Allogeneic Adipose-Derived Mesenchymal Stem Cells (Horse Allo 20) for the Treatment of Osteoarthritis-Associated Lameness in Horses: Characterization,Safety,and Efficacy of Intra-Articular Treatment

Abstract

Osteoarthritis commonly causes lameness in the horse and has a great impact in performance animals. Due to the limitations of current medical therapies, allogenic mesenchymal stem cells (MSCs) may become an alternative method to control inflammation, reduce tissue damage and pain, and therefore improve lameness. We present the results of a regulatory clinical trial testing adipose-derived MSCs (Horse Allo 20) in veterinary (Agencia Española del Medicamento y Productos Sanitarios, Spanish Medicines Agency, Reference number 325/ECV) involving a total number of 80 participants and with 90 days of follow-up period. The manufacturing process of Horse Allo 20 was robust with no influence of the adipose tissue donor (gender, age, or breed), sample origin (intraperitoneal or subcutaneous), or storage conditions (fresh vs. frozen product presentations) on the quality, safety, and efficacy of the drug product. An in vivo safety study showed that local and systemic tolerance was safe even after repeated intra-articular administration (three injections). An in vivo efficacy study demonstrated the efficacy of the treatment after one or two injections by a reduction in lameness (P < 0.05) for an extended period of time (90 days), decreasing the need for prolonged local and/or systemic anti-inflammatory therapies and their well-known deleterious effects and toxicities.

Introduction

Osteoarthritis (OA) is characterized by synovial membrane inflammation, a deterioration of articular cartilage, and changes in the bone and soft tissues of the joint causing pain, deformity, loss of motion, decreased function, and lameness. Secreted inflammatory factors, such as proinflammatory cytokines, are critical mediators of the disturbed metabolism and enhanced catabolism of osteoarthritic joints [1].

In young horses, OA is predominantly trauma related with acute inflammation, while in elderly equines, it is the result of a chronic degenerative disease. Early treatment of inflamed joints could prevent or delay the OA onset in geriatric horses.

Common medication for OA treatment includes intra-articular (IA) use of corticosteroids. This medication has some drawbacks related to the possible joint deterioration by cartilage damage [2]. Systemic nonsteroidal anti-inflammatory drugs (NSAIDs) are another treatment to inhibit the disease progression and degenerative changes to the cartilage surface. Nevertheless, toxic effects such as gastric ulceration, right dorsal colitis, and renal injury have been described [3]. Alternative treatments based on IA application of mesenchymal stem cells (MSCs) are being developed based on their capacity to reduce inflammation and associated pain without side effects [4].

Treatment with allogeneic MSCs offers the advantage of being immediately available for therapy without the delay associated with the culture and expansion of autologous MSCs [5]. This is possible because allogeneic MSCs are immune evasive and their expression of major histocompatibility complex (MHC) class II molecules is negligible [5].

The naturally occurring OA in horses is similar to that of humans. It is often used as a model to investigate the pathogenesis and treatment of the disease [6], and could help to define the potential therapeutic efficacy and safety of stem cell therapies for human use.

The International Society for Cellular Therapy (ISCT) established the minimal criteria to define human MSCs to allow the comparison of results among different studies: (i) MSCs must be plastic adherent when maintained in standard culture conditions; (ii) MSCs must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14, or CD11b, CD79a, or CD19 and HLA-DR surface molecules; and (iii) MSCs must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro [7,8]. In addition, CD29 and CD44 were consistently detected in horse MSCs [9,10]. Despite the widespread use of MSCs from non-humans, there are no established minimal criteria for the identification of MSCs in other species, given that not all express the same panel of surface antigens and lack of available antibodies. Most non-human MSCs express CD29 and CD44; however, the expression of CD73, CD90, and CD105 varies depending on species and strain [11 –13]. Regarding the negative markers, CD34 and CD45 seems to be widespread. In addition, MSCs were shown to possess potent immunomodulatory properties and the ability to alter the function of immune cells [14].

Due to the novelty of allogeneic MSC clinical treatments, issues have been raised about their safety when injected in the recipient. Safety concerns represent a significant barrier to the successful translation of MSCs into an acceptable clinical therapy, although numerous studies have reported the safety of the use of allogeneic MSCs in horses [15,16], including IA administration [17,18]. According to the literature, no ectopic tissue or tumor formation [19 –23], and no cell biodistribution or persistence were found after, at least, 120 days [21,24 –27].

Although the majority of clinical trials listed on www.clinicaltrials.gov test the efficacy of autologous MSCs (more than 300) in humans, the number of clinical trials testing allogeneic MSC efficacy has increased, reaching more than 200 studies. Despite the capacity of MSCs to differentiate, growing evidence suggests that disease-modifying activity of MSCs is due to different products secreted by the cells in response to environmental niche stimuli [28,29]. This paracrine signaling mechanism by MSCs might be more important than differentiation in stimulating repair responses [30] and makes allogeneic MSCs a suitable therapy for many diseases, due to the possibility of infusion without a substantial risk of immune rejection. If MSCs differentiate to specialized cell types, they will change their phenotype, making them prone to be detected and deleted by the host immune system. Since paracrine mechanism is active in a stemness state, it is not affected by the host immune system.

IA administration of autologous MSCs has proven beneficial in experimentally induced and naturally occurring OA both in humans and horses [31,32]. It has been reported that MSCs significantly improve the rate of return to athletic performance in horses with stifle (knee) injuries compared to surgical repair alone [33], and enhance cartilage repair when combined with microfracture alone [34]. A recently published preliminary study showed a significant clinical improvement in horses with OA after IA administration of chondrogenic induced MSCs combined with platelet-rich plasma [35]. However, the previously mentioned references are framed within the research field, and they are not regulated clinical trials. In fact, up to the date, no scientific publications exist with data based in randomized clinical trials with large sample numbers and double-blind evaluation. That is the novelty of this work; we present the results of a regulatory clinical trial testing MSCs in veterinary approved by the Agencia Española del Medicamento y Productos Sanitarios, (AEMPS, Spanish Medicines Agency), with the reference trial number 325/ECV.

Thus, the objective of this study was triple: (i) to establish the robustness of the manufacturing process of MSCs by means of phenotyping the cells by flow cytometry (FC), obtained from different horse adipose tissue sources and donors; (ii) to check the safety (local and systemic tolerance) after repeated administration of allogeneic MSC IA injection in healthy horses in a safety study; and finally, (iii) to check the efficacy of MSC IA treatment for equine lameness improvement using a randomized, multicenter placebo-controlled double-blind study.

Materials and Methods

Cell isolation and culture

Subcutaneous and intraperitoneal equine adipose tissue samples were taken from four horses with age ranging from <1 to 6 years (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/scd). For subcutaneous adipose tissue extraction, the region was aseptically prepared (eg, dock region = base tail) and skin incisions were made under the influence of an adequate anesthetic [36]. Underlying tissues were harvested by a veterinary surgeon. For intra-abdominal adipose tissue extraction, the samples from omentum or hepatic falciform ligament were immediately aseptically collected by authorized personal in euthanized horses at the slaughterhouse. Adipose samples from both regions were placed in a sterile container, covered with sterile saline, and stored at room temperature. After that, the container was introduced in a biological safety bag and next introduced in a tertiary container (a box). The transport was performed at room temperature by company staff or by an authorized transport company. The maximum time allowed for the reception of the sample after the extraction was set in 24 h based on the literature [37]. About 12–60 g of adipose tissue was aseptically collected by surgical excision and they were digested with collagenase IV (Serva Electrophoresis) at 0.2 U/g over 1 h at 37°C. The resultant vascular stromal fraction was cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine (Biowest). After 24 h, cultures were washed with phosphate-buffered saline (PBS) to remove nonadherent cells and fresh medium was added. Cells were detached when confluence was over 75% and subcultured until passage (P) 3 was reached. Cultures were then detached and cryopreserved in FBS with 10% dimethyl sulfoxide (DMSO; Sigma Aldrich).

