2D and 3D Mechanobiology in Human and Nonhuman Systems

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2D and 3D Mechanobiology in Human and Nonhuman Systems Kristin M. Warren,†,§ Md. Mydul Islam,‡,§ Philip R. LeDuc,*,† and Robert Steward, Jr.*,‡ †

Departments of Mechanical Engineering, Biomedical Engineering, Computational Biology, and Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ‡ Department of Mechanical and Aerospace Engineering and Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida 32827, United States ABSTRACT: Mechanobiology involves the investigation of mechanical forces and their effect on the development, physiology, and pathology of biological systems. The human body has garnered much attention from many groups in the field, as mechanical forces have been shown to influence almost all aspects of human life ranging from breathing to cancer metastasis. Beyond being influential in human systems, mechanical forces have also been shown to impact nonhuman systems such as algae and zebrafish. Studies of nonhuman and human systems at the cellular level have primarily been done in two-dimensional (2D) environments, but most of these systems reside in three-dimensional (3D) environments. Furthermore, outcomes obtained from 3D studies are often quite different than those from 2D studies. We present here an overview of a select group of human and nonhuman systems in 2D and 3D environments. We also highlight mechanobiological approaches and their respective implications for human and nonhuman physiology. KEYWORDS: biomechanics, fluid flow, stretch, biofilms, mammalian cells, developmental biology

1. INTRODUCTION 1.1. Two-Dimensional versus Three-Dimensional Systems. At the cellular level, most human and nonhuman systems reside in a 3D environment. A tremendous number of examples of human and nonhuman systems in 3D environments may be found within nature, ranging from the arteries and lungs of the human body to zebrafish embryos or microbial biofilms. However, most studies probing these 3D systems have been done in 2D environments, which often consist of single or multicellular systems cultured on flat substrates.1,2 While 2D studies have been primarily used in the past because of their greater accessibility and decreased technical difficulties relative to 3D studies,1,2 advances in the fields of photolithography, 3D printing, soft lithography, and microcontact printing have enabled researchers to address some of the technical challenges associated with 3D studies. This has allowed researchers to begin to mimic organ-level functions and the complex geometric constraints experienced by both human and nonhuman systems. For example, Huh et al.3 used microfabrication approaches to develop a biomimetic system that mimicks the alveolar−capillary interface of the human lung. There has been a considerable increase in 3D studies, and the results obtained from these studies have been shown to be unique compared with their 2D counterparts. For example, Hakkinen et al.4 showed fibroblasts cultured in a 3D environment to be more spindle-shaped and to exhibit fewer protrusions and actin fibers compared with fibroblasts in a 2D environment. Imamura et al.5 showed that BT-549, BT-474, and T-47D cancer cell lines cultured in a 3D environment to © XXXX American Chemical Society

exhibit increased resistance to known cancer drugs compared with those in a 2D environment. To further understand the differences between 2D and 3D environments, comparing and contrasting the responses of human and nonhuman biological systems is essential, as this will allow researchers to learn a tremendous amount about human and nonhuman physiology, especially in mechanobiology. 1.2. 2D and 3D Biomechanics. While a variety of influences on biological systems exist, including chemical, electrical, and thermal effects, mechanical effects have gained greater attention recently, prompting more in-depth investigations into the field of biomechanics.6−9 Biomechanics can be defined as the study of mechanics applied to biology. Within the area of mechanics, subjects may include mechanical responses and properties such as stress, strain, strength, pressure, and structure. Applying these topics to biology helps to determine functionality within organisms, predict effects due to changes within the organism, and engineer solutions or methodologies for advancing the field. Y. C. Fung, who wrote one of the first well-known books on biomechanics, defined generalized biomechanics problems into prescribed steps that can be applied to a diversity of human and Special Issue: Interfaces for Mechanobiology and Mechanochemistry: From 2-D to 3-D Platforms Received: December 11, 2015 Accepted: April 21, 2016

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DOI: 10.1021/acsami.5b12064 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces nonhuman systems in 2D and 3D environments.10 Those steps include (1) observing the anatomy, organization, and morphology of the organism; (2) determining the material’s mechanical properties; (3) deriving the governing equations of the system; (4) creating boundary conditions that accurately depict the material’s native environment; (5) analytically, experimentally, or numerically solving the boundary-value problem; (6) testing the solutions obtained from the boundary-value problem and making adjustments as needed; (7) validating the theoretical results with the experimental ones; and (8) using the validated theory to understand, predict, and explore similar problems for use in practical applications. As mentioned by Humphrey,10 Fung’s approach to biomechanics is utilized by many researchers in the field for the investigation of human and nonhuman systems. In addition to being important in nonhuman systems, examining biomechanics in human systems is equally as important, as mechanics has major implications in cardiovascular, neurological, and musculoskeletal disease.11,12 Within this field, however, the range of biomechanics topics is vast, and a thorough exploration of all these disciplines in both human and nonhuman systems is far beyond the scope of this paper. Therefore, we describe below a few specific examples of biomechanics that are particularly critical to biology, including biofluid mechanics, biotribology, injury biomechanics, and plant mechanics. 1.2.1. Biofluid Mechanics. Biofluid mechanics plays a major role in a wide range of applications, including advancing research in the analysis, diagnosis, and treatment of many cardiovascular diseases and complications. As one of the most studied biofluids, blood and its flow through the body continue to be investigated through many methods, including computational models that are used to develop artificial heart pumps, valves, and dialysis machines.13−15 A specific biofluid of interest is sickle-celled blood. Sickle cell anemia is an inherited blood disorder in which a patient’s red blood cells can be misshapen into a sickle shape, causing hemolysis (rupture) of these cells. Studying sickle cell flow and deformation may aid in the identification and evaluation of an effective therapy, thus preventing hemolysis.16 Sickle cell rheological studies, which utilize step 2 of Fung’s biomechanics problem-solving approach, have been performed using many biomechanical approaches to understand how sickle cells become misshapen (Figure 1). A primary cause of the sickling (or distortion) of red blood cells is hemoglobin S (HbS) polymerization.16 HbS molecular polymerization occurs once the cell is deoxygenated and hydrophobic residues are exposed and self-assemble into other hydrophobic fibers.16 Although this disfigurement is reversible, the cell membrane and cytoskeleton undergo significant structural damage in cycling from the normal shape to the sickled shape. This causes permanent deformation and changes in the rheological properties of the red blood cells,16 subsequently changing the overall mechanical properties of blood. For example, patients with sickle cell anemia have been shown to have increased blood viscosity, which has been suggested to be a major determining factor in the increased peripheral vascular resistance commonly observed in these patients.17 In addition, the physiological interactions between red blood cells and fluid flow may yield multiple 3D structural configurations, yet studies probing these interactions are often done in 2D environments. This is important as 2D biofluid mechanics studies may yield results that are in contrast or underestimated relative to 3D studies. For example, a tumor−

