A composite hydrogel and its preparation method and application

Abstract

The invention discloses a composite hydrogel and a preparation method and application thereof, belonging to the field of medical technology, and comprising the following steps: culturing MSCs, preparing a solution and preparing a hydrogel; the preparation method is simple and easy, and is conducive to large-scale production. The composite hydrogel disclosed by the invention is loaded with mesenchymal stem cells from placental tissue, the hydrogel has good cell compatibility, high cell survival rate, can adapt to wounds, has close contact with tissues, does not require additional auxiliary materials, and has a good effect on tissue repair.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a composite hydrogel and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells with the potential for self-renewal and multidirectional differentiation. MSCs have the functions of promoting tissue repair and regulating immunity, and are not tumorigenic. They are safe for human use and have been widely used to treat dozens of diseases such as tissue damage, nerve damage, osteoarthritis, and neurodegenerative diseases. MSCs are easy to obtain and expand in vitro, have low immunogenicity, and are not subject to ethical restrictions, making them the most widely used stem cells.

[0003] There are still many problems to be solved in the widespread use of MSCs in the clinical treatment of DFU. The first is the selection of the source of MSCs; the second is how to enhance the colonization rate, survival rate and repair function of MSCs after transplantation. These are the difficulties and challenges of using MSCs in the clinical treatment of DFU. Placental MSCs have strong proliferation ability and low immunogenicity. Because they are derived from vascular-rich placental tissue, they have strong angiogenesis ability and are very suitable for the treatment of DFU.

[0004] Hydrogel biomaterials can provide cells with a three-dimensional mesh structure, increase the retention and implantation of transplanted cells, and support the adhesion, proliferation and differentiation of stem cells. They are good carriers for improving the colonization rate, survival rate and repair function of MSCs. At present, the research on various types of hydrogels has become a hot issue in biomaterials. Hydrogels can be divided into two types: natural and synthetic hydrogels. Natural hydrogels, such as collagen, gelatin, hyaluronic acid (HA), chitosan, etc., have the advantages of good biocompatibility and biodegradability, but poor mechanical properties such as rigidity and stretchability. Synthetic hydrogels (such as PEG derivatives, polycaprolactone (PCL), polyvinyl alcohol (PVA), etc.) can produce chemical cross-linking and have relatively high mechanical properties. Although they have poor biological activity, unsatisfactory biocompatibility and slow degradation, they can withstand strong mechanical loads and have certain advantages in certain applications. Most of the existing MSCs-loaded hydrogels use synthetic hydrogels such as PEG, sodium alginate, and PVA to ensure the rigidity of the hydrogel's shape. Tests have shown that highly rigid hydrogels are not suitable for skin wounds. Problems include: they cannot adapt to wounds of different sizes, shapes, and areas; they cannot come into close contact with wound tissues; they degrade slowly and may even hinder wound healing. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a composite hydrogel, which solves the problems of the hydrogel's loose contact with wounds of different sizes, shapes and areas and poor biocompatibility with loaded stem cells.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a composite hydrogel, comprising the following steps:

[0007] S1: Cultivate MSCs;

[0008] S2: solution preparation;

[0009] S3: Preparation of hydrogel;

[0010] The culturing of MSCs specifically comprises:

[0011] S11: Aspirate the old culture medium of cells in the culture flask and wash twice with PBS solution;

[0012] S12: Add trypsin-EDTA solution and incubate at 37°C for 5-7 minutes until the cells are about to separate and appear as round pellets. Then add fresh culture medium and centrifuge at 1200 g for 5 minutes. Discard the supernatant to obtain the precipitated cells.

[0013] S13: Add fresh culture medium to the precipitated cells and culture the cells at 37°C until the cell concentration is 2500-125000 cells / 1ul;

[0014] The solution configuration includes an initiator solution configuration and a GelMA solution configuration;

[0015] The preparation of the hydrogel comprises:

[0016] S31: PBS solution, collagen, GelMA solution and initiator solution are mixed evenly, and then cultured MSCs are added and mixed continuously to obtain a crude product;

[0017] S32: irradiating the crude product with light to gelate it;

[0018] S33: Mix the gelled substance with culture medium, culture at 37°C for 4 to 8 minutes, and then remove the culture medium to obtain the product.