Horse Allo 20 genetic stability, phenotyping by FC, and viability

The genetic stability of MSCs during the manufacturing process was checked through the microscopic study of metaphases (karyotyping) and in vitro transformation test. The karyotype is used to detect changes in the chromosomal number (aneuploidies), deletions, translocations and other types of chromosomal rearrangements starting from different samples. This test was performed by Laboratorios Echevarne, Barcelona, Spain. The cell transformation assay is used to confirm that MSCs are nontransformed cells by analyzing in vitro their anchorage-independent growth capacity in a semisolid medium, one of the hallmarks of transformation. This test was performed by Instituto de Salud Carlos III, Majadahonda-Madrid, Spain.

For the FC assays, the antibodies selected were based on the minimal surface markers panel proposed by the ISCT [7] and others [9,10], in the availability of specific antibodies manufactured for horse or with described cross-reactivity. Identity panel was composed of surface biomarkers specific for MSCs, while purity panel was composed of markers of other cell types considered impurities. The acceptance criteria established for MSCs as constituent of Horse Allo 20 were (i) for identity: CD29, CD44, CD90, and CD105 of positive cells and (ii) for purity: MHC-II, CD34, CD45, and CD79a of negative cells. The antibodies were used following manufacturer's instructions for dilutions: CD29 (mouse anti-human CD29, phycoerythrin labeled, clone TS2/16; BioLegend^®), CD34 (mouse anti-dog, phycoerythrin labeled, clone 1H6; BD Pharmigen^™), CD44 (rat anti-mouse, phycoerythrin labeled, clone IM7; BD Biosciences), CD45 (mouse anti-human, peridinin-chlorophyll protein-C Cyanine 5.5 labeled, clone 2D1; BD Biosciences), CD79a (mouse anti-human, phycoerythrin labeled, clone HM47; Exbio), CD90 (mouse anti-human, fluorescein isothiocyanate labeled, clone 5E10; Exbio), CD105 (mouse anti-human, Alexa Fluor 488 labeled, clone SN6; Bio-Rad), and MHC-II (mouse anti-horse, phycoerythrin labeled, clone CVS20; Bio-Rad).

Interassay and intra-assay coefficient of variation (CV) were established for each marker. For this purpose, assays were performed with three different cell culture samples in P4, obtained from three donors (H071, H062, and H25) and two adipose tissue origins (subcutaneous and intraperitoneal). Three independent experiments were performed and measured by FC in triplicate (Acquisition 1, 2, and 3).

Cells in P3 were thawed and cultured until confluence >75% in P4. Cell culture was then detached, counted, and stained following manufacturer's instructions. Data acquisition was performed by using a BD FACSCalibur^™ flow cytometer (BD Biosciences) and data analysis was done employing the FlowJo^® software (FlowJo LLC). Mean, standard deviation, and CV (%) for intra-assay and interassay precision were checked. The acceptance criteria were: (i) <10% CV desirable for all methods, (ii) <20%–25% CV acceptable for immunoassays per Fit-for-Purpose article, and (iii) <30% CV may be acceptable for rare event detection [38].

Alternatively, cells were cryostored in DMEM + 10% DMSO for up to 1 year, thawed, and cell viability was checked by Trypan Blue vital dye counting. Viability was expressed as percentage of viable cells (%).

Horse Allo 20 preparation for IA treatment

Cells in P3 were thawed and cultured for 7 days (or until confluence >75%). Cell cultures were then detached and prepared in two presentations: fresh, consisting in a borosilicate syringe prefilled with 2 mL suspension in DMEM (10 × 10⁶ cell/mL), and frozen, consisting in a cryovial with 2 mL suspension in DMEM +10% DMSO (10 × 10⁶ cell/mL). The fresh presentation was directly sent to each trial site by urgent shipping to assure that delivery was completed before 24 h. Viability was <70% when arriving at the veterinary hospital (internal company tests). The frozen presentation was cryostored until been sent to the clinics for treatment.

In vivo safety study

Study design and participants

The study was carried out as a single-center, blind with randomized placebo-control joint assignment safety study (Fig. 1). All animal procedures and protocols were conducted by licensed veterinary surgeons and comply with both national and European legislation (Spanish Royal Decree RD1201/2005 and EU Directive 86/609/CEE as modified by 2003/65/CE, respectively) for the protection of animals used for research experimentation and other scientific purposes. The animals included one male and seven nonpregnant females between 2 and 20 years of age, with no specification of breed or weight and free of active inflammatory or infectious processes. The patients showed a lameness grade ≤1 according to the American Association of Equine Practitioners (AAEP) guidelines. Exclusion criteria included horses suffering from OA in more than one joint of the forelimbs or hindlimbs and horses that have been treated with systemic or local anti-inflammatory drugs (NSAIDs or steroidal) in the 2 weeks before the date of inclusion in this study. Animal welfare was the first priority during the study; so patients could be removed after enrolment based on the veterinary professional decision.

FIG. 1.

Safety study: scheduled data collection, timeline, and treatment allocation. (A) List with the tasks to be performed and data to be collected in each study day. (B) Timeline detailing the study days and follow-up periods. (C) Treatment allocation in study days 0 and 15. MSCs derived from adipose tissue of various donors (hoATMSC001 or H062) and in different presentations (fresh or frozen) or control (PBS or DMEM + DMSO) were applied to the participants (S1–S8). (D) Treatment allocation on study day 30. MSCs derived from adipose tissue of various donors (hoATMSC001 or H062) and in different presentations (fresh or frozen) or control (PBS or DMEM + DMSO) were applied to the participants (S1–S8). MSC, mesenchymal stem cell; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. Color images available online at www.liebertpub.com/scd

Procedures

The patients were numbered randomly from S1 to S8. Distal interphalangeal or metatarsophalangeal joints from right or left limbs of the first animal were randomly assigned to Treatment (Horse Allo 20 fresh or frozen presentation) or Control (PBS or DMEM + DMSO). The sides of the rest of the animals were assigned consecutively alternating control and treatment sides. All horses received Horse Allo 20 in two different joints on study day 0, a retreatment on the same joints on day 15, and a third treatment on day 30. Treatments on days 0 and 15 were from the same donor, and treatment on day 30 was from a different donor (Fig. 1C, D).

Control joints received 2 mL IA dose of PBS or DMEM + 10% DMSO. Treatment joints received 2 mL IA dose of Horse Allo 20 in a fresh or frozen presentation. The treatments were allocated as (i) Treatment side of the horses: Horse Allo 20 or (ii) Control side of the horses (contralateral): PBS or DMEM + DMSO. The needle size employed was from 20 to 22 gauges and 1–1.5 inches length. IA injections of Horse Allo 20 were made following standard arthrocentesis procedures, ensuring the correct positioning of the needle into the synovial space to avoid cartilage damage.

