Low-dose xenogeneic mesenchymal stem cells target canine osteoarthritis through systemic immunomodulation and homing.

BACKGROUND: As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. METHODS: This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. RESULTS: The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. CONCLUSION: This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

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.

Homing of radiolabelled xenogeneic equine peripheral blood-derived MSCs towards a joint lesion in a dog.

Osteoarthritis (OA) is a highly prevalent condition in dogs, causing a substantial reduction in quality of life and welfare of the animals. Current disease management focusses on pain relief but does not stop the progression of the disease. Therefore, mesenchymal stem cells (MSCs) could offer a promising disease modifying alternative. However, little is known about the behavior and the mode of action of MSCs following their administration. In the current case report, 99mTechnetium labelled xenogeneic equine peripheral blood-derived MSCs were intravenously injected in a 9 year old dog suffering from a natural occurring cranial cruciate ligament rupture. The biodistribution of the MSCs was evaluated during a 6-h follow-up period, using a full body scintigraphy imaging technique. No clinical abnormalities or ectopic tissue formations were detected throughout the study. A radiopharmaceutical uptake was present in the liver, heart, lung, spleen, kidneys and bladder of the dog. Furthermore, homing of the radiolabelled MSCs to the injured joint was observed, with 40.61 % higher uptake in the affected joint in comparison with the healthy contralateral joint. Finally, a local radioactive hotspot was seen at a part of the tail of the dog that had been injured recently. The current study is the first to confirm the homing of xenogeneic MSCs to a naturally occurring joint lesion after IV administration.

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.

Scintigraphic tracking of 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells after intravenous, intramuscular, and subcutaneous injection in healthy dogs.

BACKGROUND: Mesenchymal stem cell treatments in dogs have been investigated as a potential innovative alternative to current conventional therapies for a variety of conditions. So far, the precise mode of action of the MSCs has yet to be determined. The aim of this study was to gain more insights into the pharmacokinetics of MSCs by evaluating their biodistribution in healthy dogs after different injection routes. METHODS: Three different studies were performed in healthy dogs to evaluate the biodistribution pattern of radiolabelled equine peripheral blood-derived mesenchymal stem cells following intravenous, intramuscular and subcutaneous administration in comparison with free 99mTechnetium. The labelling of the equine peripheral blood-derived mesenchymal stem cells was performed using stannous chloride as a reducing agent. Whole-body scans were obtained using a gamma camera during a 24-h follow-up. RESULTS: The labelling efficiency ranged between 59.58 and 83.82%. Free 99mTechnetium accumulation was predominantly observed in the stomach, thyroid, bladder and salivary glands, while following intravenous injection, the 99mTechnetium-labelled equine peripheral blood-derived mesenchymal stem cells majorly accumulated in the liver throughout the follow-up period. After intramuscular and subcutaneous injection, the injected dose percentage remained very high at the injection site. CONCLUSIONS: A distinct difference was noted in the biodistribution pattern of the radiolabelled equine peripheral blood-derived mesenchymal stem cells compared to free 99mTechnetium indicating equine peripheral blood-derived mesenchymal stem cells have a specific pharmacokinetic pattern after systemic administration in healthy dogs. Furthermore, the biodistribution pattern of the used xenogeneic equine peripheral blood-derived mesenchymal stem cells appeared to be different from previously reported experiments using different sources of mesenchymal stem cells.

The Evaluation of Equine Allogeneic Tenogenic Primed Mesenchymal Stem Cells in a Surgically Induced Superficial Digital Flexor Tendon Lesion Model.

Background: Tendon injuries are very common in horses and jeopardize the athletic performance, and due to the high risk of reinjury may lead to early retirement. The use of mesenchymal stem cells for the treatment of equine tendon disease is widely investigated because of their regenerative potential. The objective of this study is to investigate the safety and efficacy of equine allogeneic tenogenic primed mesenchymal stem cells (tpMSCs) for the management of tendinitis in horses. Methods: A core lesion was surgically induced in the superficial digital flexor tendon of both forelimbs of eight horses. After 7 days, one forelimb was treated with tpMSCs, while the contralateral forelimb served as an intra-individual control and was treated with saline. A prescribed exercise program was started. All horses underwent a daily clinical evaluation throughout the entire study period of 112 days. Blood samples were taken at different time points for hematological and biochemical analysis. Tendon assessment, lameness examination, ultrasound assessment and ultrasound tissue characterization (UTC) were performed at regular time intervals. At the end of the study period, the superficial digital flexor tendons were evaluated macroscopically and histologically. Results: No suspected or serious adverse events occurred during the entire study period. There was no difference in local effects including heat and pain to pressure between a single intralesional injection of allogeneic tpMSCs and a single intralesional injection with saline. A transient moderate local swelling was noted in the tpMSC treated limbs, which dissipated by day 11. Starting at a different time point depending on the parameter, a significant improvement was observed in the tpMSC treated limbs compared to the placebo for echogenicity score, fiber alignment score, anterior-posterior thickness of the tendon and echo type by UTC assessment. Immunohistochemistry 112 days post-injection revealed that the amount of collagen type I and Von Willebrand factor were significantly higher in the tendon tissue of the tpMSC group, while the amount of collagen type III and smooth muscle actin was significantly lower. Conclusion: Equine allogeneic tenogenic primed mesenchymal stem cells were shown to be well-tolerated and may be effective for the management of tendon injuries.

