Modified collagen, photo-crosslinked biomaterials and preparation methods and applications thereof

Abstract

The present invention relates to modified collagen, photo-crosslinked biomaterials, and preparation methods and applications thereof. The modified collagen includes collagen grafted with a group shown in formula (A). The photo-crosslinked biomaterial includes a hydrogel formed by curing the modified collagen. The photo-crosslinked biomaterial of the present invention has good biocompatibility and cell compatibility, and has important application value in the fields of in vivo or in vitro tissue and organ repair and reconstruction, tissue engineering, 3D printing substrates, and cell culture carriers.

Description

Technical Field

[0001] The invention relates to modified collagen, photo-crosslinked biomaterials and preparation methods and applications thereof. Background Art

[0002] Biomaterials are one of the main components of organisms. They have excellent biocompatibility and can assist and regulate biological tissues in vivo or in vitro. Among the tissue components of the human body, collagen is the main component of the extracellular matrix. Its unique biological activity and three-dimensional structure play an important role in the normal physiological function and injury repair of tissues and organs. Therefore, it has great potential in the field of biomedical materials.

[0003] At present, the main methods for preparing hydrogels are physical cross-linking by repeated freezing and thawing, or chemical cross-linking by cross-linking agents. However, the repeated freezing and thawing process is cumbersome and has high requirements on equipment, and the cross-linking agent method may have concerns about toxic residues. Summary of the invention

[0004] In order to solve one of the above technical problems existing in the prior art, the present invention provides a photo-crosslinked biomaterial with good biocompatibility and cell compatibility, which has important application value in the fields of in vivo or in vitro tissue and organ repair and reconstruction, tissue engineering, 3D printing substrates, and cell culture carriers. The present invention also provides a preparation method of the photo-crosslinked biomaterial, which has a fast curing time, a simple process, and is not prone to toxic residues.

[0005] In a first aspect, the present invention provides a modified collagen, comprising collagen grafted with a group represented by formula (A),

[0006]

[0007] In formula (A), R1 and R2 are the same or different and are independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl or halogen, It represents the position where the group represented by formula (A) is connected to the collagen.

[0008] In some embodiments, in formula (A), R1 and R2 are the same or different, and are each independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C6-C8 aryl, chlorine, bromine or iodine. In some embodiments, in formula (A), R1 and R2 are the same or different, and are each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl or phenyl.

[0009] In some embodiments, the group represented by formula (A) is grafted onto the free amino group of the collagen.

[0010] In some embodiments, the grafting rate of the modified collagen is 40% or more, for example, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99% and any value therebetween. In some preferred embodiments, the grafting rate of the modified collagen is 90% or more, more preferably 95% or more.

[0011] In a second aspect, the present invention provides a method for preparing modified collagen, comprising subjecting a raw material solution I containing collagen to a modification reaction with a raw material solution II containing a compound of formula (B),

[0012]

[0013] In formula (B), R1 and R2 are the same or different, and are independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C6-C8 aryl or halogen.

[0014] In some embodiments, in formula (B), R1 and R2 are the same or different, each independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C6-C8 aryl, chlorine, bromine or iodine. In some embodiments, in formula (B), R1 and R2 are the same or different, each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl or phenyl. In some embodiments, the compound of formula (B) is maleic anhydride.

[0015] In some embodiments, the collagen includes at least one of collagen extracted from animal tissue and recombinant collagen. For example, the collagen may be collagen extracted from cowhide or beef tendon, or recombinant collagen prepared using yeast as a genetic engineering vector.

[0016] In some embodiments, the collagen is in a solid and / or liquid form. For example, the collagen may be a collagen liquid dissolved in a solvent (liquid form), a freeze-dried sponge form (solid form), or a fibrous collagen slurry (solid-liquid mixture).

[0017] In some embodiments, the raw material solution I is obtained by diluting collagen into a solvent, and the solvent is selected from at least one of an organic acid and an inorganic acid. Therefore, in some embodiments, the raw material solution I also includes an organic acid and / or an inorganic acid. Preferably, the organic acid includes at least one of acetic acid and citric acid. Preferably, the inorganic acid includes at least one of hydrochloric acid, sulfuric acid and nitric acid.

