Novel collagen and application thereof

Abstract

The invention discloses novel collagen and application thereof. The preparation method of the novel collagen comprises the following steps: pre-freezing a collagen solution to obtain porous collagen; then, in a freezing environment, soaking the porous collagen in a solution containing a cross-linking agent to obtain collagen gel; the cross-linking agent is EDC and NHS (N-Hydroxysuccinimide); and then freeze-drying the soaked collagen material. Compared with the existing commercial dressing, the novel collagen disclosed by the invention is excellent in liquid export speed, water absorption performance, biocompatibility and exudate management performance when being used as the dressing. In addition, the novel collagen cross-linked with DNA has the effects of regulating CD4 + T cells, chemotactizing the CD4 + T cells, regulating immune cells to play a secretion function, inhibiting scars, promoting wound healing capacity and repairing skin.

Description

Technical Field

[0001] The invention relates to medical material technology, and in particular to a novel collagen and its application as a dressing, especially a wound dressing for trauma and burns. Background Art

[0002] Collagen dressings play a certain role in promoting the healing of burn wounds. However, when facing severe burn wounds, collagen dressings still have shortcomings such as low efficiency in draining excess exudate, weak immune regulation ability, and limited ability to promote scarless healing. Therefore, it is necessary to modify collagen dressings to enhance their functional properties. Summary of the invention

[0003] In view of the defects or shortcomings of the prior art, the present invention provides a new type of collagen.

[0004] To this end, the novel collagen proposed in the present invention is prepared by the following method:

[0005] The collagen solution is pre-frozen to obtain porous collagen;

[0006] Then, in a freezing environment, the porous collagen is immersed in a solution containing a cross-linking agent to obtain a collagen gel; the cross-linking agent is EDC and NHS;

[0007] Then the collagen gel is freeze-dried to prepare the novel collagen.

[0008] An optional solution is that the pre-freezing condition is to cool down from room temperature to freezing temperature at a uniform rate. A further optional solution is that the pre-freezing condition is to cool down from room temperature to freezing temperature of -196 to -180°C at a uniform rate.

[0009] An optional solution is that the freezing environment is -30 to -10°C.

[0010] An optional solution is that the pre-freezing time is 2 to 5 minutes; the soaking treatment time is 12 to 24 hours; and the freeze-drying time is 24 to 48 hours.

[0011] An optional solution is to soak the porous collagen in a solution containing a cross-linking agent under a freezing environment, and then soak it in water to obtain a collagen gel.

[0012] In a further embodiment, DNA is cross-linked to the novel collagen. In a specific embodiment, DNA can be cross-linked to the novel collagen material using existing methods.

[0013] Compared with existing commercial dressings, the novel collagen of the present invention is excellent in liquid extraction speed, water absorption performance, biocompatibility and exudate management performance as a dressing.

[0014] In addition, the novel collagen cross-linked with DNA of the present invention has the functions of regulating CD4+T cells, chemotactic CD4+T cells, regulating the secretion function of immune cells, inhibiting scars, promoting wound healing and repairing skin.

[0015] Based on this, the present invention also provides the use of the above-mentioned novel collagen for preparing medical dressings including traumatic wound dressings or burn wound dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are scanning electron microscope microscopic morphology images of ACol prepared in Example 1 of the present invention. Figure A is a scanning electron microscope morphology image of ACol with different directional pore widths and a collagen dressing with random pores; Figures B and B are respectively: the cross-sectional porosity and pore width or pore size were calculated using ImageJ software based on the scanning electron microscope images.

[0017] Figure 2 These are scanning electron microscope microscopic morphology images of Cryo-ACol and DCA prepared in Example 1 of the present invention. From left to right, the columns of images are: surface microscopic morphologies of Cryo-ACol and DCA observed under low, medium, and high magnification scanning electron microscopes.

[0018] Figure 3 Energy dispersive X-ray spectroscopy characterization of Cryo-ACol and DCA of different concentrations prepared in Example 1 of the present invention.

[0019] Figure 4 This is the distribution of cross-linked DNA in the DCA prepared in Example 1 of the present invention.

[0020] Figure 5 This is a characterization of the amount of DNA cross-linking in the DCA prepared in Example 1 of the present invention.

