A highly biocompatible and stable collagen fiber, its preparation method and application.

Abstract

This invention belongs to the field of biomedical materials technology, specifically relating to a highly biocompatible and highly stable collagen fiber, its preparation method, and its application. The method includes the following steps: (1) constructing collagen fibers; (2) cross-linking collagen fibers with a cross-linking agent; (3) dialysis, homogenization, centrifugation, and collection of the precipitate. The prepared cross-linked collagen fibers are tightly packed and highly cross-linked, exhibiting good thermal stability and resistance to enzymatic degradation. The cross-linked collagen fibers have high biocompatibility, promoting fibroblast proliferation; they have high biocompatibility, and the inflammatory response caused after implantation is significantly mild and quickly eliminated. No calcification nodes appear after implantation of the cross-linked collagen fibers, demonstrating excellent anti-calcification effects. The cross-linked collagen fiber implant provided by this invention significantly promotes the production of new collagen and can be applied in fields such as implants, artificial skin, hemostatic sponges, scaffold materials, and medical devices, with broad application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a collagen fiber with high biocompatibility and stability, a preparation method and application. BACKGROUND

[0002] Collagen is the main structural component of the dermis. With the growth of age, the content of collagen in the skin will decrease significantly, leading to various aging phenomena such as skin wrinkles. Therefore, collagen fibers are widely used in the field of dermatology to prepare injectable collagen implants. However, uncrosslinked collagen implants have poor stability and are rapidly degraded by collagenase in the body, which seriously affects their use effect.

[0003] Chemical crosslinking is currently the main method for crosslinking collagen. Chinese patent CN113384748A discloses a method for preparing collagen implants by crosslinking collagen hydrogel with glutaraldehyde; Chinese patent CN109385682A discloses a method for preparing collagen fibers with good tensile strength by crosslinking with EDC / NHS. Chinese patent CN101234216B discloses a method for preparing collagen gels with good thermal stability by crosslinking with aldehyde polysaccharides. However, the residues of glutaraldehyde and other crosslinking agents in these patents pose a health risk, and the stability of the prepared crosslinked collagen needs to be further improved.

[0004] To solve the above technical problems, the inventors have unexpectedly found a method for preparing collagen fibers with high biocompatibility and stability. The crosslinked collagen fibers prepared by the method are tightly packed and highly crosslinked, have good thermal stability and anti-enzymatic ability; the crosslinked collagen fibers have high biocompatibility and can significantly promote the proliferation of fibroblasts; the crosslinked collagen fibers do not form calcified nodes after implantation and have excellent anti-calcification effect; the crosslinked collagen fiber implants provided by the present application can significantly promote collagen regeneration and can be applied in the fields of implants, artificial skin, hemostatic sponges, scaffold materials, medical devices, etc., and have a wide application prospect. SUMMARY

[0005] To solve the above technical problems, the present application provides a method for preparing collagen fibers, comprising the following steps: (1) constructing collagen fibers; (2) crosslinking the collagen fibers with a crosslinking agent; (3) dialysis, uniformity and collection of the precipitate;

[0006] The method for constructing collagen fibers in step (1) is as follows:

[0007] The collagen solution with a concentration of 0.1-5 mg / mL is prepared, 10 mM-100 mM sodium phosphate dibasic / sodium phosphate monobasic solution (pH 6-8) is added, and the precipitate is collected by centrifugation; the obtained precipitate is uniformly dispersed in 20 mM sodium phosphate dibasic / sodium phosphate monobasic solution (pH 6-9) to make the concentration of collagen 3 mg / mL, and then incubated at 17-25 °C for 8-19 h; the incubated collagen is homogenized at 4 °C and 10 000 rpm for 20 min; and the collagen fibers are obtained by centrifugation.

[0008] Preferably, the cross-linking agent in step (2) is one or more of carbodiimide, epichlorohydrin, sodium metaphosphate, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, proanthocyanidin, hesperidin, tannic acid, genipin, riboflavin, naringenin, quercetin, epigallocatechin gallate, oleuropein, L-threose (LT), phytic acid, isocyanate, acyl azide, diphenyl phosphate, and bis(N-hydroxysuccinimidyl) adipate (NHS-SA).

