Hydrogel with skin injury repairing effect

Abstract

The invention belongs to the field of 3D biological printing, and particularly relates to hydrogel with a skin injury repairing effect. Aiming at the problem of functional modification of sodium alginate in the existing 3D biological printing technology, the invention provides a preparation method of Eu < 3 + >-mediated collagen polypeptide / sodium alginate composite hydrogel, and the Eu < 3 + >-mediated collagen polypeptide / sodium alginate composite hydrogel is successfully applied to a 3D printing skin wound dressing. The calcium alginate is used as biological ink, the skin full-thickness injury dressing is prepared through a 3D printing technology, and finally the stability of the calcium alginate is enhanced through coordination between Ca < 2 + > and sodium alginate. The 3D printing skin wound dressing constructed by the invention has a good function of promoting skin full-thickness injury repair, and has application potential in the fields of tissue engineering and regenerative medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biofunctional materials, and particularly relates to a hydrogel with a skin injury repair effect. Background Art

[0002] Skin injury will damage the protective function of the skin on the human internal environment, and severe skin trauma is difficult to heal under natural conditions. The skin is the largest organ of the human body and plays an important role in protecting the human body from the influence of the external environment and maintaining internal balance. After the skin is damaged, it will cause serious health problems such as viral infection, blood and tissue fluid loss, and damage to the tissue structure and function. Skin wound repair is a complex physiological process, mainly including four stages: hemostasis stage, inflammation stage, proliferation stage, and remodeling stage. Large-area full-thickness skin defects will damage the epidermis, dermis, and subcutaneous tissue, and this kind of skin trauma is difficult to heal spontaneously, so clinical treatment is needed to promote its repair. Skin wound dressings can prevent skin wounds from being further damaged, and at the same time provide a good environment for wound healing, thus effectively promoting the healing of full-thickness skin trauma.

[0003] 3D printing technology can prepare materials that can fit the shape and depth of wounds, and this technology has received more and more attention in the preparation of skin wound dressings. Sodium alginate (SA) is a natural polymer extracted from brown algae, which can coordinate with metal ions to form a gel at room temperature, and is a widely used 3D printing bioink. Patent CN107139447A discloses a 3D printing strategy that uses sol-like sodium alginate as the printing raw material, and after extrusion, it is cured by a Ca 2+ solution to prepare a hydrogel model with a three-dimensional structure. Patent CN114470336A discloses a mixed hydrogel composed of sodium alginate and gelatin or methacrylamide-based gelatin. Using it as a 3D printing bioink can print out a scaffold structure, and then through Ca 2+ crosslinking to prepare a hydrogel scaffold. However, SA itself does not have biological activity, and it is urgent to construct functionalized SA for 3D printing of biofunctional materials. Summary of the Invention

[0004] Aiming at the above technical problems, the present invention aims at the problem of functional modification of sodium alginate in the existing 3D bioprinting technology, and proposes a preparation method of Eu 3+ -mediated collagen polypeptide / sodium alginate composite hydrogel, and uses it as a bioink. Through 3D printing technology, a full-thickness skin injury dressing is prepared, and finally the coordination between Ca 2+ and sodium alginate is used to enhance its stability. The 3D printed skin wound dressing constructed by the present invention has a good function of promoting the repair of full-thickness skin injury and has application potential in the fields of tissue engineering and regenerative medicine. Specifically, it includes the following contents:

[0005] In a first aspect, the present invention provides a hydrogel having a skin injury repair effect, and the hydrogel comprises the following components: sodium alginate, europium ion (Eu 3+ ), and collagen polypeptide; the sequence of the collagen polypeptide is:

[0006] (G-X-Y) m1 -collagen functional sequence-(G-X-Y) m2 -(D) n ;

[0007] wherein, G is glycine, and D is aspartic acid;

[0008] X and Y are selected from the same or different amino acids;

[0009] m1 and m2 are both any integers greater than or equal to 1, and the sum of m1 and m2 is greater than or equal to 6;

[0010] n is any integer greater than or equal to 2;

[0011] The collagen functional sequence is selected from any one of integrin binding site GFOGER, integrin binding site GROGER, integrin binding site GLOGER, integrin binding site GMOGER, integrin binding site GAOGER, integrin binding site GLKGEN, integrin binding site GLOGEN, DDR binding site GVMGFO, MMP cleavage site GPLGIAGITGAR, MMP cleavage site GPQGIAGQRGVV, MMP cleavage site GPQGLLGAOGIL, MMP cleavage site GPQGLAGQRGIV, heparin binding site GRPGKRGKQGQK, and fibronectin binding site GLPGQRGER.

[0012] Preferably, X is proline P and Y is hydroxyproline O; or both X and Y are hydroxyproline O.

[0013] Preferably, the sequence of the collagen polypeptide is: (G-P-O) 3 -GFOGER-(G-P-O) 3 -(D) 2 .

[0014] Preferably, the concentration ratio of the sodium alginate, europium ion (Eu 3+ ) and collagen polypeptide is: 40 mg / mL: 2 mM: 6 mM.

[0015] Preferably, the preparation method of the hydrogel is: dissolving sodium alginate in the collagen polypeptide solution to obtain a mixed solution of collagen polypeptide and sodium alginate; mixing this mixture with Eu3+ The solutions are mixed in equal volumes to prepare the hydrogel.

[0016] In a second aspect, the present invention provides a skin wound dressing, which is obtained by preparing the hydrogel described in the first aspect above through 3D printing technology.

[0017] In a third aspect, the present invention provides a method for preparing the skin wound dressing described in the second aspect above, the method comprising the following steps:

[0018] (1) Prepare the hydrogel described in the first aspect above;

[0019] (2) 3D printing: Using the hydrogel prepared in step (1) as bioink, printing out a gel material through 3D printing technology, and atomizing and dropping a CaCl 2 solution onto the surface of the 3D printed gel material to obtain the skin wound dressing.

[0020] Preferably, the 3D printing technology in step (2) uses an extrusion 3D printer.

[0021] Preferably, the concentration of the CaCl 2 solution used in step (2) is 170 mg / mL.

[0022] Preferably, the skin wound dressing is a disc-shaped material with a diameter of 15 mm and a height of 2.5 mm.

[0023] In a fourth aspect, the present invention provides the use of the hydrogel described in the first aspect above or the skin wound dressing described in the second aspect above in the preparation of skin injury repair products.

[0024] Preferably, the skin injury is a full-thickness skin injury.

[0025] The beneficial effects of the present invention are as follows: ① Sodium alginate is a widely used 3D printing ink, but it does not have biological functions. In view of the problem of functional modification of sodium alginate in the existing 3D bioprinting technology, the present invention proposes a preparation method of Eu 3+ -mediated collagen polypeptide / sodium alginate composite hydrogel, in which the collagen polypeptide functionalizes and modifies sodium alginate to form a composite hydrogel with biological functions; ② Sodium alginate cannot print out a model with fine structure, while the composite hydrogel provided by the present invention improves its 3D printability and successfully applies it to 3D printing of skin wound dressings; ③ The skin wound dressing prepared by using the composite hydrogel as bioink through 3D printing technology effectively promotes the healing of full-thickness skin defects in SD rats; ④ The prepared full-thickness skin wound dressing has no risk of virus transmission and immunogenicity and has good biocompatibility; ⑤ The preparation process of the composite hydrogel is simple and the reaction conditions are mild. Description of the Drawings

[0026] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0027] Figure 1 is Eu 3+ 3D printability results of the collagen polypeptide / sodium alginate hydrogel mediated by; wherein A is a photo and SEM image of the grid material printed based on gels SA-C, SA-E-C, and SA-E-D-C; B is a hollow cylinder printed based on gel SA-E-D-C; C is a star printed based on gel SA-E-D-C; D is a meniscus printed based on gel SA-E-D-C; E is a model diagram, top view, and side view of an ear printed based on gel SA-E-D-C;

[0028] Figure 2 is Eu 3+ and Ca 2+ Characterization results of the physicochemical properties of the collagen polypeptide / sodium alginate hydrogel mediated by; wherein A-C are SEM images of gels SA-C, SA-E-C, and SA-E-D-C respectively; D is the porosity of gels SA-C, SA-E-C, and SA-E-D-C; E is the compressive modulus of the gels; F is the curve of the swelling ratio of the gels varying with time; G is the frequency scanning curve of the gels; H is the strain scanning curve; I is the variation of G' and G" with time under alternating scanning at 1% and 80% strain;

[0029] Figure 3 Shows the healing conditions of full-thickness skin wounds of SD rats at various time points after applying different dressings; wherein A is a photo of the skin wound of SD rats; B is the change in the wound contour; C is the wound healing rate;

[0030] Figure 4 Are the H&E and Masson staining results of the skin tissues of SD rats in each group at different time points;

[0031] Figure 5 Is the collagen fiber expression generation rate at the skin wound site of SD rats in each group at different time points. Detailed implementation manners

[0032] The technical solution of the present invention will be further described below in detail with reference to embodiments, but the protection scope of the present invention is not limited to the following.

