A polydopamine and collagen composite scaffold and a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of polydopamine and collagen composite scaffold and its preparation method and application, preparation method includes the following steps: (1) dopamine hydrochloride is added to the deionized water containing appropriate amount of ammonia and anhydrous ethanol, stirring at room temperature, to obtain crude polydopamine. (2) the crude polydopamine is centrifuged at low speed, and then high-speed centrifugation is carried out, and the precipitate is collected;After cleaning, freeze-drying is carried out, to obtain purified polydopamine. (3) then weigh appropriate amount of polydopamine and add to collagen solution, adjust pH, stirring at room temperature, freeze-drying, to obtain polydopamine and collagen composite scaffold. The polydopamine and collagen composite scaffold of the present application are prepared by physical blending rather than chemical modification strategy, which not only maintains the biocompatibility of the composite scaffold but also greatly reduces the production cost, and the simple preparation process provides feasibility guarantee for clinical conversion, and the composite scaffold can be used for the treatment of diabetic wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine technology, and particularly relates to a polydopamine and collagen composite scaffold as well as a preparation method and application thereof. BACKGROUND

[0002] Diabetic ulcer (DU) is the most intractable chronic complication in the course of diabetes, and its difficult-to-heal characteristics are mainly attributed to the complex interweaving of multiple pathological mechanisms and the superposition of self-strengthening effect. In-depth analysis of its pathological nature can find that the sustained high glucose microenvironment triggers oxidative stress, chronic inflammation, angiogenesis disorder and extracellular matrix metabolism imbalance, and these processes are interrelated and form a vicious cycle that is difficult to break.

[0003] In the face of such a complex pathological network, the limitations of current clinical routine treatment methods are increasingly prominent: 1) surgical debridement can remove necrotic tissue but cannot improve the pathological microenvironment; 2) growth factor drugs are limited by rapid degradation in vivo and resistance to treatment; 3) traditional collagen dressings can only provide a passive physical barrier and lack the function of dynamically adjusting the microenvironment. More attention should be paid to the fact that some high-end dressings containing bioactive ingredients have high costs and the risk of immune rejection, which seriously restricts their clinical in-depth promotion and application.

[0004] Polydopamine (PDA) is a biomimetic polymer material formed by the self-polymerization of dopamine under weak alkaline conditions, and its unique catechol and amino structures endow it with strong adhesion, biocompatibility, photothermal effect and surface functionalization ability, etc. Collagen is the main structural component of the extracellular matrix and plays an irreplaceable role in the normal wound repair process. Its natural three-dimensional porous structure provides an ideal physical space for cell migration and vascular ingrowth, and the specific bioactive sequence on the surface can effectively promote cell proliferation and differentiation. In addition, the controllable degradation characteristics of collagen enable it to maintain a dynamic balance in the process of tissue regeneration. However, in the specific pathological microenvironment of diabetes, the advantages of collagen materials are difficult to fully exert, for example, oxidative stress easily leads to damage to the structure of collagen fibers, chronic inflammation easily causes a large number of pro-inflammatory factors to be adsorbed on the surface of the material, and cell signal transduction abnormalities caused by high glucose state easily weaken the bioactivity function of collagen.

[0005] To overcome these limitations, this study attempts to improve the pathological adaptability of collagen scaffolds through material modification. Although early chemical modification strategies partially improved material performance, they often sacrificed the natural biological activity of collagen. In addition, the design of introducing exogenous bioactive factors into the scaffold system shows therapeutic potential, but faces the problems of complex preparation process and cost control. The above difficulties prompt researchers to turn their attention to natural biomimetic materials with multiple biological activities, among which polydopamine has attracted attention due to its unique molecular characteristics. SUMMARY

[0006] In order to overcome the shortcomings and deficiencies existing in the prior art, the purpose of the present application is to provide a polydopamine and collagen composite scaffold and a preparation method and application thereof.

[0007] The purpose of the present application is achieved by the following technical solution: a preparation method of a polydopamine and collagen composite scaffold, comprising the following steps:

[0008] (1) First, add an appropriate amount of anhydrous ethanol to deionized water, then add an appropriate amount of ammonia water, stir at room temperature to obtain solution A; then dissolve an appropriate amount of dopamine hydrochloride in deionized water to obtain solution B; then add solution B to A, stir at room temperature to obtain crude polydopamine;

[0009] (2) Centrifuge the crude polydopamine at low speed to remove insoluble impurities or large particle materials, then centrifuge at high speed to collect the precipitate; then wash the precipitate with ethanol first, then with deionized water, collect the precipitate after washing and freeze-drying to obtain purified polydopamine;

[0010] (3) Then weigh an appropriate amount of polydopamine and add it to a collagen solution, adjust the pH to 5.5-6.5, stir at room temperature, and freeze-dry to obtain a polydopamine and collagen composite scaffold.

[0011] Preferably, step (1) is specifically: first add 30-50 mL of anhydrous ethanol to 80-100 mL of deionized water, then add 1-3 mL of ammonia water, stir at room temperature to obtain solution A; then dissolve 400-600 mg of dopamine hydrochloride in 8-12 mL of deionized water to obtain solution B; then add solution B to A, stir at room temperature for 8-16 h to obtain crude polydopamine.

[0012] Preferably, in step (2), the first centrifugation speed is 1500-2500 rpm, the centrifugation time is 3-7 min, the second centrifugation speed is 10000-12000 rpm, the centrifugation time is 10-20 min, and the mass fraction of ethanol is 25%-35%.

[0013] Preferably, in the step (3), the mass concentration of collagen in the collagen solution is 0.01-1.0 mg / mL, the mass concentration of polydopamine is 0.5-50 μg / mL, and the stirring time is 8-16 h.

[0014] A polydopamine and collagen composite scaffold prepared according to the preparation method described above.

[0015] The polydopamine and collagen composite scaffold is used in the preparation of biological products for promoting the healing of diabetic wounds.

[0016] Preferably, the composite scaffold, the polydopamine and collagen composite scaffold, can promote cell proliferation, facilitate cell migration, and be conducive to the expression of CD31 and VEGFA proteins by HUVEC cells.

[0017] Preferably, the composite scaffold, the polydopamine and collagen composite scaffold, can up-regulate the CD31 and VEGFA proteins of HUVEC cells under a high-sugar and inflammatory environment, while reducing iNOs cytokines, and is conducive to the formation of new blood vessels.

[0018] Preferably, the composite scaffold, the polydopamine and collagen composite scaffold, can reduce the expression of inflammatory factors of RAW264.7 cells by interfering with the receptors TNFR1 and TLR4.

[0019] Preferably, the composite scaffold, the polydopamine and collagen composite scaffold, can inhibit the phosphorylation of the MAPK / NF-κB signal pathway.

[0020] The polydopamine and collagen composite scaffold of the present application breaks through the limitations of traditional single-function materials, realizes the multifunctional integration of antioxidant, immunomodulation and structural support, and embodies the treatment concept of "pathological microenvironment adaptation". The preparation strategy of physical blending instead of chemical modification not only maintains the biocompatibility of the composite scaffold but also greatly reduces the production cost, and the simple preparation process provides feasibility guarantee for clinical transformation. Molecular mechanism research shows that the synergistic activation of cell signal pathways by the surface characteristics of the composite scaffold has an important influence, and the combination of specific bioactive sequences and functional groups can enhance the response efficiency of cells to growth signals. The stage-responsive functional release characteristics form a precise match with the dynamic pathological process of diabetic ulcers.

[0021] The present application constructs a polydopamine and collagen composite scaffold for the treatment of diabetic wounds, proposes an innovative treatment concept of multi-stage synergistic repair, and discusses the mechanism of action. The treatment concept mainly shows that: 1) in the early stage of wound healing, the composite scaffold quickly responds to high oxidative stress, effectively removes excess free radicals and inhibits the excessive activation of inflammatory signals. The functional intervention in this stage creates favorable conditions for breaking the "oxidation-inflammation" vicious cycle. 2) As the treatment progresses, the composite scaffold continuously releases active ingredients to regulate the polarization direction of macrophages, promoting the transformation of the inflammatory microenvironment to the repair type. 3) In the tissue reconstruction stage, the collagen network with complete biological activity provides structural support for cell migration and matrix deposition, and its degradation products promote angiogenesis and collagen ordered arrangement through endogenous signaling. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a PDA, PDA / Col composite scaffold physical map; wherein, Figure A is PDA nanoparticles, black powder; Figure B is Col and PDA / Col freeze-dried form map.

[0023] Figure 2 It is the particle size detection and Zeta potential determination of PDA; wherein, A is the particle size determination result of PDA; B is the Zeta potential determination result of PDA.

[0024] Figure 3 It is the scanning electron microscopy results of PDA, Col and PDA / Col composite scaffold; wherein, A, D are the scanning electron microscopy results of PDA at different magnifications; B, E are the scanning electron microscopy results of Col at different magnifications; C, F are the scanning electron microscopy results of PDA / Col at different magnifications.

