Projection type light-cured biological 3D printing method

Abstract

The application provides a projection type light-curing biological 3D printing method, which is based on full analysis of a biological ink curing process and rheological characteristics, and a principle of cell damage caused by free radicals, uses a cross-linking strategy of irradiation with different intensities of light before and after a gel point of the biological ink, controls a free radical flux, reduces a time of direct contact of the free radicals with cells, and improves a survival rate of the cells in a printing process; meanwhile, the use of large light intensity irradiation before the gel point makes a printing piece preliminarily formed, can greatly shorten light irradiation time and printing time consumption, avoids refraction and scattering of a light source, and greatly improves printing efficiency and printing resolution.

Description

Technical Field

[0001] This invention belongs to the field of photopolymer 3D printing and bio-3D printing technology, specifically relating to a projection-type photopolymer bio-3D printing method. Background Technology

[0002] Bioprinting refers to a manufacturing method that uses bio-inks to create personalized biological functional structures according to biomimetic morphology, biological function, and cell growth microenvironment requirements. As a powerful cell assembly technology, bioprinting has been widely applied in biomedical fields such as tissue repair, disease models, drug screening, and clinical treatment. Due to the advantages of high resolution, high printing efficiency, and high cell viability of projection-based photopolymerization 3D printing, it has shown great potential in bioprinting and has attracted increasing attention in the biomedical field. Projection-based photopolymerization bioprinting can rapidly manufacture personalized biological functional structures according to biomimetic morphology, biological function, and cell growth microenvironment requirements.

[0003] The basic principle of projection-based photopolymer 3D printing technology is to use a computer-controlled printing light source to illuminate a photosensitive material according to a designed pattern. The illuminated photosensitive material undergoes a cross-linking polymerization reaction to form a solid. Then, the printing platform rises to a certain height to perform the next layer of photopolymerization printing, and this process is repeated to complete the printing of the three-dimensional object. Currently, projection-based photopolymer 3D printing is mainly based on non-biological materials such as photosensitive resins, while bio-3D printing requires the use of biological materials to ensure cell survival. Biological materials and non-biological materials have drastically different characteristics during the cross-linking polymerization process. Existing projection-based photopolymer bio-3D printing does not take this into account, still printing biological inks according to traditional projection-based photopolymer 3D printing methods, which is very detrimental to cell survival.

[0004] Compared with traditional photosensitive resins, bio-inks have the following unique characteristics:

[0005] Non-biological materials such as photosensitive resins are usually small molecule monomers, while biological materials are mainly large molecule monomers such as proteins, sugars, and lipids.

[0006] Photosensitive resins have a high content of photosensitive functional groups, typically 70-90%, while biomaterials have a very low content of photosensitive functional groups, typically less than 20%.

[0007] Biomaterials need to contain as few chemicals as possible to ensure high biocompatibility; therefore, diluents, stabilizers, modifiers, and many other additives that promote polymerization cannot be used in bio-inks.

[0008] Bio-inks contain a large amount of water, and their biomaterial concentration is usually less than 20%. The large amount of dissolved oxygen in the water will further hinder the cross-linking of biomaterial monomers.

[0009] Biomaterial monomers have complex functional groups, relatively high solution viscosity, low molecular diffusion rate, and low cross-linking efficiency.

[0010] Based on the above characteristics, bio-inks exhibit poor formability and printability, making traditional projection-based photopolymerization 3D printing methods using photosensitive resins unsuitable for photopolymerization printing of biomaterials. Therefore, developing a projection-based photopolymerization bio-3D printing method based on the properties of bio-inks is crucial for improving printing efficiency and quality. Summary of the Invention

[0011] To address the problems existing in the prior art, this invention provides a projection-based photopolymerization biological 3D printing method that is suitable for biomaterials, has high printing efficiency, and good cell activity.

[0012] A projection-based photopolymerization bio-3D printing method includes the following steps:

[0013] (1) Using cell-loaded bio-ink as the printing material, 3D printing technology was used to print layer by layer, and high light intensity was used to irradiate the bio-ink until it reached the gel point to obtain a pre-cured printed part.

[0014] (2) Remove the pre-cured print and remove any residual bio-ink and light absorber;

[0015] (3) The pre-cured printed part after step (2) is immersed in a low concentration photoinitiator solution and post-processed with low light intensity. After the post-processing is completed, the cell-loaded printed part is obtained.

[0016] The gel point refers to the point at which multifunctional monomers polymerize to a certain degree, at which the viscosity suddenly increases, the mechanical properties of the system change abruptly, and gelation occurs. It's important to note that the gel point is not the endpoint of complete solidification of biomaterials; the degree of cross-linking at the gel point is 40-60%.

