Collagen composition and preparation method and use thereof

Abstract

The present disclosure provides a collagen composition and a preparation method and use thereof, wherein the injection comprises a first modified collagen with a cross-linking degree of 30%-50%, a second modified collagen with a cross-linking degree greater than 50% and less than 70%, a third modified collagen with a cross-linking degree greater than 70% and less than 80%, and pharmaceutically acceptable excipients. The collagen composition can be used for human tissue filling to reduce skin wrinkles and depressions at the injection site.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a collagen composition and a preparation method and application thereof. Background Art

[0002] The structure of soft tissue in the human body is maintained by proteins such as collagen elastin and extracellular matrix containing glycosaminoglycans. Various factors can cause soft tissue defects, which can usually be restored or corrected by surgical or non-surgical injection of biological tissues or synthetic compounds. At present, there are many types of fillers commonly used in the field of cosmetic surgery, especially facial fillers, such as autologous fat, hyaluronic acid, hyaluronic acid, etc. Autologous fat is non-toxic and harmless, and will not produce immune reactions and rejection reactions, but it will cause unevenness and even the formation of nodules in the filled area; while hyaluronic acid and hyaluronic acid are prone to rejection reactions, and thus inflammation and scar formation.

[0003] Collagen is the most abundant and widely distributed functional protein in mammals. It is a filler and support for tissues, accounting for 25%-30% of the total protein in the body. Due to its good biocompatibility and biodegradability, it is increasingly used in plastic surgery, such as filling soft tissue to remove wrinkles and improving the contour of the injection site. Gels directly prepared from natural collagen materials have defects such as fast degradation and poor mechanical properties, which limit their application. In the prior art, collagen is generally treated by chemical cross-linking to improve its mechanical properties and increase degradation time.

[0004] Cross-linking 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, which leads to biological toxicity and allergenicity. Chinese patent document CN101648989A discloses a method for manufacturing long-acting collagen, which reduces the amount of glutaraldehyde residue by cross-linking collagen with low concentrations of glutaraldehyde. However, glutaraldehyde-cross-linked collagen usually appears yellow and is prone to calcification after implantation in human tissue, which leads to undesirable hardening or degradation of the implant.

[0005] In addition, after chemical cross-linking, the viscoelasticity and mechanical properties of collagen gel are improved, which not only increases the difficulty of industrial preparation, but also increases the difficulty of injecting collagen into tissues, which is not conducive to clinical injection operations.

[0006] Therefore, it is necessary to provide a collagen composition for tissue augmentation. Summary of the invention

[0007] The present disclosure provides a collagen composition, a preparation method and use thereof. The collagen composition comprises collagens with different degrees of cross-linking, so that the collagen composition has good tissue filling effect, degradation cycle and operability, and improves the satisfaction of test patients.

[0008] The first aspect of the present disclosure provides a collagen composition comprising:

[0009] (1) a first modified collagen having a cross-linking degree of 30% to 50%,

[0010] (2) a second modified collagen having a cross-linking degree greater than 50% and less than 70%,

[0011] (3) a third modified collagen having a cross-linking degree greater than 70% and less than 80%, and

[0012] (4) pharmaceutically acceptable excipients;

[0013] Wherein, based on the total mass of the collagen composition,

[0014] The mass proportion of the first modified collagen in the collagen composition is 0.1%-2.0%;

[0015] The mass proportion of the second modified collagen in the collagen composition is 0.5%-4.0%;

[0016] The mass proportion of the third modified collagen in the collagen composition is 0.1%-2.0%.

[0017] In any embodiment, in the collagen composition, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.2%-2.0%; the mass proportion of the second modified collagen in the collagen composition is 0.6%-3.6%; the mass proportion of the third modified collagen in the collagen composition is 0.25%-1.75%.

[0018] In any embodiment, in the collagen composition, the mass concentration of the first modified collagen in the collagen composition is 2-20 mg / mL; the mass concentration of the second modified collagen in the collagen composition is 5-40 mg / mL, optionally 6 mg / mL-36 mg / mL; and / or the mass concentration of the third modified collagen in the collagen composition is 2 mg / mL-25 mg / mL, optionally 2.5 mg / mL-17.5 mg / mL.

[0019] In any embodiment, the collagen composition includes an anesthetic, and the anesthetic includes lidocaine. In any embodiment, the mass proportion of the anesthetic in the collagen composition is 0.1%-0.5%, and can be 0.2%-0.4%. In any embodiment, the mass concentration of the anesthetic in the collagen composition is 1mg / mL-5mg / mL, and can be 2mg / mL-4mg / mL. The anesthetic in the collagen composition helps to reduce the pain of the subject when receiving tissue injection.

[0020] In any embodiment, the pharmaceutically acceptable excipients include water for injection and sodium chloride. Optionally, the collagen composition is an isotonic solution, which is beneficial to reduce the impact of the injection solution on cells in the blood at the injection site and reduce adverse reactions caused by cell atrophy or rupture.

[0021] In any embodiment, the average cross-linking degree of the collagen composition is 30%-80%, and optionally 50%-70%, which helps to further improve the filling effect, degradation cycle and operability of the collagen composition.

[0022] In any embodiment, in the collagen composition, the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) does not exceed 0.5 μg / g, optionally, does not exceed 0.4 μg / g. In any embodiment, the mass concentration of N-hydroxysuccinimide (NHS) does not exceed 3 μg / g, optionally, does not exceed 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 improve the safety of the collagen composition.

[0023] In any embodiment, the thermal denaturation temperature (DSC) of the collagen composition is 50°C-75°C, and can be optionally 51.6°C-73.5°C. Thus, the molecular weight and cross-linking degree of the modified collagen in the collagen composition can be further adjusted so that the collagen composition has a good degradation cycle and operability.

[0024] In any embodiment, the viscosity of the collagen composition is 8000mPa.s-11000mPa.s, and can be optionally 8000mPa.s-10500mPa.s, which can further improve the fluidity of the collagen composition, thereby improving the operability and filling effect of the collagen composition.

[0025] In any embodiment, the pushing force of the collagen composition is 3N-10N, and can be optionally 3N-8.5N, which can further improve the operability of the collagen composition and facilitate clinical use.

[0026] In any embodiment, the first modified collagen, the second modified collagen and the third modified collagen are each independently selected from type I collagen, and can be modified type I collagen, which contains a triple helix structure that improves the filling and supporting effect of the collagen composition.

[0027] The second aspect of the present disclosure provides a method for preparing modified collagen, which comprises at least the following two cross-linking steps:

[0028] (1) Physical cross-linking: providing a buffer solution containing type I collagen, adjusting the pH value to 6.0-9.0 to perform a cross-linking reaction, and obtaining a physically cross-linked product;

[0029] (2) Chemical crosslinking: The physically crosslinked product prepared in (1) is placed in a crosslinking solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to undergo a crosslinking reaction to obtain a modified collagen with a crosslinking degree of 30%-80%.

[0030] The method is prepared by using a cross-linking solution including EDC and NHS, avoiding the use of a glutaraldehyde cross-linking agent, thereby avoiding the toxicity problem caused by glutaraldehyde residues, and also overcoming the problem of yellowing of collagen caused by glutaraldehyde as a cross-linking agent.

[0031] In any embodiment, in step (1), the pH value is adjusted to 7.0-7.5.

[0032] In any embodiment, in the chemical cross-linking reaction,

[0033] The mass concentration of EDC in the cross-linking solution is 20 mg / mL-160 mg / mL;

[0034] The mass concentration of NHS in the cross-linking solution is 10 mg / mL-120 mg / mL;

[0035] The mass concentration ratio of EDC to NHS in the cross-linking solution is 1.1-2.0; and / or

[0036] The pH value of the cross-linking solution is 5.5-7.0;

[0037] The chemical cross-linking reaction time is 2h-12h;

[0038] The temperature of the chemical cross-linking reaction is 20°C-25°C.

[0039] In any embodiment, the method comprises at least one, two or three of step A of preparing a first modified collagen with a cross-linking degree of 30%-50%, step B of preparing a second modified collagen with a cross-linking degree greater than 50% and less than 70%, and step C of preparing a third modified collagen with a cross-linking degree greater than 70% and less than 80%.

[0040] In the chemical crosslinking reaction of step A, the mass concentration of EDC in the chemical crosslinking reaction system is 20 mg / mL-80 mg / mL; the mass concentration of NHS in the chemical crosslinking reaction system is 10 mg / mL-70 mg / mL; the time of the chemical crosslinking reaction is 2h-4h; the temperature of the chemical crosslinking reaction is 20°C-25°C; in the chemical crosslinking reaction of step B, the mass concentration of EDC in the chemical crosslinking reaction system is 60 mg / mL-140 mg / mL; the mass concentration of NHS in the chemical crosslinking reaction system is 10 mg / mL-70 mg / mL; the time of the chemical crosslinking reaction is 2h-4h; the temperature of the chemical crosslinking reaction is 20°C-25°C; The mass concentration in the chemical crosslinking reaction system is 50mg / mL-100mg / mL; the time of the chemical crosslinking reaction is 6h-8h; the temperature of the chemical crosslinking reaction is 20℃-25℃; in the chemical crosslinking reaction of step C, the mass concentration of EDC in the chemical crosslinking reaction system is 110mg / mL-160mg / mL; the mass concentration of NHS in the chemical crosslinking reaction system is 80mg / mL-120mg / mL; the time of the chemical crosslinking reaction is 10h-12h; the temperature of the chemical crosslinking reaction is 20℃-25℃. The method can prepare modified proteins with different crosslinking degrees.