[0033] In the following embodiments, the GPO is Gly (glycine)-Pro (proline)-Hyp (hydroxyproline); the GFOGER is Gly (glycine)-Phe (phenylalanine)-Hyp (hydroxyproline)-Gly (glycine)-Glu (glutamic acid)-Arg (arginine); the SA is sodium alginate.

[0034] In the following examples, the sequence of the collagen polypeptide is (GPO) 3 GFOGER(GPO) 3 DD for example; the collagen polypeptide contains (GPO) 3 repetitive sequence for stabilizing the triple helix structure of the polypeptide; the collagen polypeptide contains the cell integrin binding sequence GFOGER in natural collagen; the N-terminus of the collagen polypeptide contains two amino acids D, such that the amino acids at this position coordinate with Eu 3+ for coordination.

[0035] In the following examples, the method for preparing the gel is as follows: Mix the SA and Eu(NO 3 ) 3 solutions to prepare a gel formed under the mediation of Eu 3+ When preparing the gel modified with collagen polypeptide, dissolve SA with the collagen polypeptide solution and then mix it with Eu(NO 3 ) 3 solution to form a polypeptide-modified composite gel; Use an atomizer to atomize the CaCl 2 solution into droplets and cover the surfaces of the SA solution and the above two hydrogels to prepare Ca 2+ solidified gels SA-C, SA-E-C and SA-E-D-C.

[0036] In the following examples, the method for characterizing the 3D printability of the gel is as follows: Use the gel not solidified by Ca 2+ as bioink and print it into a fine grid shape and other various shapes.

[0037] In the following examples, the methods for the physicochemical properties of the composite hydrogel include: rheological test, field emission scanning electron microscope (SEM) test, anti-compression performance test and swelling performance test.

[0038] In the following examples, the method for preparing the skin wound dressing is as follows: Use the gel not solidified by Ca 2+ as bioink, use an extrusion 3D printer to print a cylindrical shape with a length and width of 15 mm and a height of 2.5 mm, and then solidify it with Ca 2+ .

[0039] In the following examples, the experimental model for verifying the efficacy of the skin wound dressing is: SD rat full

[0040] thickness skin injury model.

[0041] Example 1 3D Printability of Collagen Polypeptide / Alginate Composite Hydrogel Mediated by Eu 3+ ​

[0042] SA and Eu(NO 3 ) 3 ·6H 2 O were separately dissolved in 100 mM Hepes buffer at pH 7.0 to prepare the initial solutions of SA and Eu 3+ , with their initial concentrations being 80 mg / mL and 4 mM respectively. The initial solution of SA was diluted to 100 mg / mL using 100 mM Hepes buffer at pH 7.0 to prepare the working solution of SA. The initial solutions of SA and Eu 3+ were mixed at a volume ratio of 1:1 to form the gel SA-E. After mixing, the concentration of SA was 40 mg / mL and the concentration of Eu 3+ was 2 mM. By dissolving the polypeptide CMP-DD[(GPO) 3 GFOGER(GPO) 3 DD] in 100 mM Hepes buffer at pH 7.0 to prepare the initial solution of the polypeptide CMP-DD with an initial concentration of 12 mM, and then dissolving SA with the initial solution of the polypeptide to prepare the initial mixed solution of SA / CMP-DD, such that the initial concentration of SA was 80 mg / mL. Then, the initial mixed solution of SA / CMP-DD was mixed with the initial solution of Eu 3+ at a ratio of 1:1 to form the composite gel SA-E-D. At this time, the final concentrations of SA, Eu 3+ and the polypeptide CMP-DD were 40 mg / mL, 2 mM and 6 mM respectively. The working solution of SA, the gel SA-E and SA-E-D were used as bioinks respectively, and they were transferred into the printing syringes. Using an extrusion 3D printer, the 3D printing of the materials was carried out through a conical plastic needle to prove the printability of the SA solution, the gel SA-E and SA-E-D. Among them, the printing speed was set to 2 mm / s and the extrusion speed was 1.8 mm 3 / s. When printing the grid material, a square model was selected, and the length and width of the model were both set to 20 mm, the height was 3 mm, the layer height was 0.6 mm, and the line width was 3 mm. After the material printing was completed, droplets of a CaCl 2 solution with a concentration of 170 mg / mL were covered on the surface of the material and infiltrated for 10 minutes to prepare 3D printed materials SA-C, SA-E-C and SA-E-D-C with different shapes respectively.

