Application of III-type recombinant collagen peptide in medical beauty filling material

Abstract

The invention relates to III-type collagen, in particular to application of III-type recombinant collagen peptide in a medical beauty filling material. Compared with aerogel obtained by photo-crosslinking only by the III type collagen peptide, the aerogel obtained by photo-crosslinking the III type collagen peptide and the catechol conjugated chitosan has the advantages that the mechanical property and the biological property of the aerogel obtained by photo-crosslinking only by the III type collagen peptide are improved. The III-type collagen peptide is used as a raw material to prepare the aerogel, and the aerogel can be applied to self-filling, has a natural and lasting filling effect and strong stability, and is not easy to deform or shift. The aerogel preparation process provided by the invention is environment-friendly, does not need the steps of high-temperature heating, chemical corrosion, organic solvent elution and the like, and is relatively environment-friendly.

Description

Technical Field

[0001] The present invention relates to type III collagen, and in particular to use of type III recombinant collagen peptides in medical and aesthetic filling materials. Background Art

[0002] Type III collagen is a type of collagen, which is widely distributed in mammals, present in the skin, tendons, bones, internal organs and other parts, accounting for about 25%-30% of the total protein in mammals, and 10-20% of human skin collagen. It is dominant in embryonic skin and is also considered to be the fetal form of collagen. It is also distributed in arteries, smooth muscles, endoneurial membranes, liver, spleen, kidneys, lungs, uterus, gastrointestinal tract, etc. Type III collagen is composed of three identical polypeptide chains, presenting a unique triple helical conformation, which is relatively immature, unstable, and has low elastic tension.

[0003] Type III collagen accounts for 80% of the baby's skin, making it soft and elastic. It is spread around type I collagen like a net, connective tissue, maintain elasticity, protect blood vessels and nerves, improve cell microenvironment, promote wound healing, and reduce skin inflammation. After adulthood, this protein in the human body gradually decreases and does not regenerate. The lack of it will slow down the metabolism of the skin, make it dry and loose, reduce its ability to lock in water, become dry and rough, and breed wrinkles.

[0004] Type III collagen can be used in wound repair, hemostatic materials, artificial blood vessels, artificial cartilage, and drug sustained-release carriers.

[0005] Type III collagen can simulate the collagen structure of human beings, is similar to human beings, has low allergy and sensitization, good compatibility, few side effects, and strong effects. Some recombinant human type III collagen products are 100% homologous to humans, have no allergic symptoms, have good transdermal absorption capacity and hydrophilicity, and good water retention. Summary of the invention

[0006] However, the performance and filling effect of medical cosmetic filling materials made only of type III collagen as raw material are not ideal. The aerogel obtained by photo-crosslinking type III collagen peptide and catechol conjugated chitosan in the present invention has improved mechanical and biological properties compared with the aerogel obtained by photo-crosslinking type III collagen peptide alone.

[0007] One of the purposes of the present invention is to provide a method for preparing an aerogel. The method comprises:

[0008] Prepare type III collagen peptide solution; potassium persulfate, 10 -2The camphorquinone bond of M and catechol conjugated chitosan are added to the solution for preparing type III collagen peptide to obtain a precursor solution; the precursor solution is subjected to a photocrosslinking reaction to obtain a gel; the gel is directionally frozen to solidify the water in the gel, and then freeze-dried to obtain an aerogel for filling.

[0009] Among them, the step of preparing type III collagen peptide solution includes: adding type III collagen α1 chain to a calcium chloride-ethanol-water ternary dissolution system with a molar ratio of 1:2:8 at a solid-liquid ratio of 1 mg:20 mL, dissolving for 3 hours at 70°C, and cooling to room temperature. Then transfer the above solution to a cellulose dialysis bag (C8000-10000Da) and place it in deionized water for 3 to 4 days. Centrifuge the recombinant mouse COL3A1 solution after dialysis at 25°C and 10000rpm for 20 minutes to obtain a recombinant mouse COL3A1 solution with a mass fraction of 1.8%. The above recombinant mouse COL3A1 solution is placed in an electric hot air drying oven, dried and concentrated at 40°C to a type III collagen peptide solution with a mass fraction of 5.0%, and stored at 5°C for standby use.

