TWI720425B - Bioink containing nutritional composition and use of the nutrient composition for enhancing self-healing capability of bioink - Google Patents

Abstract

Provided is use of a nutritional composition for preparing a self-healing bioink composition, wherein the nutritional composition includes an amino acid, a vitamin, and an inorganic salt, and the bioink composition includes a biodegradable polyurethane and a gelatin. Also provided is a self-healing shape memory bioink composition including a biodegradable polyurethane, a gelatin, a methacryloyl, and the nutritional composition.

Description

Bio-ink containing nutritional composition and use of nutritional composition to enhance the self-healing power of bio-ink

The present invention relates to a novel use of a nutritional composition, in particular to a use of a nutritional composition to prepare a bio-ink composition with self-healing power, and a bio-ink composition with self-healing power and shape memory.

Hydrogel is a three-dimensional polymer network with high water content and is biocompatible because of its mechanical properties similar to those of biological soft tissues. Intelligent water glue can respond to changes in pressure, temperature, pH, electric and magnetic fields or other stimuli. Examples of smart water glues include environmentally sensitive water glue shapes, shape memory water glues, and self-healing water glues. Among environmentally sensitive hydrogels, heat-sensitive hydrogels are often used in biomedical engineering fields, such as drug carriers, biosensors, three-dimensional printing, or other fields because of their easy handling. Photosensitive hydrogels can form adjustable cross-linked structures in a fast and highly accurate manner. They are also suitable for biomedical engineering, including wound repair, minimally invasive surgery, and three-dimensional printing. Self-healing hydrogel can repair structural or functional damage by itself, and it can be divided into non-autonomous or autonomous self-repairing systems. The spontaneous self-healing system does not require additional additives or Self-repair can be completed by stimulation, so it has great potential for development in biomedical applications.

The polymers that make up the hydrogel can be classified into natural or synthetic polymers depending on the source. Natural polymers include collagen, fibrin, chitosan, and gelatin; synthetic polymers include polyethylene glycol (PEG) and polycaprolactone (PCL) , Polylactic acid (polylactide, PLA), and polyurethane (polyurethane, PU), etc. Natural polymer water glue has excellent biocompatibility, but its mechanical strength and structural stability are poor. For example, gelatin is a kind of biological It only forms a gel at a temperature lower than 28°C, but melts at the temperature of the human body. In contrast, synthetic polymer hydrogels have better mechanical properties, but often have problems of cytotoxicity, low diffusibility, and non-biodegradability.

At present, most of the water glues known to be used for 3D printing are heat-sensitive and light-sensitive water glues, but such water glues rarely have self-healing properties, such as heat-sensitive gelatin and light-sensitive methacrylate Gelatin (gelatin methacryloyl, GelMA), and heat-sensitive or light-sensitive polyurethane. On the other hand, directly using self-healing water glue for 3D printing encounters challenges such as resolution, shape fidelity, and stackability. For example, the printed product of self-healing water glue formed by benzaldehyde-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA) has the resolution and shape fidelity In order to maintain a specific shape after printing, one of the solutions is to print the self-healing water glue onto another material, but there are still poor contact between the two materials and non-biodegradable problem.

In order to prepare a 3D printing structure with high resolution, structural stability, biocompatibility, and self-healing power for application in the field of biomedicine, a self-healing 3D printing water glue was developed, especially available It is really necessary for the three-dimensional printing bio-ink to directly print living cells.

For this reason, one purpose of the present invention is to provide a nutritional composition for preparing a self-healing bio-ink composition, wherein the nutritional composition includes an amino acid, a vitamin, and an inorganic salt And the bio-ink composition includes a biodegradable polyurethane and a gelatin.

In an embodiment of the present invention, the biodegradable polyurethane comprises a hard segment and a soft segment connected to each other, the hard segment is formed by the reaction of a diisocyanate and a chain extender, and the soft segment is at least An oligomer diol, and the chain extender includes an anionic chain extender. By adjusting the soft segment composition of the biodegradable polyurethane, such as the type and proportion of oligomer diols, the aforementioned bio-ink composition can be used to print printed products with different degradation rates and mechanical properties.

In an embodiment of the present invention, the bio-ink composition includes the biodegradable polyurethane in a weight ratio of 8% to 28%, gelatin in a weight ratio of 2% to 7%, and the weight ratio of at least 0.01%. Nutritional composition.

In an embodiment of the present invention, the bio-ink composition may further include methacrylated gelatin used as a photo-crosslinking agent to facilitate the subsequent photo-curing reaction. In a preferred embodiment, the bio-ink composition contains 1% to 10% methacrylated gelatin by weight.

Another object of the present invention is to provide a bio-ink composition with self-healing power and shape memory, which contains a biodegradable polyurethane, a gelatin, a methacrylated gelatin, and a nutritional composition , Wherein the nutritional composition includes an amino acid, a vitamin, and an inorganic salt.

In an embodiment of the present invention, the bio-ink composition with self-healing power and shape memory includes the biodegradable polyurethane in a weight ratio of 8% to 28%, and gelatin in a weight ratio of 2% to 7%. The weight ratio of methacrylated gelatin is 1% to 10%, and the weight ratio of the nutritional composition is at least 0.01%.

The present invention discloses a new application of the nutritional composition supporting cell growth to make the bio-ink composition containing polyurethane and gelatin have self-repairing power. When the aforementioned bio-ink composition further includes methacrylated gelatin for photocrosslinking, it exhibits self-healing power regardless of whether it is exposed to light. Therefore, not only the structural damage caused by extrusion in the filamentous water glue formed by the extrusion of the bio-ink composition during three-dimensional printing can be repaired, but also the printed multilayer structure can be integrated without Will separate from each other, so there is no need to print the bio-ink composition on other supporting materials in order to improve structural stability or shape fidelity; at the same time, the printed product has the characteristic of spontaneously repairing internal damage even after light curing It can maintain its quality and extend its service life.

