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The hydrogel microstructures were fabricated using a 3D DLW system comprising a femtosecond (fs) laser beam operating at a wavelength of 535 nm (Fig. 1a). The absorption spectra of the HMPP, PEGda, and HMPP+PEGda solutions were measured using a UV-visible spectrometer and are shown in Fig. 1b. There is an absorption peak at the wavelength of 247.5 nm, which corresponds to the TPP of the hydrogel around a wavelength of 535 nm. The TPP of the HMPP+PEGda hydrogel for the fabrication of the hydrogel microstructures is shown in Fig. 1c. The HMPP photoinitiator absorbed two-photon energy from the laser beam and generated radicals in the focussing region, leading to cross-linked polymerisation between the PEGda molecules and the formation of larger molecules. By tracing the designed structures with the piezo stage, microstructures were fabricated in the hydrogel.
Fig. 1 HMPP+PEGda hydrogel for three-dimensional direct laser writing.
a Schematic of the experimental setup for 3D DLW of the hydrogel microstructures. b Absorption spectra of HMPP, HMPP+PEGda, and PEGda solutions obtained using a UV-visible spectrometer. c Schematic of the TPP of the HMPP+PEGda hydrogel using 3D DLW. d TPP laser threshold power as a function of the writing speed for the HMPP+PEGda hydrogel for different ratios of water content.The relationship between the polymerisation laser threshold power Pth and writing speed s was studied to experimentally characterise the polymerisation properties of the designed HMPP+PEGda hydrogel (Fig. 1d). The
$ {P}_{th} $ of the HMPP+PEGda hydrogel with different ratios of water content ranged from 2.8 mW to 9.0 mW for s ranging from 1 μm·s−1 to 100 μm·s−1. These values correspond to laser pulse energies ranging from 0.056 nJ to 0.18 nJ, which are lower than the maximum safety laser threshold for applications involving biological samples. In addition, the ratio of water content had a significant effect on the polymerisation efficiency of the hydrogel. Fig. 1d shows that for a fixed scanning speed, the hydrogel with 79% water content required much higher laser energy to polymerise compared with the hydrogels with lower water contents.In addition, the laser threshold power of the HMPP+PEGda hydrogel showed a power law relationship with the writing speed for shorter exposures (laser speed higher than 50 μm·s−1, corresponding to an exposure time of 270
$ \mathrm{m}\mathrm{s} $ )31,$$ {P}_{th}\propto C\times {s}^{1/N} $$ (1) where N is the nonlinearity exponent of the absorption for a specific photoinitiator, C is a coefficient related to the properties of the photoinitiators and precursors, and the laser threshold power
$ {P}_{th} $ is defined as the minimum laser power needed to initiate polymerisation at a given writing speed, which can be obtained experimentally. By varying the water content in the hydrogel samples from 0% to 79%, N was determined to be 2 regardless of the water content ratio, while C decreased from 0.5786 to 0.8792. For longer exposures (laser writing speed lower than 50 μm·s−1),$ {P}_{th} $ is significantly higher than the mathematical fit (straight dashed line) and becomes almost independent of the laser writing speed. This result can be attributed to a higher rate of radical generation in the TPP hydrogel compared to the rate of oxygen diffusion into the exposed volume, independent of the exposure time and laser writing speed. The influence of the water content on the polymerisation properties of the hydrogel can be understood through the polymerisation kinetics theory,31 according to which a higher water content ratio accelerates the movement of free radicals, necessitating a higher energy for polymerisation in the focal region. As a result, a high water content ratio requires a higher laser energy dose to form the polymerisation voxel in 3D DLW.To demonstrate the micrometre fabrication capability, high mechanical strength, and water-response properties of the HMPP+PEGda hydrogel, various types of microstructures were designed and fabricated using the single-line fabrication and layer-by-layer fabrication methods. These experiments illustrate the versatility and potential of the hydrogel for fabricating 3D scaffolds for biomedical applications.
