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

Optically Micro-fabricated Linear and Freeform 3-D Extracellular Matrix Scaffolds for Tissue Engineering

This work was aimed at advancing multi-photon excited, freeform fabrication technology with nano-scale and sub-micron precision as an enabler for tissue engineers to investigate cellular response to a biomimetic, bio-active extracellular matrix. We demonstrated that sub-micron and micron scale Collagen and Fibronectin structures can be fabricated via multi-photon excited photochemistry using a modified Benzophenone dimer and Rose Bengal while maintaining the biomimetic ECM structures’ bioactivity. We confirmed that three-photon excitation produces significantly smaller features at comparable excitation wavelengths as a consideration to better approach focal adhesion size. Bioactivity of MPE cross-linked FN and Collagens I and II was established via immunofluorescence and fibroblast adhesion. Additionally, the relative rates of degradation in these cross-linked matrices are consistent with the known activities of these enzymes. Morphology measurements of fibroblasts grown on these proteins include log(Area), Perimeter, Area/Perimeter2 were considered as proxies for cell response. Fibroblast perimeters are statistically different when associated with the Collagen I microenvironment. Among fibroblasts grown on MPE structures of Collagen I, Fibronectin, BSA and the BSA Monolayer, the stress fiber distributions on Collagen I (all fiber lengths) are highly significantly different (p \u3c 1x10-4) than the distribution of stress fibers of cells on BSA Lines. This suggests contact guidance only for cells on BSA Lines but yet a combination of contact guidance and chemical signaling (RGD) with cells on Collagen I Lines. This supports additional overall orientation findings based on fibroblasts’ fitted ellipse major axis direction for Collagens I, II and Fibronectin. Stress fiber distribution on BSA Monolayer differed significantly from those on BSA structures (p = 0.01). This underscores the effects of pure contact guidance alone provided by the BSA fibers compared to the combined contact guidance and ECM cues provided by the FN, and collagen structures. A method similar to rapid prototyping or three-dimensional printing was accomplished to resolve cellular response at the submicron level by fabricating biomimetic, bioactive extracellular matrices in a freeform three-dimensional (3D) manner. To the best of our knowledge, simultaneous 3D spatial and chemical control of collagen scaffold synthesis at the micrometer and sub-micrometer size scales has not been fully demonstrated

(Photo-)crosslinkable gelatin derivatives for biofabrication applications

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for bio-fabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs

3D Nanoprinting Technologies for Tissue Engineering Applications

Tissue engineering recovers an original function of tissue by replacing the damaged part with a new tissue or organ regenerated using various engineering technologies. This technology uses a scaffold to support three-dimensional (3D) tissue formation. Conventional scaffold fabrication methods do not control the architecture, pore shape, porosity, or interconnectivity of the scaffold, so it has limited ability to stimulate cell growth and to generate new tissue. 3D printing technologies may overcome these disadvantages of traditional fabrication methods. These technologies use computers to assist in design and fabrication, so the 3D scaffolds can be fabricated as designed and standardized. Particularly, because nanofabrication technology based on two-photon absorption (2PA) and on controlled electrospinning can generate structures with submicron resolution, these methods have been evaluated in various areas of tissue engineering. Recent combinations of 3D nanoprinting technologies with methods from molecular biology and cell dynamics have suggested new possibilities for improved tissue regeneration. If the interaction between cells and scaffold system with biomolecules can be understood and controlled and if an optimal 3D environment for tissue regeneration can be realized, 3D nanoprinting will become an important tool in tissue engineering

Defining cellular microenvironments using multiphoton lithography

textTo understand the chemistry of life processes in detail is largely a challenge of resolving them in their native, cellular environment. Cell culture, first developed a century ago, has proven to be an essential tool for reductionist studies of cellular biochemistry and development. However, for the technology of cell culture to move forward and address increasingly complex problems, in vitro environments must be refined to better reflect the cellular environment in vivo. This dissertation work has focused on the development of methods to define cellular microenvironments using the high resolution, 3D capabilities of multiphoton lithography. Here, site-specific photochemistry using multiphoton excitation is applied to the photocrosslinking of proteins, providing the means to organize bioactive species into well-defined 3D microenvironments. Further, conditions have been identified that enable microfabrication to be performed in the presence of cells -- allowing cell outgrowth and motility to be directed in real time. In addition to the intrinsic chemical functionality of microfabricated protein structures, 3D protein matrices are shown to respond mechanically to changes in the chemical environment, enabling new avenues for micro-scale actuation to be explored. Complex 2D and 3D protein photocrosslinking is further facilitated by integrating transparency and automated reflectance photomasks into the fabrication system. These advances could be transformative in efforts to fabricate precise cellular scaffolding that replicates the morphological (and potentially biochemical) features of in vivo tissue microenvironments. Finally, these methods are applied to the study of microorganism behavior with single-cell resolution. Microarchitectures are designed that allow the position and motion of motile bacterial to generate directional microfluidic flow -- providing a foundation to develop micro-scale devices powered by cells.Chemistry and BiochemistryBiochemistr

