Infrared Spectroscopy Can Differentiate Between Cartilage Injury Models: Implication for Assessment of Cartilage Integrity.

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.

Orthotopic equine study confirms the pivotal importance of structural reinforcement over the pre-culture of cartilage implants.

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.

Osseous pathologic changes in the lumbar region of the equine vertebral column: A descriptive post-mortem study in three breeds.

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.

3D T1 relaxation time measurements in an equine model of subtle post-traumatic osteoarthritis using MB-SWIFT.

The aim of this study is to assess whether articular cartilage changes in an equine model of post-traumatic osteoarthritis (PTOA), induced by surgical creation of standard (blunt) grooves, and very subtle sharp grooves, could be detected with ex vivo T1 relaxation time mapping utilizing three-dimensional (3D) readout sequence with zero echo time. Grooves were made on the articular surfaces of the middle carpal and radiocarpal joints of nine mature Shetland ponies and osteochondral samples were harvested at 39 weeks after being euthanized under respective ethical permissions. T1 relaxation times of the samples (n = 8 + 8 for experimental and n = 12 for contralateral controls) were measured with a variable flip angle 3D multiband-sweep imaging with Fourier transform sequence. Equilibrium and instantaneous Young's moduli and proteoglycan (PG) content from OD of Safranin-O-stained histological sections were measured and utilized as reference parameters for the T1 relaxation times. T1 relaxation time was significantly (p < 0.05) increased in both groove areas, particularly in the blunt grooves, compared with control samples, with the largest changes observed in the superficial half of the cartilage. T1 relaxation times correlated weakly (Rs ≈ 0.33) with equilibrium modulus and PG content (Rs ≈ 0.21). T1 relaxation time in the superficial articular cartilage is sensitive to changes induced by the blunt grooves but not to the much subtler sharp grooves, at the 39-week timepoint post-injury. These findings support that T1 relaxation time has potential in detection of mild PTOA, albeit the most subtle changes could not be detected.

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo.

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.

Adaptations in equine axial movement and muscle activity occur during induced fore- and hindlimb lameness: A kinematic and electromyographic evaluation during in-hand trot.

BACKGROUND: The inter-relationship between equine thoracolumbar motion and muscle activation during normal locomotion and lameness is poorly understood. OBJECTIVE: To compare thoracolumbar and pelvic kinematics and longissimus dorsi (longissimus) activity of trotting horses between baseline and induced forelimb (iFL) and hindlimb (iHL) lameness. STUDY DESIGN: Controlled experimental cross-over study. METHODS: Three-dimensional kinematic data from the thoracolumbar vertebrae and pelvis, and bilateral surface electromyography (sEMG) data from longissimus at T14 and L1, were collected synchronously from clinically nonlame horses (n = 8) trotting overground during a baseline evaluation, and during iFL and iHL conditions (2-3/5 AAEP), induced on separate days using a lameness model (modified horseshoe). Motion asymmetry parameters, maximal thoracolumbar flexion/extension and lateral bending angles, and pelvis range of motion (ROM) were calculated from kinematic data. Normalised average rectified value (ARV) and muscle activation onset, offset and activity duration were calculated from sEMG signals. Mixed model analysis and statistical parametric mapping compared discrete and continuous variables between conditions (α = 0.05). RESULTS: Asymmetry parameters reflected the degree of iFL and iHL. Maximal thoracolumbar flexion and pelvis pitch ROM increased significantly following iFL and iHL. During iHL, peak lateral bending increased towards the nonlame side (NLS) and decreased towards the lame side (LS). Longissimus ARV significantly increased bilaterally at T14 and L1 for iHL, but only at LS L1 for iFL. Longissimus activation was significantly delayed on the NLS and precipitated on the LS during iHL, but these clear phasic shifts were not observed in iFL. MAIN LIMITATIONS: Findings should be confirmed in clinical cases. CONCLUSIONS: Distinctive, significant adaptations in thoracolumbar and pelvic motion and underlying longissimus activity occur during iFL and iHL and are detectable using combined motion capture and sEMG. For iFL, these adaptations occur primarily in a cranio-caudal direction, whereas for iHL, lateral bending and axial rotation are also involved.

