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The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.
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Artrite Reumatoide , Vesículas Extracelulares , Lipidômica , Neutrófilos , Líquido Sinovial , Líquido Sinovial/metabolismo , Humanos , Animais , Vesículas Extracelulares/metabolismo , Cavalos , Neutrófilos/metabolismo , Neutrófilos/patologia , Lipidômica/métodos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Masculino , Inflamação/metabolismo , Inflamação/patologia , Feminino , Lactosilceramidas/metabolismo , Glucosilceramidas/metabolismo , Espondilartrite/metabolismo , Espondilartrite/patologiaRESUMO
In order to improve the ability of clinical diagnosis to differentiate articular cartilage (AC) injury of different origins, this study explores the sensitivity of mid-infrared (MIR) spectroscopy for detecting structural, compositional, and functional changes in AC resulting from two injury types. Three grooves (two in parallel in the palmar-dorsal direction and one in the mediolateral direction) were made via arthrotomy in the AC of the radial facet of the third carpal bone (middle carpal joint) and of the intermediate carpal bone (the radiocarpal joint) of nine healthy adult female Shetland ponies (age = 6.8 ± 2.6 years; range 4-13 years) using blunt and sharp tools. The defects were randomly assigned to each of the two joints. Ponies underwent a 3-week box rest followed by 8 weeks of treadmill training and 26 weeks of free pasture exercise before being euthanized for osteochondral sample collection. The osteochondral samples underwent biomechanical indentation testing, followed by MIR spectroscopic assessment. Digital densitometry was conducted afterward to estimate the tissue's proteoglycan (PG) content. Subsequently, machine learning models were developed to classify the samples to estimate their biomechanical properties and PG content based on the MIR spectra according to injury type. Results show that MIR is able to discriminate healthy from injured AC (91%) and between injury types (88%). The method can also estimate AC properties with relatively low error (thickness = 12.7% mm, equilibrium modulus = 10.7% MPa, instantaneous modulus = 11.8% MPa). These findings demonstrate the potential of MIR spectroscopy as a tool for assessment of AC integrity changes that result from injury.
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Cartilagem Articular , Espectrofotometria Infravermelho , Feminino , Cartilagem Articular/lesões , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/metabolismo , Animais , Cavalos , Espectrofotometria Infravermelho/métodos , Aprendizado de Máquina , Proteoglicanas/metabolismoRESUMO
Osteoarthritis causes progressive joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Currently, osteoarthritis is diagnosed at late stages through clinical examination and radiographic imaging, hence it is challenging to address and provide timely therapeutic interventions to slow disease progression or ameliorate symptoms. Extracellular vesicles are cell-derived vesicles that play a key role in cell-to-cell communication and are potential sources for specific composite biomarker panel discovery. We here used a multi-omics strategy combining proteomics and phospholipidomics in an integral approach to identify composite biomarkers associated to purified extracellular vesicles from synovial fluid of healthy, mildly and severely osteoarthritic equine joints. Although the number of extracellular vesicles was unaffected by osteoarthritis, proteome profiling of extracellular vesicles by mass spectrometry identified 40 differentially expressed proteins (non-adjusted p < 0.05) in osteoarthritic joints associated with 7 significant canonical pathways in osteoarthritis. Moreover, pathway analysis unveiled changes in disease and molecular functions during osteoarthritis development. Phospholipidome profiling by mass spectrometry showed a relative increase in sphingomyelin and a decrease in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine in extracellular vesicles derived from osteoarthritic joints compared to healthy joints. Unsupervised data integration revealed positive correlations between the proteome and the phospholipidome. Comprehensive analysis showed that some phospholipids and their related proteins increased as the severity of osteoarthritis progressed, while others decreased or remained stable. Altogether our data show interrelationships between synovial fluid extracellular vesicle-associated phospholipids and proteins responding to osteoarthritis pathology and which could be explored as potential composite diagnostic biomarkers of disease.
