Intra-articular bone marrow mononuclear cell therapy improves lameness from naturally occurring equine osteoarthritis.

Osteoarthritis (OA) can be debilitating and is related to impaired resolution of synovial inflammation. Current treatments offer temporary relief of clinical signs, but have potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that have the ability to reduce OA symptoms in people and inflammation in experimentally-induced synovitis in horses. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatment groups: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Lameness was evaluated subjectively and objectively, joint circumference measured, and synovial fluid collected for cytology and growth factor/cytokine quantification at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at p < 0.05. There were no adverse effects noted in any treatment group. There was a significant increase in synovial fluid total nucleated cell count in the BMNC-treated group on day 7 (median 440; range 20-1920 cells/uL) compared to day 0. Mononuclear cells were the predominant cell type across treatments at all time points. Joint circumference decreased significantly in the BMNC-treated group from days 7 to 21 and was significantly lower at day 21 in the BMNC-treated group compared to the saline-treated group. Median objective lameness improved significantly in the BMNC group between days 7 and 21. GM-CSF, IL-1ra, IGF-1, and TNF-α were below detectable limits and IL-6, IL-1ß, FGF-2 were detectable in a limited number of synovial fluid samples. Inconsistent and limited differences were detected over time and between treatment groups for synovial fluid PGE2, SDF-1, MCP-1 and IL-10. Decreased lameness and joint circumference, coupled with a lack of adverse effects following BMNC treatment, support a larger clinical trial using BMNC therapy to treat OA in horses.

Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis.

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1ß staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair.

Osteoarthritis (OA) is the most prevalent joint disease causing major disability and medical expenditures. Synovitis is a central feature of OA and is primarily driven by macrophages. Synovial macrophages not only drive inflammation but also its resolution, through a coordinated, simultaneous expression of pro- and anti-inflammatory mechanisms that are essential to counteract damage and recover homeostasis. Current OA therapies are largely based on anti-inflammatory principles and therefore block pro-inflammatory mechanisms such as prostaglandin E2 and Nuclear factor-kappa B signaling pathways. However, such mechanisms are also innately required for mounting a pro-resolving response, and their blockage often results in chronic low-grade inflammation. Following minor injury, macrophages shield the damaged area and drive tissue repair. If the damage is more extensive, macrophages incite inflammation recruiting more macrophages from the bone marrow to maximize tissue repair and ultimately resolve inflammation. However, sustained damage and inflammation often overwhelms pro-resolving mechanisms of synovial macrophages leading to the chronic inflammation and related tissue degeneration observed in OA. Recently, experimental and clinical studies have shown that joint injection with autologous bone marrow mononuclear cells replenishes inflamed joints with macrophage and hematopoietic progenitors, enhancing mechanisms of inflammation resolution, providing remarkable and long-lasting effects. Besides creating an ideal environment for resolution with high concentrations of interleukin-10 and anabolic growth factors, macrophage progenitors also have a direct role in tissue repair. Macrophages constitute a large part of the early granulation tissue, and further transdifferentiate from myeloid into a mesenchymal phenotype. These cells, characterized as fibrocytes, are essential for repairing osteochondral defects. Ongoing "omics" studies focused on identifying key drivers of macrophage-mediated resolution of joint inflammation and those required for efficient osteochondral repair, have the potential to uncover ways for developing engineered macrophages or off-the-shelf pro-resolving therapies that can benefit patients suffering from many types of arthropaties, not only OA.

A novel murine muscle loading model to investigate Achilles musculotendinous adaptation.

Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Here, we introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This model includes the application of tissue (muscle and tendon)-loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human clinical protocols. Under computer control, the foot was rotated through the entire ankle joint range of motion while the plantar flexors simultaneously contracted to simulate body mass loading, consistent with human therapeutic exercises. This approach achieved two key components of the heel drop and raise movement: ankle range of motion coupled with body mass loading. Model development entailed the tuning of parameters such as footplate speed, number of repetitions, number of sets of repetitions, treatment frequency, treatment duration, and treatment timing. Initial model development was carried out on uninjured mice to define a protocol that was well tolerated and nondeleterious to tendon biomechanical function. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared with untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.NEW & NOTEWORTHY We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

Fibrous osteodystrophy in a dromedary camel.

A 6-y-old female dromedary camel (Camelus dromedarius L.) was presented for assessment of firm, bilateral swellings rostral and ventral to the eyes. Serum biochemistry revealed hyperglycemia (28.5 mmol/L), hypocalcemia (1.27 mmol/L), hyperphosphatemia (3.39 mmol/L), hypoproteinemia (total protein 50 g/L), and hypoalbuminemia (20 g/L). Based on the poor prognosis associated with the presumptive diagnosis of fibrous osteodystrophy, the camel was euthanized. Gross postmortem findings revealed expanded fibrous tissue replacing the maxilla and mandible, and bilaterally prominent parathyroid glands. Histology of the maxilla revealed proliferative loose fibrous tissue with widely scattered, regularly spaced, small spicules of mineralized bone. The parathyroid glands were prominent bilaterally; the internal and external parathyroid glands were composed of plump cells with abundant pale basophilic cytoplasm and open nuclei. The pathologic findings were consistent with the antemortem diagnosis of fibrous osteodystrophy. The camel's diet, which was not specifically balanced for a camel, included grass hay, sweet feed, and alfalfa pellets. The camel's caregivers reported feeding her treats of cookies. A feed analysis was not available. The biochemistry abnormalities and clinical and postmortem findings, along with a diet that was not balanced for a camel, are consistent with a diagnosis of nutritional secondary hyperparathyroidism.

Macrophage Activation in the Synovium of Healthy and Osteoarthritic Equine Joints.

Synovitis is a major component of osteoarthritis and is driven primarily by macrophages. Synovial macrophages are crucial for joint homeostasis (M2-like phenotype), but induce inflammation (M1-like) when regulatory functions become overwhelmed. Macrophage phenotypes in synovium from osteoarthritic and healthy joints are poorly characterized; however, comparative knowledge of their phenotypes during health and disease is paramount for developing targeted treatments. This study compared patterns of macrophage activation in healthy and osteoarthritic equine synovium and correlated histology with cytokine/chemokine profiles in synovial fluid. Synovial histology and immunohistochemistry for M1-like (CD86), M2-like (CD206, IL-10), and pan macrophage (CD14) markers were performed on biopsies from 29 healthy and 26 osteoarthritic equine joints. Synovial fluid cytokines (MCP-1, IL-10, PGE2, IL-1ß, IL-6, TNF-α, IL-1ra) and growth factors (GM-CSF, SDF-1α+ß, IGF-1, and FGF-2) were quantified. Macrophage phenotypes were not as clearly defined in vivo as they are in vitro. All macrophage markers were expressed with minimal differences between OA and normal joints. Expression for all markers increased proportionate to synovial inflammation, especially CD86. Synovial fluid MCP-1 was higher in osteoarthritic joints while SDF-1 and IL-10 were lower, and PGE2 concentrations did not differ between groups. Increased CD14/CD86/CD206/IL-10 expression was associated with synovial hyperplasia, consistent with macrophage recruitment and activation in response to injury. Lower synovial fluid IL-10 could suggest that homeostatic mechanisms from synovial macrophages became overwhelmed preventing inflammation resolution, resulting in chronic inflammation and OA. Further investigations into mechanisms of arthritis resolution are warranted. Developing pro-resolving therapies may provide superior results in the treatment of OA.

Inflamed synovial fluid induces a homeostatic response in bone marrow mononuclear cells in vitro: Implications for joint therapy.