In addition to the preday 0 physical examination, safety evaluation of horses included a physical examination on days 0, 15, and 45 (Fig. 1A for detailed information). Body temperature was taken by using a digital rectal thermometer. Joint temperature was taken by using a noncontact infrared thermometer. Blood samples were also taken on those days and analyzed for the following variables: general hematology and biochemistry parameters, including Ultra-sensitive C-reactive protein (CRP). Synovial fluid sample was obtained on days 0, 15, 30, and 45, or if two or more signs of inflammation appeared, and the next variables were analyzed: microscopic examination, diagnosis, appearance, red blood cells, leukocytes, polynuclear, mononuclear, and mesothelial cells, total protein, and microbiological culture in samples from inflamed joints. Reference values for the equine parameters were based in the literature [39]. Specifically, for total protein, the reference value for normal horses has been documented as 18.3 g/L, and, in general, a value <20 g/L is considered normal [40]. For the diagnosis of infectious arthritis, the reference value is 40 g/L [41,42] and levels >60 g/L are recognized in some chronically infected joints [40]. Besides, false-positive increases in the total protein concentration secondary to excessive ethylenediaminetetraaceticacid (EDTA) can be suspected in EDTA tubes that are not adequately filled [43], which should be taken into consideration with small volume samples. A table representing the reference values in the horse is provided as Supplementary Table S2.

The needle size employed in all the arthroscopic procedures was from 20 to 22 gauges and 1–1.5 inches length. IA injections were made following standard arthrocentesis procedures, ensuring the correct positioning of the needle into the synovial space to avoid cartilage damage and aspirating a few drops of joint fluid. Synovial sample was collected in a microtube 1.3 mL, with screw cap, K3 EDTA (SARSTEDT AG and Co. KG, Germany).

A 24-h period of exercise restriction in a box was established for the safety study.

Outcomes

Safety was evaluated during the scheduled visits or when necessary for animal welfare. Each joint was considered an experimental unit. Reference values for synovial fluid parameters in horses are presented as Supplementary Table S2 [44].

In vivo efficacy study

Study design and participants

It was carried out as a parallel-group, blind, randomized and controlled (placebo-control group) clinical trial (Consort diagram for the study design in Fig. 2). All animal procedures and protocols were conducted by licensed veterinary surgeons and comply with both national and European legislation (Spanish Royal Decree RD1201/2005 and EU Directive 86/609/CEE as modified by 2003/65/CE, respectively) for the protection of animals used for research experimentation and other scientific purposes. The clinical trial (Reference number 325/ECV) was ethically approved by the AEMPS (Spanish Medicines Agency) and monitored by an external Contract Research Organization (CRO). The CRO also conducted the training of the seven veterinary professionals who participated in the study to standardize procedures. In addition, an external expert in pharmacovigilance performed the review of all the adverse events (AEs) recorded to establish a single evaluation criterion and a causality assessment.

FIG. 2.

Efficacy study design. Consolidated Standards of Reporting Trials diagram displaying the progress of all the participants in the efficacy study. Color images available online at www.liebertpub.com/scd

A total of 72 horses from seven equine clinics and breeding and training facilities were randomized for this trial. Inclusion criteria were healthy equine males or nonpregnant females over 2 years of age, suffering from OA in one or more joints of the fore or hind limbs, which produced lameness grade 2–4 (inclusive) based on the guidelines of the AAEP. OA must have been diagnosed by imaging techniques (radiology, magnetic resonance imaging, or ultrasound) in the affected joint selected for treatment. The origin of lameness must have been located in the selected joint by IA analgesic block. Low motion joints (tarsometatarsal, distal intertarsal, and proximal interphalangeal joints) were anesthetized with low volumes (3–5 mL) of local anesthetics. High motion joints (distal interphalangeal, metacarpophalangeal, radiocarpal, and tarsocrural joints) were anesthetized with higher volumes (10–30 mL) of local anesthetics. Twenty to twenty-two gauges and 1–1.5inches length with mepivacaine or bupivacaine were used.

Exclusion criteria included treatment with systemic, local, or IA anti-inflammatory drugs or vaccination in the 2 weeks before the date of inclusion in this study, as well as horses that, due to severe OA (grade 5 lameness) or concomitant diseases, would be required to be treated with systemic or local anti-inflammatory drugs or any compound that could interfere with the lameness evaluation (listed as not permitted) during the study. Females intended for breeding during the study and horses that would not be available for the entire duration of the study or enrolled in another study were also excluded.

Animal welfare was the first priority during the study; so patients could be removed after enrolment based on the veterinary professional decision. In addition, animals could be withdrawn in the case of a protocol violation.

Randomization and masking

Patients were randomly assigned (1:1) to control (CP) or experimental (Horse Allo 20 fresh presentation) group according to a random list of numbers generated using the SPSS Statistical Package in the specified ratio. Randomization and Blinding was ensured during the trial, in which neither the patient owner nor any of the investigators or staff involved in the treatment or clinical evaluation of the patients or CRO monitors were aware of the treatment received. Each study site was supplied with study test articles in identically appearing packaging.

Procedures

Animals received either an IA dose of Horse Allo 20 (fresh presentation only) or CP on day 0. When upon clinical examination on day 45 ± 2 the animal was classified as nonresponder, a second dose of the same type was administered. No post-IA injection rest was indicated in the protocol for the efficacy study. The application of motion restriction was left to the discretion of the veterinary surgeon.

Horses were clinically evaluated in the inclusion visit and treatment visit or day 0 and after treatment administration (days 15 ± 2, 45 ± 2, 60 ± 2, and 90 ± 2), and AEs or concomitant treatments were recorded. An AE was considered any untoward veterinary occurrence that does not necessarily have a causal relationship with the treatment. If the relationship with Horse Allo 20 administration was considered probable after an independent analysis, then it was treated as an adverse reaction (AR).

A summary of the procedures scheduled during each visit is shown in Supplementary Table S3.

Outcomes

The primary efficacy endpoint was the comparison of the percentage of responders versus no responders between treatments. A responder was defined as an animal with a reduction in one or more grades on day 45 ± 2 lameness evaluation grade versus its preday 0 grade.

There were three secondary endpoints: (i) the percentage of responders on day 15, 60, and 90, (ii) lameness grades (Between Treatments Mean grades comparisons and Within Treatment Mean grade reductions), and finally, (iii) the percentage of animals requiring a second treatment administration.

Each joint of the horse was considered an experimental unit. The hypothesis to be tested was that there is a significant difference between the number of responders to one or two doses of Horse Allo 20 administered for the treatment of naturally occurring equine OA, in relation to treatment with CP for the primary efficacy endpoint.

Statistical analysis

Seventy-five patients were randomly assigned to treatment or placebo group. The analysis datasets were classified into Safety population, Intention-to-treat (ITT) population, and per protocol (PP) population as described in the “Guideline on Statistical Principles for Clinical Trials for Veterinary Medicinal Products (pharmaceuticals)” [45]. To ensure basal homogeneity between groups, a baseline analysis was performed. Response parameters obtained at baseline, explanatory variables, and other variables with potential influence on the main response variable were compared (Supplementary Table S4).

