Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 157-168).

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Gabor Forgacs. Vita. Includes bibliographical references.

Engineering a complex tissue that exceeds 100-200 [mu]m requires a vascular connection. Methods to enhance vascularization include the delivery of angiogenic factors, and the use of scaffolds that encourage vascular ingrowth. However, these techniques rely on the host to vascularize the construct upon implantation, which is often too slow to provide nutrients to the entire construct. Hence, recent research has focused on creating de novo vascular networks prior to implantation. Such technologies would enable faster anastomosis with the host vascular system, as well as fully perfused constructs that can increase cell viability. Many techniques have been investigated to create de novo vascular networks with varying levels of success. Our approach was to utilize native vascular extracellular matrix (ECM) obtained from decellularizing highly vascularized tissue as a substrate for re-endothelialization and thus to create a three-dimensional vascular bed for ultimate use with various implant and tissue engineering applications. We have demonstrated a method of chemical decellularization that effectively removes cellular material while leaving behind an organized patent vascular network down to the capillary scale. Standard histological methods, DNA quantification, as well as vascular corrosion casting demonstrated this efficacy. Subsequent subcutaneous implantation then explantation of the scaffold at 7 and 28 days was used to assess the immunogenicity of the graft by analyzing the presence of immune cells. This scaffold was then re-endothelialized with human dermal microvascular endothelial cells (HDMECs) and conditioned with peristaltic flow for 60 hours to help improve vascular patency. Cellular distribution was determined qualitatively by first incubating the HDMECs with gold nanotracers, then imaging their presence upon implantation through ultrasound-guided photoacoustic (US/PA) imaging. Following the culture process, the scaffolds were analyzed for vascular patency through vascular corrosion casting, and cellular phenotype through histological methods---demonstrating a decrease in vascular damage. The re-endothelialized scaffolds were then assessed for functional vascular performance by perfusing whole blood through them. Results demonstrated better blood clearance in re-endothelialized scaffolds compared to scaffolds without cells. These results point to the ability of the optimized acellular (OA) scaffold to be used in future experiments focused on vascular and tissue engineering. / text

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Intervertebral disc tissue engineering is challenging because it involves the integration of multiple tissues with distinct structures and compositions such as lamellar annulus fibrosus, gel?like nucleus pulposus and cartilage endplate. Each of them has different compositions and different structures. It is hypothesized that integration of tissues can be enhanced with appropriate mechanical and biological stimuli. Meanwhile, effect of torsional stimulus on cell re?orientation in mesenchymal stem cell?collagen tubular constructs is investigated in this study. Furthermore, it is proposed that these findings can be used to fabricate a multicomponent unit for intervertebral disc tissue engineering. It has been demonstrated that mechanical and biological stimuli can stabilize the interface between osteogenic and chondrogenic differentiated constructs with enhanced ultimate tensile stress while the phenotype of osteogenic and chondrogenic differentiated constructs were maintained. Scanning electronic microscopic images have shown aligned collagen fibrils and presence of calcium at the interface, indicating the possibility of the formation of a calcified zone. In addition, it is proven that torsional stimulus triggered re?orientation of mesenchymal stem cells in collagen lamellae towards a preferred angle. Cell alignments were confirmed by using a MatLab?based program to analyze the actin filament and the cell alignment via Phalloidin and Hematoxylin staining, respectively. Cells and actin filaments were inclined around 30o from the vertical axis, while cells and filaments in the control group (static loading) aligned along the vertical axis. Furthermore, a double?layers bioengineered unit was fabricated, with intact osteogenic differentiated parts at both ends. Comparatively higher cell density was observed at the interface between layers, demonstrating the interactions between layers, while the phenotype of each part was maintained in 14 days culture. This study concludes that a multi?components bioengineered unit with preferred cell alignments can be fabricated. This provides new insights to future development of bioengineered spinal motion segment for treating late stage disc degeneration. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

