Polypeptide-modified mesenchymal stem cell-loaded hydrogel composition as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide modified and mesenchymal stem cell loaded hydrogel composition as well as a preparation method and application thereof, and particularly discloses a polypeptide modified and mesenchymal stem cell loaded hydrogel composition which comprises a polypeptide modified dual-network hydrogel carrier and mesenchymal stem cells, the dual-network hydrogel carrier comprises hydrogel formed by hyaluronic acid and a polypeptide graft shown as SEQ ID NO.1, and hydrogel formed by oxidized dextran. According to the hydrogel composition, the hydrogel and the stem cells have a synergistic effect, the number of skin hair follicles can be increased, scar formation can be reduced, meanwhile, secretion of type I collagen is promoted, the epidermis thickness and inflammatory cell infiltration conditions are reduced, and the hydrogel composition has application prospects in the aspects of treating skin injuries and promoting wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of skin repair, and particularly relates to a polypeptide-modified hydrogel composition loaded with mesenchymal stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] As the largest organ of the human body, the skin is often damaged due to factors such as diseases, burns, accidental traumas, and surgical operations. Delayed healing may lead to wound infections or scars. However, wound healing is a complex process involving multiple factors, including inflammation, proliferation, and tissue remodeling. Although many new technologies (including growth factors and skin substitutes) have been widely used to promote wound healing, in most cases, existing treatment methods can only achieve 50%-60% wound healing. Therefore, shortening the wound healing time after skin injury and restoring its structural integrity and function are urgent problems to be solved clinically.

[0003] Mesenchymal stem cells can differentiate into skin tissue cells, increase the content of skin fibroblasts, and the cytokines and growth factors secreted by them can regulate inflammatory responses, induce angiogenesis, enhance epithelial cell growth, and accelerate the formation of granulation tissue and extracellular matrix. A large number of preclinical and clinical trials have shown that mesenchymal stem cell therapy can accelerate wound healing, bringing hope for the treatment of various chronic wounds that are difficult to heal. Systemic or local application of mesenchymal stem cells to the wound can promote the repair of damaged skin, improve the healing quality, enhance angiogenesis during the wound healing process, and shorten the patient's recovery time.

[0004] In recent years, polypeptide hydrogels have characteristics such as biocompatibility, biodegradability, easily adjustable hydrophilicity / hydrophobicity, providing a moist environment to promote wound repair, and preventing further bacterial infection. They have been widely used in the fields of tissue engineering, drug delivery, 3D bioprinting, and wound healing. Therefore, how to make full use of the role of mesenchymal stem cells in wound healing and design a hydrogel with related functions is expected to become a breakthrough in the development of wound healing products. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art, and thus provide a preparation method and an application of a composition of mesenchymal stem cells and a polypeptide hydrogel. The present invention prepares a polypeptide hydrogel using hyaluronic acid, oxidized dextran, and a specific polypeptide as materials. The polypeptide hydrogel has good biocompatibility, can promote cell adhesion, and by loading mesenchymal stem cells, the wound healing effect is increased.

[0006] One aspect of the present invention provides a polypeptide-modified hydrogel composition loaded with mesenchymal stem cells, which includes a polypeptide-modified double-network hydrogel carrier and mesenchymal stem cells;

[0007] The double-network hydrogel carrier includes a hydrogel formed by a graft of hyaluronic acid and a polypeptide shown in SEQ ID NO.1 and a hydrogel formed by oxidized dextran.

[0008] Further, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, and amniotic mesenchymal stem cells.

[0009] Further, the concentration of mesenchymal stem cells in the hydrogel composition is 1×10⁶ cells / 1.15 mL hydrogel or more, preferably 8×10⁵ cells / 1.15 mL hydrogel - 1.2×10⁷ cells / 1.15 mL hydrogel.

[0010] Further, the hydrogel composition is prepared by the following method:

[0011] S1) Prepare the hyaluronic acid-polypeptide graft: Couple the carboxyl group of hyaluronic acid with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1 to obtain the hyaluronic acid-polypeptide graft;

[0012] S2) Prepare oxidized dextran: Prepare oxidized dextran by oxidizing dextran;

[0013] S3) Prepare the hydrogel precursor solutions of the hyaluronic acid-polypeptide graft and oxidized dextran respectively, and then mix the two hydrogel precursor solutions and mesenchymal stem cells evenly to obtain the hydrogel composition.

[0014] Further, in S1), the coupling method is to activate the carboxyl group on hyaluronic acid and then react with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1.

[0015] Further, in S1), the molar ratio of the carboxyl group on hyaluronic acid to the polypeptide shown in SEQ ID NO.1 (GFOGER) is 3 - 5:1, preferably 4:1.

[0016] Further, in S1), the molecular weight of hyaluronic acid is 100 kDa - 200 kDa, preferably 110 kDa - 150 kDa.

[0017] Further, in S2), the molecular weight of dextran is 50 kDa - 100 kDa, preferably 60 kDa - 80 kDa.

