Individuals with spinal cord injuries (SCI) are at increased risk for cardiopulmonary and cardiovascular disease. The GameCycleTM exercise system integrates arm-ergometry and video gaming with the goal of providing a fun and motivational exercise platform. For this thesis research, two studies were conducted to evaluate the design of the GameCycleTM and its efficacy as an exercise platform. The objective of the first study was to teach subjects how to safely and effectively use the GameCycleTM, to determine if subjects are able to learn how to operate the GameCycleTM in an acceptable time period, to learn whether they are able to reach their target heart rate zone using the GameCycleTM, and to obtain feedback from new users regarding features of the new system. Participants included 14 subjects with SCI (11 men and 3 women, 37.5 +/- 6.5 years). Subjects were trained to use the GameCycleTM and were required to complete a timed demonstration. Metabolic data were collected over a 14 minute exercise bout while playing the GameCycleTM. All subjects were able to complete training successfully and 12 (86%) of the subjects were able to reach their target heart rate zones. All of the participants conveyed that the GameCycleTM was easy to learn, operate, and has easily adjustable settings to suit their needs. 86% of participants found the GameCycleTM to be enjoyable and that it was likely motivate manual wheelchair users to exercise regularly. This study indicates that the GameCycleTM is easy to use and confirms previous findings that aerobic training zones can be reached. The goal of the In-home Phase was to evaluate the effectiveness of the GameCycleTM as compared to standard ergometry. Nine persons with SCI (1 woman, 8 men, 36.2 +/- 5.5 years) completed a four-month in-home trial in which they were asked to exercise with the GameCycleTM for two months and a standard arm-ergometer for two months. Results indicate that subjects exercised for significantly longer durations (p=0.035) with the GameCycleTM. This suggests that the GameCycleTM is more enjoyable and will increase exercise dosage for longterm exercise compares to standard arm-ergometry.

Slip and fall accidents are a serious occupational and public health problem. While shoe-floor-contaminant friction is known to be critical to slip risk, no method of measuring shoe-floor-contaminant friction is widely accepted as being relevant to human slips. In addition, the tribological mechanisms of the shoe-floor-contaminant interface are poorly understood. This dissertation studies slips and falls from a biomechanical and tribological perspective. Heel contact control was investigated during human slipping experiments. Knee joint torques were found to be the primary contributor to heel acceleration during contact with the floor. For the tribology portion of this research, experimental testing was performed using a novel whole shoe slip testing method and a pin-on-disk tribometer. The experiments revealed that shoe-floor-contaminant friction could be described with the theoretical Stribeck curve. The lubrication regime that was determined to be most relevant to shoe-floor-contaminant friction was the mixed-lubrication regime. A computational model was developed to describe this mixed-lubrication regime, simulating the hydrodynamic and contacting pressures at the shoe-floor-contaminant interface applied to pin-on-disk experiments. The model-predicted friction values showed good agreement with experimental data. Because the custom code was limited to simple geometries, FEA was examined for its ability to simulate mixed-lubrication of an entire shoe heel against a floor surface. Limitations were discovered in current FEA software packages that prevented their use in shoe-floor-contaminant friction modeling. Therefore, a hybrid model that used FEA software to simulate the contact and custom modeling to simulate the lubrication effect was proposed. The research presented in this dissertation may be the first step towards developing a comprehensive shoe-floor-contaminant friction model, which will be useful for evaluating slip potential of shoes and flooring, designing safer shoes and floor surfaces, and understanding the biomechanics of slipping.

