Extracellular matrix composition analysis of human articular cartilage for the development of organ-on-a-chip

Abstract

IntroductionArticular cartilage has a complex extracellular matrix (ECM) that provides it a defined architecture for its load-bearing properties. The complete understanding of ECM components is imperative for developing biomimetic organ-on-a-chip tissue construct. ObjectiveThis study aimed to decellularize and characterize the ECM for its protein profiling to generate a niche for enhanced chondrocyte proliferation. MethodsArticular cartilage scrapings were subjected to mechanical and collagenase digestion, followed by sodium dodecyl sulfate (SDS) treatment for 8 h and 16 h. The de-cellularization efficiency was confirmed by hematoxylin & eosin, alcian blue, masson's trichrome staining, and scanning electron microscopy (SEM). The ECM protein profile was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a bottom–up approach. ResultsHistological characterization revealed void lacunae that lacked staining for cellular components. The ECM, sulfated glycosaminoglycan content, and collagen fibers were preserved after 8 h and 16 h of de-cellularization. The SEM ultrastructure images showed that few chondrocytes adhered to the ECM after 8 h and cell–free ECM after 16 h of de-cellularization. LC-MS/MS analysis identified 66 proteins with heterotypic collagen types COL1A1-COL6A1, COL14A1, COL22A1 and COL25A1 showed moderate fold change and expression levels, while COL18A1, COL26A1, chondroitin sulfate, matrix metalloproteinase-9 (MMP9), fibronectin, platelet glycoprotein 1 beta alpha (GP1BA), vimentin, bone morphogenetic protein 6 (BMP6), fibroblast growth factor 4 (FGF4) and growth hormone receptor (GHR) showed maximum fold change and expression levels. ConclusionsThe standardized de-cellularization process could preserve majority of ECM components, providing structural integrity and architecture to the ECM. The Identified proteins quantified for their expression levels provided insight into engineering the ECM composition for developing cartilage-on-a-chip.