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BACKGROUND: The modified Meek technique is not commonly used in equine wound management, despite the consistent reliable and superior results compared with other grafting techniques. Major drawbacks are the need for specialised, expensive equipment and general anaesthesia. OBJECTIVES: To describe adjustments of the modified Meek technique enabling use in the standing horse without the need for the full equipment. This implied the use of full-thickness skin grafts manually harvested from the pectoral area and manually cut into micrografts. Graft acceptance; healing progress; and final functional and cosmetic result were outcome parameters. STUDY DESIGN: Descriptive case series. METHODS: Eight horses with traumatic wounds at the dorsal side of the carpus or tarsus, healing by second intention, were treated. Original wound areas and areas of graft acceptance and rejection were determined from post-processing of digital photographs and percentage acceptance, wound contraction and epithelialisation were calculated. RESULTS: The initial mean wound area was 55.4 cm2 . Graft acceptance was 95.3 ± 2.5%. Wound closure was due to 46.0 ± 25.6% wound contraction and 54.0 ± 25.6% epithelialisation and resulted in 96.8 ± 1.9% reduction of the initial wound area 28.0 ± 8.5 days after grafting. The scar was flat, flexible and functional, usually with thin and regular hair growth. The adapted procedure was fast and efficient, with a learning curve for the increased manual work. MAIN LIMITATIONS: Small study population. CONCLUSIONS: This adapted modified Meek technique can successfully be performed in the standing horse and obviates the need for the full expensive equipment and general anaesthesia. The acceptance of the full-thickness grafts is excellent resulting in fast and satisfactory healing.
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In articular cartilage (AC), the collagen arcades provide the tissue with its extraordinary mechanical properties. As these structures cannot be restored once damaged, functional restoration of AC defects remains a major challenge. We report that the use of a converged bioprinted, osteochondral implant, based on a gelatin methacryloyl cartilage phase, reinforced with precisely patterned melt electrowritten polycaprolactone micrometer-scale fibers in a zonal fashion, inspired by native collagen architecture, can provide long-term mechanically stable neo-tissue in an orthotopic large animal model. The design of this novel implant was achieved via state-of-the-art converging of extrusion-based ceramic printing, melt electrowriting, and extrusion-based bioprinting. Interestingly, the cell-free implants, used as a control in this study, showed abundant cell ingrowth and similar favorable results as the cell-containing implants. Our findings underscore the hypothesis that mechanical stability is more determining for the successful survival of the implant than the presence of cells and pre-cultured extracellular matrix. This observation is of great translational importance and highlights the aptness of advanced 3D (bio)fabrication technologies for functional tissue restoration in the harsh articular joint mechanical environment.
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BACKGROUND: Data on equine lumbar pathology hardly exist in breeds other than Thoroughbreds. OBJECTIVES: To describe pathological changes of the osseous lumbar vertebral column in Warmblood horses, Shetland ponies and Konik horses. STUDY DESIGN: Descriptive post-mortem study. METHODS: The lumbar vertebral columns of 34 Warmblood horses, 28 Shetland ponies, and 18 Konik horses were examined by computed tomography (CT). Osteoarthritis (OA) of articular processes (APJs), OA of intertransverse joints (ITJs), intervertebral disc (IVD) mineralisation, impingement of spinous (SPs) and transverse (TPs) processes and spondylosis were scored. Breed differences in prevalence and severity of pathologies were analysed by linear regression analysis and by calculating Spearman's rank correlation coefficients (rs). RESULTS: In Warmblood horses, the prevalence of OA of APJs, impingement of SPs and TPs was respectively 90%, 36%, and 35%, significantly higher than in the other breeds (p < 0.001). In Konik horses, IVD mineralisation (40%) and spondylosis (10%) were more frequent than in Warmbloods and Shetland ponies (p = 0.03). Severity score for OA of ITJs was highest in Shetland ponies (p < 0.001). For impingement of SPs, severity score was highest in Warmbloods (p = 0.03), and of TPs lowest in Shetland ponies (p = 0.003). For all parameters, except for spondylosis in Shetland ponies, there was a positive correlation between percentage of vertebrae affected and age, with IVD mineralisation scores increasing faster in Konik horses (p < 0.001). In all breeds, there was also a positive relation between scores of severity and age for OA of APJs and ITJs and for IVD mineralisation, with severity scores increasing faster in Shetland ponies (p = 0.04). Strong left/right correlations of the severity scores were seen for OA of the APJ, ITJ, impingement of TPs, and paramedian spondylosis (rs = 0.74-0.86, all p < 0.001). MAIN LIMITATIONS: Clinical histories were not available. CONCLUSIONS: There are distinct breed differences between prevalence and severity of osseous pathologies of the lumbar spine. Warmblood horses have higher scores for most pathologies with IVD mineralisation being more important in Konik horses and OA of ITJs in Shetland ponies.
