Collagen implant and preparation method therefor

Abstract

The present invention relates to the technical field of medical instruments, and particularly disclosed are a collagen implant and a preparation method therefor. After special cross-linking treatment (the cross-linking degree can reach up to about 90%), the degradation rate of collagen can be regulated and controlled, so that the in-vivo retention time of collagen can be greatly prolonged. After the above collagen implant is injected, no significant adverse reaction occurs at the injection area, and HE staining of tissue sections showed no significant inflammatory reaction. Six months after the implantation, the implant is still not completely degraded, showing good resistance to biodegradation. Therefore, the implant exhibits good biocompatibility and can be maintained in vivo for at least six months after implantation, and thus has good application value.

Description

Collagen implant and preparation method thereof Technical Field

[0001] The present application relates to the field of medical device technology, and in particular to a collagen implant and a preparation method thereof. Background Art

[0002] Collagen, a natural protein with excellent biocompatibility, has been widely used as a soft tissue filler in medical plastic surgery and has established relevant industry standards and technical review points. Collagen sources include collagen extracted from animal tissues and recombinant humanized collagen. Extracted collagen is primarily obtained from bovine and porcine skin and Achilles tendon tissues.

[0003] Collagen implants are made by suspending collagen in a buffer, sterilizing the product, filling it into a syringe, and injecting it through a needle. Currently, the main methods for sterilizing the product include collagen filtration sterilization followed by aseptic production, and terminal irradiation sterilization. Filtration sterilization requires concentration, cross-linking, crushing, and filling under aseptic process conditions after filtration sterilization, resulting in a long cycle, high costs, and low production capacity. Irradiation sterilization can cross-link the aqueous collagen, altering its physical properties and potentially clogging the injection needle.

[0004] Furthermore, most collagen implants currently available in the art contain uncross-linked or low-cross-linked collagen. However, uncross-linked or low-cross-linked collagen degrades quickly after subcutaneous injection, has a short retention time, and requires multiple uses. Therefore, there is still room for improvement. Technical issues

[0005] The purpose of this application is to overcome the shortcomings of the above-mentioned existing technologies and provide a collagen implant and a preparation method thereof. The collagen in this application, after undergoing a special cross-linking treatment (the cross-linking degree can reach up to about 90%), can regulate the degradation rate and significantly increase its retention time in the body. Technical Solutions

[0006] To achieve the above objectives, the technical solutions adopted in this application are:

[0007] The present application provides a method for preparing a collagen implant, comprising the following steps:

[0008] S1. Take animal tissue slices, soak them in an acid solution to swell them, grind them into a paste, add protease to obtain an enzymatic hydrolysis product, and then use a salt solution to salt out the enzymatic hydrolysis product to obtain collagen precipitate;

[0009] S2. At a pH of 7-8, reacting the collagen precipitate obtained in step S1 with an active agent, and then adding a strong hydrogen bonding reagent to react to obtain an antigen-removed collagen gel;

[0010] S3, mixing the collagen gel obtained in step S2 with a cross-linking agent and cross-linking the mixture at a pH value of 5 to 10 to obtain a cross-linked product;

[0011] S4, washing the cross-linked product of step S3, then homogenizing under high pressure, and sterilizing by irradiation to obtain a collagen implant;

[0012] The cross-linking agent includes at least one of epoxide, diamide, diisocyanate, polyethylene glycol and carbodiimide.

[0013] After extensive research and testing, the inventors of this application have discovered that the collagen in the collagen implant of this application has been cross-linked, with a cross-linking degree of up to approximately 90%, exhibiting strong resistance to degradation and remaining subcutaneously for more than six months. Furthermore, collagen has the ability to induce autologous tissue regeneration, with new tissue filling wrinkles and depressions.

[0014] The collagen implants involved in this application are mostly highly cross-linked products, which swell and dissolve less in acidic or alkaline environments. There is no need to adjust the pH to its isoelectric point (pH 7-8) during cross-linking agent treatment, and there is no effect on subsequent centrifugation and precipitation.