[0043] The results are as Figure 1 shown. The photos of the printed models and the SEM characterization structures indicate that the grid structure printed by the SA solution is prone to collapse and cannot form a regular shape; using the gel SA / Eu 3+It is possible to print a grid with fine and regular structure. When using the gel SA-E-D modified with polypeptide CMP-DD as the bioink, its printing accuracy is the same as that of the gel SA-E ( Figure 1 as shown in A in Figure 1 ). We further printed hollow cylinders ( Figure 1 as shown in B in Figure 1 ), stars ( Figure 1 as shown in C in + ). From the above results, it can be seen that the gel SA-E

[0044] Example 2 Eu 3+ and Ca 2+ Characterization of the physicochemical properties of the collagen polypeptide / sodium alginate hydrogel mediated

[0045] 1. Field emission scanning electron microscope (SEM) and porosity characterization of the composite gel

[0046] Prepare the SA working solution, gel SA-E and SA-E-D using the method in Example 1. Then, use an atomizer to atomize the CaCl 2 solution into droplets and cover the surfaces of the SA working solution, gel SA-E and SA-E-D to prepare gel SA-C, SA-E-C and SA-E-D-C respectively. Freeze-dry the composite gels SA-C, SA-E-C and SA-E-D-C using a freeze dryer. Cut the freeze-dried samples into thin slices with a thickness of 2-3 mm, stick them on the conductive glue on the surface of the metal disc, and sputter gold for 2 minutes under vacuum conditions. Use a field emission scanning electron microscope to characterize their morphology.

[0047] Mark the masses of the freeze-dried gels SA-C, SA-E-C and SA-E-D-C as m 1 , and mark the volumes as V 1 . Moisten the materials with ultrapure water for 10 minutes, and record the wet weights of the materials as m 2 , and record the weight of water in the materials as m 3 (m 3 = m 2 - m 1 ), and record the volume of water as V 2 (V 2 = m 3 / ρ). The porosity P = V 2 / V 1 × 100%. Each sample is measured in parallel three times.

[0048] The results are as shown in Figure 2As shown in A - D, all three gels formed porous structures. Gel SA - C formed pores with irregular shapes and had the largest pore size; the pores of gel SA - E - C were regular in shape and the pore size decreased; while the composite gel SA - E - D - C formed a regular porous structure with a smaller pore size. The porosity results showed that gel SA - C had the largest porosity (207%), the porosity of gel SA - E - C decreased to 187%, and the porosity of SA - E - D - C further decreased to 131%. The above results indicate that Eu 3+ After binding with polypeptides CMP and SA, they both made it form a more regular porous structure.

[0049] 2. Characterization of the anti - compression performance of the composite gel

[0050] Prepare SA working solution, gel SA - E and SA - E - D using the method in Example 1. Respectively take SA working solution, gel SA - E and SA - E - D as bio - inks, transfer them into a printing syringe, and use an extrusion - type 3D printer to print out cylindrical models. The printing speed is set to 2 mm / s, the extrusion speed is 1.8 mm 3 / s. Set the length and width of the model to be 10 mm, the height to be 6 mm, the layer height to be 0.6 mm, and the line width to be 1 mm. After the material printing is completed, use an atomizer to cover the surface of the material with droplets of a CaCl 2 solution with a concentration of 170 mg / mL, and soak for about 10 minutes. Conduct a compression test using a universal testing machine, with a compression rate of 1 mm / min and a strain of 70%. Calculate the compression modulus through the average slope of the stress - strain curve at 15% strain.

