Use of type iii recombinant collagen peptides in medical aesthetic filling materials

Abstract

The present application relates to type III collagen, and particularly to the use of type III recombinant collagen peptide in medical and cosmetic filling materials. The aerogel obtained by photo-crosslinking of type III collagen peptide and catechol conjugated chitosan has improved mechanical properties and biological properties compared to the aerogel obtained by photo-crosslinking of type III collagen peptide alone. The aerogel prepared from type III collagen peptide as a raw material can be used for autologous filling, and has natural and long-lasting filling effect, strong stability, and is not prone to deformation or displacement. The preparation process of the aerogel is environmentally friendly, and does not require high-temperature heating, chemical corrosion, organic solvent elution and other steps, and is relatively environmentally friendly.

Description

Technical Field

[0001] This invention relates to type III collagen, and more specifically to the use of type III recombinant collagen peptides in medical aesthetic filler materials. Background Technology

[0002] Type III collagen is a type of collagen widely distributed in mammals, found in skin, tendons, bones, and internal organs, accounting for approximately 25%-30% of total mammalian protein, and 10-20% of collagen in human skin. It is dominant in the skin during the embryonic period and is considered the fetal form of collagen. It is also found in arteries, smooth muscle, nerve endometrium, liver, spleen, kidneys, lungs, uterus, and gastrointestinal tract. Type III collagen consists of three identical polypeptide chains, exhibiting a unique triple helix conformation. It is relatively immature, unstable, and has low elastic tension.

[0003] Infant skin contains 80% type III collagen, making it soft and supple. It spreads like a network around type I collagen, connecting tissues, maintaining elasticity, protecting blood vessels and nerves, improving the cellular microenvironment, promoting wound healing, and reducing skin inflammation. In adulthood, this protein gradually decreases in the body and does not regenerate. Its deficiency slows skin metabolism, causes dryness and sagging, reduces the skin's ability to retain moisture, leading to dryness, roughness, and 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 mimic the structure of human collagen, is similar to human collagen, has low allergenicity and sensitization, good biocompatibility, few side effects, and strong effects. Some recombinant human type III collagen products are 100% homologous to human collagen, have no allergic symptoms, good transdermal absorption and hydrophilicity, and good water retention. Summary of the Invention

[0006] However, the performance and filling effect of cosmetic fillers made solely from type III collagen are not ideal. This invention presents an aerogel obtained by photocrosslinking type III collagen peptides and catechol-conjugated chitosan. Compared to aerogels obtained by photocrosslinking type III collagen peptides alone, this aerogel exhibits improved mechanical and biological properties.

[0007] One objective of this invention is to provide a method for preparing aerogels. This method includes:

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

[0009] The steps for preparing the type III collagen peptide solution include: adding type III collagen α1 chains to a calcium chloride-ethanol-water ternary dissolution system with a solid-liquid ratio of 1 mg:20 mL at a molar ratio of 1:2:8, dissolving at 70°C for 3 hours, and then cooling to room temperature. The solution is then transferred to a cellulose dialysis bag (C8000-10000 Da) and dialyzed in deionized water for 3–4 days. After dialysis, the recombinant mouse COL3A1 solution is centrifuged at 25°C and 10000 rpm for 20 minutes to obtain a 1.8% recombinant mouse COL3A1 solution. This recombinant mouse COL3A1 solution is then placed in an electrically heated drying oven and concentrated at 40°C to a 5.0% type III collagen peptide solution, which is then stored at 5°C for later use.

[0010] In the precursor solution, the final concentration of camphorquinone is 1–2 × 10⁻⁶. -2 The final concentration of M, catechol conjugated chitosan is 2.5–7 wt%, and the final concentration of type III collagen peptide is 5.0%.

[0011] In this process, the precursor solution was irradiated under a 50W 467nm blue LED at room temperature (25℃) for 10 hours to crosslink and obtain a gel.

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

[0013] One objective of this invention is to provide a method for preparing a medical aesthetic filler material. The method includes:

[0014] Dermal extracellular matrix was prepared by cutting the dermal extracellular matrix into small pieces, freeze-drying it, grinding it into powder, adding it to a hydrochloric acid-pepsinogen mixed solution, stirring and digesting it at room temperature, filtering it, adjusting the pH to 7.4, adding 10×PBS to balance the osmotic pressure, and storing it at 4℃ to obtain a dermal extracellular matrix solution. The aerogel prepared by the above method was added to the dermal extracellular matrix solution, dialyzed, and freeze-dried to obtain the filling material.

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

[0016] In the hydrochloric acid-pepsinogen mixed solution, the concentration of hydrochloric acid was 0.01 mol / L, the concentration of pepsinogen was 1 g / L, and the concentration of dermal extracellular matrix was 10 g / L.

[0017] In this method, 0.5g of aerogel was added to 50mL of dermal extracellular matrix solution.

[0018] One of the objectives of this invention is to provide a medical aesthetic filler material prepared by the above method.

[0019] One of the objectives of this invention is to provide the use of type III collagen peptides in medical aesthetic filler materials.

[0020] Beneficial effects:

[0021] 1. The present invention provides an aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan. This aerogel has a lower bulk density compared to aerogels obtained by photocrosslinking type III collagen peptides alone or aerogels obtained by photocrosslinking only type III collagen peptides.

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

[0023] 3. The aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention has significantly improved compressive elasticity and higher stability compared to aerogels obtained by photocrosslinking type III collagen peptides alone or aerogels obtained by photocrosslinking only type III collagen peptides. This indicates that the mechanical properties of the aerogel prepared by the method provided in this invention are significantly improved.

[0024] 4. The aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention has improved adsorption performance and stability compared to aerogels obtained by photocrosslinking type III collagen peptides alone or aerogels obtained by photocrosslinking only type III collagen peptides.

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

[0026] 6. The aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention has a faster healing rate for in vivo wounds compared to aerogels obtained by photocrosslinking type III collagen peptides alone or aerogels obtained by photocrosslinking only type III collagen peptides.

[0027] 7. The aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention, compared with aerogels obtained by photocrosslinking type III collagen peptides alone and aerogels obtained by photocrosslinking only type III collagen peptides, can rapidly induce vascular endothelial cells to sprout and form new vascular network structures, while maintaining the integrity and stability of the vascular network structure.

[0028] 8. The aerogel provided by this invention can be applied to autologous filling, with a natural and long-lasting filling effect, strong stability, and is not easily deformed or displaced.

[0029] 9. The aerogel preparation process provided by this invention is environmentally friendly, requiring no high-temperature heating, chemical corrosion, organic solvent elution, or other steps, making it more environmentally friendly. Attached Figure Description

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

[0031] Figure 2 The compression-rebound curves are for the aerogels of Examples 1-3.

[0032] Figure 3 The compression-rebound curves are for the aerogels of Comparative Examples 1 and 2.

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

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

[0035] Figure 6 The aerogels used in Examples 1-3 and Comparative Examples 1-2 are used to promote the proliferation of BMSCs.

[0036] Figure 7 The graph shows the wound healing rate of rats with abdominal wall defects using the aerogel-based filling materials prepared in Examples 1-3 and Comparative Examples 1-2.

[0037] Figure 8 The expression levels of Ang-related mRNAs in the wound area tissue of rats with abdominal wall defects were measured using filling materials prepared based on aerogels from Examples 1-3 and Comparative Examples 1-2.

[0038] Figure 9 SEM images (50 μm) of the aerogels of Examples 1-3 and Comparative Examples 1-2. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this invention are all conventional reagents and are commercially available; methods not specifically 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] Recombinant mouse COL3A1 (type III collagen α1 chain, Bio-Rad, JN1232-RIC) was added to a ternary dissolution system of calcium chloride-ethanol-water with a molar ratio of 1:2:8 and a mixing ratio of 1:20. The solution was dissolved at 70°C for 3 hours and then 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. After dialysis, the recombinant mouse COL3A1 solution was centrifuged at 25°C and 10000 rpm for 20 minutes to obtain a 1.8% (w / w) recombinant mouse COL3A1 solution. This solution was then dried and concentrated in an electric heating oven at 40°C to a 5.0% (w / w) type III collagen peptide solution, and stored at 5°C for later use.

[0043] 2. Preparation of type III collagen photocrosslinking precursor solution and aerogel

[0044] Potassium persulfate with a final concentration of 1 mM and a final concentration of 10... -2 Camphorquinone M and catechol conjugated chitosan at a final concentration of 2.5 wt% were added to a type III collagen peptide solution. After being dissolved by sonication and removing air bubbles, the solution was stored in the dark at 3°C ​​to obtain the precursor solution.

