Polydopamine and collagen composite scaffold as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a polydopamine and collagen composite scaffold as well as a preparation method and application thereof, and the preparation method comprises the following steps: (1) adding dopamine hydrochloride into deionized water containing a proper amount of ammonia water and absolute ethyl alcohol, and stirring at room temperature to obtain crude polydopamine; (2) carrying out low-speed centrifugation on the crude polydopamine, then carrying out high-speed centrifugation, and collecting the precipitate; and cleaning and freeze-drying to obtain purified polydopamine. And (3) weighing a proper amount of polydopamine, adding the polydopamine into the collagen solution, adjusting the pH value, stirring at room temperature, and freeze-drying to obtain the polydopamine and collagen composite scaffold. According to the polydopamine and collagen composite scaffold, through a preparation strategy of physical blending instead of chemical modification, the biocompatibility of the composite scaffold is kept, the production cost is greatly reduced, a simple preparation process provides feasibility guarantee for clinical transformation, and the composite scaffold can be used for treating diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to a polydopamine and collagen composite scaffold, and a preparation method and application thereof. Background Art

[0002] Diabetic ulcer (DU), as the most intractable chronic complication in the course of diabetes, its characteristic of being difficult to heal is mainly attributed to the complex interweaving of multiple pathological mechanisms and the superposition of self-reinforcing effects. By deeply analyzing its pathological essence, it can be found that the continuous hyperglycemic 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] Facing such a complex pathological network, the limitations of current clinical conventional treatment methods are becoming increasingly prominent: 1) Although surgical debridement can remove necrotic tissue, it cannot improve the pathological microenvironment; 2) Growth factor drugs are limited by the problems of rapid degradation in vivo and treatment resistance; 3) Traditional collagen dressings can only provide a passive physical barrier and lack the function of dynamically regulating the microenvironment. More worthy of attention is that some high-end dressings containing bioactive components have high costs and risks of immune rejection, and these factors seriously restrict their in-depth clinical promotion and application.

[0004] Polydopamine (PDA) is a biomimetic polymer material formed by the oxidative self-polymerization of dopamine under weak alkaline conditions. Its unique catechol and amino structures endow it with physical and chemical properties such as strong adhesion, biocompatibility, photothermal effect and surface functionalization ability. 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 blood vessel ingrowth, and specific bioactive sequences on the surface can effectively promote cell proliferation and differentiation. In addition, the controllable degradation characteristics of collagen enable it to maintain dynamic balance during tissue regeneration. However, in the specific pathological microenvironment of diabetes, the advantages of collagen materials are difficult to be fully exerted. For example, oxidative stress easily causes damage to the collagen fiber structure, chronic inflammation easily makes a large amount of pro-inflammatory factors adsorbed on the material surface, and abnormal cell signal transduction caused by the hyperglycemic state easily weakens the biological activity function of collagen.

[0005] To address these limitations, this study attempts to enhance the pathological adaptability of collagen scaffolds through material modification. Although early chemical modification strategies partially improved material properties, they often came at the cost of sacrificing the natural bioactivity of collagen. Additionally, while the design of introducing exogenous bioactive factors into the scaffold system demonstrated therapeutic potential, it faced challenges in complex preparation processes and cost control. These dilemmas have prompted researchers to turn their attention to natural biomimetic materials with multiple bioactivities, among which polydopamine has attracted much attention due to its unique molecular properties. Summary of the Invention

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

[0007] The purpose of the present invention is achieved through the following technical solutions: A preparation method of a polydopamine and collagen composite scaffold, comprising the following steps:

[0008] (1) First, add an appropriate amount of absolute ethanol to deionized water, then dropwise add an appropriate amount of ammonia water, and 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 and stir at room temperature to obtain crude polydopamine.

[0009] (2) Centrifuge the crude polydopamine at a low speed to remove insoluble impurities or large particulate matter, then centrifuge at a high speed and collect the precipitate; first wash it once with ethanol, then wash it with deionized water, and after washing clean, collect the precipitate and freeze-dry it to obtain purified polydopamine.

[0010] (3) Then weigh an appropriate amount of polydopamine and add it to the 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 absolute ethanol to 80 - 100 mL of deionized water, then dropwise add 1 - 3 mL of ammonia water, and 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 and 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, which is prepared according to the above-mentioned preparation method.

[0015] An application of a polydopamine and collagen composite scaffold in the preparation of a biological product for promoting the healing of diabetic wounds.

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

[0017] Preferably, the polydopamine and collagen composite scaffold of the composite scaffold can up-regulate the CD31 and VEGFA proteins of HUVEC cells under high glucose and inflammatory environments, and at the same time reduce the iNOs cytokine, which is beneficial to the formation of new blood vessels.

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

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

[0020] The beneficial effects of the present invention are as follows: The polydopamine and collagen composite scaffold of the present invention breaks through the limitations of traditional single-functional materials, realizes the multi-functional integration of antioxidant, immune regulation and structural support, and embodies the treatment concept of "pathological microenvironment adaptation". Through a preparation strategy of physical blending rather than chemical modification, the biocompatibility of the composite scaffold is maintained while the production cost is greatly reduced, and the simple preparation process provides a feasibility guarantee for clinical transformation. Molecular mechanism research shows that the surface characteristics of the composite scaffold have an important impact on the synergistic activation of cell signaling pathways, and the combination of specific bioactive sequences and functional groups can enhance the response efficiency of cells to growth signals. Its stage-responsive function release characteristics are precisely matched with the dynamic pathological process of diabetic ulcers.

[0021] The present invention constructs a polydopamine and collagen composite scaffold for the treatment of diabetic wounds, proposes an innovative treatment concept of multi-stage synergistic repair, and explores its mechanism of action. This treatment concept is mainly manifested as follows: 1) In the initial stage of wound healing, the composite scaffold rapidly responds to the high oxidative stress state, effectively scavenges excessive free radicals and inhibits the over-activation of inflammatory signals. The functional intervention at 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 into a reparative type. 3) In the tissue reconstruction stage, the collagen network with intact biological activity provides structural support for cell migration and matrix deposition, while its degradation products promote angiogenesis and orderly collagen arrangement through endogenous signal transduction. Description of the Drawings

[0022] Figure 1 Figures are the physical pictures of PDA and PDA / Col composite scaffolds; among them, Figure A is PDA nanoparticles, in the form of black powder; Figure B is the freeze-dried morphology of Col and PDA / Col.

[0023] Figure 2 Figures are for the particle size detection and Zeta potential determination of PDA; among them, A is the result of PDA nanoparticle size determination; B is the result of PDA Zeta potential determination.

[0024] Figure 3 Figures are the scanning electron microscope results of PDA, Col and PDA / Col composite scaffolds; among them, A and D are the scanning electron microscope results of PDA at different magnifications; B and E are the scanning electron microscope results of Col at different magnifications; C and F are the scanning electron microscope results of PDA / Col at different magnifications.

[0025] Figure 4 Figures are for the active structure detection of Col and PDA / Col composite scaffolds; among them, A is the circular dichroism spectrum result of Col and PDA / Col composite scaffolds; B is the result of the active structure detection kit of Col and PDA / Col composite scaffolds; C is the SDS-PAGE electrophoresis result of Col and PDA / Col composite scaffolds.

[0026] Figure 5 Figures are the infrared scanning spectrum results of PDA, Col and PDA / Col composite scaffolds and the ultraviolet absorption spectrum result of PDA; among them, A is the ultraviolet absorption spectrum result of PDA; B is the infrared scanning spectrum result of PDA, Col and PDA / Col composite scaffolds.

[0027] Figure 6CCK-8 results of PDA, Col, and PDA / Col composite scaffolds on different cells; among them, A shows the cell viability results of different cells at different concentrations of Col; B shows the cell viability results of different cells at different concentrations of PDA; C shows the viability results of different cells when cultured on the composite scaffold.

[0028] Figure 7 Collagen forms gels with DMEM medium at different concentrations.

