Summary
This study uses temperature and material composition to control the yield stress properties of yield stress fluids. The solid-like state of the ink can protect the printing structure, and the liquid-like state can continuously fill the printing position, realizing the digital light processing 3D printing of extremely soft bioinks.
Abstract
Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing parameters of digital light processing (DLP). However, the acquisition of working curves wastes materials and requires high formability of materials, which are not suitable for biomaterials. In addition, the reduction of cell activity due to multiple exposures and the failure of structural formation due to repeated positioning are both unavoidable problems in conventional DLP bioprinting. This work introduces a new method of obtaining the working curve and the improvement process of continuous DLP printing technology based on such a working curve. This method of obtaining the working curve is based on the absorbance and photorheological properties of the biomaterials, which do not depend on the formability of the biomaterials. The continuous DLP printing process, obtained from improving the printing process by analyzing the working curve, increases the printing efficiency more than tenfold and greatly improves the activity and functionality of cells, which is beneficial to the development of tissue engineering.
Introduction
Tissue engineering1 is important in the field of organ repair. Due to the lack of organ donation, some diseases, such as liver failure and kidney failure, cannot be cured well, and many patients do not receive timely treatment2. Organoids with the required function of the organs may solve the problem caused by the lack of organ donation. The construction of organoids depends on the progress and development of bioprinting technology3.
Compared with extrusion-type bioprinting4 and inkjet-type bioprinting5, the printing speed and printing accuracy of the digital light processing (DLP) bioprinting method are higher6,7. The printing module of the extrusion-type method is line-by-line, while the printing module of the inkjet-type method is dot-by-dot, which is less efficient than the layer-by-layer printing module of DLP bioprinting. The modulated ultraviolet (UV) light exposure to a whole layer of material to cure a layer in DLP bioprinting and the feature size of the image determines the accuracy of DLP printing. This makes DLP technology very efficient8,9,10. Due to overcuring of the UV light, the precise relationship between the curing time and the printing size is important for high-accuracy DLP bioprinting. Furthermore, continuous DLP printing is a modification of DLP printing method that can greatly improve the printing efficiency11,12,13. For continuous DLP printing, precise printing conditions are the most important factors.
The relationship between the curing time and the printing size is called the Jacobs working curve, which is widely used in DLP printing14,15,16. The traditional method to obtain the relationship is to expose the material for a certain time and measure the curing thickness to obtain a data point about the exposure time and curing thickness. Repeating this operation at least five times and fitting the data points obtains the Jacobs working curve. However, this method has obvious disadvantages; it needs to consume a lot of material to achieve the curing, the results are highly dependent on the printing conditions, the bioinks used in DLP bioprinting are expensive and rare, and the formability of the bioinks is usually not good, which can lead to inaccurate measurements of curing thickness.
This article provides a new method to obtain the curing relationship according to the physical properties of the bioink. Using this theory can optimize continuous DLP printing. This method can be used to obtain the curing relationship more quickly and accurately; the continuous DLP curing can therefore be better determined.
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Protocol
1. Theoretical preparation
- Define three parameters: liquid absorbance (Al), solid absorbance (As), and threshold time (tT)17.
- Rewrite the traditional Jacobs working curve using these three parameters17 according to Equation 1:
(Equation 1)
Here, tH is the curing time of one single layer, and H is the height of one single layer.
(Equation 1)2. Parameter acquisition
- Measure the threshold time of the bioink using a rheometer equipped with an element for temperature control.
- Use a 365 nm light source to expose the testing platform of the rheometer and make the light intensity at a certain value.
- Set the rheometer to get the Time-Moduli data during a period of 300 s, and take each data point every 0.3 s through the Time Settings options in the rheometer software.Click the Start Test button of the rheometer to start the test, and at the same time, click the Start Button of the light source.
- Counting from the start of exposure, when the storage modulus data is equal to the loss modulus data, the corresponding time is recognized as the threshold time. Record manually.
- Build the absorbance test equipment as shown in the previous work17. Use two upper and lower glass slides to clamp the ring-shaped printed structure (5 mm inner diameter, 10 mm outer diameter) with a thickness of 500µm so that the inner circle of the ring forms a chamber. Place the chamber on the test area of the light intensity meter and set the light source to expose the chamber area.
NOTE: Figure 1 shows the schematic diagram of photorheological test results and data processing results, and the absorbance testing equipment.- Measure the incident light intensity (Ii) when the test chamber is not filled with material from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment.
- Fill the test chamber with 10 µLof bioink.
- Expose the test chamber with bioink to UV light at 365 nm. Obtain the light intensity (Ilh) from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment.
- Obtain the light intensity when the bioink is cured (Ish) from the absorbance test equipment by reading the display of the light intensity meter of the testing equipment when the value no longer changes. This value is the solid absorbance, Ish.
- Calculate the liquid absorbance and solid absorbance using Equations 2 and 3:
Equation 2
Equation 3
- Obtain the Jacobs working curve according to the obtained parameters.
Equation 2
Equation 3
Figure 1: Test results and equipment. (A) Schematic diagram of photorheological test results and data processing results. (B) Absorbance testing equipment. This figure has been modified with permission from Li et al.17. Please click here to view a larger version of this figure.
3. Continuous DLP printing parameter settings
- Use DLP software to achieve DLP printing, and the set of printing parameters in the software as follows.