[0045] The precursor solution was irradiated under a 50W 467nm blue LED at room temperature (25℃) for 10 hours to crosslink and obtain a gel.

[0046] The gel was placed in liquid nitrogen for directional freezing to solidify the water content, and then freeze-dried for 30 hours to obtain a filling aerogel. Figure 9 ).

[0047] Example 2: Preparation of Aerogel

[0048] Type III collagen peptide solution and precursor solution were prepared according to Example 1. The precursor solution contained a final concentration of 1.5 × 10⁻⁶. -2 The aerogel was obtained by conjugating camphor quinone (M) and 5 wt% catechol-chitosan, following the same steps as in Example 1. Figure 9 ).

[0049] Example 3: Preparation of Aerogel

[0050] Type III collagen peptide solution and precursor solution were prepared according to Example 1. The precursor solution contained a final concentration of 2.0 × 10⁻⁶. -2 The aerogel was obtained by conjugating camphor quinone (M) and 7 wt% catechol-chitosan, following the same steps as in Example 1. Figure 9 ).

[0051] Comparative Example 1: Preparation of Aerogels

[0052] A type III collagen peptide solution was prepared according to Example 1. The gel was then placed in liquid nitrogen for directional freezing to solidify the water content in the gel. After freeze-drying for 30 hours, a filling aerogel was obtained. Figure 9 ).

[0053] Comparative Example 2: Preparation of Aerogels

[0054] A type III collagen peptide solution was prepared according to Example 1 and stored in the dark at 3°C. Potassium persulfate with a final concentration of 1 mM and a final concentration of 10... -2Camphorquinone M was added to a type III collagen peptide solution, dissolved by sonication and degassed, and then stored in the dark at 3°C ​​to obtain a precursor solution. The precursor solution was then irradiated under a 50W 467nm blue LED at room temperature (25°C) for 2–10 hours to crosslink, resulting in a gel. The gel was then freeze-dried in liquid nitrogen to solidify the water content, followed by freeze-drying for 30 hours to obtain a filling aerogel. Figure 9 ).

[0055] Test example: Aerogel bulk density detection

[0056] The aerogel densities obtained in Examples 1-3 and Comparative Examples 1-2 were tested using GB / T 5480. The results showed that the bulk density of the pure III collagen aerogel obtained in Comparative Example 1 was 45.2 mg / cm³. 3 The aerogel prepared in Comparative Example 2 had a bulk density of 27.6 mg / cm³. 3 The aerogels prepared in Examples 1-3 had a bulk density of 21.9 mg / cm³. 3 18.2 mg / cm 3 20.8 mg / cm 3 This indicates that the aerogels prepared in Examples 1-3 have a lower volume density than those in Comparative Examples 1-2.

[0057] Test example: Aerogel infrared detection

[0058] The aerogels prepared in Examples 1-3 and Comparative Examples 1-2 were characterized using FTIR. The samples were dried in a vacuum oven at 40°C for 24 hours to remove moisture before testing. The testing range was 4000–500 cm⁻¹. -1 .

[0059] like Figure 2 It can be seen that the three-dimensional aerogel prepared in Comparative Example 1 has a density of 3328 cm⁻¹. -1 An absorption peak for the NH stretching vibration appears at 2925 cm⁻¹. Simultaneously, an absorption peak for the NH stretching vibration appears at 2925 cm⁻¹. -1 1652cm -1 1550cm -1 The presence of characteristic absorption peaks representing amide bonds (amide B, amide I, and amide II) at the specified locations demonstrates that the triple helix structure of collagen is well maintained in the collagen aerogel.

[0060] Compared with the FTIR spectrum of the aerogel prepared in Comparative Example 1, the aerogels prepared in Examples 1-3 respectively exhibit a significantly broader absorption peak at 3400 cm⁻¹, and a peak at 1652 cm⁻¹. -1 1550cm -1The characteristic absorption peaks of amide I and amide II bonds appearing at the FTIR values ​​are enhanced, corresponding to the NH bending vibration and CN stretching vibration of the unacetylated amino groups in the chitosan molecule. Furthermore, the FTIR curves of Examples 1-3 show an absorption peak for the CH bending vibration of chitosan at 1375 cm⁻¹, and a C=O stretching vibration of the amide bond formed through conjugation reactions such as the formation of amide bonds between the carboxyl and amino groups at 1720 cm⁻¹. This indicates that catechol conjugated chitosan is coupled into collagen, forming an integral three-dimensional structure.

