Recombinant collagen filler as well as preparation method and application thereof

Abstract

The invention provides a recombinant collagen filler as well as a preparation method and application thereof, the recombinant collagen filler comprises the following components: recombinant collagen gel and coral hydroxyapatite particles, and the mass ratio of the recombinant collagen gel to the coral hydroxyapatite particles is (2.1-11): 1; the filling agent also selectively comprises a first diluent, and the content of the first diluent is 32.5-45.5% by taking the total mass of the recombinant collagen filling agent as 100%; wherein the coral hydroxyapatite particles are uniformly distributed in the recombinant collagen gel; wherein the recombinant collagen gel is prepared from recombinant collagen microspheres and a second diluent. The filler disclosed by the invention is low in cost, high in cost performance, rapid and convenient in injection process, long in acting time, free of repeated injection and capable of removing wrinkles for a long time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, relates to the field of injectables for aesthetic medicine filling, and particularly relates to a recombinant collagen filler and its preparation method and application. Background Art

[0002] With the improvement of living standards, people's demand for beauty is increasing day by day. As a safe and effective way, injectable fillers are increasingly favored by consumers. The injectable filling technology is simple to operate, and the effect can be immediately shown only after a few minutes of injection. It is painless, does not affect work and life, is very convenient, leaves no trace, and has good confidentiality, protecting the privacy of customers. However, an ideal injectable soft tissue filler should simultaneously have biocompatibility, safety, easy operability, anchoring property and durability, which has always been a very challenging topic for scholars researching and developing plastic and aesthetic products.

[0003] At present, injectable light aesthetic medicine injection materials include materials such as hyaluronic acid, botulinum toxin, regenerative injectables, and collagen. Their main function is to fill and shape, so as to achieve the elimination of facial wrinkles and beauty anti-aging.

[0004] Currently, the commonly used injectable light aesthetic medicine fillers in the world are as follows:

[0005] 1. Hyaluronic acid filler: It has comprehensive facial applicability. Medium and large molecular hyaluronic acid has strong supportability and is suitable for filling and shaping. There is basically no recovery period after injection. In addition, hyaluronic acid can be completely absorbed, so it is also a good choice for areas such as the cranial top and the back of the head. However, the maintenance time is relatively short, and it needs to be replenished regularly about half a year later. There will be obvious swelling locally after injection, but it can be quickly reduced by applying ice packs for cold compress.

[0006] 2. Botulinum toxin: It has significant advantages in the fields of beauty and medicine, such as safety, rapidity, and remarkable effect. However, it also has some disadvantages, such as timeliness, side effects, and unnatural facial expressions.

[0007] 3. Regenerative injectables: Regenerative injectables are favored in the field of aesthetic medicine filling for their natural, lasting, and safe characteristics. However, their high cost and technical requirements also limit their wide application. With the progress of technology and the gradual acceptance of the market, regenerative injectables are expected to become the mainstream anti-aging products in the future.

[0008] 4. Collagen: The commonly seen collagen injectables on the market at present are bovine collagen and porcine collagen, which can be absorbed or degraded by the human body and have significant repair, regeneration, and whitening effects in the field of medical beauty. However, bovine / porcine collagen belongs to heterologous protein, and only patients who pass the skin allergy test are allowed to use it.

[0009] In summary, after injection of the current injectable materials on the market, as the injected materials gradually degrade, the filling effect also gradually dissipates, and the durability of the filling effect is poor. Periodic injections need to be carried out repeatedly, which only treats the symptoms but not the root cause. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a recombinant collagen filler, its preparation method and application, aiming at the problems of poor filling durability of existing filling materials, induration and Tyndall effect in tissues after use.

[0011] To achieve the above object, on the one hand, the present invention provides a recombinant collagen filler, the components of which include a recombinant collagen gel and coral hydroxyapatite particles, and the mass ratio of the recombinant collagen gel to the coral hydroxyapatite particles is (2.1-11):1.

[0012] The filler also selectively includes a first diluent. Based on the total mass of the recombinant collagen filler being 100%, the content of the first diluent is 32.5-45.5%;

[0013] Among them, the coral hydroxyapatite particles are uniformly distributed in the recombinant collagen gel;

[0014] Among them, the recombinant collagen gel is prepared from recombinant collagen particles and a second diluent.

[0015] According to a specific embodiment of the present invention, preferably, based on the total mass of the recombinant collagen filler being 100%, the content of the first diluent is 35-45%.

[0016] According to a specific embodiment of the present invention, preferably, the mass ratio of the recombinant collagen gel to the coral hydroxyapatite particles is (4-10):1

[0017] The recombinant collagen filler provided by the present invention is an injectable cosmetic plastic material. After filling, the recombinant collagen filler can be durable, have a relatively high mechanical strength, and can achieve a more natural effect after injection, avoiding the Tyndall effect around the eyes, and the tissue is soft without induration.

[0018] During the frequency sweep test using a rheometer at 37°C and 1% strain from 0.1 to 100 Hz, G′ (storage modulus) is always greater than G″ (loss modulus), and the elastic part is dominant, which is defined as a gel. Among them, G' represents the elastic part, that is, the part stored in the deformation ability; G″ represents the viscous part, that is, the part lost in the deformation ability. G″ < G', and the elastic part is dominant, which is defined as a gel.