[0041] In any embodiment, the mass concentration of type I collagen in the buffer is 2 mg / mL-3 mg / mL.

[0042] In any embodiment, the mass proportion of impurity proteins in the type I collagen does not exceed 0.3%, and optionally, does not exceed 0.2%, which is helpful for preparing modified collagen with good quality.

[0043] In any embodiment, the type I collagen is prepared by a method comprising the following steps: pre-treating, enzymatically hydrolyzing, crudely extracting, purifying and filtering and sterilizing cowhide and / or cow Achilles tendon to obtain type I collagen.

[0044] The third aspect of the present disclosure provides a method for preparing a collagen composition, comprising: mixing a first modified collagen, a second modified collagen and a third modified collagen, and then homogenizing them to obtain a collagen composition. The preparation method has industrial feasibility and can realize industrialized production.

[0045] In any embodiment, the first modified collagen is prepared as a first protein liquid, which contains the first modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; the second modified collagen is prepared as a second protein liquid, which contains the second modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; the third modified collagen is prepared as a third protein liquid, which contains the third modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; the first protein liquid, the second protein liquid and the third protein liquid are mixed to obtain a total liquid, wherein the mass proportion of the first protein liquid in the total liquid is 10%-40%; the mass proportion of the second protein liquid in the total liquid is 30%-80%; the mass proportion of the third protein liquid in the total liquid is 10%-50%. By preparing modified collagens with different cross-linking degrees into solutions and then mixing them, the difficulty of mixing modified collagens with different cross-linking degrees can be reduced; at the same time, in the prepared collagen composition, collagens with different cross-linking degrees have different degradation times, supporting effects and fluidity, which is conducive to obtaining a collagen composition that can take into account degradation cycle, filling effect and operability.

[0046] In any embodiment, the mass concentration of the first modified collagen in the first protein solution is 35 mg / mL±3.5 mg / 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.

[0047] In any embodiment, the mass proportion of the anesthetic in the first protein solution, the second protein solution or the third protein solution is 0.1%-0.5%, and optionally 0.2%-0.4%. In any embodiment, the anesthetic includes lidocaine.

[0048] In any embodiment, using physiological saline to prepare the first protein solution, the second protein solution and the third protein solution can reduce the difference in osmotic pressure between the collagen composition and human blood, thereby reducing adverse reactions caused thereby.

[0049] In any embodiment, the homogenization process using a homogenizer with a pore size of 1000 μm, 500 μm and / or 100 μm is helpful to improve the dispersion uniformity of the modified collagen.

[0050] The fourth aspect of the present disclosure provides use of the collagen composition of the first aspect, the modified collagen prepared by the method of the second aspect, and the collagen composition prepared by the method of the third aspect in preparing an injection for human tissue filling.

[0051] In any embodiment, the tissue augmentation comprises facial tissue augmentation.

[0052] In any embodiment, the use includes use in preparing an injection for improving facial wrinkles and depressions.

[0053] The fifth aspect of the present disclosure provides the use of the collagen composition of the first aspect, the modified collagen prepared by the method described in the second aspect, and the collagen composition prepared by the method described in the third aspect in preparing a filler for human body cosmetic surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown are pathological sections of rabbits after one month of collagen injection;

[0055] Figure 2 Shown are pathological sections of rabbits 6 months after receiving collagen injection;

[0056] Figure 3 Shown are pathological sections of rabbits 12 months after receiving collagen injection;

[0057] Figure 4 A photograph showing the appearance of modified collagen D8 placed in a syringe;

[0058] Figure 5 The comparative photos of the appearance of the collagen injection extrudates prepared in Example 9 and Comparative Example 4 are shown;

[0059] Figure 6 Comparative photos showing the uniformity of the collagen injection solutions prepared in Example 9 and Comparative Example 4. DETAILED DESCRIPTION

[0060] Unless otherwise indicated, all numbers used in this specification and claims to indicate content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. should be understood as modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the attached claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through the present disclosure, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0061] Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximate, the numerical values ​​set forth in the specific embodiments are provided as accurately as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include each narrower numerical range that falls within the broader numerical range, just as if these narrower numerical ranges were all clearly written herein.

[0062] 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. The meanings of similar expressions can be deduced analogously.

[0063] [Collagen composition]

[0064] The first aspect of the present disclosure provides a collagen composition, comprising:

[0065] (1) a first modified collagen having a cross-linking degree of 30% to 50%,

[0066] (2) a second modified collagen having a cross-linking degree greater than 50% and less than 70%,

[0067] (3) a third modified collagen having a cross-linking degree greater than 70% and less than 80%, and

[0068] (4) Pharmaceutically acceptable excipients.

[0069] The collagen composition provided by the present disclosure can be used for tissue filling to achieve body shaping and cosmetic surgery. Optionally, the collagen composition is in liquid form or semi-solid form. In some embodiments, the collagen composition is in gel form. In some embodiments, the collagen composition is in hydrogel form.

[0070] In this article, "modified collagen" refers to collagen that has been modified. Compared with natural collagen, the amino and / or carboxyl groups in the collagen molecules form stable connections within or between collagen peptide chains, increasing the cross-linking degree of collagen, thereby improving the mechanical strength, elastic modulus or heat resistance of collagen.

[0071] In some embodiments, in the collagen composition, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.1%-2.0%; the mass proportion of the second modified collagen in the collagen composition is 0.5%-4.0%; the mass proportion of the third modified collagen in the collagen composition is 0.1%-2.0%.

[0072] Modified collagen with different cross-linking degrees has different degradation times after implantation into a subject (e.g., human). Generally, modified collagen with a low cross-linking degree has a shorter degradation time, but the hardness of modified collagen with a low cross-linking degree is also lower than that of modified collagen with a high cross-linking degree, resulting in poor support.

[0073] In the collagen composition provided by the present disclosure, the first modified collagen has good fluidity, which can be completely degraded in 1-2 months, preventing local collapse of the injection site caused by excessive degradation, and improving the shaping effect. Therefore, the first modified collagen can improve the fluidity and softening filling support effect of the composition; but the degradation cycle of the first modified collagen is short, which is not conducive to reducing the frequency of surgery. The degradation cycle of the second modified collagen is higher than that of the first modified collagen. When the second modified collagen degrades in the body, the natural regeneration of collagen in the subject's body is accompanied, which can at least partially offset the deterioration of the filling support effect caused by the degradation of the collagen composition; in addition, the second modified collagen has good fluidity. Adding the second modified collagen to the collagen composition helps the collagen composition to take into account the filling support effect, fluidity and good degradation time. The third modified collagen has a large molecular weight and a large hardness, so it can provide excellent support and a long-lasting degradation cycle, but the edge of the injection site is obvious and the shaping effect is poor. In addition, the viscosity of the third modified collagen is increased, the fluidity is deteriorated, and it is not conducive to clinical operation. In the collagen composition provided by the present invention, modified collagens with different degrees of cross-linking are used in a coordinated manner, so that the collagen composition can have good filling and supporting effects, clinical operability and degradation cycle, which not only improves the plastic surgery effect, but also reduces the pain of the patients and improves their satisfaction.

[0074] In some embodiments, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.2%-2.0%, 0.4%-2.0%, 0.6%-2.0% or 0.2%-1.5%. In some embodiments, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.5%, 0.7%, 0.9%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0% or the range between any two of the above values. This helps to further improve the fluidity of the collagen composition, improve the softening effect of cosmetic shaping and reduce the pushing force of the composition during injection. In some embodiments, based on the total mass of the collagen composition, the mass proportion of the second modified collagen in the collagen composition is 0.6%-3.6%, 0.8%-3.6%, 0.6%-3.0% or 1.0%-3.6%. In some embodiments, based on the total mass of the collagen composition, the mass proportion of the second modified collagen in the collagen composition is 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 the range between any two of the above values. This helps to further improve the support performance and fluidity of the collagen composition, while having a good degradation cycle.

[0075] In some embodiments, based on the total mass of the collagen composition, the mass proportion of the third modified collagen in the collagen composition is 0.25%-1.75%, 0.4%-1.75%, 0.5%-1.5% or 0.5%-1.75%. In some embodiments, based on the total mass of the collagen composition, the mass proportion of the third modified collagen in the collagen composition is 0.5%, 0.7%, 0.9%, 1.0%, 1.3%, 1.5%, 1.6% or the range between any two of the above values, which helps to further improve the degradation cycle and support performance of the composition.

[0076] In some embodiments, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.2%-2.0%; the mass proportion of the second modified collagen in the collagen composition is 0.6%-3.6%; the mass proportion of the third modified collagen in the collagen composition is 0.25%-1.75%, which helps to further balance the supporting performance and fluidity of the collagen composition and extend the degradation cycle.

[0077] In some embodiments, in the collagen composition, based on the total mass of the collagen composition, the mass proportion of the first modified collagen in the collagen composition is 0.2%-1.2%; the mass proportion of the second modified collagen in the collagen composition is 0.6%-3.6%; the mass proportion of the third modified collagen in the collagen composition is 0.25%-1.20%.

[0078] In some embodiments, in the collagen composition, the mass concentration of the first modified collagen in the collagen composition is 2mg / mL-20mg / mL. In some embodiments, the mass concentration of the first modified collagen in the collagen composition is 2mg / mL-18mg / mL, 2mg / mL-15mg / mL, 2mg / mL-13mg / mL, 2mg / mL-10mg / mL or 5mg / mL-20mg / mL. In some embodiments, the mass concentration of the first modified collagen in the collagen composition is 3mg / mL, 5mg / mL, 7mg / mL, 9mg / mL, 12mg / mL, 15mg / mL, 17mg / mL, 19mg / mL or a range between any two of the above values.