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

[0030] Figure 9 Antioxidant performance tests of PDA, Col, and PDA / Col composite scaffolds; among them, A and B show the ROS scavenging effects and scavenging rate calculations of PDA, Col, and PDA / Col composite scaffolds; C and D show the quantitative results of the scavenging of ABTS radicals and DPPH radicals by PDA, Col, and PDA / Col composite scaffolds respectively.

[0031] Figure 10 EdU detection experimental results of PDA, Col, and PDA / Col composite scaffolds; among them, A shows the EdU detection results of L929 cells in each component; B shows the corresponding quantitative statistics.

[0032] Figure 11 Cell migration promotion experimental results of PDA, Col, and PDA / Col composite scaffolds; among them, A and B show the migration results and quantitative statistics of HUVEC cells in different culture environments; C and D show the Transwell results and quantitative statistics of L929 cells.

[0033] Figure 12 Protein regulation results of PDA, Col, and PDA / Col composite scaffolds on HUVEC cells; among them, A shows the Western blot experimental results of HUVEC cells for CD31 and VEGFA; B and C show the quantitative results of CD31 and VEGFA.

[0034] Figure 13 Immunofluorescence results of PDA, Col, and PDA / Col composite scaffolds in the hyperglycemic and inflammatory models of HUVEC cells; among them, A shows the cell immunofluorescence results; B shows the immunofluorescence quantitative results; C shows the cell RT-qPCR results.

[0035] Figure 14 Tube formation and Western blot assays of PDA, Col, and PDA / Col composite scaffolds in a hyperglycemic and inflammatory model of HUVEC cells. Among them, A shows that the composite scaffold can reduce the expression of iNOS in HUVEC cells under hyperglycemic and inflammatory conditions, and B shows the quantitative results; C shows the angiogenesis-promoting function of the composite scaffold, and D-G show the quantitative results; the red arrows indicate the areas where the lumen formation of HUVEC cells is poor under inflammatory and hyperglycemic conditions.

[0036] Figure 15 In an inflammatory model of RAW264.7 cells, Western blot was used to detect the expression of different proteins. Among them, A and F show the Western blot assays after culturing RAW264.7 cells in an inflammatory model with PDA, Col, and PDA / Col composite scaffolds for 12 h; B-E and G-J show the quantitative analysis of the Western blot assay blots.

[0037] Figure 16 Western blot results after co-culturing RAW264.7 and L929 cells. Among them, A shows the Western blot results after co-culturing RAW264.7 and L929 cells for 12 h, and B-G show the quantification.

[0038] Figure 17 After culturing RAW264.7 cells in an inflammatory model with PDA, Col, and PDA / Col composite scaffolds for 12 h, Western blot was used to detect the phosphorylation levels of different proteins. Among them, A and F show the Western blot results; B-E and G-J show the quantitative analysis of the Western blot assay blots, respectively.

[0039] Figure 18 Results of immunofluorescence, flow cytometry, and RT-qPCR assays in a bone marrow-derived macrophage (BMDM) inflammatory model. Among them, A shows BMDM immunocytochemistry, and B-C show the quantitative results; D shows the expression of FITC-CD86 and APC-CD206 in BMDM cells detected by flow cytometry; E-I show the expression of mRNA in BMDM cells detected by RT-qPCR.

[0040] Figure 19 Wound healing in mice at 14 days. Among them, A shows the evolution of wound healing within 14 days, and B shows the quantitative analysis of the wound healing process beyond 14 days.

[0041] Figure 20 Protein detection in wound tissues of mice at day 7. Among them, A shows the Western blot assay, and B-D show the quantitative protein expression of iNOS, TNF-α, and α-SMA, respectively.

[0042] Figure 21 On the 21st day, HE and Masson staining were performed to observe the degree of skin structure recovery.

[0043] Figure 22 For immunohistochemical analysis of the tissue recovery status at the wound closure site; among them, A is the immunohistochemical staining of each group on the 21st day; B - D are the quantification of immunohistochemical α - SMA, COL1A1, and COL3A1 respectively. Specific implementation manners

[0044] For the convenience of those skilled in the art to understand, the following combines examples and attached Figures 1 - 22 to further illustrate the present invention. The content mentioned in the implementation manners does not limit the present invention.

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

[0046] I. Experimental methods

[0047] 1.1 Preparation of PDA / Col composite scaffold

[0048] First, add 40 mL of absolute ethanol to 90 mL of deionized water, then dropwise add 2 mL of ammonia water, and stir at room temperature for 10 min to obtain solution A. Then dissolve 500 mg of dopamine hydrochloride in 10 mL of deionized water to obtain solution B, and then add solution B to A, and stir at room temperature for 12 h to obtain crude polydopamine. Next, perform purification: centrifuge the crude polydopamine at 2000 rpm for 5 min to remove insoluble impurities or large - particle substances, then centrifuge at 11000 rpm for 15 min, and collect the precipitate. Then, wash it once with 30% ethanol and then with deionized water. After washing clean, collect the precipitate and perform freeze - drying to obtain purified polydopamine. Then weigh 1 mg of polydopamine and add it to 100 mL of 0.1 mg / mL collagen solution, adjust the pH to 6.0, stir at room temperature for 12 h, and perform freeze - drying to obtain a polydopamine and collagen (PDA / Col) composite scaffold.

[0049] 1.2 Determination of PDA particle size and Zeta potential

[0050] Use the nanoparticle measuring instrument of HORIBA company to measure the particle size distribution of PDA. The specific steps are as follows: Use the obtained polydopamine freeze - dried powder above to prepare a 0.1 mg / mL aqueous solution of polydopamine. After fully mixing and ultrasonically treating for 10 minutes, add it to a disposable cuvette with light transmission on all four sides. Before measurement, adjust the instrument so that the dispersion system is water, the test sample is polydopamine, and the number of measurements n = 5. Similarly, use an electrokinetic cell, select the Zeta potential detection mode, detect the potential of PDA, and the number of measurements n = 5.

[0051] 1.3. Observation of the morphology of PDA by scanning electron microscopy

[0052] Ensure that the sample is completely dry to avoid interference from volatiles under vacuum. Coat PDA on a conductive substrate and sputter a 3 - 5 nm gold or platinum layer on the surface of PDA using an ion sputtering instrument to reduce the charging effect. For high - resolution observation, a thinner platinum / palladium coating is recommended. Firmly paste the sample on the sample stage with conductive glue to avoid charge accumulation. Set the acceleration voltage to 5 - 10 kV to reduce the damage of the electron beam to PDA (polymers are easily decomposed). Adjust the working distance to 5 - 10 mm to balance the depth of field and resolution. Locate the target area at low magnification (500 - 2,000×) and gradually magnify to 10,000 - 50,000× to observe the surface details. If abnormal bright lines or distortions appear in the image, re - coat or reduce the acceleration voltage. Wear gloves throughout the sample handling process to avoid grease contamination.

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

[0054] Use the collagen active structure detection kit from Dongguan Collagen Biotechnology Co., Ltd. The collagen concentration is about 0.5 mg / mL. Detect the sample according to the instructions, that is, at room temperature, directly drop 100 μL of the sample solution into the detection reagent and invert it 5 - 10 times. Observe whether there are red flocculants in the reagent. The presence of red flocculants indicates that the collagen in the sample has a triple - helix structure that maintains its activity, otherwise it does 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 collagen in a weakly acidic buffer (pH 4.0) or a low - ionic - strength solution (avoid phosphate because it has strong absorption in the far - ultraviolet region), and the final concentration is about 0.1 - 0.2 mg / mL. Centrifuge (4℃, 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 injecting the sample to avoid air bubble residues. Scan the baseline with pure buffer (under the same conditions as the sample) in the wavelength range of 170 - 250 nm to deduct background interference. Inject the collagen solution into the cuvette, avoiding air bubble generation. Scan 3 times and take the average to reduce noise. If necessary, purge with nitrogen to exclude oxygen interference.