[0010] Among them, the final concentration of camphorquinone in the precursor solution is 1-2×10 -2 M, the final concentration of catechol-conjugated chitosan is 2.5-7wt%, and the final concentration of type III collagen peptide is 5.0%.

[0011] The precursor solution was placed under 50W 467nm blue light LED for 10 hours of irradiation at room temperature (25°C) to cross-link and obtain a gel.

[0012] One of the objectives of the present invention is to provide an aerogel prepared by the above method.

[0013] One of the purposes of the present invention is to provide a method for preparing a medical cosmetic filling material. The method comprises:

[0014] Prepare dermal extracellular matrix; cut the dermal extracellular matrix into pieces and freeze-dry it, grind it into powder and add it to a hydrochloric acid-pepsinogen mixed solution, stir and digest it at room temperature and then filter it, adjust the pH value to 7.4, add 10×PBS to balance the osmotic pressure, store it at 4°C, and obtain a dermal extracellular matrix solution; add the aerogel prepared by the above method to the dermal extracellular matrix solution, dialyze it, and freeze-dry it to obtain a filling material.

[0015] The steps of preparing the dermal extracellular matrix include: removing epithelial tissue and fat from fresh pig skin and digesting it with 0.25% trypsin for 6 hours; rinsing with deionized water and soaking it with 70% ethanol by volume for 10-12 hours; treating it with 3% H2O2 by volume for 15 minutes and washing it with deionized water again; acting it with 0.26% EDTA / 0.69% Tris solution containing 1% TritonX-100 for 12 hours; washing it with deionized water and soaking it with 0.1% peracetic acid / 4% ethanol by volume for 2 hours; and storing it in PBS at 4°C after washing it with deionized water.

[0016] Wherein, in the hydrochloric acid-pepsinogen mixed solution, the hydrochloric acid concentration is 0.01 mol / L, the pepsinogen mass concentration is 1 g / L, and the mass concentration of the dermal extracellular matrix is ​​10 g / L.

[0017] Among them, 0.5 g of aerogel was added into 50 mL of dermal extracellular matrix solution.

[0018] One of the objectives of the present invention is to provide a medical cosmetic filling material prepared by the above method.

[0019] One of the purposes of the present invention is to provide the use of type III collagen peptide in medical aesthetic filling materials.

[0020] Beneficial effects:

[0021] 1. The present invention obtains an aerogel by photocrosslinking type III collagen peptide and catechol-conjugated chitosan. Compared with an aerogel obtained by photocrosslinking type III collagen peptide alone and an aerogel obtained by photocrosslinking only type III collagen peptide, the volume density of the aerogel is lower.

[0022] 2. The aerogel obtained by photocrosslinking type III collagen peptide and catechol conjugated chitosan in the present invention not only retains the three-dimensional structure of type III collagen, but also realizes the coupling of catechol conjugated chitosan, compared with the aerogel obtained by photocrosslinking type III collagen peptide alone and the aerogel obtained by photocrosslinking only type III collagen peptide.

[0023] 3. The aerogel obtained by photocrosslinking type III collagen peptide and catechol conjugated chitosan in the present invention has significantly improved compressive elasticity compared with the aerogel obtained by photocrosslinking type III collagen peptide alone and the aerogel obtained by photocrosslinking only type III collagen peptide, and the stability of its compressive elastic performance is higher, indicating that the mechanical properties of the aerogel prepared by the method provided by the present invention are significantly improved.

[0024] 4. The aerogel obtained by photocrosslinking type III collagen peptide and catechol-conjugated chitosan in the present invention has improved adsorption performance and stability of adsorption performance compared with aerogel obtained by photocrosslinking type III collagen peptide alone and aerogel obtained by photocrosslinking only type III collagen peptide.

