CN117899268A

CN117899268A - Collagen composition, preparation method and application thereof

Info

Publication number: CN117899268A
Application number: CN202410081888.XA
Authority: CN
Inventors: 孙冰冰; 唐冬慧; 张伟; 李晓静; 黄果; 田翔宇; 张娈
Original assignee: Beijing Tianxinfu Medical Appliance Co Ltd
Current assignee: Beijing Tianxinfu Medical Appliance Co Ltd
Priority date: 2023-06-05
Filing date: 2024-01-19
Publication date: 2024-04-19
Also published as: CN116444826A

Abstract

The present disclosure provides a collagen composition, a preparation method and an application thereof, wherein the injection comprises a first modified collagen with a crosslinking degree of 30% -50%, a second modified collagen with a crosslinking degree of more than 50% and less than 70%, a third modified collagen with a crosslinking degree of more than 70% and less than 80%, and pharmaceutically acceptable auxiliary materials. The collagen composition may be used for human tissue augmentation to reduce skin wrinkles and sagging at the injection site.

Description

Collagen composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of medical biological materials, in particular to a collagen composition, a preparation method and application thereof.

Background

The structure of soft tissue in the human body is maintained by proteins such as collagen elastin and the like and an extracellular matrix containing glycosaminoglycans, and various factors may cause soft tissue defects, which can be recovered or corrected, typically by surgical or non-surgical injection of biological tissue or synthetic compounds. At present, various types of fillers, particularly facial fillers, are commonly used in the cosmetic and cosmetic arts, such as autologous fat, hyaluronic acid, and the like. The autologous fat is nontoxic and harmless, does not generate immune reaction and rejection reaction, but can cause the phenomenon that the filling part is rugged and even hard-combined; whereas hyaluronic acid, hyaluronic acid and the like are liable to cause rejection reactions, and thus inflammatory reactions and scar formation occur.

Collagen is the most abundant and widely distributed functional protein in mammals, is a tissue filler and support, accounting for 25% -30% of the total protein in the body, and is increasingly used in cosmetic applications due to its good biocompatibility and biodegradability, such as filling soft tissues to remove wrinkles and improve injection site contours. The gel prepared directly from the natural collagen material has the defects of high degradation speed, poor mechanical property and the like, so that the application of the gel is limited. In the prior art, collagen is generally treated by chemical crosslinking to improve the mechanical properties and increase the degradation time.

Crosslinking collagen with glutaraldehyde can enhance the mechanical properties of collagen and prolong the degradation time of collagen, however glutaraldehyde treatment of collagen usually leaves high concentrations of glutaraldehyde, resulting in biotoxic and allergenic properties. Chinese patent document CN101648989a discloses a method for producing long-acting collagen, which reduces glutaraldehyde residue by crosslinking collagen with low concentration glutaraldehyde. Glutaraldehyde-crosslinked collagen, however, is often yellow in color and is subject to calcification after implantation into human tissue, resulting in undesirable hardening or degradation of the implant.

In addition, after chemical crosslinking, the viscoelasticity and mechanical properties of the collagen gel are improved, so that the industrial preparation difficulty is improved, the difficulty of injecting the collagen into tissues is also increased, and the clinical injection operation is not facilitated.

Accordingly, there is a need to provide a collagen composition for tissue augmentation.

Disclosure of Invention

The collagen composition comprises collagen with different crosslinking degrees, so that the collagen composition has good tissue filling effect, degradation period and operability, and the satisfaction degree of a tested patient is improved.

The collagen composition provided in the first aspect of the present disclosure includes:

(1) A first modified collagen having a degree of crosslinking of 30% to 50%,

(2) A second modified collagen having a degree of crosslinking of greater than 50% and less than 70%,

(3) A third modified collagen having a degree of crosslinking of more than 70% and 80% or less, and

(4) Pharmaceutically acceptable auxiliary materials;

wherein, based on the total mass of the collagen composition,

The mass ratio of the first modified collagen in the collagen composition is 0.1-2.0%;

The mass ratio of the second modified collagen in the collagen composition is 0.5-4.0%;

The mass ratio of the third modified collagen in the collagen composition is 0.1% -2.0%.

In any embodiment, the first modified collagen comprises 0.2% to 2.0% by mass of the collagen composition based on the total mass of the collagen composition; the mass ratio of the second modified collagen in the collagen composition is 0.6-3.6%; the weight percentage of the third modified collagen in the collagen composition is 0.25-1.75%.

In any embodiment, the collagen composition comprises a first modified collagen at a mass concentration of 2-20mg/mL in the collagen composition; the mass concentration of the second modified collagen in the collagen composition is 5-40mg/mL, and can be selected to be 6-36 mg/mL; and/or the mass concentration of the third modified collagen in the collagen composition is 2mg/mL to 25mg/mL, optionally 2.5mg/mL to 17.5mg/mL.

In any embodiment, the collagen composition comprises an anesthetic comprising lidocaine. In any embodiment, the anesthetic is present in the collagen composition in an amount of 0.1% to 0.5%, alternatively 0.2% to 0.4% by mass. In any embodiment, the anesthetic is present in the collagen composition at a mass concentration of 1mg/mL-5mg/mL, optionally 2mg/mL-4mg/mL. The anesthetic in the collagen composition helps to reduce pain in the subject when receiving tissue injections.

In any embodiment, the pharmaceutically acceptable auxiliary materials comprise water for injection and sodium chloride, and optionally, the collagen composition is an isotonic solution, so that the influence of the injection on cells in blood of an injection site is reduced, and adverse reactions caused by cell atrophy or rupture are reduced.

In any embodiment, the collagen composition has an average degree of crosslinking of 30% to 80%, optionally 50% to 70%, which helps to further improve the filling effect, degradation cycle and handleability of the collagen composition.

In any embodiment, the collagen composition has a mass concentration of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) of no more than 0.5 μg/g, alternatively, no more than 0.4 μg/g. In any embodiment, the mass concentration of N-hydroxysuccinimide (NHS) is no more than 3 μg/g, alternatively no more than 2.5 μg/g. Reducing the mass concentration of the residual cross-linking agent in the collagen composition helps to improve the quality of the collagen composition and improves the safety of the collagen composition.

In any embodiment, the thermal denaturation temperature (DSC) of the collagen composition is 50 ℃ to 75 ℃ and optionally 51.6 ℃ to 73.5 ℃, thereby further adjusting the molecular weight and crosslinking degree of the modified collagen in the collagen composition to provide the collagen composition with good degradation cycle and operability.

In any embodiment, the viscosity of the collagen composition is in the range of 800mpa.s to 11000mpa.s, optionally in the range of 800mpa.s to 10500mpa.s, and the fluidity of the collagen composition can be further improved, thereby improving the handleability and filling effect of the collagen composition.

In any embodiment, the pushing force of the collagen composition is 3N-10N, optionally 3N-8.5N, which can further improve the operability of the collagen composition and is beneficial to clinical use.

In any embodiment, the first modified collagen, the second modified collagen, and the third modified collagen are each independently selected from the group consisting of type I collagen, optionally modified type I collagen, having a triple helical structure that improves the filling and supporting effects of the collagen composition.

The second aspect of the present disclosure provides a method for preparing a modified collagen, which at least comprises the following two crosslinking steps:

(1) Physical crosslinking: providing a buffer solution containing type I collagen, regulating the pH value to 6.0-9.0, and performing a crosslinking reaction to obtain a physical crosslinking product;

(2) Chemical crosslinking: and (3) placing the physical crosslinking product prepared in the step (1) into a crosslinking liquid containing 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) for crosslinking reaction, so as to obtain the modified collagen with the crosslinking degree of 30-80%.

The method is prepared by using a crosslinking liquid comprising EDC and NHS, and avoids using glutaraldehyde crosslinking agents, so that the toxicity problem caused by glutaraldehyde residues is avoided, and the problem of collagen yellowing caused by glutaraldehyde serving as the crosslinking agent is also overcome.

In any embodiment, in step (1), the pH is adjusted to 7.0-7.5.

In any embodiment, in the chemical crosslinking reaction,

The mass concentration of the EDC in the crosslinking liquid is 20mg/mL-160mg/mL;

The mass concentration of NHS in the crosslinking liquid is 10mg/mL-120mg/mL;

the mass concentration ratio of EDC to NHS in the crosslinking liquid is 1.1-2.0; and/or

The pH value of the crosslinking liquid is 5.5-7.0;

the time of the chemical crosslinking reaction is 2-12 h;

The temperature of the chemical crosslinking reaction is 20-25 ℃.

In any embodiment, the method at least comprises a step A of preparing a first modified collagen with a crosslinking degree of 30% -50%, a step B of preparing a second modified collagen with a crosslinking degree of more than 50% and less than 70%, a step C of preparing a third modified collagen with a crosslinking degree of more than 70% and less than 80%, two or three steps,

In the chemical crosslinking reaction, the mass concentration of EDC in a chemical crosslinking reaction system is 20mg/mL-80mg/mL; the mass concentration of the NHS in the chemical crosslinking reaction system is 10mg/mL-70mg/mL; the time of the chemical crosslinking reaction is 2-4 h; the temperature of the chemical crosslinking reaction is 20-25 ℃; in the step B, in the chemical crosslinking reaction, the mass concentration of EDC in a chemical crosslinking reaction system is 60mg/mL-140mg/mL; the mass concentration of the NHS in the chemical crosslinking reaction system is 50mg/mL-100mg/mL; the time of the chemical crosslinking reaction is 6-8 hours; the temperature of the chemical crosslinking reaction is 20-25 ℃; in the chemical crosslinking reaction, the mass concentration of EDC in a chemical crosslinking reaction system is 110mg/mL-160mg/mL; the mass concentration of the NHS in the chemical crosslinking reaction system is 80mg/mL-120mg/mL; the time of the chemical crosslinking reaction is 10-12 h; the temperature of the chemical crosslinking reaction is 20-25 ℃. The method can prepare modified proteins with different crosslinking degrees.

