Preparation method and application of hydrogel with stem cell three-dimensional culture and tissue repair functions

Abstract

The preparation method comprises the following steps: dissolving methylacryloylated recombinant human XVII type collagen in a blue light initiator solution, filtering to obtain methylacryloylated recombinant human XVII type collagen hydrogel precursor liquid, irradiating under blue light to obtain the hydrogel with the stem cell three-dimensional culture and tissue repair functions, and drying to obtain the hydrogel with the stem cell three-dimensional culture and tissue repair functions. The hydrogel with the stem cell three-dimensional culture and tissue repair functions is obtained. The hydrogel with the stem cell three-dimensional culture and tissue repair functions prepared by the invention can support the three-dimensional culture of stem cells; the hydrogel can be quickly digested and the adipose-derived stem cells can be extracted in an enzymolysis manner; under the in-vitro culture condition, the stem cells can grow, proliferate and migrate in the hydrogel, and the dryness can still be reserved; the hydrogel and the hydrogel loaded with the stem cells can also promote wound repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of gel preparation, and in particular relates to a preparation method and application of a hydrogel with stem cell three-dimensional culture and tissue repair functions. Background Art

[0002] While existing two-dimensional cell culture techniques are simple to operate, they struggle to simulate the complex in vivo microenvironment, limiting their application in drug screening and disease modeling. Hydrogels, with their three-dimensional network structure, can highly mimic the extracellular matrix (ECM). By manipulating physicochemical properties (such as hardness and porosity), they can precisely guide cell behavior, making them ideal vehicles for tissue engineering and regenerative medicine. However, traditional hydrogel materials have limitations: synthetic materials offer controllable mechanical properties but lack biocompatibility; while natural materials (such as collagen) possess excellent bioactivity, animal-derived materials carry the risk of viral contamination and immune rejection.

[0003] Recombinant collagen technology, particularly type XVII collagen, has overcome these bottlenecks through synthetic biology. Produced using a eukaryotic expression system, it boasts high water solubility, processability, and batch stability, circumventing the drawbacks of animal-derived products and demonstrating outstanding performance in promoting stem cell recruitment and wound healing. In response to the clinical needs of full-thickness skin defects and cartilage repair, stem cell-loaded hydrogels demonstrate significant potential. Among these, photocurable hydrogels have become a research hotspot in the field of wound repair dressings and tissue regeneration, as they can adapt to wound shape in situ, provide a three-dimensional scaffold for cell growth, and simultaneously achieve controlled degradation.

[0004] Currently, hydrogels are used in the fields of three-dimensional stem cell culture, full-thickness skin defect wound treatment, and cartilage defect repair, and related patents have also been authorized. The following are patents related to recombinant human collagen hydrogels:

[0005] Patent application publication number CN118718128A discloses a method for preparing a hydrogel with tissue repair and fibrosis prevention functions, and the hydrogel itself. The hydrogel comprises recombinant human type I and / or type III collagen (unstructured), a solution of methacryloylated chitosan containing a photoinitiator, and astragalus polysaccharide. The hydrogel exhibits antibacterial properties and prevents intrauterine adhesions.

[0006] Patent application with publication number CN118340946A discloses a composite hydrogel, its preparation method and application. The composite hydrogel is loaded with mesenchymal stem cells from placental tissue. The hydrogel has good cell compatibility, high cell survival rate, can adapt to wounds, and has close contact with tissues. It does not require additional auxiliary materials and has a good effect on tissue repair.

[0007] There are currently no reports on the preparation of type XVII collagen into hydrogels suitable for stem cell culture and tissue repair. At the same time, there are no reports that stem cells can grow, proliferate, migrate, and retain their stemness in hydrogels containing type XVII collagen under in vitro culture conditions. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing a hydrogel with three-dimensional stem cell culture and tissue repair functions.

[0009] The second object of the present invention is to provide a hydrogel prepared by the method and having the functions of three-dimensional stem cell culture and tissue repair.

[0010] The third object of the present invention is to provide a use of the hydrogel in preparing a product for culturing adipose-derived mesenchymal stem cells.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] The first aspect of the present invention provides a method for preparing a hydrogel having three-dimensional stem cell culture and tissue repair functions, comprising the following steps:

[0013] The first step is the preparation of methacryloylated recombinant human type XVII collagen (rhCol-MA) The chemical modification reagent is added dropwise to a recombinant human type XVII collagen solution having a concentration of 0.05-0.2 g / mL (preferably 0.1 g / mL), wherein the mass ratio of recombinant human type XVII collagen to the chemical modification reagent is 1-15:1 (preferably 12:1, 6:1, or 3:1). The addition time is controlled to be 5-20 min (preferably 10 min). A NaOH solution having a concentration of 0.5-2 mol / L (preferably 1 mol / L) is added dropwise until the pH of the solution is 8-9. The mixture is stirred at a temperature of 50-70°C (preferably 60°C) for 0.5-5 h (preferably 2 h). PBS buffer (pH = 7.4) is added to the solution to dilute the reaction solution and continuously stirred to terminate the reaction. The solution is dialyzed against deionized water at room temperature for 3-5 days (preferably 4 days). The dialyzed solution is centrifuged, the supernatant is collected, and freeze-dried to obtain the methacryloylated recombinant human type XVII collagen (rhCol-MA).