[0025] 5. The aerogel obtained by photocrosslinking type III collagen peptide and catechol-conjugated chitosan in the present invention has improved biocompatibility compared with the aerogel obtained by photocrosslinking type III collagen peptide alone and the aerogel obtained by photocrosslinking only type III collagen peptide, and can promote the proliferation of rat bone marrow mesenchymal stem cells.

[0026] 6. The aerogel obtained by photocrosslinking type III collagen peptide and catechol-conjugated chitosan in the present invention has a faster healing rate for in vivo wounds than the aerogel obtained by photocrosslinking type III collagen peptide alone and the aerogel obtained by photocrosslinking only type III collagen peptide.

[0027] 7. The aerogel obtained by photocrosslinking type III collagen peptide and catechol-conjugated chitosan in the present invention can quickly induce the sprouting of vascular endothelial cells to form a new vascular network structure and maintain the integrity and stability of the vascular network structure, compared with the aerogel obtained by photocrosslinking type III collagen peptide alone and the aerogel obtained by photocrosslinking only type III collagen peptide.

[0028] 8. The aerogel provided by the present invention can be used for self-filling, and the filling effect is natural and long-lasting, with strong stability and not easy to deform or shift.

[0029] 9. The aerogel preparation process provided by the present invention is environmentally friendly and does not require high-temperature heating, chemical corrosion, organic solvent elution and other steps, and is relatively environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The infrared images are, from top to bottom, infrared images of aerogels provided by Comparative Example 1 and Comparative Example 2, and Examples 1 to 3.

[0031] Figure 2 Compression-rebound curves of the aerogels of Examples 1 to 3.

[0032] Figure 3 It is the compression-rebound curve of the aerogel of comparative examples 1-2.

[0033] Figure 4 The adsorption amounts of water, crude oil and diesel by the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 are respectively.

[0034] Figure 5 The adsorption amounts of water, crude oil and diesel by the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 after 200 cycles of adsorption are shown respectively.

[0035] Figure 6 The graphs show the proliferation effects of the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 on BMSCs.

[0036] Figure 7 This is a graph showing the wound healing rate of the filling materials prepared based on the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 on abdominal wall defect model rats.

[0037] Figure 8 The filling materials prepared based on the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 affect the expression of Ang-related mRNA in the wound area tissue of rats with abdominal wall defect models.

[0038] Fig. 9 The SEM images (50 μm) of the aerogels of Examples 1 to 3 and Comparative Examples 1 to 2 are shown. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0040] Example 1: Preparation of aerogel

[0041] 1. Preparation of type III collagen peptide solution

[0042] The recombinant mouse COL3A1 (type III collagen α1 chain, Biolab, JN1232-RIC) was put into a ternary dissolution system of calcium chloride-ethanol-water with a molar ratio of 1:2:8 and a mixing ratio of 1:20. It was dissolved at 70°C for 3 hours and cooled to room temperature. The above solution was then transferred to a cellulose dialysis bag (C8000-10000Da) and dialyzed in deionized water for 3 to 4 days. The recombinant mouse COL3A1 solution after dialysis was centrifuged at 25°C and 10000rpm for 20 minutes to obtain a recombinant mouse COL3A1 solution with a mass fraction of 1.8%. The above recombinant mouse COL3A1 solution was placed in an electric hot air drying oven, dried and concentrated at 40°C to a type III collagen peptide solution with a mass fraction of 5.0%, and stored at 5°C for standby use.

[0043] 2. Preparation of Type III Collagen Photocrosslinking Precursor and Aerogel

[0044] Potassium persulfate at a final concentration of 1 mM and potassium persulfate at a final concentration of 10 -2 Camphorquinone with a final concentration of 2.5 wt % and catechol-conjugated chitosan with a final concentration of 2.5 wt % were added to the type III collagen peptide solution, dissolved by ultrasound and bubbles removed, and then stored in a dark environment at 3° C. to obtain a precursor solution.

