Three dimensional imaging of tissue engineering scaffold

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

Focused ion beam analysis of cell growth in 3D interconnected porous structure scaffold

Hydrogels are synthetic or natural polymer networks that have emerged as promising candidates for 3D tissue engineering scaffolds. In the past several years, research interest has shifted from hydrogel implants to injectable formulations, which have the advantage that cells and bioactive compounds can be mixed easily with precursor solutions prior to gelation to give homogeneously loaded gels. In addition, in situ gelation allows the formation of complex shapes and can be applied using minimally invasive surgery. However, electron imaging of cell growth in situ to understand cell behaviour and activity is still challenging, especially when cells are growing in porous extracellular matrix (ECM)-like structures. 3D porous hydrogels of high permeability and biocompatible structure can mimic the microenvironment of ECM, but for high resolution imaging, there are still obstacles to overcome. Porous microstructures, with or without residing cells, are not appropriate for microtomy, and thus transmission electron microcopy (TEM) imaging is extremely difficult to apply for study of cell-hydrogel interfaces. The other alternative, scanning electron microscopy (SEM), is limited to observation of the surface region only and is not suitable for probing 3D scaffolds. In this study, we obtained images of the cellhydrogel interface by exposing and probing target samples using focused ion beam (FIB) milling. Hydrogels were prepared and mixed with African green monkey kidney cell

Bridging structure and mechanics of three-dimensional porous hydrogel with X-ray ultramicroscopy and atomic force microscopy

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. As a promising candidate for engineered complex 3D tissue scaffolds, hydrogels have emerged as synthetic or natural polymers with 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, hydroxypropyl cellulose methacrylate (HPC-MA) hydrogels were examined 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 μm 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. By further incorporating Atomic Force Microscopy (AFM), the elastic modulus of the hydrogel was determined and mechanical modelling of individual pores and the bulk scaffold also proved to be feasible. 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

Tuning the surface properties of hydrogel at the nanoscale with focused ion irradiation

With the site-specific machining capability of Focused Ion Beam (FIB) irradiation, we aim to tailor the surface morphology and physical attributes of biocompatible hydrogel at the nano/micro scale particularly for tissue engineering and other biomedical studies. Thin films of Gtn-HPA/CMC-Tyr hydrogels were deposited on a gold-coated substrate and were subjected to irradiation with a kiloelectronvolt (keV) gallium ion beam. The sputtering yield, surface morphology and mechanical property changes were investigated using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Monte Carlo simulations. The sputtering yield of the hydrogel was found to be approximately 0.47 µm3 nC-1 compared with Monte-Carlo simulation results of 0.09 µm3 nC-1. Compared to the surface roughness of the pristine hydrogel at approximately 2 nm, the average surface roughness significantly increased with the increase of ion fluence with measurements extended to 20 nm at 100 pC µm-2. Highly packed submicron porous patterns were also revealed with AFM, while significantly decreased pore sizes and increased porosity were found with ion irradiation at oblique incidence. The Young's modulus of irradiated hydrogel determined using AFM force spectroscopy was revealed to be dependent on ion fluence. Compared to the original Young's modulus value of 20 MPa, irradiation elevated the value to 250 MPa and 350 MPa at 1 pC µm-2 and 100 pC µm-2, respectively. Cell culture studies confirmed that the irradiated hydrogel samples were biocompatible, and the generated nanoscale patterns remained stable under physiological conditions