A MnO2-protein fiber composite particle gel ink and its preparation and application

Abstract

The invention belongs to the technical field of gel ink and discloses a MnO 2 ‑protein fiber composite particle gel ink and its preparation and application. Method: 1) Using GelMA, MEO 2 MA and UPyMA react under the action of KPS to prepare GMU particle gel; 2) prepare manganese dioxide modified amyloid protein fiber solution; 3) GMU particle gel and manganese dioxide modified amyloid protein fiber solution are mixed in PBS solution to obtain MnO 2 Protein fiber composite particle gel ink. The ink of the present invention has a good effect of promoting cell osteogenic differentiation while maintaining good cell compatibility and maintaining the stemness of mesenchymal stem cells. The ink of the present invention has good mechanical properties after molding, ensures the macroporous structure required in the spatial structure, and at the same time gives the porous particle gel system good three-dimensional stem cell culture performance. The ink of the present invention is used to prepare bone repair materials or three-dimensional culture of stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel ink, and specifically relates to a MnO2-protein fiber composite particle gel ink and a preparation method and application thereof. Background Art

[0002] The synthesis of injectable hydrogel matrices for cell delivery can use physical or chemical cross-linking methods to bridge polymer chains. Chemically cross-linked gels are usually formed by free radical polymerization using cross-linkers and initiators, which may expose cells to cytotoxic chemicals, free radicals or light irradiation. Once the network is permanently cross-linked, it is usually difficult to obtain good injectability. In addition, modified natural polymers with functional groups that interact non-covalently usually have excellent cell compatibility. When developing injectable hydrogels with these polymers, no other chemical reactions are required, which can avoid the introduction of small molecules or free radicals that damage cells.

[0003] Related studies have shown that ROS plays a vital role in the survival, proliferation and terminal differentiation of MSCs (mesenchymal stem cells). The oxidative environment has different regulatory effects on stem cell osteogenesis or adipogenesis. Most transplanted MSCs experience oxidative stress, and excessive ROS produced by host tissues or MSCs themselves are the main cause of cell cycle arrest and cell death. Therefore, the osteogenic differentiation of bone marrow mesenchymal stem cells can be promoted by regulating the balance of ROS [Martin JR, Patil P, Yu F, Gupta MK, Duvall CL. Enhanced stem cell retention andantioxidativeprotection with injectable, ROS-degradable PEG hydrogels[J]. Biomaterials. 2020, 263: 120377].

[0004] How to obtain a hydrogel with good mechanical properties, high cell survival rate, long-term cell stemness maintenance and osteodifferentiation performance is one of the problems that people need to solve. Summary of the invention

[0005] In order to solve the problems of insufficient mechanical properties of gel materials used for cell delivery, low cell survival rate, insufficient maintenance of cell stemness, etc., while taking into account biological properties such as promoting bone differentiation, the present invention provides a MnO2-protein fiber composite particle gel ink and a preparation method thereof. The gel ink of the present invention is a 3D printing gel ink.

[0006] Another object of the present invention is to provide an application of the gel ink. The gel ink is used to prepare a gel product that has the function of regulating the active oxygen microenvironment and cooperating with mesenchymal stem cells to promote osteogenesis. Alternatively, the gel ink is used to prepare a bone repair material or a three-dimensional culture scaffold material for stem cells.

[0007] The object of the present invention is achieved through the following solutions.

