ArticleLiterature Review

Concepts and challenges in the use of mesenchymal stem cells as a treatment for cartilage damage in the horse

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Abstract

Osteoarthritis (OA), the most common form of joint disease affecting humans and horses, is characterized by the advance and decline of cartilage and loss of function of the affected joint. The progression of OA is steadily accompanied with biochemical events, which interfere with the cytokines and proteolytic enzymes responsible for progress of the disease. Recently, regenerative therapies have been used with an assumption that mesenchymal stem cells (MSCs) possess the potential to prevent the advancement of cartilage damage and potentially regenerate the injured tissue with an ultimate goal of preventing OA. We believe that despite various challenges, the use of allogenic versus autologous MSCs in cartilage regeneration, is a major issue which can directly or indirectly affect the other factors including, the timing of implantation, dose or cell numbers for implantation, and the source of MSCs. Current knowledge reporting some of these challenges that the clinicians might face in the treatment of cartilage damage in horses are presented. In this regard we conducted two independent studies. In the first study we compared donor matched bone marrow and synovial fluid - derived equine MSCs in vitro, and showed that the SFMSCs were similar to the BMMSCs in their proliferation, expression of CD29, CD44 and CD90, but, exhibited a significantly different chondrogenesis. Additionally, 3.2-21% of all SFMSCs were positive for MHC II, whereas, BMMSCs were negative. In the second study we observed that injection of both the autologous and allogenic SFMSCs into the tarsocrural joint resulted in elevated levels of total protein and total nucleated cell counts. Further experiments to evaluate the in vivo acute or chronic response to allogenic or autologous MSCs are imperative.

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... tendons can improve the morphological orientation of the affected fibres. 1 In addition, MSCs have anti-inflammatory and immunomodulatory properties. 3 MSCs can regulate the production of tumour necrosis factor-alpha (TNFα) to mediate inflammation and suppress T-cell proliferation. 4 Paracrine mechanisms in MSCs are important for the activation of their immunomodulatory potential. ...
... Synovial fluid in joints provides growth factors and nutrients for chondrogenesis of articular cartilage, 3 and it is known that SF cells synthesise collagen. 28 ...
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Background Mesenchymal stem cells (MSCs) have been used therapeutically in equine medicine. MSCs release extracellular vesicles (EVs), which affect cell processes by inhibiting cell apoptosis and regulating inflammation. To date, little is known about equine EVs and their regenerative properties. Objectives To characterise equine MSC‐derived extracellular vesicles (EVs) and evaluate their effect on equine chondrocytes treated with pro‐inflammatory cytokines in vitro. Study design In vitro experiments with randomised complete block design. Methods Mesenchymal stem cells from bone marrow, adipose tissue, and synovial fluid were cultured in vitro. The MSC culture medium was centrifuged and filtered. Isolated particles were analysed for size and concentration (total number of particles per mL). Transmission electron microscopy analysis was performed to evaluate the morphology and CD9 expression of the particles. Chondrocytes from healthy equines were treated with the inflammatory cytokines interleukin (IL)‐1β and tumour necrosis factor‐alpha. MSC‐derived EVs from bone marrow and synovial fluid cells were added as co‐treatments in vitro. Gene expression analysis by real‐time PCR was performed to evaluate the effects of EVs. Results The particles isolated from MSCs derived from different tissues did not differ significantly in size and concentration. The particles had a round‐like shape and positively expressed CD9. EVs from bone marrow cells displayed reduced expression of metalloproteinase‐13. Main limitations Sample size and characterisation of the content of EVs. Conclusions EVs isolated from equine bone marrow MSCs reduced metalloproteinase 13 gene expression; this gene encodes an enzyme related to cartilage degradation in inflamed chondrocytes in vitro. EVs derived from MSCs can reduce inflammation and could potentially be used as an adjuvant treatment to improve tissue and cartilage repair in the articular pathologies.
... 11(1): 128-134 132 considered to be better than that of the SVF of adipose tissue (SVF-AT). The use of MSCs-BM in comparison with SVF-AT leads to better clinical, biochemical, and histological results in the treatment of horse joints with OA on day 70 following treatment (Frisbie et al., 2009 (Zayed et al., 2018). ...
... Long-term observation of animals after MSCs transplantation (1 year) showed that a significant number of horses subjected to cell therapy worked at the training level or returned to the previous level(Broeckx Sarah et al., 2019). Due to the presence of a considerable number of sources of MSCs,Zayed et al. (2018) conducted comparative studies of MSCs-BM and MSCs from synovial fluid (MSCs-SF) of horses in vitro. The results showed that MSCs-SF had similar proliferative activities to MSCs-BM and expressed the membrane markers CD29, CD44, and CD90, but demonstrated different chondrogenesis characteristics. ...
Article
One of the major problems observed in veterinary practice is articular cartilage injuries in animals. In terms of agriculture, it leads to their culling from the herd, even if they are highly productive animals. With companion animals, owners usually have to decide between euthanasia or long-term sometimes lifelong treatment of the injury by a veterinarian. The use of mesenchymal stem cells (MSCs) for the treatment of cartilage injury in veterinary medicine is based on the good results observed in preclinical studies, where large animals have been used as experimental models to study the regenerative activity of MSCs. According to the literature, MSCs in veterinary medicine have been used to treat cartilage injury of dogs and horses, whereas sheep and goats are generally models for reproducing the disease in preclinical experimental studies.
... 11(1): 128-134 132 considered to be better than that of the SVF of adipose tissue (SVF-AT). The use of MSCs-BM in comparison with SVF-AT leads to better clinical, biochemical, and histological results in the treatment of horse joints with OA on day 70 following treatment (Frisbie et al., 2009 (Zayed et al., 2018). ...
... Long-term observation of animals after MSCs transplantation (1 year) showed that a significant number of horses subjected to cell therapy worked at the training level or returned to the previous level(Broeckx Sarah et al., 2019). Due to the presence of a considerable number of sources of MSCs,Zayed et al. (2018) conducted comparative studies of MSCs-BM and MSCs from synovial fluid (MSCs-SF) of horses in vitro. The results showed that MSCs-SF had similar proliferative activities to MSCs-BM and expressed the membrane markers CD29, CD44, and CD90, but demonstrated different chondrogenesis characteristics. ...
Article
Full-text available
One of the major problems observed in veterinary practice is articular cartilage injuries in animals. In terms of agriculture, it leads to their culling from the herd, even if they are highly productive animals. With companion animals, owners usually have to decide between euthanasia or long-term sometimes lifelong treatment of the injury by a veterinarian. The use of mesenchymal stem cells (MSCs) for the treatment of cartilage injury in veterinary medicine is based on the good results observed in preclinical studies, where large animals have been used as experimental models to study the regenerative activity of MSCs. According to the literature, MSCs in veterinary medicine have been used to treat cartilage injury of dogs and horses, whereas sheep and goats are generally models for reproducing the disease in preclinical experimental studies.
... These disorders often result from overuse injuries, muscle fatigue, inflammation of the tendon structure, or intervertebral disk degeneration of the vertebral column. Examples of MSDs include osteoarthritis (OA), tendon ligament injury (TLI), and intervertebral disk degeneration (IVDD) [57]. ...
... Up to now, treatment options for MSDs include systemic or intra-articular administration of anti-inflammatory drugs, hyaluronic acid (HA), cells-based products including platelet-rich plasma (PRP), and autologous/ allogeneic cells implantation [57,58]. Since MSDs have a high prevalence, regenerative therapies, including the use of MSCs, have been brought to the attention of veterinary practitioners as an alternative to the more traditional treatments [54,59,60]. ...
Article
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Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.
... Treatment of musculoskeletal disorders involves the use of systemic or intra-articular antiinflammatory drugs, hyaluronic acid (HA), platelet-rich plasma (PRP), as well as autologous and allogeneic stem cell transplantation. [14] Examples of musculoskeletal disorders, osteoarthritis (OA), tendon ligament injury, and intervertebral disk degeneration (IVDD) can be given. ...
Article
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Today, with the advancement of technology, medical science has shifted its focus to regeneration. Regenerative medicine aims to restore irreparable tissues and organs to their normal function by stimulating the body's repair mechanisms using different approaches. Regenerative medicine is currently one of the important research areas in both human and veterinary medicine. This field of medicine provides diversity in veterinary medicine due to the abundance of animal species and anatomical and physiological differences, but the abundance of variables requires more meticulous work. Due to the difficulty in translating clinical results from one species to another, the scope of the work is quite limited. Today, stem cell applications are widely used in veterinary medicine, particularly in horses and dogs with muscle and skeletal system diseases. This chapter evaluates mesenchymal stem cells, their production, the use of mesenchymal stem cell therapies in horses and dogs with muscle-skeletal system diseases, and the use of mammary stem cells and stem cells in reproductive medicine.
... In general, it varies between 10-30 × 10 6 and depends on the clinical condition, the disease's specificity, the size of the lesion, and the application type (if they are applied subcutaneously, intravenously or intra-articular, for example) [3,79,83]. For example, in horses, the recommended dosage for intra-articular application for osteoarthritis is 20 × 10 6 MSCs [84]. ...