[0045] The precursor solution was placed under 50W 467nm blue light LED irradiation at room temperature (25°C) for 10 hours to cross-link and obtain a gel.

[0046] The gel was frozen in liquid nitrogen to solidify the water in the gel, and then freeze-dried for 30 hours to obtain a filling aerogel ( Fig. 9 ).

[0047] Example 2: Preparation of aerogel

[0048] Prepare type III collagen peptide solution and precursor solution with reference to Example 1. The precursor solution contains a final concentration of 1.5×10 -2 M of camphorquinone and 5wt% of catechol conjugated chitosan, and the same steps as in Example 1 were followed to obtain an aerogel ( Fig. 9 ).

[0049] Example 3: Preparation of aerogel

[0050] Prepare type III collagen peptide solution and precursor solution with reference to Example 1, and add the precursor solution containing a final concentration of 2.0×10 -2 M of camphorquinone and 7wt% of catechol conjugated chitosan, and the same steps as in Example 1 were followed to obtain an aerogel ( Fig. 9 ).

[0051] Comparative Example 1: Preparation of aerogel

[0052] The type III collagen peptide solution was prepared by referring to Example 1, and the gel was frozen in liquid nitrogen to solidify the water in the gel, and then freeze-dried for 30 hours to obtain a filling aerogel ( Fig. 9 ).

[0053] Comparative Example 2: Preparation of aerogel

[0054] Prepare type III collagen peptide solution according to Example 1, store it in a dark place at 3°C, add potassium persulfate with a final concentration of 1 mM and -2M camphorquinone was added to the type III collagen peptide solution, dissolved by ultrasound and bubbles were removed, and then stored in a dark environment at 3°C ​​to obtain a precursor solution. The precursor solution was placed under a 50W 467nm blue light LED for 2 to 10 hours of irradiation reaction at room temperature (25°C) to obtain a gel. The gel was frozen in liquid nitrogen to solidify the water in the gel, and then freeze-dried for 30 hours to obtain a filling aerogel ( Fig. 9 ).

[0055] Test example: Aerogel volume density detection

[0056] GB / T5480 was used to test the density of the aerogels prepared in Examples 1 to 3 and Comparative Examples 1 to 2. The results show that the volume density of the pure III collagen aerogel prepared in Comparative Example 1 is 45.2 mg / cm 3 The volume density of the aerogel prepared in Comparative Example 2 is 27.6 mg / cm 3 The volume density of the aerogels prepared in Examples 1 to 3 is 21.9 mg / cm 3 、18.2mg / cm 3 、20.8mg / cm 3 , indicating that the volume density of aerogels prepared in Examples 1 to 3 is lower than that in Comparative Examples 1 to 2.

[0057] Test example: Aerogel infrared detection

[0058] The aerogels prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were characterized by FTIR. The samples were placed in a vacuum oven at 40°C and dried for 24 hours to remove the water, and then tested. The test range was 4000 to 500 cm -1 .

[0059] like Figure 2 It can be seen that the three-dimensional aerogel prepared in Comparative Example 1 has a -1 At 2925cm -1 、1652cm -1 、1550cm -1 The characteristic absorption peaks of amide bonds representing amide B, amide I and amide II appeared at the bottom, proving that the triple helix structure of collagen is well maintained in the collagen aerogel.

[0060] Compared with the FTIR graph of the aerogel prepared in Comparative Example 1, the aerogels prepared in Examples 1 to 3 have an obvious broad absorption peak at 3400 cm-1 and a -1 、1550cm -1The characteristic absorption peaks of the amide bond of amide I and amide II appearing at 1375 cm-1 are enhanced, corresponding to the NH bending vibration and CN stretching vibration of the unacetylated amino group in the chitosan molecule. In addition, the FTIR curves of Examples 1 to 3 respectively show the absorption peaks of CH bending vibration in chitosan at 1375 cm-1, and the C=O stretching vibration of the amide bond formed by the conjugation reaction of the carboxyl group and the amino group to form an amide bond at 1720 cm-1, which indicates that the catechol conjugated chitosan is coupled into the collagen to form an overall three-dimensional structure.

