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Three dimensional imaging of tissue engineering scaffold
thesisposted on 22.02.2017, 03:37 by Abuelfilat, Ala' Yasin Raji
The state of the art newly emerged field of tissue engineering is presenting a real life-saving solution to patients suffering from organ failure or tissue loss and is overcoming the series burden associated with the shortage of tissues and organ donors for transplantation. It is solving a major health care problem which is one of the most frequently tragic and costly problems in health care as it causes tremendous distress to patients and their communities. In fact, researchers worldwide have gathered their diverse expertise in so many research areas to try and overcome this problem through focusing their research effort on regenerating these diseased tissues or organs by utilising the principles of tissue engineering to restore, maintain or even improve tissue functionality. The basic principle involves transplantation of cells onto a scaffolding material to form a tissue engineered construct which can mimic the in vivo microenvironment of cells and hence promote cell growth. However, the need for an in vitro 3D scaffold that can substitute specific tissue-types is becoming increasingly prevalent in tissue engineering and stem cell research. Also, in order for this regeneration to occur successfully and for cell-material to be further enhanced, the choice of the material and its design properties such as porosity and interconnectivity is crucial to resemble the native ECM environment. In fact, hydrogels are promising candidates for engineered complex 3D tissue scaffolds since they have tissue-like stiffness, biocompatibility and high permeability for oxygen, nutrients and other water-soluble metabolites, similar to the native extracellular matrix. However, high-resolution characterization of hydrogels and their three-dimensional porous structures still remains a challenge. In this research we aimed at exploring a new highly spatial resolution X-ray Ultramicroscopy (XuM) imaging technique to provide a fast three dimensional visualisation of biocompatible porous hydrogel structures. We also aim to demonstrate the capabilities of XuM imaging technique to reconstruct the three dimensional porous hydrogel models and quantitatively analyse the geometry of individual pore sizes, their spatial distribution and interconnectivity. The nanomechanics of the hydrogel samples will be further investigated by Atomic Force Microscopy (AFM) force spectroscopy through obtaining their elastic modulus and the reconstruction of their three dimensional porous structures, which will allow for the mechanical modelling and simulation of individual pores. The bulk scaffold will also be proven to be feasible, thereby establishing a rational approach for exploring structuremechanics relationships. In this study we have examined the hydroxypropyl cellulose methacrylate (HPC-MA) hydrogels for the first time through X-ray ultramicroscopy (XuM), an imaging technique based on phase contrast and with high spatial resolution, to visualise, reconstruct and analyse 3D porous structures. This Scanning Electron Microscopy (SEM) based X-ray system produced projection images of 1.67 J.lm pixel size, with distinguishable hydrogel membrane structures. In addition, reconstruction of the tomographic series provides the complete geometry of individual pores and their spatial distribution and interconnectivity, which play vital roles in accurate prediction of the hydrogel's porous structure prior to and during its implantation in vivo. Furthermore the elastic modulus of the hydrogels was determined and mechanical modelling of individual pores and the bulk scaffold also proved to be feasible by incorporating the Atomic Force Microscopy (AFM) technique. The commercialised platform we utilised offers prompt visualization and specialized simulation of customized 3D scaffolds for cell growth, which will be a unique application of tissue engineering in future personalized medicine.