From Academia: 3D Printed Lens, Silk as Biomaterial, Aspiration-assisted freeform bioprinting

Category: Blog,From Academia
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From Academia” features recent, relevant, close to commercialization academic publications in the space of healthcare 3D printing, 3D bioprinting, and related emerging technologies. In this issue, we included an article focusing on how a 3D printed lens using two-photon lithography can help with SRS microscopy, an imaging technique for living cells; an article on how silk nanofiber can improve extrusion-based 3D bioprinting; an article focusing on aspiration-assisted freeform bioprinting of cellular spheroid in yield stress gel.

Email: Rance Tino (tino.rance@gmail.com) if you want to pen an Expert Corner blog for us or want to share relevant academic publications with us.

3D-Printed high-NA catadioptric thin lens for suppression of XPM background in Stimulated Raman Scattering microscopy

Authored by Andrea Bertoncini, Sergey P. Laptenok, Luca Genchi, Vijayakumar P. Rajamanickam, Carlo Liberale. Journal of Biophotonics, 9 July 2020 

Images of the 3D printed lens (left). Optical image of the lens 3D printed on a glass substrate (microscope coverslip #1,5) (right). SEM image of the central part of the lens, showing the central Fresnel-lens-like refractive part and the outer reflective part. Copyright. Journal of Biophotonics
Images of the 3D printed lens (left). Optical image of the lens 3D printed on a glass substrate (microscope coverslip #1,5) (right). SEM image of the central part of the lens, showing the central Fresnel-lens-like refractive part and the outer reflective part. Copyright. Journal of Biophotonics

Stimulated Raman Scattering (SRS) imaging with different collection optics. A, SRS images of a IP-S 3D printed grid at a nonresonant wavenumber (2550 cm−1). A strong Cross Phase Modulation (XPM)-related background is visible with the 0.4 NA lens, while it is suppressed with the 3D printed lens and the high NA microscope objective. B, SRS images of the same grid as above at 2950 cm−1 wavenumber, where the photopolymer Raman spectrum has a peak. C, SRS images of a HepG2 cancer cell at 2850 cm−1, highlighting lipid droplets in the cell. The suppression of XPM artifacts with 3D printed lens is comparable to the high-NA microscope objective. Excitation was done with the 1.27 NA microscope objective. Copyright. Journal of Biophotonics
Stimulated Raman Scattering (SRS) imaging with different collection optics. A, SRS images of a IP-S 3D printed grid at a nonresonant wavenumber (2550 cm−1). A strong Cross Phase Modulation (XPM)-related background is visible with the 0.4 NA lens, while it is suppressed with the 3D printed lens and the high NA microscope objective. B, SRS images of the same grid as above at 2950 cm−1 wavenumber, where the photopolymer Raman spectrum has a peak. C, SRS images of a HepG2 cancer cell at 2850 cm−1, highlighting lipid droplets in the cell. The suppression of XPM artifacts with 3D printed lens is comparable to the high-NA microscope objective. Excitation was done with the 1.27 NA microscope objective. Copyright. Journal of Biophotonics

Abstract: 

Stimulated Raman Scattering (SRS) is a fast chemical imaging technique with remarkable bioscience applications. Cross Phase Modulation (XPM) is a ubiquitous nonlinear phenomenon that can create spurious background signals that render difficult a high‐contrast imaging in SRS measurements. The XPM‐induced signal is usually suppressed using high numerical aperture (NA) microscope objectives or condensers to collect the transmitted excitation beam. However, these high NA optics feature short working distances, hence they are not compatible with stage‐top incubators, that are necessary to perform live‐cell time‐lapse experiments in controlled environments. Here, we show a 3D printed high NA compact catadioptric lens that fits inside stage‐top incubators and allows the collection of XPM‐free SRS signals. The lens delivers SRS images and spectra with a quality comparable to a signal collection with a high‐NA microscope objective. We also demonstrate the compatibility of the 3D printed lens with other nonlinear microscopies usually associated with SRS in multimodal microscopes.

Silk fibroin nanofibers: a promising ink additive for extrusion three-dimensional bioprinting

Authored by S.Sakai, A.Yoshii, S.Sakurai, K.Horii, O.Nagasuna. Materials Today Bio. September 2020

Images of silkworm cocoon, degummed silk fibroin fibers, silk fibroin nanofibers (SFNFs) obtained by grinding of silk fibroin fibers dispersed in water, and ink-containing SFNFs used for 3D bioprinting. Copyright. Materials Today Bio
Images of silkworm cocoon, degummed silk fibroin fibers, silk fibroin nanofibers (SFNFs) obtained by grinding of silk fibroin fibers dispersed in water, and ink-containing SFNFs used for 3D bioprinting.Copyright. Materials Today Bio
a) Effects of SFNF addition on the width of hydrogel filament obtained by extruding 1.5 w/v% HA-Ph (HA-Ph) and 0.1w/v% HA-Phþ0.9 w/v% HAþ1.0 w/v%gelatin-Ph (HA-Phþgelatin-Ph) inks free ofSFNFs and containing 1.0 w/v% SFNFs(þ). Bars: mean±S.D. (n¼3). Blue prints and the blueprint-based hydrogels with b) lattice, c) nose, and d) ear shape obtained from 1.5 w/v% HA-Ph inks free of SFNFs[SFNFs] and containing 1.0 w/v% SFNFs[SFNFs (þ)]. The content of HRP in the inks was 5 U/mL. HA, hyaluronic acid; HRP, horseradish peroxidase; Ph, phenolic hydroxyl; SFNFs, silk fibroin nanofibers; S.D.,standard deviation. Copyright. Materials Today Bio
a) Effects of SFNF addition on the width of hydrogel filament obtained by extruding 1.5 w/v% HA-Ph (HA-Ph) and 0.1w/v% HA-Phþ0.9 w/v% HAþ1.0 w/v%gelatin-Ph (HA-Phþgelatin-Ph) inks free ofSFNFs  and containing 1.0 w/v% SFNFs(þ). Bars: mean±S.D. (n¼3). Blue prints and the blueprint-based hydrogels with b) lattice, c) nose, and d) ear shape obtained from 1.5 w/v% HA-Ph inks free of SFNFs[SFNFs] and containing 1.0 w/v% SFNFs[SFNFs (þ)]. The content of HRP in the inks was 5 U/mL. HA, hyaluronic acid; HRP, horseradish peroxidase; Ph, phenolic hydroxyl; SFNFs, silk fibroin nanofibers; S.D.,standard deviation.Copyright. Materials Today Bio

