Synthetic and Natural Bioinks

Category: Blog,From Academia
blank blank May 03, 2021

In this “From Academia” blog, we focus on a key ingredient for successful bioprinting, the bioinks. The first article is a recently published review article that will lay the foundation of various bioprinting methods as well as a special focus on the natural, synthetic, or hybrid materials used as bioinks. This article also addresses the challenges, limitations, and future directions concerning the bioprinting technique. This second article shows how bioprinting and organoid technology can be merged to generate centimeter-scale tissues that have self-organized features including lumens, branched vasculatures, and tubular intestinal epithelia with in vivo-like crypts and villus domains. This method could potentially be used to produce larger functional tissue with more geometry and cellular control. The third article introduced a new hydrogel bioink composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The researchers show that 3D printed constructs with this new bioink demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue. “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.

Natural and Synthetic Bioinks for 3D Bioprinting

– Authored by Dr. Roghayeh Khoeini  Dr. Hamed Nosrati  Dr. Abolfazl Akbarzadeh  Dr. Aziz Eftekhari  Dr. Taras Kavetskyy  Prof. Rovshan Khalilov  Dr. Elham Ahmadian  Dr. Aygun Nasibova  Dr. Pallab Datta  Dr. Leila Roshangar  Dr. Dante C. Deluca  Dr. Soodabeh Davaran  Prof. Magali Cucchiarini  Prof. Ibrahim T. Ozbolat. Advanced Nanobiomed Research. March 30 2021

Abstract:

Bioprinting offers tremendous potential in the fabrication of functional tissue constructs for replacement of damaged or diseased tissues. Among other fabrication methods used in tissue engineering, bioprinting provides accurate control over the construct’s geometric and compositional attributes using an automated approach. 

bioprinting technique
Extrusion-based bioprinting. (A) Piston-driven dispension, (B) screw-driven dispension, and (C) 2 pneumatic dispensing. Copyright. Advanced Nanobiomed Research.

Bioinks are composed of the hydrogel material and living cells that are critical process variables in the fabrication of functional, mechanically robust constructs. Appropriate cells can be encapsulated in bioinks to create functional tissue structures. Ideal bioinks are required to undergo a sol‐gel transition consuming minimal processing time, and a plethora of chemical and physical crosslinking mechanisms are generally exploited to achieve high shape fidelity and construct stability. On the other hand, crosslinking of hydrogel material at rapid rates can cause nozzle clogging, and hence, optimization of the bioink is often necessary. Bioinks can be formulated using natural or synthetic biomaterials, alone or in combination of these biomaterials. 

bioprinting technique
Inkjet bioprinting including thermal and piezoelectric inkjet bioprinting. Copyright. Advanced Nanobiomed Research.

In this review, we discuss the various bioprinting methods and analyze the natural, synthetic, or hybrid materials used as bioinks and appraise the challenges, limitations, and future directions concerning the bioprinting technique.

bioprinting technique
Laser assisted bioprinting. Copyright. Advanced Nanobiomed Research.

Keywords: bioink, bioprinting, hybrid materials, tissue engineering

Recapitulating macro-scale tissue self-organization through organoid bioprinting

– Authored by Jonathan A. Brassard, Mike Nikolaev, Tania Hübscher, Moritz Hofer & Matthias P. Lutolf. Nature Materials, September 21 2020

Synthetic and Natural Bioinks
Illustration of the BATE (bioprinting-assisted tissue emergence)concept using spontaneously self-organizing building blocks to create large-scale tissues. Copyright. Nature Materials

Abstract:

Bioprinting promises enormous control over the spatial deposition of cells in three dimensions but current approaches have had limited success at reproducing the intricate micro-architecture, cell-type diversity and function of native tissues formed through cellular self-organization. We introduce a three-dimensional bioprinting concept that uses organoid-forming stem cells as building blocks that can be deposited directly into extracellular matrices conducive to spontaneous self-organization. By controlling the geometry and cellular density, we generated centimetre-scale tissues that comprise self-organized features such as lumens, branched vasculature and tubular intestinal epithelia with in vivo-like crypts and villus domains. Supporting cells were deposited to modulate morphogenesis in space and time, and different epithelial cells were printed sequentially to mimic the organ boundaries present in the gastrointestinal tract. We thus show how biofabrication and organoid technology can be merged to control tissue self-organization from millimetre to centimetre scales, opening new avenues for drug discovery, diagnostics and regenerative medicine.

