CN117618671A

CN117618671A - Collagen filler, preparation method and application thereof

Info

Publication number: CN117618671A
Application number: CN202311711140.5A
Authority: CN
Inventors: 郭立英; 黄兆辉; 张絮然; 傅维擎; 张安琪; 赵霄雨; 白露; 冯娇艳
Original assignee: Yantai Desheng Marine Biotechnology Co ltd
Current assignee: Yantai Desheng Marine Biotechnology Co ltd
Priority date: 2023-12-13
Filing date: 2023-12-13
Publication date: 2024-03-01

Abstract

The invention relates to a collagen bulking agent with controllable pore structure, wherein the collagen bulking agent has an average pore size D ₅₀ The invention also relates to a preparation method of the collagen filler and application of the collagen filler, wherein the porosity is 80% -99.5%, the crosslinking degree is 10% -70%, and the in-vitro enzymolysis loss rate is less than 40%.

Description

Collagen filler, preparation method and application thereof

Technical Field

The invention relates to the field of medical biological materials, in particular to a collagen filler with a controllable pore structure and application thereof. The invention also relates to a preparation method of the collagen with the controllable pore structure and application of the collagen with the controllable pore structure.

Background

Collagen is a natural protein, is used as a main component of skin tissues, and is an ideal material for correcting and repairing soft tissue defects. Collagen exists in the form of fibers, is a ubiquitous component of connective tissue, accounts for about 30% of total proteins in the human body, and is an essential matrix skeleton for organs and tissues. Collagen fibers are used as attachment and scaffold for cell growth, can induce migration, proliferation and differentiation of fibroblasts, epithelial cells and the like, and are partially involved in nutrient transport and metabolism of tissues and organs. In addition, as the technology of extracting and purifying collagen is mature, the immunogenicity and other risks are reduced to a low level, and even negligible. Therefore, compared with other filling materials, the collagen has the advantages of incomparable physical properties, biological properties and the like. However, in the market at present, the filler mainly composed of collagen generally has an undesirable supporting effect after being implanted into a human body, is easily displaced, is easily degraded in a short period of time to lose tissue filling capacity, or causes a series of toxicity risks due to excessive crosslinking.

Aiming at the technical problem of the collagen in the field of filling agents, CN113768815A discloses a collagen implant and a preparation method thereof, wherein the purity of the collagen prepared by the method is up to 99.82 percent, the collagen is higher in activity and safety, a fiber structure with a distinct shape is formed after collagen molecules are self-aggregated, each fiber is completely exposed and mutually staggered to form a fiber web, the fiber thickness is uniform, and the better elastic strength is formed. Then, since the implant is not crosslinked to increase the maintenance time, the degradation loss rate of the implant at 3 months is about 40%, and the degradation loss rate of the implant is far higher than that of the crosslinked collagen implant although the product has a certain advantage of enzymolysis resistance compared with other uncrosslinked collagen implants.

CN114288469a discloses a composition and a preparation method of a filler of double-crosslinked collagen, which double-crosslinks collagen fibers by reducing monosaccharides and carbodiimides, and data show that the melting point is improved after double-crosslinking compared with single-stage crosslinking, but the melting point at 66.6 ℃ is still at a medium level in crosslinked collagen implants. In addition, the homogenization process of the implant is freeze ball milling, so that the homogenization efficiency is improved, and meanwhile, the network structure overlapped by the collagen fibers can be damaged to a certain extent, so that the supporting effect and the tissue metabolism efficiency of the filler after the filler is implanted into tissues are affected.

CN102924731B discloses a triple-crosslinked collagen, a manufacturing method and an application thereof, wherein the triple-crosslinked collagen suspension is crosslinked step by step for three times, different types of crosslinking agents are used at each stage, and the product after the outstanding crosslinking has high crosslinking degree, high melting point and low enzymolysis rate, so that the product is maintained for a longer time in the filling and repairing process. However, triple crosslinking also carries the risk of more crosslinker residue on the product, and the use of multiple crosslinkers in one product carries the risk of more priming or potential toxicity to clinical patients. In addition, in the cleaning process after crosslinking, PBS with the volume being more than 10 times is used for cleaning for three times, namely, the concentration of the crosslinking agent is reduced to one thousandth of the original total amount in the cleaning process, so that the risk of more residual crosslinking agent caused by incomplete cleaning is increased, and the toxicity risk of the product in the application of the product is triggered.

For the above reasons, there is a need for a tissue filler, such as a dermal tissue filler, that has good support shaping ability, longer maintenance time, and excellent biocompatibility and safety synergy. The collagen filler provided by the invention has controllable pore structure, good mechanical property and more consistent maintenance time with the expected filling effect, and the preparation method of the collagen filler can provide a product with better and safer performance for the current collagen filler products.

Disclosure of Invention

The collagen bulking agent disclosed herein overcomes the shortcomings of known implants. One of the purposes of the invention is to provide a collagen filler which has a controllable pore structure, good supporting and shaping effects, higher dynamic viscosity and maintenance time and meets the expected purpose.

The collagen filler disclosed by the invention has the advantages of larger pore diameter, higher porosity, lower crosslinking degree and better enzymolysis resistance, can be maintained for a longer time in-vitro and in-vivo degradation tests, and can maintain longer filling time. Due to the structure of the product, the method is not only beneficial to the growth, proliferation and differentiation of cells, but also provides more convenience for the transportation and metabolism of nutrient substances in tissues so as to synergistically promote the regeneration of collagen fibers; in the aspect of safety and effectiveness of the product, the mild crosslinking process ensures that the product keeps lower crosslinking degree and higher bioactivity, and particularly, the sufficient and thorough crosslinking efficiency also improves the enzymolysis resistance of the collagen and prolongs the maintenance time of the product, thereby achieving the synergistic effect in the aspects of safety and effectiveness.

According to one embodiment of the invention, the collagen bulking agent of the present invention has an average pore size D _s0 From 50 μm to 500. Mu.m, preferably from 80 μm to 400. Mu.m, even from 90 μm to 300. Mu.m, in particular from 100 μm to 250. Mu.m, especially from 120 μm to 200. Mu.m.

According to another embodiment of the invention, the collagen filler according to the invention has a porosity of 80% to 99.5%, even 85% to 98%, in particular 90% to 97%.

According to another embodiment of the invention, the collagen bulking agent of the present invention has a dynamic viscosity of 90000mpa.s to 300000mpa.s, preferably 95000mpa.s to 280000mpa.s, even 98000mpa.s to 270000mpa.s, in particular 100000mpa.s to 260000mpa.s, in particular 120000mpa.s to 250000mpa.s, at a shear rate of 2Hz and a temperature of 25±1 ℃.

According to another embodiment of the invention, the collagen bulking agent of the present invention has a melting point of 55 to 85 ℃, preferably 58 to 80 ℃, even 60 to 78 ℃, especially 62 to 75 ℃, especially more than 65 ℃, or less than 72 ℃.

