Biomedical Engineering - Theses
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The dynamics and characterization of self-assembly in biopolymers and biosystems
Biological self-assembly is the foundation of the strength and elasticity which characterizes nature-derived biomaterials, including the cell’s architecture. Thus, an in-depth understanding of the mechanics behind this process can open the doors to various biomedical applications. For this purpose, this research employed novel experimental and characterization techniques to study self-assembly in biopolymers and biosystems. Initially, a droplet dissolution technique in liquid crystalline stages was used to analyze the process of self-assembly in two important biopolymers, silk and cellulose. Here, we report an effective and simple approach based on droplet dissolution in a liquid binary phase for the formation of silk fibroin transparent spheres as well as cellulose microbeads, both of which can span several hundred micrometers in diameter. The microstructure of the spheres formed at different ethanol concentrations was characterized by electron microscopy. High concentrations of ethanol caused droplets to be encased in a thin shell which collapses once it is taken out of the liquid phase. Generally, low ethanol concentrations produce transparent silk spheres and solid cellulose microbeads. This work on biopolymers demonstrates that controlled droplet dissolution self-assembling may be explored as a novel and effective way to tailor the microstructures of nature-derived biomaterials. The spheres generated in this manner have several different characteristics which can have multiple potential uses, such as templates for scaffolds, microcarriers, as well as photonics and nano-technological applications. The second part of this thesis investigated self-assembling in biosystems. Cell aggregates are an important tool in studying tissue remodelling, extracellular matrix formation, cell-cell interaction, and last but not the least, tissue-like biomechanical properties. A medium-throughput method was designed to characterize the mechanical properties of mesenchymal cell aggregates. This study was the first to present a precise and fast method to determine the Young’s modulus of mesenchymal cell aggregates, utilizing a step-by step aspiration technique. We were also able to recreate conditions that very closely resemble the in vivo environment, where the cells were found to be stretched, and the spheroids are soft and elastic Finally, potential applications of the self-assembled cell aggregates were explored in lung disease study and drug screening, specifically for Idiopathic Pulmonary Fibrosis (IPF). We demonstrated that the cell aggregates from IPF patients show an increase in stiffness, therefore mechanical testing of spheroids is an effective technique to study this disease. It was also found that a novel compound, PF670462, modulates the effect of TGF-beta and inhibits the fibrotic response of IPF cell aggregates. That is, this drug softens IPF spheroids and downregulates fibrogenic gene expression, therefore providing basis for the potential use of PF670462 in IPF treatment.
Objective measures of dyskinesia: Towards a better understanding of the neuromotor control in children with dyskinetic cerebral palsy
Dyskinetic cerebral palsy is characterised by variable involuntary movements resulting in sustained twisting and jerky movements often causing functional limitations. Assessing dyskinesia using observation-based clinical tools with sufficient reliability and sensitivity can be challenging. However, accurate assessment of dyskinesia is necessary for effective management and development of novel therapies to treat dyskinesia in children with cerebral palsy. In this dissertation, techniques in motion analysis are investigated to explore objective methods that can detect, differentiate and grade the severity of dyskinesia in children with cerebral palsy. In addition, mobile tools such as inertial measurement units are investigated as a possible strategy to address concerns in clinical translation. Three upper limb activities were performed by six typically developing children and twelve children with cerebral palsy. Upper limb movements were recorded using a marker-based optoelectronic motion capture system, inertial measurement units and electromyography. Features of kinematic joint angles, joint centre trajectories and muscle activity were extracted and analysed against clinical assessments of dyskinesia. Kinematic studies using a marker-based optoelectronic system are recommended over inertial measurement units and electromyography for assessing dyskinesia in children with cerebral palsy. Features of volitional and non-volitional movement identified differences between typically developing upper limbs and upper limbs with dyskinesia, albeit more consistently across activities on the non-volitional side. In contrast, differentiating upper limbs with spasticity from dyskinesia in children with cerebral palsy was challenging with only a small number of features demonstrating significant differences. Finally, kinematic-based objective measures capturing smoothness of movement during the reach-and-hold activity provided the best prospect of assessing the severity of dyskinesia in children with cerebral palsy. This dissertation provides encouraging results that support the use of objective measures to assess dyskinesia in children with cerebral palsy. However, there are clear technology barriers that need to be addressed to translate these assessment techniques into clinical practice. With continued research into improving the accuracy of inertial measurement units and other technological advances in markerless video tracking, the future remains promising for the clinical use of objective measures to assess dyskinesia in children with cerebral palsy.
