Tissue regeneration promoting filling material and preparation method thereof

Abstract

The invention discloses a filling material for promoting tissue regeneration and a preparation method thereof. The filling material is prepared by using type II collagen as a microsphere wall material and hydroxyapatite as a core material and dispersing composite microspheres in type I collagen gel. The filling material for promoting tissue regeneration prepared by the invention is used for filling parts such as eyebrow lines, eyebrow subcutaneous support, zygomatic fat and cheek fat pads, lacrimal ducts, nasolabial folds, noses, lips, chin, mandible periosteum and the like, has an obvious mechanical support effect, continuously stimulates the formation and rearrangement of endogenous collagen on the basis of extrinsic collagen supply, and can be used for promoting tissue regeneration. The medical and aesthetic plastic requirements are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of beauty material preparation, and particularly relates to a tissue regeneration promoting filling material and a preparation method thereof. Background Art

[0002] Collagen is the most widely distributed protein in the human body and animals, and its content accounts for about 25% - 30% of the total protein content in the body. Collagen is a biological macromolecule in the extracellular matrix (ECM), and its basic structure consists of three polypeptide chains coiled around each other into a superhelical structure. Collagen is a filler and support for tissues, maintaining the integrity of cell structure and other biological functions, and defining the shape and form of tissues to endow bones, tendons, cartilage and skin with certain mechanical strength. It has high biological activity and biological functionality, and can participate in the migration, proliferation and differentiation of various types of cells, and perform its special biological functions.

[0003] In recent years, collagen can provide a regeneration scaffold for cells, induce the neogenesis of its own collagen, reduce skin inflammation, effectively reconstruct the skin barrier, and at the same time the degraded amino acids can effectively inhibit the activity of tyrosinase and reduce melanin production, so it has a wide range of applications in cosmetic dermatology.

[0004] With the increase of age, a large amount of collagen in the human body will gradually be lost, which is the main reason for skin aging. Skin aging will lead to a series of negative reactions such as dull and dry skin, so it is necessary to continuously supplement collagen to keep the skin young. Oral collagen has little effect on improving the skin. Only by injecting it into the deep dermis can it play a role in cosmetic plastic surgery. Medical cosmetic collagen is generally highly purified human or animal collagen, which has good tissue compatibility and can be naturally absorbed or degraded by the skin, and can stimulate dermal fibroblasts to newly produce collagen, and then regenerate and repair cells and skin tissues. At present, collagen is the most widely used biomedical material in plastic and cosmetic surgery. However, for plastic and cosmetic projects with certain requirements for mechanical support, such as glabellar lines, subcutaneous support of eyebrows, zygomatic and buccal fat pads, tear troughs, nasolabial folds, etc., simple collagen products cannot achieve good filling effects. In order to achieve a long-term and certain mechanical support effect, relevant research on cross-linking modification of collagen, adding PLLA microspheres, PCL microspheres, etc. has emerged, which can achieve a certain effect of enhancing mechanical strength. However, cross-linked modified collagen will cause a "puffy" phenomenon to a certain extent due to the presence of cross-linking agents, and the false face is obvious. PLLA microspheres and PCL microspheres are synthetic polymer materials. Although they can stimulate the regeneration of dermal collagen, they may also cause adverse reactions including allergic reactions, nodules, uneven distribution, etc.

[0005] Hydroxyapatite is a safe inorganic material composed of calcium and phosphorus. It is the main inorganic component of human and animal bones, has a certain solubility in the body, is close to the human body pH value, has high biocompatibility, can participate in the body's metabolism, and has guaranteed safety. Moreover, hydroxyapatite is widely used in the overseas medical aesthetic market. Its clinical use is second only to hyaluronic acid, and it is a product material recognized by many beauty seekers and medical aesthetic practitioners. When hydroxyapatite microparticles or microspheres are injected under the dermis, there are certain requirements for their particle size. If the particle size is too low, the microparticles will enter the blood vessels, posing a potential risk of blood vessel thrombosis. If the particle size is too high, problems such as injection needle blockage and nodules after injection will occur. Hydroxyapatite microspheres solve the above problems to a certain extent, but due to the relatively large density of hydroxyapatite, it is not evenly dispersed in the gel medium. Summary of the Invention

[0006] The purpose of the present invention is to provide a tissue regeneration promoting filling material and a preparation method thereof.