[0051] The results are as shown in Figure 2 E. The compression modulus of gel SA - C was only 10.5 KPa. After introducing Eu 3+ into the gel system, the combined action of Eu 3+ and Ca 2+ improved the gel - forming performance of SA. Therefore, the compression moduli of gels SA - E - C and SA - E - D - C increased to 16.5 KPa and 13.8 KPa respectively. The above results indicate that the SA composite hydrogel formed under the mediation of Eu 3 and Ca 2+ has good anti - compression performance.

[0052] 3. Characterization of the swelling performance of the composite gel

[0053] Prepare gels SA - C, SA - E - C and SA - E - D - C using the method in 1 above; weigh the mass M of gels SA - C, SA - E - C and SA - E - D - C 0(g), then soak them in ultrapure water, ensuring that the water completely submerges the gel, and place it in a 37 °C constant temperature water bath. Weigh the gel at certain time points and record its mass as M t , dry the moisture on the surface of the gel before weighing, and calculate the swelling ratio SR of the composite gel [SR = (M t - M 0 ) / M 0 .

[0054] The results are as shown in F in Figure 2 . The swelling ratios of these three gels all reached equilibrium around 2 h, and the swelling ratio of gel SA-C reached 116.8 ± 4.3%. After introducing Eu 3+ into the gel system, the combined action of Eu 3+ and Ca 2+ improved the mechanical properties of SA. Therefore, the swelling ratios of gels SA-E-C and SA-E-D-C decreased by 73.7 ± 2.1% and 81.2 ± 10.9% respectively. The above results indicate that the SA composite hydrogel formed under the mediation of Eu 3+ and Ca 2+ has good swelling properties and can effectively absorb wound exudate, thus promoting wound healing.

[0055] 4. Rheological testing of composite gels and characterization of self-healing properties

[0056] Prepare gels SA-C, SA-E-C, and SA-E-D-C using the method in 1 above; place the composite gels SA-C, SA-E-C, and SA-E-D-C on the rheometer sample stage respectively, set the shear strain to 1%, and perform frequency scanning by testing the storage modulus G’ (Pa) and loss modulus G” (Pa) of the gel within the angular frequency range of 1 - 100 rad / s; then fix the angular frequency at 1 rad / s and detect the changes in G’ (Pa) and G” (Pa) of the gel within the shear strain range of 1 - 100%. Alternate scanning is performed at shear strains of 1% and 80% with a fixed angular frequency of 1 rad / s to detect the self-healing properties of the gel.

[0057] The frequency scanning is as shown in Figure 2As shown in Figure G, the results indicate that the storage moduli (G’) of gels SA-C, SA-E-C, and SA-E-D-C are all greater than the loss moduli (G”), and their G’ values show an upward trend with the increase in frequency, presenting typical rheological properties of hydrogel materials. When the shear frequency reaches 80 rad / s and higher, the G’ of gel SA-C reaches around 6300 Pa, while the G’ of gels SA-E-C and SA-E-D-C increase to around 9800 Pa and 7200 Pa respectively, demonstrating that gels SA-E-C and SA-E-D-C have stronger gel-forming properties and mechanical strength than SA-C. The strain sweep is shown in Figure 2 As shown in Figure H, the results indicate that with the increase in strain, the G’ of gels SA-C, SA-E-C, and SA-E-D-C all gradually decrease, while G” continuously increases. When the strain is greater than 30%, the value of G” exceeds G’. This proves that these three gels have typical shear-thinning behavior. The results of the alternating sweep of gels SA-C, SA-E-C, and SA-E-D-C at a low strain of 1% and a high strain of 80% are shown in Figure 2 As shown in Figure I, it shows that when the strain is 1%, the G’ and G” of gels SA-C, SA-E-C, and SA-E-D-C are respectively around 5000 Pa and 600 Pa, and at this time they all maintain gel characteristics. However, when the gels are under a high-amplitude oscillatory shear of 80%, the G’ all decreases to around 480 Pa, and at this time G” is greater than G’, so the gels all transform into a quasi-liquid state. After the shear strain returns to 1% again, the G’ and G” values of the gels return to the initial state, and this process can be repeated for several cycles, proving that these three gels all have good self-healing properties.