Article
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Simple Summary Athletic horses are often exposed to traumatic injuries, resulting in severe financial losses. Adipose tissue possesses a high potential as an easily accessible source and provides a higher yield of mesenchymal stem cells for various applications in regenerative medicine. Concerning the identification of the stemness features of isolated cells, some of the most commonly applied standards are not applicable because of the species-specific responses to the differentiation protocols. In many cases, the cells cannot reveal their multipotent properties, so their stemness features remain questionable. The adaptation, optimization, and standardization of equine-specific protocols for cell isolation and culture conditions are also discussed. The presented new approaches elucidate the possibility of the transition from cell-based to cell-free therapy with regenerative purposes in horses as an alternative treatment to cellular therapy. The current review summarizes aspects of the specificity of equine adipose stem cells concerning their features, immunophenotyping, secretome profile, differentiation abilities, culturing conditions, and consequent possibilities for clinical application in some equine-specific disorders. Abstract Adipose tissue is recognized as the major endocrine organ, potentially acting as a source of mesenchymal stem cells for various applications in regenerative medicine. Athletic horses are often exposed to traumatic injuries, resulting in severe financial losses. The development of adipose-derived stem cells’ regenerative potential depends on many factors. The extraction of stem cells from subcutaneous adipose tissue is non-invasive, non-traumatic, cheaper, and safer than other sources. Since there is a lack of unique standards for identification, the isolated cells and applied differentiation protocols are often not species-specific; therefore, the cells cannot reveal their multipotent properties, so their stemness features remain questionable. The current review discusses some aspects of the specificity of equine adipose stem cells concerning their features, immunophenotyping, secretome profile, differentiation abilities, culturing conditions, and consequent possibilities for clinical application in concrete disorders. The presented new approaches elucidate the possibility of the transition from cell-based to cell-free therapy with regenerative purposes in horses as an alternative treatment to cellular therapy. In conclusion, their clinical benefits should not be underestimated due to the higher yield and the physiological properties of adipose-derived stem cells that facilitate the healing and tissue regeneration process and the ability to amplify the effects of traditional treatments. More profound studies are necessary to apply these innovative approaches when treating traumatic disorders in racing horses.
... However, there are several methods used to treat cartilage damage, containing microfracture, mosaicplasty, autologous chondrocyte implantation (ACI) [5], and novel biomedical techniques (e.g., stem cell therapy and tissue engineering). Although these techniques can be used as treatments, there are still some challenges, such as not fully understanding cartilage aetiology and pathogenesis, lags of diagnosis due to the aneural nature of the tissue, and difficulties of drug and biomolecule delivery because of being avascular [2,6,7]. Tissue engineering (TE) with a combination of cell, scaffold, and signaling (mechanical, chemical, and electrical signals) offers a promising approach for the regeneration of cartilage, which can overcome the present issues [8][9][10]. ...
Article
Stem cell therapy is a promising strategy for cartilage tissue engineering, and cell transplantation using polymeric scaffolds has recently gained attention. Herein, we encapsulated human adipose-derived stem cells (hASCs) within the alginate sulfate hydrogel and then added them to polycaprolactone/gelatin electrospun nanofibers and extracellular matrix (ECM) powders to mimic the cartilage structure and characteristic. The composite hydrogel scaffolds were developed to evaluate the relevant factors and conditions in mechanical properties, cell proliferation, and differentiation to enhance cartilage regeneration. For this purpose, different concentrations (1-5 % w/v) of ECM powder were initially loaded within an alginate sulfate solution to optimize the best composition for encapsulated hASCs viability. Adding 4 % w/v of ECM resulted in optimal mechanical and rheological properties and better cell viability. In the next step, electrospun nanofibrous layers were added to the alginate sulfate/ECM composite to prepare different layered hydrogel-nanofiber (2, 3, and 5-layer) structures with the ability to mimic the cartilage structure and function. The 3-layer structure was selected as the optimum layered composite scaffold, considering cell viability, mechanical properties, swelling, and biodegradation behavior; moreover, the chondrogenesis potential was assessed, and the results showed promising features for cartilage tissue engineering application.
... MSCs represent a high therapeutic potential in veterinary medicine for several species [25,26]. However, numerous factors remain to be determined to define the best MSCs-based OA therapy, such as the dose, the time of implantation, the tissue of origin, the health status of the donor [27], the number of injections required [28], the use of allogenic or autologous MSCs [29,30], or the combination with a complementary therapeutic approach [24]. ...
Article
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Background Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. Methods An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC co-culture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO 2 ⁻ production. To identify the underlying molecular actors, stable isotope-labeling by amino acids in cell culture based secreted protein analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. Results Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture conditioned medium, the abundance of decorin and matrix metalloproteinase 3 was significantly modified, as confirmed by western blot analyses. Conclusions These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNFα and IL-1β on cartilage explant by decreasing the secretion and activity of matrix metalloproteinase 3 and increasing the decorin secretion. Graphical abstract mdMSCs capacity to reduce the catabolic consequences of cartilage exposure to pro-inflammatory cytokines. These effects can be explained by mdMSC-secreted bioactive such as TIMP-1 and decorin, known as an inhibitor of MMP3 and an anti-inflammatory protein, respectively.
... MSCs are multipotent stromal cells of either adult or embryologic origin that can differentiate into multiple tissue types (bone, cartilage, and fat). Methods for isolating and characterizing MSCs have been enumerated elsewhere and are beyond the scope of this review [103,104]. MSCs have not been demonstrated to have anti-inflammatory effects and indeed tend to induce transient inflammatory reactions [105][106][107]. Interestingly, this inflammatory reaction has been shown to differ between MSCs of different tissue origins [107]. ...
Article
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Osteoarthritis is a common and debilitating disease affecting horses across breeds and disciplines. Although the cornerstone of therapy among equine practitioners remains systemic and local anti-inflammatory medications, this approach only addresses the symptoms of osteoarthritis, rather than modifying the progression of the disease itself. There has been great interest in various biologic and cell-based therapies, such as autologous conditioned serum, platelet-rich plasma, and mesenchymal stem cells, as potentially being disease-modifying osteoarthritis drugs. In vitro and experimental results for these novel modalities are promising. However, although the use of these therapies is now widespread, scientific evidence supporting their efficacy in clinical cases is limited to date. Gene therapy for delivery of anti-inflammatory cytokines or growth factors has also been investigated experimentally with good results but has not entered widespread clinical practice. Standardized definitions of disease and large randomized controlled trials, organized across institutions, are needed improve evidence-based recommendations for osteoarthritis treatment. This review provides a brief overview of what is known about the pathophysiology of osteoarthritis and addresses the current literature for medical treatment of osteoarthritis in the horse.
... Because of the prevalence of intraarticular injections of BMMSCs in equine regenerative medicine, we decided to use these cells in our study as a comparison with SFMSCs [33]. Furthermore, in order to improve the feasibility of using these in the clinic, we chose to evaluate MSCs derived from the same recipient donor (autologous) or another donor (allogeneic) [34]. Autologous MSCs have a less immunologic reaction; however, using these MSCs has proven to be costly and time-consuming in vitro [17]. ...
Article
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Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.
... Pezzanite et al. showed that allogenic transplantation triggers a higher immune response than autologous transplantation [22]. Immune responses that could generate inflammation are a big concern following SF-MSCs transplantation as they could lead to cartilage damage [31]. To reduce at minimum the immunological responses and consequent rejection of autologous or allogeneic grafts, different therapeutic strategies must be investigated. ...
... The higher or lower differentiation potential in cells might vary among donors (Zayed et al., 2017). Moreover, in the case of equine synovial fluid cells, even if they might represent a less invasive MSCs harvesting site in equines, there is still the challenge of further evaluating their use in intra-articular injections i.e. osteoarthritis to support their clinical application (Zayed et al., 2018a). ...
Article
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Mesenchymal stem cells (MSCs) have been used in equines as an alternative therapy. A comparative study about the phenotype and in vitro performance of different MSCs tissue sources in adult equines was needed. This study might serve to provide the knowledge to select a valuable harvesting source of MSCs. Bone marrow, synovial and adipose (mesenteric, neck and tail fat) tissues were collected from adult equines. Cell surface markers expression (CD11α/CD18, CD45, CD79α, CD90, CD105 and MHC II) and in vitro differentiation assays were made. In vitro cell migration, cell growth and wound healing capacity tests helped to study their behavior and properties. MSCs phenotype was positively confirmed by the cell surfaces markers and a tri-lineage differentiation profile. Bone marrow cells showed the highest migration capacity, while synovial fluid cells displayed the highest cell growth. Bone marrow cells showed a better wound healing when compared with all the different MSCs. We conclude that bone marrow, synovial and adipose tissue derived from adult equines are a good source for cell therapy but they conserve different functional properties: bone marrow showed an interesting migration and wound healing capacity while synovial fluid cells and their highest cell growth suggest that these MSCs would yield higher cell numbers in a shorter time.