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Doenças dos Cavalos , Vértebras Lombares , Animais , Cavalos , Doenças dos Cavalos/patologia , Vértebras Lombares/patologia , Masculino , Feminino , Especificidade da Espécie , Doenças da Coluna Vertebral/veterinária , Doenças da Coluna Vertebral/patologiaRESUMO
During evolution, animals have returned from land to water, adapting with morphological modifications to life in an aquatic environment. We compared the osteochondral units of the humeral head of marine and terrestrial mammals across species spanning a wide range of body weights, focusing on microstructural organization and biomechanical performance. Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of the microarchitecture-function relationship is pivotal for understanding articular biology and, hence, for the development of durable functional regenerative approaches for treatment of joint damage, which are thus far lacking.
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Cartilagem Articular , Mamíferos , Animais , Matriz Extracelular , PeleRESUMO
This study investigates repeated low-dose lipopolysaccharide (LPS) injections in equine joints as a model for recurrent joint inflammation and its impact on animal welfare. Joint inflammation was induced in eight horses by injecting 0.25 ng of LPS three times at two-week intervals. Welfare scores and clinical parameters were recorded at baseline and over 168 h post-injection. Serial synoviocentesis was performed for the analysis of a panel of synovial fluid biomarkers of inflammation and cartilage turnover. Clinical parameters and a final synoviocentesis were also performed eight weeks after the last sampling point to assess the recovery of normal joint homeostasis. Statistical methods were used to compare the magnitude of response to each of the 3 LPS inductions and to compare the baseline and final measurements. Each LPS injection produced consistent clinical and biomarker responses, with minimal changes in welfare scores. General matrix metalloproteinase (MMP) activity and joint circumference showed greater response to the second LPS induction, but response to the third was comparable to the first. Gylcosaminoglycans (GAG) levels showed a significantly decreased response with each induction, while collagen-cleavage neoepitope of type II collagen (C2C) and carboxypropetide of type II collagen epitope (CPII) showed quicker responses to the second and third inductions. All parameters were comparable to baseline values at the final timepoint. In conclusion, a consistent, reliable intra-articular inflammatory response can be achieved with repeated injections of 0.25 ng LPS, with minimal impact on animal welfare, suggesting potential as a refined translational model of recurrent joint inflammation.
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Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8 h post-injection, which started to decline at 24 h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0 h), SF-EVs collected at 5, 24, and 48 h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5 h and 24 h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation.
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Líquido Sinovial , Sinovite , Animais , Cavalos , Fosfolipídeos , Lipopolissacarídeos/efeitos adversos , Sinovite/induzido quimicamente , Sinovite/veterinária , Inflamação/induzido quimicamenteRESUMO
BACKGROUND: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. OBJECTIVES: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. STUDY DESIGN: Pre-clinical experimental study. METHODS: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = -1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. RESULTS: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. MAIN LIMITATIONS: Ex vivo study protocol and a low number of investigated joints. CONCLUSIONS: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.
