3D microfabrication of biological machines

The burgeoning field of additive manufacturing, or “3D printing”, centers on the idea of creating three-dimensional objects from digital models. While conventional manufacturing approaches rely on modifying a base material via subtractive processes such as drilling or cutting, 3D printing creates three-dimensional objects through successive deposition of two- dimensional layers. By enabling rapid fabrication of complex objects, 3D printing is revolutionizing the fields of engineering design and manufacturing. This thesis details the development of a projection-based stereolithographic 3D printing apparatus capable of high- resolution patterning of living cells and cell signals dispersed in an absorbent hydrogel polymer matrix in vitro. This novel enabling technology can be used to create model cellular systems that lead to a quantitative understanding of the way cells sense, process, and respond to signals in their environment. The ability to pattern cells and instructive biomaterials into complex 3D patterns has many applications in the field of tissue engineering, or “reverse engineering” of cellular systems that replicate the structure and function of native tissue. While the goal of reverse engineering native tissue is promising for medical applications, this idea of building with biological components concurrently brings about a new discipline: “forward engineering” of biological machines and systems. In addition to rebuilding existing systems with cells, this technology enables the design and forward engineering of novel systems that harness the innate dynamic abilities of cells to self-organize, self-heal, and self-replicate in response to environmental cues. This thesis details the development of skeletal and cardiac muscle based bioactuators that can sense external electrical and optical signals and demonstrate controlled locomotive behavior in response to them. Such machines, which can sense, process, and respond to signals in a dynamic environment, have a myriad array of applications including toxin neutralization and high throughput drug testing in vitro and drug delivery and programmable tissue engineered implants in vivo. A synthesis of two fields, 3D printing and tissue engineering, has brought about a new discipline: using microfabrication technologies to forward engineer biological machines and systems capable of complex functional behavior. By introducing a new set of “building blocks” into the engineer’s toolbox, this new era of design and manufacturing promises to open up a field of research that will redefine our world

Recent advances in 3D printing of biomaterials.

3D Printing promises to produce complex biomedical devices according to computer design using patient-specific anatomical data. Since its initial use as pre-surgical visualization models and tooling molds, 3D Printing has slowly evolved to create one-of-a-kind devices, implants, scaffolds for tissue engineering, diagnostic platforms, and drug delivery systems. Fueled by the recent explosion in public interest and access to affordable printers, there is renewed interest to combine stem cells with custom 3D scaffolds for personalized regenerative medicine. Before 3D Printing can be used routinely for the regeneration of complex tissues (e.g. bone, cartilage, muscles, vessels, nerves in the craniomaxillofacial complex), and complex organs with intricate 3D microarchitecture (e.g. liver, lymphoid organs), several technological limitations must be addressed. In this review, the major materials and technology advances within the last five years for each of the common 3D Printing technologies (Three Dimensional Printing, Fused Deposition Modeling, Selective Laser Sintering, Stereolithography, and 3D Plotting/Direct-Write/Bioprinting) are described. Examples are highlighted to illustrate progress of each technology in tissue engineering, and key limitations are identified to motivate future research and advance this fascinating field of advanced manufacturing

3D printed muscle-powered bio-bots

Complex biological systems sense, process, and respond to a range of environmental signals in real-time. The ability of such systems to adapt their functional response to dynamic external signals motivates the use of biological materials in other engineering applications. Recent advances in 3D printing have enabled the manufacture of complex structures from biological materials. We have developed a projection stereolithographic 3D printing apparatus capable of patterning cells and biocompatible polymers at physiologically relevant length scales, on the order of single cells. This enables reverse engineering in vitro model systems that recreate the structure and function of native tissue for applications ranging from high-throughput drug testing to regenerative medicine. While reverse engineering native tissues and organs has important implications in biomedical engineering, the ability to “build with biology” presents the next generation of engineers with both a unique design challenge and opportunity. Specifically, we now have the ability to forward engineer bio-hybrid machines and robots (bio-bots) that harness the adaptive functionalities of biological materials to achieve more complex functional behaviors than machines composed of synthetic materials alone. Perhaps the most intuitive demonstration of a “living machine” is a system that can generate force and produce motion. To that end, we have designed and 3D printed locomotive bio-bots, powered by external electrical and optical stimuli. In addition to being the first demonstrations of untethered locomotion in skeletal musclepowered soft robots, these bio-hybrid machines have served as meso-scale models for studying tissue self-assembly, maturation, damage, remodeling, and healing in vitro. Bio-hybrid machines that can dynamically sense and adaptively respond to a range of environmental signals have broad applicability in healthcare applications such as dynamic implants or targeted drug delivery. Advanced research in exoskeletons and hyper-natural functionality could even extend the useful application of such machines to national defense and environmental cleanup. We have developed a modular skeletal muscle bioactuator that can serve as a fundamental building block for such machines, setting the stage for future generations of bio-hybrid machines that can self-assemble, self-heal, and perhaps even self-replicate to target grand engineering challenges. Furthermore, we present a robust optimized protocol for manufacturing 3D printed muscle-powered biological machines, and a mechanism to incorporate biological “building blocks” into the toolbox of the next generation of engineers and scientists

