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.

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro.

High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed.

Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.