[0021] Figure 6 The characteristic peaks of phosphorus element characterized by X-ray photoelectron spectrometer in DCA prepared in Example 1 of the present invention.

[0022] Figure 7 Fourier transform infrared spectroscopy characterization of Cryo-ACol prepared in Example 1 of the present invention and DCA of different concentrations.

[0023] Figure 8 This is the characterization of the liquid absorption speed of ACol prepared in Example 1 of the present invention.

[0024] Fig. 9 This is a characterization of the liquid absorption rate of Cryo-ACol and DCA prepared in Example 1 of the present invention.

[0025] Fig.10Characterization of the liquid absorption of ACol, Cryo-ACol and DCA prepared in Example 1 of the present invention; Figure A shows the maximum liquid absorption of ACol with different directional channel widths, collagen dressings with chaotic pores, and commercial collagen dressings; the two figures in B show the maximum liquid absorption of ACol and Cryo-ACol with a directional channel width of 120 μm; and the maximum liquid absorption of DCA dressings with different concentrations.

[0026] Fig.11 The biocompatibility characterization of Cryo-ACol and DCA prepared in Example 1 of the present invention; Figure A shows live-dead staining of cells after co-culture of Cryo-ACol, DCA and mouse fibroblasts, and Figure B shows the cell proliferation rate after co-culture of Cryo-ACol, DCA and mouse fibroblasts.

[0027] Fig.12 The in vitro simulated skin method of Cryo-ACol, DCA, commercial collagen dressing (Commercial), and ACol in Example 2 of the present invention is used to characterize the exudate intervention characteristics.

[0028] Fig.13 This is a finite element simulation of the exudate intervention characteristics of DCA and commercial collagen dressings in Example 2 of the present invention.

[0029] Fig.14 The exudate management ability of DCA was tested by the in vivo fluorescent labeled simulated exudate method of Cryo-ACol, DCA, commercial collagen dressing, and ACol in Example 2 of the present invention.

[0030] Fig.15 It is a transcriptome sequencing characterization of the immunoregulatory ability of collagen dressing and DCA in Example 3 of the present invention; Figure A is a differential gene heat map; Figure B is a KEGG enrichment map of differential genes, in which the ordinate is: different signal pathways enriched by KEGG; Figure C is a GO enrichment chord diagram, in which the circumferential labels represent: T cell-related genes and their corresponding GO entries; Figure D is genes related to CD4+T cells, in which Figure i is a GO enrichment map, and Figure ii is a specific gene expression heat map.

[0031] Fig.16 This is the immunofluorescence characterization of the chemotaxis of CD4+T cells by collagen dressing and DCA in Example 3 of the present invention.

[0032] Fig.17 This is an ELISA test of the collagen dressing and DCA on cytokines secreted by CD4+T cells in Example 3 of the present invention.

[0033] Fig.18 This is the in vitro scar inhibition test of the collagen dressing and DCA in Example 3 of the present invention.

[0034] Fig.19 The photographic representation of the healing of deep II degree burns in rats by the commercial collagen dressing, DCA, Cryo-ACol, and DNA-crosslinked commercial collagen dressing in Example 4 of the present invention, Com represents: commercial collagen dressing (commercial), DNA+Com represents: commercial collagen dressing treated with DNA crosslinking.

[0035] Fig. 20 The present invention shows histological staining to characterize the healing of deep II degree burns in rats by the commercial collagen dressing, DCA, Cryo-ACol, and DNA cross-linked commercial collagen dressing in Example 4 of the present invention.

[0036] Fig.21 The immunoregulatory characterization of the collagen dressing and DCA on rat burn wounds in Example 4 of the present invention; A and B are the distribution and relative expression levels of CD4 (red), IL-10 (green), and cell nucleus (blue), respectively.

[0037] Fig. 22 This is a characterization of the regulation of the secretion of α-SMA, a scar marker, on rat burn wounds by collagen dressing and DCA in Example 4 of the present invention; A and B are: immunofluorescence staining of α-SMA and its relative expression level, respectively.