[0014] In the first step, the chemical modification reagent is selected from methacrylic anhydride and glycidyl methacrylate.

[0015] The method for preparing the recombinant human type XVII collagen solution in the first step comprises the following steps:

[0016] Recombinant human type XVII collagen is added to PBS buffer to a concentration of 0.05-0.2 g / mL (preferably 0.1 g / mL), and stirred at 50-70°C (preferably 60°C) until completely dissolved to obtain the recombinant human type XVII collagen solution.

[0017] The dialysis in the first step uses a dialysis bag with a molecular weight cut-off of 12-14 kDa (preferably 14,000).

[0018] The centrifugal conditions in the first step were: centrifugation at 3000 rpm for 15 min.

[0019] The freeze-drying time in the first step is 24 to 72 hours (preferably 48 hours) and the temperature is -65°C.

[0020] The pH of the PBS buffer in the first step is 7.4.

[0021] The grafting rate of the methacryloyl recombinant human type XVII collagen (rhCol-MA) is 8-60% (preferably 9-53%, more preferably 15%-53%, and most preferably 9.95%, 28.23%, and 52.79%).

[0022] The second step is the preparation of hydrogels with three-dimensional stem cell culture and tissue repair functions:

[0023] Methacryl-modified recombinant human type XVII collagen (rhCol-MA) is dissolved in a blue light initiator solution with a concentration of 1-5 mg / mL (preferably 2.5 mg / mL), filtered to obtain a methacrylylated recombinant human type XVII collagen hydrogel precursor solution, and irradiated under blue light (preferably for 1 minute) to obtain the hydrogel with three-dimensional stem cell culture and tissue repair functions.

[0024] In the second step, the blue light initiator is selected from lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (abbreviated as LAP) and I2959 ultraviolet light initiator.

[0025] The preparation method of the blue light initiator solution in the second step comprises the following steps:

[0026] The blue light initiator is dissolved in PBS buffer to obtain a blue light initiator solution with a concentration of 1-5 mg / mL (preferably 2.5 mg / mL).

[0027] In the second step, a sterile syringe filter with a diameter of 0.22 μm is used for filtration.

[0028] In the second step, the blue light uses an LED blue light lamp with a power of 16-20W and a wavelength of 405nm.

[0029] The pH of the PBS buffer in the second step is 7.4.

[0030] The mass fraction of the methacryloyl recombinant human type XVII collagen hydrogel precursor solution is 5-15% (preferably 7.5%-15%, most preferably 7.5%, 12.5%, 15%).

[0031] The second aspect of the present invention provides a hydrogel prepared by the method and having the functions of three-dimensional stem cell culture and tissue repair.

[0032] A third aspect of the present invention provides a use of the hydrogel in preparing a product for culturing adipose-derived mesenchymal stem cells. In preliminary experiments, a comparison was made between a gelatin-derived photocurable hydrogel and a recombinant human type I collagen-derived photocurable hydrogel. It was found that after culturing adipose-derived stem cells for 7 days using a recombinant human type XVII collagen-derived photocurable hydrogel, their stemness remained. Flow cytometry was used to detect stemness-related positive and negative markers to confirm whether the cell phenotype met the standards defined by the International Society for Stem Cell Therapy (ISCT).

[0033] The fourth aspect of the present invention provides a use of the hydrogel in preparing a wound repair product.

[0034] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0035] The hydrogel, prepared by the present invention, supports three-dimensional stem cell culture and tissue repair. It can be rapidly digested and extracted from adipose-derived mesenchymal stem cells through enzymatic hydrolysis. Under in vitro culture conditions, the stem cells can grow, proliferate, and migrate within the hydrogel while retaining their stemness. Furthermore, due to the hydrogel's excellent biocompatibility, the stem cell-loaded hydrogel can firmly adhere to the wound surface, promoting the repair and regeneration of full-thickness skin defect wounds.

[0036] The invention points of the present invention are as follows:

[0037] Recombinant human type XVII collagen was constructed using a eukaryotic yeast expression system and, after methacrylation modification, can form a hydrogel under blue light (365-405 nm) initiation. Compared with traditional methods, this invention has the following advantages: (1) gelation can be achieved without the addition of exogenous polymer materials; (2) a safe and non-toxic photoinitiator system is used instead of a chemical crosslinker; (3) the product is purified and has a clear composition, avoiding the immunological risks of animal-derived materials; (4) the mechanical properties of the gel are adjustable (modulus is controlled by protein concentration), adapting to the needs of skin / cartilage tissue repair; (5) it supports enzymatic cell recovery, facilitating subsequent research. Compared with commercial matrix gel, this product has the characteristics of high batch stability and convenient digestion, showing significant application value in the fields of three-dimensional cell culture and tissue regeneration medicine. Animal experiments have confirmed its good tissue repair effect and biocompatibility, and can support cell survival culture for up to 4 weeks.