Carpal Kinematics In the Normal, Scapholunate Deficient and Surgically Reconstructed Wrist
Normal wrist function is ensured by a delicate interplay between bony architecture and soft-tissue constraints. Disruptions of this balance may alter carpal kinematics and joint contact, impairing the normal wrist physiology and biomechanics. The scapholunate interosseous ligament (SLIL), whose integrity is pivotal to normal scapholunate motion and that of the entire wrist, has been recognised as the most commonly injured carpal ligament. If left untreated, prolonged exposure to the altered joint motion may induce attrition of the secondary soft-tissue stabilisers, setting the stage for degenerative osteoarthritis. However, the exact role of these ligamentous stabilisers in maintaining normal scapholunate motion remains poorly understood. At present, many surgical procedures have been developed to treat scapholunate instability, but very few of them have been experimentally validated for their efficacy. Hence the aim of this investigation was twofold. Firstly, to evaluate the effects of different stages of scapholunate instability on wrist kinematics, and establish the relative contribution of various secondary scapholunate stabilisers to scapholunate motion and; secondly, to biomechanically evaluate the outcome of two reconstructive surgeries in treating scapholunate instability. As these experiments were highly invasive, a novel cadaveric wrist testing platform (CWTP), consisting of a computer-controlled dynamic wrist cadaver simulation apparatus (DWSA) and a biplane X-ray fluoroscopy system, was developed. A comprehensive set of validation experiments were carried out to assess the performance of the CWTP in carrying out in-vitro wrist simulations. It was shown that the DWSA was capable of achieving highly accurate and repeatable wrist movements, with robust control performance maintained even in the presence of severe ligament injuries to the wrist. The measurement accuracy and precision of the custom-built biplane X-ray fluoroscopy system were also evaluated. And the results demonstrated that the system was capable of resolving rigid body motion to a sub-degree and sub-millimetre accuracy. Dynamic wrist simulations, including flexion-extension (FEM), radial-ulnar deviation (RUD) and the dart-thrower's motion (DTM), were then performed in seven cadaveric wrist specimens. A four-stage ligament division protocol was developed based on a previous study that correlated specific anatomic lesions of the scapholunate supporting ligaments with different Geissler grades of scapholunate instability, as well as the widely-accepted injury mechanism of hyperextension, ulnar deviation and intercarpal supination. This included sequentially dividing the volar (v-) and the membranous (m-) SLIL in Stage I; the dorsal (d-) SLIL in Stage II; the radioscaphocapitate (RSC) and the long radiolunate (LRL) ligaments in Stage III; and the dorsal intercarpal (DIC) ligament, the scaphotrapeziotrapezoid (STT) ligament, and about 40% of the dorsal radiocarpal (DRC) ligament in Stage IV. Reconstructive surgeries were then performed with the goal of re-stabilising the severely disrupted scapholunate joint. Two different techniques, including a novel dorsal transarticular loop tenodesis (DTLT) procedure and the widely-used three-ligament tenodesis (3LT) procedure, were assessed for their efficacy in restoring the normal scapholunate biomechanics. With incremental ligament division, the scaphoid became progressively more flexed, ulnarly deviated, pronated and radially translated. The largest and the second-largest changes in the scaphoid kinematics were observed after the Stage II and IV ligament divisions, respectively. The motion of the dissociated scaphoid is likely to be primarily dictated by the bony geometry. Dividing the v- and m-SLIL, or the RSC and LRL ligaments, however, did not induce appreciable alterations. The lunate was found to be progressively more extended, supinated, and translated towards the volar and ulnar aspect of the radius. The most considerable changes in the lunate kinematics were noted after the complete division of the SLIL in Stage II. Its dissociation from the scaphoid was immediately evident, as the lunate rotation in the sagittal plane was observed to be significantly attenuated. Dividing the volar extrinsic ligaments in Stage III caused the lunate to pronate instead of rotating into further supination, as well as to become ulnarly translated. In comparison, apart from causing the lunate to become statistically more supinated, dividing the dorsal carpal