Tendon function is essential for quality of life, yet the pathogenesis and healing of tendinopathy remains poorly understood compared to other musculoskeletal disorders. The aim of regenerative medicine is to replace traditional tissue and organ transplantation by harnessing the developmental potential of stem cells to restore structure and function to damaged tissues. The recently discovered interdependency of cell phenotype and biophysical environment has created a paradigm shift in cell biology. This dissertation introduces a dynamic in vitro model for tendon function, dysfunction and development, engineered to characterize the mechanobiological relationships dictating stem cell fate decisions so that they may be therapeutically exploited for tendon healing. Cells respond to mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. A naturally-derived decellularized tendon scaffold (DTS) was invented to serve as a biomimetic tissue culture platform, preserving the structure and function of native extracellular matrix. DTS in concert with a newly designed dynamic mechanical strain system comprises a tendon bioreactor that is able to emulate the three-dimensional topography, extracellular matrix proteins, and mechanical strain that cells would experience in vivo. Mesenchymal stem cells seeded on decellularized tendon scaffolds subject to cyclic mechanical deformation developed strain-dependent alterations in phenotype and measurably improved tissue mechanical properties. The relative tenogenic efficacies of adult stem cells derived from bone marrow, adipose and tendon were then compared in this system, revealing characteristics suggesting tendon-derived mesenchymal stem cells are predisposed to differentiate toward tendon better than other cell sources in this model. The results of the described experiments have demonstrated that adult mesenchymal stem cells are responsive to mechanical stimulation and, while exhibiting heterogeneity based on donor tissue, are broadly capable of tenocytic differentiation and tissue neogenesis in response to specific ultrastructural and biomechanical cues. This knowledge of cellular mechanotransduction has direct clinical implications for how we treat, rehabilitate and engineer tendon after injury. / Ph. D.

Nanoparticles can be used as building blocks of materials. Properties of such materials depend on the organization of the constituent particles. Thus, control over particle organization enables control over material properties. However, robust and scalable methods for arranging nanoparticles are still lacking. This dissertation explores the use of an externally applied magnetic field to organize magnetic nanoparticles into microstructures of desired shape. It extends to proofs of concept towards applications in material design and tissue engineering. First, external control over dipolar self-assembly of magnetic nanoparticles (MNPs) in a liquid dispersion is investigated experimentally. Scaling laws are derived to explain experimental observations, correlating process control variables to microstructure morphology. Implications of morphology on magnetic properties of such structures are then explored computationally. Specifically, a method is proposed wherein superparamangetic nanoparticles, having no residual magnetization, can be organized into anisotropic structures with remanence. Another application explores the use of magnetic forces in organizing human cells into three-dimensional (3D) structures of desired shape and size. When magnetized cells are held in place for several days, they are seen to form inter-cellular contacts and organize themselves into tight clusters. This provides a method for 3D tissue culture without the use of artificial scaffolding materials. Finally, a method to pattern heterogeneities in the stiffness of an elastomer is developed. This makes use of selective inhibition of the catalyst of crosslinking reactions by magnetite nanoparticles. The last chapter discusses future possibilities. / Ph. D.

High levels of ionizing radiation are known to cause degradation and/or cross-linking in polymers. Lower levels of ionizing radiation, such as x-rays, are commonly used in the treatment of cancers. Material characterization has not been fully explored for polymeric materials exposed to therapeutic radiation levels. This study investigated the effects of therapeutic radiation on three porous scaffolds: polycaprolactone (PCL), polyurethane (PU) and gelatin. Porous scaffolds were fabricated using solvent casting and/or salt leaching techniques. Scaffolds were placed in phosphate buffered saline (PBS) and exposed to a typical cancer radiotherapy schedule. A total dose of 50 Gy was broken into 25 dosages over a three-month period. PBS was collected over time and tested for polymer degradation through high performance liquid chromatography (HPLC) and bicinchoninic acid (BCA) protein assay. Scaffolds were characterized by changes in microstructure using Scanning Electron Microscopy (SEM), and crystallization using Differential Scanning Calorimetry (DSC). Additionally, gelatin ε-amine content was analyzed using Trinitrobenzene Sulfonic Acid Assay (TNBSA). Gelatin scaffolds immersed in PBS for three months without radiation served as a control. Each scaffold responded differently to radiation. PCL showed no change in molecular weight or microstructure. However, the degree of crystallinity decreased 32% from the non-irradiated control. PU displayed both changes in microstructure and a decrease in crystallinity (85.15%). Gelatin scaffolds responded the most dramatically to radiotherapy. Samples were observed to swell, yet maintain shape after exposure. As gelatin was considered a tissue equivalent, further studies on tissues are needed to better understand the effects of radiotherapy. / Master of Science