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Doenças das Cartilagens , Cartilagem Articular , Doenças dos Cavalos , Animais , Cavalos , Microtomografia por Raio-X/veterinária , Ácido Ioxáglico , Meios de Contraste , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Doenças das Cartilagens/diagnóstico por imagem , Doenças das Cartilagens/veterinária , Doenças dos Cavalos/diagnóstico por imagem , Doenças dos Cavalos/patologiaRESUMO
In this study, we mapped and quantified changes of proteoglycan (PG) content and biomechanical properties in articular cartilage in which either blunt or sharp grooves had been made, both close to the groove and more remote of it, and at the opposing joint surface (kissing site) in equine carpal joints. In nine adult Shetland ponies, standardized blunt and sharp grooves were surgically made in the radiocarpal and middle carpal joints of a randomly chosen front limb. The contralateral control limb was sham-operated. At 39 weeks after surgery, ponies were euthanized. In 10 regions of interest (ROIs) (six remote from the grooves and four directly around the grooves), PG content as a function of tissue-depth and distance-to-groove was estimated using digital densitometry. Biomechanical properties of the cartilage were evaluated in the six ROIs remote from the grooves. Compared to control joints, whole tissue depth PG loss was found in sites adjacent to sharp and, to a larger extent, blunt grooves. Also, superficial PG loss of the surgically untouched kissing cartilage layers was observed. Significant PG loss was observed up to 300 µm (sharp) and at 500 µm (blunt) from the groove into the surrounding tissue. Equilibrium modulus was lower in grooved cartilage than in controls. Grooves, in particular blunt grooves, gave rise to severe PG loss close to the grooved sites and to mild degeneration more remote from the grooves in both sharply and bluntly grooved cartilage and at the kissing sites, resulting in loss of mechanical strength over the 9-month period.
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Cartilagem Articular , Cavalos , Animais , ProteoglicanasRESUMO
OBJECTIVE: Folate receptor beta (FR-ß) has been used as a clinical marker and target in multiple inflammatory diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). However, the conditions under which FR-ß+ macrophages arise remain unclear and could be affected by corticosteroids. Therefore, we studied FR-ß expression in vitro in macrophage subtypes and determined their response to triamcinolone acetonide (TA), a clinically often-used corticosteroid. DESIGN: Human monocyte-derived macrophages were differentiated to the known M0, M1, or M2 macrophage phenotypes. The phenotype and FR-ß expression and plasticity of the macrophage subtypes were determined using flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: FR-ß expression was low in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated (M1-like) macrophages and high in macrophage colony-stimulating factor (M-CSF)-generated (M0 and M2-like) macrophages. FR-ß expression remained high once the M0 or M2 macrophages were stimulated with pro-inflammatory stimuli (interferon-γ plus lipopolysaccharide) to induce M1-like macrophages. On the contrary, anti-inflammatory TA treatment skewed GM-CSF macrophage differentiation toward an M2 and FR-ß+ phenotype. CONCLUSIONS: As corticosteroids skewed monocytes toward an FR-ß-expressing, anti-inflammatory phenotype, even in an M1 priming GM-CSF environment, FR-ß has potential as a biomarker to monitor success of treatment with corticosteroids. Without corticosteroid treatment, M-CSF alone induces high FR-ß expression which remains high under pro-inflammatory conditions. This explains why pro-inflammatory FR-ß+ macrophages (exposed to M-CSF) are observed in arthritis patients and correlate with disease severity.
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Corticosteroides , Receptor 2 de Folato , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Macrófagos , Biomarcadores/metabolismo , Receptor 2 de Folato/metabolismo , Ácido Fólico/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologiaRESUMO
BACKGROUND: Gait kinematics measured during equine gait analysis are typically evaluated by analysing (asymmetry-based) discrete variables (eg, peak values) obtained from continuous kinematic signals (eg, timeseries of datapoints). However, when used for the assessment of complex cases of lameness, such as bilateral lameness, discrete variable analysis might overlook relevant functional adaptations. OBJECTIVES: The overall aim of this paper is to compare continuous and discrete data analysis techniques to evaluate kinematic gait adaptations to lameness. STUDY DESIGN: Method comparison. METHODS: Sixteen healthy Shetland ponies, enrolled in a research programme in which osteochondral defects were created on the medial trochlear ridges of both femurs, were used in this study. Kinematic data were collected at trot on a treadmill before and at 3 and 6 months after surgical intervention. Statistical parametric mapping and linear mixed models were used to compare kinematic variables between and within timepoints. RESULTS: Both continuous and discrete data analyses identified changes in pelvis and forelimb kinematics. Discrete data analyses showed significant changes in hindlimb and back kinematics, where such differences were not found to be significant by continuous data analysis. In contrast, continuous data analysis provided additional information on the timing and duration of the differences found. MAIN LIMITATIONS: A limited number of ponies were included. CONCLUSIONS: The use of continuous data provides additional information regarding gait adaptations to bilateral lameness that is complementary to the analysis of discrete variables. The main advantage lies in the additional information regarding time dependence and duration of adaptations, which offers the opportunity to identify functional adaptations during all phases of the stride cycle, not just the events related to peak values.
