Symposium S.SM09—Advances in 3D Printing for Medical Applications

Viscoelastic complex fluids exhibit rheological nonlinearity at a high shear rate. Although typical nonlinear effects, shear thinning and shear thickening, have been usually understood by variation of intrinsic quantities such as viscosity, one still requires a better understanding of the microscopic origins, currently under debate, especially on the shear-thickening mechanism. We present accurate measurements of shear stress in the bound hydration water layer using noncontact dynamic force microscopy. We find shear thickening occurs above 10^6 1/s shear rate beyond 0.3-nm layer thickness, which is attributed to the nonviscous, elasticity associated fluidic instability via fluctuation correlation. Such a nonlinear fluidic transition is observed due to the long relaxation time (1 us) of water available in the nanoconfined hydration layer, which indicates the onset of elastic turbulence at nanoscale, elucidating the interplay between relaxation and shear motion, which also indicates the onset of elastic turbulence at nanoscale above a universal shear velocity of 1 mm/s. This extensive layer-by-layer control paves the way for fundamental studies of nonlinear nanorheology and nanoscale hydrodynamics, as well as provides novel insights on viscoelastic dynamics of interfacial water. We will also discuss the tip-based microfluidic characterization and manipulation of liquids.

As additive manufacturing (AM, also known as 3D printing) technologies have proliferated, approaches based on solidification of photosensitive liquid resins have showed great promise due to their superior resolution and precision. However, these techniques have been largely limited to prototyping applications due to the constraints on available materials and their mechanical properties, as well as slow builds and poor surface quality resulting from layer-wise fabrication. With the advent of volumetric AM (VAM), complete 3D structures with complex geometries can now be produced in a single step, leaping over the traditional limitations of layer-by-layer approaches to 3D printing.
Volumetric 3D printing generates a 3D distribution of absorbed optical energy within a volume of photosensitive material, concurrently curing all points within a target geometry, on a timescale of 10s to 100s of seconds. No substrate is required as the part forms unsupported in the resin container. The most versatile implementation of VAM is known as computed axial lithography (CAL) which adapts the principles of computed tomography (CT) to generate a sequence of intensity-modulated projections, which are then beamed sequentially into a rotating resin container via a DLP projector to create the required energy dose. Because there is no fluid motion, and no hydrodynamic forces during a build, this approach is particularly compelling for very soft materials such as the hydrogels widely used in tissue engineering and regenerative medicine applications. The absence of support structure also enables complex geometries such as vasculature to be built.
Successful volumetric 3D printing requires spatio-temporal control over the polymerization reaction at all points within the fabrication volume. This demands a more detailed understanding of a host of process parameters, compared with traditional layered approaches. Perhaps the most important such parameters are the absorbed volumetric energy dose E_VOL, as well as the rate at which this dose is delivered. The progress of the photochemical reaction may then be characterized in terms of a variety of metrics, such as double-bond conversion, or the evolution of mechanical and optical properties of the material. We investigate the inter-relationship of these properties, obtained from ex-situ measurements such as real-time FTIR spectroscopy, photo-rheology, and mechanical testing, developing a quantitative framework for predicting volumetric structure formation. An additional resin characteristic critical for CAL printing is nonlinear threshold behavior. This allows resin that receives a sub-threshold dose to remain liquid, and derives from the interaction of the generated radicals and inhibitory species present in the resin. For some chemistries the inhibitory effect is provided by dissolved molecular oxygen, but for other formulations, deliberately including another inhibitor is necessary. We discuss the implications of these interacting factors for photo-resin design within the volumetric AM paradigm, as well as more broadly in photopolymer-based AM methods. We also discuss several material classes for VAM, including high-performance engineering materials, as well as cell-culture compatible hydrogels.

References

Kelly, B. E., Bhattacharya, I., Heidari, H., Shusteff, M., Spadaccini, C. M., Taylor, H. K., “Volumetric additive manufacturing via tomographic reconstruction” Science 2019, 363, 1075-1079.

Borosilicate glass is a type of glass material consisting SiO₂ and B₂O₃ as network formers and alkali oxide modifiers. Their excellent optical transparency, chemical resistance, low thermal expansivity and tunable chemical composition make them useful for a wide range of applications such as lab ware, medical implants, microelectromechanical systems (MEMS) , sensing and detection etc. Conventional glass fabrication techniques involve melting, casting and use of undesirable chemicals and these imply that there is little room for flexibility in shape and resolution. Recent surge in additive manufacturing efforts now show that ceramic glasses can now be fabricated via stereolithography. However this has not been shown possible for borosilicates due to their low softening temperatures.
Herein, we report a simple additive manufacturing protocol for fabricating 3D low softening temperature borosilicate glass. It entails first fabricating a liquid glass composite, printing on a layer by layer basis to form a 'green ensemble'. This ‘green ensemble’ must then be debound to yield a 'brown ensemble' and sintered to yield transparent and amorphous glass ensembles. The properties of the printed glass depend on thermal processing parameters (temperature, time and environment) and these can be readily tuned and optimized to yield 3D glass for a wide range of applications. The printed glass show good optical properties, show no devitrification and exhibit minimal surface roughness requiring no further polishing and finishing typically associated with other glass fabrication techniques.

Two photon polymerization is an additive manufacturing method that offers many advantages over conventional processes for scalable mass production of medical devices, including microneedles and other medical devices with small-scale features. First, the raw materials (e.g., inorganic-organic hybrid materials and acrylate-based polymers) used in two photon polymerization can be purchased at low cost. Second, two photon polymerization can be established in a conventional environment; no cleanroom facilities or other types of specialized facilities are required. Third, two photon polymerization is a straightforward approach for creating medical devices with complex and small-scale features. Two photon polymerization has been used to create hollow microneedles with a larger range of shapes and dimensions than conventional microneedle fabrication methods. We have prepared several types of hollow microneedle-based biosensors using microneedles that were fabricated using either two photon polymerization or digital micromirror device-based stereolithography. In these biosensors, the sensors (either colorimetric or electrochemical) are located within the bores of the microneedles or underneath the microneedles. Application-specific testing and functional testing of the microneedle-based biosensors will be considered.

Hydrogels have long been considered a significantly promising class of biomaterials. While the chemomechanical tunability of a multitude of hydrogels have been demonstrated in vitro, the ability to deliver these materials in a minimally invasive manner to avoid iatrogenic damages has yet to be fully explored. In the present work, we demonstrate a method of in-situ extrusion of hydrogels with compositions and chemomechanical properties that can be dynamically adjusted “on-the-fly” using a custom catheter that is utilized intravascularly. A custom UV-integrated, multi-lumen microcatheter (inner/outer lumen diameters: 0.6/1.28 mm) was assembled and a suite of low viscosity, shear thinning gelatin-based precursors were formulated for delivery. While the shear thinning property makes this precursor a suitable choice for injection through a catheter with extended length and small diameter, intensity modulation of UV to cross-link the precursor aids greatly in tuning the stiffness at the catheter tip. We show that by modulating the absolute and relative flow rates as well as the UV intensity (0~150 µW output from a 105µm core multimode fibre), we can extrude hydrogels with stiffnesses dynamically varying from <10 to 80 kPa. In addition, through the dual luminal architecture, it is also possible to sequentially switch or coaxially coextrude two types of precursor compositions. The concentrations and diffusivities of additives (as preliminarily demonstrated using food dyes) within the extruded scaffold can also be dynamically altered by 10³ and 10² orders respectively, in a single piece of extruded hydrogel fibre, therefore allowing applications a highly customizable drug depot to be deployed using this technique. To demonstrate the initial utility of this system, we successfully performed embolization of a saccular aneurysm model (diameter ~ 12 mm) and a capillary vasculature model (diameter <1 mm) using a pulsatile vascular flow phantom. The former required the extruded hydrogels to be relatively stiffer via increased photo-cross linking to fill the aneurysm while avoiding leakage into the parent vessel. In capillary embolization, the precursor was injected with lower crosslinking leading to increased viscosity to penetrate further into the vessels to fully embolize the structure. These findings yield direct application ideas in therapeutics such as vascular embolization in a variety of disease states, including cerebral aneurysms, arterio-venous malformations, vascularized tumors, and hemorrhagic vessels. The system can also be utilized for intravascular drug delivery and targeted cell therapies, which may require the physician to constantly adapt to the state of the patient. Further, such a dynamically tunable hydrogel delivery method may be able to serve as a supporting host matrix for functional sensing devices such as microelectrode arrays and quantum dot based chemical sensors.