[0017] Before the gel point, the reaction system contains a large number of uncrosslinked biomaterial monomers and has a low viscosity, so the growth reaction dominates. After the gel point, the number of uncrosslinked monomers in the reaction system decreases and the viscosity of the system increases significantly, so the termination reaction dominates.

[0018] Using higher irradiation intensity before the gel point has little effect on the degree of crosslinking of hydrogels. However, after the gel point, since the termination reaction itself becomes dominant, using higher irradiation intensity is not conducive to improving the degree of crosslinking of hydrogels. Therefore, it is necessary to change the exposure strategy.

[0019] Before the gel point, the reaction system is liquid, and cells are in direct contact with free radicals. Minimizing the contact time between cells and free radicals can effectively reduce cell death. After the gel point, the reaction system is nearly solid, and the biomaterial provides some protection for the cells. The low concentration of free radicals helps the biomaterial to further solidify without affecting cell viability. Therefore, this invention uses high-intensity light irradiation before the gel point to photocur the bio-ink with the shortest possible exposure time until the gel point is reached. After the gel point, low-intensity light irradiation is used to reduce free radical damage to cells.

[0020] Preferably, the irradiance of the high-intensity light is 10–40 mW / cm². 2 A further preferred value is 20–35 W / cm². 2 .

[0021] Preferably, the high-intensity light exposure time is 5–12 seconds. The specific duration of high-intensity light exposure is selected based on the gel point time of the bio-ink.

[0022] Preferably, the irradiance of the low-intensity light is 0.1–5 mW / cm². 2 As a further preferred option, 0.1–1 mW / cm² 2 .

[0023] Preferably, the irradiation time with low light intensity is 20–60 min. More preferably, it is 20–40 min.

[0024] To ensure cell survival, the wavelengths of the high-intensity light source and the low-intensity light source are preferably 405nm.

[0025] Preferably, the printing temperature is 37°C.

[0026] Preferably, in step (3), the photoinitiator solution is a PBS solution of photoinitiator, wherein the concentration of photoinitiator is 0.05 to 0.1% w / v.

[0027] Preferably, in step (2), PBS solution is used to remove residual bio-ink and light absorber by first cleaning and then soaking the pre-cured print.

[0028] Specifically, the pre-cured printed parts are placed in a PBS solution at 37°C to wash away the residual bio-ink, and then soaked in another PBS solution for 1-2 hours to fully remove the light absorbers.

[0029] Preferably, the bio-ink uses PBS solution as a solvent and includes 200,000 to 2,000,000 live cells / mL, 10 to 15% w / v of biomaterial, 0.2 to 0.5% w / v of photoinitiator, and 0.05 to 0.1% w / v of light absorber.

[0030] Biomaterials are characterized by good bioactivity, good formability, and certain mechanical properties. As a further preferred option, the biomaterial is one or more of the following: methacrylamide gelatin (GelMA), methacrylamide silk fibroin, methacrylamide hyaluronic acid, methacrylamide sericin, polyethylene glycol diacrylate, methacrylamide chitosan, polyether F127 diacrylate, methacrylamide sodium alginate, methacrylamide chondroitin sulfate, and tetra-arm polyethylene glycol acrylate.

[0031] It should be noted that biomaterials modified with fluorescence, functional proteins, or structures, as long as their main structure is the aforementioned biomaterials, are still within the scope of protection of this invention.

[0032] Photoinitiators are compounds that can absorb energy of a certain wavelength, generate free radicals, and thus initiate monomer polymerization, cross-linking, and curing. More preferably, the photoinitiator is one or more of the following: 2,4,6-trimethylbenzoyldiphenoxyphosphine, ethyl 2,4,6-trimethylbenzoylphosphonate, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, diazonium salts, diaryliodomonium salts, triarylthionium salts, alkylthionium salts, iron aromatic salts, sulfonyloxyketones, and triarylsiloxanes.

[0033] A light absorber is a compound that can absorb light of a specific wavelength, thereby preventing monomer polymerization. Light absorbers are commonly used in 3D printing to prevent over-curing of the printing material due to light scattering. Preferably, the light absorber is one or more of pigments, ultraviolet light absorbers, quenchers, and free radical scavengers.

[0034] As a further preferred embodiment, the light absorber is one or more of the following: iron oxide, carbon black, annatto, tartrazine, phenyl benzoate, 2,4-dihydroxybenzophenone, organonickel chelate, and hindered amine light stabilizer.

[0035] Preferably, the bio-ink may further include biological factors. Further, the biological factors include, but are not limited to, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, transforming growth factor, nerve growth factor, interleukin-like growth factor, bone morphogenetic protein, platelet-derived proliferation factor, growth hormone-releasing inhibitory factor, osteosarcoma-derived growth factor, erythropoietin, colony-stimulating factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor.

