Injectable filler as well as preparation method and application thereof

Abstract

The invention provides an injectable filler as well as a preparation method and application thereof. On the basis of systematically screening the influence of parameters such as collagen concentration, buffer solution type, preparation method, concentration and the like in the filler on the performance of the filler, the injectable filler with excellent performance in various aspects is obtained through optimization. The invention also scientifically screens the filtering and degerming process parameters and conditions of the collagen, and systematically researches the influence of a plurality of parameters on the activity maintenance of the collagen in the process. The sterilization method obtained through optimization is easy and convenient to operate, economical, environmentally friendly and good in repeatability, damage of dry heat sterilization, moist heat sterilization, irradiation sterilization and the like to the structure and activity of the collagen is avoided, in a word, the sterilization effect can be guaranteed, and the three-dimensional structure of the collagen can be kept. The composition can continuously stimulate regeneration of the collagen on the basis of supplementing the collagen in the initial stage, and the composite effect of short-term supporting and long-term repairing is achieved on the whole.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly relates to an injectable filler, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is an ideal biomedical material with a unique triple helix structure, having characteristics such as high tensile strength, low antigenic activity, low irritation, and low cytotoxicity. It can promote cell growth and adhesion, and cooperate with new cells and tissues to repair wounds. In the field of medical aesthetics, collagen can be injected into facial tissues as a tissue filler to play a supporting and filling role, thereby achieving the purpose of correcting wrinkles. However, after the collagen cosmetic filler prepared by the prior art is injected into the body, the effect is not lasting, and new fillers need to be continuously supplemented to maintain its effect.

[0003] On the other hand, collagen was approved as a filler in the field of medical aesthetics earlier than hyaluronic acid, but the clinical application process is slower than that of hyaluronic acid. One important factor is that due to the easy destruction of the collagen structure, traditional sterilization methods will cause different degrees of damage to the triple helix structure of collagen and lose its biological activity. The sterilization process of collagen has always been a major problem in this field, which greatly limits its wide application. Summary of the Invention

[0004] An object of one aspect of the present invention is to provide an injectable filler, which contains 30 - 55 mg / mL of collagen, 30 - 55 mg / mL of PLLA microspheres, and PBS buffer solution.

[0005] In a preferred embodiment of the present invention, the collagen in the filler is obtained through sterilization, and the sterilization includes the following steps: dissolving collagen in 0.01M - 0.05M phosphoric acid or hydrochloric acid at 15 - 25°C, filtering to obtain a filtrate, adjusting the pH value of the filtrate to 4 - 5 with 0.01M - 0.05M alkali solution to obtain sterile collagen, and the concentration of collagen dissolved in the acid solution is 1 - 25 mg / mL.

[0006] In a preferred embodiment of the present invention, the filtration is secondary filtration, and the pore sizes of the filter membranes used for filtration are 0.45 μm and 0.22 μm respectively.

[0007] In a preferred embodiment of the present invention, the alkali solution is 0.01M sodium hydroxide or potassium hydroxide.

[0008] In a preferred embodiment of the present invention, collagen is dissolved in 0.01M phosphoric acid or hydrochloric acid.

[0009] In a preferred embodiment of the present invention, the PBS buffer solution in the injectable filler is prepared by adding a certain amount of NaCl to the PB buffer solution.

[0010] In a preferred embodiment of the present invention, the PBS buffer solution in the injectable filler is prepared by adding a certain amount of NaCl to a PB buffer solution with a concentration of 0.03 - 0.04 mol / L.

[0011] In a preferred embodiment of the present invention, the weight fraction of the PB buffer solution in the PBS buffer solution of the injectable filler is 99.5 - 99.99%, and the weight fraction of NaCl is 0.01 - 0.5%.

[0012] In a preferred embodiment of the present invention, the weight fraction of the PB buffer solution in the PBS buffer solution of the injectable filler is 99.8 - 99.99%, and the weight fraction of NaCl is 0.01 - 0.2%.

[0013] In a preferred embodiment of the present invention, the osmotic pressure of the PBS buffer solution in the injectable filler is 90 - 105 mosmol / kg.

[0014] In a preferred embodiment of the present invention, the osmotic pressure of the PBS buffer solution in the injectable filler is 95 - 100 mosmol / kg.