[0008] A method for preparing a MnO2-protein fiber composite particle gel ink comprises the following steps:

[0009] 1) Using a solvent to prepare a solution of methacrylic acid modified gelatin (GelMA); mixing the methacrylic acid modified gelatin solution with MEO2MA (di(ethylene glycol) methyl ether methacrylate) and KPS potassium persulfate, adding 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidine-2)ureido)ethyl methacrylate (UPyMA) and stirring evenly, deoxygenating, adding N,N,N',N'-tetramethylethylenediamine, standing at room temperature for 8-12 hours, dialyzing, and freeze-drying to obtain GMU particle gel; the solvent is a mixture of water and ethanol, and the volume ratio of water to ethanol is (2-4):1;

[0010] The structure of UPyMA is ;

[0011] The structure of MEO2MA is , n is 2;

[0012] The UPyMA was dissolved in an alkaline aqueous solution with a pH of 13-14 before use; the dialysis temperature was 25°C-37°C; the mass volume ratio of the double bond modified gelatin (GelMA): MEO2MA was 0.2g:0.2mL, and the mass ratio of GelMA: UPyMA was 0.1:0.01; the dialysis time was 5-7 d; the mass ratio of GelMA: KPS was 0.1: (0.001-0.01);

[0013] 2) Under stirring conditions, KMnO4 solution is added dropwise to the amyloid fiber AF solution, and the reaction is carried out for 3-5 hours after the addition is completed to obtain a manganese dioxide-modified amyloid fiber (MnO2-AF) solution; the concentration of the amyloid fiber AF solution is 0.1-1wt%, and the concentration of the KMnO4 solution is 1-20 mg / mL; the dropping speed is 8-12 μL / second; the mass ratio of amyloid fiber AF to potassium permanganate is 1:(10-50); the stirring speed is 300-500 rpm;

[0014] 3) The GMU particle gel and manganese dioxide modified amyloid protein fiber (MnO2-AF) solution were mixed in PBS solution to obtain MnO2-protein fiber composite particle gel ink.

[0015] The concentration of the GMU particle gel in the ink is 5-50 mg / mL.

[0016] The mass ratio of GMU particle gel and manganese dioxide-modified amyloid protein fibers was 5:(0.1~1).

[0017] The solution of amyloid fiber AF was obtained by the following method:

[0018] The hen egg white lysozyme is dissolved in an acid solution with a pH value of 2, and the solution is incubated at 85 to 95°C for 8 to 12 hours to obtain an AF solution. The mass volume ratio of the hen egg white lysozyme to the acid solution with a pH value of 2 is 0.5 g: (4 to 5) mL.

[0019] The MnO2-protein fiber composite particle gel ink is prepared by the above method.

[0020] The MnO2-protein fiber composite particle gel ink is used to prepare a gel product that can regulate the active oxygen microenvironment and cooperate with mesenchymal stem cells to promote bone formation. Alternatively, the gel ink is used to prepare bone repair materials or three-dimensional culture scaffold materials for stem cells.

[0021] The present invention uses GMU particle gel as the organic phase and MnO2-protein fiber as the fiber matrix for regulating the active oxygen microenvironment, and the released manganese particles can further serve as bone induction active components to construct a particle gel ink that can regulate the active oxygen microenvironment and cooperate with mesenchymal stem cells to promote osteogenesis.

[0022] The MnO2-protein fiber composite particle gel ink is a 3D printing gel ink, which can be used for 3D printing of scaffold materials.

[0023] The ink of the present invention is a nanofiber composite particle gel ink that can respond to the cell active oxygen microenvironment, maintain cell stemness and have a certain protective function for cells, avoiding the problems of cell immunogenicity and low survival rate involved in current stem cell delivery therapy; at the same time, the manganese particles released after manganese dioxide consumes active oxygen can effectively enhance the osteogenic activity of the material, and it is expected to better meet the clinical treatment needs of bone defects.

