Mechanics of Biological Systems and Materials and the Mechanics of Composite, Hybrid & Multifunctional Materials, Vol. 3

Conference Proceedings of the Society for Experimental Mechanics Series Series Editor Kristin B. Zimmerman Society for Experimental Mechanics, Inc., Bethel, USA i

The Conference Proceedings of the Society for Experimental Mechanics Series presents early findings and case studies from a wide range of fundamental and applied work across the broad range of fields that comprise Experimental Mechanics. Series volumes follow the principle tracks or focus topics featured in each of the Society’s two annual conferences: IMAC, A Conference and Exposition on Structural Dynamics, and the Society’s Annual Conference & Exposition and will address critical areas of interest to researchers and design engineers working in all areas of Structural Dynamics, Solid Mechanics and Materials Research. ii

Karen Kasza • Jonathan B Estrada • Alexander McGhee • Kunal Mishra • Michael Keller Editors Mechanics of Biological Systems and Materials and the Mechanics of Composite, Hybrid & Multifunctional Materials, Vol. 3 Proceedings of the 2025 Annual Conference on Experimental and Applied Mechanics River Publishers

Published, sold and distributed by: River Publishers Broagervej 10 9260 Gistrup Denmark www.riverpublishers.com ISBN 97887-438-0829-9 (Hardback) ISBN 97887-438-0834-3 (eBook) https://doi.org/10.13052/97887-438-0829-9 Conference Proceedings of the Society for Experimental Mechanics An imprint of River Publishers © The Society for Experimental Mechanics, Inc. 2025 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, or reproduction in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Preface Mechanics of Biological Systems and Materials and the Mechanics of Composite, Hybrid & Multifunctional Materials represents one of five volumes of technical papers presented at the 2025 SEM Annual Conference & Exposition on Experimental and Applied Mechanics organized by the Society for Experimental Mechanics and held in Milwaukee, WI, June 2-5, 2025. The complete Proceedings also includes volumes on: Dynamic Behavior of Materials; Advancement of Optical Methods & Digital Image Correlation in Experimental Mechanics; Fracture, Fatigue, Failure, Damage Evolution and Thermomechanics & Infrared Imaging; and Mechanics of Additive & Advanced Manufacturing, Inverse Methods and Machine Learning. Each collection presents early findings from experimental and computational investigations on an important area within Experimental Mechanics, the Mechanics of Biological Systems and Materials, Micro-and Nanomechanics and other experimental and applied mechanics such as research in progress. The Biological Systems and Materials segment of this volume summarizes the exchange of ideas and information among scientists and engineers involved in the research and analysis of how mechanical loads interact with the structure, properties and function of living organisms and their tissues. The scope includes experimental, imaging, numerical and mathematical techniques and tools spanning various length and time scales. This symposium at the Annual Meeting of the Society for Experimental Mechanics provides a venue where state-of-the-art experimental methods can be leveraged in the study of biological and bio-inspired materials, traumatic brain injury, cell mechanics and biomechanics in general. A major goal of the symposium was for participants to collaborate in the asking of fundamental questions and the development of new techniques to address bio-inspired problems in society, human health, and the natural world. The 2025 Symposium is the 15th International Symposium on the Mechanics of Biological Systems and Materials. The organizers would like to thank all the speakers and staff at SEM for enabling a successful program. This volume also includes papers presented from the 11th International Symposium on the mechanics of composite, hybrid & multifunctional materials. These papers highlight how the commercial market for composite continues to expand with a wide range of applications from sporting equipment to aerospace vehicles. This growth has been fueled by new material developments, greater understanding of material behaviors, novel design solutions, and improved manufacturing techniques. The broad range of applications and the associated technical challenges require an increasingly multidisciplinary and collaborative approach between the mechanical, chemical, and physical sciences to sustain and enhance the positive impact of composites on the commercial and military sectors. New materials are being developed from recycled source materials, leading to composites with unique properties and more sustainable sources. Existing materials are also being used in new and critical applications, which requires a deeper understanding of material behaviors and failure mechanisms on multiple length and time scales. In addition, the unique properties of composites present many challenges in manufacturing and in joining with other materials. New testing methods must be developed to characterize the novel composite properties, to evaluate application and product life cycle performance, as well as to evaluate impacts and merits of new manufacturing methods. This segment of the volume presents early research findings from experimental and computational investigations related to the processing, characterization, and testing of composite, hybrid, and multifunctional materials. Editors: Karen Kasza, Columbia University, NY, USA; Jonathan B Estrada, University of Michigan, MI, USA; Alexander McGhee, University of Arizona, AZ, USA; Kunal Mishra, Corning Incorporated, NY, USA; Michael Keller, University of Tulsa, OK, USA v