CONTEXTO: O relacionamento entre a movimentação toracolombar e a ativação muscular durante a locomoção normal e quando há claudicação é pouco compreendido. OBJETIVOS: Comparar a cinemática toracolombar e pélvica e a atividade do músculo longissimus dorsi (longissimus) em cavalos ao trote entre o momento inicial (baseline) e claudicação induzida no membro torácico (iFL) e pélvico (iHL). DELINEAMENTO DO ESTUDO: Estudo experimental controlado cruzado. METODOLOGIA: Dados cinemáticos tridimensionais das vertebras toracolombar e pelve, e eletromiografia de superfície (sEMG) bilateral do longissimus na T14 e L1 foram coletados de forma síncrona de cavalos clinicamente não claudicantes (n = 8) trotando no momento inicial (baseline), e durante iFL e iHL (2-3/5 AAEP), induzidos separadamente em dias distintos utilizando um modelo de claudicação (ferradura modificada). Parâmetros de movimentação assimétrica, flexão/extensão máxima da toracolombar e ângulos de virada lateral, e amplitude de movimento da pelve (ROM) foram calculados a partir dos dados de cinemática. O valor médio normalizado retificado (ARV) e início da ativação muscular, e término e duração da atividade foram calculados utilizando sinais de sEMG. Análise de modelo misto e mapeamento paramétrico estatístico compararam variáveis discretas e contínuas entre condições (α=0.05). RESULTADOS: Parâmetros de assimetria refletiram o nível de iFL e iHL. A flexão toracolombar máxima e a ROM da pelve aumentaram significativamente com iFL e iHL. Durante iHL, o pico de flexão lateral aumentou em direção ao lado não-claudicante (NSL) e diminuiu em direção ao lado claudicante (LS). Longissimus ARV aumentou significativamente para ambos os lados na T14 e L1 para iHL, mas apenas no LS para iFL. A ativação do longissimus foi significativamente retardado no NLS e precipitado no LS durante iHL, mas essa mudança de fase clara não foi observada no iFL. PRINCIPAIS LIMITAÇÕES: Esses achados precisam ser confirmados em casos clínicos. CONCLUSÕES: Adaptações significantes e distintas na movimentação toracolombar e pélvica e atividade do músculo longissimus ocorre durante iFL e iHL e são detectadas utilizando captura de movimento e sEMG. Para iFL, essas adaptações ocorrem primariamente na direção cranio-caudal, enquanto que em iHL, movimento lateral e rotação axial também estão envolvidos.

Site- and Zone-Dependent Changes in Proteoglycan Content and Biomechanical Properties of Bluntly and Sharply Grooved Equine Articular Cartilage.

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.

Dual-contrast computed tomography enables detection of equine posttraumatic osteoarthritis in vitro.

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.

Penetration of topically administered dexamethasone disodium phosphate and prednisolone acetate into the normal equine ocular fluids.

BACKGROUND: Topical dexamethasone and prednisolone are currently the mainstay treatment for equine ophthalmic inflammatory diseases, such as equine recurrent uveitis. Comparative pharmacokinetic studies in horses are lacking and current guidelines are mainly based on empirical data and extrapolation from other species. OBJECTIVES: To investigate the penetration and local concentrations of topically applied dexamethasone and prednisolone in normal equine ocular fluids and serum. STUDY DESIGN: Prospective randomised experimental pharmacokinetic study. METHODS: Twenty-one Shetland ponies without ophthalmic disease were treated bilaterally topically every 2 hours during 24 hours to obtain steady state drug concentrations. One eye was treated with 0.15 mg of dexamethasone disodium phosphate (0.1%), and the other eye was simultaneously treated with 1.5 mg of prednisolone acetate (1%). Serum samples were taken prior to the induction of general anaesthesia. Aqueous and vitreous humour samples were taken during euthanasia at time points after administration of the last dose (t = 5 min, t = 15 min, t = 30 min, t = 60 min, t = 90 min, t = 120 min, t = 180 min). Each pony was randomly assigned to one time point, and three ponies were sampled per time point. Dexamethasone and prednisolone concentrations were measured by liquid chromatography-mass spectrometry. RESULTS: The mean dexamethasone concentration in aqueous humour was 32.4 ng/mL (standard deviation [SD] 10.9) and the mean prednisolone concentration was 321.6 ng/mL (SD 96.0). In the vitreous and in serum samples concentrations of both corticosteroids were below the limit of detection (LOD 2.5 ng/mL). MAIN LIMITATIONS: The study group was limited to subjects without evidence of current ophthalmic disease. A limited number of time points were measured. CONCLUSIONS: Potentially effective dexamethasone and prednisolone concentrations were measured in the anterior chamber, but vitreal concentrations were negligible. Systemic uptake was low. Therefore, treatment with only topically administered corticosteroids is deemed insufficient in horses in cases of posterior uveitis. Further studies evaluating other routes of administration are warranted.

Continuous versus discrete data analysis for gait evaluation of horses with induced bilateral hindlimb lameness.

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.

A comparative study of breed differences in the anatomical configuration of the equine vertebral column.

