A tissue regeneration promoting filler material and its preparation method

Abstract

This invention discloses a tissue regeneration-promoting filler material and its preparation method. The filler material is made by dispersing composite microspheres in a type I collagen gel, using type II collagen as the microsphere wall material and hydroxyapatite as the core material. The tissue regeneration-promoting filler material prepared by this invention is used for filling areas such as eyebrow wrinkles, eyebrow subcutaneous support, malar fat and buccal fat pads, tear troughs, nasolabial folds, nose, lips, chin, and mandibular periosteum. It provides significant mechanical support and, based on exogenous collagen supply, continuously stimulates the formation and rearrangement of endogenous collagen, meeting the needs of cosmetic surgery.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic material preparation technology, specifically relating to a tissue regeneration-promoting filler material and its preparation method. Background Technology

[0002] Collagen is the most widely distributed protein in humans and animals, accounting for approximately 25% to 30% of the total protein content in the body. Collagen is a biological macromolecule in the extracellular matrix (ECM), its basic structure consisting of three polypeptide chains intertwined in a superhelical structure. Collagen acts as a filler and support for tissues, maintaining cellular structural integrity and other biological functions, defining the shape and form of tissues, and providing bones, tendons, cartilage, and skin with a certain mechanical strength. It possesses high biological activity and functionality, participating in the migration, proliferation, and differentiation of many types of cells, and performing its unique biological functions.

[0003] In recent years, collagen has been widely used in cosmetic dermatology because it can provide a scaffold for cell regeneration, induce the regeneration of its own collagen, reduce skin inflammation, and effectively rebuild the skin barrier. At the same time, the degraded amino acids can effectively inhibit tyrosinase activity and reduce melanin production.

[0004] As we age, the body gradually loses a significant amount of collagen, which is the primary cause of skin aging. Skin aging leads to a series of negative reactions such as dullness and dryness, necessitating continuous collagen replenishment to maintain youthful skin. Oral collagen has almost no effect on improving skin; only injection into the deep dermis can achieve cosmetic results. Medical cosmetic collagen is generally highly purified human or animal collagen, possessing excellent tissue compatibility. It can be naturally absorbed or degraded by the skin, stimulating dermal fibroblasts to produce new collagen, thereby regenerating and repairing cells and skin tissue. Currently, collagen is the most widely used biomedical material in cosmetic surgery. However, for cosmetic procedures requiring specific mechanical support, such as frown lines, subcutaneous eyebrow support, cheek and buccal fat pads, tear troughs, and nasolabial folds, simple collagen products cannot achieve satisfactory filling effects. To achieve long-lasting effects with certain mechanical support, research has emerged on cross-linking modification of collagen, the addition of PLLA microspheres, PCL microspheres, etc., which can achieve a certain degree of enhanced mechanical strength. However, cross-linked modified collagen may cause a "masking" phenomenon to some extent due to the presence of cross-linking agents, resulting in obvious mask-like appearance. PLLA microspheres and PCL microspheres are synthetic polymers, and although they can stimulate the regeneration of collagen in the dermis, they may also produce adverse reactions, including allergic reactions, nodules, and uneven distribution.

[0005] Hydroxyapatite is a safe inorganic material composed of calcium and phosphorus, the main inorganic components of human and animal bones. It has a certain solubility in the body, a pH value close to that of the human body, high biocompatibility, and can participate in the body's metabolism, ensuring its safety. Furthermore, hydroxyapatite is widely used in overseas medical aesthetic markets, second only to hyaluronic acid in clinical use, and is a product material recognized by many beauty seekers and medical professionals. When hydroxyapatite microparticles or microspheres are injected into the dermis, there are specific requirements for their particle size. If the particle size is too small, the microparticles may enter blood vessels, posing a potential risk of vascular thrombosis; if the particle size is too large, it may cause injection blockage and post-injection nodules. Hydroxyapatite microspheres have solved these problems to some extent, but due to the high density of hydroxyapatite, it is not uniformly dispersed in gel media. Summary of the Invention

[0006] The purpose of this invention is to provide a tissue regeneration-promoting filler material and its preparation method.

