Relaxin is a peptide hormone emanating from the corpus luteum of the ovary which circulates during pregnancy. Traditionally, the hormone has been associated with female reproductive processes but recent evidence has suggested relaxin may play a vital role in regulating renal and cardiovascular function. Analogous to pregnancy, chronic administration of recombinant human relaxin (rhRLX) to nonpregnant female or male rats induces renal vasodilation and hyperfiltration, as well as reduces the myogenic reactivity of small renal arteries. Additionally, elimination of circulating relaxin in pregnant rats using relaxin neutralizing antibodies or ovariectomy prevents the pregnancy associated changes in the renal circulation. Based on these findings we postulated that relaxin exerts similar vasodilatory effects in the systemic circulation. We conducted several studies examining the role of relaxin in modulating systemic hemodynamics and vascular wall mechanical properties. Analogous to pregnancy, administration of rhRLX to nonpregnant female or male (normotensive and hypertensive) rats elicited increases in cardiac output and global arterial compliance, as well a decrease in systemic vascular resistance. By neutralizing circulating relaxin in midpregnant rats with specific antibodies, we determined that the hormone was essential for the transition of the systemic circulation from the virgin to the pregnant state. In order to understand the mechanistic bases for relaxin-induced increase in global arterial compliance, we examined the hormones effects on vascular wall geometric and compositional remodeling. From a geometric perspective, small renal arteries isolated from rhRLX-treated rats and mice were characterized by larger arterial wall area when compared to control counterparts. This resulted primarily from a relaxin-mediated increase in smooth muscle cell hyperplasia, as indicated by increased smooth muscle cell density, not hypertrophy. From a compositional perspective, these arteries were characterized by decreased collagen with no change in elastin content. In contrast, external iliac arteries from rhRLX-treated mice did not exhibit any alterations in biochemical composition or smooth muscle cell density when compared to control mice. Comparable results were observed in relaxin knock-out (Rlx-/-) and wild-type (Rlx+/+) mice, with small renal arteries from Rlx-/- mice exhibiting increased arterial collagen and decreased smooth muscle cell density. Finally, from a tissue mechanics perspective, small renal arteries from rhRlx-treated mice exhibited a strain-dependent reduction in tissue strain energy such that the greatest reduction was observed at the highest circumferential and axial strains. We conclude relaxin exerts systemic vasodilatory effects in both pregnant and nonpregnant states and in a gender-independent manner. Our findings indicate that the relaxin-induced increase in global arterial compliance is, at least in part, due to vascular wall compositional and geometric remodeling. Further, the vascular wall remodeling effects of relaxin appear to be artery-type specific. Finally, our data indicate that the relaxin-induced compositional remodeling of small renal arteries contributes to tissue mechanical properties under conditions of high circumferential and axial loads.

Numerous pathologies that affect the urinary bladder, such as spinal cord injury (SCI), bladder outlet obstruction, or diabetes, cause bladder wall remodeling. Bladder remodeling is marked by changes in the extracellular matrix (ECM) proteins collagen and elastin as well as alterations in bladder smooth muscle cell (BSMC) hypertrophy and phenotype. Previous work has examined the bladder following SCI and found an early increase in elastin and hypertrophy followed by longer term fibrosis. These studies indicated that the mediating factor in bladder remodeling in response to early stage SCI and other pathologies such as obstruction and diabetes is strain. In the SCI bladder, the strain history is changed from normal filling and voiding to over distension and intermittent smooth muscle cell contraction. The goals of this study were to utilize an ex vivo organ culture model to examine the effects of strain on bladder smooth muscle remodeling, examine the effects of TGF-ß1, found to be up regulated in the SCI bladder, and to utilize a tissue engineering methodology to examine the effects of strain in cell-seeded biologic scaffolds. The first aim from this study showed that abnormal strain frequency profoundly induces elastogenesis in the ex vivo bladder. Further examination with the addition of TGF-ß1 with and without mechanical stimulation showed that mechanical stretch of the ex vivo bladder mimics the early stage SCI bladder in remodeling and cell phenotype, and the addition of TGF-ß1 alters this phenotype. Additionally, it was found that TGF-ß1 added to culture of BSMC on collagen gels decreases gel contraction but increases collagen organization of the gels. Finally, in a tissue engineered construct it was found that the growth factors VEGF and FGF-2 promote penetration of BSMC into small intestinal submucosa and that strain frequency alters the ECM proteins that the BSMC produce with a frequency of 0.1 Hz promoting elastogenesis and a frequency of 0.5 Hz promoting collagen production. The information gained in this study gives further insight into the role of strain in pathological remodeling of the bladder, and it provides a basis for tissue engineering constructs with controlled BSMC penetration and ECM composition.