Two-photon sensitive biomaterials for dynamic control of cellular microenvironments

Two-photon (2P) activable photocleavable protecting groups (PPGs) can be introduced in polymer networks as photodegradation sites or as blocking groups for active sites, which enable the alternation of mechanical properties and biochemical signals and allow to study consequent cell response in a spatiotemporal controlled manner. So far, the design of high efficient 2P activable hydrogels is challenging. This Thesis presents novel designs of photodegradable hydrogels that contain the 4’-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl (PMNB) PPG. PMNB-gels formed under physiological conditions and showed tuneable hydrolytic stability and adequate rate for cell encapsulation. Moreover, PMNB-gels can be photodegraded efficiently upon 2P excitation (λ = 740 nm). Preliminary experiments of PMNB-gels as 4D matrices for the investigation of cell response are presented. In a second part, a 2P-activatable PPGs endowed with an extended π conjugation was demonstrated and introduced to yield the RGD cell adhesive peptide. The targeted peptide is obtained but only in low yield due to its low stability. The results of this Thesis provide new tools for instructing cells in 3D cultures using 2P-activated processes and demonstrate the potential of photochemistry for the realization of 4D biomaterials.Zwei-Photonen-(2P)-aktivierbare photolytisch spaltbare Schutzgruppen (PPGs) können in Polymernetzwerke als photokysestellen oder als Schutzgruppen für aktive Stellen eingeführt werden, das Alternieren von mechanischen Eigenschaften und biochemischen Signalen ermöglichen und es erlauben, die daraus resultierende Zellreaktion in einer räumlich-zeitlich kontrollierten Weise zu untersuchen. Bisher ist das Design von hocheffizienten 2P-aktivierbaren Hydrogelen eine Herausforderung. In dieser Arbeit werden neuartige Designs von photodegradierbaren Hydrogelen vorgestellt, die 4'-Methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl (PMNB) PPG enthalten. PMNB-Gele bildeten sich unter physiologischen Bedingungen und zeigten eine einstellbare hydrolytische Stabilität und eine angemessene Geschwindigkeit für die Immobilisierung von Zellen. Darüber hinaus können PMNB-Gele bei 2P-Anregung (λ = 740 nm) effizient photolytisch abgebaut werden. Es werden erste Experimente mit PMNB-Gelen als 4D-Matrizen für die Untersuchung der Zellreaktion vorgestellt. In einem zweiten Teil wurde ein eine 2P-aktivierbares PPGs mit einer verlängerten π-Konjugation demonstriert und eingeführt, um das zelladhäsive RGD-Peptid zu erhalten. Das angestrebte Peptid wurde gewonnen, allerdings aufgrund seiner geringen Stabilität nur in geringer Ausbeute. Die Ergebnisse dieser Arbeit liefern neue Werkzeuge für die Steuerung von Zellen in 3D-Kulturen mit Hilfe von 2P-aktivierbaren Prozessen und zeigen das Potenzial der Photochemie für die Realisierung von 4D-Biomaterialien

Physical Aspects of Cell Culture Substrates: Topography, Roughness, and Elasticity

The cellular environment impacts a myriad of cellular functions by providing signals that can modulate cell phenotype and function. Physical cues such as topography, roughness, gradients, and elasticity are of particular importance. Thus, synthetic substrates can be potentially useful tools for exploring the influence of the aforementioned physical properties on cellular function. Many micro‐ and nanofabrication processes have been employed to control substrate characteristics in both 2D and 3D environments. This review highlights strategies for modulating the physical properties of surfaces, the influence of these changes on cell responses, and the promise and limitations of these surfaces in in‐vitro settings. While both hard and soft materials are discussed, emphasis is placed on soft substrates. Moreover, methods for creating synthetic substrates for cell studies, substrate properties, and impact of substrate properties on cell behavior are the main focus of this review. The cellular environment plays a significant role in cell phenotype and function. As such, physical properties of cell culture substrates including topography, roughness, and elasticity may be utilized to investigate the influence of these physical cues on the cellular response. In this review, strategies for modulating the physical properties of surfaces, the influence of these changes on cell responses, and the promise and limitations of these surfaces in in‐vitro settings are highlighted, with a particular emphasis on elastic substrates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90132/1/336_ftp.pd