[0007] A tissue regeneration-promoting filler material, wherein the filler material is made by dispersing composite microspheres in a type I collagen gel, with type II collagen as the microsphere wall material and hydroxyapatite as the core material.

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

[0009] (1) Type I collagen extraction: animal-derived soft tissue is used as raw material. It is degreased and determinated to remove immunogenicity. It is then depyrogenated and decontaminated with proteins, and finally purified.

[0010] (2) Type II collagen extraction: animal cartilage tissue is used as raw material. The tissue is defatted and fascia is removed. The immunogenicity is removed by determinate peptide treatment and finally purified.

[0011] (3) Type II collagen-hydroxyapatite composite microspheres were prepared by microfluidic method and mixed with purified type I collagen and cytokines at a mass ratio of (2-5):(7-8):(0.01-0.1) to form a tissue regeneration filling material.

[0012] The extraction steps for type I collagen are as follows: Select fresh animal skin, remove surface fat and meat scraps, soak in purified water to remove blood, wash, cut into pieces, and pulverize in a tissue grinder. Wash the pulverized tissue with deionized water; use 0.1-0.3M... Soak the tissue in NaOH at 20-30℃ with shaking for 20-28 hours. Wash 2-4 times with 75% ethanol at a material-to-liquid ratio of 1g:5ml, and then wash 3-5 times with deionized water. Place the pulverized tissue in Tris-HCl buffer and soak at 4℃ for 20-28 hours. Centrifuge to collect the precipitate and wash with deionized water. Place the pulverized tissue in 0.005-0.015N hydrochloric acid and add 3-7% pepsin (pepsin activity 8000-12000 IU / g). Enzymatically hydrolyze at 26-30℃ for 42-56 hours. Centrifuge to collect the supernatant. Adjust the pH to neutral with 4-6M NaOH and salt out with saturated NaCl at 4℃ for 8-12 hours. Centrifuge and wash the precipitate 2-4 times with saturated NaCl. Dialyze the precipitate with 0.3-0.7M acetic acid for 20-28 hours, and then dialyze with deionized water for 60-80 hours to obtain type I collagen gel.

[0013] The extraction steps for type II collagen are as follows: Select fresh animal cartilage, remove surface fat and fascia, wash with physiological saline, cut into pieces, and pulverize in a tissue grinder. Wash the pulverized tissue with deionized water. Add 0.05-0.15 mol / L NaOH to the pulverized tissue at a material-to-liquid ratio of 1g:20ml, and ultrasonically clean for 0.3-0.7 hours each time, for 2-4 times. Then add deionized water and ultrasonically clean for 10-30 minutes each time, for 2-4 times. Add 0.05-0.15 mol / L NaOH to the pulverized tissue. In NaOH solution, sonicate for 10-14 hours, discard the NaOH solution, and add deionized water at a material-to-liquid ratio of 1g:20ml for washing, 10-20 minutes each time, 3-5 times, until the pH of the washing solution is neutral. Then, add 0.05-0.15mol / L hydrochloric acid at a material-to-liquid ratio of 1g:20ml to the pulverized tissue for decalcification, treat at 25℃ for 3-5 hours, discard the decalcification solution, and add deionized water for washing, 10-30 minutes each time. Wash 3-5 times until the pH of the washing solution is neutral; place the pulverized tissue in 0.005-0.015N hydrochloric acid and add 8-12% pepsin (pepsin activity 8000-12000 IU / g), and enzymatically hydrolyze at 26-30℃ for 60-80 hours. Centrifuge and collect the supernatant, adjust to neutral with 4-6M NaOH, and then add NaCl to make the final NaCl concentration 4M. Salt out at 4℃ for 8-12 hours, 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 in question is a pig, cow, sheep, or horse.