[0061] Test Example: Mechanical Stability

[0062] The compressive elastic properties of pure collagen aerogel and collagen / cellulose aerogel samples were tested using a universal electronic testing machine. The variation law of mechanical properties of the samples was analyzed, and the influence of cellulose and UV irradiation crosslinking time on the mechanical properties of aerogels was explored. The compression and recovery rates were set at 5 mm / min, the stress was controlled at 0.1 MPa, the ambient temperature was 25℃, and the number of cycles ranged from 1 to 200.

[0063] Figure 2 The compression rebound curves are shown for the aerogels prepared in Examples 1-3, respectively. Figure 3 The figures show the compression rebound curves of the aerogels prepared in Comparative Examples 1 and 2, respectively. As can be seen from the figures, the maximum stress and maximum yield stress of the aerogels prepared in Examples 1-3 are higher than those in Comparative Examples 1-2 after 1, 100, and 200 cycles, respectively. Furthermore, the reduction in maximum stress and maximum yield stress after 1, 100, and 200 cycles is less than that in Comparative Examples 1-2. This indicates that the aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention significantly improves compressive elasticity compared to the aerogel obtained by photocrosslinking type III collagen peptides alone, and its compressive elasticity performance is more stable. This demonstrates that the mechanical properties of the aerogel prepared using the method provided by this invention are significantly improved.

[0064] Test Example: Adsorption Performance Test

[0065] The dried aerogel (with a porosity of 98%) was completely immersed in water or different oils. After adsorption saturation, it was taken out and weighed. This process was repeated three times to calculate the average adsorption amount. The adsorption amount is 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 fuel as the adsorbate model to characterize the aerogel's ability to repeatedly absorb oil. First, the oil absorption ratio of the dried aerogel is tested. Then, the oil-absorbed aerogel is placed in a centrifuge to remove as much of the absorbed oil as possible. Finally, the aerogel is removed, and its oil absorption ratio is tested again. This process is repeated several times, and a graph showing the change in oil absorption ratio with the number of absorption cycles is plotted, thus summarizing the aerogel's cyclic oil absorption performance.

[0067] The results show that the aerogels provided in each embodiment and comparative example all exhibit adsorption properties for water, crude oil, and diesel oil, with the adsorption capacity decreasing sequentially. Figure 4 It can be seen that the aerogels of Examples 1-3 have higher adsorption capacities for water, crude oil, and diesel oil than those of Comparative Examples 1-2. For example... Figure 5 It can be seen that after 200 cycles of adsorption, the adsorption capacity of the aerogels in Examples 1-3 did not decrease significantly, while the adsorption capacity of the aerogels in Comparative Examples 1-2 all decreased. This indicates that the aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention has improved adsorption performance and stability compared to the aerogel obtained by photocrosslinking type III collagen peptides alone.

[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 20-25g were sacrificed by cervical dislocation and immersed in 75% ethanol for 15 minutes. Bone marrow was harvested from the femur and tibia under aseptic conditions and treated with a mixture containing double antibiotics (10...). 5 The cells were cultured in MEM-α medium containing U / L penicillin, 100 mg / L streptomycin, and 20% fetal bovine serum at 37°C with 5% CO2, with the medium changed every 3 days. Once the cell count reached approximately 80%, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:2 or 1:3. The cells were then cultured in MEM-α medium containing 10% fetal bovine serum, with the medium changed every 2 days. This process was repeated until the cells reached 2-4 passages, at which point they were ready for use.