Slips and falls are serious public health concerns in older populations. Understanding relationships between propensity to slip and biomechanical and physiological characteristics is important to identify factors responsible for slip-initiated falls and to improve slip/fall prevention. Thus, the first goal of this thesis was to investigate the relationship between knee flexion/extension strength and slip severity. Reduced muscle strength is associated with aging and falls. Knee corrective moments generated during slipping assist in balance recovery. Isometric knee flexion/extension peak torque, rate of torque development (RTD), and angular impulse were measured in 30 young and 28 older subjects. Motion data were collected for an unexpected slip during self-paced walking. Slips were characterized as non-hazardous or hazardous based on a 1.0 m/s peak slip velocity threshold measured at the slipping heel. Within-gender regressions relating strength to slip hazardousness and age group revealed significantly greater left knee extension RTD and angular impulse in young males experiencing non-hazardous versus hazardous slips. Findings were not evident in older males, who perhaps implement cautious walking styles, allowing less reliance on post-slip recovery reactions. Other strength variables were not associated with hazardousness. Thus, rapid knee extension force generation may assist balance recovery from hazardous slips. Decreased postural stability is also associated with aging and falls. Therefore, the second goal of this project was to investigate the association between ability to integrate sensory information important for balance and slip severity. The Sensory Organization Test (SOT) was administered and COP standard deviation (COP ST DEV) and path length (PATH LENGTH) were calculated for each condition. COP ST DEV, PATH LENGTH, and variable ratios were regressed on age group and hazardousness within condition. Significantly greater PATH LENGTH and its subsequent effects on ratio variables associated with Condition 4, in which somatosensation was rendered inaccurate, were evident in individuals experiencing hazardous versus non-hazardous slips. Conditions in which vestibular or visual information was rendered inaccurate or missing were not associated with hazardousness. Somatosensory channels detect slips first at the shoe-floor interface and thus may be especially important in early detection and response to a slip.

Liver disease is a leading cause of mortality in the United States. Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid artificial liver-assist devices or long-term therapy by replacing the diseased liver with functional tissue-engineered constructs that utilize a combination of cells, scaffolds and bioactive factors. A rate-limiting step for TE&RM technologies has been the loss of hepatocyte-specific functions in vitro after hepatocytes are isolated from their highly specialized in vivo microenvironment. The identification of a biologic substrate that can maintain a functional hepatocyte differentiation profile during in vitro culture would advance potential TE&RM therapeutic strategies. The present study is predicated upon the hypothesis that the substrate upon which hepatocytes are seeded is a critical determinant of cell phenotype and function. Biologic scaffolds composed of extracellular matrix (ECM) derived from mammalian organs have been used to maintain cell phenotype in vitro. Two studies were performed to evaluate the ability of ECM scaffolds to maintain primary human hepatocyte (PHH) phenotype in vitro. The first study evaluated the effect of ECM scaffolds derived from porcine liver (PLECM) and human liver (HLECM) upon maintainence of PHH specific functions in vitro. Cytochrome P450 activity and albumin secretion were used as indicators of hepatocyte functionality. No differences in hepatoycte-specific functions were measured when comparing PHH cultured with PLECM and HLECM. From a clinical perspective, the results are appealing because of the limited availability of human liver tissue compared to porcine liver tissue for the manufacture of ECM scaffolds. The second study cultured PHH between two layers of PLECM and compared with Matrigel double gel cultures and adsorbed type-1 collagen. Albumin secretion, hepatic transport activity and ammonia metabolism were used as markers of hepatocyte functionality. PHH cultured between two layers of porcine liver ECM had significantly higher levels of albumin secretion, hepatic transport activity, and ammonia metabolism compared to PHH cultured on collagen.