... Nevertheless, as previously described in Wistar rats knees by Leijs [34], describing no significant difference in human bone MSCs attachment to synovium between OA (MIA) and healthy rat knees, this may suggest that a pre- vious synovitis is not an obligatory prerequisite to activate exogenous MSC's trophic properties after intra-articular injection or implantation. Additionally, the number of MSCs injected have been determined according to the lit- erature, although there is no real consensus on the ideal dose and timing of injections [72]. After intra-articular in- jection and homing, MSCs probably exert their symptom- atic beneficial analgesic properties [60,73], at least, by their paracrine anti-inflammatory and anti-apoptotic ac- tivities, rather than a structural articular protection. ...
Preprint
Background: Mesenchymal stem cells (MSCs) are found in synovial fluid (SF) and can easily be harvested during arthrocentesis or arthroscopy. However, SF-MSC characterization and chondrogenicity in collagen sponges have been poorly documented as well as their hypothetical in vivo chondroprotective properties with intra-articular injections during experimental osteoarthritis (OA). Methods: SF-MSCs were isolated from human SF aspirates in patients suffering from advanced OA undergoing total knee joint replacements. SF-MSCs at passage 2 (P2) were characterized by flow cytometry for epitope profiling. SF-MSCs at P2 were subsequently cultured in vitro to assess their multilineage potentials. To assess their chondrogenicity, SF-MSCs at P4 were seeded in collagen sponges for 4 weeks under various oxygen tensions and growth factors combinations to estimate their gene profile and matrix production. Also, SF-MSCs were injected into the joints in a nude rat anterior cruciate ligament transection (ACLT) to macroscopically and histologically assess their possible chondroprotective properties,.
... Nevertheless, as previously described in Wistar rats knees by Leijs [34], describing no significant difference in human bone MSCs attachment to synovium between OA (MIA) and healthy rat knees, this may suggest that a previous synovitis is not an obligatory prerequisite to activate exogenous MSC's trophic properties after intra-articular injection or implantation. Additionally, the number of MSCs injected have been determined according to the literature, although there is no real consensus on the ideal dose and timing of injections [72]. After intra-articular injection and homing, MSCs probably exert their symptomatic beneficial analgesic properties [60,73], at least, by their paracrine anti-inflammatory and anti-apoptotic activities, rather than a structural articular protection. ...
Article
Full-text available
Background Mesenchymal stem cells (MSCs) are found in synovial fluid (SF) and can easily be harvested during arthrocentesis or arthroscopy. However, SF-MSC characterization and chondrogenicity in collagen sponges have been poorly documented as well as their hypothetical in vivo chondroprotective properties with intra-articular injections during experimental osteoarthritis (OA). Methods SF-MSCs were isolated from human SF aspirates in patients suffering from advanced OA undergoing total knee joint replacements. SF-MSCs at passage 2 (P2) were characterized by flow cytometry for epitope profiling. SF-MSCs at P2 were subsequently cultured in vitro to assess their multilineage potentials. To assess their chondrogenicity, SF-MSCs at P4 were seeded in collagen sponges for 4 weeks under various oxygen tensions and growth factors combinations to estimate their gene profile and matrix production. Also, SF-MSCs were injected into the joints in a nude rat anterior cruciate ligament transection (ACLT) to macroscopically and histologically assess their possible chondroprotective properties,. Results We characterized the stemness (CD73+, CD90+, CD105+, CD34−, CD45−) and demonstrated the multilineage potency of SF-MSCs in vitro. Furthermore, the chondrogenic induction (TGF-ß1 ± BMP-2) of these SF-MSCs in collagen sponges demonstrated a good capacity of chondrogenic gene induction and extracellular matrix synthesis. Surprisingly, hypoxia did not enhance matrix synthesis, although it boosted chondrogenic gene expression (ACAN, SOX9, COL2A1). Besides, intra-articular injections of xenogenic SF-MSCs did exert neither chondroprotection nor inflammation in ACLT-induced OA in the rat knee. Conclusions Advanced OA SF-MSCs seem better candidates for cell-based constructs conceived for cartilage defects rather than intra-articular injections for diffuse OA.
... Most of the available literature on equine soft tissue lesions (mostly tendons) reported MSC numbers ranging between 10 Â 10 6 and 30 Â 10 6 , with 10 Â 10 6 cells being most commonly used (Schnabel et al., 2009;Godwin et al., 2012). Furthermore, the current dose recommendation for equine OA by direct IA injection is 20 Â 10 6 MSCs (Schnabel et al., 2013;Zayed et al., 2018). ...
Article
Since the clinical use of mesenchymal stem cells (MSCs) for treating musculoskeletal injuries is gaining popularity, practitioners should be aware of the factors that may affect MSCs from tissue harvesting for MSC isolation to cell delivery into the injury site. This review provides equine practitioners with up-to-date, practical knowledge for the treatment of equine patients using MSCs. A brief overview of laboratory procedures affecting MSCs is provided, but the main focus is on shipping conditions, routes of administration, injection methods, and which commonly used products can be combined with MSCs and which products should be avoided as they have deleterious effects on cells. There are still several knowledge gaps regarding MSC-based therapies in horses. Therefore, it is important to properly manage the factors which are currently known to affect MSCs, to further strengthen the evidence basis of this treatment.
Article
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There is an unmet need for novel and efficacious therapeutics for regenerating injured articular cartilage in progressive osteoarthritis (OA) and/or trauma. Mesenchymal stem cells (MSCs) are particularly promising for their chondrogenic differentiation, local healing environment modulation, and tissue- and organism-specific activity; however, despite early in vivo success, MSCs require further investigation in highly-translatable models prior to disseminated clinical usage. Large animal models, such as canine, porcine, ruminant, and equine models, are particularly valuable for studying allogenic and xenogenic human MSCs in a human-like osteochondral microenvironment, and thus play a critical role in identifying promising approaches for subsequent clinical investigation. In this mini-review, we focus on [1] considerations for MSC-harnessing studies in each large animal model, [2] source tissues and organisms of MSCs for large animal studies, and [3] tissue engineering strategies for optimizing MSC-based cartilage regeneration in large animal models, with a focus on research published within the last 5 years. We also highlight the dearth of standard assessments and protocols regarding several crucial aspects of MSC-harnessing cartilage regeneration in large animal models, and call for further research to maximize the translatability of future MSC findings.
Article
Zusammenfassung In den letzten Jahren haben zellbasierte Therapeutika zur Behandlung von Osteoarthritiden in der Pferdemedizin einen regelrechten Boom erlebt. In der Praxis werden diese Therapeutika in Eigenverantwortung des Tierarztes aus Patientenblut oder anderen körpereigenen Geweben wie Fettgewebe oder Knochenmark hergestellt. Auch wenn diesen zellbasierten Therapiemethoden das einheitliche therapeutische Konzept der regenerativen Medizin gemein ist, unterscheiden sie sich maßgeblich hinsichtlich Herstellungsverfahren, Inhaltsstoffen und Funktionsweisen. Grundlegendes Wissen hierzu ermöglicht es dem praktizierenden Tierarzt, das für ihn und seine Pferdepatienten geeignete Produkt auszuwählen und bestmögliche Behandlungsstrategien zu erstellen.
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Recent studies have shown that mesenchymal stem cells (MSCs) can play a restorative role against degenerative joint diseases in horses. The purpose of this study was to investigate whether fetal bone marrow-derived cells (BMC)-derived nanoparticles (BMC-NPs) can stimulate the survival of equine chondrocytes. Equine fetal BMCs were isolated and characterized, and the role of BMC-NPs s in equine chondrocytes undergoing inflammatory cell death was examined. BMCs have several characteristics, such as the potential to differentiate into chondrocytes and osteocytes. Additionally, BMCs expressed immunoregulatory genes in response to treatment with tumor necrosis factor-alpha (TNF-α) and Interleukin 1 beta (IL-1β). We found that BMC-NPs were taken up by equine chondrocytes. Functionally, BMC-NPs promoted the growth of chondrocytes, and reduced apoptosis induced by inflammatory cytokines. Furthermore, we observed that BMC-NPs upregulated the phosphorylation of protein kinase B (Akt) in the presence of IL-1β, and reduced the phosphorylation of TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the chondrocytes. Cumulatively, our study demonstrated that equine fetal BMC-NPs have the potential to stimulate the survival of chondrocytes damaged by inflammatory cytokines. Thus, BMC-NPs may become an alternative cell-free allogenic therapeutic for degenerative joint diseases in horses.
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Background: Autologous and allogeneic adult mesenchymal stem/stromal cells (MSCs) are increasingly being investigated for treating a wide range of clinical diseases. Allogeneic MSCs are especially attractive due to their potential to provide immediate care at the time of tissue injury or disease diagnosis. The prevailing dogma has been that allogeneic MSCs are immune privileged, but there have been very few studies that control for matched or mismatched major histocompatibility complex (MHC) molecule expression and that examine immunogenicity in vivo. Studies that control for MHC expression have reported both cell-mediated and humoral immune responses to MHC-mismatched MSCs. The clinical implications of immune responses to MHC-mismatched MSCs are still unknown. Pre-clinical and clinical studies that document the MHC haplotype of donors and recipients and measure immune responses following MSC treatment are necessary to answer this critical question. Conclusions: This review details what is currently known about the immunogenicity of allogeneic MSCs and suggests contemporary assays that could be utilized in future studies to appropriately identify and measure immune responses to MHC-mismatched MSCs.
