Department of Biomedical Engineering, University of California Irvine, Irvine, CA 92697-2715, United States.
Department of Chemical and Biomolecular Engineering, University of California Irvine, Irvine, CA 92697-2580, United States.
Acta Biomater. 2024 Nov;189:439-448. doi: 10.1016/j.actbio.2024.09.050. Epub 2024 Oct 3.
We introduce a method utilizing single laser-generated cavitation bubbles to stimulate cellular mechanotransduction in dermal fibroblasts embedded within 3D hydrogels. We demonstrate that fibroblasts embedded in either amorphous or fibrillar hydrogels engage in Ca signaling following exposure to an impulsive mechanical stimulus provided by a single 250 µm diameter laser-generated cavitation bubble. We find that the spatial extent of the cellular signaling is larger for cells embedded within a fibrous collagen hydrogel as compared to those embedded within an amorphous polyvinyl alcohol polymer (SLO-PVA) hydrogel. Additionally, for fibroblasts embedded in collagen, we find an increased range of cellular mechanosensitivity for cells that are polarized relative to the radial axis as compared to the circumferential axis. By contrast, fibroblasts embedded within SLO-PVA did not display orientation-dependent mechanosensitivity. Fibroblasts embedded in hydrogels and cultured in calcium-free media did not show cavitation-induced mechanotransduction; implicating calcium signaling based on transmembrane Ca transport. This study demonstrates the utility of single laser-generated cavitation bubbles to provide local non-invasive impulsive mechanical stimuli within 3D hydrogel tissue models with concurrent imaging using optical microscopy. STATEMENT OF SIGNIFICANCE: Currently, there are limited methods for the non-invasive real-time assessment of cellular sensitivity to mechanical stimuli within 3D tissue scaffolds. We describe an original approach that utilizes a pulsed laser microbeam within a standard laser scanning microscope system to generate single cavitation bubbles to provide impulsive mechanostimulation to cells within 3D fibrillar and amorphous hydrogels. Using this technique, we measure the cellular mechanosensitivity of primary human dermal fibroblasts embedded in amorphous and fibrillar hydrogels, thereby providing a useful method to examine cellular mechanotransduction in 3D biomaterials. Moreover, the implementation of our method within a standard optical microscope makes it suitable for broad adoption by cellular mechanotransduction researchers and opens the possibility of high-throughput evaluation of biomaterials with respect to cellular mechanosignaling.
我们介绍了一种利用单个激光生成的空化泡来刺激真皮成纤维细胞中细胞力学转导的方法，这些细胞嵌入在 3D 水凝胶中。我们证明，在单个 250 µm 直径的激光生成空化泡提供的脉冲机械刺激下，嵌入无定形或纤维状水凝胶中的成纤维细胞会发生 Ca 信号转导。我们发现，与嵌入无定形聚乙醇酸聚合物（SLO-PVA）水凝胶中的细胞相比，嵌入纤维胶原水凝胶中的细胞的细胞信号转导空间范围更大。此外，对于嵌入胶原中的成纤维细胞，我们发现与径向轴相比，相对于圆周轴极化的细胞具有更高的细胞机械敏感性范围。相比之下，嵌入 SLO-PVA 中的成纤维细胞没有显示出与方向有关的机械敏感性。嵌入水凝胶并在无钙培养基中培养的成纤维细胞没有显示出空化诱导的力学转导；这暗示了基于跨膜 Ca 转运的钙信号转导。这项研究证明了单个激光生成的空化泡在具有光学显微镜的同时进行成像的 3D 水凝胶组织模型中提供局部非侵入性脉冲机械刺激的实用性。 意义声明：目前，对于 3D 组织支架内细胞对机械刺激的敏感性进行非侵入性实时评估的方法有限。我们描述了一种原始方法，该方法利用标准激光扫描显微镜系统中的脉冲激光微束生成单个空化泡，以向 3D 纤维状和无定形水凝胶中的细胞提供脉冲机械刺激。使用该技术，我们测量了嵌入无定形和纤维状水凝胶中的原代人真皮成纤维细胞的细胞机械敏感性，从而提供了一种有用的方法来研究 3D 生物材料中的细胞力学转导。此外，我们的方法在标准光学显微镜中的实施使其适合细胞力学转导研究人员广泛采用，并为基于细胞机械信号的生物材料的高通量评估开辟了可能性。
