Alternative Biofabrication Methods: Sonolithography, Single Cell Bioprinting, Melt Electrowriting

Category: Blog,From Academia
blank blank May 04, 2021

While FDM, SLA, two-photon laser, drop on demand are some of the most popular biofabrication methods, this “From Academia” issue includes three less well know but trending methods that could be complementary or alternative to the typical 3D bioprinting process. The first article introduces a biofabrication method using ultrasound waves, also known as sonolithography. This gentle method can rapidly generate 2D cell patterns for a variety of materials, as well as act as a complementary technique to additive manufacturing where surface patterning combined with layer‐by‐layer fabrication can facilitate the generation of structures with more internal complexity. However, this method does not allow selectively targeting and manipulating individual cells. The second article addresses exactly that problem with a single cell bioprinting method using short laser pulses, which allow for the precise and efficient selection and positioning of individual mammalian cells, as well as transferring of specific cell/cells to a target surface with precision and high cell viability. The third publication was written in 2017, but we are anticipating the author’s upcoming paper in a few weeks. This paper reviews the principles behind a fabrication technique called melt eletrowriting (or electrostatic writing) and also compares it to an adjacent technique called eletrospinning. The author also lists potential biomedical applications of this technique.

From Academia” features recent, relevant, close to commercialization academic publications. Subjects include but not limited to healthcare 3D printing, 3D bioprinting, and related emerging technologies.

Email: Rance Tino (tino.rance@gmail.com) if you want to share relevant academic publications with us.

Sonolithography: In‐Air Ultrasonic Particulate and Droplet Manipulation for Multiscale Surface Patterning

– Authored by Jenna M. Shapiro  Bruce W. Drinkwater  Adam W. Perriman  Mike Fraser. Advanced Materials Technologies. December 2 2020

Biofabrication
Figure 1. Working principles of sonolithography. a) Schematic representation of the sonolithography process. In the case of liquid material, droplets are generated, pass through an acoustic pressure field generated by ultrasonic standing waves, and are deposited into a pattern onto a substrate. Nodal localization is shown for larger blue particles, at the minimum amplitude points. b) The pattern can be predicted through simulation (details in the Supporting Information) of the acoustic radiation forces. Here, the simulated pressures are shown for four transducer pairs, arranged in an octagon with 5λ (43 mm) spacing between the transducers. A 25 mm × 25 mm square region of interest in the center is outlined in green, corresponding to experimental images. Zero acoustic pressure areas (nodes) are black and maximum acoustic pressure areas (antinodes) are white. c) A still from a video (Video S1, Supporting Information) of nebulized water being patterned onto water-sensitive paper using the octagonal array, taken at t = 15 s, where the patterning has become clear. Contrast has been enhanced for ease of visualization. d) Nebulized water ( 1–5 µm), water dispensed from a droplet‐on‐demand (DOD) generator ( 25 µm), and colored sand ( 0.5–1 mm) have been patterned with the same octagonal array. Nebulized water localizes to antinodes, whereas DOD water and sand localize to nodes. For the combined water image (bottom left), both nebulized water and DOD-generated water (here, ∅ 80 µm) have been patterned consecutively onto the same piece of water-sensitive paper. The photographs of sand (recolored to red) and nebulized water (in greyscale) have been contrast-enhanced and overlaid (bottom right) to demonstrate the different physical arrangements of these particles. e) Image analysis was performed to compare the deposition patterns of the water and sand with the simulated pressures in (b). The radial average pixel intensity of greyscaled photos of the nebulized (green) and DOD water (blue) and sand (red) was plotted against the distance from the center. Pixel intensity has been normalized to the maximum intensity at the darkest regions, such that peaks correspond to the areas with the greatest density of patterned material. Also shown is the simulated acoustic pressures (black dashed), where the peaks shown correspond to the antinodes, and zero values to the nodes.