[0038] The hydrogel with three-dimensional stem cell culture and tissue repair functions prepared by the present invention has the following social effects:

[0039] Medical innovation: Break through the limitations of traditional materials and accelerate tissue regeneration by developing new materials that precisely adapt to skin repair needs.

[0040] Research-driven: Establishing the first type XVII collagen-based three-dimensional culture system, providing innovative tools for stem cell research and complex disease treatment.

[0041] The hydrogel with three-dimensional stem cell culture and tissue repair functions prepared by the present invention has the following economic effects:

[0042] Reduced costs: By optimizing the preparation process, the hydrogel preparation process of the present invention is relatively simple, reducing the use of traditional chemical cross-linking agents (such as glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), etc.), with low toxicity and low production cost.

[0043] Promote market growth: With the aging population and rising health awareness, the demand for efficient and safe tissue repair materials is growing. The commercial application of this invention will stimulate market demand and drive the development of upstream and downstream industry chains.

[0044] Create economic value: Given its wide application potential in the medical field, the promotion and use of this invention will directly create huge economic benefits and bring considerable economic returns to medical companies, scientific research institutions and investors.

[0045] The hydrogel with three-dimensional stem cell culture and tissue repair functions prepared by the present invention has the following technical effects:

[0046] Efficient curing: Under blue light irradiation, the methacrylated recombinant human type XVII collagen solution containing a photoinitiator (photoinitiator concentration is 2.5 mg / ml) can be rapidly cured within 1 minute, significantly improving surgical efficiency and ease of operation.

[0047] Stem cell culture support: Experiments have shown that stem cells can grow, proliferate, and survive in hydrogels for up to 4 weeks, providing a stable microenvironment for long-term in vitro culture and subsequent treatment.

[0048] Enzymatic hydrolysis: The hydrogel can be rapidly digested by enzymatic hydrolysis, facilitating cell recovery and subsequent processing.

[0049] Tissue repair effect: Animal experiments show that this hydrogel and hydrogel loaded with adipose stem cells can effectively promote the repair and regeneration of full-thickness skin defect wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the preparation of recombinant human type 17 collagen hydrogel using EDC / NHS as chemical cross-linking agents.

[0051] Figure 2 Schematic diagram of the results of measuring the cytotoxicity of the hydrogel extract using the CCK-8 method.

[0052] Figure 3 Schematic diagram of the chemical modification principle of methacrylylated recombinant human type XVII collagen.

[0053] Figure 4 Schematic diagram of methacryloylated recombinant human type XVII collagen freeze-dried sample.

[0054] Figure 5 Schematic diagram of the nuclear magnetic resonance hydrogen spectra of recombinant human type XVII collagen before and after modification and methacryloylation recombinant human type XVII collagen with different degrees of modification.

[0055] Figure 6 Schematic diagram of rhCol-MA hydrogel precursor solution and photocuring of recombinant human type XVII collagen hydrogel after blue light irradiation.

[0056] Figure 7 Schematic diagram of the hydrogel scanning electron microscopy results.

[0057] Figure 8 Schematic diagram of the live-dead staining results at different time points after three-dimensional culture of adipose-derived mesenchymal stem cells.

[0058] Figure 9 Schematic diagram of cell counting after hydrogel enzymatic digestion.

[0059] Figure 10Schematic diagram of flow cytometry results of adipose-derived mesenchymal stem cells after 7 days of 2D culture in culture dishes and 3D culture in hydrogels.

[0060] Figure 11 This is a general schematic diagram of the healing of acute full-thickness wounds in nude mice.

[0061] Figure 12 This is a statistical analysis chart of the phase change rate of wound area of ​​acute full-thickness wounds in nude mice.

[0062] Figure 13 H&E staining images of wound tissue after treatment with hydrogel and stem cell-loaded hydrogel.

[0063] Figure 14 Schematic diagram of the preparation and application of light-cured recombinant human type XVII collagen hydrogel. DETAILED DESCRIPTION

[0064] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0065] In the existing technology, there are usually four conventional methods for preparing hydrogels containing recombinant human collagen. The first is to use physical methods without adding auxiliary reagents to induce collagen to spontaneously form a gel by changing the temperature of the solution. However, the molecular weight of recombinant human type XVII collagen is only 23.8 KD and it has good water solubility. Compared with large molecular collagen, the feasibility of this method is poor; the second is to use other components as carriers of the hydrogel and mix the recombinant human collagen into the hydrogel as an active ingredient. For example, the patent applications with publication numbers CN118750659A, CN118750660A, and CN118718128A all use methacryloyl chitosan as a carrier of the hydrogel. The hydrogel prepared by this method has complex components, which is not conducive to exploring and explaining the biological effects of recombinant human type I and (or) type III collagen; the third is to chemically modify the recombinant human collagen, for example, methacrylic anhydride can be used. Modify recombinant human collagen by grafting methacryloyl groups onto the recombinant human collagen. After purification, the modified product can undergo a free radical chain reaction under the irradiation of a photoinitiator and a specific light source, turning the solution from a liquid into a hydrogel. The hydrogel prepared in this way has the advantages of rapid gelation, adjustable hardness, and in situ formation. The fourth method is to add a cross-linking agent (for example, glutaraldehyde, EDC / NHS) to the recombinant human collagen sol. The cross-linking agent has certain cytotoxicity. After using the cross-linker to prepare the recombinant human collagen hydrogel, dialysis is often required to remove the unreacted cross-linking agent. This method takes a long time to cross-link and cannot achieve three-dimensional cell culture.