[0019] The recombinant collagen filler provided by the present invention has a uniform appearance, without stratification, precipitation or suspension. The coral hydroxyapatite particles are evenly distributed in the gel and are evenly wrapped by the gel. When the filler is placed in a container and tilted or inverted, it does not flow. When the filler is placed in a container and tilted at 30°-360°, the filler body does not flow along the bottle wall or slowly flow down, being in a non-flowing state. According to a specific embodiment of the present invention, preferably, the pushing force of the recombinant collagen filler through a 27G needle is 25-35N, more preferably 28-32N, and further preferably 31N.

[0020] In the present invention, the conditions for measuring the pushing force are as follows: filling 2 mL of the recombinant collagen filler sample, installing a 27G needle, and pushing at a constant speed of 10 mm / min until the sample is completely pushed out with a constant force value, and the experiment ends. During the experiment, the maximum force value measured is the pushing force.

[0021] According to a specific embodiment of the present invention, preferably, the pushing force of the recombinant collagen filler through a 27G needle is about 31N.

[0022] According to a specific embodiment of the present invention, preferably, in the frequency sweep test of the recombinant collagen filler at 37°C and 1% strain from 0.1 to 100 Hz, G′ (storage modulus) is always greater than G″ (loss modulus), and elasticity accounts for the main part, being a gel.

[0023] In the present invention, a rheometer is used to characterize its storage modulus G' and loss modulus G. G' represents the elastic part, that is, the part stored in the deformation ability; G″ represents the viscous part, that is, the part lost in the deformation ability. When G″ < G', elasticity accounts for the main part, being a gel; when G″ > G', viscosity accounts for the main part, being a sol.

[0024] According to a specific embodiment of the present invention, the recombinant collagen gel is obtained by physically cross-linking recombinant collagen microspheres to obtain physically cross-linked recombinant collagen microspheres, and then adding them to a second diluent and stirring and swelling.

[0025] According to a specific embodiment of the present invention, the recombinant collagen microspheres are formed by spray granulation of a recombinant human-derived collagen solution.

[0026] According to a specific embodiment of the present invention, preferably, in the step of spray granulation, the spray temperature is 100-260°C, the feeding speed is 200-600 ml / h, and the nozzle diameter is 0.75-2 mm.

[0027] According to a specific embodiment of the present invention, preferably, in the recombinant human-derived collagen solution, the mass fraction of recombinant human-derived collagen is 10%-20%.

[0028] The recombinant human collagen in the present invention is the recombinant human collagen disclosed in CN108070032B [A Purification Method of Recombinant Human Collagen].

[0029] According to a specific embodiment of the present invention, the recombinant human collagen has the amino acid sequence shown in SEQ ID No: 1.

[0030] According to a specific embodiment of the present invention, preferably, the cross-linking method of the physical cross-linking is thermal cross-linking.

[0031] According to a specific embodiment of the present invention, preferably, the cross-linking temperature of the thermal cross-linking is 120 - 180 °C, and the cross-linking time is 1 - 6 h.

[0032] According to a specific embodiment of the present invention, preferably, the concentration of the recombinant collagen microspheres in the second diluent is 8% - 30% (w / v), more preferably 10% - 30% (w / v).

[0033] According to a specific embodiment of the present invention, preferably, the rotation speed of the stirring is 100 - 150 r / min, and the stirring time is 1 - 4 h.

[0034] According to a specific embodiment of the present invention, preferably, the temperature of the swelling is 2 - 8 °C, and the swelling time is 16 - 24 h.

[0035] The recombinant collagen gel of the present invention is obtained by physically cross-linking and swelling the recombinant collagen microspheres, and it is a gel that is uniform, without stratification, precipitation, or suspended particles. The present invention controls the quality of the recombinant collagen gel by controlling the stirring time and the swelling time. If the stirring time is too short and the swelling time is too short, it cannot be dissolved well. If the stirring time is too long and the swelling time is too long, it will increase the risk of exceeding the microbial limit. Therefore, in the present invention, it is stirred at a rotation speed of 100 - 150 r / min for 1 - 4 h, and then swollen at 2 - 8 °C for 16 - 24 h. The obtained recombinant collagen gel is a mashed potato-like gel, which does not flow when tilted or inverted in a container, and is visually uniform, without stratification, precipitation, suspension, etc.

[0036] According to a specific embodiment of the present invention, preferably, the first diluent includes one or a combination of two or more of water for injection, purified water, and phosphate buffer solution.

[0037] According to a specific embodiment of the present invention, preferably, the second diluent includes one or a combination of two or more of water for injection, purified water, and phosphate buffer solution.

[0038] According to a specific embodiment of the present invention, preferably, the water for injection includes sterile physiological saline.

[0039] According to a specific embodiment of the present invention, preferably, the particle size of the recombinant collagen microspheres is 2 - 10 μm.

[0040] According to a specific embodiment of the present invention, preferably, the molecular weight of the recombinant collagen microspheres is 30 - 90 kDa.

[0041] According to a specific embodiment of the present invention, preferably, the coral hydroxyapatite particles are prepared by subjecting coral raw materials to pretreatment, crushing and granulation, and hydrothermal exchange.