Abstract:

Acoustic fields are increasingly being used in material handling applications for gentle, noncontact manipulation of particles in fluids. Sonolithography is based on the application of acoustic radiation forces arising from the interference of ultrasonic standing waves to direct airborne particle/droplet accumulation in defined spatial regions. This approach enables reliable and repeatable patterning of materials onto a substrate to provide spatially localized topographical or biochemical cues, structural features, or other functionalities that are relevant to biofabrication and tissue engineering applications.

The technique capitalizes on inexpensive, commercially available transducers and electronics. Sonolithography is capable of rapidly patterning micrometer to millimeter scale materials onto a wide variety of substrates over a macroscale (cm2) surface area and can be used for both indirect and direct cell patterning.

Alternative Biofabrication Methods
Sonolithography as a tool for indirect and direct cell patterning. a) Tile‐scan of GFP‐HUVECs, one week after seeding onto a type I collagen‐patterned substrate. b) A higher magnification of the region marked by a white box in (a) to show individual cells in the center antinode and the first antinode ring. c) A magnified image of the center node of an untreated petri dish substrate immediately after patterning GFP‐HUVECs using the 80 µm DOD dispenser. Individual cells can be observed as dark spheres within the droplets; examples are indicated by red arrows. d) A tile scan is taken of cells deposited via DOD generator onto a type I collagen‐coated dish, 1 d after patterning. Cell‐containing droplets spread when deposited on a type I collagen‐coated dish. The acoustic field deforms the surface of the liquid layer that is formed. An increase in cell density corresponds to regions in this dish where the liquid layer was deformed. Copyright. Advanced Materials Technologies

Keywords: acoustic patterning, acoustophoresis, biofabrication, surface patterning, ultrasound

Single Cell Bioprinting with Ultrashort Laser Pulses

– Authored by Jun Zhang  Patrick Byers  Amelie Erben  Christine Frank  Levin Schulte‐Spechtel  Michael Heymann  Denitsa Docheva  Heinz P. Huber  Stefanie Sudhop  Hauke Clausen‐Schaumann. Advanced Functional Materials. March 26 2021

Single Cell Bioprinting
Laser‐based transfer of single cells and of cell clusters. a) Laser‐based cell‐transfer setup combined with inverted optical microscopy for cell selection. b) Upright optical configuration, as used for time‐resolved analysis of the transfer process shown in Figures 2a,c and 3e, and in Figures S1, S2, and S7, Supporting Information. c) Single (upper panel) and multiple (lower panel) B16F1 cells are identified with an optical microscope (bright field, 32× objective, NA = 0.6), selected and transferred from a hydrogel reservoir to a target substrate. Red crosses mark the center of the bright field images, which correspond to the laser focus position in the reservoir (left images). The laser pulse energy is 3 µJ and the focus depth is 70 µm. Scale bar for all micrographs = 50 µm. Copyright. Advanced Functional Materials

Abstract:

Tissue engineering requires the precise positioning of mammalian cells and biomaterials on substrate surfaces or in preprocessed scaffolds.

Although the development of 2D and 3D bioprinting technologies has made substantial progress in recent years, precise, cell‐friendly, easy to use, and fast technologies for selecting and positioning mammalian cells with single-cell precision are still in need.

A new laser‐based bioprinting approach is therefore presented, which allows the selection of individual cells from complex cell mixtures based on morphology or fluorescence and their transfer onto a 2D target substrate or a preprocessed 3D scaffold with single-cell precision and high cell viability (93–99% cell survival, depending on cell type and substrate).

In addition to precise cell positioning, this approach can also be used for the generation of 3D structures by transferring and depositing multiple hydrogel droplets. By further automating and combining this approach with other 3D printing technologies, such as two‐photon stereolithography, it has a high potential of becoming a fast and versatile technology for the 2D and 3D bioprinting of mammalian cells with single-cell resolution.