[0066] The present invention has found through preliminary experiments that it is difficult to make recombinant human collagen solution spontaneously gel by freezing induction. Therefore, recombinant human type 17 collagen hydrogel is prepared using EDC / NHS as a chemical crosslinking agent. The method is as follows: 4 mL of recombinant human type 17 collagen solution with a concentration of 150 mg / mL is prepared with deionized water, the pH of the solution is adjusted to between 5-6 with 1 mmol / L hydrochloric acid, 1 mL of EDC / NHS mixed solution (containing 47.9 mg EDC and 57.6 mg NHS) is added, and the mixture is stirred at 37°C for 1 hour. The results are as follows: Figure 1 As shown, Figure 1 Schematic diagram of the preparation of recombinant human type 17 collagen hydrogels using EDC / NHS as chemical crosslinkers. The solution is fluid before crosslinking (left), while the gel is non-fluid after crosslinking (right).

[0067] According to the national standard GB / T 16886.5 "Biological evaluation of medical devices Part 5: In vitro cytotoxicity test", the cell compatibility of the two hydrogels was evaluated. 100 mg of methacryloylation recombinant human type XVII collagen (rhCol-MA) and recombinant human type 17 collagen hydrogel (rhcol-EN) were weighed, immersed in 5 mL of complete culture medium, and incubated at 37°C for 24 h to obtain hydrogel extracts with a concentration of 20 mg / mL. L929 cells were digested, counted using a cell counter, centrifuged (1000 rpm, 5 min), the supernatant discarded, and supplemented with complete culture medium to a cell density of 2.5×10 4 200 μL of cell suspension was added to each 96-well plate and cultured in a cell culture incubator (37°C, 5% CO2). After the cells adhered to the wall, the original culture medium was discarded and complete culture medium containing the hydrogel extract was added. The cells were co-cultured with L929 cells for 24 h. Cell proliferation was then detected using the CCK-8 kit. The results were as follows: Figure 2 As shown, Figure 2 Schematic diagram of the cytotoxicity of hydrogel extracts measured using the CCK-8 assay. Compared to the control group, the extract of the hydrogel (rhcol-EN) cross-linked with EDC / NHS exhibited significant cytotoxicity. Table 1 compares the hydrogels prepared by the two methods. As can be seen from Table 1, the chemically modified hydrogels of the present invention exhibit greater biosafety and have the potential to enable three-dimensional cell culture.

[0068] Table 1

[0069] Example 1

[0070] A method for preparing a hydrogel with three-dimensional stem cell culture and tissue repair functions, comprising the following steps:

[0071] (I) Preparation of methacryloylated recombinant human type XVII collagen (rhCol-MA)

[0072] In the first step, 12 g of recombinant human type XVII collagen (rhCol, Jiangsu Chuangjian Medical Technology Co., Ltd., catalog number: 170801) was added to a round-bottom flask containing 120 mL of PBS buffer (pH = 7.4) and stirred at 60°C and 240 rpm until completely dissolved to obtain a recombinant human type XVII collagen solution with a concentration of 0.1 g / mL.

[0073] In the second step, 1 mL of methacrylic anhydride (purchased from Sigma-Aldrich, catalog number: 276685) was added dropwise to the above solution. The mass ratio of recombinant human type XVII collagen to methacrylic anhydride was 12:1, the initial concentration of methacrylic anhydride was 55 mmol / L (abbreviated as 55 mM), and the addition time was controlled to be 10 min. A 1 mol / L NaOH solution was added dropwise until the pH of the above solution was adjusted to 8-9. The reaction was then carried out in a constant temperature water bath at 60°C with magnetic stirring for 2 h. The reaction route of methacrylation of recombinant human type XVII collagen is as follows: Figure 3 As shown, Figure 3 Schematic diagram of the chemical modification principle of methacrylylated recombinant human type XVII collagen.

[0074] Methacrylic anhydride is liquid at room temperature, with a purity of ≥97% and a density of 1.04g / ml.

[0075] PBS buffer, also known as phosphate buffered saline, has a density of 1 g / ml and was purchased from Wuhan Saiweier Biotechnology Co., Ltd. with the product number G4250-500ML.

[0076] Step 3: Dilute the reaction solution by adding 120 mL of PBS buffer (pH 7.4) to the above solution and continue stirring to terminate the reaction. Place the above solution in a dialysis bag (brand: Viskase, molecular weight cutoff: 14,000) and dialyze against deionized water at room temperature for 4 days.