[0056] 1.5. Determination of the molecular weight and ultraviolet absorption of Col and PDA / Col composite scaffolds

[0057] Molecular weight detection was analyzed by SDS-PAGE electrophoresis method, and the electrophoresis apparatus used the supporting tools of Bio-Rad company (the power supply is the basic model). The sample and 5×SDS loading buffer (containing 2% SDS, 5% β-mercaptoethanol) were mixed at a volume ratio of 4:1, the final protein concentration was 1 mg / mL, and heated at 100 °C for 10 minutes (collagen has high thermal stability, and 10 minutes is to ensure sufficient denaturation). The Tris-glycine electrophoresis formulation was used: 18.77 g of glycine, 3.03 g of Tris, and 1.0 g of SDS were formulated into 1 L of solution. The 8% PAGE gel and protein marker were from Shanghai YaMei Biotechnology Co., Ltd., and the electrophoresis was carried out at 80 V throughout the process. After the marker between 100-250 kDa was separated, the electrophoresis was stopped. Stained with Coomassie Brilliant Blue R-250 solution at room temperature for 2 h, and then decolorized until the gel was nearly transparent and the blue bands were clearly visible. Take pictures and record against a white background, and observe the position of each sample band.

[0058] Using a UV-visible spectrophotometer, different concentrations of PDA aqueous dispersion (about 3.2-3.5 mL) were added to a constant quartz cuvette, and in the wavelength range of 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. Detection of functional groups of samples by Fourier transform infrared spectrometer

[0060] Weigh 1-2 mg of the sample and 100-200 mg of dry KBr powder into an agate mortar and grind them evenly until they become fine powder. Gently press the powder with the convex surface of a stainless steel spoon to roughly judge the grinding degree. Wait until the powder is smooth and delicate before proceeding with the tablet pressing treatment. Ensure that the sample is completely dry, or make it completely dry during the grinding process before detection. Use the instrument with a scanning range of 4000-400 cm -1 (mid-infrared region), the resolution is set to 4 cm -1 , and the number of scans is 8.

[0061] 1.7. Data statistics and analysis

[0062] All the data obtained above were statistically analyzed using Origin2024 software. Each group had at least 3 parallel results, and for some (such as the ultraviolet absorption measurement results and infrared spectra), one of them was selected for display. The significant differences were p<0.05 (*), p<0.01 (**), p<0.001 (***).

[0063] II. Experimental results

[0064] 2.1. Preparation of PDA / Col composite scaffolds

[0065] According to the above experimental method, the following was obtained Figure 1The black powder of A was preliminarily determined to be PDA based on its color and powder state, combined with existing reference data. Further compounded with collagen to form a composite scaffold, the pure white collagen freeze-dried sponge turned light gray by adjusting the pH, as Figure 1 shown in B. It can be seen that the collagen has loaded polydopamine nanoparticles.

[0066] 2.2 Particle size, Zeta potential and scanning electron microscopy of PDA

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

[0068] 2.3 Detection of the active structure and molecular weight of Col and PDA / Col composite scaffolds

[0069] As Figure 4 shown in A, the circular dichroism results show that the tested sample has a negative peak at 198 nm and a positive peak at about 220 nm, meeting the requirements of the standard circular dichroism spectrum of collagen. As Figure 4 shown in B, the collagen detection kit shows that red flocculents appear both before and after the combination of collagen and polydopamine, indicating that the collagen maintains a relatively stable triple helix structure both before and after binding to polydopamine, which is also the active structure of collagen. As Figure 4 shown in C, after the combination of collagen and polydopamine, there is an increase in molecular weight, indicating that the binding 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 scaffolds

[0071] As Figure 5 shown in A, when detecting the polydopamine aqueous dispersion by ultraviolet-visible absorption spectroscopy (UV-Vis), the detection results indicate that polydopamine has full-wavelength absorption within the wavelength range of 200 - 800 nm and there is no characteristic absorption peak. It can be seen that UV detection is not suitable for the quantification of polydopamine.

[0072] As Figure 5 shown in B, analyzing the infrared spectrum results, for polydopamine, at 3300 - 3500 cm -1 : N-H / O-H stretching vibration (amino group, phenolic hydroxyl group and adsorbed water). At 1600 - 1650 cm -1 : Aromatic ring C=C stretching vibration (benzene ring conjugated structure). At 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 : Out-of-plane bending vibration of aromatic ring C-H (ortho-substituted benzene ring). After dopamine polymerization, 1600 cm -1 and 1510 cm -1 The doublet at is the signature signal of PDA. However, the 1600 - 1650 cm -1 peak is broadened, possibly due to cross-linking leading to structural disorder. Compared with dopamine (DA), polydopamine has a higher peak intensity ratio at 1600 cm -1 and 1510 cm -1 , and the more perfect the conjugated aromatic ring structure.

[0073] For the infrared spectral analysis of collagen, 3300 cm -1 : N-H stretching vibration (amide A band, overlapping with O-H). 3080 cm -1 : Amide B band (N-H stretching vibration). 1650 cm -1 : Amide I band (C=O stretching vibration, α-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 (side chains of glycine and proline). The peak positions and ratios of amide I band (1650 cm -1 ) and amide II band (1550 cm -1 ) can reflect the integrity of the collagen triple helix structure.

[0074] Appearance of new peaks: 1700 - 1750 cm -1 : May be due to enhanced ester bond (C=O) or quinone structure. 1220 - 1260 cm -1 : Enhanced C-N stretching vibration (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] This research project mainly uses L929, RAW264.7, HUVEC, HaCaT and BMDM cells for experiments. 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 a dedicated 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 resuscitation: Preheat the medium, trypsin, etc. in a 37°C water bath in advance. Take out the cryopreservation tube from the liquid nitrogen tank or -80°C refrigerator and immediately place it in a 37°C water bath. Gently shake it quickly until the ice crystals are almost completely melted (about 1.5 - 2 minutes). Avoid prolonged water bath, which may enhance the toxicity of DMSO. Transfer the cell suspension to a 10 mL centrifuge tube containing 5 mL of cold 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 preheated complete medium, transfer them to a T25 culture flask, and add medium to 5 mL. It is recommended to inoculate at the same density as before cryopreservation. Place it in a 37°C, 5% CO2 incubator. Change the medium for the first time after 24 hours to remove dead cells and residual DMSO. Observe that the cells adhere and have a normal morphology. The adherent cells should be extended without excessive debris. Confirm that the cell density reaches 80 - 90% confluence, with good condition, no vacuoles or debris. Then cell passage and cell experiments can be carried out.

[0080] Cell passage and cell cryopreservation: Preheat PBS, trypsin (0.25% Trypsin-EDTA), and complete medium. Aspirate and discard the old medium, add 3 mL of PBS and gently wash once to remove the serum that inhibits trypsin activity. Then add 1 mL of trypsin (for T25 culture flask), gently shake to cover the cell layer, and incubate and digest at 37°C. Immediately terminate when the cell gaps increase and the edges curl. Add 2 mL of complete medium (containing FBS), pipette the cell layer until it completely detaches, and transfer it to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Add an appropriate amount of fresh medium and gently pipette to form a single-cell suspension. Adjust according to the cell type, inoculate the cell suspension in proportion into a new culture dish, and add medium to the standard volume (such as adding 5 mL to a T25 flask). At the same time, record the passage number to avoid using high-passage cells. Generally, primary cells are passaged ≤5 times, and immortalized cells ≤20 times.

[0081] When cryopreserving cells, prepare a cryopreservation solution (90% FBS + 10% DMSO) or a commercial cryopreservation solution. Note that after centrifuging and collecting the cells, resuspend them with the cryopreservation solution. Aliquot the cell suspension into cryotubes and perform a programmed cooling procedure (4°C for 30 min → -20°C for 2 h → -80°C overnight → long-term storage in liquid nitrogen).

[0082] Macrophage polarization induction: After normal culture, seed RAW264.7 cells into 6-well plates at a density of 300,000 cells per well. After the cells adhere, add 100 ng / mL LPS and 20 ng / mL IFN-γ to the M1 pro-inflammatory phenotype group, and add 20 ng / mL IL-4 and the same dose of IL-13 to the M2 anti-inflammatory phenotype group. The experimental groups are collagen, polydopamine, and their complexes, and the blank control group adds an equal volume of PBS. Incubate at 37°C in 5% CO2 for 12 h.