[0058] Example 3 Full-thickness skin injury repair function of 3D-printed skin dressings

[0059] Using the method in Example 1, SA working solution, gels SA-E, and SA-E-D were prepared and used as bioinks. A cylindrical model was printed using an extrusion 3D printer, where the printing speed was set at 2 mm / s, the extrusion speed was 1.8 mm 3 / s, the length and width of the model were both set at 15 mm, the height was 2.5 mm, the layer height was 0.6 mm, and the line width was 1 mm. After the material printing was completed, droplets of a CaCl 2 solution with a concentration of 170 mg / mL were covered on the surface of the material and infiltrated for about 10 minutes to prepare cylindrical gel materials SA-C, SA-E-C, and SA-E-D-C, which were placed at 4 °C for standby.

[0060] Select SD female rats weighing 150 - 200 g. Use 10% chloral hydrate and inject the rats intraperitoneally at a dose of 0.3 mL / 100 g to anesthetize them. Use a hair clipper to shave the hair on the rats' backs, then use depilatory cream to remove the remaining hair, and then wash the rats' backs thoroughly with 0.9% sodium chloride injection. Disinfect the exposed skin on their backs with iodophor, then use the mouth of a 15 mL centrifuge tube to mark a circular area with a diameter of 1.5 cm on the exposed skin. Use surgical scissors to completely cut off the upper skin tissue to form a circular wound area, with the wound depth reaching the fascia, and stop bleeding. Then randomly divide the rats into 5 groups. Among them, there is no dressing on the skin wound of the blank group, the skin wounds of the control group rats are dressed with the gel material SA - C, and the skin wounds of the experimental group rats are treated with the gel dressings SA - E - C and SA - E - D - C respectively. The dressings are fixed with band - aids and elastic bandages. Each group of rats is raised separately, and the wound status is observed every day. Take pictures of the wounds on the 4th, 7th, 11th, and 21st days after surgery. At the same time, place a ruler with precise graduations at the wound site to calibrate the magnification of the pictures. Then decapitate the rats to death by cervical dislocation, remove the tissue at the wound edge, fix it with 10% formaldehyde, perform routine paraffin embedding, sectioning, and H&E staining and Masson staining. The full - thickness skin wound pictures of each group of rats at each time point are obtained by splicing using Adobe Photoshop software. Use Image J software to calculate the area of the rats' skin wounds and analyze the generation rate of collagen fiber expression in the pictures of the Masson staining results. Calculate the wound healing rate and the collagen fiber generation rate (Collagen Fiber, CF%) of the rats' skin wounds through the following formulas:

[0061] Wound healing rate = Wound area at each time point / Initial wound area;

[0062] CF% = A C / A 0 ;

[0063] where A C is the area of collagen fibers, and A 0 is the total area of skin tissue.

[0064] The healing results of the full - thickness skin wounds of SD rats at each time point after applying different dressings are as Figure 3 shown. The photos of the skin wounds of SD rats (as Figure 3As shown in Figure A, on the 4th day, the wounds on the skin of rats in the control group, SA-C group, and SA-E-C group were bright red and there was bleeding. Among them, the wound areas in the control group and SA-C group were larger, and there was some infection at the edges, showing redness and swelling. The skin wound area in the SA-E-C group was smaller and there was no infection. Moreover, the wound area in the SA-E-C group further shrank, and a scab tissue appeared at the wound, making it relatively smooth. On the 7th day, the skin wound areas of rats in all groups decreased and scab tissues were formed. The scab tissue areas at the wound sites in the control group and SA-C group were larger, the surfaces were uneven, and the colors were darker. The scab areas of the skin wounds in the SA-E-C group and SA-E-D-C group were smaller and the surfaces were flat. Among them, the scab color in the SA-E-C group was darker and the shape was irregular. The scab of the skin wound in the SA-E-D-C group was a regular shape with a lighter color. On the 11th day, the scab of the wound of the rats in the SA-E-D-C group fell off, the wound became flat and the color became lighter. There was still darker scab tissue at the wound sites in the control group and SA-C group. Among them, the scab areas in the control group and SA-C group were larger than those in the SA-E-C group. On the 21st day, there were still obvious sunken areas on the skin of the rats in the control group and SA-C group, and the color of this area was a darker red. There was a depression on the skin of the SA-E-C group, but the color of this part was light pink. The skin trauma of the rats in the SA-E-D-C group was basically healed, the skin was relatively smooth, and the original wound site on the skin showed white and a small amount of light pink. The changes in the edges and areas of the skin wounds of rats at different time periods (as shown in Figure 3 Figure B) showed that compared with the control group and SA-C group, the reduction rate of the skin wound area of rats in the SA-E-C and SA-E-D-C groups was significantly faster, and the wound healing rate of the SA-E-D-C group was also faster than that of the SA-E-C group. On the 21st day, the wound area of the SA-E-D-C group decreased from 268.13 mm 2 to 2.40 mm 2 , which was significantly smaller than that of the SA-E-C group (decreased from 245.33 mm 2 by 14.07 mm 2 ), the SA-C group (decreased from 248.13 mm 2 by 19.40 mm 2 ), and the control group (decreased from 239.55 mm 2 to 15.27 mm 2 ). The results of the wound healing rate are shown in Figure 3 Figure C, indicating that the wound healing rates of the skin wounds of rats in the SA-E-C and SA-E-D-C groups were significantly higher than those in the control group and SA-C group, and the wound healing rate of the SA-E-D-C group was the highest. The above results prove that the scaffold material SA-E-D-C has the most obvious effect on promoting wound closure.