[0077] Step 4: Pour the dialyzed solution into a centrifuge tube and centrifuge at 3000 rpm for 15 min. Take the supernatant and place it in a -80°C refrigerator and freeze overnight. In the fifth step, the supernatant was lyophilized in a freeze dryer for 48 h at -65 °C to obtain 10.5 g of methacryloyl-recombinant human type XVII collagen with a yield of 87.5%. Figure 4 As shown, Figure 4 Schematic diagram of methacryloylated recombinant human type XVII collagen freeze-dried sample.

[0078] In the second step, 1 mL of methacrylic anhydride was replaced with 2 mL and 4 mL of methacrylic anhydride. At this time, the initial concentrations of methacrylic anhydride in the reaction system were 110 mmol / L and 220 mmol / L, respectively. Other conditions remained unchanged, and 9.9 g and 9.5 g of methacryloylated recombinant human type XVII collagen were obtained, with yields of 82.5% and 79.17%, respectively.

[0079] (II) Characterization of methacryloylated recombinant human type XVII collagen (rhCol-MA)

[0080] The molecular structure before and after the grafting reaction was determined by nuclear magnetic resonance spectroscopy, the degree of modification was quantified, and the grafting rate was calculated. The samples of collagen before and after modification were subjected to nuclear magnetic resonance testing and the molecular structure of collagen before and after modification was compared and analyzed. The results are as follows: Figure 5 As shown, Figure 5 Schematic diagram of the H NMR spectra of recombinant human type XVII collagen before and after modification, and of methacrylylated recombinant human type XVII collagen with varying degrees of modification. After modification, the signal from lysine residues decreases (2.9 ppm), the signal from the methyl group increases (1.8 ppm), and the signal characteristic of the acrylic acid proton double bond of methacrylamide increases (5.3-5.5 ppm). This trend increases with increasing methacrylic anhydride dosage. The reactant feed ratios and grafting yields are shown in Table 2.

[0081] Table 2

[0082] (III) Preparation of light-cured recombinant human type XVII collagen hydrogel

[0083] In the first step, 25 mg of blue light initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) was dissolved in 10 ml of PBS buffer (pH = 7.4) to obtain a blue light initiator solution with a concentration of 2.5 mg / mL.

[0084] In the second step, methacryloyl-recombinant human type XVII collagen with a grafting rate of 28.23% in Table 2 was selected, and 75 mg of the rhCol-MA freeze-dried sample was fully dissolved in 1 ml of the above-prepared blue light initiator solution with a concentration of 2.5 mg / mL. The solution was filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a hydrogel precursor solution with a mass fraction of 7.5% rhCol-MA.

[0085] In the third step, methacryloyl-recombinant human type XVII collagen with a grafting rate of 28.23% in Table 2 was selected, and 150 mg of the rhCol-MA freeze-dried sample was fully dissolved in 1 ml of the above-prepared blue light initiator solution with a concentration of 2.5 mg / mL. The solution was filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a hydrogel precursor solution with a mass fraction of 15% rhCol-MA.

[0086] In the fourth step, 800 μl of the rhCol-MA hydrogel precursor solution prepared in the second and third steps were respectively drawn into a glass bottle and irradiated under an LED blue light (power 16-20W, wavelength 405nm) for 1 minute to obtain a light-cured recombinant human type XVII collagen hydrogel. Figure 6 As shown, Figure 6 Schematic diagrams of the rhCol-MA hydrogel precursor solution and the photocured recombinant human type XVII collagen hydrogel after blue light irradiation. A is a schematic diagram of the rhCol-MA hydrogel precursor solution prepared in the third step, and a is a schematic diagram of the photocured recombinant human type XVII collagen hydrogel obtained after blue light irradiation of the rhCol-MA hydrogel precursor solution prepared in the third step. B is a schematic diagram of the rhCol-MA hydrogel precursor solution prepared in the second step, and b is a schematic diagram of the photocured recombinant human type XVII collagen hydrogel obtained after blue light irradiation of the rhCol-MA hydrogel precursor solution prepared in the second step. As can be seen from the figure, the rhCol-MA hydrogel precursor solutions prepared in the second and third steps are both liquids. After blue light irradiation, they are cured to obtain the photocured recombinant human type XVII collagen hydrogel.

[0087] In the present invention, methacryloyl-recombinant human type XVII collagen with grafting rates of 9.95%, 28.23%, and 52.79% were respectively prepared into rhCol-MA hydrogel precursor solutions with mass fractions of 5%, 7.5%, 12.5%, and 15%, and then light-cured recombinant human type XVII collagen hydrogels were prepared according to the above steps, as shown in Table 3:

[0088] Table 3

[0089] As shown in Table 3, hydrogel precursors containing 5%, 7.5%, 12.5%, and 15% rhCol-MA by mass were prepared from recombinant human type XVII collagen with a 9.95% grafting ratio. However, the 5% and 7.5% rhCol-MA hydrogel precursors failed to gel after blue light irradiation. Hydrogel precursors containing 5%, 7.5%, 12.5%, and 15% rhCol-MA by mass were prepared from recombinant human type XVII collagen with a 28.23% grafting ratio. The 5% rhCol-MA hydrogel precursor failed to gel after blue light irradiation, while the other hydrogel precursors successfully formed corresponding hydrogels. In subsequent testing and application, the 28.23% grafting ratio was used to prepare hydrogels.