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

[0084] For 6-8-week-old male C57BL / J mice, after cervical dislocation, immerse them in 75% ethanol for 5 minutes. Then remove the hind limbs, remove the muscle, immerse the femurs in 75% ethanol for 5 minutes, and wash them with cold PBS. After cutting off both ends, use BMDM induction medium (high-glucose DMEM medium containing 15% L929 supernatant and 10% FBS) to blow and wash the bone marrow until there is no red substance in the cavity. Then filter through a 70 μm cell strainer, centrifuge at 1500 rpm for 3 minutes to collect the precipitate, and then perform red blood cell lysis. Centrifuge again to collect the precipitate, which can be washed once with PBS, and then directly counted with complete medium and seeded for culture, and can be directly used for subsequent experiments.

[0085] When polarizing BMDM cells, use a method similar to that of RAW264.7, with the difference that the doses of IL-4 and IL-13 added during M2 phenotype induction are 10 ng / mL.

[0086] 1.3 Detection of the effect of samples on cell viability by CCK-8 method

[0087] Culture L929, RAW264.7, HUVEC, and HaCaT cells under normal conditions. Seed the cells into 96-well plates one night in advance, with approximately 6000 cells per well. The next day, first remove the medium in the wells, then add complete medium containing the sample to be tested as the experimental group, and complete medium without the sample to be tested as the control group. After incubating for 24 h, remove the medium again. Add serum-free medium containing CCK-8 reagent to all cell culture wells, and select another cell-free culture well as the blank group. Incubate for 1 h, measure the absorbance at 450 nm, and use Graphpad Prism 9.5 to perform statistical analysis on the obtained data to calculate cell viability. The calculation formula is:

[0088]

[0089] Among them, 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] Collect New Zealand rabbit blood using an anticoagulant tube, defibrinate it, and centrifuge at 3500 rpm for 15 minutes. Then use normal saline or PBS as the washing solution and centrifuge at 3500 rpm for 5 minutes to wash and purify the red blood cells, and finally prepare a 4% red blood cell solution. Next, prepare a 2% TritonX-100 solution and a sample solution to be tested with twice the concentration using the same washing solution. Subsequently, the red blood cell solution is mixed with normal saline / PBS (negative control group), 2% TritonX-100 solution (positive control group), or the sample solution to be tested (experimental group) at a ratio of 1:1, incubated in a water bath at 37°C for 1 h, and finally centrifuged at 1200 rpm for 3 minutes. Absorb the supernatant and measure the absorbance at 450 nm, and calculate the hemolysis rate. The calculation formula is:

[0092]

[0093] Among them, Ax is the absorbance of the experimental group; A0 is the absorbance of the negative control group; 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] Use L929 cells. The cells are cultured in a well plate until they reach about 70%-80%. In the experimental group, add a basal medium containing the drug, and in the control group, use only the basal medium, and culture for 2 h. Then add EdU reagent (20 μM) to all wells and incubate for 6 h. Remove the medium, add 1 mL of PBS containing 4% paraformaldehyde to each well, incubate at room temperature for 15 min, and then remove the PBS containing 4% paraformaldehyde. Add 1 mL of PBS containing 3% BSA to each well and wash thoroughly 3 times, 5 min each time. Discard the supernatant, add 1 mL of PBS containing 0.3% TritonX-100 to each well, and incubate at room temperature for 20 min. Discard the supernatant and then wash. Prepare the Click reaction solution according to the number of samples and the instructions of the reagent supplier. Discard the supernatant, add 500 μL of the Click reaction solution prepared as above to each well, gently shake to ensure that the Click reaction solution evenly covers the cells, and incubate at room temperature in the dark for 30 min. Discard the supernatant and then wash. Add 500 μL of DAPI working solution to each well, and incubate at room temperature in the dark for 5-10 min. After washing, observe and photograph using a confocal microscope.

[0096] 1.6 Scratch experiment and Transwell experiment to investigate the effect of the composite scaffold on cell migration

[0097] Scratch assay: The scratch assay is usually performed using a 6-well plate. Therefore, the cells are seeded on the 6-well plate. Before the scratch operation, the cells must be confluent. The visual standard is that there are no obvious cell gaps under a 40X field of view and the cell state is good. Taking L929 cells as an example, the number of cells seeded per well is generally 200,000 - 250,000. A 200 μL pipette tip is selected for the scratch operation, so that the scratch width is appropriate. After the scratch is completed, each well is washed 3 times with 2 mL of PBS to prevent floating cells from adhering in the scratch area. The control group is a separate serum-free medium, while the experimental group is a basal medium containing the test sample.

[0098] Transwell assay: After digesting and centrifuging L929 cells, they are resuspended in serum-free medium and the density is adjusted to 1.0x10 5 -5x10 5 cells / mL. The Transwell chamber (pore size 8 μm) is placed in a 24-well plate. The lower chamber is filled with 500 μL of basal medium containing the test sample as the experimental group, and the control group is a basal medium without anything. 200 μL of cell suspension resuspended in basal medium is added to the upper chamber. Incubate in a 37 °C, 5% CO2 incubator for 12 h to wait for cell migration. Take out the chamber, gently rinse with PBS, wipe the non-migrated cells on the surface of the upper chamber with a cotton swab, and fix the lower chamber membrane with methanol or paraformaldehyde for 30 minutes. Stain with 0.1% crystal violet (solution prepared with 20% ethanol) for 30 minutes, rinse with PBS multiple times, air dry, place the membrane on a glass slide, and randomly select fields of view under the microscope to count the migrated cells.

[0099] 1.7. Investigation of ROS Scavenging and Free Radical Scavenging Rate

[0100] Accurately weigh 6.25 mg of ABTS1 and 2.82 mg of K2S2O8, and dissolve them separately in 4 mL of deionized water to prepare stock solutions. Then mix the two in a 1:1 ratio and slowly shake in the dark at room temperature for 6 h to obtain a solution rich in nitrogen free radicals. Adjust the absorbance of the solution to 0.8, and use this as the final concentration to measure the test sample. ABTS solution volume: test sample volume = 200 μL: 20 μL, incubate at room temperature for 10 minutes, and measure the absorbance at 734 nm. Similarly, prepare a 50 μg / mL DPPH solution and perform the experiment according to the above steps. The difference is that the incubation time is extended to 30 minutes, and the absorbance at 519 nm is measured.

[0101] In addition, L929 cells were co-incubated with a ROS inducer (when using 3% H2O2, diluted 1000-fold to reach the final concentration, 882 μM H2O2) in a carbon dioxide incubator for 6 h, then the test substances (PDA, Col and their complexes) were added and incubated for 2 h, and washed 3 times with PBS. Subsequently, DCFH-DA was used to capture the generation of ROS, incubated in the incubator for 30 minutes, and also washed 3 times with PBS, and the change in fluorescence intensity was detected using a live cell imager; by comparing the signal differences between the experimental group and the control group, the ROS scavenging rate was calculated. A blank control (without inducer) was set in the experiment 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. Angiogenesis assay

[0103] Consumables such as 48-well plates and pipette tips were pre-frozen at -20 °C 12 h in advance, the Matrigel was thawed in advance, diluted 1:1 with DMEM (high glucose) medium, and stored at 4 °C. The next day, 100 μL of Matrigel was added to each well of the pre-frozen 48-well plate, then placed in the incubator for 1 h. After the Matrigel was completely solidified, it was taken out, and HUVEC cells with or without the test drug prepared in advance were carefully added to the wells coated with Matrigel. After 10 h, the tube formation of endothelial cells was recorded. Quantification was performed using ImageJ, and the obtained data were statistically analyzed using Graphpad Prism 9.5.

