Factors Affecting Dimensional Accuracy of 3-D Printed Anatomical Structures Derived from CT Data.

Additive manufacturing and bio-printing, with the potential for direct fabrication of complex patient-specific anatomies derived from medical scan data, are having an ever-increasing impact on the practice of medicine. Anatomic structures are typically derived from CT or MRI scans, and there are multiple steps in the model derivation process that influence the geometric accuracy of the printed constructs. In this work, we compare the dimensional accuracy of 3-D printed constructs of an L1 vertebra derived from CT data for an ex vivo cadaver T-L spine with the original vertebra. Processing of segmented structures using binary median filters and various surface extraction algorithms is evaluated for the effect on model dimensions. We investigate the effects of changing CT reconstruction kernels by scanning simple geometric objects and measuring the impact on the derived model dimensions. We also investigate if there are significant differences between physical and virtual model measurements. The 3-D models were printed using a commercial 3-D printer, the Replicator 2 (MakerBot, Brooklyn, NY) using polylactic acid (PLA) filament. We found that changing parameters during the scan reconstruction, segmentation, filtering, and surface extraction steps will have an effect on the dimensions of the final model. These effects need to be quantified for specific situations that rely on the accuracy of 3-D printed models used in medicine or tissue engineering applications.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in digital light projection(DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks and model cells. A multiphase many-body dissipative particle dynamics model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4-3 kPa) with a typical layer thickness of 50µm, an XY resolution of 38 ± 1.5µm and Z resolution of 237 ± 5.4µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel networks lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Tuning the Poisson's Ratio of Biomaterials for Investigating Cellular Response.

Cells sense and respond to mechanical forces, regardless of whether the source is from a normal tissue matrix, an adjacent cell or a synthetic substrate. In recent years, cell response to surface rigidity has been extensively studied by modulating the elastic modulus of poly(ethylene glycol) (PEG)-based hydrogels. In the context of biomaterials, Poisson's ratio, another fundamental material property parameter has not been explored, primarily because of challenges involved in tuning the Poisson's ratio in biological scaffolds. Two-photon polymerization is used to fabricate suspended web structures that exhibit positive and negative Poisson's ratio (NPR), based on analytical models. NPR webs demonstrate biaxial expansion/compression behavior, as one or multiple cells apply local forces and move the structures. Unusual cell division on NPR structures is also demonstrated. This methodology can be used to tune the Poisson's ratio of several photocurable biomaterials and could have potential implications in the field of mechanobiology.

Digital microfabrication of user-defined 3D microstructures in cell-laden hydrogels.

Complex 3D interfacial arrangements of cells are found in several in vivo biosystems such as blood vasculature, renal glomeruli, and intestinal villi. Current tissue engineering techniques fail to develop suitable 3D microenvironments to evaluate the concurrent effects of complex topography and cell encapsulation. There is a need to develop new fabrication approaches that control cell density and distribution within complex 3D features. In this work, we present a dynamic projection printing process that allows rapid construction of complex 3D structures using custom-defined computer-aided-design (CAD) files. Gelatin-methacrylate (GelMA) constructs featuring user-defined spiral, pyramid, flower, and dome micro-geometries were fabricated with and without encapsulated cells. Encapsulated cells demonstrate good cell viability across all geometries both on the scaffold surface and internal to the structures. Cells respond to geometric cues individually as well as collectively throughout the larger-scale patterns. Time-lapse observations also reveal the dynamic nature of mechanical interactions between cells and micro-geometry. When compared to conventional cell-seeding, cell encapsulation within complex 3D patterned scaffolds provides long-term control over proliferation, cell morphology, and geometric guidance. Overall, this biofabrication technique offers a flexible platform to evaluate cell interactions with complex 3D micro-features, with the ability to scale-up towards high-throughput screening platforms.

Printing Parameters of Fused Filament Fabrication Affect Key Properties of Four-Dimensional Printed Shape-Memory Polymers.

