CN118241483A - Ribose cross-linked collagen fiber, and preparation and application thereof - Google Patents

Abstract

The invention provides a ribose cross-linked collagen fiber, which is formed by cross-linking collagen fiber with ribose as a cross-linking agent, wherein the diameter of the collagen fiber is 15-80 nm; the diameter of the ribose cross-linked collagen fiber is between 150 and 200 nm; the crosslinking degree of the ribose crosslinked collagen fiber is between 40.7 and 56.1 percent; tm value = 58.54 ℃ -66.46 ℃. The invention also provides a preparation method and application of the ribose cross-linked collagen fiber. The ribose cross-linked collagen fiber provided by the invention has lower thrust and collagenase degradation resistance. The crosslinking agent used in the invention is natural ribose without any toxic side effect, and has better biocompatibility compared with other crosslinking forms.

Description

Ribose cross-linked collagen fibers and their preparation and application

Technical Field

The invention relates to ribose cross-linked collagen fiber and the preparation and application thereof.

Background Art

As we age, the collagen content in the body gradually decreases, and problems such as sagging skin, increased wrinkles, and joint pain will occur. Therefore, how to supplement collagen has always been a concern in the field of beauty and health care. High-purity animal-derived collagen has good biocompatibility and can be used as a tissue filler. However, due to the presence of collagenase in the body, clinical use has found that natural collagen only exists in the body for 3-6 months. In order to increase the retention time of collagen fillers in the body, glutaraldehyde, reducing sugars, epoxide crosslinkers and imine crosslinkers have been used to crosslink collagen to prepare more stable collagen fillers with longer retention time in the body. The earliest cross-linked collagen filler product used in clinical wrinkle removal Collagen implants and the cross-linked collagen product "Fu Li Mei" by Shuangmei Technology Co., Ltd., which has been approved for marketing, use glutaraldehyde as a cross-linking agent. Other approved cross-linked collagen filler products include Col Bar Life Science Ltd., which uses ribose as a cross-linking agent. Currently, no corresponding products of other types of cross-linking agents have been approved, and their biosafety needs to be further verified in clinical trials. A common feature of these products is that they first prepare collagen fibers by fibrosis under neutral conditions and then undergo cross-linking reactions. Among them, reducing sugar cross-linking is a cross-linking and stabilization reaction of collagen fibers caused by the interaction between collagen fibers and reducing glucose under simulated physiological conditions. Reducing sugars form Schiff bases between the ε-amino groups on the lysine and hydroxylysine residues of collagen fibers. The Schiff bases undergo Amadori rearrangement and a series of further reactions to form irreversible stable cross-links between collagen molecules. Since ribose cross-linkers are natural reducing sugars without any toxic side effects, they have better biocompatibility than other cross-linking forms. However, ribose cross-linking of collagen is inefficient and takes a long time to react (US 6,682,760 B2). We believe that the cross-linking efficiency and resistance to collagenase degradation of ribose cross-linked collagen fibers can be improved by regulating the fibrosis process and the assembly and arrangement of collagen molecules.

Summary of the invention

In order to improve the cross-linking efficiency and resistance to collagenase degradation of ribose cross-linked collagen fibers, and increase the cross-linking degree and melting point of the cross-linked collagen fibers, the present invention provides a new ribose cross-linked collagen fiber that can be used as a filler.

As one aspect of the present invention, ribose cross-linked collagen fibers are formed by cross-linking collagen fibers using ribose as a cross-linking agent; the diameter of the collagen fibers is between 15-80nm; the diameter of the ribose cross-linked collagen fibers is between 150-200nm; the cross-linking degree of the ribose cross-linked collagen fibers is between 40.7%-56.1%, preferably 48.6%-56.1%; the melting point Tm value = 58.54℃-66.46℃, preferably Tm value = 59.59℃-66.46℃.

As another aspect of the present invention, it relates to a cross-linked collagen fiber filler, wherein the filler contains the above-mentioned ribose cross-linked collagen fiber and may further contain lidocaine.