ligaments and the STT ligament in Stage IV induced no noticeable alterations in the lunate kinematics. The DTLT resulted in a slightly insufficient reduction of the scaphoid in both the sagittal and transverse planes. And the procedure was found to induce a substantial amount of unphysiological volar translation of the scaphoid during ulnar deviation of the wrist. In contrast, the 3LT resulted in a much better correction of the scaphoid flexion; however, the scaphoid still appeared malrotated in pronation in the transverse plane. Furthermore, the pathological scaphoid supination was still present during wrist extension after the 3LT procedure, suggesting that the reconstruction was not capable in preventing the volar opening of the scapholunate interval. For the lunate, poor results were obtained after the 3LT, as the lunate remained collapsed in significant extension and supination. And since the tendon graft was tightened towards the ulnar side of the wrist, the 3LT not only did not correct the ulnar translation of the carpus, the condition was exacerbated by the procedure. The DTLT, on the other hand, led to a successful reduction of the dorsally collapsed lunate, which in turn restored its dynamic extension. The abnormal lunate supination was also corrected. However, cases of overcorrections were observed in these two planes. The results suggested that apart from the SLIL, the dorsal carpal and the STT ligaments might have a vital contribution to the scaphoid stability, and the volar extrinsic ligaments are essential in preventing ulnar translation of the carpus. For the DTLT procedure, a complementary dorsal capsulodesis might be beneficial in correcting the residual scaphoid flexion. And the non-physiological volar translation of the scaphoid during wrist ulnar deviation might be due to the passive stretching of the radially situated ECR-B graft used by the procedure, pronating the scaphoid and lunate as a unit towards the volar aspect of the radius. For the 3LT procedure, its inefficacy in preventing the volar opening of the scapholunate interval might be attributed to the ulnar and dorsal directed pull of the tendon graft, which might not be adequate for counteracting the supination tendency experienced by the scaphoid during wrist extension. Therefore it may be beneficial for a volar reconstruction of the scapholunate joint to be carried out in conjunction with the 3LT procedure. Furthermore, given the unsuccessful reduction of the lunate after 3LT, a transosseous passage of the tendon graft might be considered, instead of simply attaching it to the dorsum of the lunate. In summary, this study advances the understanding of normal and pathological wrist function, and provide a foundation for improved clinical practice in the surgical treatment of scapholunate injuries of the wrist.
Surface Acoustic Wave Microfluidic Platform for Cell Mechanical Measurement
Cells are dynamic, living structures that remodel themselves in response to stimuli from environment or in relation to cellular processes such as cell growth, proliferation, differentiation, migration and death. The change of cell mechanical property can be a biophysical indicator in response to the abnormal alteration in cell functionality under pathological conditions. The advances in tool development for cell mechanical measurement have facilitated in-depth discussion of cell mechanics, but heavily limited by low throughput and high cost. The emerging lab-on-a-chip microfluidic methods provide a promising solution due to the miniaturisation, among which the acoustofluidic method (the fusion of acoustics and microfluidics) appears to be advantageous due to its tunability, biocompatibility and acousto-mechanical nature. In this dissertation, I explored the application of surface acoustic wave (SAW) microfluidics in the area of cell mechanics, including establishing SAW devices for cell mechanical measurement, comparing SAW-based measurement with the benchmark from a conventional method, investigating the impacts on cell mechanical characteristic, and extending the concept to a high-throughput cytometry comparable to the real-world need. The results show that the SAW microfluidic method can provide an effective measurement on cell mechanical characteristics and probe the impact of cellular interior structure or cellular phenotype. It is consistent with the conventional benchmark and can be a complement for some cellular structures of interest. At last, it can operate as a continuous-flow high-throughput cytometry, which could be exploited in future studies related to cell mechanics.