Furthermore, the present invention discloses that the bio-ink composition comprising polyurethane, gelatin, methacrylated gelatin, and nutritional composition still has many advantages, including high biocompatibility, printability and stackability, and after light curing Long-term structural stability, good flexibility and shape memory, so it is suitable for high-resolution, high-fidelity, and multi-layer stacked three-dimensional bioprinting, and even for four-dimensional bioprinting that includes time parameters for expansion Increase the application method of printing the finished product. In practical applications, the bio-ink composition of the present invention is particularly suitable for preparing artificial tissues or bionic scaffolds.

The following will further illustrate the embodiments of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent scope.

The photos at different times in Figure 1 show that after the solid water gel formed by the PCL100/Gel/DMEM-LG bio-ink is divided into several pieces, the pieces will be inseparable once again in contact with each other at 25°C for a period of time.

The photos at different times in Fig. 2 show that after the solid water gel formed by the PCL80DL20/Gel/DMEM-LG bio-ink is divided into two pieces, the pieces will be inseparable once again in contact with each other at 25°C for a period of time.

Figure 3 shows the storage modulus (G') and loss modulus (G") of PCL80DL20/Gel/DMEM-LG bio-ink at 1% and 140% strain in the repair-destruction cycle test.

The photos at different times in Figure 4 show that the cracks in the solid water gel formed by the PCL70DL20HB10-5H/Gel/DMEM-HG bio-ink healed and shrunk with time at 22°C.

Figure 5 shows the storage modulus and loss modulus of PCL70DL20HB10-5H/Gel/DMEM-HG bio-ink in the repair-destruction cycle test at 1% and 300% strain.

The photos at different times in Figure 6 show that the cracks in the solid water gel formed by the PCL70DL10HB20/Gel/DMEM-HG bio-ink heal and shrink with time at 20°C.

Figure 7 shows the storage modulus and loss modulus of PCL70DL10HB20/Gel/DMEM-HG bio-ink in the repair-destruction cycle test at 1% and 1000% strain.

The photos at different times in Fig. 8 show that the cracks in the solid water glue formed by the PCL70DL20HB10-5H/Gel bio-ink did not heal and shrink with time at 20°C.

The photos at different times in Fig. 9 show that the solid water glue formed by PCL80DL20/Gel/GelMA/DMEM-LG (also referred to as PUG) bio-ink is divided into several pieces, and these pieces will be in contact with each other for a period of time at 20℃. Separated unity.

Figure 10 shows the storage modulus and loss modulus of PUG bio-ink at 1% and 500% strain in the repair-destruction cycle test.

Figure 11 shows the viscosity change of PUG bio-ink at 25°C and ^{shear rate range of 0.1-1000s -1.}

Fig. 12A is a photograph of the silk-like water glue extruded from the 160μm nozzle of a 3D printing device with PUG bio-ink.

The photo of FIG. 12B shows the process of the PUG filamentous water glue of FIG. 12A being rotated and stacked to form a tubular structure.

Fig. 12C is a photograph of the 50-layer tubular structure printed with the PUG filament water glue of Fig. 12A.

Figure 13A is a photo of a grid-like four-layer structure printed with PUG bio-ink.

Fig. 13B is a photograph of the structure of Fig. 13A after being irradiated with 5W ultraviolet light for 1 minute.

Fig. 13C is a photomicrograph of the structure of Fig. 13B after soaking in phosphate buffer saline (PBS) at 37°C for 5 days; the scale bar represents 500 μm.

Figure 14A is a photo of a square four-layer structure printed with PUG bio-ink after being irradiated with a 5W ultraviolet lamp for 1 minute.

The photograph of Fig. 14B shows that the structure of Fig. 14A is cut into two separate parts.

The photo in FIG. 14C shows that the two parts of the structure in FIG. 14B contact each other for a period of time and then form an inseparable unity again.

Figure 15 shows the storage modulus and loss modulus at 1% and 300% strain of the PUG bio-ink irradiated with ultraviolet light in the repair-destruction cycle test.

Figure 16A is a top view photograph of a rectangular parallelepiped structure printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute.

Fig. 16B is a side view photograph of the structure of Fig. 16A along the long side.

The photograph of Figure 16C shows the construction of Figure 16A being bent with tweezers at room temperature.

The photograph of Fig. 16D shows that the curved structure in Fig. 16C returns to the state shown in Fig. 16B.

Figure 17A is a photo of a tubular twenty-five-layer structure printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute.

The photograph of Figure 17B shows the construction of Figure 17A being compressed with tweezers at room temperature.

The photo in FIG. 17C shows that the compressed structure in FIG. 17B returns to the state shown in FIG. 17A.

Figure 18A is a top view photograph of a six-layer cuboid structure printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute.

The photograph of Fig. 18B shows that the structure of Fig. 18A is bent with tweezers and the shape is fixed at -20°C.

Fig. 18C is a photograph of the bent structure in Fig. 18B after detaching from the tweezers.

Figure 18D is a photograph of the structure of Figure 18C after being placed in 37°C water for 1 minute.

Figure 19A is a top view photograph of a honeycomb-shaped three-layer structure printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute.

The photograph of Fig. 19B shows the compressed structure obtained by compressing the structure of Fig. 19A with tweezers and fixing the shape at -20°C.

Figure 19C is a photograph of the structure of Figure 19B after being placed in water at 37°C for 1 minute.