Using the single-line fabrication method, 3D woodpile microstructures were fabricated in an aqueous environment to illustrate the micrometre fabrication capability of the TPP hydrogel (Fig. 2a). As shown in Fig. 2b, the 3D woodpile structures fabricated with different periodicities
$ a $ within a volume of 50 × 50 × 10 μm3 in the HMPP+PEGda hydrogel exhibited a highly uniform clearance similar to the size of the cell scaffold used in various works32. The normalised profiles of the woodpile structures are shown in Fig. 2b. The periods of the woodpile structures ranged from 1 μm to 2 μm, corresponding to a two-line resolution of 1 μm, and confirm the micrometre fabrication capability of the hydrogel. The 3D integrity and openness of the woodpile structures were verified by imaging the woodpile structure with a periodicity of 1 µm in water using confocal reflection mode microscopy. A green laser beam (488.5 nm) was used to scan and image the microstructure. The volumetric image of the woodpile microstructure is shown in Fig. 2c (left). Confocal images were also acquired at various focussing heights (top, middle, and bottom) in the centre of the microstructure. The images clearly show that the periodicity and the openness of the woodpile structure were maintained throughout the structure. Although the feature size from TPP fabrication using 535 nm light can be further reduced to approximately 190 nm in theory30, it is very challenging in practice to reduce the microstructure feature size because of the instability of the microstructures and the optimised fabrication conditions required for specific fabrication tasks4.Fig. 2 Single-line 3D DLW and characterisation of 3D woodpile structures with varied lattice periods.
a CAD design of the woodpile structure. b Bright-field micrographs of the woodpile structures with varied periodicities fabricated by 3D DLW with a = 1 µm, 1.5 µm, and 2 µm, and the profiles of the fabricated woodpile structures determined using ImageJ, corresponding to s1, s2, and s3 shown in the figures. The longitudinal feature size of the single-line fabrication is estimated to be 2-3 times the linewidth. c Left: Volumetric confocal image of woodpile microstructure in water with periodicity a = 1 µm. Right: Confocal images in the centre of the woodpile microstructure obtained at different focussing heights at the top, middle, and bottom. The scale bar is 4 µm in the right. The confocal images were acquired using a Nikon confocal microscope under the confocal reflection mode using a 488.5 nm laser.Based on the polymerisation properties of the hydrogel, microcubic structures were fabricated by layer-by-layer fabrication (Fig. 3a and 3b). The microcubic structures were fabricated with a layer distance of 500 nm and size of 60 × 60 × 5 μm3 under various laser powers.
Fig. 3 Fabrication of 3D microstructures using a layer-by-layer laser writing process.
a Layer-by-layer laser writing scheme. b Measured Young′s modulus of the TPP hydrogel in air as a function of the fabrication laser power, inset: optical micrograph of the microcubic structure for the measurement of the Young′s modulus. The scale bar is 20 µm. c and d 3D DLW of fractal tree variation patterns in the hydrogel. c Left: biological neuron structure. Right: Schematic design of the fractal tree structure. d Bright-field microscope images of the fabricated fractal tree structures compared with their corresponding CAD designs. The scale bar is 4 µm.The hardness of the microcubic structures in air fabricated by different laser powers is shown in Fig. 3b, and a top-view transmissive optical image of a 3D cubic microstructure fabricated with a laser power of 3.6 mW, a writing speed of 50 μm·s−1, and a layer distance of 500 nm is shown in the inset. As shown in Fig. 3b, when the laser power was lower than 3.6 mW, 3D microcubic structures could not be fabricated because of the insufficient number of free radicals generated and subsequently, the inadequate polymerisation in the exposed area. When the laser power was higher than 4.0 mW, local explosions occurred because of the excessive heat within the exposed area of the resin. The explosions consequently caused bubbles and polymer decomposition. When the energy per unit area increased, more free radicals could be generated. Hence, the crosslink density of the polymer increased, and the obtained microstructure became harder to deform. As a result, the hardness of the hydrogel increased from approximately 6 MPa to 11 MPa.
