A preparation method of modified collagen scaffold and its product and application

Abstract

The present invention discloses a preparation method of a modified collagen scaffold, its product and application, comprising the following steps: step 1, cross-linking a collagen solution with a glycosylation reagent to obtain a first cross-linked product; step 2, first infiltrating the first cross-linked product in an ultraviolet photosensitive reagent, and then performing ultraviolet cross-linking to obtain a second cross-linked product; step 3, performing low-dose irradiation cross-linking on the second cross-linked product to complete the preparation of a collagen modified scaffold. The present invention proposes a composite physical cross-linking method, which can reduce costs and risks and realize large-scale production, and can protect the natural structure and activity of collagen to the greatest extent. The modified collagen scaffold prepared by the present invention has stable properties, a relatively stable degradation curve and enzyme tolerance, and good physiological hemostatic function.

Description

Technical Field

[0001] The present invention relates to the field of biological scaffold materials, and in particular to a preparation method of a modified collagen scaffold, a product thereof and an application thereof. Background Art

[0002] Collagen is an ancient structural protein with a highly conserved sequence and good biocompatibility, low antigenicity, and degradability. Collagen scaffold materials are often used to promote cell adhesion, tissue regeneration, and repair. When collagen is used as a tissue engineering scaffold, its structure plays a vital role in controlling the mechanical properties of the scaffold and the interaction between cells and the scaffold. However, when the uncross-linked collagen freeze-dried scaffold encounters tissue fluid and blood, the scaffold structure is not strong and cannot resist the local tension and compression of tissue fluid, which causes the scaffold material to collapse or become thinner easily. Then the pore structure of the material is blocked by the tension of water, which cannot provide cells with favorable adhesion, crawling, and growth space under physiological conditions, and may even cause cell death; it is often necessary to perform certain modification treatments to enhance its stability so that it can exist in the human body for a certain period of time before being degraded.

[0003] At present, collagen is mainly modified to increase its cross-linking degree and improve its stability. Existing collagen modification methods are mainly divided into physical cross-linking and chemical cross-linking. Physical cross-linking methods include irradiation, dehydration, ultraviolet cross-linking, etc. However, the ultraviolet cross-linking method has a low cross-linking degree and cannot solve the fundamental problem of mechanical strength and the support and defect problems in clinical use; the dose of the irradiation method cannot be accurately controlled, and it is easy to have uneven strength after irradiation, resulting in unstable biological activity and performance of the scaffold, and may even cause damage or degradation to the peptide chain structure, which in turn leads to a decrease in mechanical strength. The chemical cross-linking method often uses glutaraldehyde, formaldehyde, and carbodiimide for cross-linking, which can lead to potential cytotoxicity, local tissue calcification, and reagent residues; the enzymatic cross-linking method faces the problem that it is difficult for the enzyme to cross-link with collagen whose molecular structure remains intact because the cross-linking sites are mainly present inside the helical structure. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a method for preparing a modified collagen scaffold, which can solve the problems of poor effect of collagen modification methods, destruction of collagen activity and biological toxicity.

[0005] The second object of the present invention is to provide a modified collagen scaffold, which can solve the problems of low mechanical strength and poor enzyme tolerance of the collagen scaffold.

[0006] The third object of the present invention is to provide an application of a modified collagen scaffold, which can solve the problem of preparing physiological hemostatic scaffold materials.

[0007] The first object of the present invention is achieved by the following technical solutions:

[0008] A method for preparing a modified collagen scaffold comprises the following steps:

[0009] Step 1, cross-linking the collagen solution with a glycosylation reagent to obtain a first cross-linked product;

[0010] Step 2, soaking the first cross-linked product in an ultraviolet photosensitizer, and then performing ultraviolet cross-linking to obtain a second cross-linked product;

[0011] Step 3: performing multiple low-dose radiation cross-linking on the second cross-linked product to complete the preparation of a collagen modified scaffold.

[0012] Furthermore, in step 1, the glycosylation reagent is one of D-ribose or D-glucose; the glycosylation reagent is dissolved in a buffer solution at a concentration of 0.02-1.5 mol / L.

[0013] Furthermore, in step 1, after the collagen solution and the glycosylation reagent are mixed and stirred, the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution, and the first cross-linked product is obtained after freeze-drying; or the collagen solution is first freeze-dried, and then mixed and stirred with the glycosylation reagent, the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution to obtain the first cross-linked product.