[0018] In some embodiments, the feed solution II further comprises an organic solvent. Preferably, the organic solvent comprises a C2-C6 ketone compound, preferably acetone.

[0019] In some embodiments, the concentration of collagen in the raw material solution I is 2-20 mg / g. Since collagen has viscosity after dissolution, if the concentration is above 20 mg / g, the viscosity of the solution will be too high, resulting in poor operability. If the concentration is below 2 mg / g, it will be difficult to form a gel because there are too few collagen molecules.

[0020] In some embodiments, the concentration of the compound of formula (B) in the raw material solution II is 0.1-5 M. If the concentration is lower than 0.1 M, too much organic solvent such as acetone will over-dilute the collagen solution, which will cause difficulties in subsequent experimental operations; on the other hand, if the concentration is higher than 5 M, the compound of formula (B) such as maleic anhydride will not be completely dissolved, which will affect the quality of the final product.

[0021] In some embodiments, the weight ratio of the compound of formula (B) in the raw material solution II to the collagen in the raw material solution I is (10-30): 100. In the present invention, the weight ratio of the collagen in the raw material solution I to the compound of formula (B) in the raw material solution II is within this range, and a good grafting rate after modification can be obtained, and the physical strength of the obtained collagen hydrogel is better. If the ratio is lower than 10:100, the grafting rate after modification is poor, and the collagen hydrogel cannot be well formed, and its physical strength performance is weakened, and the practical applicability is not good. On the other hand, even if the amount of the compound of formula (B) is further increased, that is, the ratio is higher than 30:100, no better effect can be obtained, but it will increase the burden on the cost side.

[0022] In some embodiments, the temperature of the modification reaction is 0-25°C, for example, 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 15°C, 20°C, 25°C and any value therebetween. In some preferred embodiments, the temperature of the modification reaction is 4-8°C.

[0023] In some embodiments, the time of the modification reaction is 8-24 hours, for example, it can be 8 hours, 12 hours, 16 hours, 20 hours, 24 hours and any value therebetween. In the present invention, the time of the modification reaction falls within this range, and then it can be fully modified. If it is less than 8 hours, it is impossible to fully modify. In addition, even if the modification time is higher than 24 hours, it is impossible to obtain better results, but it will increase the burden on the time cost.

[0024] In some embodiments, the preparation method further comprises performing solid-liquid separation and / or purification treatment on the product after the modification reaction.

[0025] Preferably, the solid-liquid separation is performed by filtration or centrifugation, preferably by centrifugation. The present invention uses centrifugation for solid-liquid separation, and the obtained solids are compact, have low water content, and have little loss; in contrast, the general solid-liquid separation method will easily obtain relatively loose solids, have high water content, and have high loss.

[0026] Preferably, the purification treatment is performed by washing or dialysis, preferably by dialysis. In the present invention, when removing impurities, because the dialysis method can utilize the selectivity of the dialysis membrane for molecular size, while the impurities are diffused and removed, the large molecular collagen is also trapped in the dialysis membrane, thereby obtaining collagen with higher purity.

[0027] In a third aspect, the present invention provides a photo-crosslinked biomaterial, comprising a hydrogel formed by curing the aforementioned modified collagen or the modified collagen obtained according to the aforementioned preparation method.

[0028] In some embodiments, the Young's modulus of the photocrosslinked biomaterial is 0.15-0.5 kPa, preferably 0.3-0.5 kPa.

[0029] In some embodiments, the bulk modulus of the photocrosslinked biomaterial is 2-20 kPa, preferably 5-10 kPa.

[0030] In some embodiments, the photocrosslinked biomaterial has a yield strength of 25-200 kPa, preferably 90-150 kPa.

[0031] In a fourth aspect, the present invention provides a method for preparing a photo-crosslinked biomaterial, comprising photo-crosslinking and curing the aforementioned modified collagen or the modified collagen obtained according to the aforementioned preparation method with a photoinitiator under light.

[0032] In some embodiments, the photoinitiator is selected from 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) and / or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). The present invention uses Irgacure 2959 and LAP as photoinitiators, which has the advantage of lower cytotoxicity; on the contrary, if other photoinitiators are used, the cytotoxicity will be higher, which is less ideal.

