1. Introduction
Tendonitis and desmitis are defying clinical challenges in equine patients requiring long recovery periods, and ineffective tendon repair entails their sportive careers. Tendons operate near its functional limit during maximal exercise. Their ability to adapt to stress and self-repair is limited. A controlled exercise program alone or in combination with a variety of conservative treatments, such as corrective shoeing and nonsteroidal anti-inflammatory drugs (NSAIDs), is still the gold-standard therapy for equine tendon disease [
1]. Current treatments often do not fully repair or regenerate the injured or affected tendon nor lead to its total functional recovery [
1,
2].
The aim on tendinopathy treatment is the achievement of tissue regeneration to provide return to complete organ function and performance. Tissue engineering has gained a special interest over the last years for tissue repair. Among this, the development of mesenchymal stem cell-based therapies has boosted, being a promising approach to tissue repair and regeneration including tendinopathy and desmitis [
1,
3,
4,
5,
6].
Mesenchymal Stem Cells (MSCs) can be isolated from several tissue sources, such as bone marrow, peripheral blood, dental pulp, umbilical cord, and amniotic fluid [
7]. MSCs characteristics have been defined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT), and those include being plastic-adherent when maintained in standard culture conditions, expressing cluster of differentiation (CD), such as CD44, CD90 and CD105 and no expression of major histocompatibility complex (MHC)-class II markers and of hematopoietic-related markers (CD45 and CD34) [
8]. Finally, MSCs must be able to differentiate in vitro into, at least, osteoblasts, adipocytes and chondroblasts, in the presence of adequate differentiation culture media [
8].
Synovial membrane mesenchymal stem cells (SM-MSCs) were initially isolated, in 2001 by
De Bari et al. [
9], from human knee joints showing a great proliferative ability in culture, even after passage 10 (P10), and multilineage differentiation potential in vitro [
9]. These cells represent a good source of MSCs and a promising therapeutic tool mostly for musculoskeletal pathologies [
10]. Sakagushi
et al., compared the properties of different sources of human stem cells - bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue- and observed the superiority of synovium as a MSCs source for treatment of musculoskeletal pathologies as they had more ability to chondrogenesis. Pellets of synovium derived stem cells were larger and more stained for cartilage matrix [
11].
SM-MSCs chondrogenic capacity is higher than other studied sources of MSCs, such as bone marrow (BM-MSCs) [
12,
13]. Cartilage pellets from SM-MSCs were significantly larger than those from BM-MSCs [
12]. SM-MSCs have a higher production of Uridine Diphosphate Glucose Dehydrogenase (UDPGD) [
13], an enzyme that converts UDP-glucose into UDP-glucuronate, one of the two substrates required by hyaluronan synthase for hyaluronan polymer assembly. Besides, Sox-9, collagen type -II (Col-II), aggrecan, specific markers for chondrogenesis, as well as cartilage-specific molecules such as cartilage oligomeric matrix protein (COMP), were also found in high amount on equine synovial fluid derived MSCs and extracellular matrix respectively by reverse transcription polymerase chain reaction (RT-PCR) [
13].
In a recent study, using a rabbit model,
Bami et al., highlight the superiority of SM-MSC’s in terms of chondrogenesis, osteogenesis, myogenesis and tenogenesis [
14]. Also, a study of xenogenic implantation of SM-MSCs in equine articular defects confirmed a better healing of the cartilage of affected knees as well as a higher expression of collagen type II, indicating the presence of hyaline cartilage in the healed defect [
15].
SM-MSCs were defined as MSCs due to their phenotypic profile and differentiation potential. Even though there are no specific antibodies markers to identify these MSCs, there is a general agreement that MSCs should be negative to hematopoietic markers CD34 e CD45 and positive to CD44, CD73, CD90 and CD105 [
16].
Mochizuki et al., found that SM-MSCs maintain their proliferative ability, despite the part of the synovium they are collected from [
17].
In 2003,
Fickert et al., reported that the markers CD9, CD44, CD54, CD90, and CD166 can be used to identify MSCs isolated from the synovium of human patients with osteoarthritis (OA), and also confirmed that CD9/CD90/CD166 triple-positive cell subgroups have obvious chondrogenic and osteogenic differentiation ability [
18].
Prado et al., confirmed the mesenchymal nature of equine synovial membrane and fluid-derived stem cells through the expression of significant markers of hematopoietic (CD45, CD34, CD117 and CD133) and mesenchymal (CD105, CD90), pluripotency (Oct3/4 and Nanog), embryonic (Tra-1-81), inflammatory and angiogenesis markers (vascular endothelial growth factor (VEGF-R1) and LY6a) [
19]. Although the presence of hematopoietic and inflammatory markers was not expected, variations may occur and must be considered the influence of acute or chronic stages of osteochondrosis expression and/or inflammatory events [
19,
20].