In any embodiment, the type I collagen is present in the buffer at a mass concentration of 2mg/mL to 3mg/mL.

In any embodiment, the amount of the hetero-protein in the type I collagen is not more than 0.3%, alternatively not more than 0.2%, which contributes to the preparation of a modified collagen of good quality.

In any embodiment, the type I collagen is prepared by a method comprising the steps of: and (3) performing pretreatment, enzymolysis, crude extraction, purification and filtration sterilization treatment on the cow leather and/or the cow achilles tendon to obtain the type I collagen.

A third aspect of the present disclosure provides a method of preparing a collagen composition, comprising: and mixing the first modified collagen, the second modified collagen and the third modified collagen, and homogenizing to obtain the collagen composition. The preparation method has industrial feasibility and can realize industrialized production.

In any embodiment, preparing a first modified collagen into a first protein solution, wherein the first protein solution comprises the first modified collagen with the mass concentration of 20mg/mL-50mg/mL and an anesthetic with the mass concentration of 1mg/mL-5 mg/mL; preparing second modified collagen into a second protein liquid, wherein the second protein liquid comprises second modified collagen with the mass concentration of 20mg/mL-50mg/mL and anesthetic with the mass concentration of 1mg/mL-5 mg/mL; preparing a third modified collagen into a third protein liquid, wherein the third protein liquid comprises the third modified collagen with the mass concentration of 20mg/mL-50mg/mL and an anesthetic with the mass concentration of 1mg/mL-5 mg/mL; mixing the first protein liquid, the second protein liquid and the third protein liquid to obtain total liquid, wherein the mass ratio of the first protein liquid in the total liquid is 10% -40%; the mass ratio of the second protein liquid in the total liquid is 30% -80%; the mass ratio of the third protein liquid in the total liquid is 10% -50%. The modified collagen with different crosslinking degrees is prepared into a solution and then mixed, so that the mixing difficulty of the modified collagen with different crosslinking degrees can be reduced; meanwhile, in the prepared collagen composition, collagen with different crosslinking degrees has different degradation time, supporting effect and fluidity, and is beneficial to obtaining the collagen composition which can take the degradation period, filling effect and operability into consideration.

In any embodiment, the mass concentration of the first modified collagen in the first protein solution is 35mg/ml±3.5mg/mL; the mass concentration of the second modified collagen in the second protein solution is 35 mg/mL+/-3.5 mg/mL; and/or the mass concentration of the third modified collagen in the third protein solution is 35 mg/mL+/-3.5 mg/mL.

In any embodiment, the anesthetic is present in the first protein solution, the second protein solution, or the third protein solution in an amount of 0.1% to 0.5%, alternatively 0.2% to 0.4% by mass. In any embodiment, the anesthetic comprises lidocaine.

In any embodiment, the first protein solution, the second protein solution and the third protein solution are prepared by using physiological saline, so that the difference between the collagen composition and the osmotic pressure of human blood can be reduced, and adverse reactions caused by the difference can be reduced.

In any embodiment, the homogenization treatment is performed using a homogenizer having a pore size of 1000 μm, 500 μm and/or 100 μm, which helps to improve the dispersion uniformity of the modified collagen.

A fourth aspect of the present disclosure provides a collagen composition according to the first aspect, a modified collagen prepared by the method according to the second aspect, and the use of a collagen composition prepared by the method according to the third aspect in the preparation of an injection for filling human tissue.

In any embodiment, the tissue augmentation comprises facial tissue augmentation.

In any embodiment, the use includes use in the preparation of an injection for improving facial wrinkles and pits.

A fifth aspect of the present disclosure provides a collagen composition according to the first aspect, a modified collagen prepared by the method according to the second aspect, and the use of a collagen composition prepared by the method according to the third aspect in the preparation of a filler for cosmetic shaping of a human body.

Drawings

FIG. 1 shows pathological sections of rabbits after 1 month of collagen injection administration;

FIG. 2 shows pathological sections of rabbits after 6 months of collagen injection administration;

FIG. 3 shows pathological sections of rabbits after 12 months of administration of collagen injections;

FIG. 4 shows a photograph of the appearance of modified collagen D8 placed in a syringe;

fig. 5 shows a comparative photograph of the appearance of the collagen injection extrudates prepared in example 9 and comparative example 4;

fig. 6 shows a comparison photograph of the homogeneity of the collagen injection prepared in example 9 and comparative example 4.

Detailed Description

Unless otherwise indicated, all numbers expressing quantities, concentrations, proportions, masses, volumes, times, temperatures, thicknesses, technical effects, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about" or "approximately". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. It will be appreciated by those skilled in the art that each numerical parameter should be construed in light of the number of significant digits and conventional rounding techniques, or in a manner well understood by those skilled in the art, depending upon the desired properties and effects sought to be obtained by the present disclosure.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein, the expression "a and/or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and/or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. Similar expressions may be used in this sense.

[ Collagen composition ]

A first aspect of the present disclosure provides a collagen composition comprising:

(1) A first modified collagen having a degree of crosslinking of 30% to 50%,

(4) Pharmaceutically acceptable auxiliary materials.

The collagen composition provided by the present disclosure may be used for tissue augmentation to achieve a shaped cosmetic appearance, optionally in a liquid or semi-solid form. In some embodiments, the collagen composition is in the form of a gel. In some embodiments, the collagen composition is in the form of a hydrogel.

Herein, "modified collagen" is collagen subjected to modification treatment. Compared with natural collagen, amino and/or carboxyl in the collagen molecule form stable connection in or among collagen peptide chains, so that the crosslinking degree of the collagen is improved, and the mechanical strength, elastic modulus or heat resistance of the collagen is further improved.

In some embodiments, the first modified collagen comprises 0.1% to 2.0% by mass of the collagen composition based on the total mass of the collagen composition; the mass ratio of the second modified collagen in the collagen composition is 0.5-4.0%; the mass ratio of the third modified collagen in the collagen composition is 0.1% -2.0%.

Modified collagen with different degrees of cross-linking have different degradation times after implantation into a subject (e.g., a human) and, in general, modified collagen with low degrees of cross-linking have shorter degradation times, but the hardness of modified collagen with low degrees of cross-linking is also lower than that of modified collagen with high degrees of cross-linking, resulting in poor supporting effect.

In the collagen composition provided by the disclosure, the first modified collagen has good fluidity, can be completely degraded in 1-2 months, and can prevent local collapse of an injection part caused by excessively rapid degradation and improve the shaping effect, so that the first modified collagen can improve the flowability and soft filling and supporting effects of the composition; however, the degradation period of the first modified collagen is short, which is not beneficial to reducing the operation frequency. The degradation period of the second modified collagen is higher than that of the first modified collagen, and when the second modified collagen is degraded in vivo, the second modified collagen is simultaneously regenerated naturally in the body of the subject, so that the degradation of the filling and supporting effect caused by the degradation of the collagen composition can be at least partially counteracted; in addition, the second modified collagen has good fluidity. The addition of the second modified collagen to the collagen composition helps the collagen composition to achieve a good packing and supporting effect, flow properties and good degradation time. The third modified collagen has a large molecular weight and a large hardness, so that the third modified collagen can provide excellent supporting effect and lasting degradation period, but has obvious edge of an injection part and poor shaping effect. In addition, the viscosity of the third modified collagen is increased, the fluidity is deteriorated, and the clinical operation is not facilitated. In the collagen composition provided by the disclosure, the modified collagen with different crosslinking degrees are used cooperatively, so that the collagen composition has good filling and supporting effects, clinical operability and degradation period, not only improves the shaping and beautifying effects, but also reduces the pain of tested patients and improves the satisfaction.

In some embodiments, the first modified collagen is present in the collagen composition in a mass ratio of 0.2% to 2.0%, 0.4% to 2.0%, 0.6% to 2.0%, or 0.2% to 1.5% based on the total mass of the collagen composition. In some embodiments, the first modified collagen is present in the collagen composition in a mass ratio of 0.5%, 0.7%, 0.9%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, or a range between any two of the foregoing, based on the total mass of the collagen composition. This helps to further improve the flowability of the collagen composition, enhance the softening effect of cosmetic shaping and reduce the push force of the composition in injectable use. In some embodiments, the second modified collagen is present in the collagen composition in a mass ratio of 0.6% to 3.6%, 0.8% to 3.6%, 0.6% to 3.0%, or 1.0% to 3.6% based on the total mass of the collagen composition. In some embodiments, the second modified collagen is present in the collagen composition in a mass ratio of 0.7%, 0.9%, 1.2%, 1.5%, 1.8%, 2.0%, 2.3%, 2.5%, 2.8%, 3.0%, 3.2%, 3.5%, or a range between any two of the foregoing, based on the total mass of the collagen composition. This helps to further improve the propping properties and flowability of the collagen composition while having a good degradation cycle.

In some embodiments, the third modified collagen is present in the collagen composition in a mass ratio of 0.25% to 1.75%, 0.4% to 1.75%, 0.5% to 1.5%, or 0.5% to 1.75% based on the total mass of the collagen composition. In some embodiments, the third modified collagen is present in the collagen composition in a mass ratio of 0.5%, 0.7%, 0.9%, 1.0%, 1.3%, 1.5%, 1.6% or in a range between any two of the foregoing values, based on the total mass of the collagen composition, which helps to further improve the degradation cycle and the proppability of the composition.