[0033] In some embodiments, the concentration of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone is 0.01-1% (w / v). If the concentration is lower than 0.01%, effective gelation cannot be achieved, and if the concentration is higher than 1%, cytotoxicity may be increased.

[0034] In some embodiments, the concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.01-2% (w / v). If the concentration is lower than 0.01%, effective gelation cannot be achieved, and if the concentration is higher than 2%, cytotoxicity may be increased.

[0035] In some embodiments, the modified collagen is resuspended in a solvent and then photo-crosslinked and cured. Preferably, the solvent is water or phosphate buffer. In some embodiments, the modified collagen is purified and then resuspended in a solvent and then photo-crosslinked and cured. The amount of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone used is 0.01 to 1% (w / v) of the volume of the purified collagen solution; and / or the amount of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate used is 0.01 to 2% (w / v) of the volume of the purified collagen solution.

[0036] In some embodiments, the photo-crosslinking curing is performed using ultraviolet light.

[0037] In some embodiments, the wavelength of the ultraviolet light is 365nm-405nm. The present invention uses ultraviolet light within this range, so the photoinitiator can effectively absorb it.

[0038] In some embodiments, the UV irradiation time is more than 15 seconds, preferably 15 seconds to 300 seconds, for example, 15 seconds, 30 seconds, 50 seconds, 100 seconds, 150 seconds, 200 seconds, 250 seconds and any value therebetween. In the present invention, the UV irradiation time is within the above range, and the irradiation can be terminated by visually observing that the collagen is gelled and has no fluidity. If it is less than 15 seconds, it cannot be effectively gelled; and if the irradiation time is continued for a longer time, no better effect can be obtained, but the time cost will be increased.

[0039] In the present invention, the compound of formula (B) is used, for example, maleic anhydride is specifically used to modify collagen. If conventional acid is used instead of anhydride, the modification cannot be carried out smoothly. In addition, as for anhydride, if other anhydrides such as succinic anhydride are used for modification, the modified collagen has poor reactivity and cannot gel under the action of ultraviolet light. In addition, the inventors of the present invention have found through research that cross-linking and curing by ultraviolet light does not require the addition of a cross-linking agent, and the curing speed is fast, the process is simple, and it is not easy to have toxic residues, and it can achieve in-situ cross-linking, which solves the defects of traditional hydrogel preparation.

[0040] In a fifth aspect, the present invention provides a photo-crosslinked biomaterial obtained according to the aforementioned preparation method or the use of the aforementioned photo-crosslinked material in the field of biomedical materials.

[0041] In some embodiments, the present invention provides the use of the aforementioned photo-crosslinked biomaterial in the repair and reconstruction of tissues and organs, tissue engineering, 3D printing substrates or cell culture carriers.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] (1) Collagen is a heat-sensitive substance. If the temperature during the preparation process is too high, its triple helical structure will be destroyed and lose its original efficacy. The preparation methods in the prior art usually require the use of a high temperature environment, but the preparation process of the present invention does not require the use of a high temperature environment, so the triple helical structure in the collagen can be prevented from being destroyed and the integrity of the collagen can be retained.

[0044] (2) The collagen hydrogel prepared according to the present invention has good biocompatibility because no chemical cross-linking agent needs to be added during the preparation process and no toxic residue is generated.

[0045] (3) The method for preparing collagen hydrogel of the present invention is photo-crosslinking curing, which has a short curing time, low cost, and can achieve in-situ crosslinking, so it has wider applicability.

[0046] (4) According to the preparation method of the present invention, a collagen hydrogel with excellent physical strength can be prepared in a manner with an extremely high grafting rate.

[0047] In addition, the in situ cross-linked hydrogel of the present invention has the following benefits:

[0048] Traditional hydrogels are applied to the affected area after gelation. Their shape is fixed and cannot change with the affected area, so it is difficult to closely bond with surrounding tissues, and their applications are relatively limited. In situ cross-linked hydrogels generally have no fixed shape. After injection in a relatively low viscosity state, their physical form changes and forms a hydrogel that is fixed in the tissue gap. Therefore, they can more closely bond with biological tissues such as the affected area than traditional hydrogels.