Differences between groups for qualitative variables were tested using a chi-square test for homogeneity if application conditions were satisfied; alternatively, Fisher's exact test or likelihood ratio test was used. For quantitative variables, differences between groups were tested using a Student's t-test if application conditions were satisfied; otherwise, Wilcoxon rank-sum test was used. Application conditions were tested using Levene's Test for homogeneity of variances and Kolmogorov-Smirnov's Test for normality of data distribution for each treatment group.

The statistical analysis was performed using SAS System v9.4 (SAS Institute, Inc., Cary, NC). For all statistical tests, a nominal significance level of 5% (P < 0.05) was applied. No adjustment for multiple tests was performed.

Results

Horse Allo 20 phenotyping

Identity and purity

Data from the FC analysis are shown in Table 1 and Fig. 3.

FIG. 3.

Horse Allo 20 characterization by flow cytometry: identity and purity panels. (A) Graphical representation of positive cells for each biomarker detected by flow cytometry. The measurements correspond to interassay and intra-assay tests and to prethawing and post-thawing storage. Data are represented as % mean ± SD. Color legend for each bar is explained in the plot. (B) Expression of the different biomarkers on adipose tissue-derived MSCs (Horse Allo 20) at passage 4 was assessed by flow cytometry. MSCs were positive for the identity panel of biomarkers (CD29, CD44, CD90, and CD105) and negative for the purity panel of biomarkers (CD34, CD45, CD79, and MHC-II). SD, standard deviation; MHC, major histocompatibility complex.

Table 1.

Horse Allo 20 Phenotyping by Flow Cytometry: Intra-Assay, Interassay, and Prethawing /Post-Thawing Effect

	Intra-assay variation (Acquisitions)				Interassay variation (Experiments)				P4			P4 post-thawing
	1	2	3	CV (%)	1	2	3	CV (%)	H071	S4	S7	H071	S4	S7
Identity
CD29	99.07	98.99	98.97	<10	99.17	99.13	98.73	<10	99.02	98.13	97.67	79.35	99.90	97.77
CD44	99.72	99.74	99.70	<10	99.75	99.65	99.77	<20–25^a	99.44	98.43	98.90	95.40	99.83	99.13
CD90	99.95	99.93	99.42	<10	99.96	99.42	99.92	<10	99.52	100.00	99.93	100.00	100.00	99.90
CD105	22.65	22.03	21.87	>30^b	24.25	21.37	20.92	<10	47.20	39.23	42.73	21.30	74.40	33.67
Purity
CD34	1.84	2.00	2.14	>30^b	2.28	2.18	1.52	<20–25^a	2.22	0.42	1.42	0.07	0.80	0.65
CD45	0.65	0.65	0.71	>30^b	0.82	0.60	0.59	<10	0.72	3.06	0.48	0.00	0.37	0.24
CD79a	1.03	0.96	1.04	>30^b	0.99	1.11	0.94	<10	0.70	0.09	0.84	0.05	0.42	0.43
MHC-II	0.55	0.49	0.46	>30^b	0.45	0.50	0.55	>30^b	0.56	0.05	0.63	0.05	0.42	0.36

Numbers in the table represent the mean of % positive cells for each biomarker assessed.

Acceptable for immunoassays per Fit-for-Purpose article.

Acceptable for rare event detection.

CV, coefficient of variation; P, passage; MHC, major histocompatibility complex.

Interassay and intra-assay CV were <10% or acceptable for identity panel of biomarkers, with the exception of CD105 that showed an intra-assay variation >30%. For the purity panel of biomarkers, interassay and intra-assay CV were mostly >30%. These results were considered acceptable due to the nature of the Fit-for-purpose immunoassay. In addition, purity biomarkers are characterized by very low% of positive cells measured by FC (<10% positive cells), which makes minor differences among acquisitions or experiments trigger higher CV.

It can be concluded that no significant differences existed among experiments, acquisitions, and tissue sample origin from different donors.

Genetic stability

MSCs at P4 and P6 (beyond the product release) were analyzed and no changes in karyotype and/or anchorage-independent growth in soft agar were detected, thus confirming the genetic stability of the MSCs. Supplementary Figure S1 document contains example images of the results for the karyotyping and anchorage-independent growth.

Freezing thawing effect

MSC viability (%) prethawing and post-thawing was 98.04 and 92.45, respectively, with no significant changes. In addition, no significant changes in phenotype between prethawing and post-thawing were observed (Table 1; Fig. 3).

In vivo safety of Horse Allo 20 IA administration

A total number of eight horses were employed for the safety study according to the criteria described in the experimental “Procedure” section.

Clinical parameters

Daily rectal temperature showed no significant alterations or fever symptoms in any patient, suggesting no systemic effects of Horse Allo 20 injection in body temperature. Regarding the joint temperature, no significant differences were observed when comparing the following: (i) DMEM + 10% DMSO versus PBS buffer, (ii) Horse Allo 20 versus any of the controls, and (iii) between fresh and frozen presentations of Horse Allo 20 (Supplementary Fig. S2).

No local reactions were observed after the first injection with Horse Allo 20. After the second and third injection, some of the joints injected with Horse Allo 20 showed signs of swelling or joint flare (pain, deformation, and synovitis) with or without lameness, but all of them resolved spontaneously in 24–48 h without further intervention.

Hematological and biochemistry parameters

All the biochemical or hematological parameters analyzed, including CRP, remained within ranges considered normal. Some parameters showed interindividual variability, but all of them remained stable during the procedures.

Synovial fluid parameters

No xanthochromia associated to a chronic process in the joint was observed. Given the small size joints selected, iatrogenic hemorrhage disturbed the erythrocytes counts in some samples.

The number of nucleated cells remained in normal values during the study follow-up. Even after repeated sampling to characterize inflammatory signs, none of the samples showed results higher than 100,000 cells, which is established as the reference value to consider an inflammatory reaction.

Polymorphonuclear cell counting was normal in all the samples. Even in the samples taken after the second and third administration of the product, the polymorphonuclear/nucleated cell ratio never exceeded 25%, which is considered the limit to determine the presence of inflammation.

No mesothelial cells were found in any of the samples.

The values obtained for the erythrocyte counting, total protein assessment, leukocyte counting, and polynuclear cell counting in all the animals can be seen in Supplementary Tables S5–S8.

In vivo efficacy of Horse Allo 20 IA treatment

A total of 76 horses were enrolled according to the inclusion criteria described in the experimental “Procedure” section, 72 were randomized and 70 completed the study on day 90 (±2). Basal homogeneity was confirmed by comparing all the variables between CP and Horse Allo 20 groups (Supplementary Table S4).

Efficacy results were virtually identical for the ITT analysis and the PP analysis; therefore, only the ITT results are presented.

Primary efficacy endpoint

Results for the comparison of the percentage of responders on day 45 are presented in Fig. 4A. The stratified analysis by age showed that treatment with Horse Allo 20 was statistically significantly superior (P < 0.05) to treatment with CP for the primary variable in animals younger than 20 years (n = 62).

FIG. 4.

