published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

　　　Strontium (Sr) is not only widely studied for its compound as a drug for treating osteoporosis, but there is also a growing interest of its addition in orthopaedic biomaterials. Over the years, the development of orthopaedic biomaterials has already advanced to a new era in the search of resorbable and/or bioactive materials. Due to its anabolic and anti-resportive properties of Sr on bone regeneration as a drug, strontium has been extensively investigated for its potential in other orthopaedic applications. The purposes of this study were to investigate strontium containing hydroxyapatite (Sr-HA) bone cement for the delivery of gentamicin and the effects of Sr incorporation in coral and borosilicate glass as bone engineering scaffolds. 　　　 　　　Three types of Sr incorporated materials are reported here, in which these include an applied study of the drug elution property of previously published bone cement and two initial studies of the biological properties of newly developed coral and borosilicate scaffolds. Firstly, the gentamicin release, bioactivity and mechanical property of bioactive bone cement filler based on Sr-HA were compared to a commercially available gentamicin-loaded poly(methyl methacrylate) (PMMA). Over the study period of 30 days, the cumulative gentamicin release from Sr-HA bone cement was much greater than PMMA bone cement (+ 34%); better bioactivity of Sr-HA was also confirmed with the apatite formation after simulated body fluid immersion. Goniopora, a highly interconnected porous coral, was hydrothermally converted to coralline hydroxyapatite (CHA) or coated with hydroxyapatite and incorporated with Sr. As the first report of incorporating Sr into coral with the structure remained, about 4-16% Sr was detected on CHA. Sr-HA coated coral was studied in vitro and in vivo (ovariectomized rat model) resulting in better cell proliferation and higher scaffold volume retention (+40%). Finally, the development of Sr incorporated borosilicate (SrB) glass scaffold explored a new material for bone tissue engineering, but more importantly, it introduced a phenomenal idea of the stimulatory effect of a local alkaline microenvironment on bone regeneration. Detections of an exceedingly high pH (~ pH 8.6) condition on the material surface and release of Sr, Si and B ions during the degradation of scaffold SrB were confirmed to stimulate osteoblasts and facilitate apatite formation. Although new bone was observed on both scaffolds, higher bone area/tissue area (B.Ar/T.Ar) on scaffold SrB indicated more new bone formation over borosilicate scaffold without Sr addition. 　　　 　　　The significance of this study is to explore and develop three orthopaedic biomaterials advancing the stimulatory effects of Sr on bone regeneration. The drug elution properties of Sr-HA bone cement provides a fascinating alternative for treating osteomyelitis. Furthermore, by incorporating Sr into CHA and borosilicate scaffold, it brings out the importance on the readiness of the Sr release of the materials in order to deliver the stimulatory effects. Subsequently, a localized pH micro-environment arisen by material degradation is emphasized as a controlling factor in bone regeneration on biomaterials. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Stem cell-based therapies are an important developing technology for treating cardiovascular ischemic disease, including subsequent co-morbidities such as ulcerative wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment wound healing and neovascularization processes and have been more recently investigated for their endothelial-like behavior. This doctoral work aims to understand mechanisms underlying matrix-driven MSC tubulogenesis within PEGylated fibrin gels, specifically (1) why this behavior occurs and (2) if this behavior has clinical utility. Briefly, a three-dimensional morphological quantification pipeline was first developed for analyzing the maturity of vascular networks (Chapter 2). This method was applied in later studies that examined the full spectrum of MSC behavior in PEGylated fibrin gels, linking biomaterial properties with network development (Chapter 3). Mechanisms underlying the cell-matrix relationship were more fully clarified through gain-of-function cell studies. These studies indicated that PEGylated fibrin promotes endothelial-like MSC behavior through a combination of hypoxic stress and bioactive fibrin cues (Chapter 4). Notably, this endothelial-like MSC behavior closely mirrored vasculogenic mimicry, a process whereby tumors establish non-endothelialized vasculature in response to hypoxic stress. The functionality of these tumor vessels suggests that mature endothelial differentiation of MSCs may not be necessary to achieve therapeutically beneficial tissue perfusion. This hypothesis opens up new mechanisms for exploitation in vascular tissue engineering strategies. / text