Figure 20 shows the survival rate of mouse neural stem cells in polyurethane dispersion, PUG bio-ink, or cell culture medium.

Figure 21 shows the relative proliferation rate of mouse neural stem cells after culturing in a polyurethane dispersion or PUG bio-ink printed constructs for a specified number of days.

The present invention provides a use of a nutritional composition for preparing a self-healing bio-ink composition, wherein the nutritional composition includes an amino acid, a vitamin, and an inorganic salt, and the bio-ink composition Contains a biodegradable polyurethane and a gelatin. The present invention also provides a bio-ink composition with self-healing power and shape memory, which contains a biodegradable polyurethane, a gelatin, a methacrylated gelatin, and the aforementioned nutritional composition. The following examples illustrate the preparation method and characteristics of the biological ink water glue of the present invention, especially the self-healing power by macro-damage repair and micro-rheological analysis, and by using the biological ink for general three-dimensional printing or Printing together with living cells illustrates the printability, stackability, and high biocompatibility of the bio-ink water glue of the present invention, as well as the flexibility and shape memory of the solid water glue printed from it.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The term "nutrient composition" as used herein refers to a combination of substances required for the survival, growth, or proliferation of mammalian cells, such as commercially available cell culture media, including but not limited to basal medium Eagle (BME for short). Medium), minimum basal medium (Minimal Essential Medium, MEM medium for short), Dulbecco's modified Eagle Medium (DMEM medium for short), and Roswell Park Memorial Institute 1640 Medium (RPMI for short) -1640 medium). The nutritional composition includes amino acids, vitamins, and inorganic salts, and may further include monosaccharides and other compounds.

The "photo-initiator" referred to herein is a compound that forms free radicals under light (including ultraviolet light or visible light), which can be used to cure a photosensitive polymer. Examples of photoinitiators include water-soluble VA-086, VA-044, and V-50, and water-insoluble Irgacure 1870, Irgacure 2959 and the like.

Materials and methods Preparation of biodegradable polyurethane

In all the embodiments herein, the biodegradable polyurethane (herein also referred to as polyurethane or PU) is a polyurethane synthesized by an aqueous process, which has heat sensitivity or light sensitivity. The biodegradable polyurethane has a main chain, and the main chain includes a hard segment and a soft segment that are connected. The hard segment is formed by the reaction of diisocyanate and a chain extender; the diisocyanate can be isophorone diisocyanate (IPDI); the chain extender includes an anionic chain extender Agent and a second chain extender. An example of the anionic chain extender is dimethylolpropionic acid (2,2-bis(hydroxymethyl)propionic acid, DMPA); an example of the second chain extender is ethylenediamine with short chain and high reactivity (ethylenediamine, EDA). The soft segment contains at least one oligomer diol, such as poly(ε-caprolactone)diol (poly(ε-caprolactone)diol, PCL diol for short), DL type polylactic acid diol (poly(D, L-lactide)diol, PDLLA diol for short), LL-type poly(L,L-lactide)diol (PLLA diol for short), poly(hydroxybutyrate) diol (poly(3) -hydroxybutyrate, PHB diol for short), polyethylene butylene adipate diol (PEBA diol for short), or a combination thereof. The characteristics of the biodegradable polyurethane can be adjusted by changing the composition of the hard segment and the soft segment.

In one embodiment, the biodegradable polyurethane is formed by the reaction of oligomer diol, IPDI, DMPA, and EDA at an immolar ratio of 1:3~4:1:1~2, wherein the oligomer is binary Alcohol can be completely composed of PCL diol, or formed by the reaction of PCL diol with other oligomer diols in different molar ratios. As an example, the following briefly describes the synthesis method of the biodegradable polyurethane (PCL80DL20 polyurethane) containing the soft segment formed by the reaction of PCL diol and PDLLA diol at 80:20 percent by mole. Add PCL glycol (molecular weight approximately 2000g/mol; purchased from Sigma-Aldrich) and PDLLA glycol (molecular weight approximately 1500g/mol; purchased from Sigma-Aldrich) into a four-necked reaction flask at a molar ratio of 4:1 , Stirring at a speed of 180 rpm for 30 minutes at 95°C and full nitrogen filling, and then adding the catalyst stannous dioctanoate (tin(II)2-ethylhexanoate, T-9 for short; purchased from Alfa Aesar) and IPDI (available from Acros) Organics) react for about 3 hours. After that, DMPA (purchased from Sigma-Aldrich) and methyl ethyl ketone (MEK; purchased from JTBaker) as a solvent were added to a four-necked reaction flask, and the reaction was stirred at 75°C for about 1 hour, and the temperature was lowered to 50°C. Then, triethylamine (TEA for short; purchased from RDH) was added to neutralize the reaction. Disperse the aforementioned reaction mixture in deionized water at a speed of 1100 rpm, and then add ethylene diamine (purchased from Tedia) to obtain a biodegradable polyurethane dispersion (solid content about 30 wt%). That disperse The residual solvent in the liquid can be removed by distillation under reduced pressure. The heat-sensitive biodegradable polyurethane can form a solid hydrogel at 37°C.

The other types of biodegradable polyurethane used in the following examples are prepared in a similar manner as described above, including PCL100 polyurethane, whose soft segment is entirely composed of PCL diol; PCL70LL30 polyurethane, whose soft segment is composed of PCL diol and PLLA binary The percentage of emolal alcohol is formed by 70:30; PCL70DL20HB10-5H polyurethane, the soft segment is formed by the reaction of PCL diol, PDLLA diol, and the percentage of PHB diol, 70:20:10, and The preparation process uses 2-hydroxyethyl methacrylate (HEMA) to replace half of the EDA; and PCL70DL10HB20 polyurethane, the soft segment of which is composed of PCL diol, PDLLA diol, and PHB diol The percentage of Irmol is 70:10:20.