[0079] In some embodiments, in the collagen composition, the mass concentration of the second modified collagen in the collagen composition is 4mg / mL-40mg / mL. In some embodiments, the mass concentration of the second modified collagen in the collagen composition is 5mg / mL-40mg / mL, 10mg / mL-40mg / mL, 4mg / mL-35mg / mL, 4mg / mL-30mg / mL or 10mg / mL-35mg / mL. In some embodiments, the mass concentration 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 the range between any two of the above values.

[0080] In some embodiments, in the collagen composition, the mass concentration of the third modified collagen in the collagen composition is 2 mg / mL-25 mg / mL. In some embodiments, the mass concentration of the third modified collagen in the collagen composition is 5 mg / mL-25 mg / mL, 10 mg / mL-25 mg / mL, 2 mg / mL-20 mg / mL, 5 mg / mL-20 mg / mL or 2 mg / mL-15 mg / mL. In some embodiments, the mass concentration of the third modified collagen in the collagen composition is 3 mg / mL, 6 mg / mL, 9 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 22 mg / mL or a range between any two of the above values.

[0081] The concentration of modified collagen can be determined using any method known in the art, such as the Kjeldahl method.

[0082] The mass concentration and mass ratio of the first modified collagen, the second modified collagen and the third modified collagen within the above range further improves the flow properties, degradation cycle and filling and supporting effect of the collagen composition.

[0083] In some embodiments, the collagen composition comprises an anesthetic, and the anesthetic comprises lidocaine.

[0084] In some embodiments, the mass proportion of the anesthetic in the collagen composition is 0.1%-0.5%, and optionally 0.2%-0.4%. The anesthetic in the collagen composition helps to reduce the pain felt by the subject when receiving tissue injection.

[0085] In some embodiments, the mass concentration of the anesthetic in the collagen composition is 1 mg / mL-5 mg / mL, and can be 2 mg / mL-4 mg / mL. In some embodiments, the mass concentration of the anesthetic in the collagen composition is 3 mg / mL, which can not only reduce the pain of the subject during surgery, but also reduce the adverse reactions caused by excessive anesthetic, such as cardiovascular side effects and allergic reactions.

[0086] In some embodiments, the pharmaceutically acceptable excipients in the collagen composition include water for injection and sodium chloride, and the sodium chloride is dissolved in the water for injection and dispersed in the collagen composition. In some embodiments, the collagen composition is an isotonic solution, and optionally, the mass concentration of the sodium chloride in the collagen composition is 0.9 g / 100 mL, which is conducive to reducing the effect of the collagen composition on blood cells at the injection site and reducing adverse reactions caused by cell atrophy or rupture.

[0087] In some embodiments, the average cross-linking degree of the collagen composition is 30%-80%. In some embodiments, the average cross-linking degree of the collagen composition is 35%-70%, 55%-70%, 40%-80% or 35%-80%. In some embodiments, the average cross-linking degree 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 operability of the collagen composition.

[0088] The collagen composition is prepared using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as chemical crosslinking agents, avoiding the use of glutaraldehyde crosslinking agents commonly used in the prior art, making the collagen composition provided by the present disclosure whiter and more beautiful. At the same time, the toxic reaction caused by glutaraldehyde residue is also avoided, thereby improving the safety of the collagen composition.

[0089] The collagen composition has a lower residual content of cross-linking agent, thereby improving the safety and quality of the collagen composition. In some embodiments, the mass concentration of EDC in the collagen composition does not exceed 0.5 μg / g. In some embodiments, the mass concentration of EDC in the collagen composition does not exceed 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 does not exceed 3 μg / g. In some embodiments, the mass concentration of NHS in the collagen composition does not exceed 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.

[0090] In some embodiments, the thermal denaturation temperature (DSC) of the collagen composition is 50°C-75°C. In some embodiments, the DSC of the collagen composition is 51.6°C-73.5°C. In some embodiments, the DSC of the collagen composition is 55°C, 60°C, 65°C, 70°C, 75°C or a range between any two of the above values. The thermal denaturation temperature can reflect the thermal stability of the collagen composition. The higher the thermal denaturation temperature, the stronger the heat resistance of the collagen composition, the less likely the microstructure of the collagen molecule (for example, the triple helix structure) will change, the more conducive to the water absorption of the collagen molecule itself, and the collagen composition. Maintain good elasticity, thereby improving the shaping effect of the composition.

[0091] The thermal denaturation temperature (DSC) has a well-known meaning in the art, and can be measured using methods known in the art. As an example, it can be measured by referring to the melting point determination method in Appendix D of YY / T 0954-2015 "Passive Surgical Implant Type I Collagen Implant".

[0092] In some embodiments, the viscosity of the collagen composition is 8000mPa.s-11000mPa.s. In some embodiments, the viscosity of the collagen composition is 8000mPa.s-10500mPa.s, 8050mPa.s-10500mPa.s, 8300mPa.s-10000mPa.s, 9000mPa.s-10500mPa.s. In some embodiments, the viscosity of the collagen composition is 8000mPa.s, 8500mPa.s, 9000mPa.s, 95000mPa.s, 10000mPa.s, 10500mPa.s, or a range between any two of the above values, which can further improve the fluidity of the collagen composition, thereby improving the operability and filling effect of the collagen composition.

[0093] Viscosity has a well-known meaning in the art, reflecting the viscosity of a fluid. The viscosity of the collagen composition can be measured using methods known in the art. For example, a viscometer can be used to measure the viscosity according to the method disclosed in Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0094] The collagen composition provided by the present disclosure has good pushing force, which can reflect the operability of the collagen composition when used. In the field of medical beauty, a 27G or 30G syringe needle is generally used for tissue injection, especially facial injection filling, to reduce facial wounds. The inner diameter of the syringe needle is small, so the magnitude of the pushing force can directly affect the operability of clinical use.

[0095] In some embodiments, the pushing force of the collagen composition is 3N-10N. In some embodiments, the pushing force of the collagen composition is 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 can further improve the fluidity of the collagen composition, thereby improving the intraoperative operability of the collagen composition and improving clinical convenience.

[0096] 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 type I collagen helps to absorb water, retain moisture, and repair damage, which is beneficial to improving the filling and supporting effect of the collagen composition in the body and promoting wound repair.

[0097] [Method for preparing modified collagen]

[0098] The second aspect of the present disclosure provides a method for preparing modified collagen, the method comprising at least the following two cross-linking steps:

[0099] (1) Physical cross-linking: providing a buffer solution containing type I collagen, adjusting the pH value to 6.0-9.0 to perform a cross-linking reaction, and obtaining a physically cross-linked product;

[0100] (2) Chemical cross-linking: The physically cross-linked product prepared in (1) is placed in a cross-linking solution containing EDC and NHS to carry out a cross-linking reaction, so as to obtain a modified collagen with a cross-linking degree of 30%-80%.

[0101] The method is prepared by using a cross-linking solution including EDC and NHS, avoiding the use of a glutaraldehyde cross-linking agent, thereby avoiding the toxicity problem caused by glutaraldehyde residues and the problem of yellowing of the collagen appearance.

[0102] In some embodiments, in step (1), the mass concentration of type I collagen in the buffer solution is 2 mg / mL-3 mg / mL, which is beneficial to improve the reaction yield.

[0103] Type I collagen can be obtained by using commercial products or by extraction using methods in the prior art. In some embodiments, the type I collagen is obtained by a method comprising the following steps: pre-treating cowhide and / or cow Achilles tendon, enzymolysis, crude extraction, purification, and filtration sterilization to obtain type I collagen.

[0104] During the extraction process, other proteins other than type I collagen, i.e., impurities, may remain in type I collagen, affecting the quality of collagen. In some embodiments, the weight percentage of impurities in type I collagen does not exceed 0.3%. In some embodiments, the weight percentage of impurities in type I collagen does not exceed 0.25%, 0.20%, 0.15% or 0.10%, which helps to prepare modified collagen with good quality.

[0105] In some embodiments, the buffer in step (1) can be a phosphate buffered saline (PBS), such as a PBS buffer of pH 6.8, a PBS buffer of pH 7.0, or a PBS buffer of pH 7.3. The buffer can be prepared using a method commonly used in the art, for example, with reference to the method disclosed in Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0106] In some embodiments, in step (1), an acidic or alkaline solution can be used to adjust the pH value of the buffer (or reaction system). In some embodiments, an alkaline solution can be used to adjust the pH value of the physical crosslinking buffer (or reaction system) to 7-7.5. The alkaline solution can use any alkaline solution in the prior art, preferably an alkali metal salt solution, such as sodium hydroxide solution, potassium hydroxide solution.

[0107] In some embodiments, the reaction time of physical crosslinking of type I collagen in a buffer is 30 min-120 min, optionally, 30 min-100 min, 30 min-80 min, 30 min-60 min. In some embodiments, the reaction time of physical crosslinking of type I collagen in a buffer is 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min or the range between any two of the above values.

[0108] Through physical cross-linking, collagen fibers can be polymerized, which does not involve covalent bonding and can be reversibly polymerized and depolymerized. Physical cross-linking can reduce the distance between collagen fibers, help promote the chemical cross-linking reaction between collagen fibers, and improve the cross-linking degree and reaction efficiency of the chemical cross-linking reaction.

[0109] In some embodiments, in the chemical cross-linking reaction, the mass concentration of EDC in the cross-linking solution is 20 mg / mL-160 mg / mL. In some embodiments, the mass concentration of EDC in the cross-linking solution is 40 mg / mL-160 mg / mL, 60 mg / mL-160 mg / mL or 100 mg / mL-160 mg / mL. In some embodiments, the mass concentration of EDC in the cross-linking solution is 30 mg / mL, 50 mg / mL, 70 mg / mL, 90 mg / mL, 120 mg / mL, 150 mg / mL or a range between any two of the above values.