[0071] 2. Cell compatibility testing

[0072] Cell proliferation of BMSCs seeded into aerogels prepared in Examples 1-3 and Comparative Examples 1-2 was determined using a CCK-8 assay kit. Cylindrical aerogels (6 mm in diameter and 1 mm thick) were prepared, with 12 aerogels per group. After sterilization, four aerogels from each group were placed in three 96-well plates. BMSCs were seeded into the plates, with three replicates at each time point. The control group consisted only of BMSCs. The plates were incubated at 37°C for 1, 3, and 7 days. A 1:10 mixture of CCK-8 reagent and MEM-α was added to each well, and the plates were incubated at 37°C for another 2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0073] like Figure 6 As shown, after co-culturing the aerogels provided in Examples 1-3 with BMSCs, their absorbance values ​​were significantly increased compared to the control group and Comparative Examples 1-2. This indicates that the aerogels obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention have improved biocompatibility compared to aerogels obtained by photocrosslinking type III collagen peptides alone, 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] Porcine dermis was decellularized using 0.25% trypsin / 1% Triton X-100. The main process was as follows: Fresh porcine 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 for 10-12 hours; treated with 3% H2O2 for 15 minutes, and washed again with deionized water; treated with a 0.26% EDTA / 0.69% Tris solution containing 1% Triton X-100 for 12 hours; washed with deionized water and soaked in 0.1% peracetic acid / 4% ethanol for 2 hours; washed with deionized water and stored in PBS at 4°C.

[0077] 2. Preparation of filler material

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

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

[0080] 3. The repair effect of aerogel on abdominal wall defects

[0081] Twelve SD rats were randomly divided into a control group and an experimental group, with six rats in each group. Both groups of rats were fasted for 12 hours before surgery. After anesthesia induced by isoflurane inhalation, the skin and muscles on the right side of the abdomen were removed with a sterile scalpel to create a defect with a diameter of 1.5 cm, thus establishing a rat model of abdominal wall defect preservation (Zheng Cui, Qiao Jing, Zhang Wei, et al. Study on rat model of partial abdominal wall defect repair by chitosan hernia patch [J]. Chinese Marine Drugs, 2018, 37(3):18-24.).

[0082] Rats with abdominal wall defects were divided into a model group, a test group, and a control group. The model group was not to be treated. The control group had the defect filled with polypropylene material (WS-P15×15 hernia repair material (Changzhou Medical Device Co., Ltd., registration certificate number: 20163130370)) and fixed between the skin and muscle layers with sutures. The test group had the defect filled with a filling material based on aerogel provided in Examples 1-3 and Comparative Examples 1-2, and fixed between the skin and muscle layers with sutures. All rats in each group had the wound treated on the skin surface and fixed with silk sutures, covered with sterile gauze, and the wound dressing was changed regularly.

[0083] 4. Wound healing rate

[0084] The wound healing status of each group was observed, and the wound healing rate was analyzed using ImageJ software. The wound healing rate was calculated as (1 - area of ​​unhealed wound / area of ​​original wound) × 100%. The wound healing rate when the wound healing rate reached 90% was calculated as 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 aerogel-based filling materials provided in Examples 1-3 exhibited the fastest wound healing rate in rats with abdominal wall defects, significantly higher than those in Comparative Examples 1-2 and the control group. This demonstrates that the aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan, compared to the aerogel obtained by photocrosslinking type III collagen peptides alone, results in a faster wound healing rate in vivo when used as a filling material.

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

[0087] cDNA was obtained by reverse transcription using the GoScript reverse transcription system (A5000, A5001, Prometheus (Beijing) Biotechnology Co., Ltd.) (sample loading was performed on ice). PCR primer design: upstream primer 5'-CAAACACTTCCTGACCCAAC-3'; downstream primer 5'-CCTTGATGCTGCCCTTGT-3'. A certain amount of template RNA was added to the primers; the template RNA and primer mixture was pre-denatured at 70℃ for 5 min, and then placed on ice; RT-Mix was prepared, and 10 μl was added to each sample tube; the reverse transcription program was set. cDNA was obtained after the program was completed; RT-PCR experiments using the qPCRMasterMix dye method: After thawing at room temperature, qPCRMasterMix was placed on ice. Before use, it was vortexed to mix thoroughly, then collected by gentle centrifugation. A template-free reaction mixture was prepared, and after gentle vortexing, 18 μL of the mixture was aliquoted into each well. 2 μL each of diluted standard DNA template and sample DNA were added to the corresponding wells. The reaction plate was sealed, and the entire reaction mixture was gently centrifuged until all components reached the bottom of the tube. Air bubbles were removed, and the PCR reaction was observed using an RT-PCR amplification instrument. After the reaction, the PCR products were collected and detected by agarose gel electrophoresis. Finally, an image analysis system was used to perform grayscale analysis of the PCR products.

