Cartilage permeability assessment based on proteomic analysis of plasma protein penetration

Purpose: Articular cartilage contains proteins belonging to the insoluble collagen superfamily and collagen-associated proteins which form the structural framework of cartilage. Proteoglycans and other important proteins, albeit in minor amounts, together fill in the mesh. The highly cross-linked nature of the cartilage restricts, to a degree, access and infiltration of non-cartilage proteins from synovial fluid into cartilage. In this study, we utilized proteomic methodology to comprehensively monitor the degree of plasma protein penetration into cartilage in health and disease. We hypothesized that aging and joint damage would alter the degree and depth of plasma protein infiltration in cartilage thereby providing an index of cartilage permeability. The goal of the project was to evaluate cartilage permeability in different joints and disease and also to identify plasma proteins with special binding patterns. Methods: We collected hip and knee cartilage samples (both healthy and osteoarthritic) as surgical waste from surgical repair of trauma or joint replacement. Serial transverse frozen sections of 12μm thickness were generated at different depths (superficial, middle and deep layers) from the cartilage surface. To avoid the confounding background by intracellular proteins, chondrocytes were depleted by performing one rapid freeze/thaw cycle in hypotonic solution. We performed chaotropic quanidine-HCl (Gu-HCl) extraction to obtain extractable proteins from these cartilage sections. All batches of extracts were then processed for mass spectrometry analysis. The experiments were performed with an EasyLC nanoflow HPLC (Proxeon Biosystems, Odense, Denmark) connected to a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) equipped with a nanoEasy spray ion source (Proxeon Biosystems, Odense, Denmark). Results: Analysis of extracts produced from tissue harvested at different depths within cartilage revealed that non-matrix proteins, originating in the plasma, could be detected within the cartilage tissue. Plasma proteins could be found even in the deep region of cartilage. Interestingly, immunoglobulins and albumin could be found in greater amounts in arthritic knee cartilage compared with non-arthritic knee cartilage (Figure 1). Although these two proteins were less abundant in deep hip cartilage compared with non-arthritic hip cartilage the amount of both proteins declined with the distance from the cartilage surface in all types of cartilage. This suggested, as expected, that cartilage was more permeable in the superficial compared with deeper layers. These results also suggested that these proteins likely do not specifically cross-link to cartilage matrix proteins since their distribution pattern followed the intrinsic nature of cartilage permeability. In contrast, fibrinogens were less abundant in knee arthritic cartilage tissues compared to healthy cartilage, especially in the deep region (Figure 2). Generally, the amount of fibrinogen declined with the distance from the superficial layer. However, in healthy hip cartilage, this pattern was reversed; the amount of fibrinogen increased with the distance from the surface. In hip OA cartilage, fibrinogen was only detected in the superficial layer but was undetectable in the deeper region. Conclusions: These results demonstrate that cartilage permeability is altered as a result of cartilage degradation in OA. This alteration differed by joint site. Plasma proteins that are enriched in the deeper regions of cartilage may be involved in specific interactions with matrix proteins and thus remain in the deeper region. Further targeted quantitative proteomic analysis with larger sample number can improve the understanding of plasma protein distribution. This experimental system could lead to new insights into the feasibility of plasma proteins to enhance or inhibit cartilage degradation. These results would also have important implications for drug penetration into cartilage.Figure 2. Fibrinogen identified in cartilage. Rank: a higher ranking represent higher levels in comparison to the other proteins within that particular sample (i.e. ranking over total number of proteins identified). NA: not detected. SUP: superficial; INM: intermediate; DEP: deep layer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)