[0110] In some embodiments, in the chemical cross-linking reaction, the mass concentration of NHS in the cross-linking solution is 10 mg / mL-120 mg / mL. In some embodiments, the mass concentration of NHS in the cross-linking solution is 30 mg / mL-120 mg / mL, 50 mg / mL-120 mg / mL, 70 mg / mL-120 mg / mL or 90 mg / mL-120 mg / mL. In some embodiments, the mass concentration of NHS in the cross-linking solution is 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, 115 mg / mL or a range between any two of the above values.

[0111] In some embodiments, the mass concentration ratio of EDC to NHS in the cross-linking solution is 1.1-2.0. In some embodiments, the mass concentration ratio of EDC to NHS in the cross-linking solution is 1.1-1.5. In some embodiments, the mass concentration ratio of EDC to NHS in the cross-linking solution 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 above values. The amount and amount ratio of EDC to NHS in a suitable range helps promote the chemical cross-linking reaction.

[0112] In some embodiments, the pH value of the cross-linking solution is 5.5-7.0. In some embodiments, the pH value of the cross-linking solution is 6.0, 6.5, 7.0 or a range between any two of the above values.

[0113] In some embodiments, the time of the chemical crosslinking reaction is 2h-12h. In some embodiments, the time of the chemical crosslinking reaction is 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h or a range between any two of the above values. In some embodiments, the temperature of the chemical crosslinking reaction is 20°C-25°C.

[0114] Chemical cross-linking reaction can promote the full progress of the reaction under appropriate pH conditions, reaction temperature and reaction time, and improve the cross-linking degree and reaction efficiency of the modified collagen.

[0115] In some embodiments, the method includes at least one, two or three of the following steps: step A for preparing a first modified collagen with a cross-linking degree of 30%-50%, step B for preparing a second modified collagen with a cross-linking degree greater than 50% and less than 70%, and step C for preparing a third modified collagen with a cross-linking degree greater than 70% and less than 80%.

[0116] In some embodiments, in the chemical cross-linking reaction of step A, the mass concentration of EDC in the chemical cross-linking reaction system is 20 mg / mL-80 mg / mL; the mass concentration of NHS in the chemical cross-linking reaction system is 10 mg / mL-70 mg / mL; the time of the chemical cross-linking reaction is 2h-4h; and the temperature of the chemical cross-linking reaction is 20°C-25°C.

[0117] In some embodiments, in the chemical cross-linking reaction of step A, the mass concentration of EDC in the chemical cross-linking reaction system is 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL or a range between any two of the above values.

[0118] In some embodiments, in the chemical cross-linking reaction of step A, the mass concentration of NHS in the chemical cross-linking reaction system is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL or a range between any two of the above values.

[0119] In some embodiments, in the chemical cross-linking reaction of step A, the time of the chemical cross-linking reaction is 2 h, 2.5 h, 3 h, 3.5 h, 4 h or a range between any two of the above values.

[0120] The preparation method including step A can obtain a first modified collagen with a cross-linking degree of 30%-50%.

[0121] In some embodiments, in the chemical cross-linking reaction of step B, the mass concentration of EDC in the chemical cross-linking reaction system is 60 mg / mL-140 mg / mL; the mass concentration of NHS in the chemical cross-linking reaction system is 50 mg / mL-100 mg / mL; the time of the chemical cross-linking reaction is 6h-8h; and the temperature of the chemical cross-linking reaction is 20°C-25°C.

[0122] In some embodiments, in the chemical crosslinking reaction of step B, the mass concentration of EDC in the chemical crosslinking reaction system is 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL or a range between any two of the above values.

[0123] In some embodiments, in the chemical cross-linking reaction of step B, the mass concentration of NHS in the chemical cross-linking reaction system is 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL or a range between any two of the above values.

[0124] In some embodiments, in the chemical cross-linking reaction of step B, the time of the chemical cross-linking reaction is 6 h, 6.5 h, 7 h, 7.5 h, 8 h or a range between any two of the above values.

[0125] The production method including step B can obtain a second modified collagen having a cross-linking degree of greater than 50% and less than 70%.

[0126] In some embodiments, in the chemical cross-linking reaction of step C, the mass concentration of EDC in the chemical cross-linking reaction system is 110 mg / mL-160 mg / mL; the mass concentration of NHS in the chemical cross-linking reaction system is 80 mg / mL-120 mg / mL; the time of the chemical cross-linking reaction is 10h-12h; and the temperature of the chemical cross-linking reaction is 20°C-25°C.

[0127] In some embodiments, in the chemical crosslinking reaction in step C, the mass concentration of EDC in the chemical crosslinking reaction system is 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL or a range between any two of the above values.

[0128] In some embodiments, in the chemical cross-linking reaction of step C, the mass concentration of NHS in the chemical cross-linking reaction system is 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL or a range between any two of the above values.

[0129] In some embodiments, in the chemical cross-linking reaction of step C, the time of the chemical cross-linking reaction is 10 h, 10.5 h, 11 h, 11.5 h, 12 h or a range between any two of the above values.

[0130] The production method including step C can obtain a third modified collagen having a cross-linking degree of greater than 70% and less than 80%.

[0131] By the preparation method including step A, step B and / or step C, modified collagen or collagen composition having different cross-linking degrees can be obtained.

[0132] The preparation method provided by the present disclosure has mild reaction conditions, short reaction cycle, high efficiency, and is suitable for industrial production of modified collagen. The prepared modified collagen has excellent quality and is more beautiful.

[0133] [Method for preparing collagen composition]

[0134] The third aspect of the present disclosure provides a method for preparing a collagen composition, comprising: mixing a first modified collagen, a second modified collagen and a third modified collagen, and then homogenizing them to obtain the collagen composition.

[0135] In some embodiments, the first modified collagen, the second modified collagen, and the third modified collagen are prepared as solutions respectively and then homogenized, which is beneficial to improving the dispersion uniformity of modified collagens with different cross-linking degrees.

[0136] In some embodiments, the first modified collagen is prepared as a first protein solution, wherein the first protein solution comprises the first modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL.

[0137] In some embodiments, the mass concentration of the first modified collagen in the first protein solution is 25 mg / mL-45 mg / mL, 20 mg / mL-40 mg / mL or 30 mg / mL-50 mg / mL. In some embodiments, the mass concentration of the first modified collagen in the first protein solution is 22 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or a range between any two of the above values.

[0138] In some embodiments, the mass concentration of the anesthetic in the first protein solution is 2 mg / mL-4 mg / mL. In some embodiments, the mass concentration of the anesthetic in the first protein solution is 3 mg / mL.

[0139] In some embodiments, the first protein solution comprises a first modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL.

[0140] In some embodiments, the second modified collagen is prepared as a second protein solution, wherein the second protein solution comprises the second modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL.

[0141] In some embodiments, the mass concentration of the second modified collagen in the second protein solution is 25 mg / mL-45 mg / mL, 20 mg / mL-40 mg / mL or 30 mg / mL-50 mg / mL. In some embodiments, the mass concentration of the second modified collagen in the second protein solution is 22 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or a range between any two of the above values.

[0142] In some embodiments, the mass concentration of the anesthetic in the second protein solution is 2 mg / mL-4 mg / mL. In some embodiments, the mass concentration of the anesthetic in the second protein solution is 3 mg / mL.

[0143] In some embodiments, the second protein solution comprises a second modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL.

[0144] In some embodiments, the third modified collagen is prepared as a third protein solution, wherein the third protein solution comprises the third modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and the anesthetic with a mass concentration of 1 mg / mL-5 mg / mL.

[0145] In some embodiments, the mass concentration of the third modified collagen in the third protein solution is 25 mg / mL-45 mg / mL, 20 mg / mL-40 mg / mL or 30 mg / mL-50 mg / mL. In some embodiments, the mass concentration of the third modified collagen in the third protein solution is 22 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or a range between any two of the above values.

[0146] In some embodiments, the mass concentration of the anesthetic in the third protein solution is 2 mg / mL-4 mg / mL. In some embodiments, the mass concentration of the anesthetic in the third protein solution is 3 mg / mL.

[0147] In some embodiments, the third protein solution comprises a third modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL.

[0148] In some embodiments, the first protein solution comprises a first modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL, the second protein solution comprises a second modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL, and the third protein solution comprises a third modified collagen with a mass concentration of (35±3.5) mg / mL and an anesthetic with a mass concentration of (3±0.3) mg / mL.

[0149] The first protein liquid, the second protein liquid and the third protein liquid are mixed to obtain a total liquid, and then homogenized. In some embodiments, the mass proportion of the first protein liquid in the total liquid is 10%-40%; the mass proportion of the second protein liquid in the total liquid is 30%-80%; and the mass proportion of the third protein liquid in the total liquid is 10%-50%.

[0150] The first protein solution, the second protein solution and the third protein solution are prepared and mixed separately, which helps to improve the uniformity of modified collagen with different cross-linking degrees and can make the anesthetic fully dispersed in the collagen injection solution.

[0151] In some embodiments, the mass proportion of the first protein solution in the total solution is 10%, 15%, 20%, 25%, 30%, 35%, 40% or a range between any two of the above values.

[0152] In some embodiments, the mass proportion 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 values.

[0153] In some embodiments, the mass proportion of the third protein solution in the total solution is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or a range between any two of the above values.