According to another embodiment of the invention, the collagen bulking agent of the present invention has a degree of crosslinking of 10% to 70%, preferably 12% to 65%, even 15% to 60%, especially 18% to 55%, especially 20% to 50%.

According to another embodiment of the invention, the collagen bulking agent of the present invention has a degradation loss of less than 40%, preferably less than 35%, even less than 30%, especially less than 25%, especially less than 20% under certain enzymatic conditions.

According to another embodiment of the invention, the collagen bulking agent of the present invention has a melting point of 55 to 85 ℃, preferably 58 to 80 ℃, even 60 to 78 ℃, especially 62 to 75 ℃, especially more than 65 ℃, or less than 73 ℃.

According to another embodiment of the invention, the collagen bulking agent of the present invention comprises a combination of different pore sizes, preferably a combination of one or more pore sizes of 50 μm to 120 μm with one or more pore sizes of 120 μm to 500 μm, even a combination of one or more pore sizes of 50 μm to 100 μm with one or more pore sizes of 120 μm to 450 μm, in particular a combination of one or more pore sizes of 50 μm to 90 μm with one or more pore sizes of 130 μm to 420 μm, or a combination of one or more pore sizes of 50 μm to 80 μm with one or more pore sizes of 140 μm to 400 μm.

According to another embodiment of the present invention, the collagen bulking agent of the present invention has an average pore size D ₅₀ From 50 μm to 500 μm, a porosity of from 80% to 99.5%, a degree of crosslinking of from 10% to 70%, and an in vitro enzymatic hydrolysis loss of less than 40%.

The collagen filler is prepared by adopting a dynamic template method, on one hand, the relative distance between collagen fiber bundles in the crosslinking process is controlled by controlling the pore size taking microspheres as templates, and then the pore size of the collagen fiber bundles after forming a crosslinked framework is controlled; on the other hand, the mechanical stirring and microsphere adding modes are controlled to realize the crosslinking process of a dynamic single template (or multiple templates), so that each collagen fiber bundle can obtain the same crosslinking opportunity, and the more thorough and uniform crosslinking effect of the collagen implant is realized.

According to another embodiment of the invention, the method for preparing the collagen filler regulates and controls the hierarchical porous structure inside the collagen filler by controlling the adding sequence of microspheres with different pore diameters in the process of pore forming of a template. For example, when the cross-linking process is started, firstly adding the microspheres with large aperture to carry out cross-linking reaction for a period of time, then separating out the microspheres with large aperture, then adding the microspheres with smaller aperture as a pore-forming template to continuously participate in the cross-linking reaction, and repeating the steps according to the aperture required by the product, thereby preparing the fiber mesh hierarchical porous structure with one or more small apertures (or containing small apertures) sleeved by the large aperture. Generally, pore sizes of 50 to 400 μm are advantageous for cell migration and tissue ingrowth. Wherein, the pore wall formed by the crosslinked reticular fiber has higher mechanical property, which is favorable for cell adhesion; the perforated macroporous sleeve pores (or pores) and the reticular fiber interweaved microporous structure endow the collagen filler with higher porosity, and are beneficial to the transportation of nutrient substances and the discharge of cell metabolites, thereby providing more convenient space topology structure for tissue regeneration. The collagen filler prepared by the invention can construct a pore structure suitable for tissue defect by adjusting the size, the number and the step-by-step addition sequence time of the pore-forming template, thereby constructing a tissue filler which is more matched with tissue repair and regeneration, and being convenient for adapting to different use conditions.

The collagen filler of the present invention prepared according to the method of the present invention may have a multi-stage pore size, for example, a multi-stage pore size of a combination of two or more of a larger pore size and a smaller pore size in a range of 50 μm to 500 μm, for example, a multi-stage pore size of a combination of two or more of a smaller pore size of 50 μm to 120 μm and a larger pore size of 120 μm to 400 μm. The collagen filler with the multi-level pore diameter also has excellent performance in mechanics and higher dynamic viscosity, which is probably beneficial to the fact that the pores are connected through crosslinked collagen network fibers, so that the collagen filler still keeps higher viscosity under the action of shearing force, the displacement risk of the product after being implanted into tissues is greatly reduced, and the collagen filler is more suitable for filling and repairing moderately severe gravity wrinkles.

According to another embodiment of the present invention, the method for preparing the collagen bulking agent of the present invention comprises the steps of:

(1) Providing collagen and dissolving the collagen, optionally followed by filter sterilization;

(2) Adding buffer solution, adjusting pH to 6-8, stirring to form collagen suspension;

(3) Adding the microspheres, and stirring until uniform;

(4) Adding a cross-linking agent, regulating the temperature to 10-40 ℃, and carrying out cross-linking reaction under the constant temperature condition;

(5) The microspheres in the suspension were removed.

According to another embodiment of the invention, in step (1), the concentration of the collagen solution after solubilization is 0.5-15mg/mL, preferably 0.8-12mg/mL, even 1-10mg/mL, especially 1.5-8mg/mL, especially 2-6mg/mL.

According to another embodiment of the invention, in step (1), the collagen is dissolved in an acid solution of 0.001-0.1mol/L, such as an inorganic or organic acid, in particular selected from hydrochloric acid, phosphoric acid, acetic acid, citric acid, or mixtures thereof.

According to another embodiment of the invention, in step (1), the dissolving of collagen is performed under stirring, for example for 1h to 5 days, even 6h to 4 days, especially 12h to 3 days, especially 24h to 2 days.

According to yet another embodiment of the invention, in step (1), the temperature at which the collagen is dissolved is from 0 ℃ to 25 ℃, even from 1 ℃ to 20 ℃, especially from 2 ℃ to 10 ℃, especially from 4 ℃ to 8 ℃.

According to another embodiment of the invention, in step (2), the buffer solution is selected from phosphate buffer solutions, such as phosphate/hydrogen phosphate buffer solution, citrate buffer solution, tris buffer, monobasic potassium phosphate sodium hydroxide buffer, dibasic sodium phosphate-citric acid buffer.

According to another embodiment of the invention, in step (2), the treatment temperature is from 20 ℃ to 37 ℃, even from 25 ℃ to 35 ℃, especially from 27 ℃ to 34 ℃, especially from 30 ℃ to 33 ℃.

According to another embodiment of the invention, in step (2), the buffer solution has a concentration of 0.05 to 0.5mol/L, preferably a concentration of 0.06 to 0.3mol/L, in particular 0.08 to 0.25mol/L, especially 0.09 to 0.2mol/L.

According to another embodiment of the invention, in step (2), the volume ratio of collagen solution to buffer solution is 20:1 to 5:1, even 15:1 to 6:1, especially 12:1 to 7:1, especially 10:1 to 8:1.

According to another embodiment of the invention, in step (2), the pH is adjusted to 6.0 to 8.0, even 6.5 to 7.5, especially 6.8 to 7.3, especially 6.9 to 7.2.