In some embodiments, the first modified collagen is present in the collagen composition in a mass ratio of 0.2% to 2.0% based on the total mass of the collagen composition; the mass ratio of the second modified collagen in the collagen composition is 0.6-3.6%; the weight ratio of the third modified collagen in the collagen composition is 0.25-1.75%, which is helpful for further considering the supporting performance and fluidity of the collagen composition and prolonging the degradation period.

In some embodiments, the first modified collagen comprises from 0.2% to 1.2% by mass of the collagen composition based on the total mass of the collagen composition; the mass ratio of the second modified collagen in the collagen composition is 0.6-3.6%; the weight percentage of the third modified collagen in the collagen composition is 0.25-1.20%.

In some embodiments, the collagen composition comprises a first modified collagen at a mass concentration of 2mg/mL to 20mg/mL in the collagen composition. In some embodiments, the first modified collagen is present in the collagen composition at a mass concentration of 2mg/mL to 18mg/mL, 2mg/mL to 15mg/mL, 2mg/mL to 13mg/mL, 2mg/mL to 10mg/mL, or 5mg/mL to 20mg/mL. In some embodiments, the first modified collagen is present in the collagen composition at a mass concentration of 3mg/mL, 5mg/mL, 7mg/mL, 9mg/mL, 12mg/mL, 15mg/mL, 17mg/mL, 19mg/mL, or a range between any two of the foregoing.

In some embodiments, the collagen composition comprises a concentration of the second modified collagen in the collagen composition of 4mg/mL to 40mg/mL by mass. In some embodiments, the mass concentration of the second modified collagen in the collagen composition is 5mg/mL to 40mg/mL, 10mg/mL to 40mg/mL, 4mg/mL to 35mg/mL, 4mg/mL to 30mg/mL, or 10mg/mL to 35mg/mL. In some embodiments, the concentration by mass of the second modified collagen in the collagen composition is 6mg/mL, 9mg/mL, 12mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 28mg/mL, 32mg/mL, 38mg/mL, or a range between any two of the foregoing.

In some embodiments, the collagen composition comprises a third modified collagen at a mass concentration of 2mg/mL to 25mg/mL in the collagen composition. In some embodiments, the third modified collagen is present in the collagen composition at a mass concentration of 5mg/mL to 25mg/mL, 10mg/mL to 25mg/mL, 2mg/mL to 20mg/mL, 5mg/mL to 20mg/mL, or 2mg/mL to 15mg/mL. In some embodiments, the third modified collagen is present in the collagen composition at a mass concentration of 3mg/mL, 6mg/mL, 9mg/mL, 12mg/mL, 15mg/mL, 18mg/mL, 22mg/mL, or a range between any two of the foregoing.

The determination of the modified collagen mass concentration may be performed using any method known in the art, as an example, the kjeldahl method.

The mass concentration and mass ratio of the first modified collagen, the second modified collagen and the third modified collagen within the above ranges further improve the flowability, degradation period and filling and supporting effects of the collagen composition.

In some embodiments, the collagen composition comprises an anesthetic comprising lidocaine.

In some embodiments, the anesthetic is present in the collagen composition at a mass ratio of 0.1% to 0.5%, alternatively 0.2% to 0.4%. The anesthetic in the collagen composition helps to reduce pain in the subject when receiving tissue injections.

In some embodiments, the anesthetic is present in the collagen composition at a mass concentration of 1mg/mL to 5mg/mL, optionally 2mg/mL to 4mg/mL. In some embodiments, the anesthetic is present in the collagen composition at a mass concentration of 3mg/mL, which not only reduces the subject's intraoperative pain, but also reduces adverse effects caused by excessive amounts of anesthetic, such as cardiovascular side effects, allergic reactions.

In some embodiments, pharmaceutically acceptable excipients in the collagen composition include water for injection and sodium chloride, the sodium chloride being dissolved in the water for injection and dispersed in the collagen composition. In some embodiments, the collagen composition is an isotonic solution, optionally, the sodium chloride is present in the collagen composition at a mass concentration of 0.9g/100mL, which advantageously reduces the effect of the collagen composition on blood cells at the injection site, reducing adverse effects caused by cell atrophy or rupture.

In some embodiments, the collagen composition has an average degree of crosslinking of 30% to 80%. In some embodiments, the collagen composition has an average degree of crosslinking of 35% to 70%, 55% to 70%, 40% to 80%, or 35% to 80%. In some embodiments, the average degree of crosslinking of the collagen composition is 33%, 37%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or a range between any two of the above values, which helps to further improve the filling effect, degradation cycle and handleability of the collagen composition.

The collagen composition is prepared by using 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as chemical crosslinking agents, and glutaraldehyde crosslinking agents commonly used in the prior art are avoided, so that the collagen composition provided by the present disclosure is whiter and more attractive. Simultaneously, toxic reaction caused by glutaraldehyde residue is avoided, and the safety of the collagen composition is improved.

The collagen composition has lower residual content of the crosslinking agent, so that the safety and the quality of the collagen composition are improved. In some embodiments, the mass concentration of EDC in the collagen composition is no more than 0.5 μg/g. In some embodiments, the mass concentration of EDC in the collagen composition is no more than 0.4 μg/g, 0.35 μg/g, 0.3 μg/g, 0.25 μg/g, 0.2 μg/g, 0.15 μg/g, or 0.1 μg/g. In some embodiments, the mass concentration of NHS in the collagen composition is no more than 3 μg/g. In some embodiments, the mass concentration of NHS in the collagen composition is no more than 2.5 μg/g, 2.0 μg/g, 1.5 μg/g, 1.0 μg/g, 0.5 μg/g, or 0.1 μg/g.

In some embodiments, the collagen composition has a thermal denaturation temperature (DSC) of 50 ℃ to 75 ℃. In some embodiments, the collagen composition has a DSC of 51.6 ℃ to 73.5 ℃. In some embodiments, the collagen composition has a DSC of 55 ℃, 60 ℃, 65 ℃,70 ℃, 75 ℃, or a range between any two of the above. The thermal denaturation temperature can reflect the thermal stability of the collagen composition, and the higher the thermal denaturation temperature is, the stronger the heat resistance of the collagen composition is, the less the microstructure (for example, a triple helix structure) of the collagen molecule is easy to change, the more the water absorption of the collagen molecule is facilitated, the good elasticity of the collagen composition is maintained, and the shaping effect of the composition is improved.

Heat denaturation temperature (DSC) has a meaning well known in the art and can be determined using methods known in the art, as exemplified by the annex D melting point assay in YY/T0954-2015 passive surgical implant type I collagen implant.

In some embodiments, the collagen composition has a viscosity of 8000 Pa.s to 11000mPa.s. In some embodiments, the collagen composition has a viscosity of 8000 Pa.s-10500mPa.s, 8050mPa.s-10500mPa.s, 8300mPa.s-10000 Pa.s, 9000mPa.s-10500mPa.s. In some embodiments, the viscosity of the collagen composition is in the range of 800 mpa.s, 8500mpa.s, 9000mpa.s, 95000mpa.s, 10000mpa.s, 10500mpa.s, or any range therebetween, and the flowability of the collagen composition can be further improved, thereby improving the handleability and filling effect of the collagen composition.

Viscosity has a meaning well known in the art, reflecting the magnitude of fluid viscosity. The viscosity of the collagen composition may be determined using methods known in the art, and by way of example, may be determined using a viscometer according to the methods disclosed in the fourth section of the chinese pharmacopoeia 2020.

The collagen composition provided by the present disclosure has a good push force, which can reflect the operability of the collagen composition when in use. In the medical field, a syringe needle with the specification of 27G or 30G is generally adopted for tissue injection, particularly facial injection filling, so as to reduce facial wound, and the inner diameter of the syringe needle is smaller, so that the magnitude of pushing force can directly influence the operability of clinical use.

In some embodiments, the collagen composition has a push force of 3N to 10N. In some embodiments, the collagen composition has a push force of 3N-6N, 3N-9N, 3N-8N, or 3N-7N. In some embodiments, the pushing force of the collagen composition is 3.5N, 4N, 4.5N, 5N, 5.5N, 6N, 6.5N, 7N, 7.5N, 8N, 8.5N, 9N, 9.5N or a range between any two of the above values, which may further improve the mobility of the collagen composition, thereby improving the intraoperative operability of the collagen composition, and improving clinical convenience.

In some embodiments, the first modified collagen, the second modified collagen, and the third modified collagen are each independently type I collagen. In some embodiments, the first modified collagen, the second modified collagen, and the third modified collagen are each independently selected from modified type I collagen. The triple helix structure contained in the type I collagen is beneficial to absorbing water, preserving moisture and repairing injury, improving the filling and supporting effects of the collagen composition in the body and promoting wound repair.

[ Method for producing modified collagen ]

The second aspect of the present disclosure provides a method for preparing modified collagen, the method at least comprising the following two crosslinking steps:

(2) Chemical crosslinking: and (3) placing the physical crosslinking product prepared in the step (1) into crosslinking liquid containing EDC and NHS to carry out crosslinking reaction, so that the modified collagen with the crosslinking degree of 30-80% is obtained.

The method is prepared by using a crosslinking liquid comprising EDC and NHS, and avoids using glutaraldehyde crosslinking agent, thereby avoiding toxicity caused by glutaraldehyde residue and yellowing of collagen appearance.

In some embodiments, in step (1), the type I collagen is present in the buffer at a concentration of 2mg/mL to 3mg/mL by mass, which is advantageous for increasing the reaction yield.

