Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.

Effective tissue engineering scaffolds should mimic the physical and chemical attributes of native tissue. Native tissues have intricate patterns, a multitude of porosities, and large water contents that are each directly associated with their ability to regulate and support life function. Therefore, the physical architecture of scaffolds intended to mimic these tissues for engineering applications plays an important role in scaffold performance both in vitro and in vivo. Self-assembling molecules organize into intricate patterns with a complexity that resembles that of native tissue. Hyaluronic acid (HA) hydrogels are widely used in tissue engineering for a variety of applications but fail to offer physical architecture beyond the inherent hydrogel porosity. To address this issue, a novel method to impose architecture within thin HA-based films using crystal nucleation was developed in the Schmidt lab [1]. The work described herein extends this method for use in three-dimensional matrices, with the main vii goal being the creation of hydrogels with a complex macroarchitecture. Four in situ self-assembling molecules were used: glycine, guanidine, urea and potassium dihydrogen phosphate. The crystallization of each molecule creates a specific porous network within the hydrogel that is the negative imprint of the crystalline geometry. The novel restriction of aqueous polymer into the molecule interstitial crystalline space allows hydrogels to embody complex geometric lumen architectures. The hydrogels were characterized in terms of their internal architectures, swelling, bulk moduli, biodegradability, cytotoxicity and in vitro cellular response. The unique structure-property relationships displayed by hydrogels templated by each of the crystallizing molecules were characterized in regards to mechanical properties. The need for complex microscopic architecture is conserved over many tissue engineering applications and templated scaffolds were evaluated for two unique applications. Crystal-templated hydrogels were investigated for use as an artificial stem cell niche environment to expand undifferentiated neural progenitor cells. Additionally, the templated hydrogels were evaluated for the in vitro study of myelin expression from Schwann cells. A hydrogel that combines the biocompatible properties of HA and the architectural complexity of native tissue may prove beneficial for biomedical applications. / text

A major challenge facing the tissue engineering discipline is cartilage tissue repair and engineering, because of the highly specialized structure and limited repair capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified about a decade ago as a potential candidate for cell based therapy and tissue engineering applications. The present study aimed to utilize gene therapy with isolated DPSCs to induce chondrogenic transgene expression and chondrogenic lineage differentiation, with the ultimate goal of engineering cartilage tissue-like constructs. We isolated DPSCs from human teeth extracted for orthodontic treatment. We further enriched the isolated population using immunomagnetic bead selection, which increased stem cell markers: Stro-1 and CD146, compared to unselected population. The DPSCs showed the ability to differentiate into the chondrogenic lineage when induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3 transgene. We successfully constructed the recombinant adeno-associated viral vector encoding the human TGFβ-3, and determined the best multiplicity of infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to 60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan was verified by immunohistochemistry and mRNA. We successfully fabricated an electrospun nano-fiber scaffold upon which morphology, proliferation and viability of the DPSCs were examined. DPSCs attached and proliferated on nano-fiber scaffolds demonstrating better viability compared to micro-fiber scaffolds. Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber scaffolds showed higher expression levels compared to micro-fiber scaffolds or culture plate. Scaffolds seeded with DPSCs were implanted in nude mice. Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan. Semi-thin sections of the transduced DPSCs constructs examined by transmission electron microscopy (TEM) showed chondrocytic cellular and intra-cellular features, as well as extracellular matrix formation (n=2/group). In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSCs constructs (n=5/group). Western blot analysis confirmed this expression pattern on the protein level (n=3/group). Engineered constructs mechanical properties were examined and compared to patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced constructs. Micro-fiber scaffolds constructs showed a higher elastic modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber scaffolds was lower. As an alternative gene delivery method, electroporation parameters for DPSCs transfection were optimized, and compared to commonly used chemical transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher relative TGFβ-3 mRNA and protein expression compared to non transfected control and to eGFP transfected DPSCs. Transfected DPSCs showed increased relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan, compared to non transfected DPSCs. Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3 transgene, and seeding them on PLLA/PGA scaffolds makes it a potential candidate for cartilage tissue engineering and cell based therapy. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Regeneration of the tissues to replace diseased, missing and traumatized dentin/pulp requires combining the recent progress in stem cell and tissue engineering research. Dental pulp stem cells (DPSCs) are considered as a promising population of cells in regenerative dentistry and shown to be able to produce dentin/pulp-like tissues following implantation in-vivo. Securing a good blood supply is critical in pulp regeneration, however, this is a challenging task due to the unique structure of the tooth, the anatomy of which permits only a microcirculatory system via a very small apical opening (<0.3-1mm). This limitation raises the need to develop novel methods to enhance angiogenesis during pulp regeneration. It was shown that DPSCs reside in the microvasculature region of the dental pulp and interact with perivascular cells. Therefore, endothelial cells could be a major source of modulators of pulp-dentin development and angiogenesis. If a pulp tissue substitute with pre-formed endothelial network could be engineered in-vitro, it would not only gain rapid anastomosis with host vasculature but also regulate DPSC function in pulp regeneration. In this study, for the first time, synergistic effects of DPSCs and human umbilical vein endothelial cells (HUVECs) on osteo/odontogenic differentiation and angiogenesis were investigated using two-dimensional and three-dimensional direct co-culture systems. Furthermore, the potential of three-dimensional DPSC constructs prevascularized with HUVECs in dental pulp regeneration in-vivo was exmined. HUVECs promoted odonto/osteogenic differentiation of DPSCs in direct two-dimensional co-cultures in-vitro. Further, addition of DPSCs stabilized the pre-existing vessel-like structures formed by HUVECs and increased the longevity of these structures on matrigel in-vitro. Using two different systems, scaffold-free self-assembling microtissue spheroids and peptide hydrogel scaffold, the interactions of DPSCs and HUVECs in three-dimensional cultures were investigated. The results demonstrated that DPSCs can self assemble into three-dimensional microtissue spheroids when cultured alone or with HUVECs. DPSCs promoted survival and vascular structure formation by HUVECs both in scaffold-free microtissue spheroids and peptide hydrogel scaffold. In contrast, HUVECs, when cultured alone, neither formed vascular structures nor survived in either of the 3D systems. The latter phenomenon was attributable to vascular endothelial growth factor secreted by DPSCs, a major factor responsible for endothelial function. Co-cultures also showed enhanced odonto/osteogenic differentiation in both three-dimensional microtissue spheroid and peptide hydrogel scaffold systems. Following implantation of tooth-root fragments filled with three-dimensional DPSC constructs into the subcutaneous space of immunodefficient mice, vascularised pulp-like tissue was regenerated within the root canals. Compared to DPSC-only group, DPSC/HUVEC co-culture groups showed higher vascularisation, extracellular matrix formation and mineralization in regenerated tissue. More importantly, HUVEC-lined vascular lumens were observed in regenerated tissues suggesting the successful integration of in-vitro formed pre-vascular structures to the host vasculature. In summary, the findings suggest that DPSCs and HUVECs display significant synergy during odonto/osteogenic differentiation and angiogenesis when co-cultured either in two-dimensional or three-dimensional culture systems. Unravelling these fundamental behavioural patterns of DPSCs provides novel insights into the process of pulp regeneration, leading to new avenues for more effective therapies in regenerative endodontics. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Regenerative medicine is an interdisciplinary field which has advanced with the use of biotechnologies related to biomaterials, growth factors, and stem cells to replace or restore damaged cells, tissues, and organs. Among various therapeutic approaches, cell-based therapy is most challenging and exciting for both scientists and clinicians pursuing regenerative medicine. Specifically, stem cells, including mesenchymal stem cells and adipose-derived stem cells, are promising candidate cell types for cell-based therapy because they can differentiate into multiple cell types for tissue regeneration and stimulate other cells through neovascularization or paracrine signaling. Also, for effective treatment using stem cells, the tissue engineered constructs, such as bioactive degradable scaffolds, that provide the physical and chemical cues to guide their differentiation are incorporated with stem cells before implantation. Also, it was previously demonstrated that tissue-engineered matrices can promote tubulogenesis and differentiation of stem cells to vascular cell phenotypes. Hence, during tissue regeneration after stem cell therapy, there are numerous factors that need to be monitored. As a result, imaging-based stem cell tracking is essential to evaluate the distribution of stem cells as well as to monitor proliferation, differentiation, and interaction with the microenvironment. Therefore, there is a need for a stem cell imaging technique that is not only noninvasive, sensitive, and easy to operate, but also capable of quantitatively assessing stem cell behaviors in the long term with high spatial resolution. Therefore, the overall goal of this research is to demonstrate a novel imaging method capable of continuous in vitro assessment of stem cells as prepared with tissue engineered constructs and noninvasive longitudinal in vivo monitoring of stem cell behaviors and tissue regeneration after stem cell implantation. In order to accomplish this, gold nanoparticles are demonstrated as photoacoustic imaging contrasts to label stem cells. In addition, ultrasound and photoacoustic imaging was utilized to monitor stem cells and neovascularization in the injured rat tissue. Therefore, using these methods, tissue regeneration can be promoted and noninvasively monitored, resulting in a better understanding of the tissue repair mechanisms following tissue injury. / text