[0007] A tissue regeneration promoting filling material, wherein the filling material is made by using type II collagen as the microsphere wall material, hydroxyapatite as the core material, and the composite microspheres are dispersed in type I collagen gel.

[0008] The preparation method of the tissue regeneration promoting filling material is carried out according to the following steps:

[0009] (1) Extraction of type I collagen: Using animal-derived soft tissue as the raw material, through degreasing, de-terminal peptide treatment to remove immunogenicity, and then through pyrogen removal and impurity protein removal treatment, and finally purified.

[0010] (2) Extraction of type II collagen: Using animal-derived cartilage tissue as the raw material, degreasing and removing fascia, through de-terminal peptide treatment to remove immunogenicity, and finally purified.

[0011] (3) Prepare composite microspheres of type II collagen-hydroxyapatite by microfluidics method, and mix them evenly with purified type I collagen and cytokines according to the mass ratio of (2-5):(7-8):(0.01-0.1) to make a tissue regeneration promoting filling material.

[0012] The operating steps for extracting type I collagen are as follows: Select fresh animal skins, remove the surface fat and minced meat, soak in purified water to remove blood, wash and cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; Wash the pulverized tissue by shaking and soaking with 0.1 - 0.3M NaOH at 20 - 30°C for 20 - 28h, with a material - liquid ratio of 1g:5ml, wash with 75% ethanol 2 - 4 times, and then wash with deionized water 3 - 5 times; Place the pulverized tissue in Tris - HCl buffer solution, soak at 4°C for 20 - 28h, centrifuge to collect the precipitate, and wash with deionized water until clean; Place the pulverized tissue in 0.005 - 0.015N hydrochloric acid, add 3 - 7% pepsin (pepsin activity 8000 - 12000IU / g), enzymatically hydrolyze at 26 - 30°C for 42 - 56h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 4 - 6M NaOH, salting - out at 4°C with saturated NaCl for 8 - 12h, centrifuge, wash the precipitate with saturated NaCl 2 - 4 times, first dialyze the precipitate against 0.3 - 0.7M acetic acid for 20 - 28h, and then dialyze against deionized water for 60 - 80h to obtain type I collagen gel.

[0013] The operating steps for extracting type II collagen are as follows: Select fresh animal cartilages, remove the surface fat and fascia, wash with physiological saline, cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; According to the material - liquid ratio of 1g:20ml, add 0.05 - 0.15mol / L NaOH to the pulverized tissue, perform ultrasonic cleaning, each cleaning for 0.3 - 0.7h, and clean 2 - 4 times; Then add deionized water and perform ultrasonic cleaning, each cleaning for 10 - 30min, and clean 2 - 4 times; Add the pulverized tissue to 0.05 - 0.15mol / L NaOH solution, perform ultrasonic treatment for 10 - 14h, discard the NaOH solution, add deionized water for cleaning according to the material - liquid ratio of 1g:20ml, each cleaning for 10 - 20min, and clean 3 - 5 times until the pH value of the cleaning solution is neutral; According to the material - liquid ratio of 1g:20ml, add 0.05 - 0.15mol / L hydrochloric acid to the pulverized tissue for decalcification, treat at 25°C for 3 - 5h, discard the decalcification solution, add deionized water for cleaning, each cleaning for 10 - 30min, and clean 3 - 5 times until the pH value of the cleaning solution is neutral; Place the pulverized tissue in 0.005 - 0.015N hydrochloric acid, add 8 - 12% pepsin (pepsin activity 8000 - 12000IU / g), enzymatically hydrolyze at 26 - 30°C for 60 - 80h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 4 - 6M NaOH, then add NaCl to make the final concentration of NaCl 4M, salting - out at 4°C for 8 - 12h, centrifuge, wash the precipitate with 3 - 5M NaCl 2 - 4 times, and dialyze the precipitate against deionized water to obtain type II collagen gel.

[0014] The animal is pig, cattle, sheep or horse.