The type I collagen can be obtained by commercially available products or by extraction using a method in the prior art. In some embodiments, the type I collagen is made by a method comprising the steps of: and (3) performing pretreatment, enzymolysis, crude extraction, purification and filtration sterilization treatment on the cow leather and/or the cow achilles tendon to obtain the type I collagen.

In the extraction process, other proteins except the type I collagen, i.e. hybrid proteins, possibly remain in the type I collagen, which affects the quality of the collagen. In some embodiments, the amount of the hetero-protein in the type I collagen is no more than 0.3%. In some embodiments, the amount of the hetero-protein in the type I collagen is not more than 0.25%, 0.20%, 0.15% or 0.10% by weight, which is useful for preparing a modified collagen with excellent quality.

In some embodiments, the buffer in step (1) may be Phosphate Buffered Saline (PBS), e.g., PBS buffer at ph6.8, PBS buffer at ph7.0, PBS buffer at ph 7.3. The buffers may be formulated using methods commonly used in the art, for example with reference to methods disclosed in the fourth section of the chinese pharmacopoeia 2020 edition.

In some embodiments, in step (1), an acidic or basic solution may be used to adjust the pH of the buffer (or reaction system). In some embodiments, an alkaline solution may be used to adjust the pH of the physical crosslinking buffer (or reaction system) to 7-7.5. The alkaline solution may be any alkaline solution known in the art, and preferably an alkali metal salt solution such as sodium hydroxide solution or potassium hydroxide solution is used.

In some embodiments, the reaction time for physical cross-linking of type I collagen in buffer is 30min-120min, alternatively 30min-100min, 30min-80min, 30min-60min. In some embodiments, the reaction time for physical cross-linking of type I collagen in a buffer is 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, or a range between any two of the foregoing.

By physical crosslinking, the collagen fibers can be polymerized, which does not involve covalent attachment, and can be reversibly polymerized and depolymerized. The physical crosslinking can reduce the distance between the collagen fibers, is favorable for promoting the chemical crosslinking reaction between the collagen fibers, and improves the crosslinking degree and the reaction efficiency of the chemical crosslinking reaction.

In some embodiments, the EDC is present in the crosslinking solution at a mass concentration of 20mg/mL to 160mg/mL in the chemical crosslinking reaction. In some embodiments, the EDC is present in the crosslinking fluid at a mass concentration of 40mg/mL-160mg/mL, 60mg/mL-160mg/mL, or 100mg/mL-160mg/mL. In some embodiments, the EDC is present in the crosslinking fluid at a mass concentration of 30mg/mL, 50mg/mL, 70mg/mL, 90mg/mL, 120mg/mL, 150mg/mL, or a range between any two of the foregoing.

In some embodiments, the mass concentration of NHS in the crosslinking solution is 10mg/mL-120mg/mL in the chemical crosslinking reaction. In some embodiments, the mass concentration of NHS in the cross-linking solution is 30mg/mL-120mg/mL, 50mg/mL-120mg/mL, 70mg/mL-120mg/mL, or 90mg/mL-120mg/mL. In some embodiments, the mass concentration of NHS in the cross-linking solution is 20mg/mL, 40mg/mL, 60mg/mL, 80mg/mL, 100mg/mL, 115mg/mL, or a range between any two of the foregoing.

In some embodiments, the EDC to NHS mass concentration ratio in the crosslinking solution is 1.1 to 2.0. In some embodiments, the EDC to NHS mass concentration ratio in the crosslinking solution is 1.1 to 1.5. In some embodiments, the mass concentration ratio of EDC to NHS in the crosslinking liquid is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or a range between any two of the foregoing values. The EDC to NHS amounts and ratios of amounts in the appropriate ranges help to promote the chemical crosslinking reaction.

In some embodiments, the cross-linking liquid has a pH of 5.5 to 7.0. In some embodiments, the cross-linking liquid has a pH of 6.0, 6.5, 7.0 or a range between any two of the foregoing values.

In some embodiments, the chemical crosslinking reaction is for a period of time ranging from 2 hours to 12 hours. In some embodiments, the chemical crosslinking reaction is performed for a period of time of 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, or a range between any two of the foregoing. In some embodiments, the chemical crosslinking reaction is at a temperature of 20 ℃ to 25 ℃.

Under proper pH condition, reaction temperature and reaction time, the chemical crosslinking reaction can promote the reaction to proceed fully, and the crosslinking degree and reaction efficiency of the modified collagen are improved.

In some embodiments, the method comprises at least one, two or three steps of step a for preparing a first modified collagen having a degree of cross-linking of 30% -50%, step B for preparing a second modified collagen having a degree of cross-linking of greater than 50% and less than 70%, and step C for preparing a third modified collagen having a degree of cross-linking of greater than 70% and less than 80%.

In some embodiments, the process a provides a mass concentration of EDC in the chemical crosslinking reaction system of 20mg/mL to 80mg/mL in the chemical crosslinking reaction; the mass concentration of the NHS in the chemical crosslinking reaction system is 10mg/mL-70mg/mL; the time of the chemical crosslinking reaction is 2-4 h; the temperature of the chemical crosslinking reaction is 20-25 ℃.

In some embodiments, the mass concentration of EDC in the chemical cross-linking reaction system in the chemical cross-linking reaction of step a is 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, or a range between any two of the above.

In some embodiments, in the chemical crosslinking reaction, the mass concentration of NHS in the chemical crosslinking reaction system is 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, or a range between any two of the foregoing.

In some embodiments, the step a is performed in a chemical crosslinking reaction for a period of time of 2h, 2.5h, 3h, 3.5h, 4h, or a range between any two of the foregoing values.

The first modified collagen with the crosslinking degree of 30-50% can be obtained by the preparation method comprising the procedure A.

In some embodiments, the process B provides a mass concentration of EDC in the chemical crosslinking reaction system of 60mg/mL to 140mg/mL in the chemical crosslinking reaction; the mass concentration of the NHS in the chemical crosslinking reaction system is 50mg/mL-100mg/mL; the time of the chemical crosslinking reaction is 6-8 hours; the temperature of the chemical crosslinking reaction is 20-25 ℃.

In some embodiments, in the chemical crosslinking reaction, the mass concentration of EDC in the chemical crosslinking reaction system is 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, or a range between any two of the foregoing values.

In some embodiments, in the chemical crosslinking reaction, the mass concentration of NHS in the chemical crosslinking reaction system is 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, or a range between any two of the foregoing.

In some embodiments, the step B is performed in a chemical crosslinking reaction for a period of time of 6h, 6.5h, 7h, 7.5h, 8h, or a range between any two of the foregoing values.

The second modified collagen having a degree of crosslinking of more than 50% and 70% or less can be obtained by the production method including the step B.

In some embodiments, the process C is performed in a chemical crosslinking reaction, such that the EDC has a mass concentration in the chemical crosslinking reaction system of 110mg/mL to 160mg/mL; the mass concentration of the NHS in the chemical crosslinking reaction system is 80mg/mL-120mg/mL; the time of the chemical crosslinking reaction is 10-12 h; the temperature of the chemical crosslinking reaction is 20-25 ℃.

In some embodiments, in the chemical crosslinking reaction, the mass concentration of EDC in the chemical crosslinking reaction system is 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, or a range between any two of the foregoing values.

In some embodiments, in the chemical crosslinking reaction, the mass concentration of NHS in the chemical crosslinking reaction system is 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, or a range between any two of the foregoing.

In some embodiments, the step C is performed in a chemical crosslinking reaction for a period of time of 10h, 10.5h, 11h, 11.5h, 12h, or a range between any two of the foregoing.

The preparation method comprising the step C can obtain the third modified collagen with the crosslinking degree of more than 70% and less than 80%.

By the preparation method comprising the steps a, B and/or C, modified collagen or collagen compositions having different degrees of cross-linking can be obtained.

The preparation method provided by the disclosure has the advantages of mild reaction conditions, short reaction period and high efficiency, and is suitable for the industrialized production of the modified collagen. The prepared modified collagen has excellent quality and is more attractive.

[ Process for producing collagen composition ]

A third aspect of the present disclosure provides a method of preparing a collagen composition, comprising: mixing the first modified collagen, the second modified collagen and the third modified collagen, and homogenizing to obtain the collagen composition.

In some embodiments, the first modified collagen, the second modified collagen and the third modified collagen are prepared into solutions respectively and then subjected to homogenization treatment, which is beneficial to improving the dispersion uniformity of the modified collagen with different crosslinking degrees.

In some embodiments, the first modified collagen is prepared as a first protein solution comprising a first modified collagen mass concentration of 20mg/mL to 50mg/mL and an anesthetic mass concentration of 1mg/mL to 5 mg/mL.

In some embodiments, the first modified collagen in the first protein solution has a mass concentration of 25mg/mL-45mg/mL, 20mg/mL-40mg/mL, or 30mg/mL-50mg/mL. In some embodiments, the first modified collagen in the first protein solution has a mass concentration of 22mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, or a range between any two of the foregoing.

In some embodiments, the anesthetic is present in the first protein solution at a mass concentration of 2mg/mL to 4mg/mL. In some embodiments, the mass concentration of anesthetic in the first protein solution is 3mg/mL.

In some embodiments, the first protein solution comprises a first modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL.

In some embodiments, the second modified collagen is prepared as a second protein solution comprising a second modified collagen mass concentration of 20mg/mL to 50mg/mL and an anesthetic mass concentration of 1mg/mL to 5 mg/mL.

In some embodiments, the mass concentration of the second modified collagen in the second protein solution is 25mg/mL to 45mg/mL, 20mg/mL to 40mg/mL, or 30mg/mL to 50mg/mL. In some embodiments, the mass concentration of the second modified collagen in the second protein solution is 22mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, or a range between any two of the foregoing.

