Articular cartilage has a limited regeneration capacity, and damage of cartilage often results in the onset of degenerative disease such as osteoarthritis (OA). MRI and CT imaging of cartilage and subchondral bone are becoming increasingly important in early detection and treatment of OA as well as for quantifying quality of tissue-engineered samples. Non-invasive CT scanners have been used to image cartilage tissue with the help of contrast agents. However, since only one energy source is available, imaging information of multiple soft and hard tissues is lost given that the overall x-ray attenuation is measured. Medipix All Resolution System (MARS) CT offers the possibility of applying more than one energy source. It is able to measure the energy of each photon individually and therefore determines the characteristics of attenuation.

In this study, an ionic contrast agent (Hexabrix) was used to image the negatively charged extra-cellular matrix component, glycosaminoglycan (GAG), which is abundantly found in the middle and lower layers of healthy cartilage tissue. GAG distribution in the cartilage tissue could be imaged using an inverse relationship with Hexabrix signal (i.e. high signal represents low GAG content). Eight bovine cartilage-bone explants (3mm × 5mm) were incubated in 4 different Hexabrix concentrations ranging from 20% to 50% in PBS. Sections were imaged using the MARS scanner at high and low energies (13.32 keV and 30.84 keV). Images were pre-processed, reconstructed and colour-coded using different enhancement techniques and virtual experimental software. Histological (Safranin-O) staining and quantitative biochemical analysis of GAG content (DMMB dye assay) was performed to correlate GAG distribution and content with MARS-CT images.

High resolution images of both cartilage and bone regions were obtained, with contrast enhanced CT of cartilage correlating well with histological staining. X-ray attenuation was high in regions poor in GAG content, whereas attenuation was low in GAG rich regions. Furthermore, there was a direct inverse correlation between Hexabrix signal and GAG content as measured in superficial (2.9 μg/mg) and middle/deep regions (10.6 μg/mg) in cartilage explants.

It can be concluded that the MARS technique can be used to image GAG distribution and GAG content, and therefore could be used clinically to assess quality of healthy or osteoarthritic cartilage, as well as non-destructive imaging of GAG content in engineered tissues.

Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function to damaged articular cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells, but more importantly, the generation of engineered tissue of sufficient quality of clinically relevant size. We describe a novel approach to engineer cartilage grafts using pre-differentiated micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds.

Expanded (P2) human nasal chondrocytes (HNCs) or bone marrow-derived mesenchymal stem cells (MSCs) from 3 donors (age 47–62 years) were micro-mass cell pellet cultivated at 5 × 105 cells/pellet for 4 days. Subsequently, pellets were integrated into degradable 3D Printed polymer (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a total of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression.

After 4 days, micro-mass cultured pellets could be successfully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seeded constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis observed within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively).

We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency (∼100%), as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Furthermore, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage.