published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type II collagen is the major extracellular matrix (ECM) protein present in cartilage and is detected in non-chondrogenic tissues such as the heart and the neural tube during developmental stages involving rapid tissue morphogenesis indicating an active role played by the collagen in embryogenesis. Type II collagen is synthesized as a procollagen precursor which has amino- and carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central triple helical domain. Two isoforms of type II procollagen are generated by alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous to those present in regulators of the bone morphogenetic protein (BMP) signaling such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv). Previous in vitro binding assays and overexpression studies in frog embryo suggest that the CR domain of IIA antagonized BMP signaling. In order to give a better understanding of the function of IIA in embryonic development and cellular signaling, several approaches including expression pattern analyses, phenotypic analyses of null mutant and gain of function studies are employed in this study. Expression studies of IIA mRNA in early postimplantation mouse embryos find that it is present in the axial mesendoderm (including the anterior definitive endoderm [ADE] and the prechordal plate) which is a critical head organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II collagen (Col2a1) gene by homologous recombination, indeed reveals that the anterior-most neural tissue is deficient at early somitogenesis denoted by reduction/loss of the forebrain/optic cup markers. Marker studies indicate that the ADE may already be affected at the neural plate stage in IIA-/-. The neural phenotype of IIA-/- displays significant similarities with mutants deficient in BMP pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting that IIA plays a role in maintaining the specification and/or regulating the signaling properties of the anterior midline tissue which involves regulation of BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos suggest that IIA regulate BMP and the related Nodal signaling pathways in a context dependent manner which has significant implications in normal anterior neural plate development. Based on the work described in this thesis and the body of existing evidence, a model is presented which suggests that IIA promote/maintain anterior neural plate development by regulating the range and extent of BMP signaling in the anterior neural plate. This study sheds light on the role of an ECM component in regulating tissue patterning and cellular signaling during early mouse development and also provides putative function for the CR domain of other fibrillar procollagens including type I, III and V which is poorly understood currently. This work will provide the framework for the design of subsequent studies in re-examining the role of these fibrillar procollagens in embryogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

The corneal wound healing response involves the activation of keratocytes to proliferate from a quiescent phenotype. The mitogens that cause the initial transformation of the quiescent keratocytes to the active phenotype have not been identified. Even though serum is commonly used to replicate this in vitro, the cornea is avascular and therefore likely not exposed to serum. In the first part of this dissertation, a DMEM/F12 extract of corneal stromas was made and shown to stimulate keratocyte proliferation in both a dose-dependent and cell-density dependent manner. This extract contains mitogens that differ from the mitogens present in serum based on their effect on keratocytes and their biochemical characteristics. Culture in extract replicates in vitro the changes observed during the activation of keratocytes in the wound-healing phase.The corneal stroma contains an extensive extracellular matrix that consists primarily of collagens and proteolgycans. This matrix is maintained and secreted by the keratocytes, cells with unique characteristics lost during the activation observed at wound healing. The second part of this dissertation aims to develop a defined culture medium that maintains the keratocyte phenotype during proliferation. Keratocytes were cultured in serum-free medium and the effect of the growth factors on the markers for the keratocyte phenotype determined. Only insulin was shown to stimulate cell proliferation in a consistent manner, while maintaining commonly accepted keratocyte markers. When this defined culture medium was supplemented with ascorbic acid to study collagen synthesis, a marked increase in both collagen synthesis and keratan sulfate proteoglycan accumulation was measured. This newly developed culture medium, containing insulin and ascorbate, allows for cell growth, maintains the keratocyte markers, and could be used to study the native, non-activated keratocyte phenotype in culture.This dissertation shows that the culture media described herein replicate in vitro all the phenotypes observed during the corneal wound healing response in vivo. These culture media, in turn, could be used to obtain more knowledge about the different keratocyte phenotypes, and how they could be manipulated in culture. (329 words)

In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Orthopaedic Surgery / Master / Master of Philosophy

A proteomic procedure for analyzing mouse left ventricular collagen by mass spectrometry has been developed. The procedure involves a purification step that removes non-collagenous cellular components from the collagen extracellular matrix, a step that solubilizes the collagen in aqueous solvents before it is proteolytically digested for analysis with ESI-LCMS/MS. Collagen from healthy and lathrytic mice has been positively identified by applying the SEQUEST database search algorithm to spectra from the collagen prepared using this procedure. Analysis shows that the relative percentage of collagen peptides detected in lathrytic tissue is significantly greater than that of the healthy tissue. These preliminary results suggest that the percentage of cross-linked collagen is lower in the lathrytic tissue as indicated by the greater protein sequence coverage obtained for this tissue. This procedure lays the ground work for future experimentation that has the ability to allow for the identification and quantification of cross-linked peptides.