According to another embodiment of the present invention, the material of the microspheres is biomedical inert material, preferably the microspheres are inert microspheres, and are selected from inert bioceramic microspheres, medical metal and alloy microspheres, inert polymer material microspheres, such as alumina ceramic microspheres, titanium alloy microspheres, polytetrafluoroethylene microspheres, alumina ceramic microspheres, titanium silicon microspheres, arteFill (polymethyl methacrylate microspheres, PMMA), radio (hydroxyapatite microspheres, caHA), sculptra (poly l-lactic acid microspheres, PLLA), elanse (polycaprolactone microspheres, PCL), etc.

According to another embodiment of the invention, the microspheres have an average particle size of 50 μm to 500. Mu.m, preferably 80 μm to 400 μm, even 90 μm to 300. Mu.m, especially 100 μm to 250. Mu.m, especially 120 μm to 200. Mu.m. Alternatively, steps (3) to (5) may be repeated as many times, in particular 2-5 times, even 2 or 3 times, as required, wherein the average particle size of the microspheres differs, wherein the particle size of the microspheres differs, preferably one or more particle sizes 50 μm to 120 μm in combination with one or more particle sizes 120 μm to 500 μm, even one or more particle sizes 50 μm to 100 μm in combination with one or more particle sizes 120 μm to 450 μm, in particular one or more particle sizes 50 μm to 90 μm in combination with one or more particle sizes 130 μm to 420 μm, or one or more particle sizes 50 μm to 80 μm in combination with one or more particle sizes 140 μm to 400 μm. Optionally, one or more microspheres with different diameters can be added into the collagen suspension according to the filling requirement, and the pore forming effect of the collagen implant is further controlled by controlling the diameters of the microspheres, so that the filling requirements of different facial depressions are finally met.

According to another embodiment of the invention, the microspheres have a bulk volume of 0.1% to 10%, preferably 0.2% to 8%, even 0.3% to 6%, especially 0.4% to 5%, especially 0.5% to 4%, or 1-3% relative to the total volume of the collagen suspension. The bulk value of the microspheres directly determines the pore structure of the collagen during the crosslinking process. That is, when the volume of the microspheres accumulated in the collagen suspension is relatively large, it is shown that the larger added amount of the microspheres fills more microspheres in the collagen suspension under the condition that the diameters of the microspheres are the same, so that more pores are formed in the crosslinked framework, and a larger crosslinked network is formed.

According to another embodiment of the invention, in step (3), the treatment temperature is from 20 ℃ to 37 ℃, even from 25 ℃ to 35 ℃, especially from 27 ℃ to 34 ℃, especially from 30 ℃ to 33 ℃.

According to another embodiment of the invention, in step (4), the cross-linking agent is any one or more of epoxide cross-linking agents, such as selected from sorbitol, glycerol, 1,4 butanediol diglycidyl ether.

According to another embodiment of the invention, in step (4), the cross-linking agent concentration is 0.005% to 3.0%, even 0.01% to 2.0%, especially 0.015% to 1.5%, especially 0.02% to 1.0%, or 0.025% to 0.5%.

According to another embodiment of the invention, in step (4), the pH is adjusted to 8 to 12, even 8.5 to 11.5, especially 9.0 to 11.0, especially 9.5 to 10.5.

According to another embodiment of the invention, in step (4), the reaction temperature is 20-40 ℃, even 25 ℃ to 37 ℃, especially 28 ℃ to 36 ℃, especially 30 ℃ to 35 ℃.

According to another embodiment of the invention, in step (4), the reaction time is from 6h to 8 days, even from 12h to 6 days, in particular from 36h to 5 days, especially from 2 days to 4 days.

According to another embodiment of the present invention, in the step (5), the microspheres are removed by sieving, for example, the specific operation may be that a sieve is arranged at the outlet of the reaction tank, and sieving is performed while stirring, so as to prevent the microspheres from blocking the sieve, and the pore size of the sieve is adjusted according to the specific microsphere size.

According to another embodiment of the invention, in the purification step the product is collected by centrifugation at > 5000rpm and then repeatedly washed more than 5 times (containing 5 times) with 5-50 volumes of phosphate buffer, for example at a concentration of 0.05-0.3mol/L, preferably at a concentration of 0.08-0.20mol/L, especially 0.09-0.15mol/L, especially 0.10-0.12mol/L, to remove the cross-linking agent.

According to another embodiment of the invention, in the purification step, the amount of phosphate buffer added each time is 6-10 times that of the centrifugal precipitation, and the washing is performed more than or equal to 5 times to remove the cross-linking agent from the cross-linked collagen suspension.

According to another embodiment of the invention, the process of the invention further comprises a homogenization step, in which the product is poured into a homogenizer and homogenized to homogeneity, for example, cyclic homogenization greater than or equal to 2 times, even greater than or equal to 3 times.

According to another embodiment of the present invention, the preparation method of the collagen filler further comprises a filling step, wherein the homogenized sample is filled in a syringe, and the collagen filler is obtained after internal packaging and external packaging.

According to another embodiment of the present invention, the preparation method of the collagen bulking agent of the present invention is sequentially performed according to the following steps:

(1) Dissolving collagen extracted from the tissue in an acid solution of 0.001mol/L to 0.1mol/L, and then filtering and sterilizing;

(2) Adding phosphate buffer solution, regulating pH to 6-8, stirring at a certain temperature until collagen is separated out to form white suspension;

(3) Adding 0.1% -10% (v/v) microsphere, and stirring at a certain temperature until it is uniform;

(4) Adding a cross-linking agent, regulating the temperature to 20-40 ℃, and reacting for 3-10 days under the constant temperature condition;

(5) Sieving the suspension while stirring, wherein the mesh size is exchanged according to the microsphere size (mesh size < microsphere diameter) to remove all microspheres in the suspension;

(6) Centrifuging to collect a product, and repeatedly cleaning the product with phosphate buffer solution to remove the cross-linking agent;

(7) Homogenizing: pouring the product into a homogenizer, homogenizing until uniform;

(8) Optionally, filling: filling the homogenized sample into a syringe, and obtaining the collagen filler after internal packaging and external packaging.

According to another embodiment of the invention, the temperature of step (2) and step (3) is between 20 and 40 c,

for example 25-37 ℃.

The collagen protein is extracted from animal, such as animal tissue, and can be injected into the middle layer to deep layer of dermis to fill skin defect caused by soft tissue defect.

According to another embodiment of the present invention, the collagen from which the collagen bulking agent of the present invention is prepared is collagen extracted from animal tissue, comprising one or more of bovine, equine, ovine, porcine, fish-derived atelopeptide collagen type I, type II or type III, collagen-enriched decellularized human skin tissue, decellularized pig skin tissue or bovine skin tissue; more preferably porcine-derived atelopeptide collagen type I or type III.