[0042] According to a specific embodiment of the present invention, preferably, the pretreatment step includes ultrasonically cleaning the coral raw materials to wash away surface impurities, dust, etc., then soaking them in a sodium hypochlorite solution or a hydrogen peroxide solution, and then washing them with purified water and drying for standby.

[0043] Through hydrothermal exchange, the mineral components in natural coral stones are converted into natural coral hydroxyapatite with a "nano - flower" structure. Further, the obtained natural coral hydroxyapatite particles are screened to obtain coral hydroxyapatite particles meeting the requirements of the present invention.

[0044] According to a specific embodiment of the present invention, preferably, the particle size distribution range of the coral hydroxyapatite particles is 20 - 50 μm.

[0045] According to a specific embodiment of the present invention, preferably, the porosity of the coral hydroxyapatite particles is 50 - 70%;

[0046] According to a specific embodiment of the present invention, preferably, the conversion rate (hydroxyapatite content) of the coral hydroxyapatite particles is ≥80%.

[0047] According to a specific embodiment of the present invention, the raw materials of the coral hydroxyapatite particles include natural coral;

[0048] According to a specific embodiment of the present invention, preferably, the natural coral includes Porites and / or Goniopora; more preferably Porites.

[0049] The injectable recombinant collagen filler gel of the present invention utilizes the tight combination of "nano - flower" - structured natural coral hydroxyapatite particles and a recombinant collagen gel formed by the swelling of physically cross - linked recombinant collagen microspheres to obtain an injectable recombinant collagen filler. Among them, the recombinant collagen gel is safe, non - toxic, biodegradable, has excellent biocompatibility, and has no virus risk. The natural coral hydroxyapatite particles have a porosity of 50% - 70%, can load growth factors and grow in, and the degradation time is controllable, and can maintain a satisfactory degradation performance.

[0050] The recombinant collagen filler for injection provided by the present invention. The recombinant collagen microspheres in the material are obtained by spray-drying a recombinant human collagen solution to form recombinant collagen microspheres with a diameter of 2-10 μm. Then, the above-mentioned recombinant collagen microspheres are physically crosslinked at a temperature of 120-180 °C for 1-6 h.

[0051] The physically crosslinked recombinant collagen microspheres with a diameter of 2-10 μm used in the present invention have the following advantages:

[0052] The recombinant collagen microspheres obtained by this method are milky white powders, which are safe, non-toxic, biodegradable, have excellent biocompatibility, and have no virus risk. Compared with the collagen powder obtained by crushing collagen sponges, the recombinant collagen microspheres have a smaller particle size and swell more uniformly, which is beneficial for injection. The gel formed by the recombinant collagen microspheres can uniformly wrap the "nano-flower" structured natural coral hydroxyapatite particles, avoiding the aggregation of natural coral hydroxyapatite particles.

[0053] In the present invention, the mass ratio of the physically crosslinked recombinant collagen gel to the natural coral hydroxyapatite particles is (2.1-11):1. In the material prepared with this mass ratio, the gel formed by the physically crosslinked recombinant collagen microspheres can uniformly wrap the "nano-flower" structured natural coral hydroxyapatite particles, avoiding the aggregation of natural coral hydroxyapatite particles. Even when used after 24 months of storage, the injection effect can still be maintained. In addition, if the amount of recombinant collagen microspheres is too large and the amount of natural coral hydroxyapatite particles is too small, it may cause low support and too fast degradation time; it cannot or is insufficient to stimulate the generation of enough fibrous connective tissue in a timely manner; if the amount of recombinant collagen microspheres is too small and the amount of natural coral hydroxyapatite particles is too large, it may lead to easy needle clogging during the injection process and too strong mechanical properties, resulting in too long a degradation time in the later stage and too short a time to maintain the initial support effect, and the injection site may collapse before sufficient fibrous connective tissue of the human body can be stimulated to replace its support effect.

[0054] The natural coral hydroxyapatite particles with a diameter of 20-50 μm used in the present invention have the following advantages:

[0055] When hydroxyapatite is used in injectable plastic materials, it is often ground into powder, and generally, the finer the particle size, the better, so that it can easily enter human tissues during powder injection. The natural coral hydroxyapatite particles of the present invention have a particle size of 20-50 μm, have a "nano-flower" surface microstructure, can well support the skin, have a porosity of 50%-70%, can load growth factors to grow in, and have a good degradation time. The recombinant collagen filler obtained by wrapping the natural coral hydroxyapatite particles with a "nano-flower" structure with the recombinant collagen gel of the present invention has good support, a high elastic modulus, can achieve the most natural effect after injection, the tissue at the injection site is soft, without induration, and the particle size of the particles can not only ensure that the filler can be easily injected subcutaneously with a fine needle, but also ensure that the particles remain in the treatment site permanently.

[0056] On the other hand, the present invention also provides a preparation method of the above-mentioned recombinant collagen filler, wherein the preparation method includes:

[0057] (1) Mix and stir the coral hydroxyapatite particles with a first diluent to obtain a coral hydroxyapatite particle solution;

[0058] (2) Mix, stir, swell and sterilize the recombinant collagen gel and the coral hydroxyapatite particle solution to obtain the recombinant collagen filler.

[0059] In the above preparation method, preferably, the concentration of the coral hydroxyapatite particles in the first diluent is 10%-40% (w / v), preferably 16%-24%.