[0019] The beneficial effects of the present invention are as follows: a hydrogel based on a gelatin and collagen formula is constructed, which has certain flexibility, fluidity and viscosity, and is helpful to closely adhere to the wound surface, not only directly protecting the wound, but also providing an extracellular skeleton-like environment for the loaded MSCs, which is beneficial to the survival, proliferation, differentiation and release of exocrine components of MSCs; and the method provided by the present invention has simple steps, is easy to implement, and has good application prospects.

[0020] Based on the above technical solution, the present invention can also be improved as follows.

[0021] Furthermore, the MSCs are UC-MSCs, AM-MSCs or CP-MSCs.

[0022] The beneficial effect of adopting a further technical solution is that these MSCs have a good therapeutic effect on skin damage.

[0023] Furthermore, the initiator solution is a photoinitiator LAP solution, and the steps of preparing the photoinitiator LAP solution include:

[0024] Mix the photoinitiator LAP with the PBS solution in the dark, dissolve at 40-50°C for 12-18 minutes, and shake for 5-7 times during the process.

[0025] Furthermore, the concentration of the photoinitiator LAP solution is 2.5 g / L.

[0026] The beneficial effect of adopting further technical solutions is that the initiator has precise light response control, which ensures the repeatability and accuracy of the experiment, and 2.5g / L is the standard concentration, which can improve the efficiency of the experiment and ensure the consistency and comparability of the experimental results.

[0027] Further, the GelMA solution preparation steps include:

[0028] The GelMA is mixed with the photoinitiator LAP solution, and heated to dissolve at 60-70° C. in the dark for 20-30 min, during which time the mixture is shaken 5-7 times to obtain the product.

[0029] Furthermore, the solid-liquid ratio of GelMA to photoinitiator LAP solution was 1.5 g:10 mL.

[0030] The beneficial effects of adopting the further technical solution are: the GelMA solution has adjustable gel properties, good biocompatibility, water solubility and controllable release performance, and the mechanical properties and release properties of the gel can be adjusted according to specific needs at a suitable concentration.

[0031] Furthermore, the volume percentages of the PBS solution, collagen, GelMA solution and initiator solution are 1% to 70%: 10% to 50%: 13% to 70%: 1 to 4%.

[0032] The beneficial effect of adopting a further technical solution is that the raw material ratio of the hydrogel can be finely adjusted according to the different loads of MSCs and different tissue wounds, and the collagen in the raw materials plays an important role in maintaining skin and wound healing.

[0033] Furthermore, the light irradiation is performed by irradiating with a light source of 405 nm for 10 to 30 seconds.

[0034] The beneficial effect of adopting the further technical solution is that the raw materials can be gelled quickly.

[0035] The invention discloses a composite hydrogel prepared by a method for preparing a composite hydrogel.

[0036] The invention also discloses the application of the composite hydrogel in preparing tissue damage repair products.

[0037] The beneficial effects of the present invention are as follows: the composite hydrogel provided by the present invention has good cell compatibility, high cell survival rate, can adapt to wounds, has close contact with tissues, does not require additional auxiliary materials, and has a good effect on tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a picture of hair loss wounds in transgenic diabetic mice (db / db);

[0039] Figure 2 The photos are recorded for Examples 1 to 3, Comparative Examples of Wound Repair and Natural Wound Repair;

[0040] Figure 3 It is a line graph of Examples 1 to 3, comparative example wound repair and natural wound repair;

[0041] Figure 4 This is a fluorescence detection image of wound slices at 7 days of wound repair in Example 1;

[0042] Figure 5 This is a fluorescence detection image of wound slices at 7 days of wound repair in Example 2;

[0043] Figure 6 This is a fluorescence detection image of the wound slice at 7 days of wound repair in Example 3. DETAILED DESCRIPTION

[0044] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0045] Example 1

[0046] A method for preparing a composite hydrogel comprises the following steps:

[0047] S1: Cultivating MSCs, comprising the following steps:

[0048] S11: remove the old culture medium of UC-MSCs in the culture bottle and wash twice with PBS solution;

[0049] S12: Add trypsin-EDTA solution and incubate at 37°C for 5 min. Observe under an inverted microscope. When the cells are about to separate and appear as round pellets, add Mesenchymal Stem Cell Medium (Cat No. 7501, ScienCell. Inc) to terminate the trypsin action. Centrifuge at 1200 g for 5 min. Discard the supernatant to obtain the precipitated cells.

