The administration of pharmaceutical aerosols to infants on mechanical ventilation needs to be improved by increasing the efficiency of delivery devices and creating better ways of evaluating potential therapies. Aerosolized medicines such as surfactants have been administered to ventilated infants with mixed results, but studies have shown improvement in respiratory function with a much lower dose than with liquid instillation through an endotracheal tube (ETT). An aerosolized medicine must be transported through the ventilation tubing and deposit in the lungs to have the desired therapeutic response. This work has taken a systematic approach to (i) develop new devices for the efficient production of small sized charged pharmaceutical aerosols, (ii) adapt a lead device to an infant ventilation system, (iii) develop a novel breathing infant lung (BIL) in vitro model capable of capturing lung delivery efficiency in an infant without the need for human subjects testing, and (iv) evaluate the hypothesis that small sized charged pharmaceutical aerosols can improve drug delivery efficiency to the lungs of a ventilated infant. Three new devices were developed and screened for the efficient generation of small sized charged pharmaceutical aerosols, which were: wick electrospray, condensational vapor, and a modified vibrating mesh nebulizer in a streamlined low flow induction charger (LF-IC). Of these devices, only the LF-IC produced a small [mean(SD) = 1.6(0.1) micrometers] and charged (1/100 Rayleigh limit) aerosol at a pharmaceutically relevant production rate [mean(SD) = 183(9) micrograms per minute]. The LF-IC was selected as a lead device and adapted for use in an infant ventilation system, which produced an increase in in vitro lung filter deposition efficiency from 1.3% with the commercial system to 34% under cyclic ventilation conditions. The BIL model was first shown to produce a realistic pressure-volume response curve when exposed to mechanical ventilation. The optimized LF-IC was then implemented in the BIL model to demonstrate superior reduction in inspiratory resistance when surfactant was delivered as an aerosol compared to liquid instillation. For the delivery of an aerosolized medication, the lung deposition efficiency increased from a mean(SD) 0.4(0.1)% when using the conventional delivery system to 21.3(2.4)% using the LF-IC in the BIL model, a 59-fold increase. The charged aerosol produced by the LF-IC was shown to have more depositional loss in the LF-IC than an uncharged aerosol, but the charge decreased the exhaled fraction of aerosol by 17%, which needs additional study to achieve statistical significance. Completion of this work has produced a device that can achieve lung delivery efficiency that is 59-fold greater than aerosols from conventional vibrating mesh nebulizers in invasively ventilated infants using a combination of small particle size, synchronization with inspiration and appropriate charge. The BIL model produced in this work can be used to test clinically relevant methods of administering medications to infants and can be used to provide more accurate delivery estimates for development of new nebulizers and inhalers. The LF-IC developed in this work could be used for controlled and efficient delivery of aerosolized antibiotics, steroids, non-steroidal anti-inflammatories, surfactants, and vasodilators.

Several surgically corrective procedures are considered to treat Adult Acquired Flatfoot Deformity (AAFD) patients, relieve pain, and restore function. Procedure selection is based on best practices and surgeon preference. Recent research created patient specific models of Adult Acquired Flatfoot Deformity (AAFD) to explore their predictive capabilities and examine effectiveness of the surgical procedure used to treat the deformity. The models’ behavior was governed solely by patient bodyweight, soft tissue constraints, and joint contact without the assumption of idealized joints. The current work expanded those models to determine if an alternate procedure would be more effective for the individual. These procedures included one hindfoot procedure, the Medializing Calcaneal Osteotomy (MCO), and one of three lateral column procedures: Evans osteotomy, Calcaneocuboid Distraction Arthrodesis (CCDA), Z osteotomy and the combination procedures MCO & Evans osteotomy, MCO & CCDA, and MCO & Z osteotomy all used in combination with a tendon transfer. The combination MCO & Evans and MCO & Z procedures were shown to provide the greatest amount of correction for both forefoot abduction and hindfoot valgus. However, these two procedures significantly increased the joint contact force, specifically at the calcaneocuboid joint, and ground reaction force along the lateral column. With exception to the lateral bands of the plantar fascia and middle spring ligament, the strain present in the plantar fascia, spring, and deltoid ligaments decreased after all procedures. The use of patient specific computational models provided the ability to investigate effects of alternate surgical corrections on restoring biomechanical function in flatfoot patients.