Effect of perturbations on the biomechanics of lower limb in relation to anterior cruciate ligament (ACL) injuries
The knee is the most injured joint in the body with injuries often caused by a tear in the anterior cruciate ligament (ACL). Over 70% of ACL injuries are non-contact, meaning that an excessive amount of force or moment is generated by the individuals themselves, which induces the tear. Non-contact ACL injuries most commonly occur following a perturbation. Perturbations make the athlete unbalanced or at loss of control, which ultimately alters their normal neuromuscular control and can lead to injury. In order to understand the effect of this unconscious neuromuscular response, this study aimed to induce unanticipated perturbations during walking in 10 male and 10 female athletes. Moreover, the effect of prophylactic knee braces was examined during these perturbations. Musculoskeletal modelling in OpenSim was used to calculate kinematics, kinetics, and muscle forces during these perturbations. Females portrayed muscle force patterns during perturbations, that are likely to increase the risk of ACL injury. Furthermore, females exhibited a larger difference in muscle forces between their dominant and non-dominant limbs. Finally, prophylactic knee braces significantly reduced peak quadriceps and soleus muscle forces during perturbations. Unlike planned movements in laboratory studies, unexpected perturbations provide with deeper insight in the mechanism of ACL injuries. Although ACL injuries do not typically occur during walking, potentially injurious movement patterns during a disturbance to natural balance while walking, could provide insight on what may be reproduced, on a higher scale, during high impact and high-speed athletic tasks that can effectively tear the ACL.
Mechanical behaviour of Subchondral Bone: Elastic Moduli and Energy Dissipation in Subchondral Bone under Impact and Repetitive Loads
The damage to osteochondral tissue can develop degenerative joint diseases such as subchondral bone necrosis, osteochondral fracture, and osteoarthritis. In the development of these joint diseases, the subchondral bone plays a crucial role due to several risk factors: obesity, hereditary, aging, gender, and intense mechanical loads. The intense mechanical loads include both trauma/impact and repetitive loads which occur during high-intensity training and have been identified as the primary risk factor in the development of joint diseases. In this dissertation, we utilized mechanical tests combined with digital image correlation and micro-computed tomography to study the mechanical behaviour (in particular, Elastic Modulus and Energy Dissipation) of subchondral bone under both trauma/impact and repetitive loads. Results suggest that the presence of severe cartilage lesion in osteochondral tissue reduces the stiffness of cartilage and increases the compressive strain in the subchondral bone under trauma/impact loads. Moreover, the application of simulated physiological repetitive loads on subchondral bone has demonstrated the importance of the highest speeds in the development of the joint disease, consistent with the observations made in equine athletes. The results also exhibit the influence of microstructural properties on the mechanical behaviour of subchondral bone under repetitive loads. The results and approach of simulating physiological loading may be useful in optimizing training protocol and prevent joint diseases.
An integrated circuit towards improved management of epilepsy and other neurological conditions
Epilepsy is the second-most common neurological disorder, with an estimated prevalence of 1% worldwide. Antiepileptic drugs (AEDs) are the frontline treatment but, despite more than 25 agents available, there can be significant difficulties in finding a treatment plan that controls seizures without unacceptable side effects. Furthermore, approximately 30% of patients do not obtain full control of seizures with the available AEDs. Therefore, there exists a need for novel therapies. Therapeutic electrical stimulation of the brain and peripheral nervous system has shown great promise in treating those with refractory epilepsy, but still little is known about the mechanisms underlying this. Recent work looking at prolonged recordings of the brain has revealed a deep rhythmicity to seizures that may provide a method of developing patient-specific prediction algorithms that could be used to titrate electrical stimulation therapies. What is missing in research and in clinical practise is an implantable device which provides enough flexibility to explore new paradigms and adapt to suit the needs of the patient. Safety considerations of implantable devices necessarily precedes large-scale clinical evaluation, and this has prevented exploration of speculative higher density arrays, which come at a cost to power, complexity and mechanical reliability with no guarantee of improvement to efficacy. Rather than redesign the system from scratch, this PhD thesis asks, “how can existing implantable technology be leveraged to create flexible high-density brain recording and stimulation systems?” In response to this question, an integrated circuit called XPAND was designed to enable seamless expansions of electrode count for a wide variety of commercial stimulators. Comprising a 6 x 64 element high voltage crosspoint switch powered and configured entirely using biphasic stimulation pulses, XPAND was designed with clinical safety in mind. Fabricated in Austria Microsystems H35 high voltage CMOS process, key modules within the chip were characterised, and the crosspoint switch shown to simultaneously route high voltage stimulation and ECG signals.