[0015] After high-pressure homogenization, the collagen implant of the present application can have a particle size of 0.1 μm to 100 μm. After irradiation sterilization, the prepared collagen implant can smoothly pass through 27G, 30G and other needles.

[0016] As a preferred embodiment of the method for preparing the collagen implant described in the present application, when the cross-linking agent is epoxide, the pH value of the cross-linking in step S3 is 9-10; when the cross-linking agent is carbodiimide, the pH value of the cross-linking in step S3 is 5-6.

[0017] In the technical solution of the present application, the sample is treated with an epoxide cross-linking agent, and the pH value is 9~10, which is conducive to the preparation of highly cross-linked samples, and the obtained highly cross-linked samples have a higher and more stable degree of cross-linking. Experiments have found that when other treatment conditions are the same, the pH of the epoxide cross-linking agent affects the degree of cross-linking. When the pH is around 7~8 (neutral environment), the sample treated with a low concentration of epoxide cross-linking agent has a low degree of cross-linking, less than 20%, and the degree of cross-linking is unstable, which is not suitable for the preparation of highly cross-linked samples, such as Comparative Example 1.

[0018] When treating samples with carbodiimide crosslinker, there is no need to adjust the pH value of the crosslinker, which is 5~6. The pH value of the crosslinker after preparation does not need to be adjusted to neutral, and highly cross-linked samples can be prepared. At the same time, experiments have found that changing the crosslinker frequently can greatly increase the crosslinking rate. A 2% concentration of carbodiimide can reach a crosslinking degree of 80-90% after three days of treatment, greatly shortening the production cycle.

[0019] Principle of carbodiimide crosslinker: Carbodiimide (EDC) couples with the carboxyl groups of glutamic acid and aspartic acid in collagen under acidic conditions to form an intermediate product with an O-isoacylurea structure. Under the attack of collagen amino groups, this intermediate product can form amide bonds within the collagen molecule and between adjacent collagen molecules, thereby completing intramolecular and intermolecular amide crosslinking.

[0020] Epoxide crosslinker principle: Epoxides undergo a condensation reaction with the amino groups in collagen molecules. Under alkaline conditions, the epoxy groups preferentially react with the amino groups in collagen molecules (collagen molecules contain both acidic carboxyl groups and basic amino groups).

[0021] Under alkaline conditions, preferably, the pH value of the cross-linking of the present application is 9-10, which can prepare a stable highly cross-linked sample.

[0022] As a preferred embodiment of the method for preparing the collagen implant described in the present application, the concentration of the cross-linking agent is 0.1-5.0%, preferably 1-4%.

[0023] When the crosslinking agent is used at the above preferred concentration, the prepared collagen implant has a high degree of crosslinking. When the epoxide crosslinking agent is used at a low concentration (<1%), the sample obtained has a low degree of crosslinking, less than 20%, and the degree of crosslinking is unstable.

[0024] As a preferred embodiment of the method for preparing the collagen implant described in the present application, in step S3, the cross-linking time is 1 to 12 days, preferably 3 to 12 days.

[0025] Low-cross-linked (cross-linked for 1 day) collagen implants can only last for 1-3 months after subcutaneous injection, which is a short time.

[0026] This study investigated the effect of crosslinking time on the degree of crosslinking using an epoxide crosslinker. For example, in Example 1, crosslinking was performed for 3, 6, 9, and 12 days, with a crosslinking degree of 80-90% achieved at 12 days. Carbodiimide crosslinkers, on the other hand, were more efficient. By optimizing experimental conditions, a 2% carbodiimide concentration was found to achieve a crosslinking degree of 80-90% in just 3 days, significantly shortening the production cycle.

[0027] As a preferred embodiment of the method for preparing the collagen implant described in the present application, in step S4, the conditions of the high-pressure homogenization are controlled as follows: temperature 2-10° C., pressure 1-100 MPa, and homogenization 1-8 times.