The goal of this study was to evaluate biomechanical differences between healthy subjects and those with ankle instability during the gradual onset of lower extremity fatigue from a landing activity. An understanding of these differences is needed in order to prevent future injury to or further debilitation in individuals with ankle instability. A functional fatiguing activity was designed to focus fatigue on the quadriceps muscles, as those are the muscles most frequently fatigued during sport. Measures were taken throughout the progression of fatigue with a force plate and a motion tracking system and included vertical ground reaction force and lower extremity kinetics, kinematics, and energetics. The time required to reach self-reported fatigue and a balance assessment, the Star Excursion Balance Test, before and after the onset of fatigue was also recorded. Significant differences were observed between groups in peak ground reaction force, ground reaction force impulse, and frontal plane ankle joint impulse. Results indicated that subjects with ankle instability not only exhibited a different baseline for most measurements than normal subjects, but also managed the progression of fatigue differently. With this information and information from further studies, recommendations and/ or training schemes could be made and implemented to help those with ankle instability avoid recurrent injuries.

Spider silk is a material that both has impressive mechanical properties and is also environmentally friendly. Though there are limitless potential engineering applications for such materials, industrial production of spider silk has proven to be challenging. Farming silk from spiders, as is done with silkworms, is not a viable option for large-scale production of spider silk due to the venomous and predatory nature of spiders. Here, an attempt is made to express synthetic spider silk minifibroins heterologously in Escherichia coli, to purify the recombinant spidroins from cell lysate, and to spin them into artificial fibers through a biomimetic process. Silk minifibroins were designed to be similar to Major Ampullate Spidroin 1 from Latrodectus hesperus. Synthetic fibers were examined by scanning electron and light microscopy, and their mechanical properties were tested by a tensometer. Properties of synthetic silk were compared to those of native dragline silk from the same species from which their design was inspired, revealing synthetic silk fibers with lower breaking stress and breaking strain.

Management and control of the low back pain as an important health problem in the industrial societies necessitates to investigate how the risk of this disease is affected by aging. Since the abnormalities of the lumbopelvic kinematics are related to the existence or risk of low back injuries, the objective of this dissertation was set to find the age-related differences in lumbopelvic kinematics when performing basic trunk motions reaching to range of motion in different anatomical planes. A cross-sectional study was designed where sixty asymptomatic individuals between 20–70 years old with no confounding health condition, no current or previous highly physically demanding occupation and a body mass index between 22 and 30, were divided in five equally-sized and gender-balanced age groups, and attended two sessions of data collection to perform three repetitions of self-selected slow and fast trunk forward bending and backward return, as well as one left and right lateral bending and axial twist. Following an extensive literature review, the lumbar contribution (LC) to the trunk motion, the mean absolute relative phase (MARP) between the thoracic and pelvic motions as well as variation in MARP under repetitive motions, denoted by deviation phase (DP) were selected and used for the assessment of age-related differences in lumbopelvic kinematics during forward bending and backward return tasks. Lumbopelvic kinematics during the lateral bending and axial twist tasks were assessed using the lumbar and pelvic ranges of motion (ROMs) and coupled motion ratios (CMRs) as respectively the maximum flexion/rotation in the primary (i.e., intended) and the secondary (i.e., coupled) planes of trunk motion, where the latter was normalized to the conjugate ROM for better comparison. The results showed age-related differences between the age groups above and under 50 years of age generally. A smaller LC during the forward bending and backward return tasks were observed in the older versus younger age groups, suggesting that the synergy between the active and passive lower back tissues is different between the older and younger people, which may affect the lower back mechanics. Also, smaller MARP and DP suggesting a more in-phase and more stable lumbopelvic rhythm were observed in the older versus younger age groups, which may be a neuromuscular strategy to protect the lower back tissues from excessive strain, in order to reduce the risk of injury. Furthermore, the coupled motion of lumbar spine in the transverse plane during the lateral bending to the left, and the coupled motion of pelvis in the sagittal plane during the axial twist to the right were larger in older versus younger age groups. In summary, the lumbopelvic kinematics changes with aging, especially after the age of 50 which implies alterations in the active and passive tissue responses to the task demands, as well as the neuromuscular control patterns. Drawing a conclusion regarding ii the effect of aging on the risk of low back pain from these results requires a further detailed knowledge on age-related differences in spinal active and passive tissue properties.