[0036] Free radicals are reactive substances generated when photoinitiators are exposed to specific wavelengths of light. They can promote cross-linking reactions, but also cause significant damage to cells. The longer the cells are in direct contact with free radicals, the higher the cell death rate; the higher the concentration of free radicals the cells are exposed to, the higher the cell death rate.

[0037] Bio-ink cross-linking and curing has the following characteristics:

[0038] The polymerization reaction during monomer crosslinking can be divided into two typical reaction types: propagation reaction and termination reaction. Propagation reaction refers to the continuous chain polymerization reaction between monomers; termination reaction refers to the unsustainable polymerization reaction between monomers and free radicals.

[0039] The growth reaction is a first-order reaction with respect to the free radical concentration, meaning the growth reaction rate is directly proportional to the first power of the free radical concentration; the termination reaction is a second-order reaction with respect to the free radical concentration, meaning the termination reaction rate is directly proportional to the square power of the free radical concentration.

[0040] The degree of crosslinking in bio-inks is related to the relative proportion of growth and termination reactions; that is, the higher the proportion of growth reactions and the lower the proportion of termination reactions, the higher the degree of crosslinking in the bio-ink; conversely, the lower the proportion of growth reactions and the higher the proportion of termination reactions, the lower the degree of crosslinking in the bio-ink. Under fully crosslinked conditions, the relative proportion of growth and termination reactions is inversely proportional to the free radical concentration, meaning the degree of crosslinking in bio-inks is inversely proportional to the free radical concentration in the system.

[0041] The concentration of free radicals in the system can be adjusted by changing the concentration and type of photoinitiator, the concentration and type of photoabsorber, and the irradiation power of the light source.

[0042] Preferably, in bio-3D printing, the concentration of free radicals in the system is adjusted by changing the irradiation power of the light source. That is, under the same total irradiation energy and sufficient cross-linking, bio-inks cured by low-intensity long-term irradiation have a higher degree of cross-linking, while bio-inks cured by high-intensity short-term irradiation have a lower degree of cross-linking.

[0043] Furthermore, the printing method of the present invention is applicable to various types of projection-type photopolymerization 3D printers commonly found on the market. When using the printing method of the present invention, only the light intensity of the light source needs to be adjusted, and no other hardware of the printer needs to be changed. It has the advantages of being convenient, easy to implement, and easy to promote.

[0044] The projection-based photopolymerization bio-3D printing method of this invention, based on a thorough analysis of the curing process and rheological characteristics of bio-ink, as well as the principle of free radical damage to cells, controls the free radical flux by using a crosslinking strategy of irradiating with light of different intensities before and after the gel point of the bio-ink. This reduces the time that free radicals directly contact cells and improves the cell survival rate during the printing process. At the same time, the use of high-intensity light irradiation before the gel point to pre-form the printed part can greatly shorten the irradiation time and printing time, avoid the refraction and scattering of the light source, and significantly improve printing efficiency and printing resolution.

[0045] This invention, based on chemical reaction kinetics and considering the different characteristics of bio-inks and traditional photosensitive resins, optimizes the photocrosslinking and rheological processes of bio-inks. It is applicable to various types of bio-inks and can be directly applied without significant modifications to existing projection-type photopolymerization printers. More importantly, this invention significantly improves printing efficiency, print quality, and cell viability compared to traditional methods.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] I. The projection-type photopolymerization bio-3D printing method of the present invention fully considers the different characteristics of bio-inks and traditional photosensitive resins. Based on the photocrosslinking process and rheological process of bio-inks, the curing conditions of bio-inks have been optimized, making it more suitable for printing bio-inks with soft texture and high water content.

[0048] II. Regarding the materials used for printing, the projection-type photopolymerization biological 3D printing method of the present invention can print bio-inks made of various biological materials, such as methacrylamide gelatin, methacrylamide silk fibroin, methacrylamide hyaluronic acid, etc.

[0049] Third, the high-intensity rapid cross-linking printing proposed in this invention can greatly shorten printing time and illumination time, avoid refraction and scattering of light sources, and significantly improve printing efficiency and printing resolution. It has important value for the mass production and standardized production of projection-type photopolymerization biological 3D printing.

[0050] IV. Regarding the printing performance of bio-inks, the printing method proposed in this invention, by employing different cross-linking strategies before and after the gel point, can more easily adjust the degree of cross-linking and mechanical properties of the printed bio-ink. In practical bio-3D printing, it is often necessary to adjust the mechanical properties of the printed biomaterials according to requirements such as biomimetic morphology, biological function, and cell growth microenvironment. The printing method of this invention can provide a wider mechanical property window, which has potential value for the development of bio-3D printing in the fields of tissue engineering and regenerative medicine.