[0009] Preferably, the cross-linking agent in step (2) is bis(N-hydroxysuccinimidyl) adipate.

[0010] Preferably, the concentration of the cross-linking agent in step (2) is 0.01-5 mg / mL.

[0011] Preferably, the amount of the cross-linking agent added in step (2) is 4-5 mg / mL.

[0012] Preferably, the reaction temperature of the cross-linking agent in step (2) is 4-25 °C, and the reaction time is 12-24 h.

[0013] Preferably, the reaction temperature of the cross-linking agent in step (2) is 8-25 °C.

[0014] Preferably, the reaction temperature of the cross-linking agent in step (2) is 8 °C.

[0015] Preferably, the reaction time of the cross-linking agent in step (2) is 24 h.

[0016] Preferably, the collagen in step (1) is animal collagen, including type I, type II, and type III.

[0017] Preferably, the collagen in step (1) is type I collagen.

[0018] Preferably, the dialysis in step (3) is carried out by adding glycine after cross-linking, and the precipitate is collected by centrifugation at 4-25 °C and 10 000 rpm for 20-30 min after dialysis in a buffer at 4-25 °C for 48-72 h.

[0019] The second object of the present application is to provide the collagen fiber prepared by the preparation method.

[0020] The third object of the present application is to provide the application of the collagen fiber in the preparation of implants, artificial skin, hemostatic sponge, scaffold material and medical devices.

[0021] The fourth object of the present application is to provide a collagen fiber implant prepared by the collagen fiber.

[0022] The fifth object of the present application is to provide the application of bis(N-hydroxysuccinimide)suberate as a collagen crosslinking agent.

[0023] The beneficial effects of the present application are: (1) the present application provides a preparation method of collagen fiber, the method is simple and convenient, and the crosslinked collagen fiber prepared by the method is tightly packed and highly crosslinked, has good thermal stability and anti-enzymatic ability; (2) the crosslinked collagen fiber has high biocompatibility, can significantly promote the proliferation of fibroblasts; the crosslinked collagen fiber has high biological safety, the inflammatory reaction caused after implantation is significantly slight and quickly eliminated; (3) the crosslinked collagen fiber does not appear calcification nodes after implantation, has excellent anti-calcification effect; (4) the crosslinked collagen fiber implant significantly promotes collagen regeneration, can be applied in the fields of implants, artificial skin, hemostatic sponge, scaffold material and medical devices, and has wide application prospect; (5) the present application also provides a new use of bis(N-hydroxysuccinimide)suberate as a collagen crosslinking agent, and a collagen fiber crosslinked by bis(N-hydroxysuccinimide)suberate. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Effects of collagen fibers crosslinked by NHS-SA with different concentrations

[0025] Figure 2 Scanning electron microscope images of collagen fibers crosslinked by NHS-SA at different temperatures

[0026] Figure 3 Anti-enzymatic ability of collagen fibers crosslinked by NHS-SA

[0027] Figure 4 Denaturation detection of collagen fibers crosslinked by NHS-SA

[0028] Figure 5 Thermal stability of collagen fibers crosslinked by NHS-SA

[0029] Figure 6 Cell activity of collagen fibers crosslinked by NHS-SA

[0030] Figure 7 Live / dead staining of NHS-SA crosslinked collagen fibers

[0031] Figure 8 In vivo photo of NHS-SA crosslinked collagen fibers implanted in mouse skin

[0032] Figure 9 Histological HE staining of NHS-SA crosslinked collagen fibers implanted in mouse skin

[0033] Figure 10 Histological Masson staining of NHS-SA crosslinked collagen fibers implanted in mouse skin

[0034] Figure 11 Histological alizarin red staining of NHS-SA crosslinked collagen fibers implanted in mouse skin DETAILED DESCRIPTION

[0035] The application will be described in detail below with specific examples. Any technical solutions that can be thought of by any person skilled in the art on the basis of the application and in combination with common knowledge in the art shall fall within the protection scope of the application.