Lameness between Horse Allo 20 and placebo groups. (A) Percentage of responders comparing Horse Allo 20 versus control treatment. Y axis represents the study day. Day 45 < 20 corresponds to the stratified analysis of horses <20 years old. Data are represented as % mean ± 95% of confidence interval. Differences were considered significant when P < 0.05. Legend for each line is explained in the plot. (B) Lameness mean score reduction comparing Horse Allo 20 versus control treatment. Data are represented as lameness mean score reduction ± SD. Differences were considered significant when P < 0.05. Legend for each line is explained in the plot. (C) Lameness mean score from day 0 to 90 comparing Horse Allo 20 versus control treatment. Data are represented as lameness mean score, upper and lower limits.

Secondary efficacy endpoints

Mean grade reduction

Statistical analysis demonstrated that the grade of reduction of lameness was significantly greater from day 60 onward (day 60 and 90) in animals treated with Horse Allo 20, compared with animals treated with CP (Fig. 4B).

The percentage of responders was calculated for each treatment on days 15, 45, 60, and 90 showing that the percentage of responders in the Horse Allo 20 group was significantly greater on days 15, 60, and 90 compared with CP animals (Fig. 4A); 78.40% of the horses in the Horse Allo 20 group received a second injection on day 45. The percentage of responders in the experimental group from the ITT population on day 45 (51.4%) increased to 70.30% on day 90, thus confirming the safety (two doses received with no noticeable AEs) and efficacy (reduction of lameness compared to day 0) of the treatment. Twenty-six out of 37 horses in the experimental group were responders on day 90. Considering the 26 responders, 18 out of 26 were responders on day 45 and 8 of 26 responded after receiving the second injection of Horse Allo 20. It implies that 30.77% of the horses needed a second dose to improve lameness grade. When one dose was not effective, a second dose was administered, resulting in an improvement in lameness grade.

Mean lameness grades

Mean lameness grades were lower in animals treated with Horse Allo 20 compared with CP on days 15, 60, and 90. Statistical comparison of treatment groups revealed that mean lameness grades were significantly lower (P < 0.05) in animals treated with Horse Allo 20 on day 90 (Fig. 4C).

Distribution of lameness grades

The distribution of the lameness grades over time was analyzed and compared between treatment groups. The statistical analysis revealed significant differences between treatment groups on days 60 and 90 in the distribution of grades of lameness, with greater percentages of animals treated with Horse Allo 20 in the lower grades compared with animals that received CP (P < 0.05 for day 60 and 90). Overall, the number and percentage of animals receiving a second treatment on day 45 were analyzed and compared between treatments. The majority of animals in both treatment groups received a second treatment administration on day 45.

Statistical analysis of all the safety variables evaluated during the physical examinations performed at predefined time points did not show significant differences between treatments in any of the variables and confirmed the general good health of the animals during the study.

The study of hematological and biochemical variables showed no evidence of immune alterations in any of the animals. Interestingly, although inside the normal range, the number of eosinophils was higher in the group of treated animals with good response to Horse Allo 20 treatment.

Adverse events

Any abnormal symptom, including laboratory parameters, was recorded by the investigators, and considered AEs. An external expert in pharmacovigilance established the causality of the AE, and if it was probably related to the administration of Horse Allo 20, it was considered an AR.

Statistical comparison of the AE in Horse Allo 20 and CP groups revealed that the number of animals with AEs was significantly higher (P < 0.05) in animals treated with Horse Allo 20.

Based on the causality analysis, the incidence of AR was low (10.4%) with no evidence of an immunogenic origin. The most reported AR was transient swelling or heat that resolved spontaneously within 24–48 h. No relationship between the incidence of an AR and lack of efficacy for Horse Allo 20 could be determined.

Discussion

In this study, we report the results of a regulatory clinical trial using horse adipose-derived allogeneic MSCs for the treatment of equine OA. Before the trial, the robustness of the manufacturing process for Horse Allo 20 was demonstrated, with no differences in product characteristics with independence of the donor, adipose tissue sample anatomic origin, or storage methodology employed. The safety and efficacy of Horse Allo 20 have been proved in two studies involving a total number of 80 participants. Eventually, a mode of action for MSCs in OA has been proposed.

While the characterization of human MSCs is well established [7], there is a lack of standardized markers to phenotype equine MSCs in veterinary medicine. Based on the indications given by other authors [7,9,46], the acceptance criteria finally established for MSCs as constituent of Horse Allo 20 were CD29 > 70%, CD44 > 70%, CD90 > 70%, and CD105 20%–30% for identity and MHC-II <10%, CD34 < 10%, CD45 < 10%, and CD79a <10% for purity. The results of our study showed a consistent expression of the identity and purity panels independent of the donor (sex, age, and breed) and adipose tissue source (intraperitoneal or subcutaneous).

It is generally recognized that allogeneic cells are deleted by host immune responses, but MSCs do not follow the rule of allogeneic rejection, thus escaping the normal process of alloantigen recognition [5]. The absence of MHC class II gives MSCs the potential to escape recognition by alloreactive CD4+ T cells. Nevertheless, a variation in MHC class II expression of MSCs not associated to their ability to maintain their stemness or undergo trilineage differentiation [47] has been described. In another work, horses receiving MHC-II-positive MSCs did not develop adverse joint reactions [48]. Anyhow, our MHC-II measurements are consistent and negative for all the samples employed during the studies; so no AR or antigen recognition was expected.

According to the ISCT criteria, the expression of CD105 on human MSCs must exceed 95% [7]. However, the results of this study showed a lower expression and this outcome was in concordance with a previous study pointing to a variable CD105 expression on MSCs depending on different sources [9]. In addition, the low CD105 expression might be due to the use of a CD105 antibody for human epitopes [49]. Also, it has been previously published that enzymatic methods (such as the use of trypsins for detaching cells from culture flasks) might modify some protein epitopes, preventing antibody attachment and posterior detection by FC techniques [50]. Anyhow, the obtained percentage of CD105-positive cells in this work (20%–25%) was homogeneous among donors and tissue samples. This is in accordance with a recent study with adipose-derived equine MSCs [51] that employed the same CD105 clone (SN6).

Besides, no genetic alterations were observed in P4 and P6 MSCs, thus assuring the genetic stability of Horse Allo 20. This result is in concordance with previous literature showing the genetic stability of MSCs [52].

Some works have suggested that cryopreservation of cells could alter their immunomodulatory characteristics [53]. Because of this, we have compared immunophenotype markers before and after cryogenic process. No differences in the expression of identity and purity markers were observed in our study. These results are supported by the literature, where fresh and frozen MSCs exhibited similar phenotypes and gene expression pattern [54].

Overall, it can be concluded that the manufacturing process for Horse Allo 20 is robust and no influence of sex, age, source of adipose tissue, freezing, or thawing is observed in the MSC proprieties. Both treatments of Horse Allo 20, fresh and frozen, have similar attributes and consequently considered comparable, and no adverse impact on safety or efficacy was seen.

To establish the safety profile of the product, eight healthy horses were repeatedly injected with the product. Control joints were injected with PBS or DMEM + 10% DMSO. Treated joints were injected with two presentations of Horse Allo 20, fresh and frozen. Overall, no systemic reactions were found after the repeated injection of the control and treated joints; all the parameters remained within normal ranges, including acute phase proteins like CRP. Besides, local reactions after the second and third injection resolved spontaneously without further intervention.