[0015] In a preferred embodiment of the present invention, the molecular weight of the PLLA microspheres in the injectable filler is 10,000 - 150,000 Da, preferably 30,000 - 70,000 Da, and the particle size is 20 - 50 μm, preferably 30 - 40 μm.

[0016] In a preferred embodiment of the present invention, the weight ratio of collagen to PLLA microspheres in the injectable filler is 1:0.25 - 4.

[0017] Another object of the present invention is to provide a method for preparing an injectable filler, the filler comprising 30 - 55 mg / mL of collagen, 30 - 55 mg / mL of PLLA microspheres and a PBS buffer solution, and the preparation method is: mixing collagen with the PBS buffer solution, stirring at room temperature until evenly dispersed to obtain a mixed solution, adding PLLA microspheres, and stirring and dispersing evenly to obtain a milky white liquid, thus obtaining the product.

[0018] In a preferred embodiment of the present invention, the preparation method of the injectable filler is aseptic operation throughout the process.

[0019] Another object of the present invention is to provide the application of the injectable filler of the present invention in aesthetic medical products.

[0020] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest are weight / weight percentages.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention has developed an injectable filler that can sustainably stimulate collagen regeneration. The inventors have found in practice that the collagen concentration in the filler, as well as the type, preparation method, concentration, etc. of the buffer solution, will affect the osmotic pressure, ignition residue, and properties of the filler. The present invention has scientifically selected an injectable filler with excellent performance in all aspects.

[0023] The present invention has scientifically screened the process parameters and conditions for the filtration and sterilization of collagen, and systematically studied the effects of multiple parameters in this process on the structure and activity retention of collagen. The sterilization method of the present invention is simple to operate, economical, environmentally friendly, and has good repeatability, avoiding the damage to the structure and activity of collagen caused by dry heat sterilization, moist heat sterilization, and irradiation sterilization. In short, the method of the present invention can maintain the three-dimensional structure of collagen while ensuring the sterilization effect.

[0024] The filler of the present invention can, on the basis of initially supplementing collagen, continuously stimulate the regeneration of collagen, achieving a composite effect of short-term support and long-term repair overall. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the effect diagram of different-dose irradiation sterilization for Comparative Example 1.

[0026] Figure 2 It is the electrophoresis effect diagram of the sample of Example 1.

[0027] Figure 3 It is the electrophoresis effect diagram of the sample of Example 2.

[0028] Figure 4 It is the electrophoresis effect diagram of the sample of Example 3.

[0029] Figure 5 It is the electrophoresis effect diagram of the sample of Example 4.

[0030] Figure 6 It is the electrophoresis effect diagram of the sample of Example 5.

[0031] Figure 7 It is the electrophoresis effect diagram of the sample of Example 6.

[0032] Figure 8Appearance characteristics diagram of sample No. 27 in Example 6.

[0033] Figure 9 Appearance characteristics diagram of sample No. 28 in Example 6.

[0034] Figure 10 Electrophoresis effect diagram of the sample in Example 7.

[0035] Figure 11 Circular dichroism spectrum diagram of the sample in Example 8.

[0036] Figure 12 Sterilization qualification test effect diagram of Example 9.

[0037] Figure 13 Sirius red staining effect diagram 6 months after the experiment in Example 22, experimental group.

[0038] Figure 14 Sirius red staining effect diagram 6 months after the experiment in Example 22, control group. Detailed implementation manners

[0039] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other arbitrarily. Those not specially described in the embodiments are all conventional products that can be obtained by commercial purchase.

[0040] The electrophoresis experiment refers to YY / T 1805.2-2021 "Tissue engineering medical device products - Collagen - Part 2: Detection of the molecular weight of type I collagen - Sodium dodecyl sulfate polyacrylamide gel electrophoresis method".

[0041] The lyophilization process of the embodiments of the present invention is as follows:

[0042] Freezing process Temperature (°C) Set time (min) Holding time (min) Vacuum degree (mbar) Primary freezing -40 30 150 - First sublimation -5 30 720 0.1 Second sublimation 25 30 1320 0.1 Total duration - - 40h -

[0043] The secondary filter heads in the embodiments are respectively: hydrophilic polyethersulfone needle filters (PES, 0.45μm), sterile hydrophilic polyethersulfone needle filters (PES, 0.22μm). Unless otherwise specified, the samples in the embodiments are all filtered by secondary filtration.

[0044] Prepare sterile collagen by the preferred method of the present invention and prepare the injectable filler of the present invention.