[0014] Furthermore, in step 1, the cross-linking reaction time of the collagen solution and the glycosylation reagent is 48-72 hours; the cross-linking reaction time of the freeze-dried collagen solution and the glycosylation reagent is 48-120 hours.

[0015] Furthermore, in step 1, the concentration of the collagen solution is 1-80 mg / ml; the addition ratio of the collagen solution and the glycosylation reagent is 1:(0.05-2); and the final concentration of the glycosylation reagent after the reaction is 0.05-1.5 mol / L.

[0016] Furthermore, in step 2, the UV crosslinking time is 15-240 min; the UV crosslinking illumination is 800-1400 μw / cm 2 ; The wavelength of the ultraviolet light for ultraviolet cross-linking is 190-280nm; the light source power for ultraviolet cross-linking is 5-15W.

[0017] Furthermore, the UV crosslinking in step 2 is carried out in an upper and lower UV light box, the carrier is a quartz glass plate, and the distance between the UV lamp and the quartz glass plate is 10-30 cm.

[0018] Furthermore, in step 3, the low-dose irradiation ray is one of gamma rays, X-rays or high-speed electron flow; the low-dose irradiation range is 5-10 kGy and / or 10-18 kGy; the number of low-dose irradiation times is 1-5 times.

[0019] The second object of the present invention is achieved by the following technical solutions:

[0020] A modified collagen scaffold is prepared by a modified collagen scaffold preparation method.

[0021] The third object of the present invention is achieved by the following technical solutions:

[0022] Application of a method for preparing a modified collagen scaffold, wherein the modified collagen scaffold is used for preparing medical materials

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The invention of the present invention is a method for preparing a collagen modified scaffold, which can be cross-linked by a physical cross-linking method combined with a biological cross-linking method, wherein a glycosylation reagent is cross-linked with a collagen solution, the structure is more compact and non-biotoxic, ultraviolet cross-linking can increase the mechanical strength of the scaffold, and low-dose irradiation can avoid the destruction of protein peptide chains, thereby ensuring the integrity of the collagen matrix. The preparation method of the present invention can be easily realized at a relatively low cost and risk, is convenient and easy to operate, easy to realize, low cost, low risk, and can protect the natural structure and activity of collagen to the greatest extent. The physical cross-linking method is combined with non-enzymatic glycosylation cross-linking, which will not destroy the collagen triple helix structure, while strengthening the cross-linking strength and effect, making the cross-linking more uniform.

[0025] 2. The modified collagen scaffold obtained by the preparation method of the present invention has stable properties, good mechanical strength, a stable degradation curve and enzyme tolerance, and no foreign body reaction or local inflammatory reaction after implantation in the human body, and is safe for the human body. The modified collagen scaffold obtained by the preparation method of the present invention has good physiological hemostasis function and can be widely used in the field of clinical medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis diagram of the collagen scaffold protein prepared by the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] A method for preparing a modified collagen scaffold comprises the following steps:

[0029] Step 1, cross-linking the collagen solution with a glycosylation reagent to obtain a first cross-linked product;

[0030] Step 2, soaking the first cross-linked product in an ultraviolet photosensitizer, and then performing ultraviolet cross-linking to obtain a second cross-linked product;

[0031] Step 3: performing multiple low-dose radiation cross-linking on the second cross-linked product to complete the preparation of a collagen modified scaffold.

[0032] After the collagen solution reacts with ribose, it is irradiated multiple times at low doses, which can enhance the sterilization effect and enhance the protective effect on the protein; multiple irradiations at low doses can also avoid the destruction of the protein peptide chain structure caused by irradiation, the decrease in mechanical strength, and ensure the integrity of the matrix structure. D-ribose and D-glucose are natural small molecule monosaccharides, non-toxic, and can avoid the adverse consequences caused by cross-linking agents such as aldehydes; when D-ribose is cross-linked with the collagen solution, the collagen macromolecules will lose water bound by hydrogen bonds. After losing the water molecules, the collagen membrane structure is more compact and has better mechanical strength, and is also more conducive to subsequent physical cross-linking reactions.

[0033] The collagen solution may be derived from mammals, such as collagen from cattle and pigs, or from marine organisms, such as collagen from fish, jellyfish, abalone, etc.

[0034] Furthermore, in step 1, the glycosylation agent is D-ribose or D-glucose; the D-ribose is dissolved in a buffer solution at a concentration of 0.05-1.5 mol / L. The buffer solution is a PBS buffer solution. Preferably, the glycosylation agent is D-ribose.