Extrusion-based (fused filament fabrication) three-dimensional (3D) printing of shape-memory polymers (SMPs) has the potential to rapidly produce highly customized smart-material parts. Yet, the effects of printing parameters on the shape-memory properties of printed SMPs remain poorly understood. To study the extent to which the 3D printing process affects the shape-memory properties of a printed SMP part, here temperature, extrusion rate multiplier, and fiber orientation were systematically varied, and their effect on shape-memory fixing and recovery ratios was evaluated. Fiber orientation, as determined by print path relative to the direction(s) of loading during shape-memory programming, was found to significantly impact the fixing ratio and the recovery ratio. Temperature and multiplier had little effect on either fixing ratio or recovery ratio. To facilitate the use of printed SMP parts in biomedical applications, a cell viability assay was performed on 3D-printed samples prepared using varied temperature and multiplier. Reduction in multiplier was found to increase cell viability. The results indicate that fiber orientation can critically impact the shape-memory functionality of 3D-printed SMP parts, and that multiplier can affect cytocompatibility of those parts. Thus, researchers and manufacturers employing SMPs in 3D-printed parts and devices could achieve improved part functionality if print paths are designed to align fiber direction with the axis(es) in which strain will be programmed and recovered and if the multiplier is optimized in biomedical applications in which a part will contact cells.

Printing Double-Network Tough Hydrogels Using Temperature-Controlled Projection Stereolithography (TOPS).

We report a new method to shape double-network (DN) hydrogels into customized 3D structures that exhibit superior mechanical properties in both tension and compression. A one-pot prepolymer formulation containing photo-cross-linkable acrylamide and thermoreversible sol-gel κ-carrageenan with a suitable cross-linker and photoinitiators/absorbers is optimized. A new TOPS system is utilized to photopolymerize the primary acrylamide network into a 3D structure above the sol-gel transition of κ-carrageenan (80 °C), while cooling down generates the secondary physical κ-carrageenan network to realize tough DN hydrogel structures. 3D structures, printed with high lateral (37 µm) and vertical (180 µm) resolutions and superior 3D design freedoms (internal voids), exhibit ultimate stress and strain of 200 kPa and 2400%, respectively, under tension and simultaneously exhibit a high compression stress of 15 MPa with a strain of 95%, both with high recovery rates. The roles of swelling, necking, self-healing, cyclic loading, dehydration, and rehydration on the mechanical properties of printed structures are also investigated. To demonstrate the potential of this technology to make mechanically reconfigurable flexible devices, we print an axicon lens and show that a Bessel beam can be dynamically tuned via user-defined tensile stretching of the device. This technique can be broadly applied to other hydrogels to make novel smart multifunctional devices for a range of applications.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in Digital Light Processing (DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks. A multiphase many-body dissipative particle dynamics (mDPD) model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4 to 3 kPa) with an XY resolution of 38 ± 1.5 µm and Z resolution of 237±5.4 µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel network lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

A Three-dimensional Polymer Scaffolding Material Exhibiting a Zero Poisson's Ratio.

Poisson's ratio describes the degree to which a material contracts (expands) transversally when axially strained. A material with a zero Poisson's ratio does not transversally deform in response to an axial strain (stretching). In tissue engineering applications, scaffolding having a zero Poisson's ratio (ZPR) may be more suitable for emulating the behavior of native tissues and accommodating and transmitting forces to the host tissue site during wound healing (or tissue regrowth). For example, scaffolding with a zero Poisson's ratio may be beneficial in the engineering of cartilage, ligament, corneal, and brain tissues, which are known to possess Poisson's ratios of nearly zero. Here, we report a 3D biomaterial constructed from polyethylene glycol (PEG) exhibiting in-plane Poisson's ratios of zero for large values of axial strain. We use digital micro-mirror device projection printing (DMD-PP) to create single- and double-layer scaffolds composed of semi re-entrant pores whose arrangement and deformation mechanisms contribute the zero Poisson's ratio. Strain experiments prove the zero Poisson's behavior of the scaffolds and that the addition of layers does not change the Poisson's ratio. Human mesenchymal stem cells (hMSCs) cultured on biomaterials with zero Poisson's ratio demonstrate the feasibility of utilizing these novel materials for biological applications which require little to no transverse deformations resulting from axial strains. Techniques used in this work allow Poisson's ratio to be both scale-independent and independent of the choice of strut material for strains in the elastic regime, and therefore ZPR behavior can be imparted to a variety of photocurable biomaterial.

Three-Dimensional Printing of Double-Network Hydrogels: Recent Progress, Challenges, and Future Outlook.