Contents 1 Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells 1 Carmine Pappalettere and Giovanni Pappalettera 2 A Bone Remodeling Lab-on-a-Chip to Study Periprosthetic Osteolysis: Early Evidence that Osteocyte Signaling Contributes to Increased Osteoclast Resorption 9 M. M. Saunders 3 The Stress Relaxation Response of the Porcine Descending Aorta under Combined Normal and Torsional Loadings 19 Luc Nguyen, Abdelrahman Youssef, Calvin Nguyen, Kirtan Patel, Jack Luce, Benjamin Tijerina, and Chandler C. Benjamin 4 Investigating the Interphase in Hydroxyl-Terminated Polybutadiene (HTPB) Composites via Dynamic Mechanical Analysis and Atomic Force Microscopy 29 Jarred G. Tramell, Emily K. Hockey, Marcel M. Hatter, and Jesus O. Mares 5 Novel Experiment Design to Investigate High-Rate Shear Fracture of Composites 47 Elias Gerstein, Tyler Robertson, Andrew Baumgardner, Paul Custodio, and Nathan Spulak 6 Experimental and Numerical Analysis of Dynamic Behavior of Cork Material 51 Veronica Ilari, Carlo Sabbatini, and Marco Sasso 7 Unexpected Time Dependence in Nanofibrillated Cellulose Gels 59 Samir Patel and Jacob Notbohm 8 Biofilm Adhesion on TiAl6V4 using Laser Spallation Technique 65 Sahar Afshari and Martha E. Grady 9 Towards Single Particle Tracking in Simulated Microgravity-Grown Biofilms 69 William T. Anderson, Ross Rodriguez, Austin Stallings, Tony Butera, Emmabeth Parrish Vaughn, and Martha E. Grady vii

Chapter 1 Chapter 1 On the Detection and Quantification of Nonlinearity via Statistics of the Gradients of a Black-Box Model Georgios Tsialiamanis and Charles R. Farrar Abstrac t Detection and identification of nonlinearity is a task of high importance for structural dynamics. On the one hand, identifying nonlinearity in a structure would allow one to build more accurate models of the structure. On the other hand, detecting nonlinearity in a structure, which has been designed to operate in its linear region, might indicate the existence of damage within the structure. Common damage cases which cause nonlinear behaviour are breathing cracks and points where some material may have reached its plastic region. Therefore, it is important, even for safety reasons, to detect when a structure exhibits nonlinear behaviour. In the current work, a method to detect nonlinearity is proposed, based on the distribution of the gradients of a data-driven model, which is fitted on data acquired from the structure of interest. The data-driven model selected for the current application is a neural network. The selection of such a type of model was done in order to not allow the user to decide how linear or nonlinear the model shall be, but to let the training algorithm of the neural network shape the level of nonlinearity according to the training data. The neural network is trained to predict the accelerations of the structure for a time-instant using as input accelerations of previous time-instants, i.e. one-step-ahead predictions. Afterwards, the gradients of the output of the neural network with respect to its inputs are calculated. Given that the structure is linear, the distribution of the aforementioned gradients should be unimodal and quite peaked, while in the case of a structure with nonlinearities, the distribution of the gradients shall be more spread and, potentially, multimodal. To test the above assumption, data from an experimental structure are considered. The structure is tested under different scenarios, some of which are linear and some of which are nonlinear. More specifically, the nonlinearity is introduced as a column-bumper nonlinearity, aimed at simulating the effects of a breathing crack and at different levels, i.e. different values of the initial gap between the bumper and the column. Following the proposed method, the statistics of the distributions of the gradients for the different scenarios can indeed be used to identify cases where nonlinearity is present. Moreover, via the proposed method one is able to quantify the nonlinearity by observing higher values of standard deviation of the distribution of the gradients for lower values of the initial column-bumper gap, i.e. for “more nonlinear” scenarios. Keyword s Structural health monitoring (SHM) · Structural dynamics · Nonlinear dynamics · Machine learning · Neural networks 1.1 Introduction In the pursuit of making everyday life safer, humans have extensively tried to model the environment around them. Structures are an important part of the environment, in which humans live. They are man-made and should be safe throughout their lifetime. Structures are exposed to numerous environmental factors, which may cause them to fail. Moreover, during operation, structures are subjected to dynamic loads, which, in time, may cause failure. Such failures will most probably result in economic damage to society and may even result in loss of human lives. Therefore, for the purpose of maintaining structures safe, the field of structural health monitoring (SHM) [1] has emerged. G. Tsialiamanis ( ) Dynamics Research Group, Department of Mechanical Engineering, University of Sheffield, Sheffield, UK e-mail: g.tsialiamanis@sheffield.ac.uk C. R. Farrar Engineering Institute, MS T-001, Los Alamos National