[0050] S13: tap the culture bottle to make the precipitated cells fall off by themselves, add Mesenchymal Stem Cell Medium (Cat No. 7501, ScienCell.inc), and culture the cells at 37°C until the cell concentration reaches 20,000 cells / ul;

[0051] S2: preparing the solution, comprising the following steps:

[0052] S21: Take 20 ml of PBS and add it into a brown bottle containing 0.05 g of photoinitiator LAP;

[0053] S22: Dissolve by heating in a water bath at 45°C for 15 min, shaking 6 times during the process, to obtain a photoinitiator LAP solution with a concentration of 2.5 g / L;

[0054] S23: Weigh 1.5 g of GelMA (methacrylated gelatin) and put it into a centrifuge tube, add 10 ml of the photoinitiator LAP solution prepared in S22, and shake to fully infiltrate the GelMA;

[0055] S24: Dissolve by heating in a water bath at 65°C for 25 min, shaking for 6 times to obtain the GelMA solution;

[0056] S3: preparing a hydrogel, comprising the following steps:

[0057] S31: PBS solution, collagen CORNING 354236, GelMA solution and photoinitiator LAP solution were prepared into a mixed solution of 500ul at a volume percentage of 36%:50%:13%:1%, and then 19μl of cultured UC-MSCs were added and mixed to obtain a crude product;

[0058] S32: Take 150ul of the crude product, add it to a culture dish, and irradiate it with a 405nm ultraviolet light source for 20s to gel;

[0059] S33: Add Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to the culture dish, incubate at 37°C for 5 minutes, wash the gelled crude product, remove the culture medium, and then add fresh Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to culture for 24 hours to obtain a composite hydrogel.

[0060] Example 2

[0061] A method for preparing a composite hydrogel comprises the following steps:

[0062] S1: Cultivating MSCs, comprising the following steps:

[0063] S11: Aspirate the old culture medium of AM-MSCs in the culture bottle and wash twice with PBS solution;

[0064] S12: Add trypsin-EDTA solution and incubate at 37°C for 6 min. Observe under an inverted microscope. When the cells are about to separate and appear as round pellets, add Mesenchymal Stem Cell Medium (Cat No. 7501, ScienCell. Inc) to terminate the trypsin action. Centrifuge at 1200 g for 5 min. Discard the supernatant to obtain the precipitated cells.

[0065] S13: tap the culture flask to make the precipitated cells fall off by themselves, add Mesenchymal Stem Cell Medium (Cat No. 7501, ScienCell.inc), and culture the cells at 37°C until the cell concentration reaches 2500 cells / ul;

[0066] S2: preparing the solution, comprising the following steps:

[0067] S21: Take 20 ml of PBS and add it into a brown bottle containing 0.05 g of photoinitiator LAP;

[0068] S22: Dissolve by heating in a water bath at 40°C for 18 min, shaking 5 times during the process, to obtain a photoinitiator LAP solution with a concentration of 2.5 g / L;

[0069] S23: Weigh 1.5 g of GelMA (methacrylated gelatin) and put it into a centrifuge tube, add 10 ml of the photoinitiator LAP solution prepared in S22, and shake to fully infiltrate the GelMA;

[0070] S24: Dissolve by heating in a water bath at 60°C for 30 min, shaking 5 times during the process, to obtain the GelMA solution;

[0071] S3: preparing a hydrogel, comprising the following steps:

[0072] S31: PBS solution, collagen CORNING 354236, GelMA solution and photoinitiator LAP solution were prepared into a mixed solution of 500ul at a volume percentage of 16%:10%:70%:4%, and then 19μl of cultured AM-MSCs were added and mixed to obtain a crude product;

[0073] S32: Take 150ul of the crude product, add it to a culture dish, and irradiate it with a 405nm ultraviolet light source for 10s to gel;

[0074] S33: Add Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to the culture dish, incubate at 37°C for 4 minutes, wash the gelled crude product, remove the culture medium, and then add fresh Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to culture for 24 hours to obtain a composite hydrogel.