[0090] (IV) Morphological analysis of light-cured recombinant human type XVII collagen hydrogel The hydrogel precursor solution containing 7.5% rhCol-MA by mass was prepared in the second step of preparing the photocurable recombinant human type XVII collagen hydrogel. A cylindrical hydrogel sample with a diameter of 11.2 mm and a height of 5 mm was prepared. The sample was placed in a -80°C refrigerator, frozen overnight, and then freeze-dried. The morphological characteristics of the hydrogel were photographed under the same magnification (scale bar 30 μm) using a scanning electron microscope (SEM). Figure 7 As shown, Figure 7 The figure shows the results of scanning electron microscopy of hydrogels. As can be seen from the figure, the hydrogels all have a loose and porous three-dimensional network structure.

[0091] (V) Hydrogels for the culture of adipose-derived mesenchymal stem cells In the first step, 1 ml of the hydrogel precursor solution containing 7.5% rhCol-MA prepared in the second step of light-cured recombinant human type XVII collagen hydrogel preparation was selected. Adipose-derived mesenchymal stem cells (ADSCs) at the fourth generation with a confluence of 70%-80% cultured in a 6-well plate were digested and centrifuged, and the supernatant was removed. The cells were resuspended in 1 ml of the hydrogel precursor solution.

[0092] In the second step, 300µl of hydrogel precursor solution mixed with stem cells was pipetted into a 12-well plate and irradiated under LED blue light (405nm) for 1 minute to obtain a hydrogel encapsulating ADSCs. Human adipose-derived mesenchymal stem cell complete culture medium was added and the medium was changed every 2 days. The survival status of the stem cells in the hydrogel was observed on the 7th, 14th, and 28th days using live-dead staining.

[0093] In the third step, at a specific time point, the old culture medium in the well plate was removed, the well plate was washed three times with PBS, and the cells were stained using a live-dead staining kit (purchased from Thermo Fisher Scientific). The results were as follows: Figure 8 As shown, Figure 8 This is a schematic diagram of the live-dead staining results of adipose-derived mesenchymal stem cells at different time points after three-dimensional culture. Green fluorescence marks live cells, and red fluorescence marks dead cells. As can be seen from the figure, ① with the increase in culture time, the number of green-fluorescent-labeled ADSCs gradually increases, indicating that the hydrogel can provide a microenvironment for cell proliferation; ② after 28 days of culture, a large number of green-fluorescent-labeled ADSCs are still present in the hydrogel, indicating that ADSCs can survive for up to 4 weeks in the hydrogel; ③ no obvious red fluorescence was seen in the live-dead staining results on days 7, 14, and 28, indicating that the hydrogel has low cytotoxicity. In summary, the hydrogel is conducive to the growth and proliferation of ADSCs and has good biocompatibility.

[0094] (VI) Extraction of cells and RNA from light-cured recombinant human type XVII collagen hydrogel Digestion is a common method for extracting tissue cells. A similar digestion strategy can be used for recombinant human type XVII collagen hydrogels loaded with adipose-derived stem cells. Preliminary experiments revealed that trypsin has a stronger effect and can easily damage cells. Collagenases (types I, II, III, IV, and V) have a more moderate effect and can all digest recombinant human type XVII collagen hydrogels, causing cell damage. However, type I collagenase is able to degrade recombinant human type XVII collagen hydrogels and isolate cells in a relatively short period of time, causing minimal damage to cells. This helps maintain cell integrity and activity, and can provide high-quality cell samples for subsequent experiments or treatments.

[0095] The specific operation process is as follows: In the first step, 5 mg of type I collagenase (purchased from Sigma-Aldrich, Cat. No. C0130) was dissolved in 5 ml of sterile PBS buffer and filtered through a sterile syringe filter (0.22 µm diameter) to obtain a collagenase solution with a concentration of 1 mg / ml. Second, on the seventh day of cell 3D culture, remove the cell culture medium in step (V) using a transfer pipette and wash twice with sterile PBS. The third step is to use a sterile pipette to bluntly separate the hydrogel containing ADSCs into 3-4 mm 3 Transfer the hydrogel blocks to a 50 ml centrifuge tube and add 5 ml of 1 mg / ml collagenase solution to the hydrogel blocks containing ADSCs to fully submerge them. Step 4: Place the centrifuge tube in a 37°C water bath or incubator and shake it every 3 minutes. For easily digestible hydrogels, digestion at 37°C with shaking for 10-30 minutes is recommended, but this can be adjusted based on the specific situation. If the hydrogel has dispersed and lost its lumpy shape and breaks apart upon shaking, digestion is considered complete. Filter the dispersed cells through a 100µm diameter cell strainer and wash the collected cells several times with collagenase-free PBS to obtain a cell suspension. (The PBS buffer can also be replaced with HBSS (Hank's Balanced Salt Solution) or DPBS (Dulbecco's Phosphate Buffered Saline).