[0025] Figure 4 It is the active structure detection of Col and PDA / Col composite scaffold; wherein, A is the circular dichroism results of Col and PDA / Col composite scaffold; B is the active structure detection kit results of Col and PDA / Col composite scaffold; C is the SDS-PAGE electrophoresis results of Col and PDA / Col composite scaffold.

[0026] Figure 5 It is the infrared scanning spectrum of PDA, Col and PDA / Col composite scaffold and the ultraviolet absorption spectrum results of PDA; wherein, A is the ultraviolet absorption spectrum results of PDA; B is the infrared scanning spectrum results of PDA, Col and PDA / Col composite scaffold.

[0027] Figure 6CCK-8 results of PDA, Col and PDA / Col composite scaffold on different cells; wherein, A is the cell activity results of different cells under different concentrations of Col; B is the cell activity results of different cells under different concentrations of PDA; C is the activity results of different cells when cultured in composite scaffold.

[0028] Figure 7 Collagen and DMEM medium under different concentrations to form a gel.

[0029] Figure 8 Hemolysis test results of PDA, Col and PDA / Col composite scaffold; wherein, A, D are the hemolysis results and quantitative statistics of different concentrations of PDA; B, E are the hemolysis results and quantitative statistics of different concentrations of Col; C, F are the hemolysis results and quantitative statistics of different concentrations of PDA / Col composite scaffold.

[0030] Figure 9 Antioxidant performance test of PDA, Col and PDA / Col composite scaffold; wherein, A, B are the ROS scavenging effect of PDA, Col and PDA / Col composite scaffold and the calculation of scavenging rate; C, D are the quantitative results of PDA, Col and PDA / Col composite scaffold on ABTS free radicals and DPPH free radicals.

[0031] Figure 10 EdU detection experiment results of PDA, Col and PDA / Col composite scaffold; wherein, A is the EdU detection results of L929 cells in each component; B is the corresponding quantitative statistics.

[0032] Figure 11 PDA, Col and PDA / Col composite scaffold promote cell migration experiment results; wherein, A, B are the migration results and quantitative statistics of HUVEC cells under different culture environments; C, D are the Transwell results and quantitative statistics of L929 cells.

[0033] Figure 12 PDA, Col and PDA / Col composite scaffold protein regulation results on HUVEC cells; wherein, A is the Western blot experiment results of HUVEC cells on CD31 and VEGFA; B, C are the quantitative results of CD31 and VEGFA.

[0034] Figure 13 PDA, Col and PDA / Col composite scaffold immunofluorescence results under HUVEC cell high glucose and inflammation model; wherein, A is the cell immunofluorescence results; B is the immunofluorescence quantitative results; C is the cell RT-qPCR results.

[0035] Figure 14 Tube formation and Western blotting of PDA, Col and PDA / Col composite scaffolds in HUVEC cell high glucose and inflammation model; wherein, A is that the composite scaffold can reduce the expression of iNOS in HUVEC cells under high glucose and inflammation environment, and B is the quantitative result thereof; C is the proangiogenic function of the composite scaffold, and D-G are the quantitative results thereof; the red arrow is the poor site of tube lumen formation of HUVEC cells under inflammation and high glucose environment.

[0036] Figure 15 Western blotting for detecting the expression of different proteins in RAW264.7 cell inflammation model; wherein, A and F are Western blotting experiments after using PDA, Col and PDA / Col composite scaffolds to culture RAW264.7 cells for 12h under inflammation model; B-E and G-J are Western blotting experiment band quantification.

[0037] Figure 16 Western blotting results of RAW264.7 and L929 cells after co-culture; wherein, A is the Western blotting results of RAW264.7 and L929 cells after co-culture for 12h, and B-G are the quantitative results thereof.

[0038] Figure 17 Western blotting for detecting the phosphorylation level of different proteins after PDA, Col and PDA / Col composite scaffolds culture RAW264.7 cell inflammation model for 12h; wherein, A and F are Western blotting results; B-E and G-J are Western blotting experiment band quantification, respectively.

[0039] Figure 18 BMDM inflammation model immunofluorescence, flow cytometry and RT-qPCR experiment results; wherein, A is BMDM immunocytochemistry, and B-C are the quantitative results thereof; D is flow cytometry for detecting the expression of FITC-CD86 and APC-CD206 in BMDM cells; E-I are RT-qPCR for detecting the mRNA expression in BMDM cells.

[0040] Figure 19 Wound healing of mice for 14 days; wherein, A is the evolution of wound healing within 14d; B is the quantitative analysis of wound healing process for more than 14d.

[0041] Figure 20 Protein detection of wound tissue of mice for 7 days; wherein, A is Western blotting experiment; B-D are the protein expression quantification of iNOS, TNF-α and α-SMA, respectively.

[0042] Figure 21 HE, Masson staining was performed on day 21 to observe the degree of skin structure recovery.

[0043] Figure 22 Immunohistochemical analysis of the tissue recovery state of the wound closure site; wherein A is the immunohistochemical staining of each group on day 21; B-D are the quantification of immunohistochemical α-SMA, COL1A1, and COL3A1, respectively. DETAILED DESCRIPTION

[0044] For the convenience of those skilled in the art, the following will be combined with the examples and the accompanying drawings to facilitate the understanding of the present application. Figures 1 to 22 The present application is further described, and the content mentioned in the embodiments is not a limitation of the present application.

[0045] Example 1 Preparation and characterization of PDA / Col composite scaffold

[0046] I. Experimental method

[0047] 1.1 Preparation of PDA / Col composite scaffold

[0048] First, 40 mL of anhydrous ethanol was added to 90 mL of deionized water, and then 2 mL of ammonia was added dropwise, and the solution was stirred at room temperature for 10 min to obtain solution A. Then 500 mg of dopamine hydrochloride was dissolved in 10 mL of deionized water to obtain solution B, and then solution B was added to A, and stirred at room temperature for 12 h to obtain crude polydopamine. Then, the crude polydopamine was centrifuged at 2000 rpm for 5 min to remove insoluble impurities or large particle materials, and then centrifuged at 11000 rpm for 15 min to collect the precipitate. Then, the precipitate was washed with 30% ethanol first, and then with deionized water, and after washing, the precipitate was collected and freeze-dried to obtain purified polydopamine. Then, 1 mg of polydopamine was added to 100 mL of 0.1 mg / mL collagen solution, the pH was adjusted to 6.0, and the mixture was stirred at room temperature for 12 h, and then freeze-dried to obtain a polydopamine and collagen (PDA / Col) composite scaffold.

[0049] 1.2 Measurement of PDA particle size and zeta potential

[0050] The particle size distribution of PDA was measured using a HORIBA nanoparticle measurement instrument, and the specific steps were as follows: 0.1 mg / mL polydopamine aqueous solution was prepared using the above obtained polydopamine freeze-dried powder, and after being fully mixed and ultrasonicated for 10 minutes, it was added to a four-side light-transmitting disposable cuvette. Before measurement, the instrument was adjusted with water as the dispersion system, polydopamine as the test sample, and the measurement number n = 5. Similarly, the potential cuvette was used to select the Zeta potential detection mode to detect the potential of PDA, and the measurement number n = 5.

[0051] 1.3. SEM observation of PDA morphology

[0052] Make sure the sample is completely dry to avoid interference from volatiles under vacuum. Coat the PDA on a conductive substrate and sputter a 3-5 nm layer of gold or platinum on the PDA surface using an ion sputter to reduce the charging effect. Higher resolution observation suggests a thinner layer of platinum / palladium coating. Securely attach the sample to the sample holder with conductive glue to avoid charge accumulation. Set the acceleration voltage to 5-10 kV to reduce the damage of the electron beam to the PDA (polymers are prone to decomposition). Adjust the working distance to 5-10 mm to balance the depth of field and resolution. Position the target area under low magnification (500-2,000x) and gradually zoom in to 10,000-50,000x to observe the surface details. If abnormal bright lines or distortions appear in the image, re-coat the sample or reduce the acceleration voltage. Wear gloves throughout the sample handling process to avoid grease contamination.

[0053] 1.4. Detection of active structure of Col and PDA / Col composite scaffold

[0054] Use the collagen active structure detection kit from Dongguan Collagen Biotechnology Co., Ltd. with a collagen concentration of about 0.5 mg / mL. According to the instructions, detect the sample by adding 100 μL of sample solution directly into the detection reagent at room temperature, invert it 5-10 times, and observe whether red flocculent material is produced in the reagent. The presence of red flocculent material indicates that the collagen in the sample has a triple helix structure that maintains its activity, otherwise not.

