Label‐free relative quantification of secreted proteins as a non‐invasive method for the quality control of chondrogenesis in bioengineered substitutes for cartilage repair

Abstract

Cartilage tissue engineering is making progress, but the competing available strategies still leave room for improvement and consensual overviews regarding the best combinations of scaffolds and cell sources are limited by the capacity to compare them directly. In addition, because most strategies involve autologous cell transfer, once these are optimized, the resulting implants require individual quality control prior to grafting in order to emphasize patient-to-patient differential responsiveness to engineering processes. Here, cartilage substitutes prepared from human mesenchymal stem cells undergoing chondrogenic differentiation within distinct scaffolds were used as pilot samples to investigate the pertinence of a novel method with the aim of characterizing the implants. The limits and advantages of analysing, by label-free liquid chromatography-coupled matrix-assisted laser desorption and ionization (LC-MALDI) mass spectrometry, the secreted proteome released into culture medium by engineered cartilage tissues were investigated and compared with more classically used methods for biomaterial characterization. This method did not require sacrificing the biomaterials and robustly evidenced their chondrogenic statuses. In more detail, the method highlighted differences between batches prepared from distinct donors. It was adapted to distinct scaffolds and allowed a comparison of the influence of individual engineering steps, such as growth factor combinations and oxygen tension. Finally, it evidenced subtle changes between replicate substitutes within a series, thereby distinguishing the least and most accomplished ones. We conclude that relative quantification of secreted proteins through label-free LC-MALDI will be useful, not only to orientate engineering methodologies, but also to ultimately provide non-invasive quality control of engineered tissue substitutes for the repair of cartilage and possibly other connective tissues.

HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

“…5 μm sections were stained with Alcian blue staining to visualize proteoglycan content. To visualize collagen II content, immunohistochemical analysis was performed as described previously ( 56 ). The stained slides were observed and recorded by light microscopy (DMD108, Leica) at 4× original magnification.…”

Understanding interactions between biomaterials and biological systems using proteomics

“…In another study, Henrionnet et al [84] used a label-free LC-MALDI technique to perform relative quantification of secreted proteins in tissue-engineered cartilage constructs, consisting of human mesenchymal stem cells (hMSCs) undergoing chondrogenic differentiation inside collagen-based scaffolds. This study emphasised the potential power of secretome analysis as a noninvasive testing/validation tool for the quality of such tissueengineered constructs, but could also be used as an in vitro biomaterial evaluation tool.…”

Integrative Metabolic Pathway Analysis Reveals Novel Therapeutic Targets in Osteoarthritis

“…Proteomic approaches have been applied to increase knowledge of the differentiation processes of MSCs obtained from different tissues (9). For instance, label-free relative quantification strategies have recently been performed for the secretome and proteome by guest on September 6, 2020 characterization of hBMSCs undergoing chondrogenesis (10,11). However, label-free proteomic methods are less accurate, less precise, and less reproducible for protein quantification compared to those based on stable isotope labeling such as stable isotope labeling by amino acids in cell culture (SILAC) (12,13).…”