According to another embodiment of the present invention, the collagen from which the collagen bulking agent of the present invention is prepared is modified collagen obtained by a chemical crosslinking process, and the degree of enzymolysis is not more than 40% after the in vitro enzymolysis test, for example, enzymolysis for a certain time at 37 ℃ and a collagenase concentration of 1 mg/ml.

According to another embodiment of the present invention, the collagen used for preparing the collagen filler of the present invention is modified collagen obtained by chemical crosslinking, and after the crosslinking process is completed, the modified collagen is cleaned to remove the crosslinking agent which does not participate in the reaction, thereby ensuring the safety of the collagen filler. The residual amount of the crosslinking agent of the collagen filler prepared by the present invention was detected by gas chromatography, and as a result, was not detected. And is far lower than the control amount of other facial fillers for the index (less than or equal to 2.0 mug/g).

In order to achieve the aim of the invention, the collagen filling agent is prepared by adopting a dynamic template method, on one hand, the relative spacing of collagen fiber bundles in the crosslinking process is controlled by controlling the pore size taking microspheres as templates, and then the pore size of the collagen fiber bundles after forming a crosslinking framework is controlled; on the other hand, by controlling a certain stirring rotation speed, each collagen fiber bundle can obtain the same crosslinking opportunity under the dynamic state, so that the more thorough and uniform crosslinking effect of the collagen implant is realized.

The collagen filler provided by the invention has a macroporous fiber network structure, and the 'skeleton' formed by fibers has higher dynamic viscosity and shows stronger mechanical properties due to more sufficient crosslinking reaction.

According to the collagen filler, a fiber network structure with controllable pore diameter is obtained by adopting a dynamic template method, and collagen fibers with more complete crosslinking reaction are obtained by reducing the concentration of a crosslinking agent and prolonging the crosslinking time, so that the enzymolysis resistance of the filler is improved. In view of obtaining a fibrous network structure with controllable pore diameters, on one hand, microsphere templates with different diameters can prepare implant samples with different pore diameters, and the implants with different pore diameters can adapt to skin filling with different concave degrees; on the other hand, the cross-linking sites with sufficient reaction also obtain longer maintenance time, which is beneficial to obtaining longer lasting and stable filling effect of the product after implantation.

In addition, the collagen filler provided by the invention adopts a template method to build a collagen fiber network in the process of crosslinking collagen fibers, so that the implant maintains more fiber network structures, ensures the transportation of nutrient substances and metabolites after being implanted into a body, and induces cells to migrate, proliferate and differentiate into the implant, thereby enabling local fibroblast proliferation and synthesizing and secreting more type I collagen fibers.

The collagen filler provided by the invention maintains a part of complete triple helix structure of collagen, so that the active ingredients of the collagen are maintained, and the potential sensitization and poisoning risks of the crosslinking reaction are greatly reduced.

The invention also relates to a collagen composition comprising the collagen bulking agent of the present invention, optionally comprising additives such as suspending agents, antioxidants, therapeutically active agents such as pharmaceutically active agents; contains nutritional agent, proteins such as recombinant collagen, polypeptide, various amino acids, polysaccharides such as sodium hyaluronate, and chitosan; or a combination thereof.

The invention also relates to the use of the collagen bulking agent of the present invention for the preparation of a product for medical cosmetic plastic treatment, such as the treatment of fine wrinkles, facial sculptures, correction, surgical operations or surgical treatment bulking agents.

The invention also relates to a method of medical cosmetic treatment such as the treatment of fine wrinkles, facial sculptures, correction, surgery or surgical treatment comprising the step of using a collagen bulking agent according to the present invention.

Drawings

Fig. 1 shows a freeze-dried sem image of the collagen bulking agent of example 1 of the present application, which has a hierarchical porous structure.

Fig. 2 shows a scanning electron microscope image of a lyophilized product of the collagen bulking agent of comparative example 1 of the present application.

Fig. 3 shows a gas chromatogram (not detected) of the residual amount of the crosslinking agent of the collagen filler of example 4 of the present application.

Fig. 4 shows a scanning electron microscope image of a lyophilized product of the collagen bulking agent of example 6 of the present application.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. Accordingly, the detailed description of the embodiments of the invention provided below is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.

Examples 1-6 and comparative examples 1-3: preparation of collagen bulking agent

Example 1:

example 1 of the present invention implements the following steps:

(1) Collagen preparation solution is dissolved: weighing 10g of collagen, wherein the collagen raw material is purchased type I collagen (GVB-atelo-03, guangdong Shengshi) and is of pig origin. Adding 2000mL of 0.01mol/L hydrochloric acid solution, and stirring at 4 ℃ for 48 hours until complete dissolution to obtain a uniform collagen solution;

and (3) filtering and sterilizing: under the pressure of 0.25Mpa, a filtering membrane of 0.2 mu m is used for filtering the collagen solution, and the collagen solution is filtered twice in sequence, so as to achieve the aim of sterilization and obtain the sterile collagen filtrate.

(2) Collagen fiber precipitation: adding 0.1mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution, adjusting pH to 6, and stirring at room temperature until collagen fibers are separated out.

(3) And (3) hole making of the template: alumina ceramic microspheres with diameters of 50 μm and 100 μm are added respectively, the stacking volume ratio is 0.5% and 1% respectively, and stirring is continued at 25 ℃ until uniform.

(4) Crosslinking: a0.01 mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 4g of sorbitol was added, the pH was adjusted to 11, the temperature was adjusted to 30℃and the reaction was stirred for 3 days.

(5) Sieving: sieving the suspension with stirring, wherein the mesh number of the sieve is 320 meshes, so as to remove all microspheres in the suspension;

(6) Purifying: the product was collected by centrifugation at 5000rpm and the collagen suspension was repeatedly washed with 10 volumes of 0.01mol/L sterile phosphate buffer and the above procedure was repeated 5 times to remove cross-linker residues and bacterial endotoxins in the collagen.

(7) Homogenizing: pouring the purified and collected product into a homogenizer, circularly homogenizing for 3 times until the purified and collected product is uniform;

(8) And (3) filling: and filling the homogenized sample into a 1mL syringe, and then carrying out inner wrapping and outer wrapping to obtain the collagen filler.

Example 2:

example 2 of the present invention implements the following steps:

(1) Collagen preparation solution is dissolved: weighing 10g of collagen, wherein the collagen raw material is purchased type I collagen and is of pig origin. Adding 2000mL of 0.01mol/L hydrochloric acid solution, and stirring at 10 ℃ for 30h until complete dissolution to obtain a uniform collagen solution;

and (3) filtering and sterilizing: under the pressure of 0.25Mpa, a filtering membrane with the thickness of 0.2 mu m is used for filtering the collagen solution, and the collagen solution is filtered twice in sequence, so as to achieve the aim of sterilization, and the sterile collagen filtrate is obtained.

(2) Collagen fiber precipitation: to the mixture was added 0.1mol/L of a phosphate alkaline (0.1 mol/LNaOH) buffer solution, the pH was adjusted to 6, and the mixture was stirred at room temperature until collagen fibers were precipitated.