[0051] Fifth, regarding cell survival rate during printing, the printing method proposed in this invention takes into account the principle of free radical damage to cells. By controlling the free radical flux, the time for free radicals to directly contact cells is reduced, effectively improving cell survival rate during the printing process. This provides assistance for research on projection-based photopolymerization 3D printing in the biomedical field.

[0052] VI. By adjusting the light intensity and duration of post-treatment (low-intensity irradiation), the dynamic properties of constructed biological functional structures can be regulated. Changes in the mechanical properties of the cell's microenvironment can regulate cell behavior during culture, such as inducing cell differentiation and migration, which is beneficial for life science and clinical medical research and applications.

[0053] In summary, the projection-based photopolymerization bio-3D printing method of the present invention has been optimized based on the characteristics of bio-inks, making it applicable to various types of bio-inks. At the same time, it can be directly applied without requiring extensive modifications to existing projection-based photopolymerization printers, thereby improving the printing efficiency and quality of projection-based photopolymerization bio-3D printing and ensuring cell survival rate. Attached Figure Description

[0054] Figure 1 This is a schematic flowchart of the projection-type photopolymerization bio-3D printing method according to an embodiment of the present invention;

[0055] Figure 2 Photocrosslinking-rheological properties test diagram of bio-ink during traditional printing methods;

[0056] Figure 3 The trend of crosslinking degree during the reaction process of different printing methods;

[0057] Figure 4 These are photographs showing the state of the printed parts at various stages in the embodiments of the present invention;

[0058] Figure 5 This is a comparison chart of the printing performance of the printing method of this invention and the conventional method;

[0059] Figure 6 Photographs of a bionic ear and a perforated support structure printed using the printing method of this invention.

[0060] Figure 7 This is a diagram illustrating the analysis of cell apoptosis under different conditions.

[0061] Figure 8 This is a comparison chart of cell apoptosis rates printed using the printing method of this invention and conventional methods. Detailed Implementation

[0062] To make the objectives, features, and advantages of this invention more apparent and understandable, the technical solutions of this invention will be described in detail below with reference to the accompanying drawings. Several embodiments of this invention are shown in the accompanying drawings. However, this invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to make the disclosure of the technical solutions of this invention more thorough and comprehensive.

[0063] The following describes the technical solution of the present invention in further detail, using commonly used biomaterials such as methacrylamide gelatin (GelMA), lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) as the photoinitiator, lemon yellow as the light absorber, and mouse myoblast C2C12 cells as the live cells.

[0064] like Figure 1 As shown, a projection-based photopolymerization bio-3D printing method includes the following steps:

[0065] 1. Prepare GelMA as a 10-15% w / v bio-ink using phosphate-buffered saline (PBS) solution, and place it in a water bath at 40-60°C to allow the GelMA to dissolve completely;

[0066] 2. Add 0.2-0.5% w / v LAP, 0.05-0.1% w / v lemon yellow and 20-2 million / mL mouse myoblasts C2C12 to the bio-ink, stir thoroughly to dissolve, until there is no solid residue in the bio-ink;

[0067] 3. Take 3-10ml of bio-ink and put it into the ink tank of the projection-type light curing printer. To prevent the bio-ink from condensing, set the temperature of the ink tank and the printing platform to 37℃.

[0068] 4. Use a computer to slice the designed print file and send the sliced ​​file to the printer processor;

[0069] 5. To ensure cell survival, the wavelength of the light source was set to 405nm;

[0070] 6. Set the irradiance of the light source to 30mW / cm². 2Based on the gel point time of the bio-ink, the exposure time was set to 5-12s, and the printing layer height was set to 50-200um;

[0071] 7. After printing, remove the pre-cured print and place it in a 37°C PBS solution to wash away any residual bio-ink.

[0072] 8. Immerse the cleaned printouts from step 7 in a separate PBS solution at 37°C for 1-2 hours to thoroughly remove any residual lemon yellow.

[0073] 9. Prepare a low-concentration photoinitiator solution of 0.05-0.1% w / v using PBS solution;

[0074] 10. Place the printed parts, after removing residual bio-ink and lemon yellow, in a transparent container, and add a low-concentration photoinitiator solution until the printed parts are completely submerged; then place the container in an incubator at 37°C, using a 405nm wavelength light source at 0.3mW / cm². 2 The irradiation intensity is post-processed for 20-60 minutes;

[0075] 11. Remove the post-processed printout and clean it with PBS solution at 37°C to obtain a fully solidified cell-loaded printout.

[0076] The principles of the present invention will be explained in detail below.

