A stem cell injection for treating diabetic foot and preparation method thereof

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a stem cell injection for treating diabetic foot and a preparation method thereof. The present invention discloses a preparation method of a stem cell injection for treating diabetic foot, specifically: firstly, a modified hyaluronic acid dispersion is mixed with a gelatin dispersion, and a cell suspension containing human umbilical cord mesenchymal stem cells is added after emulsification and sterilization to obtain a hydrogel solution; then the hydrogel solution is dripped into sterile mineral oil, and after heat preservation, filtration, washing, and drying, hydrogel microspheres are obtained; finally, the hydrogel microspheres are mixed with deionized water and cocamidopropyl betaine to obtain. The hydrogel microspheres provided by the present invention have higher stability and a more suitable matrix environment, which can not only improve cell survival rate and integrity, improve injection administration efficiency, but also promote the proliferation of mesenchymal stem cells, cytokine secretion and directional endothelial tube differentiation, and show excellent therapeutic effects when used to treat diabetic foot.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a stem cell injection for treating diabetic foot and a preparation method thereof. Background Art

[0002] Diabetic foot is one of the serious complications of diabetic patients, manifested by ulcer formation and deep tissue destruction. The wound of diabetic foot ulcer is a typical chronic and difficult to heal wound. Its pathogenesis involves neuropathy, vascular disease and infection, and its treatment is a difficult problem in the current medical community. The clinical manifestations are diverse and complex. Common symptoms include foot infection, ulcer and (or) deep tissue destruction. In severe cases, it may manifest as osteomyelitis, tendonitis and foot gangrene. The complexity of the formation and healing mechanism of diabetic foot ulcer wounds makes clinical treatment difficult, and the prognosis is poor, the cost is high, and the disability rate is extremely high, which has a huge impact on the patient's life, psychology and socio-economics. Stem cell therapy is a treatment method for diabetic foot that has emerged in recent years. Mesenchymal stem cells are multifunctional stem cells with self-renewal and directed differentiation capabilities. They can play a role in repairing wounds when used to treat diabetic foot.

[0003] The patent with application number CN202011563933.3 provides a method for preparing a stem cell preparation for the treatment of diabetic foot. Umbilical cord mesenchymal stem cells, pancreatic islet cells, glycine and fibroblasts are dissolved in a solvent and stored at 3-6°C. The invention utilizes the omnipotence of stem cells to compound umbilical cord mesenchymal stem cells, pancreatic islet cells, glycine and fibroblasts to enhance the function of stem cells, improve the body's repair ability, improve the local microenvironment, accelerate tissue healing, and can effectively treat diabetic foot; the patent with application number CN202010641794.5 provides an umbilical cord mesenchymal stem cell preparation for the treatment of diabetic foot. The umbilical cord of a full-term healthy fetus is selected, and the umbilical cord mesenchymal stem cells are isolated. The culture system is expanded and cultured in a culture system without other animal-derived components except humans, and various indicators such as surface marker expression and in vitro differentiation ability are evaluated. Identification, in line with the basic characteristics of mesenchymal stem cells and without tumor-forming side effects, the stem cells that pass various tests and meet the standards are stored in liquid nitrogen or used for the preparation of subsequent mesenchymal stem cell preparations. The stem cell preparations provided by the invention have excellent performance and have good therapeutic effects on diabetes and its complications of lower limb ischemia syndrome; the above inventions all use umbilical cord mesenchymal stem cells to treat diabetic foot. Mesenchymal stem cells are a type of common clinical tissue stem cells with multidirectional differentiation potential, especially mesenchymal stem cells derived from the umbilical cord, which have many advantages, such as wide source, easy acquisition, low antigenicity, etc., and umbilical cord mesenchymal stem cells are closer to embryonic cells, and the effect of promoting wound healing is significantly better than adult cells. However, stem cell therapy is usually injected into the patient's body, which will lead to problems such as low cell survival rate, cell loss, and low drug administration efficiency, and repeated injections will also cause complications such as infection. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention solves the practical problems in the clinical treatment of diabetic foot ulcers, penetrates the fields of biomedical polymer materials, tissue engineering, etc., selects medical biopolymer materials hyaluronic acid and gelatin, and forms a hydrogel material with an interpenetrating network structure after functional modification. The hydrogel material forms a functionalized cell / material three-dimensional complex with umbilical cord mesenchymal stem cells, which is applied to the treatment of diabetic ulcer animal models, thereby accelerating the natural repair process of tissues, promoting the healing of refractory ulcers and effectively shortening the wound healing time.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A method for preparing a stem cell injection for treating diabetic foot comprises the following steps:

[0007] Dispersing modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid dispersion; dispersing gelatin in deionized water to obtain a gelatin dispersion; mixing the modified hyaluronic acid dispersion with the gelatin dispersion, adding a cell suspension containing human umbilical cord mesenchymal stem cells after emulsification and sterilization, and mixing evenly to obtain a hydrogel solution; dropping the hydrogel solution into sterile mineral oil, keeping it warm at 35-40° C. for 10-15 hours, filtering, washing, and drying with sterile gauze to obtain hydrogel microspheres; mixing the hydrogel microspheres with deionized water and cocamidopropyl betaine evenly to obtain a stem cell injection solution;

[0008] The preparation method of the modified hyaluronic acid is as follows:

[0009] S1. Dissolve hyaluronic acid in deionized water under stirring, add sodium periodate, raise the temperature to 30-50°C, react for 2-6 hours in the dark, then add glycerol, continue stirring for 20-40 minutes, cool to room temperature, dialyze, and freeze-dry to obtain oxidized hyaluronic acid;

[0010] S2. Dispersing the oxidized hyaluronic acid obtained in step S1 in dimethyl sulfoxide under stirring, adding p-toluenesulfonic acid and N-methyl-2-hydroxyethylamine in sequence, reacting at 60-80° C. for 4-8 hours, cooling to room temperature, dialyzing, and freeze-drying to obtain amino hyaluronic acid;

[0011] S3, under nitrogen atmosphere and stirring, dispersing the amino hyaluronic acid obtained in step S2 in dimethyl sulfoxide, adding 5-bromo-ferulic acid, reacting at 90-110° C. for 8-14 h, cooling to room temperature, dialyzing, and freeze-drying to obtain modified hyaluronic acid;

[0012] The synthesis route of hydrogel microspheres is as follows:

[0013] in It is the main chain of gelatin molecule.

[0014] The invention first uses hyaluronic acid as a raw material, and uses sodium periodate to oxidize the hyaluronic acid to obtain oxidized hyaluronic acid; then uses oxidized hyaluronic acid and N-methyl-2-hydroxyethylamine as raw materials and p-toluenesulfonic acid as an acidic catalyst to carry out an esterification reaction to obtain amino hyaluronic acid; then uses amino hyaluronic acid and 5-bromo-ferulic acid as raw materials, and carries out an N-alkylation reaction under high temperature catalysis to obtain modified hyaluronic acid; finally, the modified hyaluronic acid, gelatin and human umbilical cord mesenchymal stem cells are compounded, and subjected to a Schiff base condensation reaction to obtain hydrogel microspheres. The present invention chemically modifies hyaluronic acid to obtain modified hyaluronic acid, and uses the modified hyaluronic acid and gelatin to construct a functionalized polymer hydrogel material. Human umbilical cord mesenchymal stem cells further form hydrogel microspheres with the functionalized polymer hydrogel material. The hydrogel microspheres are three-dimensional complexes of cells and materials. The three-dimensional complex has excellent biocompatibility and a high specific surface area, which is conducive to cell adhesion and growth. The complex has a high VEGF expression level and can significantly improve the healing of diabetic ulcers in mice and rats, laying a certain foundation for further research on the treatment of diabetic foot ulcers.

[0015] In order to obtain a functional cell / material three-dimensional complex, the mass ratio of hyaluronic acid, deionized water, sodium periodate, and glycerol in step S1 is 1:90-110:0.6-0.9:0.4-0.7; the molar ratio of the carboxyl group on the oxidized hyaluronic acid, N-methyl-2-hydroxyethylamine, and p-toluenesulfonic acid in step S2 is 1:1-3:0.12-0.17, and the amount of oxidized hyaluronic acid added to dimethyl sulfoxide is 0.05-0.09 g / mL; the mass ratio of the amino hyaluronic acid to 5-bromo-ferulic acid in step S3 is 100:20-30, and the dimethyl sulfoxide is 1:1-3:0.12-0.17. The amount of amino hyaluronic acid added to sulfone is 0.06-0.1 g / mL; the volume ratio of the modified hyaluronic acid dispersion, gelatin dispersion and cell suspension is 40-50:45-55:5; the mass fraction of the modified hyaluronic acid in the modified hyaluronic acid dispersion is 0.08-0.12%, and the mass fraction of gelatin in the gelatin dispersion is 0.08-0.12%; the emulsification is performed at 1000-3000 rpm for 1-3 min; the mass ratio of the hydrogel microspheres, deionized water and cocamidopropyl betaine is 10-15:100-120:3-6.