[0104] 1.9. Total cellular RNA extraction and real-time fluorescence quantitative PCR

[0105] Total cellular RNA extraction: For the cells that had been treated with the drug, first remove the original medium, then wash 3 times with DPBS, and then perform cell lysis. For a 6-well plate, add 1 mL of Trizol reagent to each well and place on ice for 5 - 10 min. Collect the Trizol reagent with lysed cells into a 1.5 mL centrifuge tube, add 200 μL of chloroform, quickly vortex and mix until the liquid becomes milky white, and place on ice for 10 min. Subsequently, centrifuge at 4 °C, 12000 rpm × 15 min. During centrifugation, prepare a new 1.5 mL centrifuge tube and insert it into ice for pre-cooling and standby.

[0106] After centrifugation, carefully collect 300 μL of the upper clear liquid, transfer it to a clean and pre-cooled 1.5 mL centrifuge tube, and add 1 mL of isopropanol. Invert the tube manually up and down to mix well, place it on ice for 5 min, and then centrifuge again at 4 °C, 12,000 rpm for 10 min. The centrifuged RNA is a very small amount of grayish-white precipitate. Carefully pour out the isopropanol inside, and then invert the tube onto a flat piece of paper for 30 - 60 s to let the isopropanol inside flow out as much as possible. Then add 1 mL of freshly prepared 75% ethanol (anhydrous ethanol + DEPC water), invert the tube up and down to mix well for washing, and place it on ice for 5 min. Centrifuge at 4 °C, 7,500 rpm for 5 min. After centrifugation, a dandruff-like precipitate is obtained. Carefully pour out the ethanol inside, and then invert the centrifuge tube onto the paper for 5 min to air dry naturally.

[0107] Obtain a clean precipitate, add 30 - 50 μL of DEPC water (or use TAE buffer) to dissolve the RNA, mix well and place it on ice, and then perform concentration and purity determination. The RNA concentration is measured using NanoUV, and the purity is measured using agarose electrophoresis. After measuring the concentration, generally transfer a part of the RNA to a new 1.5 mL centrifuge tube for experiments, and store the remaining at -80 °C. Adjust the concentration of the experimental RNA to 100 ng / μL.

[0108] Reverse transcription and qPCR: Place the prepared 200 μL centrifuge tube on ice for pre-cooling, and operate according to the steps of the reverse transcription kit. Generally, use a 10 μL or 20 μL reaction system, and usually the RNA addition amount is 500 ng - 1 μg.

[0109] After the reaction, obtain cDNA, which should be stored on ice and its concentration is measured. The concentration of cDNA after reverse transcription of 500 μg RNA is generally about 800 ng / μL. According to needs, generally perform a 10-fold preliminary dilution first to obtain 80 ng / μL of cDNA. If there is no time for subsequent experiments, the cDNA can be stored in a -20 °C refrigerator. Generally, select a 20 μL reaction system, first prepare the cDNA at a concentration of 10 ng / μL, and the primer pair at a concentration of 2.5 μM.

[0110] Table 1 Reverse transcription reaction conditions

[0111]

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

[0113]

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

[0115]

[0116] Table 4 PCR Primer Information

[0117]

[0118] 1.10. Immunoblotting (Western blot, WB)

[0119] Take out the cultured cells, aspirate the culture medium in the well plate, wash twice with cold PBS, then add 1 mL of PBS to moisten the cells, and scrape the cells as much as possible with a cell scraper. Transfer the scraped cells into a pre-cooled 1.5 mL EP tube, centrifuge at 2000 rpm for 5 min. Discard the supernatant, add 100 μL of pre-prepared lysis buffer containing protease inhibitor, mix well, and lyse on ice for 30 - 60 min. Centrifuge at 12000 rpm for 15 min, take the supernatant, and transfer it to a new pre-cooled EP tube. Quantify the protein and adjust the protein concentration to 2 - 5 mg / mL. According to the molecular weight of the target protein, select an appropriate separating gel for protein electrophoresis. Usually, run at 80 V for 120 min, and the recommended loading amount is 10 - 20 μg, with a volume of 5 - 10 μL.

[0120] 1.11. Immunocytochemistry (ICC)

[0121] The cells for this experiment are directly seeded into a glass-bottom culture dish, treated with drugs, and then fixed with 4% paraformaldehyde solution for 10 minutes after obtaining the cells. Then, permeabilize with PBS solution containing 0.3% Triton X-100 at room temperature for 5 minutes. Block the cells with PBS containing 5% goat serum or a commercial immunofluorescence blocking solution for 1 h. After washing thoroughly, incubate with the primary antibody overnight at 4°C, and then incubate with the corresponding fluorescent secondary antibody at room temperature for 2 h. Use DAPI / Hoechst staining to identify the cell nuclei, and then perform confocal microscopy observation and recording. Use ImageJ for quantification. The calculation formula is as follows:

[0122]

[0123] Among them, 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. Cell Co-Cultivation

[0125] RAW264.7 and L929 cells were cultured until 80% confluence, and cells in the logarithmic growth phase were taken after passage. A 6-well plate was used for the experiment. RAW264.7 cells (3x105 / well) were seeded in the upper chamber, and L929 cells (3x105 / well) were seeded in the lower chamber (basal chamber). The pore size of the insert was 0.4 μm, and the material was PET. According to the above induction protocol, the cells were cultured for 12 h, and then immunoblotting experiments were performed.

[0126] 1.13 Flow cytometry

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

[0128] Take out the cells from the incubator, remove the culture medium, and gently wash twice with pre-cooled PBS. Add 0.25% trypsin (containing EDTA) to digest the cells, terminate the digestion with serum-containing culture medium, and centrifuge (1200 rpm, 3 min) to collect the cells.

[0129] Resuspend the cells with pre-cooled flow cytometry buffer, adjust the cell concentration to 1×106 / mL, and the antibodies and dyes used subsequently were also diluted with flow cytometry buffer. Add live / dead cell dye and incubate at room temperature in the dark for 30 min. Centrifuge and wash twice.

[0130] Prepare the antibodies in advance according to the instructions, then add the antibodies against membrane proteins CD86 and F4 / 80, and incubate in the dark for 30 min (4℃). Centrifuge and wash twice, and resuspend in 4% paraformaldehyde and fix at room temperature for 15 min.

[0131] Centrifuge to remove the fixing solution and wash once. Resuspend the cells with 1% Triton X-100 and incubate at room temperature in the dark for 30 min. After centrifugation, add the intracellular antibody CD206 and incubate in the dark at 4℃ for 1 h. Centrifuge and wash twice, and resuspend in flow cytometry buffer.

[0132] II. Experimental results

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

[0134] The results of the CCK-8 experiment were as Figure 6 shown. When the collagen concentration started to increase from 1.0 mg / mL, the cell viability began to be inhibited. It was found that high-concentration (>1.5 mg / mL) collagen and cell culture medium would form a weak colloidal state in a 37℃ incubator. When the concentration reached 2 mg / mL, collagen and the culture medium could form a more obvious gel state ( Figure 7)。When the collagen concentration is within 1 mg / mL, it has little effect on the cell viability of the 4 types of cells investigated.

[0135] When the collagen concentration is 0.1 mg / mL, the cell viability is close to 100%. When the collagen concentration is higher than 0.1 mg / mL, the cell viability shows a slight decrease. It should be noted that as the concentration increases, the viability decreases, but not significantly. Therefore, for the purpose of the experiment, in order to prevent collagen from forming a gel with the culture medium and affecting cell viability, resulting in inaccurate experimental results, this study selected 0.1 mg / mL as the collagen concentration in the subsequent experiments. For polydopamine, when the concentration is 50 μg / mL, the cell viability is inhibited and reduced to about 75%. Continuing to increase the concentration of polydopamine to 100 μg / mL, the cell viability inhibition rate will reach 50%. This result indicates that the safe concentration of polydopamine to be adopted in the subsequent experiments is below 50 μg / mL. In order to ensure that the cell viability is not inhibited by the composite scaffold, this study finally selected the concentration of polydopamine to be 10 μg / mL.

[0136] Finally, this study determined the final theoretical experimental concentration of PDA / Col to be 0.11 mg / mL. Through cell viability detection again, it was proved that the polydopamine and collagen composite scaffold with a concentration of 0.11 mg / mL is the most friendly to cells.

