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3D printing, UV light control thickness and crosslinking of hydrogel layers

By Frieda Wiley November 12, 2018
Scanning electron microscopy image of a three-dimensional-printed complex object. Scale bar is 500 μm. Photo courtesy of Hang Win, PhD.

Ensuring engineered tissue has the ideal integrity, or biofunctionality, that allows it to support intracellular communication is a critical element in support tissue regeneration. However, engineering a hydrogel that embodies the integrity necessary to support biofunctionality without promoting pathogenesis, or the development and progression of disease, can be difficult to achieve; but researchers at the University of Colorado, Boulder have found a way to accomplish this by employing a three-dimensional (3D) biostereolithographic printing technology in which they could control the hydrogel’s stiffness. According to their study published in a recent issue of Nature Communications, the researchers developed a multi-layer, heterogeneous hydrogel and were able to control its stiffness by using free radical polymerization (FRP), a formation that occurs when numerous free radicals unite to create large molecules, or polymers, with ultraviolet light (UV).

The research team 3D printed a hydrogel structure using a stereolithographic technique in which a computer-modulated laser builds structures by crosslinking layers sequentially—layer by layer. The UV light initiates FRP by activating an initiator within the liquid. The researchers incorporated an oxygen inhibition layer between the cured polymer structure and an oxygen-permeable window. The window was a 10-µm-thick polydimethylsiloxane (PDMS) film coated on a glass substrate. The PDMS coating is highly permeable to oxygen from the surrounding air. The freely diffusing oxygen forms chain-terminating peroxide molecules upon encountering free radicals that inhibits FRP. This process “physically limits the curing thickness during the layer-by-layer construction process in stereolithography,” the authors wrote. Ultraviolet light exposure increases the crosslinking, which subsequently increases the stiffness of the hydrogel. In their work, the researchers were able to control the degree of hydrogel crosslinking within the layer, and thereby the hydrogel stiffness, while maintaining the thickness of the layer.

“This study demonstrates that, by altering the degree of UV light exposure, you can control the degree of crosslinking within a layer,” says Juan Taboas, assistant professor with the department of oral biology in the School of Dental Medicine and the Department of Biomedical Engineering in the Swanson School of Engineering at the University of Pittsburgh. “This is a big deal because 3D printers print layer by layer and you want the layers to be of uniform thickness.”

Tissue regeneration can be a balancing act that requires the perfect blend of cells and a hydrogel matrix with specific degree of stiffness. Hydrogels that are too flexible lack the integrity to support conductive properties and facilitate intercellular communication. On the other end of the spectrum, bioengineered tissues that are too stiff can become pathogenic, or disease-causing. These rigid structures can result in conditions such as heart disease and breast cancer.

Previously considered a hindrance in free radical polymerization, oxygen inhibition under highly controlled setting can create variations in stiffness within a monolithic structure. The researchers selected polyethylene glycol dimethalcrylate (PEGDMA, Mw 750) as their hydrogel material due to its free-radical and UV-curable profile. PEGDMA is widely used in the biomedical research community for these convenient features. According to the research team, the approach should be applicable to free-radical cross-linking materials.

The UV radiation dose and depth of the solution in which the PEGDMA is bathed in the presence of a constant oxygen concentration of 0.35 mol m-3 with an oxygen diffusion rate of 2.84 × 10-11m2s-1 moderate the rate at which double bonds form in crosslinked PEGDMA. When UV exposure falls below a threshold of approximately 20 mJ cm-2 in the presence of oxygen, oxygen inhibits FRP, but double bond conversion does not occur. At higher levels of UV radiation exposure, the research team noted having observed an oxygen inhibition layer on top of the PDMS accompanied by a minimal-yet-constant decrease in layer thickness. The greater the exposure to UV light the hydrogel receives, the faster is the rate of double-bond conversion, or cross-linking.

To illustrate how 3D-printing technology can modulate stiffness and geometry independently in the presence of an oxygen inhibition layer, the research team 3D-printed a buffalo logo. This was accomplished by exposing the logo to higher doses of UV light than the logo’s background. After doing so, the research team observed different stiffness between the two regions while maintaining uniform heights in other regions.

Although further studies are needed for validation before exploring tissue regeneration application, Wei Tan, associate professor of mechanical engineering at the University of Colorado, Boulder and the study’s corresponding author, believes this study also sheds light on the utility of the often-overlooked heterogeneous structures. “These heterogeneous structures do not receive as much attention as other biomechanical structures, and I think the most important finding is that we consider heterogeneous properties that actually require biotissue regeneration,” Tan says. For example, “The vascular tissue has three layers, and each layer has a different biomechanical property,” she says.

Read the article in Nature Communications