The importance of the equine thoracolumbar vertebral column in orthopaedic disorders is well recognized and diagnostic imaging becomes more feasible, but little is known about variations in the anatomical configuration within breeds. In this descriptive post-mortem study, anatomical variations in three widely differing breeds: Warmblood horses, Shetland ponies and semi-feral Konik horses are described. The caudal cervical (C), thoracic (T), lumbar (L) and sacral (S) regions of the vertebral column of 30 Warmblood horses, 29 Shetland ponies and 18 Konik horses were examined using computed tomography and visualized by volume rendering. Homologous/morphologic variations in the caudal cervical area were frequently seen in Warmblood horses (43%), which was significantly more than in the other breeds (p < 0.001). The as standard described equine formula of 18 T, 6 L and 5 S vertebrae was seen in 78% of Konik horses, but only in 53% Warmblood horses and 38% Shetland ponies, which was significantly different (p < 0.05). Overall, Shetland ponies showed a higher tendency of thoracoization, lumbarization and more variations in the number of vertebrae and pairs of ribs. Ankylosed intertransverse joints (ITJs) between transverse processes of the lumbar vertebrae were most common between the second last and last lumbar vertebra and prevalence was significantly higher in Shetland ponies (61%), than in Warmblood horses (38%) and Konik horses (7%) (p < 0.0001). Cranial to the second last lumbar vertebra there were fewer ITJs ankylosed (14%) in Warmblood horses (p < 0.0095), and this decrease in number of ankylosed ITJs was different compared to the change in ankylosed ITJs in Shetland ponies (p < 0.005). ITJs occurred asymmetrically in 15% (12/77) of the cases. A limitation of the study was that clinical data of the horses were only incompletely available, precluding any conclusions about the potential clinical implications of anatomical variations. Knowledge of variation in osseous anatomy of the equine thoracolumbar vertebral column is important for the interpretation of diagnostic imaging. To assess the functional importance and clinical relevance of this variation, follow-up studies are necessary.

Evaluation of articular cartilage with quantitative MRI in an equine model of post-traumatic osteoarthritis.

Chondral lesions lead to degenerative changes in the surrounding cartilage tissue, increasing the risk of developing post-traumatic osteoarthritis (PTOA). This study aimed to investigate the feasibility of quantitative magnetic resonance imaging (qMRI) for evaluation of articular cartilage in PTOA. Articular explants containing surgically induced and repaired chondral lesions were obtained from the stifle joints of seven Shetland ponies (14 samples). Three age-matched nonoperated ponies served as controls (six samples). The samples were imaged at 9.4 T. The measured qMRI parameters included T1 , T2 , continuous-wave T1ρ (CWT1ρ ), adiabatic T1ρ (AdT1ρ ), and T2ρ (AdT2ρ ) and relaxation along a fictitious field (TRAFF ). For reference, cartilage equilibrium and dynamic moduli, proteoglycan content and collagen fiber orientation were determined. Mean values and profiles from full-thickness cartilage regions of interest, at increasing distances from the lesions, were used to compare experimental against control and to correlate qMRI with the references. Significant alterations were detected by qMRI parameters, including prolonged T1 , CWT1ρ , and AdT1ρ in the regions adjacent to the lesions. The changes were confirmed by the reference methods. CWT1ρ was more strongly associated with the reference measurements and prolonged in the affected regions at lower spin-locking amplitudes. Moderate to strong correlations were found between all qMRI parameters and the reference parameters (ρ = -0.531 to -0.757). T1 , low spin-lock amplitude CWT1ρ , and AdT1ρ were most responsive to changes in visually intact cartilage adjacent to the lesions. In the context of PTOA, these findings highlight the potential of T1 , CWT1ρ , and AdT1ρ in evaluation of compositional and structural changes in cartilage.

Structural, compositional, and functional effects of blunt and sharp cartilage damage on the joint: A 9-month equine groove model study.

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.

Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model.

One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr2+) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr2+ ions (MgPSr) and a medical-grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm3) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer-modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load-bearing properties of MgP-based ceramics with simultaneous induction of new bone formation.

T2* mapping in an equine articular groove model: Visualizing changes in collagen orientation.