[0028] As a preferred embodiment of the method for preparing the collagen implant described in the present application, the irradiation sterilization is sterilization using cobalt 60, and the irradiation dose is 15 to 40 kGy.

[0029] Currently, implant sterilization typically involves filtration sterilization followed by aseptic production. Filter sterilization takes a long time and requires subsequent aseptic production, which is costly. Irradiation sterilization is also used, but further processing in a sterile environment after irradiation carries the risk of microbial contamination and is also expensive.

[0030] The sterilization method used in this application is irradiation sterilization of the final product. Prior to sterilization, the collagen particles are processed to a size of 10 microns or even smaller through a high-pressure homogenization process. Even though the physical properties of the collagen implant may change after irradiation sterilization (cross-linking between molecules occurs, forming a gel state, resulting in excessive pushing force and needle blockage, etc.), the product still has a relatively suitable pushing force and can be smoothly pushed out through a 27G needle.

[0031] As a preferred embodiment of the method for preparing the collagen implant described in the present application, in step S1, the acid solution is acetic acid or hydrochloric acid.

[0032] As a preferred embodiment of the method for preparing the collagen implant described in the present application, in step S1, the protease includes at least one of pepsin, trypsin, and papain; and the saline solution is a 2-4 mol / L sodium chloride solution.

[0033] As a preferred embodiment of the method for preparing the collagen implant described in the present application, the active agent is at least one of organic acid anhydrides, acid chlorides, amides, epoxides and methyl halides.

[0034] Preferably, in step S1, the animal tissue is derived from skin, Achilles tendon and other tissues of animals such as pigs and cows.

[0035] Preferably, in step S4, ethanol or purified water is used for cleaning, and the ethanol is anhydrous ethanol or 75% ethanol.

[0036] The present application also provides a collagen implant prepared by the preparation method of the above collagen implant. Beneficial effects

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] This application provides a collagen implant and its preparation method. After undergoing a special cross-linking treatment (with a cross-linking degree of up to approximately 90%), the collagen in this application has a controlled degradation rate, significantly increasing its in vivo retention time. The collagen implant in this application has shown no significant adverse reactions in the injection area, and no significant inflammatory response was observed after anatomical HE staining. Six months after implantation, the implant remained incompletely degraded, demonstrating good biodegradability. Therefore, this implant exhibits excellent biocompatibility and maintains its in vivo survival for at least six months after implantation, demonstrating its promising application value. Best Mode for Carrying Out the Invention

[0039] This embodiment provides a method for preparing a collagen implant, comprising the following steps:

[0040] S1. Animal tissues such as skin and Achilles tendon from pigs, cattle, etc. are sliced, soaked in a dilute acetic acid solution to swell, and then ground into a paste, adjusted to a pH of about 3, and enzymatically hydrolyzed with 0.3% pepsin by mass for 24 hours to obtain an enzymatic hydrolyzate. Sodium chloride is then added to a concentration of 2 mol / L, and the enzymatic hydrolyzate is salted out and washed multiple times to obtain a collagen precipitate.

[0041] S2. The collagen precipitate obtained in step S1 was reacted with methyl halide at a pH of 7 to 8 for 40 hours, and then a strong hydrogen bonding reagent, a guanidine compound, was added and reacted for 30 hours at a pH of 8.0 to obtain an antigen-removed collagen gel;

[0042] S3, mixing the collagen gel obtained in step S2 with a cross-linking agent, cross-linking the mixture at a pH of 9 to 10, to obtain a cross-linked product; in Example 1, the cross-linking agent is an epoxide (polypropylene oxide solution) at a concentration of 1%;

[0043] S4. The cross-linked product from step S3 is washed with ethanol or pure water, and then subjected to high-pressure homogenization (temperature controlled at approximately 4°C, 5 MPa, twice). The homogenized collagen particles are evenly dispersed in phosphate buffered saline, pre-filled into syringes, and sterilized by irradiation (Cobalt 60, irradiation dose of 25 kGy) to obtain a collagen implant.