Impact of multiple scale structural mechanisms on epithelium movement
Simple epithelial tissue is formed by individual cells connecting together to form a monolayer sheet. This sheet generates the complex structures of organs by undergoing both large and small scale morphogenic events. Columnar epithelial cells have a height greater than their width and are polarised with a ring of circumferential contractile fibres localised to their apical cell-cell junctions. There has been considerable investigation into the movement of this apical network, especially using lasers to sever individual apical cell edges. From these apical investigations, a variety of mechanical mechanisms have been suggested to control epithelium movement. However, these mechanisms are contrasting, and are often derived for specific experiments, rather than general apical epithelium movement. Recently, it has been recognised that in additional to the apical network, the entire three-dimensional cell shape impacts epithelium movement. Additionally, there has been growing interest in understanding how epithelium scale movement is controlled by smaller scaler structures, such as contractile fibres. This thesis utilises both computational and experimental approaches to investigate how the mechanics of underlying structures can drive epithelium scale movement, with a focus on the contribution of individual cells and contractile fibres to apical epithelium movement upon laser severing. These investigations demonstrate that studying the mechanisms of underlying cell and sub cell structures can provide new insights into the causal mechanics of general epithelium movement. It is also shown that inclusion of these mechanisms can lead to greater prediction of epithelium movement.
Red Blood Cell Passage through Narrow Capillaries: Sensitivity to Stiffness and Shape
Red blood cells (RBCs) squeeze through narrow capillaries as they transport oxygen to tissues and carbon dioxide to the lungs. The deformability of RBCs has been shown to depend on the viscoelasticity of the cell membrane and cytoplasm as well as the surface area to volume ratio (SA:V ratio) of the cell. In certain pathological diseases such as malaria, RBCs undergo restructuring of the membrane structure and modifications to the cell shape, which significantly reduce their deformability. Nonetheless, it is still unclear which factor has the greatest impact on the passage of RBCs through small capillaries. Here, we present a systematic analysis designed to identify the individual contributions of cell stiffness and SA:V ratio to the ability of RBCs to traverse narrow capillaries in a microfluidic device. We modified cellular rigidity using glutaraldehyde fixation, changed SA:V ratio by altering the buffer osmolarity and probed RBCs passage through microchannels. Our results showed that dramatic stiffening (~8 fold) had little effect (~6% retardation) on the ability of RBCs of the same geometry to traverse the channels. On the other hand, a moderate decrease (~13%) in the SA:V ratio affected the traversal of RBCs of similar stiffness more markedly (~19% decrease). We further studied RBCs infected by two different species of malaria parasites known to affect humans, Plasmodium falciparum and knowlesi. We found that P. falciparum rigidified the host RBC, but infected RBCs penetrated into microchannels with similar efficiency to uninfected RBCs. By contrast, P. knowlesi reduced the SA:V ratio of the host RBC resulting in restricted passage. We found that the earliest stage immature RBCs (reticulocytes) exhibited a similar SA:V ratio to mature RBCs and, despite being 30% larger, travelled into microchannels as efficiently as mature cells. Our finite element (FE) model provides a coherent rationale for our experimental observations, indicating that cell stiffness changes do not significantly affect RBC traversal in small capillaries due to the highly nonlinear mechanical behaviour of the cell membrane. Our numerical simulations predict that RBCs with low SA:V ratios are more prone to trapping in small capillaries (within the physiological size range) than RBCs with high membrane stiffness. Therefore, therapies targetting surface area to volume ratio of RBCs may be more effective than those that target cell stiffness.