[0038] Fig.23 The present invention is a characterization of the correlation between the scar-free healing effect and the immune regulation performance of the DCA dressing of Example 4; A is a visual picture of the wounds treated with DCA dressings on days 0, 7, 14 and 28 after the back skin burn defects of normal mice and mice treated with Anti-CD4 antibodies; B is a statistical picture of the wound size on days 7 and 14 in Figure A; C is a statistical picture of the scar size on day 28 in Figure A; D is a HE and Masson staining of the skin sample on day 28 in Figure A; E is a statistical picture of the number of hair follicles according to Figure D. DETAILED DESCRIPTION

[0039] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0040] The initial collagen solution of the present invention can be homemade collagen fiber or commercially available product. When homemade, it can be obtained by dissolving collagen fibers in a solvent (such as acetic acid, PBS buffer, etc.). The collagen fibers can be selected from animal collagen or commercially available collagen. Animal collagen is such as rat tail collagen, bovine Achilles tendon collagen, etc. Commercially available collagen is specifically such as Bonag collagen.

[0041] The solution containing the crosslinking agent used in the present invention is specifically configured with a solvent and a crosslinking agent that can dissolve the crosslinking agent but does not affect the process and effect of the present invention. In the specific scheme, those skilled in the art can select a suitable solution concentration to achieve the effect of the present invention. The role of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in the crosslinking agent used in the present invention includes but is not limited to activating the carboxyl groups on the aspartic acid and glutamic acid residues in collagen to chemically react to form a catalyzer-unstable amine-reactive O-acylisourea intermediate; the role of N-hydroxysuccinimide (NHS) includes but is not limited to: enhancing the stability of the EDC crosslinked product by reacting with the amine-reactive O-acylisourea intermediate to form a more stable amine-reactive NHS ester.

[0042] The pre-freezing and freezing environment described in the present invention refers to a temperature environment below 0°C that affects the collagen configuration or material microstructure. Based on the disclosure of the present invention, those skilled in the art can select and optimize the specific temperature of each step in the preparation process (including the freezing temperature and freezing environment described herein). The purpose of freeze drying is to dry the material under the condition of ensuring the structure of the prepared material, and the freeze drying temperature environment can select a suitable temperature condition.

[0043] The novel collagen of the present invention is cross-linked with DNA by soaking the novel collagen material in a buffer containing DNA and a phosphate group activator, and obtaining a collagen material cross-linked with DNA after incubation. The role of the phosphate group activator used is to form a phosphate group activated intermediate ester with the phosphate group on the DNA, and to maintain the stability of the intermediate ester, so that the phosphate group on the DNA is cross-linked with the amine group on the collagen material. Those skilled in the art can select a suitable substance as a phosphate group activator for the reaction system, such as but not limited to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The buffer can be selected from commonly used buffers in the biomedical field, such as 2-(N-morpholine) ethanesulfonic acid (MES) buffer, phosphate buffer, etc. The type of DNA cross-linked in the material of the present invention is not limited, and can be: total DNA such as calf thymus DNA, salmon sperm DNA, etc., or a DNA fragment of a specific sequence.

[0044] The raw materials used in the following examples are all commercially available products.

[0045] Example 1: Preparation and characterization of DNA cross-linked directional porous collagen dressing (Cryo-Acol and DCA)

[0046] 1.1 Preparation of DCA

[0047] 1.1.1 Preparation of oriented porous collagen

[0048] Type I collagen fibers (Bonage) were dissolved in 0.1 M glacial acetic acid to prepare a 20 mg / mL collagen solution;

[0049] A copper column reaction table was placed in a low-temperature constant-temperature reaction bath and a heat-insulating material was placed around it to seal it, so as to construct a directional temperature gradient reaction system. The collagen solution was placed in a polytetrafluoroethylene tubular mold with a diameter of 1 cm, a height of 1 cm, and a thickness of 0.2 cm. The mold was placed on the copper column reaction table. Liquid nitrogen at -196°C was added to the low-temperature constant-temperature reaction bath. The relative height between the copper column reaction table and the liquid nitrogen level was adjusted to adjust the cooling rate to 4, 2, and 1°C / min, respectively, so as to adjust the growth rate of ice crystals in the collagen solution. After 3 minutes, directional porous collagen was obtained. The directional porous collagen was placed in a -80°C freeze dryer and freeze-dried for 24 hours to obtain ACol.