[0077] The characteristic of cross-linking and curing of bio-inks lies in the fact that the polymerization reaction during the monomer cross-linking process can be divided into two typical reaction types:

[0078] Growth response;

[0079] Terminate the reaction.

[0080] The growth reaction is characterized by a continuously occurring chain polymerization reaction between monomers, and its reaction equation is as follows:

[0081]

[0082] Where M is a monomer, It is a monomeric free radical or a chain free radical. It is a chain free radical.

[0083] The reaction rate v of the growth reaction p for:

[0084]

[0085] Where, k p C is the rate constant for the growth reaction. (M) Monomer concentration, This represents the concentration of free radicals.

[0086] The terminating reaction is characterized by an unsustainable polymerization reaction between the monomer and the free radical, and its reaction equation is:

[0087]

[0088] in, RM is a monomeric or chain radical. m+n It is an inert polymer.

[0089] The reaction rate v that terminates the reaction t for:

[0090]

[0091] Where, k t The rate constant for terminating the reaction, This represents the concentration of free radicals.

[0092] In summary, the growth reaction is a first-order reaction with respect to free radical concentration, meaning the growth reaction rate is directly proportional to the first power of the free radical concentration; the termination reaction is a second-order reaction with respect to free radical concentration, meaning the termination reaction rate is directly proportional to the square power of the free radical concentration.

[0093] Under fully cross-linked conditions, the degree of cross-linking of the bio-ink is related to the relative proportions of the growth and termination reactions, i.e.:

[0094]

[0095] The higher the proportion of growth reactions and the lower the proportion of termination reactions, the higher the crosslinking degree of the bio-ink; conversely, the lower the proportion of growth reactions and the higher the proportion of termination reactions, the lower the crosslinking degree of the bio-ink.

[0096] Therefore, we can draw the following conclusion: Under fully cross-linked conditions, the relative ratio of growth reaction to termination reaction is inversely proportional to the free radical concentration, that is, the free radical concentration in the cross-linked system of bio-ink is inversely proportional.

[0097] The concentration of free radicals in the system can be adjusted by changing the concentration and type of photoinitiator, the concentration and type of photoabsorber, and the irradiation power of the light source.

[0098] In bioprinting, the concentration of free radicals in the system can be adjusted by changing the irradiation power of the light source. That is, under the same total irradiation energy and sufficient cross-linking, bio-inks cured by low-intensity long-term irradiation have a higher degree of cross-linking, while bio-inks cured by high-intensity short-term irradiation have a lower degree of cross-linking.

[0099] It should be noted that during the monomer crosslinking polymerization process in bio-ink, the viscosity of the reaction system, the proportion of each substance in the reaction system, and the content of photoinitiator / photoabsorber are constantly changing dynamically, and the proportion of the two polymerization reactions is also different at different stages.

[0100] Specifically, the reaction rate constant is usually influenced by both the reactivity of the reactants and the molecular diffusion rate. The reactivity of the reactants is mainly affected by their molecular structure; during cross-linking, free radicals containing unpaired electrons are more reactive, while monomers without excess electrons are less reactive.

[0101] The molecular diffusion rate D can be described by the Stokes-Einstein equation:

[0102]

[0103] Where k is the Boltzmann constant; T is the temperature; μ is the viscosity of the solvent; and r is the radius of the diffusing molecule.

[0104] As can be seen, under constant environmental factors, the molecular diffusion rate is affected by the molecular radius. Generally speaking, bio-ink materials are biomacromolecules, while the free radicals generated by the cracking of photoinitiators usually have smaller molecular weights.

[0105] Therefore, the following conclusions can be drawn: Before the gel point, the reaction system contains a large number of uncrosslinked monomers and has a low viscosity, resulting in a fast monomer diffusion rate, so the growth reaction dominates; at the gel point, the number of uncrosslinked monomers decreases and the viscosity of the system increases significantly, resulting in a significant decrease in the monomer diffusion rate. Although the free radical diffusion rate also decreases, its diffusion rate is still relatively fast due to its small molecular weight, so the termination reaction dominates.

[0106] Therefore, before the gel point, since the growth reaction itself is dominant, using a higher irradiation intensity has little effect on the degree of crosslinking of the hydrogel. However, after the gel point, when the termination reaction is dominant, using a higher irradiation intensity still has a greater effect on the degree of crosslinking of the hydrogel.

[0107] Example

[0108] Bio-ink was prepared using PBS as solvent, with GM90 (GelMA with a substitution rate of 90%) at a concentration of 10% w / v, LAP at a concentration of 0.25% w / v, tartrazine at a concentration of 0.05% w / v, and mouse myoblast C2C12 at a concentration of 1 million cells / mL.