[0015] The specific operation steps for preparing the composite microspheres described in step (3) are as follows: The oil phase solution and the water phase solution are respectively introduced into the microfluidic chip by pressure driving or syringe pump driving. The pressures of the oil phase and the water phase are adjusted. PDMS-FF-100 chips are used to prepare the microspheres. After the microdroplets are uniformly generated, they can be received into a centrifuge tube containing the receiving phase solution, and the microdroplets are received for 20 - 40 min; the received centrifuge tube is sealed, gently shaken to accelerate the solidification of the microdroplets, and left standing for 10 - 30 min; the fluorinated oil at the bottom of the centrifuge tube is taken out with a pipette to obtain the microspheres; a demulsifier is added according to the volume ratio of microspheres to demulsifier of 1:2, and the mixture is centrifuged at 2000 - 3000 rpm for 0.5 - 1.5 min with an oscillator, the waste liquid at the bottom is taken out, and the operation is repeated 1 - 2 times, and the microspheres are collected and freeze-dried.

[0016] The demulsifier is a 50% ethanol solution.

[0017] The oil phase is: hydroxyapatite microparticles are dispersed in dimethylformamide, and the mass-to-volume ratio of hydroxyapatite microparticles to dimethylformamide is 1 g:40 ml; the water phase is: type II collagen is dissolved in 0.5 M acetic acid solution to prepare an acidic solution with a concentration of 1%.

[0018] The cytokines are transforming growth factor-β and interleukin-17, and the dosage mass ratio is 3:1.

[0019] The mixing described in step (3) is carried out using a homogenizer, homogenized for 100 - 140 min, and filled after sufficient degassing.

[0020] The beneficial effects of the present invention: The tissue regeneration-promoting filling material prepared by the present invention is used for filling parts such as the glabellar lines, subcutaneous support of the eyebrows, zygomatic and buccal fat pads, tear troughs, nasolabial folds, nose, lips, chin, and periosteum of the mandible. The mechanical support effect is obvious. On the basis of providing exogenous collagen, it continuously stimulates the formation and rearrangement of endogenous collagen to meet the needs of medical aesthetic plastic surgery. Description of the Drawings

[0021] Figure 1 It is an electron micrograph of type II collagen-hydroxyapatite composite microspheres.

[0022] Figure 2 It is the live / dead staining of L929 fibroblasts.

[0023] Figure 3 It is the Masson's trichrome staining result of the material implanted for 21 d. Detailed Embodiments

[0024] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Example 1

[0026] A preparation method of a tissue regeneration promoting filling material is carried out according to the following steps:

[0027] (1) Extraction of type I collagen: Select fresh pigskin, remove surface fat and minced meat, soak in purified water to remove blood, wash and cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; wash and soak the pulverized tissue with 0.2M NaOH at 25°C for 24h, with a material-liquid ratio of 1g:5ml, wash 3 times with 75% ethanol, and then wash 4 times with deionized water; place the pulverized tissue in Tris-HCl buffer solution, soak at 4°C for 24h, centrifuge to collect the precipitate, and wash with deionized water; place the pulverized tissue in 0.01N hydrochloric acid, add 5% pepsin (pepsin activity 10000IU / g), enzymatically hydrolyze at 28°C for 48h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 5M NaOH, salting-out at 4°C with saturated NaCl for 10h, centrifuge, wash the precipitate 3 times with saturated NaCl, dialyze the precipitate against 0.5M acetic acid for 24h first, and then dialyze against deionized water for 72h to obtain type I collagen gel.

[0028] (2) Extraction of type II collagen: Select fresh porcine cartilage, remove the surface fat and fascia, wash it with normal saline, cut it into pieces, and crush it in a tissue grinder. Wash the crushed tissue with deionized water; according to the ratio of material to liquid of 1 g:20 ml, add 0.1 mol / L NaOH to the crushed tissue, and perform ultrasonic cleaning for 0.5 h each time, for 3 times; then add deionized water and perform ultrasonic cleaning for 20 min each time, for 3 times; add the crushed tissue to 0.1 mol / L NaOH solution, perform ultrasonic treatment for 12 h, discard the NaOH solution, and wash it with deionized water according to the ratio of material to liquid of 1 g:20 ml for 15 min each time, for 4 times, until the pH value of the cleaning solution is neutral; according to the ratio of material to liquid of 1 g:20 ml, add 0.1 mol / L hydrochloric acid to the crushed tissue for decalcification, treat it at 25 °C for 4 h, discard the decalcification solution, add deionized water for cleaning, for 20 min each time, for 4 times, until the pH value of the cleaning solution is neutral; place the crushed tissue in 0.01 N hydrochloric acid, add 10% pepsin (pepsin activity 10000 IU / g), enzymatically hydrolyze at 28 °C for 72 h, centrifuge with a centrifuge to collect the supernatant, adjust it to neutral with 5 M NaOH, then add NaCl to make the final concentration of NaCl 4 M, perform salting out at 4 °C for 10 h, centrifuge, wash the precipitate with 4 M NaCl for 3 times, and dialyze the precipitate against deionized water to obtain type II collagen gel.