Preparation of methacrylated gelatin

Methacrylated gelatin can be formed by the reaction of gelatin and methacrylic anhydride (MAA), which is biocompatible and biodegradable. The preparation method of methacrylated gelatin used in the following examples is briefly described as follows. Add 10g of the gelatin produced by acid hydrolysis (with a gel strength of about 300 bloom) into 100mL of 0.25M carbonate-bicarbonate buffer (prepared from sodium carbonate and sodium bicarbonate, pH 9), and stir at 45°C until the gelatin is completely dissolved , And then add 2mL of methacrylic anhydride dropwise. After 90 minutes of reaction, adjust the pH of the aforementioned solution to about 7.4 with hydrochloric acid or sodium hydroxide to stop the reaction. The resulting solution was dialyzed against water with a dialysis membrane with a molecular weight cutoff of 12-14 kDa at 37°C for about seven days, and then the dialysate was filtered with filter paper. The obtained filtrate is freeze-dried to remove moisture, and then a dry powder of methacrylated gelatin with a degree of substitution of about 93.3% can be obtained, which is stored at -20°C.

Cell culture medium

Unless otherwise specified, the cell culture medium used in the following examples is purchased from Thermo Fisher Scientific’s Gibco DMEM medium, which contains glycine, L-arginine, and L-cystine -cystine), L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine (L-lysine), L-methionine (L-methionine), L-phenylalanine (L-phenylalanine), L-serine (L-serine), L-threonine ( Amino acids such as L-threonine, L-tryptophan, L-tyrosine, and L-valine; choline, pantothenic acid Vitamins such as calcium pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, thiamine, and inositol; chlorine Calcium, iron nitrate, Inorganic salts such as magnesium sulfate, potassium chloride, sodium chloride, and sodium dihydrogen phosphate; acid-base indicators (such as phenol red) and other compounds such as sodium pyruvate; and D-glucose, the concentration of which can be adjusted, for example 1g/L (also denoted as low glucose or LG) or 4.5g/L (also denoted as high glucose or HG).

Rheological properties measurement

Load approximately 0.6 mL of bio-ink on the sample stage of the rheometer (HR2; TA Instruments), and set its temperature to 25°C. When the bio-ink reaches 25°C, time sweep, frequency sweep at 1% strain, and strain sweep at 1 Hz are performed in sequence to confirm that the colloidal strength of the bio-ink is stable Then, the critical strain value for sol-gel conversion was determined. After that, the repair-destruction cycle test was performed by time scanning, that is, in a cycle, the storage modulus (G') and loss modulus (loss modulus, G"), after about 300 seconds, measure the G'and G" of the damaged colloid with a strain exceeding the critical strain value of the sol-gel conversion. The aforementioned repair-destruction cycle test is repeated several times, with an interval of about 600 seconds between each cycle for the gel to repair itself.

In order to check whether the bio-ink has the characteristics of shear-thinning, the viscosity of the bio-ink is measured ^{at 25°C and a flow mode with a shear rate of 0.1-1000s -1.}

Cell culture

The cell survival test uses neural stem cells isolated from the brains of adult mice. The cell line was cultured in DMEM-HG medium and Han's F-12 medium (Ham's F-12 medium; purchased from Thermo Fischer Scientific) under the conditions of 37°C and 5% carbon dioxide in a 1:1 volume ratio of DMEM/F. -12 medium with 10% fetal bovine serum (FBS; purchased from Thermo Fisher Scientific), 400 μg/ml G418 (purchased from Invitrogen), and 1% penicillin-streptomycin (penicillin-streptomycin; Available from Thermo Fisher Scientific). The cells change the medium every two days.

Statistical Analysis

The experimental data are all expressed as mean±standard deviation (S.D.). Each experiment was repeated three times independently to verify its reproducibility. The statistical differences between the experimental groups are determined by the analysis of variance and Student’s T test. A p value of less than 0.05 is regarded as a statistically significant difference between the two groups, and is marked with the symbol *.

Example 1 Cell culture medium enhances the self-healing power of polyurethane/gelatin bio-ink

In order to examine whether the nutritional composition affects the self-healing ability of the bio-ink, a variety of bio-ink compositions containing biodegradable polyurethane, gelatin and cell culture medium were first prepared, and the self-healing test was carried out. The bio-ink composition preferably contains 8% to 28% by weight of biodegradable polyurethane, 2% to 7% by weight of gelatin, and 0.01% to 5% by weight of cell culture medium.

1.1 PCL100 polyurethane/gelatin/DMEM-LG medium

A PCL100 polyurethane dispersion, a gelatin aqueous solution, and a DMEM medium aqueous solution (containing 3.7g/L sodium bicarbonate and low glucose) are uniformly mixed at 37°C to obtain a bio-ink, referred to as PCL100/Gel/DMEM-LG , Wherein the weight percentages of the PCL100 polyurethane, gelatin, and cell culture medium are about 16%, 4%, and 1%, respectively. After that, put the bio-ink into a container, such as a cell culture plate, and let it stand for a period of time at 4°C to 25°C to obtain a solid water gel. In order to test the self-healing power, as shown in Figure 1, the solid water glue is cut into multiple pieces that are completely separated. When any one piece is picked up with tweezers, the other piece cannot be lifted together, indicating that the solid water glue Not enough adhesiveness. However, when the blocks are in contact with each other and placed at 25°C for about 24 hours, it can be observed that when the edges of a block are picked up with tweezers, multiple blocks can be lifted together without shaking. Cause any lumps to detach. This result indicates that the bio-ink and the solid hydrogel formed by it have spontaneous self-healing power, which can automatically repair the damaged area in the hydrogel without additional stimulation. It is speculated that this feature is due to the microstructure reorganization of the contact interface of the hydrogel .

