No abstract

Vascular calcification is a severe pathological event in the manifestation of atherosclerosis. Pathogenic triggers mediating osteogenic differentiation of arterial smooth muscle cells (SMC) in humans remain insufficiently understood and are to a large extent investigated in animal models or cells derived thereof. Here, we describe an in vitro model based on SMC derived from healthy and diseased humans that allows to comprehensively investigate vascular calcification mechanisms. Comparing the impact of the commonly used SMC culture media VascuLife, DMEM, and M199, cells were characterised by immunofluorescence, flow cytometry, qPCR, and regarding their contractility and proliferative capacity. Irrespective of the arterial origin, the clinical background and the expansion medium used, all cells expressed typical molecular SMC marker while contractility varied between donors. Interestingly, the ability to induce an osteogenic differentiation strongly depended on the culture medium, with only SMC cultured in DMEM depositing calcified matrix upon osteogenic stimulation, which correlated with increased alkaline phosphatase activity, increased inorganic phosphate level and upregulation of osteogenic gene markers. Our optimized model is suitable for donor-oriented as well as broader screening of potential pathogenic mediators triggering vascular calcification. Translational studies aiming to identify and to evaluate therapeutic targets in a personalized fashion would be feasible. Vascular calcification due to osteogenic differentiation of arterial smooth muscle cells (SMC) is a severe pathological event in atherosclerosis. Massive, bone-like or brittle, amorphous calcifications can occlude the lumen, cause atherosclerotic plaque rupture, thrombus formation and infarction or result in failure of the arterial wall 1-6. Deposition of calcified matrix within the vessel wall was described as an active, SMC-driven process 5,7. This process is initially marked by the switch of SMC from their contractile phenotype towards a synthetic state, in which proliferation and migration is enhanced 8-12. The ability of SMC for phenotypic switching 1,9 is important during vascular repair, but makes them also susceptible to pathological changes 8,9,12-14 , including their differentiation into osteoblast-and chondrocyte-like cells or acquisition of macrophage and foam cell markers 1,11,13-22. Several studies investigated the processes and key factors involved in the osteogenic differentiation of mammalian SMC, subsequent deposition of mineralized matrix and vascular calcification 20,21,23-25. These studies provide evidence that an active inhibition of calcification and the prevention of pro-osteogenic gene expression is crucial for retention of the SMC phenotype 7,23,26-28. Conversely, it was found that SMC-mediated calcification is enhanced by an inflammatory environment, oxidative stress, hypercalcaemia and hyperphosphatemia, as well as apoptosis, and senescence 1,25,29-31. Despite the fact that these pathogenic triggers link atheroscleros...

One of the major reasons for failure after total joint arthroplasty is aseptic loosening of the implant. At articulating surfaces, defined as the interface between implant and surrounding bone cement, wear particles can be generated and released into the periprosthetic tissue, resulting in inflammation and osteolysis. The aim of the present study was to evaluate the extent to which osteoblasts and macrophages are responsible for the osteolytic and inflammatory reactions following contact with generated wear particles from Ti-6Al-7Nb and Co-28Cr-6Mo hip stems. To this end, human osteoblasts and THP-1 monocytic cells were incubated with the experimentally generated wear particles as well as reference particles (0.01 and 0.1 mg/ml) for 48 h under standard culture conditions. To evaluate the impact of these particles on the two cell types, the release of different bone matrix degrading matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and relevant cytokines were determined by multiplex enzyme-linked immunosorbent assays. Following incubation with wear particles, human osteoblasts showed a significant upregulation of MMP1 and MMP8, whereas macrophages reacted with enhanced MMP3, MMP8 and MMP10 production. Moreover, the synthesis of TIMPs 1 and 2 was inhibited. The osteoblasts and macrophages also responded with modified expression of the inflammatory mediators interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor. These results demonstrate that the release of wear particles affects the release of proinflammatory cytokines and has a negative impact on bone matrix formation during the first 48 h of particle exposure. Human osteoblasts are directly involved in the proinflammatory cascade of bone matrix degradation. The simultaneous activation and recruitment of monocytes/macrophages boosted osteolytic processes in the periprosthetic tissue. By the downregulation of TIMP production and the concomitant upregulation of MMPs as a response to particle exposure, bone formation around implants may be suppressed, resulting in implant failure.