Safety and immunomodulatory properties of equine peripheral blood-derived mesenchymal stem cells in healthy cats.

OBJECTIVE: Due to the immunomodulatory properties of mesenchymal stem cells (MSCs) through stimulation of endogenous immune cells by paracrine signals and cell contact, they have been proposed as alternative treatment option for many inflammatory and immune-mediated diseases in veterinary medicine. However, the long-term cultivation possibilities of feline MSCs are currently compromised due to a restricted proliferation capacity. Therefore, the xenogeneic use of equine peripheral blood-derived MSCs (ePB-MSCs) would present an interesting alternative thanks to their superior cultivation properties. To the authors' knowledge, there are currently no safety reports concerning the xenogeneic use of ePB-MSCs in cats. Therefore, the overall goal of this preliminary study was to investigate if ePB-MSCs can safely be administered in healthy cats and by extension evaluating their immunogenic and immunomodulatory properties. METHODS: Ten healthy cats were intravenously (i.v.) injected with 3 × 105 ePB-MSCs at three time points (T0, T1, T2). All cats were daily inspected by the caretaker and underwent a physical examination with hematological and biochemical analysis at day 0 (T0), week 2 (T1), week 4 (T2) and week 6 (T3) by a veterinarian. Furthermore, a modified mixed lymphocyte reaction (MLR) was performed at T0 and T3 for each cat in order to evaluate immunogenic and immunomodulatory properties of the ePB-MSCs RESULTS: No adverse clinical effects could be detected following repeated i.v. administration of ePB-MSCs in all cats. Significant lower protein (T1: P-value = 0.002; T2: P-value > 0.001; T3: P-value = 0.004) and albumin levels (T1: P-value = 0.003; T2: P-value = 0.001) were seen after repeated administration of ePB-MSCs, compared to T0. However, all biochemical and hematological parameters stayed within clinical acceptance level. In addition, the repeated injections did not induce a cellular immune response before and after repeated ePB-MSCs administration. Furthermore, convincing immunomodulatory properties of ePB-MSCs on feline peripheral blood mononuclear cells were confirmed in the MLR-assay CONCLUSION: This preliminary study demonstrates that ePB-MSCs can safely be administered in healthy cats and provide a promising alternative for the treatment of various inflammatory diseases in cats.

A Feasibility Study on the Use of Equine Chondrogenic Induced Mesenchymal Stem Cells as a Treatment for Natural Occurring Osteoarthritis in Dogs.

Conventional treatments of osteoarthritis (OA) reduce pain and the inflammatory response but do not repair the damaged cartilage. Xenogeneic peripheral blood-derived equine chondrogenically induced mesenchymal stem cells (ciMSC) could thus provide an interesting alternative. Six client-owned dogs with confirmed elbow OA were subjected to a baseline orthopedic examination, pressure plate analysis, general clinical examination, hematological analysis, synovial fluid sampling, and radiographic examination, and their owners completed two surveys. After all examinations, a 0.9% saline solution (placebo control product = CP) was administered intra-articularly. After 6 weeks, all examinations were repeated, owners again completed two surveys, and equine ciMSCs were administered in the same joint. After another 6 weeks, dogs were returned for a final follow-up. No serious adverse events or suspected adverse drug reactions were present during this study. No significant differences in blood analysis were noted between the CP and ciMSC treatment. Two adverse events were observed, both in the same dog, one after CP treatment and one after ciMSC treatment. The owner surveys revealed significantly less pain and lameness after ciMSC treatment compared to after CP treatment. There was no significant difference in the orthopedic examination parameters, the radiographic examination, synovial fluid sampling, and pressure plate analysis between CP treatment and ciMSC treatment. A single intra-articular administration of equine ciMSCs proved to be a well-tolerated treatment, which reduced lameness and pain according to the owner's evaluations compared to a placebo treatment.

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.