[0015] The specific steps for preparing the composite microspheres in step (3) are as follows: the oil phase solution and the aqueous phase solution are introduced into the microfluidic chip by pressure drive or injection pump drive, the pressure of the oil phase and the aqueous phase are adjusted, and the microspheres are prepared using the PDMS-FF-100 chip. After the microdroplets are uniformly generated, they can be received into a centrifuge tube containing the receiving phase solution for 20-40 minutes. The centrifuge tube is sealed, and the microdroplets are slightly oscillated to accelerate solidification. The tube is then left to stand for 10-30 minutes. The fluorinated oil at the bottom of the centrifuge tube is removed with a pipette to obtain the microspheres. The demulsifier is added at a volume ratio of 1:2 between the microspheres and the demulsifier, and the tube is centrifuged at 2000-3000 rpm for 0.5-1.5 minutes. The waste liquid at the bottom is removed. The operation is repeated 1-2 times. The microspheres are collected and freeze-dried.

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

[0017] The oil phase consists of hydroxyapatite microparticles dispersed in dimethylformamide, with a mass-to-volume ratio of hydroxyapatite microparticles to dimethylformamide of 1 g: 40 ml; the aqueous phase consists of type II collagen 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, in a mass ratio of 3:1.

[0019] The mixing in step (3) is performed using a homogenizer for 100-140 minutes to fully degas the product before filling.

[0020] The beneficial effects of this invention are as follows: The tissue regeneration-promoting filling material prepared by this invention can be used to fill areas such as eyebrow wrinkles, eyebrow subcutaneous support, malar fat and buccal fat pads, tear troughs, nasolabial folds, nose, lips, chin, and mandibular periosteum. It has a significant mechanical support effect and continuously stimulates the formation and rearrangement of endogenous collagen on the basis of providing exogenous collagen, thus meeting the needs of medical aesthetics and plastic surgery. Attached Figure Description

[0021] Figure 1 Electron micrograph of type II collagen-hydroxyapatite composite microspheres.

[0022] Figure 2 Live and dead staining of L929 fibroblasts.

[0023] Figure 3 Results of trichrome staining of the material implanted 21 days ago. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0025] Example 1

[0026] A method for preparing a tissue regeneration-promoting filler material comprises the following steps:

[0027] (1) Extraction of Type I collagen: Fresh pig skin was selected, surface fat and meat scraps were removed, blood was removed by soaking in purified water, washed and cut into pieces, and pulverized in a tissue grinder. The pulverized tissue was washed with deionized water. The pulverized tissue was washed and soaked in 0.2M NaOH at 25℃ for 24h with shaking. It was washed 3 times with 75% ethanol at a material-to-liquid ratio of 1g:5ml, and then washed 4 times with deionized water. The pulverized tissue was placed in Tris-HCl buffer and soaked at 4℃ for 24h. The precipitate was collected by centrifugation and washed with deionized water. The pulverized tissue was placed in 0.01N hydrochloric acid and 5% pepsin (pepsin activity 10000IU / g) was added. After enzymatic hydrolysis at 28℃ for 48h, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 5M NaOH and salted out with saturated NaCl at 4℃ for 10h. After centrifugation, the precipitate was washed 3 times with saturated NaCl. The precipitate was first dialyzed with 0.5M acetic acid for 24h and then dialyzed with deionized water for 72h to obtain Type I collagen gel.

[0028] (2) Type II collagen extraction: Fresh pig cartilage was selected, surface fat and fascia were removed, washed with physiological saline, cut into pieces, and pulverized in a tissue grinder. The pulverized tissue was washed with deionized water. At a material-to-liquid ratio of 1g:20ml, 0.1mol / L NaOH was added to the pulverized tissue, and ultrasonic cleaning was performed for 0.5 hours each time, for a total of 3 times. Then, deionized water was added again, and ultrasonic cleaning was performed for 20 minutes each time, for a total of 3 times. Finally, 0.1mol / L NaOH was added to the pulverized tissue. The tissue was ultrasonically treated in NaOH solution for 12 hours. The NaOH solution was discarded, and deionized water was added at a material-to-liquid ratio of 1g:20ml for washing. Each washing lasted 15 minutes, and the washing was repeated 4 times until the pH of the washing solution was neutral. Then, 0.1mol / L hydrochloric acid was added to the pulverized tissue at a material-to-liquid ratio of 1g:20ml for decalcification. The tissue was treated at 25℃ for 4 hours. The decalcification solution was discarded, and deionized water was added for washing. Each washing lasted 20 minutes, and the washing was repeated 4 times until the pH of the washing solution was neutral. The pulverized tissue was placed in 0.01N hydrochloric acid, and 10% pepsin (pepsin activity 10000 IU / g) was added. After enzymatic hydrolysis at 28℃ for 72 hours, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 5M NaOH, and then NaCl was added to bring the final NaCl concentration to 4M. Salting was performed at 4℃ for 10 hours. After centrifugation, the precipitate was washed 3 times with 4M NaCl. The precipitate was dialyzed against deionized water to obtain type II collagen gel.