No motion restriction was indicated in the protocol for the study follow-up. It was left to the discretion of the veterinary expert. It has been suggested that restricted joint movement after the treatment may be beneficial, leading to reduced clearance of the drug product and enhancing penetration of IA tissues [55]. Although there is some evidence that a period of rest facilitates improved absorption of the treatment and therapeutic efficacy, exercise per se does not promote negative effects [2].

Data from the safety study can be explained as mild swelling and synovial effusion due to a transient light synovitis, without immunogenic inflammatory signs. Comparable results have been observed after injection of autologous and allogenic MSCs in equine healthy joints [18], where moderate yet transient changes to the local synovial environment were observed.

MSCs elicit an increase of vascular fenestration of the joint, promoting edema and thus an increase in the total protein content [16]. This effect might have been potentiated and perpetuated by the repeated arthrocentesis to characterize the inflammation in those joints with signs of potential inflammation during the study.

At the end of the safety study, all horses presented values similar to basal levels, thus showing that after three infiltrations, the treatment with Horse Allo 20 is safe despite the slight transient swelling and its spontaneous resolution.

Also, a regulatory clinical trial to test the efficacy of Horse Allo 20 was performed (in vivo efficacy study). The main outcome observed was a reduction in the lameness grade when comparing experimental versus control group, not only at an early stage (15 days) but also at a later stage of follow-up (90 days) thus confirming the long-term effect of MSC application. The reduction in lameness after MSC treatment was also reported in previous studies [35,56].

The stratified analysis of the responders showed that Horse Allo 20 was significantly efficient in young horses. The cutoff point to define a horse as geriatric was established in ≥20 years in a retrospective study of 690 equine subjects [57]. This result supports the idea of a mechanism of action for MSCs by immunomodulation, as advanced age is associated with altered immune function and enhanced inflammatory responses [58]. Transforming growth factor beta (TGF-β) MSC pretreatment improves the function of MSCs in wounds [59] and mobilizes MSCs from the bone marrow to the peripheral blood by which MSCs migrate to sites of injury [27]. In addition, equine TGF-β mRNA expression decreases with advancing age [60], suggesting a mechanism of action based on TGF-β pathway (Fig. 5).

FIG. 5.

Proposed mechanism of action for Horse Allo 20 in OA joints. The levels of growth factors (VEGF, TNF-α, TGF-β, IGF-1, and IFN-γ), cytokines (IL-1β, IL-4, IL-5, IL-13, and IL-17), and metalloproteases (MMP1, MMP13, ADAMTS-4, ADAMTS-5, and aggrecan) are elevated in OA (Step B). These molecules are secreted by different cell types (apoptotic chondrocytes, B cells, macrophages, fibroblasts, and T cells) and contribute to the pathogenesis of OA through several mechanisms. These mechanisms include an increase in the vascular infiltration, bone turnover, macrophages activation, fibrosis in the synovium, cartilage erosion, apoptotic chondrocytes, and synovial inflammation, which eventually trigger a hypoxia environment. MSCs injected by intra-articular procedure keep an undifferentiated status due to the hypoxic environment and are activated by the secreted TGF-β (Step C). MSCs exert their immunomodulation role through TGF-β (Step D), decreasing the synovial inflammation, cartilage degeneration, and bone turnover. Blood flow is restored and eventually, the healthy joint status is reached (Step A). ADAMTS, a disintegrin like and metalloproteinase with thrombospondin type 1 motifs; VEGF, vascular endothelial growth factor; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase; OA, osteoarthritis; MSC, mesenchymal stem cell; TGF, transforming growth factor; TNF, tumor necrosis factor. Color images available online at www.liebertpub.com/scd

The final incidence of AR was low (10.4%) with no evidence of immunogenic origin. The most reported AR was transient swelling or heat that resolved spontaneously within 24–48 h. These results are supported by the literature where it was concluded that an IA injection of autologous adipose tissue MSCs for OA in humans was not associated with apparent AE [61]. Besides, a concise review of 70 human studies with more than 1,400 patients treated with adipose-derived cell therapy and with a follow-up ranging from less than a month to 3 years [62] showed very few AE related directly to the cell therapy. In fact, they were rather related to the harvesting of adipose tissue, trauma associated with injection, or the nature of the underlying condition being treated.

Conventional treatment of OA is based on the application of IA corticosteroids or systemic NSAIDs. According to a Cochrane systematic review, corticosteroids have a short duration of effect and safety concerns that limit the frequency of their use [63]. Among these safety concerns, it has been described that prolonged exposure to IA corticosteroids may have adverse effects causing significantly greater cartilage volume loss, accelerating the progression of OA and with no significant difference in knee pain when compared to a saline treatment [64]. Regarding the use of NSAIDs, they are used to provide analgesia and because of their anti-inflammatory effects, but long-term use of these medications can have unwanted gastrointestinal (stomach and colon ulcers) [65], liver [66], and kidney side effects [67]. These side effects are in contrast with the lack of AEs observed when treating OA with MSCs, thus highlighting the properties of MSCs as a new cell-based therapy, given their efficacy and safety.

Conclusions

We have established a robust manufacturing method for Horse Allo 20 that showed no differences in product characteristics with independence of the donor, tissue sample origin, or storage conditions. Horse Allo 20 was overall well tolerated by the target animal and had similar local AEs to other common intraarticular treatments. The administration of Horse Allo 20 effectively reduced lameness induced by OA for an extended period of time (90 days), decreasing the need for prolonged local and/or systemic anti-inflammatory therapies and their well-known deleterious effects and toxicities.

Footnotes

Acknowledgments

The work has been funded by the National Programme for the Promotion of Talent and Its Employability “Ayudas para contratos Torres Quevedo” from the Spanish Ministry of Economy, Industry and Competitiveness (grant code PTQ-14-06645) and by the Centre for the Development of Industrial Technology (CDTI) from the Ministry of Economy, Industry and Competitiveness (grant code IDI-20140599, co-funded by the Fondo Europeo de Desarrollo Regional (FEDER) from the European Union). We thank all the veterinary experts who participated during the clinical trial and the safety study: Francisco Pereira from the Clínica Equina O Cabalo (Pontevedra, Spain), Claudio Nomen from Equihealth Veterinarios (Barcelona, Spain), Ramón Herrán from the Universidad Complutense de Madrid (Madrid, Spain), Javier López from the Universidad Complutense de Madrid (Madrid, Spain), Jorge Sánchez from the Clínica Equina La Sierra (Madrid, Spain), Eduardo M. Hernández from Vet-Express Clínica Equina (Córdoba, Spain), Antonio J. Villatoro from the Universidad de Málaga (Málaga, Spain), and Herminio Pose Nieto from SERVETEQ, Servicios Veterinarios Equinos (Lugo, Spain). We also acknowledge the participation of the Contract Research Organisation (ONDAX Scientific HQ) for monitoring the field study and the collaboration of Declan O'Rourke, external expert in pharmacovigilance, for establishing the causality of the adverse events.

Author Disclosure Statement

I.R.-H. declares no competing financial interests. J.G.-C., L.M.-P., and M.H.-P. declare competing financial interests as shareholders in Centauri Biotech S.L. L.N.-N. and M.I.R.-G. are both employed by Centauri Biotech S.L.

