Among intermediate filaments populating cells, lamin filaments are of particular interest due to their universal role in the cell nucleus. Structural disruptions and mechanical alterations occurring in lamin mutations, commonly named laminopathies, result in increased cellular apoptosis and necrosis, leading to severe clinical symptoms. However, it remains quite unclear what determines the tensile stiffness and strength of alpha-helical rod domains in lamins, which are the elementary building blocks of the nuclear envelope. This study is concerned with the computation of these effective mechanical properties. Molecular dynamics simulations are used at the microscopic scale to predict the atomistic force fields of lamin fragments subjected to different axial stretches. The axial force of an equivalent continuous rod is deduced with the principle of virtual work. Using this methodology, we eventually predict the stiffness and strength of the rod for different configurations that are relevant for lamins. Results show that the tensile stiffness and strength of lamins are determined by non-local electrostatic interactions. Nonlocal interactions refer to points along the rod that are physically close in space despite not being parametrically close. Non-local electrostatic interactions result from heterogeneous distributions of electric charges, which are responsible for pretensions in lamin rods and play a major role in their tensile response. Our computational models show that mutations changing the charge of a single residue can significantly alter the individual mechanical behaviour of the lamin protein and hamper the ability of the nuclear envelop to withstand mechanical stresses. Such results pave the way towards the use of computational medicine to advance the treatments of laminopathies.

Spider silk is an important model system of a fully sustainable, natural biopolymer featuring multi-functionality and outstanding mechanical performance. Typical for a biomaterial, these properties are owed to a hierarchical structure spanning many decades from molecular to macroscopic scales. Because of its attractive characteristics, this material has been intensely studied in the past decades; nonetheless revealing its structure has proven surprisingly difficult. We combined atomic force microscopy (AFM), vibrational spectroscopy and other techniques to develop the most detailed structural model for a spider silk to date, using the recluse spider as a model system. Mechanical and nano-mechanical techniques were used to reveal macroscopic properties of the spider’s web and relate them to interactions between the constituents of the material, down to the level of specific bonds within the constituting amino acids.

At the centimeter scale, the spider web is “looped” into a 1-D meta-structure to increase toughness through a unique “strain cycling” mechanism. This meta-structure is held in place through a self-strengthening adhesive mechanism that avoids the weakness usually inherent to peeling/delamination failure. This inspired the design of a novel class of meta-materials with tunable mechanical properties. We further discovered that the ribbon silk of the recluse spider consists entirely of uniform, parallel nanofibrils about 10 nm in diameter and many microns long. The nanofibrils are relatively weakly adhering to each other, which gives the silk ribbon highly anisotropic mechanical properties. We developed a method combining AFM indentation with finite element analysis (FEA) to provide extensive characterization of the ribbon’s nanomechanical properties, including anisotropy of local stiffness and strength, as well as plastic deformation. The smallest length scale — at the level of spidroin molecules — was probed using vibrational spectroscopies: six different protein secondary structures were identified in silk nanofibrils for the first time, and their exact composition and angular orientation was determined. The level of detail revealed in our experimental studies will allow for computational models and analyses of this fascinating material in unprecedented detail. Multi-scale models can directly be validated and calibrated using experimental data at different length scales, with the prospect of developing a much better understanding of the material than before.

We further devised a disassembly process for silk fibers, which allows us to “reverse-engineer” silk fibers into their nanofibrillar components. This top-down approach allowed us to mass-produce native spider silk nanofibrils for the first time, with many applications and opportunities to further study nanofibrils. In addition, we developed a bottom-up approach where we visualize the molecular-scale assembly process of silk molecules into nanofibrils. In addition to structure, these methods also reveal details about the formation process of nanofibrils. Thus, we believe that these strategies will pave the way for synthesis of high-performance fibers and materials inspired by spider silk.