Vat photopolymerization (VP) is an additive manufacturing (AM) technology that permits the fabrication of parts with complex geometries and feature sizes as small as a few microns. These attributes make VP an attractive option for the fabrication of scaffolds for tissue engineering. However, there are few printable materials with low cytotoxicity that encourage cellular adhesion. In addition, these resins are not readily available and must be synthesized. A novel resin based on 2-acrylamido-2-methyl-1-propanesulfonic acid (NaAMPS) and poly(ethylene glycol) diacrylate (PEGDA) was formulated and printed using VP. The mechanical properties, water content, and high fidelity of the scaffold indicated promise for use in tissue engineering applications. Murine fibroblasts were observed to successfully adhere and proliferate on the scaffolds. The growth, migration, and differentiation of a cell is known to dependent heavily on its microenvironment. In engineered constructs, much of this microenvironment is provided by the tissue scaffold. The physical environment results from the scaffold's geometrical features, including pore shape and size, porosity, and overall dimensions. Each of these parameters are known to affect cell viability and proliferation, but due to the difficulty of isolating each parameter when using scaffold fabrication techniques such as porogen leaching and gas foaming, conflicting results have been reported. Scaffolds with pore sizes ranging from 200 to 600 μm were fabricated and seeded with murine fibroblasts. Other geometric parameters (e.g., pore shape) remained consistent between scaffold designs. Inhomogeneous cell distributions and fewer total cells were observed in scaffolds with smaller pore sizes (200-400 μm). Scaffolds with larger pores had higher cell densities that were homogeneously distributed. These data suggest that tissue scaffolds intended to promote fibroblast proliferation should be designed to have pore at least 500 μm in diameter. Techniques developed for selective placement of dissimilar materials within a single VP scaffold enabled spatial control over cellular adhesion and proliferation. The multi-material scaffolds were fabricated using an unmodified and commercially available VP system. The material preferences of murine fibroblasts which resulted in their inhomogeneous distribution within multi-material scaffolds were confirmed with multiple resins and geometries. These results suggest that multi-material tissue scaffolds fabricated with VP could enable multiscale organization of cells and material into engineered constructs that would mimic the function of native tissue. / Doctor of Philosophy / Vat photopolymerization (VP) is a 3D printing (or additive manufacturing) technology that is capable of fabricating parts with complex geometries with very high resolution. These features make VP an attractive option for the fabrication of scaffolds that have applications in tissue engineering. However, there are few printable materials that are biocompatible and allow cells attachment. In addition, those that have been reported cannot be obtained commercially and their synthesis requires substantial resources and expertise. A novel resin composition formulated from commercially available components was developed, characterized, and printed. Scaffolds were printed with high fidelity. The scaffolds had mechanical properties and water contents that suggested they might be suitable for use in tissue engineering. Fibroblast cells were seeded on the scaffolds and successfully adhered and proliferated on the scaffolds. The growth, migration, and differentiation of cells is influenced by the environmental stimuli they experience. In engineered constructs, the scaffold provides many of stimuli. The geometrical features of scaffolds, including how porous they are, the size and shape of their pores, and their overall size are known to affect cell growth. However, scaffolds that have a variety of pore sizes but identical pore shapes, porosities, and other geometric parameters cannot be fabricated with techniques such as porogen leaching and gas foaming. This has resulted in conflicting reports of optimal pore sizes. In this work, several scaffolds with identical pore shapes and porosities but pore sizes ranging from 200 μm to 600 μm were designed and printed using VP. After seeding with cells, scaffolds with large pores (500-600 μm) had a large number of evenly distributed cells while smaller pores resulted in fewer cells that were unevenly distributed. These results suggest that larger pore sizes are most beneficial for culturing fibroblasts. Multi-material tissue scaffolds were fabricated with VP by selectively photocuring two materials into a single part. The scaffolds, which were printed on an unmodified and commercially available VP system, were seeded with cells. The cells were observed to have attached and grown in much larger numbers in certain regions of the scaffolds which corresponded to regions built from a particular resin. By selectively patterning more than one material in the scaffold, cells could be directed towards certain regions and away from others. The ability to control the location of cells suggests that these printing techniques could be used to organize cells and materials in complex ways reminiscent of native tissue. The organization of these cells might then allow the engineered construct to mimic the function of a native tissue.

