Fabrication and basic evaluation of fabricated MAPs In this study, we fabricated and used two types of female moulds to prepare MAPs: one was a base mould without patterns. The other was the mould for the patterned MAPs (Supplementary Fig. S1).
First, we fabricated the MAPs using a mixed solution of hyaluronic acids and tattoo ink. We evaluated the feasibility of each mixing ratio and the formation of MNs in the elaborated MAPs. Here, base female moulds with no internal patterns were used for fabricating all MAPs. Fig. 2a shows the results for different mixing ratios. Among the results obtained with all mixing ratios, we obtained fragmented and cracked MAP with a 1:1 mixing ratio. Carbon black could be considered one of the constituents of tattoo ink that causes an incomplete and cracked internal structure of the MAP27,28. Except for the 1:1 mixing ratio, all MAPs with other ratios were successfully fabricated with intact MN structures. Regarding the dimension of MNs for each step, MN lengths of a metal mould, a PDMS female mould, and fabricated MNs measured 872.2±1.6, 858.0±1.2, and 829.8±2.3 μm, respectively (Fig. 2a, Supplementary Fig. S2). Approximately 1.6–3.2% shrinkage was observed at the midpoints of processes, including the curing process of PDMS mould fabrication and the drying process of MAPs. Similarly, the base lengths of the MNs decreased by approximately 1.6 and 5.3% during the female mould fabrication and drying processes, respectively (Fig. 2a, Supplementary Fig. S2).
Subsequently, the failure force of the MNs in each MAP was evaluated using a compression test (Fig. 2b). In particular, we measured the forces at which the inflexion points were observed in the force-displacement graphs (red rectangles). The average forces measured with each mixing ratio, 3, 5, 7, and 15:1, were 146.2±13.2, 302.4±20.7, 605.3±31.7, and 603±23.4 mN, respectively. Considering the mechanical strengths from previous studies, we considered that the fabricated MNs with all mixing ratios had sufficient mechanical strength (56 mN) to penetrate the skin layer29. In addition, our MNs exhibited much higher mechanical strength than hyaluronic acid-based MNs30. We attribute the carbon black pigment in tattoo inks to an increase in mechanical strength for the same reason described above. From the results, we finally chose a mixing ratio of 7:1 because the MNs have higher mechanical strength and more ink volume than those with 3:1 or 5:1 mixing ratios.
Fabrication of patterned female mould We fabricated female moulds that were used for MAPs with specific patterns. Before fabrication, we designed patterns, including letter characters, numbers, and symbols so that they could be fabricated using patterned moulds and expressed in one MAP for identification.
First, we set one MN, the inserted part of a MN during penetration, in the MN array as one dot of biotagging patterns. Thus, 169 dots, the same number as the cavities in the female base mould, existed in one MAP because the basic array of MNs was designed with a 13 × 13 size. In addition, we divided the entire array of 13 × 13 into three parts, including 6 × 13, 2 × 13, and 5 × 13, to express letters, spaces, numbers, and symbols, respectively (Fig. 3a). Then, one row at the top and bottom was blanked, considering the entire longitudinal length of the patterns. Thus, one MAP could express one English letter and number or symbol. The spaces and horizontal widths inside the array were designed such that each pattern could express a character that was sufficiently recognisable by the naked eye. In addition, various letters or symbols can be expressed by adjusting the composition of MN array and number of MNs
Inside the pattern, the spaces between each dot, both vertically and horizontally, were designed to be the same as the centre-to-centre distance of the MNs in the female base moulds. In addition, we set a virtual square block surrounding each dot pattern to realise the pillar structures of each patterned plug as a support for holding the plug shapes. These square blocks and dot patterns are indicated by red rectangles in Figure 3(a). Finally, we designed various patterns for the patterned plugs, considering the dimensions of the female base mould, processability, and readability. They included 26 English letter character patterns, 10 number patterns, 7 symbol patterns, one three-letter combination, and Chinese character patterns presenting the authors’ affiliations (Supplementary Fig. S3).
The detailed structures of the patterned plugs were designed to have pillars with pyramidal-shaped tips at the end (Fig. 3b). These pyramidal shapes can be used for positioning and filling cavities that should be left open in the base mould when fabricating the patterned moulds. In addition, the pillar structures support pyramidal tips, as described above, and secure the space for introducing a diluted PDMS mixture to fill unnecessary cavities. For dimensions, as described earlier in the Materials and Method section, pyramidal shapes at the tip of plugs were designed as 810 and 440 μm for the length and the base, respectively, so that all tips of pattern parts can fit in the cavities of a base mould. In this study, patterned plugs were fabricated with five different combinations that include the combination of a letter and number, and abbreviation letters of authors’ affiliations: A1, C3, E5, UT, and IIS.
Figure 3(c) shows plugs fabricated with different patterns. Pyramidal and supporting pillars were fabricated, as shown in the magnified inset. The pyramidal tips had a length of 600.7±25.4 μm and a base length of 365.7±1.7 μm. The pillar structures had designed lengths of 10 mm and 9.9 mm for the fabricated plugs. The vertical length was shorter than the designed value. In this regard, the dimensional changes might be caused by the time of exposure during 3D printing and the printing mechanism. The 3D printer created patterns layer by layer by utilizing UV exposure to the UV-curable resin. The exposure time for each layer significantly affected the vertical resolution and the resultant dimensions. In particular, increasing the exposure time and adjusting the designed dimensions might solve this issue. Although vertical lengths of pyramidal tips were confirmed to be smaller than those designed, the centre-to-centre distance, which is the most important for assembling a base mould and a pattern plug, was well matched: 994.5±0.6 μm for a female base mould and 993.7±0.9 μm for a fabricated pattern plug.