[0109] Add 3-10 ml of bio-ink to the ink tank of the projection-type photopolymerization 3D printer, and set the temperature of the ink tank and printing platform to 37℃; slice the designed print file and send the sliced ​​file to the printer processor; set the light source wavelength to 405 nm and the irradiation power to 30 mW / cm². 2 The exposure time for each layer is 10 seconds, and the printing layer height is 100 μm.

[0110] After printing, remove the pre-cured print and place it in a 37°C PBS solution to wash away any residual bio-ink. Then, soak the cleaned print in another 37°C PBS solution for 1 hour to thoroughly remove any residual lemon yellow.

[0111] Prepare a 0.05% w / v low-concentration photoinitiator solution from LAP using PBS solution;

[0112] The printed parts, after removing residual bio-ink and lemon yellow, were immersed in a transparent container containing a low-concentration photoinitiator solution and heated at 37°C using a 405nm wavelength light source at 0.3mW / cm². 2 The irradiation intensity was post-processed for 30 minutes;

[0113] After post-processing, remove the printed part and clean it with PBS solution at 37°C to obtain a fully solidified cell-borne printed part.

[0114] The status diagrams of the printed parts at different stages during the printing process are shown below. Figure 4 ,Depend on Figure 4 It can be seen that high-intensity rapid cross-linking printing before the gel point results in low cross-linking degree and low mechanical strength of the printed part, which cannot maintain its shape. Significant deformation and collapse occur after removal. Figure 4 (A). The deformed printed part was then immersed in a low-concentration initiator solution, and under the influence of liquid surface tension and buoyancy, the printed part recovered its initial shape. Figure 4 (B); then it undergoes a slow cross-linking post-treatment with low light intensity, at which point the degree of cross-linking increases, resulting in a final printed part with a complete structure, high mechanical strength, and high printing precision. Figure 4 C).

[0115] Comparative Example

[0116] Printing was performed using a traditional method of complete layer-by-layer curing, with the same composition of the bio-ink and layer height as in the previous example; the difference was that the light intensity was 30 mW / cm². 2 The illumination time is 25 seconds per layer; no post-processing is required after printing to obtain the cell-loaded print.

[0117] In traditional projection-based bio-3D printing, since the printed part is formed by a single illumination process, the irradiation dose is controlled within a certain range to balance printing accuracy, printing efficiency, cell viability, and mechanical strength. Figure 2 (The image shows the traditional printing window). Insufficient irradiation dose leads to poor mechanical strength, making printed parts prone to deformation and collapse; excessive irradiation dose results in reduced printing accuracy and increased cell apoptosis rate. Therefore, under normal circumstances, the total irradiation dose for traditional printing is controlled at 2-3 times the irradiation dose at the gel point.

[0118] The gel point refers to the point at which the viscosity of a multifunctional monomer polymerizes to a certain degree, causing a sudden increase and abrupt change in the mechanical properties of the system, resulting in gelation. In practical applications, a rheometer is commonly used to measure the storage modulus G′ and dissipation modulus G″ of bio-inks during photocuring to determine the gel point. The crosslinking degree and rheological properties of the bio-ink in Comparative Example 1 were tested under a light intensity of 30 mW / cm². 2 The gel point of the bio-ink was measured, and the test results are shown below. Figure 2 ;like Figure 2 As shown, before curing, G" is usually 4-6 orders of magnitude higher than G′, at which point the reaction system is closer to a liquid state. During photocrosslinking, G" usually remains basically unchanged. As the crosslinking reaction proceeds, G′ gradually increases until it exceeds G". At this point, the reaction system is closer to a gel state. The degree of reaction when G" equals G′ is usually called the gel point.

[0119] It should be noted that the gel point is not the end point of complete curing of biomaterials. The degree of cross-linking of biomaterials at the gel point is usually 40-60%. Because biomaterials are soft and brittle, have a high water content, and very low mechanical strength, they cannot maintain their own structure at the gel point and are very prone to deformation or even collapse. Therefore, further light exposure is required to fully cure them.