[0036] Referring to Chinese Patent CN 112778412A, medical-grade yak collagen is prepared as follows: yak tendons are washed to remove foreign matter and crushed into small pieces; the crushed small pieces are defatted with 10% n-butanol, and the precipitate is washed with water to neutralization; the precipitate obtained in the previous step is decalcified with 0.5M hydrochloric acid, and after decalcification, the precipitate is washed with water to neutralization; the tissue precipitate obtained in the previous step is soaked in a 0.1M sodium hydroxide solution, stirred at 25°C for 4h, and the precipitate is washed with water to neutralization; collagen is extracted with a 0.5M acetic acid solution containing 1g / L pepsin to obtain a crude collagen extract; the pH is adjusted to neutralization, and the enzyme is inactivated; dialysis is performed with an 8-14kDa dialysis bag, and after dialysis, freeze-drying is performed to obtain collagen with low endotoxin content.

[0037] Example 1, Preparation and characterization of NHS-SA crosslinked collagen fibers with different concentrations

[0038] 1.1 Preparation of collagen fibers

[0039] ① Collagen solution was prepared: collagen powder was dissolved in water, 0.5M acetic acid and / or hydrochloric acid and / or phosphoric acid solution was added, the pH was adjusted to 3.0-5.0, and a clear collagen solution was obtained, with a collagen concentration of 0.1-5mg / mL;

[0040] ②Induced self-assembly: in the prepared collagen acid solution, 10 mM-100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution (pH 6-8) was added, and after standing for 1.5 h, centrifugation was performed, and the collagen precipitate was collected;

[0041] ③Redissolution: 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution (pH 6-9) was used to uniformly disperse the obtained collagen precipitate, and the collagen concentration was 3 mg / mL;

[0042] ④Incubation: the redissolved collagen solution was incubated at 17℃-25℃ for 8-19 h;

[0043] ⑤Homogenization: the incubated collagen was homogenized at 4℃ at a speed of 10,000 rpm for 20 min;

[0044] ⑥Screening: the homogenized collagen was screened using a 40-mesh screen;

[0045] ⑦Centrifugation: the screened collagen solution was centrifuged to obtain collagen fibers.

[0046] 1.2 Preparation of collagen fibers crosslinked by different concentrations of NHS-SA

[0047] The prepared collagen fibers were taken, and 0.25, 1.25, 2.5, 4, and 5 mg / mL NHS-SA solutions were added thereto, respectively, at a concentration of 3 mg / mL. After crosslinking at 8℃ and 220 rpm for 24 h, glycine was added to remove unreacted NHS-SA, and then the sample was dialyzed against 0.02 M PBS (pH 7.4) for 72 h. After homogenization at 10,000 rpm for 20 min and centrifugation at 10,000 rpm for 30 min, the obtained precipitate was collected, which was the crosslinked collagen fibers.

[0048] 1.3 Crosslinking degree characterization

[0049] The crosslinking degree of the crosslinked collagen was detected by the TNBS method. The crosslinked and non-crosslinked collagen was freeze-dried, and 11 mg of the freeze-dried sample was taken and placed in 4% sodium bicarbonate. Then, 0.5% TNBS was added thereto, and the mixture was reacted at 40℃ for 4 h. Subsequently, 6 mol / L HCl was added thereto, and the mixture was reacted at 60℃ for 1.5 h. After dilution, anhydrous ether was added to remove unreacted TNBS. Then, the ultraviolet absorbance at 345 nm was measured, and the crosslinking degree was calculated.

[0050] Calculation of the crosslinking degree: α = (W1-W0)-(W2-W0) / (W1-W0) × 100%

[0051] Wherein a: cross-linking degree; Wo: background absorbance; W1: absorbance of uncross-linked collagen; W2: absorbance of collagen after cross-linking.

[0052] The results of UV detection are shown in Figure 1 and the cross-linking degree is calculated. With the increase of the concentration of cross-linking agent (0.25, 1.25, 2.5, 4, 5 mg / mL), the cross-linking degree of the collagen fiber cross-linked by NHS-SA gradually increases from 67% to 85%, 96%, 100% and 100%. The results show that the collagen fiber cross-linked by NHS-SA has a high cross-linking degree, and NHS-SA is a high-efficiency collagen cross-linking agent.

[0053] Example Two, Preparation and Characterization of Collagen Fiber Cross-Linked at Different Temperatures

[0054] 2.1 Preparation of Collagen Fiber

[0055] The preparation method is referred to Example One.

