Thesis (M.S.)--University of Michigan, 2003. / Includes bibliographical references.

Thesis (Ph. D.)--Medical College of Georgia, 1997. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--Medical College of Georgia, 1997. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Louisville, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Thesis (M.S.)--University of Louisville, 1998. / Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Joint disorders significantly affect quality of life and present unique challenges for tissue engineering. In the craniofacial space, and especially for the temporomandibular joint (TMJ), there is an unmet need for anatomically precise and mechanically robust cartilage and bone tissues to recapitulate native function. Current surgical reconstruction methods, whether using autologous or synthetic options, suffer from imprecision, comorbidities, complications, and frequently require subsequent operations. Furthermore, many craniofacial graft efforts have focused on improving bone without addressing cartilage, which is essential to proper TMJ function. Thus, there is a compelling need to engineer a human-sized, biologically and anatomically matched cartilage-bone TMJ replacement. This dissertation demonstrates the ability to generate such a graft with native-like properties in a human-sized large animal model by focusing on two aims: (i) establish methods to fabricate and culture anatomically specific, autologous cartilage-bone grafts (Aim 1), and (ii) show improvement of graft performance after six months implantation in vivo compared to previous methods, controls, and native tissue (Aim 2). Using Yucatan mini-pigs as a human-sized model, the ramus-condyle unit (RCU), a geometrically intricate portion of the mandible and primary load bearing section of the TMJ, was targeted for reconstruction. Scaffolds were created using computer tomography (CT) image-guided micromilling of decellularized bone matrix, then infused with autologous adipose-derived chondrogenic and osteogenic progenitors. These biological constructs were then cultured in vitro in a novel dual-perfusion bioreactor before in vivo implantation. Similar in vitro culture of representative constructs done in parallel demonstrated cell attachment and some differentiation. After six months implantation, the dual cartilage-bone RCU grafts maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a significantly greater extent than either bone-only grafts or acellular scaffolds, and showed remarkable similarity to native tissue. Furthermore, tracking of implanted cells enabled additional insights into the progression of cartilage and bone regeneration. The methods and results established in this dissertation form a promising basis for the next evolution in engineering full-sized, patient-specific, and biologically and mechanically robust TMJ replacements.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.

Directed regeneration of axons in the CNS has potential for the treatment of CNS disorders and injuries. In contrast to mammals, following optic nerve lesion zebrafish regenerate axons that navigate to their correct targets and form new synapses leading to functional recovery. Correct pathfinding is thought to rely on a range of molecular cues in the CNS which the growing axon expresses receptors for. However, the specific guidance cues are not well elucidated. It is likely that a proportion of them will be the same as during development, while some may be specific to regeneration. Alternatively, axons may simply retrace former trajectories guided by the molecular environment or mechanical constraints of degenerating tracts, as demonstrated in the mammalian PNS. To elucidate this, we investigated regeneration in the astray/robo2 knockout mutant which exhibits misprojection of optic axons during development leading to the establishment of ectopic tracts. We show that degenerating tracts do not provide a strong guidance cue for regenerating axons in the CNS as ectopic tracts in the astray mutant are not repopulated following lesion despite presenting a similar environment to entopic degenerating tracts. We also find that as astray mutant (knockout) and robo2 morphant (transient knockdown) projection and termination errors persist in the adult, it is clear that there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development. In addition, we find a reduced importance of the axon guidance receptor Robo2 and its repellent ligand Slit2 for pathfinding during regeneration as specific developmental pathfinding errors of optic axons in astray mutants are corrected during adult optic nerve regeneration and global overexpression of Slit2 elicits pathfinding defects during development but not regeneration. To address regeneration-associated gene regulation in axotomised retinal ganglion cells, we carried out a microarray analysis. We found that many genes detected as a gradient in the adult retina during regeneration are not differentially expressed in the embryonic eye, despite having distinct expression patterns in other embryonic tissues. Of the genes which exhibit strong differential expression in the retina of both regenerating adults and developing embryos, foxI1 is one of the most interesting candidates as other fox genes have been implicated in axon guidance and due to its highly restricted retinal expression pattern. Surprisingly, further investigation has revealed that foxI1 knockout mutant embryos have retinotectal projections which appear normal in terms of axon pathfinding and mapping. Another family of genes indicated by the array, which are cytosolic phosphoproteins known to be involved in the signal transduction cascade of multiple inhibitory guidance cues during axon growth, are the crmps. Knocking down crmp2 with morpholinos during development resulted in a sparser innervation of the tectum with individual axons which trend towards having less complex arbors with shorter branches and reduced overall axon length. As a whole this work adds to our current knowledge of optic axon guidance during development and regeneration and the relative importance and effect of selected potential guidance cues, which may help toward informing future mammalian CNS regeneration research.