Collagen implant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical beauty collagen implants, and particularly relates to a collagen implant as well as a preparation method and application thereof, and the preparation method comprises the following steps: S1, dissolving a collagen dry product in a hydrochloric acid solution to obtain a collagen solution; s2, adding a NaOH solution into the collagen solution, adjusting the pH value, and adding a NaCl solution to obtain a collagen suspension; s3, performing centrifugal treatment on the collagen suspension to obtain collagen; s4, directly pouring the collagen white egg to obtain a collagen implant; according to the preparation method of the collagen implant, NaOH and NaCl are used for adjusting acid-soluble collagen to be neutral for precipitation, collagen fibers are self-assembled and centrifuged to obtain a sample, washing is not needed, operation steps are reduced, the working efficiency is improved, the prepared sample is uniform, fine and smooth and small and uniform in pushing force, meanwhile, the loss of the collagen caused by washing is reduced, and the collagen implant is suitable for popularization and application. The cost is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical and cosmetic collagen implants, and particularly relates to a collagen implant and a preparation method and application thereof. BACKGROUND

[0003] Collagen is one of the most abundant structural proteins in the extracellular matrix of mammals, and is widely distributed in the connective tissue of vertebrates, including skin, bone, tendon, ligament, etc. Among them, type I collagen is the most abundant, which ensures the strength and elasticity of the skin. However, with age, the synthesis of type I collagen decreases, resulting in a decrease in skin tightness and the appearance of wrinkles. Type I collagen is mainly located in the basement membrane zone, supporting the filtration and scaffold functions of the skin. The complex balance between collagen synthesis and degradation is crucial for maintaining skin health and a youthful appearance. Due to factors such as age and ultraviolet damage, collagen fibers are damaged, and fragmented collagen fibers accumulate in the extracellular matrix. The ability of fibroblasts to synthesize collagen gradually decreases, leading to destruction and collapse of the extracellular matrix, loss of skin tissue integrity, and appearance of rough texture, wrinkles, spots, and atrophy, etc. Skin aging phenomena. Collagen has good biocompatibility, biodegradability, good mechanical properties, and other obvious advantages as a facial soft tissue filler material, and is widely used in facial wrinkle filling.

[0004] The existing collagen preparation process mostly uses high-concentration salt to precipitate collagen, and the collagen fibers need to be washed multiple times to remove the excess salt in the collagen fibers (CN113768815A uses Na2HPO4 to precipitate collagen fibers, and then uses sterilized physiological saline to wash the collagen, and CN119593080A uses physiological saline to wash the precipitated collagen fibers to remove excess polyols), and then uses PBS to disperse the collagen fibers. This makes the production process complicated, low in efficiency, increases the loss of collagen, and is high in cost.

[0005] Therefore, how to overcome the low yield of collagen precipitated by high-concentration salt is a technical problem that needs to be solved in the field.

[0006] It should be noted that the above information disclosed in the background section is only used to understand the background of the present application, and therefore, the above description is not considered to constitute prior art information. SUMMARY

[0007] The present disclosure at least provides a collagen implant and a preparation method and application thereof.

[0008] In a first aspect, the embodiments of the present disclosure provide a preparation method of collagen implant, comprising the following steps: S1, dissolving collagen dry product in hydrochloric acid solution to obtain a collagen solution; S2, adding NaOH solution to the collagen solution to adjust pH, and adding NaCl solution to obtain a collagen suspension; S3, centrifuging the collagen suspension to obtain collagen; and S4, directly perfusing the collagen to obtain the collagen implant.

[0009] In an alternative embodiment, the pH of the collagen solution is not more than 2.

[0010] In an alternative embodiment, the concentration of the NaOH solution is 0.1-1.0 M.

[0011] In an alternative embodiment, the concentration of the NaCl solution is not more than 30 mg / mL.

[0012] In an alternative embodiment, the pH of the collagen solution after adding the NaOH solution ranges from 6.0 to 8.0.

[0013] In an alternative embodiment, the final concentration of NaCl in the collagen solution after adding the NaCl solution is not more than 2.4 mg / mL.

[0014] In an alternative embodiment, the centrifugation of the collagen suspension comprises: firstly, low-speed stirring at a speed not higher than 200 rpm for not more than 30 min, and then fully standing at 2-8℃, and then centrifugation at a speed not lower than 1000 rpm for not more than 10 min.

[0015] In an alternative embodiment, the recovery rate of collagen in the preparation method is not less than 92%.

[0016] In a second aspect, the embodiments of the present disclosure also provide a collagen implant prepared by the method as described above, wherein the concentration of collagen in the collagen implant is not less than 29 mg / mL.

[0017] In a third aspect, the embodiments of the present disclosure also provide the use of the collagen implant as described above in the field of medical and beauty injection.