[0016] Ferulic acid is a derivative of cinnamic acid and a functional phenolic acid in plant cell walls. Pharmacological studies and clinical applications have shown that ferulic acid has significant antioxidant and free radical scavenging activity. It also has the effects of inhibiting the production of inflammatory mediators, anti-thrombosis and platelet aggregation, as well as protecting cardiovascular and antagonizing endothelin production. Ferulic acid has a wide range of effects on the cardiovascular and cerebrovascular systems, is easily metabolized by the human body, has low toxicity and good safety. Ferulic acid can promote the proliferation and vitality of vascular endothelial cells, and can protect vascular endothelial cells through a variety of signal transduction pathways to maintain the integrity of the vessel wall; in the treatment of diabetic foot, in addition to the homing effect, mesenchymal stem cells can also promote angiogenesis through autocrine and paracrine secretion of a variety of growth factors, improve blood flow in lesion tissues, and thus promote wound healing. Studies have shown that mesenchymal stem cells can secrete a variety of cytokines, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), etc. These factors can promote angiogenesis, enhance microhemodynamics, and promote wound healing. Among them, VEGF is the strongest and most effective one among a series of factors that regulate angiogenesis and repair. Ferulic acid has a protective effect on mesenchymal stem cells, and can enhance the proliferation of mesenchymal stem cells, secrete cytokines and directional endothelial tube differentiation, thereby achieving vascular damage repair.

[0017] The present invention has the following beneficial effects:

[0018] The present invention carries out specific chemical modification on hyaluronic acid, and introduces ferulic acid and aldehyde groups into the structure of the modified hyaluronic acid, thereby not only retaining the degradability and biocompatibility of the modified hyaluronic acid, but also making it have the biological activity of ferulic acid. By targetedly improving the structure and properties of hyaluronic acid and giving it multiple functional groups and biologically active groups, not only the application of hyaluronic acid in the fields of biomaterials, disease treatment, etc. is expanded, but also its application effect in these fields is enhanced.

[0019] The present invention uses modified hyaluronic acid and gelatin to construct a functionalized polymer hydrogel material, and further forms hydrogel microspheres with human umbilical cord mesenchymal stem cells and functionalized polymer hydrogel materials. Compared with traditional biogels, the hydrogel microspheres have higher stability and a more suitable matrix environment, can improve cell survival rate and cell integrity, improve injection efficiency, and promote mesenchymal stem cell proliferation, cytokine secretion and directional endothelial tube differentiation. The hydrogel microspheres are mixed with deionized water and cocamidopropyl betaine to prepare a stem cell injection, which can significantly improve the wound healing efficiency and shorten the wound healing cycle when used to treat diabetic foot. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the hydrogen nuclear magnetic resonance spectrum of the hyaluronic acid and modified hyaluronic acid provided in Example 1.

[0021] Figure 2 This is a SEM image of the hydrogel material prepared according to the method described in Example 1.

[0022] Figure 3 These are the test results of the cell proliferation ability of hydrogel microspheres on human umbilical cord mesenchymal stem cells.

[0023] Figure 4 It is the healing effect of hydrogel microspheres on the wound of diabetic mice.

[0024] Figure 5 It is the effect of hydrogel microspheres on VEGF protein expression. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] The raw materials used in the following examples are all common commercial products. Phosphate buffer (PBS), sterile, pH 7.4, purchased from Shanghai Yueteng Biotechnology Co., Ltd.; hyaluronic acid, pharmaceutical grade, molecular weight 10000Da, purchased from Shandong Runxin Pharmaceutical Co., Ltd.; gelatin, pharmaceutical grade, model CP2020, purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.; human umbilical cord mesenchymal stem cells, product number HUXUC-01001, cell concentration 1×10 6 cells / mL, purchased from Saiye (Guangzhou) Biotechnology Co., Ltd.; cell proliferation activity kit (CCK-8) was purchased from Takara.