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Surgery-obtained synovium specimens (SSSs) can provide a source of synovial mesenchymal stem cells (SMSCs) for experimental studies. However, these specimens contain diverse tissues, including the intima and subintima; therefore, these SMSCs are not entirely derived from the intima and their cell source is heterogeneous. The present study isolated synovial fragments (SFs) from synovial fluid dilutions extracted from patients with temporomandibular joint (TMJ) osteoarthrosis. Unlike SSSs, SFs, which are membranous and translucent, consist of only several cell layers, indicating the presence of only the intima. In the present study, SF cells (SFCs) and SSS cells (SSSCs) exhibited a homogeneous, fibroblast‑like, spindle‑shaped morphology after passaging in vitro. Furthermore, both cell types exhibited similar proliferative and differentiation potentials in vitro. However, SFCs exhibited more uniform surface markers compared with SSSCs when analysed by flow cytometry. Taken together, these results indicated that SFs contained a greater amount of unmixed intima than SSSs, and that SFCs exhibited more homogeneous characteristics than SSSCs, thereby offering an improved source of SMSCs in the TMJ.
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Mesenchymal stem cells (MSCs) were officially named more than 25 years ago to represent a class of cells from human and mammalian bone marrow and periosteum that could be isolated and expanded in culture while maintaining their in vitro capacity to be induced to form a variety of mesodermal phenotypes and tissues. The in vitro capacity to form bone, cartilage, fat, etc., became an assay for identifying this class of multipotent cells and around which several companies were formed in the 1990s to medically exploit the regenerative capabilities of MSCs. Today, there are hundreds of clinics and hundreds of clinical trials using human MSCs with very few, if any, focusing on the in vitro multipotential capacities of these cells. Unfortunately, the fact that MSCs are called "stem cells" is being used to infer that patients will receive direct medical benefit, because they imagine that these cells will differentiate into regenerating tissue-producing cells. Such a stem cell treatment will presumably cure the patient of their medically relevant difficulties ranging from osteoarthritic (bone-on-bone) knees to various neurological maladies including dementia. I now urge that we change the name of MSCs to Medicinal Signaling Cells to more accurately reflect the fact that these cells home in on sites of injury or disease and secrete bioactive factors that are immunomodulatory and trophic (regenerative) meaning that these cells make therapeutic drugs in situ that are medicinal. It is, indeed, the patient's own site-specific and tissue-specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs. Stem Cells Translational Medicine 2017.
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Background Intra-articular injection of mesenchymal stem cells (MSCs) is efficacious in osteoarthritis therapy. A direct comparison of the response of the synovial joint to intra-articular injection of autologous versus allogeneic MSCs has not been performed. The objective of this study was to assess the clinical response to repeated intra-articular injection of allogeneic versus autologous MSCs prepared in a way to minimize xeno-contaminants in a large animal model. Methods Intra-articular injections of bone marrow-derived, culture-expanded MSCs to a forelimb metacarpophalangeal joint were performed at week 0 and week 4 (six autologous; six autologous with xeno-contamination; six allogeneic). In the week following each injection, clinical and synovial cytology evaluations were performed. ResultsFollowing the first intra-articular injection, there were no differences in clinical parameters over time. Following the second intra-articular injection, there was a significant adverse response of the joint to allogeneic MSCs and autologous MSCs with xeno-contamination with elevated synovial total nucleated cell counts. There was also significantly increased pain from joints injected with autologous MSCs with xeno-contamination. Conclusions Repeated intra-articular injection of allogeneic MSCs results in an adverse clinical response, suggesting there is immune recognition of allogeneic MSCs upon a second exposure.
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Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application.
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The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared to those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. The immune suppressive activity of the two cell types was evaluated by adding autologous or allogeneic BMDMSCs to activated lymphocytes and assessing suppression of lymphocyte proliferation and IFNɣ production. 56 allogeneic and 12 autologous combinations were evaluated. Studies were also done to elucidate mechanisms by which equine MSCs suppress lymphocyte function. Potential mechanisms evaluated included production of prostaglandin E2 (PGE2), nitric oxide, transforming growth factor-beta (TGFß), and Indoleamine 2,3-dioxygenase. We found that autologous and allogeneic BMDMSCs both induced mild but equivalent levels of spontaneous lymphocyte activation in vitro. In vitro assays assessing the ability of BMDMSCs to suppress activated lymphocytes, both allogeneic and autologous BMDMSCs suppressed T cell proliferation and IFNɣ production to an equal degree. The primary mechanism of equine BMDMSCs suppression of T cells was mediated by prostaglandin E2. We concluded that allogeneic and autologous BMDMSCs are equivalent in terms of their immune modulatory properties, and stimulated PBMCs appear to trigger the immune suppressive properties of MSCs. Therefore, both cell types appear to have equal potency in modulating inflammatory processes related to acute or chronic musculoskeletal injuries in the horse. .
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Mesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer’s Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points. A mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls. Intra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases.
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Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ “super-healer” strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.
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Mesenchymal stem cells promising role in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. Six age groups from bone marrow mesenchymal stem cells of Wistar rats were studied (newborn, infant, young, pre-pubertal, pubertal and adult). Quantitative proteomic assay was performance by iTRAQ using an 8-plex iTRAQ labeling and the proteins differentially expressed were grouped in pluripotency, proliferative and metabolism processes. Proliferation makers, CD117 and Ki67 were measure by flow cytometry assay. Real time polymerase chain reaction analysis of pluripotency markers Rex1, Oct4, Sox2 and Nanog were done. Biological differentiation was realized using specific mediums for 14 days to induce osteogenesis, adipogenesis or chondrogenesis and immunostain analysis of differentiated cell resulting were done. Enzimoimmunoassay analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were also done to validate iTRAQ data. Taking together these results indicate for the first time that mesenchymal stem cells have significant differences in their proliferative, pluripotency and metabolism profiles and those differences are age depending.
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Background: Isolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were characterized by morphology and expression of specific markers for stem cells, pluripotency, inflammation, and cell cycle. Results: The medium MEM was more effective (97 % ± 2) to maintain both cultures. The cultures were composed by adherent cells with fibroblast-like shape, which had a growth pattern represented by a sigmoidal curve. After the expansion, the cells were analyzed by flow cytometry for stem cells, inflammatory, and cell cycle markers, and both lineages showed significant expression of CD45, Oct3/4, Nanog, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF, and LY6a. In contrast, there were differences in the cell cycle phases between the lineages, which was not observed in relation to the mitochondrial electrical potential. Conclusion: Given the large impact that joint pathology has on the athletic performance horses, our results suggested that the SF and SM are promising sources of stem cells with satisfactory characteristics of growth and gene expression that can be used in equine regenerative medicine.
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Multipotent mesenchymal stromal cells (MSC) have attracted interest for their cytotherapeutic potential, partly due to their immunomodulatory abilities. The aim of this study was to test the robustness of our equine cord blood (CB) MSC isolation protocol, to characterize the CB-MSC before and after cryopreservation, and to evaluate their immunosuppressive phenotype. We hypothesized that MSC can be consistently isolated from equine CB, have unique and reproducible marker expression and in vitro suppress lymphoproliferation. Preliminary investigation of constitutive cytoplasmic Toll-like receptor (TLR) 3 and 4 expression was also preformed due to their possible association with anti- or pro-inflammatory MSC phenotypes, respectively. Surface markers were assessed for antigen and mRNA expression by flow cytometry and quantitative polymerase chain reaction (qPCR). Immunomodulatory properties were evaluated in mixed lymphocyte reaction assays, and TLR3 and TLR4 expression were measured by qPCR and immunocytochemistry (ICC). CB-MSC were isolated from each off nine cord blood samples. CB-MSC highly expressed CD29, CD44, CD90, and lacked or had low expression of major histocompatibility complex (MHC) class I, MHC-II, CD4, CD8, CD11a/18 and CD73 before and after cryopreservation. CB-MSC suppressed in vitro lymphoproliferation and constitutively expressed TLR4. Our findings confirmed CB as a reliable MSC source, provides an association of surface marker phenotype and mRNA expression and suggest anti-inflammatory properties of CB-MSC. The relationship between TLRs and lymphocyte function warrants further investigation.