Polymeric microspheres have been widely investigated as delivery systems and are clinically used today. We examined the use of poly(lactide-co-glycolide) (PLGA) microspheres in delivery systems for soft tissue engineering, chemotherapeutic delivery, and cartilage tissue engineering. Soft tissue defects due to trauma or tumor removal remain a clinical challenge. We examined the use of PLGA microspheres and adipose derived stem cells (ASCs) to fill in soft tissue defects. We first demonstrated the use of PLGA microspheres to increase ASC proliferation and survival by encapsulating fibroblast growth factor-2 (FGF-2). The released FGF-2 increased ASC proliferation and survival in vitro. Addition of the FGF-2 microspheres in an in vivo study resulted in an increase in angiogenesis. We then examined the ability of released adipogenic factors to induce the differentiation of ASCs into mature adipocytes. Oil red O staining and Western blots confirmed that adipogenesis was induced by the released factors. The second goal was to examine a delivery system to reduce the risk of local recurrence in breast cancer patients following a lumpectomy. Breast cancer lumps are commonly treated by tumor removal (lumpectomy) followed by radiation or chemotherapy, and both have adverse side effects. PLGA microspheres encapsulating doxorubicin were embedded with a natural scaffold, gelatin, to locally deliver chemotherapy and maintain the breast contour. Our results demonstrated a more controlled release from microspheres embedded within gelatin compared to microspheres alone. Released doxorubicin killed tumor cells in vitro. The implantation of the scaffolds in vivo resulted in tumor ablation. Local and systemic toxicity were not observed even though a dose 60 times the normal dose was given. Our next objective was to analyze the release of TGF-beta1 (TGF-) from PLGA microspheres incorporated into a synthetic hydrogel, poly(ethylene glycol) (PEG)-genipin for cartilage repair. The release of TGF-was dependent upon the genipin concentration of the hydrogel. The released TGF- was bioactive, as demonstrated by the inhibition of mink lung cell proliferation. The final goal was to develop and characterize a hydrogel based on PEG-genipin to gel in situ. As such, we examined genipin and multi-branched aminated PEG. Gelation time was affected by pretreating genipin. Exposure of the genipin aqueous solution to air and oxygen decreased the gelation time. PEG structure also had an effect on gelation time. The gelation time was reduced by utilizing 4-arm PEG and increasing the temperature from 25oC to 37oC. The results of this thesis demonstrate the efficacy of PLGA microspheres embedded in hydrogels for use as delivery systems for soft tissue and cartilage tissue engineering. The delivery systems can be modified to tailor delivery rates, deliver multiple drugs/growth factors, tailor degradation, and promote tissue growth.

Balance is control through the integration of vestibular, visual and proprioceptive senses; however, the impact of each during walking is not fully understood. The primary aim of this thesis is to understand the impact of vestibular signals in the maintenance of balance during walking. Galvanic vestibular stimulation (GVS) was used to evoke internal perturbations of the human balance system. Subjects were exposed to GVS during gait and basic results were consistent with published literature; GVS increased the mediolateral deviation of whole body movements towards the side of the galvanic anode during gait. Results indicate that head stability is of key importance to the balance system. The head was held relatively level as compared to the thorax or pelvis throughout gait irrespective of the presence of vision or GVS. Though the pelvis experiences significant tilting due to GVS, a counter rotation of the head compensates for this tilt of lower segments to preserve the level reference frame of the head. Vision was found to be of paramount importance during gait. When walking with eyes open, GVS had a minimal effect on the gait trajectory, spatial foot placement parameters, or tilt angle of the head, thorax, and pelvis. Lastly, temporal ordering of the tilt angles of the head, thorax, and pelvis were observed depending on the perturbation according to the stance limb providing preliminary evidence that segmental tilting occurs during gait. Thus, the well published inverted pendulum model is incorrect for balance losses during gait. Future testing of a greater number of subjects with a larger range of ages and physical condition may provide a greater insight to the intricacies of the human vestibular system.

Chronic back pain has historically been treated through spinal decompression and fusion often accompanied by fixation devices. Concerns regarding the effect of rigid fixation on the surrounding tissue and vertebral levels adjacent to fusion have given rise to a new paradigm based on restoring healthy or natural motion of the operated level. This paradigm revolves around the design and implementation of so-called motion preservation devices. In vitro testing has been and will continue to be an integral step in the design and evaluation process for both rigid fixation and motion preservation devices. However, the metrics commonly used to asses the efficacy of a rigid fixation device are insufficient for the assessment of motion preservation devices. In addition, motion preservation device metrics have not been rigorously defined or characterized in the healthy human spine. The kinematic response of the human cadaveric spine to loading in a six-degree-of-freedom spine testing apparatus can be expressed in terms of Euler angles and the helical axis of motion while the viscoelastic response can be expressed in terms of the energy dissipated by each specimen during a single cycle of testing. Beyond conventional metrics, a new, noninvasive method based on applying test kinematics to a three-dimensional rigid-body model of the spine is developed and used to investigate articulation of the facet joints. Articulation is investigated based on a distance map between adjacent articular surfaces and quantified through the calculation of a parameter describing the proportion of the facet contact area. Statistically significant differences were found between the facet contact area parameter at full extension and full flexion at every level of the lumbar spine during in vitro testing (p<0.037). Additionally, significant differences were found between the mean helical axis locations of some of the levels. A significant difference was found between the anterior/posterior location of the helical axis during flexion and Extension at the L1-L2 level (p=0.003). The sensitivity of these parameters in describing differences in lumbar kinematics between levels and between different portions of the range-of-motion lends credence to their efficacy in evaluating the quality of motion achieved after implantation of a motion preservation device.