[0018] The collagen implant preparation method of the present application uses NaOH and NaCl to adjust the acid-dissolved collagen to neutral to precipitate, and the collagen fibers self-assemble, and the sample is obtained by centrifugation, and the isoelectric point characteristics of collagen are used, the collagen is dissolved under acidic conditions, when the pH of the solution is adjusted to the isoelectric point, the net charge of the collagen molecules is zero, the intermolecular repulsion is reduced, and the solubility is reduced to the minimum, so that a large amount of precipitation is obtained from the solution, and the salting-out effect of the neutral salt such as NaCl can further promote the precipitation and improve the recovery rate; the method does not need to be washed, reduces the operation steps, improves the work efficiency, and the prepared sample is uniform and delicate, the pushing force is small and uniform, and the loss of collagen caused by washing is reduced, the recovery rate is improved, and the cost is reduced.

[0019] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. The objects and other advantages of the present application will be realized and achieved by the structure particularly pointed out in the description and the drawings.

[0020] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the following preferred embodiments are specifically described, and the accompanying drawings are described in detail as follows. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0022] Figure 1 The appearance diagram of the collagen after precipitation provided by the embodiments of the present disclosure is shown in the following figure: Figure 2 The appearance diagram of the collagen after drawing provided by the embodiments of the present disclosure is shown in the following figure: Figure 3 The fiber diagram of the collagen provided by the embodiments of the present disclosure is shown in the following figure: Figure 4 The SDS-PAGE analysis diagram provided by the embodiments of the present disclosure is shown in the following figure: Figure 5 The DSC spectrum provided by the embodiments of the present disclosure is shown in the following figure. DETAILED DESCRIPTION

[0023] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the present application will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application.

[0024] As used herein, the phrases "in one embodiment", "according to one embodiment", "in some embodiments", and the like are generally intended to refer to the fact that a particular feature, structure, or characteristic described in connection with a given embodiment can be included in at least one embodiment of the disclosure. Thus, appearances of the phrases "in one embodiment", "according to one embodiment", "in some embodiments", and the like are not necessarily all referring to the same embodiment. As used herein, the terms "example", "exemplary", and the like are utilized to merely indicate that an example or an exemplary embodiment is described, but not necessarily a preferred or advantageous embodiment. Rather, the use of terms such as "example", "exemplary", and the like are intended to present concepts in a concrete manner.

[0025] In this document, example embodiments of the disclosure will be described in greater detail with reference to the accompanying drawings. As used herein, expressions such as "at least one of", when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list. For example, the expression "at least one of a, b, and c" should be understood as including only a, only b, only c, both a and b, both a and c, both b and c, or all of a, b, and c.

[0026] The terminology used herein is for the purpose of describing particular example configurations only and is not intended to be limiting. As used herein, the singular articles "a", "an", and "the" can be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "including", and "has" are inclusive and therefore specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order in which they are discussed or illustrated, unless specifically identified as an order of performance. Additional or alternative steps can be employed.

[0027] It is to be noted that like-numbers and letters on the figures identify corresponding items, and thus, once an item is defined in one figure, it is not necessarily redefined in a subsequent figure.

[0028] Some embodiments of the present application will be described in detail with reference to the drawings. The following embodiments and features of the embodiments described below can be combined with each other in the case of no conflict.

[0029] The present application provides a preparation method of collagen implant, comprising the following steps: S1, dissolving collagen dry product in hydrochloric acid solution to obtain a collagen solution; S2, adding NaOH solution to the collagen solution to adjust pH, and adding NaCl solution to obtain a collagen suspension; S3, centrifuging the collagen suspension to obtain collagen; S4, directly perfusing the collagen to obtain the collagen implant.

[0030] In some embodiments, in particular, the pH value of the collagen solution is not more than 2.

[0031] In some embodiments, in particular, the concentration of the NaOH solution is 0.1-1.0 M.

[0032] In some embodiments, in particular, the concentration of the NaCl solution is not more than 30 mg / mL.

[0033] In some embodiments, in particular, the pH value of the collagen solution after adding the NaOH solution ranges from 6.0 to 8.0.

[0034] In some embodiments, in particular, the final concentration of NaCl in the collagen solution after adding the NaCl solution is not more than 2.4 mg / mL.

[0035] In some embodiments, in particular, the centrifugation of the collagen suspension comprises: First, low-speed stirring at a speed not higher than 200 rpm for not more than 30 min, fully standing at 2-8℃, and then centrifugation at a speed not lower than 1000 rpm for not more than 10 min.

[0036] In some embodiments, in particular, the recovery rate of collagen in the preparation method is not less than 92%.

[0037] The present application also provides a collagen implant prepared by the method as described above, wherein the concentration of collagen in the collagen implant is not less than 29 mg / mL.