[0055] Use a circular dichroism spectrometer to detect whether the collagen in the sample has a triple helix structure that maintains its activity. Dissolve the collagen in a weakly acidic buffer (pH 4.0) or a low ionic strength solution (avoid phosphate, as it has strong far-UV absorption). The final concentration is about 0.1-0.2 mg / mL. Centrifuge (4°C, 12,000 rpm, 10 minutes) to remove insoluble aggregates. Use a quartz cuvette with an optical path of 0.1 cm or 0.2 cm. Rinse with ultrapure water before adding the sample to avoid air bubbles. Scan the baseline (wavelength range 170-250 nm) with pure buffer (same conditions as the sample) to remove background interference. Inject the collagen solution into the cuvette, avoiding air bubbles. Scan 3 times to take the average and reduce noise, and if necessary, pass nitrogen gas to remove oxygen interference.

[0056] 1.5. Determination of molecular weight and UV absorption of Col and PDA / Col composite scaffold

[0057] Molecular weight detection was analyzed by SDS-PAGE electrophoresis method, and the electrophoresis device used Bio-Rad company's supporting tools (power supply for basic version). According to the volume ratio of 4:1, the sample was mixed with 5x SDS loading buffer (containing 2% SDS, 5% β-mercaptoethanol), and the final concentration of protein was 1 mg / mL, 100 ℃ heating for 10 min (collagen has high thermal stability, 10 min to ensure sufficient denaturation). Tris-glycine electrophoresis formula was used: glycine 18.77 g, Tris 3.03 g, SDS 1.0 g, prepared into 1 L solution. 8% PAGE gel and protein marker were from Shanghai Yezheng Biomedicine Technology Co., Ltd., and the whole electrophoresis was 80 V. After the marker between 100-250 kDa was separated, the electrophoresis was stopped. Coomassie brilliant blue R-250 solution was stained at room temperature for 2 h, and then decolorized until the gel was nearly transparent, and the blue band was clearly visible. Photograph was recorded under white background, and the band position of each sample was observed.

[0058] Using ultraviolet visible spectrophotometer, different concentrations of PDA water dispersion (about 3.2-3.5 mL) were added into constant quartz cuvette, the wavelength range was 200-800 nm, the scanning interval was 0.5 nm, and 3 groups were detected in parallel to detect the ultraviolet absorption of polydopamine.

[0059] 1.6, Fourier transform infrared spectrometer detects functional groups of sample

[0060] Weigh 1-2 mg of sample and 100-200 mg of dry KBr powder in a marcasite mortar and grind it into fine powder. Lightly press the powder with the convex surface of the stainless steel spoon to roughly judge the grinding degree. When the powder is smooth and delicate, it can be pressed into a tablet. Ensure that the sample is completely dry, or dry it completely during the grinding process. The scanning range of the instrument is 4000-400 cm -1 (mid-infrared region), the resolution is set to 4 cm -1 , and the scanning times are 8.

[0061] 1.7, data statistics and analysis

[0062] The above data were statistically analyzed using Origin2024 software, each group had at least 3 parallel results, and part (such as ultraviolet absorption photometric determination results and infrared spectrum) selected one of them as display. Significant difference p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0063] II. Experimental results

[0064] 2.1, preparation of PDA / Col composite scaffold

[0065] According to the above experimental method, as Figure 1A, according to the color and powder state, and combined with existing reference data, it is preliminarily determined that it is PDA. Further combined with collagen as a composite scaffold, the pure white collagen freeze-dried sponge becomes light gray by adjusting the pH, as shown in Figure 1 B, it can be seen that the collagen has loaded polydopamine nanoparticles.

[0066] 2.2, PDA particle size, zeta potential and scanning electron microscope

[0067] The particle size of polydopamine (PDA) nanoparticles was determined by DLS method combined with scanning electron microscope, and the results are shown in Figure 2 and Figure 3 According to the above method, the particle size of PDA nanoparticles is about 80-180 nm, and the zeta potential is about -70 mV.

[0068] 2.3, activity structure and molecular weight detection of Col and PDA / Col composite scaffold

[0069] As shown in Figure 4 A, the circular dichroism spectrum shows that the test sample has a negative peak at 198 nm and a positive peak at about 220 nm, which meets the requirements of the standard spectrum of collagen circular dichroism. As shown in Figure 4 B, the collagen detection kit shows that red flocculent matter appears before and after the combination of collagen and polydopamine, indicating that the triple helix structure of collagen is relatively stable before and after the combination of collagen and polydopamine, which is also the active structure of collagen. As shown in Figure 4 C, after the combination of collagen and polydopamine, the molecular weight increases, indicating that the combination of Col and PDA is relatively stable and can be detected by electrophoresis without breaking.

[0070] 2.4, UV absorption and main functional groups of Col and PDA / Col composite scaffold

[0071] As shown in Figure 5 A, when the polydopamine aqueous dispersion is detected by ultraviolet-visible absorption spectrum (UV-Vis), the detection result shows that polydopamine has full wavelength absorption within 200-800 nm wavelength, and there is no characteristic absorption peak, which shows that ultraviolet detection is not suitable for the quantification of polydopamine.

[0072] As shown in Figure 5 B, analysis of infrared spectrum results, for polydopamine, in the range of 3300-3500 cm -1 : N-H / O-H stretching vibration (amino, phenolic hydroxyl and adsorbed water). 1600-1650 cm -1 : aromatic ring C=C stretching vibration (benzene ring conjugated structure). 1510-1530 cm -1: N-H bending vibration (secondary amine or indole structure). 1280-1300 cm -1 : C-O stretching vibration (phenolic hydroxyl group). 800-820 cm -1 : Aromatic ring C-H out-of-plane bending vibration (ortho-substituted benzene ring). After dopamine polymerization, 1600 cm -1 and 1510 cm -1 are the characteristic signals of PDA. However, the 1600-1650 cm -1 peak broadens, possibly due to cross-linking leading to structural disorder. Compared with dopamine (DA), the polydopamine has a higher ratio of the 1600 cm -1 to 1510 cm -1 peak intensity, indicating a more perfect conjugated aromatic ring structure.

[0073] For the infrared spectral analysis of collagen, 3300 cm -1 : N-H stretching vibration (amide A band, overlapped with O-H). 3080 cm -1 : Amide B band (N-H stretching vibration). 1650 cm -1 : Amide I band (C=O stretching vibration, alpha-helix structure). 1550 cm -1 : Amide II band (N-H bending vibration and C-N stretching vibration). 1240 cm -1 : Amide III band (C-N stretching and N-H bending). 1450 cm -1 : CH2 / CH3 bending vibration (glycine, proline side chain). The peak position and ratio of the amide I band (1650 cm -1 ) and the amide II band (1550 cm -1 ) can reflect the integrity of the collagen triple helix structure.

[0074] New peaks appear: 1700-1750 cm -1 : Possibly due to the enhancement of ester bond (C=O) or quinone-like structure. 1220-1260 cm -1 : C-N stretching vibration enhancement (Schiff base product).

[0075] Example 2 In vitro test of PDA / Col composite scaffold

[0076] I. Experimental methods

[0077] 1.1. Cell culture and macrophage polarization induction

[0078] The main research subjects of this study are L929, RAW264.7, HUVEC, HaCaT and BMDM cells. Generally, L929, RAW264.7 and HaCaT cells are cultured in high glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S), HUVEC cells are cultured in endothelial cell medium (ECM) containing 5% fetal bovine serum, 1% endothelial cell growth supplement (ECGS) and 1% penicillin / streptomycin. BMDM cells are cultured in high glucose DMEM medium containing 15% L929 cell supernatant, 10% FBS and 1% P / S.

[0079] Cell recovery: Pre-warm the culture medium, trypsin, etc. in a 37°C water bath. Take the cryopreservation tube from the liquid nitrogen tank or -80°C refrigerator and immediately place it in a 37°C water bath. Shake gently until the ice crystals are almost completely melted (about 1.5-2 minutes). Avoid long water bath to prevent DMSO toxicity. Transfer the cell suspension to a 10 mL centrifuge tube containing 5 mL of cold culture medium, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Some cells (such as primary cells HUVEC or BMDM) can be directly inoculated without centrifugation (to avoid mechanical damage). Gently resuspend the cells with 2-3 mL of pre-warmed complete medium and transfer to a T25 culture flask, add medium to 5 mL, and recommend inoculation at the same density as before freezing. Place in a 37°C, 5% CO2 incubator, and change the medium for the first time after 24 hours to remove dead cells and residual DMSO. And observe the cell adhesion and normal morphology, the adherent cells should stretch out, with no excessive debris. Confirm that the cell density reaches 80-90% confluence, with good condition, no vacuoles, and debris. Cell passage and cell experiment can be performed.

[0080] Cell passage and cell cryopreservation: Pre-warm PBS, trypsin (0.25% Trypsin-EDTA), complete medium. Discard the old culture medium, add 3 mL of PBS for gentle washing once, and remove the serum that inhibits trypsin activity. Add 1 mL of trypsin (T25 culture flask), gently shake to cover the cell layer with liquid, and incubate at 37°C. When the cell gap increases and the edge curls, terminate immediately, add 2 mL of complete medium (containing FBS), blow the cell layer to completely detach, and transfer to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant. Add an appropriate amount of fresh culture medium and gently blow to form a single cell suspension. Adjust according to the cell type, inoculate the cell suspension into a new culture dish in proportion, and add medium to the standard volume (such as 5 mL for a T25 flask). At the same time, record the passage number to avoid using high passage cells, generally primary cells ≤5 passages, and immortalized cells ≤20 passages.