[0050] 1.1.2 Preparation of directional porous collagen dressing (Cryo-ACol)

[0051] According to the preparation method of oriented frozen collagen in 1.1.1, oriented porous collagen was constructed at a cooling rate of 1°C / min;

[0052] Then, the oriented porous collagen (not frozen) was placed in a cross-linking solution at -20°C for 24 hours; the solution containing the cross-linking agent in this embodiment was prepared by mixing 75% ethanol, 200mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 50mM N-hydroxysuccinimide (NHS) at -20°C; after the cross-linking was completed, a frozen collagen gel was formed, and the collagen gel was soaked in ultrapure water overnight to exchange excess glacial acetic acid, ethanol and cross-linking agent;

[0053] Finally, the collagen was placed in a -80°C freeze dryer for 48 h to form Cryo-ACol.

[0054] 1.1.3 Preparation of DCA

[0055] 50 mg of calf thymus DNA (MilliporeSigma, Burlington, MA, USA) was dissolved in 50 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0), and then 625 mg of EDC and 750 mg of NHS were added and mixed for 30 min; EDC and NHS were selected as the phosphate group activators in this example, and EDC formed a phosphate group-activated intermediate ester with the phosphate group on the DNA, and NHS maintained the stability of the intermediate ester to ensure that the DNA collagen material was cross-linked through the phosphoamido group.

[0056] After the pH of the mixture was increased to 7.3 with phosphate buffered saline (PBS), Cryo-ACol collagen dressing with a diameter of 7 mm and a thickness of 2 mm was immersed in the mixture and incubated in the solution at room temperature for 2 hours. Phosphoamidite bonds were formed between the phosphate groups in the DNA molecules and the amino groups in the collagen molecules, so that the DNA was cross-linked to the scaffold. The collagen scaffold was rinsed with DNase-free water to remove the free DNA on the surface of the collagen scaffold to obtain the modified collagen scaffold DNA-Cryo-ACol (DCA).

[0057] 1.2 Characterization

[0058] 1.2.1 Morphological characterization

[0059] The surface morphologies of ACol, Cryo-ACol, and DCA were characterized using a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan) at 5 kV.

[0060] The samples were dehydrated in 50%, 70%, 80%, 90% and 100% ethanol solutions for 15 min respectively, placed in hexamethyldisilane for 30 min, and then air-dried in a fume hood. The dehydrated samples were sprayed with gold before testing.

[0061] The results are as follows Figure 1 As shown: Directed pore structures with different pore widths appeared in the ACol group. As the freezing rate decreased, the pore width increased from 51.7±0.78μm to 120.0±5.7μm. The average pore sizes of random pore collagen dressings were 68.0±11.8μm and 67.0±5.7μm, respectively.

[0062] Figure 2 The yellow circles represent the microporous structure on the vertical pore wall. By comparing DCA and Cryo-ACol, it can be found that the cross-linking of DNA did not change the structural characteristics of the directional porous dressing. In the high-magnification SEM image, it can be found that the collagen surface has a coating-like structure after cross-linking DNA, and the horizontal stripes are not obvious, which preliminarily verifies that the DNA is modified on the collagen surface.

[0063] 1.2.2 Characterization of cross-linking

[0064] 1.2.2.1 Energy dispersive X-ray spectroscopy

[0065] The samples were processed according to the SEM sample preparation method in 1.2.1, and elemental analysis was performed using energy dispersive X-ray spectroscopy (EDAX, S-4800, Hitachi, Tokyo, Japan).

[0066] EDAX results are as follows Figure 3As shown: the proportion of phosphorus P in the DCA surface elements increased, and increased with the increase of cross-linking concentration, which once again verified the binding of DNA to collagen. The original C, N, and O conditions did not change significantly as shown by the EDAX graph.

[0067] 1.2.2.2 Immunofluorescence staining and methyl green adsorption experiment

[0068] Type I collagen fibers in DCA were labeled with rabbit anti-mouse Anti-collagen I antibody (Ab34710, Proteintech, Wuhan, Hubei, China); SYTOX TM DNA in DCA was labeled with dapoxetine green (S34860, ThermoFisher Scientific, Waltham, MA, USA). After washing with PBS, the samples were observed with a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Hesse, Germany). The fluorescence intensity of DNA was semi-quantified using ImageJ software.

[0069] The results are as follows Figure 4 , 5 As shown in the figure, as the cross-linking concentration increases, the distribution of DNA on the collagen surface gradually becomes uniform, and its fluorescence intensity gradually increases. In addition, the DNA content in DCA was measured by methyl green adsorption experiment to quantify the DNA content in DCA, as shown in the figure. Figure 5 shown.