There is a large body of evidence supporting the theory that cell physiology largely depends on the mechanical properties of its surroundings or micro-environment. More recently studies have shown that changes to intra-cellular mechanical properties can also have a meaningful impact on cell function and in some cases lead to the progression of ailments or disease. For example, small changes to the protein sequence of a structural nuclear envelope protein called lamin-A is known to cause a variety of neurological and musculoskeletal diseases referred to as laminopathies. Currently, there is little incite into how these mutations lead to disease progression due in part to an inability to measure protein-specific mechanical changes and how these alterations may relate to disruptions in intra-cellular signaling or function. \par To improve upon the ability to measure mechanical properties inside living cells, a previously validated, genetically-encoded resonant energy transfer (FRET)-force biosensor was modified to localize to the nuclear envelope. This biosensor integrated into the nuclear envelope protein Nesprin-2G and senses small deformations that are resolved by indirect measurements of spectroscopic fluctuations in the fluorescent emission of the sensor. To accurately measure these changes, a new spectral-imaging technique named SensorFRET was developed which can resolve small changes in the FRET sensor under varying levels of fluorescent intensity and with known absolute precision. Using SensorFRET, the Nesprin-2G biosensor (Nesprin-TS) reported changes in actomyosin contractility, nuclear shape, and nuclear deformation. Using Nesprin-TS, fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome (HGPS) reported less force on Nesprin-2G molecules relative to healthy fibroblasts on average.\par To demonstrate how intra-cellular forces on the nucleus may impact normal cell physiology, bone-marrow derived mesenchymal stem cells (MSCs) were genetically modified such that the cytoskeleton was decoupled from the nucleus by saturating KASH binding proteins with a non-functional truncated protein called DN-KASH. MSCs treated with DN-KASH preferentially differentiated into osteocytes (bone cells) at a higher rate than MSCs exposed to osteogenic growth factors. This osteogenic preference after DN-KASH treatment was independent of the cell substrate topology and did not significantly alter integrin expression. However, this tendency to differentiate into osteocytes was dependent on substrate stiffness. Overall, the data imply that an intra-cellular force-dependent mechanism connected to the cell nucleus strongly influences MSC differentiation.

Lung cancer is the number one cause of cancer related death worldwide, with more than 1.6 million fatalities each year. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, with KRAS being one of the most prevalent oncogenic driver mutations. Therapeutic approaches for KRAS-mutated NSCLC have been extensively explored due to the US National Cancer Institute RAS Initiative, but methods of directly targeting KRAS or downstream effectors, such as MEK, still have poor results. Previous reports have shown that KRAS-mutated NSCLC activate distinct receptor tyrosine kinases (RTKs) depending on the epithelial or mesenchymal state. Epithelial-to-mesenchymal transition (EMT) is known to play a role in the metastasis and poor prognosis of cancer, and is induced by extracellular matrix (ECM) stiffness. Hallmarks of EMT include loss of E-Cadherin and increase in Vimentin. This research investigates the role of KRAS in EMT transition due to increased ECM stiffness in KRAS mutant NSCLC, and how this affects the efficacy of KRAS and MEK inhibition. To understand how KRAS mutations in NSCLC play a role in this stiffness induced EMT, experiments were performed to detect the gene and protein expression of EMT markers, as well as possible sources of mechanosensing, including primary cilia and receptor tyrosine kinases. We hypothesized that KRAS plays a role in activation of mechanosensors and directly correlates to EMT induced by increased mechanical forces. Results show when KRAS was inhibited and there was increased mechanical forces, either from stretch or substrate stiffness, there was a decreased activation of mechanosensors. KRAS inhibition also prevented the cells from undergoing stiffness-induced EMT. This supports our hypothesis that KRAS plays a key role in ECM stiffness induced EMT. Future studies include examining the mechanism behind this phenomenon and in vivo studies.

