Abstract
Introduction and Objective
Total joint replacement (TJR) is indicated for patients with end-stage osteoarthritis (OA) where conservative treatment has failed. Approximately 1.3 million primary hip replacement surgeries have been recorded in the United Kingdom since 2003 and this number is set to rise due to an increase in obesity as well as an ageing population. Total hip replacement (THR) has a survival rate of 85% at 20 years; the most common reason for failure is aseptic loosening which often occurs secondary to osteolysis caused by immune-mediated inflammation responses to wear debris generated from the materials used in the THR implant. Therefore, by understanding the biological steps by which biomaterials cause immune-mediated reactions it should be possible to prevent them in the future thereby reducing the number of costly revision surgeries required.
Materials and Methods
The human osteoblast-like cell line (MG-63) was seeded at a density of 100,000 cell per well of a 6-well plate and treated with and increasing doses (0.5, 5, and 50mm3 per cell) of cobalt-chromium (CoCr) particles generated on a six-station pin-on-plate wear generator or commercially available ceramic oxide nanopowders (Al2O3 and ZrO2) for 24 hours. TNF-alpha was used as a positive control and untreated cells as a negative control. Cells were then analysed by transmission electron microscopy (TEM) to determine whether the osteoblasts were capable of phagocytosing these biomaterials. MG-63 cells were used in conjunction with trypan blue and the XTT Cell Proliferation II Kit to assess cytotoxicity of the biomaterials investigated. Cells supernatants were also collected and analysed by enzyme-linked immunosorbant assay (ELISA) to investigate changes in pro-inflammatory protein secretion. Protein extracted from lysed cells was used for western blotting analysis to investigate RANKL protein expression to determine changes to osteolytic activation. Lysed cells were also used for RNA extraction and subsequent cDNA synthesis for real-time quantitative polymerase chain reaction (RT-qPCR) in order to assess changes to pro-inflammatory gene expression.
Results
There was no significant change to cellular viability or proliferation in the osteoblasts treated with CoCr, Al2O3 or ZrO2 when compared to the untreated negative control. TEM images showed clear and distinct intracellular vesicles within the cell cytoplasm which contained CoCr, Al2O3 and ZrO2. RANKL expression increased at 5 and 50mm3 per cell CoCr and 50mm3 per cell Al2O3 and ZrO2. Pro-inflammatory protein secretion of CXCL10, IL-8, and IL-6 all significantly increased at 50mm3 per cell CoCr, Al2O3, and ZrO2. Similarly to the protein secretion, CXCL10, IL-8, and IL-6 gene expression was significantly upregulated at 50mm3 per cell CoCr, Al2O3, and ZrO2.
Conclusions
Increased in vitro RANKL expression in response to CoCr, Al2O3, and ZrO2 may result in disruption of bone metabolism and lead to osteolysis which can contribute to aseptic loosening in vivo. Significant increases in IL-6 are particularly important because as well as being a pro-inflammatory cytokine, IL-6 is also secreted by osteoblasts in order to stimulate mature osteoclast formation to mediate bone breakdown. CXCL10 and IL-8 are chemotactic cytokines and increased secretion in response to implant biomaterials can contribute to ongoing pro-inflammatory responses through the recruitment of monocytes and neutrophils respectively. This is interesting as in vivo data demonstrates increased cellular infiltrate in patients experiencing responses to implant materials. Overall, these findings show clear immune activation as well as altered metabolism of MG-63 osteoblast cells in response to implant wear debris which is in agreement with in vivo clinical reports.