[0070] 1.2.2.3 X-ray photoelectron spectroscopy detection

[0071] The surface elements of Cryo-ACol and DCA were analyzed using an X-ray photoelectron spectrometer (K-Alpha, ThermoFisher Scientific, Waltham, MA, USA).

[0072] The DCA surface should have the phosphorus element in the DNA phosphate backbone. The analysis results are as follows Figure 6 As shown: The peak value of the P element on the surface of DNA-Col increased significantly, which once again verified that DNA was cross-linked to the collagen scaffold.

[0073] 1.2.2.4 Fourier transform infrared spectroscopy detection

[0074] Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR, FTIR-8400S, Japan) was used to determine the functional groups in Cryo-ACol and DCA. -1 Within the range of 4cm -1 The resolution was scanned 32 times.

[0075] The Fourier transform infrared spectroscopy results are as follows Figure 7 As shown, the wavelength is 1143–1213 cm -1 The stretching vibration of POP indicates the presence of DNA; at 950-1068cm -1 The stretching vibration of the PNC bond appeared at the position, indicating that a stable chemical bond was formed between DNA and collagen, indicating the phosphoramide bond formed between the phosphate groups in DNA and the amino groups of collagen fibrils.

[0076] 1.2.3 Characterization of water absorption performance

[0077] 1.2.3.1 Water absorption speed test

[0078] The absorption of 200 μL of blue ink by each dressing was recorded using a video camera.

[0079] The results are as follows Figure 8 As shown in the figure, as the width of the ACol channel increases, the liquid extraction speed increases. In the same way, the time from the beginning of liquid absorption to the liquid extraction to the top of the dressing is recorded, and the formula is: liquid absorption speed = 200 / liquid absorption time.

[0080] The results are as follows Fig. 9 Shown: Compared with commercial collagen dressing (Bonager TM ) (the corresponding experimental group in the figure is commercial), the liquid extraction time of Cryo-ACol is shortened by 50 times, and the liquid extraction speed of Cryo-ACol is also significantly improved compared with the 120μm group with the fastest extraction speed in ACol. In addition, DNA modification does not affect the water absorption performance of the material, and DCA also has an excellent liquid extraction speed.

[0081] 1.2.3.2 Water absorption test

[0082] The weight gain of the dressing during the absorption process was recorded with an electronic balance, and the final absorption capacity of different dressings was calculated by the formula: Absorption capacity = maximum liquid absorption volume (g) / dressing bottom area (cm 2 ).

[0083] The results are as follows Fig.10 Shown: Compared with commercial collagen dressing (Bonager TM ) compared with the commercial collagen dressing (BonagarTM ), the maximum absorption volume of Cryo-ACol doubled, and the modification of DNA did not affect the maximum absorption volume. DCA had an excellent absorption volume, which was greater than the amount of exudate produced in severe burns.

[0084] 1.2.4 Biocompatibility testing

[0085] Different concentrations of DCA dressings, Cryo-ACol, and commercial collagen dressings (Bonagar TM ) were co-cultured with mouse fibroblast L929 cell line for 48 h, and then the cell compatibility of the material was detected by live and dead cell staining and CCK8 assay.

[0086] The results are as follows Fig.11 As shown: 1 mg / mL DCA has the best biocompatibility, and this concentration of DCA was used for subsequent experiments.

[0087] Example 2: Testing of exudate management performance of DCA

[0088] 2.1 In vitro simulated skin method to detect DCA exudate management ability

[0089] The pig skin was cut into 2 cm × 2 cm × 0.4 cm blocks, and a drop of blue ink was dripped on the surface of the pig skin block; a commercial collagen dressing (Bonager TM ), ACol, Cryo-ACol, and DCA dressings were placed on droplets on the surface of pig skin to absorb the droplets; the dressing absorption process was recorded with a camera, and one minute later, the dressing was removed; and the ink remaining on the surface of the pig skin was photographed.