Breast cancer and prostate cancer tend to spread to the bone. Once they reach the bone site, cancer cells colonize and start to grow, which can have a negative impact on the bone. We have developed the first in vitro testbeds for studying breast cancer and prostate cancer metastasis to bone. Our research has shown that cancer cells undergo significant morphological changes as they cluster together and form tumors at the bone metastases site. These changes are related to the cancer phenotype and can dramatically alter the mechanical properties of the cancer cells extracted from the tumors. Eukaryotic cells consist of the cytoskeleton responsible for the cell's shape, internal organization, and mechanical rigidity. The cytoskeleton consists of actin microfilaments, microtubules, and intermediate filaments. Our studies on nanomechanical testing of bone metastatic breast and prostate cancer tumors using in vitro models indicate a progressive softening of the cells with metastasis progression. Confocal imaging of cancer tumors during progression has revealed quantitatively and spatially significant actin dynamics. Experiments showed the reduction and reorganization of actin filaments during metastasis progression. This work describes steered molecular dynamics (SMD) simulations to evaluate actin dynamics. Molecular nuances such as conformational locks and nonbonded interactions at the inter-strand and intra-strand interfaces regulate F-actin dynamics. We have observed significant changes in gene expressions related to actin and actin depolymerization factor cofilin (ADF/cofilin) associated with these changes Actin-related genes are downregulated, and cofilin genes are upregulated during metastasis. Steered molecular dynamics simulations of actin and actin with ADF/cofilin describe the mechanisms of the resilience of actin molecules and depolymerization of actin by ADF/cofilin.

Additionally, tumorigenesis occurs through integrin activation, a critical step in initiating the colonization of cancer cells at the bone site. Integrin protein acts as a mechanotransducer that establishes mechanical reciprocity between the extracellular matrix (ECM) and cells at integrin-based adhesion sites. This protein plays a critical role in cell-ECM adhesion and cellular signaling. As new biomaterials are being developed for tissue engineering applications, understanding cellular adhesion to engineered surfaces mediated by integrins is critical. We conducted steered molecular dynamics (SMD) simulations to investigate the mechanical responses of integrin αvβ3 with and without ligand binding for tensile, bending, and torsional loading conditions. The ligand-binding integrin confirmed the integrin activation during equilibration by opening the hinge between βA and the hybrid domain. This activation of liganded αvβ3 integrin influenced the molecule's stiffness observed during tensile loading. Furthermore, we observed that the interface interaction between β-tail, hybrid, and epidermal growth factor domains altered integrin dynamics. The deformation of extended integrin models in the bending and unbending directions of integrin reveals the integrin molecule's stored folding energy and directionally dependent stiffnesses. Along with available experimental data, SMD simulation results were used to predict the mechanical properties of integrin and reveal underlying mechanisms of integrin-based adhesion on polymer clay nanocomposite-based biomaterials. The evolution of actin dynamics and integrin activation is critical to metastasis and constructing a mechanobiological cell model of metastasis.

Globally, an estimate of 3.5 billion people is affected by oral diseases. Oral diseases disproportionally affect the most vulnerable and disadvantage populations and remains among the most common noncommunicable diseases. The range of diseases include dental caries, periodontal disease, tooth loss, oral cancer among many others. Untreated dental caries is the most common global health condition, affecting permanent teeth of 2 billion people and primary tooth of more than 500 million children. An estimated 800 million resin composite, 100 million amalgam, and millions of glass ionomer cement restorations are placed each year and are one of the most prevalent medical interventions in the human body, not to mention the over five million implants placed in the United States each year. The cost for these therapies is immense. The combined complexity and prevalence of dental diseases requires well engineered and effective interventions to optimize patient outcomes. Our particular focus has been on exploring biomimetic approaches that can impart the key biological activity toward prevention of dental and oral diseases as well as restoration of oral health.

Biomolecules are essential in physiological processes, and smaller molecules such as peptides offer to mimic their function to be uniquely versatile molecular tools in coupling biologically instructive roles in material systems. We have been exploring peptide-based approaches including repairing mineralized tissues of teeth damaged due to caries, trauma or periodontal diseases and derivatizing dental materials with wide range of bioactivities including antimicrobial activity toward preventing infection. We identified several intrinsically disordered peptides and utilized the resulting sequence information to recapitulate the functional domains of native proteins. Several of these peptides demonstrated a rich conformational propensity that can be further tunable at the bio-hybrid material interfacial design. To address the different unmet health care need, we designed peptides with different properties ranging from self-assembly to mediating mineralization or catalytic function. By adapting experimental and computational approaches combined with transparent machine learning (ML) methods, we continue our search for the peptides that present desired function. In our approach, we expanded our predictions from single function to multifunctional peptides to control their properties at the complex materials to dental tissue interfaces. In this presentation, we will discuss our approach for next generation treatments that are composed of peptides, peptide-mimetics and peptide-polymer hybrids. We designed antimicrobial peptides (AMPs) and developed localized delivery approaches for their easy deployments on the sites. We developed machine learning approaches to classify antimicrobial peptides and enrich sequence domains for effective antimicrobial search with enhanced properties. By incorporating multi-pronged strategy to repair and protect exposed dentin and collagen, we combined different peptide, peptide-mimetic and peptide-polymer hybrid to treat different lesion types. Our peptide design and engineering approach targeting biohybrid interfaces provides an alternative delivery strategy to deploy peptides on the sites and increases their availability and preserving their efficacy. Peptide hybrids could offer multi-pronged and microinvasive strategies to overcome the limitations of current approaches as well as address the high prevalence of root caries and dental erosion in the aging population.