[0060] In the above preparation method, preferably, in step (1), the rotation speed of the stirring is 100-150 r / min, and the stirring time is 30-60 min to obtain a white coral hydroxyapatite particle solution.

[0061] In the above preparation method, preferably, in step (2), the rotation speed of the stirring is 100-150 r / min, and the stirring time is 1-4 h.

[0062] In the above preparation method, preferably, in step (2), the swelling temperature is 2-8 °C, and the swelling time is 16-24 h.

[0063] In the above preparation method, preferably, in step (2), the ratio of the recombinant collagen gel to the coral hydroxyapatite particles is (2.1-11):1.

[0064] To ensure that the material can be used for human injection, it is crucial to ensure aseptic conditions during the preparation process. In the present invention, the above-mentioned recombinant collagen gel is mixed with the coral hydroxyapatite particle solution, and then subjected to moist heat sterilization or irradiation sterilization to obtain a recombinant collagen filler.

[0065] In the above preparation method, preferably, in step (2), the sterilization is moist heat sterilization and / or irradiation sterilization; more preferably, the temperature of the moist heat sterilization is 121 - 124 °C, and the time is 15 - 30 min; the effective dose of the irradiation sterilization is 15 - 35 kGy.

[0066] In addition, during the specific preparation process, all the equipment and utensils used also need to be sterilized. The parts of the equipment that come into contact with the product and the utensils used, such as scissors, forceps, etc., are wrapped separately and labeled with moist heat sterilization indicator stickers, and then placed in a steam sterilizer for moist heat sterilization at 121 °C for 30 min.

[0067] The above preparation method only illustrates how to prepare the injectable recombinant collagen filler provided by the present invention. If this material is packaged into a finished product using a syringe, the subsequent packaging steps specifically include:

[0068] Filling step:

[0069] Assemble the filling machine on the ultra-clean bench to ensure aseptic operation and change gloves in a timely manner. Transfer the prepared material to the filling machine, open the lower discharge port, and adjust the filling machine so that the filling volume is 1.0 - 1.5 ml.

[0070] Open the package of the disposable syringe in the ultra-clean operation bench, connect it to the screw port at the outlet of the filling machine, turn on the main switch of the air compressor (outside the clean workshop), and step on the filling switch of the filling machine to fill the material into the syringe.

[0071] Inner packaging step:

[0072] After filling, tighten the screw cap on the ultra-clean bench, then place it in a blister shell and seal it on a medical heat sealer to complete the inner packaging.

[0073] On the other hand, the present invention also provides an application of the above-mentioned recombinant collagen filler or the above-mentioned preparation method in the preparation of products for repairing skin defects.

[0074] In some specific embodiments, preferably, the product includes products for wrinkle correction and / or tissue filling.

[0075] The above technical solutions of the present invention have the following advantages:

[0076] First, it is a recombinant collagen gel formed by physical cross-linking of recombinant collagen microspheres. The recombinant collagen gel encapsulating the "nano-flower" structure of coral hydroxyapatite is milky white and opaque, avoiding the Tyndall effect around the eyes after injection. The recombinant collagen gel evenly encapsulates natural coral hydroxyapatite particles to obtain a recombinant collagen filler. The particle size of the filler is suitable for being easily injected subcutaneously with a needle, and the effect can be seen immediately after injection. As the collagen degrades, it stimulates the production of the skin's own collagen, and the degradation products can provide raw materials for the production of skin collagen. Subsequently, the newly generated collagen occupies the injection site and releases coral hydroxyapatite. The continuous stimulation causes the skin to produce fibrous connective tissue to achieve the purpose of repairing skin defects, with a long action time and no need for multiple injections.

[0077] Second, it has a long-lasting effect in removing wrinkles. Most people only need to be injected 1 - 2 times.

[0078] Third, it is fast. The injection process is convenient, only taking 3 - 5 minutes, and normal activities can be resumed immediately after injection.

[0079] Fourth, it is safe. Collagen is needed by the human body and can be absorbed by the human body to achieve the purpose of removing wrinkles. Description of the Drawings

[0080] Figure 1 It is a scanning electron micrograph of the recombinant collagen gel prepared in Example 1 (where a is 200 times and b is 1000 times).

[0081] Figure 2 It is a scanning electron micrograph of natural coral hydroxyapatite with a size of 20 - 50μm (where a is 100 times and b is 10000 times).

[0082] Figure 3 It is a scanning electron micrograph of the recombinant collagen filler prepared in Example 1 (where a is 1000 times and b is 5000 times).

[0083] Figure 4 It is a test result graph of the fluidity appearance of the recombinant collagen filler prepared in Example 1 (where a: tilted; b: inverted).

[0084] Figure 5 It is a test result graph of the rheological mechanics (viscoelasticity) of the recombinant collagen filler prepared in Example 1.

[0085] Figure 6 It is the test result of the injection force of the recombinant collagen filler prepared in Example 1.

[0086] Figure 7 It is the cell migration result of the recombinant collagen filler of Examples 1 - 3.

[0087] Figure 8It is a hematoxylin-eosin staining picture of the recombinant collagen filler prepared in Example 1 implanted into the subcutaneous tissue for 52 weeks. Detailed implementation manners

[0088] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention will now be described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0089] In actual production, those skilled in the art can understand that, on the premise of obtaining qualified products, some related process steps can be adjusted or increased or decreased.