[0065] The H&E staining results of the skin tissues of SD rats are as follows Figure 4 shown. On the 4th day, there were inflammatory cell infiltrations in the skin wound tissues of each group. The dermis of the skin in the control group and the SA-C group was thinner, the granulation tissue was sparse, and the wound depression was severe. Granulation tissue appeared in the skin of the SA-E-C and SA-E-D-C groups to fill the wound, making its surface flat. Among them, scab tissue appeared at the wound of the SA-E-D-C group, and the density of its granulation tissue was significantly higher than that of the SA-E-C group. Moreover, the dermal tissue of this group of skin began to form. On the 11th day, scab tissue appeared in the skin of the control group and the SA-C group. A relatively thick epidermal tissue began to form in the SA-E-C group. The density of the granulation tissue of the skin in these three groups increased, but there were still inflammatory cell infiltrations, and the arrangement of tissue fibers was relatively disordered. The skin of the SA-E-D-C group formed a relatively thin epidermal tissue, and its tissue fibers were regularly and densely arranged along the epidermal growth direction. At the same time, the inflammatory cells decreased significantly. On the 21st day, a relatively thick epidermal layer appeared in the skin of the control and SA-C groups, and the arrangement of tissue fibers began to become regular. The thickness of the epidermal layer of the skin in the SA-E-C and SA-E-D-C groups further decreased, and the density of the fibrous tissue further increased, and was higher than that of the control and SA-C groups. At the same time, a relatively mature dermis was formed. Among them, the epidermal layer of the skin in the SA-E-D-C group was the most regular, blood vessels appeared in the dermis, the density of its tissue fibers was significantly higher than that of the SA-E-C group, and showed a bundle shape and was regularly arranged along the epidermal growth direction. The above results indicate that the wound dressings SA-E-C and SA-E-D-C have the function of promoting granulation and epidermal regeneration, and can inhibit epidermal hyperplasia at the same time, and the effect of SA-E-D-C is the most obvious.

[0066] The Masson staining results of the skin tissues of SD rats are as follows Figure 4As shown, on the 4th day, the collagen fibers in the skin of the control group and the SA-C group were arranged in a scattered and sparse manner, and even showed fractures of the collagen fibers. Denser collagen fibers began to appear in the dermis of the skin in the SA-E-C group and the SA-E-D-C group, and the number of collagen fibers in the skin of the SA-E-D-C group was significantly more than that of other groups. On the 11th day, the number of collagen fibers in the skin of the control group and the SA-C group further increased, but was still significantly less than that in the SA-E-C group and the SA-E-D-C group. Coarser fiber bundles were formed by the collagen fibers in the skin of the latter two groups and were arranged densely and regularly. The collagen fibers in the skin of the SA-E-D-C group intertwined to form a network and were arranged parallel to the epidermal direction. On the 21st day, the collagen fibers in the skin of the control group and the SA-C group were arranged relatively scattered. The density of the collagen fibers in the skin of the SA-E-C group and the SA-E-D-C group was significantly greater than that of the other two groups and were arranged regularly under the epidermis. Among them, the collagen fibers in the SA-E-D-C group formed coarser fiber bundles and showed wavy undulations. The above results prove that the skin dressings SA-E-C and SA-E-D-C can promote the formation of collagen fibers at the skin trauma site, and the effect of SA-E-D-C is more significant.