[0154] By preparing modified collagens with different cross-linking degrees into solutions and then mixing them, the difficulty of mixing modified collagens with different cross-linking degrees can be reduced, and the dispersion uniformity of the modified collagen can be improved. The preparation method reduces the difficulty of industrial production of modified collagen compositions and can realize industrial production of modified collagen.

[0155] At the same time, by adjusting the mass ratio of the first protein liquid, the second protein liquid and the third protein liquid, the composition of collagens with different degrees of cross-linking in the prepared collagen composition can be adjusted, so as to adjust the degradation time, supporting effect and fluidity of the collagen composition, and obtain a collagen composition that can take into account the degradation cycle, filling effect and operability.

[0156] In some embodiments, the mass proportion of the anesthetic in the first protein solution, the second protein solution or the third protein solution is 0.1%-0.5%, optionally 0.2%-0.4%, for example, 0.3%.

[0157] In some embodiments, the anesthetic includes lidocaine, which has a rapid onset of action and high safety, and is beneficial for reducing pain felt by the subject during surgery.

[0158] In some embodiments, physiological saline or 0.9% sodium chloride injection can be used to prepare the first protein solution, the second protein solution and the third protein solution, which can reduce the difference in osmotic pressure between the collagen composition and human blood, and reduce the adverse reactions and irritation to tissue sites caused thereby.

[0159] In some embodiments, homogenization using a homogenizer with a pore size of 1000 μm, 500 μm and / or 100 μm helps to improve the dispersion uniformity of the modified collagen.

[0160] [use]

[0161] The fourth aspect of the present disclosure provides use of the collagen composition of the first aspect, the modified collagen prepared by the method of the second aspect, and the collagen composition prepared by the method of the third aspect in preparing an injection for human tissue filling.

[0162] In some embodiments, the collagen composition can be used as a filler for human tissue filling. In some embodiments, the collagen composition can be used for body shaping and cosmetic surgery to improve skin wrinkles and depressions. In some embodiments, the tissue filling includes facial tissue filling.

[0163] In some embodiments, the use includes the use of the collagen composition in the preparation of a filler for human body shaping and cosmetic surgery. In some embodiments, the use includes the use of the collagen composition in the preparation of a filler for human facial shaping and cosmetic surgery. The filler can be used in the form of an injectable preparation.

[0164] In some embodiments, the use includes use in the preparation of an injection for improving facial wrinkles and depressions.

[0165] In clinical application, the collagen composition provided by the present disclosure can be injected into a preset human body part, such as the face, by a syringe. Generally, the collagen composition can be filled into the dermis and / or subcutaneous tissue layer of the skin tissue. The filling amount of the collagen composition is usually changed according to the physical condition of the subject and the clinical experience of the clinician.

[0166] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by such combination are all considered as part of the disclosure of this application.

[0167] The technical solution of the present disclosure will be more clearly and explicitly described in the following examples in an illustrative manner. It should be understood that these examples are only for illustrative purposes and are by no means intended to limit the scope of protection of the present disclosure. The scope of protection of the present disclosure is limited only by the claims.

[0168] Example

[0169] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0170] Test Method

[0171] (1) Test of free amino group ratio and cross-linking degree

[0172] 1.1 Calculation of free amino content

[0173] The hydrate of collagen and ninhydrin is heated in a weakly acidic solution, causing oxidation and deamination reactions. The product is bluish purple with maximum absorption at 570nm.

[0174] A glycine gradient solution was prepared, and the absorbance at 570 nm was measured by UV-visible spectrophotometry. The linear regression equation was obtained by linear regression analysis.

[0175] Weigh the test sample (cross-linked modified collagen, type I collagen raw material solution), add a known amount of purified water, and treat it in a boiling water bath for 20 minutes. The protein in the test sample is hydrolyzed by heat to release favorable amino groups. Take the supernatant and measure the absorbance. Use the linear regression equation to calculate the content C (μg / mL) of free amino groups in the test sample.

[0176] 1.2 Calculation of free amino group ratio

[0177] K1 and K2 are the proportions of free amino groups in the prepared cross-linked modified collagen and type I collagen raw material solutions (μg / mg), respectively, and the calculation formula is as follows:

[0178]

[0179] Where: C1 and C2 are the contents of free amino groups in the cross-linked modified collagen and type I collagen raw material solutions (μg / mL), respectively;

[0180] M1 and M2 are the weights (mg) of the cross-linked modified collagen and type I collagen raw material solutions, respectively;

[0181] A1 and A2 are the collagen contents (mg / mL) in the cross-linked modified collagen and type I collagen raw material solutions, respectively;

[0182] ρ1 and ρ2 are the densities of the cross-linked modified collagen and type I collagen raw material solutions (1000 mg / mL), respectively;

[0183] v1 and v2 are the volumes of the cross-linked modified collagen and type I collagen raw material solutions (1 mL), respectively.

[0184] 1.3 Calculation of cross-linking degree

[0185] Crosslinking degree (S) calculation formula

[0186]

[0187] Where: K1 and K2 are the means of no less than three samples.

[0188] (2) Test of EDC residue in collagen injection

[0189] Dimethylbarbituric acid test solution reacts with carbodiimide (EDC) to form a purple-red complex, and the absorbance is measured at 599 nm by UV-visible spectrophotometry. Weigh the EDC reference substance and prepare a gradient solution, measure the absorbance, and obtain the linear regression equation by linear regression analysis.

[0190] Weigh 1g of the test sample collagen injection, centrifuge at 14000r / min for 10min, take the supernatant, filter it through a 0.22 supernatant filter membrane, and measure the absorbance. Substitute the absorbance into the linear regression equation to calculate the mass concentration of EDC in the test sample.

[0191] (3) Testing of NHS residues in collagen injection

[0192] Weigh 2g of collagen injection solution, add one volume of 0.9% NaCl solution to dilute, centrifuge at 14000r / min for 10min, take the supernatant, filter through 0.22% solution, vibrate at 40Hz for 20min under 25% conditions, and use high performance liquid chromatography (reference: Determination of N-hydroxysuccinimide content in collagen implants based on high performance liquid chromatography. Yu Jianyong, Wang Shudong, Gu Jianhua, etc. "Chinese Journal of Aesthetic Medicine", 2019, 28(1): 29-32) to determine the mass concentration of NHS in the solution. The mass concentration multiplied by 2 is the residual NHS content (μg / mL) of collagen injection solution.

[0193] (4) Test of thermal denaturation temperature of collagen injection

[0194] The process of characterizing changes in the physical or chemical properties of a substance by measuring its thermodynamic changes.

[0195] Referring to the melting point determination method in Appendix D of YY / T 0954-2015 “Passive Surgical Implants - Type I Collagen Implants”, about 20 mg of sample was placed in a sealed crucible (liquid crucible) for testing.

[0196] 1.2 Results Analysis

[0197] In a nitrogen atmosphere of 50 mL / min, the temperature was scanned in the range of 40°C to 100°C at a heating rate of 2°C / min, and the sample absorption peak was recorded as the thermal denaturation temperature.

[0198] (5) Test of viscosity of collagen injection solution

[0199] A rotational viscometer (NDJ-9S) was used according to the second method of Appendix VIG of the Pharmacopoeia of the People's Republic of China (Part II) (2010 edition).

[0200] The sample was squeezed into a 50 mL beaker, and a No. 3 rotor was used, with the speed set at 6 rpm. After stabilization, three consecutive measurements were performed and the average value was taken.

[0201] (6) Test of the pushing force of collagen injection

[0202] Place the test sample at room temperature (20℃-25℃) for more than 30 minutes until the sample is balanced to room temperature, and measure with a 27G needle (inner diameter 0.21mm). The injection end of the syringe is fixed downward on the fixture, the pushing speed is 25mm / min, and the pushing length is 20mm. Record the maximum continuous force value after the starting point as the pushing force test result.

[0203] (7) Test of modified collagen concentration and mass ratio

[0204] Prepare a gradient concentration solution of hydroxyproline reference substance, measure the absorbance at 560nm by UV-visible spectrophotometry, and obtain the linear regression equation by linear regression. Weigh the collagen injection sample m (mg), hydrolyze it and dilute it to 50mL, measure the absorbance, calculate the hydroxyproline concentration C (μg / mL) in the sample using the linear regression equation, and calculate the collagen content according to the following formula:

[0205]

[0206] Hydroxyproline accounts for about 12.77% of collagen, and the detection sample volume is 0.1 mL; V represents the volume of the detection solution (1 mL), and ρ represents the sample density (1 g / mL).

[0207] The mass proportion of modified collagen = the mass of modified collagen / the mass of collagen injection.

[0208] Preparation of cross-linked modified collagen

[0209] Preparation of type I collagen solution:

[0210] Pretreatment: Wash and chop the cowhide and soak it in sodium sulfate solution for later use. Alkalinize it in sodium hydroxide solution at low temperature (0℃-20℃), take out the precipitate, wash it with purified water until pH<8, and set aside.

[0211] Enzymatic hydrolysis: the pre-treated product was added into 0.5M glacial acetic acid solution to fully dissolve, pepsin was added for enzymatic hydrolysis to remove the terminal peptide of collagen molecule, centrifuged, and the supernatant was collected for later use.

[0212] Salting out: Add sodium chloride solution to the collected supernatant to precipitate collagen, collect the collagen precipitate, wash and remove impurities, and then dissolve it in 3% sodium acetate solution for later use.

[0213] Purification: The salting-out product was treated by ion exchange chromatography using a cationic resin.

[0214] Sterile filtration: The purified product was concentrated by ultrafiltration and then sterilized by filtration with a capsule filter, wherein the pore size of the filter membrane used was a double-layer hydrophilic polyethersulfone filter membrane of 0.45 μm + 0.22 μm, to obtain type I collagen preservation solution, wherein the mass proportion of impurity proteins was 0.21%.

