Abstract
Articular cartilage has a limited regenerative capacity. Tissue engineering strategies adopting seeding and differentiation of individual chondrocytes on porous 3D scaffolds of clinically relevant size remains a considerable challenge. A well documented method to produce small samples of differentiated cartilage tissue in vitro is via micro-mass (pellet) culture, whereby, high concentrations of chondrocytes coalesce to form. a spherical tissue pellet. However, pellet culture techniques are not applied clinically as it is only possible to produce small amounts of tissue (1–2mm). The aims of this study were to develop a method for mass-production of pellets, and investigate whether an alternative “pellet seeding” approach using smart 3D scaffold design would allow large numbers of spherical pellets to be fixed in place.
Chondrocytes were isolated from bovine articular cartilage via enzymatic digestion. Freshly isolated and expanded (passage 2) chondrocytes were placed in 96-well plates with round- or v-shaped wells at a range of densities from 0.1, 0.25 and 0.5 million cells per pellet, and centrifuged at 500g for 2 min. In order to assess pellet forming conditions, cells were treated with or without 300 mg/mL fibronectin (FN, Sigma) to improve cell-cell adhesion. Wells were also coated with or without silicone (Sigmacote) to prevent cell adhesion to wells. Pellets were cultured in vitro for up to 14 days and were assessed at various timepoints for size, shape, cell number (DNA assay) and cell differentiation capacity (histology). A robotic Bioplotter device was used to produce porous, biodegradable scaffolds by plotting −250μm polymer (PEGT/PBT) fibres in a layer-by-layer process. Scaffolds with specific 3D pore architecture were produced to allow spherical pellets to be press-fit in each pore thereby fixing them in place throughout the scaffold.
Primary and expanded chondrocytes plated at a density of 0.25 million cell/pellet in v-shaped 96-well plates without both FN and silicone treatment produced pellets with consistently better spherical shape and total cell number (as determined via DNA). Under these conditions, cell (re)differentiation and cartilage extracellular matrix formation was observed via positive staining for safranin-O. Mass production of pellets was achieved by culturing multiple 96-well plates in parallel. FN treatment promoted cell-cell adhesion, but also cell adhesion to well plates, irrespective of silicone treatment, resulting in irregular shaped pellets, as did the use of round-bottom shaped wells.
Smart scaffold design and layer-by-layer fabrication process allowed direct control over the fibre spacing and pore size (1.0–1.25mm). Multiple layers of spherical pellets (1.25–1.5mm) were press-fit in place, thereby limiting the need for direct cell adhesion to the scaffold. Continued culture of constructs containing pellets resulted in consistent tissue formation throughout the scaffold.
In this study, we describe an alternative approach to the design and seeding of scaffolds for cartilage tissue engineering. Current limitations involved with adherence and de-differentiation of single cell populations were avoided by taking advantage of smart 3D scaffold design and pellet-seeding and culture techniques. Further optimisation and automation of the process is necessary, however, such strategies could be beneficial for future scaffold-based cell therapies for repairing articular cartilage defects.
Correspondence should be addressed to Associate Professor N. Susan Stott at Orthopaedic Department, Starship Children’s Hospital, Private Bag 92024, Auckland, New Zealand