Bioink extrusion-printing, while capable of creating macroscopically accurate anatomic models, fails to replicate the microstructure of the natural tissue due to resolution limitation of this bottom-up fabrication approach. We re-engineer the extrusion process to co-extrude a fiber device in line with the hydrogel bioink. This allows deviation from a pure bottom-up towards a combined approach, where a bottom-up extrusion process creates the macrostructure, while the fiber device in a top-down fashion subsequently promotes the growth of natural microstructure from within the volume of the print.

Fibers can embed a range of bio-functional and biosensing devices and systems. We aim to incorporate microfluidic, ultrasonic, and shape-memory-alloy (SMA) structures mimicking the functionalities of microvasculature, innervation, and musculature, respectively, into a single fiber-device. Those functional structures are created by a thermal draw of fibers from multimaterial preforms – a process familiar from an optical communication fiber fabrication. In this process, the macroscopic preform – a thick and short rod, cross-sectionally structured to meet the desired fiber functionality – is heated to become a viscous liquid and scaled by a thermal draw into a thin and long fiber, preserving the cross-sectional geometry of the preform. Then the fiber cores are patterned axially by a spatially coherent material selective capillary instability to form arrays of ultrasonic transducers contacted in parallel and microchannels with periodic outlets spanning the entire fiber length ^[1].

In the extrusion process, the fiber is coated with hydrogel, contains tissue cells, extracellular matrix material, nutrients and growth-factors, forming a scaffolded print-line that is raster-layed into a printed model cross section additively constructing the final model in a layer-by layer manner. Subsequently, the fiber-embedded microchannel structure is used as artificial vasculature supplying building material and nutrients into surrounding bioink to ignite the creation of natural vasculature and promote the proliferation of the bioink-contained tissue cells with their subsequent maturation into a natural-tissue-like structure. The ultrasonic transducers embedded in the fiber are used to sense the changes in the surrounding material density, monitoring cell-proliferation process, thus serving as artificial tactile nerves. SMA cores, contracting upon transduction of electric current, mimic the function of muscle yarns.

The proposed bioprinting approach will likely deliver a biosynthetic tissue with natural-like microstructure opening new routes in regenerative medicine, such as wound infilling and organ replacement. Moreover, it would allow creating lab-bench anatomical models of realistic tissue, in which bio-material delivery and biosensing is performed volumetrically with a microscale resolution, thus forming a novel stand-alone platform for investigation of micrometabolic processes. Such a platform would be a valuable asset to drug toxicity investigation, and real-time survivability monitoring of such artificial tissues and organs both in-vitro and in animal subjects.

[1] C. Faccini de Lima, L. van der Elst et al., “Towards Digital Manufacturing of Smart Multimaterial Fibers,” Nanoscale Res. Lett. 14, 209 1-6 (2019).

The use of drop-on-demand (DOD) bioprinting has attracted numerous attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach to facilitate important cell-cell and cell-matrix interactions. In this work, a HP d300e Digital Dispenser will be used for precise deposition of nano-liter droplets. Here, we propose the use of a general purpose polyvinylpyrrolidone (PVP)-based bio-inks for printing of nano-liter cell droplets. Different PVP-based bio-inks (0 – 3% w/v) are prepared and evaluated for their printability in term of droplet formation and droplet impact on substrate surface; the short-term and long-term viability of printed cells (primary human skin fibroblasts) are also evaluated. We will discuss the range of printable cell concentration and its corresponding droplet resolution, cell viability and proliferation over time post-printing. Furthermore, we also will demonstrate different DOD printing strategies to initiate the hydrogel cross-linking mechanism and achieve tunable material properties within the hydrogel constructs.

Since the discovery of chemotherapy in the beginning of the 20^thcentury, researchers around the world have been actively developing new and more effective chemotherapeutic agents to better treat cancer. Traditionally, chemotherapeutic agents work by interfering with cell division. However, by virtue of their mechanism of action, healthy normal cells can also be targeted and destroyed. As a result, while chemotherapy is an effective way of managing cancer, the resulting side effects limits its use. One approach currently taken to reduce these side effects is to deliver the chemotherapy drugs directly to the tumor via transarterial chemoembolization, or other similar procedures. While this has been effective in reducing systemic toxicity, more can be done to improve this. Ideally, a device that could sequester any unreacted chemotherapy agents could be installed "downstream" of the tumor prior to them entering systemic circulation. Such drug-capture materials have yet to be realized due to the difficulty in achieving materials that have the right surface chemistry and geometry for blood flow.

Working together with medical doctors, computational fluid dynamics experts, chemists, and materials scientists, we report the fabrication of DNA functionalized 3D printed porous materials that can be used to capture doxorubicin and cisplatin, two commonly used DNA-targeting chemotherapy agents. We discuss the concept behind the device, the use of 3D printed materials as an ideal substrate, and the chemistries considered in drug binding. To achieve scalability of these devices, we developed a method of attaching cheaply available genomic DNA to these materials, a departure from commonly used synthetic DNA. We characterize the surface of the structure and verify the binding of DNA to the surface via XPS, EDS and the use of chemical assays. The efficacy of these functionalized materials were demonstrated in PBS, where we observed a >70% reduction in doxorubicin concentration over a period of 20 minutes, highlighting the viability of this as a method of drug capture.

Additive manufacturing, popularly known as “3D printing”, includes an array of technologies such as fused deposition modelling (FDM), selective laser sintering (SLS) and inkjet printing. Compared with conventional manufacturing technologies, 3D printing has many distinctive advantages, including the construction of 3D structures with complex shapes and functionalities. Therefore, 3D printing technologies have seen increasing popularity in many areas, such as product design and development, industrial production, consumer goods, aerospace, and education. 3D printing has already made a tremendous impact in our society. The exciting TED Talk in 2013 by Tibbits of MIT ushered in a new era in additive manufacturing: 4D printing. With 4D printing, 3D printed static objects will change their shapes over time, i.e., 4D printing uses 3D printing technologies to produce shape-morphing objects. Such objects can meet the demanding requirements in particularly applications. The concept of 4D printing has been evolving and one popular definition of 4D printing is that the shape, property and functionality of a 3D printed object can evolve with time in predefined and programmable designs. There have been numerous investigations into the application of 3D printing in biomedical engineering. So far, the greatest biomedical applications of 3D printing are in the tissue engineering field. Tissue engineering offers a new approach to treat difficult problems in human tissue repair. It involves using live cells to form implantable devices for body tissue regeneration. In scaffold-based tissue engineering, a scaffold provides a microenvironment for cells to adhere, proliferate and differentiate and a structural framework for new tissue formation. 3D printing has many advantages in scaffold fabrication, such as control of pore size, porosity, etc. Furthermore, 3D printing can make multilayered scaffolds with different layer characteristics. Most human body tissues are complex and hierarchical and their regeneration requires structurally complex scaffolds that resemble tissue structures and can provide biochemical cues such as growth factors (GFs). Incorporating GFs and even live cells in scaffolds can greatly facilitate tissue regeneration. Since 2004, we have been investigating 3D biomedical printing and have used different 3D printing techniques for developing bone tissue engineering scaffolds (e.g., S.H.Lee, W.Y.Zhou, W.L.Cheung, M.Wang, “Producing Polymeric Scaffolds for Bone Tissue Engineering Using the Selective Laser Sintering Technique”, Transactions of the Society For Biomaterials 30^th Annual Meeting, Memphis, TN, USA, 2005, 348). We have also been exploring 4D printing in tissue engineering (e.g., C.Wang, Y.Zhou, M.Wang, “In situ Delivery of rhBMP-2 in Surface Porous Shape Memory Scaffolds Developed through Cryogenic 3D Plotting”, Materials Letters, Vol.189 (2017), 140-143). This talk will give an overview of our work in 3D/4D printing of scaffolds for regenerating tissues such as bone and blood vessels. It will focus on the design and 4D printing of shape-morphing and GF-delivering scaffolds for tissue engineering.