The domestic dog, Canis lupus familiaris, plays many roles in the lives of humans. Additionally, the dog is recognized for its potential as a model for many human hereditary diseases. Thus, the genetics and genomics of the dog are being studied extensively in order to facilitate its use as a model, as well as to help the dog for its own sake. As part of this research effort, our laboratory has added type I markers (i.e., the acidic and basic keratins, c-kit, type I and IV collagens, and the gene encoding uromodulin) to the emerging map of the canine genome. The mapping of genes, particularly those in large gene families such as the collagens, is valuable because it rapidly increases the density of gene loci on the map and provides insight regarding conservation of synteny between the dog and other mammals. The major focus of work reported here is the genetics of X-linked Alport syndrome (XLAS), a terminal renal disease that affects the human and the dog. The disease results from mutations in COL4A5, a type IV collagen gene. Reported here are the 1) sequencing and mapping of the canine cDNA encoding uromodulin, 2) mapping of the type I and type IV collagen genes, 3) sequencing of the full-length cDNA of canine COL4A5, 4) identification of a 10 bp deletion in COL4A5, causative for XLAS in our colony of mixed breed dogs, 5) development of a genetic test for identification of affected and carrier dogs in the colony and 6) assessment of gene expression in the kidneys of normal and XLAS-dogs. This assessment was performed using a canine-specific oligonucleotide microarray. XLAS dogs demonstrated up-regulation of many genes involved in extracellular matrix reorganization, cell structure, and immune response, as expected in a glomerulopathy with tubulointerstitial nephritis. Trends were verified by quantitative RT-PCR. A review of the current status of canine genetics research, and current understanding of hereditary diseases in the dog, concludes this dissertation.

Bacteria have been confirmed as the main etiological factor for root canal infection as well as for root canal treatment failure. Thus the success of endodontic treatment depends on the complete elimination of bacteria and prevention of bacterial recolonization in the root canal system. The major challenge for conventional root canal disinfection strategies is the ability of bacteria to persist as biofilms within the anatomical complexities of the root canal system. In addition, the alterations in the ultrastructure of dentin tissue results in compromised structural integrity of root dentin leading to higher risk of fracture in root-filled teeth. The objectives of this study are twofold: 1) develop and test functionalized nanoparticles to eliminate biofilm bacteria and, 2) to stabilize and strengthen the dentin organic matrix by crosslinking collagen fibrils in the presence of biopolymeric nanoparticles. A bioactive polymeric nanoparticle functionalized with a photosensitizer may present as a single step treatment to achieve both the objectives. Chitosan a bioactive polymer was used owing to their inherent antibacterial and biocompatible characteristics. Chitosan micro-/nanoparticles were synthesized as well as functionalized with photosensitizer (rose bengal) for photodynamic activation. Bioactive chitosan nanoparticle functionalized with a rose bengal is expected to combine the properties of chitosan i.e., polycationic with higher affinity to bacterial cell wall and alter membrane integrity; that of a photosensitizer i.e., to generate singlet oxygen when photoactivated; and the nano-form further potentiate these specific properties. These photodynamically activable chitosan nanoparticles showed the distinct characteristics of chitosan and rose bengal. The synergistic effect of the chitosan conjugated nanoparticles was able to eliminate monospecies and multi-species bacterial biofilms with complete disruption of the biofilm structure. The singlet oxygen generated during photoactivation produced photochemical crosslinking of dentin collagen and infiltration of chitosan nanoparticles. Following crosslinking the dentin collagen showed significantly improved mechanical properties (ultimate tensile strength and toughness) and improved resistance to degradation by bacterial collagenase. In conclusion, this study presents a potential photosensitizer functionalized chitosan nanoparticles based treatment strategy to improve the success of endodontic treatment to achieve complete disinfection of the root canal system and enhanced the mechanical/ structural integrity of the root-filled teeth.