[0215] Preparation of cross-linked modified collagen H1:

[0216] Take type I collagen preservation solution, add phosphate buffer (pH 6.8) to the preservation solution to adjust the concentration of type I collagen, obtain type I collagen raw material solution, the concentration of which is 2.2 mg / mL, add sodium hydroxide solution to adjust the solution pH to 6.0, react at 37°C for 50 minutes, separate the physical cross-linked product and place it in a chemical cross-linking solution of pH 6.0, carry out chemical cross-linking reaction at 20°C-25°C for 2 hours, centrifuge to obtain modified collagen crude product. Soak the gel crude product in PBS solution and purified water for washing, centrifuge, take the precipitate, and wash it with Na2HPO4 and purified water respectively to obtain cross-linked modified collagen H1.

[0217] In the cross-linking solution, the concentration of EDC was 20 mg / mL, the concentration of NHS was 10 mg / mL, and the solvent was 2-(N-morpholino)ethanesulfonic acid buffer (MES).

[0218] Preparation of cross-linked modified collagen H2-H6 and D1-D8:

[0219] The preparation methods of cross-linked modified collagens H2-H6 and cross-linked modified collagens D1-D8 are basically the same as the preparation method of cross-linked modified collagen H1, but the preparation parameters are adjusted, see Table 1 for details.

[0220] Table 1

[0221]

[0222] The prepared cross-linked modified collagens H1-H6 and D1-D6 are in the form of milky white gels with elasticity; the cross-linked modified collagen D7 is in the form of a slightly transparent gel with low elasticity; and the cross-linked modified collagen D8 is in the form of a slightly transparent gel sheet with strong elasticity and toughness.

[0223] The free amino group ratio and cross-linking degree of the cross-linked modified collagens H1-H6 and D1-D8 were tested using the above method. The test results are shown in Table 2.

[0224] Table 2

[0225]

[0226]

[0227] Example 1

[0228] Preparation of the first protein solution: Use physiological saline to dissolve the first modified collagen (cross-linked modified collagen H1) and lidocaine, so that the mass concentration of the cross-linked modified collagen H1 in the first protein solution is 35 mg / mL±3.5 mg / mL, and the mass concentration of lidocaine is 3 mg / mL.

[0229] Preparation of the second protein solution: Use physiological saline to dissolve the second modified collagen (cross-linked modified collagen H3) and lidocaine, so that the concentration of cross-linked modified collagen H3 in the first protein solution is 35 mg / mL±3.5 mg / mL, and the mass concentration of lidocaine is 3 mg / mL.

[0230] Preparation of the third protein solution: Use physiological saline to dissolve the third modified collagen (cross-linked modified collagen H5) and lidocaine, so that the concentration of cross-linked modified collagen H5 in the first protein solution is 35 mg / mL±3.5 mg / mL, and the mass concentration of lidocaine is 3 mg / mL.

[0231] The first protein solution, the second protein solution and the third protein solution were mixed in a weight ratio (mass ratio) of 1:6:3, and homogenized using homogenizers with pore sizes of 1000 μm, 500 μm and 100 μm respectively to obtain a collagen injection solution.

[0232] Example 2-Example 9

[0233] The preparation methods of Examples 2 to 9 are similar to the preparation method of Example 1, see Table 3 for details.

[0234] Comparative Example 1-Comparative Example 5

[0235] The preparation method of Comparative Example 1 is similar to that of Example 1, except that only the first modified collagen (cross-linked modified collagen H1) is used to prepare the injection solution.

[0236] The preparation method of Comparative Example 2 is similar to that of Example 1, except that only the second modified collagen (cross-linked modified collagen H3) is used to prepare the injection solution.

[0237] The preparation method of Comparative Example 3 is similar to that of Example 1, except that only the third modified collagen (cross-linked modified collagen H5) is used to prepare the injection solution.

[0238] The preparation method of Comparative Example 4 is similar to that of Example 1, except that only the first modified collagen (cross-linked modified collagen D7) is used to prepare the injection solution.

[0239] The preparation method of Comparative Example 5 is similar to that of Example 1, except that only the first modified collagen (cross-linked modified collagen D8) is used to prepare the injection solution, but the cross-linked modified collagen D8 is flaky and has a very high hardness, high elasticity and toughness, and cannot be homogenized. Figure 4 shown.

[0240] Comparative Example 6-Comparative Example 8

[0241] The preparation methods of Comparative Examples 6 to 8 are similar to the preparation method of Example 1, see Table 3 for details.

[0242] Table 3

[0243] serial number First protein liquid Weight Second protein solution Weight Third protein liquid Weight 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

[0244] The collagen injections prepared in Examples 1-9, Comparative Examples 1-3 and Comparative Examples 6-8 were milky white, non-transparent, and uniform in texture; the collagen injection prepared in Comparative Example 4 was slightly transparent, and uneven floccules were visible to the naked eye (see Figure 6 ).

[0245] Using the above test method, the mass proportion and mass concentration of the modified collagen in the injections prepared in Examples 1-9, Comparative Examples 1-4 and Comparative Examples 6-8 were determined. The results are shown in Table 4.

[0246] Table 4

[0247]

[0248]

[0249] The above method was used to test the EDC and NHS residues in the injection, as well as the average cross-linking degree, and the thermodynamic properties, mechanical properties, degradation properties and safety performance of the injection. The results are shown in Table 5.

[0250] Table 5

[0251]

[0252] As shown in Table 5, the collagen injections prepared in Examples 1-9 have low residual crosslinking agent content and good safety; the thermal denaturation temperature is in the range of 50°C-75°C, indicating that the collagen injections have good thermodynamic properties; the average crosslinking degree of the collagen injections is in the range of 50%-70%, and the viscosity is in the range of 8000mPa.s-11000mPa.s, indicating that the collagen injections have excellent mechanical properties and support properties, as well as a good degradation cycle; the pushing force of the injection does not exceed 10N, indicating that the injection has excellent fluidity. Therefore, the collagen injections prepared in Examples 1-9 have both good support properties and fluidity, and have improved cosmetic shaping effects and a lasting degradation cycle after injection filling.

[0253] It can be seen from Comparative Example 1 that the injection prepared by the modified cross-linked protein H1 has a lower degree of cross-linking and viscosity, indicating that the mechanical properties of the modified cross-linked protein are poor, which is not conducive to the filling support performance and the degradation cycle is short.

[0254] It can be seen from Comparative Example 2 and Example 6 that, by compounding three collagens with different cross-linking degrees, although the average cross-linking degrees and thermal denaturation temperatures of the two are basically similar, the viscosity and pushing force of the compounded injection are higher, indicating that the compounded injection has better viscoelastic properties, which can be understood to have a more ideal filling effect and a longer-lasting tissue support period. However, the injection prepared in Comparative Example 2 with a single cross-linking degree cannot take into account the degradation period, filling effect and clinical operability, and its clinical use is limited.

[0255] It can be seen from Comparative Example 3 that the injection prepared by the modified cross-linked protein H5 has a higher viscosity and pushing force, which is not conducive to the operability of the injection and the convenience in clinical practice.

[0256] From Comparative Example 4, it can be seen that the injection prepared by D7 has a lower crosslinking degree and viscosity, indicating that the mechanical properties of the modified crosslinked protein are poor, which is not conducive to the filling support performance; at the same time, the injection is in a transparent non-uniform gel state, and the product quality is poor. The injection prepared by Comparative Example 4 and Example 9 is filled into a syringe, Figure 6The gels on the left and right are the products prepared in Comparative Example 4 and Example 9, respectively. It can be seen that the injection prepared in Comparative Example 4 is slightly transparent gel-like, and the properties are uniform as seen by the naked eye; the injection prepared in Example 9 has a uniform texture and is uniformly milky white. The two are squeezed out on a culture dish, Figure 5 The gels on the left and right sides are the products prepared in Comparative Example 4 and Example 9, respectively. The injection prepared in Comparative Example 4 has liquid precipitation, slight fluidity, and the extrusion lines disappear quickly, but are not completely leveled; the injection prepared in Example 9 has no liquid precipitation, has excellent supporting force, and can keep the extrusion lines from disappearing and collapsing.

[0257] It can be seen from Comparative Example 6 that when the dosage of modified cross-linked protein H1 is increased, the cross-linking degree and viscosity of the injection solution decrease accordingly. Although the fluidity is improved, the mechanical properties are reduced, and the degradation cycle is short.

[0258] It can be seen from Comparative Example 7 that after the dosage of modified cross-linked protein H3 is reduced, the dosage of the third protein solution needs to be increased to improve the tissue filling effect, but this significantly increases the viscosity of the injection solution, reduces its fluidity and clinical operability, and limits its clinical use. In addition, after tissue filling, the injection solution of Comparative Example 7 has poor supporting performance and fast degradation of the first modified collagen, and the third modified collagen with a long degradation period remains in the filled area, resulting in poor aesthetics of cosmetic shaping and an extremely short maintenance period.

[0259] It can be seen from Comparative Example 8 that when the dosage of modified cross-linked protein H5 is increased, the viscosity of the injection solution increases accordingly, the supporting performance is significantly improved, and the degradation cycle is prolonged, but the injection filling effect is sharpened, which is not conducive to the aesthetics of cosmetic shaping.

[0260] Degradation test of collagen injection

[0261] The collagen injection prepared in Example 9 was also subjected to animal experiments to investigate the degradation of the collagen injection 1 month, 6 months and 12 months after surgery, as follows:

[0262] Test subjects: New Zealand rabbits, male, weighing 2.5kg-3.0kg, housed in single cages.