[0090] The results are as follows Fig.12 As shown: the characteristics of different dressings absorbing liquid on the pig skin surface: ① Different directions: the direction of liquid absorption of the random pore dressing is from the center to the surroundings (i pink triangle), while the directional pore structure is from the surroundings to the center (ii-iv green triangle); ② Whether liquid impregnation occurs: the random pore dressing will cause the droplets to squeeze to the edge and impregnate (i yellow arrow), while the directional pore structure is not obvious (ii-iv red arrow); ③ Different liquid residues: According to the depth of the color of the residual liquid, it can be found that both the random pore dressing and the simple directional ACol have more residual liquid, while the DCA group has no obvious liquid residue. This shows that DCA has special exudate intervention characteristics, which can give the dressing the advantages of reducing liquid impregnation, improving liquid extraction efficiency, and increasing liquid extraction.

[0091] 2.2 Verification of DCA exudate absorption characteristics by finite element simulation

[0092] The finite element software simulated the viscosity (1.9 mPa·s) and density (1.03 g / cm3 ) and depict the flow of exudate within the dressing.

[0093] The results are as follows Fig.13 As shown: There is no difference in the liquid flow rate in each direction of the commercial collagen dressing group at the moment of liquid entry, but as time goes by, the flow rate in the middle is higher than that at the edge; while in DCA, the flow rate around it is higher than that in the center at the moment of liquid entry, and this phenomenon becomes more obvious as time goes by. In addition, the porous structure can promote the flow of liquid between different channels, suggesting that this flow characteristic may be related to its special directional channels and microporous structure.

[0094] 2.3 In vivo fluorescent labeling simulated exudate method to detect DCA exudate management ability

[0095] A full-thickness wound defect model was established using 6-week-old C57BL / 6J mice. General anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and ibuprofen was given to mice for postoperative analgesia from 24 h before surgery to 10 days after surgery. In vivo, a full-thickness wound defect model with a circular skin injury (7 mm in diameter) was constructed on the back of the mouse. Fetal bovine serum labeled with rhodamine 123 was used as an exudate simulating agent, and 30 μL of the exudate simulating agent was dropped into the mouse skin defect. Photos were taken before and after dressing treatment under ultraviolet irradiation (λ=365 nm).

[0096] The results are as follows Fig.14 As shown: Commercial collagen dressings will cause exudate residue and infiltration (white arrow + white dotted line), simple oriented ACol will have liquid residue (white arrow), and the oriented porous group has neither of the above two phenomena, indicating that DCA can not only reduce the residue of exudate but also inhibit the outward infiltration of exudate.

[0097] Example 3: Characterization of the immunomodulatory ability of DCA

[0098] 3.1 Transcriptome sequencing and analysis

[0099] According to the "Burn (Scald) Animal Model Preparation Specification", a desktop ultra-temperature controlled scald device (YLS-5Q, Anhui Zhenghua Bio-Instrument Co., Ltd., China) was used to prepare a deep second-degree burn wound model on the back of mice. Collagen dressings and DCA dressings were applied. Total RNA was extracted from the wound 7 days later, and the gene expression profile was obtained using the Illumina HiSeq sequencing platform. RNA quality control was performed and gene location distribution was determined. FPKM (fragments per kilobase of transcript per million, DNA fragments per million mapped reads per kilobase of transcript) values ​​were analyzed using R software for the relative abundance of expressed genes. Next, principal component analysis was performed on the RNA data to further verify the biological repetition between specimens.

[0100] The above results were obtained using R software ( https: / / www.r-project.org / )Analyze samples based on gene expression levels.

[0101] Using DESeq2( https: / / bioconductor.org / ) were used for differential gene expression analysis. If P < 0.05 and log2 (foldchange) > |1|, the results were considered to have changed significantly. Hierarchical cluster analysis was performed using R software.

[0102] The ggplot2 R package was used to draw a volcano plot to show the overall distribution of differentially expressed genes. GO (gene ontology) enrichment analysis was performed on the differentially expressed genes using R software.

[0103] The results are as follows Fig.15 As shown: Transcriptome sequencing found that DCA can significantly upregulate immune-related genes, and it is mainly enriched in the biological functions of CD4+T cells in T cells, which preliminarily proves that DCA has the ability to regulate CD4+T cells.

