Developing platforms for tissue engineering of the airways

Understanding airway dynamics has been under rigorous study for past decades due to its implication in health and disease, where in vitro, in vivo and human subjects are continuously being investigated for causation of diseased pathways. However, limitations arise as the variability in the models are unavoidable, and many factors such as cell microenvironments are usually uncontrollable. To surpass this problem, tissue engineering offers a platform to create systematically a controlled environment for the cells to grow. This discipline is used in order to observe cause-and effect scenarios that elucidates physiological processes and pathological dysregulation within the biology of interest. A platform that allows factors to be tuned to mimic biological conditions of the airways will be truly helpful in unlocking information on how airway cells behave and respond to their environment. In this work, the tissue engineering central dogma of cells, biomaterials and bioreactor was utilised to setup an observable environment for the cells to be studied. Specifically, airway smooth muscle and epithelial cells were cultured on biomimetic scaffolds that can be tuned mechanically and chemically. Airway smooth muscle cells cultured on stiffening scaffolds showed an asthma-like phenotype, displaying elevated marker for contractility, cell size, and proliferation capacity. For the airway epithelium, proliferation was also increased in increasing matrix stiffness, and augmenting the scaffold with a functional group similar to the native epithelium further supported its growth. Lastly, a double-chambered bioreactor was designed to support culture of the airways, in which its evaluation and performance was assessed computationally and experimentally to obtain optimum parameters for airway culture. It is hoped that the platforms developed in this thesis for the tissue engineering of the airways will elucidate pathways on how disease processes occur without the need for in vivo models. Such model may be used not just on understanding of the biology of the airways, but also a platform to evaluate therapeutic options for alleviation of chronic and lethal airway diseases

Growth‐Factor Free Multicomponent Nanocomposite Hydrogels That Stimulate Bone Formation

Synthetic osteo‐promoting materials that are able to stimulate and accelerate bone formation without the addition of exogenous cells or growth factors represent a major opportunity for an aging world population. A co‐assembling system that integrates hyaluronic acid tyramine (HA‐Tyr), bioactive peptide amphiphiles (GHK‐Cu2+), and Laponite (Lap) to engineer hydrogels with physical, mechanical, and biomolecular signals that can be tuned to enhance bone regeneration is reported. The central design element of the multicomponent hydrogels is the integration of self‐assembly and enzyme‐mediated oxidative coupling to optimize structure and mechanical properties in combination with the incorporation of an osteo‐ and angio‐promoting segments to facilitate signaling. Spectroscopic techniques are used to confirm the interplay of orthogonal covalent and supramolecular interactions in multicomponent hydrogel formation. Furthermore, physico‐mechanical characterizations reveal that the multicomponent hydrogels exhibit improved compressive strength, stress relaxation profile, low swelling ratio, and retarded enzymatic degradation compared to the single component hydrogels. Applicability is validated in vitro using human mesenchymal stem cells and human umbilical vein endothelial cells, and in vivo using a rabbit maxillary sinus floor reconstruction model. Animals treated with the HA‐Tyr‐HA‐Tyr‐GHK‐Cu2+ hydrogels exhibit significantly enhanced bone formation relative to controls including the commercially available Bio‐Oss

Developing platforms for tissue engineering of the airways

Understanding airway dynamics has been under rigorous study for past decades due to its implication in health and disease, where in vitro, in vivo and human subjects are continuously being investigated for causation of diseased pathways. However, limitations arise as the variability in the models are unavoidable, and many factors such as cell microenvironments are usually uncontrollable. To surpass this problem, tissue engineering offers a platform to create systematically a controlled environment for the cells to grow. This discipline is used in order to observe cause-and effect scenarios that elucidates physiological processes and pathological dysregulation within the biology of interest. A platform that allows factors to be tuned to mimic biological conditions of the airways will be truly helpful in unlocking information on how airway cells behave and respond to their environment. In this work, the tissue engineering central dogma of cells, biomaterials and bioreactor was utilised to setup an observable environment for the cells to be studied. Specifically, airway smooth muscle and epithelial cells were cultured on biomimetic scaffolds that can be tuned mechanically and chemically. Airway smooth muscle cells cultured on stiffening scaffolds showed an asthma-like phenotype, displaying elevated marker for contractility, cell size, and proliferation capacity. For the airway epithelium, proliferation was also increased in increasing matrix stiffness, and augmenting the scaffold with a functional group similar to the native epithelium further supported its growth. Lastly, a double-chambered bioreactor was designed to support culture of the airways, in which its evaluation and performance was assessed computationally and experimentally to obtain optimum parameters for airway culture. It is hoped that the platforms developed in this thesis for the tissue engineering of the airways will elucidate pathways on how disease processes occur without the need for in vivo models. Such model may be used not just on understanding of the biology of the airways, but also a platform to evaluate therapeutic options for alleviation of chronic and lethal airway diseases

Characterization and multi-response morphological optimization for preparation of defect-free electrospun nanofibers using the Taguchi method

