Tissue engineering is a field devoted to the design and creation of replacement tissues with the ultimate goal of one day providing replacement organs. Traditional strategies to accomplish this through the bulk seeding of cells onto a single monolithic porous bio-scaffold are unable to realise a precise architecture, thus the inability to mimic the cells natural micro-environment found within the body. Bio-printing approaches are the current state of the art with the ability to accurately mimic the complex 3D hierarchical structure of tissue. However, a functional construct also requires high strength to provide adequate support in load bearing applications such as bone and cartilage tissue engineering, and to maintain the open geometry of a large intricate channel network, which is crucial for the transport of nutrients and wastes. Typical approaches utilise materials which have processing parameters more amendable for cell incorporation, thus they can be simultaneously deposited with scaffolding material. However, the resulting construct is typically of low strength. This thesis explores the automation of a printing and “tissue assembly” process with the ability to incorporate delicate cell aggregates or spheroids within a high strength bio-scaffold requiring harsh processing parameters, at precise locations. The 3D printed bio-scaffold has a lattice architecture which enables a frictional fit to be formed between the particle and scaffold, thus preventing egress. To achieve this the pore must be expanded before the delivery of a single 1mm particle. Novel subsystems were developed to automate this process and provide the ability to achieve scalable, flexible, complex constructs with accurate architecture. A system architecture employing the benefits of modularity was devised. The main subsystems developed were the singulation device, to ensure the separation of a single particle; the injection device, to deliver and seed particles into the scaffold, and the control system, to facilitate the operation of the devices. Three generations of singulation devices have been developed ranging from mechanical to fluid manipulation methods alone. The first prototype utilised mechanical methods, with simple control methods. However the inability to correctly position the lead particle within the singulation chamber, resulted in damage to the test alginate particles. In the second prototype a fully fluidics based device utilised two trapping sites to capture the leading particles. Singulation success rates of up to 88% was achieved. Higher rates were limited by the trapped particle’s interaction with the lagging particles during capture. In a similar concept to the second prototype, the third prototype utilised only a single trapped particle, and achieved much higher throughput, and 100% singulation accuracy. The injection device, utilised a conical expanding rod within a thin outer sheath. It was able to expand the pore, with minimal damage to the scaffold, providing an unobstructed path for the delivery of the particle into the pore. A decentralised control system was devised to integrate the process operation for the electro-mechanical devices. Separate microcontrollers were able to sense, interact and communicate with one another, and the master control PC, to execute specific tasks to automate the process. The development of systems to automate the process has addressed the ability to accurately incorporate delicate cells with a high strength bio-scaffold, and will enable the realisation and investigation of intricate complex constructs, unachievable with current manual processes. Thus features found within the body may be more closely mimicked and functionalised, which may provide the necessary signals, micro-environment and infrastructure to correctly regulate the formation of complex functional tissue, supported by the adequate mass transport of nutrients and wastes. This may one day lead to 3D printing or assembly of viable replacement tissue, accurate in vitro model systems for laboratory testing, or even whole organs.

Tissue regeneration using in-vitro scaffold becomes a vital mean to mimic the in-vivo counterpart due to the insufficiency of animal models to predict the applicability of drug and other physiological behavior. Three-dimensional (3D) bio-printing is an emerging technology to reproduce living tissue through controlled allocation of biomaterial and cell. Due to its bio-compatibility, natural hydrogels are commonly considered as the scaffold material in bio-printing process. However, repeatable scaffold structure with good printability and shape fidelity is a challenge with hydrogel material due to weak bonding in polymer chain. Additionally, there are intrinsic limitations for bio-printing of hydrogels due to limited cell proliferation and colonization while cells are immobilized within hydrogels and don’t spread, stretch and migrate to generate new tissue. The goal of this research is to develop a bio-ink suitable for extrusion-based bio-printing process to construct 3D scaffold. In this research, a novel hybrid hydrogel, is designed and systematic quantitative characterization are conducted to validate its printability, shape fidelity and cell viability. The outcomes are measured and quantified which demonstrate the favorable printability and shape fidelity of our proposed material. The research focuses on factors associated with pre-printing, printing and post-printing behavior of bio-ink and their biology. With the proposed hybrid hydrogel, 2 cm tall acellular 3D scaffold is fabricated with proper shape fidelity. Cell viability of the proposed material are tested with multiple cell lines i.e. BxPC3, prostate stem cancer cell, HEK 293, and Porc1 cell and about 90% viability after 15-day incubation have been achieved. The designed hybrid hydrogel demonstrate excellent behavior as bio-ink for bio-printing process which can reproduce scaffold with proper printability, shape fidelity and higher cell survivability. Additionally, the outlined characterization techniques proposed here open-up a novel avenue for quantifiable bio-ink assessment framework in lieu of their qualitative evaluation.

Fabrication of scaffold is the key for bone tissue engineering, which is commonly regarded as the most potential route for repairing bone defects. Previously, porous ceramic scaffolds were fabricated through a variety of traditional methods, like moulding and casting, but most of them cannot produce customised tissue-engineered scaffolds. Therefore, 3D printing methods are gaining more attention and are currently being explored and developed to make scaffolds with acceptable biocompatibility. With the considerable development of bone tissue engineering, the bioactivity of scaffolds is becoming increasingly demanded, which leads to new methods and techniques to produce highly biomimetic bone scaffolds. In this study, a new fabrication process to optimise the structures of scaffolds was developed, and intensive researches were performed on the porous scaffolds to confirm their advantages in biological performance. Specifically, by combination of motor assisted extrusion deposition and gas-foaming (graphite as the porogen) technique, hierarchically porous scaffolds with improved microstructures, i.e. multi-scaled pores from nanometre to millimetre (nm-μm-mm), was successfully developed. In this thesis, the optimal content of porogen for scaffolds was studied in terms of compressive strength and in-rod porosities. The most concerned physicochemical properties of scaffolds were carefully examined and the results revealed that such scaffolds exhibit excellent physicochemical properties owing to hierarchically porous structures. Due to additional in-rod micropores and increased specific surface area, along with better hydrophilicity, hierarchically porous scaffolds exerted complete superiority in biological activity, including promoting cellular proliferation of osteoblasts, adhesion and spreading status, as well as the ability to induce cellular differentiation.