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This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application. Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsatellite typing and used as allogeneic MHC-mismatched MSC recipients. MHC homozygote horses of known ELA-A2 haplotype were used as MSC and peripheral blood leukocyte (PBL) donors. One MHC homozygote horse of the ELA-A2 haplotype was the recipient of ELA-A2 donor MSCs as an MHC-matched control. Donor MSCs, which were previously isolated and immunophenotyped, were thawed and culture expanded to achieve between 30x10(6) and 50x10(6) cells for intradermal injection into the recipient's neck. Recipient serum was collected and tested for the presence of anti-donor antibodies prior to MSC injection and every 7 days after MSC injection for the duration of the 8-week study using the standard two-stage lymphocyte microcytotoxicity dye-exclusion test. In addition to anti-ELA-A2 antibodies, recipient serum was examined for the presence of cross-reactive antibodies including anti-ELA-A3 and anti-RBC antibodies. All MHC-mismatched recipient horses produced anti-ELA-A2 antibodies following injection of ELA-A2 MSCs and developed a wheal at the injection site that persisted for the duration of the experiment. Anti-ELA-A2 antibody responses were varied both in terms of strength and timing. Four recipient horses had high-titered anti-ELA-A2 antibody responses resulting in greater than 80% donor PBL death in the microcytotoxicity assays and one of these horses also developed antibodies that cross-reacted when tested on lymphocyte targets from a horse with an unrelated MHC type. Allogeneic MSCs are capable of eliciting antibody responses in vivo that can be strong and also cross-reactive with MHC types other than that of the donor. Such responses could limit the effectiveness of repeated allogeneic MSC use in a single horse, and could also result in untoward inflammatory responses in recipients.
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To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model. BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O'driscoll score. Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O'driscoll score was higher than other groups. The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo.
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Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellular matrices after induction of differentiation. The total number of SF-derived mesenchymal stromal cells reached >1 × 107 by the fourth passage. SF-derived cells were strongly positive (>90% cells positive) for CD44, CD90 and major histocompatibility complex (MHC) class I, and moderately positive (60–80% cells positive) for CD11a/CD18, CD105 and MHC class II by flow cytometry. SF-derived cells were negative for CD34 and CD45. Under specific nutrient conditions, SF-derived cells differentiated into osteogenic, chondrogenic, adipogenic and tenogenic lineages, as indicated by the expression of specific marker genes and by the production of specific extracellular matrices. Chondrogenic induction in culture resulted in a change in cell shape to a ‘stone-wall’ appearance and formation of a gelatinous sheet that was intensely stained with Alcian blue. SF may be a novel source of multipotent mesenchymal stem cells with the ability to regenerate chondrocytes.
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Introduction Autologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells’ immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues. Methods Mitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA). Results Co-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α. Conclusions Equine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.
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Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, BM-MSCs and AT-MSCs showed fastest in vitro differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes.
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Background Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties. Methods BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells. Results BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs. Conclusions By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.
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Introduction Platelet-rich plasma (PRP) is nowadays widely applied in different clinical scenarios, such as orthopedics, ophthalmology and healing therapies, as a growth factor pool for improving tissue regeneration. Studies into its clinical efficiency are not conclusive and one of the main reasons for this is that different PRP preparations are used, eliciting different responses that cannot be compared. Platelet quantification and the growth factor content definition must be defined in order to understand molecular mechanisms behind PRP regenerative strength. Standardization of PRP preparations is thus urgently needed. Methods PRP was prepared by centrifugation varying the relative centrifugal force, temperature, and time. Having quantified platelet recovery and yield, the two-step procedure that rendered the highest output was chosen and further analyzed. Cytokine content was determined in different fractions obtained throughout the whole centrifugation procedure. Results Our method showed reproducibility when applied to different blood donors. We recovered 46.9 to 69.5% of total initial platelets and the procedure resulted in a 5.4-fold to 7.3-fold increase in platelet concentration (1.4 × 106 to 1.9 × 106 platelets/μl). Platelets were highly purified, because only <0.3% from the initial red blood cells and leukocytes was present in the final PRP preparation. We also quantified growth factors, cytokines and chemokines secreted by the concentrated platelets after activation with calcium and calcium/thrombin. High concentrations of platelet-derived growth factor, endothelial growth factor and transforming growth factor (TGF) were secreted, together with the anti-inflammatory and proinflammatory cytokines interleukin (IL)-4, IL-8, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-α. No cytokines were secreted before platelet activation. TGF-β3 and IFNγ were not detected in any studied fraction. Clots obtained after platelet coagulation retained a high concentration of several growth factors, including platelet-derived growth factor and TGF. Conclusions Our study resulted in a consistent PRP preparation method that yielded a cytokine and growth factor pool from different donors with high reproducibility. These findings support the use of PRP in therapies aiming for tissue regeneration, and its content characterization will allow us to understand and improve the clinical outcomes.
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Although bone marrow is the main source, mesenchymal stem cells have already been isolated from various other tissues, such as the liver, pancreas, adipose tissue, peripheral blood and dental pulp. These plastic adherent cells are morphologically similar to fibroblasts and have a high proliferative potential. This special group of cells possesses two essential characteristics: self-renewal and differentiation, with appropriate stimuli, into various cell types. Mesenchymal stem cells are considered immunologically privileged, since they do not express costimulatory molecules, required for complete T cell activation, on their surface. Several studies have shown that these cells exert an immunosuppressive effect on cells from both innate and acquired immunity systems. Mesenchymal stem cells can regulate the immune response in vitro by inhibiting the maturation of dendritic cells, as well as by suppressing the proliferation and function of T and B lymphocytes and natural killer cells. These special properties of mesenchymal stem cells make them a promising strategy in the treatment of immune mediated disorders, such as graft-versus-host disease and autoimmune diseases, as well as in regenerative medicine. The understanding of immune regulation mechanisms of mesenchymal stem cells, and also those involved in the differentiation of these cells in various lineages is primordial for their successful and safe application in different areas of medicine.
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Background: Specific growth factors have been proposed as therapeutic proteins for cartilage repair. Hypothesis: Platelet-rich plasma (PRP) provides symptomatic relief in early osteoarthritis (OA) of the knee. Study design: Randomized controlled trial; Level of evidence, 1. Methods: A total of 78 patients (156 knees) with bilateral OA were divided randomly into 3 groups. Group A (52 knees) received a single injection of PRP, group B (50 knees) received 2 injections of PRP 3 weeks apart, and group C (46 knees) received a single injection of normal saline. White blood cell (WBC)-filtered PRP with a platelet count 3 times that of baseline (PRP type 4B) was administered in all. All the groups were homogeneous and comparable in baseline characteristics. Clinical outcome was evaluated using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire before treatment and at 6 weeks, 3 months, and 6 months after treatment. They were also evaluated for pain by a visual analog scale, and overall satisfaction with the procedure and complications were noted. Results: Statistically significant improvement in all WOMAC parameters was noted in groups A and B within 2 to 3 weeks and lasting until the final follow-up at 6 months, with slight worsening at the 6-month follow-up. The mean WOMAC scores (pain, stiffness, physical function, and total score) for group A at baseline were 10.18, 3.12, 36.56, and 49.86, respectively, and at final follow-up were 5.00, 2.10, 20.08, and 27.18, respectively, showing significant improvement. Similar improvement was noted in group B (mean WOMAC scores at baseline: 10.62, 3.50, 39.10, and 53.20, respectively; mean WOMAC scores at final follow-up: 6.18, 1.88, 22.40, and 30.48, respectively). In group C, the mean WOMAC scores deteriorated from baseline (9.04, 2.70, 33.80, and 45.54, respectively) to final follow-up (10.87, 2.76, 39.46, and 53.09, respectively). The 3 groups were compared with each other, and no improvement was noted in group C as compared with groups A and B (P < .001). There was no difference between groups A and B, and there was no influence of age, sex, weight, or body mass index on the outcome. Knees with Ahlback grade 1 fared better than those with grade 2. Mild complications such as nausea and dizziness, which were of short duration, were observed in 6 patients (22.2%) in group A and 11 patients (44%) in group B. Conclusion: A single dose of WBC-filtered PRP in concentrations of 10 times the normal amount is as effective as 2 injections to alleviate symptoms in early knee OA. The results, however, deteriorate after 6 months. Both groups treated with PRP had better results than did the group injected with saline only.
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Multipotent mesenchymal stromal cells (MSCs) have generated considerable interest in the fields of regenerative medicine, cell therapy and immune modulation. Over the past 5 years, the initial observations that MSCs could enhance regeneration and modulate immune responses have been significantly advanced and we now have a clearer picture of the effects that MSCs have on the immune system particularly in the context of inflammatory-mediated disorders. A number of mechanisms of action have been reported in MSC immunomodulation, which encompass the secretion of soluble factors, induction of anergy, apoptosis, regulatory T cells and tolerogenic dendritic cells. It is clear that MSCs modulate both innate and adaptive responses and evidence is now emerging that the local microenvironment is key in the activation or licensing of MSCs to become immunosuppressive. More recently, studies have suggested that MSCs have the capacity to sense their environment and have a role in pathogen clearance in conjunction with the resolution of insult or injury. This review focuses on the mechanisms of MSC immunomodulation discussing the multistep process of MSC localisation at sites of inflammation, the cross talk between MSCs and the local microenvironment as well as the subsequent mechanisms of action used to resolve inflammation.Immunology and Cell Biology advance online publication, 23 October 2012; doi:10.1038/icb.2012.56.