[0018] Further, in S1), the coupling method is to disperse hyaluronic acid in an acidic buffer solution, add a catalyst for the carboxyl activation reaction, react until complete, adjust the pH value to above 7 to end the reaction, and obtain carboxyl-activated hyaluronic acid; react the carboxyl-activated hyaluronic acid with the polypeptide shown in SEQ ID NO.1 until the reaction is complete.

[0019] Further, in S1), the coupling method is to disperse hyaluronic acid in an aqueous solution, add 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution to adjust the pH value to 5-6, add a catalyst for the carboxyl activation reaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, react until complete, add carbonate / bicarbonate buffer solution to adjust the pH to 7-7.5, and obtain carboxyl-activated hyaluronic acid; disperse the polypeptide shown in SEQ ID NO.1 in a gelatin solution, and then react the carboxyl-activated hyaluronic acid with the polypeptide shown in SEQ ID NO.1 until the reaction is complete, and purify it by dialysis.

[0020] Further, in S2), the oxidation method is to oxidize dextran with an oxidizing agent NaIO4.

[0021] Further, in S2), the oxidation method is to dissolve dextran in water to obtain a dextran solution; dropwise add a solution containing NaIO4, and react in the dark until complete; add ethylene glycol to terminate the reaction.

[0022] Further, in S3), the hyaluronic acid-polypeptide graft is mixed with 8-10 times its mass of water to obtain a hyaluronic acid-polypeptide graft hydrogel precursor solution. Preferably, it is 9 times its mass of water.

[0023] Further, in S3), the oxidized dextran is mixed with 8-10 times its mass of water to obtain an oxidized dextran hydrogel precursor solution. Preferably, it is 9 times its mass of water.

[0024] Further, in S3), the hyaluronic acid-polypeptide graft and / or the oxidized dextran are promoted to be uniformly dispersed in water by mechanical stirring and / or heating.

[0025] Further, in S3), the mixing ratio between the hyaluronic acid-polypeptide graft hydrogel precursor and the oxidized dextran hydrogel precursor is a volume ratio of 10:3-1, preferably a volume ratio of 10:1.8-1.2, and more preferably 10:1.5.

[0026] Further, in S3), the mixing order of the hyaluronic acid-polypeptide graft hydrogel precursor, the oxidized dextran hydrogel precursor, and the mesenchymal stem cells is to first mix the oxidized dextran hydrogel precursor and the mesenchymal stem cells evenly, and then mix them evenly with the hyaluronic acid-polypeptide graft hydrogel precursor.

[0027] Another aspect of the present invention provides the use of a polypeptide-modified hydrogel composition loaded with mesenchymal stem cells in the preparation of a drug for treating skin injuries and promoting wound healing.

[0028] Further, the treatment of skin injuries or promotion of wound healing is to increase the wound healing rate, restore the number of hair follicles in the damaged skin, reduce the scars generated during skin injury or wound healing, reduce the inflammatory reaction during skin injury or wound healing, and increase the synthesis and secretion of collagen at the skin injury or wound site.

[0029] Another aspect of the present invention provides a pharmaceutical composition for treating skin injuries, and the pharmaceutical composition uses the above-mentioned polypeptide-modified hydrogel composition loaded with mesenchymal stem cells as an active ingredient.

[0030] Another aspect of the present invention provides a preparation method of a polypeptide-modified hydrogel composition loaded with mesenchymal stem cells, which includes the following steps:

[0031] S1) Prepare a hyaluronic acid-polypeptide graft: Couple the carboxyl group of hyaluronic acid with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1 to obtain a hyaluronic acid-polypeptide graft;

[0032] S2) Prepare oxidized dextran: Prepare oxidized dextran by oxidizing dextran;

[0033] S3) Prepare the hyaluronic acid-polypeptide graft and oxidized dextran into hydrogel precursor solutions respectively, and then mix the two hydrogel precursor solutions with mesenchymal stem cells evenly to obtain the hydrogel composition.

[0034] Further, in S1), the coupling method is to activate the carboxyl group on hyaluronic acid and then react with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1.

[0035] Further, in S1), the molar ratio of the carboxyl group on hyaluronic acid to the polypeptide shown in SEQ ID NO.1 is 3-5:1, preferably 4:1.

[0036] Further, in S1), the molecular weight of hyaluronic acid is 100 kDa - 200 kDa, preferably 110 kDa - 150 kDa.

[0037] Further, in S2), the molecular weight of dextran is 50 kDa - 100 kDa, preferably 60 kDa - 80 kDa.

[0038] Further, in S1), the coupling method is to disperse hyaluronic acid in an acidic buffer solution, add a catalyst for carboxyl activation reaction, react until complete, adjust the pH value to above 7 to end the reaction, and obtain carboxyl-activated hyaluronic acid; react the carboxyl-activated hyaluronic acid with the polypeptide shown in SEQ ID NO.1 until the reaction is complete.