[0061] Test case: Mechanical stability

[0062] The compression elastic properties of the prepared pure collagen aerogel and collagen / cellulose aerogel samples were tested using a universal electronic testing machine, the change law of the mechanical properties of the samples was analyzed, and the influence of cellulose and UV irradiation cross-linking time on the mechanical properties of aerogels was discussed. The compression and recovery rates were set to 5 mm / min, the stress control was 0.1 MPa, the ambient temperature was 25°C, and the number of cycles was 1 to 200.

[0063] Figure 2 are the compression-rebound curves of the aerogels prepared in Examples 1 to 3, respectively. Figure 3 The compression rebound curves of the aerogels prepared in Comparative Examples 1 and 2 are shown in the figure. As shown in the figure, the maximum stress and maximum yield stress of the compression rebound curves of the aerogels prepared in Examples 1 to 3 are higher than those in Comparative Examples 1 to 2 at 1 cycle, 100 cycles and 200 cycles, and the reduction in the maximum stress and maximum yield stress at 1 cycle, 100 cycles and 200 cycles is less than that in Comparative Examples 1 to 2, indicating that the aerogel obtained by photo-crosslinking type III collagen peptide and catechol conjugated chitosan in the present invention has significantly improved compression elasticity compared to the aerogel obtained by photo-crosslinking type III collagen peptide alone, and the stability of its compression elasticity performance is higher, indicating that the mechanical properties of the aerogel obtained by the method provided by the present invention are significantly improved.

[0064] Test example: Adsorption performance test

[0065] The dried aerogel (porosity of 98%) was completely immersed in water or different oils. After saturation with adsorption, it was taken out and weighed. This was repeated three times to calculate the average adsorption amount, which was the ratio of the mass difference of the aerogel before and after adsorption to the mass of the aerogel before adsorption.

[0066] The cyclic adsorption test uses diesel as the adsorbent model to characterize the aerogel's ability to repeatedly absorb oil. First, the oil absorption rate of the aerogel after drying is tested, and then the aerogel after oil absorption is placed in a centrifuge to throw out the absorbed oil as much as possible. Finally, the aerogel is taken out and the oil absorption rate of the aerogel is tested again. Repeat the above steps several times, and make a schematic diagram of the oil absorption rate changing with the number of oil absorption times, so as to summarize the cyclic oil absorption performance of the aerogel.

[0067] The results show that the aerogels provided in the examples and comparative examples all have adsorption effects on water, crude oil and diesel, and the adsorption amounts decrease in sequence. Figure 4 It can be seen that the aerogels of Examples 1 to 3 have higher adsorption capacities for water, crude oil and diesel than those of Comparative Examples 1 to 2. Figure 5 It can be seen that after 200 cycles of adsorption, the adsorption amount of the aerogels of Examples 1 to 3 did not decrease significantly, while the adsorption amount of the aerogels of Comparative Examples 1 to 2 decreased. This shows that the aerogel obtained by photo-crosslinking type III collagen peptide and catechol conjugated chitosan in the present invention has improved adsorption performance compared to the aerogel obtained by photo-crosslinking using only type III collagen peptide, and the stability of the adsorption performance is also improved.

[0068] Test example: Cell experiment

[0069] 1. Isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs)

[0070] SD rats aged 2-4 weeks and weighing 20-25 g were selected and killed by cervical dislocation and immersed in 75% ethanol for 15 min. Bone marrow was obtained from the femur and tibia under sterile conditions and the mixture was purified by using a double antibody (10 5 U / L penicillin, 100mg / L streptomycin) and 20% fetal bovine serum MEM-α culture medium is placed in an incubator at 37°C and 5% CO2 for culture, and the medium is changed every 3 days; when the number of cells reaches about 80%, digest with 0.25% trypsin digestion solution, pass at a ratio of 1:2 or 1:3, and culture with MEM-α culture medium containing 10% fetal bovine serum, and the culture medium is changed every 2 days. Repeat the above operation until the cells are passed to 2-4 generations and then set aside.