[0027] Example 1

[0028] A method for preparing a stem cell injection solution comprises the following steps:

[0029] S1. Under stirring at 180 rpm, dissolve hyaluronic acid in deionized water, add sodium periodate, heat to 40°C, react in the dark for 4 hours, then add glycerol, continue stirring for 30 minutes, cool to room temperature to obtain a reaction mixture, put the reaction mixture into a dialysis bag with a cutoff of 4000 Daltons, place it in deionized water for 96 hours (deionized water is replaced every 12 hours for the first 48 hours, and every 24 hours for the next 48 hours), collect the liquid in the bag after the dialysis is completed, freeze-dry it to obtain oxidized hyaluronic acid, and measure the aldehyde content in the oxidized hyaluronic acid by sodium hydroxide-methanol solution titration method, which is 2.58 mmol / g; wherein the mass ratio of hyaluronic acid, deionized water, sodium periodate, and glycerol is 1:100:0.8:0.6;

[0030] S2. Disperse the oxidized hyaluronic acid obtained in step S1 in dimethyl sulfoxide under stirring at 180 rpm, add p-toluenesulfonic acid and N-methyl-2-hydroxyethylamine in sequence, react at 70° C. for 6 h, cool to room temperature to obtain a reaction mixture, put the reaction mixture into a dialysis bag with a cutoff of 4000 Daltons, and dialyze in deionized water for 96 h (deionized water is replaced every 12 h for the first 48 h, and every 24 h for the next 48 h). After the dialysis is completed, collect the liquid in the bag and freeze-dry it to obtain amino hyaluronic acid, and the amino grafting rate is 50.6% (the ratio of the number of carboxyl groups participating in the reaction in the hyaluronic acid structure to the number of carboxyl groups theoretically in the hyaluronic acid structure); wherein the molar ratio of carboxyl groups, N-methyl-2-hydroxyethylamine, and p-toluenesulfonic acid on the oxidized hyaluronic acid is 1:2:0.15, and the amount of oxidized hyaluronic acid added to dimethyl sulfoxide is 0.07 g / mL;

[0031] S3, under nitrogen atmosphere and stirring at 180 rpm, dispersing the amino hyaluronic acid obtained in step S2 in dimethyl sulfoxide, adding 5-bromo-ferulic acid, reacting at 100° C. for 12 h, cooling to room temperature to obtain a reaction mixture, placing the reaction mixture in a dialysis bag with a cutoff of 4000 Daltons, and dialyzing in deionized water for 96 h (deionized water was replaced every 12 h for the first 48 h, and every 24 h for the next 48 h), collecting the liquid in the bag after the dialysis was completed, and freeze-drying to obtain modified hyaluronic acid, wherein the grafting rate of ferulic acid was 12.8%; wherein the mass ratio of the amino hyaluronic acid to the 5-bromo-ferulic acid was 100:25, and the amount of the amino hyaluronic acid added to the dimethyl sulfoxide was 0.08 g / mL;

[0032] S4. Disperse the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid dispersion with a mass fraction of 0.1%; disperse gelatin in deionized water (stir at 60°C for 30 minutes and then cool to 35-40°C for insulation) to obtain a gelatin dispersion with a mass fraction of 0.1%; mix the modified hyaluronic acid dispersion with the gelatin dispersion, emulsify at 2000 rpm for 2 minutes, and add a cell suspension containing human umbilical cord mesenchymal stem cells (the cell concentration in the cell suspension is 2×10 7 cells / mL, commercially available human umbilical cord mesenchymal stem cells were centrifuged and resuspended in sterile PBS buffer), mixed evenly to obtain a hydrogel solution; the hydrogel solution was dripped into sterile mineral oil using a syringe, kept warm at 37°C for 12 hours, solids were collected by filtration, washed with sterile phosphate buffer, and the surface moisture was wiped dry with sterile gauze to obtain hydrogel microspheres (the average particle size of the hydrogel microspheres was measured to be 75 μm using a particle size distribution laser diffraction analyzer Mastersizer 2000); the hydrogel microspheres were mixed evenly with deionized water and cocamidopropyl betaine to obtain a stem cell injection, and the injection method was subcutaneous injection; wherein the volume ratio of the modified hyaluronic acid dispersion, the gelatin dispersion, and the cell suspension was 45:50:5, and the mass ratio of the hydrogel microspheres, deionized water, and cocamidopropyl betaine was 13:110:4.