(3) And (3) hole making of the template: alumina ceramic microspheres with a diameter of 100 μm were added to the mixture in a bulk ratio of 1%, and stirring was continued at 25℃until uniform.

(4) Crosslinking: a0.01 mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 6g of sorbitol was added, the pH was adjusted to 11, the temperature was adjusted to 35℃and the reaction was stirred for 6 days.

(5) Sieving: sieving the suspension with stirring, wherein the mesh number of the sieve is 160 meshes, so as to remove all microspheres in the suspension;

Example 3:

example 3 of the present invention implements the following steps:

(1) Collagen preparation solution is dissolved: 10g of collagen is weighed, and the collagen raw material is type I collagen purchased by the company and is pig source. Adding 2000mL of 0.01mol/L hydrochloric acid solution, and stirring at 10 ℃ for 30h until complete dissolution to obtain a uniform collagen solution;

(2) Collagen precipitation: adding 0.1mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution into the sterile collagen filtrate obtained in the step (2), adjusting the pH to 7, and stirring at room temperature until collagen fibers are separated out.

(3) And (3) hole making of the template: polytetrafluoroethylene microspheres with a diameter of 100 μm were added to a bulk volume of 2%, and stirring was continued at 25 ℃ until uniform.

(6) Purifying: the product was collected by centrifugation at 6000rpm and the collagen suspension was repeatedly washed with 8 volumes of 0.01mol/L sterile phosphate buffer and the above procedure was repeated 6 times to remove the cross-linker residue and bacterial endotoxin in the collagen.

(7) Homogenizing: pouring the purified and collected product into a homogenizer, and circularly homogenizing for 3 times until the purified and collected product is uniform;

Example 4:

example 4 of the present invention implements the following steps:

(1) Collagen preparation solution is dissolved: 10g of collagen is weighed, and the collagen raw material is type I collagen purchased by the company and is pig source. Adding 0.01mol/L hydrochloric acid solution 4000mL, and stirring at 10 ℃ for 18h until complete dissolution to obtain a uniform collagen solution;

and (3) filtering and sterilizing: under the pressure of 0.2Mpa, a filtering membrane with the thickness of 0.2 mu m is used for filtering the collagen solution, and the collagen solution is filtered twice in sequence, so that the aim of sterilization is fulfilled, and the sterile collagen filtrate is obtained.

(2) Collagen precipitation: adding 0.1mol/L alkaline (0.1 mol/LNaOH) phosphate buffer solution into the sterile collagen filtrate obtained in the step (2), adjusting the pH to 7, and stirring at room temperature until collagen fibers are separated out.

(3) And (3) hole making of the template: polytetrafluoroethylene microspheres with a diameter of 200 μm were added to a bulk volume of 2%, and stirring was continued at 25 ℃ until uniform.

(4) Crosslinking: 0.01mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution containing 6g 1,4 butanediol glycidyl ether (purity: 95%) was added, pH was adjusted to 9, temperature was adjusted to 30℃and the reaction was stirred for 3 days.

(5) Sieving: sieving the suspension with stirring, wherein the mesh number of the sieve is 90 meshes, so as to remove all microspheres in the suspension;

(6) Purifying: the product was collected by centrifugation at 10000rpm and the collagen suspension was repeatedly washed with 8 volumes of 0.01mol/L sterile phosphate buffer and the above procedure was repeated 6 times to remove the cross-linker residue and bacterial endotoxin in the collagen.

(7) Homogenizing: the purified and collected product is poured into a homogenizer and circularly homogenized for 3 times until uniform.

Example 5:

example 5 of the present invention implements the following steps:

(1) Collagen preparation solution is dissolved: 10g of collagen is weighed, and the collagen raw material is type I collagen purchased by the company and is pig source. Adding 0.01mol/L hydrochloric acid solution 3000mL, and stirring at 10deg.C for 16h until dissolution is complete to obtain uniform collagen solution;

(2) Collagen precipitation: adding 0.1mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution into the sterile collagen filtrate obtained in the step (2), adjusting the pH to 8, and stirring at room temperature until collagen fibers are separated out.

(3) And (3) hole making of the template: polytetrafluoroethylene microspheres with the diameter of 200 mu m are added, the stacking volume ratio is 1%, and stirring is continued to be uniform at normal temperature.

(6) Purifying: the product was collected by centrifugation at 10000rpm and the collagen suspension was repeatedly washed with 10 volumes of 0.01mol/L sterile phosphate buffer, and the above procedure was repeated 5 times to remove the cross-linker residue and bacterial endotoxin in the collagen.

Example 6:

example 6 of the present invention implements the following steps:

And (3) filtering and sterilizing: the collagen solution was filtered using a 0.2 μm filter membrane under a pressure of 0.25 Mpa.

(2) And filtering twice successively to reach the aim of sterilization and obtain the sterile collagen filtrate.

Collagen precipitation: adding 0.1mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution into the sterile collagen filtrate obtained in the step (2), regulating the pH to 8, and stirring at room temperature until collagen fibers are separated out.

(3.1) first template hole making: adding polytetrafluoroethylene microspheres with the diameter of 200 mu m, wherein the stacking volume ratio of the polytetrafluoroethylene microspheres is 0.5%, and continuously stirring the microspheres at normal temperature until the microspheres are uniform;

(4.1) crosslinking: 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 6g 1,4 butanediol glycidyl ether (purity: 95%) is added, pH is adjusted to 9, temperature is adjusted to 35 ℃, and stirring reaction is carried out for 2 days.

(5.1) sieving: sieving the suspension with stirring, wherein the mesh number of the sieve is 90 meshes, so as to remove all microspheres in the suspension;

(3.2) second template hole making: adding polytetrafluoroethylene microspheres with the diameter of 50 mu m, wherein the stacking volume ratio of the polytetrafluoroethylene microspheres is 0.5%, and continuously stirring the microspheres at normal temperature until the microspheres are uniform;

(4.2) crosslinking: the crosslinking temperature was adjusted to 35℃and the reaction was stirred for 2 days.