Nevertheless, the immunophenotype characterization of equine MSCs (eMSCs), as well as in other veterinary species, is not yet completely established [
19]. This is a major challenge as the expression of certain adult stem cell markers may differ between species. For that reason, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of eMSCs [
8,
20].
Horses are high performance athletes prone to musculoskeletal diseases – osteoarticular, tendon/ligament lesions and fractures - of various degrees due to sport and age-related injuries. These pathologies resemble human musculoskeletal conditions, turning horses into a valuable animal model for assessing stem cell and cell-based therapies prior to the translation of results into humans [
21]. The use of a therapy able to regenerate these structures and restoring their complete functionality instead of an ordinary healing is the aim of our study and of the equine practitioners among the world.
Recent studies suggest that MSCs can self-renewal, to migrate to injury sites (homing), to perform multilineage differentiation and to secrete bioactive factors, increasing proliferation and migration of tendon stem/progenitor cells via paracrine signaling and increasing regeneration ability of tissues with poor aptitude [
1,
3,
4,
5,
22,
23].
In fact, the knowledge of the importance of this paracrine action has opened doors to cell-free therapeutic strategies in regenerative medicine. The soluble factors (cytokines, chemokines and growth factors) and non-soluble factors (extracellular vesicles and exosomes) released in the extracellular space by MSC’s, commonly known as secretome, became the focus of the novel therapeutic approaches due to their key role in cell to cell communication, their active influence on immune-modulation and pro-regenerative capacity both in vitro and in vivo [
23]. For this reason, in this study, secretome was also analysed with the prospect of being used therapeutically, in the future, in similar clinical cases.
In the present study, equines used as show jumping and dressage athletes, as well as leisure horses with acute and chronic lesions, were treated with intralesional administrations of the considered combination – autologous serum and eSM-MSC’s. This treatment consisted of two injections, 15 days apart. Pre- and post-treatment evaluations consisted of clinical, orthopedic and tendon/ligament ultrasound exams. None of the selected patients have received any other regenerative treatment before.
4. Discussion
Over the last years, eSM-MSCs have become an interesting subject for those who study cellular and cell-based therapies due to their promising ability to promote tissue regeneration with high capacity of regeneration of articular structures, tendon and ligaments. Regarding the collection, isolation, expansion, freezing and thawing protocols used in this clinical trial, it was possible to use these cells in equine tendon regenerative treatments. The fully characterization of eSM-MSCs presents itself a big challenge since eSM-MSCs are not as studied as MSCs from other species, namely human MSCs. However, in this study, their stemness and origin has been confirmed through different processes: trilineage differentiation, karyotype, secretome and immunohistochemistry. All SM-MSCs cultures presented monolayer culture, plastic-adherence capacity and fibroblast-like shape [
40,
41,
42,
43], accomplishing some of the minimal criteria defined by ISCT. Successful osteogenic, chondrogenic and adipogenic differentiation has also been demonstrated.
De Bari et al., [
9] were to first group to isolate MSCs from synovial tissues.
Karyotype presented some genomic variations when the number of passages was increased. That is consistent with some studies regarding genomic variations along cell passages. [
44,
45,
46,
47,
48] DNA replication is a critical event for timely genome duplication. Errors in replication lead to genomic instability across evolution [
49]. Prieto Gonzalez et al., consider that genomic instability, incurred during the process of stem cell isolation, culture expansion, and reprogramming, might be the most critical point of a stem cell-based therapeutic approach as a viable option in the clinical perspective [
50]. Peterson et al., highlighted there is very little evidence linking genomic abnormalities, for example, in human Pluripotent Stem Cell (hPSC’s) with tumorigeneses. [
44] The frequency and effects of variations is increasing with the development of even more sensitive methods for detecting genomic variation [
45].
As reported by
Simona Neri, interpretation of genetic instability and senescence of cultured MSCs is controversial, but the increasing incidence of genetic alterations at advanced culture times clearly indicates that few culture passages correspond to a reduced chance to harbour dangerous alterations. Therefore, a prudential behaviour is desirable with reduction of culture times as much as possible to avoid safety concerns [
51]. More studies must be performed in this area.
During the last decade it has been shown that MSCs therapeutic effectiveness is due mainly to the release of paracrine factors, namely CM, composed of soluble (cytokines, chemokines and growth factors) and non-soluble factors (extracellular vesicles) that are primarily secreted in the extracellular space by the stem cells [
52]. CM’s paracrine signaling can be considered as the primary mechanism by which MSCs contribute to healing processes, becoming their study an interesting subject [
53,
54].