[0045] The molecular weight of the PLLA microspheres of the present invention is 10,000 - 150,000 Da, and the particle size is 20 - 50 μm. The preparation method is as follows: ① Aqueous phase preparation: Add an appropriate amount of purified water to a container, slowly add polyvinyl alcohol, and stir until the polyvinyl alcohol is completely dissolved to obtain an aqueous phase solution; ② Organic phase preparation: Add an appropriate amount of dichloromethane to a container, weigh 10,000 - 150,000 Da of poly-L-lactic acid and add it to the container, and stir until it is completely dissolved to obtain an organic phase solution; ③ Emulsification: Slowly pour the above organic phase into the aqueous phase, and use a membrane emulsifier to emulsify the liquid; ④ Solidification: Stir and volatilize the emulsified emulsion until the dichloromethane is completely volatilized; ⑤ Washing and collection: Centrifuge the suspension, collect the solid component, dry it, and sterilize it to obtain the microsphere product.

[0046] The PB buffer solution in the present invention is a phosphate buffer solution, and the PBS buffer solution is a phosphate buffered saline. Sterilize the buffer solution in the composition before preparing the injectable filler.

[0047] The ignited residue of the composition of the present invention: According to item 7.2.5.5 Ignited Residue of YYT0954 Passive Surgical Implants Type I Collagen Implants, the ignited residue of the implant should not be more than 15 mg / g, and the detection method is determined with reference to the General Principles of the Chinese Pharmacopoeia 2020 Edition, Volume IV, Method for the Examination of Ignited Residue <0841>.

[0048] Irradiation sterilization of collagen in Comparative Example 1

[0049] Put 0.05 M PBS buffer solution into a beaker. Weigh a certain amount of collagen, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker to make the final concentration of collagen 10 mg / mL. Stir at room temperature until it is evenly dispersed, and let it stand to defoam until the bubbles disappear. Treat the above-prepared collagen solution by irradiation sterilization. The results show that: compared with the non-irradiated group, the collagen in the irradiated group undergoes chain breakage, and the appearance property changes, and the degree of denaturation increases with the increase of the irradiation dose. The results of different irradiation doses are as Figure 1 shown.

[0050] Bacterial filtration of collagen in Comparative Example 2

[0051] Put 0.05 M PBS buffer solution into a beaker. Weigh a certain amount of collagen, use sterilized scissors to cut it into several pieces, and then slowly add it to the beaker to make the final concentration of collagen 10 mg / mL. Stir at room temperature until it is evenly dispersed, and let it stand to defoam until the bubbles disappear. Filter it with a syringe equipped with a 0.45 μm + 0.22 μm secondary filter head. The results show that: the filter membrane is blocked and it is difficult to complete the filtration.

[0052] Bacterial filtration of collagen in Example 1

[0053] Preparation of phosphoric acid solution: Add 0.5 g of phosphoric acid to a 500 mL volumetric flask, and make up to the mark with ultrapure water to prepare 0.01 M dilute phosphoric acid for standby.

[0054] Preparation of hydrochloric acid solution: Add 41.67 g of 12 mol / L concentrated hydrochloric acid to a 500 mL volumetric flask, and make up to the mark with ultrapure water to prepare 1 mol / L dilute hydrochloric acid for standby. Gradually dilute 1 mol / L dilute hydrochloric acid to obtain 0.1 mol / L dilute hydrochloric acid and 0.01 mol / L dilute hydrochloric acid respectively.

[0055] Preparation of NaOH solution: Weigh 20 g of sodium hydroxide into a beaker, dissolve it with ultrapure water, transfer it to a 500 mL volumetric flask after cooling, and make up to the mark with ultrapure water, mix well to prepare 1 mol / L NaOH solution for standby. Take 5 g of 1 mol / L NaOH solution in a 500 mL volumetric flask, add ultrapure water to make up to the mark to obtain 0.01 mol / L NaOH solution; take 10 g of 1 mol / L NaOH solution in a 100 mL volumetric flask, add ultrapure water to make up to the mark to obtain 0.1 mol / L NaOH solution.