Myocardial Infarction (MI) is one of the most prevalent and deadliest diseases in the United States. Since the host myocardium becomes irreversibly damaged following MI, current research is focused on identification of novel, less invasive, and more effective treatment options for patients. Cellular cardiomyopathy, in which viable cells are transplanted into the necrotic tissue, has the potential to regenerate and integrate with the host myocardium. Stem cells, specifically pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC), are ideal candidates for this procedure because they are pluripotent; however, ESCs must be predifferentiated to avoid teratoma formation in vivo. In this dissertation, our goal was improve upon current protocols to direct differentiation of ESCs into cardiomyocytes using an embryoid body (EB) model. We immobilized pro-cardiomyogenic proteins, specifically Sonic Hedgehog (SHH) and Bone Morphogenetic Protein 4 (BMP4) to paramagnetic beads and delivered them in the interior of the EB. While lineage commitment was indiscriminate, the presence of the beads alone appeared to guide differentiation into cardiomyocytes: there were significantly more contracting areas in EBs containing beads than in the presence of SHH or BMP4. To take advantage of this result, we immobilized Arginine-Glycine-Aspartic Acid (RGD) peptides to the beads and magnetized them following incorporation into the EB. Magnetically mediated strain increased the expression of mechanochemical markers, and in combination with BMP4 increased the percentage of cardiomyocytes. Finally, PEGylated fibrin gels were used to investigate the effect of seeding method and fibrinogen concentration on cardiomyocyte behavior and maturation. Cells seeded on top of compliant hydrogels had the most contracting regions compared to stiffer PEGylated fibrin gels, whereas cardiomyocytes seeded within the hydrogels could not remodel the matrix or maintain contractility. As an alternative to 3D culture, we seeded cardiomyocytes within gel layers, which maintained viability as well as contractile activity. We observed that PEGylated fibrin gels can maintain ESC-derived cardiomyocytes; however, the ratio of cardiomyocytes and non-cardiomyocytes should be optimized to maintain contractile phenotypes. Therefore, this dissertation presents novel methods to differentiate ESCs into cardiomyocytes, and subsequently promote their maturation in vitro, for the treatment of MI. / text

The use of nanotechnology to develop methods for fabrication and characterization of organized hybrid nanostructures that include integrated polymeric, biological and inorganic compounds has increased exponentially during the last decade. Such bio-nano-composite materials could be used in solving current biomedical problems spanning from nanomedicine to tissue engineering and biosensing. In this dissertation, a systematic study has been carried out on the synthesis, characterization, of two interfaces between naturally derived and synthetic nanostructures. Carbon nanotubes and porous silicon represent the synthetic nanostructures that were developed for the purpose of interfacing with the naturally derived bovine type I collagen and respiratory syncytial virus DNA respectively. Firstly, the synthesis of collagen-carbon nanotubes by two different techniques: fibrillogenesis through slow wet fiber drawing (gelation process) and electrospinning has been highlighted. Characterization of the novel nanocomposite was conducted using electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, nanoindentation, and Raman spectroscopy. The collagen-carbon nanotube gelation process was found to have superior nanoscale surface mechanical properties that were more conducive to higher osteoblast specific protein expression such as osteocalcin. Applications of the developed nanofibers are detailed in the fields of orthopaedics and tissue engineering. Secondly, an overview of porous silicon synthesized by hydrofluoric acid is presented. A parametric study was performed to determine the optimal pore size was carried out. The use of porous silicon as a biosensor to detect RSV virus by DNA hybridization was then provided and the importance of the interface chemistry was highlighted.

Skeletal muscles are composed of thousands of muscle fibres (muscle cells), densely packed together in parallel and surrounded by connective tissue sheaths. These fibres are multinuclear in nature, which allows for the control and regulation of the highly organised, protein rich cellular interior. The primary function of skeletal muscle is to produce force, which allows for movement to occur or posture to be maintained, and the regulation of this function is in turn reliant on the expression of specific genes and proteins. Skeletal muscle exhibits a high degree of plasticity, and can adapt in response to stimuli such as increased/decreased use, metabolic perturbations or changes in the systemic environment which often occur as a result of exercise, ageing, disuse or disease. Examining responses and adaptations in skeletal muscle in vivo are challenging due to experimental restrictions, and studies are limited by ethical issues surrounding experimentation on human beings and indeed on animals following the principals of refinement, reduction and replacement. Thus in vitro studies are often conducted in order to further understand mechanisms involved in adaptation. However, the environment to which skeletal muscle cells are exposed to in vitro is far removed from that in the body, and the resulting cellular architecture is often abnormal in morphology. Tissue engineered skeletal muscle has shown much promise in rectifying these issues, as cells can be grown on/within a matrix which is biologically relevant and engineered to grow in a uniaxial manner in parallel to one another. However, this field is in its relative infancy, and to date little data exists with regards the behaviour and characteristics of human muscle derived cells (MDCs) in tissue engineered constructs. In this thesis, human skeletal MDCs were obtained, characterised and subsequently cultured in a suitable model for tissue engineering purposes. MDCs were seeded on to a fibrin based hydrogel, which self-assembled over time to form a cylindrically shaped construct held in place between two anchor points. In ii this model, the cells were shown to align uniaxially and in parallel to one another in a fascicular like structure. The model was improved in terms of biomimicity and maturation by both increasing the seeding density of the MDCs, and by increasing the ratio of myogenic to non-myogenic cells. These models appear to promote the development of a slow muscle, as evidenced by the favourably high levels of MYH7 transcription in comparison to other isoforms, and showed suggestions of sarcomeric organisation as indicated by the classically striated pattern of protein organisation when myosin heavy chain immunostaining was conducted. The work conducted in the final chapter of this thesis focussed on developing a system capable of assessing and quantifying the force produced by these tissue engineered human skeletal muscle constructs when electrically stimulated. Further work in this area should aim to determine these functional characteristics and thereafter use the model for physiological, cellular and molecular studies in exercise science.