Synovial inflammation is a central feature of osteoarthritis (OA), elicited when local regulatory macrophages (M2-like) become overwhelmed, activating an inflammatory response (M1-like). Bone marrow mononuclear cells (BMNC) are a source of naïve macrophages capable of reducing joint inflammation and producing molecules essential for cartilage metabolism. This study investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). Equine BMNC cultured in autologous SF or ISF (n = 8 horses) developed into macrophage-rich cultures with phenotypes similar to cells native to normal SF and became more confluent in ISF (~100%) than SF (~25%). BMNC cultured in SF or ISF were neither M1- nor M2-like, but exhibited aspects of both phenotypes and a regulatory immune response, characterized by increasing counts of IL-10+ macrophages, decreasing IL-1ß concentrations and progressively increasing IL-10 and IGF-1 concentrations. Changes were more marked in ISF and suggest that homeostatic mechanisms were preserved over time and were potentially favored by progressive cell proliferation. Collectively, our data suggest that intra-articular BMNC could increase synovial macrophage counts, potentiating the macrophage- and IL-10-associated mechanisms of joint homeostasis lost during the progression of OA, preserving the production of cytokines involved in tissue repair (PGE2 , IL-10) generally impaired by frequently used corticosteroids.

Autologous bone marrow mononuclear cells modulate joint homeostasis in an equine in vivo model of synovitis.

Osteoarthritis (OA) is characterized by macrophage-driven synovitis. Macrophages promote synovial health but become inflammatory when their regulatory functions are overwhelmed. Bone marrow mononuclear cells (BMNCs) are a rich source of macrophage progenitors used for treating chronic inflammation and produce essential molecules for cartilage metabolism. This study investigated the response to autologous BMNC injection in normal and inflamed joints. Synovitis was induced in both radiocarpal joints of 6 horses. After 8 h, 1 inflamed radiocarpal and 1 normal tarsocrural joint received BMNC injection. Contralateral joints were injected with saline. Synovial fluid was collected at 24, 96, and 144 h for cytology, cytokine quantification, and flow cytometry. At 144 h, horses were euthanatized, joints were evaluated, and synovium was harvested for histology and immunohistochemistry. Four days after BMNC treatment, inflamed joints had 24% higher macrophage counts with 10% more IL-10+ cells than saline-treated controls. BMNC-treated joints showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing regulatory macrophages and synovial fluid IL-10 concentrations compared with saline-treated controls. BMNC-treated joints were comparable to healthy joints histologically, which remained abnormal in saline-treated controls. Autologous BMNCs are readily available, regulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA.-Menarim, B. C., Gillis, K. H., Oliver, A., Mason, C., Ngo, Y., Werre, S. R., Barrett, S. H., Luo, X., Byron, C. R., Dahlgren, L. A. Autologous bone marrow mononuclear cells modulate joint homeostasis in an equine in vivo model of synovitis.

Urinary bladder matrix does not improve tenogenesis in an in vitro equine model.

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, MMP-13, and Scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. COMP expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, addition of UBM failed to induce greater matrix organization than tendon ECM alone. This article is protected by copyright. All rights reserved.

Urinary Bladder Matrix Does Not Improve Tenogenesis in an In Vitro Equine Model.

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1848-1859, 2019.

Adipose-Derived Stromal Vascular Fraction and Cultured Stromal Cells as Trophic Mediators for Tendon Healing.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-ß1 (TGF-ß1) and TGF-ß3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019.

Regenerative Medicine Therapies for Equine Wound Management.

Wound management in horses can strike fear in some and passion in others. Wounds are common injuries in horses of all descriptions and requires exceptional knowledge and care to achieve a successful outcome. New treatments to overcome the critical challenges with equine wounds are always desired: managing dehisced and/or nonhealing wounds, managing exuberant granulation tissue, and ultimately achieving a functional tissue coverage. Regenerative medicine represents a broad set of tools with great promise to manipulate the deficiencies recognized in equine wound healing and improve the outcome.