[0263] Experimental process: New Zealand rabbits were anesthetized, the back hair was removed and the skin was prepared, and the surgical area was disinfected with iodine before the operation; with the spine of the New Zealand rabbit as the center, two points on each side were taken, and 0.2 mL of the collagen injection prepared in Example 9 was injected into the subdermis and the carnosus with a 1 mL syringe. The tissues of the injection sites were taken 1 month, 6 months and 12 months after the operation, and the pathological sections and staining were performed to observe the degradation as follows:

[0264] One month later: Fusiform collagen-like objects can be seen gathering in almost the entire dermis, growing in an expansive manner, with a rough and curled internal structure, differentiating into many filaments that are tightly connected to each other, with a large number of bundles of thick collagen fibers extending into them, separating and enveloping them, and fibroblasts appearing inside the remaining collagen-like objects, with incomplete fiber wrapping around them. This indicates that a large amount of new collagen can be seen at the injection site, with no obvious inflammatory reaction. Figure 1 .

[0265] After 6 months: Collagen-like substances can be seen in the dermis, with a delicate internal structure and expansive growth. Some bundles of collagen fibers separate it into multiple irregular areas, surrounded by incomplete fibrous capsules. This indicates that a large amount of collagen can be seen at the injection site, which has an increasing trend compared to 1 month after surgery. Figure 2 .

[0266] After 12 months: A moderate amount of irregular gel was seen in the thin layer of squamous epithelium, in which a small amount of collagen fibers were generated, a small amount of small blood vessels and myofibroblasts grew in, no obvious tissue cells, no multinucleated giant cell reaction, and no obvious inflammatory cell infiltration. This shows that a small amount of collagen fibers can still be seen at the injection site, no obvious inflammatory reaction, and Figure 3 .

[0267] The results show that the collagen injection prepared in Example 9 has good in vivo safety and does not cause obvious inflammatory response; at the same time, it is not completely degraded 12 months after injection and has a good degradation cycle.

[0268] Safety Testing

[0269] (1) Intradermal reaction test

[0270] Testing standard: GB / T 16886.10-2017 / ISO 10993-10:2010

[0271] The collagen injection prepared in Example 9 was used as the test sample, sodium lauryl sulfate was used as the positive control, and 0.9% sodium chloride injection was used as the negative control. The test sample was prepared according to the following method:

[0272] Weigh 5 g of the test sample and place it in 25 mL of 0.9% sodium chloride injection, and mix it in a shaking incubator at 37° C. and 60 rpm for 72 h to obtain an extract.

[0273] Male New Zealand rabbits weighing 2.5kg-3.0kg were taken and kept in single cages. They were weighed 4h-18h before the test and the hair on both sides of the back spine was completely removed to form the test and observation area.

[0274] 0.2 mL of the test sample extract was intradermally injected into the first 5 points of the hairless area on the left side of the spine of each rabbit; 0.2 mL of the negative control was intradermally injected into the last 5 points on the left side;

[0275] In the same rabbit, 0.2 mL of the positive control extract was intradermally injected into the first 5 points of the hairless area on the right side of the spine; and 0.2 mL of the negative control was intradermally injected into the last 5 points on the right side.

[0276] Observe the injection site immediately after injection, and at (24±2)h, (48±2)h, and (72±2)h after injection. Score the tissue reaction of erythema and edema at each site at each time according to the scoring system, and record the scoring results. The scoring table is as follows:

[0277]

[0278] After scoring at (72±2)h, add up all the erythema and edema scores of each animal's test sample or control at (24±2)h, (48±2)h and (72±2)h, and divide by 15 to calculate the score of each animal's test sample or blank control. Add up the scores of the three animals and divide by 3 to get the total average score of each test sample and the corresponding blank control. Subtract the blank control score from the test sample score to get the final score of the test sample. The test results of the test samples are as follows:

[0279]

[0280]

[0281] The test results of the positive control are as follows:

[0282]

[0283] The results showed that the collagen injection prepared in Example 9 had good intradermal reactivity.

[0284] (2) In vitro cytotoxicity test

[0285] Testing basis: GB / T16886.5-2017 / ISO 10993-5:2009

[0286] Sample preparation:

[0287] Take 1 g of the collagen injection prepared in Example 9, add MEM culture medium containing 10% fetal bovine serum at a ratio of 0.2 g:1 mL, and incubate at 37° C. for 72 h to take the extract as a test sample.

[0288] Test group: Under aseptic operation, sterile filter paper was cut into 3 discs with a diameter of 1.0 cm, and 100% of the sample test solution was absorbed during the test.

[0289] Negative control group: High-density polyethylene (USP, R149K0) was cut into 1.0 cm diameter discs, 3 discs in total.

[0290] Positive control group: Sterile filter paper was cut into 3 discs with a diameter of 1.0 cm. During the test, MEM culture medium containing 0.1% zinc diethyldithiocarbamate (ZDEC) was absorbed.

[0291] Experimental process:

[0292] The experimental process was performed aseptically. L-929 cells were cultured in MEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The cells were dispersed in fresh medium and adjusted to 2.5×10 5 Inoculate 6-well plates with 2 mL / well of cell suspension at 37°C and 5% CO2, and culture for 24 hours in a 37°C, 5% CO2 incubator. Discard the original culture medium, add 2 mL of fresh culture medium, and then gently place the sample or negative and positive controls. Be careful not to move them after placement to avoid physical damage to the cells, and continue to culture in the incubator for 24 hours. Take out the culture plate and observe the cell morphology under a microscope.

[0293] Evaluation criteria:

[0294] The cytotoxicity ratings are shown in the table below, with reference to the standards.

[0295] Grading Degree of reaction Reaction area observation 0 none No reaction area was observed around or under the sample. 1 slight There are some deformed or degenerated cells under the sample 2 Mild The reaction area is limited to the area below the sample 3 Moderate The reaction area exceeds the sample size to 1.0 cm 4 Severe The reaction area exceeds the sample by more than 1.0 cm

[0296] The test results are as follows:

[0297]

[0298] The toxicity reaction of the test sample to L-929 cells was grade 1, indicating that the test sample had no potential cytotoxic effect on L-929 cells.

[0299] (3) Skin sensitization test

[0300] Test basis: GB / T 16886.10-2017 / ISO 10993-10:2010

[0301] 15 female albino guinea pigs weighing 300-500 g were taken. They were marked and weighed before the test and randomly divided into a sample test group and a 0.9% sodium chloride injection control group. The skin reaction of the guinea pigs at the provocation site was observed 24h and 48h after the removal of the sample, and the weight of the animals was recorded.

[0302] Intradermal induction phase:

[0303] Test sites A, B and C were set between the head and the inner part of the shoulder blade of the guinea pig, the hair was removed, and 0.1 mL was injected intradermally in pairs at the test sites of each animal.

[0304] Site A: Injection of a stable emulsion of Freund's complete adjuvant and 0.9% sodium chloride injection in a 50:50 (volume ratio).

[0305] Site B: Injection test of the collagen prepared in Example 9; animals in the control group were injected with 0.9% sodium chloride injection.

[0306] Site C: The collagen prepared in Example 9 was mixed with an emulsion prepared with Freund's complete adjuvant and solvent (50%) in a volume ratio of 50:50 and then injected intradermally; the control group was injected with an emulsion prepared with 0.9% sodium chloride injection and adjuvant.

[0307] No irritation occurred at the test site. The test area was pretreated with 10% sodium dodecyl sulfate (24±2) h before the topical application and massaged into the skin.

[0308] Local induction phase:

[0309] After the intradermal induction phase (7 ± 1) days, the selected concentration in the intradermal induction site B was used in an area of ​​about 8 cm 2 The induced injection point is covered with a water-absorbent gauze soaked in the test sample, then covered with a layer of cellophane and fixed with a non-irritating adhesive tape. The bandage and patch are removed after (48±2) hours.

[0310] Stimulation phase:

[0311] After the local induction phase (14±1) days, all test animals and control animals were stimulated with the test sample. The patch was applied topically to the untested area during the induction phase, then covered with a layer of cellophane and fixed with a non-irritating adhesive tape. The bandage and patch were removed after (24±2) hours.

[0312] Animal observation:

[0313] The skin conditions of the stimulated areas of the animals in the test group and the control group were observed at (24±2)h and (48±2)h after the patch was removed. The skin erythema and edema reactions at each stimulated area and each observation time were described and graded according to the Magnusson and Kligman grading criteria, as follows:

[0314] Patch test reaction grade No significant change 0 Scattered or macular erythema 1 Moderate confluent erythema 2 Severe erythema and / or edema 3

[0315] Evaluation criteria:

[0316] According to the Magnusson and Kligman grading standard, when the grade of the control group animals is less than 1, and the grade of the test group is greater than or equal to 1, it generally indicates sensitization. If the grade of the control group animals is greater than or equal to 1, and the reaction of the test group animals exceeds the most severe reaction in the control group, it is considered sensitization.

[0317] The test results of the collagen injection prepared in Example 9 are as follows:

[0318]

[0319] The skin reaction grading of all animals at each time period was grade 0, and the positive stimulation rate of the test group was 0%, indicating that the injection would not cause allergic reaction.

Claims

1. A collagen composition, characterized in that: include: (1) a first modified collagen having a cross-linking degree of 30% to 50%, (2) a second modified collagen having a cross-linking degree greater than 50% and less than 70%, (3) a third modified collagen having a cross-linking degree greater than 70% and less than 80%, and (4) pharmaceutically acceptable excipients; Wherein, based on the total mass of the collagen composition, The mass proportion of the first modified collagen in the collagen composition is 0.1%-2.0%; The mass proportion of the second modified collagen in the collagen composition is 0.5%-4.0%; The mass proportion of the third modified collagen in the collagen composition is 0.1%-2.0%; and Pharmaceutically acceptable excipients.