[0056] 2.2 Preparation of Collagen Fiber Cross-Linked at Different Temperatures

[0057] Take the long-acting collagen fiber prepared, configure the concentration to be 3 mg / mL, add 4 mg / mL of NHS-SA solution to it, and cross-link it at 8℃ and 25℃ respectively. After cross-linking for 24 h, add glycine to remove the unreacted NHS-SA, and then dialyze for 72 h. After homogenization at a rotation speed of 10,000 rpm for 20 min, centrifuge (10,000 rpm, 30 min) again, and collect the obtained precipitate, which is the collagen fiber after cross-linking.

[0058] 2.3 Cross-Linking Degree Characterization

[0059] Freeze-dry the samples of collagen fiber cross-linked by NHS-SA at different temperatures, take NHS-SA cross-linked collagen fiber samples respectively, and take 11 mg of freeze-dried sample of uncross-linked collagen fiber, and place it in 4% sodium bicarbonate. Add 0.5% TNBS to it, react at 40℃ for 4 h, add 6 mol / L HCl to it, react at 60℃ for 1.5 h, dilute, add anhydrous ether to remove the unreacted TNBS, and then measure the ultraviolet absorbance at 345 nm, and calculate the cross-linking degree.

[0060] Calculation of cross-linking degree: a = (W1-W0)-(W2-W0) / (W1-W0) x 100%

[0061] Wherein a: cross-linking degree; Wo: background absorbance; W1: absorbance of uncross-linked collagen; W2: absorbance of collagen after cross-linking.

[0062] The cross-linking degree was calculated, and the results are shown in Table 1. The cross-linking degree of the collagen fibers cross-linked by NHS-SA at 8°C and 25°C was 100%, indicating that NHS-SA can also achieve efficient cross-linking of collagen fibers at low temperatures.

[0063] Table 1 Cross-linking degree of collagen fibers cross-linked at different temperatures

[0064] Temperature Crosslinking degree 8℃ 100％ 25℃ 100％

[0065] 2.4 Scanning electron microscope characterization

[0066] The collagen fibers cross-linked in Example 2.2 were freeze-dried, then sliced and sampled, and then characterized by SEM after gold spraying.

[0067] The experimental results are shown in Table 1. The collagen fibers cross-linked by NHS-SA are closely arranged and form a uniform fibrous network structure. Figure 2

[0068] Preparation and characterization of glutaraldehyde cross-linked collagen fibers

[0069] 1. Preparation of glutaraldehyde cross-linked collagen fibers

[0070] Glutaraldehyde was added to a collagen fiber solution with a concentration of 3 mg / mL, and the final concentration of glutaraldehyde was 5 mg / mL. The solution was stirred at 37°C and pH 7.4 for 24 h. After the cross-linking reaction was completed, the collagen fibers were collected by centrifugation.

[0071] 2. Characterization of the cross-linking degree of glutaraldehyde cross-linked collagen fibers

[0072] 11 mg of untreated collagen, glutaraldehyde cross-linked collagen fibers of the comparative example, and NHS-SA cross-linked collagen fibers prepared in Example 3 were weighed and placed in 4% sodium bicarbonate. 0.5% TNBS was added, and the mixture was reacted at 40°C for 4 h. Then 6 mol / L HCl was added, and the mixture was reacted at 60°C for 1.5 h. After dilution, anhydrous ether was added to remove unreacted TNBS. Then the ultraviolet absorbance at 345 nm was measured, and the cross-linking degree was calculated.

[0073] Calculation of cross-linking degree: α = (W1-W0)-(W2-W0) / (W1-W0) x 100%, where α: cross-linking degree; Wo: absorbance of background; W1: absorbance of un-cross-linked collagen; W2: absorbance of cross-linked collagen.

[0074] ​The experimental results are shown in Table 2. The cross-linking degree of the collagen fibers cross-linked by glutaraldehyde is 85% when the concentration of glutaraldehyde is 5 mg / mL, and the cross-linking degree of the collagen fibers cross-linked by NHS-SA can reach 100% when the concentration of NHS-SA is 4 mg / mL, indicating that NHS-SA is a more efficient collagen cross-linking agent.