As another aspect of the present invention, it relates to a method for preparing the above-mentioned cross-linked collagen filler, characterized in that the method comprises: using physiological saline to adjust the protein concentration to 35 mg/mL and the lidocaine concentration to 0.2%, and after homogenization, aseptically filling into a 1 mL syringe and sealing.

As another aspect of the present invention, it relates to a method for preparing the above-mentioned ribose cross-linked collagen fibers, comprising:

The purified collagen molecules from which immunogenicity and virus are removed are fibrotic under acidic conditions and then cross-linked with ribose, wherein the pH value of the acidic conditions is 5.0-6.0 and the ribose concentration is 1%-5% (w:v).

It can be seen from the embodiments of the present invention that the present invention prepares collagen fibers with a smaller diameter under acidic conditions, and has good thermal stability and resistance to collagenase degradation after ribose cross-linking, and can maintain a longer degradation cycle. The comprehensive display of these properties enables the ribose cross-linked collagen fibers prepared by the present invention to be used as a filler in combination with lidocaine in the fields of plastic surgery. In addition, the ribose cross-linked collagen fibers have lower thrust and good biocompatibility, ensuring the safety of the product and being suitable for injection filling.

DETAILED DESCRIPTION

This application was jointly developed by Beijing Aibairui Biotechnology Co., Ltd. and Beijing Bairen Medical Technology Co., Ltd.

The inventor found in the process of experimenting with reference to the prior art that the collagen fiber prepared under neutral conditions has a large diameter. Although the ribose cross-linking agent can improve the melting point of the collagen fiber and enhance the ability to resist collagenase degradation, the cross-linking reaction efficiency is low and the reaction time is long (US 6,682,760 B2). CN114288469A reports that the collagen fiber prepared under neutral conditions is double-crosslinked using carbodiimide after ribose cross-linking, further improving the melting point of the cross-linked collagen fiber. However, double cross-linking reduces the biosafety of single ribose cross-linked collagen fiber. CN108752604 dissolves collagen molecules (molecular weight 5-300,000) in ionic solution by heating, and performs ribose cross-linking after cooling and regeneration, increasing collagen solubility, shortening cross-linking time, and enhancing cross-linking efficiency, but collagen is prone to protein denaturation after being treated with high-temperature ionic solution, and loses the functional activity of the protein itself. It is generally believed that the collagen fiber diameter formed under acidic conditions is relatively small and cannot meet the requirements as a filler. However, after further experiments, the inventors surprisingly found that as long as the process of preparing collagen fibers under acidic conditions is controlled, the prepared collagen fibers can react with ribose evenly to form covalent bonds, the reaction efficiency is improved, and the melting point and cross-linking degree of ribose-cross-linked collagen fibers are significantly improved. After further processing, they can be used as fillers.

The neutral conditions referred to in this application refer to a pH value of 6.5-7.5, and the acidic conditions referred to in this application refer to a pH value of 5.0-6.0.

The collagen fibers referred to in this application refer to: the product of collagen molecules after phosphate and NaCl fibrillation reaction. Under neutral conditions, the reaction has collagen fibers with a diameter of 80-200nm as the main component, and under acidic conditions, the reaction has collagen fibers with a diameter of 15-30nm as the main component.

The ribose-crosslinked collagen fibers referred to in the present application refer to products obtained by centrifugation and washing after the collagen fibers undergo ribose crosslinking reaction.

The method for testing the melting point of the prepared ribose cross-linked collagen fibers is as follows:

The ribose cross-linked collagen fibers were quantitatively transferred to the test aluminum crucible of the differential scanning calorimeter, and then sealed in the differential scanning calorimeter. The temperature was increased at 10K/min under a nitrogen atmosphere and the scanning measurement was performed in the temperature range of 25°C-100°C.