[0038] The present application also provides the use of the collagen implant as described above in the field of medical and cosmetic injection.

[0039] Embodiment 1: Take 1.0 g of dry collagen protein and dissolve it in 500 mL of 10 mmol / L hydrochloric acid solution. After complete dissolution, slowly add NaOH solution (0.5M-1.0M) to the acidic collagen protein solution with a collagen concentration of 2 mg / mL and a pH of 2. Adjust the solution pH to 6.0-8.0, slowly add 30 mg / mL NaCl solution to adjust the final concentration of NaCl to 0.3 mg / mL, stir at 200 rpm for 30 min, control the temperature at 2-8°C, stand for 24 h, centrifuge at 10000 rpm for 10 min to collect collagen.

[0040] Example 2: Take 1.0 g of dry collagen protein and dissolve it in 500 mL of 10 mmol / L hydrochloric acid solution. After complete dissolution, slowly add NaOH solution (0.5M-1.0M) to the acidic collagen protein solution with a collagen concentration of 2 mg / mL and a pH of 2. Adjust the solution pH to 6.0-8.0, slowly add 30 mg / mL NaCl solution to adjust the final concentration of NaCl to 0.3 mg / mL, stir at 200 rpm for 30 min, control the temperature at 2-8°C, stand for 24 h, centrifuge at 10000 rpm for 10 min to collect collagen.

[0041] Example 3: Take 1.0 g of dry collagen protein and dissolve it in 500 mL of 10 mmol / L hydrochloric acid solution. After complete dissolution, slowly add NaOH solution (0.5M-1.0M) to the acidic collagen protein solution with a collagen concentration of 2 mg / mL and a pH of 2. Adjust the solution pH to 6.0-8.0, slowly add 30 mg / mL NaCl solution to adjust the final concentration of NaCl to 0.3 mg / mL, stir at 200 rpm for 30 min, control the temperature at 2-8°C, stand for 24 h, centrifuge at 10000 rpm for 10 min to collect collagen.

[0042] Example 4: Take 1.0 g of dry collagen protein and dissolve it in 500 mL of 10 mmol / L hydrochloric acid solution. After complete dissolution, slowly add NaOH solution (0.5M-1.0M) to the acidic collagen protein solution with a collagen concentration of 2 mg / mL and a pH of 2. Adjust the solution pH to 6.0-8.0, slowly add 30 mg / mL NaCl solution to adjust the final concentration of NaCl to 0.3 mg / mL, stir at 200 rpm for 30 min, control the temperature at 2-8°C, stand for 24 h, centrifuge at 10000 rpm for 10 min to collect collagen.

[0043] Comparative Example 1: Dissolve 1.0 g of dry collagen in 500 mL of 10 mmol / L hydrochloric acid solution, after complete dissolution, slowly add 0.2M Na2HPO4 solution to the collagen solution with a concentration of 2 mg / mL and pH 2, adjust the pH of the solution to 6.0-8.0, the stirring speed is 200 rpm, stirring for 30 min, control the temperature at 2-8℃, stand for 24 h, centrifugal collection of collagen at 10000 rpm for 10 min, and wash the collagen precipitate with normal saline for 3 times.

[0044] 1. Material properties Please refer to Figure 1 , as Figure 1 The appearance of the collagen after precipitation is shown in the figure, examples 1-4 are in the NaCl concentration below 2.4 mg / mL, from the figure, it can be seen that the collagen presents fine fibrous, after standing, it is obviously layered, and after centrifugation, the collagen presents milky white, more viscous. The comparative example is washed with normal saline for 3 times, and after centrifugation, part of it presents a relatively sparse and transparent state.

[0045] Please refer to Figure 2 , the state of each group of materials after being pushed out by a 30G syringe needle, as Figure 2 shown, the collagen of examples 1-4 presents continuous drawing state, showing better mechanical properties and plasticity, and the mechanical properties and plasticity of comparative example 1 are worse than those of examples.

[0046] 2. Performance measurement Specifically, the performance parameters of examples 1-4 and comparative example 1 are shown in table 1.