T2* mapping is promising for the evaluation of articular cartilage collagen. In this work, a groove model in a large animal is used as a model for posttraumatic arthritis. We hypothesized that T2* mapping could be employed to differentiate between healthy and (subtly) damaged cartilage. Eight carpal joints were obtained from four adult Shetland ponies that had been included in the groove study. In this model, grooves were surgically created on the proximal articular surface of the intermediate carpal bone (radiocarpal joint) and the radial facet of the third carpal bone (middle carpal joint) by either coarse disruption or sharp incision. After 9 months, T2* mapping of the entire carpal joint was carried out on a 7.0-T whole-body magnetic resonance imaging (MRI) scanner by means of a gradient echo multi-echo sequence. Afterwards, assessment of collagen orientation was carried out based on Picrosirius Red-stained histological sections, visualized by polarized light microscopy (PLM). The average T2* relaxation time in grooved samples was lower than in contralateral control sites. Opposite to the grooved areas, the "kissing sites" had a higher average T2* relaxation time than the grooved sites. PLM showed mild changes in orientation of the collagen fibers, particularly around blunt grooves. This work shows that T2* relaxation times are different in healthy cartilage vs (early) damaged cartilage, as induced by the equine groove model. Additionally, the average T2* relaxation times are different in kissing lesions vs the grooved sites.

Arthroscopic Determination of Cartilage Proteoglycan Content and Collagen Network Structure with Near-Infrared Spectroscopy.

Conventional arthroscopic evaluation of articular cartilage is subjective and insufficient for assessing early compositional and structural changes during the progression of post-traumatic osteoarthritis. Therefore, in this study, arthroscopic near-infrared (NIR) spectroscopy is introduced, for the first time, for in vivo evaluation of articular cartilage thickness, proteoglycan (PG) content, and collagen orientation angle. NIR spectra were acquired in vivo and in vitro from equine cartilage adjacent to experimental cartilage repair sites. As reference, digital densitometry and polarized light microscopy were used to evaluate superficial and full-thickness PG content and collagen orientation angle. To relate NIR spectra and cartilage properties, ensemble neural networks, each with two different architectures, were trained and evaluated by using Spearman's correlation analysis (ρ). The ensemble networks enabled accurate predictions for full-thickness reference properties (PG content: ρin vitro, Val= 0.691, ρin vivo= 0.676; collagen orientation angle: ρin vitro, Val= 0.626, ρin vivo= 0.574) from NIR spectral data. In addition, the networks enabled reliable prediction of PG content in superficial (25%) cartilage (ρin vitro, Val= 0.650, ρin vivo= 0.613) and cartilage thickness (ρin vitro, Val= 0.797, ρin vivo= 0.596). To conclude, NIR spectroscopy could enhance the detection of initial cartilage degeneration and thus enable demarcation of the boundary between healthy and compromised cartilage tissue during arthroscopic surgery.

Critical-sized cartilage defects in the equine carpus.

AIM: The horse joint, due to its similarity with the human joint, is the ultimate model for translational articular cartilage repair studies. This study was designed to determine the critical size of cartilage defects in the equine carpus and serve as a benchmark for the evaluation of new cartilage treatment options. MATERIAL AND METHODS: Circular full-thickness cartilage defects with a diameter of 2, 4, and 8 mm were created in the left middle carpal joint and similar osteochondral (3.5 mm in depth) defects in the right middle carpal joint of 5 horses. Spontaneously formed repair tissue was examined macroscopically, with MR and µCT imaging, polarized light microscopy, standard histology, and immunohistochemistry at 12 months. RESULTS: Filling of 2 mm chondral defects was good (77.8 ± 8.5%), but proteoglycan depletion was evident in Safranin-O staining and gadolinium-enhanced MRI (T1Gd). Larger chondral defects showed poor filling (50.6 ± 2.7% in 4 mm and 31.9 ± 7.3% in 8 mm defects). Lesion filling in 2, 4, and 8 mm osteochondral defects was 82.3 ± 3.0%, 68.0 ± 4.6% and 70.8 ± 15.4%, respectively. Type II collagen staining was seen in 9/15 osteochondral defects but only in 1/15 chondral defects. Subchondral bone pathologies were evident in 14/15 osteochondral samples but only in 5/15 chondral samples. Although osteochondral lesions showed better neotissue quality than chondral lesions, the overall repair was deemed unsatisfactory because of the subchondral bone pathologies. CONCLUSION: We recommend classifying 4 mm as critical osteochondral lesion size and 2 mm as critical chondral lesion size for cartilage repair research in the equine carpal joint model.

Arthroscopic near infrared spectroscopy enables simultaneous quantitative evaluation of articular cartilage and subchondral bone in vivo.