Finally, we confirmed that the fabricated patterned plugs fit well inside the cavities of the base mould, with uniform contact with the surfaces. Moreover, unnecessary cavities were successfully filled with masking cavities for patterns (Fig. 3d). We observed that the diluted PDMS completely merged with the base mould after curing and removing the plugs. The patterned moulds were cleaned using organic solvents and stored before the fabrication of the patterned MAPs.
Fabrication of patterned MAPs using patterned moulds Using five different patterned plugs, we successfully fabricated patterned MAPs (Fig. 4). The fabrication process was the same as that for the MAPs without patterns. The final MAPs were square-shaped with a side length of approximately 20 mm. The average thickness of the back layer was approximately 413 μm. Inside the MAPs, the pyramidal tip parts protruded with a step from the back layer because the step structures were formed in patterned moulds by the plug pillars during the filling process. The representative step structures are marked with red arrows in Figure 4. The thickness of steps was 447.7±16.6 μm among fabricated MAPs. Here, we consider step structures as having both advantages and disadvantages. Steps are expected to play the role of a pedestal for MNs, resulting in improved skin penetration during MAP application31. However, step structures can affect the flexibility of MAPs as they may contribute to an increase in MAP thickness. As the step structures are formed by the residue of the diluted PDMS mixture as well as pillars of patterned plugs during the filling process, we consider that the step thickness can be decreased or adjusted by controlling the amount of diluted PDMS mixture for filling.
Animal experiments: Evaluation of MAP’s dissolution during its application Subsequently, animal experiments were performed using the fabricated patterned MAPs. Before the biotagging experiments, the application time of MAP was evaluated to determine the length of the dissolved parts and the proper time for rat experiments. Figure 5 indicates that the length of the dissolved MNs increases with the application time. The back layer began to dissolve after 7 min. We considered that the back layer was partially in contact with the liquefied portion of the dissolved MNs as the application time increased, resulting in the subsequent dissolution of its surface.
The percentage values with the length dissolved represent how much the MNs were dissolved compared to the original lengths. Consequently, we finally chose 7 min for further animal experiments considering an entire length dissolution of over 90%.
Animal experiments: Biotagging as the direct formation of identification patterns on skin As a final experiment, biotagging was performed using patterned MAPs to form identification patterns on rats’ skin directly. First, we evaluated the formation of designated patterns on the skin after applying MAPs, starting on day zero. Figure 6 illustrates the pattern-formation results obtained using the patterned MAPs. In particular, the figure shows that most of the MNs in MAPs penetrated the skin, and the ink mixture was delivered inside the skin successfully. The diameters of the dots confirmed on the surface range from 240 to 400 μm. The diameters of the dot patterns may vary according to the skin penetration depth. From day 0, when the MAPs were first applied, we confirmed that all the formed patterns were clearly visible to the naked eye. The results from day 10 also confirmed that all patterns remained without any loss or changes compared to those on day 0. Thus, we concluded that the ink mixture was delivered into the dermis layer by the MAP and settled in the layer.
In addition, the partial dissolution of the back-layer surface was confirmed by all patterns, as described in the previous section. We confirmed that removing the residue by partial dissolution did not affect pattern formation once the ink mixture was delivered into the skin (data not shown). Concerning the misalignment and distortion of character patterns, the slipping of MAPs or skin, which may occur during application, contributes to the misalignment of patterns, including tilted, moved, distorted, or stretched patterns. We considered that a fixing device, such as an applicator32, can be helpful to fix the MAP or skin stably during its application, resulting in patterns without any shape deformations.
We found that hair trimming was necessary, at least in rats, before applying MAPs to enable successful penetration into the skin. Simultaneously, the patterns were expected to be covered with hair that grew over time. We suggest that biotagging specific spots with little or no hair could be a feasible solution to hair-related issues. In addition, MAPs containing fluorescent ink that can be triggered by external stimulation, such as UV light, are considered useful for recognising patterns, even with grown hair25.
Second, we evaluated the long-term persistence of the patterns over one month with base MAPs and over three weeks with patterned MAPs (Supplementary Fig. S4). Similarly, we confirmed that all the formed patterns were retained inside the skin with clear readability. Considering the turnover time of basal cells in the epidermis layer33 and related literature34, it was expected that patterns formed by our developed MAPs would exist in the dermis layer and could play a role in identifying tags, that is, biotagging.
Finally, we harvested the dorsal skin where the MAPs were applied and performed histological analyses. We investigated two points: The first was to confirm that the ink mixture was delivered into the skin and remained inside. The second was to verify whether abnormal cell behaviour related to inflammation was observed. Figure 7 shows the histological data of the pristine dorsal skin and the MAP-applied skin on days 1, 3, and 7. We confirmed that the ink mixture was successfully delivered mainly to the dermis layers and that the ink pigments remained where delivered35. Compared to the skin without MAP application, black-coloured patterns with constant gaps, almost the same as the distance between MNs, were confirmed from the stained tissues (yellow arrows). Although the average length of MNs used in this research was over 800 μm, the deepest depth of ink pigment was approximately 380 μm. We considered that insufficient MNs’ penetration and the flexibility of the skin contributed to these results.
From the tissue sections, the mitigation of vast neutrophils was not observed near the region where the ink mixture was delivered. Thus, acute inflammation did not occur owing to the MNs penetration and delivery of the ink mixture. However, we observed that the thickness of the epidermal layer and the number of hair follicles (circular shapes with blue colour) increased. Such morphological changes might be caused by using a depilatory cream before MAP application36.