[0049] In situ cross-linked hydrogels have the following application advantages in the biomedical field: during surgery, the hydrogel is injected into the affected area and then cross-linked and solidified, which is simple to operate and less invasive; in the field of tissue engineering, the cross-linked hydrogel will closely fit the shape of the affected area and can effectively promote tissue reconstruction; in wound repair applications, in situ cross-linked hydrogels can close wounds and have the potential to replace traditional suture surgery. Other applications include drug release, 3D printing, and gene delivery.

[0050] definition

[0051] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0052] The term "collagen" used in this article refers to a family of proteins. At least more than 30 coding genes for collagen chains have been discovered, which can form more than 16 types of collagen molecules. According to their structure, they can be divided into fibrous collagen, basement membrane collagen, microfibril collagen, anchoring collagen, hexagonal reticular collagen, non-fibrous collagen, transmembrane collagen, etc. Collagen has good biocompatibility, biodegradability and biological activity. The collagen used in this article can be collagen extracted from animal tissues, such as collagen extracted from cowhide and beef tendons; or "recombinant collagen", for example, recombinant collagen produced using yeast as a genetic engineering vector.

[0053] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A flow chart showing a method for preparing a photo-crosslinked biomaterial according to some embodiments of the present invention.

[0055] Figure 2 The schematic diagram of the structure of collagen modified with maleic anhydride (hereinafter also referred to as ColMe) prepared according to the present invention is shown.

[0056] Figure 3 The schematic diagram of the structure of the collagen modified by succinic anhydride prepared according to Comparative Example 1 of the present invention is shown.

[0057] Figure 4 The FT-IR spectrum of ColMe prepared according to Example 1 of the present invention is shown.

[0058] Figure 5 The cell compatibility results of ColMe prepared according to Example 1 of the present invention are shown.

[0059] Figure 6 The figure shows the evaluation of the grafting rate after modification according to the present invention for different addition amounts of maleic anhydride.

[0060] Figure 7The photographs show the physical strength of GelMA according to Example 1 and Example 4 of the present invention and a commercial product. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts disclosed herein.

[0062] The present invention grafts anhydride with a carbon-carbon double bond structure onto the free amino group of collagen to obtain anhydride-modified collagen, and then mixes the modified collagen with a photoinitiator evenly, and reacts and cross-links and solidifies it under light conditions such as ultraviolet light, thereby preparing a photo-crosslinked biomaterial gel.

[0063] Please refer to Figure 1 , which is a flow chart of a method for preparing a photocrosslinked collagen hydrogel according to some embodiments of the present invention. Figure 1 As shown, the preparation method of the present invention comprises the following steps: providing collagen raw material (S1); diluting collagen to obtain raw material solution I (S2); preparing maleic anhydride solution to obtain raw material solution II (S3); modifying collagen (S4); collecting solids (S5); removing impurities (S6); adding a photoinitiator (S7); and photocrosslinking (S8).

[0064] In step (S1), the collagen raw material provided may be collagen extracted from animal tissues, or recombinant collagen, such as collagen extracted from cowhide or beef tendon, or recombinant collagen produced using yeast as a genetic engineering vector.

[0065] In some embodiments, the collagen raw material is in liquid form, but may also be in solid form, or in a solid-liquid mixture state, such as a collagen liquid dissolved in a solvent (liquid form), a freeze-dried sponge form (solid form), or a fibrous collagen slurry (solid-liquid mixture).

[0066] In step (S2), the solvent used to dilute the collagen raw material includes but is not limited to organic acid, inorganic acid and a combination thereof, such as acetic acid, citric acid, hydrochloric acid and the like.

[0067] In the raw material solution I, the concentration of the diluted collagen raw material is 2-20 mg / g. Since collagen has viscosity after dissolution, if the concentration is above 20 mg / g, the solution viscosity will be too high, resulting in poor operability. If the concentration is below 2 mg / g, it will be difficult to form a gel because there are too few collagen molecules.