Figure 1. Biomechanical techniques for determining rheological properties of sickle cells: (A) concentric cylinder or Couette viscometer; (B) cone-and-plate viscometer; (C) microfluidics device; (D) filtration; (E) ektacytometry; (F) micropipette aspiration; (G) parallel-plate flow chamber. Reprinted with permission from ref 16. Copyright 2010 Annual Reviews.

platelet adhesion study revealed thrombin to stimulate tumor− platelet adhesion to a maximum of 4-fold in a 2D environment and a maximum of 413-fold in a 3D environment, suggesting that the 2D study significantly underestimated the importance of thrombin in tumor−platelet aggregation in vivo.18 1.2.2. Biotribology. Biotribology is the study of friction, lubrication, and wear within biological systems. Biotribology is useful in a range of areas, including arthroplasty, a surgical procedure necessary to prevent bone surfaces from constantly sliding against each other and causing wear and friction to occur at the contact interface, resulting in deleterious responses. Such responses may include thinning of the hip cartilage, potentially leading to a total hip replacement.19 Efforts have been made to utilize biotribology to decrease wear of implanted hip replacement devices in sliding contacts by increasing the amount of lubrication within the joint. The Stribeck curve, a graph describing the properties of friction between two surfaces, is often used to determine appropriate lubrication regimes that reduce friction between sliding contacts.20 In the case of total hip replacements, texturing the surface was shown to reduce the amount of friction by up to 50% and enhance wear resistance by trapping wear debris.20 At the cellular level, biotribological properties, such as friction coefficients of vascular smooth muscle cells (VSMCs), have been investigated and shown to be influenced by cytoskeletal depolymerization and cellular cross-linking agents such as glutaraldehyde.21 Friction properties at the cellular level are important not only in normal biological functions such as respiration, cell adhesion, and cell migration but also in pathology and implants. An example of friction properties in implants involves endovascular devices, which apply shear forces on VSMCs through endothelial denudation caused from stent placement.22 Creating a device that reduces the amount of friction would B


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from recent plant biomechanics studies have been equally as useful. For example, studies of the functional and structural properties of giant reed stems and the flapping mechanism of the bird-of-paradise flower have been utilized to develop modern textile and composite fabrication methods and a hingeless flapping device, respectively.34,35 The previous and recent studies demonstrate how plant mechanics has aided and will continue to aid in the understanding and development of more complex systems and processes. Furthermore, the 3D natural environments in nonhuman systems, such as biofilms, show complex mechanobiological responses that are not found in 2D single-cell-layer responses36,37 The importance of biomechanics in human and nonsystems is especially important in 2D and 3D areas. While most biological systems reside in a 3D environment, analysis of these systems can be done from either a 2D or 3D perspective. An example of a 2D analysis could consist of analyzing the in-plane strains associated with uniaxial stretching of a monolayer of mammalian cells,38 while an example of 3D analysis could involve the use of 3D traction force microscopy, which enables analysis of the in-plane and out-of-plane traction forces exerted by a cell embedded in a 3D matrix. 39 Additionally, developmental biology organisms are often modeled as 3D biomechanical systems since they consist of multiple layers of cells that have complex 3D structures. In this review, we present select human and nonhuman systems that have been studied in 2D or 3D environments and highlight important findings from a mechanobiology perspective for each system.