[0071] 2. Cytocompatibility testing

[0072] The cell proliferation of BMSCs inoculated into the aerogels prepared in the above-mentioned Examples 1 to 3 and Comparative Examples 1 to 2 was determined by the CCK-8 kit. The cylindrical aerogels provided in Examples 1 to 3 and Comparative Examples 1 to 2 with a diameter of 6 mm and a thickness of 1 mm were prepared, with 12 in each group. After sterilization, 4 of each group were placed in 3 96-well plates, BMSCs were plated, 3 replicates were set at each time point, and only BMSCs were inoculated in the blank group. Cultured at 37°C for 1, 3, and 7 days respectively. CCK-8 reagent and MEM-α were mixed in a ratio of 1:10, 110 μl of the mixed reagent was added to each well, and the aerogels were placed in a 37°C incubator for continued culture for 2 hours, and then taken out and the absorbance value at 450nm was measured with an enzyme marker.

[0073] like Figure 6 As shown, after the aerogels provided in Examples 1 to 3 were co-cultured with BMSCs, their absorbance values ​​were significantly increased compared with those of the control group and comparative examples 1 to 2, indicating that the aerogels obtained by photo-crosslinking type III collagen peptides and catechol-conjugated chitosan in the present invention have improved biocompatibility compared with the aerogels obtained by photo-crosslinking using only type III collagen peptides, and can promote the proliferation of rat bone marrow mesenchymal stem cells.

[0074] Test example: Animal experiment

[0075] 1. Preparation of dermal extracellular matrix

[0076] 0.25% trypsin / 1% TritonX-100 was used to decellularize pig dermis. The main process is as follows: fresh pig skin was digested with 0.25% trypsin for 6 hours after removing epithelial tissue and fat; rinsed with deionized water and soaked in 70% ethanol by volume for 10-12 hours; treated with 3% H2O2 by volume for 15 minutes, and washed with deionized water again; treated with 0.26% EDTA / 0.69% Tris solution containing 1% TritonX-100 for 12 hours; washed with deionized water and soaked in 0.1% peracetic acid / 4% ethanol by volume for 2 hours; washed with deionized water and stored in PBS at 4°C.

[0077] 2. Preparation of filling materials

[0078] The dermal extracellular matrix was cut into pieces and freeze-dried, then ground into powder using a cryo-grinder, added to a hydrochloric acid-pepsinogen mixed solution (hydrochloric acid concentration 0.01 mol / L, pepsinogen mass concentration 1 g / L, dermal extracellular matrix mass concentration 10 g / L), stirred and digested at room temperature for 72 h, and then filtered; the pH value of the solution was adjusted to 7.4 with NaOH (0.1 mol / L), 10×PBS was added to balance the osmotic pressure, and stored at 4°C to obtain a dermal extracellular matrix solution.

[0079] 0.5 g of the aerogels provided in Examples 1 to 3 and Comparative Examples 1 to 2 were added to 50 mL of dermal extracellular matrix solution, dialyzed for 1 week using a dialysis bag with a molecular weight cutoff of 3500 Da, and freeze-dried at -80°C for 3 days to obtain a filling material.

[0080] 3. Aerogel repair effect on abdominal wall defects

[0081] Twelve SD rats were divided into a control group and an experimental group by random number table method, with 6 rats in each group. The rats in both groups fasted for 12 hours before surgery, and anesthetized by inhalation of isoflurane. After disinfection of the skin, a sterile scalpel was used to remove the skin and muscle on the right side of the abdomen to create a 1.5 cm diameter defect, and the rat model with preserved abdominal wall defect was established (Zheng Cui, Qiao Jing, Zhang Wei, et al. Study on the model of partial abdominal wall defect repaired by chitin hernia patch in rats [J]. Chinese Journal of Marine Drugs, 2018, 37(3): 18-24.).