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Doenças dos Cavalos , Coxeadura Animal , Animais , Fenômenos Biomecânicos , Análise de Dados , Membro Anterior , Marcha , Membro Posterior , Doenças dos Cavalos/diagnóstico , Cavalos , Coxeadura Animal/diagnósticoRESUMO
To prevent the progression of posttraumatic osteoarthritis, assessment of cartilage composition is critical for effective treatment planning. Posttraumatic changes include proteoglycan (PG) loss and elevated water content. Quantitative dual-energy computed tomography (QDECT) provides a means to diagnose these changes. Here, we determine the potential of QDECT to evaluate tissue quality surrounding cartilage lesions in an equine model, hypothesizing that QDECT allows detection of posttraumatic degeneration by providing quantitative information on PG and water contents based on the partitions of cationic and nonionic agents in a contrast mixture. Posttraumatic osteoarthritic samples were obtained from a cartilage repair study in which full-thickness chondral defects were created surgically in both stifles of seven Shetland ponies. Control samples were collected from three nonoperated ponies. The experimental (n = 14) and control samples (n = 6) were immersed in the contrast agent mixture and the distributions of the agents were determined at various diffusion time points. As a reference, equilibrium moduli, dynamic moduli, and PG content were measured. Significant differences (p < 0.05) in partitions between the experimental and control samples were demonstrated with cationic contrast agent at 30 min, 60 min, and 20 h, and with non-ionic agent at 60 and 120 min. Significant Spearman's rank correlations were obtained at 20 and 24 h (ρ = 0.482-0.693) between the partition of cationic contrast agent, cartilage biomechanical properties, and PG content. QDECT enables evaluation of posttraumatic changes surrounding a lesion and quantification of PG content, thus advancing the diagnostics of the extent and severity of cartilage injuries.
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Cartilagem Articular , Osteoartrite , Animais , Cartilagem Articular/patologia , Cátions , Meios de Contraste , Cavalos , Osteoartrite/diagnóstico por imagem , Osteoartrite/etiologia , Osteoartrite/patologia , Proteoglicanas , Tomografia Computadorizada por Raios X , ÁguaRESUMO
There is an increasing interest in controlled release systems for local therapy in the treatment of human and equine joint diseases, aiming for optimal intra-articular concentrations with no systemic side effects. In this study, the intra-articular tolerability and suitability for local and sustained release of tacrolimus (FK506) from monospheres composed of [PDLA-PEG1000]-b-PLLA multiblock copolymers were investigated. Unloaded and tacrolimus-loaded (18.4 mg tacrolimus/joint) monospheres were injected into the joints of six healthy horses, with saline and hyaluronic acid (HA) in the contralateral joints as controls. Blood and synovial fluid were analysed for the tacrolimus concentration and biomarkers for inflammation and cartilage metabolism. After an initial burst release, sustained intra-articular tacrolimus concentrations (>20 ng/mL) were observed during the 42 days follow-up. Whole-blood tacrolimus levels were below the detectable level (<0.5 ng/mL). A transient inflammatory reaction was observed for all substances, evidenced by increases of the synovial fluid white blood cell count and total protein. Prostaglandin and glycosaminoglycan release were increased in joints injected with unloaded monospheres, which was mitigated by tacrolimus. Both tacrolimus-loaded monospheres and HA transiently increased the concentration of collagen II cleavage products (C2C). A histologic evaluation of the joints at the endpoint showed no pathological changes in any of the conditions. Together, these results indicate the good biocompatibility of intra-articular applied tacrolimus-loaded monospheres combined with prolonged local drug release while minimising the risk of systemic side effects. Further evaluation in a clinical setting is needed to determine if tacrolimus-loaded monospheres can be beneficial in the treatment of inflammatory joint diseases in humans and animals.
