SummaryObjectiveCollagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC.MethodWe labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland–Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC.ResultsBoth, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4).ConclusionsPTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA.

Optical clearing is an effective method to reduce light scattering of biological tissues that provides significant enhancement of light penetration into the biological tissues making non-invasive diagnosis more feasible. In current report Optical Coherence Tomography (OCT) in conjunction with optical clearing is applied for assessment of deep cartilage layers and cartilage-bone interface. The solution of Iohexol in water has been used as an optical clearing agent. The cartilage-bone boundary becomes visible after 15 min of optical clearing that enabling non-invasive estimation of its roughness: Sa = 10 ± 1 µm. The results show that for 0.9 mm thick cartilage optical clearing is stopped after 50 min with an increase of refractive index from 1.386 ± 0.008 to 1.510 ± 0.009. Current approach enables more reliable detection of arthroscopically inaccessible regions, including cartilage-bone boundary and subchondral bone, and potentially improves accuracy of the osteoarthritis diagnosis.

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Cellulose nanofiber aerogels were used as preforms that were impregnated with a bio-epoxy resin via a widely used vacuum infusion process. The simple and straightforward nanocomposite processing approach resulted in an almost 70 % improvement in the storage modulus of the polymer with only an 11.7 wt% cellulose nanofiber content. The nanofibers were well dispersed in the polymer matrix and the fiber structures were anisotropically aligned. The impregnation time of the aerogels was also significantly lower than that of the more commonly used nanopapers. It was thus shown that environmentally friendly and mechanically robust nanocomposites could be produced by impregnating cellulose nanofiber aerogels with a thermosetting resin, using a processing approach that has a potential to be scaled up for commercial use.

Contrast-enhanced micro-computed tomography (CEμCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEμCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0–13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13–39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEμCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEμCT method for 3D characterization of articular cartilage.

s u m m a r yObjective: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (mCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). Method: Samples (n ¼ 15) used in this study were harvested from human lateral tibial plateau (n ¼ 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. Results: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (r s ¼ À0.600, P ¼ 0.018), entropy of patterns (EP) (r s ¼ À0.648, P ¼ 0.018), homogeneity index (HI) (r s ¼ 0.555, P ¼ 0.032)) and tidemark roughness (TMR) (r s ¼ À0.579, P ¼ 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (r p ¼ 0.876, P < 0.0001; r p ¼ 0.665, P ¼ 0.007, respectively) and channel density (CD) (r p ¼ 0.680, P ¼ 0.011; r p ¼ 0.582, P ¼ 0.023, respectively). TMR was associated with CAF (r p ¼ 0.926, P < 0.0001) and average channel size (r p ¼ 0.574, P ¼ 0.025). CC topography differed statistically significantly in early OA vs healthy samples. Conclusion:We introduced a m-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA.

SummaryObjectiveHistopathological grading of osteochondral (OC) tissue is widely used in osteoarthritis (OA) research, and it is relatively common in post-surgery in vitro diagnostics. However, relying on thin tissue section, this approach includes a number of limitations, such as: (1) destructiveness, (2) sample processing artefacts, (3) 2D section does not represent spatial 3D structure and composition of the tissue, and (4) the final outcome is subjective. To overcome these limitations, we recently developed a contrast-enhanced μCT (CEμCT) imaging technique to visualize the collagenous extracellular matrix (ECM) of articular cartilage (AC). In the present study, we demonstrate that histopathological scoring of OC tissue from CEμCT is feasible. Moreover, we establish a new, semi-quantitative OA μCT grading system for OC tissue.ResultsPathological features were clearly visualized in AC and subchondral bone (SB) with μCT and verified with histology, as demonstrated with image atlases. Comparison of histopathological grades (OARSI or severity (0–3)) across the characterization approaches, CEμCT and histology, excellent (0.92, 95% CI = [0.84, 0.96], n = 30) or fair (0.50, 95% CI = [0.16, 0.74], n = 27) intra-class correlations (ICC), respectively. A new μCT grading system was successfully established which achieved an excellent cross-method (μCT vs histology) reader-to-reader intra-class correlation (0.78, 95% CI = [0.58, 0.89], n = 27).ConclusionsWe demonstrated that histopathological information relevant to OA can reliably be obtained from CEμCT images. This new grading system could be used as a reference for 3D imaging and analysis techniques intended for volumetric evaluation of OA pathology in research and clinical applications.

NMR experiments carried out at magnetic fields below 1 T provide new relaxation parameters unavailable with conventional clinical scanners. Contrast of T generally becomes larger towards low fields, as slow molecular reorientation processes dominate relaxation at the corresponding Larmor frequencies. This advantage has to be considered in the context of lower sensitivity and frequently reduced spatial resolution. The layered structure of cartilage is one example where a particularly strong variation of T across the tissue occurs, being affected by degenerative diseases such as osteoarthritis (OA). Furthermore, the presence of H- N cross-relaxation, leading to so-called quadrupolar dips in the H relaxation time dispersion, provide insight into the concentration and mobility of proteoglycans and collagen in cartilage, both being affected by OA. In this study, low-field imaging and variable-field NMR relaxometry were combined for the first time for tissue samples, employing unidirectional load to probe the mechanical properties. 20 human knee cartilage samples were placed in a compression cell, and studied by determining relaxation profiles without and with applied pressure (0.6 MPa) at 50 μm in-plane resolution, and comparing with volume-averaged T dispersion. Samples were subsequently stored in formalin, prepared for histology and graded according to the Mankin score system. Quadrupolar dips and thickness change under load showed the strongest correlation with Mankin grade. Average T and change of maximum T under load, as well as its position, correlate with thickness and thickness change. Furthermore, T (ω) above 25 mT was found to correlate with thickness change. While volume-averaged T is not a suitable indicator for OA, its change due to mechanical load and its extreme values are suggested as biomarkers available in low-field MRI systems. The shape of the dispersion T (ω) represents a promising access to understanding and quantifying molecular dynamics in tissue, pointing toward future in vivo tissue studies.