Studies of hemangioblasts (a common progenitor of hematopoietic and endothelial cells) during human development are difficult due to limited access to early human embryos. To overcome this obstacle, the in vitro approach of using human embryonic stem cells (hESC) and the embryoid body (hEB) system has been invaluable to investigate the earliest events of hematopoietic and endothelial cell formation. Herein, firstly, optimal culture conditions of hEB were determined for differentiation of hESC toward hematopoietic and endothelial cell lineages and then different developmental stages of hEB were characterized for angio-hematopoietic cell markers expression. Day-8 to day-10 hEB included the highest numbers of hematopoietic and endothelial progenitor cells, and CD34+ CD45- day-10 hEB cells were sorted to evaluate their hemangioblastic cell potential. Next, we established an in vivo chick embryo system that allowed sorted candidate hemangioblast populations to proliferate, migrate, and differentiate. Different stages of hEB cells, day-10 hEB cells purified for expression of CD34, and human peripheral blood hematopoietic stem/progenitor cells were examined for their engraftment capacity in chick embryos. Meanwhile, we examined the multi-lineage differentiation potentiality of CD146+ CD34- differentiating hESC. Recently, our group characterized pericytes/perivascular cells, that displayed positive expression of CD146 (and a lack of mature endothelial cell markers) in a variety of human organisms. Differentiating hESC include a CD146+ population that concomitantly expresses endothelial progenitor cell markers (CD31, CD34, CD133, and BB9). CD146+ pericyte-like hESC were tested for their hematopoietic, myogenic, and neurogenic potential. Finally, perivascular cells were obtained from human fetal placenta villi in order to evaluate their myogenic differentiation, migration ability, and mesenchymal stem cell phenotype. Placental villi are exceptionally rich in fetal microvessels; these might prove to be a beneficial source for stem cells that reside within blood vessel walls. Both CD146+ pericytes isolated from freshly dissociated placenta and purified blood vessel vasculature of placenta were observed for their myogenic potential. In summary, this project provided approaches to understand the early events of hematopoiesis using hESC and a chick embryo model, and allowed for observation of the mesenchymal lineage potential of perivascular cells derived from hESC and human fetal placenta.

Blood soluble drag reducing polymers (DRPs) represent a potential novel treatment of hypoperfusion and other disorders. Injections of these high molecular weight viscoelastic molecules into the blood of experimental animals at sub-nanomolar concentrations were shown to increase cardiac output with no changes in blood pressure (a reduction of vascular resistance) and enhance tissue perfusion and oxygenation. The DRP intravascular phenomena have been successfully utilized in animal models of various pathologies including hemorrhagic shock, hypobaric hypoxia, coronary stenosis, and diabetes. Chronic injections of DRPs demonstrated the reduction/prevention of atherosclerosis. Two reported potential mechanisms behind the DRP intravascular effects were a decrease in flow separations at vascular bifurcations and a reduction/elimination of the Fåhraeus effect (cell-free plasma layer existing in the near-vessel-wall space) in microvessels. The latter effect may enhance blood transport efficiency and selectively implement an increased shear stress on the endothelial cells in microvessels. This work was aimed to expand the knowledge on the mechanisms behind the phenomenological effects of DRPs in the cardiovascular system and to study new biomedical applications. A rodent model of chemically-induced diabetes illustrated the potential utility of DRPs for the improvement of microcirculation impaired by disease development and implicated as an etiology of its complications. Additional rodent experiments tested and proved the absence of acute and chronic deleterious effects of hemodynamically effective concentrations of DRPs on hematological, serum chemistry, blood gas and blood coagulation parameters. Further experiments were performed to determine the DRP concentration thresholds which could be safely used intravenously. A hypothesis that DRPs affect RBC deformability was tested using bulk blood filtration and viscoelastometry techniques. The filterability of RBC suspensions with DRPs was found to be slightly increased reflecting a potential increase in RBC deformability. It was also shown that DRPs slightly decreased RBC viscoelasticity which was increased due to diabetes in rodents which also reflects a potential increase in RBC deformability. Finally, DRPs were explored in tissue engineering, demonstrating that this new microhemodynamic phenomena could be employed to retard the inflammatory response to implanted biodegradable synthetic scaffolds. This resulted in enhanced collagen structure and production in tissues that replaced the scaffold material.

Insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) have been implicated in the intrinsic response of the heart to myocardial infarction (MI), and it has been postulated that exogenous administration of one or both of these growth factors may enhance myocardial repair and regeneration. We hypothesized that a biodegradable, elastomeric poly(ester urethane)urea (PEUU) capable of sustained, local delivery of IGF-1 or HGF to ischemic myocardium would improve left ventricular (LV) dimension and function. PEUU scaffolds without growth factor or scaffolds containing either IGF-1 or HGF were applied onto the hearts of rats injured by MI. Improvement in LV function and restoration of LV wall thickness, muscle mass, and angiogenesis were assessed 8 weeks post-patch implantation. Improvement in LV dimension and function due to IGF-1-loaded patches was not significant compared to control patches, while HGF-loaded patches significantly worsened LV dimension compared to IGF-loaded and control patches. No significant differences in muscle or capillary formation or LV function were noted between groups. Although no significant improvements were noted with growth factor-loaded patches, trends towards improved LV dimension with IGF-1-loaded patches and trends towards worsened LV dimension and function with HGF-loaded patches may warrant additional investigation. Furthermore, we evaluated the ability of PEUU to deliver molecules deigned to induce cellular gene expression in a spatially-controlled manner in vitro. PEUU films and scaffolds with spatially-defined regions containing or omitting inducer molecules were fabricated, and cells transduced to express green fluorescent protein (GFP) were cultured on films and within scaffolds to evaluate spatial control of gene expression. PEUU demonstrated an extended period of controlled release of the inducer molecule as well as providing spatial control over GFP expression in both PEUU films and three-dimensional scaffolds. Hence, these scaffolds may provide a means to control progenitor cell commitment in a spatially-defined manner in vivo for tissue repair and regeneration. With an elastic scaffold delivery system, such a technique might ultimately find application in cardiovascular tissue engineering.

The shoulder is the most dislocated joint in the body, with over 80% of dislocations occurring in the anterior direction. During anterior dislocation, the glenohumeral capsule within the shoulder is the primary joint stabilizer. Physical diagnostic examinations of the capsule following dislocation are crucial for clinicians to determine the location and extent of pathology in the capsule, and diagnoses from the examinations are used to decide the type, location, and extent of surgical repair to restore capsule function. These examinations, however, are not standardized for joint position among patients. Since capsule function is dependent upon joint position and can vary among patients, the lack of standardized joint positions for the examinations leads to misdiagnoses of the location and extent of capsule pathology that subsequently result in improper surgical procedures to restore capsule function. In fact, over 50% of re-dislocations and over 80% of pain and loss of motion following surgery have been shown to result from misdiagnoses of pathology to the capsule. Therefore, the objective of the current work was to suggest joint positions where the stability provided by the capsule is consistent among patients in response to application of an anterior load. Two subject-specific finite element models of the glenohumeral joint were developed based on experimental data collected from two cadavers, and that used an isotropic hyperelastic constitutive model to represent the capsule. The models were validated by comparing predicted capsule strains in the models to experimental capsule strains in clinically relevant joint positions. The models were used to identify clinically relevant joint positions where the distributions of predicted capsule strain were correlated with an r2 value greater than 0.7, and to further identify if strain was higher in specific regions of the capsule than others at these positions. The clinically relevant joint positions resulting from application of a 25 N anterior load at 60° of glenohumeral abduction and 10° - 40° of external rotation resulted in distributions of strain that were correlated with an r2 value greater than 0.7. Of these positions, those with 20° - 40° of external rotation resulted in capsule strains that were significantly higher in the glenoid side of the anterior band of the inferior glenohumeral ligament than in other regions of the capsule. Therefore, the current work suggests that standardizing physical diagnostic examinations for anterior instability at joint positions with abduction and a mid-range of external rotation may allow clinicians to more effectively diagnose the location and extent of capsule pathology resulting from anterior dislocation, and may ultimately lead to an improvement in surgical outcomes. The current work further provides an excellent foundation to evaluate the stability provided by the capsules of multiple subject-specific finite element models of the glenohumeral joint, to identify multiple joint positions for physical examinations that can be used to diagnose pathology throughout the glenohumeral capsule.