In our study, eSM-MSCs revealed a CM with a high level of KC/GRO, MCP-1, Il-6, FGF-2, G-CSF, GM-CSF and IL-8. This highlights the fibroblast intense activity, producing KC/GRO that is chemotaxic for neutrophils during inflammation. MCP-1 is essential for reperfusion and the successful completion of musculoskeletal tissue after ischemic injury [
55]. Macrophages are tissue resident cells involved in tissue regeneration beside their inflammatory and infection response [
56]. IL-6 is a pro-inflammatory and angiogenic interleukin capable of increasing the expression of growth factors, reactivating for example, intrinsic growth programs of neurons, promoting axonal regrowth, creating a link between inflammation and tissue regeneration [
57,
58]. FGF-2 is a recognized GF responsible for proliferation of tenogenic stem cells. FGF-2 signaling has been reported to produce a tendon progenitor population that expressed scleraxis during somite development [
59]. FGF-2 plays a crucial role in cell proliferation and collagen production, becoming a useful GF for tissue regeneration by promoting stem cell proliferation [
60]. G-CSF is a cytokine that mobilizes bone marrow-derived cells (BM-DCs) to peripheral blood. A study suggests that injection of G-CSF promoting BM-DCs release in the target area - rotator cuff - effectively enhanced rotator cuff healing by promoting tenocyte and cartilage matrix production [
61].
Wright et al., presented a study confirming skeletal muscle damage, including that following strenuous exercise, induces an elevation in plasma G-CSF, implicating it as a potential mediator of skeletal muscle repair [
62]. Recent human trials have shown the benefits of G-CSF administration as a treatment for neuromuscular diseases, considering that G-CSF affects skeletal muscle, leading to functional improvements [
63,
64,
65,
66,
67,
68]. GM-CSF is an hematopoietic growth factor with pro-inflammatory functions [
69]. Major sources of GM-CSF are T and B cells, monocyte/macrophage endothelial cells, and fibroblasts. Neutrophils, eosinophils, epithelial cells, mesothelial cells, Paneth cells, chondrocytes, and tumor cells can also produce GM-CSF [
70].
Paredes et al., evidenced that elevated levels of pro-inflammatory factors such as those found at these cells CM (GM-CSF, G-CSF, Il-6, IL-8 and IL-17), were implicated in the activation of resident tendon cells for effective healing, stimulating tendon cell proliferation [
71,
72]. IL-8 is one of the major mediators of inflammatory response and is a potent angiogenic factor. This is similar to IL-6 but has a longer half-life [
73].
A recent study highlights hematopoietic factors promote tendon healing in aged mouse tendons. Histochemical results demonstrated vascularization of the injury site was significantly elevated. It was concluded that vascular endothelial growth factor (VEGF) not only plays an important role in decreasing adipocyte accumulation but also improves vascularization of the tendon during aged tendon healing. Active regulation of VEGF may improve the treatment of age-related tendon diseases and tendon injuries [
74].
Studies with human BM-MSCs using a human-specific proteome profiler array with different angiogenic factors such as VEGF-A, IL-6, IL-8, platelet-derived growth factor A (PDGF-A), endothelin-1 (ET1), and urokinase plasminogen activator (uPA), which has not been previously reported in the CM of human MSCs, has also been identified in the equine one, confirming what we found in this study [
75]. This factor has been proposed as a modulator of the different neovascularization stages, through the enhancement of VEGF gene promotor activity [
75,
76].
Schokry et al., [
77] reported that BM-MSCs therapies have recovery times of 3-6 months and conservative therapeutic methods allow recovery in 12-18 months without regeneration but formation of fibrous scar tissue. Retrospectively, no re-injuries of tendons have occurred in horses treated with this new approach, during the study frame time. In the literature [
78],
Smith et al., referred as a low percentage re-injury rate 27% for SFD tendonitis treated with bone marrow stem cells. Horses returned to “full function” as defined by
Cook et al. and modified by
Guest et al., [
33,
79].
A study using a murine osteoarthritis (OA) model demonstrated that an injection of MSCs CM, similarly to injection of MSCs, resulted in early pain reduction and had a protective effect on the development of cartilage damage in a murine OA model, by using the regenerative capacities of the MSCs-secreted factors [
80].
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 MSCs-CM has been proposed. Even though there are very few clinical reports to refer to in veterinary medicine, recent acquisitions suggest that MSCs-derived products may have major advantages compared to the related cells, e.g., they are considered safer and less immunogenic [
52]. As evidenced before, eSM-MSCs CM factors are able to promote tendon healing by reducing inflammation and fatty infiltration, stimulating cell proliferation and tenogenic differentiation [
81].
In this study we used a cell-based therapy instead of CM itself, but we are aware of its effect and potential on cell-based therapies, its advantages and therapeutic effects, reason why this study was performed.