[0029] (3) The oil phase solution and aqueous phase solution are introduced into the microfluidic chip by pressure drive or injection pump drive. The pressure of the oil phase and aqueous phase is adjusted. Microspheres are prepared using PDMS-FF-100 chip. After the microdroplets are uniformly generated, they can be received into the centrifuge tube containing the receiving phase solution for 30 min. The receiving centrifuge tube is sealed and gently shaken to accelerate the solidification of the microdroplets. It is then left to stand for 20 min. The fluorinated oil at the bottom of the centrifuge tube is removed with a pipette to obtain microspheres. Demulsifier is added at a volume ratio of 1:2 between microspheres and demulsifier and the mixture is centrifuged at 2500 rpm for 1 min. The waste liquid at the bottom is removed. The operation is repeated once. The microspheres are collected and freeze-dried to obtain porous composite microspheres. These microspheres are mixed with purified type I collagen and cytokines at a mass ratio of 4:7:0.05. The mixing is performed using a homogenizer for 120 min. After thorough degassing, the microspheres are filled into containers. The demulsifier is a 50% ethanol solution; the oil phase consists of hydroxyapatite microparticles dispersed in dimethylformamide, with a mass-to-volume ratio of hydroxyapatite microparticles to dimethylformamide of 1g:40ml; the aqueous phase consists of type II collagen dissolved in 0.5M acetic acid solution to prepare a 1% acidic solution; the cytokines are transforming growth factor-β and interleukin-17, used in a mass ratio of 3:1.

[0030] Example 2

[0031] A method for preparing a tissue regeneration-promoting filler material comprises the following steps:

[0032] (1) Extraction of Type I Collagen: Fresh cowhide was selected, surface fat and meat scraps were removed, blood was removed by soaking in purified water, washed and cut into pieces, and crushed in a tissue grinder. The crushed tissue was washed with deionized water. The crushed tissue was washed and soaked in 0.1M NaOH at 20℃ for 28h with shaking. It was washed twice with 75% ethanol at a ratio of 1g:5ml, and then washed three times with deionized water. The crushed tissue was placed in Tris-HCl buffer and soaked at 4℃ for 20h. The precipitate was collected by centrifugation and washed with deionized water. The crushed tissue was placed in 0.008N hydrochloric acid and 4% pepsin (pepsin activity 9000IU / g) was added. After enzymatic hydrolysis at 27℃ for 45h, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 4M NaOH and salted out with saturated NaCl at 4℃ for 8h. After centrifugation, the precipitate was washed twice with saturated NaCl. The precipitate was first dialyzed with 0.4M acetic acid for 20h and then dialyzed with deionized water for 60h to obtain Type I collagen gel.