Diamond is quickly becoming an interesting material for medical implants with evidence that it provides not only a good biointerface but also an antimicrobial surface. Previously ultrananocrystalline diamond was shown to be a good bionic implant material [1] and our group have shown that a polycrystalline diamond coating can be produced on a 3D printed titanium substrate [2]. However up until now, all diamond implants have been as a coating material, with limitations as to its capacity to be used as a three dimensional surface. Here we present our recent results fabricating diamond implants using additive manufacturing including presenting for the first time a hybrid 3D diamond-titanium printed material which was recently published [3]. Diamond implants fabricated via using three different techniques (i) nanodiamond seeding, (ii) polycrystalline diamond coating and (iii) composite diamond-titanium biomaterial all showed superior cellular proliferation compared to the as-printed SLM titanium.as well as a decrease in microbial activity. The possibility of 3D diamond opens up large possibilities for new diamond technologies
[1] K Ganesan, DJ Garrett, A Ahnood, M Shivdasani, W Tong, A Turnley, K Fox, H Meffin and S Prawer (2014) “An all-diamond, hermetic electrical feedthrough array for a retinal prosthesis” Biomaterials Vol. 35 (3) 908-915.
[2] A Rifai, D W. Lau, N Tran, H Zhan, A Sarker, A D Stacey, E Mayes, A D. Greentree, E Pirogova, K Fox, (2018) “Development
of 3D polycrystalline diamond as a coating material for biomedical applications” ACS Materials and Interfaces 10 (10), 8474-8484.
[3] K Fox, A Rifai, N Mani, P Reineck, A Jones, PA. Tran, A Ramezannejad, M Brandt, BC. Gibson, AD. Greentree and N Tran<span style="font-size:10.8333px"> (2019) "</span>3D-Printed Diamond-Titanium Composite: A New Material for Implant Engineering" ACS Applied Bio Materials https://doi.org/10.1021/acsabm.9b00801

It is now more than 30 years since the priciples of Additive Manufacturing or 3D Printing moved from a concept to practical implementation and over 20 years since the first publications on its use to fabricate implanrable medical devices. In this period it has rapidly progressed from being seen as a topic for Science Fiction to now a method for the production of proto- or in vitro model tissues for tissue-on-a-chip applications and laboratory animal studies. Indeed there are several commercial organizations that have developed and market commercial Bioprinters. However, despite these not inconsiderable advances, the original target of printed cell-laden implants or tissue patches have still not been realized.
A key obstacle to the use of 3D printing for the fabrication of cell-laden constraucts is the need to provide a functional vasculature for the maintenance of cell viability through the provision of oxygen and nutrients, as well as a pathway for waste removal. It is well known that the diffusion limit in the 3D culture of cells limits spheroid size to a few hundred μm and that the capillaries in healthy tissue have a diameter of 5 – 10 μm and are typically spaced around 50 μm. However, current additive manufacturing routes that are in widespread use in biofabriction have a spatial resolution > 100 μm. Hence, new approaches to producing high resolution vascular structures and models for angiogenesis are required.
Here we describe the use of ultra-high resolution inkjet printing (UHIJP) as a route to fabricate vascular structures in gelatine methacrylate (GelMA) hydrogels. This technique is related to electrospraying and electrospinning in that a local electric field is used to form a "Taylor Cone" with the meniscus of a fluid at a fine nozzle. Through using a controlled electrical pulse, individual mono-sized droplets are ejected with volume down to a single femtolitre (10^-18 m³) with droplet diameters in the range 1 - 3 μm. UHIJP has been used to print structures from a thermoreversible hydrogel based on triblock polymers of polyethylene oxide and polypropylene oxide (Pluronic F127). These are subsequently encapsulated in GelMA and photoplymerised before removal of the Pluronic and replacement with an aqueous fluid. The exposed internal surface of the vascular structure can be modified with proteins such as laminin or fibrinogen to promote cell adhesion and this has been demonstrated through seeding with human umbilical vein endothelial cells (HUVECs) to allow fully endotheliized structures with vascular diameter as small as 20 μm.
In order to ensure continuous tubular structures without obstruction, more complex vascular designs have been fabricated with minimum diameters of approximately 60 μm and seeded with HUVECs. Confocal microscopy shows that these structures are fully endothelized and act as an effective barrier to labelled dextran probes. As a further demonstration of the utility of the structures, the GelMA matrix has been seeded with human dermal fibroblasts and this has been co-cultured with the HUVEC lined vasculature demonstrating cell viability is maintained after several days in culture.

3D Printing has been a highly successful method to bring new materials and complexities in design through digital manufacturing. It has enabled advancement from rapid prototyping through actual production. Because of its mechanical, chemical and biological properties, 3D-printed polyether ether ketone (PEEK) has great potential as a customized bone replacement. Its density and mechanical properties are closer to bone compared to most Ti-based alloys. In this study, PEEK samples were printed using fused deposition modeling (FDM) and evaluated in terms of their dimensional accuracy, crystallinity, and mechanical properties. The dimensional integrity and surface quality of the printed materials are important. Crystallinity and mechanical properties increased with elevated chamber temperature and post-printing annealing. In this study, we also utilized three available commercial printers. Variations of material properties from three printers are evident. Many factors affect the quality of 3D-printed PEEK. Future FDA regulations for 3D-printed products are needed for this highly customizable manufacturing process to ensure safety and effectiveness for biomedical applications. Future work will report on nanocomposite fabrication and testing.

3D printing is a promising approach for designer biomaterial architectures with information-rich structure and dynamic functionality. In particular, stimuli-responsive hydrogels consisting of crosslinked, hydrophilic polymers could be used for tissue engineering, drug delivery and other biomedical applications. One design consideration is that these biomaterials must be responsive to at least two physicochemical stimuli – the first to pattern desired structures and additional orthogonal stimuli to trigger dynamic behaviors such as degradation, actuation, or self-adhesion. Here, we show that light-directed 3D printing based on ionic crosslinking enables smart materials with biologically-inspired functionalities. First, we demonstrate reversible 3D printing of alginate hydrogel microstructures to template hydrogel microfluidics and pattern collective cell migration. Second, we add graphene oxide into these alginate hydrogels to enhance mechanical properties and oil repulsion in seawater-like conditions. Finally, we prepare double network hydrogels that stimuli-responsive and self-adhesive as a simple “do-it-yourself” construction set for soft machines and microfluidic devices. This modular approach enables “plug-and-play” hydrogel parts for ionic soft machines that emulate actuation, sensing, and fluid transport in living systems.

Bioprinting of autologous tissues encompasses many different steps, and accelerating the process can be very critical for patients with an urgent need of organs. The first step for the transition from “trial and error” to “predict and control” phase of the bioprinting is to understand the underlying science of this unique technology. The physical aspects of self-assembly and the dynamics of multicellular systems used with printing technologies for tissue fabrication are therefore very important. We have developed a microscopic and mathematical method to study tissue fusion, an essential element of self-assembly phenomena during tissue maturation in the post-bioprinting procedure. The model is generalized for different bioink geometries. A mathematical method is introduced, and microscopic interactions at the cellular level in the maturation of bioprinted tissues have been studied. The fusion of tissue-like cell aggregates (cellular bioinks) is elaborated with the help of bonds among their adhesion molecules. The statistical mechanics is used to describe the fusion procedure. Consequently, the partition function for the model and the system total energy are calculated and compared to understand the effect of bioink geometries on the acceleration of the maturation of bioprinted tissues. We identified the driving force and energy of the self-assembly in the maturation of bioprinted tissues. The force and energy are not only critical to control bioprinted final product but also significant for other multicellular interactions such as cancer metastasis and migrations, in which decelerating the procedure is important. Based on our microscopic and mathematical method, we introduce some topological criteria for bioinks for faster maturation of bioprinted tissues.