Abducens motor neurons (ABD) are known to receive oculomotor signals via the excitatory and inhibitory burst neurons (BN) as well as head velocity related signals via the vestibular nucleus (VN). If the oculomotor input to the ABD was the same, would there be a difference in the properties of the observed eye movement between head restrained (HR) and head unrestrained (HU) gaze shifts? To answer this question, the activity of 22 BN was recorded during HR and HU visual motor tasks performed by non human primates. A template matching algorithm was used to find a pair of trials (HR, HU) with matching BN activity. This guaranteed that the oculomotor input to the ABD was the same. Matched trials were found to have similar gaze amplitudes, but the peak eye velocity of HU movement was lower than HR movement. A time varying gain of the head velocity input was calculated as the ratio of the difference between the eye velocities over the head velocity. This yielded a gain that was high at the onset of the movement, decreased through out the gaze shift and plateau at one after gaze shift offset. Thus the head movement was highly inhibiting the eye movement at HU gaze onset which decreased throughout the gaze shift until it reached and remained at one at the end of the gaze shift. Finally a computer simulation was used to check if the difference in the eye velocities could be explained by VN inputs. The simulation modeled the difference in ABD firing rate (ÄABD) between HR and HU matched trials as the weighted sum of the difference in the firing rate of VN (ÄPVPc, ÄPVPi and ÄEHc) input. The BN input was not used in this model because for matched trials the input was the same, thus the difference was equal to zero. The simulation showed that the weight of EHc cells input was the highest, thus accounting for most of the difference in the ABD. This lead to the conclusion that EHc cells played a major role in reducing the eye velocity during head unrestrained gaze shifts.

Inhaled antibiotics are frequently used for treating infections associated with cystic fibrosis (CF) and are under development for other uses including hospital-acquired pneumonias. However, the long-term efficacy of inhaled anti-infectives depends largely on the uniformity of pulmonary drug deposition, which is variable in diseased lungs. In surfactant replacement therapy (SRT), premature infants who lack adequate levels of surfactant receive a bolus of exogenous surfactant through an endotracheal tube. The success of this therapy is due to the pulmonary dispersion of surfactant from convective flows generated by surface tension gradients along the airway surface. Based on this same mechanism, aerosolized surfactant drug carriers have been proposed as a potential means of augmenting drug distribution in diseased lungs. However, little experimental evidence exists to support this. The goal of this study was to assess the potential for aerosolized surfactant drug carriers to improve the dispersion of medications in the lungs following aerosol deposition. This study included the design of an aerosol delivery system that produced an aerosol of respirable size that was delivered onto two realistic in vitro models of the airway surface. The first model incorporated porcine gastric mucin (PGM) and the second utilized human bronchial epithelial (HBE) cell cultures, including CF and non-CF cells. Differences in the dispersion of various surfactants (cationic, anionic, and non-ionic) vs. saline (control) were quantified using fluorescence microscopy and different sized fluorescent tags acting as drug analogs. The tags spanned a size range from the molecular level to a 1.0 micron polystyrene sphere. On the PGM model, surfactants enhanced dispersion by 2-20 fold vs. saline with fairly uniform dispersion for all sized tags. When sufficiently hydrated, the HBE cell cultures, both CF and non-CF, also demonstrated significant surfactant spreading compared to saline, with similar areas and patterns for all sized tags. This study demonstrates the potential for aerosolized surfactant carriers to improve the uniformity of pulmonary drug distribution following deposition. Further studies are required to demonstrate their efficacy in these in vitro models and for in vivo drug applications.