Arthroscopic assessment of articular tissues is highly subjective and poorly reproducible. To ensure optimal patient care, quantitative techniques (e.g., near infrared spectroscopy (NIRS)) could substantially enhance arthroscopic diagnosis of initial signs of post-traumatic osteoarthritis (PTOA). Here, we demonstrate, for the first time, the potential of arthroscopic NIRS to simultaneously monitor progressive degeneration of cartilage and subchondral bone in vivo in Shetland ponies undergoing different experimental cartilage repair procedures. Osteochondral tissues adjacent to the repair sites were evaluated using an arthroscopic NIRS probe and significant (p < 0.05) degenerative changes were observed in the tissue properties when compared with tissues from healthy joints. Artificial neural networks (ANN) enabled reliable (ρ = 0.63-0.87, NMRSE = 8.5-17.2%, RPIQ = 1.93-3.03) estimation of articular cartilage biomechanical properties, subchondral bone plate thickness and bone mineral density (BMD), and subchondral trabecular bone thickness, bone volume fraction (BV), BMD, and structure model index (SMI) from in vitro spectral data. The trained ANNs also reliably predicted the properties of an independent in vitro test group (ρ = 0.54-0.91, NMRSE = 5.9-17.6%, RPIQ = 1.68-3.36). However, predictions based on arthroscopic NIR spectra were less reliable (ρ = 0.27-0.74, NMRSE = 14.5-24.0%, RPIQ = 1.35-1.70), possibly due to errors introduced during arthroscopic spectral acquisition. Adaptation of NIRS could address the limitations of conventional arthroscopy through quantitative assessment of lesion severity and extent, thereby enhancing detection of initial signs of PTOA. This would be of high clinical significance, for example, when conducting orthopaedic repair surgeries.

Combination of optical coherence tomography and near infrared spectroscopy enhances determination of articular cartilage composition and structure.

Conventional arthroscopic evaluation of articular cartilage is subjective and poorly reproducible. Therefore, implementation of quantitative diagnostic techniques, such as near infrared spectroscopy (NIRS) and optical coherence tomography (OCT), is essential. Locations (n = 44) with various cartilage conditions were selected from mature equine fetlock joints (n = 5). These locations and their surroundings were measured with NIRS and OCT (n = 530). As a reference, cartilage proteoglycan (PG) and collagen contents, and collagen network organization were determined using quantitative microscopy. Additionally, lesion severity visualized in OCT images was graded with an automatic algorithm according to International Cartilage Research Society (ICRS) scoring system. Artificial neural network with variable selection was then employed to predict cartilage composition in the superficial and deep zones from NIRS data, and the performance of two models, generalized (including all samples) and condition-specific models (based on ICRS-grades), was compared. Spectral data correlated significantly (p < 0.002) with PG and collagen contents, and collagen orientation in the superficial and deep zones. The combination of NIRS and OCT provided the most reliable outcome, with condition-specific models having lower prediction errors (9.2%) compared to generalized models (10.4%). Therefore, the results highlight the potential of combining both modalities for comprehensive evaluation of cartilage during arthroscopy.

Fixation of Hydrogel Constructs for Cartilage Repair in the Equine Model: A Challenging Issue.

OBJECTIVE: To report on the experiences with the use of commercial and autologous fibrin glue (AFG) and of an alternative method based on a 3D-printed polycaprolactone (PCL) anchor for the fixation of hydrogel-based scaffolds in an equine model for cartilage repair. METHODS: In a first study, three different hydrogel-based materials were orthotopically implanted in nine horses for 1-4 weeks in 6 mm diameter full-thickness cartilage defects in the medial femoral trochlear ridge and fixated with commercially available fibrin glue (CFG). One defect was filled with CFG only as a control. In a second study, CFG and AFG were compared in an ectopic equine model. The third study compared the efficacy of AFG and a 3D-printed PCL-based osteal anchor for fixation of PCL-reinforced hydrogels in three horses for 2 weeks, with a 4-week follow-up to evaluate integration of bone with the PCL anchor. Short-term scaffold integration and cell infiltration were evaluated by microcomputed tomography and histology as outcome parameters. RESULTS: The first study showed signs of subchondral bone resorption in all defects, including the controls filled with CFG only, with significant infiltration of neutrophils. Ectopically, CFG induced clear inflammation with strong neutrophil accumulation; AFG was less reactive, showing fibroblast infiltration only. In the third study the fixation potential for PCL-reinforced hydrogels of AFG was inferior to the PCL anchor. PCL reinforcement had disappeared from two defects and showed signs of dislodging in the remaining four. All six constructs fixated with the PCL anchor were still in place after 2 weeks. At 4 weeks, the PCL anchor showed good integration and signs of new bone formation. CONCLUSIONS: The use of AFG should be preferred to xenogeneic products in the horse, but AFG is subject to individual variations and laborious to make. The PCL anchor provides the best fixation; however, this technique involves the whole osteochondral unit, which entails a different conceptual approach to cartilage repair.