Influencing or ideally directing the innate immune response after implantation remains one of the major challenges in the development of biomaterials and the design of three dimensional (3D) scaffolds [1]. Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful in early phases, a prolonged inflam-mation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization is precise control over biomaterial geometry. For example, a concave-structured poly(2-hydroxyethyl methacrylate) hydrogel with a highly ordered architec-ture and exactly equally-sized pores of up to 40 μm, showed a pronounced infiltration of murine macrophages being mainly directed towards the healing phenotype in vivo [2].
For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. The additive manufacturing technique of melt elec-trowriting (MEW) is an especially suitable and advantageous approach in this context as it enables the production of highly defined scaffold geometries built of fibers with diameters in the lower micrometer range [3]. Our group has demonstrated the ability of cells to attach, infil-trate, and proliferate upon seeding onto MEW scaffolds [4], and that the surface modification of MEW scaffolds results in specific interaction with cells [5, 6].
This talk will present our findings in applying MEW for the fabrication of fibrous 3D scaffolds made from poly(ε-caprolactone) (PCL) with precisely defined inter-fiber spacing from 100 μm down to 40 μm in a variety of pore geometries (rectangular, triangular and round). These scaf-folds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for box-shaped pores with 40 μm inter-fiber spacing [7]. Moreover, a novel way of producing double-hierarchical scaffolds by MEW will be pre-sented that induce an even stronger M2-type differentiation stimulus for human macrophages.
Literature
[1] A. Vishwakarma, et al: Engineering Immunomodulatory Biomaterials To Tune the Inflammatory Re-sponse. Trends Biotechnol 2016, 34, 470-82.
[2] J. D. Bryers, et al: Engineering biomaterials to integrate and heal: the biocompatibility paradigm shifts. Biotechnol Bioeng 2012, 109, 1898-911.
[3] T.D. Brown, et al: Direct writing by way of melt electrospinning. Advanced Materials 2011, 23(47), 5651-7.
[4] G. Hochleitner, et al: Additive manufacturing of scaffolds with sub-micron filaments via melt electro-spinning writing. Biofabrication 2015, 7, 035002.
[5] S. Bertlein, et al: Permanent Hydrophilization and Generic Bioactivation of Melt Electrowritten Scaf-folds. Advanced Healthcare Materials, 2019, 1801544.
[6] C. Blum, et al: Extracellular matrix-modified fiber scaffolds as a pro-adipogenic mesenchymal stromal cell delivery platform. ACS Biomaterials Science & Engineering, 2019, 5, 12, 6655-6666.
[7] T. Tylek, et al: Precisely Defined Fiber Scaffolds with 40 μm Porosity Induce Elongation Driven M2-like Polarization of Human Macrophages. Biofabrication, available online: https://doi.org/10.1088/1758-5090/ab5f4e

Elastomer based microfluidic devices microfabricated via conventional methodologies, such as soft lithography and stereolithography, have been used for applications such as chemical species mixing, fast bio-chemical assays, controlled cell culturing etc. However, several challenges exist for these methods, including long fabrication times, limited throughput, complex manual alignment steps, and difficulty integrating functional elements such as electrodes, valves, sensing elements, and/or 3-D structures for mixing or sorting. A more freeform fabrication methodology is demanded to enable new applications in microfluidics. Here, we demonstrate a novel 3D printing strategy that can effectively overcome the above issues via precisely controlling the extrusion of polymeric inks into self-supporting microfluidic structures. Specifically, extruded silicone filaments are stacked in an orientation such that adhesion and gravity are balanced during printing, eliminating the need for sacrificial materials. This creates interconnected hollow structures, such as micro chambers and channels, with a minimum inner diameter of 100 micron. The advantage of 3D printed self-supporting microfluidic structures are further demonstrated via several types of devices. First, chemical species mixers are printed with silicone channels, embedded with micro herringbone ridge arrays that are printed with the biocompatible material, polycaprolactone (PCL). Optimization of the mixing effect is conducted via facile adjustment of the geometric profile of the herringbone ridges. Next, a multi-channel salinity sensor is created via directly printing silicone channels and chambers aligned onto prefabricated electronic circuits. A seamless and robust bonding is formed between the microfluidic structures and the substrate as the material cures. We also demonstrate that, with this printing strategy, functional microfluidic valves and pumps can be readily printed with overlapping silicone channels and UV-curable encapsulating polymers. Finally, going beyond the planar morphology, branching microfluidic channels are printed onto a spherical surface, demonstrating the potential application of 3D printed microfluidics onto curved surfaces. In the future, this may even be the human body as directly printed physiological sensors. The demonstrated methods overcome current limitations by enabling rapid, high-throughput, high-resolution fabrication of elastomeric microfluidic devices on planar and non-planar geometries with integrated multi-material functional elements for diverse applications.

Calcium phosphates is the main material found in the bone; for this reason, they are used in the fabrication of scaffolds in tissue engineering applications. However, the application of these materials depends not only the formulation but also on the geometry of the bone to be replaced. 3D printing is positioned as a powerful tool for obtaining specific pieces for high-performance custom applications. In this study, we employed hot melt extrusion in order to manufacture Ceramic-Polymer filament (Calcium Phosphates- Polylactic acid). The obtained filaments exhibited fragility but were suitable for use in the scaffolds fabrication by 3D filament printing technique. The filaments were subjected to biological tests and showed non-cytotoxic behavior, deposition of apatite phases on their surface, adequate cell proliferation, and cell adhesion. Previous results in vivo showed that the anchor between the bone and the implant plays a vital role in order to avoid implant failure.

Scaffold-based tissue engineering is a major approach in tissue engineering and cells and/or biomolecules can be loaded into the matrix or onto the surface of 3D scaffolds for enhancing tissue regeneration. Scaffolds mimicking the natural extracellular matrix of human tissues can serve very well as a substrate for cell attachment, proliferation, and differentiation and thus facilitate new tissue formation in vivo. The architectures (pore size, shape, interconnectivity, porosity, etc.) and properties (biological and mechanical) of scaffolds should be carefully controlled to match the defect shape and size and features of the target tissue. 3D printing technologies enable us to fabricate porous scaffolds with accurate control over their architectures and properties. On the other hand, 3D printing of shape morphing polymers has attracted great attentions in tissue engineering as scaffolds with shape morphing ability can reshape themselves after implantation to match the defect and anatomy of host tissues. Biomolecules such as growth factors have been often used in tissue engineering to accelerate tissue regeneration; and vascular endothelial growth factors (VEGF) is used in the regeneration of gastrointestinal tract and vasculature. In this study, an extrusion-based 3D printing system was used to construct porous scaffolds which have abilities of self-folding upon heating to the body temperature and controlled release of VEGF. For 3D printed scaffolds, the self-folding ability was achieved by using a shape memory polymer poly(D, L-lactide-co-trimethylene carbonate) (PDLLA-TMC) which could change shape at a temperature greater than 37°C, while the controlled release of growth factor was achieved by using gelatin methacrylate (GelMA) as the functional layer to load VEGF. In scaffold fabrication, a planar porous structure was firstly fabricated by accurate deposition of viscous PDLLA-TMC solution. The permanent tubular shape of PDLLA-TMC was made by folding printed planar porous structure into a tube on a glass stick at 80°C. Then PDLLA-TMC tubular porous structure was flattened at 25°C. Finally, GelMA loaded with VEGF was printed onto the PDLLA-TMC planar porous scaffold layer and crosslinked by UV. Shape morphing of PDLLA-TMC/GelMA-VEGF scaffolds was studied by immersing scaffolds in water at 37°C. Planar porous scaffolds could fold automatically into tubes within 60s. SEM examinations showed that interconnected macropores were regularly arranged on the scaffolds. In vitro release tests revealed a sustained and steady release of VEGF in the 21 day tests. For rat mesenchymal stem cells (rMSCs) seeded on scaffolds, LIVE/DEAD assay results indicated very good cell viability. MTT assay results showed good cell proliferation after 1, 4, 7-day culture. This study has demonstrated a good strategy to develop unique scaffolds for tubular tissues or organs such as vasculature and gastrointestinal tract.