[0039] Further, in S1), the coupling method is to disperse hyaluronic acid in an aqueous solution, add 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution to adjust the pH value to 5-6, add a catalyst for carboxyl activation reaction 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, react until complete, add carbonate / bicarbonate buffer solution to adjust the pH to 7-7.5, and obtain carboxyl-activated hyaluronic acid; disperse the polypeptide shown in SEQ ID NO.1 in a gelatin solution, and then react the carboxyl-activated hyaluronic acid with the polypeptide shown in SEQ ID NO.1 until the reaction is complete, and purify it by dialysis.

[0040] Further, in S2), the oxidation method is to oxidize dextran with an oxidizing agent NaIO4.

[0041] Further, in S2), the oxidation method is to dissolve dextran in water to obtain a dextran solution; dropwise add a solution containing NaIO4, and react in the dark until complete; add ethylene glycol to terminate the reaction.

[0042] Further, in S3), the hyaluronic acid-polypeptide graft is mixed with 8-10 times its mass of water to obtain a hyaluronic acid-polypeptide graft hydrogel precursor solution, preferably 9 times its mass of water.

[0043] Further, in S3), the oxidized dextran is mixed with 8-10 times its mass of water to obtain an oxidized dextran hydrogel precursor solution, preferably 9 times its mass of water.

[0044] Further, in S3), the hyaluronic acid-polypeptide graft and / or the oxidized dextran are promoted to be uniformly dispersed in water by mechanical stirring and / or heating.

[0045] Further, in S3), the mixing ratio between the hyaluronic acid-polypeptide graft hydrogel precursor and the oxidized dextran hydrogel precursor is 10:3-1 in volume ratio, preferably 10:1.8-1.2 in volume ratio, and more preferably 10:1.5.

[0046] Further, in S3), the mixing order of the hyaluronic acid-polypeptide graft hydrogel precursor, the oxidized dextran hydrogel precursor and the mesenchymal stem cells is to first mix the oxidized dextran hydrogel precursor and the mesenchymal stem cells evenly, and then mix them evenly with the hyaluronic acid-polypeptide graft hydrogel precursor.

[0047] The beneficial effects of the present invention are as follows:

[0048] The dual-network hydrogel modified with polypeptide (HA-Gel-GFOGER-ODex) prepared by the present invention, wherein the GFOGER sequence is derived from the key integrin-binding site of collagen and can specifically bind to integrin on the cell surface. This modification can enhance the adhesion of cells in the hydrogel on the one hand, and effectively promote cell proliferation, differentiation and migration on the other hand, making the material closer to the biological function of the natural extracellular matrix (ECM). In terms of chemical composition, the hydrogel selects hyaluronic acid and gelatin that can bind to cell surface receptors as the main components, and further modifies the GFOGER polypeptide sequence on this basis to provide biochemical signals. The combined action of chemical and biological signals regulates cell behavior, more precisely simulates the in vivo environment, and optimizes the growth and differentiation conditions of cells. The dual-network hydrogel (HA-Gel-GFOGER-ODex) mixed with mesenchymal stem cells can promote skin injury repair, and the combination of the two has a synergistic effect, which can increase the number of skin hair follicles, reduce scar formation, promote the secretion of type I collagen at the same time, and reduce the epidermal thickness and inflammatory cell infiltration.

[0049] The present invention realizes the simplification of the preparation process and the significant reduction of costs by using natural polymer raw materials such as hyaluronic acid, gelatin and dextran through amidation and imidation reactions under mild conditions, which is beneficial to large-scale production and application promotion. Description of the Drawings

[0050] Figure 1 Where A is the precursor solution of HA-Gel-GFOGER hydrogel; B is the precursor solution of ODex hydrogel.

[0051] Figure 2 Where A is the effect diagram of the healing of mouse skin wounds at different time periods; B is the statistical chart of the proportion of the remaining wound area of the mouse.

[0052] Figure 3 It is the result diagram of the number of hair follicles in the HE staining of mouse skin tissue in different groups. Where A is the photo of the staining result, and B is the comparison diagram of the number of hair follicles.

[0053] Figure 4 It is the result diagram of the epidermal thickness in the HE staining of mouse skin tissue in different groups. Where A is the photo of the staining result, and B is the comparison diagram of the epidermal thickness data.

[0054] Figure 5 It is the result diagram of inflammatory cell infiltration in the HE staining of mouse skin tissue in different groups.

[0055] Figure 6 It is the detection of the cell proliferation marker Ki67 by IF staining of mouse skin tissue. Where A is the IF staining photo, and B is the comparison result of the marker data.

[0056] Figure 7 IF staining was performed on mouse skin tissue to detect the vascular endothelial cell marker CD31; where A is the IF staining photo and B is the comparison result of the marker data.

