Committee:
Robert Guldberg, Ph.D. (Georgia Institute of Technology)
Todd McDevitt, Ph.D. (Georgia Institute of Technology)
Raymond Vito, Ph.D. (Georgia Institute of Technology)
Jaroslava Halper, M.D., Ph.D. (University of Georgia)

Tissue engineering holds promise to produce functional tissue replacements suitable for implantation and thereby represents a potential long-term strategy for cartilage repair. The interplay between environmental factors, however, gives rise to complex culture conditions that influence the development of tissue-engineered constructs. A fibrous capsule that is composed of abundant type I collagen molecules and resembles fibrocartilage usually forms at the outer edge of tissue-engineered cartilage, yet the understanding of its modulation by environmental cues is still limited. Therefore, this dissertation was aimed to characterize the capsule formation, development and function through manipulation of biochemical parameters present in a hydrodynamic environment while a chemically reliable media preparation protocol for hydrodynamic cultivation of neocartilage was established. To this end, a novel wavy-wall bioreactor that imparts turbulent flow-induced shear stress was employed as the model system and chondrocyte-seed constructs were cultivated under varied biochemical conditions.

Our results demonstrated that tissue morphology, biochemical composition and mechanical strength of hydrodynamically engineered cartilage were maintained as the serum content decreased by 80% (from 10% to 2%). Transient exposure of the low-serum constructs to exogenous insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1) further accelerated their development in comparison with continuous treatment with the same bioactive molecules. The process of the capsule formation was found to be activated and modulated by the concentration of serum which contains soluble factors that are able to induce fibrotic processes and the capsule development was further promoted by fluid shear stress. Moreover, the capsule formation in hydrodynamic cultures was identified as a potential biphasic process in response to concentrations of fibrosis-promoting molecules such as TGF-β. Finally, the presence of the fibrous capsule at the construct periphery was shown to effectively improve the ability of engineered cartilage to integrate with native cartilage tissues, but evidently compromise its tissue homogeneity.

Characterization of the fibrous capsule and elucidation of the conditions under which it is formed provide important insights for the development of tissue engineering strategies to fabricate clinically relevant cartilage tissue replacements that possess optimized tissue homogeneity and properties while retaining a minimal capsule thickness required to enhance tissue integration.

Committee:
Ravi V. Bellamkonda, Department of Biomedical Engineering, Georgia Institute of Technology
Shawn R. Gilbert, School of Medicine, University of Alabama at Birmingham
W. Robert Taylor, School of Medicine, Emory University
Johnna S. Temenoff, Department of Biomedical Engineering, Georgia Institute of Technology

Severe extremity trauma often involves significant damage to multiple tissue types, including bones, skeletal muscles, peripheral nerves, and blood vessels.  Such injuries present unique challenges for reconstruction, and improving structural and functional outcomes of intervention remains a pressing, unmet clinical need.  While tissue engineering/regenerative medicine (TE/RM) therapeutics offer promising potential to overcome the status quo limitations of surgical reconstruction, very few products have transitioned to clinical practice.  Improving treatment options will likely require advancing our understanding of the biological interactions occurring in the repair of damaged tissues.

Bone tissue is known to be innervated and highly vascularized, and both tissue types are involved in normal bone physiology.  However, the degree to which these tissue relationships influence the repair of large, multi-tissue defects remains unknown.  Accordingly, the goal of this thesis was to investigate tissue regeneration in two novel composite injury models.  First, we characterized healing in a composite bone and nerve injury model where a segmental bone defect was combined with a peripheral nerve gap.  Our results indicated that although tissue regeneration was not impaired, the composite injury group experienced a marked functional deficit in the operated limb compared to single-tissue injury.  Second, we developed a model of composite bone and vascular extremity trauma by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia to evaluate the effects on BMP-2-mediated bone repair.  Interestingly, our results demonstrated a stimulatory effect of the recovery response to ischemia on bone regeneration.  Finally, we investigated early vascular growth and gene expression as potential mechanisms coupling the response to ischemia with bone defect repair.  Although the response to ischemia promoted robust vascular growth in the thigh, it did not directly augment vascularization at the site of bone regeneration.  In addition, the stimulatory effects of ischemia on bone regeneration could not be explained by gene expression alone based on the genes and time points investigated.

Taken together, this thesis presents pioneering work on a new thrust of TE/RM research – tissue regeneration in models of composite injury.  This work has provided new insights on the complexity of composite tissue repair, specifically in regard to the relationship between vascular tissue growth and bone healing.  Going forward, successful leverage of models of composite tissue injuries will provide valuable test beds to screen new technologies, advance the understanding of tissue repair biology, and ultimately, may produce new therapeutic interventions for limb salvage and reconstruction that improve outcomes for extremity trauma patients.