To better characterize the cells under study, we carried out immunohistochemistry assays. The markers choice was based on a previous work [
8] and include several of the criteria used for humans as determined by the ISCT. Results of our study demonstrated the presence of embryonic stem cell markers – Oct 4 and Nanog. Detection of these markers was previously described
by Beltrami et al., in multipotent adult stem cells (HMASC) from bone marrow human [
82], as well as, by
Riekstina et al., who also demonstrated the presence of these markers in HMASC derived from bone marrow, adipose tissue, heart and dermis [
83]. Greco et al., also evidenced elevated expression of Oct 4 in P3 MSCs and hypothesized OCT 4 expression could be an indicator of MSC differentiation potential in clinical diagnostics [
84]. In equine characterization of synovial fluid and membrane-derived MSCs, Prado et al., also evidenced the presence of Nanog and Oct 4 markers [
19]. In contrast, Fulber et al., had no positive results for these two markers, in equine mesenchymal stem cell of synovial tissues [
43]. Vimentin, a mesenchymal stem cell marker was also detected, suggesting mesenchymal origin. The presence of Lysozyme confirms the synovial origin of the cells as stated by
Fulber et al. [
43].
Immunohistochemistry analysis showed absence of expression of CD31, sinaptophysine and Pan-cytokeratin, discarding the vascular, neuronal and epithelial origin of our cells. GFAP was weakly expressed, being less expressed in P3 than in P0 cells. CD31 was performed to investigate the presence of hematopoietic cells in eSM-MSCs. The expression of VEGF was not found, being this results similar with those from
Fulber et al., and other authors that evidenced the absence of hematopoietic markers [
43,
85]. The absence of neuronal and dermal markers was also consistent with other studies [
19,
43].
In our clinical trial, we manage to treat mainly early acute lesions. 87.5% of the cases were acute lesions of tendons or ligaments. This concerns with having a master bank cell of allogenic eSM-MSCs that allows treatments in early acute phases versus treatments with autologous cells where time of tissue collection, preparation and cell culture need to be considered. Furthermore, cells harvesting for autologous treatment is an invasive procedure unnecessary with this new product. The possibility of having a master cell bank enables faster healing of the organ and a quicker return to sportive life. Horses spend less time in recovery time and have a regenerated tissue instead of a fibrotic tissue. These are some advantages of this mixture. Another one concerns with the fact that in early stages of lesion there is inflammatory phase, the paracrine factors released by eSM-MSC’s also have anti-inflammatory action, reducing inflammation.
Chronic cases represented 12.5% of the cases, involving 4 structures. Three of them recovered in 30 days and one of them had a delayed recovery time.
The delayed recovery time in 20% of the structures, meaning 12.5% of the animals, was due to, in case 6, increased number of involved structures (more than one tendon or ligament) and foot conformation abnormality, the horse had a fetlock hyperextension that was disabling the correct tendon healing. This was corrected with special shoeing. Inappropriate rehabilitation program (case number 7) was another cause of delayed recovery time. As soon as the corrective shoeing was performed, ligament regeneration started.
Kamm et al., 2021, conclude that based on the evidence to date, tendons appear to have improved healing when treated with allogeneic MSCs, and the use of these treatments in equine tendon and ligament lesions is warranted [
86]. Colbath et al., 2020, claimed that some of the advantages of using allogenic stem cells, include the ability to bank cells and reduce the time to treatment, to collect MSCs from younger donor animals and the ability to manipulate banked cells prior to administration [
87]. Some of disadvantages focused on the risk of immunological reactions. However, nowadays there are several studies in horses accumulating evidence that allogeneic MSCs maybe a safe alternative to autologous MSCs [
87]. Nevertheless, donor’s health must always be taken in attention as well as his age [
88].
Author Contributions
Conceptualization, I.L.R, B.L, P.S, L.M.A, J.M.S, and A.C.M.; methodology, I.L.R, B.L, P.S, A.C.S., M.V.B, A.R.C, S.S.P, A.R, B.P, I.A, R.D.A, J.M.S and A,C,M; software, I.L.R, A.C.S., M.V.B, R.D.A and J.M.S.; validation, I.L.R, A.C.S, R.D.A. M.V.B., B.L, P.S, A.R, A.R.C, S.S.P, L.M.A, B.P, C.O, I.A, J.M.S, A.C.M.; formal analysis, I.L.R.; investigation, I.L.R, B.L, P.S, A.C.S., M.V.B, A.R.C, S.S.P, A.R, L.M.A, B.P, C.O, I.A, R.D.A, J.M.S, and A.C.M..; re-sources, J.M.S and A.C.M; data curation, I.L.R, P.S, B.L.; writing—original draft preparation, I.R.L, B.L, P.S,.; writing—review and editing, I.L.R, B.L, P.S; visualization, I.R.L, B.L, P.S, A.C.S, M.V.B, A.R.C, S.S.P, A.R, L.M.A, B.P, C.O, I.A, R.D.A, J.M.S and A.C.M.; supervision, L.M.A, J.M.S and A.C.M; project administration, A.C.M..; funding acquisition, A.C.M. All authors reviewed the final work and approved its submission. All authors agreed to be personally accountable for the author’s own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Laboratory: arrival and preparation of the fresh tissue to start digestion, isolation and expansion. (a) Tissue collected in the field and (b) isolated synovial membrane.