[0067] The formation rate of collagen fibers in the Masson staining results is as Figure 5 shown. The formation rates of collagen fibers in the skin of the SA-E-C group and the SA-E-D-C group at each time point were significantly greater than those of the blank group and the SA-C group, and the collagen formation rate of the SA-E-D-C group was the highest. On the 4th day, the collagen expression and formation rates of the blank group and the SA-C group were both lower than 10%, while the collagen formation rates of the skin in the SA-E-C group and the SA-E-D-C group reached about 20% and 40% respectively. On the 11th day, the collagen formation rates of the skin in each group showed an upward trend, but the collagen fiber formation rates of the blank group and the SA-C group were significantly lower than those of the SA-E-C group and the SA-E-D-C group. Among them, the collagen formation rate of the SA-E-D-C group was significantly higher than that of the other three groups. On the 21st day, the collagen expression and formation rates of the blank group and the SA-C group were around 65%, while the collagen expression and formation rates of the skin in the SA-E-C group and the SA-E-D-C group were close to 75% and 80% respectively. The above results prove that the skin dressings SA-E-C and SA-E-D-C can significantly increase the formation rate of collagen fibers at the skin trauma site, and the promoting effect of SA-E-D-C is more obvious.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention. In the specific implementation and application process of the present invention, those skilled in the art can make changes to the technical solutions according to specific experimental conditions and requirements, and are not used to limit the present invention. All those within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A hydrogel with the function of repairing skin damage, Characterized in that, The hydrogel comprises the following components: sodium alginate, europium ion (Eu 3+ ), and collagen polypeptide; the sequence of the collagen polypeptide is: (G-X-Y) m1 -Collagen functional sequence-(G-X-Y) m2 -(D) n ; wherein, G is glycine and D is aspartic acid; X and Y are selected from the same or different amino acids; m1 and m2 are both any integers greater than or equal to 1, and the sum of m1 and m2 is greater than or equal to 6; n is any integer greater than or equal to 2; The collagen functional sequence is selected from any one of integrin binding site GFOGER, integrin binding site GROGER, integrin binding site GLOGER, integrin binding site GMOGER, integrin binding site GAOGER, integrin binding site GLKGEN, integrin binding site GLOGEN, DDR binding site GVMGFO, MMP cleavage site GPLGIAGITGAR, MMP cleavage site GPQGIAGQRGVV, MMP cleavage site GPQGLLGA OGIL, MMP cleavage site GPQGLAGQRGIV, heparin binding site GRPGKRGKQGQK, fibronectin binding site GLPGQRGER.

2. The hydrogel according to claim 1, Characterized in that, X is proline P and Y is hydroxyproline O; or both X and Y are hydroxyproline O.

3. The hydrogel according to claim 2, Characterized in that, The sequence of the collagen polypeptide is: (G-P-O) 3 -GFOGER-(G-P-O) 3 -(D) 2 .

4. The hydrogel according to claim 3, Characterized in that, The concentration ratio of the sodium alginate, europium ion (Eu 3+ ) and collagen polypeptide is: 40 mg / mL: 2 mM: 6 mM.

5. The hydrogel according to claim 4, Characterized in that, The preparation method of the hydrogel is as follows: Sodium alginate is dissolved in the collagen polypeptide solution to obtain a mixed solution of collagen polypeptide and sodium alginate; the mixed solution is mixed with Eu 3+ solution in equal volume to prepare the hydrogel.

6. A skin wound dressing, Characterized in that, The skin wound dressing is prepared by the 3D printing technique from any one of the hydrogels described in claims 1 - 5.

7. The preparation method of the skin wound dressing according to claim 6, Characterized in that, The method comprises the following steps: (1) Prepare any one of the hydrogels described in claims 1 - 5; (2) 3D printing: Using the hydrogel prepared in step (1) as a bioink, printing out a gel material through 3D printing technology, and atomizing and dropping a CaCl 2 solution onto the surface of the 3D-printed gel material to obtain a skin wound dressing.

8. The preparation method of the skin wound dressing according to claim 7, Characterized in that, In step (2), an extrusion 3D printer is used for the 3D printing technique.

9. The preparation method of the skin wound dressing according to claim 7, Characterized in that, The CaCl used in the step (2) 2 has a solution concentration of 170 mg / mL.

10. The application of the hydrogel according to claim 1 or the skin wound dressing according to claim 6 in the preparation of skin damage repair products.

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