Composition analysis of proteoglycans synthesized in vitro by chondrocytes of various origins

Introduction . The use of cellular constructs based on human pluripotent stem cells (hPSCs) is associated with a number of challenges, including the need to standardize methods for cultivating chondrocyte-like hPSCs derivatives to produce a cartilage tissue similar to natural hyaline cartilage. Glycosaminoglycans (GAGs) are the basis of the extracellular matrix (ECM) of cartilage tissue; therefore, a qualitative and quantitative analysis of the GAG composition of cartilage-like tissue engineering structures is an important step in the final assessment of their potential therapeutic effectiveness. Objective . To determine the composition of GAGs synthesized in vitro by chondrocytes of various origins using enzyme-linked immunoassay (ELISA) and liquid chromatography with tandem mass spectrometry (LC–MS/MS), as well as to evaluate the effect of 2D and 3D culturing on their synthesis. Materials and methods . We analyzed the GAG levels in 2D and 3D tissue-engineered structures obtained from the cartilage tissue of five donors, chondrocyte-like cells differentiated from two hPSC lines. Cellular spheroids were obtained by aggregation in microlunar plates and cultured in mini-bioreactors. The analysis of the GAG content in cell culture samples and spheroids was carried out using ELISA and LC–MS/MS. The Kruskal–Wallis and Dunn tests were used to assess the statistical significance of the differences between the samples. Results . The ELISA study revealed statistically significant differences (p < 0.0021), confirming higher levels of GAGs synthesized in 3D cultures of native chondrocytes compared to 2D cultures (108.67 ng/mL and 1099.87 ng/mL, respectively). The average number of spectra of the chondroitin sulfate proteoglycan 4 protein, determined using LC–MS/MS, was also higher in 3D cultures, amounting to 41.75 spectra compared to 2.24 spectra in 2D culture samples. The levels of aggrecan, biglican, and decorin did not differ between cultures. 3D cultures of chondrocyte-like cells from hPSC showed no significant differences in the content of GAG compared to 2D cultures, which indicates the need to optimize the conditions for their differentiation. Conclusions . In our study, the composition of the GAGs synthesized in vitro by chondrocytes of various origins was determined using ELISA and LC–MS/MS. The the effect of 2D and 3D cultivation on their synthesis was evaluated. The results showed that 3D culture media create favorable conditions for a more complete formation of chondrocytic ECM in native chondrocyte samples. Despite this, the obtained spheroids of chondrocyte-like hPSCs derivatives fail to achieve functional identity with natural cartilage tissue, even after completion of differentiation protocols, thus not representing optimal tissue engineering structures for correcting cartilage defects.