Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

STATISTICAL METHODS FOR ADVANCED ECONOMETRIC MODELS WITH APPLICATIONS TO VEHICLE HOLDING AND SPEED QUANTILES DISTRIBUTION

This dissertation proposes statistical methods to formulate, estimate and apply complex transportation models. Two main problems are part of the analyses conducted and presented in this dissertation. The first method solves an econometric problem and is concerned with the joint estimation of models that contain both discrete and continuous decision variables. The use of ordered models along with a regression is proposed and their effectiveness is evaluated with respect to unordered models. Procedure to calculate and optimize the log-likelihood functions of both discrete-continuous approaches are derived, and difficulties associated with the estimation of unordered models explained. Numerical approximation methods based on the Genz algortithm are implemented in order to solve the multidimensional integral associated with the unordered modeling structure. The problems deriving from the lack of smoothness of the probit model around the maximum of the log-likelihood function, which makes the optimization and the calculation of standard deviations very difficult, are carefully analyzed. A methodology to perform out-of-sample validation in the context of a joint model is proposed. Comprehensive numerical experiments have been conducted on both simulated and real data. In particular, the discrete-continuous models are estimated and applied to vehicle ownership and use models on data extracted from the 2009 National Household Travel Survey. The second part of this work offers a comprehensive statistical analysis of free-flow speed distribution; the method is applied to data collected on a sample of roads in Italy. A linear mixed model that includes speed quantiles in its predictors is estimated. Results show that there is no road effect in the analysis of free-flow speeds, which is particularly important for model transferability. A very general framework to predict random effects with few observations and incomplete access to model covariates is formulated and applied to predict the distribution of free-flow speed quantiles. The speed distribution of most road sections is successfully predicted; jack-knife estimates are calculated and used to explain why some sections are poorly predicted. Eventually, this work contributes to the literature in transportation modeling by proposing econometric model formulations for discrete-continuous variables, more efficient methods for the calculation of multivariate normal probabilities, and random effects models for free-flow speed estimation that takes into account the survey design. All methods are rigorously validated on both real and simulated data.

Soft computing based parameter identification in pavements and geomechanical systems

Accurate estimation of road pavement geometry and layer material properties through the use of proper nondestructive testing and sensor technologies is essential for evaluating pavement’s structural condition and determining options for maintenance and rehabilitation. For these purposes, pavement deflection basins produced by the nondestructive Falling Weight Deflectometer (FWD) test data are commonly used. The nondestructive FWD test drops weights on the pavement to simulate traffic loads and measures the created pavement deflection basins. Backcalculation of pavement geometry and layer properties using FWD deflections is a difficult inverse problem, and the solution with conventional mathematical methods is often challenging due to the ill-posed nature of the problem. In this dissertation, a hybrid algorithm was developed to seek robust and fast solutions to this inverse problem. The algorithm is based on soft computing techniques, mainly Artificial Neural Networks (ANNs) and Genetic Algorithms (GAs) as well as the use of numerical analysis techniques to properly simulate the geomechanical system. A widely used pavement layered analysis program ILLI-PAVE was employed in the analyses of flexible pavements of various pavement types; including full-depth asphalt and conventional flexible pavements, were built on either lime stabilized soils or untreated subgrade. Nonlinear properties of the subgrade soil and the base course aggregate as transportation geomaterials were also considered. A computer program, Soft Computing Based System Identifier or SOFTSYS, was developed. In SOFTSYS, ANNs were used as surrogate models to provide faster solutions of the nonlinear finite element program ILLI-PAVE. The deflections obtained from FWD tests in the field were matched with the predictions obtained from the numerical simulations to develop SOFTSYS models. The solution to the inverse problem for multi-layered pavements is computationally hard to achieve and is often not feasible due to field variability and quality of the collected data. The primary difficulty in the analysis arises from the substantial increase in the degree of non-uniqueness of the mapping from the pavement layer parameters to the FWD deflections. The insensitivity of some layer properties lowered SOFTSYS model performances. Still, SOFTSYS models were shown to work effectively with the synthetic data obtained from ILLI-PAVE finite element solutions. In general, SOFTSYS solutions very closely matched the ILLI-PAVE mechanistic pavement analysis results. For SOFTSYS validation, field collected FWD data were successfully used to predict pavement layer thicknesses and layer moduli of in-service flexible pavements. Some of the very promising SOFTSYS results indicated average absolute errors on the order of 2%, 7%, and 4% for the Hot Mix Asphalt (HMA) thickness estimation of full-depth asphalt pavements, full-depth pavements on lime stabilized soils and conventional flexible pavements, respectively. The field validations of SOFTSYS data also produced meaningful results. The thickness data obtained from Ground Penetrating Radar testing matched reasonably well with predictions from SOFTSYS models. The differences observed in the HMA and lime stabilized soil layer thicknesses observed were attributed to deflection data variability from FWD tests. The backcalculated asphalt concrete layer thickness results matched better in the case of full-depth asphalt flexible pavements built on lime stabilized soils compared to conventional flexible pavements. Overall, SOFTSYS was capable of producing reliable thickness estimates despite the variability of field constructed asphalt layer thicknesses.

The choice of spatial scales and spatial smoothness priors for various spatial patterns

Given the drawbacks for using geo-political areas in mapping outcomes unrelated to geo-politics, a compromise is to aggregate and analyse data at the grid level. This has the advantage of allowing spatial smoothing and modelling at a biologically or physically relevant scale. This article addresses two consequent issues: the choice of the spatial smoothness prior and the scale of the grid. Firstly, we describe several spatial smoothness priors applicable for grid data and discuss the contexts in which these priors can be employed based on different aims. Two such aims are considered, i.e., to identify regions with clustering and to model spatial dependence in the data. Secondly, the choice of the grid size is shown to depend largely on the spatial patterns. We present a guide on the selection of spatial scales and smoothness priors for various point patterns based on the two aims for spatial smoothing.

