The overall goal of this research project is to develop methods for producing a tissue engineered ligament. The envisioned tissue engineering strategy involves three steps: seeding bone marrow stromal cells (BMSCs) onto electrospun scaffolds, processing them into cords that allow cell infiltration, and conditioning them with uniaxial cyclic stretch. These steps were addressed in three complimentary studies to establish new methods to engineer a tissue with ligament-like cells depositing organized extracellular matrix (ECM). In the first study scaffold topographies were systematically varied to determine topographies that induce cells to orient and differentiate into ligament-like cells in static culture. Scaffolds — electrospun from poly (ester-urethane urea) (PEUUR) with different fiber diameters degrees of fiber alignments — were biocompatible and supported cell growth. Topographic cues guided cell alignment, and cell elongation increased with increasing fiber alignment. Finally, expression of the ligament-like markers collagen type I and decorin were enhanced on the smallest fiber diameters compared to larger diameters. In the second study BMSCs — seeded onto aligned electrospun PEUUR scaffolds — were cyclically stretched to determine the effect of dynamic mechanical stimulation on BMSC alignment and differentiation. BMSCs remained aligned parallel to the direction of fiber alignment and expressed ligament markers (e.g. collagen type I, decorin, scleraxis, and tenomodulin) on electrospun scaffolds after the application of stretch. However, the cyclic stretch regimen was not able to enhance expression of ECM components. In the third study techniques were developed to produce more clinically relevant constructs with improved cell infiltration. Specifically, a co-electrospun scaffold composed of two well integrated components was developed to create larger pores. The scaffold was also embedding in a photo-crosslinkable hydrogel to prevent the fibers from collapsing. These results demonstrate the feasibility of making a tissue engineered ligament by seeding BMSCs on an aligned, co-electrospun scaffold with submicron diameter fibers and then applying cyclic mechanical stretch. Future work will involve combining these three steps to achieve materials suitable for in vivo testing. / Ph. D.

Studies regarding the feasibility of ovalbumin (OVA) as a bone scaffold material have found its cost, availability, interaction with cells, and ability to degrade in the body into safe byproducts to be ideal for such an application. However, weak mechanical properties cause hesitation in the use of OVA as a scaffolding material in much stronger native tissue. To enhance the mechanical strength of the OVA scaffolds without compromising in vitro cellular performance, Ca-P crystals were grown on unmodified OVA and phosphonated OVA (p-OVA) samples via biomineralization processes using 5x-concentrated simulated body fluid (5x SBF). Electron microscopy (ESEM/EDS) data confirm the formation of Ca-P crystals on the surface of OVA and p-OVA scaffolds. Mechanically, rheology data measured a minimum of a three-fold increase in each mineralized scaffold's complex shear modulus over unmineralized counterparts. Degradation in a PBS+collagenase XI environment showed that mineralization extended total time to degradation. It was also shown that the formation of the Ca-P crystals had no negative effects on in vitro cell studies. To measure cellular response, a live/dead assay was conducted to confirm cell viability after 24 hours. In conclusion, improvements were made to mechanical strength without compromising in vitro cell-scaffold response. While it remains unknown whether the increase in strength is adequate for use as a bone scaffold, future work should focus on gathering necessary information to study OVA scaffolds in animal models for eventual consideration as a bone graft substitute material. / Master of Science

Biodegradable polyesters, such as poly(DL-lactic-co-glycolic acid) (PLGA), have been used to fabricate porous bone scaffolds to support bone tissue development. These scaffolds allow for cell seeding, attachment, growth and extracellular matrix production in vitro and are replaced by new bone tissue when implanted into bone sites in vivo. Hydroxyapatite (HAP) and μ-tricalcium phosphate (μ-TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, μ-TCP, and biodegradable polyesters are osteoconductive, there is no evidence that these scaffold materials are osteoinductive (support cell differentiation). Calcium and phosphate ions, in contrast, have been postulated to be osteogenic factors that enhance osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized which permits controlled release of calcium and phosphate ions and thus is hypothesized to be osteoinductive. Incorporation of Zr-ACP into a highly porous poly(DL lactic-co-glycolic acid) (PLGA) scaffold could potentially increase the osteoinductivity of the scaffold and therefore promote osteogenesis when implanted in vivo. To determine the osteoinductivity of Zr-ACP, a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line was used to measure cell response to Zr-ACP. To accomplish this objective, Zr-ACP was added to cell culture at different stages in cell maturation (days 0, 4 and 11). DNA synthesis, alkaline phosphatase (ALP) activity, osteopontin synthesis and collagen synthesis were determined. Results indicate that culture in the presence of Zr-ACP significantly increased cell proliferation, ALP activity and osteopontin synthesis but not collagen synthesis. To determine the feasibility of incorporating Zr-ACP into a PLGA scaffold, PLGA/Zr-ACP composite foams (5% or 10% (w/v) polymer:solvent with 25 wt% or 50 wt% Zr-ACP) were fabricated using a thermal phase inversion technique. Scanning electron microscopy revealed a highly porous structure with pores ranging in size from a few microns to about 100 μm. The amorphous structure of the Zr-ACP was maintained during composite fabrication as confirmed by X-ray diffraction measurements. Composite scaffolds also showed significantly greater compressive yield strengths and moduli as compared to pure polymer scaffolds. The results of this study indicate that Zr-ACP enhances the osteoblastic phenotype of MC3T3-E1 cells in vitro and can be incorporated into a porous PLGA scaffold. Porous PLGA/Zr-ACP composites are promising for use as bone scaffolds to heal bone defects. / Master of Science

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.

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in ful lment of the requirements for the degree of Master of Science in Engineering. Johannesburg, 2015 / This research investigates the e ects of the frequency components of the lightning First Short Stroke (FSS) on the current pathway through human tissues using frequency domain analysis. A Double Exponential Function (DEF) is developed to model the FSS with frequency components in the range 10 Hz 100 kHz. Human tissues are simulated using Finite Element Analysis (FEA) in COMSOL and comprises of two types of models: Single Layer Cylindrical Model (SLCM) and Multi-layered Cylindrical Model (MLCM). The SLCM models 54 human tissues independently and the MLCM models the human leg with ve tissue layers: bone marrow, cortical bone, muscle, blood and fat. Three aspects are analysed: current density, complex impedance and power dissipation. From the SLCM results, aqueous tissues have the lowest impedances and tissue heat dissipation is proportional to tissue impedance. Results from the MLCM show that 85% of the FSS current ows through muscle, 11% ows through blood, 3:5% through fat and the rest through cortical bone and bone marrow. From the results, frequency dependent equivalent circuit models consisting of resistors and capacitors connected in series are proposed. The simulation results are correlated with three main clinical symptoms of lightning injuries: neurological, cardiovascular and external burns. The results of this work are applicable to the analysis of High Voltage (HV) injuries at power frequencies. / MT2017