Despite the recent advances in high-sensitivity lab-on-chip assays, adapting them for point-of-care settings remains a challenge due to the complexities in fabrication, and the need for an external measurement system. In this paper, I present a novel layer-sandwiching approach with an integrated Surface-Mount-Device (SMD) based on-chip absorbance measurement system, which allows for rapid prototyping of Point-of-Care (POC) assays.

In this approach, the layers are 3D printed using a 3DS Projet MJP Multijet printer which allows for the fabrication of 100 μm thickness flexible layers and sub-100 micron width channels. While the optical transparency of the material allows for on-chip signal acquisition, the use of SMD LEDs enables low power operation and the availability of a wide range of narrow emission spectrums. The sandwiched device has the middle layer as the flow layer and is sandwiched between two layers of parafilm. These three layers are then further sandwiched between an optical readout system. The hydrophilic nature of the 3D printed flow layer allows for a capillary-driven flow of reagents and the two parafilm layers prevent leakage. Because of the stacked nature of the layers, the device could also be disassembled, cleaned and reused.

The system was tested by performing MTT assay on-chip. For this test, human prostate cancer cells were treated with 20 μM Camptothecin in a standard 96 well plate. The cells were incubated for 24 hrs at 5% CO₂ and 37°C. Then MTT dye was added and after 4 hrs of incubation, DMSO was added for dissolving the formazan crystals. Absorbance measurement for each well was performed separately on a spectrophotometer, and on the 3D printed device with 595 nm emission SMD-LEDs, and a strong correlation was found.

The proposed fabrication approach is far simpler than conventional methods. The high sensitivity, capillary-driven flow and the low cost of manufacturing make this system ideal for performing POC diagnostics assays, and other field assays where it’s not possible to carry the bulky and expensive measurement systems.

While additive manufacturing (AM) is capable of providing a method of producing geometrically complex and highly detailed structures that conventional manufacturing methods cannot, the generation of residual stress in many AM processes can negatively impact properties and performance. This research aims to address knowledge gaps in understanding how residual stresses are distributed at the grain and subgrain scale, the dislocation structures that accommodate these stresses, and how post-processing stress relief strategies effect this. Utilizing the selective laser melting (SLM) process, spinal cage implants were manufactured from ELI Ti64 powder which were then subjected to stress relieving heat treatment cycles above and below the Ti64 β transus as a function of time and cooling rate. Residual stress was than quantitatively measured via HR-EBSD and bulk properties informed via compression testing. It was found that grain scale stress decreased at an inversely proportional rate with heat treatment time, while the retention of β phase significantly changed the stress and dislocation distribution.

Biofabrication is a young field of research that aims at the automated generation of hierarchical tissue-like structures from cells and materials through Bioprinting or Bioassembly [1]. However, the lack of variety in printable hydrogel systems, which are the mainly used materials for the formulation of bioinks [2], is one major drawback for the advancement of the complete field [3]. Gelatin is often adopted for this purpose, usually modified with (meth-)acryloyl functionalities for post-fabrication curing by free radical photo-polymerization, resulting in a hydrogel that is cross-linked via non-degradable polymer chains of uncontrolled length.
We have introduced GelAGE as a new thiol-ene clickable and broadly applicable alternative for gelatin-based bioinks [4]. The advantage of this system is the absence of non-degradable polymeric components after cross-linking compared to the free radical polymerization, as well as a better control over cross-linking density, and compatibility with Vis-light addressable initiator systems such as (Ru/SPS). Here we present the evolution of this system by stepwise altering the cross-linker from DTT to PEG-dithiol and star shaped 8-armed PEG-octathiol to multi-thiolated hyaluronic acid and finally a Hyaluronan-AGE / thiolated-Hyaluronan bioink as comparison. This led to a continuous improvement of the printability in terms of shape fidelity with decreasing overall polymer concentrations at constant cytocompatibility. We also present biological evaluation of this system for cartilage engineering.
References
1. J. Groll, et al: Biofabrication, 8 (2016) 013001.
2. J. Groll, et al: Biofabrication, 11 (2019) 013001.
3. T. Jüngst, et al: Chemical Reviews, 116 (2016) 1496.
4. S. Bertlein, et al: Advanced Materials, 29 (2017) 1703404.

Viscoelastic complex fluids exhibit rheological nonlinearity at a high shear rate. Although typical nonlinear effects, shear thinning and shear thickening, have been usually understood by variation of intrinsic quantities such as viscosity, one still requires a better understanding of the microscopic origins, currently under debate, especially on the shear-thickening mechanism. We present accurate measurements of shear stress in the bound hydration water layer using noncontact dynamic force microscopy. We find shear thickening occurs above 10^6 1/s shear rate beyond 0.3-nm layer thickness, which is attributed to the nonviscous, elasticity associated fluidic instability via fluctuation correlation. Such a nonlinear fluidic transition is observed due to the long relaxation time (1 us) of water available in the nanoconfined hydration layer, which indicates the onset of elastic turbulence at nanoscale, elucidating the interplay between relaxation and shear motion, which also indicates the onset of elastic turbulence at nanoscale above a universal shear velocity of 1 mm/s. This extensive layer-by-layer control paves the way for fundamental studies of nonlinear nanorheology and nanoscale hydrodynamics, as well as provides novel insights on viscoelastic dynamics of interfacial water. We will also discuss the tip-based microfluidic characterization and manipulation of liquids.

As additive manufacturing (AM, also known as 3D printing) technologies have proliferated, approaches based on solidification of photosensitive liquid resins have showed great promise due to their superior resolution and precision. However, these techniques have been largely limited to prototyping applications due to the constraints on available materials and their mechanical properties, as well as slow builds and poor surface quality resulting from layer-wise fabrication. With the advent of volumetric AM (VAM), complete 3D structures with complex geometries can now be produced in a single step, leaping over the traditional limitations of layer-by-layer approaches to 3D printing.
Volumetric 3D printing generates a 3D distribution of absorbed optical energy within a volume of photosensitive material, concurrently curing all points within a target geometry, on a timescale of 10s to 100s of seconds. No substrate is required as the part forms unsupported in the resin container. The most versatile implementation of VAM is known as computed axial lithography (CAL) which adapts the principles of computed tomography (CT) to generate a sequence of intensity-modulated projections, which are then beamed sequentially into a rotating resin container via a DLP projector to create the required energy dose. Because there is no fluid motion, and no hydrodynamic forces during a build, this approach is particularly compelling for very soft materials such as the hydrogels widely used in tissue engineering and regenerative medicine applications. The absence of support structure also enables complex geometries such as vasculature to be built.
Successful volumetric 3D printing requires spatio-temporal control over the polymerization reaction at all points within the fabrication volume. This demands a more detailed understanding of a host of process parameters, compared with traditional layered approaches. Perhaps the most important such parameters are the absorbed volumetric energy dose E_VOL, as well as the rate at which this dose is delivered. The progress of the photochemical reaction may then be characterized in terms of a variety of metrics, such as double-bond conversion, or the evolution of mechanical and optical properties of the material. We investigate the inter-relationship of these properties, obtained from ex-situ measurements such as real-time FTIR spectroscopy, photo-rheology, and mechanical testing, developing a quantitative framework for predicting volumetric structure formation. An additional resin characteristic critical for CAL printing is nonlinear threshold behavior. This allows resin that receives a sub-threshold dose to remain liquid, and derives from the interaction of the generated radicals and inhibitory species present in the resin. For some chemistries the inhibitory effect is provided by dissolved molecular oxygen, but for other formulations, deliberately including another inhibitor is necessary. We discuss the implications of these interacting factors for photo-resin design within the volumetric AM paradigm, as well as more broadly in photopolymer-based AM methods. We also discuss several material classes for VAM, including high-performance engineering materials, as well as cell-culture compatible hydrogels.