increase the ease of implantation. These interactions between cells and devices are 3D in terms of the cellular organization but have often been investigated through 2D modeling and culture approaches. However, despite the possible differences between 2D and 3D biotribology, 2D and 3D studies have been shown surprisingly to yield comparable results. For example, utilizing a novel microtribometer device, Cobb et al.22 determined the coefficient of friction for cultured human corneal epithelial cells to be about 0.05 ± 0.02, while Dunn et al.23 determined the coefficient of friction for a living mouse eye to be approximately 0.068 using a custom portable reciprocating microtribometer. 1.2.3. Injury Biomechanics. The field of injury biomechanics focuses on injuries from physical contact.10,24,25 After injury, biomechanics is critical for understanding human pain tolerance, increasing safety standards, reducing the number of serious injuries in society, and rehabilitating the patient.26,27 Injury biomechanics generally involves studying the mechanical response of cells and tissue but may also involve treatment. While there are a host of biomechanical injuries that can occur, we will focus the present discussion on traumatic brain injury (TBI), since this is among the leading causes of death and disability among young people, affecting over 1 million people annually and having a peak incidence between the ages of 15 and 24.28 TBI occurs structurally in three dimensions in terms of its force propagation through cells and tissues, but it has primarily been investigated using 2D model systems. One example of this is the investigation of TBI within rats, where 2D deformation has been analyzed using strain images obtained from magnetic resonance imaging.29 This study indicated that information derived from this 2D model could provide good indicators of the 3D strain field. In the case of TBI, 2D studies of biomechanics are crucial since shear stress is a primary failure mode at the cellular level.30 However, 2D TBI studies have failed to recapitulate the complex geometry of the brain, supporting the need for 3D TBI studies. 3D TBI studies have been conducted using brain tissue from human cadavers, small primates, and other small mammals. It should be noted that the tissue samples previously mentioned are not easily accessible to many laboratories. In addition, there are a limited number of approaches to reliably interrogate and interpret the effects of mechanics on the brain in vitro and in vivo. This is the case since in vivo samples of the brain are often difficult to obtain because of the lack of muscle tonus and the reduced volume of cerebrospinal fluid. Additionally, the mechanical stress state of the tissue is dependent on the strain magnitude and its tensorial nature. 1.2.4. Plant Mechanics. In addition to being important in human systems, biomechanics is also essential in nonhuman systems, including bacteria and plants. For example, most plants use osmotic pressure to transport water from their roots many meters below the ground surface to their leaves. Additionally, these plants must be able to withstand static and dynamic loads from weather conditions and animals.31 Knowledge gained from nature outside of human systems has led to the advancement of many bioinspired technologies. For example, Leonardo da Vinci’s designs of the first parachute were inspired by dandelion pappus.32 Furthermore, his observation of the tree trunk cross-sectional area and its relation to the branches spurred his interest in fluid mechanics. Ultimately, da Vinci’s study of fluid mechanics yielded the first flow visualizations of a mammalian cardiovascular system.33 While da Vinci’s findings illustrate past studies in plant biomechanics, results derived

2. MECHANICS IN 2D AND 3D HUMAN SYSTEMS The human body comprises many diverse and complex biological systems. While structure and function of the human body are organized through tissues and organs at one scale, the cells within those tissues and organs heavily influence human physiology and pathology at an even smaller scale. Therefore, a deeper understanding of human physiology and pathology requires investigations of the human body at multiple length scales, especially at the cellular level. However, information derived from cell-based studies will depend on the in vitro models available and their ability to mimic the chemical, electrical, mechanical, and geometrical constraints that cells experience in vivo. In this section of the review, we focus on mechanical inputs in 2D and 3D in vitro models in human systems designed to mimic in vivo mechanical perturbations, including fluid shear stress and stretch, and discuss the mechanobiological implications of their outcomes. 2.1. 2D and 3D Mechanics: Fluid Shear Stress. Fluid flow acting upon the apical surface of the cell body exerts a fluid shear stress. The frequency, magnitude, and direction of fluid shear stress are known to affect human health, as an aberrant response to fluid shear stress by the cells lining the heart’s major arteries may yield atherosclerotic plaques or even a heart attack if left undetected.40 The impact of fluid shear stress on the arteries of the heart is important at the cellular level since endothelial cells lining the inside of blood vessels are constantly exposed to a blood-flow induced fluid shear stress. Fluid shear stress can influence the progression of cardiovascular disease, leading to many exciting research directions.12,40−42 Under fluid flow, endothelial cells experience a variety of morphological changes.40,43 Such changes include a deviation from their characteristic cobblestone morphology under mechanically unstimulated conditions toward a more elongated shape aligned along the direction of fluid flow after prolonged C


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Figure 2. Fluid shear stress in 2D and 3D human systems. (A) Normal intercellular stress distribution and intercellular stress orientation of endothelial cells in a 2D environment under fluid shear stress. (B) 3D schematic of normal intercellular stress of endothelial cells before and 24 h after exposure to fluid shear stress. (C) Schematic of a 3D tumor vascular model. (D) Fluorescent image of cancer cell and endothelial cell coculture. Reprinted with permission from refs 52 (copyright 2015 American Physiological Society) and 139.

exposure to a laminar, unidirectional fluid shear stress. In contrast, endothelial cells exposed to fluid flow that is nonuniform in direction and magnitude (i.e., disturbed flow) maintain their cobblestone morphology and exhibit no preferred orientation. These endothelial cell responses occur regardless of whether the endothelial cells reside in a 2D or 3D environment.40,43−45 While the morphological and structural changes induced by fluid shear stress represent a common endothelial cell response, a less common response that has received attention from investigators is the change in intercellular stresses.46−49 Intercellular stresses are the mechanical stresses that adherent cells within a confluent 2D monolayer or a cluster of cells within a 3D cluster exert upon their neighbors. These mechanical stresses are transmitted through cell−cell junctions, and the ability to resolve these stresses in two and three dimensions has been recently demonstrated.46,49−51 Monolayer stress microscopy has been used to measure and visualize intercellular stresses generated by mammalian cells in a 2D environment and represents step 2 of Fung’s problem-solving approach.10 Furthermore, intercellular stresses generated by the endothelial monolayer in a 2D environment have been demonstrated to decrease in magnitude and guide endothelial cell alignment under laminar fluid shear stress52 (Figure 2A,B). Fluorescence resonance energy transfer (FRET) has also been used to demonstrate that intercellular stresses decrease under fluid shear.53 Studies of intercellular stresses of endothelial cells in a 3D environment still remain a challenge, but Hur et al.50 developed a methodology to measure both out-of-plane and in-plane intercellular stresses of an endothelial monolayer in a 2D environment. This method could be extended to measure intercellular stresses of endothelial cells as well as all anchorage-dependent cells in a 3D environment. The morphological and intercellular stress changes induced on endothelial cells by fluid shear stress have been studied in a 2D environment, but studies in a 3D environment likely would provide additional and complementary findings for a deeper