Committtee: 
Andres Garcia, PhD, School of Mechanical Engineering, GT
Thomas Barker, PhD, School of Biomedical Engineering, GT
Richard Cummings, PhD, Department of Biochemistry, Emory University
Daniel Ratner, PhD, Department of Bioengineering, University of Washington
John Kauh, MD, Department of Hematology and Medical Oncology, Emory University

Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of their glycan receptors (C-type lectin Receptors (CLRs)) has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory.  Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. However, optimal factors for DC phenotype modulation by surface presented glycans are unknown.  Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. 

The purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. Primary human DCs were exposed to a variety of engineered, adsorbed, glycoconjugates and their subsequent phenotype assessed via a novel, in-house developed, high throughput assay.  A multivariate model was then used to determine optimal factors for glycan presentation from non-phagocytosable surfaces.  To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads for DC treatment. 

High isoelectric point and density glycoconjugates presented from non-phagocytosable displays modulated DC phenotype toward a pro-inflammatory phenotype to the greatest extent.  Additionally, DC response to glycoconjugates was found to be significantly different for each modality of glycan display.  This work indicates that different mechanisms are involved in DC response to glycoconjugate display modality.  These results provide indications for the future design of glycan microarray systems, biomaterials and combination products.

Committee:
Luke Brewster, M.D., Ph.D. (Emory University School of Medicine)
Robert Nerem, Ph.D. (Georgia Institute of Technology)
Manu Platt, Ph.D. (Georgia Institute of Technology)
Alexander Rachev, Ph.D. (Georgia Institute of Technology)

The development of small diameter tissue engineered blood vessels (TEBVs) with low thrombogenicity, low immunogenicity, suitable mechanical properties, and a capacity to remodel to their environment could significantly advance the treatment of coronary and peripheral artery disease. Despite significant advances in the field of tissue engineering, autologous vessels are still primarily utilized as grafts during bypass surgeries. However, undamaged autologous tissue may not always be available due to disease or prior surgery. TEBVs lack long-term efficacy due to a variety of types of failures including aneurysmal dilations, thrombosis, and rupture; the mechanisms of these failures are not well understood. In vitro mechanical testing may help the understanding of these failure mechanisms. The typical mechanical tests lack standardized methodologies; thus, results vary widely. 

The overall goal of this study is to develop novel experimental and mathematical models to study the mechanical properties and failure mechanisms of TEBVs. Our results suggest that burst pressure tests, the current standard, are not sufficient to assess a TEBVs’ suitability as a coronary substitute; creep and/or cyclic loading tests are also required. Results from this model can help identify the most insightful experiments and quantities to be measured – ultimately reducing the overall number of experimental iterations. Improving the testing and characterization of TEBVs is critically important in decreasing the time necessary to validate the mechanical and functional responses of TEBVs over time, thus quickly moving TEBVs from the benchtop to the patient.

COMMITTEE MEMBERS:
Tom Burkholder – Applied Physiology, GA Tech
Daniel Goldman – Physics, GA Tech
Karen Liu – Computer Science, GA Tech
Randy Trumbower – Rehabilitation Medicine, Emory

Interactions between the neural and musculoskeletal systems are a prerequisite for the production of robust movement. In spite of this, the neural control and musculoskeletal structure underlying biological movements are typically studied independently, with little attention paid to how changes in one may affect the other. Understanding these interactions may be critical to improving current rehabilitation technologies and therapy methods. As an example, balance disorders are multifactorial in nature and identifying whether biomechanical or neural changes are the source of instability remains an unanswered question.

I have used a combined experimental and modeling approach to understand neural and biomechanical interactions governing human balance control. I developed a simple four-bar linkage model with delayed feedback to investigate frontal-plane standing balance. Using methods from time-delay systems I present evidence from this model that biomechanical structure is important for behavioral function and show that neural control and biomechanical structure co-vary for stable human balance. Predictions from the model were tested experimentally to dissociate the effects of inertia and postural configuration on balance. In addition, I applied robust control methods to solve the difficult problem of comparing the relative performance between neuromechanical systems that differ in parameter values and predicted a common mechanism to explain changes in neural control across biomechanical contexts.

In the future, the analytical tools and simulation methods I have developed can be generalized to investigate changes in neuromechanical interactions of various deficits in biomechanics (ACL rupture, amputation) and neural control (Parkinson’s disease, stroke).

Furthermore, this approach can be used to explain how neural control and biomechanical structure relate to the diversity of animal form and function, as well as suggest biomimetic control policies for robotics.