Yes / Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. / Engineering and Physical Sciences Research Council. Grant Number: EPSRC EP/F500513/1

Yes / Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Im-munohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrody-namic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of a-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

Yes / Complex biological tissues are highly viscoelastic and dynamic. Efforts to repair or replace cartilage, tendon, muscle, and vasculature using materials that facilitate repair and regeneration have been ongoing for decades. However, materials that possess the mechanical, chemical and resorption characteristics necessary to recapitulate these tissues have been difficult to mimic using synthetic resorbable biomaterials. Herein, we report a series of resorbable elastomer-like materials that are compositionally identical and possess varying ratios of cis:trans double bonds in the backbone. These features afford concomitant control over the mechanical and surface eroding degradation properties of these materials. We show the materials can be functionalized post-polymerization with bioactive species and enhance cell adhesion. Furthermore, an in vivo rat model demonstrates that degradation and resorption are dependent on succinate stoichiometry in the elastomers and the results show limited inflammation highlighting their potential for use in soft tissue regeneration and drug delivery.

Yes / Fabrication of extracellular matrix (ECM)-like scaffolds (in terms of structural-functional) is the main challenge in skin tissue engineering. Herein, inspired by macromolecular components of ECM, a novel hybrid scaffold suggested which includes silk/hyaluronan (SF/HA) bio-complex modified by PCP: [polyethylene glycol/chitosan/poly(ɛ-caprolactone)] copolymer containing collagen to differentiate human-adipose-derived stem cells into keratinocytes. In followed by, different weight ratios (wt%) of SF/HA (S1:100/0, S2:80/20, S3:50/50) were applied to study the role of SF/HA in the improvement of physicochemical and biological functions of scaffolds. Notably, the combination of electrospinning-like and freeze-drying methods was also utilized as a new method to create a coherent 3D-network. The results indicated this novel technique was led to ~8% improvement of the scaffold's ductility and ~17% decrease in mean pore diameter, compared to the freeze-drying method. Moreover, the increase of HA (>20wt%) increased porosity to 99%, however, higher tensile strength, modulus, and water absorption% were related to S2 (38.1, 0.32 MPa, 75.3%). More expression of keratinocytes along with growth pattern similar to skin was also observed on S2. This study showed control of HA content creates a microporous-environment with proper modulus and swelling%, although, the role of collagen/PCP as base biocomposite and fabrication technique was undeniable on the inductive signaling of cells. Such a scaffold can mimic skin properties and act as the growth factor through inducing keratinocytes differentiation.

Yes / Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.

Significant advancements have been made within the field of regenerative medicine over the last few decades with the goal of creating biological substitutes to mimic tissue for research and wound healing purposes. Simply put, regenerative medicine works by understanding and then manipulating the processes by which cells communicate and proliferate for healing purposes. Before valuable progress can be made in regenerative medicine, smaller steps need to be taken first, like understanding the biomaterials that are used within regenerative medicine research. Biomaterials, which are materials that interact with cells and perform a function, are used to mimic the native extracellular matrix of cell scaffolding in regenerative medicine research. Numerous types of biomaterials exist, and it is important to choose the most appropriate material for the goal at hand. Therefore, biomaterials need to be characterized before useful research with the materials can be done. An important aspect of biomaterials that can be characterized is fluid flow through the biomaterials. This is important because adequate transport of oxygen, nutrients, waste, and soluble factors are required for cell proliferation and survival.[1] Biomaterials can be characterized based on their chemical, physical, and mechanical characteristics via many different characterization methods that are discussed in this paper. The overall goal of this research is to characterize the fluid flow metrics through Micro-porous Annealed Particle (MAP) hydrogels and others using Dynamic Contrast Enhanced – Magnetic Resonance Imaging (DCE-MRI) and computational analysis of the images via MATLAB. The analysis was utilized to analyze the fluid flow through several different biomaterial types, allowing for observational comparison between biomaterial groups. Overall, this method for characterizing fluid flow through biomaterials shows promise for future use and further understanding of biomaterials' roles in regenerative medicine. / Master of Science / Regenerative medicine encompasses the use of scientific knowledge and tools to determine novel methods for generating functioning tissues and organs. Commonly, biomaterials are used to assist in this process. Biomaterials frequently function as a solid structure that houses cells and encourages cell growth, eventually leading to tissue formation. Many different types of biomaterials exist, so it is important to determine the most suitable biomaterial for each project to improve efficiency and experiment outcomes. Biomaterial properties, like stiffness or flexibility, can be determined through various scientific testing methods. An important property of biomaterials is the fluid flow through the biomaterials. Cells housed inside biomaterials require oxygen and nutrients to grow, so it is important that fluids carrying these molecules can flow through biomaterials to provide support for the cells. This paper utilizes a computational analysis method to analyze Magnetic Resonance Imaging (MRI) images of fluid flow through biomaterials. The analysis provides information on fluid flow metrics through the biomaterials, like fluid flow velocity and direction. This analysis provides a new method for understanding biomaterial properties and provides the analysis for several different biomaterials.