[0033] (2) Type II collagen extraction: Fresh bovine cartilage was selected, surface fat and fascia were removed, and the cartilage was washed with physiological saline, cut into pieces, and pulverized in a tissue grinder. The pulverized tissue was washed with deionized water. At a material-to-liquid ratio of 1g:20ml, 0.08mol / L NaOH was added to the pulverized tissue, and ultrasonic cleaning was performed for 0.4h each time, for a total of 4 times. Then, deionized water was added again, and ultrasonic cleaning was performed for 10min each time, for a total of 4 times. 0.08mol / L NaOH was added to the pulverized tissue. The tissue was ultrasonically treated in NaOH solution for 10 hours. The NaOH solution was discarded, and deionized water was added at a material-to-liquid ratio of 1g:20ml for washing. Each washing lasted 10 minutes, and the washing was repeated 3 times until the pH of the washing solution was neutral. Then, 0.08mol / L hydrochloric acid was added to the pulverized tissue at a material-to-liquid ratio of 1g:20ml for decalcification. The tissue was treated at 25℃ for 3 hours. The decalcification solution was discarded, and deionized water was added for washing. Each washing lasted 10 minutes, and the washing was repeated 3 times until the pH of the washing solution was neutral. The pulverized tissue was placed in 0.008N hydrochloric acid, and 8% pepsin (pepsin activity 9000 IU / g) was added. After enzymatic hydrolysis at 27℃ for 60 hours, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 4M NaOH, and then NaCl was added to make the final NaCl concentration 4M. Salting was carried out at 4℃ for 8 hours. After centrifugation, the precipitate was washed twice with 3M NaCl. The precipitate was dialyzed against deionized water to obtain type II collagen gel.

[0034] (3) The oil phase solution and aqueous phase solution are introduced into the microfluidic chip by pressure drive or injection pump drive. The pressure of the oil phase and aqueous phase is adjusted. Microspheres are prepared using PDMS-FF-100 chip. After the microdroplets are uniformly generated, they can be received into a centrifuge tube containing the receiving phase solution. The microdroplets are received for 20 min. The centrifuge tube is sealed and gently shaken to accelerate the solidification of the microdroplets. It is then left to stand for 10 min. The fluorinated oil at the bottom of the centrifuge tube is removed with a pipette to obtain microspheres. Demulsifier is added at a volume ratio of 1:2 between microspheres and demulsifier and the mixture is centrifuged at 2000 rpm for 1.5 min. The waste liquid at the bottom is removed. The operation is repeated twice. The microspheres are collected and freeze-dried to obtain porous composite microspheres. These microspheres are mixed with purified type I collagen and cytokines at a mass ratio of (2:7:0.02). The mixing is performed using a homogenizer for 100 min. After thorough degassing, the microspheres are filled into containers. The demulsifier is a 50% ethanol solution; the oil phase consists of hydroxyapatite microparticles dispersed in dimethylformamide, with a mass-to-volume ratio of hydroxyapatite microparticles to dimethylformamide of 1g:40ml; the aqueous phase consists of type II collagen dissolved in 0.5M acetic acid solution to prepare a 1% acidic solution; the cytokines are transforming growth factor-β and interleukin-17, used in a mass ratio of 3:1.

[0035] Example 3

[0036] A method for preparing a tissue regeneration-promoting filler material comprises the following steps:

[0037] (1) Extraction of Type I Collagen: Fresh sheepskin was selected, surface fat and meat scraps were removed, and blood was removed by soaking in purified water. The skin was washed, cut into pieces, and crushed in a tissue grinder. The crushed tissue was washed with deionized water. The crushed tissue was washed and soaked in 0.3M NaOH at 30℃ for 20h with shaking. It was washed 4 times with 75% ethanol at a material-to-liquid ratio of 1g:5ml, and then washed 5 times with deionized water. The crushed tissue was placed in Tris-HCl buffer and soaked at 4℃ for 28h. The precipitate was collected by centrifugation and washed with deionized water. The crushed tissue was placed in 0.012N hydrochloric acid and 6% pepsin (pepsin activity 11000IU / g) was added. After enzymatic hydrolysis at 30℃ for 42h, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 6M NaOH and salted out with saturated NaCl at 4℃ for 12h. After centrifugation, the precipitate was washed 4 times with saturated NaCl. The precipitate was first dialyzed with 0.6M acetic acid for 28h and then dialyzed with deionized water for 80h to obtain Type I collagen gel.