Laboratory, Los Alamos, NM, USA e-mail: farrar@lanl.gov © The Society for Experimental Mechanics, Inc. 2024 M. R. W. Brake et al. (eds.), Nonlinear Structures & Systems, Volume 1, Conference Proceedings of the Society for Experimental Mechanics Series, https://doi.org/10.1007/978-3-031-36999-5_1 1 Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells Carmine Pappalettere and Giovanni Pappalettera Abstract In recent years, cancer research has advanced significantly, resulting in better treatments for various tumour types and extended patient life expectancy. Nevertheless, there is still considerable interest in alternative clinical approaches. A major challenge is creating treatments that precisely target tumours while preserving healthy tissue. Research has shown that low-intensity ultrasound can selectively harm tumour cells due to their unique mechanical properties compared to healthy cells. This paves the way for a technique called Onco-Ultrasound-Tripsy, OUT, which involves using ultrasound vibrations to induce selective mechanical damage to tumour cells. Many advancements have been observed in this context and this survey aims to present several results related to the employment of such approach with applications to lymphoma, breast cancer, osteosarcoma and melanoma cells. Keywords Cancer treatment · Ultrasound· Cellular fatigue · Selective cellular damage · Onco-Ultrasound-Tripsy Introduction The use of ultrasound in medicine dates back to 1942 when Karl and Friederich Dussik attempted to adapt industrial techniques for detecting metal flow to visualize intracranial structures in real time [1]. Around the same time, Raimar Pohlman, influenced by Paul Langevin’s discovery that fish reacted strongly to ultrasound, developed a physiotherapy protocol at Charite´ in Berlin [2]. This protocol set a power density limit of 5 W/cm2 and required continuous transducer movement during treatment. In 1942, Lynn & Putnam [3] pioneered the use of ultrasound for targeting brain tissue in animals, marking an early milestone in therapeutic ultrasound. Their equipment leveraged the piezoelectric properties of crystals first discovered by Pierre and Jacques Curie sixty years earlier [4]. They also applied Gruetzmacher’s technique, which involved grounding the surface of a vibrating quartz plate to create a concave curvature, similar to a mirror, allowing ultrasound waves to be focused on a precise location. Since then, ultrasound has been utilized in treating various conditions, including bone fractures, osteoporosis, thrombosis, glaucoma, nerve injuries, skin wounds, and cancer either as a standalone therapy or in combination with drugs [5]. Over time, ultrasound technology has seen major advancements, including miniaturization, improved functionality, more sophisticated control systems, and a wider array of probes and actuators to support expanding medical applications [6]. Some treatments have been approved for clinical use, while others remain in development. Significant progress has been made in tumor treatment using High-Intensity Focused Ultrasound, HIFU [7, 8]. HIFU directs high-energy ultrasound waves to a small target area, preserving the surrounding tissue. The ablated tissue volume typically measures 1–3 mm (transverse) ×8–15 mm (along the beam axis) after a single exposure. The two primary mechanisms behind HIFU-induced tissue damage are mechanical energy conversion into heat and cavitation. Additionally, low-intensity ultrasound, with drug assistance, has been investigated for anti-tumor applications, including sonodynamic therapy, which combines low-intensity ultrasound with a chemotherapeutic agent (sonosensitizer) [9]. This approach (called LIPUS) holds promise due to its ability to selectively target cancer cells while preserving healthy tissue. Carmine Pappalettere · Giovanni Pappalettera Department of Mechanics, Mathematics and Management, Polytechnic University of Bari, 70126 Bari, Italy e-mail: carmine.pappalettere@poliba.it; giovanni.pappalettera@poliba.it © The Author(s), under exclusive license to River Publishers 2025 Karen Kasza et al. (eds.), Mechanics of Biological Systems and Materials and the Mechanics of Composite, Hybrid & Multifunctional Materials, Vol. 3, Conference Proceedings of the Society for Experimental Mechanics Series, https://doi.org/10.13052/97887-438-0829-9 1

2 C. Pappalettere and G. Pappalettera In general, when ultrasound interacts with tissue, it produces three main effects: cavitation, hyperthermia, and resonant excitation [10, 11]. Unlike cavitation and thermal effects, resonant excitation operates based on the different resonance frequencies of healthy and cancerous cells, allowing for targeted therapeutic action. The so called Onco-Ultrasound-Tripsy approach (OUT) aims to exploit the mechanical properties of cancer cells for selective damage, emphasizing the potential of low-intensity ultrasound to inhibit cavitation and thermal effects while promoting resonant and fatigue ones. Changes in resonance frequency due to modifications in mechanical properties, such as a demonstrated reduction of up to 70% in the rigidity of cancer cells [12], support this approach. After having had the idea many years ago and having studied since the 90s the possibility of selectively killing tumor cells by exploiting their different resonance frequency compared to healthy cells, Pappalettere and his collaborators from Polytechnic University of Bari began 20 years ago to deepen the research on the