Equine allogeneic tenogenic primed mesenchymal stem cells: A clinical field study in horses suffering from naturally occurring superficial digital flexor tendon and suspensory ligament injuries.

BACKGROUND: Mesenchymal stem cells are an innovative therapeutic for various equine orthopaedic diseases, including soft tissue injuries. OBJECTIVES: To evaluate the safety and efficacy of tenogenic primed equine allogeneic peripheral blood-derived mesenchymal stem cells (tpMSCs) in horses with naturally occurring superficial digital flexor tendon (SDFT) and suspensory ligament (SL) injuries. STUDY DESIGN: Multicentre, blinded, randomised, placebo-controlled clinical trial. METHODS: One hundred client-owned horses with SDFT and SL injuries were randomised to receive an intralesional tpMSC (66) or saline (34) injection. Clinical and ultrasonographic evaluation was performed before treatment and on Days 56 ± 3 and 112 ± 3 after treatment. Long-term data on re-injury was collected up to 2 years after treatment. RESULTS: Significantly more tpMSC-treated horses achieved improvement in fibre alignment score (FAS) (100% vs. 54.5%, p < 0.001) and echogenicity (97.0% vs. 57.6%, p < 0.001) on Day 112 ± 3, and their lesion size decreased significantly (-27.6 ± 25.91 vs. -4.6 ± 26.64 mm2 , p < 0.001) compared to the placebo group. A FAS = 0 was achieved in 65% of tpMSC-treated horses, as compared to 9% of placebo-treated horses at Day 112 ± 3. The attending veterinarians reported no re-injury in 41 of 53 tpMSC and in 2 of 26 saline-treated horses available for long-term follow-up (p < 0.001). MAIN LIMITATIONS: As this study consisted of client-owned horses, no samples for histology were collected. Long-term follow-up was only available for a subset of enrolled horses. CONCLUSIONS: The intralesional administration of tpMSCs was safe and improved the quality of healing and long-term outcomes in sports horses with naturally occurring SDFT and suspensory injuries.

Repeated intra-articular administration of equine allogeneic peripheral blood-derived mesenchymal stem cells does not induce a cellular and humoral immune response in horses.

OBJECTIVE: The use of mesenchymal stem cells (MSCs) for the treatment of equine joint disease is widely investigated because of their regenerative and immunomodulatory potential. Allogeneic MSCs provide a promising alternative to autologous MSCs, since the former are immediately available and enable a thorough donor screening. However, questions have been raised concerning the immunogenic potential of allogeneic MSCs, especially after repeated administration. METHODS: Current retrospective study assessed the cellular and humoral immunogenicity of ten jumping and dressage horses with naturally occurring degenerative joint disease which were treated 3 times intra-articularly with a 1 mL stem cell suspension containing 1.4-2.5 million chondrogenic induced equine allogeneic peripheral blood-derived MSCs (ciMSCs) combined with 1 mL equine allogeneic plasma. Stem cells from 2 donor horses were used. Horses were clinically evaluated for joint effusion, presence of pain to palpation and skin surface temperature at the local injection site, joint range of motion, occurrence of adverse events and the presence of ectopic tissue. The cellular immune response was analyzed using a modified mixed lymphocyte reaction and the humoral immune response was investigated using a flow cytometric crossmatch assay by which the presence of alloantibodies against the ciMSCs was evaluated. Presence of anti-bovine serum albumin antibodies was detected via ELISA. RESULTS: Clinical evaluation of the horses revealed no serious adverse effects or suspected adverse drug reactions and no ectopic tissue formation at the local injection site or in other areas of the body. Generally, repeated administration led to a decrease of horses with joint effusion of the affected joint. Pain to palpation, skin surface temperature and joint range of motion did not increase or even decreased after treatment administration. Allogeneic ciMSCs did not induce a cellular immune response and no alloantibodies were detected in the recipients' serum, regardless the presence of BSA antibodies in 70 % of the horses. CONCLUSION: Repeated intra-articular injections with allogeneic equine ciMSCs did not elicit clinically relevant adverse events. Furthermore, current study indicates the absence of a cellular or a humoral immune response following repeated intra-articular injections.

Equine Allogeneic Chondrogenic Induced Mesenchymal Stem Cells Are an Effective Treatment for Degenerative Joint Disease in Horses.