[0044] After treatment with a 1% epoxide (polypropylene glycol solution), the cross-linking status of the collagen implants was examined after cross-linking for 1, 3, 6, 9, and 12 days (hereinafter referred to as the degree of cross-linking %), as shown in Table 1.

[0045] Table 1

[0046] Example 1 Cross-linking for 1 day Cross-linking for 3 days Cross-linking for 6 days Cross-linking for 9 days Cross-linking for 12 days Cross-linking degree% 17% 41% 64.01% 76.82% 83.53% Modes for Carrying Out the Invention

[0047] Similar to Example 1, except that the crosslinking agent is 2% epoxide (polypropylene oxide solution). The crosslinking conditions of the collagen implants crosslinked for 3 and 9 days were tested, as shown in Table 2.

[0048] Table 2

[0049] Example 2 Crosslinking for 3 days Crosslinking for 9 days Crosslinking degree % 57% 84% Industrial Applicability

[0050] This application provides a collagen implant and its preparation method. After undergoing a special cross-linking treatment (with a cross-linking degree of up to approximately 90%), the collagen in this application has a controlled degradation rate, significantly increasing its in vivo retention time. The collagen implant in this application has shown no significant adverse reactions in the injection area, and no significant inflammatory response was observed after anatomical HE staining. Six months after implantation, the implant remained incompletely degraded, demonstrating good biodegradability. Therefore, this implant exhibits excellent biocompatibility and maintains its in vivo survival for at least six months after implantation, demonstrating its promising application value.

Claims

1. A preparation method of a collagen implant, characterized in that, It includes the following steps: S1. Take an animal tissue section, soak it in an acid solution to swell, crush it into a paste, then add a protease to obtain an enzymolysis product, and then use a salt solution to salting-out the enzymolysis product to obtain a collagen precipitate; S2. Under the condition of pH 7-8, react the collagen precipitate obtained in step S1 with an active agent, and then add a strong hydrogen bond reagent to react to obtain an antigen-removed collagen gel; S3. Mix and crosslink the collagen gel obtained in step S2 with a crosslinking agent, and the pH value of crosslinking is 5-10 to obtain a crosslinked product; S4. Wash the product crosslinked in step S3, then perform high-pressure homogenization and irradiation sterilization to obtain a collagen implant; The crosslinking agent includes at least one of epoxide, diacyl diamine, diisocyanate, polyethylene glycol and carbodiimide.

2. The preparation method of the collagen implant as described in claim 1, wherein, When the crosslinking agent is epoxide, the pH value of crosslinking in step S3 is 9-10; when the crosslinking agent is carbodiimide, the pH value of crosslinking in step S3 is 5-6.

3. The preparation method of the collagen implant as described in claim 1, characterized in that, The concentration of the crosslinking agent is 0.1-5.0%.

4. The preparation method of the collagen implant as claimed in claim 1, wherein In step S3, the crosslinking time is 1-12 days, preferably 3-12 days.

5. The preparation method of the collagen implant according to claim 1, wherein, In step S4, the conditions of high-pressure homogenization are controlled as follows: temperature 2-10°C, pressure 1-100 MPa, and homogenization 1-8 times.

6. The preparation method of the collagen implant as described in claim 1, wherein, The irradiation sterilization is carried out using cobalt 60 sterilization, and the irradiation dose is 15-40 kGy.

7. The preparation method of the collagen implant according to claim 1, characterized in that, In step S1, the acid solution is acetic acid or hydrochloric acid with a concentration of 1-10%.

8. The preparation method of the collagen implant as described in claim 1, characterized in that, In step S1, the protease includes at least one of pepsin, trypsin, and papain; the salt solution is a sodium chloride solution with a concentration of 2-4 mol / L.

9. The preparation method of the collagen implant as claimed in claim 1, wherein, The active agent is at least one of organic acid anhydride, acyl chloride, amide, epoxide, and halomethane.

10. A collagen implant prepared by the preparation method of the collagen implant according to any one of claims 1-9.