Synthetic and Natural Bioinks
Synthetic and Natural Bioinks
Fig. 2 | Macroscopic intestinal tube printing. a, Illustration of BATE applied to intestinal tissue engineering. Robust control over cellular density and tissue geometry can be achieved directly inside environments permissive to multicellular self-organization. b, Bright-field images of tube evolution. Insets show the dense condensation phase (day 2), the formation of a lumen and appearance of budding structures (day 6) and the formation of crypts with dark Paneth cells (day 9). Images are representative of n> 10 biologically independent experiments. Scale bars, 200 μm. c, Intestinal tube after 21 days of culture showing an intact epithelium despite large accumulation of dead cells (right) and classical organoid culture before passaging (right) for size
comparison. Scale bars, 200 μm. d, For two tubes, mean tube diameter and total length versus number of days in culture. Standard deviation of the mean along the tube’s length is indicated for the diameter. e, Influence of bioink cell density on tube diameter after 6 days of culture. Results of three different experiments are shown with two tubes for each. **P= 0.0074 (left) and 0.0022 (right), ****P< 0.0001, determined by one-way ANOVA with Tukey’s multiple comparisons test. f, Histological cross-section stained with Hematoxylin and Eosin to visualize the continuous lumen and cellular organization. Images are representative of n= 3 biologically independent experiments. Scale bar, 200 μm. g, Macroscopic images of intestinal tube spanning >15 mm. Images are representative of n= 3 biologically independent experiments.

Keywords: Assay systems, bioinspired materials, stem-cell biotechnology, tissue engineering, tissues

3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix

– Authored by Yu Jung Shin, Ryan T. Shafranek, Jonathan H. Tsui, Jelisha Walcott, Alshakim Nelson, Deok-Ho Kim. Acta Biomaterialia. January 1 2021

Synthetic and Natural Bioinks
Schematic overview of the cdECM bioink preparation and fabrication of 3D bio-printed constructs. Schematic overview of the fabrication and printing of engineered cdECM bioinks. Fabrication of the bioink involved decellularization and solubilization of cardiac tissues into pre-gels which were modified with Laponite to afford a viscoelastic bioink capable of providing a high-fidelity print. Incorporation of the cross-linker PEG-DA and a photo-initiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, allowed tunable mechanical properties by changing PEG-DA concentration. Copyright. Acta Biomaterialia

Abstract:

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. 

Bio-based materials that support cellular adhesion, differentiation, and proliferation – including gelatin, collagen, hyaluronic acid, and alginate – have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). 

Synthetic and Natural Bioinks
Demonstration of 3D printability of cdECM composite bioink in the z-direction. 3D printing of multiple stacks of square prints was conducted to demonstrate the ability to fabricate a mechanically stable, layered 3D construct in the (vertical) z-direction. (a) Schematic of the 3DP process involved in an 18-layered tower. Each print was conducted using a 25G nozzle at 9 psi or a 22G nozzle at 8 psi with the low-modulus bioink (6.0% PEG-DA) and immediately cross-linked using blue-light (405 nm) irradiation for 30 s. This process was repeated 18 times to create a stable, 18-layered construct. (b) Picture of an 18-layered construct printed with a 22G nozzle at 8 psi. (c) Analysis of the linewidth quantified for different heights of the 18-layered construct printed with a 25G nozzle at 9 psi post-print (n = 4) and (d) top view of the construct printed with a 25G nozzle at 9 psi. The measurements were taken using calipers every 1 mm until the final layer. Copyright. Acta Biomaterialia

The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states.

 The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Keywords: Direct-ink writing,Bioinks,Decellularized extracellular matrix,Cardiac tissue engineering

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