1.2 PCL80DL20 polyurethane/gelatin/DMEM-LG medium

A PCL80DL20 polyurethane dispersion, a gelatin aqueous solution, and a DMEM medium aqueous solution (containing 3.7g/L sodium bicarbonate and low glucose) are uniformly mixed at 37°C to obtain a biological ink, referred to as PCL80DL20/Gel/DMEM-LG , Wherein the weight percentages of the PCL80DL20 polyurethane, gelatin, and cell culture medium are about 16%, 4%, and 1%, respectively. After that, the bio-ink is poured into a cell culture plate, and left to stand for a period of time at 4°C to 25°C to obtain a solid water gel. To test the self-healing power, as shown in Figure 2, the solid water glue was cut into two completely separated pieces. When the blocks are in contact with each other and placed at 25°C for about 24 hours, it can be observed that when the edge of one block is picked up with tweezers, the other block can be lifted together, showing the bio-ink and its formation The solid water glue has spontaneous self-healing power.

In order to evaluate the self-repairing effect of the bio-ink, the aforementioned bio-ink was subjected to a repair-destruction cycle test using a rheometer at 25°C. The results are shown in Figure 3. At 1% strain, G'and G" are fixed values and G'is greater than G", indicating that the bio-ink has a stable gel state. When the strain increases to 140% (over After passing the critical strain of sol-gel conversion of PCL80DL20/Gel/DMEM-LG bio-ink), it is observed that G" is greater than G', indicating that the gel is destroyed and transformed into sol. However, after a recovery time of about 600 seconds, The bio-ink exhibits a stable gel state again at 1% strain, indicating that it has self-healing power. Figure 3 also shows that the bio-ink still exhibits nearly 100% of the initial gel strength after several repair-destruction cycles. It shows that this self-healing power is repeatable.

1.3 PCL70DL20HB10-5H polyurethane/gelatin/DMEM-HG medium

Mix a PCL70DL20HB10-5H polyurethane dispersion, a gelatin aqueous solution, and a DMEM medium aqueous solution (containing 3.7g/L sodium bicarbonate and high glucose) at 37℃ to obtain a biological ink, referred to as PCL70DL20HB10-5H/Gel /DMEM-HG, wherein the weight percentages of the PCL70DL20HB10-5H polyurethane, gelatin, and cell culture medium are about 16%, 4%, and 1%, respectively. After that, the bio-ink is poured into a cell culture plate, and left to stand for a period of time at 4°C to 25°C to obtain a solid water gel. To test the self-healing power, as shown in Figure 4, the solid water glue was split and dug to form several cracks. When the solid water glue is placed at 22°C for about 14 hours, the healing and shrinkage of the aforementioned cracks can be observed, indicating that the bio-ink and the formed solid water glue have spontaneous self-healing power.

The repair-destruction cycle test of the aforementioned biological ink was carried out using a rheometer at 25°C, and the results are shown in Figure 5. At 1% strain, G'and G" are fixed values and G'is greater than G", indicating that the bio-ink has a stable gel state. When the strain increases to 300% (more than the critical strain of sol-gel conversion of PCL70DL20HB10-5H/Gel/DMEM-HG bio-ink), it is observed that G" is greater than G', indicating that the gel is destroyed and transformed into a sol. But after After a recovery time of about 600 seconds, the bio-ink once again showed a stable gel state at 1% strain, indicating that it has self-healing power. Figure 5 also shows that the bio-ink still exhibits a near-distance performance after several repair-destruction cycles. The initial gel strength of 100% indicates that the self-healing power is repeatable.

1.4 PCL70DL10HB20 polyurethane/gelatin/DMEM-HG medium

A PCL70DL20HB10 polyurethane dispersion, a gelatin aqueous solution, and a DMEM medium aqueous solution (containing 3.7g/L sodium bicarbonate and high glucose) are uniformly mixed at 37°C to obtain a biological ink, referred to as PCL70DL10HB20/Gel/DMEM-HG , Wherein the weight percentages of PCL70DL10HB20 polyurethane, gelatin, and cell culture medium are about 16%, 4%, and 1%, respectively. After that, the bio-ink is poured into a cell culture plate, and left to stand for a period of time at 4°C to 25°C to obtain a solid water gel. In order to test the self-healing power, as shown in Figure 6, the solid water glue was split and dug to form several cracks. When the solid water glue is placed at 20°C for about 12 hours, the healing of the aforementioned cracks can be observed, indicating that the bio-ink and the solid water glue formed have spontaneous self-healing power.

The repair-destruction cycle test of the aforementioned biological ink was carried out using a rheometer at 25°C, and the results are shown in Figure 7. At 1% strain, G'and G" are fixed values and G'is greater than G", indicating that the bio-ink has a stable gel state. When the strain increases to 1000% (more than the critical strain of sol-gel conversion of PCL70DL10HB20/Gel/DMEM-HG bio-ink), it is observed that G" is greater than G', indicating that the gel is destroyed and transformed into a sol. But after about 600 After a recovery time of 2 seconds, the bio-ink exhibited a stable gel state again at 1% strain, indicating that it has self-healing power. Figure 7 also shows that the bio-ink still exhibits close to 100 after several repair-destruction cycles % Of the initial colloidal strength indicates that the self-healing power is repeatable.

