RESUMO
Implant surface topography can modulate macrophage behavior during wound healing by the production of proinflammatory cytokines. This study investigated the activation of FAK, Src, and ERK1/2 signaling intermediates of the proinflammatory ERK1/2 pathway in RAW 264.7 macrophages in response to polished (P), coarse-grit-blasted (B), acid etched (E), and grit-blasted and etched (SLA) surface topographies. In addition, the effects of these topographies on cell spreading, vinculin organization, and viability were determined. Macrophages on the SLA surface changed from predominantly well-spread cells to ones with a more spherical morphology over time. In contrast, macrophages on the P surface changed from being predominantly spherical cells to well spread. The morphological changes were associated with changes in the distribution of vinculin. The overall patterns of the pFAK, pSrc, pERK1/2 levels as well as pERK1/2 nuclear translocation associated with cell shape with greater activation being seen with a more spread morphology. These results suggest that surface topography differentially activates signaling pathways that affect cell function and raise the possibility that topographies can be designed to optimize desired cell responses.
Assuntos
Forma Celular/fisiologia , Quinase 2 de Adesão Focal/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/fisiologia , Quinases da Família src/fisiologia , Animais , Western Blotting , Contagem de Células , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Imuno-Histoquímica , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Fosforilação , Propriedades de Superfície , Vinculina/metabolismoRESUMO
Implanted rough surfaces have long been associated with the accumulation of macrophages and other cells of the monocytic lineage such as foreign body giant cells and osteoclasts. As cells of the moncytic lineage are part of the immune system, the response of this cell family to biomaterials has attracted wide concern. This study compared events at the interface of implant surface topographies with varied roughness in a rat subcutaneous model. Titanium-coated epoxy replicas of machined, etched, blasted, titanium-plasma-sprayed (TPS), sandblasted-and-etched (SLA), micromachined, and polished surfaces were implanted for up to 11 weeks, and processed for light or electron microscopy or immunohistochemistry for ED1, a marker for recruited macrophages. Initially, healing appeared similar among all surfaces, the frequency of mineralization followed the order of SLA, micromachined, TPS, machined, etched, blasted, and polished surfaces. On the SLA surface macrophages, as identified by both ultrastructural morphology and immunohistochemistry were the predominant cell type at 1 week and persisted until mineralization occurred as early as 2 weeks. On smoother surfaces collagenous matrix predominated at 2 weeks and subsequently increased with time. There, thus, appears to be two routes to bone-like tissue formation on Ti implants in this rat subcutaneous model; macrophage-mediated and macrophage-independent dense collagenous-matrix-associated.
Assuntos
Implantes Experimentais , Macrófagos/imunologia , Osteogênese/fisiologia , Titânio/química , Animais , Materiais Biocompatíveis/química , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Macrófagos/ultraestrutura , Masculino , Teste de Materiais , Ratos , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired.