title: Investigating the effects of nanovibrational stimulation on dermal fibroblasts
Creators: name: Antanaviciute, Egle Morta
Publisher: name: University of Glasgow
Year: 2023

description: Vibration with 1000 Hz frequency, 0.12 g acceleration and 30 nm displacement has been demonstrated to promote mesenchymal stem cell osteogenesis in 2D and 3D cultures without requiring supplementation with biochemical osteogenic fate inducers. This indicates that it could potentially be applied as an in vivo mechanotherapy to promote bone regeneration and treat delayed fracture unions or bone tissue disorders, such as osteoporosis. Applied to the exterior of the body, nanoscale vibrational waves would propagate through the soft tissues surrounding the target bone site, stimulating the cells within those tissues. However, these vibration parameters are highly unusual in the context of other vibrational stimulation studies, and little is known about how they may affect other cell types. Fibroblasts are the most common cell type in the soft connective tissues, where they play a vital role in tissue repair after injury. Fibroblast behaviour during the process of wound healing is regulated by complex interactions between signalling pathways stimulated by biochemical and biomechanical factors. Applied mechanical stimulation could interfere with these pathways and potentially lead to undesirable outcomes, such as impaired wound healing or excessive matrix deposition and fibrosis. This thesis aims to investigate how fibroblasts respond to nanovibrational stimulation in 2D and 3D culture conditions and in presence and absence of pro-fibrotic growth factor transforming growth factor-β1 (TGFβ1). The features important for fibroblast function during wound healing were examined, including proliferation, contractility, synthesis of collagen I, collagen III and αsmooth muscle actin, as well as gene expression of endogenous TGFβ1 and inflammatory factors interleukin-6 and osteopontin. A liquid chromatography-mass spectrometry-based metabolomics experiment was performed to detect any alterations in fibroblast metabolome in response to nanovibrational stimulation, followed by RNA sequencing to evaluate its effects on gene transcription. Finally, levels of reactive oxygen species, apoptosis, and DNA damage, all of which play a role in the development of fibrotic diseases, were investigated using flow cytometry and alkaline comet assay. It was discovered that fibroblasts are sensitive to nanovibrational stimulation, but the effects under the basal conditions are subtle and unlikely to cause undesirable fibroblast activity in healthy tissues. However, nanovibration had potentially negative outcomes on the fibroblast phenotype in 3D when applied in the presence of TGFβ1, increasing DNA damage and gene expression of inflammatory factor osteopontin. These outcomes were not observed in 2D cultures, where indications of anti-fibrotic effects of nanovibration were observed, suggesting that the effects of nanovibrational stimulation in pro-fibrotic conditions may depend on the mechanical properties of the matrix. Overall, this work presents the first investigation of the fibroblast response to nanovibrational stimulation and highlights the potential benefits and detriments of the treatment.