Figure 1.
Laboratory: arrival and preparation of the fresh tissue to start digestion, isolation and expansion. (a) Tissue collected in the field and (b) isolated synovial membrane.
Figure 2.
Schematic representation of event sequence from the collection of synovial membrane to the administration of the therapeutic combination. After the collection, the synovial membrane is transported to the laboratory where it is separated from the whole tissue, decontaminated, incubated and digested. Then cells are cultured and expanded and finally cryopreserved in a cell bank. When needed for treatment, cells are prepared with autologous serum and then applied in selected equine patient.
Figure 2.
Schematic representation of event sequence from the collection of synovial membrane to the administration of the therapeutic combination. After the collection, the synovial membrane is transported to the laboratory where it is separated from the whole tissue, decontaminated, incubated and digested. Then cells are cultured and expanded and finally cryopreserved in a cell bank. When needed for treatment, cells are prepared with autologous serum and then applied in selected equine patient.
Figure 3.
Timeline of eSM-MSCs treatment protocol and rehabilitation program. The day before the first treatment (T0), blood from the patient was collected to prepare autologous serum. At T0 the mixture of autologous serum and eSM-MSC’s was injected intralesional after a clinical and ultrasound examination. After 15 days the same procedure was repeated. At day 30 (T2), a clinical and ultrasound examination was performed and if a favorable outcome was considered, the animal progressed to a physical rehabilitation program. During the physical rehabilitation program, the patient was also re-evaluated at days 60 and 90.
Figure 3.
Timeline of eSM-MSCs treatment protocol and rehabilitation program. The day before the first treatment (T0), blood from the patient was collected to prepare autologous serum. At T0 the mixture of autologous serum and eSM-MSC’s was injected intralesional after a clinical and ultrasound examination. After 15 days the same procedure was repeated. At day 30 (T2), a clinical and ultrasound examination was performed and if a favorable outcome was considered, the animal progressed to a physical rehabilitation program. During the physical rehabilitation program, the patient was also re-evaluated at days 60 and 90.
Figure 4.
eSM-MSCs in culture, isolated through enzymatic digestion. (a) - Passage 0 (P0) and (b) - Passage 1 (P1). Plastic adhesion, monolayer and fibroblast-like shape of eSM-MSC’s may be observed. Magnification: 100x.
Figure 4.
eSM-MSCs in culture, isolated through enzymatic digestion. (a) - Passage 0 (P0) and (b) - Passage 1 (P1). Plastic adhesion, monolayer and fibroblast-like shape of eSM-MSC’s may be observed. Magnification: 100x.
Figure 8.
Immunolabelling of eSM-MSCs P0 and P3. Magnification 600x. Images present positive Ag expression to Oct-4 and Nanog confirming stem cell origin, to Vimentin confirming non-epithelial origin of the cells and to Lysozyme confirming synovial origin of the cells. Positive expression was revealed by cytoplasmatic staining of the cells. CD31, Synaptophysin and Pan-cytokeratin had negative expression, did not stain, and confirmed these cells had no vascular, neuronal or epithelial origin. GFAP represents a neuronal origin and had a weak expression in P0, which reduced in P3.
Figure 8.
Immunolabelling of eSM-MSCs P0 and P3. Magnification 600x. Images present positive Ag expression to Oct-4 and Nanog confirming stem cell origin, to Vimentin confirming non-epithelial origin of the cells and to Lysozyme confirming synovial origin of the cells. Positive expression was revealed by cytoplasmatic staining of the cells. CD31, Synaptophysin and Pan-cytokeratin had negative expression, did not stain, and confirmed these cells had no vascular, neuronal or epithelial origin. GFAP represents a neuronal origin and had a weak expression in P0, which reduced in P3.
Figure 10.
Ultrasound images of cases number: 4, 14 and 16 that represent the clinical cases concerning Superficial Digital Flexor Tendon (SDFT). Transversal and longitudinal images at day 1 and day 30 after first treatment with eSM-MSCs. These ultrasounds are representative of the cases and very illustrative of good fiber alignment and cross-sectional area reduction, evidencing tissue regeneration.
Figure 10.