Additive Manufacturing and Physicomechanical Characteristics of PEGDA Hydrogels: Recent Advances and Perspective for Tissue Engineering

In this brief review, we discuss the recent advancements in using poly(ethylene glycol) diacrylate (PEGDA) hydrogels for tissue engineering applications. PEGDA hydrogels are highly attractive in biomedical and biotechnology fields due to their soft and hydrated properties that can replicate living tissues. These hydrogels can be manipulated using light, heat, and cross-linkers to achieve desirable functionalities. Unlike previous reviews that focused solely on material design and fabrication of bioactive hydrogels and their cell viability and interactions with the extracellular matrix (ECM), we compare the traditional bulk photo-crosslinking method with the latest three-dimensional (3D) printing of PEGDA hydrogels. We present detailed evidence combining the physical, chemical, bulk, and localized mechanical characteristics, including their composition, fabrication methods, experimental conditions, and reported mechanical properties of bulk and 3D printed PEGDA hydrogels. Furthermore, we highlight the current state of biomedical applications of 3D PEGDA hydrogels in tissue engineering and organ-on-chip devices over the last 20 years. Finally, we delve into the current obstacles and future possibilities in the field of engineering 3D layer-by-layer (LbL) PEGDA hydrogels for tissue engineering and organ-on-chip devices

Optical Printing of Multiscale Hydrogel Structures

Hydrogel has been a promising candidate to recapitulate the chemical, physical and mechanical properties of natural extracellular matrix (ECM), and they have been widely used for tissue engineering, lab on a chip and biophotonics applications. A range of optical fabrication technologies such as photolithography, digital projection stereolithography and laser direct writing have been used to shape hydrogels into structurally complex functional devices and constructs. However, it is still greatly challenging for researchers to design and fabricate multiscale hydrogel structures using a single fabrication technology. To address this challenge, the goal of this work is the design and develop novel multimode optical 3D printing technology capable of printing hydrogels with multiscale features ranging from centimeter to micrometer sizes and in the process transforming simple hydrogels into functional devices for many biomedical applications. Chapter 2 presents a new multimode optical printing technology that synergistically combined large-scale additive manufacturing with small-scale additive/subtractive manufacturing. This multiscale fabrication capability was used to (i) align cells using laser induced densification in Chapter 3, (ii) develop diffractive optics based on changes in refractive indices in Chapter 4, (iii) print diffractive optical elements in Chapter 5, and (iv) digitally print complex microfluidic devices and other 3D constructs in Chapter 6. Overall, this work open doors to a new world of fabrication where multiscale functional hydrogel structures are possible for a range biomedical application

Microfabrication of spatially-patterned, polymer scaffolds for applications in stem cell and tissue engineering

textTissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures.Biomedical Engineerin

Topology Optimization and 3D printing of Large Deformation Compliant Mechanisms for Straining Biological Tissues

This paper presents a synthesis approach in a density-based topology optimization setting to design large deformation compliant mechanisms for inducing desired strains in biological tissues. The modelling is based on geometrical nonlinearity together with a suitably chosen hypereleastic material model, wherein the mechanical equilibrium equations are solved using the total Lagrangian finite element formulation. An objective based on least-square error with respect to target strains is formulated and minimized with the given set of constraints and the appropriate surroundings of the tissues. To circumvent numerical instabilities arising due to large deformation in low stiffness design regions during topology optimization, a strain-energy based interpolation scheme is employed. The approach uses an extended robust formulation i.e. the eroded, intermediate and dilated projections for the design description as well as variation in tissue stiffness. Efficacy of the synthesis approach is demonstrated by designing various compliant mechanisms for providing different target strains in biological tissue constructs. Optimized compliant mechanisms are 3D-printed and their performances are recorded in a simplified experiment and compared with simulation results obtained by a commercial software.Comment: 23 pages, 14 figure