Double bundle anterior cruciate ligament reconstruction (DB-ACLR) has recently gained popularity in Europe and Japan. This procedure utilizes two separate tissue grafts to replicate the two functional bundles of the intact anterior cruciate ligament (ACL). Therefore it is believed that the two grafts will be able to restore both the anterior and rotatory laxity to that of an intact knee. However, as in the case of a traditional single bundle ACL reconstruction, there are several variables that can affect the outcome. The knee flexion angle at which each of the two grafts are fixed, is one such variable. Since it is understood that an improper force distribution among the two grafts could lead to the failure one or both of the grafts, it is important to fix the grafts, such that the in situ force of each graft does not exceed that of their respective intact bundle. Therefore, one of the objectives of this thesis is to study if and how the knee flexion angle for graft fixation affects the force distribution of the two grafts in DB-ACLR. A second concern regarding DB-ACLR is related to the complications of drilling a second femoral tunnel. Not only can tunnel placement become more complex, but more problems may also arise in the event of a revision surgery. Therefore, a DB-ACLR procedure that utilizes only a single tibial and femoral tunnel will be investigated. In this procedure, a single femoral tunnel will be created for the PL graft, while the second graft will be fixed on the lateral femoral epicondyle via a staple (fixation protocol PL+OTT). In order to study the effect of the knee flexion angle of graft fixation, as well as the PL+OTT procedure, knee kinematics will be collected for the intact, ACL(-), and reconstructed knees under both a 134 N anterior tibial load, as well as a combined rotatory load of 10 N-m valgus, and 5 N-m internal/external tibial rotation. Lastly, the in situ force of the intact ACL, as well as the intact bundles will be determined, and compared with the in situ force of the grafts.

Manual wheelchair uses rely on their upper limbs for mobility and activities of daily living. Unfortunately more than half of manual wheelchair users will experience shoulder pain, due in part to repetitive loading during wheelchair propulsion and transfers. While chronic upper extremity pathology has been well documented, no research has investigated acute rotator cuff changes that occur as a result of wheelchair propulsion. Ultrasound is a non-invasive, convenient method to examine soft tissue structures of the shoulder, but tendinosis is rated subjectively by the operator. Here we apply image analysis techniques to quantify tendon size, echogenicity, and greyscale texture. We have developed a standardized protocol, and custom reference marker, to maximize reliability of these measures. Further, content validity was established by relating greyscale-based quantitative ultrasound measures to known risk factors for shoulder pain and pathology including increased age, duration of wheelchair use, and body weight. Quantitative ultrasound measures also correlated to clinically graded tendinosis and discriminated between people with and without symptoms on physical examination. Sixty-seven manual wheelchair users underwent quantitative ultrasound examinations of the biceps and supraspinatus tendons before and after an intense wheelchair propulsion task. Biceps tendon greyscale texture post-propulsion was significantly impacted by clinically graded tendinopathy, duration of wheelchair use, resultant force, and stroke frequency when controlling for pre-propulsion ultrasound image texture. Subjects with tendinopathy or a longer duration of wheelchair use tended to have a darker, less organized tendon microstructure following propulsion likely due to the presence of inflammatory factors or other fluid. In contrast, subjects who used a higher stroke frequency or resultant force showed a brighter, more aligned tendon fibrillar structure due to mechanical loading of the tendon. In a subsample of subjects, we found that increased shoulder forces and moments during propulsion correlated with more severe supraspinatus tendinopathy. These subjects also experienced a larger decrease in supraspinatus tendon width and greyscale variance following the intense propulsion task. Quantitative ultrasound measures describe tendon microstructure and are sensitive to risk factors for shoulder pain and pathology. This technique may help identify the best interventions to reduce an individuals risk of developing upper limb pathology.

Cellular therapy holds tremendous potential in regeneration of mineralized tissues such as bones and teeth. We have characterized and identified pericytes as a unique population of dental pulp stem cells (DPSCs) that can be sorted by CD146+CD34-CD45-CD56-, expanded in culture, and differentiated into osteogenic, chondrogenic, and adipogenic lineages. A well-characterized stem cell source and an appropriate microenvironment containing growth factors and/or extracellular matrix (ECM) proteins to stimulate differentiation and mineralization are required for successful cellular therapies. To understand cell-ECM protein interaction, we studied the signaling role of phosphophoryn (PP), an ECM protein found in dentin and bone. PP signals through integrins, mitogen activated protein kinase (MAPK), and Smad pathways. There is also signaling crosstalk between the MAPK and Smad pathways. To better understand the complex signaling pathways involved in stem cell differentiation during dentin or bone formation, we have utilized quantitative proteomic strategies to study stem cell differentiation triggered by PP and BMP-2. Proteins upregulated and downregulated during differentiation were identified by mass spectrometry. With the ultimate goal of better enabling the regeneration of diseased or damaged mineralized tissue, our findings in this study have enhanced our understanding in stem cell differentiation to the osteoblastic/odontoblastic lineages and lay foundations for the development of future craniofacial regeneration.