[0057] Figure 8 IF staining was performed on mouse skin tissue to detect the collagen fiber marker Collagne I; where A is the IF staining photo and B is the comparison result of the marker data. Detailed implementation manners

[0058] To better understand the present invention, the present invention will be further described below with reference to the following embodiments and drawings. The embodiments are only for explanation and do not limit the present invention in any way. In the embodiments, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well known in the art, or the conditions recommended by the instrument manufacturer.

[0059] Example 1: Preparation method of a polypeptide-modified and mesenchymal stem cell-loaded hydrogel composition

[0060] This example provides a preparation method of a polypeptide hydrogel (HA-Gel-GFOGER-ODex) and mesenchymal stem cell composition, including the following steps:

[0061] Step 1) Synthesis of HA-Gel-GFOGER

[0062] Weigh 1 g of 130 kDa hyaluronic acid and dissolve it in 200 mL of deionized water to obtain a hyaluronic acid solution containing 2.64 mM carboxyl groups; add 2-morpholinoethanesulfonic acid (MES) buffer (0.05 M) to adjust the pH of the reaction solution to 5.4; add 10.56 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10.56 mM of N-hydroxysuccinimide (NHS) as catalysts, stir for 2 hours, and carry out the reaction at room temperature; add an appropriate amount of carbonate / bicarbonate buffer to adjust the pH to 7 - 7.5 to obtain an activated carboxyl hyaluronic acid solution;

[0063] Add 120 mg of polypeptide GFOGER (SEQ ID NO.1) to 100 mL of a 10% gelatin solution by mass, and then add the GFOGER gelatin solution to the activated carboxyl hyaluronic acid solution, and carry out the reaction at 37 °C, stirring overnight;

[0064] After the reaction is completed, dialyze with 0.1×PBS (i.e., 0.1-fold concentration of phosphate buffer solution), and freeze-dry to obtain a white foamy product HA-Gel-GFOGER, that is, a mixture of hyaluronic acid and gelatin modified with polypeptide GFOGER;

[0065] Step 2) Synthesis of oxidized dextran (ODex)

[0066] Weigh 5 g of 70 kDa dextran and dissolve it thoroughly in 500 mL of deionized water to obtain a dextran solution containing 12.61 mM repeating units; add a NaIO4 solution containing 12.61 mM to the reaction flask, stir in the dark for 4 h, and react at room temperature; add 3 mL of ethylene glycol to terminate the reaction;

[0067] After the reaction, dialyze with 0.1×PBS and freeze-dry to obtain the white product oxidized dextran (ODex);

[0068] Step 3) Preparation of HA-Gel-GFOGER-ODex

[0069] Weigh appropriate amounts of the HA-Gel-GFOGER obtained in Step 1 and the ODex lyophilized powder obtained in Step 2 and place them in clean 50 mL centrifuge tubes respectively. Add deionized water equivalent to 9 times the mass of the lyophilized powder to each tube and mix evenly ( Figure 1 ) to obtain the HA-Gel-GFOGER hydrogel precursor solution and the ODex hydrogel precursor solution; stir to promote the uniform dispersion of the solutes. The mass fraction of both obtained hydrogel precursor solutions is 10%.

[0070] Put the HA-Gel-GFOGER hydrogel precursor solution into an oven at 65°C and heat for 2 hours;

[0071] And heat the HA-Gel-GFOGER hydrogel precursor solution in a thermostatic water bath to 35 - 45°C, then mix it with the ODex hydrogel precursor solution at a volume ratio of 10:1.5, stir evenly, and let it stand to obtain the transparent HA-Gel-GFOGER-ODex hydrogel;

[0072] Step 4) Synthesis of the polypeptide-modified double-network hydrogel and mesenchymal stem cell composition

[0073] Resuscitate the umbilical cord mesenchymal stem cells and culture them in DMEM high-glucose medium for 3 - 5 days. Observe the cell state every day. When the cell density reaches more than 80% under the microscope, subculture can be carried out; then use 0.25% trypsin to digest for 3 min and carry out 2 - 3 subcultures; aliquot the mesenchymal stem cells into 1×10 5 cells / tube, 1×10 6 cells / tube, 1×10 7Cells / tubes were centrifuged at 300 g for 5 min, and the supernatant was discarded. First, mesenchymal stem cells with different densities were separately mixed with HA-Gel-GFOGER. The ODex component was heated to 35 - 45 °C in a constant temperature water bath to reduce its viscosity, and then mixed with mesenchymal stem cells and HA-Gel-GFOGER. A 1 mL pipette was used to slowly pipette and mix well to avoid generating bubbles. Finally, the cell concentrations in the hydrogel were 1×10⁵ cells / 1.15 mL hydrogel, 1×10⁶ cells / 1.15 mL hydrogel, and 1×10⁷ cells / 1.15 mL hydrogel, respectively.

[0074] Example 2: Construction of a full-thickness skin injury model in mice

[0075] Step 1) A full-thickness skin injury model was constructed using 5 - 6-week-old male balb / c mice.

[0076] Step 2) The mice were anesthetized by intraperitoneal injection of 2.5% tribromoethanol. After successful anesthesia, the hair on the back of the mice was shaved off.