[0075] Table 2 Comparison of cross-linking degrees of collagen fibers cross-linked by glutaraldehyde and NHS-SA

[0076] Type Crosslinking degree Glutaraldehyde crosslinking 85％ NHS-SA crosslinking agent crosslinking 100％

[0077] The control group in the following examples uses the collagen fibers prepared by the comparative example.

[0078] Example Three, Preparation and Characterization of NHS-SA Cross-Linked Collagen Fibers

[0079] 1. Preparation of NHS-SA Cross-Linked Collagen Fibers

[0080] Take the prepared long-acting collagen fibers, configure the concentration to be 3 mg / mL, add 4 mg / mL of NHS-SA solution, cross-link at 8°C for 24 h, then add glycine to remove unreacted NHS-SA, and then dialyze for 72 h. After homogenization at a speed of 10,000 rpm for 20 min, centrifugation (10,000 rpm, 30 min) is performed again, and the obtained precipitate is collected, which is the cross-linked collagen fibers.

[0081] The collagen fibers used in the subsequent examples of the embodiments are prepared by the method described above in this example.

[0082] 2. Enzymatic Hydrolysis Experiment

[0083] 10 mg of untreated collagen fibers were weighed as a blank, and the glutaraldehyde cross-linked collagen fibers prepared by the comparative example and the NHS-SA cross-linked collagen fibers prepared by Example Three were weighed, and the initial dry weight (about 10 mg) was recorded. Collagenase was prepared in a buffer solution (TES, 1 mM CaCl2, pH 7.4), and the concentration of collagenase was 5 U / mL. The enzymatic hydrolysis experiment was carried out at 37°C and 220 rpm. After the same time of enzymatic hydrolysis, the samples were washed with water and centrifuged. The samples obtained after centrifugation were freeze-dried and weighed, and the enzymatic hydrolysis rate was calculated.

[0084] AW% = (Wo-W) / W x 100%

[0085] Where AW%: enzymatic hydrolysis rate; Wo: initial weight of the sample before enzymatic hydrolysis; W: weight of the sample after enzymatic hydrolysis.

[0086] The experimental results are shown in Table 3. Figure 3As shown, the enzymatic hydrolysis rates of the blank group (untreated collagen fibers) on days 1, 2, 3, and 7 were 25%, 94%, 98%, and 100%, respectively; the enzymatic hydrolysis rates of the comparative group (prepared glutaraldehyde-crosslinked collagen fibers) on days 1, 2, 3, and 7 were 38%, 41%, 45%, and 47%, respectively; and the enzymatic hydrolysis rates of the experimental group (NHS-SA crosslinked collagen fibers prepared in Example 3) on days 1, 2, 3, and 7 were 4%, 11%, 14%, and 23%, respectively. The results indicate that glutaraldehyde crosslinking can improve the enzymatic hydrolysis resistance of collagen fibers, but the NHS-SA crosslinked collagen fibers exhibit a more significant resistance to enzymatic hydrolysis.

[0087] 3. Denaturation detection

[0088] Take 0.01 mg / mL of uncrosslinked collagen fiber extract and 0.01 mg / mL of NHS-SA crosslinked collagen fiber extract prepared in Example 3; prepare 0.01 mg / mL of boil-denatured type I collagen and 0.01 mg / mL of gelatin, and add 100 μL of each to a 96-well plate. Perform five replicates for each sample. After incubating in the dark at 4°C for 12 hours, aspirate the supernatant. Add 20 μM of the denatured collagen-targeting fluorescent probe FAM-(GPO). 10 After heating at 90℃ for 20 min, ice water was immediately added for cooling. 100 μL of each sample was added to each well. After 4 h in a dark environment at 4℃, each sample was washed three times with 200 μL of 10 mM PBS (pH 7.4) and the fluorescence intensity was measured using an ELISA reader.

[0089] The results are as follows Figure 4 As shown, boil-denatured type I collagen and gelatin react with the denatured collagen-targeting fluorescent probe FAM-(GPO). 10 After binding, they exhibited very strong fluorescence intensity (42754 and 35839); while uncrosslinked, intact triple-helix collagen fibers and NHS-SA crosslinked collagen fibers showed very strong fluorescence intensity when bound to the denatured collagen-targeting fluorescent probe FAM-(GPO). 10 After binding, they exhibited similar, extremely weak fluorescence signals. The results indicate that the NHS-SA cross-linked collagen fibers were prepared under low-temperature, mild conditions, maintaining the complete triple helix structure and completely avoiding the potential denaturation risks during collagen cross-linking.