understanding in many mechanics-related pathologies. For example, fluid shear stress has been suggested to significantly influence endothelial cell barrier permeability and tumor angiogenesis, in part by enhancing exposure of endothelial cells to vascular endothelial growth factor (VEGF) and facilitating and stimulating endothelial cell sprouting, proliferation, and tube formation.54,55 Since angiogenesis is a 3D process, Buchanan et al.56 cocultured breast cancer tumor cells (MDA-MB-231) and endothelial cells in a 3D collagen-based, microfluidic tumor vascular model57 (Figure 2C,D). Using this model, they studied the effects of normal, low, and high fluid shear stress (4, 1, and 10 dyn/cm2, respectively) on tumor− endothelial cell paracrine signaling associated with angiogenesis and found that high fluid shear stress downregulates MDA-MB231-expressed angiogenic factors. From these results it was concluded that fluid forces regulate endothelial cell and cancer cell behavior. 2.2. 2D and 3D Mechanics: Stretch. Mechanical stretch represents another mechanical cue that plays a key regulatory role in the maintenance and development of the organs and tissues of the human body in vivo.58 The range of effective stretch magnitudes varies depending on cell type, loading mode, and cell developmental stage, which has led to many studies investigating the cellular response in a 2D environment.59−61 While there are many systems that experience mechanical stretch within the body, a selected cell type is presented here in more detail. Fibroblasts are a diverse and robust cell type that form the connective tissues of the body and are constantly exposed to diverse loading regimes of mechanical tension. Fibroblasts under stretch have been extensively studied in 2D environments, and their responses have been suggested to have implications in physiology and pathology.62−64 For example, tendon fibroblasts in a 2D environment align parallel to the stretch direction under uniaxial tension and display a contrasting random orientation without stretch.11,65 This is significant because aligned fibroblasts have been observed in intact tendons while D


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Figure 3. Mechanical stretch in 2D and 3D human systems. (A) Device used to stretch fibroblasts in 2D environments. (B) Schematic showing the device in the stretched and unstretched states. (C) Cancer cell migration increases in the presence of stretched fibroblasts. (D) Fibroblast extracellular matrix under control (unstimulated) and stretched conditions. (E) Custom-built device used to stretch fibroblasts in a 3D matrix. (F, G) Fibroblast retraction and reinforcement cytoskeletal responses, respectively, after application of stretch. Reprinted with permission from refs 62 and 66.

randomly oriented fibroblasts have been observed in healing tendons in vivo, suggesting that fibroblast alignment with mechanical tension plays a significant role in maintaining tissue structural integrity.11,65 From an alternative perspective, fibroblast response to tension in 2D environments also influences cancer cell migration. Ao et al.62 developed a microfluidic cell stretching device to investigate how stretched normal tissue-associated fibroblasts (NAFs) affect cancer cell migration compared with unstretched cancer-associated fibroblasts (CAFs), which are known to increase cancer cell migration under mechanically unstimulated conditions (Figure 3A−D). In this study, human prostatic NAFs were prestretched for approximately 24 h and subsequently cocultured with SCC61 head and neck squamous carcinoma cells, while mechanically unstimulated human prostatic CAFs were cocultured with SCC61 cells in separate experiments. NAFs increased SCC61 migration at levels comparable to CAFinduced cancer cell migration through increased extracellular matrix secretion.62 Stretch was therefore suggested to play a critical role in cancer cell migration through stromal tissues primarily composed of fibroblasts.62 The above study illustrates step 4 of Fung’s approach since one of the major purposes of this study was to design a system with in vitro boundary conditions that closely mimic the in vivo boundary conditions of cancer cell migration. While 2D tensile studies are currently more diverse in terms of stretch magnitude, frequency, and direction and have revealed important information about tissue physiology and pathology, 3D tensile studies have been more focused on areas such as engineering of functional tissues. For example, chicken embryo fibroblast-populated engineered tissue constructs (ETCs) have been developed by culturing fibroblasts in a more structurally physiologically relevant 3D collagen-based environment mounted on a custom cell stretcher (Figure 3E− G). The stretching system consisted of a force transducer, a mounting stand, and a stepper motor to stretch the fibroblast

ETCs via various stretch regimes.66 Mechanical stretch induced both reinforcement and retraction responses of the actin cytoskeleton. Retraction responses included retraction of cellular protrusions and formation of filamentous actin (Factin) reservoirs, while reinforcement responses involved polymerization and thickening of actin stress fibers, extension of cellular protrusions, and decreases in the size and number of F-actin reservoirs.66 Furthermore, actin reinforcement responses occurred along the stretch direction at all stretch magnitudes, while retraction responses displayed no preferred orientation and occurred at most stretch magnitudes. This implied that a critical stretch threshold value is required for retraction responses and, more importantly, illustrated the potential role of stretch in tissue remodeling in 3D as well as 2D environments.66 2.3. 2D and 3D Mechanics: Combined Fluid Shear Stress and Stretch. As previously mentioned, endothelial cells line the intraluminal layer of all blood vessels within the body and are continuously exposed to blood-flow-induced fluid shear stress. However, endothelial cells within the arteries of the heart also experience blood-pressure-induced cyclic stretch as well.67,68 The combined effect of these hemodynamic forces on arterial endothelial cells is extremely important in cardiovascular physiology and pathology. As a result, researchers have attempted to develop in vitro models that mimic the in vivo physiological mechanical loading conditions; an ideal in vitro model should mimic both the fluid shear stress and stretch loading regimes experienced by endothelial cells. Moore et al.69 developed one of the first in vitro models to mimic such hemodynamic forces. This model consisted of compliant silicone tubing designed to function as an artificial blood vessel. The inside of the vessel was coated with fibronectin and subsequently seeded with calf pulmonary artery endothelial cells. A pulsatile pressure gradient was used to expose endothelial cells to both pulsatile fluid shear stress and cyclic stretch. This model also enabled the investigation of E