In biomedical engineering, fractal structures are of great interest because of their unique geomaterial arrangement and mechanical properties33. The fabrication of fractal structures for cell growth and cell proliferation has attracted much interest in biomedical engineering. Therefore, in this work, neuron-inspired fractal tree structures were designed and fabricated by layer-by-layer fabrication in the TPP hydrogel to further demonstrate its potential for future applications in neural tissue engineering. Fractal patterns have been found in various natural systems, such as the bifurcation in lungs34, vasculature networks35, and biological neural networks36 (as shown in Fig. 3c, left) 33.
The mathematical model of the fractal tree structure was based on Murray′s laws37, which describe a fractal branching network. The model includes the description of the geometrical parameters of a neural network (i.e., the length of each branch, its radius, and its branching angle). For a branch with a parent branch of length
$ {l}_{i} $ and right-hand daughter branch of length$ {l}_{i+1} $ (at a branching angle of$ \theta $ ) that in turn has a daughter of length$ {l}_{i+2} $ branching off to the right at angle θ as shown in the right of Fig. 3c, Murray proposed the relation$ {l}_{i+1}=1/2\;\times \;{l}_{i} $ . By increasing the branch or fractal order$ n $ , different neuron-inspired fractal structures can be generated. Four neuron-inspired fractal tree structures were designed and fabricated in the experiment. As shown in Fig. 3d, we chose the branching angle to be$ {30}^\circ $ and the branching order n to range from 1 to 4. The optical images in Fig. 3d show that the fabricated microstructures maintained a very uniform geometry that is consistent with the computer-aided design (CAD) model.Along with features such as a low laser threshold power, micrometre resolution, and relatively high mechanical strength, the TPP hydrogel also exhibits a water-responsive shape-memory effect due to its high water content ratio.
This feature was demonstrated by designing and fabricating a structure named ‘octagons to squares’ using 3D DLW and experimentally characterising its swelling and shrinking behaviours. An array of 4 × 4 identical octagons was arranged on a two-dimensional thin substrate (approximately 1 μm) and mutually connected with fixed joints (the red dots shown in Fig. 4a (right)). Each octagon has a size of d = 40 μm defined as the distance between the two farthest free joints (blue dots shown in Fig. 4a), and a thickness of z = 5.5 μm. Owing to the unique dynamic change between the octagon and square shapes36, the designed structures were able to shrink and swell, and thereby switch between the octagon and square shapes. The evaporation of water in the hydrogel microstructures caused the structures to shrink from octagons to squares. On the other hand, increasing the water content in the hydrogel microstructures caused the designed structures to swell from squares back to octagons.
Fig. 4 3D DLW and characterisation of reversible shape-memory structure ‘octagons to squares’.
