S.S. Karhula

Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography

Summary Objective Collagen 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. Method We 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. Results Both, 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). Conclusions PTA-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.

Imaging of subchondral bone by optical coherence tomography upon optical clearing of articular cartilage

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.

Micro-CT Analysis of Bone Healing in Rabbit Calvarial Critical-Sized Defects with Solid Bioactive Glass, Tricalcium Phosphate Granules or Autogenous Bone

ABSTRACT Objectives The purpose of the present study was to evaluate bone healing in rabbit critical-sized calvarial defects using two different synthetic scaffold materials, solid biodegradable bioactive glass and tricalcium phosphate granules alongside solid and particulated autogenous bone grafts. Material and Methods Bilateral full thickness critical-sized calvarial defects were created in 15 New Zealand white adult male rabbits. Ten defects were filled with solid scaffolds made of bioactive glass or with porous tricalcium phosphate granules. The healing of the biomaterial-filled defects was compared at the 6 week time point to the healing of autologous bone grafted defects filled with a solid cranial bone block in 5 defects and with particulated bone combined with fibrin glue in 10 defects. In 5 animals one defect was left unfilled as a negative control. Micro-computed tomography (micro-CT) was used to analyze healing of the defects. Results Micro-CT analysis revealed that defects filled with tricalcium phosphate granules showed new bone formation in the order of 3.89 (SD 1.17)% whereas defects treated with solid bioactive glass scaffolds showed 0.21 (SD 0.16)%, new bone formation. In the empty negative control defects there was an average new bone formation of 21.8 (SD 23.7)%. Conclusions According to findings in this study, tricalcium phosphate granules have osteogenic potential superior to bioactive glass, though both particulated bone with fibrin glue and solid bone block were superior defect filling materials.

Effects of articular cartilage constituents on phosphotungstic acid enhanced micro-computed tomography

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.

3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography

Summary Objective Histopathological 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. Results Pathological 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). Conclusions We 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.

Correlations of low‐field NMR and variable‐field NMR parameters with osteoarthritis in human articular cartilage under load

NMR experiments carried out at magnetic fields below 1 T provide new relaxation parameters unavailable with conventional clinical scanners. Contrast of T1 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 T1 across the tissue occurs, being affected by degenerative diseases such as osteoarthritis (OA). Furthermore, the presence of 1H‐14 N cross‐relaxation, leading to so‐called quadrupolar dips in the 1H relaxation time dispersion, provide insight into the concentration and mobility of proteoglycans and collagen in cartilage, both being affected by OA.

Optical clearing of articular cartilage: a comparison of clearing agents

Optical clearing technique was applied to the problem of OCT imaging of articular cartilage and subchondral bone. We show that optical clearing significantly enhances visualization of articular cartilage and cartilage-bone interface. The effect of different clearing agents was analyzed. For the clearing, iohexol solution and propylene glycol (PG) were used. Clearing was performed in vitro at room temperature by immersion method. Cylindrical osteochondral samples (d=4.8mm) were drilled from bovine lateral femur and stored in phosphate-buffered saline at -20°C until clearing. Monitoring of clearing process was performed using high-speed spectral-domain OCT system providing axial resolution of 5.8μm at 930nm. Total duration of experiment was 90-100min to ensure saturation of clearing. We have shown that iohexol solution and PG are capable to optically clear articular cartilage enabling reliable characterization of cartilagebone interface with OCT. Being a low osmolarity agent, iohexol provides minimal changes to the thickness of cartilage sample. Clearing saturation time for the cartilage sample with the thickness of 0.9 mm measured with OCT is of 50 min. However, less than 15 min is enough to reliably detect the rear cartilage boundary. Alternatively, PG significantly (60%) reduces the cartilage thickness enabling better visualization of subchondral bone. It was observed that PG has higher clearing rate. The clearing saturation time is of 30 min, however less than 5 min is enough to detect cartilage-bone interface. We conclude that iohexol solution is superior for OCT imaging of cartilage and cartilage-bone interface, while PG suits better for subhondral bone visualization.