Vascular endothelial cells (VEC) assemble into capillary-like structures during angiogenesis, and this neovascularization process plays an important role in a wide range of physiological and pathological scenarios. Based on significant upregulation of its expression in VEC during capillary morphogenesis, profilin-1 (Pfn1 - a ubiquitously expressed actin-binding protein) was previously implicated in capillary morphogenesis of VEC. The overall objective of the present study was to investigate whether and how loss of Pfn1 function affects a) the various cellular functions that are important for capillary morphogenesis such as VEC migration, invasion and proliferation, and b) the overall capillary forming ability of VEC. Loss of Pfn1 function in VEC was achieved either by suppressing the overall expression of Pfn1 by RNA interference method or selectively abrogating specific ligand-interactions (actin, proline-rich ligands) of Pfn1 by expressing various point-mutants of Pfn1 in a near-null endogenous Pfn1 background (knockdown and knock-in approach). Loss of Pfn1 expression causes a major change in actin cytoskeleton in VEC. Particularly, there is a significant depletion of actin filaments and focal adhesions in VEC when Pfn1 expression was silenced. Silencing of Pfn1 expression also significantly impairs the migratory ability of VEC. Analyses of leading edge dynamics revealed that Pfn1 depletion results in decreased velocity and frequency of lamellipodial protrusion. Further experiments with point-mutants of Pfn1 showed that both actin and polyproline interactions of Pfn1 are required for efficient lamellipodial protrusion and overall migration of VEC. Loss of Pfn1 expression is associated with reduced dynamics of VE-cadherin dependent cell-cell adhesion, which was also found to be correlated with increased nuclear accumulation of p27 Kip1 (a major cell-cycle inhibitor) and reduced VEC proliferation. Finally, we found that loss of overall expression of Pfn1 significantly impairs collagen gel invasion and three-dimensional (3-D) capillary morphogenesis of VEC. Abolishing either of actin or polyproline interactions of Pfn1 also leads to a dramatic inhibition of capillary mophogenesis of VEC. Taken together, these results demonstrate that Pfn1 plays a critical role in capillary morphogenesis of VEC through its interactions with both actin and polyproline ligands. This study may further imply that Pfn1 could be a novel angiogenesis target.

Heart diseases are leading causes of death in the world. The inability of heart muscles to regenerate or restore the lost function makes the use of stem cells an attractive therapeutic option for patients with heart diseases. Stromal cells from fat tissue, namely adipose-derived stem cells (ADSCs), exhibit properties of mesenchymal stem cells and can be differentiated towards several cell types. That adipose tissue is abundant and easily harvested from patient¡¯s own body makes it an ideal source for patient specific stem cells. Yet, less is known about the capacity for self-renewal and stability properties of mesenchymal ADSCs during expansion in culture. Furthermore, efficient differentiation of these cells towards cardiogenic lineage has not been established. We first characterized human ADSCs cultured for varying times. Flow cytometry indicated that ADSCs maintained expression of mesenchymal markers (CD29, CD44 and CD90) for extended expansion over 20 passages. In contrast, mRNAs for pluripotent markers, such as Nanog, Oct4 and Sox2, were significant only in early passages, but were dramatically declined during culture. Serum removal increased mRNA levels for various cardiogenic genes, such as Mef2C, cardiac actin and troponin. Moreover, the protein kinase activator phorbol ester (phorbol myristate acetate PMA, 10 nM) caused further increases in these cardiac mRNAs. The upregulation of cardiac mRNAs by serum removal and PKC activation were constant in ADSCs cultured for various times. The use of various inhibitors specific for PKC subtypes suggested that the novel PKC theta/delta isoforms mediate this upregulation. RT-PCR revealed that ADSCs express significant mRNA for PKC delta, but not theta isoform. Overexpression of cDNA for PKC delta resulted in marked increases in cardiac mRNA expression. These results indicate that activation of PKC delta induces expression of multiple cardiogenic genes freshly-prepared and expanded ADSCs. These findings demonstrated that human ADSCs maintain the expression of mesenchymal markers and the ability to exhibit cardiac gene expression for extended expansion. They also suggest that a specific signaling molecule is involved in the transdifferentiation towards cardiogenic lineage.