Comparative example 1 Bio-ink without cell culture medium

A PCL70DL20HB10-5H polyurethane dispersion and a gelatin aqueous solution are uniformly mixed at 37°C to obtain a biological ink, referred to as PCL70DL20HB10-5H/Gel, wherein the weight percentages of the PCL70DL20HB10-5H polyurethane and gelatin are about 16% and 4% respectively . After that, the bio-ink is poured into a cell culture plate and left to stand for a period of time at 4°C to 25°C to obtain a solid water gel. In order to test the self-healing power, as shown in Figure 8, the solid water glue was split and dug to form several cracks. When the solid hydrogel was placed at 20°C for 20 hours, it was observed that the aforementioned crack did not heal or shrink significantly, indicating that the bio-ink without cell culture medium and the solid hydrogel formed by it had no self-healing power.

The same results were also seen in bio-inks prepared from PCL70DL10HB20 or PCL70LL30 polyurethane dispersions and an aqueous gelatin solution (data not shown).

Example 2 Cell culture medium enhances the self-healing power of polyurethane/gelatin/methacrylated gelatin bio-ink

A PCL80DL20 polyurethane dispersion, a gelatin and methacrylated gelatin aqueous solution, a cell culture medium aqueous solution (containing 3.7g/L sodium bicarbonate and low glucose), and a photoinitiator VA-086 (purchased from Wako Chemicals) Company) uniformly mix at 37℃ to obtain a bio-ink, referred to as PCL80DL20/Gel/GelMA/DMEM-LG or PUG, where the weight percentage of PCL80DL20 polyurethane, gelatin, methacrylated gelatin, and cell culture medium is about 12 %, 3%, 4%, and 1%, and the photoinitiator accounts for 1.5% of the total solid content of the bio-ink. After that, the bio-ink is poured into a cell culture plate and left to stand for a period of time at 4°C to 25°C to obtain a solid water gel. To test the self-healing power, as shown in Figure 9, the solid water glue was cut into two completely separated pieces. When the blocks are in contact with each other and placed at 20°C for 12 hours, it can be observed that the edge of the block can be picked up with tweezers. And lifted up another block, showing that the bio-ink and the solid water glue formed by it has spontaneous self-healing power. Alternatively, the same result can be observed when the block obtained according to the aforementioned steps is placed at 4°C for 24 hours (data not shown).

In order to evaluate the self-repairing effect of the bio-ink, the PUG bio-ink was subjected to a repair-destruction cycle test using a rheometer at 25°C. The results are shown in Figure 10. At 1% strain, G'and G" are fixed values and G'is greater than G", indicating that the bio-ink has a stable gel state. When the strain increases to 500% (exceeding the critical strain for sol-gel conversion of PUG bio-ink), it is observed that G" is greater than G', indicating that the gel is destroyed and transformed into a sol. However, after a recovery time of about 600 seconds, The bio-ink exhibits a stable gel state again at 1% strain, indicating that it has self-healing power. Figure 10 also shows that the bio-ink still exhibits more than 80% of the initial gel strength after several repair-destruction cycles. This self-healing power is repeatable.

Example 3 The characteristics of PUG bio-ink

In order to explore the feasibility of the polyurethane/gelatin/methacrylated gelatin bio-ink of the nutritional composition of the present invention for three-dimensional printing, the PUG bio-ink described in Example 2 was used for rheological property analysis and three-dimensional printing.

3.1 Printability and stackability of bio-ink

Figure 11 shows the change in viscosity of the PUG bio-ink at 25°C and a ^{shear rate range of 0.1-1000s -1 measured by a rheometer.} According to the figure, the viscosity of the bio-ink decreases as the shear rate increases (the slope is about -1.36). This shear-thinning characteristic indicates that the bio-ink has good printability.

When printing, take a pre-designed three-dimensional pattern as a blueprint, and use a three-dimensional printing device connected to a computer to extrude the PUG bio-ink through a syringe to form a silk-like water glue, which is stacked on a printing platform into one A structure with a predetermined structure. The bio-ink is printed under the following conditions: the temperature of the printing platform is between 4℃ and 25℃, which can be adjusted according to the requirements of the resolution of the structure. The diameter of the nozzle outlet connected to the syringe is 160μm, and the nozzle temperature is between 20℃ and 30℃. °C, preferably 24°C to 27°C. As shown in Figure 12A, the filamentous water glue extruded from the nozzle is continuous and has no regional expansion, indicating that the bio-ink has good printability. As shown in FIGS. 12B and 12C, the filamentous water glue can be rotated and stacked fifty layers to form a tubular structure that can be picked up by tweezers and has a stable structure, indicating that the bio-ink is stackable.

3.2 The structural stability of the structure after being irradiated with ultraviolet light

Figure 13A is a photo of a grid-like four-layer structure printed with PUG bio-ink; the printing conditions of the structure are as follows: the printing platform temperature is 4°C, the nozzle diameter is 160μm, and the nozzle temperature is 26°C. Figure 13B is a photograph of the structure after being irradiated with 5W ultraviolet light (wavelength: 365nm) about 0.5 cm apart for 1 minute. Since the PUG bio-ink contains methacrylated gelatin for light crosslinking and a photoinitiator, the printed product has increased mechanical strength due to the crosslinking reaction between methacrylated gelatin molecules after being irradiated with ultraviolet light, so The structure of Figure 13B can be clamped and moved to a cell culture plate for subsequent processing. Fig. 13C is a photomicrograph of the structure irradiated with ultraviolet light after being soaked in a PBS solution at 37°C for 5 days, which shows that the specific grid structure of the structure can be maintained in a physiological environment for at least 5 days. The width of the linear water glue between the grids is about 195μm.