[0068] In step (S3), the solvent used in preparing the maleic anhydride solution is an organic solvent, preferably acetone. Although water is a very common solvent, it is not suitable to be used as a solvent because the anhydride is easily decomposed into acid in water.

[0069] In raw material solution II, the concentration of the prepared maleic anhydride solution is 0.1-5M. If it is lower than the lower limit, too much acetone will over-dilute the collagen solution, which will cause difficulties in subsequent experimental operations; on the other hand, if it is higher than the upper limit, the maleic anhydride will not be completely dissolved, which will affect the quality of the final product.

[0070] In step (S4), if Figure 2 As shown, maleic anhydride is used to modify collagen. If conventional acid is used instead of anhydride, the modification cannot be carried out smoothly. In addition, as for anhydride, if conventional succinic anhydride is used for modification, it is found that it can effectively lower the isoelectric point of collagen, allowing it to dissolve in a neutral environment. However, the structure of succinic anhydride lacks a double bond compared to maleic anhydride, so the modified collagen has poor reactivity and cannot form a gel under the action of ultraviolet light.

[0071] The amount of maleic anhydride solution added is 10%-30% (W maleic anhydride / W collagen raw material) by weight percentage relative to the collagen raw material. If the amount added falls within this range, a good grafting rate after modification can be obtained, and the physical strength of the obtained collagen hydrogel is excellent. If the amount added is less than 10%, the grafting rate after modification is poor, and the collagen hydrogel cannot be well formed, and its physical strength is poor and the practical applicability is not good. On the other hand, even if the amount added is higher than 30%, no better effect can be obtained, but it will increase the burden on the cost side.

[0072] The time for modifying the diluted collagen raw material with maleic anhydride solution is 8-24 hours. If the modification time falls within this range, the modification can be fully performed. If it is less than 8 hours, the modification cannot be fully performed. In addition, even if the modification time is longer than 24 hours, no better effect can be obtained, but the burden on time cost will increase.

[0073] In step (S5), the modified solids can be collected by conventional methods with filtering effects, including but not limited to centrifugal collection, filter collection (filter collection), or a combination thereof. Among them, centrifugal collection is more ideal. Using centrifugal collection, the solids obtained are compact, have low water content, and have little loss; while other conventional collection methods tend to obtain loose solids, have high water content, and have high loss.

[0074] In step (S6), the method for removing impurities can be dialysis or liquid washing. When removing impurities, dialysis can utilize the selectivity of the dialysis membrane for molecular size, and while the impurities are diffused and removed, the large molecular collagen is also trapped in the dialysis membrane, so that collagen with higher purity can be obtained, which is more ideal.

[0075] In step (S7), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (hereinafter referred to as Irgacure 2959, manufactured by Sigma-Aldrich) or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (hereinafter referred to as LAP, manufactured by Sigma-Aldrich) can be used as a photoinitiator. Using Irgacure 2959 and LAP as photoinitiators has the advantage of lower cytotoxicity; on the contrary, if other photoinitiators are used, the cytotoxicity will be higher, which is less ideal.

[0076] When using Irgacure 2959, the dosage is 0.01-1% (w / v) of the volume of the collagen solution after removing impurities. If it is lower than 0.01%, it will not form gel effectively, and if it is higher than 1%, there will be concerns about excessive cytotoxicity.

[0077] When using LAP, the usage amount is 0.01-2% (w / v) of the volume of the collagen solution after removing impurities. If it is lower than 0.01%, it will not be able to form gel effectively, and if it is higher than 2%, there will be concerns about excessive cytotoxicity.

[0078] In step (S8), ultraviolet light is used as the light source for photo-crosslinking and curing, and its wavelength is 365nm-405nm. If it falls within this range, the photoinitiator can be effectively absorbed. The ultraviolet light is irradiated for more than 15 seconds, for example, between 15 seconds and 300 seconds, and the irradiation can be terminated by visually observing that the collagen is gelled and has no fluidity. Even if the irradiation is continued for a longer time, better results cannot be obtained, but time costs will be increased, and there is a risk of unexpected situations. On the contrary, if it is less than 15 seconds, gelation cannot be effectively achieved.

[0079] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts disclosed herein.