[0090] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0091] The amino acid sequence of the recombinant human-derived collagen used in the following examples is: GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP (SEQ ID No:1).

[0092] Example 1

[0093] 1. Preparation method of recombinant collagen gel for recombinant collagen filler:

[0094] Step 1: Prepare a 15% (wt) recombinant human collagen solution by mixing recombinant human collagen (a protein with the amino acid sequence of SEQ ID No: 1 in the sequence listing) with sterile normal saline;

[0095] Step 2: Perform spray granulation on the recombinant human collagen solution from Step 1 on a spray dryer. Observe that the recombinant collagen microspheres ejected into the collection bottle of the spray dryer are white powder, and the particle size of the recombinant collagen microspheres is 2 - 10 μm;

[0096] Step 3: For the preparation method of the above-mentioned recombinant collagen microsphere spray granulation, the spray temperature is 140 °C, the feeding rate is 300 ml / h, and the nozzle diameter is 0.75 mm.

[0097] Step 4: Physically crosslink the recombinant collagen microspheres from Step 2, with a crosslinking temperature of 170 °C and a time of 2 h;

[0098] Step 5: After mixing the crosslinked recombinant collagen microspheres from Step 4 with sterile normal saline, stir at a speed of 100 r / min for 1 h, and then swell in a 2 - 8 °C refrigerator for 16 h to obtain a recombinant collagen gel; wherein, the concentration of the recombinant human collagen microspheres in the physiological saline is 16% (w / v), that is, recombinant human collagen microspheres / (recombinant human collagen microspheres + normal saline) × 100% = 16%.

[0099] 2. Preparation method of natural coral hydroxyapatite microspheres with a particle size of 20 - 50 μm for recombinant collagen fillers:

[0100] Step 1: Ultrasonically clean natural high-quality Porites coral stones to wash away surface impurities, dust, etc., then soak them in a 5% sodium hypochlorite solution, and then wash them with purified water and dry for later use;

[0101] Step 2: Through the "hydrothermal exchange" method, convert the mineral components in natural coral stones into natural coral hydroxyapatite;

[0102] Step 3: Screen the above-treated natural coral hydroxyapatite to obtain the coral hydroxyapatite particles, wherein the particle size distribution range of the coral hydroxyapatite particles is 20 - 50 μm, and the porosity and conversion rate data are shown in Table 2.

[0103] Step 4: Mix the natural coral hydroxyapatite particles evenly with sterile normal saline and stir at a rotation speed of 100 r / min for 30 min to obtain a natural coral hydroxyapatite particle solution. Among them, the concentration of natural coral hydroxyapatite particles in normal saline is 16% (w / v), that is, natural coral hydroxyapatite particles / (natural coral hydroxyapatite particles + normal saline) × 100% = 16%.

[0104] 3. Preparation method for the recombinant collagen filler:

[0105] Mix the above-mentioned recombinant collagen gel with the natural coral hydroxyapatite particle solution. Among them, the mass ratio of the recombinant collagen gel to the natural coral hydroxyapatite particles is 25:4. Stir at a rotation speed of 100 r / min for 1 h, then swell in a refrigerator at 2 - 8 °C for 16 h, and then perform moist heat sterilization at 121 °C for 30 min to obtain the recombinant collagen filler.

[0106] Example 2

[0107] Other conditions are the same as those in Example 1, and the differences are:

[0108] 1. Dissolve recombinant human collagen (a protein with the amino acid sequence of SEQ ID No: 1 in the sequence listing) in water to prepare a 12% (wt) recombinant human collagen solution;

[0109] 2. Preparation method for spray granulation of recombinant collagen microspheres, the spray temperature is 170 °C, the feeding speed is 600 ml / h, and the nozzle diameter is 1.5 mm; the particle size of the recombinant collagen microspheres is 2 - 10 μm;

[0110] 3. Physically crosslink the recombinant collagen microspheres, the crosslinking temperature is 170 °C, and the time is 3 h.

[0111] Example 3

[0112] Other conditions are the same as those in Example 1, and the differences are:

[0113] 1. Mix the natural coral hydroxyapatite particles evenly with sterile normal saline and stir at a rotation speed of 100 r / min for 30 min to obtain a natural hydroxyapatite particle solution. Among them, the concentration of natural coral hydroxyapatite particles in normal saline is 24% (w / v);

[0114] 2. The mass ratio of the above-mentioned recombinant collagen gel to the natural coral hydroxyapatite particles is 4:1.

[0115] Comparative Example 1

[0116] Other conditions were the same as those in Example 1, with the differences being: 1. Purchased commercially available nano-hydroxyapatite (CAS No.: 719892-61-2); 2. The concentration of the nano-hydroxyapatite in the physiological saline was 24% (w / v); 3. The mass ratio of the recombinant collagen gel to the nano-hydroxyapatite particles was 4:1.

[0117] Comparative Example 2

[0118] Other conditions were the same as those in Example 1, with the differences being: 1. Instead of spray granulation, the recombinant human-derived collagen solution was freeze-dried into a recombinant collagen sponge and then physically cross-linked; 2. The recombinant collagen sponge was pulverized into powder using a pulverizer with a rotation speed of 5000 rpm and a time setting of 5 s to obtain recombinant collagen powder, which was mixed with sterile physiological saline to obtain a recombinant collagen gel.