(5.2) sieving: sieving the suspension with stirring, wherein the mesh number of the sieve is 320 meshes, so as to remove all microspheres in the suspension;

Comparative example 1: the procedure is as in example 1, except that the preparation is carried out by triple crosslinking without using template microspheres, the following steps being carried out:

and (3) filtering and sterilizing: filtering the collagen solution with a 0.2 μm filter membrane under 0.25Mpa, filtering twice successively to achieve the aim of sterilization, obtaining sterile collagen filtrate,

(2) Collagen fiber precipitation: adding 0.1mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution, adjusting pH to 6, stirring at room temperature until collagen fiber is separated out,

(3) First cross-linking: adding 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 0.3g glutaraldehyde, adjusting pH to 7, adjusting temperature to 30 ℃, stirring and reacting for 16h,

(4) Centrifuging to collect precipitate to obtain crosslinked collagen fiber after first crosslinking, dispersing in phosphate buffer solution,

(5) Second crosslinking: adding 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 5g of 1, 4-butanediol diglycidyl ether into the obtained crosslinked collagen fiber, adjusting pH to 11, adjusting temperature to 40 ℃, stirring and reacting for 16h,

(6) Centrifuging and collecting the precipitate to obtain crosslinked collagen fiber after the second crosslinking, dispersing in phosphate buffer solution,

(7) Third crosslinking: adding 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 5g carbodiimide into the obtained crosslinked collagen fiber, adjusting pH to 5, adjusting temperature to 35 ℃, stirring and reacting for 16h,

(8) Centrifuging and collecting the precipitate to obtain crosslinked collagen fiber after the third crosslinking,

(9) Purifying: and centrifuging at 5000rpm to collect the product to obtain crosslinked collagen fiber after the third crosslinking, repeatedly cleaning with 10 times of 0.01mol/L sterile phosphate buffer solution, and repeating the above operation for 5 times to remove the crosslinking agent residue and bacterial endotoxin in the collagen.

(10) Homogenizing: pouring the purified and collected product into a homogenizer, circularly homogenizing for 3 times until the purified and collected product is uniform;

(11) And (3) filling: and filling the homogenized sample into a 1mL syringe, and then carrying out inner wrapping and outer wrapping to obtain the collagen filler.

According to FIG. 1, after the collagen bulking agent is subjected to freeze-drying treatment to remove water, the collagen bulking agent presents a hierarchical porous structure of 50-300 mu m, and the pore walls are fiber net-shaped pore walls formed by crosslinking collagen fibers.

The collagen bulking agent of example 1 has a larger and more abundant pore size than the collagen bulking agent of comparative example 1 (shown in fig. 2), and this hierarchical porous structure facilitates the growth of fibroblasts and the transport of nutrients and metabolites after the material is implanted into the human body to promote regeneration and remodeling of the collagen fiber network. According to fig. 3, the collagen filler of the invention has no crosslinking agent residue detected, and the residual quantity is 0, which indicates that the preparation process related to the invention thoroughly removes the crosslinking agent, and further proves that the collagen filler of the invention has higher safety.

Comparative example 2: the procedure is as in example 1, except that comparative example 2 is prepared using a double crosslinking method and without using template microspheres, the following steps are performed:

(3) First cross-linking: adding 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 20g glucose, adjusting pH to 7, adjusting temperature to 30 ℃, stirring and reacting for 7 days,

(5) Second crosslinking: adding 0.01mol/L phosphate alkaline (0.1 mol/L NaOH) buffer solution containing 10g of carbodiimide and 2g of N-hydroxysuccinimide (NHS) into the obtained crosslinked collagen fiber, adjusting the pH to 5.5, adjusting the temperature to room temperature, stirring and reacting for 16 hours,

(6) Purifying: and centrifuging at 5000rpm to collect the product to obtain crosslinked collagen fiber after the second crosslinking, repeatedly cleaning with 10 times of 0.01mol/L sterile phosphate buffer solution, and repeating the above operation for 5 times to remove the crosslinking agent residue and bacterial endotoxin in the collagen.

Comparative example 3: the procedure is as in example 1, except that comparative example 3 is prepared using a method that does not use template microspheres, the following steps are performed:

(3) Crosslinking: adding 0.01mol/L phosphate alkaline (0.1 mol/LNaOH) buffer solution containing 4g sorbitol, adjusting pH to 11, adjusting temperature to 30 ℃, stirring and reacting for 3 days,

(4) Purifying: centrifuging at 5000rpm to collect the product, repeatedly cleaning the collagen suspension with 10 times of 0.01mol/L sterile phosphate buffer solution, and repeating the above operation for 5 times to remove crosslinking agent residue and bacterial endotoxin in collagen;

(5) Homogenizing: pouring the purified and collected product into a homogenizer, circularly homogenizing for 3 times until the purified and collected product is uniform;

(6) And (3) filling: and filling the homogenized sample into a 1mL syringe, and then carrying out inner wrapping and outer wrapping to obtain the collagen filler.

Examples 7 to 10: results of properties of collagen bulking agent

Example 7: dynamic viscosity of collagen bulking agent

The present example detects the dynamic viscosity of collagen fillers.

The detection method comprises the following steps: the experiment is carried out according to the third method of pharmacopoeia of the people's republic of China (edition 2020), and specific test parameters are referred to YY 0954-2015 passive surgical implant type I collagen implant. The test samples were examples 1 to 6 and comparative examples 1 to 3.

TABLE 1 dynamic viscosity detection results of collagen bulking agent

From the dynamic viscosity test data in Table 1 above, the dynamic viscosity of the collagen fillers of examples 1-6 of the present invention is significantly higher than that of comparative examples 1-3. Without wishing to be bound by theory, the reasons for this may include the following two points: first, the collagen filler prepared by the dynamic template method has larger pore size and is shown as a larger strain range. And secondly, after the sufficient crosslinking reaction, the crosslinking site of the crosslinked fiber improves the overall stability of the reticular fiber. The combined action of the two points ensures that the collagen filler in the invention shows higher dynamic viscosity when resisting continuous shearing force, thus indicating that the collagen filler has more excellent plasticity.

Example 8: degree of crosslinking of collagen bulking agent

In this example, the degree of crosslinking (amino substitution) of the collagen filler was examined.

The detection method comprises the following steps: in the experimental example, the content of ninhydrin is detected to carry out the crosslinking degree analysis of the collagen filler.

< experimental group 1>

1.0g of the sample prepared in examples 1 to 6 was weighed, 3mL of purified water and 2mL of ninhydrin reaction solution were added successively, the mixture was uniformly mixed, heated at 100℃for 30 minutes, cooled to room temperature by an ice-water bath, diluted to 25mL with 50% isopropanol, and the absorbance value was measured at 570 nm.

< experimental group 2>

The procedure of experiment group 2 was identical to that of experiment 1, except that the samples used in experiment group 2 were collagen implants prepared at the same concentration as the uncrosslinked samples.

< control group >

The control group procedure was identical to that of experiment 1, except that the sample was replaced with an equal amount of purified water.

< Standard Curve drawing >

And drawing a standard curve by taking L-glutamic acid as a standard solution. Specifically, 10mmol/L L-glutamic acid standard solution L-glutamic acid standard solution 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL are respectively sucked into 25mL colorimetric tubes with plugs (corresponding L-glutamic acid is respectively 0, 2.0, 4.0, 6.0, 8.0 and 10.0 mmol), then 1.0, 0.8, 0.6, 0.4, 0.2 and 0mL of purified water are sequentially added, and the following operations are carried out from the step of adding 3mL of purified water sequentially in the experimental group 1 until detection is completed.

