapies for osteoarthritis, tendonitis, and intervertebral disk diseases are actively investigated in veterinary medicine, providing useful therapeutic protocols for human medicine [106]. A further advantage of the use of spontaneous diseases to assess the efficacy of MSCs treatment is not only that experimental animals are preserved but affected companion animals enrolled in the studies can receive up-to-date therapeutic opportunity and long-term follow-up.

Secretome and Extracellular Vesicles as a Potential Therapy for Different Disease Areas
During the last decade, it has been demonstrated that MSCs therapeutic effectiveness is mainly due to the release of paracrine factors, named secretome, composed of free soluble factors (including cytokines, chemokines, and growth factors) and non-soluble nano/microstructured extracellular vesicles (EVs) [107,108]. Several studies support the hypothesis that the EVs fraction alone may be sufficient to heal the injured tissue or prevent tissue damage in several contexts [49]. EVs isolated from MSCs exhibit the same functions as stem cells, for example, their anti-inflammatory and pro-regenerative activity [2,109,110]; but they also can elide some issues related to the usage of the whole stem cell. In detail, they have lower immunogenicity, smaller size, and so they could represent a safer alternative compared to the cell injection [111][112][113]. Despite these advantages, more studies need to be conducted to elucidate kinetics, mechanisms of action, bioavailability (including factors like dosage and frequency), and route of administration. EVs delivery could be provided by using suitable materials and methods such as the encapsulation within hydrogel or scaffold, improving EVs immobilization plus frequency and dosage of administration.
However, this approach, especially in the veterinary sphere, is still in its early stages. Only recently, the therapeutic use of the MSCs-secretome, instead of the parental cells, has been proposed for veterinary applications, also overcoming the practical difficulties related to stem cell application. In detail, based on the hypothesis that cells may promote tendon repair via paracrine factors, an interesting study has been recently published regarding the secretome profile in eight dogs [114]. In detail, exosomes and soluble factor derived from BM-MSCs and AD-MSCs of the same canine donor have been described and compared for the first time, paying attention also to their immunomodulatory capacity. Both cells types share analogy as morphology, but they also have their biological features such as gene expression and proliferation and differentiation. Outcomes showed a higher proliferation rate for AD-MSCs, whereas the production of exosomes and soluble factors, comprising several cytokines, was more active in BM-MSCs. These results were also confirmed by the proteomic analysis. The limitation of this work was the small number and size of the animal model; nevertheless, it is the first step toward secretome characterisation and application [115]. In this contest, El-Tookhy et al. investigated the exosome and microvesicles role in wounds healing process of experimentally induced critical size defects in six dogs. Wound reduction size was observed in 14 days comparing skin area treated with exosomes and the one treated with PBS as a control group. As expected, photographs and histopathological evaluation showed better and faster endpoints of the healing process in dogs treated with exosomes, in particular about the formation of extracellular matrix, angiogenesis, and re-epithelization. The findings underline the efficacy and safety of cell-free therapy in wound repair, paving the way for future new therapy approach that overcome the limitations associated with the use of cells implantation [116].
As far as we know, Lange-Consiglio et al. were the first group to test in vivo the immunomodulatory features of conditioned medium from amniotic membrane-derived MSCs (AMC-CM) evaluating the beneficial effects in thirteen horses affected by spontaneous tendon or ligament injuries [117,118]. All horses included in the study had lesions graded from four to six, presenting an advanced injury stage. Intralesional treatment was well-tolerated, no adverse reactions to the AMC-CM injections occurred, and no soreness was noticed, thus confirming that the treatment was well tolerated in the short-term period. Moreover, the absence of abnormal fibrotic and metaplastic tissue at any time post-injection in the treated area underlines the safety of the secretome administration. Treatment outcome was successful for all the patients considered; in detail, six out of thirteen horses were healed entirely and resumed their previous activity within 4-5 months post-treatment. This latter study suggests a new approach of cell-free treatment for tendon and ligament diseases, obtaining advantages with respect to cell therapy. Apart from a higher safety profile of their administration and their lower immunogenicity, such EVs benefits occur because of the possibility of an easy production of large quantities, and a high storage efficacy, thanks to the possibility to prepare freeze-dried secretome [2,109,119]. Indeed, the lyophilization process allows obtaining a ready-to-use product, causing minimal damage to the substances sensitive to the high temperature and increasing the shelf life of the products.
All the previous considerations lead to a statement: secretome is taking on the role of a new potential therapeutic strategy in different diseases [120,121]. Avoiding the necessity of living cell implantation, secretome shows promising outlook to be prepared as pharmaceutical products suitable for regenerative medicine [122,123]. In particular, EVs are considered a new therapeutic tool having a prominent role in joint and musculoskeletal disorders ( Figure 2). Besides all these considerations, up to now, in veterinary medicine, the clinical use of MSC-EVs, although very promising, is still in its infancy, despite numerous pre-clinical models have been proposed. A key aspect which is missing for EVs and/or secretome pharmaceuticalization is a standardised, reproducible and GMP-compliant production process, in order to obtain validate products suitable for future clinical trials. Isolation method and characterization process are reported in human literature, but unfortunately, in the veterinary field, more evidence is required to confirm the feasibility of this innovative cell-free treatment [2].

Conclusions
MSCs belong to body repair system and are defined as multipotent progenitor cells able to self-replicate and to differentiate into different cell types, demonstrating significant potential in clinical use. In the veterinary field, many studies have been conducted for the development of the most effective procedures for MSC-based treatment, taking into consideration cell tissue sources, isolation process, culturing conditions, cryopreservation, cell dosage, administration route, and frequency. As a matter of fact, in the last decade, MSCs have emerged as a promising therapeutic tool for the treatment of musculoskeletal pathologies in veterinary medicine. Interestingly, the results accumulated so far have provided evidence that veterinary patients affected by naturally occurring diseases should provide more reliable outcomes of cell therapy than laboratory animals, thus allowing translating potential therapies to the human field. More recently, a cell-free therapy based on MSC-secretome has been proposed. Even though there are very few clinical reports to refer to in veterinary medicine, recent acquisitions suggest that MSC-derived products may have major advantages compared to the related cells, e.g., they are considered safer and less immunogenic. Although several studies propose secretome as a novel therapeutic biological product, a better understanding of the nature, bioavailability, and the mechanisms of action responsible for the beneficial effects of the paracrine molecules is needed. Based on these sceneries, a secretome GMP-compliant production process is needed for veterinary clinical use and further studies on in vivo animal models; indeed, a veterinary medicinal product based on stable secretome formulation represents a valid strategy for MSDs therapy.