[0056] Preparation of Sample 1: Take 300 mL of 0.01 M dilute phosphoric acid and put it into a beaker. Weigh 9 g of collagen, cut it into several pieces with a disinfected scissors, and then slowly add it to the beaker. The final concentration of collagen is 30 mg / mL. Stir at 37 °C until completely dissolved, and let it stand to defoam. After the bubbles disappear, use a syringe equipped with a 0.45 μm + 0.22 μm secondary filter head to filter the collagen solution into a clean beaker to obtain the filtrate. Take the above filtrate, pour it into a freeze-drying tray, and freeze-dry to obtain a sterile collagen dry product.

[0057] Preparation of the remaining samples refers to Sample 1. Among them, Samples 1 and 4 are directly freeze-dried from the filtrate after filtering the collagen solution, and Samples 2-3 and 5-6 are freeze-dried after adjusting the pH of the filtrate after filtering the collagen solution. The electrophoresis results of the freeze-dried products of each sample are shown in Table 1 below.

[0058] Table 1

[0059]

[0060] The electrophoresis results are shown in Figure 2 . The results show that: ① There is little difference in the results when phosphoric acid and hydrochloric acid are used as solvents; ② The activity of collagen can be partially restored after the filtrate is neutralized with an alkali solution; ③ The concentration of the alkali solution used to adjust the pH of the filtrate has an impact on the activity of collagen.

[0061] Sterilization filtration of collagen in Example 2

[0062] The preparation of the samples refers to Sample 1 above. All the samples are the filtrates of the collagen solutions after filtration, with the pH adjusted and then freeze-dried. The electrophoresis results of the freeze-dried products of each sample are shown in Table 2 below.

[0063] Table 2

[0064]

[0065] The electrophoresis results are shown in Figure 3 . The results show that: ① Using high-concentration hydrochloric acid as a solvent will destroy the activity of collagen, and ② A decrease in the collagen concentration is beneficial to maintaining the activity.

[0066] Example 3 Sterilization Filtration of Collagen

[0067] The preparation of each sample refers to Sample 1 above. Among them, Sample 10 is freeze-dried from the acid-dissolved solution without filtration, Samples 11 - 13 are freeze-dried from the filtrates of the collagen solutions after filtration, and Samples 14 - 15 are freeze-dried from the filtrates of the collagen solutions after adjusting the pH. The electrophoresis results of the freeze-dried products of each sample are shown in Table 3 below.

[0068] Table 3

[0069]

[0070] The electrophoresis results are shown in Figure 4 . The results show that: ① Further verification shows that adjusting the pH of the filtrate with an alkaline solution can partially restore the activity of collagen, and ② The filtration process has little impact on collagen. In addition, it was also observed that adjusting the pH to 4 - 5 can make the freeze-dried product have better toughness and appearance compared to adjusting the pH to neutral.

[0071] Example 4 Sterilization Filtration of Collagen

[0072] The sample preparation refers to Sample 1 above. Among them, Samples 16, 18, 20, 22 are directly freeze-dried from the filtrates of the collagen solutions after filtration, and Samples 17, 19, 21, 23 are freeze-dried from the filtrates of the collagen solutions after adjusting the pH. The electrophoresis results of the freeze-dried products of each sample are shown in Table 4 below.

[0073] Table 4

[0074]

[0075] The electrophoresis results are shown in Figure 5 . The results show that: ① A decrease in the collagen concentration is beneficial to maintaining the activity, and ② A decrease in the collagen dissolution temperature is beneficial to maintaining the activity.

[0076] Example 5 Sterilization Filtration of Collagen

[0077] The sample preparation refers to Sample 1 above. Among them, Sample 24 is freeze-dried from the unfiltered acid dissolution solution, Sample 25 is freeze-dried from the filtrate after filtering the collagen solution, and Sample 26 is freeze-dried after adjusting the pH of the filtrate of the collagen solution. The electrophoresis results of the freeze-dried products of each sample are shown in Table 5 below.

[0078] Table 5

[0079]

[0080]

[0081] The electrophoresis results are shown in Figure 6 . The results show that: ① More activity is lost after dissolving high-concentration collagen. ② It is further verified that the filtration process has basically no effect on collagen. ③ It is further verified that adjusting the pH of the filtrate with an alkali solution can partially restore the activity of collagen.

[0082] Example 6 Sterilization Filtration of Collagen

[0083] The preparation of each sample refers to Sample 1 above. The samples are all freeze-dried after adjusting the pH of the filtrate after filtering the collagen solution. The electrophoresis results of the freeze-dried products of each sample are shown in Table 6 below.