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Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes. Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes. Controlled laboratory study. Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFκB) activation were studied. Platelet-rich plasma releasate diminished IL-1 beta-induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta-induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta-induced NFκB activation to control levels containing no IL-1 beta. Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta-mediated effects on human osteoarthritic chondrocytes, including inhibition of NFκB activation. Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFκB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.
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Platelet-rich plasma (PRP) has been increasingly used in sports medicine applications. Platelets are thought to release growth factors important in wound healing, including transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF-AB), and vascular endothelial growth factor (VEGF). However, little is known about the effect of platelet activator choice on growth factor release kinetics. The choice of platelet activator would affect the timing and level of growth factor release from PRP. Controlled laboratory study. Platelet-rich plasma aliquots were activated with either thrombin or collagen. A control group of whole blood aliquots was clotted with thrombin. Supernatant containing the released growth factors was collected daily for 1 week. Levels of TGF-β1, PDGF-AB, and VEGF were measured using enzyme-linked immunosorbent assay (ELISA). The use of thrombin as an activator resulted in immediate release of TGF-β1 and PDGF-AB, while the collagen-activated PRP clots released similar amounts each day for 5 days. The use of collagen as an activator resulted in an 80% greater cumulative release of TGF-β1 from the PRP aliquots over 7 days (P < .001). Concentrating platelets to 3 times the systemic blood level resulted in a 3-fold higher release of TGF-β1, 2.5-fold greater release of PDGF, and 5-fold greater release of VEGF (all P < .0001) when compared with whole blood control clots, but no significant differences in the timing of release were noted. These experiments demonstrated that the choice of platelet activator can significantly influence the release kinetics of cytokines from PRP, with thrombin resulting in an immediate release and collagen having a more sustained release pattern. The level and rate of growth factor release depends on the selected platelet activator, a factor that should be considered when selecting a PRP system for a given application.
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The stem cell field in veterinary medicine continues to evolve rapidly both experimentally and clinically. Stem cells are most commonly used in clinical veterinary medicine in therapeutic applications for the treatment of musculoskeletal injuries in horses and dogs. New technologies of assisted reproduction are being developed to apply the properties of spermatogonial stem cells to preserve endangered animal species. The same methods can be used to generate transgenic animals for production of pharmaceuticals or for use as biomedical models. Small and large animal species serve as valuable models for preclinical evaluation of stem cell applications in human beings and in veterinary patients in areas such as spinal cord injury and myocardial infarction. However, these applications have not been implemented in the clinical treatment of veterinary patients. Reviews on the use of animal models for stem cell research have been published recently. Therefore, in this review, animal model research will be reviewed only in the context of supporting the current clinical application of stem cells in veterinary medicine.
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Equine adipose tissue-derived mesenchymal stem cells (ASCs) have only recently been investigated for their adipogenic, chondrogenic, and osteogenic differentiation potential. This chapter will briefly outline the molecular mechanisms leading to adipogenesis and the methods of equine adipose tissue harvest, ASC isolation, and adipogenic differentiation. The reader is also directed to other reported methods of adipogenesis for ASCs and mesenchymal stem cells (MSCs) from other tissues.
Article
Chondral and osteochondral lesions due to injury or other pathology are highly prevalent conditions in horses (and humans) and commonly result in the development of osteoarthritis and progression of joint deterioration. Regenerative medicine of articular cartilage is an emerging clinical treatment option for patients with articular cartilage injury or disease. Functional articular cartilage restoration, however, remains a major challenge, but the field is progressing rapidly and there is an increasing body of supportive clinical and scientific evidence. This review gives an overview of the established and emerging surgical techniques employed for cartilage repair in horses. Through a growing insight in surgical cartilage repair possibilities, surgeons might be more stimulated to explore novel techniques in a clinical setting.
Article
The influence of graphene quantum dots (GQDs) on key characteristics of bone marrow derived mesenchymal stem cells (MSCs) phenotype (i.e., self-renewal, differentiation potential, and pluripotency) is systematically investigated in this work. First, the viability and impact of GQDs on the self-renewal potential of MSCs is evaluated in order to determine a threshold for the exposing dose. Second, GQDs uptake by MSCs is confirmed due to the excellent fluorescent properties of the particles. They exhibit a homogenous cytoplasmatic distribution that increases with the time and concentration. Third, the impact of GQDs on the osteogenic differentiation of MSCs is deeply characterized. An enhanced activity of alkaline phosphatase promoted by GQDs indicates early activation of osteogenesis. This is also confirmed upon GQD-induced up-regulation of phenotypically related osteogenic genes (Runx2, osteopontin, and osteocalcin) and specific biomarkers expression (osteopontin and osteocalcin). GQDs also effectively enhance the formation of calcium-rich deposits characteristics of osteoblasts. Furthermore, genes microarray results indicate that the enhanced osteogenic differentiation of MSCs by GQDs is in progress through a bone morphogenetic protein and transforming growth factor-β relative signaling pathways. Finally, intracytoplasmatic lipid detection shows that GQDs can also promote the adipogenic differentiation of MSCs, thus confirming the prevalence of their pluripotency potential.
Article
Background: The chondrogenic potential of culture-expanded bone-marrow-derived mesenchymal stem cells (BMDMSCs) is well described. Numerous studies have also shown enhanced repair when BMDMSCs, scaffolds, and growth factors are placed into chondral defects. Platelets provide a rich milieu of growth factors and, along with fibrin, are readily available for clinical use. The objective of this study was to determine if the addition of BMDMSCs to an autologous platelet-enriched fibrin (APEF) scaffold enhances chondral repair compared with APEF alone. Methods: A 15-mm-diameter full-thickness chondral defect was created on the lateral trochlear ridge of both stifle joints of twelve adult horses. In each animal, one defect was randomly assigned to receive APEF+BMDMSCs and the contralateral defect received APEF alone. Repair tissues were evaluated one year later with arthroscopy, histological examination, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and biomechanical testing. Results: The arthroscopic findings, MRI T2 map, histological scores, structural stiffness, and material stiffness were similar (p > 0.05) between the APEF and APEF+BMDMSC-treated repairs at one year. Ectopic bone was observed within the repair tissue in four of twelve APEF+BMDMSC-treated defects. Defects repaired with APEF alone had less trabecular bone edema (as seen on MRI) compared with defects repaired with APEF+BMDMSCs. Micro-CT analysis showed thinner repair tissue in defects repaired with APEF+BMDMSCs than in those treated with APEF alone (p < 0.05). Conclusions: APEF alone resulted in thicker repair tissue than was seen with APEF+BMDMSCs. The addition of BMDMSCs to APEF did not enhance cartilage repair and stimulated bone formation in some cartilage defects. Clinical relevance: APEF supported repair of critical-size full-thickness chondral defects in horses, which was not improved by the addition of BMDMSCs. This work supports further investigation to determine whether APEF enhances cartilage repair in humans.
Article
Improvement has been reported following intra-articular (IA) injection of mesenchymal stromal cells (MSC) in several species. These observations have led to use of IA MSC in equine practice with little understanding of the mechanisms by which perceived improvement occurs. To evaluate the effect of IA allogeneic umbilical-cord-blood- (CB-) derived MSC using lipopolysaccharide (LPS) induced synovitis model. We hypothesised IA CB-MSC would decrease inflammatory response associated with LPS injection. Randomised, blinded experimental study. Feasibility studies evaluated IA LPS or CB-MSC alone into the tarsocrural joint. Principal study middle carpal joint LPS synovitis was induced bilaterally then CB-MSC were injected into one middle carpal joint. Lameness, routine synovial fluid (SF) analysis, and SF biomarkers were evaluated at 0, 8, 24, 48, and 72 h. LPS injection alone resulted in transient lameness and signs of inflammation. In joints untreated with LPS, injection of 30-million CB-MSC resulted in mild synovitis that resolved without treatment. Mild (grade 1-2) lameness in the CB-MSC-treated limb was observed in 2 horses, severe lameness (grade 4) in the third 24h post-injection. Lameness did not correlate with synovitis induced by CB-MSC injection. Simultaneous injection of LPS and CB-MSC resulted in significant reduction in SF total nucleated, neutrophil, and mononuclear cell numbers compared to contralateral LPS-only joints. No difference was detected in other parameters associated with SF analysis or in SF biomarkers. The incidence of lameness was only different from baseline at 8 h, where horses were lame in CB-MSC limbs. Allogeneic CB-MSC reduced SF cell populations and stimulated mild self-limiting inflammation in the synovitis model. Continued evaluation of the effects of IA CB-MSC therapy on synovitis in horses is needed to evaluate anti- and pro-inflammatory properties of CB-MSC. Immediate interests are dose, timing of treatment, and treatment frequency. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Over 23,000 people per day require treatment for ankle sprains. Platelet-rich plasma (PRP) is an autologous concentration of platelets that is thought to improve healing by promoting inflammation through growth factor and cytokine release. Studies to date have shown mixed results, with few randomized trials. To determine patient function among patients randomized to receive standard therapy plus PRP, compared to patients who receive standard therapy plus sham injection (placebo). Prospective, randomized, double-blinded, placebo-controlled trial. Patients with severe ankle sprains were randomized. Severity was graded on degree of swelling, ecchymosis, and ability to bear weight. PRP with lidocaine and bupivacaine was injected at the point of maximum tenderness by a blinded physician under ultrasound guidance. The control group was injected in a similar fashion with sterile 0.9% saline. Both groups had visual analog scale (VAS) pain scores and Lower Extremity Functional Scale (LEFS) on days 0, 3, and 8. LEFS and a numeric pain score were obtained via phone call on day 30. All participants were splinted, given crutches, and instructed to not bear weight for 3 days; at this time patients were reevaluated. There were 1156 patients screened and 37 were enrolled. Four withdrew before PRP injection was complete; 18 were randomized to PRP and 15 to placebo. There was no statistically significant difference in VAS and LEFS scores between groups. In this small study, PRP did not provide benefit in either pain control or function over placebo. Copyright © 2015 Elsevier Inc. All rights reserved.
