Abstract
ObjectiveDespite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs.DesignArticular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young’s modulus.ResultsAddition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young’s modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface.ConclusionScaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.
Highlights
Patients with focal chondral defects can be treated with surgical techniques such as autologous chondrocyte implantation or microfracture
Chondrocytes were cultured in the presence of bFGF in medium containing either human platelet lysate plasma (hPLP) or autologous serum
Cells expanded in medium supplemented with autologous serum and redifferentiated in the absence of bone morphogenetic protein 2 (BMP2) usually attached to the membrane, but the discs were thin and frail
Summary
Patients with focal chondral defects can be treated with surgical techniques such as autologous chondrocyte implantation or microfracture. These methods improve functionality and provide pain relief, but result in imperfect tissue repair.[1,2] Several strategies to redifferentiate chondrocytes in 3-dimensional structures such as pellet cultures or biomaterial matrices have been studied. Pellet cultures serve as a study model but require a large number of cells to generate cartilage constructs of sufficient size.[3] Biomaterials, on the other hand, are used for the fabrication of scaffolds to be applied in next-generation autologous chondrocyte implantation and tissue engineering. Drawbacks can be lack of attachment sites for cells, unwanted mechanical properties, insufficient biocompatibility and integrity, or unpredictable biodegradation of the material.[4,5,6]
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