Muscle and joint functions during walking in individuals with transfermoral amputation
Individuals with unilateral transfemoral amputation depend on compensatory muscle and joint function to generate motion of the lower limbs, which can produce gait asymmetry. Osseointegration is an alternative technique to socket-based prostheses that is used for reducing socket-skin contact problems. However, between-limb differences in joint kinematics and net joint moments may lead to abnormal hip joint contact behavior and muscle function. The aim of this dissertation is to investigate gait compensatory mechanism in individuals with transfemoral amputations fitted with socket (TFA) and bone-anchored prostheses using osseointegrated implants (BAP). In this study, two experimental and computational approaches were used to quantify the contributions of the intact and residual limb’s contralateral muscles to body center of mass acceleration and hip joint contact forces during walking. In the first approach, kinematics and kinetics data were collected from 6 TFAs and 4 BAPs performing over-ground self-selected walking task. In the second approach, a processing framework was employed using OpenSim software and MATLAB API scripting for developing three-dimensional musculoskeletal models and then to predict muscle forces and muscle contribution to waling and hip joint reaction forces. It was found that the intact limb hip muscles contributed more to body center of mass acceleration and hip contact forces than those in the residual limb. The results also suggest that osseointegrated amputees could quantify to decrease the asymmetries in the biomechanical measures between the intact and residual limbs than socket-based prosthesis amputees. The findings of this study would be useful in developing rehabilitation training programs and design of prostheses to improve gait symmetry and mitigate post-operative musculoskeletal pathology.
Cerebral perfusion markers in full-term neonates as a measure of Hypoxic-Ischemic Encephalopathy during and immediately following Hypothermia Treatment
Hypoxic-Ischaemic Encephalopathy (HIE) is a leading cause of neonatal mortality and morbidity. Therapeutic Hypothermia (TH) is the only treatment for HIE, which has reduced the rate of mortality and morbidity although still around fifty per cent of infants affected die or have a neurodevelopmental disability, such as cerebral palsy. Every infant with HIE receives the same TH protocol: 72 hours at 33.5 degrees Celsius, before being rewarmed slowly over 12 hours. The infants with moderate-severe HIE are at the highest risk of disability or death. Should it be possible to identify those infants at greatest risk during the TH, they could be targeted for modified TH treatment or for additional adjuvant treatments such as allopurinol, melatonin or ethyropeitin. The severity of the HIE, and prognosis on outcome, is challenging while the infant is cooled, as the cooling itself reduces the prognostic power of many of the previously accepted assessment methods. MRI (Magnetic Resonance Imaging) is the gold standard, but moving infants to an MRI whilst cooled is problematic, and the HIE injury to the brain may not be evident for several days. The objective of this research study was to find a clinical parameter which would identify those infants at highest risk of a poor outcome. HIE is characterised by hyper-perfusion, cerebral blood flow (CBF) exceeding metabolic demand, consequent from a physiological response preferentially redirecting blood flow to the brain during a hypoxic episode. Thus, a marker of cerebral perfusion that could classify HIE infants by the degree of hyper-perfusion was sought. Initially the Resisitive Index (RI), as determined by measuring the Cerebral Blood Flow Velocities (CBFV’s) using Doppler Ultrasound (DUS), was investigated as a possible cerebral perfusion marker. The RI is an accepted measure of the resistance of the cerebral arteries and an RI < 0.55 is considered to be indicative of a previous hypoxic event. In the first part of this study, the RI was evaluated in 80 healthy and 18 HIE infants and found to be unreliable; some infants with HIE and a poor outcome did not have an RI < 0.55, whereas some infants without HIE did. Moreover, DUS is not continuous; it is a one-off measurement requiring a skilled operator. A pursuit for another cerebral perfusion marker ensued. Frequency-Domain Near Infrared Spectroscopy (FD-NIRS) promised absolute measurements of oxy- and deoxy-Haemoglobin (HbO and HbR) in the brain tissue. Increased CBF was expected to result in a higher than normal concentration of blood and therefore Haemoglobin in the brain tissue. The results from the second part of this research study of 40 healthy and 6 HIE infants suggest that hyper-perfusion is expressed in the concentration of HbO in the brain tissue, and that this is most related to poor outcome. The limited HIE population in this study prevents generalisation from this finding, though suggests that there is justification for further research using FD-NIRS to monitor infants with HIE.