References

Kelly, B. E., Bhattacharya, I., Heidari, H., Shusteff, M., Spadaccini, C. M., Taylor, H. K., “Volumetric additive manufacturing via tomographic reconstruction” Science 2019, 363, 1075-1079.

Borosilicate glass is a type of glass material consisting SiO₂ and B₂O₃ as network formers and alkali oxide modifiers. Their excellent optical transparency, chemical resistance, low thermal expansivity and tunable chemical composition make them useful for a wide range of applications such as lab ware, medical implants, microelectromechanical systems (MEMS) , sensing and detection etc. Conventional glass fabrication techniques involve melting, casting and use of undesirable chemicals and these imply that there is little room for flexibility in shape and resolution. Recent surge in additive manufacturing efforts now show that ceramic glasses can now be fabricated via stereolithography. However this has not been shown possible for borosilicates due to their low softening temperatures.
Herein, we report a simple additive manufacturing protocol for fabricating 3D low softening temperature borosilicate glass. It entails first fabricating a liquid glass composite, printing on a layer by layer basis to form a 'green ensemble'. This ‘green ensemble’ must then be debound to yield a 'brown ensemble' and sintered to yield transparent and amorphous glass ensembles. The properties of the printed glass depend on thermal processing parameters (temperature, time and environment) and these can be readily tuned and optimized to yield 3D glass for a wide range of applications. The printed glass show good optical properties, show no devitrification and exhibit minimal surface roughness requiring no further polishing and finishing typically associated with other glass fabrication techniques.

Two photon polymerization is an additive manufacturing method that offers many advantages over conventional processes for scalable mass production of medical devices, including microneedles and other medical devices with small-scale features. First, the raw materials (e.g., inorganic-organic hybrid materials and acrylate-based polymers) used in two photon polymerization can be purchased at low cost. Second, two photon polymerization can be established in a conventional environment; no cleanroom facilities or other types of specialized facilities are required. Third, two photon polymerization is a straightforward approach for creating medical devices with complex and small-scale features. Two photon polymerization has been used to create hollow microneedles with a larger range of shapes and dimensions than conventional microneedle fabrication methods. We have prepared several types of hollow microneedle-based biosensors using microneedles that were fabricated using either two photon polymerization or digital micromirror device-based stereolithography. In these biosensors, the sensors (either colorimetric or electrochemical) are located within the bores of the microneedles or underneath the microneedles. Application-specific testing and functional testing of the microneedle-based biosensors will be considered.

Hydrogels have long been considered a significantly promising class of biomaterials. While the chemomechanical tunability of a multitude of hydrogels have been demonstrated in vitro, the ability to deliver these materials in a minimally invasive manner to avoid iatrogenic damages has yet to be fully explored. In the present work, we demonstrate a method of in-situ extrusion of hydrogels with compositions and chemomechanical properties that can be dynamically adjusted “on-the-fly” using a custom catheter that is utilized intravascularly. A custom UV-integrated, multi-lumen microcatheter (inner/outer lumen diameters: 0.6/1.28 mm) was assembled and a suite of low viscosity, shear thinning gelatin-based precursors were formulated for delivery. While the shear thinning property makes this precursor a suitable choice for injection through a catheter with extended length and small diameter, intensity modulation of UV to cross-link the precursor aids greatly in tuning the stiffness at the catheter tip. We show that by modulating the absolute and relative flow rates as well as the UV intensity (0~150 µW output from a 105µm core multimode fibre), we can extrude hydrogels with stiffnesses dynamically varying from <10 to 80 kPa. In addition, through the dual luminal architecture, it is also possible to sequentially switch or coaxially coextrude two types of precursor compositions. The concentrations and diffusivities of additives (as preliminarily demonstrated using food dyes) within the extruded scaffold can also be dynamically altered by 10³ and 10² orders respectively, in a single piece of extruded hydrogel fibre, therefore allowing applications a highly customizable drug depot to be deployed using this technique. To demonstrate the initial utility of this system, we successfully performed embolization of a saccular aneurysm model (diameter ~ 12 mm) and a capillary vasculature model (diameter <1 mm) using a pulsatile vascular flow phantom. The former required the extruded hydrogels to be relatively stiffer via increased photo-cross linking to fill the aneurysm while avoiding leakage into the parent vessel. In capillary embolization, the precursor was injected with lower crosslinking leading to increased viscosity to penetrate further into the vessels to fully embolize the structure. These findings yield direct application ideas in therapeutics such as vascular embolization in a variety of disease states, including cerebral aneurysms, arterio-venous malformations, vascularized tumors, and hemorrhagic vessels. The system can also be utilized for intravascular drug delivery and targeted cell therapies, which may require the physician to constantly adapt to the state of the patient. Further, such a dynamically tunable hydrogel delivery method may be able to serve as a supporting host matrix for functional sensing devices such as microelectrode arrays and quantum dot based chemical sensors.

Bioink extrusion-printing, while capable of creating macroscopically accurate anatomic models, fails to replicate the microstructure of the natural tissue due to resolution limitation of this bottom-up fabrication approach. We re-engineer the extrusion process to co-extrude a fiber device in line with the hydrogel bioink. This allows deviation from a pure bottom-up towards a combined approach, where a bottom-up extrusion process creates the macrostructure, while the fiber device in a top-down fashion subsequently promotes the growth of natural microstructure from within the volume of the print.

Fibers can embed a range of bio-functional and biosensing devices and systems. We aim to incorporate microfluidic, ultrasonic, and shape-memory-alloy (SMA) structures mimicking the functionalities of microvasculature, innervation, and musculature, respectively, into a single fiber-device. Those functional structures are created by a thermal draw of fibers from multimaterial preforms – a process familiar from an optical communication fiber fabrication. In this process, the macroscopic preform – a thick and short rod, cross-sectionally structured to meet the desired fiber functionality – is heated to become a viscous liquid and scaled by a thermal draw into a thin and long fiber, preserving the cross-sectional geometry of the preform. Then the fiber cores are patterned axially by a spatially coherent material selective capillary instability to form arrays of ultrasonic transducers contacted in parallel and microchannels with periodic outlets spanning the entire fiber length ^[1].

In the extrusion process, the fiber is coated with hydrogel, contains tissue cells, extracellular matrix material, nutrients and growth-factors, forming a scaffolded print-line that is raster-layed into a printed model cross section additively constructing the final model in a layer-by layer manner. Subsequently, the fiber-embedded microchannel structure is used as artificial vasculature supplying building material and nutrients into surrounding bioink to ignite the creation of natural vasculature and promote the proliferation of the bioink-contained tissue cells with their subsequent maturation into a natural-tissue-like structure. The ultrasonic transducers embedded in the fiber are used to sense the changes in the surrounding material density, monitoring cell-proliferation process, thus serving as artificial tactile nerves. SMA cores, contracting upon transduction of electric current, mimic the function of muscle yarns.

The proposed bioprinting approach will likely deliver a biosynthetic tissue with natural-like microstructure opening new routes in regenerative medicine, such as wound infilling and organ replacement. Moreover, it would allow creating lab-bench anatomical models of realistic tissue, in which bio-material delivery and biosensing is performed volumetrically with a microscale resolution, thus forming a novel stand-alone platform for investigation of micrometabolic processes. Such a platform would be a valuable asset to drug toxicity investigation, and real-time survivability monitoring of such artificial tissues and organs both in-vitro and in animal subjects.

[1] C. Faccini de Lima, L. van der Elst et al., “Towards Digital Manufacturing of Smart Multimaterial Fibers,” Nanoscale Res. Lett. 14, 209 1-6 (2019).

Since the discovery of chemotherapy in the beginning of the 20^thcentury, researchers around the world have been actively developing new and more effective chemotherapeutic agents to better treat cancer. Traditionally, chemotherapeutic agents work by interfering with cell division. However, by virtue of their mechanism of action, healthy normal cells can also be targeted and destroyed. As a result, while chemotherapy is an effective way of managing cancer, the resulting side effects limits its use. One approach currently taken to reduce these side effects is to deliver the chemotherapy drugs directly to the tumor via transarterial chemoembolization, or other similar procedures. While this has been effective in reducing systemic toxicity, more can be done to improve this. Ideally, a device that could sequester any unreacted chemotherapy agents could be installed "downstream" of the tumor prior to them entering systemic circulation. Such drug-capture materials have yet to be realized due to the difficulty in achieving materials that have the right surface chemistry and geometry for blood flow.