[0084] Table 6

[0085]

[0086] The electrophoresis results are shown in Figure 7 . The results show that there is little difference in the activity of collagen when using 0.01M phosphoric acid or 0.01M hydrochloric acid as the solvent. In addition, the appearance properties of Samples 27 and 28 are shown in Figure 8 and Figure 9 .

[0087] Example 7 Sterilization Filtration of Collagen

[0088] Preparation of 0.01M acetic acid solution: Add 2.86 mL of glacial acetic acid to a 500 mL volumetric flask, and make up to the scale line with ultrapure water to prepare 0.1 mol / L acetic acid solution for standby. Take 50 mL of 0.1 mol / L acetic acid solution in a 500 mL volumetric flask, and make up to the scale line with ultrapure water to obtain 0.01 mol / L acetic acid solution.

[0089] Preparation of 0.01M citric acid solution: Weigh 1.05 g of citric acid in a beaker, dissolve it with ultrapure water, transfer it to a 500 mL volumetric flask after cooling, make up to the scale line with ultrapure water, and mix well to prepare 0.01 mol / L citric acid solution for standby.

[0090] The sample preparation process refers to Sample 1. Each sample is the filtrate of the collagen solution after filtration, and the pH is adjusted before freeze-drying. The electrophoresis results of each freeze-dried sample are shown in Table 7 below.

[0091] Table 7

[0092]

[0093]

[0094] The electrophoresis results are shown in Figure 10 . The results show that neither acetic acid nor citric acid is suitable as a solvent for the sterilization filtration of collagen.

[0095] Example 8 Circular Dichroism Spectrum of Collagen

[0096] Principle of characterizing the three-dimensional structure of collagen by circular dichroism spectroscopy: The special right-handed superhelical structure of type I collagen endows it with a special circular dichroism spectrum. Usually, a negative absorption peak at about 194 nm and a positive absorption peak at about 220 nm are the unique circular dichroism spectra of collagen. Studies have shown that when collagen denatures or the triple helix structure is damaged, the circular dichroism spectrum of collagen will show a red shift of the negative peak and the disappearance of the positive peak. The height of the positive absorption peak can also characterize the amount of the triple helix structure.

[0097] Instruments and reagents: Glacial acetic acid (analytical grade), deionized water, Chirascan circular dichroism spectrometer

[0098] Steps: 1. Preparation of collagen solution: Using 0.5 mol / L acetic acid solution as the solvent, prepare 10 mL of collagen sample solution with a concentration of 0.1 mg / mL, dissolve it thoroughly, equilibrate for 12 h, and centrifuge to take the supernatant.

[0099] 2. Instrument settings: Set the wavelength range from 190 nm to 260 nm, the optical path of the cuvette is 0.5 mm, and the scanning speed is 0.5 nm / s; the detection environment is 25 °C and nitrogen. Use 0.5 mol / L acetic acid solution as the blank control.

[0100] 3. Scan the sample 3 times repeatedly and the blank 2 times. Measure the blank control once for each measurement of the sample.

[0101] Select samples 27, 28, 29 and the collagen raw material for circular dichroism analysis. The results are shown in Figure 11 . The results show that Sample 28 is the best, Sample 27 is the second best, and Sample 29 does not meet the requirements.

[0102] Example 9 Sterilization Qualification Test of Collagen

[0103] Sterility Test of Collagen Sponge

[0104] 1. Instruments and equipment: Clean bench, biological safety cabinet, autoclave, biochemical incubator

[0105] 2. Reagents and solutions: Fluid thioglycollate medium, Trypticase soy broth (TSB), sterile sodium chloride - peptone buffer solution with pH 7.0

[0106] 3. Preparation of bacterial suspension: Inoculate the fresh culture of Staphylococcus aureus into Trypticase soy broth, incubate at 30 - 35 °C for 18 - 24 hours, and then dilute it with sterile sodium chloride - peptone buffer solution with pH 7.0 to an appropriate concentration of bacterial suspension.

[0107] 4. Preparation before the test

[0108] 5. Clean bench: Wipe the surface of the workbench with 75% alcohol cotton before the experiment. Turn on the ultraviolet lamp and irradiate for half an hour, then turn off the ultraviolet lamp and turn on the fan for another half an hour before entering the laboratory.

[0109] 6. Transfer of test items and equipment: Transfer the required test items and equipment into the transfer window before the test, and turn on the ultraviolet lamp for disinfection for more than 30 minutes.