3.3 The self-healing power of the structure after being irradiated with ultraviolet light

Figure 14A is a photo of a square four-layer structure printed with PUG bio-ink after being irradiated with a 5W UV lamp for 1 minute; the printing conditions of the structure are as follows: the printing platform temperature is 20°C, the nozzle diameter is 160μm, The nozzle temperature is 26°C. Cut the structure into two completely separated parts as shown in Fig. 14B. After contacting the two parts with each other and placing them at 25°C for 24 hours, it can be observed that when the edge of one part is picked up with tweezers, the other part can be lifted together. , And shaking will not cause any part to detach, as shown in Figure 14C. This result shows that the structure still has self-healing power after UV irradiation.

In order to test the self-repairing effect of the PUG bio-ink after being irradiated with ultraviolet light, a rheometer was used to perform a repair-destruction cycle test on the aforementioned biological ink irradiated with 5W ultraviolet light for 1 minute at 25°C. The results are shown in Figure 15. At 1% strain, G'and G" are fixed values and G'is greater than G", indicating that the bio-ink has a stable gel state. When the strain increases to 300% (exceeding the critical strain of the sol-gel transition of the bio-ink), it is observed that G" is greater than G', indicating that the gel is destroyed and transformed into a sol. However, after a recovery time of about 600 seconds, The bio-ink exhibits a stable gel state again at 1% strain, indicating that it has self-healing power. Figure 15 also shows that the bio-ink still exhibits more than 95% of the initial gel strength after several repair-destruction cycles. Its self-healing power is repeatable.

According to the results of Example 2 and Examples 3.1 to 3.3, the exemplary bio-ink of the present invention has appropriate viscoelasticity and printability, so it can be used with a nozzle with a diameter of about 160μm for high resolution and high shape fidelity. Three-dimensional printing. At the same time, the preliminary printed product of the bio-ink has sufficient mechanical strength and stackability before being irradiated with ultraviolet light, and has the characteristics of layer-to-layer integration due to the self-healing power of the material, so there is no need to worry about the separation of layers from each other. In addition, when irradiated with UV light for a short period of time, the The structure formed by biological ink not only exhibits long-term structural stability due to the increase in mechanical strength, but also retains self-healing power. Therefore, the bio-ink of the present invention is suitable for printing multi-layer stacked high-resolution structures.

3.4 Elasticity of the structure after being irradiated with ultraviolet light

Figure 16A is a top view photo of a rectangular parallelepiped structure (10mm long side, 5mm short side, and about 1.5mm height) printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute; the printing conditions of the structure are as follows : The temperature of the printing platform is 20°C, the nozzle diameter is 160μm, and the nozzle temperature is 26°C. Figure 16B is a side view photo of the structure along the long side. After bending the structure with tweezers as shown in FIG. 16C at room temperature for a period of time, the structure can return to its original state by itself, as shown in FIG. 16D.

Figure 17A is a top view photo of a tubular twenty-five-layer structure (outer diameter about 6.5mm) printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute; the printing conditions of the structure are as follows: Print platform The temperature is 20°C, the nozzle diameter is 160μm, and the nozzle temperature is 26°C. After compressing the structure with tweezers as shown in FIG. 17B at room temperature for a period of time, the structure can return to its original shape by itself, as shown in FIG. 17C, and the structure still has a restoring force after repeated compressions.

The foregoing results indicate that the printed product of the exemplary bio-ink of the present invention has good flexibility at room temperature, so the bio-ink of the present invention is suitable for printing structures with flexible requirements, such as bionic stents.

3.5 The shape memory of the structure after being irradiated with ultraviolet light

Figure 18A is a top view of a six-layer cuboid structure printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute; the printing conditions of the structure are as follows: the printing platform temperature is 20℃, and the nozzle diameter is 160μm , The nozzle temperature is 26°C. To evaluate the shape memory of the structure, the structure was bent 180° with tweezers as shown in Figure 18B and the curved shape was fixed at -20°C for 20 minutes, and then the structure was placed on a plane at -20°C. According to Fig. 18C, the structure maintains a bend of about 164° after being separated from the tweezers, so its shape fixation rate is about 91%. After placing the curved structure in water at 37°C for 1 minute, it was observed that it returned to the flat state as shown in Figure 18D. Comparing FIGS. 18A and 18D, it can be seen that the shape recovery rate of the structure is 100%.

Figure 19A is a top view photograph of a honeycomb-shaped three-layer structure (side length 12mm) printed with PUG bio-ink after being irradiated with 5W ultraviolet light for 1 minute; the printing conditions of the structure are as follows: the printing platform temperature is 20 ℃, the nozzle diameter is 160μm, and the nozzle temperature is 26℃. Compress the structure at -20°C for 20 minutes, and then place the structure on a plane at -20°C. It can be measured that its side length becomes 5mm, as shown in the figure. Shown in 19B. After placing the compressed structure in water at 37°C for 1 minute, it can be observed that it returns to an uncompressed state with a side length of approximately 12.2 mm as shown in Figure 19C.

The foregoing results indicate that the exemplary bio-ink of the present invention has shape memory, which can be changed into different shapes under different temperature conditions. Therefore, the bio-ink of the present invention is suitable for printing structures that require shape changes, for example, artificial tissues whose final shape is to be used in minimally invasive surgery is larger than the wound.