Ultrasound images of cases number: 4, 14 and 16 that represent the clinical cases concerning Superficial Digital Flexor Tendon (SDFT). Transversal and longitudinal images at day 1 and day 30 after first treatment with eSM-MSCs. These ultrasounds are representative of the cases and very illustrative of good fiber alignment and cross-sectional area reduction, evidencing tissue regeneration.
Table 1.
Score systems used by the veterinary surgeon to assess lameness, response to flexion test and pain to pressure [
25,
26].
Table 1.
Score systems used by the veterinary surgeon to assess lameness, response to flexion test and pain to pressure [
25,
26].
Parameter |
Score |
Clinical implication |
AAEP Grading |
0 |
No Lameness |
1 |
Lameness not consistent |
2 |
Lameness consistent under certain circumstances |
3 |
Lameness consistently observable on a straight line. |
4 |
Obvious lameness at walk: marked nodding or shortened stride |
5 |
Minimal weight bearing lameness in motion or at rest |
Flexion Test |
0 |
No flexion response |
1 |
Mild flexion response |
2 |
Moderate flexion response |
3 |
Severe flexion response |
Pain to pressure |
0 |
No pain to pressure |
1 |
Mild pain to pressure |
2 |
Moderate pain to pressure |
3 |
Severe pain to pressure |
Table 2.
List of antibodies investigated, dilutions, and antigen retrieval methods applied in the immunohistochemical analysis.
Table 2.
List of antibodies investigated, dilutions, and antigen retrieval methods applied in the immunohistochemical analysis.
Marker |
Type/Clone |
Supplier |
Dilution / Incubation period |
Antigen unmasking |
Positive control |
Cells of interest |
Reference |
Oct-4 |
Polyclonal |
Abcam |
1/100 ON |
RS/WB |
Canine mast cell tumour |
Stem cells |
Ab18976 |
Nanog |
Clone Mab |
ABGENT |
1/10 ON |
RS/WB |
Canine testicular carcinoma |
Stem cells |
AM1486b |
c-Kit (CD117) |
Polyclonal |
Dako Denmark |
1/450 ON |
RS/WB |
Canine mast cell tumour |
Stem cells |
A4502 |
Lysozyme |
Polyclonal |
Dako Denmark |
1/400 ON |
RS/WB |
Canine synovial membrane |
Synovial cells |
A0099 |
Vimentin |
Clone V9 |
Dako Denmark |
1/500 ON |
RS/WB |
Canine mammary gland |
Non-epithelial cells |
M0725 |
Pan-cytokeratin |
Cocktail AE1/AE3 |
Thermo Scientific |
1/300 ON |
RS/WB |
Canine mammary gland |
Epithelial cells |
M3-343-P1 |
GFAP |
Polyclonal |
Merck Millipore |
1/2000 ON |
RS/WB |
Mouse brain tissue |
Neuronal cells |
AB5804 |
Sinaptophysine |
Clone SP11 |
Thermo Scientific |
1/150 ON |
RS/WB |
Mouse brain tissue |
Neuronal cells |
RM-9111-S |
CD31 |
Clone JC70A |
Dako Denmark |
1/50 ON |
Pepsine |
Canine spleen |
Platelet endothelial cells |
M0823 |
Table 3.
Lesion type casuistic.
Table 3.
Lesion type casuistic.
Lesion type |
Nº clinical cases |
Total number (2019) |
Tendonitis |
16 |
20 |
Desmitis |
4 |
Table 4.
Physical rehabilitation program. After eSM-MSCs treatment, all horses were submitted to a rehabilitation program consisting of two days of box rest followed by 13 days of 10 minutes hand-walk. Bandage applied on treatment day was removed 24h after treatment. At day 15 the second treatment was performed followed by another 15 days of rehabilitation, until day 30. Between day 30 and day 45 the work consisted of 20 min hand-walking, between day 45 and day 60 the work was 30 minutes of hand-walking, between day 60 and day 75, 30 minutes of hand walking plus 5 minutes trot and finally between day 75 and day 90, the patient was submitted to 30 minutes of hand-walking plus 10 minutes of trot. After this the patient could return to full work.
Table 4.
Physical rehabilitation program. After eSM-MSCs treatment, all horses were submitted to a rehabilitation program consisting of two days of box rest followed by 13 days of 10 minutes hand-walk. Bandage applied on treatment day was removed 24h after treatment. At day 15 the second treatment was performed followed by another 15 days of rehabilitation, until day 30. Between day 30 and day 45 the work consisted of 20 min hand-walking, between day 45 and day 60 the work was 30 minutes of hand-walking, between day 60 and day 75, 30 minutes of hand walking plus 5 minutes trot and finally between day 75 and day 90, the patient was submitted to 30 minutes of hand-walking plus 10 minutes of trot. After this the patient could return to full work.