Dynamic stability and variability of perturbed walking in young adults
Falls are the third major cause of inadvertent injury in young Australian adults aged between 18-35 years. The inability of an individual to respond to external perturbations due to walking on an inclined surface, or internal perturbations such as dual-task walking, are known to be associated with significantly higher risk of balance loss. Significant factors known to increase risk of balance loss during walking include performing an additional task requiring high motor-cognitive, sensory or cognitive load (internal perturbations), and walking on uneven surfaces such as sloped terrain (external perturbations). At present, however, dynamic balance of the entire body and the risk of balance loss during walking under such perturbations is not well understood. The objective of this study was to investigate dynamic stability and variability of the human body during walking, and assess the influence of external, motor-cognitive, sensory and cognitive perturbations on dynamic balance, including surface inclination, use of a cell phone, auditory and visual stimulation, and mental calculation. Nineteen healthy young adult males were recruited. Three-dimensional joint kinematics were obtained using an optical motion capturing system as subjects walked at their self-selected speed on an instrumented treadmill. Dual-tasking was simulated by subjecting participants to motor-cognitive, visual, cognitive and auditory perturbations during walking including cell phone usage (talking, texting and reading), watching a video clip, listening to music, and performing numeric calculations mentally. External perturbations were also applied through alteration of surface inclination. Variability analysis was performed on spatiotemporal gait parameters using Detrended Fluctuation Analysis (DFA) and Standard Deviation. Dynamic stability was subsequently estimated for the entire body as well as the head, trunk and lower extremity joints using linear and nonlinear measures including Margin of Stability (MoS), Lyapunov Exponent (LyE) and Maximum Floquet Multipliers (MaxFM). A novel method was devised to assess stability using Margin of Stability at heel contact (HC) and minimum foot clearance (MFC), gait events associated with backward and forward balance loss, respectively. Slip and trip propensity estimated using Required Coefficient of Friction (RCoF) and MFC height, respectively. Finally, the most destabilizing additional task while walking was determined using deviation of MoS and trip propensity values during dual-task trials from the corresponding values during baseline walking. The results showed that dual-tasking during walking adversely affects balance in a direction specific-manner. Specifically, cell phone texting and reading while walking reduces balance in the mediolateral direction, while cell phone talking increases the risk of tripping in the anteroposterior direction. Upslope terrain increased the risk of balance loss in the anteroposterior and vertical directions and did not affect gait balance in the mediolateral direction, while walking down was associated with greater stability in the anteroposterior direction. Cognitive and sensory perturbations affected gait balance mostly in the anteroposterior and vertical directions rather than the mediolateral direction. Analysis of trip propensity showed that motor-cognitive dual-tasking due to cell phone usage while walking, cognitive and sensory perturbations due to performing additional auditory and visual tasks while walking are associated with greater risk of tripping, as measured by a lower MFC height. Particularly, talking while walking, and cognitive and sensory dual-tasking while walking may ultimately lead to an increase in risk of tripping in young adults. However, the risk of tripping in young individuals is not sensitive to external perturbations caused by sloped terrains. Participants mostly changed their step length and step time during walking under perturbations. Among the various measures used to determine the most destabilizing secondary task while walking, MFC height was more sensitive to the applied perturbations. Talking while walking was associated with the largest deviation from baseline condition. The findings of this investigation confirmed that head stabilization during ambulation has higher priority compared to other segments, and individuals try to adopt different strategies to attenuate perturbations from the lower body to the head. The current data highlighted the importance of arm swing in balance maintenance during walking under perturbations, and demonstrated that individuals try to compensate restricted arm swing during walking by modulating step width. With respect to gait adaptations, the results of this research support the idea that individual’s response to applied perturbations through dual-tasking while walking depend on the magnitude of the applied perturbation. The evidence from this study suggests that talking while walking is the most challenging secondary task during locomotion among the applied perturbations in this study, and additional sensory tasks are the least challenging one. These findings have significant implications for development of a gait training protocol for more frail people to successfully address common perturbations arising from daily living activities during everyday life. These data also suggest that MFC height analysis and local stability analysis of the lower body should be performed to gain better understanding on the effect of additional concurrent task while walking on the risk of tripping and gait stability, respectively. The analysis of MoS presented extends knowledge of step-to-step balance changes during walking at different gait events associated with common fall patterns occurring at HC and MFC. Further work needs to be done to analyse the influence of similar attention-demanding secondary tasks or ‘distractions’ in more vulnerable populations, including the elderly, fallers, and individuals with sensory or motor impairments that affect locomotor control.