[0029] (3) Respectively introduce the oil-phase solution and the water-phase solution into the microfluidic chip by pressure driving or syringe pump driving, adjust the pressure of the oil phase and the water phase, use the PDMS-FF-100 chip to prepare microspheres. After the microdroplets are generated evenly, start to receive them into a centrifuge tube containing the receiving-phase solution, and receive the microdroplets for 30 min; seal the received centrifuge tube, gently oscillate to accelerate the solidification of the microdroplets, and let it stand for 20 min; use a pipette to take out the fluorinated oil at the bottom of the centrifuge tube to obtain microspheres; add a demulsifier according to the volume ratio of microspheres to demulsifier of 1:2 and centrifuge at 2500 rpm with an oscillator for 1 min, take out the waste liquid at the bottom, repeat the operation once, collect the microspheres and freeze-dry them to obtain porous composite microspheres, and mix them evenly with purified type I collagen and cytokines according to the mass ratio of 4:7:0.05. The mixing is carried out using a homogenizer and homogenized for 120 min, and then filled after sufficient defoaming. The demulsifier is a 50% ethanol solution; the oil phase is: hydroxyapatite particles dispersed in dimethylformamide, and the mass-volume ratio of hydroxyapatite particles to dimethylformamide is 1 g:40 ml; the water phase is: type II collagen dissolved in 0.5 M acetic acid solution to prepare an acidic solution with a concentration of 1%; the cytokines are transforming growth factor-β and interleukin-17, and the dosage mass ratio is 3:1.

[0030] Example 2

[0031] A preparation method of a tissue regeneration-promoting filling material is carried out according to the following steps:

[0032] (1) Extraction of type I collagen: Select fresh cowhide, remove the surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, pulverize in a tissue grinder, and wash the pulverized tissue with deionized water; wash the pulverized tissue with 0.1M NaOH by shaking and soaking at 20°C for 28h, with a material-liquid ratio of 1g:5ml, wash twice with 75% ethanol, and then wash three times with deionized water; place the pulverized tissue in Tris-HCl buffer solution and soak at 4°C for 20h, centrifuge to collect the precipitate, and wash with deionized water; place the pulverized tissue in 0.008N hydrochloric acid, add 4% pepsin (pepsin activity 9000IU / g), enzymatically hydrolyze at 27°C for 45h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 4M NaOH, salting out at 4°C with saturated NaCl for 8h, centrifuge, wash the precipitate twice with saturated NaCl, dialyze the precipitate against 0.4M acetic acid for 20h first, and then dialyze against deionized water for 60h to obtain type I collagen gel.

[0033] (2) Extraction of type II collagen: Select fresh bovine cartilage, remove the surface fat and fascia, wash and cut into pieces with physiological saline, pulverize in a tissue grinder, and wash the pulverized tissue with deionized water; according to the material-liquid ratio of 1g:20ml, add 0.08mol / L NaOH to the pulverized tissue, ultrasonically wash, each time for 0.4h, wash 4 times; then add deionized water and ultrasonically wash, each time for 10min, wash 4 times; add the pulverized tissue to 0.08mol / L NaOH solution, ultrasonically treat for 10h, discard the NaOH solution, wash according to the material-liquid ratio of 1g:20ml with deionized water, each time for 10min, wash 3 times until the pH value of the washing solution is neutral; according to the material-liquid ratio of 1g:20ml, add 0.08mol / L hydrochloric acid to the pulverized tissue for decalcification, treat at 25°C for 3h, discard the decalcification solution, add deionized water for washing, each time for 10min, wash 3 times until the pH value of the washing solution is neutral; place the pulverized tissue in 0.008N hydrochloric acid, add 8% pepsin (pepsin activity 9000IU / g), enzymatically hydrolyze at 27°C for 60h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 4M NaOH, then add NaCl to make the final concentration of NaCl 4M, salting out at 4°C for 8h, centrifuge, wash the precipitate twice with 3M NaCl, and dialyze the precipitate against deionized water to obtain type II collagen gel.