To develop or engineer artificial tissues in tissue engineering, a detailed knowledge of the in vitro culture process including cell and tissue growth inside porous scaffolds, nutrient transport, and the shear stress acting on the cells is of great advantage. It has been shown that obtaining such information by means of experimental techniques is exceedingly difficult and in some ways impossible. Mathematical modelling and computational simulation based on computational fluid dynamics (CFD) has emerged recently to be a promising tool to characterize the culture process. However, due to the complicated structure of porous scaffolds, modelling and simulation of the in vitro cell culture process has been shown to be a challenging task. Furthermore, due to the cell growth during the culture process, the geometry of the scaffold structure is not constant, but changes with time, which makes the task even more challenging. To overcome these challenges, the research presented in this thesis is aimed at developing a CFD-based mathematical model and multi-time scale computational framework for culturing cell-scaffold constructs placed in perfusion bioreactors. To predict the three-dimensional (3D) cell growth in a porous tissue scaffold placed inside a perfusion bioreactor, a model is developed based on the continuity and momentum equations, a convection-diffusion equation and a suitable cell growth equation, which characterize the fluid flow, nutrient transport and cell growth, respectively. To solve these equations in a coupled fashion, an in-house FORTRAN code is developed based on the multiple relaxation time lattice Boltzmann method (MRT LBM), where the D3Q19 MRT LBM and D3Q7 MRT LBM models have been used for the fluid flow and mass transfer simulation, respectively. In the model cell growth equation, the transport of nutrients, i.e. oxygen and glucose, as well as the shear stress induced on the cells are considered for predicting the cell growth rate. In the developed model and computational framework, the influence of the dynamic strand surface on the local flow and nutrient concentration has been addressed by using a two-way coupling between the cell growth and local flow field and nutrient concentration, where a control-volume method within the LBM framework is applied. The simulation results provide quantification of the biomechanical environment, i.e. fluid velocity, shear stress and nutrient concentration inside the bioreactor. The final simulation applied the cell growth model to the culture of a three-zone tissue scaffold where the scaffold strands were initially seeded with cells. The prediction for the 3D cell growth rate indicates that the increase in the cell volume fraction is much higher in the front region of the scaffold due to the higher nutrient supply. The higher cell growth in the front zone reduces the permeability of the porous scaffold and significantly reduces the nutrient supply to the middle and rear regions of the scaffold, which in turn limit the cell growth in those regions. However, implementation of a bi-directional perfusion approach, which reverses the flow direction for second half of the culture period, is shown to significantly improve the nutrient transport inside the scaffold and increase the cell growth in the rear zone of the scaffold. The results in this study also demonstrate that the developed mathematical model and computational framework are capable of realistically simulating the 3D cell growth over extended culture periods. As such, they represent a promising tool for enhancing the growth of tissues in perfusion bioreactors.

Nanoscale materials enable unique opportunities at the interface between physical and life sciences. The interface between nanoelectronic devices and biological systems makes possible communication between these two diverse systems at the length scale relevant to biological functions. The development of a bottom-up paradigm allows the nanoelectronic units to be synthesized and patterned on unconventional substrates. In this thesis, I will focus on the development of three-dimensional (3D) nanoelectronics, which mimics the structure of porous biomaterials to explore new methods for seamless integration of electronics with other materials, with a special focus on biological tissue. / Chemistry and Chemical Biology

I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation. / text

This thesis presents a foundation for developing a business case for companies interested in the reconstructive and cosmetic procedure markets. The focus is on reviewing adipose tissue engineering research and proposing technology opportunities that could be applied to challenging soft tissue reconstruction cases and adjacently applied to cosmetic applications. To establish the foundation for this type of program, this thesis includes an evaluation of the reconstructive and cosmetic procedure markets, current practices in these markets and their constraints, as well as a literature review of research in adipose tissue engineering and its potential clinical applications. Additionally it captures the competitive landscape of major players in the reconstructive market as well as up-and-coming players in the adipose tissue engineering field. Technology development opportunities with associated customer and business value are discussed with a recommendation for the development of a detailed business case to evaluate specific product development opportunities in these markets. / text