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.

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.

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.

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.

How exercise influences equine joint homeostasis.

The maintenance of joint homeostasis is integral to joint health. Knowledge of the influence of exercise on joint homeostasis is not only relevant for determining sustainable levels of equine athletic training, but also for the study of early development of osteoarthritis or cartilage repair in animal models. This review provides an overview of findings derived from in vivo studies and postmortem analyses investigating exercise effects on various joint tissue components in the horse, supplemented where appropriate with data from small animal models. The concept of joint homeostasis and possible methods to quantify this are also discussed, with special attention to the potential benefits and pitfalls of biomarker analysis in synovial fluid. The main conclusion is that biomechanical loading in the form of deliberate exercise has a major influence on the delicate homeostatic balance within the tissues constituting the diarthrodial joint and on their interactions, which is crucial for proper and durable joint function. The amount and intensity of exercise can have a lasting effect on tissue characteristics in juvenile animals, but affects joint homeostasis in mature animals and can affect the delicate balance between physiologic adaptation and development of pathology. Biomarkers in synovial fluid can be helpful in assessing joint homeostasis, but their use and interpretation require caution and are often far from straightforward.

Optical coherence tomography enables accurate measurement of equine cartilage thickness for determination of speed of sound.

Background and purpose - Arthroscopic estimation of articular cartilage thickness is important for scoring of lesion severity, and measurement of cartilage speed of sound (SOS)-a sensitive index of changes in cartilage composition. We investigated the accuracy of optical coherence tomography (OCT) in measurements of cartilage thickness and determined SOS by combining OCT thickness and ultrasound (US) time-of-flight (TOF) measurements. Material and methods - Cartilage thickness measurements from OCT and microscopy images of 94 equine osteochondral samples were compared. Then, SOS in cartilage was determined using simultaneous OCT thickness and US TOF measurements. SOS was then compared with the compositional, structural, and mechanical properties of cartilage. Results - Measurements of non-calcified cartilage thickness using OCT and microscopy were significantly correlated (ρ = 0.92; p < 0.001). With calcified cartilage included, the correlation was ρ = 0.85 (p < 0.001). The mean cartilage SOS (1,636 m/s) was in agreement with the literature. However, SOS and the other properties of cartilage lacked any statistically significant correlation. Interpretation - OCT can give an accurate measurement of articular cartilage thickness. Although SOS measurements lacked accuracy in thin equine cartilage, the concept of SOS measurement using OCT appears promising.

Crosslinkable hydrogels derived from cartilage, meniscus, and tendon tissue.

Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.

Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints.

BACKGROUND: Arthroscopy is widely used in various equine joints for diagnostic and surgical purposes. However, accuracy of defining the extent of cartilage lesions and reproducibility in grading of lesions are not optimal. Therefore, there is a need for new, more quantitative arthroscopic methods. Arthroscopic optical coherence tomography (OCT) imaging is a promising tool introduced for quantitative detection of cartilage degeneration and scoring of the severity of chondral lesions. The aim of this study was to evaluate the inter-investigator agreement and inter-method agreement in grading cartilage lesions by means of conventional arthroscopy and with OCT technique. For this aim, 41 cartilage lesions based on findings in conventional and OCT arthroscopy in 14 equine joints were imaged, blind coded and independently ICRS (International Cartilage Repair Society) scored by three surgeons and one PhD-student. RESULTS: The intra- and inter-investigator percentages of agreement by means of OCT (68.9% and 43.9%, respectively) were higher than those based on conventional arthroscopic imaging (56.7% and 31.7%, respectively). The intra-investigator Kappa coefficients were 0.709 and 0.565 for OCT and arthroscopy, respectively. Inter-investigator Kappa coefficients were 0.538 and 0.408 for OCT and arthroscopy, respectively. CONCLUSIONS: OCT can enhance reproducibility of arthroscopic evaluation of equine joints.