[0077] Step 3) A circular full-thickness excision wound was made on the back of the mice using a circular skin punch with a diameter of 10 mm. The punch was rotated several times on the back skin of the mice to leave a circular incision mark, and surgical scissors were used to cut along the incision to form a uniform circular incision with a size of 10 mm.

[0078] Example 3: Repair effect of the polypeptide hydrogel and mesenchymal stem cell composition on full-thickness skin injury in mice

[0079] The polypeptide hydrogel and mesenchymal stem cell composition in Case 1 above was used to repair the full-thickness skin injury model in mice constructed in Example 2, specifically including:

[0080] Step 1) The mice were randomly divided into 7 groups, with 5 mice in each group, namely the control group (without any treatment), the injury group (without treatment after modeling), the gelatin group (drug administration group: gel formed by gelatin), the HA-Gel-GFOGER-ODex group (drug administration group: the product obtained in Step 3 of Example 1), the 1×10 5 Mesenchymal stem cells and HA-Gel-GFOGER-ODex group (L-MSCs-HA-Gel-GFOGER-ODe) (drug administration group: the product obtained in Step 4 of Example 1), the 1×10 6 Mesenchymal stem cells and HA-Gel-GFOGER-ODex group (M-MSCs-HA-Gel-GFOGER-ODex) (drug administration group: the product obtained in Step 4 of Example 1), the 1×10 7Mesenchymal stem cells and the product obtained from the HA-Gel-GFOGER-ODex group (H-MSCs-HA-Gel-GFOGER-ODex) (administration group: step 4 of Example 1);

[0081] Step 2) Use a 1 mL pipette to aspirate the hydrogel and drop the hydrogel onto the mouse wound. The hydrogel flattens the wound depression to the optimal usage amount;

[0082] Step 3) Take pictures of the mouse wound healing area at D0, D4, D7, and D10 respectively, record and statistically analyze the wound area. Use Image J to quantitatively analyze the mouse wound area and evaluate the wound healing. The wound healing situation is represented by the visible wound area ratio, and the wound area percentage (%) = wound area at Dn / wound area at D0 × 100%;

[0083] Step 4) At D10, sample the healed skin tissue of each group of mice and immerse it in 4% PFA for 24 h;

[0084] As shown in the results Figure 2 It is shown that by statistically analyzing the mouse wound area with Image J, the results show that the wound area of the gelatin group continuously decreases with the increase of days, and there is no significant difference from the injury group; compared with the injury group, the wound area percentage of the HA-Gel-GFOGER-ODex group significantly decreases on the 4th, 7th, and 10th days, showing a significant difference; when mesenchymal stem cells are added to HA-Gel-GFOGER-ODex, there are significant differences between the L-MSCs-HA-Gel-GFOGER-ODex, H-MSCs-HA-Gel-GFOGER-ODex, and H-MSCs-HA-Gel-GFOGER-ODex groups and the injury group on the 4th, 7th, and 10th days, and with the increase of the cell number of mesenchymal stem cells, the mouse wound healing rate significantly increases. The above results indicate that the polypeptide hydrogel and the mesenchymal stem cell composition can accelerate the wound healing rate, which is significantly better than the HA-Gel-GFOGER-ODex group, producing an unexpected technical effect.

[0085] Example 4: HE staining of mouse healed skin tissue

[0086] Perform HE staining on the mouse skin tissue collected in Example 3, specifically including:

[0087] Step 1) Paraffin embedding of skin tissue;

[0088] Preheat the embedding machine to 65 °C in advance;

[0089] Fixation: Add 200 μL of 4% PFA to the EP tube and fix at room temperature for 20 min;

[0090] Pre-staining of skin tissue: Carefully aspirate PFA with a 100 μL pipette, add 20 μL of eosin solution, and stain at room temperature for 5 min.

[0091] Dehydration of skin tissue: Add 10 times the volume of 75% ethanol and dehydrate at room temperature for 5 min; aspirate the 75% ethanol, add 10 times the volume of 90% ethanol, and dehydrate at room temperature for 5 min.

[0092] Counterstaining of skin tissue: Add 20 μL of eosin solution and stain at room temperature for 5 min. (Counterstaining can be performed if the decolorization is severe);

[0093] Dehydration of skin tissue: Add 10 times the volume of absolute ethanol and dehydrate at room temperature for 5 min, repeat three times;

[0094] Clearing of skin tissue: Add 10 times the volume of xylene, soak at room temperature for 5 min, repeat three times;

[0095] Infiltration of skin tissue with wax: Take a white embedding cassette, remove the lid, and label the official batch number on the cassette. Wait for the skin tissue to sink to the bottom, carefully aspirate the upper layer of xylene (leave 50 μL), use a 1 mL pipette tip with the tip cut off to aspirate the sample, transfer the sample to a paraffin mold, add paraffin, soak at 65 °C for 10 min; tilt the mold to discard the paraffin, add paraffin again and soak for 10 min, gently place the white embedding cassette on the paraffin mold, let it stand at 65 °C for 10 min, wait for the paraffin to solidify, and store the wax block at room temperature;

