Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level

Abstract

Objective: High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. Design: Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. Results: The highest quality images were obtained using propagation phase-contrast imaging and PTAstaining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p < 0.0001 to p ¼ 0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. Conclusions: Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

Exploratory Full-Field Mechanical Analysis across the Osteochondral Tissue—Biomaterial Interface in an Ovine Model

“…Although standard protocol was followed including staining, the cartilage of this study did not appear to show obvious anatomical features as documented in other species [ 26 ]. This is a likely explanation of why the cartilage incurred the highest errors in the constant strain study, and further work is required to improve the cartilage imaging.…”

“…Strain errors were approximately similar between all components of the system except for cartilage which incurred errors 2–4 times higher than the other components. Further work is required to optimise the sample preparation [ 26 ] and micro-CT scanning protocols to ensure sufficient features are imaged. Strain errors of approximately 0.1% strain or less were possible with a subvolume size of 80 voxels, providing a spatial resolution of 360 µm.…”

“…Studies of large animal models inherently involve small sample sizes, and, likewise, DVC studies are usually limited to small sample sizes [ 15 , 22 ]. With optimisation the method is compatible with histological analysis which could be carried out destructively after in situ testing [ 26 ]. The developed technique ensures maximum data can be collected from each sample to inform development of successful devices.…”

“…A limitation is that this was not included as part of the presented study. Our previously documented success with cartilage imaging were carried out on smaller tissue explants (3 mm diameter) in different species to that of this trial [ 26 ]. The same protocol was followed, yet further optimisation is required to ensure successful imaging.…”

“…Prior to in situ testing sample diameter was further reduced to approximately 10 mm using a precision sectioning saw (IsoMet, Buehler, Lake Bluff, IL, USA) to improve scan quality. The sample was stained in 1% w / v phosphotungstic acid ( w / v , H 3 PW 12 O 40 , PTA, #79690 Sigma-Aldrich, St. Louis, MO, USA) solution in 70% ethanol for 21 h following established protocol [ 26 ]. The sample was mounted to the bottom polyoxymethylene platen with polymethyl methacrylate bone cement (Simplex, Kemdent, Swindon, UK).…”