[0081] When the cells are frozen, the freezing solution (90% FBS + 10% DMSO) or the commercial freezing solution is prepared. Note that after the cells are centrifuged and collected, the freezing solution is used for resuspension. The cell suspension is aliquoted into a freezing tube, and the procedure is cooled (4°C for 30 min→-20°C for 2 h→-80°C overnight→long-term storage in liquid nitrogen).

[0082] Induction of macrophage polarization: After normal culture of RAW264.7 cells, 300,000 cells were seeded in a 6-well plate. After adhesion, 100 ng / mL of LPS and 20 ng / mL of IFN-γ were added to the M1 pro-inflammatory phenotype group, 20 ng / mL of IL-4 and the same dose of IL-13 were added to the M2 anti-inflammatory phenotype group, and the experimental group was collagen, polydopamine, and their complexes. The blank control group was added with the same volume of PBS. Incubation was performed at 37°C, 5% CO2 for 12 h.

[0083] 1.2, Extraction and induction of bone marrow-derived macrophages

[0084] 6-8 week C57BL / J male mice were sacrificed by cervical dislocation and soaked in 75% ethanol for 5 minutes. Then the hind limbs were removed, the muscles were removed, the femur was soaked in 75% ethanol for 5 minutes, and the cold PBS was used for washing. After cutting off both ends, the bone marrow was blown and washed with BMDM induction medium (high-sugar DMEM medium containing 15% L929 supernatant and 10% FBS) until there was no red material in the cavity. Then filter with a 70 μm cell filter, centrifuge at 1500 rpm for 3 minutes to collect the precipitate, and then perform red blood cell lysis. The precipitate was collected by centrifugation again, and then washed once with PBS. Then, the complete medium was directly counted and plated for culture, which can be directly used for subsequent experiments.

[0085] When the BMDM cells are polarized, the method is similar to that of RAW264.7, except that the dose of IL-4 and IL-13 added during M2 phenotype induction is 10 ng / mL.

[0086] 1.3, CCK-8 method for detecting the effect of samples on cell activity

[0087] L929, RAW264.7, HUVEC, and HaCaT cells were cultured under normal conditions, and the cells were seeded in a 96-well plate the night before, with about 6000 cells per well. The next day, the culture medium in the wells was removed, and complete culture medium containing the sample to be tested was added as the experimental group, and complete culture medium without the sample to be tested was added as the control group. After incubation for 24 h, the culture medium was removed, serum-free medium containing CCK-8 reagent was added to all cell culture wells, and another group of cell-free culture wells was selected as a blank group. Incubation was performed for 1 h, and the absorbance at 450 nm was measured. The data obtained were statistically analyzed using Graphpad Prism 9.5, and the cell activity was calculated. The calculation formula is:

[0088]

[0089] Wherein, Ae is the experimental group, Ac is the control group, and Ab is the blank group.

[0090] 1.4, Hemolysis experiment of Col and PDA

[0091] The blood of New Zealand rabbits was collected in an anticoagulant tube, defibered, and centrifuged at 3500 rpm for 15 minutes. Then, the red blood cells were washed and purified by using normal saline or PBS as a washing liquid, centrifuged at 3500 rpm for 5 minutes, and finally prepared into a 4% red blood cell solution. Then, a 2% Triton X-100 solution and a 2-fold concentration sample solution to be tested were prepared using the same washing liquid. Subsequently, the red blood cell solution was mixed with normal saline / PBS (negative control group), 2% Triton X-100 solution (positive control group), or sample solution to be tested (experimental group) at a ratio of 1:1, incubated in a water bath at 37°C for 1 hour, and finally centrifuged at 1200 rpm for 3 minutes. The supernatant was aspirated to determine the absorbance at 450 nm, and the hemolysis rate was calculated. The calculation formula is as follows:

[0092]

[0093] Wherein, Ax is the absorbance of the experimental group; A0 is the absorbance of the negative control group; and As is the absorbance of the positive control group.

[0094] 1.5, Determination of the effect of the composite scaffold on cell proliferation by EdU method

[0095] L929 cells were used, and the cells were cultured in a well plate to about 70%-80%. The experimental group was added with a drug-containing basic culture medium, and the control group was simply added with a basic culture medium, and cultured for 2 hours. Then, EdU reagent (20 μM) was added for incubation for 6 hours. The culture medium was removed, 1 mL of PBS containing 4% paraformaldehyde was added to each well, and after incubation at room temperature for 15 minutes, the PBS containing 4% paraformaldehyde was removed. 1 mL of PBS containing 3% BSA was added to each well, and washed thoroughly for 3 times, each time for 5 minutes. The supernatant was discarded, and 1 mL of PBS containing 0.3% Triton X-100 was added to each well, and incubated at room temperature for 20 minutes. The supernatant was discarded, and then washed. The Click reaction liquid was prepared according to the number of samples and the instructions of the reagent. The supernatant was discarded, 500 μL of the Click reaction liquid prepared in the above table was added to each well, and gently shaken to ensure that the Click reaction liquid uniformly covered the cells, and incubated at room temperature for 30 minutes in the dark. The supernatant was discarded, and then washed. 500 μL of the working solution of DAPl was added to each well, and incubated at room temperature for 5-10 minutes in the dark. After washing, a confocal microscope was used to observe and record the photographs.

[0096] 1.6, Investigation of the effect of the composite scaffold on cell migration by scratch test and Transwell test

[0097] Scratch assay: Scratch assay is usually performed in 6-well plate, so cells are seeded in 6-well plate. Before scratch, cells must be grown to confluence, the visual standard is: no obvious gap between cells, and cells are in good condition under 40X field. The number of cells seeded is 200-250K per well for L929 cells, and 200μL tip is chosen for scratch, which is moderate in width. After scratch, each well is washed with 2mL PBS for 3 times to avoid the floating cells adhering to the scratch area. The control group is serum-free medium alone, and the experimental group is the basal medium containing the experimental sample.

[0098] Transwell assay: L929 cells are digested and resuspended in serum-free medium after centrifugation, and the density is adjusted to 1.0x10 5 -5x10 5 Transwell chamber (pore size 8μm) is placed in a 24-well plate, 500μL of basal medium containing experimental sample is added to the lower chamber as the experimental group, and the control group is basal medium without anything, and 200μL of cell suspension resuspended in basal medium is added to the upper chamber. Incubate at 37℃, 5% CO2 incubator for 12h, and wait for cell migration. Remove the chamber, gently rinse with PBS, and wipe the surface of the upper chamber with a cotton swab to remove the cells that have not migrated. Methanol or paraformaldehyde is used to fix the lower chamber membrane for 30 minutes. Stain with 0.1% crystal violet (prepared with 20% ethanol) for 30 minutes, rinse with PBS several times, and air dry. Place the membrane on a glass slide and count the migrated cells under a microscope.

[0099] 1.7, ROS scavenging and free radical scavenging rate

[0100] Accurately weigh 6.25mg ABTS and 2.82mg K2S2O8, respectively dissolved in 4mL deionized water to prepare stock solutions. Then mix them 1:1, slowly shake at room temperature for 6h in the dark, and get a solution rich in nitrogen radicals. Adjust the absorbance of the solution to 0.8, and use it as the final concentration to determine the test sample. ABTS solution volume: test sample volume = 200μL:20μL, incubate at room temperature for 10 minutes, and measure the absorbance at 734nm. Similarly, prepare a DPPH solution of 50μg / mL, and perform the experiment according to the above steps, except that the incubation time is extended to 30 minutes, and the absorbance at 519nm is measured.

[0101] In addition, L929 cells were incubated with ROS inducers (diluted 1000 times to reach a final concentration of 882 μM H2O2 when using 3% H2O2) in a carbon dioxide incubator for 6 h, then the test substance (PDA, Col and its complexes) was added for 2 h, and washed with PBS for 3 times. Then DCFH-DA was used to capture the generation of ROS, incubated in the incubator for 30 min, washed with PBS for 3 times, and the change of fluorescence intensity was detected by live cell imaging instrument; by comparing the signal difference between the experimental group and the control group, the ROS clearance rate was calculated. In the experiment, a blank control (without inducer) was set, and repeated three times to ensure the reliability of the data. The experimental results were quantified using ImageJ, and the obtained data were statistically analyzed using Graphpad Prism 9.5.