[0035] Furthermore, in step 1, after the collagen solution and the glycosylation reagent are mixed and stirred, the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution, and the first cross-linked product is obtained after freeze-drying; or the collagen solution is first freeze-dried, and then mixed and stirred with the glycosylation reagent, the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution to obtain the first cross-linked product.

[0036] Furthermore, in step 1, the cross-linking reaction time of the collagen solution and the glycosylation reagent is 48-72 hours, preferably 60 hours; the cross-linking reaction time of the freeze-dried collagen solution and the glycosylation reagent is 48-120 hours, preferably 60 hours.

[0037] Furthermore, in step 1, the concentration of the collagen solution is 1-80 mg / ml; preferably, the concentration of the collagen solution is 3-10 mg / mL; more preferably, the concentration of the collagen solution is 7 mg / mL.

[0038] Furthermore, the ratio of the collagen solution to the glycosylation agent is 1:(0.05-2); preferably, the ratio of the collagen solution to the glycosylation agent is 1:(0.05-1); more preferably, the ratio of the collagen solution to the glycosylation agent is 1:(0.1-0.2).

[0039] Furthermore, after the non-enzymatic glycosylation reaction in step 1, the concentration of the glycosylation reagent is finally controlled within the range of 0.05-1.5 mol / L.

[0040] Furthermore, in step 2, the UV crosslinking time is 15-240 min; the UV crosslinking illumination is 800-1400 μw / cm 2 ; The wavelength of the ultraviolet light for ultraviolet cross-linking is 254nm; the light source power for ultraviolet cross-linking is 5-15W.

[0041] Preferably, the illumination of UV cross-linking is 900-1200 μW / cm 2 ; preferably 1107μW / cm 2 .

[0042] Furthermore, the UV crosslinking in step 2 is carried out in an upper and lower UV light box, the carrier is a quartz glass plate, and the distance between the UV lamp and the quartz glass plate is 10-30 cm.

[0043] Furthermore, in step 3, the low-dose irradiation ray is one of gamma rays, X-rays or high-speed electron flow; the low-dose irradiation range is 5-10 kGy and 10-18 kGy; the low-dose irradiation times are 1-5 times. Preferably, the low-dose irradiation range is 7 kGy and 15 kGy; the low-dose irradiation times are 2 times.

[0044] A modified collagen scaffold is prepared by a modified collagen scaffold preparation method.

[0045] A method for preparing a modified collagen scaffold is used for preparing medical materials.

[0046] Below, the present invention is further described in conjunction with the accompanying drawings and specific embodiments:

[0047] Examples 1-16

[0048] D-ribose is dissolved in PBS buffer at a concentration of 0.2 mol / L; the concentrations of collagen solutions are 3, 5, 7, and 10 mg / mL, respectively. After the collagen solution and D-ribose buffer are mixed and stirred for 60 hours, the pH is adjusted to 7.0 with 1% ammonia water, and then 10% acetone is added for elution according to the volume of the reaction system. After freeze-drying, the first cross-linked product is obtained. The first cross-linked product is soaked in an ultraviolet photosensitive reagent, and then ultraviolet cross-linking is performed in the upper and lower layers of ultraviolet light boxes. The carrier of ultraviolet cross-linking is a quartz glass plate; the distance between the ultraviolet lamp and the quartz glass plate is 20 cm, the ultraviolet cross-linking time is 30 min, the ultraviolet wavelength is 254 nm, and the light source power is 15 w. The ultraviolet illumination of each embodiment is shown in Table 1. After completing the ultraviolet cross-linking, low-dose irradiation cross-linking is performed, and the irradiation ray is γ-ray. The irradiation dose of each embodiment is shown in Table 1. Subsequently, the performance of Examples 1-16 was tested.

[0049] Table 1 Test parameters of Examples 1-16 and Comparative Examples 1-8

[0050]

[0051]

[0052] Embodiment 17

[0053] First, freeze-dry the collagen solution with a concentration of 7 mg / mL, then soak it in a D-ribose buffer with a concentration of 0.2 mol / L, mix and stir for 72 hours, adjust the pH and dehydrate, and perform ultraviolet cross-linking and low-dose radiation cross-linking. The conditions and methods of ultraviolet cross-linking and low-dose radiation cross-linking are the same as those in Example 1-16.