[0096] Step 2) Section HE staining

[0097] Section the paraffin-embedded block on a microtome, with a thickness of 5 μm;

[0098] Deparaffinize the sections to water: Place the sections in xylene I for 5 min, xylene II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and wash with distilled water in sequence;

[0099] Stain the cell nuclei with hematoxylin: Preheat the water bath to 60 °C in advance, soak the sections in hematoxylin and stain at 60 °C for 3 - 5 min, soak in tap water for a while to remove the excess hematoxylin, differentiate with 1% acidic ethanol differentiation solution for 20 s, wash with tap water, and blue with 1% ammonia water for 20 s, then wash with tap water;

[0100] Stain the cytoplasm with eosin: Soak the sections in eosin staining solution and stain for 30 s;

[0101] Dehydration and coverslipping: Immerse the sections successively in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes for dehydration and clearing. Take the sections out of the xylene. Place a clean blotting paper on the table, take out the glass slide from the xylene and place it on the paper (with the side of the section facing up). Quickly drop a drop of mounting medium in the center of the section. Hold the forceps and gently clamp the right side of the cover slip, tilt it slightly so that its left side touches the mounting medium, and then slowly lower the cover slip to reduce or avoid generating air bubbles. Then perform microscopic examination and image acquisition and analysis.

[0102] On the 10th day, the HE staining of the skin tissues of each group was used to evaluate the repair effect of the full-thickness skin injury in mice by the number of hair follicles, the thickness of the epidermis, and the infiltration of inflammatory cells; the more the number of hair follicles, the thinner the epidermis, and the fewer the number of inflammatory cells, indicating a better wound recovery effect. Among them, the hair follicle is an important accessory structure of the skin. During the skin injury repair process, the regeneration of hair follicles is a key link. The dynamic changes in the number of inflammatory cells can reflect the process of skin injury repair.

[0103] The HE staining results showed that in terms of the number of hair follicles, compared with the injury group, the number of increased hair follicles in the gelatin group and the HA-Gel-GFOGER-ODex group showed an upward trend, but there was no significant difference; compared with the injury group, in the L-MSCs-HA-Gel-GFOGER-ODex, H-MSCs-HA-Gel-GFOGER-ODex, and H-MSCs-HA-Gel-GFOGER-ODex groups, the number of hair follicles increased significantly with the increase of mesenchymal stem cells, ( Figure 3 ). An increase in the number of hair follicles usually means a stronger regeneration ability of the skin tissue, and the damaged area can better reconstruct the accessory structures of the skin, reflecting good cell proliferation and differentiation abilities during the skin repair process, which is an important sign of the positive development of skin injury repair.

[0104] In terms of the thickness of the epidermis, compared with the control group, the thickness of the epidermis in the injury group increased by 5.4 times, the thickness of the epidermis in the gelatin group and the HA-Gel-GFOGER-ODex group increased by 3.8 times and 3.6 times respectively, and the thickness of the epidermis in the L-MSCs-HA-Gel-GFOGER-ODex, H-MSCs-HA-Gel-GFOGER-ODex, and H-MSCs-HA-Gel-GFOGER-ODex groups increased by 2.6 times, 2.4 times, and 1.4 times respectively; there was no significant difference in the thickness of the epidermis in the H-MSCs-HA-Gel-GFOGER-ODex group compared with the control group, indicating that the H-MSCs-HA-Gel-GFOGER-ODex group had recovered to a level similar to that of the control group; compared with the injury group, the thickness of the epidermis in the remaining groups decreased significantly (Figure 4 ). When the epidermis is too thick after repair, it indicates excessive cell proliferation or abnormal differentiation, and there may be problems such as scar formation. The product of the present invention can make the thickness of the epidermis after recovery similar to that of the control group, indicating a balanced state of proliferation and differentiation, and no scar tissue is formed.

[0105] In terms of inflammatory cell infiltration, compared with the injury group, the inflammatory cell infiltration in the gelatin group decreased slightly, and the inflammatory cell infiltration in the HA-Gel-GFOGER-ODex group decreased again, and its healing effect was better than that in the gelatin group; in the polypeptide hydrogel and mesenchymal stem cell group, as the number of mesenchymal stem cells increased, the inflammatory cell infiltration decreased significantly, and its healing effect was better than that in the HA-Gel-GFOGER-ODex group ( Figure 5 ).

[0106] In summary, HA-Gel-GFOGER-ODex can promote the repair of full-thickness skin damage in mice. The combined use of mesenchymal stem cells and HA-Gel-GFOGER-ODex has a synergistic effect, which can increase the number of hair follicles, reduce scar formation, and reduce epidermal thickness and inflammatory cell infiltration.