[0102] 1.8, Vessel formation experiment

[0103] The 48-well plate and the required consumables such as suction heads were pre-frozen in a -20°C environment 12 h in advance, the Matrigel was thawed in advance, diluted 1:1 with DMEM (high sugar) medium, and stored at 4°C. The next day, 100 μL of Matrigel per well was added to the pre-frozen 48-well plate, then placed in the incubator for 1 h, and after the Matrigel was completely solidified, the pre-configured HUVEC cells with or without the test drug were carefully added to the Matrigel-coated wells, and after 10 h, the tube formation of endothelial cells was recorded. ImageJ was used for quantification, and the obtained data were statistically analyzed using Graphpad Prism 9.5.

[0104] 1.9, Total RNA extraction and real-time fluorescent quantitative PCR

[0105] Total RNA extraction: the cells treated with drugs were first removed from the original culture medium, then washed with DPBS for 3 times, and then lysed. 1 mL of Trizol reagent was added to each well of a 6-well plate and placed on ice for 5-10 min. The lysed Trizol reagent was collected in a 1.5 mL centrifuge tube, 200 μL of chloroform was added, and the mixture was vortexed quickly until the liquid was milky white. It was placed on ice for 10 min. Then centrifuged at 4°C, 12000 rpm x 15 min, and a new 1.5 mL centrifuge tube was prepared and inserted into the ice for pre-cooling.

[0106] After centrifugation, carefully collect 300 μL of the upper clear liquid, transfer to a clean pre-cooled 1.5 mL centrifuge tube, and add 1 mL of isopropanol, manually invert up and down, mix thoroughly, place on ice for 5 min, and centrifuge at 4°C, 12000 rpm x 10 min. After centrifugation, the RNA is a grayish-white small amount of precipitate, carefully pour out the isopropanol inside, then pour it upside down on a flat paper for 30-60 s to let the isopropanol inside flow out as much as possible. Add 1 mL of freshly prepared 75% ethanol (absolute ethanol + DEPC water), mix well by inverting up and down, and place on ice for 5 min. Centrifuge at 4°C, 7500 rpm x 5 min, and after centrifugation, get the dandruff-like precipitate, carefully pour out the ethanol inside, then pour the centrifuge tube upside down on the paper for 5 min and dry naturally.

[0107] Get clean precipitate, add 30-50 μL of DEPC water (or use TAE buffer) to reconstitute the RNA, mix and place on ice, and then determine the concentration and purity. The concentration of RNA is determined using NanoUV, and the purity is determined using agarose electrophoresis. After determining the concentration, generally first transfer a portion of the RNA to a new 1.5 mL centrifuge tube for experiments, and the rest is stored at -80°C. The experimental RNA is adjusted to 100 ng / μL.

[0108] Reverse transcription and qPCR: Place the prepared 200 μL centrifuge tube on ice to pre-cool, and follow the reverse transcription kit procedure. Generally use 10 μL or 20 μL reaction system, and usually add 500 ng-1 μg of RNA.

[0109] After the reaction, cDNA is obtained, which should be stored on ice, and the concentration is determined. The concentration of cDNA after reverse transcription of 500 μg of RNA is generally about 800 ng / μL. According to the need, generally first perform a 10-fold preliminary dilution to obtain cDNA of 80 ng / μL. If the subsequent test is not in time, the cDNA can be stored in a -20°C refrigerator. The reaction system is generally 20 μL, and the cDNA is prepared to a concentration of 10 ng / μL, and the primer pair is prepared to a concentration of 2.5 μM.

[0110] Table 1 Reverse transcription reaction conditions

[0111]

[0112] Table 2 Real-time fluorescent quantitative PCR reagent preparation

[0113]

[0114] Table 3 Real-time fluorescent quantitative PCR reaction conditions

[0115]

[0116] Table 4 PCR primer information

[0117]

[0118] 1.10, Western blot (WB)

[0119] The cultured cells were removed, the culture medium in the well plate was aspirated, and cold PBS was washed twice. Then, 1 mL of PBS was added to moisten the cells, and the cells were scraped as much as possible using a cell scraper. The scraped cells were transferred to a 1.5 mL EP tube (pre-cooled) and centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and 100 μL of pre-prepared lysis solution containing protease inhibitors was added. The mixture was thoroughly mixed and lysed on ice for 30-60 min. Centrifugation was performed at 12000 rpm for 15 min, and the supernatant was transferred to a new pre-cooled EP tube. Protein quantification was performed to adjust the protein concentration to 2-5 mg / mL. According to the molecular weight of the target protein, the appropriate separation gel was selected for electrophoretic protein separation. Typically, 80 V was applied for 120 min, and the recommended loading amount was 10-20 μg with a volume of 5-10 μL.

[0120] 1.11, Immunocytochemistry (ICC)

[0121] The cells in this experiment were directly seeded in glass-bottomed culture dishes and treated with drugs. After obtaining the cells, they were fixed with 4% paraformaldehyde solution for 10 minutes, and then permeabilized with 0.3% Triton X-100 in PBS solution at room temperature for 5 minutes. The cells were blocked with 5% goat serum in PBS or commercial immunological blocking solution for 1 hour. After washing, the primary antibody was incubated overnight at 4°C, and then the corresponding fluorescent secondary antibody was incubated at room temperature for 2 hours. DAPI / Hoechst staining was used to identify the cell nucleus, and then confocal microscopy was used for observation and recording. ImageJ was used for quantification. The calculation formula is:

[0122]

[0123] wherein Fx is the protein quantification value of the experimental group, F0 is the protein quantification value of the blank control group, and Fs is the protein quantification value of the positive control group.

[0124] 1.12, Co-cultivation

[0125] RAW264.7 and L929 cells were cultured to 80% confluence, and the logarithmic growth phase cells were taken after passage. The experiment was performed using a 6-well plate, RAW264.7 cells (3x105 / well) were inoculated in the upper chamber, and L929 cells (3x105 / well) were inoculated in the lower chamber (basal chamber), the chamber aperture was 0.4 μm, and the material was PET. According to the induction scheme described above, the cells were cultured for 12 h, and then Western blotting was performed.

[0126] 1.13, Flow cytometry

[0127] RAW264.7 cells were cultured, polarization induction and composite scaffold were added, and incubated for 12 h, and then cell staining was performed. The proteins labeled in this experiment were CD86 and CD206, and F4 / 80 was used as a macrophage screening. The specific steps are as follows:

[0128] The cells were taken out of the incubator, the culture medium was removed, and the cells were gently washed twice with pre-cooled PBS. The cells were digested with 0.25% trypsin (containing EDTA), and the digestion was terminated with serum-containing medium, and the cells were collected by centrifugation (1200 rpm, 3 min).

[0129] The cells were resuspended with pre-cooled flow buffer, the cell concentration was adjusted to 1x106 / mL, and the subsequent antibodies and dyes used were also flow buffer. Dead and live cell dye was added, and incubated at room temperature for 30 min in the dark. Centrifugation and washing twice.

[0130] The antibodies were prepared in advance according to the instructions, and then the membrane proteins CD86 and F4 / 80 antibodies were added, and incubated in the dark (4°C) for 30 min. Centrifugation and washing twice, resuspended in 4% paraformaldehyde, and fixed at room temperature for 15 min.

[0131] The fixing solution was removed by centrifugation, and washed once. The cells were resuspended with 1% Triton X-100, and incubated at room temperature for 30 min in the dark. After centrifugation, the intramembrane antibody CD206 was added, and incubated at 4°C for 1 h in the dark. Centrifugation and washing twice, resuspended in flow buffer.

[0132] II. Experimental results

[0133] 2.1, Effect of PDA / Col composite scaffold on cell activity

[0134] The CCK-8 experimental results are shown in Figure 6 When the collagen concentration was increased from 1.0 mg / mL, the activity of the cells began to be inhibited. It was found that high concentration (>1.5 mg / mL) of collagen and cell culture medium could form a weak colloidal state in a 37°C incubator. When the concentration reached 2 mg / mL, the collagen and the culture medium could form a more obvious gel state Figure 7). While the collagen concentration within 1 mg / mL has little effect on the cell activity of the four cells studied.

[0135] When the collagen concentration is 0.1 mg / mL, the cell activity is close to 100%. When the collagen concentration is higher than 0.1 mg / mL, the cell activity appears to be slightly reduced. It should be noted that with the increase of concentration, the activity is reduced, but not obvious. Therefore, according to the purpose of the experiment, in order to prevent the formation of collagen and culture medium gel from affecting cell activity, leading to inaccurate experimental results, this study selects 0.1 mg / mL as the concentration of collagen in subsequent experiments. For polydopamine, when the concentration is 50 μg / mL, the activity of the cells is inhibited, and the activity is reduced to about 75%. Continue to increase the concentration of polydopamine to 100 μg / mL, the cell activity inhibition rate will reach 50%. The results suggest that the safe concentration of polydopamine in subsequent experiments will be 50 μg / mL or less. In order to ensure that the cell activity is not inhibited by the composite scaffold, the concentration of polydopamine in this study is finally selected as 10 μg / mL.