[0033] Figure 1 The hydrogen NMR spectra of the hyaluronic acid and modified hyaluronic acid provided in this embodiment, wherein Figure (a) is the hydrogen NMR spectrum of hyaluronic acid, and Figure (b) is the hydrogen NMR spectrum of modified hyaluronic acid. By comparing Figure (a) with Figure (b), it is found that in Figure (b), a proton peak of the benzene ring appears at δ of 6.1-6.4ppm, a proton peak of the phenolic hydroxyl group appears at δ of 9.58ppm, and a proton peak of the aldehyde group appears at δ of 9.72ppm, indicating that ferulic acid and aldehyde groups are successfully introduced into the hyaluronic acid structure.

[0034] The modified hyaluronic acid was dispersed in deionized water to obtain a modified hyaluronic acid dispersion with a mass fraction of 0.1%; gelatin was dispersed in deionized water (stirred at 60°C for 30 minutes and then cooled to 35-40°C for insulation) to obtain a gelatin dispersion with a mass fraction of 0.1%; the modified hyaluronic acid dispersion and the gelatin dispersion were mixed, emulsified at 2000rpm for 2 minutes, poured into a mold, and kept warm at 37°C for 12 hours to obtain a hydrogel material; the hydrogel material was subjected to scanning electron microscopy (SEM) analysis, the sample was freeze-dried and then extracted in a liquid nitrogen environment, coated on a conductive silica gel, and after gold spraying, the morphology of the sample was observed using a JSM-7800F field emission scanning electron microscope. The results are as follows: Figure 2As shown, the hydrogel material has a porous structure and a high specific surface area. As a cell carrier, it can not only provide a 3D culture microenvironment for cells and support cell viability and function, but also achieve efficient cell loading and enhance the therapeutic effect of stem cells.

[0035] Example 2

[0036] A method for preparing a stem cell injection is prepared according to the method described in Example 1, except that: in step S4, the volume ratio of the modified hyaluronic acid dispersion, the gelatin dispersion, and the cell suspension is 40:45:5; and the average particle size of the hydrogel microspheres is 72 μm.

[0037] Example 3

[0038] A method for preparing a stem cell injection is prepared according to the method described in Example 1, except that: in step S4, the volume ratio of the modified hyaluronic acid dispersion, the gelatin dispersion, and the cell suspension is 50:55:5; and the average particle size of the hydrogel microspheres is 76 μm.

[0039] Example 4

[0040] A method for preparing a stem cell injection is prepared according to the method described in Example 1, except that: in step S4, the mass fraction of modified hyaluronic acid in the modified hyaluronic acid dispersion is 0.08%, the mass fraction of gelatin in the gelatin dispersion is 0.12%; and the average particle size of the hydrogel microspheres is 69 μm.

[0041] Comparative Example 1

[0042] A method for preparing a stem cell injection is prepared according to the method described in Example 1, except that in step S4, the volume ratio of the modified hyaluronic acid dispersion, the gelatin dispersion, and the cell suspension is 30:50:5.

[0043] Comparative Example 2

[0044] A method for preparing a stem cell injection is prepared according to the method described in Example 1, except that steps S2 and S3 are omitted, and the modified hyaluronic acid in step S4 is replaced by oxidized hyaluronic acid.

[0045] The hydrogel microspheres prepared in Examples 1-4 were subjected to cytotoxicity analysis. The hydrogel microspheres prepared in Examples 1-4 were placed in DMEM culture medium containing 10% FBS, and the cell density was adjusted to 1.0×10 6cells / mL, 200 μL of sample was added to each well, and the cells were cultured at 37°C and 5% CO2 for 24 h. A CCK-8 kit was used according to the manufacturer's instructions, 10 μL of CCK-8 reagent was added to each well, and the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader after incubation for 2 h. Five replicate wells were set in each group, and the average value was taken to calculate the cell survival rate. The results showed that the cell survival rate of human umbilical cord mesenchymal stem cells in the hydrogel microspheres provided in Examples 1-4 was high, all above 97%, indicating that the hydrogel microspheres provided by the present invention have low cytotoxicity.