[0038] (2) Type II collagen extraction: Fresh sheep cartilage was selected, surface fat and fascia were removed, washed with physiological saline, cut into pieces, and pulverized in a tissue grinder. The pulverized tissue was washed with deionized water. At a material-to-liquid ratio of 1g:20ml, 0.12mol / L NaOH was added to the pulverized tissue, and ultrasonic cleaning was performed for 0.6 hours each time, for a total of 4 times. Then, deionized water was added again, and ultrasonic cleaning was performed for 30 minutes each time, for a total of 4 times. 0.12mol / L NaOH was added to the pulverized tissue. The tissue was ultrasonically treated in NaOH solution for 14 hours. The NaOH solution was discarded, and deionized water was added at a material-to-liquid ratio of 1g:20ml for washing. Each washing lasted 20 minutes, and the washing was repeated 5 times until the pH of the washing solution was neutral. Then, 0.12mol / L hydrochloric acid was added to the pulverized tissue at a material-to-liquid ratio of 1g:20ml for decalcification. The tissue was treated at 25℃ for 5 hours. The decalcification solution was discarded, and deionized water was added for washing. Each washing lasted 30 minutes, and the washing was repeated 5 times until the pH of the washing solution was neutral. The pulverized tissue was placed in 0.012N hydrochloric acid, and 12% pepsin (pepsin activity 11000 IU / g) was added. After enzymatic hydrolysis at 30℃ for 80 hours, the supernatant was collected by centrifugation. The supernatant was adjusted to neutral with 6M NaOH, and then NaCl was added to bring the final NaCl concentration to 4M. Salting was performed at 4℃ for 12 hours. After centrifugation, the precipitate was washed 4 times with 5M NaCl. The precipitate was dialyzed against deionized water to obtain type II collagen gel.

[0039] (3) The oil phase solution and aqueous phase solution are introduced into the microfluidic chip by pressure drive or injection pump drive. The pressure of the oil phase and aqueous phase is adjusted. Microspheres are prepared using PDMS-FF-100 chip. After the microdroplets are uniformly generated, they can be received into a centrifuge tube containing the receiving phase solution. The microdroplets are received for 40 min. The centrifuge tube is sealed and gently shaken to accelerate the solidification of the microdroplets. It is then left to stand for 30 min. The fluorinated oil at the bottom of the centrifuge tube is removed with a pipette to obtain microspheres. Demulsifier is added at a volume ratio of 1:2 between microspheres and demulsifier and the mixture is centrifuged at 3000 rpm for 0.5 min. The waste liquid at the bottom is removed. The operation is repeated twice. The microspheres are collected and freeze-dried to obtain porous composite microspheres. These microspheres are mixed with purified type I collagen and cytokines at a mass ratio of 5:8:0.08. The mixing is performed using a homogenizer and homogenized for 140 min. After thorough degassing, the microspheres are filled into containers. The demulsifier is a 50% ethanol solution; the oil phase consists of hydroxyapatite microparticles dispersed in dimethylformamide, with a mass-to-volume ratio of hydroxyapatite microparticles to dimethylformamide of 1 g: 40 ml; the aqueous phase consists of type II collagen dissolved in 0.5 M acetic acid solution to prepare a 1% acidic solution; the cytokines are transforming growth factor-β and interleukin-17, used in a mass ratio of 3:1.

[0040] Comparative Example 1

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

[0042] Comparative Example 2

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

[0044] Experimental example:

[0045] (1) Electron micrograph of the type II collagen-hydroxyapatite composite microspheres prepared in Example 1 is shown below. Figure 1 As shown.

[0046] (2) L929 fibroblast survival: Fibroblasts were viable at 2 × 10⁻⁶ mm² on the filling materials prepared in Example 1, Comparative Example 1, and Comparative Example 2. 4 L929 cells were seeded at a ratio of 1 cell / mL, and the 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. The cells were then washed three times with PBS for 3 min each time. The cells were photographed under a fluorescence microscope to evaluate the effect of different materials on the biocompatibility of L929.

[0047] The results are as follows Figure 2 As shown, the surface of the Example 1 group exhibited good biocompatibility. Compared with Comparative Example 1 and Comparative Example 2, the surface of the filling material in Example 1 group had more cells and a larger cell surface area, indicating that the material has better biocompatibility and is more conducive to cell growth and proliferation.