[0047] Table 1 Collagen concentration Kinetic viscosity Appearance Pushing force Recovery rate Example 1 29.85 ± 0.17 mg / mL 251800 ± 215 mpas Slightly white gel 11.4±0.5 94.1%±2.1% Example 2 37.46 ± 0.21 mg / mL 304300 ± 326 mpas Slightly white gel 10.9±0.4 95.2%±1.3% Example 3 47.31 ± 0.24 mg / mL 368500 ± 334 mpas Creamy white paste 12.1±0.6 95.8%±2.1% Example 4 29.41 ± 0.19 mg / mL 270100 ± 127 mpas Creamy white gel 12.3±0.3 94.1%±1.5% Comparative Example 1 14.07 ± 0.18 mg / mL 84600 ± 121 mpas Slightly white gel 17.5±1.7 83.3%±1.1% Please refer to Figure 3 , from Figure 3 and table 1, it can be seen that the collagen of example 3 presents obvious fibrous, and the concentration can reach 50 mg / mL, through self-assembly, a large number of collagen molecules are aggregated to generate collagen fibers, which can provide better support for in vivo filling, at the same time, the method can directly obtain collagen for filling injection in one step, without washing, and the collagen recovery rate reaches more than 95%. Due to washing for 3 times, the collagen loss of comparative example 1 increases, and the recovery rate is reduced by about 10% compared with examples. It is found through 30G needle measurement that the pushing force of example sample is below 12N, the sample is uniform and delicate, and it can provide better injection experience for filling injection.

[0048] Specifically, as CN113768815A adopts high concentration NaH2PO4 to adjust precipitation, the reaction system will be expanded by more than 1 times due to large amount of NaH2PO4, and at the same time, PBS three times washing will cause at least 10% collagen loss, and in the present scheme, the part of loss can be effectively avoided due to no washing; at the same time, the collagen fiber prepared by the collagen prepared by the present scheme has larger pushing force, and the sample is finally filled in a syringe, and the user needs to use how much force to push the syringe when injecting the sample, the force is quantified by the pushing force, the larger the pushing force, the larger the force required, if a sudden increasing peak appears on the pushing force diagram, it means that small particles in the sample block the needle, and the pressure needs to be increased to push it, which is converted into the user to push with more force, if the pushing force is greater than 20N, the user may not be able to push the syringe, or cause uneven injection.

[0049] 3. Natural activity Please refer to Figure 4 and Figure 5 As shown in Figure 4 and Figure 5 , it is found by SDS-PAGE detection that the collagen protein molecular band of the example keeps consistent with the control, and no degradation occurs, the method can keep the natural structure of the collagen protein from being destroyed, and maintain its biological activity, and the melting point detection finds that the sample of the example has no obvious difference with the control, which further proves that the spatial structure is not affected, and the biological activity is maintained.

[0050] In summary, the preparation method of the collagen implant uses NaOH and NaCl to adjust the acid-soluble collagen to neutral for precipitation, and the collagen fibers are self-assembled, and the sample is obtained by centrifugation, the isoelectric point characteristics of collagen protein are used, the collagen protein is dissolved under acidic conditions, when the pH of the solution is adjusted to the isoelectric point, the net charge of the collagen protein molecule is zero, the intermolecular repulsion is reduced, and the solubility is reduced to the minimum, so that a large amount of precipitation is obtained from the solution, and the salting-out effect of neutral salt such as NaCl can further promote precipitation and improve recovery rate; the present method does not need washing, reduces the operation steps, improves the work efficiency, and the prepared sample is uniform and delicate, the pushing force is small and uniform, at the same time, the collagen loss caused by washing is reduced, the recovery rate is improved, and the cost is reduced.

[0051] Based on the above ideal embodiments according to the present application, through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the contents in the specification, and the technical scope must be determined according to the scope of claims.

Claims

1. A method for preparing a collagen implant, characterized in that, Includes the following steps: S1, Dissolve dried collagen in hydrochloric acid solution to obtain collagen solution; S2, add NaOH solution to collagen solution to adjust pH, add NaCl solution to obtain collagen suspension; S3, centrifuge the collagen suspension to obtain collagen; S4, collagen white protein is directly injected to obtain collagen implant.

2. The preparation method according to claim 1, characterized in that, The pH value of the collagen solution is not greater than 2.

3. The preparation method according to claim 1, characterized in that, The concentration of the NaOH solution is 0.1–1.0 M.

4. The preparation method according to claim 1, characterized in that, The concentration of the NaCl solution is no greater than 30 mg / mL.

5. The preparation method according to claim 1, characterized in that, The collagen solution has a pH range of 6.0 to 8.0 after the addition of NaOH solution.

6. The preparation method according to claim 1, characterized in that, After adding NaCl solution to the collagen solution, the final concentration of NaCl is no greater than 2.4 mg / mL.

7. The preparation method according to claim 1, characterized in that, The centrifugation treatment of the collagen suspension includes: First, stir at a low speed of no more than 200 rpm for no more than 30 minutes, let it stand at 2-8℃ for a while, and then centrifuge at a speed of no less than 1000 rpm for no more than 10 minutes.

8. The preparation method according to claim 1, characterized in that, The collagen recovery rate in the preparation method is no less than 92%.

9. A collagen implant prepared by the method according to any one of claims 1-8, characterized in that, The collagen concentration in the collagen implant is not less than 29 mg / ml.

10. The application of the collagen implant as described in claim 9 in the field of cosmetic injection.

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