Iterative and discrete reconstruction in the evaluation of the rabbit model of osteoarthritis

Micro-computed tomography (µCT) is a standard method for bone morphometric evaluation. However, the scan time can be long and the radiation dose during the scan may have adverse effects on test subjects, therefore both of them should be minimized. This could be achieved by applying iterative reconstruction (IR) on sparse projection data, as IR is capable of producing reconstructions of sufficient image quality with less projection data than the traditional algorithm requires. In this work, the performance of three IR algorithms was assessed for quantitative bone imaging from low-resolution data in the evaluation of the rabbit model of osteoarthritis. Subchondral bone images were reconstructed with a conjugate gradient least squares algorithm, a total variation regularization scheme, and a discrete algebraic reconstruction technique to obtain quantitative bone morphometry, and the results obtained in this manner were compared with those obtained from the reference reconstruction. Our approaches were sufficient to identify changes in bone structure in early osteoarthritis, and these changes were preserved even when minimal data were provided for the reconstruction. Thus, our results suggest that IR algorithms give reliable performance with sparse projection data, thereby recommending them for use in µCT studies where time and radiation exposure are preferably minimized.

In vitro method for 3D morphometry of human articular cartilage chondrons based on micro-computed tomography

Summary Objective The aims of this study were: to 1) develop a novel sample processing protocol to visualize human articular cartilage (AC) chondrons using micro-computed tomography (μCT), 2) develop and validate an algorithm to quantify the chondron morphology in 3D, and 3) compare the differences in chondron morphology between intact and osteoarthritic AC. Method The developed protocol is based on the dehydration of samples with hexamethyldisilazane (HMDS), followed by imaging with a desktop μCT. Chondron density and depth, as well as volume and sphericity, were calculated in 3D with a custom-made and validated algorithm employing semi-automatic chondron selection and segmentation. The quantitative parameters were analyzed at three AC depth zones (zone 1: 0–10%; zone 2: 10–40%; zone 3: 40–100%) and grouped by the OARSI histological grades (OARSI grades 0–1.0, n = 6; OARSI grades 3.0–3.5, n = 6). Results After semi-automatic chondron selection and segmentation, 1510 chondrons were approved for 3D morphometric analyses. The chondrons especially in the deeper tissue (zones 2 and 3) were significantly larger (P < 0.001) and less spherical (P < 0.001), respectively, in the OARSI grade 3–3.5 group compared to the OARSI grade 0–1.0 group. No statistically significant difference in chondron density between the OARSI grade groups was observed at different depths. Conclusion We have developed a novel sample processing protocol for chondron imaging in 3D, as well as a high-throughput algorithm to semi-automatically quantify chondron/chondrocyte 3D morphology in AC. Our results also suggest that 3D chondron morphology is affected by the progression of osteoarthritis (OA).

Low-Field NMR Relaxation Times Distributions and Their Magnetic Field Dependence as a Possible Biomarker in Cartilage

The dependence of the proton NMR relaxation times on field strength and on location within the tissue has been determined for a number of bovine and human articular cartilage samples. While the strong variation of T2 across the triple-layered cartilage structure as well as its orientation dependence are well known from clinical and laboratory high-field studies, T1 shows similar behavior only in low magnetic fields. At 0.27 T, the ratio of longest to shortest T1 has been found to cover a ratio of about 3-5 in healthy tissue. At the same time, the average T1 was found to be strongly field dependent in the range down to 0.25 mT, but no spatially resolved data are available under these conditions. By correlating the spatially resolved T1 distribution obtained at field strengths of 0.27 T with mathematical decompositions of the signal recovery function into multiexponential components, an attempt is made to quantify the width of P(T1) for variable field strengths, and to identify the field value where this distribution is widest, being optimally situated as a biomarker for laboratory studies or preclinical low-field investigations where spatial resolution is absent or insufficient to resolve the cartilage layer structure.