The method for testing the cross-linking degree of the prepared ribose cross-linked collagen fibers is as follows:

Quantitatively transfer ribose cross-linked collagen fibers to a microcentrifuge tube, prepare 0.1M pH 8.5 NaHCO ₃ solution and 0.5% (v:v) TNBS solution, take 1mL of each of the two prepared solutions and add them to the ribose cross-linked collagen fibers, and react at 40°C for 2 hours. After the reaction is completed, add 3mL, 6M HCl, react at 60°C for 1.5 hours, cool to room temperature after the reaction, add 5mL of deionized water and mix evenly. Take out 5mL of the mixed solution and add ether, shake it and let it stand to separate and take out the ether. After adding new ether three times, wait for the ether in the reaction solution to completely evaporate, take out 200μL of the reaction solution and add 400μL of deionized water, mix well, and use an enzyme reader to test the 345nm absorbance value, and subtract the absorbance value of the blank group to obtain the absorbance value A. Take an equal amount of uncross-linked collagen fibers and repeat the above steps to obtain the absorbance value B, and calculate the cross-linking degree of ribose cross-linked collagen fibers using the following formula:

The method of the present invention for conducting an in vitro collagenase degradation test on the prepared ribose cross-linked collagen fibers is as follows:

Quantitatively transfer a certain amount of ribose cross-linked collagen fibers to a microcentrifuge tube, prepare 50mM pH 7.4 TES collagenase reaction buffer, and then prepare the ribose cross-linked collagen fibers into a 2-5mg/ml suspension in the TES solution. Add collagenase solution according to the total amount of ribose cross-linked collagen fibers, and the addition ratio is 1.25U/mg. Digest at 37°C for 24 hours, centrifuge the sample to collect the precipitate and supernatant, and calculate the collagen content in the precipitate and supernatant by measuring the hydroxyproline content with an ELISA reader. Referring to McPherson et al. (1986) Journal of Biomedical Material Research, 20: 79-92. The in vitro collagenase degradation rate of ribose cross-linked collagen fibers was calculated using the following formula:

Example 1: Preparation of ribose cross-linked collagen fibers under neutral conditions

(1) Animal-derived tissue pretreatment (this step can be implemented with reference to the prior art): Take fresh pig skin tissue and scrape off the surface fat, then soak it in an acetone solution at room temperature for 16 hours. Wash the treated material with deionized water for 3-5 times to remove excess acetone, and then put the material into a 2.0M NaOH solution at room temperature with stirring for 16 hours. After the treatment, soak the material in deionized water and wash it until it is neutral to achieve fat removal and virus inactivation.

(2) Extraction (this step can be implemented with reference to the prior art): The cleaned material is placed in 0.5M acetic acid for crushing and homogenization. The homogenized tissue is subjected to collagen molecule extraction at a ratio of 20:1 (tissue weight/pepsin weight), the temperature is controlled at 18°C, and the extraction is stirred for 72 hours. The extract is centrifuged to collect the supernatant, and after stirring evenly, it is clarified and filtered, and the turbidity is controlled to be less than 30 to obtain a crude collagen molecule extract.

(3) Ultrafiltration purification (this step can be implemented with reference to the prior art): Use a tangential flow system to ultrafilter the crude collagen molecule extract, concentrate it to 3 mg/ml, and then start dialysis with 20 mM acetic acid solution. After dialysis of 20 times the volume, the collagen molecule is obtained.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules and add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust the pH to 7.0, and place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have a diameter between 80-200 nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 0.5% (w:v) D-ribose-phosphate buffer are added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 70% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

(6) Filling: Take a certain volume of ribose cross-linked collagen fibers and centrifuge at 8000 rpm for 30 min. After adding physiological saline to the obtained precipitate for homogenization, adjust the concentration to 35 mg/ml. Then, aseptically fill the ribose cross-linked collagen fibers with the adjusted concentration into a 1 mL syringe and seal it to obtain the ribose cross-linked collagen fiber filler.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, with a Tm value of 56.05°C and a cross-linking degree of 30.4%. The degradation rate after in vitro collagenase treatment was 85%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, with a thrust of 6.13N.