Degenerative joint disease is one of the main causes of equine early retirement from pleasure riding or a performance career. The disease is initially triggered by an abnormal loading of normal cartilage or a normal loading of abnormal cartilage. This primary insult is accompanied with joint inflammation, which leads to further progressive degeneration of the articular cartilage and changes in the surrounding tissues. Therefore, in search for an effective treatment, 75 adult horses with early signs of degenerative fetlock joint disease were enrolled in a randomized, multicenter, double-blinded, and placebo-controlled study. Fifty animals were injected intra-articularly with the investigational veterinary product (IVP) consisting of allogeneic chondrogenic induced mesenchymal stem cells (ciMSCs) with equine allogeneic plasma, and 25 horses were injected with 0.9% NaCl (saline) control product. From week 3 to 18 after treatment, lameness scores (P < 0.001), flexion test responses (P < 0.034), and joint effusion scores (P < 0.001) were remarkably superior in IVP-treated horses. Besides nasal discharge in both treatment groups, no adverse events were observed during the entire study period. On long-term follow-up (1 year), significantly more investigational product-treated horses were working at training level or were returned to their previous level of work (P < 0.001).

Tenogenically Induced Allogeneic Peripheral Blood Mesenchymal Stem Cells in Allogeneic Platelet-Rich Plasma: 2-Year Follow-up after Tendon or Ligament Treatment in Horses.

Poor healing of tendon and ligament lesions often results in early retirement of sport horses. Therefore, regenerative therapies are being explored as potentially promising treatment for these injuries. In this study, an intralesional injection was performed with allogeneic tenogenically induced mesenchymal stem cells and platelet-rich plasma 5-6 days after diagnosis of suspensory ligament (SL) (n = 68) or superficial digital flexor tendon (SDFT) (n = 36) lesion. Clinical, lameness and ultrasonographic evaluation was performed at 6 and 12 weeks. Moreover, a survey was performed 12 and 24 months after treatment to determine how many horses were competing at original level and how many were re-injured. At 6 weeks, 88.2% of SL (n = 68) and 97.3% of SDFT lesions (n = 36) demonstrated moderate ultrasonographic improvement. At 12 weeks, 93.1% of SL (n = 29) and 95.5% of SDFT lesions (n = 22) improved convincingly. Moreover, lameness was abolished in 78.6% of SL (n = 28) and 85.7% (n = 7) of SDFT horses at 12 weeks. After 12 months (n = 92), 11.8% of SL and 12.5% of SDFT horses were re-injured, whereas 83.8 of SL and 79.2% of SDFT returned to previous performance level. At 24 months (n = 89) after treatment, 82.4 (SL) and 85.7% (SDFT) of the horses returned to previous level of performance. A meta-analysis was performed on relevant published evidence evaluating re-injury 24 months after stem cell-based [17.6% of the SL and 14.3% of the SDFT group (n = 89)] versus conventional therapies. Cell therapies resulted in a significantly lower re-injury rate of 18% [95% confidence interval (CI), 0.11-0.25] 2 years after treatment compared to the 44% re-injury rate with conventional treatments (95% CI, 0.37-0.51) based on literature data (P < 0.0001).

Tenogenically Induced Allogeneic Mesenchymal Stem Cells for the Treatment of Proximal Suspensory Ligament Desmitis in a Horse.

Suspensory ligament injuries are a common injury in sport horses, especially in competing dressage horses. Because of the poor healing of chronic recalcitrant tendon injuries, this represents a major problem in the rehabilitation of sport horses and often compromises the return to the initial performance level. Stem cells are considered as a novel treatment for different pathologies in horses and humans. Autologous mesenchymal stem cells (MSCs) are well known for their use in the treatment of tendinopathies; however, recent studies report a safe use of allogeneic MSCs for different orthopedic applications in horses. Moreover, it has been reported that pre-differentiation of MSCs prior to injection might result in improved clinical outcomes. For all these reasons, the present case report describes the use of allogeneic tenogenically induced peripheral blood-derived MSCs for the treatment of a proximal suspensory ligament injury. During conservative management for 4 months, the horse demonstrated no improvement of a right front lameness with a Grade 2/5 on the American Association of Equine Practitioners (AAEP) scale and a clear hypo-echoic area detectable in 30% of the cross sectional area. From 4 weeks after treatment, the lameness reduced to an AAEP Grade 1/5 and a clear filling of the lesion could be noticed on ultrasound. At 12 weeks (T 4) after the first injection, a second intralesional injection with allogeneic tenogenically induced MSCs and platelet-rich plasma was given and at 4 weeks after the second injection (T 5), the horse trotted sound under all circumstances with a close to total fiber alignment. The horse went back to previous performance level at 32 weeks after the first regenerative therapy and is currently still doing so (i.e., 20 weeks later or 1 year after the first stem cell treatment). In conclusion, the present case report demonstrated a positive evolution of proximal suspensory ligament desmitis after treatment with allogeneic tenogenically induced MSCs.