[0075] Example 3

[0076] A method for preparing a composite hydrogel comprises the following steps:

[0077] S1: Cultivating MSCs, comprising the following steps:

[0078] S11: remove the old culture medium of CP-MSCs in the culture bottle and wash twice with PBS solution;

[0079] S12: Add trypsin-EDTA solution and incubate at 37°C for 7 min. Observe under an inverted microscope. When the cells are about to separate and appear as round pellets, add Mesenchymal Stem Cell Medium (Cat No. 7501, ScienCell. Inc) to terminate the trypsin action. Centrifuge at 1200 g for 5 min. Discard the supernatant to obtain the precipitated cells.

[0080] S13: tap the culture bottle to make the precipitated cells fall off by themselves, add fresh Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc), and culture the cells at 37°C until the cell concentration reaches 125,000 cells / ul;

[0081] S2: preparing the solution, comprising the following steps:

[0082] S21: Take 20 ml of PBS and add it into a brown bottle containing 0.05 g of photoinitiator LAP;

[0083] S22: Dissolve by heating in a 50°C water bath for 12 min, shaking 7 times during the process, to obtain a photoinitiator LAP solution with a concentration of 2.5 g / L;

[0084] S23: Weigh 1.5 g of GelMA (methacrylated gelatin) and put it into a centrifuge tube, add 10 ml of the photoinitiator LAP solution prepared in S22, and shake to fully infiltrate the GelMA;

[0085] S24: Dissolve by heating in a 70°C water bath for 20 min, shaking 7 times during the process, to obtain the GelMA solution;

[0086] S3: preparing a hydrogel, comprising the following steps:

[0087] S31: PBS solution, collagen CORNING 354236, GelMA solution and photoinitiator LAP solution were prepared into a mixed solution of 500ul at a volume percentage of 70%:12%:15%:3%, and then 19μl of cultured CP-MSCs were added and mixed to obtain a crude product;

[0088] S32: Take 150ul of the crude product, add it to a culture dish, and irradiate it with a 405nm UV light source for 30s to gel;

[0089] S33: Add Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to the culture dish, incubate at 37°C for 8 minutes, wash the gelled crude product, remove the culture medium, and then add fresh Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to culture for 24 hours to obtain a composite hydrogel.

[0090] Comparative Example

[0091] A method for preparing a composite hydrogel comprises the following steps:

[0092] S1: Prepare the solution, including the following steps:

[0093] S11: Take 20 ml of PBS and add it into a brown bottle containing 0.05 g of photoinitiator LAP;

[0094] S12: Heat and dissolve in a 45°C water bath for 15 min, shaking 6 times during the process, to obtain a photoinitiator LAP solution with a concentration of 2.5 g / L;

[0095] S13: Weigh 1.5 g of GelMA (methacrylated gelatin) and put it into a centrifuge tube, add 10 ml of the photoinitiator LAP solution prepared in S22, and shake to fully infiltrate the GelMA;

[0096] S14: Dissolve by heating in a water bath at 65°C for 25 min, shaking for 6 times to obtain the GelMA solution;

[0097] S2: preparing a hydrogel, comprising the following steps:

[0098] S21: PBS solution, collagen CORNING 354236, GelMA solution and photoinitiator LAP solution were prepared into a mixed solution of 500 ul at a volume percentage of 1%:43%:55%:1% to obtain a crude product;

[0099] S22: Take 150ul of the crude product, add it to a culture dish, and irradiate it with a 405nm ultraviolet light source for 20s to gel;

[0100] S23: Add Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) to the culture dish, incubate at 37°C for 5 minutes, wash the gelled crude product, remove the culture medium, and then add fresh Mesenchymal Stem Cell Medium (Cat No.7501, ScienCell.inc) and culture for 24 hours to obtain a composite hydrogel.

[0101] Experimental example

[0102] Tissue wound repair experiment: This experiment uses a spontaneous diabetic mouse model, whose wound healing process is similar to that of human diabetic foot ulcers.

