Biofilm Adhesion on TiAl6V4 using Laser Spallation Technique 67 Results revealed that the adoption of the substrate assembly for bulk materials such as Ti64 and AM Ti64 has successfully generated a high compressive stress wave for cell and biofilm adhesion measurement studies. This effective implementation of the substrate assembly was achieved by applying a controlled confining layer on the Ti64 substrate to generate sufficient interface stress, compared to the common multilayer substrate assembly that includes a confining layer, an absorbing layer, a substrate, and a thin film. Both the loaded S. mutans and S. aureus on different substrates exhibited a similar failure area. However, to accurately measure the interfacial adhesion of S. aureus on Ti64, further experiments under varying laser fluences should be conducted. In the preliminary applications of the laser spallation technique for the biological cell adhesion measurement, studies quantified the interfacial adhesion strength of cells and biofilms on substrates by identifying the onset of detachment as the critical threshold, where the minimum laser fluence required to detach cells from the substrate is determined as the critical laser fluence [15, 17, 18]. More recently, the 50% adhesion strength has been reported as the interfacial adhesion strength of the cells and biofilms by researchers [16, 19]. Because of the non-uniform nature of the biological film, instead of a particular laser fluence, a range of fluences is contributed to the onset of failure adhesion [19]. Thus, this 50% adhesion failure represents the adhesion failure of cells and biofilm more effectively. Although the appearance of a dark spallation area has been identified as the failed test in other studies [16, 19], similar to the failed biofilm depicted in Fig. 1 (d), results indicated that the appearance of the bare implant surface may provide a more accurate determination of failure, which may appear lighter in color depending on the tilt of the sample when viewing, as shown in Fig. 1 (c). Fig. 1 (a) Experimental setup of laser spallation technique for cell and biofilm adhesion measurement. (b) Cell and biofilm culture substrate assembly consisted of Ti64 substrate and a waterglass layer as the confining layer. (c) S. aureus grown on Ti64 before and after loading at an iso-high fluence experiment, substrate assembly consisted of 1 mm Ti64 and 12µmthick waterglass (d) S. mutans grown on smooth titanium before and after loading across varying laser fluences. (d) Fluorescence microscopy images of spalled biofilms at increasing laser fluence from 37.46 to 75.7 mJ/mm2, captured using a 4×objective. S. mutans biofilm was stained with Syto 9 before loading. The scale bar is 0.5 mm, and the applied laser spot size is 2 mm
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