Regenerative Cartilage made by Fusion of Cartilage Elements derived from Chondrocyte Sheets prepared in Temperature-Responsive Culture Dishes

Abstract

To establish a large regenerative cartilage without any scaffolds, we cultured chondrocytes on NIsopropylacrylamide (NIPPAM)-coated dishes with 3 × 3 mm 2 grids, producing many cell sheets. We then attempted to have the cell sheets form into pellets, which we term cartilage elements, and have the cartilage elements fuse with each other. Finally, we regenerated the large cartilaginous constructs with some stiffness. The NIPPAM coating on culture dishes, which enables to detach cultured chondrocytes only by the decrease in incubation temperature, was useful for preparing many chondrocyte sheets with a homogenous size. The cell sheets could provide effective matrix production and become spherical to form the cartilage elements. For their fusion, those elements were jammed into an agarose mold and were cultured for 3 weeks. The regenerative constructs made by fusing the cartilage elements derived from human or beagle chondrocytes were subcutaneously transplanted into nude mice and the cell-donor beagles, respectively, showing fair cartilage regeneration 2 months after transplantation. They could also prevent severe foreign-body reactions that often occur when using some scaffolds. Thus, by mass production of homogenous cartilage elements using the NIPPAM-coated dishes and their mutual fusion in the agarose mold, mature cartilage was three-dimensionally regenerated without any scaffolds.

A modular approach to creating large engineered cartilage surfaces

“…Cell infiltration into the bond site was observed histologically, suggesting that cell migration occurred in proximity of neighboring constructs as has been previously seen (Bhumiratana et al 2014, Mori et al 2014, Dikina et al 2015). Raman spectroscopy of the bond site and construct was similar to spectra for human cartilage (Kunstar et al 2012, Esmonde-White 2014, Gamsjaeger et al 2014).…”

“…Modular tissue engineering is a technique of initially culturing smaller components, which are combined at a later time point to create larger surfaces (Nichol and Khademhosseini 2009). This approach has been used to combine cultured rings into tube-like constructs for tracheal reconstruction and to combine pellet cultures into larger sheets of engineered cartilage (Bhumiratana et al 2014, Mori et al 2014, Dikina et al 2015, Nover et al 2017). These platforms take advantage of the inherent adhesivity of de novo extracellular matrix.…”

Overcoming Challenges in Engineering Large, Scaffold-Free Neocartilage with Functional Properties

“…14 Even tissue-engineered cartilage products currently in clinical trials, representing the next-generation therapies, limit their clinical indications to focal defects less than 7.5 cm 2 . [15][16][17][18][19][20][21][22][23][24] Large, nonisolated defects generally receive palliative treatment until total or partial knee arthroplasty is indicated. Therefore, a tissue engineering approach that bridges the treatment of focal defects and total joint arthroplasty remains an unmet need for the foreseeable future.…”