[0082] Rats with abdominal wall defect model were divided into model group, test group and control group. The model group was not going to be treated. The control group filled the defect with polypropylene material (hernia repair material WS-P15×15 (Changzhou Medical Equipment Co., Ltd., registration number is National Medical Device Registration No. 20163130370)), and fixed it between the skin and muscle layer with sutures. The test group filled the defect with a filling material based on the aerogel provided in the above Examples 1 to 3 and Comparative Examples 1 to 2, and fixed it between the skin and muscle layer with sutures. All groups of rats were used on the surface of the wound skin and fixed with silk thread, covered with sterile gauze, and the wound gauze dressing was changed regularly.

[0083] 4. Wound healing rate

[0084] The wound healing conditions of each group were observed, and the wound healing rate was analyzed using ImageJ software. Wound healing rate = (1-unhealed wound area / original wound area) × 100%. The wound healing rate was calculated when the wound healing rate reached 90%. The wound healing rate was the ratio of the wound healing rate when the wound healing rate reached 90% to the postoperative time.

[0085] like Figure 7 As shown, the filling materials prepared based on the aerogels provided in Examples 1 to 3 have the fastest wound healing rate for the abdominal wall defect model rats, and are significantly higher than those in Comparative Examples 1 to 2 and the control group. This shows that the aerogel obtained by photo-crosslinking type III collagen peptides and catechol conjugated chitosan in the present invention is used to prepare filling materials, and the healing rate of the wound in vivo is faster than that of the aerogel obtained by photo-crosslinking type III collagen peptides alone.

[0086] 5. RT-PCR detection of Ang gene mRNA expression in the wound area of ​​the transplanted skin graft

[0087] The GoScript reverse transcription system (A5000, A5001, Promega (Beijing) Biotechnology Co., Ltd.) was used to reverse transcribe cDNA (the sample loading operation was completed on ice). PCR primer design: upstream primer 5'-CAAACACTTCCTGACCCAAC-3'; downstream primer 5'-CCTTGATGCTGCCCTTGT-3'. Take a certain amount of template RNA and add primers; pre-denature the template RNA and primer mixture at 70°C for 5 minutes, then take it out and place it on ice; configure RT-Mix and add 10μl to each sample tube; set up the reverse transcription program. After the program is completed, cDNA is obtained; use qPCRMasterMix dye method for RT-PCR experiments: qPCRMasterMix was melted at room temperature and placed on ice. It was shaken and mixed before use. It was collected by centrifugation and used. A reaction mixture without template was prepared. It was shaken and mixed slightly. 18μL of the reaction mixture was distributed to each reaction well. 2μL of the diluted standard DNA template and sample DNA were added to the corresponding reaction wells. The reaction plate was sealed and centrifuged slightly to centrifuge all the reaction components to the bottom of the tube to remove bubbles. The PCR reaction was observed using an RT-PCR amplification instrument. After the reaction was completed, the PCR product was taken and detected by agarose gel electrophoresis. Finally, the image analysis system was used to perform grayscale analysis on the PCR product.

[0088] Figure 8 It is the expression level of Ang-related mRNA in the wound area tissue when the wound healing rate reaches 90%. Figure 8 It can be seen that the filling materials prepared based on aerogels provided in Examples 1 to 3 have higher Ang-related mRNA expression levels in abdominal wall defect model rats than those in Comparative Examples 1 to 2 and the model group.

[0089] Studies have shown that Ang can regulate many molecular proteins related to angiogenesis and is an important hub for angiogenesis. It can promote the formation of lumen-like structures by endothelial cells, enhance the interaction between endothelial cells and perivascular cells, promote the maturation of blood vessels and maintain their integrity. The reconstruction of blood vessels after skin grafting generally requires two processes: the first 48 hours after skin grafting is the plasma nutrition period, which is mainly to produce endogenous fixation of the transplanted skin graft; 48 hours after the filling material of the skin graft is transplanted, it is the establishment period of angiogenesis and blood circulation, and vascular buds actively grow between the skin graft and the recipient tissue to form a lumen, which is generally completed within 4-5 days after surgery.