Example 2: Preparation of ribose cross-linked collagen fibers under neutral conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules and add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust the pH to 7.0, and place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have a diameter between 80-200 nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 times the volume of 1% (w:v) D-ribose-phosphate buffer is added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a network structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 56.68°C, and the cross-linking degree was 31.2%. The degradation rate after in vitro collagenase treatment was 82%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 6.26N.

Example 3: Preparation of ribose cross-linked collagen fibers under neutral conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules and add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust the pH to 7.0, and place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have a diameter between 80-200 nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 3% (w:v) D-ribose-phosphate buffer are added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 90% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 57.28°C, and the cross-linking degree was 34%. The degradation rate after in vitro collagenase treatment was 73%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 6.89N.

Example 4: Preparation of ribose cross-linked collagen fibers under neutral conditions

Steps (1) to (3) are the same as in Example 1.

Collagen molecule fibrillation: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust pH to 7.0, place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have a diameter between 80-200nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 5% (w:v) D-ribose-phosphate buffer solution are added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 90% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, with a Tm value of 56.76°C and a cross-linking degree of 32.8%. The degradation rate after in vitro collagenase treatment was 75%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, with a thrust of 6.43N.

Example 5: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules and add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust the pH to 6.0, and place at 30°C for 6 hours to obtain collagen fibers. 70% of the collagen fibers have a diameter of 50-80nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 times the volume of 1% (w:v) D-ribose-phosphate buffer is added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a network structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 58.54°C, and the cross-linking degree was 40.7%. The degradation rate after in vitro collagenase treatment was 58%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 5.68N.

Example 6: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules and add 1/10 volume of phosphate buffer (0.2M Na ₂ HPO ₄ :0.2M NaH ₂ PO ₄ =7:3, v:v). Mix well and adjust the pH to 5.0, and place at 30°C for 6 hours to obtain collagen fibers. 70% of the collagen fibers have a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 times the volume of 1% (w:v) D-ribose-phosphate buffer is added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a network structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 59.12°C, and the cross-linking degree was 46.9%. The degradation rate after in vitro collagenase treatment was 56%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 4.12N.

Example 7: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M _{Na2HPO4 :} 0.2M _NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have _a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 times the volume of 1% (w:v) D-ribose-phosphate buffer is added, mixed and placed at 37°C for 15 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a network structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, with a Tm value of 62.09°C and a cross-linking degree of 54.3%. The degradation rate after in vitro collagenase treatment was 42%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, with a push force of 4.32N.

Example 8: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M _{Na2HPO4 :} 0.2M _NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have _a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 1% (w:v) D-ribose-phosphate buffer are added, mixed and placed at 37°C for 12 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 60.74°C, and the cross-linking degree was 52%. The degradation rate after in vitro collagenase treatment was 49%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 3.87N.

Example 9: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen fibrosis: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M _Na2HPO4 : 0.2M _NaH2PO4 = ₇ :3, v:v), then add 0.15M NaCl and mix well to adjust the pH to 5.0. Place at 30°C for 6 hours to obtain collagen fibers. ₉₀ % of the collagen fibers have a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 1% (w:v) D-ribose-phosphate buffer solution are added, mixed and placed at 37°C for 10 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 80% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, with a Tm value of 59.59°C and a cross-linking degree of 48.6%. The degradation rate after in vitro collagenase treatment was 55%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, with a thrust of 3.74N.

Example 10: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M _{Na2HPO4 :} 0.2M _NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have _a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 times the volume of 3% (w:v) D-ribose-phosphate buffer is added, mixed and placed at 37°C for 10 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 90% ribose cross-linked collagen fibers is between 150-200 nm and forms a network structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimeter and microplate reader, and the Tm value was 66.46°C, and the cross-linking degree was 56.1%. The degradation rate after in vitro collagenase treatment was 39%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 4.87N.