[0054] Embodiment 18

[0055] First, the collagen solution with a concentration of 7 mg / mL was freeze-dried, then immersed in a D-glucose buffer with a concentration of 0.2 mol / L, mixed and stirred for 72 hours, and then the pH was adjusted and dehydrated, and ultraviolet cross-linking and low-dose radiation cross-linking were performed. The conditions and methods of ultraviolet cross-linking and low-dose radiation cross-linking were the same as those in Example 1-16.

[0056] Performance Testing

[0057] 1. Basic performance evaluation test

[0058] Performance evaluation tests were performed on Examples 1-16, including mass change, water absorption, porosity, and enzyme digestion experiments. The enzyme digestion test method is as follows:

[0059] The cross-linked scaffolds were digested with collagenase, and the hydroxyproline content of the digested products was tested to evaluate the effects of different cross-linking modification methods on the enzyme resistance of the scaffolds. If the resistance is enhanced, it means that cross-linking can prolong the retention time of the material in tissues in the body.

[0060] Take a scaffold sample with a mass of (15±0.5) mg and put it into a centrifuge tube. Add 5 ml of 0.5% type I collagenase to each tube, digest it at 37°C, 60 rpm for 1, 2, and 4 hours, and then centrifuge it at 4°C, 4000 rpm for 20 minutes. Take 2 ml of the supernatant and add 2 ml of 5 mol / L hydrochloric acid to seal it. Hydrolyze it in a boiling water bath for 2 hours, and determine the hydroxyproline content in the hydrolyzate using the standard curve prepared with the hydroxyproline standard. The performance evaluation test results are shown in Table 2.

[0061] Table 2 Performance evaluation test results of collagen scaffolds after cross-linking

[0062]

[0063] 2. Water absorption test after UV crosslinking and different doses of irradiation crosslinking

[0064] The examples 1-16 and comparative examples 1-8 that received 7 kGy irradiation were divided into three groups: A, B, and C according to the intensity of ultraviolet irradiation. Group A received ultraviolet irradiation with an intensity of 1107 (μW / cm 2 ) embodiment, group B is subjected to ultraviolet irradiation with an intensity of 1320 (μW / cm 2 ) embodiment, group C received ultraviolet irradiation with an intensity of 853 (μW / cm 2 ). Groups A, B, and C were subjected to cross-linking treatment with an irradiation dose of 7 kGy or 15 kGy. The test indicators were the multiples of the weight after liquid absorption and the dry weight, as well as the appearance of the material after absorbing the liquid. The results are shown in Table 3.

[0065] Table 3 Water absorption test results after UV crosslinking and different doses of irradiation crosslinking

[0066]

[0067] According to the results in Table 2 and Table 3, the most suitable process parameters are collagen concentration of 7 mg / ml, UV cross-linking time of 30 min, UV illumination of 1107 (μW / cm 2 ), 7kGy low-dose irradiation treatment. 2 The water absorption performance of the cross-linked protein scaffolds irradiated with low doses of radiation (7 kGy or 15 kGy) for 30 min was compared with that of the positive control. The results are shown in Table 4.

[0068] Table 4 UV illumination is 1107 μW / cm 2 Comparison of protein scaffold performance

[0069]

[0070]

[0071] According to Table 4, it is shown that the UV cross-linking combined with low-dose irradiation cross-linking has good liquid absorption performance, and the time required for the liquid to be fully absorbed is short. Moreover, the collagen scaffold modified by this method can maintain its intact structure and size after absorbing the liquid.

[0072] 3. Collagen electrophoresis verification after cross-linking

[0073] SDS-PAGE protein electrophoresis was performed on Example 10 and Example 12 (UV+15 kGy irradiation group) with Comparative Examples 1-6 and the positive control. The results are as follows Figure 1 Lanes 1-10 are positive control (Comparative Example 8), non-lyophilized Comparative Example 1, Comparative Examples 1-6, Example 10 and Example 12, respectively.

[0074] from Figure 1 It can be seen that collagen that has not been cross-linked will show 4-6 typical electrophoresis bands. The cross-linking treatment will form new chemical bonds or covalent bonds between the collagen polypeptide chains, and the electrophoresis process can no longer be separated by SDS denaturation treatment. Obviously, compared with the comparative example 1 and the positive control that have not been cross-linked, Examples 10 and 12 have no obvious electrophoresis bands. Compared with the negative control and the positive control, the typical electrophoresis bands of the bands that have only been cross-linked or irradiated with a low dose are more diffuse.