The mission of tissue engineering is to recapitulate the natural process of tissue formation by assembling cells into synthetic scaffold. This relies on the understanding of the functions and properties of the tissue microenvironment (TME), the specific extracellular environment within endogenous tissues. Although existing studies demonstrated the effect of each of the topographical, mechanical and biochemical properties on cell behaviors in isolation, the effect of these properties within the native TME are complicated and ill defined. This thesis aims to investigate how topographical, mechanical and biochemical features of natural TME contribute to the modulation of the biochemistry, morphology and functions of cells, and to translate this knowledge into the fabrication of biomaterials. Tissue cryosections as a cell culture model system was established. It allowed robust assessment of cell phenotypes in a near-natural TME. Mesenchymal stem cells (MSC) cultured on bone, cartilage and tendon cryosections adopted different morphology, supporting the idea that tissue cryosections forms a robust platform for cell-TME studies. Then, Achilles tendon TME was chosen for proof of concept. This tendon cryosection induced different cell types to adopt different morphologies, indicating that the effect of TME is cell type specific. The proliferation of MSC cultured on cryosection was suppressed, however it was instructed to commit tenogenic differentiation. Then, the necessity of TME topographical properties in forming this instruction was delineated by seeding MSC onto cross-sectional tendon cryosection. Although this surface contained native biomechanical and biochemical cues, it could not promote differentiation. This highlighted the necessity of topographical cues within the TME. Next, nano-grooved titanium surface that resembles the topographical cues of tendon TME was used to replicate the function of TME. This surface successfully promoted morphogenesis of MSC but not differentiation. This implicated that biomechanical and biochemical cues are both necessary for instructing desired cell phenotypes. The proteomes of MSC cultured on nanogrooved and planar surfaces were then studied using quantitative proteomics. This revealed some expected changes such as up regulation of cytoskeleton and cell-adhesion proteins, suggesting mechanotransduction events might have been induced by nano-grooved surface. However, expressions of RNA-binding proteins were also regulated, representing novel findings. These proteins were also found in the proteome of cellmicroenvironment interface identified through the use of subcellularfractionation and proteomics. This consolidated their involvement in cellmatrix interactions. The topographical and mechanical properties of cryosection were replicated by using bioimprinting. This imprint induced the morphogenesis of MSC, but tenocytic differentiation was induced only when collagen 1 was coated. However incorrect mechanical properties would abolish such phenotypic guidance. This suggests that topographical, mechanical and biochemical information in a TME are individually indispensable, and it is possible to functionally reconstruct a TME by bioimprinting and ECM protein coating. In summary, this study investigated the topographical, mechanical and biochemical properties in tendon TME and their combined effect on controlling cell phenotypes. It illustrates that biomimetic approach that mimics these three properties of a tissue can effectively control cell phenotypes. Further investigation on better biomimetic methods and its molecular mechanisms will help establishing strategies for constructing functional tissues. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Electrospinning has attracted great attention in the fields of tissue engineering and controlled release of drugs/biomolecules. The aim of this project was to investigate electrospinning of nanofibers with core-shell structures using emulsion electrospinning, the formation of monolithic and core-shell structured nanofibrous drug/biomolecule delivery vehicles using polymers such as poly(D,L-lactic acid) (PDLLA) and poly(lactic-co-glycolic acid) (PLGA), and the formation of multicomponent bone tissue engineering scaffolds with angiogenic property, osteoinductivity and osteoconductivity. The foundation of this project was laid by investigating monocomponent scaffolds. First, effects of properties of polymer solutions and water-in-oil (w/o) emulsions and electrospinning parameters on the morphology, diameter and structure of fibers were systematically investigated. Second, drugs (vancomycin and rifamycin) and a model protein (bovine serum albumin) were incorporated in monolithic or core-shell nanofibers via blend electrospinning or emulsion electrospinning to form single or dual delivery systems, providing fundamental understandings. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic-fibroblast growth factor (b-FGF) were then incorporated in PLGA or PDLLA nanofibrous delivery vehicles. The in vitro release behaviour of drugs and biomolecules was studied. Third, calcium phosphate (Ca-P) nanoparticles were synthesized and used for fabricating Ca-P/PLGA and Ca-P/PDLLA nanocomposite scaffolds. Homogeneous distribution of Ca-P in fibrous scaffolds could be achieved. With the assistance of emulsion electrospinning and nanocomposite electrospinning, bicomponent scaffolds containing rhBMP-2 and Ca-P nanoparticles were fabricated using dual-source dual-power electrospinning. The fibrous component ratio could be varied by using multiple syringes for electrospinning fibers. The structure and properties, including in vitro release behaviour, of mono- and bicomponent scaffolds were studied in detail. Tricomponent scaffolds incorporated with recombinant human vein endothelial growth factor (rhVEGF), rhBMP-2 and Ca-P nanoparticles were subsequently fabricated using multi-source dual-power electrospinning. To achieve a sequential release of firstly rhVEGF and then rhBMP-2, PLGA/polyethylene glycol (PEG) blends and PLGA were used for incorporating rhVEGF and rhBMP-2, respectively. For tricomponent scaffolds with different component ratios, different release amounts but similar release profiles could be achieved for the growth factors. In vitro biological investigations were conducted for mono-, bi- and tricomponent scaffolds. Pre-osteoblast cells (MC3T3-E1) were found to attach, spread, proliferate and express alkaline phosphatase (ALP) activity on rhBMP-2 and Ca-P nanoparticle incorporated bicomponent scaffolds. Calcium deposition was also observed in cells cultured with bicomponent scaffolds. Human umbilical vein endothelial cells (HUVECs) were found to attach, spread, proliferate on tricomponent scaffolds and rhVEGF released from mono-, bi- and tricomponent scaffolds could facilitate cell proliferation and migration, indicating released rhVEGF could promote angiogenesis. C3H10T1/2 cell line and human bone marrow derived mesenchymal stem cells (hBMSCs) were found to attach, spread and proliferate on bi- and tricomponent scaffolds. As compared with cells seeded on monocomponent scaffolds, C3H10T1/2 cells and hBMSCs on bi- and tricomponent scaffolds expressed higher ALP activity. Enhanced mineralization was observed for C3H10T1/2 cells and hBMSCs seeded bicomponent scaffolds comprising rhBMP-2/PLGA and Ca-P/PLGA fibers and also tricomponent scaffolds. hBMSCs seeded on rhBMP-2/PLGA and Ca-P/PLGA monocomponent scaffolds expressed abundant F-actin and vinculin, while bicomponent and tricomponent scaffolds induced much more F-actin and vinculin expression. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

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published_or_final_version / abstract / toc / Orthopaedics and Traumatology / Master / Master of Philosophy