[0104] 3.2 Immunofluorescence detection of CD4+ T cell chemotaxis

[0105] The mouse burn wound model was treated with untreated collagen dressings and DCA dressings; the dressings were harvested 3 days later and fixed with 4% paraformaldehyde; the fixed specimens were embedded in paraffin, sliced ​​into 4 μm thick slices, and permeabilized with 0.15% Triton X-100; CD4+ T cells were stained with fluorescein isothiocyanate-conjugated rat anti-mouse CD4 antibody (96127S, Cell Signaling Technology, MA, USA); all specimens were stained with 4′, 6-diamino-2-phenylindole (DAPI; Invitrogen, ThermoFisher Scientific, MA, USA); images were captured using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Hesse, Germany); and relative fluorescence intensity was calculated using ImageJ software.

[0106] The results are as follows Fig.16 As shown: The number of CD4+T cells in DCA dressing increased significantly, proving that DCA can chemotactic CD4+T cells.

[0107] 3.3 Enzyme-linked immunosorbent assay

[0108] CD4 + T cells were isolated from the spleen of blank mice and co-cultured with dermal explants treated with DCA or Cryo-Acol for 24 h. The supernatant was collected after centrifugation, and the secretion of IL-10, TGF-β, IL-17, and IFN-γ was detected using ELISA kits (E-EL-M0046, E-EL-0162, E-EL-M0047, E-MSEL-M0007, Elabscience, Wuhan, China).

[0109] The results are as follows Fig.17 As shown: DCA can promote the secretion of IL-10 and IFN-γ, especially the secretion of IL-10, which is significantly enhanced, and there is no significant difference in the effect on TGF-β and IL-17, indicating that the dressing has the function of regulating the secretion function of immune cells.

[0110] 3.4 DCA Inhibits Scar Detection in Vitro

[0111] 929 mouse fibroblasts (CL-0137, Pricella, Wuhan, Hubei, China) were activated by TGF-β (15 ng / mL, HZ-1011, Proteintech, Wuhan, China); activated fibroblasts were co-cultured with Cryo-ACol, DCA, Cryo-ACol and chemotactic T cells (Cryo-ACol+T), and DCA and chemotactic T cells (DCA+T) for 24 h. Then, cells were fixed with 4% paraformaldehyde (n=3) and permeabilized in 0.15% Triton X-100; then, cells were incubated with anti-α-sma antibody (1:500, GTX100034, Genetex, CA, USA) at 4°C overnight; primary antibody was labeled with Alexa Fluor488 goat anti-rabbit secondary antibody (33106ES60, easen Biotechnology, Shanghai, China); images were captured using confocal laser scanning microscopy.

[0112] The results are as follows Fig.18 As shown in the figure, DCA-induced CD4+T cells can inhibit the expression of scar marker α-SMA by fibroblasts, indicating that DCA regulation of CD4+T cells can inhibit scar formation in vitro, which may be related to the increased secretion of IL-10.

[0113] Example 4: Detection of the repairing effect of DCA on deep II degree burn wounds

[0114] 4.1 Construction of rat deep second-degree burn model

[0115] A deep second-degree burn wound model was established on the back of rats using a desktop ultra-temperature controlled scalding device (YLS-5Q, Anhui Zhenghua Bio-Instrument Co., Ltd., China). General anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and ibuprofen was given to rats for postoperative analgesia from 24 h before surgery to 10 days after surgery. TM ), Cryo-ACol, DCA, and DNA-crosslinked commercial collagen dressings using the same crosslinking method were placed on the wound surface; the wounds were photographed with a digital camera on days 0, 3, 7, 14, and 21, and the photos were analyzed using ImageJ software to determine the wound area, the area associated with moist dermatitis caused by exudate immersion, and the scar area.

[0116] Fig.19 The camera captured the healing process of the burn wound after treatment with different dressings, as well as the comparison of wound area at different time periods. As shown: In the rat burn skin defect model, DCA showed a significant ability to promote wound closure compared with commercial collagen dressings (pink dotted line in the figure). The comparison between DCA and commercial collagen dressings cross-linked with DNA in the green dotted line in the figure showed that DCA had a better ability to promote wound healing at 3 days and 7 days. In addition, Fig.19 The blue arrow in the middle shows moist dermatitis caused by exudate immersion. It can be found that the area of ​​moist dermatitis in the DCA group was significantly reduced.