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Figure 4. Mechanical stretch and fluid shear in 2D and 3D human systems. (A) Vector logic gate used to analyze endothelial cell responses to stretch and fluid shear stress in a 2D environment. (B) Endothelial cell alignment after exposure to a constant stretch and an increasing fluid shear stress. C) Endothelial cell alignment after exposure to a constant fluid shear stress and increasing stretch. (D) Schematic depicting the application of fluid shear stress and stretch to an epithelial monolayer. (E) Phase images, fluorescent occludin images, and vertical cross-section images of epithelium under static, fluid shear, and fluid shear plus stretch conditions. Reprinted with permission from refs 71 (copyright 2015 Elsevier) and 75 (copyright 2012 Royal Society of Chemistry).

direction of stretch and fluid shear stress, respectively. However, when fluid shear stress and stretch were combined, endothelial cells aligned diagonal to both force directions. This was found to depend on both the mechanical force direction and magnitude71 (Figure 4A−C). These responses were also characterized using a vector logic gate approach, which combined Boolean logic and vector notation to understand cellular structural reorganization.71 The examples presented above describe studies that have investigated the combined effects of both fluid shear stress and stretch on mammalian cells in 2D environments, but studies in 3D environments can lead to increased understanding of human physiology and pathology. In pursuit of this, organ-on-achip studies have increasingly become a method of choice for many researchers, as this technique has allowed researchers to more closely mimic organ-level function in vitro. Organ-on-achip studies generally utilize microfabrication approaches to develop mini-bioreactors that mimic the structure and function of organs within the human body. One goal for these smallscale devices is for them to eventually be used for developmental drug testing, potentially eliminating or significantly reducing the need for costly and labor-intensive animal and human testing.3,72 This technique gives researchers the potential to achieve more complex in vivo 3D geometrical environments while simultaneously mimicking the dynamic biochemical and biomechanical in vivo tissue environment.73,74 For example, a gut-on-a-chip model was developed by Kim et al.75 to mimic the physiological properties of the human intestines (Figure 4D,E). This model consisted of human intestinal epithelial cells cultured in a microfluidic device. This two-layer device also utilized a syringe pump and vacuum pump to simulate intraluminal flow and cyclic stretch experienced by the intestinal epithelial layer, respectively.75 This gut-on-a-chip model was reported to simulate human intestine function, and

cyclic stretch and pulsatile fluid shear stress independently. Using this model, Moore et al. observed that endothelial cells subjected to fluid shear stress and stretch exhibit enhanced Factin alignment and elongation along the fluid shear stress direction, suggesting that stretch reinforces the endothelial cell response to fluid shear stress.69 Toda et al.70 used a similar apparatus to study the effects of fluid shear stress and stretch on endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured inside silicone tubing and exposed to fluid shear stress and/or stretch (cyclic or static). HUVECs exhibited morphological changes similar to the outcomes reported by Moore et al.69 Gene expression analysis also revealed that only cyclic stretch increased mRNA expression levels of the potent vasoconstrictor endothelin 1 (ET-1). Fluid shear stress decreased ET-1 levels, whereas a combination of fluid shear stress and stretch induced ET-1 expression levels, making it almost indistinguishable from mechanically unstimulated conditions.70 ET-1 gene expression in the vascular endothelium in vivo was therefore suggested to be maintained at a specific fluid shear stress and stretch magnitude range. While the previous studies suggest that endothelial cell gene expression and structural reorganization depend partially on a balance between fluid shear stress and stretch magnitude and direction, a recent 2D in vitro study investigated the coupled effects of fluid shear stress and uniaxial stretch on endothelial cell structural reorganization.71 In this study, HUVECs were exposed to various mechanical stimulation regimes in which either the fluid shear stress or stretch magnitude was held constant while the opposing mechanical input was varied along a range of mechanical force magnitudes. It should be noted that these mechanical forces were perpendicular in direction, which is one of the complex mechanical force interactions endothelial cells commonly experience in many cardiovascular pathological conditions. When the forces were applied independently, endothelial cells aligned perpendicular and parallel to the F


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Figure 5. Monolayer and multilayer biofilms. (A, C) Fluorescent images of Vibrio cholerae. (B, D) Schematics of mono- and multilayer biofilms corresponding to (A) and (C). In the monolayer, cell−substrate bonding is most important, and the microbes are distributed on the substrate in a single layer. Contrastingly, in multilayer biofilms, both cell−substrate and cell−cell bonding are imperative. Pillars of layered microbes are enclosed in extracellular matrix on the substrate. Reprinted with permission from ref 76. Copyright 2009 American Society for Microbiology.

fluid shear stress and stretch were observed to alter epithelial shape and polarity.75