[0046] The hydrogel microspheres prepared in Examples 1-4 and Comparative Examples 1-2 were subjected to cell proliferation analysis. The hydrogel microspheres prepared in Examples 1-4 and Comparative Examples 1-2 were placed in DMEM culture medium containing 10% FBS, and the cell density was adjusted to 1.0×10 6 cells / mL, and the sample was prepared. The sample containing only human umbilical cord mesenchymal stem cells (commercially available human umbilical cord mesenchymal stem cells were centrifuged and resuspended in DMEM culture medium containing 10% FBS) was used as the control group. 100 μL of the sample was added to each well of a 96-well plate, and cultured at 37°C and 5% CO2 for 72 hours. According to the instructions of the CCK-8 cell counting kit, 10 μL of CCK-8 reagent was added to each well. After incubation for 4 hours, the absorbance at 450nm (i.e., OD value) was measured with an enzyme reader. Three replicate wells were set for each group. The quantitative data were measured in SPSS 23.0 statistical software was used to test the normality and homogeneity of variance of each group of data, and the independent sample t test was used for inter-group comparison. P < 0.05 was considered statistically significant. Figure 3 As shown, the absorbance of the hydrogel microspheres prepared in Examples 1-4 is significantly better than that in Comparative Examples 1-2 and the control group. The absorbance values ​​of the hydrogels prepared in Examples 1-4 are all above 0.62, among which the absorbance value of the hydrogel prepared in Example 1 is the highest, which is (0.67±0.05), while the absorbance value of Comparative Examples 1-2 and the control group is all below 0.54, indicating that compared with Comparative Examples 1-2 and the control group, the hydrogel microspheres prepared in Examples 1-4 have excellent cell proliferation ability, and the difference is statistically significant (P<0.05).

[0047] In vivo experiments were conducted on the stem cell injections prepared in Example 1 and Comparative Examples 1-2. Twenty 8-week-old female db / db mice (provided by the Animal Experiment Center of Tongji University) were selected and fed with a normal diet under a standard environment for one week. The random blood glucose of the mice was tested with a blood glucose meter (the average blood glucose was 30.45±3.31mmoL / L, and all exceeded 16.6mmoL / L, and the weight of the mice was 22.14±1.70g). After the mice were anesthetized, the hair on the back was shaved with an animal shaver, and 75% alcohol was used for local disinfection. A full-thickness skin injury wound of 1.5cm×1.5cm in size was formed on the back with sterile ophthalmic scissors, deep to the fascia, and sterile gauze was used for hemostasis. After the modeling was completed, it was bandaged with a 3M transparent dressing. Within 2 hours after the modeling, the stem cell injections obtained in Example 1 and Comparative Examples 1-2 (containing 1×10 6 cells), and the control group was injected with an equal volume of sterile PBS buffer. After surgery, the mice were returned to the cages for single-cage feeding and allowed to eat freely. Determination of wound healing rate: On the 3rd, 7th, and 14th days after surgery, the wound healing conditions of each group were recorded with a digital camera, and the wound area was measured with ImagePro Plus 6.0 image analysis software, and the wound healing rate was calculated. The calculation formula was: Wound healing rate = [(modeling wound area - area measured at a specific time point) / modeling wound area] × 100%; Western blot detection of VEGF protein expression: On the 7th and 14th days after surgery, the mice were anesthetized and the new skin tissue on the wound and a small amount of normal skin around were taken, and 1% PMSF RIPA lysis buffer was fully homogenized on ice, and the supernatant was collected by centrifugation at 4°C. The total protein concentration was determined by BCA method, and the protein was separated by polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 hour, and VEGF rabbit anti-mouse antibody (antibody dilution 1:300) and β-actin rabbit anti-mouse antibody (antibody dilution 1:1000) were added dropwise, incubated at 4°C overnight, rinsed with PBST for 5 minutes, repeated three times, and HRP-labeled goat anti-rabbit secondary antibody (antibody dilution 1:5000) was added, incubated at 37°C for 1 hour, rinsed with PBST for 5 minutes, repeated three times, and ECL chemiluminescent solution was added dropwise. The gel was developed by gel imaging system, and semi-quantitative analysis was performed using Quantity One software. Quantitative data were expressed as follows: SPSS 23.0 statistical software was used to test the normality and homogeneity of variance of each group of data. The independent sample t test was used for inter-group comparison. P < 0.05 was considered statistically significant. Figure 4 , Figure 5 As shown, from Figure 4It can be seen that the wound healing rates of the Example 1 group on the 3rd, 7th and 14th days after surgery were significantly higher than those of the Comparative Examples 1-2 and the control group (the difference was statistically significant (P < 0.05)). The wound healing rates of the Example 1 group on the 3rd, 7th and 14th days after surgery were (22.1 ± 2.0)%, (55.3 ± 3.3)% and (93.4 ± 4.7)%, respectively. Figure 5 It can be seen that the expression of VEGF (vascular endothelial growth factor) in the Example 1 group was higher than that in the Comparative Examples 1-2 and the control group in the same period (the difference was statistically significant (P < 0.05)). The VEGF expression of the Example 1 group on the 7th and 14th days after surgery was (1.01 ± 0.07) and (1.62 ± 0.08), respectively. VEGF can promote the growth of vascular endothelial cells and promote angiogenesis. It can be seen that the hydrogel microspheres provided by the present invention can promote the growth of vascular endothelial cells, thereby promoting the growth of new capillaries.