Example 11: Preparation of ribose cross-linked collagen fibers under acidic conditions

Steps (1) to (3) are the same as in Example 1.

(4) Collagen molecule fibrillation: Take a certain volume of purified collagen molecules, add 1/10 volume of phosphate buffer (0.2M _{Na2HPO4 :} 0.2M _NaH2PO4 = 7:3, v:v), then add 0.15M NaCl, mix well and adjust the pH to 5.0. Place at 30°C for 6 hours to obtain collagen fibers. 90% of the collagen fibers have _a diameter of 15-30nm.

(5) Collagen fiber cross-linking: After the collagen fibers are centrifuged to obtain a precipitate, 10 volumes of 5% (w:v) D-ribose-phosphate buffer are added, mixed and placed at 37°C for 10 days. The precipitate is collected by centrifugation and washed three times with pH 7.0 phosphate buffer to obtain ribose cross-linked collagen fibers. The diameter of 90% ribose cross-linked collagen fibers is between 150-200 nm and forms a mesh structure.

Step (6) is the same as in Example 1.

The ribose cross-linked collagen fibers obtained in this example were tested by differential scanning calorimetry and microplate reader, and the Tm value was 59.71°C, and the cross-linking degree was 50.2%. The degradation rate after in vitro collagenase treatment was 47%. The physical properties were further tested by a push-pull tester: 27G needle, tested at a speed of 1mL/min, and the thrust was 5.56N.

In the above embodiments, steps (1)-(3) are the same, mainly to remove fat and sterilize viruses from the materials and to extract and purify collagen molecules. Step (4) is to perform fibrosis treatment on the purified collagen molecules, and step (5) is to perform ribose cross-linking treatment on the collagen fibers. Examples 1-4 were subjected to fibrosis treatment under neutral conditions of pH 7.0, followed by cross-linking treatment for 15 days using 0.5%, 1%, 2%, and 5% D-ribose-phosphate buffer. Examples 5 and 6 were subjected to fibrosis treatment under acidic conditions of pH 6.0 and pH 5.0, followed by cross-linking treatment for 15 days using 1% D-ribose-phosphate buffer. In Examples 7-11, acidic conditions of pH 5.0 were used for fibrosis treatment and NaCl was added to the fibrosis reaction solution: Na ₂ HPO ₄ and NaH ₂ PO ₄ solution. Then, different concentrations of D-ribose-phosphate buffer were used for cross-linking treatment. In Examples 7-9, 1% ribose was used for cross-linking for 15, 12, and 10 days; and in Examples 9-11, 1%, 3%, and 5% ribose were used for cross-linking for 10 days.

The melting point, cross-linking degree, degradation rate and thrust of the ribose cross-linked collagen fibers obtained in all examples were measured. Examples 1-3 show that as the concentration of ribose increases, the melting point and cross-linking degree of the ribose cross-linked collagen fibers increase, and the collagenase degradation rate decreases. However, Example 4 shows that as the concentration of ribose continues to increase, the cross-linking degree and resistance to collagenase degradation decrease, which is consistent with the change in degradation rate with concentration after fibrosis under neutral conditions described in US 6682769. Examples 2, 5, and 6 show that the ribose cross-linked collagen fibers obtained under low ribose concentration and acidic conditions have increased thermal stability, cross-linking degree, and resistance to collagenase degradation as the pH value decreases, and the thrust decreases as the reaction pH value decreases, compared to the ribose cross-linked collagen fibers obtained under neutral conditions. Examples 7, 8, and 9 show that adding NaCl to the fibrosis reaction solution: Na ₂ HPO ₄ and NaH ₂ PO ₄ solution under acidic conditions is conducive to fiber formation under acidic conditions, and reducing the cross-linking time can still obtain ribose cross-linked collagen fibers with high melting point and high cross-linking degree. Examples 9, 10, and 11 show that reducing the cross-linking time while increasing the ribose concentration can increase the cross-linking degree and Tm value of the ribose cross-linked collagen fibers, and improve the resistance to collagenase degradation. In particular, the ribose cross-linked collagen fibers obtained in Example 10 have the best effect. The single ribose cross-linking of collagen fibers under acidic conditions is equivalent to the effect of the double cross-linking of ribose and carbodiimide reported in CN114288469A. Tm = 66.64 ° C has good thermal stability and a cross-linking degree of 56.1%, and the resistance to collagenase degradation is significantly improved; the pushing force is 4.87N, which is more conducive to injection filling; the ribose cross-linking reaction time is shortened, and it is suitable for large-scale production and preparation.