[0119] Comparative Example 3

[0120] Other conditions were the same as those in Example 1, with the differences being: 1. The recombinant collagen microspheres after spray granulation were not physically cross-linked; 2. The recombinant collagen microspheres after spray granulation were mixed with sterile physiological saline to obtain a recombinant collagen gel.

[0121] Comparative Example 4

[0122] Other conditions were the same as those in Example 1, with the difference being: 1. The particle size distribution range of the natural coral hydroxyapatite particles was 80 - 250 μm.

[0123] Comparative Example 5

[0124] Other conditions were the same as those in Example 1, with the differences being: 1. The concentration of the recombinant collagen microspheres in the physiological saline was 8% (w / v); 2. The concentration of the natural coral hydroxyapatite particles in the physiological saline was 43% (w / v); 3. The mass ratio of the recombinant collagen gel to the nano-hydroxyapatite particles was 3:2.

[0125] Comparative Example 6

[0126] Other conditions were the same as those in Example 1, with the differences being: 1. The concentration of the recombinant collagen microspheres in the physiological saline was 50% (w / v); 2. The concentration of the natural coral hydroxyapatite particles in the physiological saline was 10% (w / v); 3. The mass ratio of the recombinant collagen gel to the nano-hydroxyapatite particles was 12:1.

[0127] Effect Example

[0128] The following performance tests were carried out on the recombinant collagen fillers prepared in the above examples and comparative examples:

[0129] 1. Appearance test method: Randomly take out 2 mL of the materials prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6, pre-fill them in a syringe, and observe whether there are unswollen collagen particles and hydroxyapatite particles in the materials. The results are shown in Table 1.

[0130] 2. Scanning electron microscopy: After freeze-drying the recombinant collagen gel and recombinant collagen filler material in Example 1, place them in liquid nitrogen for 3 - 5 minutes and then break them apart. Use scanning electron microscopy to observe the microscopic morphology of their cross-sections and natural coral hydroxyapatite particles. The results are as Figure 1 , Figure 2 , Figure 3 shown.

[0131] 3. Microbial limit: Randomly take out the materials in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 and determine them according to the microbial limit inspection of non-sterile products: microbial counting method in the fourth part of the Chinese Pharmacopoeia (General Principles 1105) 2020 edition. The results are shown in Table 1.

[0132] 4. Fluidity:

[0133] Randomly take out the materials in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6, pre-fill them in a syringe, inject them into a transparent colorless glass bottle, and tilt it at 30° - 360°. Observe whether the material flows along the bottle wall or slowly drips down. The results are shown in Table 2, Figure 4 (where a: tilt; b: invert).

[0134] 5. Rheological mechanics:

[0135] Use a cone plate with a diameter of 20 mm and an angle of 1°. Adjust the diameter and thickness of the sample in Example 1 to 20 mm and 37 μm. Select the oscillation mode. Conduct a frequency sweep test at 37°C and 1% strain, with the frequency: 0.1 - 100 Hz. Use a rheometer to characterize its storage modulus G' and loss modulus G. G' represents the elastic part, that is, the part stored in the deformation ability; G″ represents the viscous part, that is, the part lost in the deformation ability. When G″ < G', the elastic part is the main part, which is a gel; when G″ > G', the viscous part is the main part, which is a sol. The results are as Figure 5 shown.

[0136] 6. The test was carried out on a universal mechanical testing machine for injection force. The 2 mL sample after filling in Example 1 was equipped with a 27G needle, fixed on the mechanical testing machine, and injected at a constant speed of 10 mm / min until the sample was pushed out with a constant force value to end the experiment. The curve of the injection force changing with time during the injection process of the sample was recorded. The results are shown in Table 2. Figure 6 。

[0137] 7. Biological evaluation:

[0138] The test was carried out according to GB / T 16886: The samples prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 were used for cytotoxicity experiments, and Example 1, Example 2, and Example 3 were used for cell migration experiments. The results are shown in Table 3 and Table 4 and Figure 7 。

[0139] 8. Porosity test:

[0140] The materials in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 were freeze-dried and sputter-coated with gold, and tested by scanning electron microscopy. The results are shown in Table 2.

[0141] 9. Conversion rate:

[0142] The proportion of the natural coral mineral components converted into hydroxyapatite was measured by a thermogravimetric analyzer for the samples prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6. The results are shown in Table 2.

[0143] Table 1 Appearance test and microbial limit results

[0144]

[0145] As can be seen from the above table, by comparing Example 1 and Example 3, it was found that the stirring time and swelling time of the prepared materials were at the maximum values of the parameters. Although the results of the microbial limit were qualified, there was an increased risk of exceeding the microbial limit. Therefore, during the material preparation process, it was more appropriate to keep the stirring time and swelling time below the maximum values.

[0146] As can be seen from the above table, by comparing Example 1 and Comparative Example 2, it was found that the sample prepared in Comparative Example 2 was uneven, layered, and had suspended collagen particles. The reason was analyzed that after freeze-drying, the recombinant collagen sponge was crushed by a crusher, and the collagen particle size was not delicate. During the compounding process with hydroxyapatite, the swelling was not as good as that of the recombinant collagen powder after spray granulation, and the swelling effect was good.