[0034] (3) The oil-phase solution and the water-phase solution are respectively introduced into the microfluidic chip by pressure driving or syringe pump driving. The pressures of the oil phase and the water phase are adjusted. The PDMS-FF-100 chip is used to prepare microspheres. After the microdroplets are generated uniformly, they can be collected into a centrifuge tube containing the receiving-phase solution, and the microdroplets are received for 20 min. The received centrifuge tube is sealed, and gently shaken to accelerate the solidification of the microdroplets, and then left standing for 10 min. The fluorinated oil at the bottom of the centrifuge tube is taken out with a pipette to obtain microspheres. The demulsifier is added according to the volume ratio of microspheres to demulsifier of 1:2, and the mixture is centrifuged at 2000 rpm for 1.5 min with an oscillator. The waste liquid at the bottom is taken out, and the operation is repeated 2 times. The microspheres are collected and freeze-dried to obtain porous composite microspheres, which are mixed evenly with purified type I collagen and cytokines according to the mass ratio (2:7:0.02). The mixing is carried out using a homogenizer, and homogenization treatment is carried out for 100 min. After sufficient degassing, it is filled. The demulsifier is a 50% ethanol solution; the oil phase is: hydroxyapatite particles are dispersed in dimethylformamide, and the mass-volume ratio of hydroxyapatite particles to dimethylformamide is 1 g:40 ml; the water phase is: type II collagen is dissolved in 0.5 M acetic acid solution to prepare an acidic solution with a concentration of 1%; the cytokines are transforming growth factor-β and interleukin-17, and the dosage mass ratio is 3:1.

[0035] Example 3

[0036] A preparation method of a tissue regeneration-promoting filling material is carried out according to the following steps:

[0037] (1) Extraction of type I collagen: Select fresh sheepskin, remove the surface fat and minced meat, soak it in pure water to remove blood, wash it, cut it into pieces, and crush it in a tissue grinder. The crushed tissue is washed with deionized water; the crushed tissue is washed and soaked in 0.3 M NaOH at 30 °C for 20 h with a material-liquid ratio of 1 g:5 ml, washed 4 times with 75% ethanol, and then washed 5 times with deionized water; the crushed tissue is placed in Tris-HCl buffer solution and soaked at 4 °C for 28 h, and the precipitate is collected by centrifugation and washed with deionized water; the crushed tissue is placed in 0.012 N hydrochloric acid, and 6% pepsin (pepsin activity 11000 IU / g) is added. After enzymatic hydrolysis at 30 °C for 42 h, the supernatant is collected by centrifugation with a centrifuge, adjusted to neutral with 6 M NaOH, salted out at 4 °C with saturated NaCl for 12 h, centrifuged, and the precipitate is washed 4 times with saturated NaCl. The precipitate is first dialyzed against 0.6 M acetic acid for 28 h, and then dialyzed against deionized water for 80 h to obtain type I collagen gel.

[0038] (2) Extraction of type II collagen: Select fresh sheep cartilage, remove the surface fat and fascia, wash it with normal saline, cut it into pieces, pulverize it in a tissue grinder, and wash the pulverized tissue with deionized water; according to the ratio of material to liquid of 1 g:20 ml, add 0.12 mol / L NaOH to the pulverized tissue, and perform ultrasonic cleaning for 0.6 h each time, cleaning 4 times; then add deionized water and perform ultrasonic cleaning for 30 min each time, cleaning 4 times; add the pulverized tissue to 0.12 mol / L NaOH solution, perform ultrasonic treatment for 14 h, discard the NaOH solution, add deionized water for cleaning according to the ratio of material to liquid of 1 g:20 ml, clean for 20 min each time, and clean 5 times until the pH value of the cleaning solution is neutral; according to the ratio of material to liquid of 1 g:20 ml, add 0.12 mol / L hydrochloric acid to the pulverized tissue for decalcification, treat at 25 °C for 5 h, discard the decalcification solution, add deionized water for cleaning, clean for 30 min each time, and clean 5 times until the pH value of the cleaning solution is neutral; place the pulverized tissue in 0.012 N hydrochloric acid, add 12% pepsin (pepsin activity 11000 IU / g), enzymatically hydrolyze at 30 °C for 80 h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 6 M NaOH, then add NaCl to make the final concentration of NaCl 4 M, salting out at 4 °C for 12 h, centrifuge, wash the precipitate with 5 M NaCl 4 times, and dialyze the precipitate against deionized water to obtain type II collagen gel.