In some embodiments, the mass concentration of anesthetic in the second protein solution is 2mg/mL to 4mg/mL. In some embodiments, the mass concentration of anesthetic in the second protein solution is 3mg/mL.

In some embodiments, the second protein solution comprises a second modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL.

In some embodiments, the third modified collagen is prepared as a third protein solution comprising a third modified collagen mass concentration of 20mg/mL to 50mg/mL and an anesthetic mass concentration of 1mg/mL to 5 mg/mL.

In some embodiments, the mass concentration of the third modified collagen in the third protein solution is 25mg/mL to 45mg/mL, 20mg/mL to 40mg/mL, or 30mg/mL to 50mg/mL. In some embodiments, the third modified collagen in the third protein solution has a mass concentration of 22mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, or a range between any two of the foregoing.

In some embodiments, the mass concentration of anesthetic in the third protein solution is 2mg/mL to 4mg/mL. In some embodiments, the mass concentration of anesthetic in the third protein solution is 3mg/mL.

In some embodiments, the third protein solution comprises a third modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL.

In some embodiments, the first protein solution comprises a first modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL, the second protein solution comprises a second modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL, and the third protein solution comprises a third modified collagen at a mass concentration of (35±3.5) mg/mL and an anesthetic at a mass concentration of (3±0.3) mg/mL.

Mixing the first protein liquid, the second protein liquid and the third protein liquid to obtain a total liquid, and homogenizing the total liquid after mixing. In some embodiments, the first protein solution comprises 10% to 40% by mass of the total solution; the mass ratio of the second protein liquid in the total liquid is 30% -80%; the mass ratio of the third protein liquid in the total liquid is 10% -50%.

The first protein liquid, the second protein liquid and the third protein liquid are respectively prepared and mixed, which is favorable for improving the uniformity of the modified collagen with different crosslinking degrees and can fully disperse the anesthetic in the collagen injection.

In some embodiments, the first protein solution comprises 10%, 15%, 20%, 25%, 30%, 35%, 40% or a range between any two of the above by mass of the total solution.

In some embodiments, the mass ratio of the second protein solution in the total solution is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or a range between any two of the above.

In some embodiments, the third protein solution comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or a range between any two of the above in the total solution by mass.

The modified collagen with different crosslinking degrees is prepared into the solution and then mixed, so that the mixing difficulty of the modified collagen with different crosslinking degrees can be reduced, and the dispersion uniformity of the modified collagen is improved. The preparation method reduces the industrial production difficulty of the modified collagen composition and can realize the industrialized production of the modified collagen.

Meanwhile, the composition of collagen with different crosslinking degrees in the prepared collagen composition can be adjusted by adjusting the mass ratio of the first protein liquid to the second protein liquid to the third protein liquid so as to adjust the degradation time, the supporting effect and the fluidity of the collagen composition, and the collagen composition which can take the degradation period, the filling effect and the operability into consideration is obtained.

In some embodiments, the anesthetic is present in the first, second, or third protein solutions in a mass ratio of 0.1% to 0.5%, alternatively 0.2% to 0.4%, and illustratively 0.3%.

In some embodiments, the anesthetic comprises lidocaine, which is fast acting and safe, which is beneficial for reducing pain sensation in the subject during surgery.

In some embodiments, the first protein solution, the second protein solution, and the third protein solution may be prepared using physiological saline or 0.9% sodium chloride injection, and the difference between the collagen composition and the osmotic pressure of human blood may be reduced, thereby reducing adverse reactions and irritation to tissue sites.

In some embodiments, homogenization treatment using a homogenizer having a pore size of 1000 μm, 500 μm, and/or 100 μm helps to improve the uniformity of dispersion of the modified collagen.

[ Use ]

In some embodiments, the collagen composition may be used as a bulking agent for human tissue bulking. In some embodiments, the collagen composition may be used to shape a cosmetic appearance to improve skin wrinkles and sagging. In some embodiments, the tissue augmentation comprises facial tissue augmentation.

In some embodiments, the use includes use of a collagen composition in the preparation of a filler for shaping a human body. In some embodiments, the use includes use of a collagen composition in the preparation of a filler for shaping facial makeup of a human. The filler may be used in the form of an injection preparation.

In some embodiments, the use includes use in the preparation of an injection for improving facial wrinkles and pits.

In clinical applications, the collagen composition provided by the present disclosure may be injected into a predetermined human body part, such as the face, by a syringe. In general, the collagen composition may be filled into a dermis layer and/or subcutaneous tissue layer of skin tissue, for example. The loading of the collagen composition generally varies according to the physical condition of the subject and the clinical experience of the clinician.

The various embodiments and preferences disclosed above may be combined with one another (as long as they are not inherently contradictory to one another) and the various embodiments resulting from such combination are considered to be part of the disclosure of the present application.

The technical aspects of the present disclosure will be more clearly and clearly illustrated below by way of example in conjunction with examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. The scope of the present disclosure is limited only by the claims.

Examples

Hereinafter, embodiments of the present application are described. The following examples are illustrative only and are not to be construed as limiting the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Test method

(1) Free amino ratio and degree of crosslinking

1.1 Calculation of free amino content

The hydrate of collagen and ninhydrin is heated in weak acid solution to cause oxidation and deamination, and the product has bluish violet color and maximum absorption at 570 nm.

Preparing glycine gradient solution, measuring absorbance at 570nm by ultraviolet-visible spectrophotometry, and obtaining linear regression equation by linear regression analysis.

Weighing a detection sample (crosslinked modified collagen and type I collagen raw material liquid), adding a known amount of purified water, treating for 20min in a boiling water bath, heating and hydrolyzing protein in the detection sample to release favorable amino groups, taking supernatant, measuring absorbance, and calculating the content C (mug/mL) of free amino groups in the detection sample by using a linear regression equation.

1.2 Calculation of free amino ratio

K ₁ and K ₂ are the free amino group ratio (mug/mg) in the prepared crosslinking modified collagen and type I collagen raw material liquid respectively, and the calculation formula is as follows:

Wherein: c ₁、C₂ is the content (mug/mL) of free amino in the raw material liquid of the crosslinking modified collagen and the type I collagen respectively;

M ₁、M₂ is the weight (mg) of the raw material liquid of the crosslinking modified collagen and the type I collagen respectively;

A ₁、A₂ is the content (mg/mL) of collagen in the raw material liquid of the crosslinking modified collagen and the type I collagen respectively;

ρ ₁、ρ₂ is the density (1000 mg/mL) of the cross-linked modified collagen and type I collagen feed solution, respectively;

v1 and v2 are the volumes (1 mL) of the raw material liquid of the crosslinking modified collagen and the type I collagen respectively.

1.3 Calculation of the degree of crosslinking

Calculation formula of crosslinking degree (S)

Wherein: k ₁、K₂ is the mean value of not less than three samples.

(2) Testing of EDC residue in collagen injection

The dimethylbarbituric acid solution was reacted with carbodiimide (EDC) to form a mauve complex, and the absorbance was measured at 599nm using uv-vis spectrophotometry. And weighing the EDC reference substance, preparing a gradient solution, measuring absorbance, and obtaining a linear regression equation through linear regression analysis.

1G of the collagen injection of the detection sample is weighed, centrifuged at 14000r/min for 10min, the supernatant is taken and filtered by a 0.22 supernatant filter membrane, and the absorbance is measured. Substituting absorbance into a linear regression equation, and calculating to obtain the mass concentration of EDC in the test sample.

(3) Testing of NHS residue in collagen injection

2G of collagen injection is weighed, diluted by adding one volume of 0.9% NaCl solution, centrifuged at 14000r/min for 10min, the supernatant is filtered by a 0.22 liquid and subjected to filter membrane, vibration is carried out at a frequency of 40Hz for 20min under the condition of 25 times, and the mass concentration of NHS in the solution is determined by adopting high performance liquid chromatography (refer to N-hydroxysuccinimide content determination in collagen implant based on high performance liquid chromatography, yu Jianyong, wang Shudong, gu Jianhua and the like, china beauty medicine, 2019, 28 (1): 29-32). The mass concentration multiplied by 2 is the NHS residue (mug/mL) of the collagen injection.

(4) Testing of thermal denaturation temperature of collagen injection

The process of a change in a physical or chemical property of a substance is characterized by measuring its thermodynamic change.

Referring to the melting point assay of appendix D of YY/T0954-2015, passive surgical implant type I collagen implant, about 20mg of the sample was taken and placed in a sealed crucible (liquid crucible) for detection.

1.2 Analysis of results

Scanning is carried out in a nitrogen atmosphere with the speed of 2 ℃/min and the temperature range of 40-100 ℃ in the nitrogen atmosphere, and the absorption peak value of the sample is recorded as the thermal denaturation temperature.

(5) Testing of viscosity of collagen injection

The method is carried out by adopting a rotary viscometer (NDJ-9S) according to the second method of annex VI G of the pharmacopoeia of the people' S republic of China (edition 2010).

The sample was extruded into a 50mL beaker, and the sample was measured three times continuously after stabilization using a No. 3 rotor, set at a speed of 6rpm, and averaged.

(6) Testing of push force of collagen injection

The test sample was allowed to stand at room temperature (20 ℃ C. -25 ℃ C.) for more than 30 minutes until the sample equilibrated to room temperature, and was measured with a 27G needle (inner diameter 0.21 mm). The injection end of the injector is downwards fixed on the clamp, the pushing speed is 25mm/min, and the pushing length is 20mm. And recording the maximum continuous force value after the starting point as a push force detection result.