[0096] In the fifth step, the cell suspension was transferred into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes to obtain stem cells cultured on the light-cured recombinant human type XVII collagen hydrogel.

[0097] Step 6: Resuspend the cells obtained in step 5 with 1 ml of sterile PBS buffer and count the cells using a cell counter (purchased from Thermo Fisher Scientific). Figure 9 Schematic diagram of cell counting after hydrogel enzymatic hydrolysis. The results show that the survival rate of ADSCs obtained after the above steps is 95%, and the cells using this method have a high survival rate.

[0098] In step 7, ADSCs at passage 4 with a confluence of 70%-80% that were passaged normally in cell culture dishes and not cultured in three dimensions were used as the control group (2D culture dish group). They were washed with sterile PBS buffer, digested with type I collagenase, centrifuged, and the supernatant removed. The cells were resuspended in 1 ml of sterile PBS buffer and counted. The cell suspension was prepared at the same density as the cell count in step 6. 600 µl of the prepared cell suspension was divided into 6 equal parts. APC anti-human CD31, FITC anti-human CD45, PE anti-human CD73, FITC anti-human CD90, APC anti-human CD105, and PE anti-human CD235a were added respectively. After incubation on ice in the dark for 30 minutes, centrifugation was performed, the supernatant removed, and the cells were resuspended in 100 µl of PBS and centrifuged three times. The cells were resuspended in 200 µl of 4°C pre-cooled PBS and cell surface markers were detected using a flow cytometer and the data were analyzed.

[0099] In step 8, 600 µl of the cell suspension obtained in step 6 was taken as the experimental group (hydrogel 3D culture group) and divided into 6 equal parts. APC anti-human CD31, FITC anti-human CD45, PE anti-human CD73, FITC anti-human CD90, APC anti-human CD105, and PE anti-human CD235a were added respectively. After incubation on ice in the dark for 30 minutes, centrifugation was performed, and the supernatant was removed. The cells were resuspended in 100 µl PBS and centrifuged three times. After resuspending the cells in 200 µl 4°C pre-cooled PBS, cell surface markers were detected using a flow cytometer and the data were analyzed.

[0100] The results are as follows Figure 10 As shown, Figure 10 The figure shows the flow cytometry results of adipose-derived mesenchymal stem cells after 7 days of 2D culture in a culture dish and 3D culture in a hydrogel. The flow cytometry results showed that the cell surface markers CD73, CD90, and CD105 were strongly positive in both the control and experimental groups, with expression rates greater than 95%, while the cell surface markers CD31, CD235a, and CD45 were negative in both the control and experimental groups, with expression rates less than 1%, which is consistent with the phenotypic characteristics of hADSCs. This indicates that the ADSCs in the control and experimental groups retain the stemness characteristics of mesenchymal stem cells. Compared with 2D culture in a culture dish, the stemness of adipose-derived mesenchymal stem cells remains after 3D culture. In addition, the present invention compared gelatin-derived photocurable hydrogels with recombinant human type I collagen-derived photocurable hydrogels and found that after 7 days of culturing adipose-derived stem cells with recombinant human type XVII collagen-derived photocurable hydrogels, the expression levels of genes involved in the three-lineage differentiation were the lowest, indicating that photocurable recombinant human type XVII collagen hydrogels are most effective in maintaining cell stemness.

[0101] (VII) Stem cell-loaded photocurable recombinant human type XVII collagen hydrogel promotes burn wound repair 1 ml of rhCol-MA hydrogel precursor solution with a mass fraction of 7.5% was prepared in the second step of preparing light-cured recombinant human type XVII collagen hydrogel. An acute full-thickness wound model of nude mice was constructed (Effects of porcine acellular dermal matrix with human epidermal stem cells on the healing of full-thickness skin defect wounds in nude mice [J]. Chinese Journal of Burns and Wound Repair, 2022, 38(01): 45-56.). The construction method is as follows: nude mice were fasted for 12 hours and anesthetized with isoflurane inhalation. The back skin of nude mice was disinfected with 75% alcohol. Then, a circular skin punch with a diameter of 8 mm was used to punch a hole in the back skin of the mice. The hole was covered with gauze and transparent film to complete the model. Twelve nude mice were randomly divided into three groups (control group, hydrogel group, and hydrogel + adipose stem cell group), with 4 mice in each group. The control group (Con group) was not treated, while the hydrogel group (H group) and the hydrogel + adipose stem cell group (HC group) were dripped with 20µl of hydrogel precursor solution and irradiated with blue light for 1 minute. The wound surface was observed every 3 days and photographed with a digital camera to record the wound healing status. On the 12th day, the wound surface was sampled and the healing quality was evaluated by HE and other pathological techniques.