References

Wen

, Lu

and Chiu

. (2014). Importance of subchondral bone in the pathogenesis and management of osteoarthritis from bench to bed. J Orthop Transl, 2:16–25.

McIlwraith

. (2010). The use of intra-articular corticosteroids in the horse: what is known on a scientific basis?. Equine Vet J, 42:563–571.

Goodrich

and Nixon

. (2006). Medical treatment of osteoarthritis in the horse—a review. Vet J, 171:51–69.

Wehling

, Evans

, Wehling

and Maixner

. (2017). Effectiveness of intra-articular therapies in osteoarthritis: a literature review. Ther Adv Musculoskelet Dis, 9:183–196.

Ankrum

, Ong

and Karp

. (2014). Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol, 32:252–260.

Schlueter

and Orth

. (2004). Equine osteoarthritis: a brief review of the disease and its causes. Equine Comp Exerc Physiol. 1:221–231.

Dominici

, Le Blanc

, Mueller

, Slaper-Cortenbach

, Marini

, Krause

, Deans

, Keating

, Prockop

and Horwitz

. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8:315–317.

De Schauwer

, Meyer

, Van de Walle

and Van Soom

. (2011). Markers of stemness in equine mesenchymal stem cells: a plea for uniformity. Theriogenology, 75:1431–1443.

De Schauwer

, Piepers

, Van de Walle

, Demeyere

, Hoogewijs

, Govaere

JLJ

, Braeckmans

, Van Soom

and Meyer

. (2012). In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry. Cytometry A, 81:312–323.

10.

Paebst

, Piehler

, Brehm

, Heller

, Schroeck

, Tárnok

and Burk

. (2014). Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: an approach toward a standardized definition. Cytometry A, 85:678–687.

11.

Rozemuller

, Prins

H-J

, Naaijkens

, Staal

, Bühring

and Martens

. (2010). Prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti-human monoclonal antibodies. Stem Cells Dev, 19:1911–1921.

12.

Calloni

, Viegas

, Türck

, Bonatto

and Pegas Henriques

. (2014). Mesenchymal stromal cells from unconventional model organisms. Cytotherapy, 16:3–16.

13.

Burk

, Badylak

, Kelly

and Brehm

. (2013). Equine cellular therapy—from stall to bench to bedside?. Cytometry A, 83:103–113.

14.

Krampera

, Glennie

, Dyson

, Scott

, Laylor

, Simpson

and Dazzi

. (2003). Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood, 101:3722–3729.

15.

Broeckx

, Borena

, Zimmerman

, Mariën

, Seys

, Suls

, Duchateau

and Spaas

. (2014). Intravenous application of allogenic peripheral blood-derived mesenchymal stem cells: a safety assessment in 291 equine recipients. Curr Stem Cell Res Ther, 9:452–457.

16.

Carrade

, Owens

, Galuppo

, Vidal

, Ferraro

, Librach

, Buerchler

, Friedman

, Walker

and Borjesson

. (2011). Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses. Cytotherapy, 13:419–430.

17.

Kol

, Wood

, Carrade Holt

, Gillette

, Bohannon-Worsley

, Puchalski

, Walker

, Clark

, Watson

and Borjesson

. (2015). Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses. Stem Cell Res Ther, 6:73.

18.

Ardanaz

, Vázquez

, Romero

, Remacha

, Barrachina

, Sanz

, Ranera

, Vitoria

, Albareda

, et al. (2016). Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints. BMC Vet Res, 12:65.

19.

von Bahr

, Batsis

, Moll

, Hägg

, Szakos

, Sundberg

, Uzunel

, Ringden

and Le Blanc

. (2012). Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem Cells, 30:1575–1578.

20.

Le Blanc

, Frassoni

, Ball

, Locatelli

, Roelofs

, Lewis

, Lanino

, Sundberg

, Bernardo

, et al. (2008). Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet, 371:1579–1586.

21.

, Luo

, Lv

, Liu

, Zhao

, Zhang

, Cao

, Wang

and Wang

. (2016). In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular delivery in a rat osteoarthritis model. Stem Cell Res Ther, 7:160.

22.

Fülber

, Maria

, da Silva

LCLC

, Massoco

, Agreste

and Baccarin

. (2016). Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment. Stem Cell Res Ther, 7:35.

23.

de Windt

, Vonk

, Slaper-Cortenbach

, van den Broek

, Nizak

, van Rijen

MHP

, de Weger

, Dhert

and Saris

DBF

. (2017). Allogeneic mesenchymal stem cells stimulate cartilage regeneration and are safe for single-stage cartilage repair in humans upon mixture with recycled autologous chondrons. Stem Cells, 35:256–264.

24.

de Windt

, Vonk

, Slaper-Cortenbach

, Nizak

, van Rijen

MHP

and Saris

DBF

. (2017). Allogeneic MSCs and recycled autologous chondrons mixed in a one-stage cartilage cell transplantion: a first-in-man trial in 35 patients. Stem Cells, 35:1984–1993.

25.

Oliveira

, Chagastelles

, Sesterheim

and Pranke

. (2017). In vivo immunogenic response to allogeneic mesenchymal stem cells and the role of preactivated mesenchymal stem cells cotransplanted with allogeneic islets. Stem Cells Int, 2017:9824698.

26.

Shim

, Lee

, Han

, Kim

, Jin

, Miao

, Yi

T-G

, Cho

, Song

and Oh

Y-K

. (2015). Pharmacokinetics and in vivo fate of intra-articularly transplanted human bone marrow-derived clonal mesenchymal stem cells. Stem Cells Dev, 24:1124–1132.

27.

Zhao

, Wang

, Liu

, Wei

, Duan

, Liu

, Li

, Zou

, Zhao

, et al. (2016). Effect of TGF-β1 on the migration and recruitment of mesenchymal stem cells after vascular balloon injury: involvement of matrix metalloproteinase-14. Sci Rep, 6:21176.

28.

Lange-Consiglio

, Rossi

, Tassan

, Perego

, Cremonesi

and Parolini

. (2013). Conditioned medium from horse amniotic membrane-derived multipotent progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and ligament injuries in vivo. Stem Cells Dev, 22:3015–3024.

29.

Zhang

, Qiu

, Shindel

, Ning

, Ferretti

, Jin

, Lin

C-S

and Lue

. (2012). Adipose tissue-derived stem cells ameliorate diabetic bladder dysfunction in a type II diabetic rat model. Stem Cells Dev, 21:1391–1400.

30.

Barry

and Murphy

. (2013). Mesenchymal stem cells in joint disease and repair. Nat Rev Rheumatol, 9:584–594.

31.

McIlwraith

, Frisbie

and Kawcak

. (2012). The horse as a model of naturally occurring osteoarthritis. Bone Joint Res, 1:297–309.

32.

Spaas

, Guest

and Van de Walle

. (2012). Tendon regeneration in human and equine athletes. Sports Med, 42:871–890.

33.

Ferris

, Frisbie

, Kisiday

, McIlwraith

, Hague

, Major

, Schneider

, Zubrod

, Kawcak

and Goodrich

. (2014). Clinical outcome after intra-articular administration of bone marrow derived mesenchymal stem cells in 33 horses with stifle injury. Vet Surg, 43:255–265.

34.

McIlwraith

, Frisbie

, Rodkey

, Kisiday

, Werpy

, Kawcak

and Steadman

. (2011). Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects. Arthroscopy, 27:1552–1561.