The crosslinking degree calculation formula:

wherein a is the amino group corresponding to the absorbance value of the experimental group 1 subtracted from the absorbance value of the control group, and b is the amino group corresponding to the absorbance value of the experimental group 2 subtracted from the absorbance value of the control group.

TABLE 2 results of measurement of degree of crosslinking of collagen fillers

	Degree of crosslinking
		Example 1	20.04％
Example 2	43.76％
		Example 3	49.22％
Example 4	24.70％
		Example 5	30.26％
Example 6	21.36％
		Comparative example 1	75.62％
Comparative example 2	62.30％
		Comparative example 3	54.16％

According to the crosslinking degree test data in Table 2 above, the crosslinking degree of the collagen fillers prepared by the methods of examples 1 to 6 of the present invention is significantly lower than that of the comparative examples, probably because the crosslinking reaction of the collagen fibers prepared by the methods of the present invention mostly occurs on the surface of the collagen fiber bundle, and a part of the natural collagen fibers remain inside, which is critical for the maintenance of the collagen activity.

Example 9: degree of enzymolysis of collagen bulking agent

In this example, the degree of enzymolysis of the collagen filler was measured.

The detection method comprises the following steps: the experimental example is used for detecting the ratio of the content of hydroxyproline in the enzymolysis supernatant of the test sample to the content of hydroxyproline in the non-enzymolysis test sample, so as to reflect the enzymolysis degree of the test sample in a certain time.

And detecting the content of hydroxyproline in the enzymolysis supernatant of the test sample and the content of hydroxyproline in the non-enzymolysis test sample according to YY/T1151-2017 collagen sponge appendix B.

The enzymatic treatment of the samples was as follows:

< experimental group 1>

1.0g of the samples prepared in examples 1-6 and the comparative example were weighed out, respectively, and 9mL of collagenase solution (pH 7.4, containing 0.1mmol/L CaCl was added ₂ The concentration of collagenase is 1 mg/mL), and the mixture is placed into a water bath at 37 ℃ for incubation for 2h. After centrifugation, 1mL of supernatant was taken for hydroxyproline content detection.

< experimental group 2>

1.0g of the samples prepared in examples 1-6 were weighed correspondingly and tested according to YY/T1151-2017 collagen sponge appendix B.

< control group >

The crosslinking degree calculation formula:

wherein, A is the hydroxyproline content corresponding to the absorbance value of the experimental group 1 subtracted from the absorbance value of the control group, and B is the hydroxyproline content corresponding to the absorbance value of the experimental group 2 subtracted from the absorbance value of the control group.

TABLE 3 results of detection of the degree of enzymolysis of collagen bulking agent

	Degree of enzymolysis
		Example 1	39.25％
Example 2	18.00％
		Example 3	36.10％
Example 4	38.45％
		Example 5	32.66％
Example 6	20.67％
		Comparative example 1	46.49％
Comparative example 2	48.67％
		Comparative example 3	54.18％

According to the enzymolysis degree detection data in Table 3, the enzymolysis degree of the collagen fillers prepared by the methods of examples 1 to 6 of the present invention is significantly lower than that of the comparative examples. The reason is mainly that epoxide is adopted in the crosslinking reaction, the crosslinking reaction speed of the epoxide and collagen is slower than that of glutaraldehyde, which provides more sufficient reaction conditions for active groups on the surface of the collagen fiber, and ensures that the crosslinking reaction in the whole collagen fiber is uniformly and completely distributed. In addition, on one hand, the full degree of the collagen crosslinking reaction can be improved by adjusting the reaction temperature, the concentration of the crosslinking agent and the reaction pH, so that the sample can still keep lower enzymolysis degree under the condition of low crosslinking degree; on the other hand, the cross-linked network structure is adjusted by adjusting the diameter size and the stacking volume ratio of the dynamic template so as to obtain collagen filling agents with different void structures, so that the sample has more application scenes.

Example 10: average pore size and porosity of collagen bulking agent

Under the same conditions, each test piece was injected into the same cylindrical mold and freeze-dried.

(1) Average pore diameter: taking a center line section of a sample, horizontally attaching the section to one side of a section sample table, observing by using a scanning electron microscope, counting the aperture size, and calculating an average value.

(2) Porosity: the test article (dried) was measured by the pycnometer method, specifically as follows:

under constant temperature conditions, the precisely weighed test sample (Ws) was immersed in a gravity flask filled with ethanol, which was pre-weighed as W1. Vacuum was applied to degas the sample until the sample was completely saturated with ethanol, then the pycnometer was refilled with ethanol and the total weight of the system was recorded as W2. Then, the sample containing ethanol was taken out, and the weight of the pycnometer and the total weight of ethanol remaining in the pycnometer were recorded as W3. Thus, the porosity (. Epsilon.) of the test sample is calculated as follows:

ε＝VP/(Vp+Vs)＝(W2-W3-Ws)/(W1-W3)

where Vs is the volume of the sample (excluding internal pores) and Vp is the pore volume inside the sample.

(3) Cell proliferation assay: CCK-8 was used to detect relative numbers during cell proliferation.

The method comprises the following specific steps:

a) 0.1g of the sample was injected into a 48-well plate, and the cell suspension was taken at 5X 10 ³ Inoculating rBMSCs on the surface of a sample by using a TCPS group as a control group, transferring the culture plate into an incubator, continuously supplementing 500 mu L of alpha-MEM for culture after cells are attached for 2 hours, and placing the incubator for continuous culture; the cells were inoculated on day 0, when the day of cell growth was noted.

b) Preparing CCK-8 solution: CCK-8 liquid and medium were mixed in a volume ratio of 1:9 preparing CCK-8 solution;

c) After the cells are respectively cultured to 1 st, 3 rd, 5 th and 7 th days in a contact culture mode, sucking out the culture medium, adding 200 mu L of CCK-8 solution into each hole, and placing the mixture into an incubator for incubation for 2 hours;

d) Taking out the incubated culture plate, sucking 100 mu L of the solution, placing the solution on an ELISA plate, selecting the wavelength of 450nm for quantitative detection of absorbance, recording the value, and calculating the proliferation rate of cells.

TABLE 4 results of average pore size, porosity and cell growth promoting rate

The results according to the average pore size and porosity of table 4 above show that the samples prepared in examples 1-6 all show a higher average pore size and a higher porosity. Example 6 obtained a hierarchical porous structure and higher porosity. In example 6, the prepared collagen filler is added sequentially by controlling the microspheres with large (200 μm) and small (50 μm) pore diameters, and the cross-linking process is mild and thorough, so that the finally prepared collagen filler has higher dynamic viscosity, porosity, proper porous structure, lower cross-linking degree and enzymolysis degree, and is more suitable for filling and repairing moderately severe gravity wrinkles. As shown in Table 4, the collagen bulking agent of the present invention has a higher cell growth promoting rate in example 6. Moreover, the cell viability of the collagen bulking agents prepared in examples 1-6 was significantly higher than that of comparative examples 1, 2, 3 and negative controls, indicating that the samples prepared in this invention were non-cytotoxic and exhibited better cell compatibility.