- Set the exposure time of the first single layer as the threshold time (tT) in the software's parameter settings.
- Calculate the exposure time of curing 10 µmthick materials according to Equation 1 and subtract the threshold time to obtain the real exposure time for curing a single layer.
- Set the time interval between adjacent layers to 0 s in the software's parameter settings.
- Start the printer by clicking the Start button in the printing software. When the printing process ends, finish printing by clicking the Stop button in the printing software.
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Representative Results
This article shows a new method to obtain curing parameters and introduces a new way to achieve continuous DLP printing, demonstrating the efficiency of this method in determining the working curve.
We used three different materials in DLP printing to verify the accuracy of the theoretical working curve obtained by the method introduced in this article. The materials are 20% (v/v) polyethylene (glycol) diacrylate (PEGDA), 0.5% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) with different concentrations of UV absorber-0.1% (w/v), 0.15% (w/v), and 0.2% (w/v) Brilliant blue. The real curing thickness data with the theoretical working curves are shown in Figure 2.
Figure 2: Comparison between the theoretical working curve and actual printing data. (A) 0.1% (w/v) absorber. (B) 0.15% (w/v) absorber. (C) 0.2% (w/v) absorber. Please click here to view a larger version of this figure.
The theoretical working curve can be used to calculate the working curve accurately. No matter what the material composition is, the high coincidence of the actual printing results and the theoretical results prove the effectiveness of the method.
We also compared the total printing time of the traditional DLP printing method with the continuous DLP printing method developed in this article. As shown in Figure 3, the smaller the printing layer thickness, the more obvious the improvement of continuous DLP printing efficiency. The curing efficiency increased more than tenfold.
Figure 3: Efficiency comparison between traditional DLP printing and continuous DLP printing. This figure has been modified with permission from Li et al.11. Please click here to view a larger version of this figure.
The acquisition of the theoretical working curve can be used to improve the DLP process and promote the progress of DLP technology, but without the acquisition of the theoretical working curve, it is impossible to accurately control the new printing method. Furthermore, the smaller the printing layer thickness, the better the printing quality, meaning that the continuous DLP printing method proposed in this article can simultaneously achieve high efficiency and high fidelity.
Figure 4: Comparison of printing results between traditional DLP printing and continuous DLP printing. (A) The cured model using the traditional method. (B) The cured model using our continuous DLP printing method. This figure has been modified with permission from Li et al.11. Please click here to view a larger version of this figure.
Unlike the traditional method that requires repeated printing experiments, this method only needs to test the relevant material properties of the material. Only a very small amount of material is needed to accurately obtain its corresponding working curve. The traditional method not only wastes material but also relies heavily on measurement methods to determine the accurate molding thickness of different exposure times. For materials with poor formability, it is difficult to accurately obtain the printing thickness, so the working curve is inaccurate.
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Discussion
The critical steps of this protocol are described in section 2. It is necessary to unify the light intensity used in the photorheology test and the printing light intensity in the actual tests. The absorbance testing equipment is the most important part. The shape of the test chamber should be the same as the photosensitive area of the light intensity meter. Due to the properties of the materials that continuously change during the whole UV light exposure process, the light intensity needs to continue to change6. According to the definition of liquid absorbance and solid absorbance in Equation 1, the curing process is simplified. Taking the data at the beginning of the exposure as liquid absorbance and the data when the light intensity is constant as the solid absorbance is the most critical operation.
It is worth noting that this method has an unavoidable limitation, which is the simplification of the curing process. Since the theoretical modeling of this method does not consider factors such as oxygen inhibition13, there are errors between the actual working curve and the theoretical working curve. Further, if the external disturbance is large, the theoretical working curve cannot be accurately used for research.
The traditional method to obtain the Jacobs working curve requires multiple printing with different exposure times15. The working curve is obtained by measuring the printing thickness corresponding to the exposure time and fitting the data. This method requires a lot of material and is very inefficient. The printing ability of the material restricts the accuracy of the working curve, and the observation and measurement of the structure also amplify the error. The method in this article to obtain the working curve can save lots of materials, accurate working curves can be obtained only through simple material property tests, and the accuracy of the working curve can be guaranteed independent of the formability of the material. In the DLP bioprinting research, when the material is very soft (E < 10 kPa) it cannot be printed well, and this will affect the printing thickness data obtained by the traditional method, thereby affecting the accuracy of the working curve18. The method mentioned in this protocol can provide a solution for the determination of the DLP printing process parameters of soft biomaterials.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
The authors gratefully acknowledge the support provided by the National Natural Science Foundation of China (Grant Nos. 12125205, 12072316, 12132014), and the China Postdoctoral Science Foundation (Grant No. 2022M712754).
Materials
| Name | Company | Catalog Number | Comments |
| Brilliant Blue | Aladdin (Shanghai, China). | 6104-59-2 | |
| DLP software | Creation Workshop | N/A | |
| Lithium phenyl-2,4,6-trimethylbenzoylphosphinate | N/A | LAP; synthesized | |
| Light source | OmniCure | https://www.excelitas.com/product-category/omnicure-s-series-lamp-spot-uv-curing-systems | 365 nm |
| Polyethylene (glycol) diacrylate | Sigma-Aldrich | 455008 | PEGDA Mw ~700 |
| Rheometer | Anton Paar, Austria | MCR302 |
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