[0024] The present invention is based on a physical cross-linking method, and a functional nanofiber material is physically embedded in the internal network of the GMU particle gel. The GelMA component in the GMU particle gel has an RGD sequence, which can promote cell adhesion; wherein the PMEO2MA chain segments are dehydrated and assembled into clusters, thereby providing a hydrophobic microenvironment, further promoting UPy dimerization to connect polymer chains, thereby forming a synergistic hydrogen bond and a hydrophobic cross-linking network. The particle gel is further compounded with MnO2-modified amyloid fibrils. The MnO2 nanoparticles can regulate the active oxygen microenvironment by reacting with ROS, thereby effectively improving the viability of MSCs, and the released Mn 2+ It can further promote the osteogenic differentiation of stem cells. Fibers can also serve as a matrix for cell adhesion and can further improve the mechanical strength and mechanical properties of the gel ink system.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The present invention is based on physical cross-linking. Through hydrogen bonds and hydrophobic interactions between particle gels, as well as the interaction between particle gels and MnO2-protein fibers, the mechanical properties of GMU particle gels are improved and the macroporous structure required in the spatial structure is ensured. At the same time, the porous particle gel system is endowed with good three-dimensional stem cell culture performance, which provides new possibilities for the research and application of gel materials in the field of stem cell delivery.

[0027] (2) The present invention controls the reaction conditions (GMU particle gel concentration and MnO2-AF concentration, etc.) so that the prepared MnO2-protein fiber composite particle gel ink that can regulate the active oxygen microenvironment has a good effect of promoting cell osteogenic differentiation while maintaining good cell compatibility and maintaining the stemness of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a fluorescent photograph of the GMU particle gel in Example 1 after being stained with fluorescein isothiocyanate (FITC);

[0029] Figure 2 is the SEM image of the GMU particle gel in Example 1;

[0030] Figure 3 TEM image of MnO2-AF in Example 2;

[0031] Figure 4 is the SEM image of the MnO2-AF / GMU particle gel in Example 2;

[0032] Figure 5 It is the time-modulus scanning curve of MnO2-AF / GMU particle gel system;

[0033] Figure 6 This is a 3D printed scaffold image of the MnO2-AF / GMU particle gel ink in Example 3;

[0034] Figure 7 The MnO2-AF / GMU particle gel ink in Example 4 is used as a platform for embedding 3D printing;

[0035] Figure 8 Mn 2+ Release curve diagram of

[0036] Fig. 9 The test results of GMU / MnO2-AF particle gel in vitro to remove reactive oxygen species;

[0037] Fig.10 The results of cell viability test after PBS (control), GMU particle gel, GMU / MnO2-AF particle gel and hBMSCs cells were co-injected and cultured for 6 h and 3 d respectively;

[0038] Fig.11 The expression level of the cell stemness gene Oct4 after the GMU and MnO2-AF / GMU particle gels were co-cultured with hBMSCs cells for 7 and 14 days in Example 1 and Example 2;

[0039] Fig.12 This is a graph showing the expression level of the cell osteogenic gene OCN after the MnO2-AF / GMU particle gel and hBMSCs cells were co-cultured in an H2O2-simulated reactive oxygen microenvironment for 7 and 14 days in Example 2. DETAILED DESCRIPTION

[0040] In order to better understand the present invention, the present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope shown in the embodiments.

[0041] In the following examples, SEM scanning is used to analyze the pore structure of the particle gel ink, and Anton Paar MCR302 rheometer is used to test the rheological properties of the particle gel ink.

[0042] Example 1

[0043] Step 1: Synthesis of modified gelatin (GelMA)

[0044] (1) Add 6 g of gelatin into 60 mL of PBS solution and stir at 40 °C until dissolved;

[0045] (2) Add 12 mL of methacrylic anhydride according to the feed ratio (gelatin: methacrylic anhydride = 1 g: 2 mL) and react at 50 °C for 5 h;

[0046] (3) Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8000 and dialyze at 40 °C for 7 days;

[0047] (4) Freeze-dry the pre-frozen dialysate to obtain the desired GelMA.

[0048] Step 2: Preparation of GMU particle gel

[0049] (1) Dissolve 0.2 g of GelMA in 2.4 mL of deionized water and 1 mL of ethanol at 40 °C.