3D responses. A biofilm has viscoelastic properties that are derived from its structure and the mechanical strength of the extracellular matrix (ECM), which is largely made of proteins and polysaccharides.36 Biofilms can respond and thrive quite well in the presence of fluid flow. This is a major issue in the piping industry, since biofilms have a tendency to become dislodged under fluid flow and enter the water supply. Biofilm structure can also become mechanically altered and will respond like a fluid when deprived of nutrients yet become gel- or solid-like as a result of overproduction of ECM.36 As shown in Figure 5A−D, biofilms can exist as mono- or multilayers. In the case of monolayers (2D), the cell−substrate interactions are very important, and the adhesive structures forming the monolayer bacterial biofilms include preformed adhesins, conditionally synthesized adhesins, and specific adhesins.76 Multilayer biofilms (3D) bind to both substrates and other cells. This cell−cell and cell−substrate binding is regulated by environmental signals and networks. Of the various signals and cues affecting biofilm formation, mechanical signaling is one of the most prominent. 2D and 3D approaches have been developed to study biofilm degradation, mechanical stress response, and fluid shear stress. One approach used a 3D finite element model to help predict biofilm detachment.79 Tension, torsion, and bending were investigated, and the biofilms responded according to the type of loading applied to their supporting substrate. Furthermore, a mathematical model was developed to understand how external forces cause biofilm detachment. This mathematical model suggests that biofilm mechanical disturbances on longer time scales results in viscous flow while, nonreversible biofilm deformation on shorter time scales results in an elastic response.78 A prominent computational 2D model was developed to describe complex biofilm morphologies seen

3. MECHANICS IN 2D AND 3D NONHUMAN SYSTEMS Mechanics lends itself to a range of scientific fields, making it an interdisciplinary approach for explaining phenomena across multiple domains. As previously discussed for human systems, an understanding of mechanics in the context of nonhuman systems can provide insight into many applications within the human body ranging from aging to pharmaceuticals as well as engineering materials and architecture. Although there are a tremendous number of fascinating nonhuman systems in which mechanics plays a role, here we present selected examples of nonhuman systems, including insects, microorganisms, and developmental biology systems, from a mechanics perspective. 3.1. 2D and 3D Mechanics in Microorganisms. 3.1.1. Microbial Biofilms. Biofilms are important in a tremendous number of areas ranging from medicine to the environment that involve the interplay of microorganisms. Biofilm formation and degradation are influenced by environmental factors and signaling pathways76,77 and are essential in many areas. For example, biofilms are important in consumable water system applications, as they are utilized for water purification and water sequestration; however, they can also be hazardous if they degrade within the water supply, leading to the spread of water-borne pathogens.78 Biofilms are also responsible for tooth decay, hospital-acquired infections, and implant infections, including those found in sutures, stents, and catheters, for example.79 Therefore, further understanding of biofilm properties could aid in predicting conditions that would result in the degradation and dislodgement of biofilms. Biofilms are formed once microbes aggregate together to create a film, and mechanics is very important in their 2D and G


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Figure 6. Responses of microorganisms. (A) Representative AFM 3D height image showing the surface topography of a Scenedesmus dimorphus cell. (B) AFM topographical image of a representative S. dimorphus cell for the determination of the Young’s modulus at subcellular points 1, 2, and 3 through nanoindentation. (C) AFM scan of Tetraselmis suecica showing ultrastructures underneath the cell membrane. (D) Transmission electron microscopy image showing the T. suecica cross section. Reprinted with permission from refs 91 (copyright 2014 AIP Publishing) and 92 (copyright 2012 Elsevier).

these macroalgal organs are independent of the 2D cells themselves. As Cooke and Lu90 discovered, cells do not act as building blocks for organs in multicellular algae as they do in mammalian organs. This is different compared with biofilms, since macroalgal structures are dictated not by aggregation of cells but rather by partitioning of the entire structure into cells. Furthermore, mechanics has not been shown to effect the shapes of planar structures or the overall shape of macroalgae as it does in mammalian morphogenesis. However, further investigation of biomechanics in microalgae is needed. In the context of Fung’s approach to biomechanics, some of the recent advancements in mechanical characterization of microalgae include determining their elastic moduli,91 force and energy requirements for lysis,92 tensile strengths,93,94 and rheological properties.95 Some of this work was done using atomic force microscopy (AFM), as shown in Figure 6. AFM can be considered both a 2D and 3D analysis method because of its inplane and out-of-plane imaging and measurement capabilities. However, because of the diversity of microalgae species, a range of reported mechanical properties for microalgae across species exists. Another example of microalgae biomechanics may be found in the biofuels community, where mechanical lysis can potentially have benefits from a chemical and environmental standpoint.96 Currently, one of the most energy-inefficient steps of biofuel production involves cell lysis, which can occur through mechanical approaches such as bead milling, mechanical pressing, osmotic shock, or sonication.97,98 Many of these approaches rely on shearing of the cell membrane to lyse the cell. Thus, understanding the mechanical properties of microalgae may be essential in developing efficient microalgae biofuel extraction processes.87,91,99 3.2. 2D and 3D Mechanics in Spider Silk. One amazing material with tremendous mechanical properties that is produced in nature is spider silk. Spider silks are protein fibers spun by spiders to create unique web structures with specific functionalities. In addition, some spiders have the ability to