[0110] 7. Test procedures:

[0111] (1) Test solution: In the clean bench, take 100 mL of fluid thioglycollate medium and Trypticase soy broth, and inoculate 0.3 g of the collagen sponge test article (prepared according to the sterilization process of sample No. 28) respectively by the direct inoculation method.

[0112] (2) Negative control: Take 1 bottle each of 100 mL of fluid thioglycollate medium and Trypticase soy broth as the negative control.

[0113] (3) Positive control: Take 1 bottle of 100 mL of fluid thioglycollate medium, inoculate 0.3 g of the test article, and add no more than 100 cfu of bacterial suspension in the biological safety cabinet.

[0114] 8. Incubation and observation

[0115] Incubate the inoculated fluid thioglycollate medium tubes at 30 - 35 °C for 14 days, the inoculated Trypticase soy broth tubes at 20 - 25 °C for 14 days, the negative control fluid thioglycollate medium group at 30 - 35 °C for 14 days, the negative control Trypticase soy broth group at 20 - 25 °C for 14 days, and the positive control tubes at 30 - 35 °C for no more than 5 days. Observe and record daily during the incubation period whether there is bacterial growth, and fill in the inspection record form.

[0116] 9. Results

[0117] See specificallyFigure 12 , the positive control group was turbid and the anaerobic layer disappeared. The test article TSB group was clear. The test article thioglycollate group was clear and the anaerobic layer was retained. The TSB negative control group was clear. The thioglycollate negative control group was clear and the anaerobic layer was retained. The test article met the requirements.

[0118] The results of the sterility qualification test of the test articles prepared by referring to the sterilization methods of samples No. 19, 21, 23, and 27 were the same as those of No. 28 above and all met the requirements.

[0119] Preparation of Injectable Filler in Example 10

[0120] Collagen was mixed with normal saline and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 50 mg / mL. PLLA microspheres were added to make its concentration 50 mg / mL, and stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 6.46, the osmotic pressure was 480 mosmol / kg, and the ignited residue was 9.12 mg / g. It was applicable to a 27G disposable injection needle, but the osmotic pressure of the composition was on the high side and did not meet the requirements.

[0121] Preparation of Injectable Filler in Example 11

[0122] Collagen was mixed with normal saline and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 25 mg / mL. PLLA microspheres were added to make its concentration 25 mg / mL, and stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 6.78, the osmotic pressure was 395 mosmol / kg, and the ignited residue was 8.35 mg / g. The sample was too thin to be applicable to a 27G disposable injection needle, and the osmotic pressure of the composition was on the high side and did not meet the requirements.

[0123] Preparation of Injectable Filler in Example 12

[0124] Collagen was mixed with 0.05 mol / L PB buffer solution (pH 7.0, osmotic pressure 118 mosmol / kg) and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 30 mg / mL. PLLA microspheres were added to make its concentration 30 mg / mL, and stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 7.09, the osmotic pressure was 321 mosmol / kg, and the ignited residue was 16.08 mg / g, which was higher than the limit value. It was applicable to a 27G disposable injection needle, and the pushing force was 15.233 ± 3.212 N.

[0125] Preparation of Injectable Filler in Example 13

[0126] Collagen was mixed with 0.04 mol / L PB buffer (pH 7.0, osmotic pressure 95 mosmol / kg), and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 40 mg / mL. PLLA microspheres were added to make its concentration 40 mg / mL, and stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.97, the osmotic pressure was 290 mosmol / kg, and the ignition residue was 15.23 mg / g, slightly higher than the limit value. It was applicable to a 27G disposable injection needle, and the pushing force was 17.652 ± 2.421 N.

[0127] Preparation of Injectable Filler in Example 14

[0128] Collagen was mixed with 0.03 mol / L PB buffer (pH 7.0, osmotic pressure 78 mosmol / kg), and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 50 mg / mL. PLLA microspheres were added to make its concentration 50 mg / mL, and stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.82, the osmotic pressure was 256 mosmol / kg, the osmotic pressure value was on the low side, and the ignition residue was 14.45 mg / g. It was applicable to a 27G disposable injection needle, and the pushing force was 20.379 ± 2.645 N.