Allogenic mesenchymal stem cells as a treatment for equine degenerative joint disease: a pilot study.

Cell-based therapies, such as treatments with mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) are thought to have beneficial effects on the clinical outcome of orthopedic injuries, but very few animal studies with large sample size are published so far. Therefore, the aim of this study was to assess the safety and report the clinical outcome of allogenic, immature or chondrogenic induced MSCs in combination with PRP for the treatment of degenerative joint disease (DJD) in 165 horses. MSCs and PRP were isolated from a 6-year-old donor horse and transplanted either in their native state or after chondrogenic induction in combination with PRP into degenerated stifle (n=30), fetlock (n=58), pastern (n=34) and coffin (n=43) joints. Safety was assessed by means of clinical evaluation and the outcome was defined as failure to return to work (score 0), rehabilitation (score 1), return to work (score 2) and return to previous level (score 3), shortly (6 weeks) after treatment or at 18 weeks for the patients that returned for long-term follow-up (n=91). No adverse effects were noticed, except for three patients who showed a moderate flare reaction within one week after treatment of the fetlock joint without long-term effects (1.8% of 165 horses). Already after 6 weeks, 45% (native MSCs) and 60% (chondrogenic induced MSCs) of the treated patients returned to work (→ score 2+3) and the beneficial effects of the treatment further increased after 18 weeks (78% for native MSCs and 86% for chondrogenic induced MSCs). With the odds ratio of 1.47 for short-term and 1.24 for long-term, higher average scores (but statistically not significant) could be noticed using chondrogenic induced MSCs as compared to native MSCs. For all three lower limb joints a higher percentage of the treated patients returned to work after chondrogenic induced MSC treatment, whereas the opposite trend could be noticed for stifle joints. Nevertheless, more protracted follow-up data should confirm the sustainability of these joints.

Intravenous application of allogenic peripheral blood-derived mesenchymal stem cells: a safety assessment in 291 equine recipients.

It has been reported that mesenchymal stem cells (MSCs) have homing capacities and immunomodulating effects after an intravenous injection. However, transplanting MSCs in murine tail veins can result in pulmonary reactions and even death of the animals. Unfortunately, only a few intravenous MSC transplantations have been reported in large animal species and these were performed in a limited number of individuals. To assess the safety of MSC transplantations, a large study on 291 recipient horses is reported here. MSCs were isolated from the peripheral blood (PB) of a 4-year-old and 6-year-old donor horse after having tested their PB for a wide range of transmittable diseases. The MSC samples from both donor horses were characterized and resuspended in 1 ml of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Dimethyl Sulfoxide (DMSO). After hand-thawing in the field, 291 horses with ages ranging from 3-months to 33-years were directly injected into their jugular vein. 281 horses (97%) received a single injection of a physiological dose of 0.2 x10(6) MSCs, 5 horses (1.7%) were re-injected after approximately 6 weeks (using the same dose and donor cells) and a single superphysiological dose of 10(6) MSCs was administered to 5 horses as well. In total, 176 recipients were injected with MSCs from the 4-year-old donor and 115 recipients received MSCs from the 6-year-old donor. From all the injected horses (n=291) no acute clinical adverse effects were noticed. Apart from one horse that died of colic 7 months after the treatment, no deaths were registered and all the horses were monitored for 1 year after the injection. In conclusion, no adverse effects were noticed in 291 recipients after an intravenous injection of allogenic PBderived MSCs. Nevertheless, further research is warranted in order to verify the immunogenic properties of these cells after allogenic transplantation into various (patho)physiological sites.

Regenerative therapies for equine degenerative joint disease: a preliminary study.

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.