experimentally in many systems using cellular automata. This model included three processes; cell growth, cell detachment, and internal and external mass transport, making it a useful tool in the analysis of biofilm development.80 In various physical experiments, there has been great interest in the degradation of ibuprofen in biofilm reactors and sewage systems.81,82 This degradation of both the pharmaceutical drug and the biofilm pose environmental risks to ecological systems. As more information regarding biofilm formation is gained from computational and experimental biomechanical studies, the removal and prevention of unwanted biofilm accumulation will be improved. 3.1.2. Algae. Algae are important in numerous fields, including biofilms, oxygen production, bioenergy, and nutraceuticals. Algae can be unicellular or multicellular. Microalgae are unicellular microorganisms that exist in freshwater or marine systems and can be considered as bacteria or plants. They can exist independently or in groups and can be grown in open systems such as ponds or closed systems such as photobioreactors.83 Through photosynthesis, microalgae release roughly 99% of all the atmosphere’s oxygen, making them an important part of the ecosystem and human health.84 Consisting of over 30 000 known species, these photosynthetic microorganisms are diverse,85 and they not only aid in carbon dioxide sequestration, which improves air quality, but also are very useful in wastewater treatment.86,87 Microalgal applications span from increasing the nutritional value of food and cosmetic manufacture to the production of pharmaceuticals and renewable energy sources.88,89 One very important facet of microalgae potential related to mechanics is the ability to mechanically extract cellular components from the microalgae. This extraction is particularly interesting because of the algal mechanics involved. In the case of macroalgae (multicellular, macroscopic algae) and many other plants, there are similarities between 2D and 3D structural approaches,90 but the shape and overall 3D form of H


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Figure 7. Mechanics in 3D Drosophila melanogaster. (A) Apical constriction and tissue bending: (a) mesoderm invagination in the D. melanogaster embryo involves furrowing of the ventral epithelium and specific cell-shape changes in apical constriction (pink) of invaginating cells (square bracket); (B) vertebrate neurulation (e.g., in the chick) proceeds by bending of the neuroepithelium (square bracket) and apical constriction in defined regions (pink); (c) schematic representation of apical constriction (pink) in epithelial cells; (d) schematic representation of bending associated with apical constriction of the epithelial tissue. (B) Micro-computed tomography scans of copulating Drosophila visualized in three dimensions. Depicted is the abdomen of a virgin female, showing details of the reproductive tract anatomy. Reprinted with permission from refs 118 (copyright 2007 Nature Publishing Group) and 120.

diseases. Major advancements in the field of developmental biology have been made possible in part through the investigation of developmental systems spanning multiple species, including Drosophila melanogaster, Danio rerio, and Xenopus laevis.110 We discuss each of these systems below with respect to mechanics. 3.3.1. Fruit Fly. Drosophila melanogaster, the common fruit fly, has been an important model system for advancing developmental biology111 for over a century. Drosophila was the first major complex organism to have its complete genome sequenced, which helped with sequencing of the human genome. Strong similarities between the human and fruit fly genomes were discovered,112 suggesting that the fruit fly could be a good model for studying developmental human physiology and pathology. The high fertility and rapid development of the fruit fly within the laboratory setting allows the fruit fly to be used in experimentation for responses to chemicals, gravity, heat, and light at different developmental stages. Studies of Drosophila have produced exciting insights into a variety of areas including mutation, cell structure and differentiation, human development, cloning, and drug discovery and delivery.113−116 Mechanical properties are very important for understanding Drosophila and how it functions. Studies of Drosophila during gastrulation along with experimentally validated in vivo computational models have provided insight into mechanical characterization of tissues within the embryo to understand how they effect furrow internalization.117 This furrow internalization, or mesoderm invagination, is driven by regulation of surface tension and surface mechanics, including apical constriction118 (Figure 7). Serving as a promoter in wound healing, the apical constriction increases the surface tension by positioning actomyosin tensile systems at cell junctions, causing invagination. Tensile forces are not the only mechanical means of tissue development or gene expression in the fruit fly. Other mechanical properties that influence cell signaling in Drosophila include viscoelasticity, stretch, deformation and strain, and fluid flow.119 In studying biomechanics of the fruit fly, advances in wound healing have also been made on the basis of the parallel to human anatomy and physiology. In the case of the fruit fly, mechanical tension is important in 3D

produce up to seven types of silk.100 The desired function of the spider silk determines the structure produced, which may include the construction of webs for catching and restraining prey, nests for offspring, and traveling. All of these constructions utilize a minimal amount of silk,101 yet this material can be stronger per unit mass than tensile steel. In the context of step 2 of Fung’s approach to biomechanics, some of the mechanical properties of spider silk investigated to date include elastic modulus, tensile strength, breaking energy,101 enthalpy, entropy,102 maximum stress and strain, toughness,103 thermal conductivity, and diffusivity.104 Because of its strength and durability, spider silk’s chemical structure has been incorporated into bioinspired materials utilized in cell culture, regenerative medicine, and coatings for implantable devices.100,105,106 With spider silk, researchers already synthesize custom cellular matrices, or scaffolds, for cell growth, differentiation, and migration. These custom bioinspired scaffolds have been used for wound healing and drug delivery, as they are biocompatible and biodegradable,107 as well as for a biochemical optical fiber sensor.108 These applications show the versatility of the 3D structure of spider silk. The 3D structure of spider silk can be tailored for specific applications with the aid of 2D scanning electron microscopy images. Initially, spider silk is in the shape of a fiber, long and tubule-shaped, and once recombinant silk proteins are created, the silk shape can be tailored for the specific application. For drug delivery, the silk is shaped into capsules or round particles, while for tissue engineering, the silk is allowed to self-assemble into a mesh, hydrogel, or foam. Spider silk’s mechanical properties make it a likely candidate in many biomedical applications, and the combination of its elasticity, strength, and toughness are highly desired compared with the natural and synthetic materials available. 3.3. 3D Mechanics in Developmental Biology. Mechanics is known to be extremely important in nonhuman developmental biology systems as well. Investigation of nonhuman developmental systems has revealed important parallels to human developmental systems.109 Therefore, examination of nonhuman developmental systems can be used to unravel mysteries that exist in a diversity of human I