[0129] Preparation of Injectable Filler in Example 15

[0130] Collagen was mixed with PBS buffer (prepared from 99.96% by weight of 0.04 mol / L PB buffer + 0.04% by weight of NaCl, pH 7.0, osmotic pressure 96 mosmol / kg), and stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The collagen concentration was 30 mg / mL. PLLA microspheres were added to make its concentration 30 mg / mL, and stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.95, the osmotic pressure was 302 mosmol / kg, and the ignition residue was 11.69 mg / g. It was applicable to a 27G disposable injection needle, and the pushing force was 15.358 ± 2.094 N. All indicators met the requirements.

[0131] Preparation of Injectable Filler in Example 16

[0132] Collagen was mixed with PBS buffer (prepared from 0.04 mol / L PB buffer with 99.96% by weight + 0.04% by weight of NaCl, pH 7.0, osmotic pressure 96 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 60 mg / mL. PLLA microspheres were added to make its concentration 60 mg / mL, and then stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 7.08, the osmotic pressure was 310 mosmol / kg, and the ignited residue was 12.53 mg / g. The sample was too viscous to be applicable to a 27G disposable injection needle.

[0133] Preparation of Injectable Filler in Example 17

[0134] Collagen was mixed with PBS buffer (prepared from 0.04 mol / L PB buffer with 99.96% by weight + 0.04% by weight of NaCl, pH 7.0, osmotic pressure 96 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 55 mg / mL. PLLA microspheres were added to make its concentration 55 mg / mL, and then stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 7.02, the osmotic pressure was 308 mosmol / kg, and the ignited residue was 11.97 mg / g. It was applicable to a 27G disposable injection needle, and the pushing force was 16.278 ± 3.125 N. All indicators met the requirements.

[0135] Preparation of Injectable Filler in Example 18

[0136] Collagen was mixed with PBS buffer (prepared from 0.03 mol / L PB buffer with 99.94% by weight + 0.06% by weight of NaCl, pH 7.0, osmotic pressure 101 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 55 mg / mL. PLLA microspheres were added to make its concentration 55 mg / mL, and then stirred and dispersed evenly to obtain a milky white liquid. The pH of this filler was measured to be 6.77, the osmotic pressure was 294 mosmol / kg, and the ignited residue was 10.56 mg / g. It was applicable to a 27G disposable injection needle, and the pushing force was 14.368 ± 1.297 N. All indicators met the requirements.

[0137] Preparation of Injectable Filler in Example 19

[0138] Collagen was mixed with PBS buffer (prepared from 0.03 mol / L PB buffer at 99.94% by weight + 0.06% by weight of NaCl, pH 7.0, osmotic pressure 101 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 30 mg / mL. PLLA microspheres were added to make its concentration 30 mg / mL, and then stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.70, the osmotic pressure was 282 mosmol / kg, and the ignited residue was 9.23 mg / g. It was applicable to a 27G disposable injection needle, and the pushing force was 11.652 ± 1.636 N. All indicators met the requirements.

[0139] Preparation of Injectable Filler in Example 20

[0140] Collagen was mixed with PBS buffer (prepared from 0.03 mol / L PB buffer at 99.94% by weight + 0.06% by weight of NaCl, pH 7.0, osmotic pressure 101 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 20 mg / mL. PLLA microspheres were added to make its concentration 20 mg / mL, and then stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.61, the osmotic pressure was 276 mosmol / kg, and the ignited residue was 9.10 mg / g. The sample was too thin to be applicable to a 27G disposable injection needle.

[0141] Preparation of Injectable Filler in Example 21

[0142] Collagen was mixed with PBS buffer (prepared from 0.01 mol / L PB buffer at 99.82% by weight + 0.18% by weight of NaCl, pH 7.0, osmotic pressure 97 mosmol / kg). The mixture was stirred at room temperature for 6 h until evenly dispersed to obtain a mixed solution. The concentration of collagen was 45 mg / mL. PLLA microspheres were added to make its concentration 45 mg / mL, and then stirred until evenly dispersed to obtain a milky white liquid. The pH of this filler was measured to be 6.52, the osmotic pressure was 293 mosmol / kg, and the ignited residue was 9.45 mg / g. The sample was too thin to be applicable to a 27G disposable injection needle.

[0143] Animal Experiment of the Injectable Filler of the Invention in Example 22

[0144] (1) Test Device

[0145] Experimental Group: Injectable Filler of Example 15

[0146] Control Group: The preparation method referred to Example 15, the difference being that no PLLA microspheres were added.

