Engineering Polymeric Scaffolds for Studying Neural Tissue Development, Pathology, and Repair

Digital light processing (DLP) 3D printing involves projecting a near-UV or visible light via an LED light source onto a digital micromirror device (DMD), which is the same device that is used in light projectors. I used this light-based bioprinting technique throughout my PhD work to develop hydrogel scaffolds to address pressing questions and challenges in in vitro neural tissue engineering and in vivo regenerative medicine applications.In Chapter 1, an overview of the development of biomaterials suited for light-based 3D-printing modalities with an emphasis on bioprinting applications is presented. The chemical mechanisms that govern photopolymerization are discussed, and the application of natural, synthetic, and composite biomaterials as 3D-printed hydrogels are highlighted. Since the quality of a 3D-printed construct is highly dependent on both the material properties and processing technique, the theoretical and practical aspects governing light-based 3D printing are also discussed. In Chapter 2, the development of a 3D printed in vitro optic nerve formation model is described. Hereditary and age-related optic nerve diseases as well as injuries can lead to vision loss and impairment which greatly reduces a person’s quality of life. In development, the optic nerve is derived from axonal projections of retinal ganglion cells in the retina. There is an unmet need in ophthalmology for a complete visual circuit in vitro model to study formation, neurodegeneration, and function of the optic nerve. We have taken the first step to create the visual circuit in vitro by developing a 3D-printed microgroove hydrogel platform for guiding axonal outgrowth from iPSC-derived retinal ganglion cell (RGC) spheroids. In Chapter 3, the development of a 3D-printed conduit for functional recovery after a peripheral nerve injury is described. Many studies have explored different materials and active cues to guide neural regeneration, with some success. However, none have demonstrated a comparable or better functional recovery than the gold standard autograft. Autografts remain insufficient for full recovery from a large injury to the sciatic nerve or brachial plexus nerves. We have designed a 3D-printed hydrogel multi-microchannel conduits with and without orthogonal micropores to guide axonal outgrowth and external vascular integration, respectively, that perform as well as the gold standard autograft therapy

High-fidelity 3D Printing using Flashing Photopolymerization.

Photopolymerization-based 3D printing has emerged as a promising technique to fabricate 3D structures. However, during the printing process, polymerized materials such as hydrogels often become highly light-scattering, thus perturbing incident light distribution and thereby deteriorating the final print resolution. To overcome this scattering-induced resolution deterioration, we developed a novel method termed flashing photopolymerization (FPP). Our FPP approach is informed by the fundamental kinetics of photopolymerization reactions, where light exposure is delivered in millisecond-scale flashes, as opposed to continuous light exposure. During the period of flash exposure, the prepolymer material negligibly scatters light. The material then polymerizes and opacifies in absence of light, therefore the exposure pattern is not perturbed by scattering. Compared to the conventional use of a continuous wave (CW) light source, the FPP fabrication resolution is improved. FPP also shows little dependency on the exposure, thus minimizing trial-and-error type optimization. Using FPP, we demonstrate its use in generating high-fidelity 3D printed constructs

X-Ray Characterization of Mesophases of Human Telomeric G-Quadruplexes and Other DNA Analogues

Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5′-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues

Compensating the cell-induced light scattering effect in light-based bioprinting using deep learning

Digital light processing (DLP)-based three-dimensional (3D) printing technology has the advantages of speed and precision comparing with other 3D printing technologies like extrusion-based 3D printing. Therefore, it is a promising biomaterial fabrication technique for tissue engineering and regenerative medicine. When printing cell-laden biomaterials, one challenge of DLP-based bioprinting is the light scattering effect of the cells in the bioink, and therefore induce unpredictable effects on the photopolymerization process. In consequence, the DLP-based bioprinting requires extra trial-and-error efforts for parameters optimization for each specific printable structure to compensate the scattering effects induced by cells, which is often difficult and time-consuming for a machine operator. Such trial-and-error style optimization for each different structure is also very wasteful for those expensive biomaterials and cell lines. Here, we use machine learning to learn from a few trial sample printings and automatically provide printer the optimal parameters to compensate the cell-induced scattering effects. We employ a deep learning method with a learning-based data augmentation which only requires a small amount of training data. After learning from the data, the algorithm can automatically generate the printer parameters to compensate the scattering effects. Our method shows strong improvement in the intra-layer printing resolution for bioprinting, which can be further extended to solve the light scattering problems in multilayer 3D bioprinting processes

3D Printing of a Biocompatible Double Network Elastomer with Digital Control of Mechanical Properties

The majority of 3D-printed biodegradable biomaterials are brittle, limiting their potential application to compliant tissues. Poly (glycerol sebacate) acrylate (PGSA) is a synthetic biodegradable and biocompatible elastomer, compatible with light-based 3D printing. In this work we employed digital-light-processing (DLP)-based 3D printing to create a complex PGSA network structure. Nature-inspired double network (DN) structures with two geometrically interconnected segments with different mechanical properties were printed from the same material in a single shot. Such capability has not been demonstrated by any other fabrication technique. The biocompatibility of PGSA after 3D printing was confirmed via cell-viability analysis. We used a finite element analysis (FEA) model to predict the failure of the DN structure under uniaxial tension. FEA confirmed the soft segments act as sacrificial elements while the hard segments retain structural integrity. The simulation demonstrated that the DN design absorbs 100% more energy before rupture than the network structure made by single exposure condition (SN), doubling the toughness of the overall structure. Using the FEA-informed design, a new DN structure was printed and the FEA predicted tensile test results agreed with tensile testing of the printed structure. This work demonstrated how geometrically-optimized material design can be easily and rapidly achieved by using DLP-based 3D printing, where well-defined patterns of different stiffnesses can be simultaneously formed using the same elastic biomaterial, and overall mechanical properties can be specifically optimized for different biomedical applications

3D printing of a biocompatible double network elastomer with digital control of mechanical properties.