[0117] 4.2 Histological staining

[0118] 4.1 Animal Experiment After 21 days of dressing placement, skin specimens were collected, fixed with 4% paraformaldehyde for 24 h, and embedded in paraffin for histological staining; paraffin sections of healing skin tissue were stained with H&E (Servicebio, Wuhan, Hubei, China), Masson (Servicebio, Wuhan, Hubei, China), and Sirius Red (Servicebio, Wuhan, Hubei, China). The stained paraffin sections were observed under a Leica MZFLIII optical microscope (Leica, Wetzlar, Germany) and a polarizing microscope (DMRX, Leica, Wetzar, Germany).

[0119] DCA was compared with commercial collagen dressings, Cryo-ACol, and DNA-crosslinked commercial collagen dressings. Fig. 20 As shown, DCA significantly promoted the regeneration of hair follicle appendages, and the collagen arrangement of the repaired skin tissue was woven and the ratio of type I / III collagen was restored.

[0120] 4.3 In vivo verification of the immunomodulatory ability of DCA

[0121] 4.3.1 Immunofluorescence staining

[0122] The skin sample sections harvested in 4.2 were permeabilized with 0.15% Triton X-100 and incubated with anti-CD4 antibody (1:50, 67786-1-Ig, Proteintech, Wuhan, China), anti-IL-10 antibody (1:50, 13356-1-AP, Proteintech, Wuhan, China), and anti-α-SMA antibody (1:50, GTX100034, Genetex, CA, USA) at 4°C overnight; the primary antibodies were labeled with Alexa Fluor 488 goat anti-rabbit secondary antibody (33106ES60, Yisen Biotech, Shanghai, China) and Alexa Fluor 594 goat anti-mouse secondary antibody (33212ES60, Yisen Biotech, Shanghai, China) for staining.

[0123] The results are as follows Fig.21 As shown: Compared with simple collagen dressings, DCA-treated healing skin tissue is enriched with more CD4+T cells.

[0124] 4.3.2 Construction of a mouse back burn model after CD4+ T cell depletion

[0125] Four-week-old C57BL / 6J mice were intravenously injected with anti-mouse CD4 antibody (100 μg / mouse, A2101, Selleck, Texas, USA) every 3 days for a total of 4 times. The control group was given a matching isotype control IgG2b (100 μg / mouse; Selleck, A2116, Selleck, Texas, USA) at the same dose. After injection, the mouse back deep II degree burn model was prepared in the same way as in 4.1 and treated with DCA dressing. The role of CD4+T cells in DCA-promoted scar-free repair was analyzed by taking pictures, histological staining, and ImageJ statistics of wound and scar area.

[0126] The results are as follows Fig. 22 As shown: After the elimination of CD4+T cells, the scarless healing effect of DCA was significantly weakened, indicating that the scarless healing effect of DCA depends on the immune regulation ability of CD4+T cells.

Claims

1. A novel collagen, characterized in that: The preparation method of the novel collagen comprises: The collagen solution is pre-frozen to obtain porous collagen; Then, in a freezing environment, the porous collagen is immersed in a solution containing a cross-linking agent to obtain a collagen gel; the cross-linking agent is EDC and NHS; Then the collagen gel is freeze-dried to prepare the novel collagen.

2. The novel collagen according to claim 1, characterized in that: The pre-freezing condition is to cool down from room temperature to freezing temperature at a uniform speed.

3. The novel collagen according to claim 1 or 2, characterized in that: The pre-freezing condition is to cool down the temperature from room temperature to a freezing temperature of -196 to -180°C at a uniform rate.

4. The novel collagen according to claim 1, characterized in that: The freezing environment is -30 to -10°C.

5. The novel collagen according to claim 1, characterized in that: The pre-freezing time is 2 to 5 minutes; the soaking time is 12 to 24 hours; and the freeze-drying time is 24 to 48 hours.

6. The novel collagen according to claim 1, characterized in that: Then, the porous collagen is immersed in a solution containing a cross-linking agent under a freezing environment, and then immersed in water to obtain a collagen gel.

7. The novel collagen according to claim 1, characterized in that: The novel collagen is cross-linked with DNA.

8. Use of the novel collagen according to any one of claims 1 to 7 for preparing medical dressings.

9. Use of the novel collagen according to any one of claims 1 to 7 for preparing traumatic wound dressings.

10. Use of the novel collagen according to any one of claims 1 to 7 for preparing burn wound dressings.

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