Article
Equine bone fractures are often catastrophic, potentially fatal, and costly to repair. Traditional methods of healing fractures have limited success, long recovery periods, and a high rate of reinjury. Current research in the equine industry has demonstrated that stem cell therapy is a promising novel therapy to improve fracture healing and reduce the incidence of reinjury; however, reports of success in horses have been variable and limited. Stem cells can be derived from embryonic, fetal, and adult tissue. Based on the ease of collection, opportunity for autologous cells, and proven success in other models, adipose- or bone marrow-derived mesenchymal stem cells (MSC) are often used in equine therapies. Methods for isolation, proliferation, and differentiation of MSC are well established in rodent and human models but are not well characterized in horses. There is recent evidence that equine bone marrow MSC are able to proliferate in culture for several passages in the presence of autologous and fetal bovine serum, which is important for expansion of cells. Mesenchymal stem cells have the capacity to differentiate into osteoblasts, the bone forming cells, and this complex process is regulated by a number of transcription factors including runt-related transcription factor 2 (Runx2) and osterix (Osx). However, it has not been well established if equine MSC are regulated in a similar manner. The data presented in this review support the view that equine bone marrow MSC are regulated by the same transcription factors that control the differentiation of rodent and human MSC into osteoblasts. Although stem cell therapy is promising in equine bone repair, additional research is needed to identify optimal methods for reintroduction and potential manipulations to improve their ability to form new bone.
Article
Recently, it has been shown that MSCs do not express the major histocompatibility complex (MHC) II antigen, and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. To evaluate the immunosuppressive capabilities of equine bone marrow derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. In vitro experiment. Phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara Ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester (CFSE)-labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and -CD8 antibodies were performed. Connemara Pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. A high proportion of CD4/CD8-double positive (DP) lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio BM-MSC: PBMC. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 DP population was similar to the PHA control. Results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments. This article is protected by copyright. All rights reserved.
Article
Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently employed in cell-based FDA-approved cartilage products, yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during post-expansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, i.e., Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, i.e., glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 wks maturation. The present study illustrates notable success in employing the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded human ACs and MSCs. Stem Cells 2014
Article
Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 10(5) MSCs; M-6: 2 × 10(6) MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.
Article
Mesenchymal stem cells (MSCs) are used in both human clinical trials and veterinary medicine for the treatment of inflammatory and immune-mediated diseases. MSCs modulate inflammation by decreasing the cells and products of the inflammatory response. Stimulated equine MSCs from bone marrow (BM), adipose tissue (AT), cord blood (CB) and umbilical cord tissue (CT) all inhibit lymphocyte proliferation and decrease inflammatory cytokine production. We hypothesized that equine MSCs inhibit T cell proliferation through secreted mediators and that MSCs from different tissue sources decrease T cell proliferation through different mechanisms. To test our hypotheses, we inhibited interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) to determine their impact on stimulated T cell proliferation. We also determined how equine MSCs modulate lymphocyte proliferation either via cell cycle arrest or apoptosis. Inhibition IL-6 or NO did not reverse the immunomodulatory effect of MSCs on activated T cells. In contrast, inhibition of PGE2 restored T cell proliferation, restored the secretion of TNF-α and IFN-γ, and increased IL-10 levels. MSCs from solid tissue-derived sources, AT and CT, inhibited T cell proliferation through induction of lymphocyte apoptosis while blood-derived MSCs, BM and CB, induced lymphocyte cell cycle arrest. Equine MSCs from different tissue sources modulated immune cell function by both overlapping and unique mechanisms. MSC tissue source may determine immunomodulatory properties of MSCs and may have very practical implications for MSC selection in the application of MSC therapy.
Article
To report outcome of horses with femorotibial lesions (meniscal, cartilage or ligamentous) treated with surgery and intra-articular administration of autologous bone marrow derived mesenchymal stem cells (BMSCs). Prospective case series. Horses (n = 33). Inclusion criteria included horses that had lameness localized to the stifle by diagnostic anesthesia, exploratory stifle arthroscopy and subsequent intra-articular administration of autologous BMSCs. Case details and follow-up were gathered from medical records, owner, trainer or veterinarian. Outcome was defined as returned to previous level of work, returned to work, or failed to return to work. Follow-up (mean, 24 months) was obtained; 43% of horses returned to previous level of work, 33% returned to work, and 24% failed to return to work. In horses with meniscal damage (n = 24) a higher percentage in the current study (75%) returned to some level of work compared to those in previous reports (60-63%) that were treated with arthroscopy alone, which resulted in a statistically significant difference between studies (P = .038). Joint flare post injection was reported in 3 horses (9.0%); however, no long-term effects were noted. Intra-articular administration of BMSC postoperatively for stifle lesions appeared to be safe, with morbidity being similar to that of other biologic agents. Improvement in ability to return to work may be realized with BMSC treatment compared to surgery alone in horses with stifle injury.
Article
Mesenchymal stem cells have demonstrated immunomodulatory capabilities as well as modest efficacy in animal models of joint injury, warranting further study as a potential treatment of joint disease. The goal of the study was to investigate the blood and synovial immune and histologic response to intra-articular injection of autologous, allogeneic, and xenogeneic bone marrow-derived mesenchymal stem cells (MSC) in horses. The study group consisted of 6 five-year-old Thoroughbred mares that had been injected previously with 15 million, genetically modified autologous, allogeneic, or xenogeneic MSC into the fetlock joints. One group of autologous cells was genetically modified to permit MSC biolocalization in the synovium. To assess response to the injection, synovial biopsies were obtained via arthroscopy 60 days after MSC injection for gross, histologic and molecular analyses. Peripheral blood mononuclear cells were isolated from each horse 120 days after MSC injection and co-cultured with a monolayer of each MSC group to permit quantification of activated CD4+ lymphocytes and cytokine release (ELISA) upon re-exposure to MSC. Arthroscopic examination revealed normal synovium with no grossly detrimental effect to the synovium or cartilage. Intra-articular MSC produced a persistent mononuclear infiltrate for at least 60 days, mostly perivascular, identified as CD3+ lymphocytes. An immune response (significant increase in CD4+ lymphocytes) was detected upon re-exposure to xenogeneic but not to allogeneic or autologous MSC. An inflammatory cytokine release from peripheral blood mononuclear cell/MSC co-cultures was present in all MSC groups but was significantly greater in the xenogeneic group. In conclusion, intra-articular injection of MSC, regardless of cell origin, incited a persistent mononuclear synovitis demonstrating a sustained biologic influence of these cells. Allogeneic cells did not elicit a detectable immune response upon re-exposure using our methods.
Article
Objectives: To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. Methods: Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. Results: There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. Clinical significance: Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.
Article
Objective. Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. Methods and Results. Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated. with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. Conclusion. Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.
Article
In the past decade, mesenchymal stem cells (MSC) have received much attention in equine veterinary medicine. The first therapeutic use of equine MSC was reported in 2003. Since then, the clinical application of MSC has been exploding with thousands of horses now treated worldwide. At present, MSC are mainly used in veterinary medicine to treat musculoskeletal diseases based on their ability to differentiate into various tissues of mesodermal origin. This is in marked contrast to human medicine, where MSC therapies are primarily focused on immune-mediated, inflammatory, and ischemic diseases. In this review, both orthopedic as well as non-orthopedic clinical applications of equine MSC are discussed. A brief overview is provided on the potential of MSC for non-orthopedic injuries with emphasis on those diseases, which occur in both humans and horses.