The majority of 3D-printed biodegradable biomaterials are brittle, limiting their potential application to compliant tissues. Poly (glycerol sebacate) acrylate (PGSA) is a synthetic biodegradable and biocompatible elastomer, compatible with light-based 3D printing. In this work we employed digital-light-processing (DLP)-based 3D printing to create a complex PGSA network structure. Nature-inspired double network (DN) structures with two geometrically interconnected segments with different mechanical properties were printed from the same material in a single shot. Such capability has not been demonstrated by any other fabrication technique. The biocompatibility of PGSA after 3D printing was confirmed via cell-viability analysis. We used a finite element analysis (FEA) model to predict the failure of the DN structure under uniaxial tension. FEA confirmed the soft segments act as sacrificial elements while the hard segments retain structural integrity. The simulation demonstrated that the DN design absorbs 100% more energy before rupture than the network structure made by single exposure condition (SN), doubling the toughness of the overall structure. Using the FEA-informed design, a new DN structure was printed and the FEA predicted tensile test results agreed with tensile testing of the printed structure. This work demonstrated how geometrically-optimized material design can be easily and rapidly achieved by using DLP-based 3D printing, where well-defined patterns of different stiffnesses can be simultaneously formed using the same elastic biomaterial, and overall mechanical properties can be specifically optimized for different biomedical applications

Photopolymerizable Biomaterials and Light-Based 3D Printing Strategies for Biomedical Applications.

Since the advent of additive manufacturing, known commonly as 3D printing, this technology has revolutionized the biofabrication landscape and driven numerous pivotal advancements in tissue engineering and regenerative medicine. Many 3D printing methods were developed in short course after Charles Hull first introduced the power of stereolithography to the world. However, materials development was not met with the same enthusiasm and remained the bottleneck in the field for some time. Only in the past decade has there been deliberate development to expand the materials toolbox for 3D printing applications to meet the true potential of 3D printing technologies. Herein, we review the development of biomaterials suited for light-based 3D printing modalities with an emphasis on bioprinting applications. We discuss the chemical mechanisms that govern photopolymerization and highlight the application of natural, synthetic, and composite biomaterials as 3D printed hydrogels. Because the quality of a 3D printed construct is highly dependent on both the material properties and processing technique, we included a final section on the theoretical and practical aspects behind light-based 3D printing as well as ways to employ that knowledge to troubleshoot and standardize the optimization of printing parameters

A sequential 3D bioprinting and orthogonal bioconjugation approach for precision tissue engineering.

Recent advances in 3D bioprinting have transformed the tissue engineering landscape by enabling the controlled placement of cells, biomaterials, and bioactive agents for the biofabrication of living tissues and organs. However, the application of 3D bioprinting is limited by the availability of cytocompatible and printable biomaterials that recapitulate properties of native tissues. Here, we developed an integrated 3D projection bioprinting and orthogonal photoconjugation platform for precision tissue engineering of tailored microenvironments. By using a photoreactive thiol-ene gelatin bioink, soft hydrogels can be bioprinted into complex geometries and photopatterned with bioactive moieties in a rapid and scalable manner via digital light projection (DLP) technology. This enables localized modulation of biophysical properties such as stiffness and microarchitecture as well as precise control over spatial distribution and concentration of immobilized functional groups. As such, well-defined properties can be directly incorporated using a single platform to produce desired tissue-specific functions within bioprinted constructs. We demonstrated high viability of encapsulated endothelial cells and human cardiomyocytes using our dual process and fabricated tissue constructs functionalized with VEGF peptide mimics to induce guided endothelial cell growth for programmable vascularization. This work represents a pivotal step in engineering multifunctional constructs with unprecedented control, precision, and versatility for the rational design of biomimetic tissues

Light-based vat-polymerization bioprinting

Light-based vat-polymerization bioprinting enables computer-aided patterning of 3D cell-laden structures in a point-by-point, layer-by-layer or volumetric manner, using vat (vats) filled with photoactivatable bioresin (bioresins). This collection of technologies — divided by their modes of operation into stereolithography, digital light processing and volumetric additive manufacturing — has been extensively developed over the past few decades, leading to broad applications in biomedicine. In this Primer, we illustrate the methodology of light-based vat-polymerization 3D bioprinting from the perspectives of hardware, software and bioresin selections. We follow with discussions on methodological variations of these technologies, including their latest advancements, as well as elaborating on key assessments utilized towards ensuring qualities of the bioprinting procedures and products. We conclude by providing insights into future directions of light-based vat-polymerization methods.ISSN:2662-844