Some remarks on the symplectic pairing on the moduli space of representations of the fundamental group of surfaces

This work grew out of an attempt to understand a conjectural remark made by Professor Kyoji Saito to the author about a possible link between the Fox-calculus description of the symplectic structure on the moduli space of representations of the fundamental group of surfaces into a Lie group and pairs of mutually dual sets of generators of the fundamental group. In fact in his paper [3] , Prof. Kyoji Saito gives an explicit description of the system of dual generators of the fundamental group.

Moduli spaces of vector bundles on a singular rational ruled surface

We study moduli spaces M-X (r, c(1), c(2)) parametrizing slope semistable vector bundles of rank r and fixed Chern classes c(1), c(2) on a ruled surface whose base is a rational nodal curve. We showthat under certain conditions, these moduli spaces are irreducible, smooth and rational (when non-empty). We also prove that they are non-empty in some cases. We show that for a rational ruled surface defined over real numbers, the moduli space M-X (r, c(1), c(2)) is rational as a variety defined over R.

Torsional impact of transversely isotropic solid with functionally graded shear moduli and a penny-shaped crack

The torsional impact response of a penny-shaped crack in an unbounded transversely isotropic solid is considered. The shear moduli are assumed to be functionally graded such that the mathematics is tractable. Laplace transform and Hankel transform are used to reduce the problem to solving a Fredholm integral equation. The crack tip stress fields are obtained. Investigated are the influence of material nonhomogeneity and orthotropy on the dynamic stress intensity factor. The peak value of the dynamic stress intensity factor can be suppressed by increasing the shear moduli's gradient and/or increasing the shear modulus in a direction perpendicular to the crack surface.

Hausdorff dimension bounds for smoothness of holonomies for codimension 1 hyperbolic dynamics

We prove that the stable holonomies of a proper codimension 1 attractor Λ, for a Cr diffeomorphism f of a surface, are not C1+θ for θ greater than the Hausdorff dimension of the stable leaves of f intersected with Λ. To prove this result we show that there are no diffeomorphisms of surfaces, with a proper codimension 1 attractor, that are affine on a neighbourhood of the attractor and have affine stable holonomies on the attractor.

Influence of inherent structure shear stress of supercooled liquids on their shear moduli

Configurations of supercooled liquids residing in their local potential minimum (i.e. in their inherent structure, IS) were found to support a non-zero shear stress. This IS stress was attributed to the constraint to the energy minimization imposed by boundary conditions, which keep size and shape of the simulation cell fixed. In this paper we further investigate the influence of these boundary conditions on the IS stress. We investigate its importance for the computation of the low frequency shear modulus of a glass obtaining a consistent picture for the low- and high frequency shear moduli over the full temperature range. Hence, we find that the IS stress corresponds to a non-thermal contribution to the fluctuation term in the Born-Green expression. This leads to an unphysical divergence of the moduli in the low temperature limit if no proper correction for this term is applied. Furthermore, we clarify the IS stress dependence on the system size and put its origin on a more formal basis.

In vitro evaluation of surface roughness of 4 resin composites after the toothbrushing process and methods to recover superficial smoothness.

OBJECTIVE: This study evaluated the efficiency of repolishing, sealing with surface sealant, and the joining of both in decreasing the surface roughness of resin-based composites after a toothbrushing process. METHOD AND MATERIALS: Ten specimens of each composite (Alert, Z100, Definite, and Prodigy Condensable), measuring 2 mm in thickness and 4 mm in diameter, were made and submitted to finishing and polishing processes on both sides of the specimens using the Sof-Lex system. The specimens were then subjected to toothbrushing (30,000 cycles), and surface roughness (Ra) was analyzed with a Surfcorder SE 1700 profilometer. The upper surface of each composite was etched with 37% phosphoric acid, and the surface-penetrating sealant Protect-it was applied on 1 surface. The roughness of these surfaces was again measured. On the other side, the surface of the specimen was repolished, and the efficiency of this procedure was measured using the profilometer. The surface roughness resulting from the joining of the 2 methods was verified by applying, in the final stage, the surface-penetrating sealant on the repolished surface. Data were analyzed with analysis of variance and Tukey test (P <.05). RESULTS: Results showed that the lowest surface roughness values were obtained for Definite, Z100, and Prodigy Condensable after the repolishing process and after the repolishing plus sealing. For Alert, the joining of repolishing plus sealing promoted the lowest values of surface roughness. CONCLUSION: Of the resin-based composites, Alert demonstrated the highest values of surface roughness in all the techniques tested.

Intersections on moduli spaces of curves

We present a new approach to perform calculations with the certain standard classes in cohomology of the moduli spaces of curves. It is based on an important lemma of Ionel relating the intersection theoriy of the moduli space of curves and that of the space of admissible coverings. As particular results, we obtain expressions of Hurwitz numbers in terms of the intersections in the tautological ring, expressions of the simplest intersection numbers in terms of Hurwitz numbers, an algorithm of calculation of certain correlators which are the subject of the Witten conjecture, an improved algorithm for intersections related to the Boussinesq hierarchy, expressions for the Hodge integrals over two-pointed ramification cycles, cut-and-join type equations for a large class of intersection numbers, etc.