[0090] 4. DSC characterization

[0091] Weigh 5 mg of the NHS-SA cross-linked collagen fiber and the freeze-dried uncross-linked collagen fiber prepared in Example 3, respectively, and then place them in a crucible under nitrogen protection. Measure their thermal stability in the range of 25-120℃ at a heating rate of 5℃ / min.

[0092] Experimental results are as followsFigure 5 As shown, the thermal denaturation temperature of NHS-SA crosslinked collagen fibers was significantly increased from 96 °C to 104 °C compared with that of non-crosslinked collagen fibers, indicating that the NHS-SA crosslinked collagen fibers had better thermal stability.

[0093] Example Four, Biological Activity and Biocompatibility of NHS-SA Crosslinked Collagen Fibers

[0094] 1. Cell Toxicity and Proliferation

[0095] To investigate the cell toxicity and proliferation of crosslinked and non-crosslinked samples, HFF-1 human fibroblasts were cultured in high glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin at 37 °C, 5% CO2. The NHS-SA crosslinked collagen fibers prepared in Example Three, glutaraldehyde crosslinked collagen fibers prepared in Comparative Example, and non-crosslinked collagen fibers were freeze-dried samples were placed in DMEM high glucose culture solution, soaked at 37 °C for 72 h, and the extract was prepared according to the standard cell toxicity test ISO 10993-5. The cells were digested with 0.25% (w / w) trypsin, seeded in a 96-well plate at a density of 7000 per well, incubated at 37 °C and 5% CO2 for 24 h, and then 100 uL / well extract of different samples was added to replace the culture medium and incubated for 24 h, 48 h, and 72 h, respectively. 10 uL Cell Counting Kit-8 (Dojindo Molecular Technologies, Japan) was added at 37 °C for 1-3 h. The base medium was blank control (n = 6), and the OD value at 450 nm was read. The cell survival rate was calculated as follows:

[0096] Cell survival rate (%) = (A-C) / (B-C) x 100%

[0097] Wherein A: OD value of different samples; B: OD value of positive control group; C: OD value of blank control group.

[0098] The experimental results are shown in Table 1. Figure 6 As shown, the cell survival rates of non-crosslinked collagen fibers at 24 h, 48 h, and 72 h were 105%, 108%, and 110%, respectively, the cell survival rates of NHS-SA crosslinked collagen fibers were 104%, 109%, and 114%, respectively, and the cell survival rates of glutaraldehyde crosslinked collagen fibers were significantly reduced, which were 90%, 61%, and 59%, respectively. The results showed that glutaraldehyde crosslinked collagen fibers had obvious cell toxicity, while NHS-SA crosslinked collagen fibers had no cell toxicity and had good biocompatibility.

[0099] 2. Live / Dead Cell Staining

[0100] To investigate cell viability in cross-linked and uncross-linked samples, HFF-1 human fibroblasts were cultured in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin at 37°C and 5% CO2. NHS-SA cross-linked collagen fibers prepared in Example 3, glutaraldehyde cross-linked collagen fibers prepared in the comparative example, and lyophilized samples of uncross-linked collagen fibers were placed in high-glucose DMEM medium and soaked at 37°C for 72 h. Extracts were prepared according to the standard cytotoxicity assay ISO 10993-5. Cells were digested with 0.25% (w / w) trypsin and seeded at a density of 40,000 cells per well in laser confocal culture dishes. The cells were incubated at 37°C and 5% CO2 for 24 h, and then incubated for 24 h and 72 h respectively with 100 μL / well of the extract from each sample. Afterward, the cells were stained with CA / PI and photographed using an Olympus laser confocal microscope.