(7) Testing of modified collagen concentration and Mass ratio

Preparing a hydroxyproline reference substance gradient concentration solution, measuring absorbance at 560nm by an ultraviolet-visible spectrophotometry, and obtaining a linear regression equation by linear regression treatment. Weighing a collagen injection sample m (mg), carrying out hydrolysis treatment, fixing the volume to 50mL, measuring absorbance, calculating the hydroxyproline concentration C (mug/mL) in the sample by using a linear regression equation, and calculating the content of collagen according to the following formula:

The hydroxyproline in the collagen accounts for about 12.77 percent, and the detection loading amount is 0.1mL; v represents the volume of the detection solution (1 mL), ρ represents the sample density (1 g/mL).

Mass ratio of modified collagen = mass of modified collagen/mass of collagen injection.

Preparation of crosslinked modified collagen

Preparation of type I collagen solution:

Pretreatment: the cowhide is washed and chopped, soaked in sodium sulfate solution for standby, alkalized in sodium hydroxide solution at low temperature (0-20 ℃), the precipitate is taken out, and purified water is washed until the pH value is less than 8 for standby.

Enzymolysis: adding the pretreatment product into 0.5M glacial acetic acid solution for full dissolution, adding pepsin for enzymolysis reaction, removing collagen molecular terminal peptide, centrifuging, and collecting supernatant for later use.

Salting out: adding sodium chloride solution into the collected supernatant to separate out collagen, collecting collagen precipitate, cleaning, removing impurities, and dissolving in 3% sodium acetate solution for use.

Purifying: and (3) carrying out ion exchange chromatography treatment on the salting-out product by adopting cationic resin.

And (3) sterilizing and filtering: and (3) filtering and sterilizing the purified product by using a capsule filter after ultrafiltration and concentration, wherein the pore diameter of the adopted filter membrane is 0.45 mu m+0.22 mu m double-layer hydrophilic polyether sulfone filter membrane, and thus the type I collagen preservation solution is obtained. Wherein the mass ratio of the hetero protein is 0.21%.

Preparation of crosslinked modified collagen H1:

Taking type I collagen preservation solution, adding phosphoric acid buffer solution (pH 6.8) into the preservation solution to adjust the concentration of type I collagen to obtain type I collagen raw material solution, wherein the concentration is 2.2mg/mL, dropwise adding sodium hydroxide solution to adjust the pH value of the solution to 6.0, reacting for 50min at 37 ℃, placing the separated physical crosslinked product into chemical crosslinked solution with pH of 6.0, performing chemical crosslinking reaction for 2h at 20-25 ℃, and centrifuging to obtain a modified collagen crude product. And (3) respectively soaking and cleaning the gel crude product in PBS solution and purified water, centrifuging, taking precipitate, and respectively cleaning the precipitate by Na ₂HPO₄ and purified water to obtain the crosslinked modified collagen H1.

In the crosslinking solution, EDC concentration is 20mg/mL, NHS concentration is 10mg/mL, and solvent is 2- (N-morpholino) ethanesulfonic acid buffer (MES).

Preparation of crosslinked modified collagens H2-H6 and D1-D8:

The preparation methods of the crosslinked modified collagens H2 to H6 and crosslinked modified collagens D1 to D8 are substantially the same as those of the crosslinked modified collagen H1, but the preparation parameters are adjusted, see specifically Table 1.

TABLE 1

The prepared cross-linked modified collagen H1-H6 and D1-D6 are in milky gel shape and have elasticity; the crosslinked modified collagen D7 is in a slightly transparent gel state, and has low elastic strength; the crosslinked modified collagen D8 is slightly transparent gel sheet-shaped and has strong elasticity and toughness.

The free amino groups of the crosslinked modified collagens H1 to H6 and D1 to D8 were measured for their ratio and degree of crosslinking by the methods described above, and the results of the measurements are shown in Table 2.

TABLE 2

Example 1

Preparation of a first protein solution: the first modified collagen (crosslinked modified collagen H1) and lidocaine were dissolved with physiological saline so that the mass concentration of crosslinked modified collagen H1 in the first protein solution was 35 mg/mL.+ -. 3.5mg/mL and the mass concentration of lidocaine was 3mg/mL.

Preparation of a second protein solution: the second modified collagen (crosslinked modified collagen H3) and lidocaine were dissolved with physiological saline so that the concentration of crosslinked modified collagen H3 in the first protein solution was 35 mg/mL.+ -. 3.5mg/mL and the mass concentration of lidocaine was 3mg/mL.

Preparation of a third protein solution: the third modified collagen (crosslinked modified collagen H5) and lidocaine were dissolved with physiological saline so that the concentration of crosslinked modified collagen H5 in the first protein solution was 35 mg/mL.+ -. 3.5mg/mL and the mass concentration of lidocaine was 3mg/mL.

Mixing the first protein liquid, the second protein liquid and the third protein liquid according to the weight ratio of 1:6:3, and sequentially homogenizing by using a homogenizer with the pore diameters of 1000 mu m, 500 mu m and 100 mu m to obtain the collagen injection.

Example 2-example 9

The preparation of examples 2-9 was similar to that of example 1, see in particular Table 3.

Comparative example 1-comparative example 5

The preparation method of comparative example 1 was similar to that of example 1, except that the injection was prepared using only the first modified collagen (crosslinked modified collagen H1).

The preparation method of comparative example 2 was similar to that of example 1, except that only the second modified collagen (crosslinked modified collagen H3) was used to prepare an injection.

The preparation method of comparative example 3 was similar to that of example 1, except that only the third modified collagen (crosslinked modified collagen H5) was used to prepare an injection.

The preparation method of comparative example 4 was similar to that of example 1, except that the injection was prepared using only the first modified collagen (crosslinked modified collagen D7).

The preparation method of comparative example 5 was similar to that of example 1 except that the injection was prepared using only the first modified collagen (crosslinked modified collagen D8), but the crosslinked modified collagen D8 was in the form of a sheet and had very high hardness, high strength of elasticity and toughness, and no homogenization treatment was performed, as shown in fig. 4.

Comparative example 6-comparative example 8

Comparative example 6-comparative example 8 was prepared in a similar manner to that of example 1, see in particular table 3.

TABLE 3 Table 3

Numbering device	First protein liquid	Parts by weight of	Second protein liquid	Parts by weight of	Third protein liquid	Parts by weight of
							Example 1	H1	1	H3	6	H5	3
Example 2	H2	4	H3	4	H5	2
							Example 3	H1	2	H4	7	H5	1
Example 4	H1	3	H4	3	H6	4
							Example 5	H2	2	H3	3	H6	5
Example 6	H1	1	H3	8	H6	1
							Example 7	H1	3	H4	6	H5	1
Example 8	H1	2	H3	5	H5	3
							Example 9	H1	2	H3	6	H6	2
Comparative example 1	H1	10	—	—	—	—
							Comparative example 2	—	—	H3	10	—	—
Comparative example 3	—	—	—	—	H5	10
							Comparative example 4	D7	10	—	—	—	—
Comparative example 5	D8	10	—	—	—	—
							Comparative example 6	H1	6	H3	3	H6	1
Comparative example 7	H1	4	H3	1	H6	5
							Comparative example 8	H1	1	H3	3	H6	6

The collagen injections prepared in examples 1 to 9, comparative examples 1 to 3 and comparative examples 6 to 8 were milky white, transparent, and uniform in texture; the collagen injection prepared in comparative example 4 had a slightly transparent feel, and was visually uneven in floc (see fig. 6).

The mass ratio and mass concentration of the modified collagen in the injections prepared in examples 1 to 9, comparative examples 1 to 4 and comparative examples 6 to 8 were measured using the above-described test methods, and the results are shown in Table 4.

TABLE 4 Table 4

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The injection was tested for EDC and NHS residues, as well as average degree of crosslinking, using the above method, and thermodynamic properties, mechanical properties, degradation properties and safety properties of the injection were tested, and the results are shown in table 5.

TABLE 5

As can be seen from Table 5, the collagen injections prepared in examples 1 to 9 have low residual content of the crosslinking agent and good safety; the thermal denaturation temperature is within the range of 50-75 ℃, which shows that the collagen injection has good thermodynamic property; the average crosslinking degree of the collagen injection is 50-70%, the viscosity is in the range of 800 Pa.s-11000mPa.s, and the collagen injection has excellent mechanical property and supporting property and good degradation period; the pushing force of the injection is not more than 10N, which indicates that the injection has excellent fluidity. Therefore, the collagen injection prepared in examples 1-9 has both good supporting performance and fluidity, and has improved cosmetic shaping effect and lasting degradation period after injection filling.

As shown in comparative example 1, the injection prepared from the modified cross-linked protein H1 has lower cross-linking degree and viscosity, which indicates that the modified cross-linked protein has poorer mechanical property, is unfavorable for filling supporting property and has short degradation period.

As can be seen from comparative example 2 and example 6, by compounding the collagen proteins with three different crosslinking degrees, the average crosslinking degree and the thermal denaturation temperature of the collagen proteins are basically similar, but the viscosity and the pushing force of the compounded injection are higher, so that the compounded injection has better viscoelastic performance, and can be understood to have more ideal filling effect and longer tissue supporting period. The injection prepared in comparative example 2 with single crosslinking degree cannot achieve the degradation period, filling effect and clinical operability, and is limited in clinical use.

As is clear from comparative example 3, the injection prepared from the modified cross-linked protein H5 has higher viscosity and pushing force, which is unfavorable for the operability and the clinical convenience of the injection.