[0048] (3) Masson's trichrome staining 21 days after material implantation: The filling materials prepared in Example 1, Comparative Example 1, and Comparative Example 2 were implanted subcutaneously into SD rats. Samples were collected on day 21, fixed, and subjected to Masson's trichrome staining. The results of the Masson's trichrome staining showed that... Figure 3 On day 21, the collagen arrangement in Example 1 was denser and the collagen tissue area was larger than that in Comparative Example 1 and Comparative Example 2, indicating that the regeneration capacity of collagen was significantly improved after the material was implanted in the experimental group.

[0049] (4) Subcutaneous injection experiment in animals: 27 healthy male SD rats, ordinary grade, 6 weeks old, weighing 250g±10g, were used. The experimental rats were randomly divided into negative control group, experimental group and positive control group, with 9 rats in each group.

[0050] Grouping: 1) Negative control group: 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 injected subcutaneously with 1 ml in each group. The injection sites were marked, and samples were collected at 1, 2, and 3 weeks for COLⅠ immunohistochemical experiments. The results were analyzed by the average optical density value analysis method of immunohistochemistry.

[0052] Immunohistochemical Mean Density Analysis: At least three 200x magnification fields of view were randomly selected from each slide within each group for imaging. During imaging, the tissue was positioned to fill the entire field of view as much as possible, ensuring consistent background lighting in each image. Image-Pro Plus 6.0 software was used to select the same brownish-yellow color as the unified standard for judging positivity in all images. Each image was analyzed to obtain the cumulative optical density (IOD) and the area per square (AREA) of the positive tissue, and the mean optical density (IOD / AREA) was calculated. The experimental results were statistically analyzed using SPSS 24.0 software. Quantitative data were analyzed using... (mean ± standard deviation) is expressed as mean. The Kolmogorov-Smirnov test was used to test the normality of the data. For normally distributed data, the t-test was used to compare the differences in means between two groups. A p-value < 0.05 was considered statistically significant. The results are shown in Table 1.

[0053] Table 1

[0054]

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

[0056] The results of immunohistochemical mean optical density analysis show that, in the same field of view, the experimental group material has a stronger collagen regeneration capacity for the skin than the pure collagen group, and can be superior to current collagen products to a certain extent. Compared with Comparative Examples 1-2, Example 1 has a higher mean optical density value, and the combination of transforming growth factor-β and interleukin-17 achieves a better regeneration effect.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a tissue regeneration promoting filler material, characterized by, The following steps are taken: (1) Collagen type I extraction: animal-derived soft tissue is used as raw material, through degreasing, removing telopeptide treatment, removing immunogenicity, and through pyrogen removal, removing impurities, and finally purified to prepare; (2) Collagen type II extraction: animal-derived cartilage tissue is used as raw material, degreasing and removing fascia, through telopeptide treatment, removing immunogenicity, and finally purified to prepare; (3) Collagen type II-hydroxyapatite composite microspheres are prepared by microfluidic method, mixed with purified collagen type I and cytokines according to the mass ratio (2-5): (7-8): (0.01-0.1) to prepare a tissue regeneration-promoting filling material; The cytokines are transforming growth factor-β and interleukin-17, and the mass ratio is 3:

1.

2. The method for preparing the tissue regeneration-promoting filler material according to claim 1, characterized in that, The operation steps of the collagen type I extraction are as follows: fresh animal skin is selected, the surface fat and meat are removed, the blood is removed by soaking in pure water, washed and cut into pieces, the tissue is crushed in a tissue crusher, and the crushed tissue is washed with deionized water; the crushed tissue is washed and soaked with 0.1-0.3M NaOH at 20-30℃ for 20-28h, the amount of liquid is 1g:5ml, 75% ethanol is washed 2-4 times, and deionized water is washed 3-5 times; the crushed tissue is placed in Tris-HCl buffer solution and soaked at 4℃ for 20-28h, the precipitate is collected by centrifugation, and the deionized water is washed; the crushed tissue is placed in 0.005-0.015N hydrochloric acid, 3-7% pepsin is added, the pepsin activity is 8000-12000 IU / g, and the enzyme is hydrolyzed at 26-30℃ for 42-56h, the supernatant is collected by centrifuge, the pH is adjusted to neutral with 4-6M NaOH, and the salt is precipitated with saturated NaCl at 4℃ for 8-12h, centrifuged, the precipitate is washed with saturated NaCl 2-4 times, the precipitate is first dialyzed against 0.3-0.7M acetic acid for 20-28h, and then dialyzed against deionized water for 60-80h to obtain collagen type I gel.