[0107] Example 5: IF detection in mouse skin tissue

[0108] The mouse skin tissue collected in Example 2 was subjected to IF detection, specifically including:

[0109] Step 1) Paraffin embedding of mouse skin tissue

[0110] The specific steps are the same as step 1 of Example 4);

[0111] Step 2) Slice IF detection

[0112] The paraffin sections were placed in a 65°C oven for 30 min-1 h until the wax dissolved;

[0113] Dewax the paraffin sections to water: sequentially place the sections in xylene I for 20 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 10 min, 95% ethanol for 10 min, 70% ethanol for 10 min, and wash with distilled water for 5 min;

[0114] Antigen repair: Place the slides in a repair box filled with EDTA antigen repair buffer (pH 8.0) in a microwave oven for antigen repair. Heat on high heat for 5 minutes, medium heat for 20 minutes, and then switch to low heat for 2 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (pH 7.4) and wash them twice on a decolorizing shaker, 5 minutes each time.

[0115] Blocking: After taking out the sections from PBS and air-drying them, use an immunohistochemical pen to draw a circle around the tissue, and add 100 μL of blocking solution (the blocking solution is PBS containing 5% BSA and 0.3% Triton X-100) to block for 1 hour;

[0116] Primary antibody incubation: Dilute the primary antibody with the blocking solution to an appropriate concentration, add 50 μL of the primary antibody to each section, place the sections flat in a wet box, and incubate overnight at 4°C;

[0117] Secondary antibody incubation: Take out the sections from the refrigerator and allow them to warm up for 20 - 30 min, wash with PBST 3 times, 10 min each time. Dilute the fluorescently labeled secondary antibody with PBS, add 50 μL of the secondary antibody to each section, and incubate at room temperature for half an hour;

[0118] Counterstaining with DAPI: Wash with PBST 3 times, 10 min each time, add the diluted DAPI, and incubate at room temperature for 10 min. Wash with PBS for 5 min;

[0119] Mounting and microscopic examination: Add about 30 μL of anti-fluorescence quencher to each section, cover with a coverslip, observe under a fluorescence microscope, and take pictures for recording.

[0120] IF was used to detect the expression of cell proliferation marker (Ki67), vascular endothelial cell (CD31) marker, and collagen fiber I (Collagen I) marker in mouse skin tissue. The test results are shown in Figures 6 - 8 。

[0121] The IF results showed that the control group expressed Ki67, CD31, and Collagen I. Since the skin of the control group was not damaged, the expression level of Ki67 was relatively low; however, in the injury group, the cell proliferation in the skin tissue increased, the blood vessels decreased, and the collagen fibers decreased; compared with the injury group, the expression level of Ki67 decreased, the expression level of CD31 increased, and the expression level of Collagen I increased in the gelatin group, showing significant differences. After repair with HA-Gel-GFOGER-ODex, the expression levels of the three also showed significant differences compared with the injury group.

[0122] In the group of polypeptide hydrogel and mesenchymal stem cells, the Ki67 expression level decreased significantly as the cell quantity increased. As the repair process progressed, the Ki67 expression level gradually recovered from the initial increase at the injury stage to a level close to normal, indicating that cell proliferation was gradually controlled and tissue repair tended to be completed. CD31, as a marker of vascular endothelial cells, an increase in CD31 expression means that the tissue repair microenvironment was improved, that is, there was a significant increase in blood vessels. The collagen fibers of Collagen I increased significantly, indicating that the composition of the present invention can significantly promote the synthesis and secretion of type I collagen, which is beneficial to the reconstruction of collagen fibers and the restoration of the skin tissue structure; among them, the H-MSCs-HA-Gel-GFOGER-ODex group had the best repair effect.

[0123] The above results show that the composition of polypeptide hydrogel and mesenchymal stem cells can significantly promote the repair of full-thickness skin injury in mice.

Claims

1. A polypeptide-modified hydrogel composition loaded with mesenchymal stem cells, characterized in that, It includes a polypeptide-modified double-network hydrogel carrier and mesenchymal stem cells; The double-network hydrogel carrier includes a hydrogel formed by a graft of hyaluronic acid and the polypeptide shown in SEQ ID NO.1 and a hydrogel formed by oxidized dextran.

2. The hydrogel composition according to claim 1, characterized in that The mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, and amniotic mesenchymal stem cells.

3. The hydrogel composition according to claim 1, characterized in that, The concentration of mesenchymal stem cells in the hydrogel composition is 1×10⁶ cells / 1.15 mL of hydrogel or more, preferably 8×10⁵ cells / 1.15 mL of hydrogel - 1×10⁷ cells / 1.15 mL of hydrogel.

4. The hydrogel composition according to claim 1, characterized in that, The hydrogel composition is obtained by the following method: S1) Prepare the hyaluronic acid-polypeptide graft: Couple the carboxyl group of hyaluronic acid and the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1 to obtain the hyaluronic acid-polypeptide graft; S2) Prepare oxidized dextran: Prepare oxidized dextran by oxidizing dextran; S3) Respectively prepare the hydrogel precursor solutions of the hyaluronic acid-polypeptide graft and oxidized dextran, and then mix the two hydrogel precursor solutions and mesenchymal stem cells evenly to obtain the hydrogel composition.