Abstract: 

Here, we investigated the usefulness of silk fibroin nanofibers obtained via mechanical grinding of degummed silkworm silk fibers as an additive in bioinks for extrusion three-dimensional (3D) bioprinting of cell-laden constructs. The nanofibers could be sterilized by autoclaving, and addition of the nanofibers improved the shear thinning of polymeric aqueous solutions, independent of electric charge and the content of cross-linkable moieties in the polymers. The addition of nanofibers to bioinks resulted in the fabrication of hydrogel constructs with higher fidelity to blueprints. Mammalian cells in the constructs showed >85% viability independent of the presence of nanofibers. The nanofibers did not affect the morphologies of enclosed cells. These results demonstrate the great potential of silk fibroin nanofibers obtained via mechanical grinding of degummed silkworm silk fibers as an additive in bioinks for extrusion 3D bioprinting.

Aspiration-assisted freeform bioprinting of pre-fabricated tissue spheroids in a yield-stress gel

Authored by Bugra Ayan, Nazmiye Celik, Zhifeng Zhang, Kui Zhou, Myoung Hwan Kim, Dishary Banerjee, Yang Wu, Francesco Costanzo & Ibrahim T. Ozbolat. Nature Communications Physics. 16 October 2020

a The bioprinting setup, where a box was filled with the yield-stress gel in one compartment and cell media in the other. b A schematic showing the process of spheroid traverse across the yield-stress gel and media compartment. c, d Schematics showing physical parameters involved in transferring of spheroids from the cell media to the yield-stress gel, FR is the magnitude of the resultant force acting on the spheroid due to its interaction with the environment, r is the nozzle’s radius, U is the bioprinting speed, and Pb, the critical aspiration pressure, is a function of R, r, U, gel properties (K, n, τ0). e Images showing a step-by-step illustration of the process. Copyright. Nature Communications Physics
a The bioprinting setup, where a box was filled with the yield-stress gel in one compartment and cell media in the other. b A schematic showing the process of spheroid traverse across the yield-stress gel and media compartment. c, d Schematics showing physical parameters involved in transferring of spheroids from the cell media to the yield-stress gel, FR is the magnitude of the resultant force acting on the spheroid due to its interaction with the environment, r is the nozzle’s radius, U is the bioprinting speed, and Pb, the critical aspiration pressure, is a function of R, r, U, gel properties (K, n, τ0). e Images showing a step-by-step illustration of the process. Copyright. Nature Communications Physics
Schematic illustration and optical photographs of 3D bioprinted a helix-shape (mesenchymal stem cell (MSC) spheroids), b initials of Penn State University (PSU, MSC spheroids), c five-layer tubular (MSC spheroids), and d double helix-shape constructs using MSC spheroids with 150 μm (F-actin) and 450 μm (Hoechst) in radius in 1.2% Carbopol yield-stress gel. The red dashed line denotes the region magnified. Copyright. Nature Communications Physics
Schematic illustration and optical photographs of 3D bioprinted a helix-shape (mesenchymal stem cell (MSC) spheroids), b initials of Penn State University (PSU, MSC spheroids), c five-layer tubular (MSC spheroids), and d double helix-shape constructs using MSC spheroids with 150 μm (F-actin) and 450 μm (Hoechst) in radius in 1.2% Carbopol yield-stress gel. The red dashed line denotes the region magnified. Copyright. Nature Communications Physics

Abstract: 

Bioprinting of cellular aggregates, such as tissue spheroids, to form three-dimensional (3D) complex-shaped arrangements, has posed a major challenge due to lack of robust, reproducible and practical bioprinting techniques. Here, we demonstrate 3D aspiration-assisted freeform bioprinting of tissue spheroids by precisely positioning them in self-healing yield-stress gels, enabling the self-assembly of spheroids for fabrication of tissues. The presented approach enables the traverse of spheroids directly from the cell media to the gel and freeform positioning of the spheroids on demand. We study the underlying physical mechanism of the approach to elucidate the interactions between the aspirated spheroids and the gel’s yield-stress during the transfer of spheroids from cell media to the gel. We further demonstrate the application of the proposed approach in the realization of various freeform shapes and self-assembly of human mesenchymal stem cell spheroids for the construction of cartilage and bone tissues.

File Name: Supplementary Movie 2 Description: Bioprinting of spheroids in yield-stress Carbopol gel (2X Speed)

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