[0120] The tests employed the printing methods of both the example and comparative versions. The trends in crosslinking degree during the reaction process under different printing methods were analyzed, and the test results are shown in [Figure number missing]. Figure 3 .like Figure 3As shown, before the gel point, due to the low viscosity of the system, the overall polymerization rate is faster, and the degree of crosslinking increases rapidly, rising from 0 to 46% at an irradiation dose of 300 mJ. Traditional projection-based photopolymerization bio-3D printing methods (traditional methods) do not consider the characteristics of bio-inks and continue to use the same irradiation parameters (high light intensity) after the gel point. It can be seen that after the gel point, due to the significant increase in system viscosity, the overall polymerization rate slows down, the degree of crosslinking increases slowly, and the rate of increase gradually decreases; even when the irradiation dose increases to 1800 mJ, the degree of crosslinking is still less than 80%, and further increases in irradiation dose result in only a small increase in crosslinking degree. In contrast, the printing method in this embodiment changes the irradiation parameters after the gel point (using low light intensity). With continuous increases in irradiation dose, its degree of crosslinking always increases at the same rate, reaching 90% when the irradiation dose increases to 1800 mJ, significantly better than the degree of crosslinking achieved by the traditional method. This indicates that, after the gel point, under the same irradiation dose, compared to continuing to use high light intensity for irradiation in traditional methods, using low light intensity for irradiation in this embodiment can better promote further cross-linking and molding of the printed parts.

[0121] The XY resolution was evaluated using a radial fringe model, and the Z resolution was evaluated using a spiral staircase model. The resolution and Young's modulus of the molded parts obtained from the example and comparative printing were tested respectively. The test results are shown in […]. Figure 5 .like Figure 5 As shown in Figures A and B, the XY resolution of the printing method in this embodiment (shown as this method in the figure) is 170µm, and the Z resolution is 190µm, while the XY resolution of the traditional method is 490µm, and the Z resolution is 820µm. Figure 5 As shown in Figure C, in terms of printing efficiency, the printing method in this embodiment takes 10 seconds to print one layer, while the traditional printing method takes 25 seconds. Figure 5 As shown in Figure D, regarding the mechanical properties of the printed parts, the Young's modulus of the printed parts obtained in this embodiment is 65 kPa, which is significantly better than the Young's modulus of 35 kPa obtained by the traditional method. In summary, compared to the traditional method, the printing method of this embodiment shows significant improvements in printing accuracy, printing efficiency, and printing quality.

[0122] like Figure 6 As shown, the printing method of this embodiment is applicable to printing various biomimetic structures, such as... Figure 6 The bionic ears of Zhong A and Figure 6 The hollow multi-hole bracket of B can be printed very well. Except for the shape and structure of the printed parts, the printing process and conditions are carried out in accordance with the embodiments.

[0123] like Figure 7 As shown, the mechanism of apoptosis under different conditions is analyzed:

[0124] A high-intensity light source with a wavelength of 405nm (30mW / cm²) is used. 2 Irradiation for 20 seconds) and a low-intensity light source (0.3mW / cm²). 2 Cells were irradiated with PBS solution containing 1 million cells / mL for 2000 seconds. Cell damage was assessed in both PBS solutions. Results are shown below. Figure 7 In sections A and B, it can be seen that without a photoinitiator to generate free radicals, simply irradiating cells with a 405nm wavelength light source, regardless of the intensity of the light source (30mW / cm²), results in significant damage. 2 It is still a low-intensity light source (0.3mW / cm²). 2 The damage to cells was less than 1%. Figure 7 The results (A and B) were essentially the same as those in the blank control group (cells not treated in any way). Figure 7 E); This indicates that the main factor damaging cells during projection-based photopolymerization bio-3D printing is not light irradiation.

[0125] Add 0.25% w / v LAP to a PBS solution with a cell concentration of 1 million cells / mL, using 30 mW / cm 2 After irradiation with a light source of 405 nm at a high irradiance for 20 seconds, the measured apoptosis rate was as high as 62% (e.g., Figure 7 As shown in C, the figure displays 30mW / cm. 2 + free radicals); This indicates that the main factor damaging cells during projection-based photopolymerization bio-3D printing is the free radicals generated by the photoinitiator.

[0126] Add 0.25% w / v LAP and 10% w / v GM90 (biological material) to a PBS solution with a cell concentration of 1 million cells / mL, and use 30 mW / cm 2 After irradiation with a light source of 405 nm for 20 seconds, the measured apoptosis rate decreased to 3.85% (e.g., ...). Figure 7 As shown in D, the value is 30 mW / cm² in the figure. 2 (+free radicals+biomaterials), which indicates that biomaterials play a good protective role for cells in photocuring engineering.

[0127] Therefore, the following conclusions can be drawn: the longer the direct contact time between cells and free radicals, the higher the cell death rate; the higher the concentration of free radicals in contact with cells, the higher the cell death rate. Before the gel point, the reaction system is liquid, and cells are in direct contact with free radicals. Minimizing the contact time between cells and free radicals can effectively reduce cell death. After the gel point, the reaction system is close to solid, and the biomaterial provides some protection for the cells. The low concentration of free radicals helps the biomaterial to further solidify without affecting cell activity.