© 2017 Trans Tech Publications Ltd, Switzerland. The study presents a method on producing defect-free polyvinyl alcohol-gelatin (PVAG) nanofibers by considering multiple morphological characteristics of the produced nanofibers using the Taguchi method. Aside from minimizing the average fiber diameter, the method was also used to produce consistent, monodispersed PVAG nanofibers without the usual defects of beading and splattering. The experiments are performed considering the effect of polymer composition (PVAG ratio and solvent ratio of water, formic acid, and acetic acid H2O:FA:HAc) and process factors (tipto-collector distance TCD and solution flow rate) on fiber morphology. Fiber morphology is measured in terms of 4 responses: average fiber diameter, standard deviation of fiber diameter, occurrence of beading, and occurrence of splattering. Results show that maximum overall desirability for electrospinning PVAG nanofibers at smallest average diameter and deviation (26.10 ± 9.88 nm) with chance of moderate beading and zero splattering is predicted at PVAG mass ratio of 6.5:3.5, H2O:FA:HAc solvent volume ratio of 4:4:2, TCD of 12.5 cm, and flow rate of 1 ml h-1. Results of confirmatory run agree with the predicted factor levels at maximum desirability, with average fiber diameter and standard deviation measured to be 26.95 ± 6.39 nm. PVAG nanofibers of the confirmatory run are also both bead- and splatter-free. Results suggest the application of Taguchi method can offer a robust and rapid approach in deriving the conditions for production of new and high-quality PVAG nanofibers for tissue engineering scaffolds

Mixed polymer and bioconjugate core/shell electrospun fibres for biphasic protein release

Effective regenerative medicine requires delivery systems which can release multiple components at appropriate levels and at different phases of tissue growth and repair. However, there are few biomaterials and encapsulation techniques that are fully suitable for the loading and controlled release of multiple proteins. In this study we describe how proteins were physically and chemically loaded into a single coaxial electrospun fibre scaffold to obtain bi-phasic release profiles. Cyto-compatible polymers were used to construct the scaffold, using polyethylene oxide (PEO) for the core and polycaprolactone (PCL) reacted or mixed with (bis-aminopropyl)polyether (Jeffamine ED2003; JFA) for the shell. Horseradish peroxidase (HRP), a model protein, was loaded in the core and functionalised onto the scaffold surface by coupling of protein carboxyl groups to the available polymer amine groups. Fibre morphologies were evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and functional group content was determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF SIMS). Hydrophobicity profiles of the fibres before and after protein loading were evaluated by water contact angle (WCA) and the mechanical properties of the electrospun scaffolds were determined by performing tensile tests. The electrospun fibre scaffolds generated by reacting PEO/PCL with 1,6-diaminohexane and those from mixing PEO/PCL with JFA were further characterised for protein conjugation and release. Fibres prepared by the mixed PEO/PCL/JFA system were found to be the most appropriate for the simultaneous release of protein from the core and the immobilisation of another protein on the shell of the same scaffold. Moreover, JFA enhanced scaffold properties in terms of porosity and elasticity. Finally, we successfully demonstrated the cytocompatibility and cell response to protein-loaded and -conjugated scaffolds using HepG2 cells. Enhanced cell attachment (2.5 fold) was demonstrated using bovine serum albumin (BSA)-conjugated scaffolds, and increased metabolic activity observed with retinoic acid (RA)-loaded scaffolds (2.7 fold)

Lysyl oxidase-like 2 is increased in asthma and contributes to asthmatic airway remodelling

Airway smooth muscle cells (ASM) are fundamental to asthma pathogenesis, influencing bronchoconstriction, airway hyper-responsiveness, and airway remodelling. Extracellular matrix (ECM) can influence tissue remodelling pathways, however, to date no study has investigated the effect of ASM ECM stiffness and crosslinking on the development of asthmatic airway remodelling. We hypothesised that TGFβ activation by ASM is influenced by ECM in asthma and sought to investigate the mechanisms involved. This study combines in vitro and in vivo approaches: human ASM cells were used in vitro to investigate basal TGFβ activation and expression of ECM crosslinking enzymes. Human bronchial biopsies from asthmatic and non-asthmatic donors were used to confirm LOXL2 expression ASM. A chronic ovalbumin model of asthma was used to study the effect of LOXL2 inhibition on airway remodelling.We found that ASM cells from asthmatics activated more TGFβ basally than non-asthmatic controls and that diseased cell-derived ECM influences levels of TGFβ activated. Our data demonstrate that the ECM crosslinking enzyme LOXL2 is increased in asthmatic ASM cells and in bronchial biopsies. Crucially, we show that LOXL2 inhibition reduces ECM stiffness and TGFβ activation in vitro, and can reduce subepithelial collagen deposition and ASM thickness, two features of airway remodelling, in an ovalbumin mouse model of asthma.These data are the first to highlight a role for LOXL2 in the development of asthmatic airway remodelling and suggest that LOXL2 inhibition warrants further r investigation as a potential therapy to reduce remodelling of the airways in severe asthma