Example 11: collagen cytotoxicity in vitro

This example examined collagen bulking agents for cytotoxicity in vitro.

The detection method comprises the following steps: the experiment is based on GB/T16886.5-2017 biological evaluation of medical instruments: part 5: the test method in vitro cytotoxicity test was performed.

TABLE 6 in vitro cytotoxicity test results of collagen bulking agent

	In vitro cytotoxicity test results (cell viability)
		Example 1	114.49±4.29％
Example 2	110.86±6.57％
		Example 3	108.79±4.13％
Example 4	118.36±10.65％
		Example 5	111.86±9.78％
Example 6	115.63±4.26％
		Comparative example 1	97.31±3.65％
Comparative example 2	96.32±1.49％
		Comparative example 3	98.45±3.67％
Blank control example	100％
		Positive control example	7.31±2.46％
Negative control example	98.61±1.68％

According to the in vitro cytotoxicity test results shown in Table 6, the samples prepared by the method show higher cell survival rate. The cell viability of the collagen bulking agents prepared in examples 1-6 was significantly higher than that of the comparative examples 1, 2, 3 and negative control, indicating that the samples prepared in this invention were non-cytotoxic and exhibited better cell compatibility.

In summary, the collagen filler prepared by the invention structurally has a fiber porous scaffold which can simulate a natural extracellular matrix, and a triple helix structure with the most characteristic of collagen molecules is partially reserved. Through full and complete crosslinking reaction, the collagen filler with obviously improved mechanical properties and greatly reduced enzymolysis degree is obtained, which is beneficial to prolonging the action time of the filler in tissues, reducing the injection frequency of the collagen filler and effectively reducing the use cost of the preparation. The collagen filler prepared according to the method of the present invention may have a multi-stage pore size, for example, a multi-stage pore size of a combination of two or more of a larger pore size and a smaller pore size in a range of 50 μm to 500 μm, for example, a multi-stage pore size of a combination of two or more of a smaller pore size of 50 μm to 120 μm and a larger pore size of 120 μm to 400 μm. The collagen filler with the multi-level pore diameter also has excellent performance in mechanics and higher dynamic viscosity, which is probably beneficial to the fact that the pores are connected through crosslinked collagen network fibers, so that the collagen filler still keeps higher viscosity under the action of shearing force, the displacement risk of the product after being implanted into tissues is greatly reduced, and the collagen filler is more suitable for filling and repairing moderately severe gravity wrinkles. In terms of biological performance, in addition to ensuring sterility and qualified endotoxin test, the cytotoxicity test results show that the collagen filling agent has a cell growth promoting rate of at least 110-120%, which shows that the filling agent has good biocompatibility, and further proves the biological safety of the collagen filling agent.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims

1. A collagen bulking agent, wherein the collagen bulking agent has an average pore size D ₅₀ From 50 μm to 500 μm, a porosity of from 80% to 99.5%, a degree of crosslinking of from 10% to 70%, and an in vitro enzymatic hydrolysis loss of less than 40%.

2. The collagen bulking agent of claim 1, wherein the collagen bulking agent has at least one of the following:

average pore diameter D ₅₀ From 80 μm to 400 μm, even from 90 μm to 300 μm, in particular from 100 μm to 250 μm, especially from 120 μm to 200 μm;

-a porosity of 80% -99%, even 85% -98%, in particular 90% -96%;

-a degree of crosslinking of 12% to 65%, even 15% to 60%, in particular 18% to 55%, in particular 20% to 50%;

-an in vitro enzymatic hydrolysis loss rate of less than 40%, preferably less than 35%, even less than 30%, in particular less than 25%, in particular less than 20%;

-a dynamic viscosity of 90000mpa.s to 300000mpa.s, preferably 95000mpa.s to 280000mpa.s, even 98000mpa.s to 270000mpa.s, in particular 100000mpa.s to 260000mpa.s, in particular 120000mpa.s to 250000mpa.s, at a shear rate of 2Hz and a temperature of 25±1 ℃;

the melting point is from 55 ℃ to 85 ℃, preferably from 58 ℃ to 80 ℃, even from 60 ℃ to 78 ℃, especially from 62 ℃ to 75 ℃, in particular greater than 65 ℃, or less than 73 ℃.

3. Collagen bulking agent of any of the preceding claims, wherein the collagen bulking agent comprises a combination of different pore sizes, preferably a combination of one or more pore sizes of 50 μm to 120 μm with one or more pore sizes of 120 μm to 500 μm, more preferably a combination of one or more pore sizes of 50 μm to 120 μm with one or more pore sizes of 120 μm to 500 μm, in particular a combination of one or more pore sizes of 50 μm to 100 μm with one or more pore sizes of 120 μm to 450 μm, even a combination of one or more pore sizes of 50 μm to 90 μm with one or more pore sizes of 130 μm to 420 μm, or a combination of one or more pore sizes of 50 μm to 80 μm with one or more pore sizes of 140 μm to 400 μm.

4. A method of preparing a collagen bulking agent of any of the preceding claims 1 to 3 comprising the steps of:

(1) Providing collagen, dissolving the collagen, and optionally filtering and sterilizing;

(3) Adding the microspheres, and stirring until uniform;

(4) Adding a cross-linking agent, regulating the temperature to 20-40 ℃, and carrying out cross-linking reaction under the constant temperature condition;

(5) Removing the microspheres from the collagen suspension;

(6) Optionally neutralisation, purification and homogenisation.

5. The method according to claim 8 or 9, wherein in step (1), the treatment temperature is 1 ℃ to 15 ℃.

6. The method according to claim 8 or 9, wherein in step (2) the volume ratio of collagen solution to buffer solution is 20:1 to 5:1, or 10:1 to 8:1.

7. The method according to claim 8 or 9, wherein in step (2), the treatment temperature is 25 ℃ to 37 ℃.

8. The method according to claim 8 or 9, wherein steps (3) to (5) are performed 2-5 times, wherein the microspheres used in each of steps (3) to (5) are performed with a different particle size, selected from the group consisting of one or more combinations of particle sizes 50 μm to 120 μm and one or more combinations of particle sizes 120 μm to 500 μm, in particular one or more combinations of particle sizes 50 μm to 100 μm and one or more combinations of particle sizes 120 μm to 400 μm, and even one or more combinations of particle sizes 50 μm to 90 μm and one or more combinations of particle sizes 150 μm to 300 μm.

9. The method of claim 8 or 9, wherein the microspheres have a bulk volume of 0.1% to 10% relative to the total volume of the collagen suspension.

10. Use of a collagen bulking agent according to any of the preceding claims 1 to 3 and a collagen bulking agent obtained according to the method of any of the preceding claims 4 to 9 for the preparation of a bulking agent product for medical cosmetic shaping.