4D Microprinting Based on Liquid Crystalline Elastomers

Two-photon laser printing (2PLP) is a disruptive three-dimensional (3D) printing technique that can afford structural fabrication at the submicrometer scale. Apart from constructing static 3D structures, research in fabricating dynamic ones, known as "4D printing”, is becoming a burgeoning field. 4D printed structures exhibit adaptability or tunability towards their environment through the control of an external stimulus. In contrast to the rapid growth in macroscale fabrication, progress in microprinted actuators has only been scarcely reported. Liquid crystal elastomer (LCE) stands out among the promising classes of smart materials for fabricating microrobotics or microactuators due to its distinct anisotropic property, which enables the printed structures to exhibit automated reversible movements upon exposure to stimuli without environmental limitations. Nevertheless, the use of 2PLP for the manufacture of 4D printed LCE microstructures with high versatility and complexity have presented some challenges, limiting their implementation in final applications. This thesis aims to overcome two main obstacles faced in this regard: first, the limitation of two-photon printable stimuli-responsive materials; and second, the lack of a facile approach for aligning liquid crystal (LC) within three dimensions. The first part of this thesis aims on expanding the library of materials used for implementing light responsiveness into LC microstructures, as light provides a higher degree of temporal and spatial control compared to other stimuli. The initial approach has involved incorporation of acrylate-functionalized photoresponsive molecules, such as azobenzene and the donor-acceptor Stenhouse adduct (DASA), into a LC ink using a conventional synthetic method. However, several challenges, such as compatibility with the LC ink, have prevented the achievement of 4D printing via 2PLP. The second approach is based on post-modifying printed LC structures and successfully fabricated microactuators with five different photoresponsive features by individually incorporating each light-absorbing molecule. Furthermore, LC microactuators that exhibit distinct actuation patterns under different colors of light were fabricated by simultaneously implementing orthogonal photoresponsive molecules. The second project presented in this thesis focuses on developing a new strategy to induce alignment domains in a more flexible manner, with the aim of spatially tailoring the LC topology of the 3D printed microstructures. This is achieved by microprinting 3D scaffolds based on polydimethylsiloxane (PDMS) to manipulate the alignment directions of LC molecules. Taking advantage of 2PLP to fabricate arbitrary scaffolds, LC alignments, including planar and radial patterns, could be introduced freely and simultaneously in three-dimensional space with varying degrees of complexity. The applicability of this alignment approach was demonstrated by fabricating responsive LC microstructures within different PDMS environments, and distinct actuation patterns were observed. Overall, these two breakthroughs have unveiled a wide array of new potentials for the utilization of responsive LC microsystems with tunable functionalities and customizable actuation responses, that can be applied across various domains and applications

Three-Dimensional Biomimetic Patterning to Guide Cellular Migration and Organization

This thesis develops a novel photopatterning strategy for biomimetic scaffolds that enables spatial and biochemical control of engineered cellular architectures, such as the microvasculature. Intricate tools that allow for the three dimensional (3D) manipulation of biomaterial microenvironments will be critical for organizing cellular behavior, directing tissue formation, and ultimately, developing functional therapeutics to treat patients with critical organ failure. Poly(ethylene glycol) (PEG) based hydrogels, which without modification naturally resist protein adsorption and cellular adhesion, were utilized in combination with a two-photon laser patterning approach to covalently immobilize specific biomolecules in custom-designed, three-dimensional (3D) micropatterns. This technique, known as two-photon laser scanning lithography (TP-LSL), was shown in this thesis to possess the capability to micropattern multiple different biomolecules at modular concentrations into a single hydrogel microenvironment over a broad range of size scales with high 3D resolution. 3D cellular adhesion and migration were then explored in detail using time-lapse confocal microscopy to follow cells as they migrated along micropatterned tracks of various 3D size and composition. Further, in a valuable modification of TP-LSL, images from the endogenous microenvironment were converted into instructions to precisely direct the laser patterning of biomolecules within PEG-based hydrogels. 3D images of endogenous microvasculature from various tissues were directly converted into 3D biomolecule patterns within the hydrogel scaffold with precise pattern fidelity. While tissue engineers have previously demonstrated the formation of vessels through the encapsulation of endothelial cells and pericyte precursor cells within PEG-based hydrogels, the vessel structure had been random, uncoordinated, and therefore, ultimately non-functional. This thesis has utilized image guided TP-LSL to pattern biomolecules into a 3D structure that directs the organization of vessels to mimic that of the endogenous tissue vasculature. TP-LSL now stands as a valuable tool to control the microstructure of engineered cellular architectures, thereby providing a critical step in the development of cellularized scaffolds into functional tissues. Ultimately, this thesis develops new technologies that advance the field of regenerative medicine towards the goal of engineering viable organs to therapeutically treat the 18 patients who die every day waiting on the organ transplant list