Do cobalt and chromium ions affect the formation of an extracellular matrix?

摘要

Background: Research regarding the biological response to metallic implant debris has a large focus on investigating the response of various cell lines upon the addition of particles and/or ions. Understanding cellular response to a material is critical in determining the pathways to negative immune response; however the majority of studies are focused on effects on 2D cell cultures in vitro, and have yet to consider the effects on the extracellular matrix; which plays a crucial role in providing the physical and chemical cues to direct cell function and fate. Collagen type 1 is the most abundant component of the organic matrix of bone which acts as a template for mineralisation and hence also for bone tissue formation. Given that the highly ordered hierarchical structure of bone is critical to maintaining bone mechanical properties and interfacial bonding, the effect of Co(Ⅱ) and Cr(Ⅲ) ions on type Ⅰcollagen could play a role in the mechanism behind aseptic loosening. Methods: Gel Preparation: Collagen gels (Cultrex Rat Collagen Ⅰ, Trevigen) were prepared in Dulbecco's Modified Eagle Medium at 1 mg/ml, with various physiologically relevant conc, of Co and Cr ions. Final pH was adjusted to 8-8.5 with sodium hydroxide to promote gelation. Analysis Techniques: Kinetic analysis of collagen fibril formation was examined (with and without Co and Cr ions) by measuring turbidity (loss of transparency) of the gelling collagen using spectrophotometry (Cecil Instruments, UK). Turbidity-time profiles were determined at an absorbance of 620nm at 37°C. In addition, collagen fibrils were left to fully form overnight at 37°C in 35mm MatTek imaging. Imaging was performed on an Olympus FV1000 confocal microscope at 488nm, using a x60 water immersion lens in reflection mode. Z-stacks were acquired for a fixed volume in each case. Results and Discussion: The characteristic curve of a turbidity graph contains a lag phase before an increase in turbidity, indicative of fibril formation, followed by a plateau phase resulting from complete fibril formation. These curves in fig 1A show the addition of Co and Cr ions does affect the fibril kinetics of collagen gels. Increasing conc, of Co ions indicate decreasing formation rates and final fibril diameters; whereas increasing conc, of Cr ions imply an apparent increase in fibril diameter, with little effect on the formation rate. Fig1B shows that the distribution of collagen fibrils in the control appears homogeneous in comparison to the Co ion-containing sample, where the density of fibrils seems reduced; and lacks any localised highly scattering regions as seen in the Cr containing sample. Preliminary micro-XRF mapping suggests the latter to be precipitation effects caused due to dehydration of the collagen. Conclusions: The presence of even low concentrations of Co and Cr ions demonstrates a significant effect on collagen fibrillogenesis. Suggesting more attention needs to paid to the Influence of implants on local matrix formation rather than simply demonstrating efficacy in in vitro monolayer cell cultures. Further work will be performed, such as circular dichroism and NMR to determine conformation of collagen molecules, transmission electron microscopy to visualize the fiber banding and rheometry to determine stiffness of the gel. These techniques will complement each other to allow determination of the mechanism of collagen-metal ion interaction, as well as evaluating the biological response to the modified collagen.