Week |
Exercise |
0-2 |
2 days: stall confinement Handwalk: 10 min Day 15: new treatment
|
3-4 |
2 days: stall confinement Handwalk: 10 min VET-CHECK – day 30
|
5 |
Handwalk: 15 min
|
6 |
Handwalk: 20 min VET-CHECK – day 45
|
7 |
Handwalk: 25 min
|
8 |
Handwalk: 30 min VET-CHECK – day 60
|
9-10 |
Handwalk: 30 min + 5min trot
|
11-12 |
Handwalk: 30 min + 10 min trot VET-CHECK - day 90
|
Table 5.
Cytogenetic analysis in passages 4 and 7 (P4 and P7). Percentage of normal cells, tetraploid cells and aneuploid cells.
Table 5.
Cytogenetic analysis in passages 4 and 7 (P4 and P7). Percentage of normal cells, tetraploid cells and aneuploid cells.
P4 |
Cytogenetic analysis |
P7 |
36% |
Normal cells 64, XY |
32% |
4% |
Tetraploid cells 128 XXYY |
8% |
60% |
Aneuploid cells:
Hipoploidy 54-63 Hiperploidy 71 |
60% |
56%
4% |
56%
4% |
Table 6.
Equine patient and Lesion characterization. Left column characterizes Equine patient: Sex - male (M) or female (F); age measured in years old (yo); sports modality- SM: Show jumping (SJ), Dressage (Dre) and Leisure (Lsr). Right column characterizes Lesion: Structure affected - Superficial Digital Flexor Tendon (SDFT), Deep Digital Flexor Tendon (DDFT) and Suspensory Ligament (SL) – Left branch (LB); affected Limb - Right Frontlimb (RF), Right Hindlimb (RH), Left Frontlimb (LF) and Left Hindlimb (LH).
Table 6.
Equine patient and Lesion characterization. Left column characterizes Equine patient: Sex - male (M) or female (F); age measured in years old (yo); sports modality- SM: Show jumping (SJ), Dressage (Dre) and Leisure (Lsr). Right column characterizes Lesion: Structure affected - Superficial Digital Flexor Tendon (SDFT), Deep Digital Flexor Tendon (DDFT) and Suspensory Ligament (SL) – Left branch (LB); affected Limb - Right Frontlimb (RF), Right Hindlimb (RH), Left Frontlimb (LF) and Left Hindlimb (LH).
Equine Patient |
Lesion: Tendonitis / Desmitis |
ID |
Sex |
Age (yo) |
SM |
Structure |
Type |
Limb |
Evolution |
1 |
M |
22 |
SJ |
SDFT DDFT |
Acute |
LF |
Favorable in 30 days |
2 |
F |
5 |
SJ |
SDFT |
Acute |
RH |
Favorable in 30 days |
3 |
F |
14 |
SJ |
SDFT |
Chronic |
RF |
Favorable in 30 days |
4 |
M |
8 |
SJ |
SDFT |
Acute |
RF |
Favorable in 30 days |
5 |
M |
7 |
SJ |
LB SL |
Acute |
LF |
Favorable in 30 days |
6 |
M |
13 |
Lsr |
SDFT DDFT SL |
Chronic |
RH |
Tendons: favorable evolution in 30 days; SL in 90 days. |
7 |
M |
15 |
SJ |
SFDT |
Acute |
RF |
Favorable in 90 days |
8 |
M |
11 |
SJ |
SDFT |
Acute |
RF |
Favorable in 30 days |
9 |
F |
10 |
SJ |
SDFT SL |
Acute |
RF |
Favorable in 30 days |
10 |
M |
9 |
SJ |
SDFT |
Acute |
LF |
Favorable in 30 days |
11 |
F |
10 |
SJ |
SDFT |
Acute |
RF |
Favorable in 30 days |
12 |
M |
12 |
Dre |
SDFT |
Acute |
LF |
Favorable in 30 days |
13 |
M |
14 |
SJ |
SL |
Acute |
LF |
Favorable in 30 days |
14 |
M |
7 |
SJ |
SDFT |
Acute |
RF |
Favorable in 30 days |
15 |
M |
12 |
SJ |
SFDT |
Acute |
LF |
Favorable in 30 days |
16 |
F |
6 |
SJ |
SFDT |
Acute |
RF |
Favorable in 30 days |
Table 7.
Ultrasonographic lesion characterization at day 1, 15 and 30. Patient identification, structure affected, lesion ultrasonographic location, cross sectional area and longitudinal fiber pattern are characterized. Assessment outcome is also evaluated. Affected structure superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT), Suspensory Ligament (SL) and Left Branch (LB). Lesion-ultrasonographic location (zones 1A-1B, 2A-2B, 3A-3B) [
27], Cross Sectional Area % (0 = 0%, 1 = <25%, 2 = >25-50%, 3 = >50-75%, 4>75%) Longitudinal Fiber Pattern (0=0%, 1=<25%, 2=>25-50%, 3=>50-75%, 4>75%) [
27] and assessment outcome (full function, acceptable function and unacceptable function) [
33].