Single Cell Bioprinting,
a) Time‐resolved fluorescence images of the transfer of individual Alexa 532‐labeled B16F1 cells (top row), unstained B16F1 cells (middle row), and pure hydrogel (bottom row). In all cases, Alexa 532 was added to the hydrogel, in order to visualize the hydrogel jet. Scale bars = 50 µm. b) Human tendon stem/progenitor cells (hTSPC) with different lateral offsets, Δx, between the cell and the focus position of the transfer laser, before (left) and after transfer (right). c) Time‐resolved images of the transfer process of single hTSPCs with different lateral offsets, Δx, between cell and transfer laser and d) close‐up images of the jet tip. Red arrows point at the transferred cells. Scale bars = 100 µm. Laser parameters (a–d): pulse energy = 2 µJ, focus depth = 52 µm. Copyright.Advanced Functional Materials

Keywords: single cell printing, single cell sorting, 3D bioprinting, three-dimensional printing, tissue engineering

Melt electrowriting with additive manufacturing principles

– Authored by Paul D. Dalton. Current Opinion in Biomedical Engineering. June 2017

Melt Electrowriting
Examples of different non-charged and charged jets. Non-charged jets include A) the column of water (red arrow) that forms when a faucet is opened or B) when honey is run as a column from a spoon. Due to Plateau-Raleigh instabilities, such jets will break up into droplets, with increasing height or lower flow rates. C) A non-charged Golden syrup jet being deposited onto a moving collector, with the speed indicated. D) Effect of applying a voltage to water when Plateau-Raleigh instabilities are occurring. When applying a voltage, the jet becomes continuous (at 10.7 kV), and then “whips” and breaks up at even higher voltages. E) Once a continuous water column is achieved with a threshold applied voltage (between 6 and 7.8 kV), the voltage can be reduced down to a level (4 kV) well below the threshold for stable jet establishment. Increasing the voltage to 17 kV shows the electrical instabilities associated with electrospinning. C is reproduced from Ref. [33], and D and E from Ref. [32] with permission. Copyright. Current Opinion in Biomedical Engine

Abstract:

The recent development of electrostatic writing (electrowriting) with molten jets provides an opportunity to tackle some significant challenges within tissue engineering.

The process uses an applied voltage to generate a stable fluid jet with a predictable path that is continuously deposited onto a collector. The fiber diameter is variable during the process and is applicable to polymers with a history of clinical use.

Melt electrowriting, therefore, has the potential for clinical translation if the biological efficacy of the implant can be improved over existing gold standards. It provides a unique opportunity for laboratories to perform low-cost, high-resolution, additive manufacturing research that is well-positioned for clinical translation, using existing regulatory frameworks.

Melt Electrowriting
MEW scaffolds made from PCL. A) With translucent, macroscopic properties, stereomicroscopy shows B) cm-range order with C) micron-scale precision. Quantitative SEM studies on MEW scaffolds show a low coefficient of variation (4%) for 15.4 ± 0.6 μm diameter deposited fibers. Depending on the “box” size, millimeter heights while maintaining accurate printing are achievable (E), shown here in a microCT scan. When MEW scaffolds are embedded within chondrocyte-containing matrices, the composite can be mechanically loaded. When large pore MEW scaffolds (98% porosity) are seeded with dividing and ECM-producing cells, a circular morphology is typically first observed (G), that later completely fills the scaffold (H). When (I) the flow rate is altered, different diameter fibers can be generated as shown here with PCL. Such different diameter fibers can be layered upon each other, shown in (J) with a false-colored SEM image. Figure A is previously unpublished, B and C reproduced from Ref. [27], D is reproduced from Ref. [49], E reproduced from Ref. [43], F reproduced from Ref. [54], G and H reproduced from Ref. [45]. All figures reproduced with permission. Copyright. Current Opinion in Biomedical Engine

Keywords: Biomaterials, Electrohydrodynamic, Scaffold, 3D printing

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