[0102] The results are as follows Figure 11 、 12 , 13, Figure 11 This is a schematic diagram of the healing of acute full-thickness wounds in nude mice. As can be seen from the figure, the wound healing rate was significantly accelerated after hydrogel treatment compared to the control group. Figure 12 This is a schematic diagram of the statistical analysis of the phase change rate of wound area in acute full-thickness wounds in nude mice. Compared with the control group, after treatment with hydrogel and hydrogel loaded with adipose stem cells, the wound closure rate was significantly accelerated starting from the 6th day, and the endpoint healing rate was higher. Figure 13 This is the H&E staining image of the wound tissue after treatment with hydrogel and hydrogel loaded with stem cells. Compared with the control group, on the 12th day after treatment with hydrogel and hydrogel loaded with adipose stem cells, the epidermis and dermis of the wound were significantly thickened, and a large number of new appendages appeared in the hydrogel group loaded with adipose stem cells, indicating that the hydrogel group loaded with adipose stem cells can promote wound repair and regeneration to a limited extent.

[0103] Figure 14 This is a schematic diagram of the preparation and application of light-cured recombinant human type XVII collagen hydrogel. Methacrylylated recombinant human type XVII collagen is obtained by chemically modifying recombinant human type XVII collagen. Under the conditions of blue light initiator and light, the methacrylylated recombinant human type XVII collagen solution can be transformed from a liquid to a gel state. The obtained light-cured recombinant human type XVII collagen hydrogel has good application prospects in the three-dimensional culture of adipose mesenchymal stem cells, wound repair and regeneration.

[0104] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A method for preparing a hydrogel with three-dimensional stem cell culture and tissue repair functions, characterized in that: The following steps are involved: The first step is to add a chemical modification reagent dropwise to a recombinant human type XVII collagen solution with a concentration of 0.05-0.2 g / mL, wherein the mass ratio of recombinant human type XVII collagen to the chemical modification reagent is 1-15:1, and the addition time is controlled to be 5-20 minutes. A NaOH solution with a concentration of 0.5-2 mol / L is added dropwise until the pH of the solution is 8-9, and the reaction is stirred at a temperature of 50-70°C for 0.5-5 hours; PBS buffer is added to the solution to dilute the reaction solution, and the reaction is terminated by continuous stirring. The solution is dialyzed with deionized water at room temperature for 3-5 days; the dialyzed solution is centrifuged, the supernatant is collected, and freeze-dried to obtain the methacryloyl recombinant human type XVII collagen; In the first step, the chemical modification reagent is selected from methacrylic anhydride and glycidyl methacrylate; In the second step, the methacrylated recombinant human type XVII collagen is dissolved in a blue light initiator solution with a concentration of 1-5 mg / mL, filtered to obtain a methacrylated recombinant human type XVII collagen hydrogel precursor solution, and irradiated under blue light to obtain the hydrogel with the function of three-dimensional stem cell culture and tissue repair; In the second step, the blue light initiator is selected from phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and I2959 ultraviolet light initiator.

2. The method for preparing a hydrogel having the function of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: The method for preparing the recombinant human type XVII collagen solution in the first step comprises the following steps: Recombinant human type XVII collagen was added to PBS buffer to a concentration of 0.05-0.2 g / mL, and stirred at 50-70° C. until completely dissolved to obtain the recombinant human type XVII collagen solution; The dialysis in the first step uses a dialysis bag with a molecular weight cut-off of 12-14 kDa; The centrifugal conditions in the first step were: centrifugation at 3000 rpm for 15 min.

3. The method for preparing a hydrogel having the function of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: The freeze-drying time in the first step is 24 to 72 hours at a temperature of -65°C.

4. The method for preparing a hydrogel having the functions of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: The pH of the PBS buffer in the first step is 7.4; The grafting rate of the methacrylylated recombinant human type XVII collagen is 8-60%.

5. The method for preparing a hydrogel having the function of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: The preparation method of the blue light initiator solution in the second step comprises the following steps: The blue light initiator was dissolved in PBS buffer to obtain a blue light initiator solution with a concentration of 1-5 mg / mL.

6. The method for preparing a hydrogel having the functions of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: In the second step, filtration was performed using a sterile syringe filter with a diameter of 0.22 µm; In the second step, the blue light uses an LED blue light lamp with a power of 16-20W and a wavelength of 405nm.

7. The method for preparing a hydrogel having the functions of three-dimensional stem cell culture and tissue repair according to claim 1, characterized in that: The pH of the PBS buffer in the second step is 7.4; The mass fraction of the methacrylylated recombinant human type XVII collagen hydrogel precursor liquid is 5-15%.

8. A hydrogel having the functions of three-dimensional stem cell culture and tissue repair, prepared by the method according to any one of claims 1 to 7.

9. Use of the hydrogel according to claim 8 in preparing a product for culturing adipose-derived mesenchymal stem cells.

10. Use of the hydrogel according to claim 8 in preparing a wound repair product.

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