Example 4 PUG bio-ink is non-cytotoxic

In order to evaluate whether the PUG bio-ink is cytotoxic, this example examines the survival rate of mouse neural stem cells treated with PCL80DL20 polyurethane dispersion or PUG bio-ink. First, 3×10 ⁵ neural stem cells were cultured at 37°C for 5 minutes with 20 μL of PCL80DL20 polyurethane dispersion, PUG bio-ink, or DMEM-HG/F-12 medium (control group). Afterwards, each group of cells were treated with propidium iodide (PI), acridine orange (AO), and VB48 fluorescent dye to label dead cells, total cells, and healthy cells, respectively, and then obtain The fluorescence micrograph was used to count the cell viability of each group.

Figure 20 shows the average survival rate of the aforementioned groups of cells. According to this figure, compared with the control group, the polyurethane dispersion treatment significantly reduced the cell survival rate to about 93.84%, but the PUG bio-ink treatment did not significantly affect the cell survival. This result indicates that the bio-ink of the present invention has no short-term cytotoxicity.

Example 5 Growth of stem cells in PUG bio-ink

This example illustrates the long-term effects of structures printed with PUG bio-ink on cell growth. First, mouse neural stem cells were added to PCL80DL20 polyurethane dispersion or PUG bio-ink at a ^{cell density of 2.5×10 5 cells/mL.} Afterwards, fill the PCL80DL20 polyurethane dispersion containing neural stem cells or PUG bio-ink into the syringe of the three-dimensional printing device, and print it on the 24-well cell culture plate according to the following conditions: the temperature of the cell culture plate is 25℃, and the nozzle diameter It is 160μm and the nozzle temperature is 26°C. After printing, irradiate the 24-well plate with 5W ultraviolet light at a distance of about 1.5 cm for 90 seconds to solidify the cell-bearing structure in the plate, and then move the 24-well plate to the 37°C incubator, and place it in the DMEM-HG/F -12 culture medium for 14 days, during which the cell proliferation degree was measured at 4 hours (day 0), day 3, day 7, and day 14. To quantify cell proliferation, the cell-containing construct was replaced with 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfobenzene)- 2H-tetrazolium monosodium salt (WST-8; purchased from Sigma) was treated, and its absorbance at 460 nm was measured using a spectrometer (SpectraMax M5; purchased from Molecular Devices).

Figure 21 shows the relative proliferation rate of the aforementioned neural stem cells after a specified number of days of culture, and its value is defined as the ratio of the absorbance value of the cells after the specified number of days of culture to the 0th day of culture; **** in the figure indicates p<0.0001. According to Figure 21, neural stem cells only slightly proliferate in the structure formed by polyurethane dispersion, and the relative proliferation rate does not increase over time. In contrast, neural stem cells continued to proliferate in the construct formed by PUG bio-ink for at least 14 days, and the relative proliferation rate on the 14th day was about 365%. This result indicates that the bio-ink of the present invention can be used to directly print structures that require cell proliferation, such as artificial tissues containing stem cells.

In summary, the foregoing examples show that the bio-ink containing polyurethane and gelatin has self-healing power due to the addition of a nutritional composition. In addition, when the aforementioned bio-ink further contains methacrylated gelatin for photocrosslinking, it exhibits self-healing power regardless of whether it is exposed to light. Furthermore, in view of the fact that the bio-ink containing polyurethane, gelatin, methacrylated gelatin, and nutritional composition still has high biocompatibility, printability and stackability, long-term structural stability after light curing, and With good flexibility and shape memory, the bio-ink is suitable for high-resolution, high-fidelity, and multi-layer stacked three-dimensional bio-printing, and even for four-dimensional bio-printing that includes time parameters.

Claims (13)

The use of a nutritional composition is to prepare a self-healing bio-ink composition, wherein the nutritional composition includes an amino acid, a vitamin, and an inorganic salt, and the bio-ink composition includes a biological ink composition. Degradable polyurethane and a gelatin. The use as described in item 1 of the scope of patent application, wherein the bio-ink composition further comprises a methacrylated gelatin. The use described in item 1 or item 2 of the scope of patent application, wherein the biodegradable polyurethane comprises a hard segment and a soft segment connected, and the hard segment is formed by the reaction of a diisocyanate and a chain extender The soft segment is at least one oligomer diol, and the chain extender includes an anionic chain extender. For the use described in item 1 or item 2 of the scope of the patent application, the bio-ink composition contains the biodegradable polyurethane in a weight ratio of 8% to 28%. For the use described in item 1 or item 2 of the scope of patent application, the bio-ink composition contains gelatin in a weight ratio of 2% to 7%. The use according to item 1 or item 2 of the scope of patent application, wherein the bio-ink composition contains the nutritional composition in a weight ratio of at least 0.01%. The use as described in item 2 of the scope of patent application, wherein the bio-ink composition contains 1% to 10% methacrylated gelatin by weight. A bio-ink composition with self-healing power and shape memory, comprising a biodegradable polyurethane, a gelatin, a methacrylated gelatin, and a nutritional composition, wherein the nutritional composition includes an amino acid, a Vitamins, and an inorganic salt. The bio-ink composition according to item 8 of the scope of patent application, wherein the biodegradable polyurethane comprises a hard segment and a soft segment connected, and the hard segment is formed by the reaction of a diisocyanate and a chain extender, The soft segment is at least one oligomer diol, and the chain extender includes an anionic chain extender. The bio-ink composition described in item 8 of the scope of patent application contains the biodegradable polyurethane in a weight ratio of 8% to 28%. The bio-ink composition described in item 8 of the scope of patent application contains gelatin in a weight ratio of 2% to 7%. The bio-ink composition described in item 8 of the scope of the patent application contains 1% to 10% methacrylated gelatin by weight. The bio-ink composition described in item 8 of the scope of the patent application includes the nutritional composition in a weight ratio of at least 0.01%.

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