[0075] 4. Hemostasis test of collagen modified scaffold

[0076] The collagen scaffolds of Examples 9-12 were subjected to a hemostasis test, and the test method was as follows:

[0077] Healthy adult New Zealand white rabbits weighing about 2.0 kg were selected, regardless of gender. The experiment will test the hemostatic effect of the material on the rabbit ear artery and liver, with 5 rabbits in each group, and one ear artery bleeding wound and liver injury wound were prepared for each animal. The collagen scaffolds treated with different cross-linking methods were the experimental group, and the gauze was used as the control group.

[0078] Wounds were artificially made on rabbit ears and livers. When blood appeared on the wounds, ordinary gauze was used to absorb the blood first, and then the hemostatic material collagen scaffold was quickly applied to the wound. At the same time, a stopwatch was started to observe the bleeding on the surface and around the hemostatic material, and the bleeding was dipped every 20 seconds with an accurately weighed ordinary gauze until the ordinary gauze could no longer dip into the bleeding. The stopwatch was stopped and the time was recorded. The average value was taken as the hemostasis time. The mass of the hemostatic material was accurately weighed with an analytical balance before hemostasis. The mass of the ordinary gauze was accurately weighed with an analytical balance before hemostasis, recorded as m1. During hemostasis, ordinary gauze was used to continuously dip into the bleeding until hemostasis was completed. After hemostasis was completed, the mass of the ordinary gauze dipped into the bleeding was accurately weighed, recorded as m2. Bleeding volume (ml) = [(weight of material after hemostasis + weight of gauze wiping blood m2)-(weight of material before hemostasis + weight of gauze m1)] / blood specific gravity. The adhesion of the collagen scaffold to the wound surface was observed, and its adhesion and application ability to the wound surface, the shrinkage or blood absorption level of the material, and the hemostasis time were evaluated. The results are shown in Table 5.

[0079] Table 5 Hemostatic effect test of Examples 9-12

[0080]

[0081]

[0082] It can be seen from Table 5 that the collagen scaffolds after D-ribose cross-linking, UV cross-linking and low-dose radiation physical cross-linking still have good physiological hemostatic function.

[0083] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a modified collagen scaffold, characterized in that: The following steps are involved: Step 1, cross-linking the collagen solution with a glycosylation reagent to obtain a first cross-linked product; Step 2, soaking the first cross-linked product in an ultraviolet photosensitizing agent, and then performing ultraviolet cross-linking to obtain a second cross-linked product; Step 3, performing multiple low-dose irradiation cross-linking on the second cross-linked product to complete the preparation of a collagen modified scaffold; In the step 1, after the collagen solution and the glycosylation agent are mixed and stirred, the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution, and the first cross-linked product is obtained after freeze-drying; or the collagen solution is first freeze-dried, and then mixed and stirred with the glycosylation agent to fully infiltrate, and the pH is adjusted to 7.2±0.2 with 1-1.5% ammonia water, and then 8-10% acetone is added according to the volume of the reaction system for elution to obtain the first cross-linked product; In step 1, the glycosylation agent is one of D-ribose or D-glucose; the glycosylation agent is dissolved in a buffer solution at a concentration of 0.02-1.5 mol / L; In step 1, the concentration of the collagen solution is 7 mg / ml; In the step 1, the addition ratio of the collagen solution and the glycosylation reagent is 1:(0.05-2); In step 2, the UV crosslinking time is 30 min, and the UV crosslinking illumination is 1107 μW / cm 2 ; In step 2, the wavelength of the ultraviolet light for ultraviolet crosslinking is 190-280nm; the power of the light source for ultraviolet crosslinking is 5-15W; In the step 3, the irradiation dose is 7 kGy, the low-dose irradiation rays are gamma rays, and the low-dose irradiation times are 2 times.

2. The method for preparing a modified collagen scaffold according to claim 1, characterized in that: In step 1, the cross-linking reaction time of the collagen solution and the glycosylation reagent is 48-72 hours; the cross-linking reaction time of the freeze-dried collagen solution and the glycosylation reagent is 48-120 hours.

3. The method for preparing a modified collagen scaffold according to claim 1, characterized in that: The ultraviolet crosslinking in step 2 is carried out in an upper and lower ultraviolet light box, the carrier is a quartz glass plate, and the distance between the ultraviolet lamp and the quartz glass plate is 10-30 cm.

4. A modified collagen scaffold, characterized in that: The modified collagen scaffold is prepared by the preparation method of any one of claims 1 to 3.

5. The use of a modified collagen scaffold according to claim 4, characterized in that: The modified collagen scaffold is used for preparing medical materials.

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