35.

Broeckx

, Zimmerman

, Crocetti

, Suls

, Mariën

, Ferguson

, Chiers

, Duchateau

, Franco-Obregón

, Wuertz

and Spaas

. (2014). Regenerative therapies for equine degenerative joint disease: a preliminary study. PLoS One, 9:e85917.

36.

Girard

, Festy

and Roche

. (2013). Local anesthetics: use and effects in autologous fat grafting. Surg Curr Res, 3:1–7.

37.

Araña

, Mazo

, Aranda

, Pelacho

and Prosper

. (2013). Adipose tissue-derived mesenchymal stem cells: isolation, expansion, and characterization. Methods Mol Biol 1036:47–61.

38.

O'Hara

, Xu

, Liang

, Reddy

, Wu

and Litwin

. (2011). Recommendations for the validation of flow cytometric testing during drug development. II assays. J Immunol Methods 363:120–134.

39.

Mahaffey

. (2002). Chapter 10. Synovial fluid. In: Diagnostic Cytology and Hematology of the Horse. Cowell

and Tyler

, eds. Mosby, St. Louis, Missouri, pp 163–170.

40.

Steel

. (2008). Equine synovial fluid analysis. Vet Clin North Am Equine Pract, 24:437–454.

41.

Francoz

, Desrochers

, Fecteau

, Desautels

, Latouche

and Fortin

. (2005). Synovial fluid changes in induced infectious arthritis in calves. J Vet Intern Med, 19:336–343.

42.

Rohde

, Anderson

, Desrochers

, St-Jean

, Hull

and Rings

. (2000). Synovial fluid analysis in cattle: a review of 130 cases. Vet Surg, 29:341–346.

43.

MacWilliams

and Friedrichs

. (2003). Laboratory evaluation and interpretation of synovial fluid. Vet Clin North Am Small Anim Pract, 33:153–178.

44.

Cowell

and Tyler

. (2002). Diagnostic Cytology and Hematology of the Horse. Mosby, St Louis, MO.

45.

European Medicine Agency. (2011). Guideline on Statistical Principles for Clinical Trials for Veterinary Medicinal Products (Pharmaceuticals). European Medicine Agency, London, United Kingdom.

46.

Bourin

, Bunnell

, Casteilla

, Dominici

, Katz

, March

, Redl

, Rubin

, Yoshimura

and Gimble

. (2013). Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy. Cytotherapy, 15:641–648.

47.

Schnabel

, Pezzanite

, Antczak

, Felippe

MJB

and Fortier

. (2014). Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro. Stem Cell Res Ther, 5:13.

48.

Joswig

A-J

, Mitchell

, Cummings

, Levine

, Gregory

, Smith

and Watts

. (2017). Repeated intra-articular injection of allogeneic mesenchymal stem cells causes an adverse response compared to autologous cells in the equine model. Stem Cell Res Ther, 8:42.

49.

Screven

, Kenyon

, Myers

, Yancy

, Skasko

, Boxer

, Bigley

, Borjesson

and Zhu

. (2014). Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow. Vet Immunol Immunopathol, 161:21–31.

50.

Gedye

, Hussain

, Paterson

, Smrke

, Saini

, Sirskyj

, Pereira

, Lobo

, Stewart

, et al. (2014). Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity. PLoS One, 9:e105602.

51.

Cabezas

, Rojas

, Navarrete

, Ortiz

, Rivera

, Saravia

, Rodriguez-Alvarez

and Castro

. (2018). Equine mesenchymal stem cells derived from endometrial or adipose tissue share significant biological properties, but have distinctive pattern of surface markers and migration. Theriogenology, 106:93–102.

52.

, Kang

, Shin

, Park

, Joo

, Kim

, Kang

B-C

, Lee

, Nakama

, et al. (2011). Stem cell treatment for patients with autoimmune disease by systemic infusion of culture-expanded autologous adipose tissue derived mesenchymal stem cells. J Transl Med, 9:181.

53.

Galipeau

, Krampera

, Barrett

, Dazzi

, Deans

, DeBruijn

, Dominici

, Fibbe

, Gee

, et al. (2016). International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials. Cytotherapy, 18:151–159.

54.

Mamidi

, Nathan

, Singh

, Thrichelvam

, Yusof

NA Mohd

, NA Fakharuzi

, Zakaria

, Bhonde

. Das and A Sen Majumdar. (2012). Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation. J Cell Biochem, 113:3153–3164.

55.

Caron

. (2005). Intra-articular injections for joint disease in horses. Vet Clin North Am Equine Pract, 21:559–573.

56.

Rodríguez-Hurtado

, Gómez-Lucas

, García-Castro

, Mariñas-Pardo

and Hermida-Prieto

. (2014). Use of allogeneic mesechymal stem cells in equine orthopaedic pathologies. Equinus, 39:48–60.

57.

Silva

and Furr

. (2013). Diagnoses, clinical pathology findings, and treatment outcome of geriatric horses: 345 cases (2006–2010). J Am Vet Med Assoc, 243:1762–1768.

58.

Horohov

, Adams

and Chambers

. (2010). Immunosenescence of the Equine Immune System. J Comp Pathol, 142:S78–S84.

59.

Ghosh

, McGrail

and Dawson

. (2017). TGF-β1 pretreatment improves the function of mesenchymal stem cells in the wound bed. Front Cell Dev Biol, 5:28.

60.

Iqbal

, Dudhia

, Bird

and Bayliss

. (2000). Age-related effects of TGF-β on proteoglycan synthesis in equine articular cartilage. Biochem Biophys Res Commun, 274:467–471.

61.

, Chai

, Jeong

, Oh

, Shin

, Shim

and Yoon

. (2017). Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a 2-year follow-up study. Am J Sports Med 45:2774–2783.

62.

Toyserkani

, Jørgensen

, Tabatabaeifar

, Jensen

, Sheikh

and Sørensen

. (2017). Concise review: a safety assessment of adipose-derived cell therapy in clinical trials: a systematic review of reported adverse events. Stem Cells Transl Med, 6:1786–1794.

63.

Bellamy

, Campbell

, Welch

, Gee

, Bourne

and Wells

. (2006). Intraarticular corticosteroid for treatment of osteoarthritis of the knee. In: Cochrane Database of Systematic Reviews. Bellamy

, ed. John Wiley and Sons, Ltd., Chichester, United Kingdom, p CD005328.

64.

McAlindon

, LaValley

, Harvey

, Price

, Driban

, Zhang

and Ward

. (2017). Effect of intra-articular triamcinolone vs saline on knee cartilage volume and pain in patients with knee osteoarthritis. JAMA, 317:1967.

65.

Marshall

and Blikslager

. (2011). The effect of nonsteroidal anti-inflammatory drugs on the equine intestine. Equine Vet J, 43:140–144.

66.

Lees

, Creed

, Gerring

, Gould

, Humphreys

, Maitho

, Michell

and Taylor

. (1983). Biochemical and haematological effects of phenylbutazone in horses. Equine Vet J, 15:158–167.

67.

Read

. (1983). Renal `medullary crest necrosis associated with phenylbutazone therapy in horses. Vet Pathol, 20:662–669.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.11 MB

0.27 MB

0.03 MB

0.02 MB

0.03 MB