[0080] Example 1

[0081] A method for preparing a photocrosslinked collagen hydrogel, the specific steps of which are as follows:

[0082] (1) A freeze-dried bovine collagen raw material in the form of a sponge is diluted with acetic acid to a concentration of 8 mg / g.

[0083] (2) Using acetone as solvent, prepare a 2.5 M maleic anhydride solution.

[0084] (3) The maleic anhydride solution obtained in step (2) is added in an amount of 30% by weight (W maleic anhydride / W collagen) relative to the collagen raw material.

[0085] (4) Modification for 24 hours at 4°C.

[0086] (5) Collect the solids by centrifugation.

[0087] (6) Dialysis was performed using phosphate buffer solution as the external dialysis solution, and the molecular weight cutoff of the dialysis membrane was 12-14 kDa.

[0088] (7) LAP was added as a photoinitiator at a concentration of 0.06% (w / v) of the volume of the collagen solution dissolved in water after dialysis.

[0089] (8) After irradiation with ultraviolet light at a wavelength of 365 nm for 60 seconds, a transparent and uniform collagen hydrogel modified with maleic anhydride (hereinafter also referred to as ColMe) was obtained.

[0090] Example 2

[0091] A method for preparing a photocrosslinked collagen hydrogel, the specific steps of which are as follows:

[0092] (1) A freeze-dried bovine collagen raw material in the form of a sponge is diluted with acetic acid to a concentration of 20 mg / g.

[0093] (2) Using acetone as solvent, prepare a 5 M maleic anhydride solution.

[0094] (3) The maleic anhydride solution obtained in step (2) was added in an amount of 18% by weight (W maleic anhydride / W collagen) relative to the collagen raw material.

[0095] (4) Modification for 8 hours at 4°C.

[0096] (5) Collect the solids by centrifugation.

[0097] (6) Dialysis was performed using phosphate buffer solution as the external dialysis solution, and the molecular weight cutoff of the dialysis membrane was 12-14 kDa.

[0098] (7) LAP was added as a photoinitiator at a concentration of 2% (w / v) of the volume of the collagen solution dissolved in water after dialysis.

[0099] (8) After irradiation with ultraviolet light at a wavelength of 365 nm for 15 seconds, a transparent and uniform ColMe hydrogel was obtained.

[0100] Example 3

[0101] A method for preparing a photocrosslinked collagen hydrogel, the specific steps of which are as follows:

[0102] (1) A freeze-dried bovine collagen raw material in the form of a sponge is diluted with acetic acid to a concentration of 2 mg / g.

[0103] (2) Using acetone as solvent, prepare a 0.1 M maleic anhydride solution.

[0104] (3) The maleic anhydride solution obtained in step (2) is added in an amount of 10% by weight (W maleic anhydride / W collagen) relative to the collagen raw material.

[0105] (4) Modification for 24 hours at 4°C.

[0106] (5) Collect the solids by centrifugation.

[0107] (6) Dialysis was performed using phosphate buffer solution as the external dialysis solution, and the molecular weight cutoff of the dialysis membrane was 12-14 kDa.

[0108] (7) Irgacrue 2959 was added as a photoinitiator at a concentration of 0.1% (w / v) of the volume of the collagen solution dissolved in water after dialysis.

[0109] (8) After irradiation with ultraviolet light at a wavelength of 365 nm for 300 seconds, a transparent and uniform ColMe hydrogel was obtained.

[0110] Example 4

[0111] A collagen hydrogel was prepared in the same manner as in Example 1 except that the amount of maleic anhydride added was changed to 9% (W maleic anhydride / W collagen).

[0112] Comparative Example 1

[0113] Please refer to Figure 3 The collagen hydrogel was prepared in the same manner as in Example 1 except that maleic anhydride was replaced by succinic anhydride. The results are shown in Table 1.