As shown in comparative example 4, the injection prepared from D7 has lower crosslinking degree and viscosity, which indicates that the modified crosslinked protein has poorer mechanical property and is unfavorable for filling supporting property; meanwhile, the injection is transparent and non-uniform gel, and the quality of the product is poor. Filling the injection prepared in comparative example 4 and example 9 into a syringe, wherein the gel on the left and right in fig. 6 is the product prepared in comparative example 4 and example 9, respectively, and it can be seen that the injection prepared in comparative example 4 is in a slightly transparent gel shape and has uniform macroscopic properties; the injection prepared in example 9 had a uniform texture and a uniform milky white color. The two were extruded onto a petri dish and the gel on the left and right in fig. 5 was the product prepared in comparative example 4 and example 9, respectively. The injection prepared in the comparative example 4 is liquid, has slight fluidity, has high vanishing speed of extrusion lines, and is not completely leveled; the injection prepared in example 9 has no liquid precipitation, has excellent supporting force, and can keep the extruded lines from disappearing and collapsing.

As is clear from comparative example 6, the injection has a reduced crosslinking degree and viscosity, improved flowability but reduced mechanical properties, and a short degradation period after the amount of the modified crosslinked protein H1 is increased.

As is clear from comparative example 7, after the amount of modified cross-linked protein H3 was reduced, the amount of the third protein solution was increased to improve tissue filling, but this significantly increased the viscosity of the injection, and the fluidity and clinical operability thereof were reduced, and the clinical use was limited. In addition, after the injection of comparative example 7 was filled in the tissue, the third modified collagen with long degradation period remained in the filling part because the first modified collagen has poor supporting property and rapid degradation, and the cosmetic appearance was poor and the retention period was extremely short.

As is clear from comparative example 8, after the dosage of the modified cross-linked protein H5 is increased, the viscosity of the injection is increased, the supporting performance is obviously improved, the degradation period is prolonged, but the injection filling effect is sharpened, which is unfavorable for the beauty of cosmetic shaping.

Degradation test of collagen injection

The collagen injection prepared in example 9 was also subjected to animal tests to examine degradation of the collagen injection at 1 month, 6 months and 12 months after operation, and is specifically as follows:

test object: new Zealand rabbits, male, weight 2.5kg-3.0kg, single cage raising.

The test process comprises the following steps: anesthetizing New Zealand rabbits, removing back hair to prepare skin, and sterilizing an operation area by using iodophor before operation; taking the spine of a New Zealand rabbit as the center, taking two points on the left side and the right side respectively, injecting 0.2mL of the collagen injection prepared in the example 9 into the hypodermis and the sarcolemma by using a 1mL syringe, taking tissue at the injection part 1 month, 6 months and 12 months after operation, pathological sections and staining, and observing degradation conditions as follows:

After 1 month: the dermis is almost the whole layer of visible spindle-shaped collagen sample aggregate, is in expansion growth, has a rough internal structure, is curled, is differentiated into more filaments, is tightly connected with each other, a large number of bundle-shaped coarse collagen fibers extend into the dermis, are divided and wrapped, the inside of the residual part of collagen sample is provided with fibroblasts, and the periphery of the residual part of collagen sample is provided with incomplete fiber wrapping. This indicates that a large amount of nascent collagen was visible at the injection site, with no apparent inflammatory response, see figure 1.

After 6 months: the dermis layer can be seen to gather collagen sample, the internal structure is fine and smooth, the swelling growth is realized, the partial bundle-shaped collagen fiber partition divides the collagen sample into a plurality of irregular areas, and the periphery is wrapped by incomplete fiber capsules. This indicates that there is a greater tendency for collagen aggregation at the injection site than 1 month post-surgery, see fig. 2.

After 12 months: medium irregular gels are seen in thin squamous epithelium, where small amounts of collagen fibril formation, small amounts of small blood vessels and myofibroblasts in-growth, no obvious tissue cells, no multinuclear giant cell reaction, no obvious inflammatory cell infiltration are seen. This indicates that small amounts of collagen fibril formation were still visible at the injection site, no significant inflammatory response was seen, see figure 3.

The results show that: the collagen injection prepared in example 9 has good in vivo safety and does not cause obvious inflammatory reaction; meanwhile, the composition still has no complete degradation after 12 months of injection, and has a good degradation period.

Safety test

(1) Intradermal reaction test

Detection standard: GB/T16886.10-2017/ISO 10993-10:2010

Using the collagen injection prepared in example 9 as a test sample, sodium dodecyl sulfate as a positive control, and 0.9% sodium chloride injection as a negative control, test samples were prepared as follows:

5g of a test sample is weighed and placed in 25mL of 0.9% sodium chloride injection, and the mixture is mixed for 72 hours at 37 ℃ and 60 revolutions per minute in a shaking incubator to obtain leaching liquor.

Taking New Zealand rabbits, male animals, and raising the rabbits in a single cage, wherein the weight of the male animals is 2.5kg-3.0 kg. Weighing 4-18 hours before the test, and thoroughly removing the fur with enough area on two sides of the back spine to be used as a test and observation position.

Injecting 0.2mL of the leaching solution of the test sample into 5 points before the dehairing area at the left side of the vertebra of each rabbit; the left post 5 points were injected intradermally with 0.2mL of negative control;

Injecting 0.2mL of positive control leaching solution into 5 points before the right dehairing area of the vertebra of the same rabbit; the 5 points after the right side were injected intradermally with 0.2mL of negative control.

Immediately after injection, and after injection (24±2) h, (48±2) h, (72±2) h, the injection site conditions were observed. According to the scoring system, the tissue response of each site in each period is scored, and the scoring results are recorded, and the scoring table is as follows:

After (72.+ -.2) h scoring, the total erythema score for (24.+ -.2), (48.+ -.2) h and (72.+ -.2) h was added to the edema score for each animal test sample or control, respectively, and divided by 15 to calculate the score for each animal test sample or control. The total average score for each test sample and corresponding blank was obtained by dividing 3 animals by 3. The final score for the test sample was obtained by subtracting the placebo score from the test sample score. The test samples were tested as follows:

The positive control was tested as follows:

the results showed that the collagen injection prepared in example 9 had good intradermal reactivity.

(2) In vitro cytotoxicity assay

The detection basis is as follows: GB/T16886.5-2017/ISO 10993-5:2009

Sample preparation:

1g of the collagen injection prepared in example 9 was taken, MEM medium containing 10% fetal bovine serum was added at a ratio of 0.2g to 1mL, and the extract was taken as a test sample at 37℃for 72 hours.

Test group: under aseptic operation, the aseptic filter paper is cut into 3 discs with the diameter of 1.0cm, and 100% of sample test solution is sucked during the test.

Negative control group: high density polyethylene (USP, R149K 0) was cut into 3 pieces of 1.0cm diameter discs.

Positive control group: the sterile filter paper was cut into 3 discs of 1.0cm diameter and the test was performed with MEM medium containing 0.1% Zinc Diethyldithiocarbamate (ZDEC).

The experimental process comprises the following steps:

The test procedure was performed aseptically. L-929 cells were cultured in MEM medium containing 10% fetal bovine serum at 37℃in a 5% CO ₂ incubator. Cells were dispersed in fresh medium, adjusted to 2.5X10 ⁵ cells/mL cell suspension, inoculated into 6-well plates, 2 mL/well, and incubated in a 5% CO ₂ incubator at 37℃for 24h. Discarding the original culture solution, adding 2mL of fresh culture medium, lightly placing the sample or negative and positive reference substances, taking care of not moving after placing, avoiding causing physical damage to cells, and placing the culture medium in an incubator for continuous culture for 24 hours. The plates were removed and the cell morphology was observed under a microscope.

Evaluation criteria:

The cytotoxicity response ratings are given in the following table, with reference to the standard.

Grading	Degree of reaction	Reaction area observations
			0	Without any means for	No reaction area was observed around and under the sample
1	Slight	Under the sample there are some malformed cells or degenerated cells
			2	Mild and mild	The reaction area is limited to the area below the sample
3	Moderate degree	The reaction area exceeded the sample size to 1.0cm
			4	Heavy weight	The reaction area exceeds the sample by more than 1.0cm

The detection results are as follows:

the test sample showed a level 1 toxic response to L-929 cells, suggesting that the test sample had no potential cytotoxic effect on L-929 cells.

(3) Skin sensitization test

The detection basis is as follows: GB/T16886.10-2017/ISO 10993-10:2010

15 Primary adult albino guinea pigs, female, 300 g-500 g, are marked and weighed before the test, and are randomly divided into a sample test group and a 0.9% sodium chloride injection control group. Skin reactions at the guinea pig challenge sites were observed 24h and 48h after removal of the sample and animal body weights were recorded.

Intradermal induction phase:

a, B and C test sites were placed between the head end of guinea pig and the inner part of scapula, the fur was removed, and 0.1mL of the test site was injected intradermally in pairs for each animal.

Part a: stable emulsifier mixed with Freund's complete adjuvant and 0.9% sodium chloride injection in a ratio of 50:50 (volume ratio) was injected.

Part B: collagen injection test prepared in example 9; animals in the control group were injected with 0.9% sodium chloride injection.

Part C: the collagen injection test prepared in example 9 was mixed with an emulsifier formulated with Freund's complete adjuvant and solvent (50%) in a volume ratio of 50:50 and then injected intradermally; the control group is injected with an emulsifying agent prepared from 0.9% sodium chloride injection and an adjuvant.

The test site did not produce irritation, and the test area was pretreated with 10% sodium dodecyl sulfate (24.+ -. 2) h before topical application, and massage was applied to the skin.

Local induction phase:

The induction injection points were covered with absorbent gauze of about 8cm ², then with a layer of cellophane, then with non-irritating adhesive tape, and after (48.+ -.2) h the bandages and patches were removed, following the intradermal induction phase (7.+ -.1) days, according to the concentration selected in the intradermal induction site B.