3. The method for preparing the tissue regeneration-promoting filler material according to claim 1, characterized in that, The operation steps of the type II collagen extraction are as follows: fresh animal cartilage is selected, the surface fat and fascia are removed, physiological saline is used for washing, and the cartilage is cut into pieces, crushed in a tissue crusher, and the crushed tissue is cleaned with deionized water; according to the ratio of 1g:20ml, 0.05-0.15mol / L NaOH is added to the crushed tissue, and ultrasonic cleaning is performed for 0.3-0.7h each time, and the cleaning is performed for 2-4 times; deionized water is added, and ultrasonic cleaning is performed for 10-30min each time, and the cleaning is performed for 2-4 times; 0.05-0.15mol / L NaOH solution is added to the crushed tissue, and ultrasonic treatment is performed for 10-14h, the NaOH solution is discarded, deionized water is added according to the ratio of 1g:20ml, and cleaning is performed for 10-20min each time, and the cleaning is performed for 3-5 times until the pH value of the cleaning solution is neutral; according to the ratio of 1g:20ml, 0.05-0.15mol / L hydrochloric acid is added to the crushed tissue for decalcification, and the treatment is performed at 25℃ for 3-5h, the decalcification solution is discarded, deionized water is added for cleaning, and the cleaning is performed for 10-30min each time, and the cleaning is performed for 3-5 times until the pH value of the cleaning solution is neutral; the crushed tissue is placed in 0.005-0.015N hydrochloric acid, and 8-12% pepsin is added, the activity of the pepsin is 8000-12000IU / g, and the enzymolysis is performed at 26-30℃ for 60-80h, the supernatant is collected by centrifugation, 4-6M NaOH is used to adjust the supernatant to neutral, NaCl is further added to the supernatant to make the final concentration of NaCl 4M, and the salt precipitation is performed at 4℃ for 8-12h, the centrifugation is performed, the precipitate is cleaned with 3-5M NaCl for 2-4 times, the precipitate is dialyzed with deionized water, and type II collagen gel is obtained.

4. The method for preparing the tissue regeneration-promoting filler material according to claim 1, characterized in that, The animal is a pig, a cow, a sheep or a horse.

5. The method for preparing the tissue regeneration-promoting filler material according to claim 1, characterized in that, The specific operation steps of the preparation of the composite microspheres 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 injection pump driving, the sizes of the oil phase and the water phase are adjusted, the PDMS-FF-100 chip is used to prepare the microspheres, after the microdroplets are uniformly generated, the microdroplets are received into the centrifuge tube containing the receiving phase solution for 20-40min, the received centrifuge tube is sealed, the microdroplets are solidified by slight oscillation, and the centrifuge tube is statically placed for 10-30min; the fluorine oil at the bottom of the centrifuge tube is taken out by using a pipette, and the microspheres are obtained; the demulsifier is added according to the volume ratio of the microspheres to the demulsifier 1:2, and the centrifuge tube is treated by oscillation at 2000-3000rpm for 0.5-1.5min, the waste liquid at the bottom is taken out, the operation is repeated for 1-2 times, the microspheres are collected, and the microspheres are freeze-dried.

6. The method for preparing the tissue regeneration-promoting filler material according to claim 5, characterized in that, The demulsifier is a 50% ethanol solution.

7. The method for preparing the tissue regeneration-promoting filler material according to claim 5, characterized in that, The oil phase is that hydroxyapatite microparticles are dispersed in dimethylformamide, and the mass-volume ratio of the hydroxyapatite microparticles to dimethylformamide is 1g:40ml; the water phase is that type II collagen is dissolved in 0.5M acetic acid solution, and an acidic solution with a concentration of 1% is configured.

8. The method for preparing the tissue regeneration-promoting filler material according to claim 1, characterized in that, In step (3), the mixing is performed by using a homogenizer, and the homogenization is performed for 100-140min, and the microspheres are filled after being fully defoamed.

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