[0048] Although the embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a stem cell injection for treating diabetic foot, characterized in that: The following steps are involved: Dispersing modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid dispersion; dispersing gelatin in deionized water to obtain a gelatin dispersion; The modified hyaluronic acid dispersion and the gelatin dispersion are mixed, and after emulsification and sterilization, a cell suspension containing human umbilical cord mesenchymal stem cells is added and mixed evenly to obtain a hydrogel solution; the hydrogel solution is dripped into sterile mineral oil, kept warm at 35-40° C. for 10-15 hours, filtered, washed, and dried with sterile gauze to obtain hydrogel microspheres; the hydrogel microspheres are mixed evenly with deionized water and cocamidopropyl betaine to obtain a stem cell injection solution; The preparation method of the modified hyaluronic acid is as follows: S1. Dissolve hyaluronic acid in deionized water under stirring, add sodium periodate, raise the temperature to 30-50°C, react for 2-6 hours in the dark, then add glycerol, continue stirring for 20-40 minutes, cool to room temperature, dialyze, and freeze-dry to obtain oxidized hyaluronic acid; S2. Dispersing the oxidized hyaluronic acid obtained in step S1 in dimethyl sulfoxide under stirring, adding p-toluenesulfonic acid and N-methyl-2-hydroxyethylamine in sequence, reacting at 60-80° C. for 4-8 hours, cooling to room temperature, dialyzing, and freeze-drying to obtain amino hyaluronic acid; S3, under nitrogen atmosphere and stirring, dispersing the amino hyaluronic acid obtained in step S2 in dimethyl sulfoxide, adding 5-bromo-ferulic acid, reacting at 90-110° C. for 8-14 h, cooling to room temperature, dialyzing, and freeze-drying to obtain modified hyaluronic acid; The structural formula of the modified hyaluronic acid is as follows: ； The volume ratio of the modified hyaluronic acid dispersion, gelatin dispersion and cell suspension is 40-50:45-55:

5.

2. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: The mass fraction of modified hyaluronic acid in the modified hyaluronic acid dispersion is 0.08-0.12%, and the mass fraction of gelatin in the gelatin dispersion is 0.08-0.12%.

3. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: The emulsification is carried out at 1000-3000 rpm for 1-3 min.

4. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: The mass ratio of the hydrogel microspheres, deionized water and cocamidopropyl betaine is 10-15:100-120:3-6.

5. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: The mass ratio of hyaluronic acid, deionized water, sodium periodate and glycerol in step S1 is 1:90-110:0.6-0.9:0.4-0.

7.

6. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: In step S2, the molar ratio of the carboxyl group on the oxidized hyaluronic acid, N-methyl-2-hydroxyethylamine, and p-toluenesulfonic acid is 1:1-3:0.12-0.17, and the amount of oxidized hyaluronic acid added to dimethyl sulfoxide is 0.05-0.09 g / mL.

7. The method for preparing a stem cell injection for treating diabetic foot according to claim 1, characterized in that: The mass ratio of the amino hyaluronic acid to 5-bromo-ferulic acid in step S3 is 100:20-30, and the amount of the amino hyaluronic acid added to dimethyl sulfoxide is 0.06-0.1 g / mL.

8. A stem cell injection for treating diabetic foot prepared by the method according to any one of claims 1 to 7.

Images