[0039] (3) Respectively introduce the oil-phase solution and the water-phase solution into the microfluidic chip by pressure driving or syringe pump driving, adjust the pressure of the oil phase and the water phase, use the PDMS-FF-100 chip to prepare microspheres. After the microdrops are generated evenly, start to receive them into a centrifuge tube containing the receiving-phase solution and receive the microdrops for 40 min; seal the received centrifuge tube, gently oscillate to accelerate the solidification of the microdrops, and let it stand for 30 min; use a pipette to take out the fluorinated oil at the bottom of the centrifuge tube to obtain microspheres; add the demulsifier according to the volume ratio of microspheres to demulsifier of 1:2 and centrifuge at 3000 rpm with an oscillator for 0.5 min, take out the waste liquid at the bottom, repeat the operation 2 times, collect the microspheres and freeze-dry them to obtain porous composite microspheres, and mix them evenly with the purified type I collagen and cytokines according to the mass ratio of 5:8:0.08. The mixing is carried out by a homogenizer and homogenized for 140 min, and then filled after sufficient defoaming. The demulsifier is a 50% ethanol solution; the oil phase is: hydroxyapatite particles dispersed in dimethylformamide, and the mass-volume ratio of hydroxyapatite particles to dimethylformamide is 1 g:40 ml; the water phase is: type II collagen dissolved in 0.5 M acetic acid solution to prepare an acidic solution with a concentration of 1%; the cytokines are transforming growth factor-β and interleukin-17, and the dosage mass ratio is 3:1.

[0040] Comparative Example 1

[0041] Other experimental conditions were the same as those in Example 1, except that the cytokine used was only transforming growth factor-β.

[0042] Comparative Example 2

[0043] Other experimental conditions were the same as those in Example 1, except that the cytokine used was only interleukin-17.

[0044] Experimental Example:

[0045] (1) The electron micrograph of the type II collagen-hydroxyapatite composite microspheres prepared in Example 1 is as Figure 1 shown.

[0046] (2) L929 fibroblast viability: L929 was inoculated on the filling materials prepared in Example 1, Comparative Example 1 and Comparative Example 2 at a ratio of 2×10 4 cell / mL. The culture medium was changed every 2 days. After 3 days of culture, the original culture medium was discarded, and the cells were gently washed twice with PBS. 150 μL of Calcein / PI reagent was added to each well, and the cells were incubated at room temperature for 10 min, then washed 3 times with PBS for 3 min each time. Photographs were taken under a fluorescence microscope to observe and evaluate the effect of different materials on the biocompatibility of L929.

[0047] The results are as Figure 2 shown. In the Example 1 group, good biocompatibility was exhibited on the surface. Compared with the Comparative Example 1 group and the Comparative Example 2 group, there were more cells on the surface of the filling material in the Example 1 group, and the cell surface area was larger, indicating that the biocompatibility of this material was better and more conducive to cell growth and proliferation.

[0048] (3) Masson's trichrome staining of the materials implanted for 21 days: The filling materials prepared in Example 1, Comparative Example 1 and Comparative Example 2 were respectively implanted subcutaneously in SD rats. On the 21st day, the samples were taken, fixed and then stained with Masson's trichrome. According to the results of Masson's trichrome staining ( Figure 3 ), on the 21st day, the collagen arrangement in the Example 1 group was denser and the collagen tissue area was larger than that in the Comparative Example 1 group and the Comparative Example 2 group, indicating that after the experimental group materials were implanted, the regenerative ability of collagen was significantly improved.