Working together with medical doctors, computational fluid dynamics experts, chemists, and materials scientists, we report the fabrication of DNA functionalized 3D printed porous materials that can be used to capture doxorubicin and cisplatin, two commonly used DNA-targeting chemotherapy agents. We discuss the concept behind the device, the use of 3D printed materials as an ideal substrate, and the chemistries considered in drug binding. To achieve scalability of these devices, we developed a method of attaching cheaply available genomic DNA to these materials, a departure from commonly used synthetic DNA. We characterize the surface of the structure and verify the binding of DNA to the surface via XPS, EDS and the use of chemical assays. The efficacy of these functionalized materials were demonstrated in PBS, where we observed a >70% reduction in doxorubicin concentration over a period of 20 minutes, highlighting the viability of this as a method of drug capture.

Additive manufacturing, popularly known as “3D printing”, includes an array of technologies such as fused deposition modelling (FDM), selective laser sintering (SLS) and inkjet printing. Compared with conventional manufacturing technologies, 3D printing has many distinctive advantages, including the construction of 3D structures with complex shapes and functionalities. Therefore, 3D printing technologies have seen increasing popularity in many areas, such as product design and development, industrial production, consumer goods, aerospace, and education. 3D printing has already made a tremendous impact in our society. The exciting TED Talk in 2013 by Tibbits of MIT ushered in a new era in additive manufacturing: 4D printing. With 4D printing, 3D printed static objects will change their shapes over time, i.e., 4D printing uses 3D printing technologies to produce shape-morphing objects. Such objects can meet the demanding requirements in particularly applications. The concept of 4D printing has been evolving and one popular definition of 4D printing is that the shape, property and functionality of a 3D printed object can evolve with time in predefined and programmable designs. There have been numerous investigations into the application of 3D printing in biomedical engineering. So far, the greatest biomedical applications of 3D printing are in the tissue engineering field. Tissue engineering offers a new approach to treat difficult problems in human tissue repair. It involves using live cells to form implantable devices for body tissue regeneration. In scaffold-based tissue engineering, a scaffold provides a microenvironment for cells to adhere, proliferate and differentiate and a structural framework for new tissue formation. 3D printing has many advantages in scaffold fabrication, such as control of pore size, porosity, etc. Furthermore, 3D printing can make multilayered scaffolds with different layer characteristics. Most human body tissues are complex and hierarchical and their regeneration requires structurally complex scaffolds that resemble tissue structures and can provide biochemical cues such as growth factors (GFs). Incorporating GFs and even live cells in scaffolds can greatly facilitate tissue regeneration. Since 2004, we have been investigating 3D biomedical printing and have used different 3D printing techniques for developing bone tissue engineering scaffolds (e.g., S.H.Lee, W.Y.Zhou, W.L.Cheung, M.Wang, “Producing Polymeric Scaffolds for Bone Tissue Engineering Using the Selective Laser Sintering Technique”, Transactions of the Society For Biomaterials 30^th Annual Meeting, Memphis, TN, USA, 2005, 348). We have also been exploring 4D printing in tissue engineering (e.g., C.Wang, Y.Zhou, M.Wang, “In situ Delivery of rhBMP-2 in Surface Porous Shape Memory Scaffolds Developed through Cryogenic 3D Plotting”, Materials Letters, Vol.189 (2017), 140-143). This talk will give an overview of our work in 3D/4D printing of scaffolds for regenerating tissues such as bone and blood vessels. It will focus on the design and 4D printing of shape-morphing and GF-delivering scaffolds for tissue engineering.

Diamond is quickly becoming an interesting material for medical implants with evidence that it provides not only a good biointerface but also an antimicrobial surface. Previously ultrananocrystalline diamond was shown to be a good bionic implant material [1] and our group have shown that a polycrystalline diamond coating can be produced on a 3D printed titanium substrate [2]. However up until now, all diamond implants have been as a coating material, with limitations as to its capacity to be used as a three dimensional surface. Here we present our recent results fabricating diamond implants using additive manufacturing including presenting for the first time a hybrid 3D diamond-titanium printed material which was recently published [3]. Diamond implants fabricated via using three different techniques (i) nanodiamond seeding, (ii) polycrystalline diamond coating and (iii) composite diamond-titanium biomaterial all showed superior cellular proliferation compared to the as-printed SLM titanium.as well as a decrease in microbial activity. The possibility of 3D diamond opens up large possibilities for new diamond technologies
[1] K Ganesan, DJ Garrett, A Ahnood, M Shivdasani, W Tong, A Turnley, K Fox, H Meffin and S Prawer (2014) “An all-diamond, hermetic electrical feedthrough array for a retinal prosthesis” Biomaterials Vol. 35 (3) 908-915.
[2] A Rifai, D W. Lau, N Tran, H Zhan, A Sarker, A D Stacey, E Mayes, A D. Greentree, E Pirogova, K Fox, (2018) “Development
of 3D polycrystalline diamond as a coating material for biomedical applications” ACS Materials and Interfaces 10 (10), 8474-8484.
[3] K Fox, A Rifai, N Mani, P Reineck, A Jones, PA. Tran, A Ramezannejad, M Brandt, BC. Gibson, AD. Greentree and N Tran<span style="font-size:10.8333px"> (2019) "</span>3D-Printed Diamond-Titanium Composite: A New Material for Implant Engineering" ACS Applied Bio Materials https://doi.org/10.1021/acsabm.9b00801

It is now more than 30 years since the priciples of Additive Manufacturing or 3D Printing moved from a concept to practical implementation and over 20 years since the first publications on its use to fabricate implanrable medical devices. In this period it has rapidly progressed from being seen as a topic for Science Fiction to now a method for the production of proto- or in vitro model tissues for tissue-on-a-chip applications and laboratory animal studies. Indeed there are several commercial organizations that have developed and market commercial Bioprinters. However, despite these not inconsiderable advances, the original target of printed cell-laden implants or tissue patches have still not been realized.
A key obstacle to the use of 3D printing for the fabrication of cell-laden constraucts is the need to provide a functional vasculature for the maintenance of cell viability through the provision of oxygen and nutrients, as well as a pathway for waste removal. It is well known that the diffusion limit in the 3D culture of cells limits spheroid size to a few hundred μm and that the capillaries in healthy tissue have a diameter of 5 – 10 μm and are typically spaced around 50 μm. However, current additive manufacturing routes that are in widespread use in biofabriction have a spatial resolution > 100 μm. Hence, new approaches to producing high resolution vascular structures and models for angiogenesis are required.
Here we describe the use of ultra-high resolution inkjet printing (UHIJP) as a route to fabricate vascular structures in gelatine methacrylate (GelMA) hydrogels. This technique is related to electrospraying and electrospinning in that a local electric field is used to form a "Taylor Cone" with the meniscus of a fluid at a fine nozzle. Through using a controlled electrical pulse, individual mono-sized droplets are ejected with volume down to a single femtolitre (10^-18 m³) with droplet diameters in the range 1 - 3 μm. UHIJP has been used to print structures from a thermoreversible hydrogel based on triblock polymers of polyethylene oxide and polypropylene oxide (Pluronic F127). These are subsequently encapsulated in GelMA and photoplymerised before removal of the Pluronic and replacement with an aqueous fluid. The exposed internal surface of the vascular structure can be modified with proteins such as laminin or fibrinogen to promote cell adhesion and this has been demonstrated through seeding with human umbilical vein endothelial cells (HUVECs) to allow fully endotheliized structures with vascular diameter as small as 20 μm.
In order to ensure continuous tubular structures without obstruction, more complex vascular designs have been fabricated with minimum diameters of approximately 60 μm and seeded with HUVECs. Confocal microscopy shows that these structures are fully endothelized and act as an effective barrier to labelled dextran probes. As a further demonstration of the utility of the structures, the GelMA matrix has been seeded with human dermal fibroblasts and this has been co-cultured with the HUVEC lined vasculature demonstrating cell viability is maintained after several days in culture.