[0136] Finally, this study determines the final theoretical experimental concentration of PDA / Col 0.11 mg / mL. Again, through cell activity detection, it is proved that the polydopamine and collagen composite scaffold of 0.11 mg / mL is the most friendly to cells.

[0137] 2.2, Hemolysis rate of PDA / Col composite scaffold

[0138] As shown in Figure 8 , this experiment explores the hemolysis rate of different concentrations of collagen and polydopamine. The results show that collagen and polydopamine exhibit extremely low hemolysis rate, even if the concentration is increased to 100 times the original concentration, there is no obvious hemolysis phenomenon. Only polydopamine will cause polydopamine to disperse in the supernatant after centrifugation after increasing the concentration, which will deepen the color of the liquid. However, when the polydopamine dispersion liquid with the same concentration is adjusted as the background, the absorbance is close to the negative control group. Therefore, it can be considered that the polydopamine and collagen (PDA / Col) composite scaffold is safe.

[0139] 2.3, Antioxidant capacity of PDA / Col composite scaffold

[0140] As shown in Figure 9As shown, by detecting the ROS, ABTS and DPPH scavenging rates of PDA / Col, it was found that the composite scaffold had the ability to target free radicals and inhibit cell oxidative stress. Collagen could remove about 50% of ROS in cells, but in the in vitro ABTS and DPPH radical scavenging test, it did not perform as expected. Therefore, this study believes that this antioxidant function is mainly exerted by polydopamine. Previous studies have shown that polydopamine has excellent antioxidant capacity, thus ensuring the effectiveness and reliability of PDA / Col composite scaffold in anti-inflammatory and promoting tissue regeneration applications.

[0141] 2.4, PDA / Col composite scaffold can promote cell proliferation

[0142] Cell proliferation experiments (EdU) can evaluate the effect of PDA / Col composite scaffold on the growth ability of key cells for wound healing, such as fibroblasts, keratinocytes or endothelial cells. As shown in Figure 10 EdU positive rate is mainly due to the addition of Col, indicating that the cell proliferation-promoting effect of the composite scaffold is mainly exerted by Col. Although PDA does not significantly promote cell proliferation, it does not inhibit cell proliferation either.

[0143] 2.5, PDA / Col composite scaffold helps cell migration

[0144] Scratch test can be used to evaluate cell migration ability, as shown in Figure 11 A-11B, under normal circumstances, collagen promotes HUVEC cell migration, while PDA has no significant effect. Transwell test analyzes the number of cells penetrating the porous membrane, however, as shown in Figure 11 C-11D, low concentration of PDA can also promote cell migration in L929 cells.

[0145] 2.6, PDA / Col composite scaffold is conducive to the expression of CD31 and VEGFA protein in HUVEC cells

[0146] One of the core mechanisms of diabetic wound difficulty is the angiogenesis disorder caused by high glucose environment, which is manifested as impaired endothelial cell function, down-regulation of pro-angiogenic factors (such as VEGF, CD31, bFGF) expression and abnormal increase of anti-angiogenic factors (such as Angiopoietin-2), thus triggering local ischemia and hypoxia and blocking nutrient transport. As shown in Figure 12 Under normal conditions, collagen can promote the expression of CD31 and VEGFA in HUVEC cells, while polydopamine has no significant effect.

[0147] 2.7, PDA / Col composite scaffold can up-regulate CD31 and VEGFA protein of HUVEC cells under high glucose and inflammatory environment, while reducing iNOs cytokines, which is conducive to the formation of new blood vessels

[0148] As Figure 13 shown, although polydopamine does not promote HUVEC cell viability under physiological conditions. However, surprisingly, when the cells are in an inflammatory environment, polydopamine can reduce the expression of iNOS in HUVEC cells, alleviate the oxidative stress state of the cells, and the expression of CD31 is also increased. HUVEC cells with alleviated oxidative stress can better enjoy the effect of collagen. This may be a manifestation of the synergistic effect of the two components of the composite scaffold.

[0149] The above results show that under normal physiological conditions, polydopamine may neither inhibit nor promote cell activity. However, when the cells are in an inflammatory or oxidative stress state, polydopamine will regulate the inflammatory response of the cells. In summary, PDA / Col makes HUVEC cells under inflammatory and high glucose environment more prone to form blood vessels Figure 14 ).

[0150] 2.8, PDA / Col composite scaffold reduces the expression of inflammatory factors of RAW264.7 cells by interfering with the inflammatory factor receptors TNFR1 and TLR4

[0151] The mechanism of polydopamine and collagen composite scaffold in treating LPS (lipopolysaccharide) induced polarized macrophages plays an anti-inflammatory role involving multiple levels of immune regulation and signal pathway intervention. As Figure 15 shown, through Western blot experiment verification, the composite scaffold can significantly reduce the level of TNF-α in the culture supernatant of macrophages stimulated by LPS, down-regulate the expression of inflammatory factors and p38MAPK phosphorylation, and at the same time enhance the expression of CD206 anti-inflammatory cytokines, and finally realize the functional conversion from "inflammation inhibition" to "repair promotion".

[0152] 2.9, cell co-culture

[0153] In order to investigate the regulation of macrophages on fibroblasts during inflammation, macrophages and fibroblasts were also co-cultured. The results are as follows Figure 16As shown, the expression of COL1A2 and COL3A1 of L929 cells was up-regulated after using PDA / Col composite scaffold under inflammatory environment. However, the effect was not ideal when using collagen alone. This indicates that the repair effect of collagen may not be good when there is no continuous inflammation in ordinary wounds, but if there is continuous inflammation, it needs to be used with anti-inflammatory substances to achieve good results. At the same time, fibroblasts also highly express MMP-1 protein under inflammatory environment, and Col, PDA and PDA / Col groups can effectively reduce its expression under inflammatory environment.

[0154] In order to continue to explore how PDA / Col composite scaffold plays an anti-inflammatory role, LPS and IFN-γ induced RAW264.7 cells and BMDM cells were used as inflammatory cell models in this study. LPS activates MyD88 and TRIF pathways through TLR4, drives transcription factors such as NF-κB, AP-1 and IRF3, and induces the expression of pro-inflammatory mediators (such as iNOS, CD86, TNF-α) and effector molecules. At the same time, remodeling metabolism (increased expression of HIF-1α, even under aerobic conditions, it turns to glycolysis for energy, supporting rapid energy demand and pro-inflammatory function) and epigenetic state, eventually polarize macrophages into M1 phenotype with antibacterial and pro-inflammatory functions. While IFN-γ often works with LPS to enhance M1 polarization through STAT1. IL-4 and IL-13, on the other hand, act as signaling molecules to induce macrophages to polarize to M2, an anti-inflammatory phenotype.

[0155] In the pro-inflammatory signal receiving stage, unlike collagen, polydopamine can reduce the expression level of TNF-R1 and TLR4 Figure 15 ), so that the sensitivity of macrophages to inflammatory cell mediators such as LPS and TNF-α is reduced, thereby reducing the M1 phenotype of macrophages. Among them, TLR4 can be specifically reduced by polydopamine, while collagen can reduce MYD88. In general, PDA / Col plays a synergistic role in inhibiting the TLR4 / MYD88 signaling pathway. Compared with collagen, polydopamine has more advantages in reducing pro-inflammatory factors such as TNF-α, iNOS, TNF-R1 and HIF-1α. However, there is no big difference between the two in the regulation of anti-inflammatory factors.

[0156] 2.10, PDA / Col composite scaffold can inhibit MAPK / NF-κB signaling pathway phosphorylation

[0157] PDA / Col composite scaffold plays a key role in diabetic wound healing by regulating MAPK / NF-κB and TLR4 / MyD88 two major inflammation-related signaling pathways. This study continues to explore how PDA / Col composite scaffold plays an anti-inflammatory role. For example Figure 17As shown, after research, it was found that polydopamine can inhibit the phosphorylation of P38, JNK1 / 2 / 3 and P65, and promote the phosphorylation of ERK1 / 2. Collagen can basically achieve similar results, the difference lies in that its regulation of P38 is not ideal. P38 factor is a master of regulating oxidative stress, cycle arrest and secretion of inflammatory factors. ERK1 / 2 is mainly to regulate cell proliferation, differentiation and activation of NF-κB.

[0158] Therefore, from a certain point of view, polydopamine can promote the proliferation and differentiation of cells in an inflammatory environment. This result echoes the previous conclusion and gives an explanation. The role of JNK is to promote AP-1 into the nucleus and promote the secretion of inflammatory factors to regulate inflammation. IKBα is an anti-inflammatory factor that binds P65 / P50 and inhibits its phosphorylation. Experiments show that at the experimental concentration, the regulation of the above factors by the two is basically equivalent. Therefore, the two have a synergistic effect in anti-inflammation.