Article
We have previously reported that a combination of transforming growth factor (TGF)-β1 and basic fibroblast growth factor (bFGF) synergistically increases the proliferation of chondrocytes obtained from knee joint immobilized for 7–14 days in male Japanese white rabbits. In the present study, we performed experiments with chondrocytes and synovial fluid obtained from rabbit knees immobilized for 0–42 days, to clarify the sequential changes in TGF-β1 and bFGF concentrations in synovial fluid and the mRNA expression of TGF-β1 receptor type I (RI) and II (RII) in chondrocytes after immobilization. The combination of TGF-β1 and bFGF had a synergistic effect on the proliferation of chondrocytes obtained from knee joints immobilized for 7–14 days. The concentration of TGF-β1 in synovial fluid was significantly higher (up to 3.6-fold) at 7–28 days after immobilization compared with that at 2 days. The mRNA for RI and RII was expressed during the whole immobilization period. The concentration of bFGF was kept at the same level at 2–7 days after immobilization, and gradually decreased thereafter. In the early stages of degenerated cartilage, up to 14 days after immobilization, the concentrations of both TGF-β1 and bFGF were higher in the synovial fluid and mRNA expression of TGF-β1 receptors in chondrocytes was kept.
Article
To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [μ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the μ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. The μ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had μ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the μ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the μ(kinetic) to a value near that of normal equine SF. In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high μ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.
Article
Reasons for performing study: Two studies report variability in proliferation and limited adipocyte differentiation of equine peripheral blood-derived adult mesenchymal stem cells, thus casting doubt on their adipogenic potential. Peripheral blood can be a valuable source of adult mesenchymal stem cells if cell culture conditions permissive for their adherence, proliferation and differentiation are defined. Hyperbaric oxygen treatment has been reported to mobilise haematopoietic progenitor stem cells into the peripheral blood in humans and mice, but similar experiments have not been done in horses. Objectives: To optimise cell culture conditions for isolation, propagation and differentiation of adult stem cells from peripheral blood and to assess the effect of hyperbaric oxygen treatment on adult stem cell concentrations. Methods: Peripheral blood was collected from the jugular vein of 6 research mares, and mononuclear cells were isolated. They were subjected to cell culture conditions that promote the adherence and proliferation of adult stem cells. The cells were characterised by their adherence, expression of cellular antigen markers, and trans-differentiation. Each horse was subjected to 3 hyperbaric oxygen treatments, and stem cells were compared before and after treatments. Stem cells derived from adipose tissue were used as controls. Results: One-third of the horses yielded viable stem cells from peripheral blood, positive for CD51, CD90 and CD105, and demonstrated osteocyte, chondrocyte and adipocyte differentiation. Hyperbaric oxygen treatment resulted in a significant increase in CD90-positive cells. Horses that did not yield any cells pretreatment did so only after 3 hyperbaric oxygen treatments. Conclusions and potential relevance: Peripheral blood can be a valuable source of adult stem cells, if one can identify reliable equine-specific markers, provide methods to increase the number of circulating progenitor cells and optimise cell culture conditions for growth and viability. Our findings are important for further studies towards technological advances in basic and clinical equine regenerative medicine.
Article
The recent emergence of autologous blood concentrates, such as platelet-rich plasma, as a treatment option for patients with orthopaedic injuries has led to an extensive debate about their clinical benefit. We conducted a systematic review and meta-analysis to determine the efficacy of autologous blood concentrates in decreasing pain and improving healing and function in patients with orthopaedic bone and soft-tissue injuries. We searched MEDLINE and Embase for randomized controlled trials or prospective cohort studies that compared autologous blood concentrates with a control therapy in patients with an orthopaedic injury. We identified additional studies by searching through the bibliographies of eligible studies as well as the archives of orthopaedic conferences and meetings. Twenty-three randomized trials and ten prospective cohort studies were identified. There was a lack of consistency in outcome measures across all studies. In six randomized controlled trials (n = 358) and three prospective cohort studies (n = 88), the authors reported visual analog scale (VAS) scores when comparing platelet-rich plasma with a control therapy across injuries to the acromion, rotator cuff, lateral humeral epicondyle, anterior cruciate ligament, patella, tibia, and spine. The use of platelet-rich plasma provided no significant benefit up to (and including) twenty-four months across the randomized trials (standardized mean difference, -0.34; 95% confidence interval [CI], -0.75 to 0.06) or the prospective cohort studies (standardized mean difference, -0.20; 95% CI, -0.64 to 0.23). Both point estimates suggested a small trend favoring platelet-rich plasma, but the associated wide confidence intervals were consistent with nonsignificant effects. The current literature is complicated by a lack of standardization of study protocols, platelet-separation techniques, and outcome measures. As a result, there is uncertainty about the evidence to support the increasing clinical use of platelet-rich plasma and autologous blood concentrates as a treatment modality for orthopaedic bone and soft-tissue injuries.
Article
The transcription factor Sox9 is necessary for early chondrogenesis, but its subsequent roles in the cartilage growth plate, a highly specialized structure that drives skeletal growth and endochondral ossification, remain unclear. Using a doxycycline-inducible Cre transgene and Sox9 conditional null alleles in the mouse, we show that Sox9 is required to maintain chondrocyte columnar proliferation and generate cell hypertrophy, two key features of functional growth plates. Sox9 keeps Runx2 expression and β-catenin signaling in check and thereby inhibits not only progression from proliferation to prehypertrophy, but also subsequent acquisition of an osteoblastic phenotype. Sox9 protein outlives Sox9 RNA in upper hypertrophic chondrocytes, where it contributes with Mef2c to directly activate the major marker of these cells, Col10a1. These findings thus reveal that Sox9 remains a central determinant of the lineage fate and multistep differentiation program of growth plate chondrocytes and thereby illuminate our understanding of key molecular mechanisms underlying skeletogenesis.
Article
There is currently great interest in the use of mesenchymal stem/stromal cells (MSCs) for the therapy of many diseases of animals and humans. However, we are still left with the serious challenges in explaining the beneficial effects of the cells. Hence, it is essential to work backward from dramatic results obtained in vivo to the cellular and molecular explanations in order to discover the secrets of MSCs. This review will focus on recent data that have changed the paradigms for understanding the therapeutic potentials of MSCs.
Article
Purpose: This study evaluated intra-articular injection of bone marrow-derived mesenchymal stem cells (BMSCs) to augment healing with microfracture compared with microfracture alone. Methods: Ten horses (aged 2.5 to 5 years) had 1-cm2 defects arthroscopically created on both medial femoral condyles of the stifle joint (analogous to the human knee). Defects were debrided to subchondral bone followed by microfracture. One month later, 1 randomly selected medial femorotibial joint in each horse received an intra-articular injection of either 20 × 10(6) BMSCs with 22 mg of hyaluronan or 22 mg of hyaluronan alone. Horses were confined for 4 months, with hand walking commencing at 2 weeks and then increasing in duration and intensity. At 4 months, horses were subjected to strenuous treadmill exercise simulating race training until completion of the study at 12 months. Horses underwent musculoskeletal and radiographic examinations bimonthly and second-look arthroscopy at 6 months. Horses were euthanized 12 months after the defects were made, and the affected joints underwent magnetic resonance imaging and gross, histologic, histomorphometric, immunohistochemical, and biochemical examinations. Results: Although there was no evidence of any clinically significant improvement in the joints injected with BMSCs, arthroscopic and gross evaluation confirmed a significant increase in repair tissue firmness and a trend for better overall repair tissue quality (cumulative score of all arthroscopic and gross grading criteria) in BMSC-treated joints. Immunohistochemical analysis showed significantly greater levels of aggrecan in repair tissue treated with BMSC injection. There were no other significant treatment effects. Conclusions: Although there was no significant difference clinically or histologically in the 2 groups, this study confirms that intra-articular BMSCs enhance cartilage repair quality with increased aggrecan content and tissue firmness. Clinical relevance: Clinical use of BMSCs in conjunction with microfracture of cartilage defects may be potentially beneficial.
Article
Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.
Article
Autologous chondrocyte implantation (ACI) has been used clinically for over 15 years and yet definitive evidence of chondrocyte persistence and direct impact on cartilage repair in full-thickness lesions is scant and no data are available on ACI in partial-thickness defects in any animal model. This study assessed the effect of chondrocytes secured using periosteal overlay in partial- and full-thickness cartilage defects in the equine model. Paired cartilage defects 15 mm in diameter were made in the patellofemoral joint of 16 horse and repaired with ACI or periosteal flap alone. Response was assessed at 8 weeks by clinical, microradiographic, and histologic appearance, and by collagen type II immunohistochemistry, and proteoglycan and DNA quantification. ACI improved histologic scores in partial- and full-thickness cartilage defects, including defect filling, attachment to the underlying subchondral bone, and presence of residual chondrocyte accumulations. For partial-thickness defects chondrocyte predominance, collagen type II content, and toluidine stained matrix were enhanced, and attachment to the surrounding cartilage improved. DNA and PG content of grafted partial-thickness defects was improved by chondrocyte implantation. Periosteal patches alone did not induce cartilage repair. This study indicated implantation of chondrocytes to cartilage defects improved healing with a combination of persisting chondrocyte regions, enhanced collagen type II formation, and better overall cartilage healing scores. Use of ACI in the more challenging partial-thickness defects also improved histologic indices and biochemical content. The equine model of cartilage healing closely resembles cartilage repair in man, and results of this study confirm cell persistence and improved early cartilage healing events after ACI.
Article
The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.