Table 7.
Ultrasonographic lesion characterization at day 1, 15 and 30. Patient identification, structure affected, lesion ultrasonographic location, cross sectional area and longitudinal fiber pattern are characterized. Assessment outcome is also evaluated. Affected structure superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT), Suspensory Ligament (SL) and Left Branch (LB). Lesion-ultrasonographic location (zones 1A-1B, 2A-2B, 3A-3B) [
27], Cross Sectional Area % (0 = 0%, 1 = <25%, 2 = >25-50%, 3 = >50-75%, 4>75%) Longitudinal Fiber Pattern (0=0%, 1=<25%, 2=>25-50%, 3=>50-75%, 4>75%) [
27] and assessment outcome (full function, acceptable function and unacceptable function) [
33].
Patient ID |
Day |
Structure |
Location |
Cross Sectional Area |
Longitudinal Fiber Pattern (%) |
Assessment Outcome |
1 |
1 |
SDFT DDFT |
1A-1B 1A-1B |
1 1 |
1 1 |
|
15 |
SDFT DDFT |
1A-1B 1A-1B |
1 1 |
1 1 |
|
30 |
SDFT DDFT |
1A-1B 1A-1B |
0 0 |
0 0 |
Full function |
2 |
1 |
SDFT |
1A-1B |
1 |
1 |
|
15 |
SDFT |
1A-1B |
1 |
1 |
|
30 |
SDFT |
1A-1B |
0 |
0 |
Full function |
3 |
1 |
SDFT |
1A-1B |
1 |
1 |
|
15 |
SDFT |
1A-1B |
1 |
1 |
|
30 |
SDFT |
1A-1B |
0 |
0 |
Full function |
4 |
1 |
SDFT |
1A-1B |
2 |
2 |
|
15 |
SDFT |
1A-1B |
2 |
2 |
|
30 |
SDFT |
1A-1B |
0 |
0 |
Full function |
5 |
1 |
LB SL |
3A-3B |
2 |
2 |
|
15 |
LB SL |
3A-3B |
2 |
2 |
|
30 |
LB SL |
3A-3B |
0 |
0 |
Full function |
6 |
1 |
SDFT DDFT SL |
2A-2B 2A-2B 2A-2B |
2 2 2 |
2 2 2 |
|
15 |
SDFT DDFT SL |
2A-2B 2A-2B 2A-2B |
1 1 2 |
1 1 2 |
|
30 |
SDFT DDFT LS |
2A-2B 2A-2B 2A-2B |
1 1 1 |
1 1 1 |
Unacceptable function. Only at day 90. |
7 |
1 |
SDFT |
2A-2B |
3 |
3 |
|
15 |
SDFT |
2A-2B |
3 |
3 |
|
30 |
SDFT |
2A-2B |
2 |
2 |
Unacceptable function. |
8 |
1 |
SDFT |
2A-2B |
2 |
3 |
|
15 |
SDFT |
2A-2B |
2 |
2 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |
9 |
1 |
SDFT |
2A-2B |
2 |
1 |
|
15 |
SDFT |
2A-2B |
1 |
1 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |
10 |
1 |
SDFT |
1A-1B |
3 |
3 |
|
15 |
SDFT |
1A-1B |
2 |
2 |
|
30 |
SDFT |
1A-1B |
0 |
0 |
Full Function |
11 |
1 |
SDFT |
1A-1B |
2 |
2 |
|
15 |
SDFT |
1A-1B |
2 |
2 |
|
30 |
SDFT |
1A-1B |
0 |
0 |
Full function |
12 |
1 |
SDFT |
2A-2B |
1 |
1 |
|
15 |
SDFT |
2A-2B |
1 |
1 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |
13 |
1 |
SL |
1A-1B |
2 |
2 |
|
15 |
SL |
1A-1B |
1 |
1 |
|
30 |
SL |
1A-1B |
0 |
0 |
Full function |
14 |
1 |
SDFT |
2A-2B |
1 |
1 |
|
15 |
SDFT |
2A-2B |
1 |
1 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |
15 |
1 |
SDFT |
2A-2B |
2 |
2 |
|
15 |
SDFT |
2A-2B |
2 |
2 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |
16 |
1 |
SDFT |
2A-2B |
2 |
2 |
|
15 |
SDFT |
2A-2B |
2 |
2 |
|
30 |
SDFT |
2A-2B |
0 |
0 |
Full function |