3D Bioprinting Systems for the Study of Mammary Development and Tumorigenesis

Understanding the microenvironmental factors that control cell function, differentiation, and stem cell renewal represent the forefront of developmental and cancer biology. To accurately recreate and model these dynamic interactions in vitro requires both precision-controlled deposition of multiple cell types and well-defined three-dimensional (3D) extracellular matrix (ECM). To achieve this goal, we hypothesized that accessible bioprinting technology would eliminate the experimental inconsistency and random cell-organoid formation associated with manual cell-matrix embedding techniques commonly used for 3D, in vitro cell cultures. The first objective of this study was to adapt a commercially-available, 3D printer into a 3D bioprinter. Goal-based computer simulations were used to identify, evaluate, and optimize the performance of a 3D bioprinting system. Implementing these findings yielded a bioprinting system with reduced needle clogging and single cell print resolution. The minimal disruption of cell function was confirmed by the retention of pluripotency marker TRA-1-81 in bioprinted human induced pluripotent stem cells (hiPSCs) 7-days post-printing. This system was then used to investigate cell behavior during the initial stages of organoid-structure formation by generating 3D bioprinted arrays of individual, mammary epithelial cell (MEC) organoid-structures. This quantifiable, 3D bioprinting approach, was able to direct the ‘self-assembly’ of large MEC structures through organoid ‘fusion’ events among individual, bioprinted organoids along the printing template. Bioprinting maintained experimental consistency among multiple 3D scaffolds and experimental conditions, and presents the capability to generate high-fidelity, 3D arrays with multiple cell types. Compared to manual matrix embedding, bioprinted, co-culture experiments, containing normal MECs and breast cancer cell lines, significantly increased the ability to generate chimeric (tumor/normal) MEC structures. Thus, bioprinting stands highly qualified to investigate the role of microenvironmental processes related to cell fate determination and tissue formation

A practical review on the measurement tools for cellular adhesion force

Cell cell and cell matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion

The mechanical control of nervous system development.

The development of the nervous system has so far, to a large extent, been considered in the context of biochemistry, molecular biology and genetics. However, there is growing evidence that many biological systems also integrate mechanical information when making decisions during differentiation, growth, proliferation, migration and general function. Based on recent findings, I hypothesize that several steps during nervous system development, including neural progenitor cell differentiation, neuronal migration, axon extension and the folding of the brain, rely on or are even driven by mechanical cues and forces.This work was supported by the Medical Research CouncilThis is the accepted version of the original publication available at http://dev.biologists.org/content/140/15/3069

Discrete element modeling of hydrogel extrusion

Hydrogels are widely used in extrusion bioprinting as bioinks. Understanding how the hydrogel microstructure affects the bioprinting process aids researchers in predicting the behavior of biological components. Current experimental tools are unable to measure internal forces and microstructure variations during the bioprinting process. In this work, discrete element modeling was used to study the internal interactions and the elastic deformation of the molecular chains within hydrogel networks during the extrusion process. Two-dimensional models of hydrogel extrusions were created in Particle Flow Code (PFC; Itasca Co., Minneapolis, MN). For our model\u27s calibration, hydrogel compression testing was used in which a cluster of particles is pushed in the vertical direction with a confined load similar to the uniaxial compression test. The parameter sensitivity study was performed using a set of parameters, e.g., coefficient of friction, restitution coefficient, and stiffness. Force distribution among the particles during the extrusion process was then predicted using the results of the study. Using this model, we analyzed the distribution of internal forces

Electrospun collagen fibers for tissue regeneration applications

Tissue engineering aims to regenerate damaged and deceased tissue by combining cells with scaffold made from an appropriate biomaterial and providing a conducive environment to guide cell growth and the formation or regeneration of new tissue or organ. While collagen, an important material of the extracellular matrix (ECM), is a natural choice as a scaffold biomaterial, the conducive environment can only be created by having the ability to control the geometry, organization, structural and mechanical properties of the scaffold. Moreover, degradability and degradation rate control of the scaffold has to be taken into consideration too. In this work, we aim at developing a scaffold that possess the geometry, organization, structural and mechanical properties of the ECM, that is also degradable with degradation rate control. We accomplish this through fabrication of scaffolds composed of collagen fibers with diameters of ~ 50 to 500 nm using electrospinning. These fibers can be organized and provide structural and mechanical support for the cells populating it. The versatile electrospinning setup not only allows mimicking the define architecture of the native ECM environment containing collagen fibers but, in the core-shell or porous structure, can also enable bioactive molecule encapsulation and their controlled release into the cell culture environment. Post fabrication processing for fiber stability via chemical or photochemical crosslinking as well as ion implantation resulted in fibers with controlled degradation rate and enhanced mechanical properties. Chemical structure characterization demonstrated close resemblance of fiber surface and native collagen. Favorable cell adhesion and proliferation demonstrated good cell compatibility using the human fetal lung (IMR-90) cells. By implementing the strategy developed in this thesis to construct scaffolds using electrospun collagen fiber that possess appropriate organization and properties to mimic the natural environment, scaffolds can be custom designed for specific tissue engineering applications with potentially improved outcome