[0128] The apoptosis rate of cells in the molded printed parts of the examples and comparative examples were tested separately, and a blank control group without any treatment was set up. The test results are shown in [the table below]. Figure 8 .like Figure 8 As shown, the cell-borne bioprinting of the embodiment (shown as the present invention in the figure) is significantly superior to traditional methods. The apoptosis rate of the printed parts obtained by the traditional method is 44.3%, while the apoptosis rate of the printed parts obtained by the embodiment is reduced to 18.1%. This is because post-treatment with low light intensity after gelation effectively promotes cross-linking and curing (see...). Figure 3 This better protects cells from damage, and low light intensity does not damage cells, effectively reducing the apoptosis rate.

[0129] In the above test data:

[0130] The apoptosis rate was tested using an apoptosis kit to stain cell motif protein-V and nucleic acids, followed by analysis using a flow cytometry. The results are as follows: Figure 7 , Figure 8 As shown, cells in the first quadrant are late-stage apoptotic cells, cells in the fourth quadrant are early-stage apoptotic cells, and cells in the third quadrant are normal cells.

[0131] The method for testing Young's modulus is as follows: The sample is tested on a mechanical testing machine using a 20N force sensor at a constant tensile rate of 10 mm / min, and the data is plotted as a tensile stress-strain curve. The slope of the linear region of the stress-strain curve corresponding to 0%-20% strain is taken as Young's modulus.

[0132] The method for testing the degree of crosslinking is as follows: The proton NMR spectra of the biomaterial before and after light exposure are measured using a nuclear magnetic resonance (NMR) spectra. The two peaks appearing in the chemical shift range of 5.2-5.7 ppm in the proton NMR spectrum correspond to the double bond content. The ratio of the double bond content after light exposure to the double bond content before light exposure is the double bond conversion rate. In this test, the double bond conversion rate is used to represent the degree of crosslinking of the biomaterial.

[0133] In summary, the method of this invention is optimized based on the characteristics of bio-inks, making it applicable to various types of bio-inks, and can be directly applied without significant modifications to existing projection-based light-curing printers. More importantly, the method of this invention significantly improves printing efficiency, printing quality, and cell viability compared to traditional methods.

[0134] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for projection photo-cured biological 3D printing, characterized in that, Includes the following steps: (1) Using cell-loaded bio-ink as the printing material, 3D printing technology is used to print layer by layer, and high light intensity is used to irradiate the bio-ink until the gel point is reached to obtain a pre-cured printed part; (2) Remove the pre-cured print and remove any residual bio-ink and light absorber; (3) Immerse the pre-cured printed part after step (2) in a low concentration photoinitiator solution and perform post-processing with low light intensity. After the post-processing is completed, the cell-loaded printed part is obtained.

2. The projection photocuring biological 3D printing method according to claim 1, characterized in that, The large light intensity irradiation intensity is 10-40 mW / cm 2 .

3. The projection photocuring biological 3D printing method according to claim 1, characterized in that, The irradiation intensity of the small light intensity is 0.1-5 mW / cm 2 , and the irradiation time is 20-60 min. 4.The projection photocuring biological 3D printing method according to claim 1, wherein, In step (3), the photoinitiator solution is a PBS solution of photoinitiator, wherein the concentration of photoinitiator is 0.05~0.1%w / v.

5. The projection photocuring biological 3D printing method according to claim 1, characterized in that, In step (2), PBS solution is used to remove residual bio-ink and light absorber by first cleaning and then soaking the pre-cured print. 6.The projection photocuring biological 3D printing method according to claim 1, wherein, The bio-ink uses PBS solution as a solvent and includes 200,000 to 2,000,000 live cells / mL, 10 to 15% w / v biomaterial monomers, 0.2 to 0.5% w / v photoinitiator, and 0.05 to 0.1% w / v light absorber. The biomaterial monomers are one or more of the following: methacrylamide gelatin, methacrylamide silk fibroin, methacrylamide hyaluronic acid, methacrylamide silk fibroin, polyethylene glycol diacrylate, methacrylamide chitosan, polyether F127 diacrylate, methacrylamide sodium alginate, methacrylamide chondroitin sulfate, and tetra-arm polyethylene glycol acrylate.

7. The projection photocuring biological 3D printing method according to claim 6, characterized in that, The photoinitiator is one or a mixture of 2,4,6-trimethylbenzoyldiphenoxyphosphine, ethyl 2,4,6-trimethylbenzoylphosphonate, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, diazonium salt, diaryliodomonium salt, triarylthionium salt, alkylthionium salt, iron aromatic salt, sulfonyloxy ketone, and triarylsiloxane.

8. The projection photocuring biological 3D printing method according to claim 6, characterized in that, The light absorber is one or more of the following: pigment, ultraviolet light absorber, quencher, and free radical scavenger.

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