[0159] It was found that there was no significant difference in the anti-inflammatory effect of PDA with a concentration of 10 μg / mL. For the inhibition of TNF-α expression, collagen showed poor performance, and the main effect was still determined by PDA. Figure 16 )。

[0160] This study uses mouse bone marrow-derived macrophages (BMDM) to better observe the macrophage inflammation model. It is found by flow cytometry and immunofluorescence that the anti-inflammatory effect of the PDA / Col composite scaffold is consistent with that described before, mainly from PDA Figure 18 A-18D). However, the results of RT-qPCR detection show that collagen also has considerable anti-inflammatory ability Figure 18 E-18I), and the final protein expression stage deviates. This may be because the collagen chamber has an anti-inflammatory effect and is regulated by multiple pathways.

[0161] Example 3 PDA / Col composite scaffold promotes diabetic wound healing animal experiment

[0162] I. Experimental method

[0163] 1.1, Construction of diabetic wound animal model

[0164] The construction of animal models of diabetic wounds usually selects streptozotocin (STZ)-induced diabetic rats or mice as research objects. Type 1 diabetes mellitus model is established by intraperitoneal injection of STZ (dose of 50-65 mg / kg, for 5 consecutive days) to destroy pancreatic beta cells. Alternatively, combined with high-sugar high-fat diet feeding (for 8 weeks) and low-dose STZ (30-40 mg / kg) to induce type 2 diabetes characteristics; after the fasting blood glucose is stable above 16.7 mmol / L and the typical symptoms of polydipsia, polyuria and weight loss appear, the area of the animal back is shaved and disinfected (commonly using isoflurane inhalation anesthesia) using a sterile biopsy punch or surgical scissors to make a full-thickness skin defect wound (diameter of 8-10 mm, deep to the fascia layer), the wound edge is smeared with iodophor and covered with sterile gauze to prevent infection, and the wound healing condition (digital camera records the wound area change, ImageJ software calculates the healing rate) and blood glucose fluctuation are monitored daily after the operation. To simulate the chronic inflammatory environment of diabetic wounds, inflammation can be further aggravated by local injection of lipopolysaccharide (LPS, 1 μg / μL) or delayed healing treatment (such as repeated mechanical damage of the wound edge); the experimental group is covered with PDA / Col composite scaffold, and the control group is covered with pure collagen scaffold, polydopamine powder gauze and normal saline gauze respectively, and the scaffold is fixed by suturing or biological glue to ensure close contact with the wound.

[0165] Postoperative tissue samples are collected regularly (such as on the 3rd, 7th, 14th, and 21st days) for histopathological analysis (H&E staining to evaluate inflammatory cell infiltration and epithelial regeneration, Masson staining to observe collagen deposition and arrangement), and immunohistochemistry / immunofluorescence to detect macrophage polarization markers, angiogenesis indicators (α-SMA / CD31), and key inflammatory pathway proteins.

[0166] All animal experiments in this study strictly follow the international 3R principle, strictly follow the international animal experiment guideline directive 2010 / 63 / EU and the NIH "Guide for the Care and Use of Laboratory Animals". 8-week-old BALB / C male mice are cultured in the SPF laboratory of the Experimental Animal Center of Guangdong Medical University. The temperature is (22±2℃), the humidity is (50±10%), the light cycle is 12h, and the food and water are freely taken. Isoflurane inhalation anesthesia is used throughout the operation, and ibuprofen is given for postoperative analgesia. The experimental endpoint is cervical dislocation to reduce animal pain.

[0167] 1.2, HE staining

[0168] HE staining procedure mainly includes tissue sample fixation, dehydration and transparency, paraffin embedding, section preparation and staining treatment: first, the collected diabetic wound tissue samples (such as 7, 14 days after operation) are immediately immersed in 4% paraformaldehyde fixing solution (pH 7.4, 4°C for 24-48h) to maintain cell morphology and terminate enzyme activity; after fixation, the sample is rinsed with running water for 12h to remove excess fixative, then sequentially immersed in gradient ethanol (70%→80%→90%→95%→100%) for dehydration (1h per concentration), then transparentized with xylene (I→II, 30 minutes each) to replace ethanol and enhance paraffin permeability; the dehydrated and transparentized tissue is immersed in molten paraffin (60-65°C) for 3 times (1h each time), embedded in a metal mold and cooled to solidify into a wax block, then continuously sectioned (thickness 4-6μm) using a rotary microtome, mounted on a polylysine-treated glass slide (40-45°C constant temperature water bath), and dried in a 60°C oven for 2h to enhance adhesion.

[0169] When staining, the section is deparaffinized with xylene I→II (10 minutes each), rehydrated with gradient ethanol (100%→95%→80%→70%) to distilled water, immersed in hematoxylin staining solution (Harris or Mayer formula) for 5-8 minutes (nuclear staining), washed with running water to remove color, then differentiated with 1% hydrochloric acid ethanol for 3-5 seconds to remove cytoplasmic non-specific coloring, and then returned to blue with 0.5% ammonia water or Scott blueing solution for 30 seconds until the nucleus is bright blue; after washing with water, the section is stained with eosin staining solution (0.5% aqueous solution, pH 4.5-5.0) for 1-2 minutes (cytoplasm and collagen staining), then rapidly dehydrated with 95% ethanol (I→II), 100% ethanol (I→II), transparentized with xylene (I→II for 5 minutes each), and mounted with neutral balsam. The analysis method requires observation under a light microscope: under 100x field, the epidermal regeneration integrity (epithelialization length and distance from wound edge), dermal layer inflammatory cell (neutrophil, lymphocyte) infiltration density (nuclear deep staining aggregation degree), granulation tissue thickness (new capillary blood vessels and fibroblast layer number), and collagen fiber arrangement state (eosin staining area continuity) are evaluated; under 400x high power lens, the epidermal keratinization layer formation, adnexal organ regeneration such as hair follicle / sebaceous gland are quantified, and the epidermal layer thickness (μm), inflammatory area area ratio (%), and granulation tissue maturity score (based on cell density and collagen deposition) are measured by image analysis software (such as ImageJ); the differences between the experimental group and the control group are compared by statistical methods to verify the promoting effect of the composite scaffold on diabetic wound tissue repair.

[0170] 1.3、Masson staining

[0171] Masson staining procedure includes tissue processing, section preparation and specific staining: first, the diabetic wound tissue samples were fixed with 4% paraformaldehyde for 24-48 h, then gradient ethanol dehydration (70%→80%→90%→95%→100%, 1 h for each concentration), xylene transparency (I→II, 30 min for each), and paraffin embedding (60-65℃ molten paraffin 3 times, 1 h for each time) were performed in sequence. After embedding, 4-6 μm thick continuous sections were prepared using a microtome, attached to polylysine glass slides and baked at 60℃ for 2 h to enhance adhesion. Before staining, the sections were dewaxed with xylene (I→II, 10 min for each) and rehydrated with gradient ethanol to distilled water, then immersed in Weigert iron hematoxylin staining solution (A solution: hematoxylin 1 g + anhydrous ethanol 100 ml; B solution: 29% ferric chloride 4 ml + concentrated hydrochloric acid 1 ml + distilled water 95 ml, mixed immediately before use) for 5-10 min to make the cell nucleus dark blue, washed with running water for 5 min, differentiated with 1% hydrochloric acid ethanol for 3-5 s to remove the cytoplasm non-specific coloration, and then returned to blue with a weak alkaline solution (such as 0.5% ammonia water) for 30 s. After water washing, the sections were immersed in the light green acid fuchsin staining solution (light green 0.7 g, acid fuchsin 0.3 g, glacial acetic acid 1 ml, distilled water 100 ml) for 5-10 min to preliminarily color the collagen fibers, washed with 0.2% aqueous solution of glacial acetic acid, and then treated with 1% aqueous solution of phosphomolybdic acid for 5-10 min to remove the red background of muscle fibers, quickly immersed in aniline blue staining solution (aniline blue 2 g, glacial acetic acid 2 ml, distilled water 100 ml) for 2-5 min to make the collagen fibers blue, and finally quickly washed with 0.2% glacial acetic acid, gradient ethanol dehydration (95%→100%, 30 s for each), xylene transparency, and neutral resin mounting.

[0172] Analysis method requires combination of microscope observation and image processing software: under 100× field of view, the overall distribution and arrangement density of collagen fibers (blue area ratio) were evaluated, and under 400× high power lens, the thickness, direction and spatial relationship with surrounding cells (red muscle fibers, dark blue-purple cell nucleus) of collagen bundles were observed; using ImageJ software to set color threshold to separate the blue collagen area, the percentage (%) of collagen area to dermal layer of wound surface was calculated, and the anisotropy index (0-1, 0 for completely disordered, 1 for highly directional) of collagen fiber arrangement was quantified by directionality analysis plug-in, to compare the maturity of new collagen (thick and tight bundle vs. thin and loose network) between experimental and control groups; at the same time, the uniformity of collagen deposition was evaluated by histopathological scoring system (such as Semi-quantitative scoring) (0-3 points: 0 = no deposition, 1 = focal, 2 = moderate continuous, 3 = extensive and uniform), and the promotion effect of PDA / Col composite scaffold on collagen remodeling of diabetic wounds was verified by statistical method, and the correlation between inflammatory factor expression and collagen maturity was related.