A Novel Bio-Inspired Insertion Method for Application to Next Generation Percutaneous Surgical Tools

The use of minimally invasive techniques can dramatically improve patient outcome from neurosurgery, with less risk, faster recovery, and better cost effectiveness when compared to conventional surgical intervention. To achieve this, innovative surgical techniques and new surgical instruments have been developed. Nevertheless, the simplest and most common interventional technique for brain surgery is needle insertion for either diagnostic or therapeutic purposes. The work presented in this thesis shows a new approach to needle insertion into soft tissue, focussing on soft tissue-needle interaction by exploiting microtextured topography and the unique mechanism of a reciprocating motion inspired by the ovipositor of certain parasitic wasps. This thesis starts by developing a brain-like phantom which I was shown to have mechanical properties similar to those of neurological tissue during needle insertion. Secondly, a proof-of-concept of the bio-inspired insertion method was undertaken. Based on this finding, the novel method of a multi-part probe able to penetrate a soft substrate by reciprocal motion of each segment is derived. The advantages of the new insertion method were investigated and compared with a conventional needle insertion in terms of needle-tissue interaction. The soft tissue deformation and damage were also measured by exploiting the method of particle image velocimetry. Finally, the thesis proposes the possible clinical application of a biologically-inspired surface topography for deep brain electrode implantation. As an adjunct to this work, the reciprocal insertion method described here fuelled the research into a novel flexible soft tissue probe for percutaneous intervention, which is able to steer along curvilinear trajectories within a compliant medium. Aspects of this multi-disciplinary research effort on steerable robotic surgery are presented, followed by a discussion of the implications of these findings within the context of future work

Electrohydrodynamic deposition and patterning of nano-hydroxyapatite for biomedical applications

Electrohydrodynamic atomisation (EHDA) spraying is a promising materials deposition technique as it allows uniform and regular deposition, and offers a range of other advantages, such as low cost compared with other current techniques, easy set-up, high deposition rate, ambient temperature processing and the capability to generate specific surface topographies. This research is aimed at using EHDA spraying to produce hydroxyapatite (HA) deposition with desirable chemical, topographical and biological characteristics for bone implant. In principle, the EHDA process involves the flow of liquid/suspension from a needle under the influence of an electric field which results jetting and droplet formation. In this work, phase-pure nano-sized hydroxyapatite (nHA) was synthesised and taken up in ethanol to prepare a suitable suspension for electrohydrodynamic flow processing. A range of key EHDA process control parameters, such as needle size, needle to substrate distance, suspension flow rate, applied voltage and spraying time, were studied and optimised. A uniform nHA coating with nanometer scale topographical features was successfully prepared on a commercially pure titanium substrate. Furthermore, due to the significance of the surface structure to the cellular response, a novel technique, namely template-assisted electrohydrodynamic atomisation (TAEA) spraying, was innovated to prepare a well-defined surface topography for guiding cell attachment, spread and growth of osteoblasts. A range of precise micro-scale uniform nHA geometries with high resolution were prepared on implants materials. Finally, to systematically investigate the effect of needle geometry to the electrospraying process, which has not been documented in the research field of this technique, an in-depth study was carried out to uncover the relationship between the needle exit angle and the droplet relic size. The droplet relic size, which is crucial for the deposition properties, has been significantly reduced via engineering the needle geometry during the electrospraying process. The results of this work have demonstrated that EHDA deposition routes show great potential for the commercial preparation of nHA coatings and patterns for bone implants with enhanced bioactivity

Attosecond physics at the nanoscale

Recently two emerging areas of research, attosecond and nanoscale physics, have started to come together. Attosecond physics deals with phenomena occurring when ultrashort laser pulses, with duration on the femto- and sub-femtosecond time scales, interact with atoms, molecules or solids. The laser-induced electron dynamics occurs natively on a timescale down to a few hundred or even tens of attoseconds, which is comparable with the optical field. On the other hand, the second branch involves the manipulation and engineering of mesoscopic systems, such as solids, metals and dielectrics, with nanometric precision. Although nano-engineering is a vast and well-established research field on its own, the merger with intense laser physics is relatively recent. In this article we present a comprehensive experimental and theoretical overview of physics that takes place when short and intense laser pulses interact with nanosystems, such as metallic and dielectric nanostructures. In particular we elucidate how the spatially inhomogeneous laser induced fields at a nanometer scale modify the laser-driven electron dynamics. Consequently, this has important impact on pivotal processes such as ATI and HHG. The deep understanding of the coupled dynamics between these spatially inhomogeneous fields and matter configures a promising way to new avenues of research and applications. Thanks to the maturity that attosecond physics has reached, together with the tremendous advance in material engineering and manipulation techniques, the age of atto-nano physics has begun, but it is in the initial stage. We present thus some of the open questions, challenges and prospects for experimental confirmation of theoretical predictions, as well as experiments aimed at characterizing the induced fields and the unique electron dynamics initiated by them with high temporal and spatial resolution