The diameter of the collagen fiber prepared under neutral conditions is relatively large, and the prepared ribose cross-linked collagen fiber has a cross-linking degree between 30.4% and 34%, and a Tm value of 56.05°C-57.28°C. Although the maintenance time of the collagen fiber as a filler is improved to a certain extent, the ability to resist collagenase degradation still cannot meet expectations. Through experiments, it is found that regulating the fibrosis process and the assembly and arrangement of collagen molecules can improve the cross-linking efficiency and the ability to resist collagenase degradation. Collagen fibers with smaller diameters are prepared under acidic conditions, which can react with the cross-linking agent fully and evenly, thereby improving the degree of cross-linking and melting point. The ribose cross-linked collagen fiber prepared by the present invention has a cross-linking degree between 40.7% and 56.1%, preferably 48.6%-56.1%, which improves the ability to resist collagenase degradation; the melting point Tm value is 58.54°C-66.46°C, preferably Tm value is 59.59.59°C-66.46°C, has good thermal stability, and can correspond to a longer degradation cycle. The comprehensive display of these properties enables the ribose cross-linked collagen fibers prepared by the present invention to be used as a filler in combination with lidocaine in fields such as plastic surgery. In addition, the cross-linked collagen fiber filler prepared using natural ribose as a cross-linking agent has a lower thrust, which greatly improves the biocompatibility and use effect of the filler.

Claims (10)

1. A ribose-crosslinked collagen fiber, which is formed by crosslinking collagen fibers using ribose as a crosslinking agent, characterized in that the diameter of the collagen fibers is between 15-80nm; the diameter of the ribose-crosslinked collagen fibers is between 150-200nm; the crosslinking degree of the ribose-crosslinked collagen fibers is between 40.7%-56.1%; and the Tm value is 58.54℃-66.46℃. 2. The ribose cross-linked collagen fiber according to claim 1, characterized in that the cross-linking degree of the ribose cross-linked collagen fiber is between 48.6% and 56.1%; and the Tm value is 59.59°C to 66.46°C. 3. The ribose cross-linked collagen fiber according to claim 2, characterized in that the cross-linking degree of the ribose cross-linked collagen fiber is 56.1% and the Tm value is 66.46°C. 4. A method for preparing the ribose-crosslinked collagen fiber according to any one of claims 1 to 3, characterized in that it comprises: pre-treating, extracting and purifying pig skin tissue, and subjecting the obtained purified collagen molecules free of immunogenicity and viruses to fibrosis under acidic conditions, followed by ribose cross-linking. 5. The method according to claim 4, characterized in that the acidic condition has a pH value of 5.0-6.0. 6. The method according to claim 4, characterized in that the mass concentration of ribose is 1%-5%. 7. The method according to claim 4, characterized in that the acidic condition is a phosphate buffer, the collagen molecule is an animal-derived collagen molecule, and the ribose is D-ribose. 8. A cross-linked collagen fiber filler, characterized in that the filler comprises the ribose cross-linked collagen fiber according to any one of claims 1 to 3. 9. The cross-linked collagen fiber filler according to claim 8, characterized in that the filler further contains lidocaine. 10. A method for preparing the cross-linked collagen filler according to claim 9, characterized in that the method comprises: using physiological saline to adjust the protein concentration to 35 mg/mL and the lidocaine concentration to 0.2%, and after homogenization, aseptically filling into a 1 mL syringe and sealing.