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BACKGROUND: There is a paucity of research describing the gait pattern of lame horses at the walk. OBJECTIVES: To describe the changes in motion pattern and vertical ground reaction forces (GRFz) in horses with induced forelimb lameness at the walk and compare those changes with the changes observed at the trot. STUDY DESIGN: Experimental study. METHODS: In 10 clinically sound Warmblood horses, moderate forelimb lameness was induced using a sole pressure model followed by trot and walk on a treadmill. Kinematic data were collected using 3D optical motion capture (OMC), and GRFz by an instrumented treadmill. Mixed models were used to compare sound baseline versus forelimb lameness (significance was set at P < .05). RESULTS: Lameness induction significantly reduced peak GRFz on the second force peak, and vertical impulse in the lame limb. Stride and stance duration in all limbs were reduced. Lameness significantly affected the vertical movement symmetry of the head and withers. Maximum limb retraction angle, fetlock extension and protraction speed were reduced in the lame limb. Body centre of mass (COM) translation was reduced in the side-to-side direction and increased in the vertical and fore-aft directions. Several compensatory kinetic and kinematic changes were observed in the nonlame limbs. The observed changes in both kinetics and kinematics were generally smaller at walk with fewer variables being affected, compared to the trot. MAIN LIMITATIONS: Only one degree and type of orthopaedic pain (sole pressure) was studied. CONCLUSIONS: Compensatory strategies of forelimb lameness at the walk include alteration of several kinetic and kinematic parameters and have some specific patterns and inter-individual differences that are not seen at the trot. However, much like at the trot, head movement and forelimb vertical force symmetry seem to be the most useful parameters to detect forelimb lameness at walk.
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Doenças dos Cavalos , Caminhada , Animais , Fenômenos Biomecânicos , Membro Anterior , Marcha , Membro Posterior , Cavalos , Coxeadura Animal/diagnósticoRESUMO
This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and euthanized at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contra-lateral sham-operated controls (p < 0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a threefold to sevenfold change in interleukin-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid extracted from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited.
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Articulações do Carpo , Doenças das Cartilagens , Cartilagem Articular , Doenças dos Cavalos , Animais , Articulações do Carpo/diagnóstico por imagem , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Cavalos , Líquido Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.
Assuntos
Bioimpressão/métodos , Cerâmica/química , Hidrogéis/química , Engenharia Tecidual/métodos , Fenômenos Biomecânicos , Bioimpressão/instrumentação , Cartilagem Articular/citologia , Proliferação de Células , Condrócitos/citologia , Condrogênese , Humanos , Células-Tronco Mesenquimais/citologia , Poliésteres/química , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Alicerces Teciduais/químicaRESUMO
Bone has great self-healing capacity, but above a certain critical size, bone defects will not heal spontaneously, requiring intervention to achieve full healing. Among the synthetic calcium phosphate (CaP) bone replacement materials, brushite (CaHPO4·2H2O)-based materials are of particular interest because of their degree of solubility and the related high potential to promote bone regeneration after dissolution. They can be produced tailor-made using modern three-dimensional (3D) printing technology. Although this type of implant has been widely tested in vitro, there are only limited in vivo data and less so in a relevant large animal model. In this study, material properties of a 3D-printed brushite-based scaffold are characterized, after which the material is tested by in vivo orthotopic implantation in the equine tuber coxae for 6 months. The implantation procedure was easy to perform and was well tolerated by the animals, which showed no detectable signs of discomfort. In vitro tests showed that compressive strength along the vertical axis of densely printed material was around 13 MPa, which was reduced to approximately 8 MPa in the cylindrical porous implant. In vivo, approximately 40% of the visible volume of the implants was degraded after 6 months and replaced by bone, showing the capacity to stimulate new bone formation. Histologically, ample bone ingrowth was observed. In contrast, empty defects were filled with fibrous tissue only, confirming the material's osteoconductive capacity. It is concluded that this study provides proof that the 3D-printed brushite implants were able to promote new bone growth after 6 months' implantation in a large animal model and that the new equine tuber coxae bone model that was used is a promising tool for bone regeneration studies.