[0114] Table 1

[0115] Example 1 Comparative Example 1 Whether it is gel yes no

[0116] Test Example 1 FT-IR test of collagen (ColMe) hydrogel cells modified with maleic anhydride

[0117] Please refer to Figure 4 FT-IR was performed on the photo-crosslinked collagen hydrogel obtained in Example 1. Comparing collagen (Col) and modified collagen (ColMe), both showed characteristic peaks of collagen, including 3370 cm -1 (amide A), 1654cm -1 (amide I), 1554cm -1 (amide II) and 1241 cm -1 (amide III). Further comparison of the characteristic peak of amide III and 1450 cm -1 The absorption value ratio of the characteristic peak (AIII / A1450), as the basis for the integrity of the collagen stranded helix structure, confirmed that the collagen modified with maleic anhydride completely preserved the structural characteristics of collagen.

[0118] Test Example 2 Evaluation of Cell Compatibility of Collagen (ColMe) Hydrogel Modified with Maleic Anhydride

[0119] The in vitro cell compatibility of the ColMe hydrogel prepared in Example 1 was tested and compared with commercially available methacrylic anhydride gelatin (GelMA). The results showed that the ColMe hydrogel had no cytotoxicity to L929 cells (see Figure 5 ), which has good cell compatibility like the commercially available GelMA.

[0120] Test Example 3 Evaluation of Grafting Rate

[0121] The grafting rate of Example 1, Example 2 and Example 4 (hereinafter referred to as ColMe_0.3, ColME_0.18 and ColMe_0.09) after modification was tested by TNBS assay. The results showed that ColMe_0.3 and ColME_0.18 both had a grafting rate of 99%, while ColMe_0.09 had a grafting rate of only 43% (see Figure 6 ).

[0122] In addition, the calculation method of the grafting rate and the information of each group are shown below.

[0123]

[0124] ColMe_0.3: W maleic anhydride / W collagen = 30% (Example 1)

[0125] ColMe_0.18: W maleic anhydride / W collagen = 18% (Example 2)

[0126] ColMe_0.09: W maleic anhydride / W collagen = 9% (Comparative Example 1)

[0127] Test Example 4 Physical Strength Evaluation

[0128] The physical strength of GelMA of Example 1, Example 4 and a commercial product was measured. The Young's modulus, bulk modulus and yield strength were measured using a rheometer. The results are shown in Table 2.

[0129] In addition, in order to more clearly understand the physical strength of Example 1, Example 4 and the commercially available GelMA, Figure 7 Their respective photos are shown.

[0130] Table 2

[0131] Example 1 Example 4 GelMA (commercially available) Young's modulus (kPa) 0.310 0.032 0.119 Bulk modulus (kPa) 8.805 0.903 1.179 Yield strength (kPa) 94.89 10.46 20.25

[0132] From Table 2 and Figure 7 The results show that the photo-crosslinked collagen hydrogel prepared by the preparation method of the present invention has more sufficient physical strength. Therefore, it can achieve better physical support in the field of tissue engineering. After gelation, it is not easy to cause damage and displacement of the hydrogel due to external factors, and can better play the functions of the hydrogel, such as releasing drugs, cell attachment, and promoting the repair of surrounding tissues.

[0133] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A photo-crosslinked biomaterial, comprising a hydrogel formed by curing modified collagen, wherein the modified collagen comprises collagen grafted with a group represented by formula (A), In formula (A), R1 and R2 are the same or different and are independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl or halogen, It represents the position where the group represented by formula (A) is connected to the collagen.

2. The photo-crosslinked biomaterial according to claim 1, characterized in that: The group represented by formula (A) is grafted onto the free amino group of the collagen.

3. The photo-crosslinked biomaterial according to claim 2, characterized in that: The grafting rate of the modified collagen is above 40%.

4. The photo-crosslinked biomaterial according to claim 2, characterized in that: The grafting rate of the modified collagen is above 90%.

5. The photo-crosslinked biomaterial according to claim 1, characterized in that: The preparation method of the modified collagen comprises the steps of subjecting a raw material solution I containing collagen to a modification reaction with a raw material solution II containing a compound of formula (B), In formula (B), R1 and R2 are the same or different, and are independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C6-C10 aryl or halogen.

6. The photo-crosslinked biomaterial according to claim 1, characterized in that: The collagen includes at least one of collagen extracted from animal tissue and recombinant collagen.