topic. Afterwards, experimental activities were conducted on U937 tumor cells of a myelocytic lymphoma at the University of Fukuoka in Japan (2015) [11, 13–15] and on MCF7 breast tumor cells MCF10 cells at the University of Dundee in Scotland (2016) [16–21] and the corresponding healthy MCF10A at the University of Bari (2019) [22]. Then they continued at the Polytechnic of Bari in collaboration with the University of Foggia on MG-63 osteosarcoma cells (2017) [23–25] and with the University of Bari both on the same U937 cells studied in Japan and on the corresponding healthy CD3/CD8+ Lymphocytes cells and on red blood cells (2023) [26, 27]. Finally, recently, experiments have been conducted on HBL melanoma cells. Some more recent studies on U937 cancer cells have been developed and validated at the California Institute of Technology (CalTech) basing on the hypothesis that cellular damage to low intensity ultrasound exposure doesn’t occur in a quasi-static/low cycle fatigue regime (cellular lysis) but in high cycle fatigue regime (apoptosis) (2020) [28, 29] while additional results can be found in [30]. The aim of this review is to provide an overview of major results obtained by this approach addressing new challenges and new open questions. Materials and Methods All the analyses have been conducted in vitro by transferring the cells grown in culture into Petri dishes and sonicating them either from the bottom or from the top of the dish, depending on whether the cells were suspended or adherent. The probe to sonicate the cells was placed in direct contact with the dish’s surfaces, coupled with gel, and the dish was filled with culture medium to facilitate wave propagation and prevent air exposure. Ultrasound waves were generated using the SonoPore KTAC-4000 device (NEPA GENE, Chiba, Japan), capable of producing frequencies between 200 kHz and 5 MHz with a fine adjustment of 1 kHz. The device allows for adjustments in pulse repetition frequency (0.5 Hz to 100 Hz), output duty cycle (0% to 100%), exposure duration, and voltage (0 V to 60 V). The voltage setting directly influences the power output, depending on the specific probe connected. A KP-S20 flat probe (NEPA GENE, Chiba, Japan) with a 20 mm emission diameter was used. For cell counting, Trypsin/EDTA was used to detach the cells from the Petri dish, and the TC20 automated cell counter (Bio-Rad, USA) was employed. Trypan blue was applied to selectively stain dead cells by permeabilizing their membranes. Following this procedure, a 100% viability rate was observed for the living cells. Additionally, the TC20 system measured the cell size, which was found to range from 10 to 15µm in diameter. Cell mortality was calculated by Eq.1 cell mortality = live cells on control-live cells after treatment live cells on control Applications of Onco-Ultrasound-Tripsy (OUT) Application on Lymphoma Cells Ultrasound exposure was applied to the U937 human histiocytic lymphoma cell line. The U937 cell line, sourced from ECACC (catalog number 85011440, UK), was originally derived from the malignant cells of a 37-year-old Caucasian male diagnosed with histiocytic lymphoma. This monocytic leukemia model retains key monocytic features, including lysozyme secretion, esterase activity, and limited phagocytic capacity, consistent with its histiocytic lineage. U937 cells, widely classified as monocytic cells, were maintained at 37◦C in a humidified 5% CO2 incubator using RPMI-1640 medium (Euroclone, Italy) supplemented with 10% fetal bovine serum, L-glutamine, penicillin G, and streptomycin. The culture medium was refreshed every 24 hours to sustain a cell density between 5.0 ×105 and 1.0 ×106 cells/mL. Cells were expanded in T25

Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells 3 Fig. 1 Analysis of the observed mortality in human leukemic U937 cells was conducted both before (untreated) and immediately after (sonicated) ultrasound treatment. (Left) treatment at sweeping frequencies. (Right) Treatment at fixed frequencies. Results for 0 KHz are referred to untreated cells. flasks, harvested on the day of the experiment, and resuspended in fresh medium at a concentration of 1 million viable cells/mL. This step was implemented to minimize CO2 accumulation, which may interfere with ultrasound energy propagation and subsequently impact cell mortality outcomes. U937 cells were seeded in 12-well plates at a density of 106 cells/mL in a total volume of 2 mL, with ultrasound exposure applied using a coupling gel. First evaluations were done by exposing cell to sweeping frequency both in descending and ascending mode [11] by sweeping frequency from 400 kHz to 620 kHz and viceversa at 0.5 Hz, 10 Hz and 50 Hz burst rate. It was observed that best results are obtained (Fig. 1 left) by working in descending mode at 10 Hz burst rate. In that situation, in fact, 79.8 % of cell mortality was observed. After these treatments the cells that remained alive were put in an incubator for 6 hours and the calculation of the living ones was re-performed. It turned out that after this time the total mortality of the U937 cancer cells rose to about 90% thus showing that the ultrasonic treatment also activated a mechanism of apoptosis of the diseased cells [11]. In a second set of experiments [26, 27] sonication of U937 cells was conducted at constant frequency values of 400, 600, 800, and 1000 kHz, employing voltage levels of 30, 40, 50, and 60 V, a 50% duty cycle, and a 10 Hz burst rate. Exposure durations of 