The expanded use of dental implants has led to an increase of peri-implant disease that shortens implant life and leads to failure. Peri-implant disease results from microbiota dysbiogenesis triggering in the host an immune inflammatory response that destroys soft and hard-tissue. At an incidence of 14.5%, and over 3 million implants placed and growing by 500,000/year, a reduced service life ending in failure will adversely impact public health and increase health care costs. Molecular studies on proteins expressed during tooth formation led to insights that can be translated to clinical care to improve health, such as development of an antimicrobial bifunctional peptide film to slow disease progression. Based on a high-affinity titanium binding peptide that anchors the anti-microbial peptide to the implant surface, greater than 98% coverage is achieved in <2 minutes even in the presence of contaminating protein, to produce a film durable to mechanical brushing that kills >90% of bacterial. Also developed was a short amelogenin peptide M59, that activates osteogenesis through the Wnt pathway. M59 can also be applied locally using the titanium binding peptide to deliver the M59 osteogenic signal where needed and on command. This non-surgical approach can improve oral health by delivering a simple to apply/reapply multiple times, using a water-delivered bifunctional peptide film serving to control microbial dysbiogenesis, reduce disease progression and induce bone formation.

According to the World Health Organization, 375,304 deaths and about 1.4M cases of prostate cancer reported in 2020. Majority of these deaths occur due to bone metastasis, or the transport of cancer from prostate to bone and subsequent skeletal failures. The highly diminished availability of bone metastasis prostate cancer samples and failure of animal models due to death preceding metastasis in animals necessitates the development of realistic prostate cancer in vitro models. A novel nanoclay-based tissue-engineered polymeric scaffold sequentially seeded with human mesenchymal cells and cancer cells recapitulates prostate cancer bone metastasis. We hypothesized that interstitial fluid flow acts as a driving force for migration of cancer cells in the vicinity of capillary pores. The fluid-flow provides unique biochemical cues for metastasis. Earlier we have reported the role of interstitial fluid in prostate cancer bone metastasis using a 3D in vitro bone metastasis testbed, with a perfusion bioreactor. However, understanding the metastatic cascade of cancer cells is essential to pave the way to discover therapies for metastatic cancers. In particular, the extravasation stage, when cancer cells start to invade into the secondary site resulting is metastatic tumors is critical. Extravasation comprises of first the transmigration across the capillary’s endothelium. Hence, we designed a bioreactor that enables interstitial fluid-flow for prostate cancer bone metastasis. We developed a novel 3D in vitro dynamic horizontal bioreactor integrated with transwell inserts, that recapitulates the in vivo microenvironment of cancer cells representing migration of cancer cells under interstitial fluid flow. Computational fluid dynamics studies, detailed biological characterizations including evaluation of gene expressions and imaging were conducted to build relationships between the fluid induced shear stresses and the progression of cancer metastasis. The computational fluid dynamics results indicate that 0.05 ml/min flow rate recapitulates the physiological condition. We evaluate the migration of the highly metastatic PC3 prostate cancer cells through the transwell insert under both dynamic and static culture conditions. While experimentally subjecting the cancer cells to the fluid derived stresses via the bioreactor experiments, we investigate the molecular mechanisms responsible for the migration of cancer cells under different culture conditions. This study demonstrates that adhesion proteins avb3 integrins play important roles in response to mechanical cues and act as mechanosensory agents that transport mechanical signals via the avb3-MMP 9 signaling axis to promote flow-induced motility of prostate cancer cells. Also, the interstitial fluid-flow does not alter the CXCR4 level, an important regulator of metastasis and invasiveness, but bone proximity upregulates CXCR4 levels enabling increased MMP-9 levels. Further, avb3 integrins and MMP-9 levels are upregulated by fluid-flow causing increased migration under fluid-flow. Overall, these studies describe the critical role of interstitial fluid-flow in prostate cancer metastasis.