[0173] 1.4, Western blot of tissue proteins

[0174] After the tissue block is removed, immediately perform grinding and grinding, and then follow the cell wb experimental scheme. If time is urgent, the tissue should be placed in liquid nitrogen in a cryogenic tube in time, and the experiment should be performed the next day. Liquid nitrogen is more conducive to tissue grinding.

[0175] 1.5, immunohistochemistry

[0176] The steps of the immunohistochemical experiment include tissue processing, antigen repair, antibody incubation, and color detection: first, the paraffin section of the diabetic wound is dewaxed with xylene (10 minutes each for I→II), and then rehydrated with gradient ethanol to distilled water, and then placed in a citrate buffer (10 mM) at pH 6.0 for heat-induced antigen repair (high-pressure pot 121℃ treatment for 15 minutes or microwave oven high heat cycle heating to boiling and maintaining for 20 minutes), and then naturally cooled to room temperature and washed with PBS (pH 7.4) for 3 times (5 minutes each time); to block the activity of endogenous peroxidase, the section is immersed in 3% H2O2 methanol solution (avoid light) for 15 minutes, and then washed with PBS and blocked with 5% normal goat serum (or serum homologous to the second antibody) for 30 minutes at room temperature to reduce non-specific binding; pour off the blocking solution, add diluted primary antibody (such as anti-COL1A1, COL3A1, α-SMA, etc., dilution ratio according to the antibody instruction, commonly 1:100-1:500) to cover the tissue area, and incubate in a wet box at 4℃ overnight (or at 37℃ for 1-2h), and then wash off the unbound antibody with PBS (3×5 minutes) after rewarming the next day; add HRP-labeled secondary antibody (such as anti-rabbit / mouse IgG, 1:200-1:1000 dilution) and incubate at room temperature for 1h, and then wash thoroughly with PBS and cover the section with DAB developing solution (TBS buffer containing 0.05% DAB and 0.03% H2O2), control the developing time under a microscope (usually 1-5 minutes, until the target area is brown and the background is not colored), and immediately stop the reaction with running water; stain the cell nucleus with hematoxylin for 1-2 minutes, differentiate with hydrochloric acid ethanol and return to blue with ammonia water, and then dehydrate with gradient ethanol, transparentize with xylene, and mount with neutral resin.

[0177] The analytical method requires the use of optical microscopy and image analysis software: Observe the distribution of positive signals (cell membrane, cytoplasm, or nuclear localization) of the target protein (e.g., α-SMA-labeled vascular endothelial cells) under 200× or 400× field of view. Use ImageJ software to set a color threshold to segment the positively stained areas (DAB brown), and calculate the percentage of positive area (% positive area) or integrated optical density (IOD) to quantify the expression level. For semi-quantitative assessment, the H-score scoring system is used (staining intensity score 0-3: 0 = no staining, 1 = weak, 2 = moderate). 3 = Strong; Positive cell percentage score 0-4: 0 = 0%, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%), multiply the two to get the H-score (0-12 points), and compare the differences between the experimental group and the control group; at the same time, a negative control should be set up (omit the primary antibody or replace it with homotype IgG) to exclude non-specific staining, and multi-parameter correlation analysis should be performed in combination with histopathological characteristics (such as the degree of inflammation in HE staining) to verify the regulatory effect of PDA / Col composite scaffold on inflammatory factors and macrophage polarization in diabetic wounds.

[0178] II. Experimental Results

[0179] 2.1 Wound healing status on day 14 and protein expression in wound tissue on day 7

[0180] like Figure 19 As shown, wounds treated with the PDA / Col composite scaffold and those treated with collagen alone achieved healing by day 14, while wounds in the model group still showed significant signs of healing. In short, the repair rates of the collagen group and the PDA / Col composite scaffold group were essentially the same. To analyze the differences between the two, this study continued with tissue staining on day 21.

[0181] like Figure 21 As shown, HE and Masson results indicate that, except for the model group, the wound healing in the other experimental groups was quite good. The PDA / Col group, Col group, and PDA group all had more collagen deposition than the model. The differences between the polydopamine group, collagen group, and composite scaffold group lay in the amount of blood vessel and hair follicle formation.

[0182] from Figure 21 The study revealed that the collagen group exhibited significant epidermal thickening with fewer hair follicles. Unlike the composite scaffold group, which showed fewer capillaries, more hair follicles, and a less thick epidermis, the composite scaffold and polydopamine groups showed greater reduction in eosinophilic infiltration compared to the collagen-only group. Only the PDA / Col composite scaffold group demonstrated the ability to rapidly heal the wound while simultaneously forming extracellular matrix within the wound tissue.

[0183] Meanwhile, the protein expression of the 7th day of the wound of the animal inflammatory tissue was investigated Figure 20 ). It was found that the inflammation of the wound of the model group still existed on the 7th day, but the expression of iNOS and TNF-α decreased after the use of collagen and polydopamine. The expression of a-SMA increased after the use of collagen. This result means that the use of the collagen composite scaffold reduces the continuous inflammation of the continuous diabetic wound, and promotes more fibroblasts to transform into myofibroblasts, which is beneficial to the contraction of the wound. This result corresponds to the wound healing cycle diagram.

[0184] 2.2, 21st day wound healing immunohistochemistry

[0185] As Figure 22 shown, the protein expression level of the 21st day wound was observed, the expression of a-SMA of the Col group was higher, and the model group was only followed. The expression of the group added with polydopamine was lower. In the immunohistochemical test, a-SMA can be used to mark capillaries, and it can be seen from the figure that the model group and the Col group have more small pores. It may be due to the fact that the newly formed capillaries in the wound healing process do not gradually disappear on the 21st day, but continue to remain to provide nutrients to the site. And, the loose type III collagen is still the main part in the process of tissue repair. The capillaries of the PDA group and the PDA / Col group are basically degenerated, and there are more type I collagen deposits.

[0186] The above embodiments are the preferred implementation of the present application, in addition to this, the present application can be implemented in other ways, without departing from the concept of the present application, any obvious replacement within the protection scope of the present application.

Claims

1. Use of a polydopamine and collagen composite scaffold in the preparation of a biological product for promoting healing of a diabetic wound, the polydopamine and collagen composite scaffold inhibiting phosphorylation of a MAPK / NF-κB signaling pathway, and the polydopamine and collagen composite scaffold being prepared by the following steps: (1) adding a proper amount of anhydrous ethanol to deionized water, then adding a proper amount of ammonia water dropwise, stirring at room temperature to obtain solution A; then dissolving a proper amount of dopamine hydrochloride in deionized water to obtain solution B; adding solution B to solution A, stirring at room temperature to obtain crude polydopamine; (2) centrifuging the crude polydopamine at a low speed to remove insoluble impurities or large particulate matter, then centrifuging at a high speed to collect the precipitate; then washing the precipitate with ethanol first, then with deionized water, collecting the precipitate after washing and drying to obtain purified polydopamine; (3) then adding a proper amount of polydopamine to a collagen solution, adjusting the pH to 5.5-6.5, stirring at room temperature, and freeze-drying to obtain a polydopamine and collagen composite scaffold; in step (3), the mass concentration of collagen in the collagen solution is 0.01-1.0 mg / mL, the mass concentration of polydopamine is 0.5-50 µg / mL, and the stirring time is 8-16 h.

2. Use according to claim 1, characterized in that: in step (1), first add 30-50 mL of anhydrous ethanol to 80-100 mL of deionized water, then add 1-3 mL of ammonia water dropwise, stir at room temperature to obtain solution A; then dissolve 400-600 mg of dopamine hydrochloride in 8-12 mL of deionized water to obtain solution B; add solution B to solution A, stir at room temperature for 8-16 h to obtain crude polydopamine.

3. Use according to claim 1, characterized in that: in step (2), the first centrifugation speed is 1500-2500 rpm, the centrifugation time is 3-7 min, the second centrifugation speed is 10000-12000 rpm, the centrifugation time is 10-20 min, and the mass fraction of ethanol is 25%-35%.

4. Use according to claim 1, characterized in that: The polydopamine and collagen composite scaffold promotes cell proliferation, facilitates cell migration, and is conducive to the expression of CD31 and VEGFA proteins by HUVEC cells.

5. The use according to claim 1, characterized in that: The polydopamine and collagen composite scaffold upregulates CD31 and VEGFA proteins of HUVEC cells in a high-sugar and inflammatory environment, while reducing iNOs cytokines, which is conducive to the formation of new blood vessels.

6. Use according to claim 1, characterized in that: The polydopamine and collagen composite scaffold reduces the expression of inflammatory factors of RAW264.7 cells by interfering with the inflammatory factor receptors TNFR1 and TLR4.

Images