In vitro effects of three equimolar concentrations of methylprednisolone acetate, triamcinolone acetonide, and isoflupredone acetate on equine articular tissue cocultures in an inflammatory environment.

OBJECTIVE To compare the effects of 3 equimolar concentrations of methylprednisolone acetate (MPA), triamcinolone acetonide (TA), and isoflupredone acetate (IPA) on equine articular tissue cocultures in an inflammatory environment. SAMPLE Synovial and osteochondral explants from the femoropatellar joints of 6 equine cadavers (age, 2 to 11 years) without evidence of musculoskeletal disease. PROCEDURES From each cadaver, synovial and osteochondral explants were harvested from 1 femoropatellar joint to create cocultures. Cocultures were incubated for 96 hours with (positive control) or without (negative control) interleukin (IL)-1ß (10 ng/mL) or with IL-1ß and MPA, TA, or IPA at a concentration of 10-4, 10-7, or 10-10M. Culture medium samples were collected from each coculture after 48 and 96 hours of incubation. Concentrations of prostaglandin E2, matrix metalloproteinase-13, lactate dehydrogenase, and glycosaminoglycan were determined and compared among treatments at each time. RESULTS In general, low concentrations (10-7 and 10-10M) of MPA, TA, and IPA mitigated the inflammatory and catabolic (as determined by prostaglandin E2 and matrix metalloproteinase-13 quantification, respectively) effects of IL-1ß in cocultures to a greater extent than the high (10-4M) concentration. Mean culture medium lactate dehydrogenase concentration for the 10-4M IPA treatment was significantly greater than that for the positive control at both times, which was suggestive of cytotoxicosis. Mean culture medium glycosaminoglycan concentration did not differ significantly. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that the in vitro effects of IPA and MPA were similar to those of TA at clinically relevant concentrations (10-7 and 10-10M).

Novel roles for scleraxis in regulating adult tenocyte function.

BACKGROUND: Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse. RESULTS: Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis. CONCLUSIONS: Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.

Transient Scleraxis Overexpression Combined with Cyclic Strain Enhances Ligament Cell Differentiation.

Efforts to generate tissue-engineered anterior cruciate ligament replacements are limited by a lack of methods to derive mature ligament cells. Viral overexpression of the tendon/ligament marker scleraxis (Scx) can drive cell differentiation; however, the use of viral vectors hampers translation to clinical use. In this study, C3H10T1/2 cells were transiently transfected with expression vectors containing the full-length murine Scx cDNA and cultured in three-dimensional collagen hydrogels under static or cyclic strain for up to 14 days. ß-galactosidase (LacZ) transfected cells served as controls. Cell morphology and gene expression for ligament-related genes, in addition to contraction (hydrogel width), mechanical properties, and glycosaminoglycan (GAG) and DNA content of hydrogels, were quantified and compared over time, between Scx and LacZ groups, and between static and cyclically strained constructs. Increased Scx expression was maintained for the entire 14-day study in both static and cyclically strained constructs. In static culture, overexpression of Scx resulted in greater cell elongation and construct contraction compared to LacZ controls. There were no differences in gene expression, DNA, or GAG content between Scx and LacZ constructs cultured under static conditions and no differences in DNA content between Scx and LacZ constructs. When exposed to cyclic strain, Scx-overexpressing cells maintained the elongated phenotype exhibited in static constructs, increased GAG production compared to static culture, and increased expression of the ligament-related genes collagen type I, decorin, and tenascin-C compared to strained LacZ controls. Cyclically strained constructs containing Scx-overexpressing cells had increased maximum load and stiffness compared to LacZ controls. The maintenance of increased Scx expression throughout the 14 day study and subsequent increases in ligament marker gene expression and mechanical properties with cyclic, but not static strain, suggest that transient transfection may be a viable alternative to viral transduction of Scx for ligament engineering studies and support a synergistic effect of Scx and mechanical strain on driving early ligament cell differentiation.