Hydrogels are widely used materials due to their biocompatibility, their ability to mimic the hydrated and porous extracellular microenvironment, as well as their ability to tune both mechanical and biochemical properties. However, most hydrogels lack mechanical toughness, and shaping them into complicated three-dimensional (3D) structures remains challenging. In the past decade, tough and stretchable double-network hydrogels (DN gels) were developed for tissue engineering, soft robotics, and applications that require a combination of high-energy dissipation and large deformations. Although DN gels were processed into simple shapes by using conventional casting and molding methods, new 3D printing methods have enabled the shaping of DN gels into structurally complex 3D geometries. This review will describe the state-of-art technologies for shaping tough and stretchable DN gels into custom geometries by using conventional molding and casting, extrusion, and optics-based 3D printing, as well as the key challenges and future outlook in this field.

Smart biomaterial platforms: Controlling and being controlled by cells.

Across diverse research and application areas, dynamic functionality-such as programmable changes in biochemical property, in mechanical property, or in microscopic or macroscopic architecture-is an increasingly common biomaterials design criterion, joining long-studied criteria such as cytocompatibility and biocompatibility, drug release kinetics, and controlled degradability or long-term stability in vivo. Despite tremendous effort, achieving dynamic functionality while simultaneously maintaining other desired design criteria remains a significant challenge. Reversible dynamic functionality, rather than one-time or one-way dynamic functionality, is of particular interest but has proven especially challenging. Such reversible functionality could enable studies that address the current gap between the dynamic nature of in vivo biological and biomechanical processes, such as cell traction, cell-extracellular matrix (ECM) interactions, and cell-mediated ECM remodeling, and the static nature of the substrates and ECM constructs used to study the processes. This review assesses dynamic materials that have traditionally been used to control cell activity and static biomaterial constructs, experimental and computational techniques, with features that may inform continued advances in reversible dynamic materials. Taken together, this review presents a perspective on combining the reversibility of smart materials and the in-depth dynamic cell behavior probed by static polymers to design smart bi-directional ECM platforms that can reversibly and repeatedly communicate with cells.

Femtosecond Laser Densification of Hydrogels to Generate Customized Volume Diffractive Gratings.

Inspired by nature's ability to shape soft biological materials to exhibit a range of optical functionalities, we report femtosecond (fs) laser-induced densification as a new method to generate volume or subsurface diffractive gratings within ordinary hydrogel materials. We characterize the processing range in terms of fs laser power, speed, and penetration depths for achieving densification within poly(ethylene glycol) diacrylate (PEGDA) hydrogel and characterize the associated change in local refractive index (RI). The RI change facilitates the fabrication of custom volume gratings (parallel line, grid, square, and ring gratings) within PEGDA. To demonstrate this method's broad applicability, fs laser densification was used to generate line gratings within the phenylboronic acid (PBA) hydrogel, which is known to be responsive to changes in pH. In the future, this technique can be used to convert ordinary hydrogels into multicomponent biophotonic systems.

In vitromodel to study confined osteocyte networks exposed to flow-induced mechanical stimuli.

Osteocytes are considered the primary mechanical sensor in bone tissue and orchestrate the coupled bone remodeling activity of adjacent osteoblast and osteoclast cells.In vivoinvestigation of mechanically induced signal propagation through networks of interconnected osteocytes is confounded by their confinement within the mineralized bone matrix, which cannot be modeled in conventional culture systems. In this study, we developed a new model that mimics thisin vivoconfinement using gelatin methacrylate (GelMA) hydrogel or GelMA mineralized using osteoblast-like model cells. This model also enables real-time optical examination of osteocyte calcium (Ca2+) signaling dynamics in response to fluid shear stimuli cultured under confined conditions. Using this system, we discovered several distinct and previously undescribed patterns of Ca2+responses that vary across networks of interconnected osteocytes as a function of space, time and connectivity. Heterogeneity in Ca2+signaling may provide new insights into bone remodeling in response to mechanical loading. Overall, such a model can be extended to study signaling dynamics within cell networks exposed to flow-induced mechanical stimuli under confined conditions.

Three-Dimensional Polymer Constructs Exhibiting a Tunable Negative Poisson's Ratio.