[0101] Experimental results are as follows Figure 7 As shown, the number of surviving cells in uncrosslinked collagen fibers at 24h and 72h was 173 cells / mm². 2 225 pieces / mm 2 The number of surviving cells in NHS-SA cross-linked collagen fibers was 184 cells / mm² at 24h and 72h. 2 206 pieces / mm 2 The number of surviving cells in glutaraldehyde-crosslinked collagen fibers was significantly reduced at 24h and 72h, to 113 cells / mm², respectively. 2 95 pieces / mm 2 The results showed that NHS-SA cross-linked collagen fibers have good biocompatibility and can provide beneficial conditions for cell survival.

[0102] Example 5: Animal Experiments with NHS-SA Cross-linked Collagen Fiber Implants

[0103] Subcutaneous implantation in mice is a common method for evaluating the biocompatibility of materials in vivo. This study used male Kunming mice (30–40 g) for in vivo evaluation. Samples before and after cross-linking were immersed in 75% (v / v) ethanol overnight and thoroughly rinsed with sterile saline. Mice that did not receive the implant served as a blank. The NHS-SA cross-linked collagen fibers prepared in Example 3 and the glutaraldehyde cross-linked collagen fibers prepared in the comparative example were injected into the subcutaneous tissue of mice, with five mice injected in parallel in each group.

[0104] 1. Implant degradation

[0105] Mice were anesthetized with 1 mL of 10% chloral hydrate at weeks 2, 4, 6, and 8. The mice were then shaved and euthanized. Skin samples were collected from the implantation site. Figure 8As shown, NHS-SA cross-linked collagen fibers showed no significant degradation within 8 weeks, while glutaraldehyde cross-linked collagen fibers and uncross-linked collagen fibers both underwent substantial degradation. The results indicate that NHS-SA cross-linked collagen fibers maintain significant anti-degradation capabilities in vivo.

[0106] 2. HE staining

[0107] Mice were anesthetized with 1 mL of 10% chloral hydrate at weeks 2, 4, 6, and 8. The mice were then shaved and euthanized. Skin samples were taken from the implantation site, and the skin was sectioned in paraffin and stained with hematoxylin and eosin (HE).

[0108] Experimental results are as follows Figure 9 As shown, when NHS-SA cross-linked collagen fibers were implanted into the subcutaneous tissue of mice, a mild inflammatory reaction was observed in the implantation site at week 2, which is a normal foreign body reaction in the early stages of implantation. Inflammatory cells significantly decreased at week 4, and almost no inflammatory cells were observed at weeks 6 and 8. Glutaraldehyde cross-linked collagen fibers, when implanted into the subcutaneous tissue, produced a severe inflammatory reaction at week 2; inflammatory cells decreased somewhat at week 4; and significant inflammatory cells remained at weeks 6 and 8. These results indicate that the biocompatibility of NHS-SA cross-linked collagen fibers is significantly better than that of glutaraldehyde cross-linked collagen fibers.

[0109] 3. Masson staining

[0110] Mice were anesthetized with 1 mL of 10% chloral hydrate at weeks 2, 4, 6, and 8. The mice were then shaved and euthanized. Skin samples were taken from the implantation site, and the skin was sectioned in paraffin and stained with Masson's stain.

[0111] Experimental results are as follows Figure 10 As shown, when NHS-SA cross-linked collagen fibers were implanted into the subcutaneous tissue of mice, fibroblasts were generated at the implantation site in the second week; new collagen fibers were observed in the fourth week; and the number of newly formed collagen fibers increased significantly in the sixth and eighth weeks. When glutaraldehyde cross-linked collagen fibers were implanted into the subcutaneous tissue of mice, obvious fibroblasts were only observed in the fourth week; and a small number of newly formed collagen fibers were only observed in the eighth week. The results indicate that NHS-SA cross-linked collagen fibers can significantly promote collagen regeneration, and their effect is significantly better than that of glutaraldehyde cross-linked collagen fibers.

[0112] 4. Alizarin Red staining

[0113] Subcutaneous implantation model of mice is a common method for evaluating the biocompatibility of materials in vivo. Male Kunming mice (30-40 g) were used for in vivo evaluation in this study. The samples before and after crosslinking were soaked in 75% (v / v) ethanol overnight and washed thoroughly with sterile normal saline. Mice without injection of implants were used as blank controls. The crosslinked collagen fibers of Example 3 and Comparative Example were injected into the subcutaneous tissue of mice, respectively, with 5 mice in each group.