Effect of a single intra-articular injection of bupivacaine on synovial fluid prostaglandin E2 concentrations in normal canine stifles.

OBJECTIVE: To identify if synovial fluid prostaglandin E2 increases in response to a single intra-articular dose of bupivacaine in the normal canine stifle. RESULTS: There were no significant differences in synovial fluid prostaglandin E2 (PGE2) concentrations between treatment groups or over time within bupivacaine or saline groups. Samples requiring ≥ 3 arthrocentesis attempts had significantly higher PGE2 concentrations compared to samples requiring 1 or 2 attempts. Following correction for number of arthrocentesis attempts, PGE2 concentrations were significantly higher than baseline at 24 and 48 h in the bupivacaine group; however there were no significant differences between the bupivacaine and saline groups. In normal dogs, a single bupivacaine injection did not cause significant synovial inflammation, as measured by PGE2 concentrations, compared to saline controls. Future research should minimize aspiration attempts and include evaluation of the synovial response to bupivacaine in clinical cases with joint disease.

Serum and Synovial Fluid Serum Amyloid A Response in Equine Models of Synovitis and Septic Arthritis.

OBJECTIVE: To investigate the serum and synovial fluid serum amyloid A (SAA) response in equine models of synovitis and septic arthritis and to compare handheld and validated immunoturbidometric assays for SAA quantification. STUDY DESIGN: Controlled, experimental study. ANIMALS: Healthy adult horses (n = 9). METHODS: Synovitis (n = 4) and septic arthritis (n = 5) were induced using lipopolysaccharide and Staphylococcus aureus, respectively, and serial serum and synovial fluid samples were collected. Serial synovial fluid cytology was performed for both models and synovial fluid from the septic arthritis model was submitted for bacterial culture. Serum and synovial fluid SAA were quantified by handheld test and immunoturbidometric assay. Cytologic and SAA data were compared within and between models (mixed model ANOVA) and results of SAA assays were compared using category-by-category analysis (weighted kappa coefficient). RESULTS: Synovial fluid total nucleated cell counts and total protein increased significantly following induction of both models. Serum and synovial fluid SAA remained normal in synovitis horses and increased significantly in septic arthritis horses. Serum SAA increased more rapidly than synovial fluid SAA. Agreement was 98% when SAA concentrations were low (<50 µg/mL) but the assays diverged when concentrations were greater than ∼100 µg/mL. Overall, there was good category-by-category agreement between SAA assays (weighted kappa = 0.824). CONCLUSION: Serum and synovial fluid SAA may be useful adjuncts in diagnosing septic arthritis in horses. SAA concentrations for the assays diverged and examination using a larger sample size is needed before direct numeric comparisons between the assays can be made.

Evaluation of a New Surgical Treatment for Equine Hind Limb Proximal Suspensory Desmitis.

OBJECTIVE: To evaluate the effects of a new microfracture and ligament splitting procedure on ligament healing and to examine the usefulness of magnetic resonance (MR) imaging for monitoring ligament healing over time using a collagenase model of hind limb proximal suspensory desmitis. STUDY DESIGN: Experimental in vivo study. ANIMALS: Healthy adult horses (n=6). METHODS: Horses were free of lameness with normal hind limb proximal suspensory ligaments (PSL). The origin of both hind limb PSL was injected with collagenase and underwent MR imaging 2 weeks later, followed by the microfracture and ligament splitting procedure on 1 limb, with the opposite limb serving as the sham-operated control. Serial lameness and MR examinations were performed. Horses were euthanatized 210 days after surgery, the PSL harvested, and histology, biochemistry, and gene expression performed on both PSL. RESULTS: Collagenase lesions viewed on MR images appeared similar to those seen clinically. Serial MR images demonstrated resolution of abnormal signal intensity and tissue formation in the microfracture sites within the third metatarsal bone. Treated limbs had histologic evidence of connective tissue appearing to originate from the small perforations and blending into the ligament but no statistical differences were identified. Gene expression for cartilage oligomeric matrix protein and decorin were significantly increased in treated compared to control limbs. CONCLUSION: The microfracture and ligament splitting procedure did incite a tissue response but further clinical investigation is necessary to determine if this tissue remodeling at the bone-ligament interface translates to improved clinical outcome. MR imaging may be useful to follow healing in horses with hind limb proximal suspensory desmitis.