a An illustration of the shrinking and swelling processes of the shape-memory microstructure (red dots represent fixed joints; blue dots represent free joints) on a thin layer of substrate. b Fabrication results and experimental observations of the reversibility of ‘octagons to squares’. c Quantitative study of the reversible behaviour of the ‘octagons to squares’ microstructure fabricated by 3D DLW. The distance between the free joints as a function of time was measured. The figure shows 2.5 cycles of the shape-change process, in which the green sections denote the swelling processes, red sections denote the shrinking processes, and the blue sections represent the saturated static state in which there is excessive water in the environment.The fabrication results and the water-responsive shape-memory effect are shown in Fig. 4b and 4c. The dynamic swelling and shrinking processes of the 3D hydrogel microstructure were quantitively characterised by measuring the distance between the two farthest joints
$ d $ (red arrow in Fig. 4b) as a function of time$ t $ . When water was added to the microstructure, the absorption of water in the hydrogel gave rise to an external force outward, relative to the fabricated structures that changed from squares to octagons (Fig. 4b (i-iii)). Quantitatively, it took the microstructure approximately 6 s to swell from a size of approximately 28.20 μm to 35.5 μm (shown in Fig. 5 with the green section, from t = 0 s to t = 6 s). When the microstructure was completely swollen, it remained in a saturated static state for a relatively long time (20-30 s) until the excess water in the microenvironment was reduced to a certain level (shown in Fig. 5 with the blue section). Compared with swelling, shrinking took a longer time (approximately 15 s). As shown in Fig. 4b (iv-vi), the evaporation of water in the microstructure resulted in an inward external force, and the octagons shrunk into squares. Therefore, the distance between the free joints in the octagons decreased from approximately 34.5 μm to 28.30 μm. The fabricated structure demonstrated a deformation ratio of 18% when swelling and shrinking. The deformation ratio is defined as the ratio of the largest distance change to the original distance, that is,$\Delta {\rm{d}} / {\rm{d}} = 18\% $ .This water-responsive shape-memory effect was further studied quantitatively by measuring the shape change multiple times. Fig. 4c shows 2.5 cycles of swelling and shrinking. Our designed hydrogel microstructure maintained its integrity after the shape changes. This indicates a relatively high degree of reversibility in the microstructure (see Movie S1 in the supporting information for the complete shape-change process). Therefore, this 3D hydrogel microstructure exhibiting a water-responsive shape-memory effect is promising for various applications in micromachines and micromanipulation.
Three-Dimensional Direct Laser Writing of PEGda Hydrogel Microstructures with Low Threshold Power using a Green Laser Beam
- Light: Advanced Manufacturing 2, Article number: (2021)
- Received: 16 March 2020
- Revised: 27 July 2020 Published online: 12 January 2021
doi: https://doi.org/10.37188/lam.2021.003
Abstract: Three-dimensional (3D) direct laser writing (DLW) based on two-photon polymerisation (TPP) is an advanced technology for fabricating precise 3D hydrogel micro- and nanostructures for applications in biomedical engineering. Particularly, the use of visible lasers for the 3D DLW of hydrogels is advantageous because it enables high fabrication resolution and promotes wound healing. Polyethylene glycol diacrylate (PEGda) has been widely used in TPP fabrication owing to its high biocompatibility. However, the high laser power required in the 3D DLW of PEGda microstructures using a visible laser in a high-water-content environment limits its applications to only those below the biological laser power safety level. In this study, a formula for a TPP hydrogel based on 2-hydroxy-2-methylpropiophenone (HMPP) and PEGda was developed for the fabrication of 3D DLW microstructures at a low threshold power (0.1 nJ per laser pulse at a writing speed of 10 μm·s−1) in a high-water-content environment (up to 79%) using a green laser beam (535 nm). This formula enables the fabrication of microstructures with micrometre fabrication resolution and high mechanical strength (megapascal level) and is suitable for the fabrication of water-responsive, shape-changing microstructures. These results will promote the utilisation of low-power 3D DLW for fabricating hydrogel microstructures using visible lasers in high-water-content environments.
Research Summary
Shape-memory hydrogel: Two-photon polymerization by green laser with low power
The fabrication of shape-memory hydrogel scaffolds not only requires biocompatibility, micrometer resolution, high mechanical strength, but also requires a low polymerisation threshold in high-water content environment to incorporate microstructures with biological tissues. Three-dimensional direct laser writing based on two-photon polymerisation is an advanced manufacturing technique to fabricate nano/microstructures in various photosensitive materials. Min Gu from China’s University of Shanghai for Science and Technology and colleagues now report development of hydrogel formula that enables two-photon polymerisation of hydrogel with low polymerization threshold (<1.5 nJ) in high water content environment (up to 79%), and strong mechanical strength (~MPa). The team also demonstrated water-responsive structures with an extraordinary shape-memory effect at a micrometer scale.
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