[0090] The aerogel obtained by photo-crosslinking type III collagen peptide and catechol-conjugated chitosan in the present invention can quickly induce vascular endothelial cells to sprout to form a new vascular network structure, and maintain the integrity and stability of the vascular network structure, compared with the aerogel obtained by photo-crosslinking type III collagen peptide alone.

[0091] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing an aerogel, comprising: preparing type III collagen peptide solution; adding potassium persulfate, camphorquinone and catechol-conjugated chitosan to the prepared type III collagen peptide solution to obtain a precursor solution; The precursor solution is subjected to a photo-crosslinking reaction to obtain a gel; The gel is directionally frozen to solidify the water in the gel, and then freeze-dried to obtain an aerogel for filling.

2. According to the method of claim 1, the step of preparing the type III collagen peptide solution comprises: Type III collagen α1 chain was added to a calcium chloride-ethanol-water ternary dissolution system with a molar ratio of 1:2:8 at a solid-liquid ratio of 1 mg:20 mL, dissolved at 70°C for 3 h, and cooled to room temperature; The solution was then transferred to a cellulose dialysis bag (C8000-10000Da) and dialyzed in deionized water for 3-4 days; The recombinant mouse COL3A1 solution after dialysis was centrifuged at 25°C and 10,000 rpm for 20 min to obtain a recombinant mouse COL3A1 solution with a mass fraction of 1.8%; The recombinant mouse COL3A1 solution was placed in an electric blast drying oven, dried and concentrated at 40°C to a type III collagen peptide solution with a mass fraction of 5.0%, and stored at 5°C for later use.

3. The method according to claim 1, wherein the final concentration of camphorquinone in the precursor solution is 1-2×10 -2 M, the final concentration of catechol-conjugated chitosan was 2.5~7wt%, and the final concentration of type III collagen peptide was 5.0%.

4. The method according to claim 1, wherein the precursor solution is placed under 50W 467nm blue light LED irradiation at room temperature (25°C) for 10 hours to cross-link and obtain a gel.

5. Aerogel prepared by any one of the methods of claims 1 to 4.

6. A method for preparing a medical cosmetic filling material, comprising: Preparation of dermal extracellular matrix; The dermal extracellular matrix was cut into pieces and freeze-dried, then ground into powder and added into a hydrochloric acid-pepsinogen mixed solution, stirred and digested at room temperature, filtered, the pH value was adjusted to 7.4, 10×PBS was added to balance the osmotic pressure, and stored at 4°C to obtain a dermal extracellular matrix solution; The aerogel prepared by the above method is added into the dermal extracellular matrix solution, dialyzed, and freeze-dried to obtain a filling material.

7. The method according to claim 6, wherein the step of preparing the dermal extracellular matrix comprises: Fresh pig skin was digested with 0.25% trypsin for 6 h after removing epithelial tissue and fat; rinsed with deionized water and soaked in 70% ethanol by volume for 10-12 h; Treat with 3% H2O2 for 15 min and wash with deionized water again; The cells were treated with a solution containing 1% TritonX-100, 0.26% EDTA, and 0.69% Tris for 12 h; After washing with deionized water, soak in 0.1% peracetic acid / 4% ethanol by volume for 2 h; After washing with deionized water, the cells were stored in PBS at 4°C.

8. The method according to claim 6, wherein in the hydrochloric acid-pepsinogen mixed solution, the hydrochloric acid concentration is 0.01 mol / L, the pepsinogen mass concentration is 1 g / L, and the mass concentration of the dermal extracellular matrix is ​​10 g / L; 0.5 g of aerogel was added to 50 mL of dermal extracellular matrix solution.

9. A medical cosmetic filling material obtained by any one of claims 6 to 8.

10. Use of type III collagen peptide in medical aesthetic filling materials.

Images