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4D-Bioprinting: a new way to develop complex architectures with evolving biomaterials

4D-Bioprinting: a new way to develop complex architectures with evolving biomaterials

Rheolution Article | May 2022

4D-Bioprinting: a new way to develop complex architectures with evolving biomaterials

by Dr. Antoine Frayssinet
Senior Application Specialist, Rheolution Inc.


3D-printed cell-seeded objects intended for implantation in the human body are designed to punctually palliate a specific degeneration or disease. However, those implants are static and inanimate in contrast to the environment of native tissues which is dynamic, where tissues are subjected over time to fluctuating biophysical, biochemical and mechanical stimuli. With 4D-bioprinting, the scientific community recently integrated “time” as the fourth dimension: scaffolds and hydrogels have the ability to evolve over time after being printed, either biologically, or with the application of an external stimulus.

Firstly, post-printing shape-shifting can be induced by subjecting the hydrogel to physical stimuli e.g. a magnetic field, a temperature change (e.g. with poly(N-isopropy-lacrylamide (PNIPAAm) or poly(ε-caprolactone) (PCL)), light irradiation or humidity (e.g. with bilayered polyethylene glycol (PEG)). Last week’s publication Article of the Month on 4D-Bioprinting presented an explicit example of an alginate-based hydrogel, sensitive to light and able to fold into complex shapes depending on the light exposure induced after printing. The complexity of designed shapes can be quite impressive, with e.g. cellulose fibrils mixed with acrylamide used by Dr. Lewis’s team to reproduce orchid flower design with biocompatible composite ink (Figure below) [3]. These extruded bulk hydrogels can thus change shape after being printed. The post-printing desired effect can however be different.

The shape can be designed during the printing process with a multi-scale approach e.g. with inkjet bioprinting, and the delivery of encapsulated drugs can be triggered afterwards. Liposomes – bilayered lipid microspheres synthetically mimicking the cell membranes – were for instance printed containing two different salt concentrations. By playing on this induced osmolarity gradient, the authors were able to shape 3D-macrospheres that could, upon temperature or pH change, release the components loaded inside each printed microsphere [4].

Another group from the ETH Zürich engineered nano-hydrogels with non-cytotoxic photo-crosslinkable methacrylated gelatin (GelMA). Specifically developed for targeted drug delivery, those nano-hydrogels, also called “microswimmers”, were rendered magnetically responsive by decorating their surface with magnetic nanoparticles [5]. Printed with a helix shape in order to optimize their swimming performances in body fluids, such microswimmers are stable drug carriers that can be directed with magnetic fields to the targeted tissus. Once in position, they can deliver the drug they were loaded with, and then rapidly (5-15 min) be degraded by collagenase (enzyme that digest collagen and gelatin), with non-cytotoxic degradation residues. In this case, the fourth dimension is expressed as the enzymatic degradation of the gel over a short period of time.

Indeed, biological signals such as glucose concentration or specific enzymes can have an impact on printed hydrogels. A joint study between the University of North Carolina, USA and Soochow University, China reported in 2018 a poly(vinyl alcohol) (PVA) based biomaterial sensitive to reactive oxygen species (ROS), biologically produced in tumor microenvironments. With this engineered biodegradation, the PVA-based hydrogel was able to enhance in vivo antitumor response and prevent tumor recurrence by delivering locally the antitumoral drug, before being degraded after 3 weeks [6]. Following the same principle, pH sensitive biomaterials such as collagen, gelatin and keratin could be of use near inflammation sites, where pH values drop.



Functionality for regenerative hydrogels can also be progressively developed with the maturation of the scaffold itself, where the seeded cells, responding to their environment, will work as a micro-factory to produce their own matrices and phenotypic profiles, adapted to the targeted degenerated tissue (see Figure above). The nature of this micro-production can be tuned with growth factors, differentiation factors (in case of seeded stem cells) or for instance with the mechanical properties of the implant [7]. Considering this cellular activity when developing a biomaterial for clinical use isn’t opposed to the biomaterial post-printing stimulation itself. Following an origami technique, Kuribayashi-Shigetomi et al. were able to harness the cell traction force of fibroblasts (fusiform common cells), to generate complex 3D-microstructures such as a cube, a sphere or a hollow tube. In this case, cells were seeded on partially fibronectin-covered glass microplates. The fourth dimension was introduced with the ability of these cells to pull on their environment over time, used to design 3D-scaffolds [8].

In recent years, 4D-Bioprinted biomaterials were investigated to develop stents – hollow fabric placed e.g. in an artery or a vein, to strengthen its structure. The clinician can easily insert the curled-up stent inside the human tissue, and then with a specific stimulus, extend it so it supports the human vessel efficiently. Successful designs were produced with a methacrylated PEG-base biomaterial, alginate or with hyaluronic acid [9][10]. To bring this short review to a close with a last application, a smart wound dressing was also developed by Dr. Akbari’s team in Canada: the dressing detects bacterial infection by monitoring the pH value (with an outside display of specific colors, visual markers dependent on the measured pH). Once the infection is detected, the printed hydrogel releases antibiotic agents at the wound site to counteract the infection [11].

With many applications, 4D-Bioprinting is a very promising technology for the biomedical field. There are still challenges remaining, such as having control over the cell alignment to allow to more closely mimic native tissues. Such an example of a micro-anisotropic cell alignment was reported in the study presented in our Article of the Month published in March. Another significant challenge is to develop bioinks with features appropriate to the targeted degenerated tissue, in terms of mechanical properties, cell population and phenotype, extracellular matrix nature and complexity. Overall, using the different 3D-bioprinting processes presented in our last Rheolution Article in April, 4D-Bioprinting optimistically progresses from “proof-of-concept” studies toward clinical applications.


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