Young's modulus and Poisson's ratio of a porous polymeric construct (scaffold) quantitatively describe how it supports and transmits external stresses to its surroundings. While Young's modulus is always non-negative and highly tunable in magnitude, Poisson's ratio can, indeed, take on negative values despite the fact that it is non-negative for virtually every naturally occurring and artificial material. In some applications, a construct having a tunable negative Poisson's ratio (an auxetic construct) may be more suitable for supporting the external forces imposed upon it by its environment. Here, three-dimensional polyethylene glycol scaffolds with tunable negative Poisson's ratios are fabricated. Digital micromirror device projection printing (DMD-PP) is used to print single-layer constructs composed of cellular structures (pores) with special geometries, arrangements, and deformation mechanisms. The presence of the unit-cellular structures tunes the magnitude and polarity (positive or negative) of Poisson's ratio. Multilayer constructs are fabricated with DMD-PP by stacking the single-layer constructs with alternating layers of vertical connecting posts. The Poisson's ratios of the single- and multilayer constructs are determined from strain experiments, which show (1) that the Poisson's ratios of the constructs are accurately predicted by analytical deformation models and (2) that no slipping occurrs between layers in the multilayer constructs and the addition of new layers does not affect Poisson's ratio.

Effects of protein solution composition on the time-dependent functional activity of fibrinogen on surfaces.

Protein function affects subsequent biological processes such as cell adhesion and thrombus formation. We have developed tools to detect the biological activity of fibrinogen using AFM techniques. In this work, we measure the effects of solution concentration, residence time, and protein competition with BSA on the time-dependent functional changes in adsorbed fibrinogen on mica surface. AFM probes were functionalized with monoclonal antibodies recognizing fibrinogen gamma 392-411, which includes the platelet binding dodecapeptide region. Results show good correlation between changes in biological activity of adsorbed fibrinogen at the molecular scale measured by AFM and platelet adhesion measured at a macroscale. Furthermore, the results show that inclusion of BSA into the solution moves the peak biological activity of fibrinogen to earlier time points. These results illustrate a complex and dynamic biological interface and offer new opportunities for improved insights into the molecular basis for the biological response to biomaterials.

Growth of high-aspect ratio horizontally-aligned ZnO nanowire arrays.

A method of fabricating horizontally-aligned zinc-oxide (ZnO) nanowire (NW) arrays with full control over the width and length is demonstrated. SEM images reveal the hexagonal structure typical of zinc oxide NWs. Arrays of high-aspect ratio horizontal ZnO NWs are fabricated by making use of the lateral overgrowth from dot patterns created by electron beam lithography (EBL). An array of patterned wires are lifted off and transferred to a flexible PDMS substrate with possible applications in several key nanotechnology areas.

Hydrogel-based diffractive optical elements (hDOEs) using rapid digital photopatterning.

Hydrogels, due to their optical transparency and biocompatibility, have emerged as an excellent alternative to conventional optical materials for biomedical applications. Advances in microfabrication techniques have helped convert conventional hydrogels into optically functional materials such as hydrogel-based diffraction optical elements (hDOEs). However, key challenges related to device customization and ease/speed of fabrication need to be addressed to enable widespread utility and acceptance of hDOEs in the field. Here, we report rapid printing of customized hDOEs on polyethylene glycol diacrylate (PEGDA) hydrogel using digital photopatterning; a novel method that combines simulated computer-generated hologram (SCGH) and projection photolithography. To showcase the versatility of this approach, a range of hDOEs are demonstrated, including 1D/2D diffraction gratings, Dammann grating, Fresnel zone plate (FZP) lens, fork-shaped grating and computer-generated hologram (CGH) of arbitrary pattern. Results demonstrate that printed hDOEs exhibit optical performance that is comparable with devices made with conventional materials. This versatile strategy can be potentially implemented with other photosensitive hydrogels to achieve user-defined hDOEs in a time-efficient and cost-effective fashion.

Direct Laser Writing of Fluorescent Silver Nanoclusters: A Review of Methods and Applications.

Metal nanoclusters (NCs) are nanomaterials of size of less than 2 nm that exhibit a set of unique physical, chemical, optical, and electronic properties. Because of recent interest in NCs, a great deal of effort is being made to develop synthetic routes that allow control over the NC size, shape, geometry, and properties. Direct laser writing is one of the few synthesis methods that allow the generation of photostable NCs with high quantum yield in a highly controlled fashion. A key advantage of laser-written NCs is the ability to create easy-to-use solid-state devices for a range of applications. This review will present necessary background and recent advances in laser writing of silver NCs and their applications in different solid-state matrixes such as glass, zeolites, and polymer substrate. This topic will be of interest to researchers in the fields of materials science, optics and photonics, chemistry, and biomedical sciences.

Perfusion-based co-culture model system for bone tissue engineering.

In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.