180 and 360 seconds were assessed to identify the optimal conditions for reducing cancer cell viability. Cell proliferation was monitored for seven days following treatment, with cell counts recorded at 0, 48, 120, and 168 hours. To ensure nutrient availability and prevent any impact on cell growth, the medium was refreshed every 48 hours. A preliminary series of experiments was performed to assess the effect of frequency variation on U937 leukemic cells. The cells were sonicated for 180 seconds at a constant voltage of 60 V, with a 50% duty cycle and a 10 Hz burst rate, using frequencies of 400, 600, 800, and 1000 kHz (Figure 1 right). Cell mortality, measured immediately after ultrasound exposure, was significantly higher in cells treated with a 1 MHz frequency (82.73%±2.34) compared to untreated controls. Notably, U937 cells exposed to 400 kHz exhibited a mortality rate approximately seven times higher than untreated cells (26.24% vs. 3.50%), indicating that even lower frequencies can negatively impact cell viability. These results suggest that among the tested sonication conditions, the 1 MHz frequency induces the highest mortality in this tumor cell type. It is important to note that mortality was calculated by Eq.1 and may be influenced by background mortality in the control group. If additional biological factors contribute to control group mortality, this could result in an apparent negative mortality rate, as observed in the case of the 600 kHz treatment. Application on Breast Cancer Cells Sonication experiments were conducted using the breast cancer cell line MCF7 [22]. The cells were cultured in plastic T-75 flasks (Sigma-Aldrich, USA) in RPMI 1640 medium (Gibco Thermo Fisher, USA), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, USA) at 37◦C in a humidified incubator with 5% CO2. To investigate the effects of intensity and frequency, cell viability after sonication was evaluated using trypan blue dye exclusion tests on macroscopic samples. For this, the cells were plated in 35 mm Petri dishes and incubated for 24 hours before sonication experiments. Prior to the experiments, MCF7 cells had a confluence of over 90%, while MCF10 cells reached around 60% confluence. After sonication at various voltage/frequency settings, the cells were immediately washed with phosphate-buffered saline (PBS) and detached from the dish bottom by treating with 0.250 mL of 0.05% trypsin for 3

4 C. Pappalettere and G. Pappalettera minutes. Then, 1.75 mL of medium was added, mixed, followed by the addition of the previous supernatant, PBS, and trypan blue and finally centrifugate at 1200 rpm for 3 mins. The following sonication conditions were chosen: a sinusoidal wave in pulsed wave mode, with a pulse repetition frequency of 10 Hz, a 50% duty cycle, and 7 fixed frequencies of 400 kHz, 436 kHz, 472 kHz, 510 kHz, 546 kHz, 582 kHz, and 620 kHz. The tests were conducted either at a constant voltage of 60 V or at a constant power of 0.1 W. This power value represents the average output power across the frequency range of 400–620 kHz and also the median power among the 7 sonication frequencies. Results in terms of observed mortality are shown in Fig. 2. Fig. 2 Response in terms of cell mortality for the fixed voltage condition (green square) and the fixed power condition (red circles) Figure 2 shows the trend of cell mortality while varying the frequency. The two tested conditions show quite similar trends indicating that cell mortality is mostly correlated with frequency. More specifically cell mortality rises 10% to 15% at 400 kHz to 55% to 60% at 620 kHz. However, this increase is concentrated in the 540–620 kHz frequency range. Application on Osteosarcoma Cells The MG-63 osteosarcoma (OS) cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) [25]. Cells were seeded at a density of 8×104 cells per T25 flask and incubated at 37◦Cin a standard environment with 95% air and 5% CO2. To detach the cells, Trypsin/EDTA was used, after which the cells were re-plated in Petri dishes and returned to the incubator. Cell confluence was monitored using a Nikon Eclipse Ti-U inverted microscope with a C1 Digital Eclipse modular confocal system. Cells were exposed for 180 s at a fixed frequency in pulse mode, with a burst rate of 10 Hz, 50% duty cycle and 60 V voltage. In an initial phase, different frequencies were tested to identify the ones able to induce the lowest % of live cancer cells after sonication. Five emission frequencies were analyzed: 400, 510, 582, 800 and 1000 kHz. The power density was, in any case, lower than 5.4 W/cm2. After these preliminary tests, two frequencies were found associated with the lower percentage of living cells after sonication: 800 and 1000 kHz. The sonication tests for these two frequencies were then repeated on two different days with three replications of the same experimental conditions for each day. Moreover, we the growth index of the cells after ultrasound treatment in real time was analyzed. This was done by using a device able to measure variation in cellular impedance. In fact, an increment of the number of adherent cells results in an increment of the measured impedance and this can be used as an indicator of the cellular growth. These growth indexes were then compared with the growth index of the untreated cells. Experiments were carried out using the xCELLigence RTCA instrument (ACEA Biosciences, Inc., CA, USA), placed in a humidified incubator maintained at 37◦C with 95% air and 5% CO2.

Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells 5 Cells were exposed for 180 seconds at a fixed frequency in pulse mode, with a burst rate of 10 Hz, a 50% duty cycle, and 60 V voltage. Initially, different frequencies were tested to identify those that caused the lowest percentage of live cancer cells post-sonication. Five emission frequencies were evaluated: 400, 510, 582, 800, and 1000 kHz, with a power density always below 5.4 W/cm2. After these preliminary tests, two frequencies (800 and 1000 kHz) were found to result in the lowest percentage of surviving cells post-sonication. These two frequencies were then tested again on two separate days, with three replicates of the same experimental conditions for each day. Additionally, the cell growth index was monitored in real time following ultrasound treatment. This was achieved by using a device that measures changes in cellular impedance, where an increase in the number of adherent cells corresponds to a rise in impedance, serving as an indicator of cell growth. These growth indices were then compared to that of untreated cells. The experiments were conducted using the xCELLigence RTCA instrument (ACEA Biosciences, Inc., CA, USA), in a humidified incubator maintained at 37◦C with 95% air and 5%CO2. Figure 3 (Left) presents the percentage of dead cells following sonication for five different tested frequencies: 400, 510, 582, 800, and 1000 kHz. The evaluation of dead cells was carried out using Trypan blue assays, as dead cells are permeable to the dye and can thus be counted. This procedure was performed immediately after the sonication process, and the number of dead cells was compared with the initial number of treated cells to calculate the percentage shown in Figure 3. The data reveals a general increase in the percentage of dead cells as the frequency rises, particularly for frequencies above 600 kHz. The results at 400 kHz should be considered an outlier and warrant further investigation. The highest cancer cell mortality was observed at the two highest frequencies: 45% of cells were dead after sonication at 800 kHz, and 60% of cells were dead at 1000 kHz. Fig. 3 (Left) Percentage of dead cells after sonication at the five selected frequencies; (Middle) follow-up of the live cells percentage two days after the sonication; (Right) Recalculation of the live cells percentage including considerations about cell/fragment diameter. The experimental results from the following two days of testing for the selected frequencies of 800 and 1000 kHz are presented in Figure 3 (Middle). The sonication experiments confirm that 1000 kHz is the frequency that results in the lowest percentage of live cancer cells post-sonication, with an average value of under 36%. Further evaluation was conducted to investigate the possibility that live cells might be present in the culture medium after treatment, potentially detached from the bottom of the dish by the ongoing pressure wave but not damaged by the ultrasound. To rule out this hypothesis, which could result in an overestimation of the number of killed cells, the culture medium was centrifuged to collect the pellet. The pellet was then cultured, and the corresponding growth index was assessed. For all the analyzed dishes, the growth index was found to be zero, indicating the absence of live cells in the medium. Results of this new calculation are presented in Fig. 3 (Right) where analysis is restricted only to cells in the range between 12 and 25µm. Application on Melanoma Cells In order to assess the effects of low-intensity ultrasound on melanoma cells, the HBL metastatic cell line was utilized, known for its high proliferative capacity. These cells were derived from human biopsies at the Department of Translational Biomedicine and Neuroscience (DiBraiN) at the University of Bari “Aldo Moro.” Cells were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM H.G.), supplemented with 10% fetal bovine serum (FBS), 1% L-Glutamine (Euroclone), and 1% Penicillin/Streptomycin (Full DMEM/HG). All cultures were maintained in an incubator at 37◦C with 95% humidity and 5% CO2.