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

[0138] As Figure 8 shown, this experiment explored the hemolysis rates of different concentrations of collagen and polydopamine. The results showed that collagen and polydopamine exhibited extremely low hemolysis rates. Even when increased to 100 times the original concentration, no obvious hemolysis phenomenon occurred. Only after increasing the concentration of polydopamine, it would cause polydopamine to disperse in the supernatant after centrifugation, making the liquid color darker. However, when the absorbance was adjusted to the background with the same concentration of polydopamine dispersion, it was close to that of 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 Figure 9As shown, in this study, by detecting the ROS, ABTS, and DPPH scavenging rates of PDA / Col, it was found that the composite scaffold has the ability to target the scavenging of free radicals and inhibit cellular oxidative stress. Collagen can scavenge about 50% of the ROS in cells, but it did not perform well in the in vitro ABTS and DPPH free radical scavenging tests. Therefore, this study believes that this antioxidant function is mainly exerted by polydopamine. Previous studies have shown that polydopamine has excellent antioxidant ability, thus ensuring the effectiveness and reliability of PDA / Col composite scaffolds in applications such as anti-inflammation and promoting tissue regeneration.

[0141] 2.4. PDA / Col composite scaffolds can promote cell proliferation

[0142] Cell proliferation experiments (EdU) can evaluate the effect of PDA / Col composite scaffolds on the growth ability of key cells for wound healing (such as fibroblasts, keratinocytes, or endothelial cells). As Figure 10 shown, the EdU positive rate mainly benefits from the addition of Col, indicating that the cell proliferation-promoting effect of this composite scaffold is mainly exerted by Col. Although PDA has no obvious promoting effect, it also does not inhibit cell proliferation.

[0143] 2.5. PDA / Col composite scaffolds are helpful for cell migration

[0144] The scratch assay can be used to evaluate cell migration ability. As Figure 11 shown in A-11B, under normal circumstances, collagen promotes the migration of HUVEC cells, while the role of PDA is not significant. The Transwell assay analyzes the number of cells migrating through the porous membrane. However, as Figure 11 shown in C-11D, in L929 cells, low concentrations of PDA can also promote cell migration.

[0145] 2.6. PDA / Col composite scaffolds are beneficial for HUVEC cells to express CD31 and VEGFA proteins

[0146] One of the core mechanisms of the difficult healing of diabetic wounds is the angiogenesis disorder caused by the hyperglycemic environment, manifested as impaired endothelial cell function, down-regulation of the expression of pro-angiogenic factors (such as VEGF, CD31, bFGF), and abnormal elevation of anti-angiogenic factors (such as Angiopoietin-2), which in turn leads to local ischemia, hypoxia, and obstruction of nutrient delivery. As Figure 12 shown, under normal conditions, collagen can promote HUVEC cells to express CD31 and VEGFA, while polydopamine does not play an obvious role.

[0147] 2.7. The PDA / Col composite scaffold can upregulate the CD31 and VEGFA proteins of HUVEC cells under high glucose and inflammatory environments, while reducing the iNOs cytokine, which is beneficial to the formation of new blood vessels.

[0148] As Figure 13 shown, although polydopamine did not promote the viability of HUVEC cells under physiological conditions. However, unexpectedly, when the cells were in an inflammatory environment, polydopamine could reduce the iNOS expression of HUVEC cells, relieve the oxidative stress state of the cells, and the expression of CD31 also increased. HUVEC cells with alleviated oxidative stress state could better enjoy the effect of collagen. This may be an embodiment of the synergistic effect of the two components of the auxiliary composite scaffold.

[0149] The above results indicate 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 cell inflammatory response. Based on the above results, PDA / Col makes HUVEC cells in inflammatory and high glucose environments more likely to form blood vessels ( Figure 14 ).

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

[0151] The mechanism by which the polydopamine and collagen composite scaffold plays an anti-inflammatory role in treating LPS (lipopolysaccharide)-induced polarized macrophages involves multi-level immune regulation and signal pathway intervention. As Figure 15 shown, verified by Western blot experiment, the composite scaffold can significantly reduce the level of TNF-α in the culture supernatant of macrophages stimulated by LPS, downregulate the cell inflammatory factors and p38MAPK phosphorylation, and at the same time enhance the expression of the anti-inflammatory cytokine CD206, ultimately realizing the functional conversion from "inflammation inhibition" to "repair promotion".

[0152] 2.9. Cell co-culture

[0153] To investigate the regulation of fibroblasts by macrophages during inflammation, this study also co-cultured macrophages with fibroblasts. The results are as Figure 16As shown, in an inflammatory environment, after using the PDA / Col composite scaffold, the expression of COL1A2 and COL3A1 in L929 cells was upregulated. However, when using collagen alone, the effect was not ideal. This indicates that the repair effect of collagen may be good in normal wounds without persistent inflammation, but if persistent inflammation occurs, it needs to be used in combination with anti-inflammatory substances to achieve better results. At the same time, in an inflammatory environment, fibroblasts also highly express MMP-1 protein, and Col, PDA, and the PDA / Col group can effectively reduce its expression in the inflammatory environment.

[0154] To further explore how the PDA / Col composite scaffold exerts its anti-inflammatory effect, this study used LPS- and IFN-γ-induced RAW264.7 cells and BMDM cells as inflammatory cell models. LPS activates the MyD88 and TRIF pathways through TLR4, driving transcription factors such as NF-κB, AP-1, and IRF3, and inducing the expression of pro-inflammatory mediators (such as iNOS, CD86, TNF-α) and effector molecules. At the same time, it remodels metabolism (the expression of HIF-1α increases, and even under aerobic conditions, it turns to glycolysis for energy supply to support rapid energy demand and pro-inflammatory functions) and epigenetic status, and finally polarizes macrophages into the M1 phenotype with antibacterial and pro-inflammatory functions. IFN-γ often synergizes with LPS to enhance M1 polarization through STAT1. IL-4 and IL-13, on the other hand, act as signaling molecules to induce macrophage polarization into the M2 phenotype, which is an anti-inflammatory phenotype.

[0155] In the pro-inflammatory signal reception stage, different from collagen, polydopamine can reduce the expression levels of TNF-R1 and TLR4 ( Figure 15 ), making macrophages less sensitive to inflammatory cell mediators such as LPS and TNF-α, thereby reducing the M1 phenotype of macrophages. Among them, TLR4 can be specifically reduced by polydopamine, and collagen can reduce MYD88. Generally speaking, the 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. In the regulation of anti-inflammatory factors, there is not much difference between the two.

[0156] 2.10. The PDA / Col composite scaffold can inhibit the phosphorylation of the MAPK / NF-κB signaling pathway

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

[0158] Therefore, from a certain perspective, polydopamine can promote cell proliferation and differentiation in an inflammatory environment. This result echoes the previous conclusion and gives an explanation. The role of JNK is to promote AP-1 to enter the nucleus, promote the secretion of inflammatory factors, and regulate inflammation. IKBα is an anti-inflammatory factor that binds to P65 / P50 and inhibits their phosphorylation. It is known from experiments that at the experimental concentration, the regulatory effects of both on the above factors are basically equivalent. Therefore, the two have a synergistic effect in anti-inflammatory.

[0159] Observing whether there are differences in the anti-inflammatory effects of different concentrations of PDA, it was found that there was no significant difference in the anti-inflammatory effect of PDA at a concentration of 10 μg / mL. For the inhibition of TNF-α expression, collagen performed poorly, and the main effect was still determined by PDA( Figure 16 ).