7. The photo-crosslinked biomaterial according to claim 6, characterized in that: The collagen includes at least one of collagen extracted from cowhide and beef tendon, or recombinant collagen prepared by using yeast as a genetic engineering vector.

8. The photo-crosslinked biomaterial according to claim 6, characterized in that: The collagen is in a solid and / or liquid state.

9. The photo-crosslinked biomaterial according to claim 5, characterized in that: In the raw material solution I, the concentration of collagen is 2-20 mg / g; and / or In the raw material solution II, the concentration of the compound of formula (B) is 0.1-5M; and / or In the raw material solution II, the weight ratio of the compound of formula (B) to the collagen in the raw material solution I is (10-30):100; and / or The raw material solution I further comprises an organic acid and / or an inorganic acid; and / or The raw material solution II also includes an organic solvent.

10. The photo-crosslinked biomaterial according to claim 9, characterized in that: The organic acid comprises at least one of acetic acid and citric acid, and the inorganic acid comprises at least one of hydrochloric acid, sulfuric acid and nitric acid; and / or The organic solvent includes a C2-C6 ketone compound.

11. The photo-crosslinked biomaterial according to claim 9, characterized in that: The organic solvent includes acetone.

12. The photo-crosslinked biomaterial according to claim 5, characterized in that: The modification reaction time is 8-24 hours, and / or the modification reaction temperature is 0-25°C.

13. The photo-crosslinked biomaterial according to claim 12, characterized in that: The temperature of the modification reaction is 4-8°C.

14. The photo-crosslinked biomaterial according to claim 5, characterized in that: The preparation method further comprises performing solid-liquid separation and / or purification treatment on the product after the modification reaction.

15. The photo-crosslinked biomaterial according to claim 14, characterized in that: The solid-liquid separation is performed by filtration or centrifugation.

16. The photo-crosslinked biomaterial according to claim 14, characterized in that: The purification treatment is performed by washing or dialysis.

17. The photo-crosslinked biomaterial according to any one of claims 1 to 16, characterized in that The photo-crosslinked biomaterial satisfies at least one of the following (a)-(c): (a) the Young's modulus of the photocrosslinked biomaterial is 0.02-0.5 kPa, (b) the bulk modulus of the photo-crosslinked biomaterial is 0.5-20 kPa, (c) The yield strength of the photo-crosslinked biomaterial is 10-200 kPa.

18. The photo-crosslinked biomaterial according to any one of claims 1 to 16, characterized in that The Young's modulus of the photo-crosslinked biomaterial is 0.3-0.5 kPa.

19. The photo-crosslinked biomaterial according to any one of claims 1 to 16, characterized in that The bulk modulus of the photo-crosslinked biomaterial is 5-10 kPa.

20. The photo-crosslinked biomaterial according to any one of claims 1 to 16, characterized in that The yield strength of the photo-crosslinked biomaterial is 90-150 kPa.

21. A method for preparing a photo-crosslinked biomaterial according to any one of claims 1 to 20, comprising photo-crosslinking and curing the modified collagen with a photoinitiator under light.

22. The preparation method according to claim 21, characterized in that: The photoinitiator is selected from 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and / or lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

23. The preparation method according to claim 22, characterized in that: 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone is used at a concentration of 0.01-1% (w / v).

24. The preparation method according to claim 22, characterized in that: The concentration of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate is 0.01-2% (w / v).

25. The preparation method according to claim 22, characterized in that: The photo-crosslinking curing is performed using ultraviolet light.

26. The preparation method according to claim 25, characterized in that: The wavelength of the ultraviolet light is 365nm-405nm.

27. The preparation method according to claim 25, characterized in that: The irradiation time of the ultraviolet light is more than 15 seconds.

28. The preparation method according to claim 25, characterized in that: The irradiation time of the ultraviolet light is 15 seconds to 300 seconds.

29. Use of the photocrosslinked material according to any one of claims 1 to 20 or the photocrosslinked biomaterial obtained according to the preparation method according to any one of claims 21 to 28 in the field of biomedical materials.

30. The use according to claim 29, characterized in that The photo-crosslinked biomaterial is used for repair and reconstruction of tissues and organs, tissue engineering, 3D printing substrates or cell culture carriers.

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