Epithelial-mesenchymal transition (EMT) regulates the cellular processes of migration, growth, and proliferation - as well as the collective cellular process of tissue remodeling - in response to mechanical and chemical stimuli in the cellular microenvironment. Cells of the epithelium form cell-cell junctions with adjacent cells to function as a barrier between the body and its environment. By distributing localized stress throughout the tissue, this mechanical coupling between cells maintains tensional homeostasis in epithelial tissue structures and provides positional information for regulating cellular processes. Whereas in vitro and in vivo models fail to capture the complex interconnectedness of EMT-associated signaling networks, previous computational models have succinctly reproduced components of the EMT program. In this work, we have developed a computational framework to evaluate the mechanochemical signaling dynamics of EMT at the molecular, cellular, and tissue scale. First, we established a model of cell-matrix and cell-cell feedback for predicting mechanical force distributions within an epithelial monolayer. These findings suggest that tensional homeostasis is the result of cytoskeletal stress distribution across cell-cell junctions, which organizes otherwise migratory cells into a stable epithelial monolayer. However, differences in phenotype-specific cell characteristics led to discrepancies in the experimental and computational observations. To better understand the role of mechanical cell-cell feedback in regulating EMT-dependent cellular processes, we introduce an EMT gene regulatory network of key epithelial and mesenchymal markers, E-cadherin and N-cadherin, coupled to a mechanically-sensitive intracellular signaling cascade. Together these signaling networks integrate mechanical cell-cell feedback with EMT-associated gene regulation. Using this approach, we demonstrate that the phenotype-specific properties collectively account for discrepancies in the computational and experimental observations. Additionally, mechanical cell-cell feedback suppresses the EMT program, which is reflected in the gene expression of the heterogeneous cell population. Together, these findings advance our understanding of the complex interplay in cell-cell and cell-matrix feedback during EMT of both normal physiological processes as well as disease progression.

Optimal management of fractures, post-traumatic arthritis and instability of the distal radioulnar joint (DRUJ) requires an understanding of the forces existing across this joint as a function of the activities of daily living. However, such knowledge is currently incomplete. The goal of this research was to quantify the loads that occur at the DRUJ during forearm rotation and to determine the effect that individual muscles have on those loads. Human and cadaver studies were used to analyze the shear (A-P), transverse (M-L) and resultant forces at the DRUJ and to determine the role that 15 individual muscles had on those forces. Data for scaling the muscles forces came from EMG analysis measuring muscle activity at nine positions of forearm rotation in volunteers during isometric pronation and supination. Muscle orientations were determined from the marked muscle origin and insertion locations of nine cadaveric arms at various stages of forearm rotation. The roles that individual muscles played in DRUJ loading were analyzed by removing the muscle of interest from the analysis and comparing the results. The EMG portion of this study found that the pronator quadratus, pronator teres, brachioradialis, flexor carpi radialis and palmaris longus contribute significantly to forearm pronation. The supinator, biceps brachii, and abductor pollicis longus were found to contribute significantly to supination. The results of the DRUJ analysis affirm that large transverse forces pass from the radius to the ulnar head at all positions of forearm rotation during pronation and supination (57.5N-181.4N). Shear forces exist at the DRUJ that act to pull the radius away from the ulna in the AP direction and are large enough to merit consideration when examining potential treatment options (7.9N-99.5N). Individual muscle analysis found that the extensor carpi radialis brevis, extensor pollicis longus, extensor carpi ulnaris, extensor indicis and palmaris longus had minimal effect on DRUJ loading. Other than the primary forearm rotators (pronator quadratus, pronator teres, supinator, biceps brachii), the muscles that exhibited the largest influence on DRUJ loading were the abductor pollicis longus, brachialis, brachioradialis, extensor carpi ulnaris, flexor carpi radialis, and flexor carpi ulnaris.

Valve replacement for critical heart valve diseases is in many cases not an option. Our clinical experience in pediatric compassionate care has shown robust function of porcine small intestinal submucosa (PSIS) valves. We assessed functional effectiveness of 4ply (~320µm) and 2ply (~166µm) PSIS mitral valves under pediatric-relevant hemodynamic pulsatile conditions. Key conclusions: (i)PSIS valves demonstrated statistically similar acute functionality in comparison to a commercially available valve. (ii)Energy losses were similar (p>0.05) under pediatric conditions which was not the case under adult aortic conditions. (iii)2ply valves were observed to be superior to 4ply, based on the robust hydrodynamic data, the mechanical properties suitable for pediatric applications and de-novo tissue replacement potential with less demand on the body. Demonstrating somatic growth, valve tissue filling matching PSIS degradation and PSIS-valve fatigue assessment are critical endeavors that need to be carried out to ensure mid to long term function of these bioscaffold mitral valves.