[0114] At 2, 4, 6, and 8 weeks, the mice were anesthetized with 10% chloral hydrate 1 mL, then shaved using depilatory cream, and sacrificed after shaving. The skin at the implantation site was sampled, and the mouse skin was paraffin sectioned after sampling, and stained with alizarin red.

[0115] The experimental results are shown in Table 1. Figure 11 As shown in Table 1, the subcutaneous tissue of mice implanted with glutaraldehyde crosslinked collagen fibers had orange-red nodes at 2 weeks; at 4-8 weeks, the calcification was more obvious, and the number of calcification nodes increased significantly. In contrast, the subcutaneous tissue of mice implanted with NHS-SA crosslinked collagen fibers had no calcification nodes during the 8-week period. The results show that the NHS-SA crosslinked collagen fibers have excellent anti-calcification ability.

[0116] In summary, the present application provides a collagen fiber with high biocompatibility and stability, a preparation method and applications. The preparation method is simple and convenient, and the crosslinked collagen fiber prepared by the method is tightly packed and highly crosslinked, has good thermal stability and anti-enzymatic ability. The crosslinked collagen fiber has high biocompatibility, can significantly promote the proliferation of fibroblasts, has high biological safety, causes significantly mild and rapid elimination of inflammatory reactions after implantation into the body, and has excellent anti-calcification effect without calcification nodes after implantation. The crosslinked collagen fiber implant provided by the present application significantly promotes the production of new collagen, can be applied in the fields of implants, artificial skin, hemostatic sponges, scaffold materials, medical devices, and has a wide application prospect. The present application also provides a new use of bis(N-hydroxysuccinimide) octanedioate as a collagen crosslinking agent, and a collagen fiber crosslinked using bis(N-hydroxysuccinimide) octanedioate.

Claims

1. A method for preparing a highly biocompatible, highly stable collagen fiber, characterized by, It comprises the following steps: (1) constructing collagen fibers; (2) cross-linking collagen fibers with a cross-linking agent; (3) dialysis, homogenization, centrifugation and collection of the precipitate; The method for constructing collagen fibers in step (1) is as follows: ① Preparing a collagen solution: dissolving collagen powder in water, adding 0.5M acetic acid and / or hydrochloric acid and / or phosphoric acid solution, adjusting the pH to 3.0-5.0 to obtain a clear collagen solution, and the collagen concentration is 0.1-5mg / mL; ② Inducing self-assembly: adding 10mM-100mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution, pH 6-8, to the prepared collagen acidic solution, standing for 1.5h, then centrifuging and collecting the collagen precipitate; ③ Resuspension: using 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution, pH 6-9, to uniformly disperse the obtained collagen precipitate, and the collagen concentration is 3mg / mL; ④ Incubation: incubating the resuspended collagen solution at 17℃-25℃ for 8-19h; ⑤ Homogenization: homogenizing the incubated collagen at 4℃ and a rotation speed of 10000rpm for 20min; ⑥ Sieving: sieving the homogenized collagen using a 40-mesh sieve; ⑦ Centrifugation: collecting the sieved collagen solution and centrifuging to obtain collagen fibers; The cross-linking agent in step (2) is bis(N-hydroxysuccinimide)suberate, and the concentration of the cross-linking agent is 4-5mg / mL; the reaction temperature of the cross-linking agent is 8-25℃, and the reaction time is 24h.

2. The production method according to claim 1, wherein The collagen in step (1) is animal collagen, including one or more of type I, type II and type III.

3. The production method according to claim 1, wherein The dialysis in step (3) is carried out after adding glycine, and the dialysis is carried out in a 4℃-25℃ buffer for 48-72h; after homogenization at 4℃-25℃, a rotation speed of 10000rpm and a time of 20-30min, the precipitate is collected by centrifugation.

4. The collagen fibers prepared by the preparation method of any one of claims 1-3.

5. The use of the collagen fibers of claim 4 in the preparation of medical devices.

6. The use according to claim 5, wherein the compound is ###0002### The medical devices include implants, artificial skin, hemostatic sponges and stent materials.

7. A collagen fiber implant, characterized by, The implants are prepared from the collagen fibers of claim 4.

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