[0049] (4) Animal subcutaneous injection experiment: Twenty-seven healthy male SD rats, ordinary grade, 6 weeks old, with a body weight of 250 g ± 10 g were taken. The experimental rats were randomly divided into a negative control group, an experimental group and a positive control group, with 9 rats in each group.

[0050] Grouping: 1) Negative control group: normal saline group; 2) Experimental group: filling materials prepared in Examples 1-3 and Comparative Examples 1-2; 3) Positive control group: collagen group.

[0051] SD rats were subcutaneously injected, with an injection volume of 1 ml for each group. The injection sites were marked, and samples were taken at 1 week, 2 weeks, and 3 weeks for COLⅠ immunohistochemistry experiments, and analyzed by the method of analyzing the average optical density value of immunohistochemistry.

[0052] Method for analyzing the average optical density value (Mean Density) of immunohistochemistry: For each group, at least 3 fields of view at 200 times magnification were randomly selected from each section for photographing. When photographing, try to make the tissue fill the entire field of view as much as possible to ensure that the background light of each photo is consistent. Using Image-Pro Plus 6.0 software, the same brownish-yellow color was selected as the unified standard for judging the positivity of all photos, and the cumulative optical density value (IOD) of the positivity of each photo and the pixel area (AREA) of the tissue were analyzed for each photo, and the average optical density value IOD / AREA was calculated. The experimental results were statistically analyzed using SPSS 24.0 software. The results of measurement data were (mean ± standard deviation), and the Kolmogorov-Smirnov test method was used for data normality test. For data that conform to the normal distribution, the t-test was used to compare the mean differences between the two groups. A P < 0.05 was considered statistically significant. The measurement results are shown in Table 1:

[0053] Table 1

[0054]

[0055] Note: * represents P < 0.05 compared with the Example 1 group, and ** represents P < 0.01.

[0056] The analysis results of the average optical density value of immunohistochemistry showed that: in the same-sized field of view, the experimental group materials had a stronger ability to promote collagen regeneration in the skin than the pure collagen group, and to a certain extent, were superior to the current collagen products. Compared with Comparative Examples 1-2, Example 1 had a higher average optical density value, and the combined use of transforming growth factor-β and interleukin-17 achieved a better regeneration-promoting effect.

[0057] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A tissue regeneration promoting filling material, characterized in that, The filling material is made by using type II collagen as the microsphere wall material, hydroxyapatite as the core material, and dispersing the composite microspheres in a type I collagen gel.

2. The preparation method of the tissue regeneration promoting filling material according to claim 1, characterized in that, The steps are as follows: (1) Extraction of type I collagen: Using soft tissues of animal origin as raw materials, through degreasing, detrminal peptide treatment to remove immunogenicity, and then through pyrogen removal and impurity protein removal treatment, and finally purified. (2) Extraction of type II collagen: Using cartilage tissues of animal origin as raw materials, degreasing and removing fascia, through detrminal peptide treatment to remove immunogenicity, and finally purified. (3) Prepare composite microspheres of type II collagen-hydroxyapatite by microfluidics method, and mix them evenly with purified type I collagen and cytokines according to the mass ratio of (2-5):(7-8):(0.01-0.1) to make a tissue regeneration promoting filling material.

3. The preparation method of the tissue regeneration promoting filling material according to claim 2, wherein, The operating steps for the extraction of type I collagen are as follows: Select fresh animal skin, remove surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, and crush in a tissue grinder. Wash the crushed tissue with deionized water; Wash the crushed tissue with 0.1-0.3M NaOH by shaking and soaking at 20-30°C for 20-28h, with a material-liquid ratio of 1g:5ml, wash with 75% ethanol 2-4 times, and then wash with deionized water 3-5 times; Place the crushed tissue in Tris-HCl buffer, soak at 4°C for 20-28h, centrifuge to collect the precipitate, and wash with deionized water; Place the crushed tissue in 0.005-0.015N hydrochloric acid, and add 3-7% pepsin (pepsin activity 8000-12000IU / g), enzymolyze at 26-30°C for 42-56h, then centrifuge to collect the supernatant, adjust to neutral with 4-6M NaOH, salting out at 4°C with saturated NaCl for 8-12h, centrifuge, wash the precipitate with saturated NaCl 2-4 times, first dialyze the precipitate against 0.3-0.7M acetic acid for 20-28h, and then dialyze against deionized water for 60-80h to obtain type I collagen gel.