Cytokine and Growth Factor Concentrations in Canine Autologous Conditioned Serum.

OBJECTIVE: To compare cytokine and growth factor concentrations in canine autologous conditioned serum (ACS) to canine plasma. STUDY DESIGN: Experimental in vivo study. ANIMALS: Client-owned, adult dogs (n=22). METHODS: Blood collected from 16 medium to large breed dogs was used to produce ACS (Orthokine(®) vet irap 10 syringes) and citrated plasma (control). Canine-specific ELISA assays were run per manufacturers' instructions for interleukin (IL)-10, IL-4, tumor necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-ß1, IL-1ß, and interleukin-1 receptor antagonist (IL-1ra). Serum, in addition to plasma and ACS, was collected from an additional 6 dogs for TNF-α, IL-1ß, and IL-1ra analysis (total of 22 dogs). Data were analyzed for differences in each cytokine concentration using pairwise comparisons between ACS, plasma, and serum using Wilcoxon signed-rank tests. Significance was set at P<.05. RESULTS: There was a large variability in growth factor and cytokine concentrations in ACS and plasma for individual dogs. There were no significant differences in IL-10, TNF-α, IGF-1, FGF-2, and TGF-ß1 concentrations between ACS, plasma, and serum. The IL-1ß concentrations in ACS (median, range 46.3 pg/mL, 0-828.8) and IL-4 (0.0 pg/mL, 0-244.1) were significantly higher than plasma (36.6 pg/mL, 0-657.1 and 0.0 pg/mL, 0-0, respectively). The IL-1ra concentration in ACS (median, range 3,458.9 pg/mL, 1,243.1-12,089.0) was significantly higher than plasma (692.3 pg/mL, 422.5-1,475.6). The IL-1ra:IL-1ß ratio in ACS was significantly higher than plasma (39.9 vs. 7.2). CONCLUSION: IL-1ra concentrations in canine ACS were comparable to those published for people and horses and pro-inflammatory cytokines remained low in canine ACS.

Fiber/collagen composites for ligament tissue engineering: influence of elastic moduli of sparse aligned fibers on mesenchymal stem cells.

Electrospun microfibers are attractive for the engineering of oriented tissues because they present instructive topographic and mechanical cues to cells. However, high-density microfiber networks are too cell-impermeable for most tissue applications. Alternatively, the distribution of sparse microfibers within a three-dimensional hydrogel could present instructive cues to guide cell organization while not inhibiting cell behavior. In this study, thin (∼5 fibers thick) layers of aligned microfibers (0.7 µm) were embedded within collagen hydrogels containing mesenchymal stem cells (MSCs), cultured for up to 14 days, and assayed for expression of ligament markers and imaged for cell organization. These microfibers were generated through the electrospinning of polycaprolactone (PCL), poly(ester-urethane) (PEUR), or a 75/25 PEUR/PCL blend to produce microfiber networks with elastic moduli of 31, 15, and 5.6 MPa, respectively. MSCs in composites containing 5.6 MPa fibers exhibited increased expression of the ligament marker scleraxis and the contractile phenotype marker α-smooth muscle actin versus the stiffer fiber composites. Additionally, cells within the 5.6 MPa microfiber composites were more oriented compared to cells within the 15 and 31 MPa microfiber composites. Together, these data indicate that the mechanical properties of microfiber/collagen composites can be tuned for the engineering of ligament and other target tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1894-1901, 2016.