6 C. Pappalettere and G. Pappalettera The culture medium, containing a pH indicator, was refreshed every two to three days based on the requirements of the cell culture. Cells were seeded in four 12-well plates at a density of 100,000 cells per well. These were incubated until reaching a confluence of 70–80%, a prerequisite for initiating ultrasound treatment. Confluence was assessed via optical microscopy. To analyze cell viability and mortality pre- and post-treatment, two control wells were included for each condition. All plates were exposed to ultrasound using the following sonication parameters: Duty Cycle 50%, Burst Rate 10 Hz, Voltage 40 V, Frequency 1 MHz, and treatment duration of 180 s. These parameters were selected based on preliminary investigations identifying the optimal frequency for inducing cell mortality. Following treatment, melanoma cells were detached from the wells, washed with 1X PBS, treated with 1X TrypsinEDTA solution (0.05%/0.02%), and centrifuged at 1100 rpm for 5 minutes. A cell count was subsequently performed for both control and treated wells using the TC-20 automated cell counter (Bio-Rad) in the presence of Trypan Blue vital dye. Cells were resuspended in 1 mL of complete DMEM/HG medium, and 10 µL of the resulting cell suspension was mixed with 10 µL of Trypan Blue (1:1 dilution). The mixture was then loaded into the counting slide chamber and analyzed using the TC-20 instrument. Cell mortality was evaluated following Eq.1 The findings for HBL melanoma cells are summarized in Figure 4. As depicted, plate #1 exhibited a mortality rate of 100%, while plate #3 and plate #4 showed mortality rates of 83.5% and 85.5%, respectively. However, plate #2 displayed a markedly different outcome, with a significantly lower mortality rate of only 3.33%. Fig. 4 Evaluation of the cell mortality in the four tested plates The results obtained for HBL melanoma cells appear somewhat inconsistent and warrant further investigation. While three of the analyzed plates exhibited significant mortality rates, plate #2 showed no such effect. This discrepancy highlights the need for additional studies to identify potential factors underlying these findings. A closer examination of plate #2’s specific conditions is necessary. As described before in this case, cells remained in culture for four days, reaching 100% confluence and displaying a slightly acidified culture medium compared to the previous day. A possible explanation for the lack of effectiveness could be alterations in the culture medium, which also serves as the propagation medium for sound waves. Such changes may have interfered with the resonance regime through absorption and dispersion mechanisms. Furthermore, additional research is required to investigate other potential contributing factors, whether biological or ultrasound-related, that may have influenced the high mortality observed in plates #1, #3, and #4. Ultimately, these findings should be validated through studies with a larger statistical sample.

Review of Recent Results of Onco-Ultrasound-Tripsy on Cancer Cells 7 Conclusion The overview presented in this paper is testifying the capability of the onco-ultrasound-tripsy approach to face with different kinds of cancer cells at least for what is related to the in-vitro operations. Even if effectiveness of the approach is strictly related to the specific targeted cancer cells in all the presented situation it was found a response in terms of mortality of the cancer cells after exposure. Even more interestingly this response exhibits a frequency dependent behaviour which is fundamental to guarantee selectivity of the approach. Starting from this a lot of efforts are still required to completely understand the whole mechanism of ultrasound-cell interaction, to model them, to understand the role of the cellular microenvironment paving the way of possible preliminary in-vivo investigations. It worth underlining, however, that first evidence of applicability in vivo were shown in [31] where it was observed the capability of the low intensity ultrasound approach in inhibiting cancer growth in mice. Acknowledgments The Authors would like to thank very much all the researchers who have contributed to the research from The University of Fukuoka (Japan), the University of Dundee (UK), The University of Foggia, The University of Bari and, in particular, from the Polytechnic University of Bari (in alphabetic order): S.N. Barile, C. Casavola, C. Cianci, M.A. Ivone and L. Lamberti. This work was partly supported by the Italian Ministry of University and Research under the Programme “Department of Excellence” Legge 232/2016 (Grant No. CUP - D93C23000100001)” References 1. Dussik KT. Weitere Ergebnisse der Ultraschalluntersuchung bei Gehirnerkrankungen. Acta Neurochir (Wien). 1952; 2:379–401. 2. Giugno A, Maugeri R, Graziano F, Gagliardo C, Franzini A, Catalano C, et al. 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Chapter 2 Chapter 1 On the Detection and Quantification of Nonlinearity via Statistics of the Gradients of a Black-Box Model Georgios Tsialiamanis and Charles R. Farrar Abstrac t Detection and identification of nonlinearity is a task of high importance for structural dynamics. On the one hand, identifying nonlinearity in a structure would allow one to build more accurate models of the structure. On the other hand, detecting nonlinearity in a structure, which has been designed to operate in its linear region, might indicate the existence of damage within the structure. Common damage cases which cause nonlinear behaviour are breathing cracks and points where some material may have reached its plastic region. Therefore, it is important, even for safety reasons, to detect when a structure exhibits nonlinear behaviour. In the current work, a method to detect nonlinearity is proposed, based on the distribution of the gradients of a data-driven model, which is fitted on data acquired from the structure of interest. The data-driven model selected for the current application is a neural network. The selection of such a type of model was done in order to not allow the user to decide how linear or nonlinear the model shall be, but to let the training algorithm of the neural network shape the level of nonlinearity according to the training data. The neural network is trained to predict the accelerations of the structure for a time-instant using as input