[0147] As can be seen from the above table, by comparing Example 1 with Comparative Example 3, it was found that the sample prepared in Comparative Example 3 was uniform, layered, had precipitation, and had suspended particles. The reason for the analysis was that the recombinant collagen gel formed by the recombinant collagen microspheres without physical crosslinking had low viscoelasticity, which was not sufficient to uniformly disperse natural coral hydroxyapatite, resulting in precipitation due to gravity and suspended particles, and agglomeration of natural coral hydroxyapatite particles occurred.

[0148] As can be seen from the above table, by comparing Example 1 with Comparative Example 5, it was found that the sample prepared in Comparative Example 5 was uniform, layered, had precipitation, and had suspended particles. The reason for the analysis was that the content of natural coral hydroxyapatite was too high, resulting in the gel formed by the recombinant collagen microspheres being unable to uniformly wrap the "nano-flower" structure natural coral hydroxyapatite particles, and the phenomenon of agglomeration of coral hydroxyapatite particles occurred.

[0149] Table 2 Detection results of recombinant collagen fillers;

[0150]

[0151] From the results of the above table, it can be seen that natural coral hydroxyapatite with a size of 20 - 50 μm has a porosity of 50% - 70%, can load growth factors and grow in, the conversion rate is greater than 80%, and it has a good degradation time.

[0152] From the results of the above table, it can be seen that the porosity of nano-hydroxyapatite in Comparative Example 1 is relatively low, the injection force is comparable to that of natural coral hydroxyapatite, the content of hydroxyapatite is high, and the degradation time is relatively long.

[0153] From the results of the above table, it can be seen that the crushed collagen powder, the particle size of coral hydroxyapatite is too large and the content is too high in Comparative Example 2, Comparative Example 4 and Comparative Example 5, which will cause the needle tube to be blocked and the clinical operation is inconvenient.

[0154] Figure 5 It is a test result graph of the rheological mechanics (viscoelasticity) of the recombinant collagen filler in Example 1.

[0155] As Figure 5 shown, when the oscillation frequency is 1 Hz, the G' of the recombinant collagen filler is 1493.7 Pa and the G'' is -111.997 Pa; when the oscillation frequency is increased to 10 Hz, the G' of the recombinant collagen filler is 1974.43 Pa and the G'' is 87.7038 Pa; when the oscillation frequency is increased to 100 Hz, the G' of the recombinant collagen filler is 109281 Pa and the G'' is -45522.4 Pa. The strain scan results are as shown above. In the strain range of the frequency scan test at 37°C and 1% strain, the G′ (storage modulus) of the recombinant collagen filler is always greater than G″ (loss modulus), and the elasticity accounts for the main part, which is a gel.

[0156] Figure 6 Test results of the injection force of the recombinant collagen filler in Example 1.

[0157] It can be seen from Figure 6 that the injection force of the recombinant collagen filler through a 27G needle is about 31 N.

[0158] The following is the biocompatibility evaluation of the recombinant collagen filler;

[0159] Table 3 Cytotoxicity results

[0160]

[0161] Table 4 shows the cell migration results;

[0162]

[0163] Figure 7 It is a display of the cell migration results.

[0164] Biological evaluation results:

[0165] The results of the cytotoxicity test are shown in Table 3. The results show that when the undiluted extract of the recombinant collagen filler acts on L929 cells for 24 h, the cell viability is above 85%, and there is no obvious cytotoxicity;

[0166] According to Figure 7 the cell migration results show that: one dosing concentration was set for each sample, and a cell scratch experiment was carried out on L929 cells. The cell migration rate of the blank control group was normalized, and the relative migration rate of each group was calculated. The test results are shown in Table 4. Figure 7 . Compared with the blank control group, the materials prepared in Example 1, Example 2, and Example 3 significantly promoted the migration of L929 cells at 48 h and 96 h;

[0167] 10. Filling effect test: 1 ml samples after filling in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 were equipped with 27G needles and 0.2 ml was respectively injected into the subcutaneous tissue of the back of SD rats. The appearance of the injection sites was observed 4 weeks, 8 weeks, 12 weeks, 26 weeks, and 52 weeks after implantation, including the presence or absence of redness, inflammation, the effect of the sample on the surrounding tissue, and the degradation of the material, as shown in Table 5.

[0168] Table 5 Gross observation of subcutaneous implantation

[0169]

[0170] As can be seen from the above table, by comparing Example 1 and Example 2, it is found that after spray granulation, the physical cross-linking time of the recombinant collagen powder increases, and the degradation time in vivo is longer; by comparing Example 1 and Example 3, it is found that the higher the content of natural coral hydroxyapatite, the longer the degradation time in vivo.

[0171] As can be seen from the above table, by comparing Example 1 with Comparative Example 1 and Comparative Example 4, it is found that using natural coral hydroxyapatite with a particle size of 20 - 50 μm is more conducive to the ingrowth of bioactive factors and collagen regeneration than nano-hydroxyapatite, and the filling effect in vivo is more obvious due to the larger pore size of natural coral hydroxyapatite.