M. Tech. Pharmaceutical Sciences. / Xenogeneic collagen has previously been reported to be a poor delivery system for bone morphogenetic proteins, because of its immunogenic effects in the host. In the laboratory the researcher uses, the problems associated with the xenogeneic bone matrix has been overcome by chemical swelling employing acetic acid followed by pepsin digestion to reduce telopeptides. This has improved host biocompatibility. The central focus of the current study was to improve the performance of the previously developed delivery system by employing a combination of BMP-2 and TGF-ß1 in a rat heterotopic assay.

In recent years, tissue engineering has shown great potential in treatment of injured tissues which aims to create artificial structures for cells to regenerate new tissues for replacing the damaged and diseased ones. The selection of scaffold materials is one of the critical factors affecting tissue healing process. Among a wide range of scaffold materials, chitosan (CS) has been demonstrated as an ideal material due to its biocompatibility, nontoxicity, biodegradability, antibacterial activity and favorable strength and stiffness. However, its insufficient mechanical properties limits its feasibility and scope for clinical application, especially for bone scaffolds. The main purpose of the study is to explore the potential of incorporation of nanofillers into CS to enhance the mechanical properties for tissue engineering. In this work, nanodiamond (ND) is applied and studied due to its high surface to volume ratio, rich surface chemistry, high mechanical strength, and excellent biocompatibility. ND/CS nanocomposites with different diamond concentration from 1wt. % to 5wt. % were synthetized through a solution casting method. The microstructure and mechanical properties of the composites were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and nanoindentation. Compared with pristine CS, the addition of ND resulted in a dramatic improvement of mechanical properties, including a 239%, 276%, 321%, 333%, and 343% increase in Young’s modulus and 68%, 96%, 114%, 118%, and 127% increase in hardness when ND amount is 1wt. %, 2wt. %, 3wt. %, 4wt. %, and 5wt. %, respectively. The strong interaction between ND surface groups and chitosan matrix is of great importance in changing polymer structure and improving mechanical properties. The cell viability and cytotoxicity of the nanocomposite were also studied using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The results show that the addition of ND has no negative effect on cell viability and the nanocomposites have no cytotoxicity.

Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.

Successful survival of large volume engineered tissues depends on the development of a vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit from such a vascularization strategy. The treatment is limited as the majority of transplanted islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue engineering is a means of designing large volume functional tissues using micron sized tissues with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells (EC) covered modules were transplanted in the omental pouch of athymic rats and human EC formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic rats with and without immunosuppressants. When the drugs were administered, endothelialized modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed vessels that integrated with the host vasculature and were perfusable until 60 days; this key result demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an allograft model. Transplantation of islets in such endothelialized modules significantly improved the vessel density around transplanted islets. Donor endothelial cells formed vessels near transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase in islet viability with transplantation of endothelialized modules in syngeneic recipients but this difference was not significant. In summary, endothelialized modules were effective in promoting stable vascularization and improving transplanted islet vascularisation. Future work should promote faster maturity of donor vessels and modulate the host immune and inflammatory responses to significantly improve transplanted islet engraftment.

Tracheal epithelium is organized along two axes: apicobasal, seen through apical ciliogenesis, and planar seen through organized ciliary beating, which moves mucus out of the airway. Diseased patients with affected ciliary motility have serious chronic respiratory infections. The standard method to construct epithelium is through air liquid interface culture which creates apicobasal polarization, not planar organization. Nanogrooved surface topography created in diffusible substrates for use in air liquid interface culture will induce planar organization of the cytoskeleton. We have created a nanogrooved gelatin device which allows basal nutrient diffusion. Multiple epithelial cells have been found to align in the direction of the nanogrooves in both sparse and confluent conditions. This device is also congruent with ALI culture as seen through formation of tight junctions and ciliogenesis. Thus, we have created nanogrooved surface topography in a diffusible substrate that induces planar alignment of epithelial cells and cytoskeleton.