[0088] Figure 8 This represents the expression level of Ang-related mRNAs in the wound area tissue when the wound healing rate reaches 90%. For example... Figure 8 It can be seen that the aerogel-based filling materials provided in Examples 1-3 showed higher levels of Ang-related mRNA expression in rats with abdominal wall defects than those in Comparative Examples 1-2 and the model group.

[0089] Studies have shown that Ang can regulate many molecular proteins related to angiogenesis and is a crucial hub for angiogenesis. It can promote the formation of lumen-like structures by endothelial cells, enhance the interaction between endothelial cells and peritubular cells, promote vascular development and maturation, and maintain vascular integrity. Vascular reconstruction after skin grafting generally involves two processes: the first 48 hours after skin grafting is the plasma nutrition period, which mainly enables the transplanted skin to achieve endogenous fixation; the next 48 hours after the grafting filler material is applied is the angiogenesis and blood circulation establishment period, during which vascular buds actively grow between the skin graft and the recipient tissue, forming lumens, which is generally completed within 4-5 days postoperatively.

[0090] The aerogel obtained by photocrosslinking type III collagen peptides and catechol conjugated chitosan in this invention can rapidly induce vascular endothelial cells to sprout and form new vascular network structures, while maintaining the integrity and stability of the vascular network structure, compared to the aerogel obtained by photocrosslinking type III collagen peptides alone.

[0091] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a medical aesthetic filler material, comprising: Preparation of dermal extracellular matrix; After the dermal extracellular matrix was cut into small pieces, freeze-dried, ground into powder, and added to a hydrochloric acid-pepsinogen mixed solution, it was stirred and digested at room temperature, filtered, the pH was adjusted to 7.4, 10×PBS was added to balance the osmotic pressure, and it was stored at 4℃ to obtain the dermal extracellular matrix solution. The aerogel prepared by the following method was added to the extracellular matrix solution of dermal cells, dialyzed, and freeze-dried to obtain the filling material; The preparation methods of aerogel include: Preparation of type III collagen peptide solution; Potassium persulfate, camphorquinone, and catechol conjugated chitosan were added to a solution for preparing type III collagen peptides to obtain a precursor solution; the final concentration of camphorquinone in the precursor solution was 1~2×10⁻⁶. -2 The final concentration of M, catechol-conjugated chitosan, is 2.5–7 wt%, and the final concentration of type III collagen peptides is 5.0 wt%. The precursor solution was irradiated under a 50W 467nm blue LED at room temperature for 10 hours to crosslink and 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. The steps for preparing type III collagen peptide solution include: Type III collagen α1 chains were added to a calcium chloride-ethanol-water ternary dissolution system with a solid-liquid ratio of 1 mg:20 mL and a molar ratio of 1:2:

8. The dissolution was carried out at 70 °C for 3 h and then cooled to room temperature. The solution obtained above is then transferred to a cellulose dialysis bag (8000-10000 Da) and dialyzed in deionized water for 3-4 days. After dialysis, the recombinant mouse COL3A1 solution was centrifuged at 25°C and 10,000 rpm for 20 min to obtain a 1.8% recombinant mouse COL3A1 solution. The centrifuged recombinant mouse COL3A1 solution was placed in an electric heating drying oven and dried and concentrated at 40°C to a 5.0% (w / w) type III collagen peptide solution. It was then stored at 5°C for later use.

2. The preparation method according to claim 1, wherein the step of preparing the dermal extracellular matrix includes: Fresh pig skin was digested with 0.25% trypsin for 6 hours after removing the epithelial tissue and fat; then rinsed with deionized water and soaked in 70% ethanol for 10-12 hours. Treat with 3% H2O2 (volume fraction) for 15 min, then wash again with deionized water; Treat with a solution containing 1% Triton X-100, 0.26% EDTA and 0.69% Tris for 12 hours; After washing with deionized water, soak in 0.1% peracetic acid / 4% ethanol for 2 hours; After washing with deionized water, store in PBS at 4°C.

3. According to the preparation method of claim 2, in the hydrochloric acid-pepsinogen mixed solution, the concentration of hydrochloric acid is 0.01 mol / L, the mass concentration of pepsinogen is 1 g / L, and the mass concentration of dermal extracellular matrix is ​​10 g / L; Add 0.5g of aerogel to 50mL of dermal extracellular matrix solution.

4. The medical aesthetic filler material prepared by the method described in claim 1.

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