4. The preparation method of the tissue regeneration promoting filling material according to claim 2, characterized in that, The operating steps for extracting type II collagen are as follows: Select fresh animal cartilage, remove the surface fat and fascia, wash it with normal saline, cut it into pieces, and crush it in a tissue grinder. Wash the crushed tissue with deionized water; according to the ratio of 1 g:20 ml of solid-liquid ratio, add 0.05 - 0.15 mol / L NaOH to the crushed tissue, and perform ultrasonic cleaning for 0.3 - 0.7 h each time, and clean 2 - 4 times; then add deionized water and perform ultrasonic cleaning for 10 - 30 min each time, and clean 2 - 4 times; add the crushed tissue into 0.05 - 0.15 mol / L NaOH solution, perform ultrasonic treatment for 10 - 14 h, discard the NaOH solution, add deionized water for cleaning according to the ratio of 1 g:20 ml of solid-liquid ratio, clean for 10 - 20 min each time, and clean 3 - 5 times until the pH value of the cleaning solution is neutral; according to the ratio of 1 g:20 ml of solid-liquid ratio, add 0.05 - 0.15 mol / L hydrochloric acid to the crushed tissue for decalcification, treat at 25 °C for 3 - 5 h, discard the decalcification solution, add deionized water for cleaning, clean for 10 - 30 min each time, and clean 3 - 5 times until the pH value of the cleaning solution is neutral; place the crushed tissue in 0.005 - 0.015 N hydrochloric acid, add 8 - 12% pepsin (pepsin activity 8000 - 12000 IU / g), enzymatically hydrolyze at 26 - 30 °C for 60 - 80 h, centrifuge with a centrifuge to collect the supernatant, adjust to neutral with 4 - 6 M NaOH, then add NaCl to make the final concentration of NaCl 4 M, perform salting out at 4 °C for 8 - 12 h, centrifuge, wash the precipitate with 3 - 5 M NaCl for 2 - 4 times, and dialyze the precipitate against deionized water to obtain type II collagen gel.

5. The preparation method of the tissue regeneration promoting filling material according to claims 3-4, characterized in that The animal is a pig, a cow, a sheep or a horse.

6. The preparation method of the tissue regeneration promoting filling material according to claim 2, characterized in that, The specific operating steps for preparing the composite microspheres in step (3) are as follows: Drive the oil phase solution and the water phase solution into the microfluidic chip respectively through pressure driving or syringe pump driving, adjust the pressure of the oil phase and the water phase, use the PDMS-FF-100 chip to prepare microspheres. After the microdroplets are generated evenly, start to receive them into a centrifuge tube containing the receiving phase solution, and receive the microdroplets for 20 - 40 min; seal the received centrifuge tube, gently oscillate to accelerate the solidification of the microdroplets, and let it stand for 10 - 30 min; use a pipette to take out the fluorinated oil at the bottom of the centrifuge tube to obtain microspheres; add the demulsifier according to the volume ratio of microspheres to demulsifier of 1:2 and centrifuge at 2000 - 3000 rpm with an oscillator for 0.5 - 1.5 min, take out the waste liquid at the bottom, repeat the operation 1 - 2 times, collect the microspheres and freeze-dry them.

7. The preparation method of the tissue regeneration promoting filling material according to claim 6, wherein, The demulsifier is a 50% ethanol solution.

8. The preparation method of the tissue regeneration promoting filling material according to claim 6, characterized in that, The oil phase is: hydroxyapatite microparticles are dispersed in dimethylformamide, and the mass-volume ratio of hydroxyapatite microparticles to dimethylformamide is 1 g:40 ml; the water phase is: type II collagen is dissolved in 0.5 M acetic acid solution to prepare an acidic solution with a concentration of 1%.

9. The preparation method of the tissue regeneration promoting filling material according to claim 2, characterized in that, The cytokine is transforming growth factor-β and interleukin-17, and the dosage mass ratio is 3:

1.

10. The preparation method of the tissue regeneration-promoting filling material according to claim 2, wherein The mixing in step (3) uses a homogenizer, homogenize for 100 - 140 min, and fill after sufficient defoaming.

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