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Figure 8. Mechanics in Xenopus laevis. (A) Generation of transgenic frogs expressing Venus and transplant engraftment of their brain tissue. Also shown are fluorescent images of transgenic animals. (B) Studies of Xenopus cell response: (a) Photograph of a microfluidic device in which a single animal cap tissue explant is being locally stimulated by the central stream bounded by culture medium (DFA). Also shown is a computational fluid dynamics simulation predicting flow patterns around a tissue model. The cells exposed to a stream of extracellular ATP contract, whereas the neighboring cells stretch. (b) Microscope image of a Xenopus embryonic tissue with a gray stream indicating the location of ATP. (c) Strain map responses of locally ATP stimulated Xenopus tissue indicating mechanical actuation. Reprinted with permission from refs 134 and 136.

biomechanics standpoint within the zebrafish embyro for comparison to other organisms’ development, specifically human development. Zebrafish organ systems that are indicative of human systems include the cardiovascular system, respiratory system, and nervous system, among others. The zebrafish nervous system is similar to that of humans in that it has a representative brain anatomy, specialized sensory organs, and the ability to exhibit “higher” behaviors. Zebrafish also serve as disease models in congenital and hereditary diseases, carcinogenesis, infections, and inflammation.131 Additionally, investigations into the mechanical material properties of the zebrafish have been conducted, including studies of the flexural stiffness of superficial neuromastsanother use of Fung’s approach to biomechanics.132 Examination of the flexural stiffness of the neuromasts could aid in understanding flow sensing on a structural and cellular level, representing an important example of 3D biomechanics in zebrafish. For example, the neuromasts detect fluid flow by being mechanically excited by fluid forces. Neuromasts are then able to control spawning, rheotaxis (the ability to hold a position within opposing fluid flow), and obstacle detection in zebrafish. 3.3.3. Xenopus. The African clawed frog, Xenopus laevis, has also been used for decades as a developmental approach in understanding many diseases and human pregnancy.110 This organism has a number of physiological and behavioral attributes that make it a desirable candidate for understanding human physiology, and 3D biomechanics plays a role in a number of those physiological attributes.110 Attributes of Xenopus include the ability to thrive in a wide spectrum of living environments, large embryonic cells, and ease of early

cell migration of the wound site to increase the rigidity of the extracellular matrix. This rigidity of larger cells at the wound site increases the mechanical stability of that area while balancing the forces that promote wound closure, including those in copulatory wounding,120 which are integrated at 3D levels121 (Figure 7B). 3.3.2. Zebrafish. Danio rerio, or zebrafish, is another great system for developmental biology, genetics, oncology, and neurophysiology for vertebrates122,123 that has also been used to reveal correlations to human development within 3D biomechanics contexts. The development of this tropical freshwater fish has been well-characterized, as its embryo and chorion are optically transparent.124 Furthermore, their small size, high fecundity, and rapid nonplacental development outside of the mother make zebrafish a useful model for human developmental systems.125 In addition, because of its high genetic conservation with humans, zebrafish are useful for disease modeling and organogenesis in humans.126−128 From a 3D biomechanics standpoint, fluid flows in these vertebrate systems and their organ development and locomotion are of great interest. In vertebrate organ development, the asymmetric positioning of internal organs and cell signaling originate from biological fluid flows.129 These fluid flows generate friction forces, tension forces, and shear stresses and transport small signaling molecules between cells that provide important information including information about chemical gradients, biomineralization, and tubulogenesis. Cilia-driven fluid flows are dominated by viscous effects and ultimately sensed by the sensory hair cells of the zebrafish, aiding in the development of the 3D inner ear and other organs.130 Researchers have been studying the development of various organs from a J


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ACS Applied Materials & Interfaces embryo manipulation.133,134 Major areas of Xenopus research include gene regulation, signal transduction, and cell-cycle control for applications such as wound healing and organogenesis, which are also known to be related to biomechanics.135 3D biomechanics plays an important role in the development and cellular response of Xenopus. Cell contractility, which regulates the anatomy and physiology of an organism, relies on 3D mechanical connections within the cell system.136−138 Xenopus strain patterns have been probed using microtechnology and computational approaches. These approaches revealed the transfer of activation signals and complex mechanical−contractile responses that control Xenopus tissue movements136 (Figure 8). The ability to investigate and use 3D biomechanics in developmental biology systems will lead to advances in many areas, including aging, cancer, organ on a chip, and regenerative medicine within human physiology.

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4. CONCLUSION We have presented an overview of the importance of biomechanics in human and nonhuman systems in 2D and 3D environments, ranging from endothelial cells and fibroblasts to microalgae and zebrafish embryos. While a majority of systems have been studied in a 2D environment, advances in 3D methods are now allowing more researchers to study human and nonhuman systems with 3D approaches that more closely resemble their native environments. These approaches have led to increased attention for 3D mechanobiology and the acceleration of new techniques and methods in 3D biomechanics that will be applied to a diversity of biological systems. Although nonhuman and human responses in 2D and 3D environments have been shown to have similar and contradicting findings, these findings will help advance our understanding of these complex biological systems. In closing, regardless of the environment or whether the biological system is human or nonhuman, mechanics plays an influential role in biological structure and function.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 407-823-5608. Fax: 407-823-0208. E-mail: rstewardjr@ ucf.edu. *Tel: 412-268-2504. Fax: 412-268-3348. E-mail: prleduc@cmu. edu. Author Contributions §

K.M.W. and M.M.I. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Gates Millennium Scholarship Program and an Alfred P. Sloan Foundation Fellowship. This work was also supported in part by the National Science Foundation (CMMI-0645124, CBET-1547810), the Air Force Office of Scientific Research (FA9550-13-1-01 08), and the Army Research Office (W911NF1510148). Additional funding supporting this work includes University of Central Florida startup resources.

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