[0103] The steps of modeling experiment are as follows:

[0104] S1: 14-16 week old db / db male mice were divided into 4 groups. After anesthesia, the backs were depilated and two symmetrical circular full skin excision wounds with a diameter of 1 cm were made. Figure 1 As shown;

[0105] S2: 24 hours later, the mice were anesthetized by gas, and one side of three groups was treated with the composite hydrogel prepared in the comparative example, and the other side was treated with the composite hydrogel prepared in Examples 1 to 3, respectively, and the last group was left untreated.

[0106] The wound diameters were measured 2, 4, 7, 10, 14, 17, and 21 days after modeling. Figure 2 As shown, the first row is the natural wound repair situation, the second row is the comparative hydrogel repair situation, and the third to fifth rows are the hydrogel repair situations of Examples 1 to 3; the repair situation line graph is shown in Figure 3 As shown, the hydrogel repair efficiency of Example 1 is the fastest, and the hydrogel repair efficiency of Example 2 is second; Example 3 has a slow repair speed in the early stage, and the repair speed increases greatly after 7 days; the repair efficiency of the comparative example is slow, but it can also repair the wound in about 17 days, indicating that the hydrogel without MSCs loading also has a certain repair ability; in summary, it takes more than 20 days to heal the wound naturally, while Examples 1 to 3 and the comparative example can repair the wound in 17 days, and the effect of Examples 1 to 2 on repairing the wound is better.

[0107] The whole skin of the wound was taken 2, 4, and 7 days after modeling and fixed for histological observation. Figures 4 to 6 As shown, in Examples 1-2, a large number of GFP-labeled stem cells can still be detected at the wound on the 7th day, indicating that the cells have good compatibility and high survival rate.

[0108] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing a composite hydrogel, characterized in that: The following steps are involved: S1: Cultivate MSCs; S2: solution preparation; S3: Preparation of hydrogel; The culturing of MSCs specifically comprises: S11: Aspirate the old culture medium of cells in the culture flask and wash twice with PBS solution; S12: Add trypsin-EDTA solution and incubate at 37°C for 5-7 minutes until the cells are about to separate and appear as round pellets. Then add fresh culture medium and centrifuge at 1200 g for 5 minutes. Discard the supernatant to obtain the precipitated cells. S13: Add fresh culture medium to the precipitated cells and culture the cells at 37°C until the cell concentration is 2500-125000 cells / ul; The solution configuration includes an initiator solution configuration and a GelMA solution configuration; The initiator solution is a photoinitiator LAP solution, and the preparation steps of the photoinitiator LAP solution include: mixing the photoinitiator LAP with a PBS solution in the dark, dissolving at 40 to 50° C. for 12 to 18 minutes, and shaking for 5 to 7 times during the mixing, to obtain the solution; The GelMA solution preparation step comprises: mixing GelMA with the photoinitiator LAP solution, heating and dissolving at 60-70° C. in the dark for 20-30 minutes, and shaking 5-7 times during the mixing, to obtain the GelMA solution; The preparation of the hydrogel comprises: S31: PBS solution, collagen, GelMA solution and initiator solution are mixed evenly, and then cultured MSCs are added and mixed continuously to obtain a crude product; S32: irradiating the crude product with light to gelate it; S33: Mix the gelled substance with culture medium, culture at 37°C for 4 to 8 minutes, and then remove the culture medium to obtain the product.

2. The method for preparing the composite hydrogel according to claim 1, characterized in that: The MSCs are UC-MSCs, AM-MSCs or CP-MSCs.

3. The method for preparing the composite hydrogel according to claim 1, characterized in that: The concentration of the photoinitiator LAP solution is 2.5 g / L.

4. The method for preparing the composite hydrogel according to claim 1, characterized in that: The solid-liquid ratio of the GelMA to the photoinitiator LAP solution is 1.5 g:10 mL.

5. The method for preparing the composite hydrogel according to claim 1, characterized in that: The volume percentages of the PBS solution, collagen, GelMA solution and initiator solution are 1% to 70%: 10% to 50%: 13% to 70%: 1 to 4%.

6. The method for preparing the composite hydrogel according to claim 1, characterized in that: The irradiation is performed by irradiating with a 405 nm light source for 10 to 30 seconds.

7. A composite hydrogel prepared by the method for preparing a composite hydrogel according to any one of claims 1 to 6.

8. Use of the composite hydrogel according to claim 7 in preparing tissue damage repair products.

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