5. The hydrogel composition according to claim 4, characterized in that, In S1), the coupling method is to activate the carboxyl group on hyaluronic acid and then react with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1; Preferably, in S1), the molar ratio of the carboxyl group on hyaluronic acid to the polypeptide shown in SEQ ID NO.1 is 3 - 5:1, preferably 4:1; Preferably, in S1), the molecular weight of hyaluronic acid is 100 kDa - 200 kDa; Preferably, in S2), the molecular weight of dextran is 50 kDa - 100 kDa.

6. The hydrogel composition according to claim 4, characterized in that, In S2), the oxidation method is to oxidize dextran with the oxidant NaIO₄; Preferably, in S2), the oxidation method is to dissolve dextran in water to obtain a dextran solution; dropwise add the NaIO₄ solution, react in the dark until complete; add ethylene glycol to terminate the reaction.

7. The hydrogel composition according to claim 4, characterized in that, In S3), the mixing ratio between the hyaluronic acid-polypeptide graft hydrogel precursor and the oxidized dextran hydrogel precursor is 10:3 - 1 by volume; Preferably, in S3), the hyaluronic acid-polypeptide graft is mixed with 8 - 10 times its mass of water to obtain the hyaluronic acid-polypeptide graft hydrogel precursor solution; Preferably, in S3), oxidized dextran is mixed with 8 - 10 times its mass of water to obtain the oxidized dextran hydrogel precursor solution; Preferably, in S3), the mixing order of the hyaluronic acid-polypeptide graft hydrogel precursor, the oxidized dextran hydrogel precursor, and mesenchymal stem cells is to first mix the hyaluronic acid-polypeptide graft hydrogel precursor and mesenchymal stem cells evenly, and then mix with the oxidized dextran hydrogel precursor evenly.

8. Use of the hydrogel composition according to any one of claims 1 - 7 in the preparation of a drug for treating skin injuries and promoting wound healing; Preferably, the treatment of skin injury or promotion of wound healing is to increase the wound healing rate, restore the number of hair follicles in the damaged skin, reduce the scar generated during skin injury or wound healing, reduce the inflammatory reaction during skin injury or wound healing, and increase the synthesis and secretion of collagen in the skin injury or wound site.

9. A pharmaceutical composition for treating skin injury, wherein the pharmaceutical composition uses the hydrogel composition according to any one of claims 1-7 as an active ingredient.

10. A method for preparing the hydrogel composition according to any one of claims 1-7, characterized in that, It comprises the following steps: S1) Prepare a hyaluronic acid-polypeptide graft: Couple the carboxyl group of hyaluronic acid with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1 to obtain a hyaluronic acid-polypeptide graft; S2) Prepare oxidized dextran: Prepare oxidized dextran by oxidizing dextran; S3) Respectively prepare hydrogel precursor solutions of the hyaluronic acid-polypeptide graft and oxidized dextran, and then uniformly mix the two hydrogel precursor solutions with mesenchymal stem cells to obtain the hydrogel composition; Preferably, in S1), the coupling method is to activate the carboxyl group on hyaluronic acid and then react with the amino group at the N-terminus of the polypeptide shown in SEQ ID NO.1; Preferably, in S1), the coupling method is to disperse hyaluronic acid in an acidic buffer solution, add a catalyst for carboxyl activation reaction, react until complete, adjust the pH value to above 7 to end the reaction, and obtain carboxyl-activated hyaluronic acid; React the carboxyl-activated hyaluronic acid with the polypeptide shown in SEQ ID NO.1 until the reaction is complete; Preferably, in S2), the oxidation method is to oxidize dextran with the oxidant NaIO4; Preferably, in S2), the oxidation method is to dissolve dextran in water to obtain a dextran solution; Dropwise add a solution containing NaIO4, react in the dark until complete; Add ethylene glycol to terminate the reaction; Preferably, in S3), the hyaluronic acid-polypeptide graft is mixed with 8-10 times its mass of water to obtain a hyaluronic acid-polypeptide graft hydrogel precursor solution; Preferably, in S3), oxidized dextran is mixed with 8-10 times its mass of water to obtain an oxidized dextran hydrogel precursor solution; Preferably, in S3), the mixing ratio between the hyaluronic acid-polypeptide graft hydrogel precursor and the oxidized dextran hydrogel precursor is a volume ratio of 10:3-1; Preferably, in S3), the mixing order of the hyaluronic acid-polypeptide graft hydrogel precursor, the oxidized dextran hydrogel precursor, and mesenchymal stem cells is to first uniformly mix the oxidized dextran hydrogel precursor with mesenchymal stem cells, and then uniformly mix with the hyaluronic acid-polypeptide graft hydrogel precursor.

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