خاصیت ویسکوالاستیکی پوست مورد مطالعه با استفاده از تکنیک های مبتنی بر میکروپروب در محیط آزمایشگاهی Skin viscoelasticity studied in vitro by microprobe-based techniques

۲٫۲٫ Histology analysis For cross-sectional histology studies, whole skin samples were embedded in an optimal-cutting-temperature compound (TissueTek, Elkhart, IN) on dry ice and kept at a temperature of
۶۲ ۱C until testing. Then, 10-μm-thick specimens were sectioned from the skin samples and stained with hematoxylin and eosin following a standard protocol. Cross-sectional imaging (Fig. 1) confirmed that other than localized damage induced by the mechanical probe during testing, removing an outer layer of a few micrometers thickness with a surgical blade and subsequently freezing the obtained sample were not destructive to the tissue histology. ۲٫۳٫ Microprobe-based mechanical testing Low-load/small-depth indentation tests were performed with a surface force microscope (SFM) described elsewhere (Supporting information). All SFM tests were performed with a conospherical diamond tip of 1 μm radius. Approximately 10 samples were used for each set of experimental conditions (i.e., 100–۵۰۰ μN maximum load, 5–۲۵ μN/s loading/unloading rate, and 10–۴۰ s hold time). High-load/large-depth indentation tests were performed with a microprobe force apparatus (MFA) described elsewhere (Supporting information). During MFA testing, the sample was kept under mild tension by a steel plate attached to the sample holder by four screws to compensate for sample shrinking by 10–۲۰% after cutting. A prestress minimizes skin deflection during indentation, reducing the variation in skin property measurement (Butz et al., 2012). All MFA tests were performed with a conospherical diamond-coated tip of 12.5 μm radius. About 10 samples were used for each set of experimental conditions (i.e., 50–۶۰۰ mN maximum load, 5–۹۰ μm/s depth rate, and 10–۶۰ s hold time). Trapezoidal load profiles were used in all tests. While the specified trapezoidal load profile was observed with relatively shallow indentations (Fig. 2a), in the case of relatively deep indentations, the profile showed nonlinear loading and unloading paths (Fig. 2c), indicating a time-dependent deformation that could not be compensated by the load transducer in a timely fashion. Thus, to maintain a constant loading and unloading rate, depth control was used during the loading and unloading phases of testing, whereas load control was used during the hold time of MFA testing. Since this problem was not encountered with the SFM load transducer, load control was used in all the SFM tests. In some MFA experiments (e.g., Fig. 2a and b), a slight decrease in maximum load was observed during the hold period because the feedback controller could not keep up with the pace of viscoelastic deformation. However, since the load change due to this effect was very small (2–۵%), it may be inferred that a constant-load condition was closely approximated during the hold period.