[0147] (2) Experimental methods

[0148] Male New Zealand rabbits (body weight: 2.3 kg - 2.5 kg) were anesthetized, and the hair on their backs was shaved and disinfected with iodophor. Subcutaneous injection was performed on the back at a total of 4 points, with an interval of 2.5 cm between every two points. 0.5 mL was injected at each point, and the injection depth at each point was the same. Then, the rabbits were raised and observed. The observation time points included 1 month, 3 months, and 6 months. At each time point, there were 3 rabbits in each of the experimental group and the control group. At the set time points, after the rabbits were euthanized, the wounds were observed and evaluated macroscopically. The obtained samples containing the implanted material and sufficient surrounding unaffected subcutaneous tissue were fixed in 10% formaldehyde solution. The fully fixed tissues were prepared into conventional pathological sections, 4-μm paraffin sections. The filler (along with the surrounding unaffected tissue) was stained with picric acid - Sirius red (PSR) to evaluate the collagen production of the tested filler.

[0149] (3) Experimental results

[0150] Six months after the filler injection, the Sirius red staining results of the experimental group were as Figure 1 shown. A large amount of newly formed collagen was visible around the microspheres, the collagen network structure was tight, and relatively thick collagen fiber tissues were formed; the Sirius red staining results of the control group were as Figure 2 shown, and there was no newly formed collagen in the implanted area.

[0151] Although the embodiments disclosed in the present invention are as above, the content described is only an implementation manner for facilitating the understanding of the present invention and is not used to limit the present invention. Any person skilled in the art within the scope of the present invention can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed by the present invention. However, the scope of patent protection of the present invention shall still be subject to the scope defined by the appended claims.

Claims

1. An injectable filler comprising 30-55 mg / mL of collagen, 30-55 mg / mL of PLLA microspheres and PBS buffer.

2. The filler according to claim 1, characterized in that The collagen in the filler is obtained by sterilization, and the sterilization comprises the following steps: dissolving the collagen in 0.01M-0.05M phosphoric acid or hydrochloric acid at 15-25°C, filtering to obtain a filtrate, and adjusting the pH value of the filtrate to 4-5 with 0.01M-0.05M alkali solution to obtain sterile collagen. The concentration of the collagen dissolved in the acid solution is 1-25mg / mL. Preferably, the filtration is a secondary filtration, and the pore sizes of the filter membranes used for filtration are 0.45μm and 0.22μm, respectively. Preferably, the alkali solution is 0.01M sodium hydroxide or potassium hydroxide. Preferably, the collagen is dissolved in 0.01M phosphoric acid or hydrochloric acid.

3. The filler according to any one of claims 1 to 2, characterized in that The PBS buffer in the injectable filler is prepared by adding a certain amount of NaCl to the PB buffer. Preferably, the PBS buffer in the injectable filler is prepared by adding a certain amount of NaCl to 0.03-0.04 mol / L PB buffer.

4. The filler according to any one of claims 1 to 3, characterized in that The weight fraction of PB buffer in the PBS buffer in the injectable filler is 99.5-99.99%, and the weight fraction of NaCl is 0.01-0.5%. Preferably, the weight fraction of PB buffer in the PBS buffer in the injectable filler is 99.8-99.99%, and the weight fraction of NaCl is 0.01-0.2%.

5. The filler according to any one of claims 1 to 4, characterized in that The osmotic pressure of the PBS buffer in the injectable filler is 90-105 mosmol / kg, preferably the osmotic pressure of the PBS buffer in the injectable filler is 95-100 mosmol / kg.

6. The filler according to any one of claims 1 to 5, characterized in that: The molecular weight of the PLLA microspheres in the injectable filler is 10,000-150,000 Da, preferably 30,000-70,000 Da, and the particle size is 20-50 μm, preferably 30-40 μm.

7. The filler according to any one of claims 1 to 6, characterized in that The weight ratio of collagen to PLLA microspheres in the injectable filler is 1:0.25-4.

8. The method for preparing the filler according to any one of claims 1 to 7, characterized in that: The preparation method comprises mixing collagen with PBS buffer, stirring at room temperature until the mixture is evenly dispersed, adding PLLA microspheres, stirring and dispersing the mixture evenly, and obtaining a milky white liquid. Preferably, the preparation method is a whole-process aseptic operation.

9. Use of the filler according to any one of claims 1 to 7 in medical beauty products.

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