[0172] As can be seen from the above table, by comparing Example 1 with Comparative Example 2 and Comparative Example 3, it is found that the material prepared from the recombinant collagen microspheres formed by spray granulation of the solution prepared with the recombinant human-derived collagen (protein having the amino acid sequence of SEQ ID No: 1 in the sequence listing) of the present invention has higher biosafety and excellent biocompatibility than the collagen powder obtained by crushing the recombinant collagen sponge after freeze-drying. Moreover, the degradation time of the recombinant collagen prepared by physical cross-linking after spray granulation is excellent, achieving a satisfactory effect.

[0173] As can be seen from the above table, by comparing Example 1 with Comparative Example 5 and Comparative Example 6, it is found that if the amount of recombinant collagen microspheres is too large and the amount of natural coral hydroxyapatite powder is too small, the supportability is low and the degradation time is too fast, insufficient to stimulate the production of enough fibrous connective tissue in a timely manner; if the amount of recombinant collagen microspheres is too small and the amount of natural coral hydroxyapatite particles is too large, it may cause the needle to be easily blocked during injection, and the mechanical properties are too strong, resulting in too long a degradation time in the later stage, and the initial support time may be too short to wait for the stimulation of sufficient proliferation of the body's own fibrous connective tissue to replace its support function, leading to collapse at the injection site.

[0174] Figure 8 It is a hematoxylin-eosin staining picture of the injection of Example 1 implanted into the subcutaneous tissue of SD rats for 52 weeks. It can be seen that there is still a small amount of material residue at the implantation site after 52 weeks of implantation of the injection, indicating that the material can be maintained in vivo for more than 1 year.

Claims

1. A recombinant collagen filler, comprising recombinant collagen gel and coral hydroxyapatite particles, wherein the mass ratio of the recombinant collagen gel to the coral hydroxyapatite particles is (2.1-11):1; The filler may further selectively include a first diluent, wherein the content of the first diluent is 32.5-45.5% based on the total mass of the recombinant collagen filler being 100%; in, The coral hydroxyapatite particles are evenly distributed in the recombinant collagen gel; Wherein, the recombinant collagen gel is prepared by recombinant collagen microspheres and a second diluent.

2. The recombinant collagen filler according to claim 1, wherein: The recombinant collagen gel is obtained by physically cross-linking recombinant collagen microspheres to obtain physically cross-linked recombinant collagen microspheres, which are then added into a second diluent and stirred and swelled.

3. The recombinant collagen filler according to claim 2, wherein: The recombinant collagen microspheres are formed by spray granulation of a recombinant human collagen solution; Preferably, in the spray granulation step, the spray temperature is 100-260°C, the feed rate is 200-600ml / h, and the nozzle diameter is 0.75-2mm; Preferably, in the recombinant human collagen solution, the mass fraction of the recombinant human collagen is 10%-20%; Preferably, the recombinant human collagen has an amino acid sequence as shown in SEQ ID No:

1.

4. The recombinant collagen filler according to claim 2, wherein: The physical cross-linking method is thermal cross-linking; Preferably, the crosslinking temperature of the thermal crosslinking is 120-180°C, and the crosslinking time is 1-6h; Preferably, the concentration of the recombinant collagen microspheres in the second diluent is 8%-30% (w / v); Preferably, the stirring speed is 100-150r / min, and the stirring time is 1-4h; Preferably, the swelling temperature is 2-8°C, and the swelling time is 16-24h.

5. The recombinant collagen filler according to claim 1, wherein: The first diluent includes one or a combination of two or more of water for injection, purified water, and phosphate buffer; Preferably, the second diluent comprises one or a combination of two or more of water for injection, purified water, and phosphate buffer.

6. The recombinant collagen filler according to claim 2, wherein: The particle size of the recombinant collagen microspheres is 2-10 μm; Preferably, the molecular weight of the recombinant collagen microspheres is 30-90 kDa.

7. The recombinant collagen filler according to claim 1, wherein: The particle size distribution range of the coral hydroxyapatite particles is 20-50 μm; Preferably, the porosity of the coral hydroxyapatite particles is 50-70%; Preferably, the conversion rate of the coral hydroxyapatite particles is ≥ 80%.

8. A method for preparing the recombinant collagen filler according to any one of claims 1 to 7, wherein: The preparation method comprises: (1) mixing and stirring the coral hydroxyapatite particles and a first diluent to obtain a coral hydroxyapatite particle solution; (2) The recombinant collagen gel and the coral hydroxyapatite particle solution are mixed, stirred, swollen, and sterilized to obtain a recombinant collagen filler.

9. The preparation method according to claim 8, wherein: The concentration of the coral hydroxyapatite particles in the first diluent is 10%-40% (w / v); Preferably, in step (1), the stirring speed is 100-150 r / min, and the stirring time is 30-60 min; Preferably, in step (2), the stirring speed is 100-150 r / min, and the stirring time is 1-4 h; Preferably, in step (2), the swelling temperature is 2-8°C and the swelling time is 16-24h; Preferably, in step (2), the sterilization is moist heat sterilization and / or irradiation sterilization; more preferably, the moist heat sterilization temperature is 121-124°C and the time is 15-30 min; the effective dose of the irradiation sterilization is 15-35 KGy.

10. Use of the recombinant collagen filler according to any one of claims 1 to 7 or the preparation method according to any one of claims 8 to 9 in preparing a product for repairing skin defects; Preferably, the product comprises a product for wrinkle correction and / or tissue augmentation.

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