2. The collagen composition according to claim 1, characterized in that Based on the total mass of the collagen composition, The mass percentage of the first modified collagen in the collagen composition is 0.2%-2.0%; The mass proportion of the second modified collagen in the collagen composition is 0.6%-3.6%; The mass proportion of the third modified collagen in the collagen composition is 0.25%-1.75%.

3. The collagen composition according to claim 1, characterized in that The mass concentration of the first modified collagen in the collagen composition is 2 mg / mL-20 mg / mL; The mass concentration of the second modified collagen in the collagen composition is 5 mg / mL-40 mg / mL; and / or The mass concentration of the third modified collagen in the collagen composition is 2 mg / mL-25 mg / mL.

4. The collagen composition according to claim 3, characterized in that The mass concentration of the second modified collagen in the collagen composition is 6 mg / mL-36 mg / mL; and / or The mass concentration of the third modified collagen in the collagen composition is 2.5 mg / mL-17.5 mg / mL.

5. The collagen composition according to any one of claims 1 to 4, characterized in that An anesthetic is included, the anesthetic comprising lidocaine.

6. The collagen composition according to claim 5, characterized in that The mass percentage of the anesthetic in the collagen composition is 0.1%-0.5%; or The mass concentration of the anesthetic in the collagen composition is 1 mg / mL-5 mg / mL.

7. The collagen composition according to claim 5, characterized in that The mass percentage of the anesthetic in the collagen composition is 0.2%-0.4%; or The mass concentration of the anesthetic in the collagen composition is 2 mg / mL-4 mg / mL.

8. The collagen composition according to any one of claims 1 to 4, characterized in that The pharmaceutically acceptable excipients include water for injection and sodium chloride.

9. The collagen composition according to any one of claims 1 to 4, characterized in that The collagen composition is an isotonic solution.

10. The collagen composition according to any one of claims 1 to 4, characterized in that The average cross-linking degree of the collagen composition is 30%-80%.

11. The collagen composition according to claim 10, characterized in that: The average cross-linking degree of the collagen composition is 50%-70%.

12. The collagen composition according to any one of claims 1 to 4, characterized in that In the collagen composition, the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is no more than 0.5 μg / g; and / or The mass concentration of N-hydroxysuccinimide (NHS) does not exceed 3 μg / g.

13. The collagen composition according to claim 12, characterized in that: In the collagen composition, the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) does not exceed 0.4 μg / g; and / or the mass concentration of N-hydroxysuccinimide (NHS) does not exceed 2.5 μg / g.

14. The collagen composition according to any one of claims 1 to 4, characterized in that The thermal denaturation temperature (DSC) of the collagen composition is 50°C-75°C.

15. The collagen composition according to claim 14, characterized in that: The thermal denaturation temperature (DSC) of the collagen composition is 51.6°C-73.5°C.

16. The collagen composition according to any one of claims 1 to 4, characterized in that The viscosity of the collagen composition is 8000mPa.s-11000mPa.s.

17. The collagen composition according to claim 16, characterized in that: The viscosity of the collagen composition is 8000mPa.s-10500mPa.s.

18. The collagen composition according to any one of claims 1 to 4, characterized in that The pushing force of the collagen composition is 3N-10N.

19. The collagen composition according to claim 18, characterized in that The pushing force of the collagen composition is 3N-8.5N.

20. The collagen composition according to any one of claims 1 to 4, characterized in that The first modified collagen, the second modified collagen and the third modified collagen are each independently type I collagen.

21. The collagen composition according to claim 20, characterized in that The first modified collagen, the second modified collagen and the third modified collagen are each independently modified type I collagen.

22. A method for preparing modified collagen, comprising at least the following two cross-linking steps: (1) Physical cross-linking: providing a buffer solution containing type I collagen, adjusting the pH value to 6.0-9.0 to perform a cross-linking reaction, and obtaining a physically cross-linked product; (2) Chemical cross-linking: placing the physically cross-linked product prepared in (1) in a cross-linking solution containing EDC and NHS to carry out a cross-linking reaction, so as to obtain a modified collagen with a cross-linking degree of 30%-80%; The mass concentration of EDC in the cross-linking solution is 20 mg / mL-160 mg / mL; The mass concentration of NHS in the cross-linking solution is 10 mg / mL-120 mg / mL; The mass concentration of type I collagen in the buffer solution is 2 mg / mL-3 mg / mL; The pH value of the cross-linking solution is 5.5-7.0; The chemical cross-linking reaction time is 2h-12h; The temperature of the chemical cross-linking reaction is 20°C-25°C.

23. The preparation method according to claim 22, characterized in that: In step (1), the pH value is adjusted to 7-7.

5.

24. The preparation method according to claim 22 or 23, characterized in that: In the chemical cross-linking reaction, The mass concentration ratio of EDC to NHS in the cross-linking solution is 1.1-2.

0.

25. The preparation method according to claim 22 or 23, characterized in that: The method comprises at least one, two or three of the following steps: step A for preparing a first modified collagen with a cross-linking degree of 30% to 50%, step B for preparing a second modified collagen with a cross-linking degree of greater than 50% and less than 70%, and step C for preparing a third modified collagen with a cross-linking degree of greater than 70% and less than 80%. In the chemical cross-linking reaction of step A, The mass concentration of EDC in the chemical crosslinking reaction system is 20 mg / mL-80 mg / mL; The mass concentration of NHS in the chemical cross-linking reaction system is 10 mg / mL-70 mg / mL; The chemical cross-linking reaction time is 2h-4h; The temperature of the chemical cross-linking reaction is 20°C-25°C; In the chemical cross-linking reaction of step B, The mass concentration of EDC in the chemical crosslinking reaction system is 60 mg / mL-140 mg / mL; The mass concentration of NHS in the chemical cross-linking reaction system is 50 mg / mL-100 mg / mL; The chemical cross-linking reaction time is 6h-8h; The temperature of the chemical cross-linking reaction is 20°C-25°C; In the chemical cross-linking reaction of step C, The mass concentration of EDC in the chemical cross-linking reaction system is 110 mg / mL-160 mg / mL; The mass concentration of NHS in the chemical cross-linking reaction system is 80 mg / mL-120 mg / mL; The chemical cross-linking reaction time is 10h-12h; The temperature of the chemical cross-linking reaction is 20°C-25°C.

26. The preparation method according to claim 22 or 23, characterized in that: The mass proportion of the impurity proteins in the type I collagen does not exceed 0.3%.

27. The preparation method according to claim 26, characterized in that: The mass proportion of the impurity proteins in the type I collagen does not exceed 0.2%.

28. The preparation method according to claim 22 or 23, characterized in that: The type I collagen is prepared by a method comprising the following steps: The cowhide and / or cow Achilles tendon is subjected to pre-treatment, enzymatic hydrolysis, crude extraction, purification and filtration sterilization to obtain type I collagen.

29. The method for preparing the collagen composition according to any one of claims 1 to 21, characterized in that: include: The first modified collagen, the second modified collagen and the third modified collagen are mixed and homogenized to obtain a collagen compound.

30. The preparation method according to claim 29, characterized in that: The first modified collagen is prepared into a first protein solution, wherein the first protein solution comprises the first modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; The second modified collagen is prepared into a second protein solution, wherein the second protein solution comprises the second modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; The third modified collagen is prepared into a third protein solution, wherein the third protein solution comprises the third modified collagen with a mass concentration of 20 mg / mL-50 mg / mL and an anesthetic with a mass concentration of 1 mg / mL-5 mg / mL; The first protein solution, the second protein solution and the third protein solution are mixed to obtain a total solution, wherein: The mass proportion of the first protein solution in the total solution is 10%-40%; The mass proportion of the second protein solution in the total solution is 30%-80%; The mass proportion of the third protein liquid in the total liquid is 10%-50%.

31. The preparation method according to claim 30, characterized in that: The mass concentration of the first modified collagen in the first protein solution is 35 mg / mL±3.5 mg / 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.

32. The preparation method according to claim 30, characterized in that: The mass proportion of the anesthetic in the first protein solution, the second protein solution or the third protein solution is independently 0.1%-0.5%; The anesthetic includes lidocaine.

33. The preparation method according to claim 32, characterized in that: The mass proportion of the anesthetic in the first protein solution, the second protein solution or the third protein solution is independently 0.2%-0.4%.

34. The preparation method according to any one of claims 30 to 33, characterized in that The first protein solution, the second protein solution and the third protein solution were prepared using physiological saline.

35. The preparation method according to any one of claims 29 to 33, characterized in that The homogenization was performed using a homogenizer having a pore size of 1000 μm, 500 μm and / or 100 μm.

36. Use of the collagen composition described in any one of claims 1 to 21, the modified collagen prepared by the method described in any one of claims 22 to 28, or the collagen composition prepared by the method described in any one of claims 29 to 35 in the preparation of an injection for human tissue filling.

37. The use according to claim 36, characterized in that The tissue augmentation includes facial tissue augmentation.

38. The use according to claim 36 or 37, characterized in that The use includes use in preparing an injection for improving facial wrinkles and depressions.

39. Use of the collagen composition described in any one of claims 1 to 21, the modified collagen prepared by the method described in any one of claims 22 to 28, and the collagen composition prepared by the method described in any one of claims 29 to 35 in the preparation of fillers for human body cosmetic surgery.

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