accelerations of previous time-instants, i.e. one-step-ahead predictions. Afterwards, the gradients of the output of the neural network with respect to its inputs are calculated. Given that the structure is linear, the distribution of the aforementioned gradients should be unimodal and quite peaked, while in the case of a structure with nonlinearities, the distribution of the gradients shall be more spread and, potentially, multimodal. To test the above assumption, data from an experimental structure are considered. The structure is tested under different scenarios, some of which are linear and some of which are nonlinear. More specifically, the nonlinearity is introduced as a column-bumper nonlinearity, aimed at simulating the effects of a breathing crack and at different levels, i.e. different values of the initial gap between the bumper and the column. Following the proposed method, the statistics of the distributions of the gradients for the different scenarios can indeed be used to identify cases where nonlinearity is present. Moreover, via the proposed method one is able to quantify the nonlinearity by observing higher values of standard deviation of the distribution of the gradients for lower values of the initial column-bumper gap, i.e. for “more nonlinear” scenarios. Keyword s Structural health monitoring (SHM) · Structural dynamics · Nonlinear dynamics · Machine learning · Neural networks 1.1 Introduction In the pursuit of making everyday life safer, humans have extensively tried to model the environment around them. Structures are an important part of the environment, in which humans live. They are man-made and should be safe throughout their lifetime. Structures are exposed to numerous environmental factors, which may cause them to fail. Moreover, during operation, structures are subjected to dynamic loads, which, in time, may cause failure. Such failures will most probably result in economic damage to society and may even result in loss of human lives. Therefore, for the purpose of maintaining structures safe, the field of structural health monitoring (SHM) [1] has emerged. G. Tsialiamanis ( ) Dynamics Research Group, Department of Mechanical Engineering, University of Sheffield, Sheffield, UK e-mail: g.tsialiamanis@sheffield.ac.uk C. R. Farrar Engineering Institute, MS T-001, Los Alamos National Laboratory, Los Alamos, NM, USA e-mail: farrar@lanl.gov © The Society for Experimental Mechanics, Inc. 2024 M. R. W. Brake et al. (eds.), Nonlinear Structures & Systems, Volume 1, Conference Proceedings of the Society for Experimental Mechanics Series, https://doi.org/10.1007/978-3-031-36999-5_1 1 A Bone Remodeling Lab-on-a-Chip to Study Periprosthetic Osteolysis: Early Evidence that Osteocyte Signaling Contributes to Increased Osteoclast Resorption M. M. Saunders Abstract Total joint replacement surgeries are highly prevalent throughout the United States. However, it is estimated that within 10 years of surgery, 20% to 30% of patients will present with a complication known as periprosthetic osteolysis (PPOL), or a degradation of the bone surrounding the implant [1]. Patients experiencing PPOL often require revision surgeries, which are associated with higher rates of morbidity and mortality [2]. Thus, there is a great need for therapeutic or pharmaceutical approaches that could delay the need for revision arthroplasties. Currently, the mechanisms that drive this osteolytic process are not fully understood. However, evidence suggests that particulate debris generated from repetitive wear of implant surfaces leads to inflammation within the joint, which ultimately disrupts bone remodeling. Remodeling is a lifelong metabolic process involving three bone cell types (osteocytes, osteoclasts, and osteoblasts) that maintains bone homeostasis by tightly coordinating bone resorption and formation in a dynamic loading environment. Furthermore, mechanical load has also been shown to play a crucial role in bone remodeling and long-term implant stability. The goal of this project was to develop a lab-on-a-chip (LOC) platform that recapitulates the bone dynamic microenvironment and allows for analysis of multicellular signaling processes that lead to PPOL. This invitrosystem will allow us to study complex signaling between osteocytes, osteoclasts and osteoblasts while eliminating confounding factors present in in vivo studies. Following development, the LOC platform was utilized to preliminarily investigate the effects of particulate debris in a dynamic loading environment on osteocyte signaling and the subsequent effect on osteoclast resorption. Keywords Lab-on-a-Chip· Periprosthetic Osteolysis · Bone Remodeling· Mechanobiology Introduction One of the leading causes of late-stage joint failure is aseptic loosening, a condition in which the bond between the bone and the implant fails in the absence of infection. This loosening is often associated with PPOL, or degradation of the bone tissue surrounding the implant. While the mechanisms that drive these processes are not fully understood, many studies have shown that particulate debris generated from wear of the implant surfaces triggers an inflammatory response within the joint, which ultimately leads to bone loss [3–4]. Additionally, long-term implant survival is dependent on the physical activity of the patient. This highlights the crucial role of mechanical load on bone metabolism and long-term implant stability. A more thorough and comprehensive understanding of the underlying mechanisms that regulate load-induced bone remodeling and implant debris-induced osteolysis may lead to novel interventions that mitigate late-stage joint failure. Background An investigation of the mechanisms responsible for PPOL requires a deeper understanding of the complex multicellular signaling cascades that regulate bone regeneration. This lifelong metabolic process, known as bone remodeling, is necessary M. M. Saunders College of Engineering and Computing, Miami University, Oxford, OH 45056 e-mail: saundem9@miamioh.edu © The Author(s), under exclusive license to River Publishers 2025 9 Karen Kasza et al. (eds.), Mechanics of Biological Systems and Materials and the Mechanics of Composite, Hybrid & Multifunctional Materials, Vol. 3, Conference Proceedings of the Society for Experimental Mechanics Series, https://doi.org/10.13052/97887-438-0829-9 2

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