3D Printing has been a highly successful method to bring new materials and complexities in design through digital manufacturing. It has enabled advancement from rapid prototyping through actual production. Because of its mechanical, chemical and biological properties, 3D-printed polyether ether ketone (PEEK) has great potential as a customized bone replacement. Its density and mechanical properties are closer to bone compared to most Ti-based alloys. In this study, PEEK samples were printed using fused deposition modeling (FDM) and evaluated in terms of their dimensional accuracy, crystallinity, and mechanical properties. The dimensional integrity and surface quality of the printed materials are important. Crystallinity and mechanical properties increased with elevated chamber temperature and post-printing annealing. In this study, we also utilized three available commercial printers. Variations of material properties from three printers are evident. Many factors affect the quality of 3D-printed PEEK. Future FDA regulations for 3D-printed products are needed for this highly customizable manufacturing process to ensure safety and effectiveness for biomedical applications. Future work will report on nanocomposite fabrication and testing.

3D printing is a promising approach for designer biomaterial architectures with information-rich structure and dynamic functionality. In particular, stimuli-responsive hydrogels consisting of crosslinked, hydrophilic polymers could be used for tissue engineering, drug delivery and other biomedical applications. One design consideration is that these biomaterials must be responsive to at least two physicochemical stimuli – the first to pattern desired structures and additional orthogonal stimuli to trigger dynamic behaviors such as degradation, actuation, or self-adhesion. Here, we show that light-directed 3D printing based on ionic crosslinking enables smart materials with biologically-inspired functionalities. First, we demonstrate reversible 3D printing of alginate hydrogel microstructures to template hydrogel microfluidics and pattern collective cell migration. Second, we add graphene oxide into these alginate hydrogels to enhance mechanical properties and oil repulsion in seawater-like conditions. Finally, we prepare double network hydrogels that stimuli-responsive and self-adhesive as a simple “do-it-yourself” construction set for soft machines and microfluidic devices. This modular approach enables “plug-and-play” hydrogel parts for ionic soft machines that emulate actuation, sensing, and fluid transport in living systems.

Bioprinting of autologous tissues encompasses many different steps, and accelerating the process can be very critical for patients with an urgent need of organs. The first step for the transition from “trial and error” to “predict and control” phase of the bioprinting is to understand the underlying science of this unique technology. The physical aspects of self-assembly and the dynamics of multicellular systems used with printing technologies for tissue fabrication are therefore very important. We have developed a microscopic and mathematical method to study tissue fusion, an essential element of self-assembly phenomena during tissue maturation in the post-bioprinting procedure. The model is generalized for different bioink geometries. A mathematical method is introduced, and microscopic interactions at the cellular level in the maturation of bioprinted tissues have been studied. The fusion of tissue-like cell aggregates (cellular bioinks) is elaborated with the help of bonds among their adhesion molecules. The statistical mechanics is used to describe the fusion procedure. Consequently, the partition function for the model and the system total energy are calculated and compared to understand the effect of bioink geometries on the acceleration of the maturation of bioprinted tissues. We identified the driving force and energy of the self-assembly in the maturation of bioprinted tissues. The force and energy are not only critical to control bioprinted final product but also significant for other multicellular interactions such as cancer metastasis and migrations, in which decelerating the procedure is important. Based on our microscopic and mathematical method, we introduce some topological criteria for bioinks for faster maturation of bioprinted tissues.

Influencing or ideally directing the innate immune response after implantation remains one of the major challenges in the development of biomaterials and the design of three dimensional (3D) scaffolds [1]. Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful in early phases, a prolonged inflam-mation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization is precise control over biomaterial geometry. For example, a concave-structured poly(2-hydroxyethyl methacrylate) hydrogel with a highly ordered architec-ture and exactly equally-sized pores of up to 40 μm, showed a pronounced infiltration of murine macrophages being mainly directed towards the healing phenotype in vivo [2].
For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. The additive manufacturing technique of melt elec-trowriting (MEW) is an especially suitable and advantageous approach in this context as it enables the production of highly defined scaffold geometries built of fibers with diameters in the lower micrometer range [3]. Our group has demonstrated the ability of cells to attach, infil-trate, and proliferate upon seeding onto MEW scaffolds [4], and that the surface modification of MEW scaffolds results in specific interaction with cells [5, 6].
This talk will present our findings in applying MEW for the fabrication of fibrous 3D scaffolds made from poly(ε-caprolactone) (PCL) with precisely defined inter-fiber spacing from 100 μm down to 40 μm in a variety of pore geometries (rectangular, triangular and round). These scaf-folds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for box-shaped pores with 40 μm inter-fiber spacing [7]. Moreover, a novel way of producing double-hierarchical scaffolds by MEW will be pre-sented that induce an even stronger M2-type differentiation stimulus for human macrophages.
Literature
[1] A. Vishwakarma, et al: Engineering Immunomodulatory Biomaterials To Tune the Inflammatory Re-sponse. Trends Biotechnol 2016, 34, 470-82.
[2] J. D. Bryers, et al: Engineering biomaterials to integrate and heal: the biocompatibility paradigm shifts. Biotechnol Bioeng 2012, 109, 1898-911.
[3] T.D. Brown, et al: Direct writing by way of melt electrospinning. Advanced Materials 2011, 23(47), 5651-7.
[4] G. Hochleitner, et al: Additive manufacturing of scaffolds with sub-micron filaments via melt electro-spinning writing. Biofabrication 2015, 7, 035002.
[5] S. Bertlein, et al: Permanent Hydrophilization and Generic Bioactivation of Melt Electrowritten Scaf-folds. Advanced Healthcare Materials, 2019, 1801544.
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[7] T. Tylek, et al: Precisely Defined Fiber Scaffolds with 40 μm Porosity Induce Elongation Driven M2-like Polarization of Human Macrophages. Biofabrication, available online: https://doi.org/10.1088/1758-5090/ab5f4e

Elastomer based microfluidic devices microfabricated via conventional methodologies, such as soft lithography and stereolithography, have been used for applications such as chemical species mixing, fast bio-chemical assays, controlled cell culturing etc. However, several challenges exist for these methods, including long fabrication times, limited throughput, complex manual alignment steps, and difficulty integrating functional elements such as electrodes, valves, sensing elements, and/or 3-D structures for mixing or sorting. A more freeform fabrication methodology is demanded to enable new applications in microfluidics. Here, we demonstrate a novel 3D printing strategy that can effectively overcome the above issues via precisely controlling the extrusion of polymeric inks into self-supporting microfluidic structures. Specifically, extruded silicone filaments are stacked in an orientation such that adhesion and gravity are balanced during printing, eliminating the need for sacrificial materials. This creates interconnected hollow structures, such as micro chambers and channels, with a minimum inner diameter of 100 micron. The advantage of 3D printed self-supporting microfluidic structures are further demonstrated via several types of devices. First, chemical species mixers are printed with silicone channels, embedded with micro herringbone ridge arrays that are printed with the biocompatible material, polycaprolactone (PCL). Optimization of the mixing effect is conducted via facile adjustment of the geometric profile of the herringbone ridges. Next, a multi-channel salinity sensor is created via directly printing silicone channels and chambers aligned onto prefabricated electronic circuits. A seamless and robust bonding is formed between the microfluidic structures and the substrate as the material cures. We also demonstrate that, with this printing strategy, functional microfluidic valves and pumps can be readily printed with overlapping silicone channels and UV-curable encapsulating polymers. Finally, going beyond the planar morphology, branching microfluidic channels are printed onto a spherical surface, demonstrating the potential application of 3D printed microfluidics onto curved surfaces. In the future, this may even be the human body as directly printed physiological sensors. The demonstrated methods overcome current limitations by enabling rapid, high-throughput, high-resolution fabrication of elastomeric microfluidic devices on planar and non-planar geometries with integrated multi-material functional elements for diverse applications.