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

[0161] Example 3 Animal Experiment on the Promotion of Diabetic Wound Healing by PDA / Col Composite Scaffold

[0162] I. Experimental Methods

[0163] 1.1 Construction of Diabetic Wound Animal Model

[0164] To construct an animal model of diabetic wounds, streptozotocin (STZ)-induced diabetic rats or mice are usually selected as the research subjects. A type 1 diabetes model is established by intraperitoneal injection of STZ (dose: 50 - 65 mg / kg, for 5 consecutive days) to destroy pancreatic islet β cells. Alternatively, a type 2 diabetes feature is induced by combining high-sugar and high-fat diet feeding (for 8 weeks) with low-dose STZ (30 - 40 mg / kg). After the fasting blood glucose is stably higher than 16.7 mmol / L and typical symptoms such as excessive drinking, polyuria, and weight loss appear, a full-thickness skin defect wound (diameter: 8 - 10 mm, depth reaching the fascia layer) is made on the shaved and disinfected area of the animal's back (usually using isoflurane inhalation anesthesia) with a sterile biopsy punch or surgical scissors. Iodophor is applied to the wound edge and sterile gauze is covered to prevent infection. The wound healing condition (the change in wound area is recorded by a digital camera, and the healing rate is calculated by ImageJ software) and blood glucose fluctuations are monitored daily after the operation. To simulate the chronic inflammatory environment of diabetic wounds, the inflammatory response can be further aggravated by local injection of lipopolysaccharide (LPS, 1 μg / μL) or delayed healing treatment (such as repeated mechanical injury to the wound edge). In the experimental group, a PDA / Col composite scaffold is covered on the wound surface, while in the control group, a pure collagen scaffold, polydopamine powder gauze, and normal saline gauze are used respectively. The scaffolds are fixed by suture or bioadhesive to ensure close adhesion to the wound surface.

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

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

[0167] 1.2. HE staining

[0168] The steps of HE staining experiment mainly include tissue sample fixation, dehydration and clearing, paraffin embedding, section preparation and staining treatment: First, the collected diabetic wound tissue samples (such as on the 7th and 14th days after surgery) are immediately immersed in 4% paraformaldehyde fixative (pH 7.4, stored at 4°C for 24 - 48 h) to maintain cell morphology and terminate enzyme activity; after fixation, the samples are rinsed with running water for 12 h to remove excess fixative, and then successively immersed in gradient ethanol (70% → 80% → 90% → 95% → 100%) for dehydration (1 h for each concentration), and then treated with xylene for clearing (Ⅰ → Ⅱ, 30 minutes each) to displace ethanol and enhance paraffin permeability; the dehydrated and cleared tissue is immersed in molten paraffin (60 - 65°C) 3 times (1 h each time), embedded in a metal mold and cooled and solidified to form a wax block, and the wax block is continuously sectioned (4 - 6 μm thick) using a rotary microtome, pasted on a polylysine-treated glass slide (spread in a 40 - 45°C constant temperature water bath), and dried in an oven at 60°C for 2 h to enhance adhesion.

[0169] During staining, the sections are dewaxed with xylene Ⅰ → Ⅱ (10 minutes each), rehydrated to distilled water with gradient ethanol (100% → 95% → 80% → 70%), stained with hematoxylin stain (Harris or Mayer formula) for 5 - 8 minutes (nuclear staining), differentiated with 1% hydrochloric acid ethanol for 3 - 5 seconds to remove non-specific cytoplasmic staining after rinsing the floating color with running water, and then blued with 0.5% ammonia water or Scott bluing solution for 30 seconds until the nucleus is bright blue; after washing with water, stained with eosin stain (0.5% aqueous solution, pH 4.5 - 5.0) for 1 - 2 minutes (cytoplasm and collagen staining), dehydrated quickly with 95% ethanol (Ⅰ → Ⅱ), 100% ethanol (Ⅰ → Ⅱ) in sequence, cleared with xylene (Ⅰ → Ⅱ, 5 minutes each), and sealed with neutral balsam. The analysis method needs to be combined with optical microscope observation: evaluate the integrity of epidermal regeneration (distance between re-epithelialization length and wound edge), infiltration density of dermal inflammatory cells (neutrophils, lymphocytes) (degree of deep staining and aggregation of cell nuclei), thickness of granulation tissue (number of layers of newly formed capillaries and fibroblasts), and arrangement state of collagen fibers (continuity of eosin-stained area) under 100× field of view; quantify the formation of epidermal stratum corneum, regeneration of appendages such as hair follicles / sweat glands, etc. under 400× high power microscope, and measure the thickness of the epidermal layer (μm), proportion of inflammatory area (%) and granulation tissue maturity score (based on cell density and collagen deposition) through image analysis software (such as ImageJ), and compare the differences between the experimental group and the control group by statistical methods to verify the promoting effect of the composite scaffold on the repair of diabetic wound tissue.

[0170] 1.3. Masson staining

[0171] The experimental steps of Masson staining include tissue processing, section preparation and specific staining: First, fix the diabetic wound tissue samples with 4% paraformaldehyde for 24 - 48 hours, and then successively perform gradient ethanol dehydration (70% → 80% → 90% → 95% → 100%, 1 hour for each concentration), xylene transparency (30 minutes for each of I and II), and paraffin impregnation and embedding (3 times with molten paraffin at 60 - 65°C, 1 hour each time). After embedding, use a microtome to prepare continuous sections with a thickness of 4 - 6 μm, attach them to polylysine-coated slides, and bake at 60°C for 2 hours to enhance adhesion; before staining, dewax the sections with xylene (10 minutes for each of I and II), rehydrate them with gradient ethanol to distilled water, and then immerse them in Weigert iron hematoxylin stain (Solution A: 1 g of hematoxylin + 100 ml of absolute ethanol; Solution B: 4 ml of 29% ferric chloride + 1 ml of concentrated hydrochloric acid + 95 ml of distilled water, mix immediately before use) and stain for 5 - 10 minutes to make the cell nuclei dark blue, rinse with running water for 5 minutes, differentiate with 1% hydrochloric acid ethanol for 3 - 5 seconds to remove non-specific cytoplasmic staining, and then blue with a weak alkaline solution (such as 0.5% ammonia water) for 30 seconds; after washing with water, immerse them successively in Ponceau acid fuchsin stain (0.7 g of Ponceau, 0.3 g of acid fuchsin, 1 ml of glacial acetic acid, 100 ml of distilled water) for 5 - 10 minutes to preliminarily stain the collagen fibers. After rinsing with 0.2% aqueous glacial acetic acid solution, treat them with 1% phosphomolybdic acid aqueous solution for 5 - 10 minutes for color separation to remove the red background of muscle fibers, and then quickly immerse them in aniline blue stain (2 g of aniline blue, 2 ml of glacial acetic acid, 100 ml of distilled water) and stain for 2 - 5 minutes to make the collagen fibers blue. Finally, quickly rinse with 0.2% glacial acetic acid, dehydrate with gradient ethanol (95% → 100%, 30 seconds each), make them transparent with xylene and seal with neutral gum.

[0172] The analysis method needs to combine microscopic observation and image processing software: Evaluate the overall distribution and arrangement density of collagen fibers (the proportion of the blue area) under a 100× field of view, and observe the thickness, orientation of collagen bundles and their spatial relationship with surrounding cells (red muscle fibers, dark blue-purple cell nuclei) under a 400× high-power microscope; Use ImageJ software to set the color threshold to separate the blue collagen area, calculate the percentage (%) of the collagen area in the dermal layer of the wound surface, and quantify the anisotropy index (0 - 1, 0 means completely disordered, 1 means highly oriented) of collagen fiber arrangement through a directionality analysis plug-in (such as Directionality), and compare the maturity of newly formed collagen in the experimental group and the control group (coarse and tight bundles vs. fine and loose networks); At the same time, combine a histopathological scoring system (such as Semi - quantitative scoring) to evaluate the uniformity of collagen deposition (0 - 3 points: 0 = no deposition, 1 = focal, 2 = moderately continuous, 3 = extensive and uniform), combine statistical methods to verify the promoting effect of the PDA / Col composite scaffold on collagen remodeling in diabetic wounds, and correlate the expression of inflammatory factors with the maturity of collagen.

[0173] 1.4, Histone Western blot

[0174] After removing the tissue block, immediately crush and grind it, and then follow the cell wb experimental protocol. If time is tight, use cryotubes to store the tissue in liquid nitrogen in a timely manner and conduct the experiment the next day. Moreover, liquid nitrogen is more conducive to tissue crushing.

