Bioprinting ink made out of pollen

Scientists at Nanyang Technological University, Singapore (NTU Singapore) have found a way to use sunflower pollen to develop a 3D printing ink material that could be used to fabricate parts useful for tissue engineering, toxicity testing and drug delivery.

Described in the journal Advanced Functional Materials, the pollen-derived ink is able to hold its shape when deposited onto a surface, making it a viable alternative to current inks used for 3D printing in the biomedical field (also known as bioprinting). Such inks are usually soft and delicate, making it a challenge to retain the final product’s desired 3D shape and structure as the bioprinter deposits the ink layer by layer. As explained by co-lead author Professor Cho Nam-Joon, “The material of the inks used is typically too soft, which means the structure of the envisioned product may collapse during printing.”

The most widely used bioprinting method today is extrusion-based bioprinting, in which inks are dispensed continuously from nozzles and deposited along digitally defined paths to fabricate 3D structures layer by layer. One of the challenges of this method is the difficulty in retaining the 3D structures and shapes of soft delicate materials like hydrogels, cells and biopolymers without additional support. A structure called a supporting matrix, within which the soft ink is deposited during the printing process, is typically used. However, this creates waste since the supporting matrix becomes unusable after printing.

“Previous research efforts were focused on developing special bioinks for efficient deposition and printability through mixing hydrogels with fibres or particles,” said co-lead author Assistant Professor Song Juha. “The main drawback of such hydrogel composite inks is nozzle clogging, which is a more significant issue in inks with a higher content of such fibres or particles. The pollen-based hybrid ink we have developed, in contrast, is mechanically strong enough to retain its structure without jamming the printer.”

The development process of the pollen-based hybrid ink starts with incubating tough sunflower pollen in an alkaline solution — an environmentally friendly process similar to soap making — for six hours to form pollen microgel particles. The pollen microgel is then mixed with hydrogels, such as alginate, a naturally occurring polymer typically obtained from brown seaweed, or hyaluronic acid, a clear, gooey substance naturally produced by the body, to form the final pollen–hydrogel hybrid ink.

Pollen-based scaffold

As a proof of concept, the scientists printed a five-layer tissue engineering scaffold, useful for culturing cells, in 12 minutes. Collagen was added to the scaffold to provide anchor points that cells can adhere to and grow. The scientists then seeded human cells on the scaffold and found it to have a high cell-seeding efficiency of 96–97% — comparable to the inverted colloidal crystal (ICC) hydrogels that are widely utilised as 3D cell culture platforms but that are time-consuming and laborious to fabricate.

Given that pollen responds to pH changes — when an environment turns acidic or alkaline — the team also tested the viability of the 3D scaffold as a stimulus-responsive drug delivery system. When a fluorescent red dye was dripped onto the scaffold, the scientists found that the pollen microgel particles released the dye into the scaffold gradually. The amount and rate of release increased with the addition of an acid; this shows that there is potential for the pollen scaffold to be used as a drug delivery system with controlled release.

“Pollen microgel particles have a hollow shell structure, which means they could potentially be used to carry drugs, cells or biomolecules in drug delivery platforms with customised 3D structures,” Prof Cho said. “We are now looking at how we can use these pollen microgel scaffolds for 3D cell culture platforms in various biomedical applications.

“There is also potential for the pollen-based scaffold to be used as a smart drug carrier, given pollen’s stimulus-responsive nature. For instance, we can further slow down the release of drugs by coating the pollen-based scaffold with a thin layer of alginate and stimulate the release by introducing an acid.”

Support structure for soft 3D printing inks

The scientists also found that the soft and flexible pollen microgel particles, derived from tough pollen grains, could potentially serve as a recyclable support matrix for use in freeform 3D printing, in which soft ink is deposited. The support matrix prevents the collapse of the printed structure as the ink cures.

The scientists fabricated a 3D printed silicon rubber mesh for the elbow using pollen microgel as the support that would retain the elbow mesh’s shape as it is being printed. After curing the printed product at 75°C for 24 hours inside the pollen microgel, the scientists found that the printed 3D silicone rubber mesh could adapt to the human elbow curvature. They also found that the mechanical properties of the silicone rubber samples printed and cured in the pollen microgel supporting matrix were similar to those of samples fabricated via the traditional casting method.

“Utilising pollen for 3D printing is a significant achievement as the process of making the pollen-based ink is sustainable and affordable,” Prof Cho said. “Given that there are numerous types of pollen species with distinct sizes, shapes and surface properties, pollen microgel suspensions could potentially be used to create a new class of eco-friendly 3D printing materials.”

The researchers are now looking to collaborate with industry to refine their 3D printing innovation and advance its commercial uptake, with Asst Prof Song envisioning potential applications such as “customised flexible membranes that fit the human skin’s contours exactly, such as wound dressing patches or facial masks”.

“Such soft and flexible membranes are usually manufactured based on flat geometry, thus resulting in problems such as fractures in the layers or a poor fit when applied on large surface areas of skin, such as the face or areas that see frequent movement like the joints,” she said. “Using our pollen-based 3D printing ink, which is biocompatible, flexible and low in cost, we can fabricate membranes that are tailored to the contours of the human skin and are capable of bending without breaking.”

Image caption: The pollen-derived ink is able to hold its shape when deposited onto a surface, making it a viable alternative to current inks used for 3D printing in the biomedical field. Image credit: NTU Singapore.

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