Influence of the cell aggregation technique on the differentiation of human articular chondrocytes in microtissues

Introduction Articular cartilage defects are a major risk factor for the development of secondary osteoarthrosis. Due to the limited self-repair capacity of this tissue, several strategies including cell-based therapies were developed to treat joint cartilage defects. However, standard cell culture results in a loss of chondrogenic properties. A 3D cell arrangement, which resembles the natural environment, is able to support the cartilage phenotype. The success of this approach depends on different factors. Hence, the aim of this study was to compare the maturation of chondrocyte-derived microtissues using different techniques.

Methods Human primary articular chondrocytes isolated from knee joints were expanded in 2D standard culture. Scaffold-free microtissues were generated using three techniques: agar overlay, v-bottom plates (pellet culture) and cell-repellent v-bottom plates. After two and four weeks, the macroscopic appearance and the size was assessed. The differentiation degree was evaluated via immunohistochemistry (IH) to detect cartilage-specific markers (collagen type II, proteoglycans, Sox9) and collagen type I and via histology to visualise typical glycosaminoglycans (Safranin O, Alcian blue) on cryosections. The metabolic activity was determined by quantifying the ATP level.

Results Microtissues were generated in all experimental setups with distinct differences in the differentiation degree. A higher differentiation level was observed in microtissues cultured in both v-bottom plates compared to microtissues on agar. This was evidenced by a higher amount of extracellular matrix resulting in bigger microtissues (diameter on v-bottom plates was 80% larger compared to agar) and increased metabolic activity. Histological analysis revealed little/local staining on agar versus an overall staining on v-bottom plates with the highest intensity on cell-repellent plates. IH analysis of cartilage-specific markers showed the same pattern. The expression level of collagen type II and proteoglycans was enhanced in microtissues from v-bottom plates. The expression of Sox9 was distributed ubiquitously in all cultivation approaches. Collagen type I was similarly expressed on a low level.

Discussion This study showed that the successful maturation of chondrocytes in microtissues is directly related to the method of induction and development of spheroids. The chondrogenic profile of microtissues in cell-repellent v-bottom plates is superior to the other techniques. Therefore, these plates are recommended for the robust generation of microtissues with a distinct chondrogenic phenotype avoiding foreign substances (agar) or physical stress (centrifugation). These microtissues seem to be quite suitable as transplants to regenerate cartilage defects.

Keywords microtissue generation, chondrogenic differentiation, cell therapy, cartilage regeneration

Address for correspondence Ursula Anderer, Brandenburg University of Technology Cottbus-Senftenberg, Institute of Biotechnology, Department of Cell Biology and Tissue Engineering, Universitaetsplatz 1, 01968 Senftenberg, Germany, E-Mail: [e-mail]

Publication History

Article published online:
08 September 2022