[0050] (2) Cool the solution in (1) to 25 °C and then add 200 μL MEO2MA and 0.01 g KPS and stir evenly;

[0051] (3) Add 400 μL UPyMA (0.05 g / mL, dissolved under alkaline conditions, pH = 13-14) and stir evenly;

[0052] (4) Then, nitrogen was passed through for 10 min to remove oxygen from the solution;

[0053] (5) Add 25 μL of N,N,N',N'-tetramethylethylenediamine and leave at room temperature overnight to form a gel;

[0054] (6) dialyzing the prepared gel in deionized water for 7 days;

[0055] (7) The dialyzed gel is crushed by a high-speed blender and then freeze-dried in a freeze dryer to obtain GMU particle gel powder.

[0056] Figure 1 This is a fluorescence photograph of the GMU particle gel taken by confocal microscopy after being stained with fluorescein isothiocyanate (FITC).

[0057] Step 3: Preparation of GMU particle gel ink

[0058] (1) Take 0.5 g of GMU particle gel powder and dissolve it in 10 mL of PBS. Stir well to obtain GMU microgel ink.

[0059] Figure 2 This is the SEM image of the GMU particle gel system after freeze-drying, which has a certain pore structure.

[0060] Example 2

[0061] Step 1: Preparation of MnO2-AF

[0062] (1) Add 0.5 g of egg white lysozyme to 4.5 mL of dilute hydrochloric acid solution with a pH of 2 and stir until dissolved;

[0063] (2) Incubate the above solution at 90 °C for 10 h to obtain the desired AF solution (concentration of 10 wt%);

[0064] (2) Take 2 mL of 10 wt% AF solution and add 17.5 mL of deionized water to adjust its concentration to 1 wt%;

[0065] (4) Add 500 μL of 20 mg / mL KMnO4 solution dropwise while stirring at a rate of 10 μL per second.

[0066] (5) After the addition is completed, the reaction is carried out in the dark for 4 h to obtain a MnO2-AF solution.

[0067] Figure 3 This is the transmission electron microscope (TEM) image of MnO2-AF.

[0068] Step 2: Preparation of MnO2-AF / GMU particle gel ink

[0069] (1) Take 0.5 g of GMU particle gel powder (gel powder prepared in Example 1) and 10 mL of MnO2-AF solution (concentration of 1 wt%), stir and mix them evenly to obtain MnO2-AF / GMU particle gel ink. Figure 4 This is the SEM photo of the MnO2-AF / GMU particle gel system after freeze-drying, which also has a certain pore structure.

[0070] Figure 5 This is the time-modulus scanning curve of the MnO2-AF / GMU particle gel system. With the increase of MnO2-AF content (the proportion of MnO2-AF solution is 0, 0.2wt%, 0.5wt%, 1.0wt%), its storage modulus increases.

[0071] Example 3

[0072] (1) Take 0.5 g of GMU particle gel powder (prepared in Example 1), 10 mL of MnO2-AF solution (in Example 2, the concentration is 1 wt%) and 50 uL of methylene blue solution (1 mg / mL), stir and mix them evenly to obtain MnO2-AF / GMU particle gel ink;

[0073] (2) The physically cross-linked particle gel ink was used to study the printing performance of the extrusion 3D printer at a printing temperature of 25 °C. Figure 6 A scaffold printed with gel ink for the particles.

[0074] Example 4

[0075] (1) Take 0.5 g of GMU particle gel powder, 10 mL of MnO2-AF solution (in Example 2, the concentration is 1 wt%) and 5 mL of PBS solution, stir and mix them evenly to obtain MnO2-AF / GMU particle gel ink;

[0076] (2) injecting the physically cross-linked particle gel ink into a circular mold to prepare a particle gel platform;

[0077] (3) Using rhodamine solution as ink, the particle gel platform embedding printing performance was studied through an extrusion 3D printer. Figure 7 Image of the particle gel ink as a platform for embedding 3D printing.