[0175] 1.5, Immunohistochemistry

[0176] The experimental steps of immunohistochemistry include tissue processing, antigen retrieval, antibody incubation, and color development detection: First, dewax the paraffin sections of diabetic wounds with xylene (10 minutes each for I→II), rehydrate with gradient ethanol to distilled water, and then place them in citrate buffer (10 mM) at pH 6.0 for heat-induced antigen retrieval (treated at 121°C for 15 minutes in a pressure cooker or heated to boiling in a microwave oven at high power and maintained for 20 minutes). After natural cooling to room temperature, rinse 3 times with PBS (pH 7.4) (5 minutes each time); to block the activity of endogenous peroxidase, immerse the sections in 3% H2O2 methanol solution (in the dark) for 15 minutes, wash with PBS, and then use 5% normal goat serum (or serum homologous to the secondary antibody) to block at room temperature for 30 minutes to reduce non-specific binding; pour off the blocking solution, add diluted primary antibody (such as anti-COL1A1, COL3A1, α-SMA, etc., the dilution ratio depends on the antibody instruction manual, usually 1:100 - 1:500) to cover the tissue area, incubate overnight at 4°C in a wet box (or for 1 - 2 hours at 37°C), and after rewarming the next day, elute the unbound antibody with PBS (3×5 minutes); add HRP-labeled secondary antibody (such as anti-rabbit / mouse IgG, diluted 1:200 - 1:1000) and incubate at room temperature for 1 hour, wash thoroughly with PBS, then cover the sections with DAB chromogenic solution (TBS buffer containing 0.05% DAB and 0.03% H2O2), control the chromogenic time under the microscope (usually 1 - 5 minutes, until the target area shows brownish-yellow color without background staining), and immediately terminate the reaction with running water; counterstain the cell nuclei with hematoxylin for 1 - 2 minutes, differentiate with hydrochloric acid ethanol and blue with ammonia water, dehydrate with gradient ethanol, clear with xylene, and seal with neutral gum.

[0177] The analysis method needs to combine an optical microscope and image analysis software: Observe the positive signal distribution (membrane, cytoplasm or nuclear localization) of the target protein (such as vascular endothelial cells labeled with α-SMA) under a 200× or 400× field of view, use ImageJ software to set the color threshold to segment the positive staining area (DAB brown), and calculate the percentage of the positive area (% positive area) or the integrated optical density (IOD) to quantitatively express the level; for semi-quantitative evaluation, use the H-score scoring system (staining intensity score 0-3: 0 = no staining, 1 = weak, 2 = medium, 3 = strong; positive cell ratio 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 (omitting the primary antibody or replacing it with isotype IgG) needs to be set to exclude non-specific staining, and multi-parameter correlation analysis is carried out in combination with tissue pathological features (such as the degree of inflammation in HE staining) to verify the regulatory effect of the PDA / Col composite scaffold on inflammatory factors and macrophage polarization in diabetic wounds.

[0178] II. Experimental results

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

[0180] As Figure 19 shown, the wounds treated with the PDA / Col composite scaffold and collagen alone achieved wound healing at day 14, while there were still obvious wounds in the model group. Briefly, the repair rates of the collagen group and the PDA / Col composite scaffold group were basically the same. To analyze the differences between the two, this study continued with tissue staining at day 21.

[0181] As Figure 21 shown, the HE and Masson results indicated that except for the model group, the wound recovery in other experimental groups was relatively satisfactory. The collagen deposition in the PDA / Col group, Col group and PDA group was more than that in the model. The difference between the polydopamine group and the collagen group and the composite scaffold group lies in the amount of blood vessel and hair follicle formation.

[0182] As Figure 21 seen, obvious thickening of the epidermis occurred in the collagen group, while there were fewer hair follicles. It was not like the composite scaffold group, where there were fewer capillaries, more hair follicles, and the epidermis was not as thick. In terms of inflammatory eosinophil infiltration, compared with the simple collagen group, the reduction in the composite scaffold and polydopamine groups was more significant. Only the PDA / Col composite scaffold group could form the extracellular matrix of tissue wounds while rapidly restoring the wound.

[0183] Meanwhile, this study investigated the protein expression in the wound of the animal inflammatory tissue on the 7th day ( Figure 20 ). This study found that on the 7th day, the wound inflammation in the model group still persisted. However, after using collagen and polydopamine, both iNOS and TNF-α decreased. After using collagen, the expression of α-SMA increased. This result indicates that the use of the collagen composite scaffold reduces the persistent inflammation of the diabetic wound while promoting more fibroblasts to transform into myofibroblasts, which is beneficial to wound contraction. This result corresponds to the wound healing cycle diagram.

[0184] 2.2 Immunohistochemistry of wound healing on the 21st day

[0185] As Figure 22 shown, observing the tissue protein expression level of the wound on the 21st day, the expression of α-SMA in the Col group was higher, and the model group was only second to it. While the expression was lower in the group with the addition of polydopamine. In the immunohistochemistry test, α-SMA can be used to label capillaries. It can be seen from the figure that there are more small pores in the model group and the Col group. It may be because on the 21st day, the newly formed capillaries in the wound healing process did not gradually disappear but continued to remain to provide nutrients to this area. Moreover, type III collagen, which is loose, is still the main component during the tissue repair process. While the capillaries in the PDA group and the PDA / Col group were basically degenerated, and at the same time, there was more deposition of type I collagen.

[0186] The above embodiments are the preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A preparation method of a polydopamine and collagen composite scaffold, characterized in that: It includes the following steps: (1) First, add an appropriate amount of absolute ethanol to deionized water, then dropwise add an appropriate amount of ammonia water, and 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 and stir at room temperature to obtain crude polydopamine; (2) Centrifuge the crude polydopamine at a low speed to remove insoluble impurities or large particle substances, then centrifuge at a high speed and collect the precipitate; then wash it once with ethanol first, and then wash it with deionized water. After washing clean, collect the precipitate and freeze-dry it to obtain purified polydopamine; (3) Then weigh an appropriate amount of polydopamine and add it to the 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.

2. The preparation method of a polydopamine and collagen composite scaffold according to claim 1, characterized in that: The specific operation of step (1) is: first add 30 - 50 mL of absolute ethanol to 80 - 100 mL of deionized water, then dropwise add 1 - 3 mL of ammonia water, and 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 and stir at room temperature for 8 - 16 h to obtain crude polydopamine.

3. The preparation method of a polydopamine and collagen composite scaffold according to claim 1, wherein: In step (2), the rotation speed of the first centrifugation is 1500 - 2500 rpm, the centrifugation time is 3 - 7 min, the rotation speed of the second centrifugation is 10000 - 12000 rpm, the centrifugation time is 10 - 20 min, and the mass fraction of ethanol is 25% - 35%.

4. The preparation method of a polydopamine and collagen composite scaffold according to claim 1, characterized in that: 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.

5. A composite scaffold of polydopamine and collagen, characterized in that: The polydopamine and collagen composite scaffold is prepared according to the preparation method described in any one of claims 1 - 4.

6. Use of a polydopamine and collagen composite scaffold as described in claim 5 in the preparation of a biological product for promoting the healing of diabetic wounds.

7. The application according to claim 6, characterized in that: The composite scaffold, namely the polydopamine and collagen composite scaffold, can promote cell proliferation, contribute to cell migration, and is beneficial to the expression of CD31 and VEGFA proteins by HUVEC cells.

8. The application according to claim 6, characterized in that: The composite scaffold, namely the polydopamine and collagen composite scaffold, can up-regulate the CD31 and VEGFA proteins of HUVEC cells under high glucose and inflammatory environments, and at the same time reduce the iNOs cytokine, which is beneficial to the formation of new blood vessels.

9. The application according to claim 6, wherein: The composite scaffold, namely the polydopamine and collagen composite scaffold, reduces the expression of inflammatory factors in RAW264.7 cells by interfering with the inflammatory factor receptors TNFR1 and TLR4.

10. The application according to claim 6, wherein: The composite scaffold, namely the polydopamine and collagen composite scaffold, can inhibit the phosphorylation of the MAPK / NF-κB signaling pathway.

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