Excitation stage:

all test animals and control animals were challenged with the test samples after the local induction phase (14±1). The preparation method comprises the steps of locally applying to the non-tested part in the induction stage, covering with a layer of cellophane, and fixing with a non-irritating adhesive tape. And removing the tape and the patch after (24.+ -.2) h.

Animal observation:

the skin conditions at the sites of challenge were observed in the animals of the test and control groups at (24.+ -. 2) h and (48.+ -. 2) h after removal of the patch. Skin erythema and edema reactions were described and graded according to Magnusson and Kligman grading standards for each site of excitation and each observation time, as follows:

Application test reaction	Grade
		No obvious change	0
Sporadic or spotted erythema	1
		Moderate fusion erythema	2
Severe erythema and/or edema	3

Evaluation criteria:

animals in the control group were rated less than 1 according to Magnusson and Kligman ratings, whereas animals in the test group rated greater than or equal to 1 generally indicated sensitization. Animals in the test group were considered sensitized when their animals in the control group had a rank of 1 or more, and their animals in the test group had responded more than the most severe of the control group.

The results of the collagen injection prepared in example 9 were as follows:

all animals had a skin response rating of 0 at each time interval, and the test group positive challenge rate was 0%, suggesting that the injection did not produce allergic reactions.

Claims

1. A collagen composition comprising:

(1) A first modified collagen having a degree of crosslinking of 30% to 50%,

(4) Pharmaceutically acceptable auxiliary materials;

wherein, based on the total mass of the collagen composition,

the mass ratio of the third modified collagen in the collagen composition is 0.1-2.0%; and

Pharmaceutically acceptable auxiliary materials.

2. The collagen composition according to claim 1, wherein the collagen composition,

Based on the total mass of the collagen composition,

The mass ratio of the first modified collagen in the collagen composition is 0.2% -2.0%;

The mass ratio of the second modified collagen in the collagen composition is 0.6-3.6%;

The weight percentage of the third modified collagen in the collagen composition is 0.25-1.75%.

3. The collagen composition according to claim 1 or 2, wherein,

The mass concentration of the first modified collagen in the collagen composition is 2mg/mL-20mg/mL;

The mass concentration of the second modified collagen in the collagen composition is 5mg/mL-40mg/mL, and can be 6mg/mL-36mg/mL; and/or

The mass concentration of the third modified collagen in the collagen composition is 2mg/mL-25mg/mL, and optionally 2.5mg/mL-17.5mg/mL.

4. A collagen composition according to any one of claims 1 to 3, comprising an anesthetic comprising lidocaine.

5. The collagen composition according to claim 4, wherein the anesthetic is present in the collagen composition in an amount of 0.1% to 0.5%, optionally 0.2% to 0.4%; or alternatively

The mass concentration of the anesthetic in the collagen composition is 1mg/mL-5mg/mL, and can be selected to be 2mg/mL-4mg/mL.

6. The collagen composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable excipients include water for injection and sodium chloride;

alternatively, the collagen composition is an isotonic solution.

7. The collagen composition according to any one of claims 1 to 6, wherein the collagen composition has an average degree of cross-linking of 30% to 80%, optionally 50% to 70%.

8. The collagen composition according to any one of claims 1 to 7, wherein the collagen composition has a mass concentration of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) of no more than 0.5 μg/g, optionally no more than 0.4 μg/g; and/or

The mass concentration of N-hydroxysuccinimide (NHS) is not more than 3. Mu.g/g, alternatively not more than 2.5. Mu.g/g.

9. The collagen composition according to any one of claims 1 to 8, wherein the collagen composition has a thermal denaturation temperature (DSC) of 50 ℃ to 75 ℃, optionally 51.6 ℃ to 73.5 ℃.

10. The collagen composition according to any one of claims 1 to 9, wherein the collagen composition has a viscosity of 800mpa.s to 11000mpa.s, optionally of 800mpa.s to 10500mpa.s.

11. Collagen composition according to any one of claims 1 to 10, wherein the collagen composition has a push force of 3N-10N, optionally 3N-8.5N.

12. The collagen composition according to any one of claims 1 to 11, wherein the first modified collagen, the second modified collagen and the third modified collagen are each independently type I collagen, optionally modified type I collagen.

13. A method for preparing modified collagen, which at least comprises the following two crosslinking steps:

14. The method according to claim 13, wherein in the step (1), the pH is adjusted to 7 to 7.5.

15. The method according to claim 13 or 14, wherein, in the chemical crosslinking reaction,

The mass concentration of NHS in the crosslinking liquid is 10mg/mL-120mg/mL;

The pH value of the crosslinking liquid is 5.5-7.0;

the time of the chemical crosslinking reaction is 2-12 h;

The temperature of the chemical crosslinking reaction is 20-25 ℃.

16. The method according to any one of claims 13 to 15, wherein the method comprises at least any one, two or three steps of step A of preparing a first modified collagen having a degree of crosslinking of 30% to 50%, step B of preparing a second modified collagen having a degree of crosslinking of more than 50% and not more than 70%, step C of preparing a third modified collagen having a degree of crosslinking of more than 70% and not more than 80%,

In the step A, in the chemical crosslinking reaction,

Enabling the mass concentration of the EDC in a chemical crosslinking reaction system to be 20mg/mL-80mg/mL;

the mass concentration of the NHS in the chemical crosslinking reaction system is 10mg/mL-70mg/mL;

The time of the chemical crosslinking reaction is 2-4 h;

The temperature of the chemical crosslinking reaction is 20-25 ℃.

In the step B, in the chemical crosslinking reaction,

Enabling the mass concentration of the EDC in a chemical crosslinking reaction system to be 60mg/mL-140mg/mL;

The mass concentration of the NHS in the chemical crosslinking reaction system is 50mg/mL-100mg/mL;

the time of the chemical crosslinking reaction is 6-8 hours;

The temperature of the chemical crosslinking reaction is 20-25 ℃.

In the step C, in the chemical crosslinking reaction,

Enabling the mass concentration of the EDC in a chemical crosslinking reaction system to be 110mg/mL-160mg/mL;

The mass concentration of the NHS in the chemical crosslinking reaction system is 80mg/mL-120mg/mL;

The time of the chemical crosslinking reaction is 10-12 h;

The temperature of the chemical crosslinking reaction is 20-25 ℃.

17. The method of any one of claims 13 to 16, wherein the type I collagen is present in the buffer at a mass concentration of 2mg/mL to 3mg/mL.

18. The method of any one of claims 13 to 17, wherein the amount of hetero-protein in the type I collagen is no more than 0.3%, optionally no more than 0.2%.

19. The method of any one of claims 13 to 18, wherein the type I collagen is prepared by a process comprising the steps of:

and (3) performing pretreatment, enzymolysis, crude extraction, purification and filtration sterilization treatment on the cow leather and/or the cow achilles tendon to obtain the type I collagen.

20. A method of preparing a collagen composition according to any one of claims 1 to 12, comprising: and mixing the first modified collagen, the second modified collagen and the third modified collagen, and homogenizing to obtain the collagen compound.

21. The method according to claim 20, wherein the first modified collagen is prepared as a first protein solution containing the first modified collagen at a mass concentration of 20mg/mL to 50mg/mL and an anesthetic at a mass concentration of 1mg/mL to 5 mg/mL;

Preparing second modified collagen into a second protein liquid, wherein the second protein liquid comprises second modified collagen with the mass concentration of 20mg/mL-50mg/mL and anesthetic with the mass concentration of 1mg/mL-5 mg/mL;

preparing a third modified collagen into a third protein liquid, wherein the third protein liquid comprises the third modified collagen with the mass concentration of 20mg/mL-50mg/mL and an anesthetic with the mass concentration of 1mg/mL-5 mg/mL;

mixing the first protein solution, the second protein solution and the third protein solution to obtain a total solution, wherein,

The mass ratio of the first protein liquid in the total liquid is 10% -40%;

the mass ratio of the second protein liquid in the total liquid is 30% -80%;

The mass ratio of the third protein liquid in the total liquid is 10% -50%.

22. The method according to claim 21, wherein the mass concentration of the first modified collagen in the first protein solution is 35mg/mL ± 3.5mg/mL;

the mass concentration of the second modified collagen in the second protein solution is 35 mg/mL+/-3.5 mg/mL; and/or

The mass concentration of the third modified collagen in the third protein solution is 35 mg/mL+/-3.5 mg/mL.

23. The method of claim 21 or 22, wherein the anesthetic is present in the first, second or third protein solutions in an amount of 0.1-0.5%, optionally 0.2-0.4% by mass;

The anesthetic agent comprises lidocaine.

24. The method according to any one of claims 21 to 23, wherein the first protein solution, the second protein solution, and the third protein solution are prepared using physiological saline.

25. The method according to any one of claims 20 to 24, wherein the homogenizing treatment is performed using a homogenizer having a pore size of 1000 μm, 500 μm and/or 100 μm.

26. Use of the collagen composition according to any one of claims 1 to 12, the modified collagen prepared by the method according to any one of claims 13 to 19, the collagen composition prepared by the method according to any one of claims 20 to 25, in the preparation of an injection for filling human tissue.

27. The use of claim 26, wherein the tissue augmentation comprises facial tissue augmentation.

28. The use according to claim 26 or 27, comprising the use in the preparation of an injection for improving facial wrinkles and depressions.

29. Use of the collagen composition according to any one of claims 1 to 12, the modified collagen prepared by the method according to any one of claims 13 to 19, the collagen composition prepared by the method according to any one of claims 20 to 25, in the preparation of a filler for cosmetic shaping of the human body.