Interactions of U937 macrophage-like cells with decellularized pericardial matrix materials: influence of crosslinking treatment.

While macrophages have been implicated in the failure of bioprosthetic heart valves, the macrophage response to crosslinked native pericardial collagen has not been previously investigated. Using decellularized bovine pericardium (DBP) as a model for native collagen, this study investigated the response of macrophage-like cells (U937s) to DBP, either: (i) untreated, or (ii) exogenously crosslinked with glutaraldehyde or 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC). We have previously validated the use of U937 cells as models for the response of human monocyte-derived macrophages to decellularized pericardial materials and, per our previous work, differentiated the U937 cells directly on the three material surfaces. After 72h in culture, the cells and medium were analyzed for DNA content, acid phosphatase activity, and cytokine and matrix metalloproteinase release. As well, cell/substrate samples were fixed for SEM. Fewer cells attached to or survived on the glutaraldehyde-treated substrate, and some showed an abnormal morphology compared to cells cultured on the other surfaces. Further, cells on glutaraldehyde-treated surfaces released more pro-inflammatory cytokines, more MMP-1 and less MMP-2 and MMP-9. The poor performance of the U937 macrophage-like cells on the glutaraldehyde-treated surfaces appears to be due to surface characteristics rather than to soluble aldehyde or other components leaching from the crosslinked material. These results provide evidence that crosslinking with glutaraldehyde is cytotoxic to macrophage-like cells, and that crosslinking with a zero-length crosslinker like EDC can be an acceptable alternative crosslinking treatment for biomaterials.

Boundary conditions during biaxial testing of planar connective tissues. Part 1: dynamic behavior.

Current mechanical testing methods used to determine the biaxial properties of planar connective tissues may lead to artifactual observations of material behavior. The method of sample gripping affects the constraint on the extracellular fibers at the bounds of the sample. This applied constraint not only affects how the load is transferred to the sample, but also how the load is transmitted throughout the rest of the material - thereby influencing the resulting mechanical behavior of the tissue. In this study, we compared the dynamic biaxial mechanical response of pericardial tissue samples under two different gripping methods: (i) the common method of suturing sample edges and (ii) a new biaxial clamping method. Tissue samples were repeatedly testing using both gripping methods under the same conditions. The tissue samples appeared to be stiffer and less extensible when mechanically tested with clamped sample edges, as opposed to when tested with sutured sample edges. Thus, the influence of the sample boundaries affected the response of the material - precisely the situation to be avoided for reliable material testing. This casts doubt on whether any in vitro mechanical testing method can used to determine the "real" properties of the tissue since the boundary conditions of the tissue in situ are presently unknown.