[0078] The present invention prepares a fiber-microgel ink modified with MnO2 nanoparticles, and promotes bone repair by regulating the reactive oxygen microenvironment and cooperating with mesenchymal stem cells. The MnO2 nanoparticles can regulate the reactive oxygen microenvironment by reacting with ROS, thereby effectively improving the viability of MSCs. Figure 8 Mn 2+ The release curve of MnO2 shows that when MnO2 is compounded onto AF, its release rate from the particle gel system is relatively slower, showing a sustained release effect. Fig. 9 The test results of GMU / MnO2-AF particle gel in vitro for removing reactive oxygen species show that with the increase of MnO2-AF content (the volume percentage of MnO2-AF solution is 0, 0.2 wt%, 0.5 wt% and 1 wt%, respectively), its efficiency in consuming reactive oxygen species is significantly improved. Fig.10 The results of cell viability tests after 6 h and 3 d of culture after co-injection of PBS (control), GMU particle gel, and GMU / MnO2-AF particle gel (in Example 2) with hBMSCs cells, respectively, indicate that the GMU particle gel has a protective effect on cells during the injection process. Fig.11 The expression level of the cell stemness gene Oct4 after 7 and 14 days of co-culture of GMU microgel, GMU / MnO2-AF particle gel (Example 2) and hBMSCs cells. Fig.12 The results are the expression levels of the osteogenic gene OCN after GMU / MnO2-AF particle gel (Example 2) and hBMSCs cells were co-cultured in the reactive oxygen microenvironment simulated by H2O2 for 7 and 14 days. The above research results show that GMU / MnO2-AF particle gel can be used as a three-dimensional culture system for hBMSCs, which can effectively maintain the activity and stemness of stem cells; and MnO2-AF can effectively regulate the reactive oxygen microenvironment, and the released Mn 2+ It can promote the upregulation of the expression level of the osteogenic gene OCN, thereby showing a synergistic stem cell osteogenic effect.

Claims

1. A method for preparing a MnO2-protein fiber composite particle gel ink, characterized in that: The following steps are involved: 1) Using a solvent to prepare methacrylic acid modified gelatin into a methacrylic acid modified gelatin solution; mixing the methacrylic acid modified gelatin solution, MEO2MA and potassium persulfate, adding UPyMA and stirring evenly, deoxygenating, adding N, N, N', N'-tetramethylethylenediamine, standing at room temperature for 8-12 hours, dialyzing, crushing, and freeze-drying to obtain GMU particle gel; the solvent is a mixture of water and ethanol, and the volume ratio of water to ethanol is (2-4):1; The structure of UPyMA is The structure of MEO2MA is , n is 2; 2) Under stirring conditions, KMnO4 solution was added dropwise to the amyloid fiber solution, and the mixture was reacted for 3-5 hours to obtain an amyloid fiber solution modified with manganese dioxide; the mass ratio of amyloid fiber to KMnO4 was 1:(10-50); 3) The GMU particle gel and the manganese dioxide modified amyloid protein fiber solution are mixed in a phosphate buffer solution to obtain a MnO2-protein fiber composite particle gel ink.

2. The method for preparing the MnO2-protein fiber composite particle gel ink according to claim 1, characterized in that: The UPyMA in step 1) is dissolved in an alkaline aqueous solution with a pH of 13-14 before use; the dialysis temperature is 25°C-37°C; The mass volume ratio of methacrylic acid modified gelatin: MEO2MA is 0.2g:0.2mL, the mass ratio of methacrylic acid modified gelatin: UPyMA is 0.1:0.01; the dialysis time is 5-7 days; the mass ratio of methacrylic acid modified gelatin: potassium persulfate is 0.1: (0.001~0.01); In step 2), the final concentration of the amyloid fiber solution in the system is 0.1-1 wt%, the concentration of the KMnO4 solution is 1-20 mg / mL; the dripping speed is 8-12 μL / sec; and the stirring speed is 300-500 rpm.

3. The method for preparing the MnO2-protein fiber composite particle gel ink according to claim 1, characterized in that: The concentration of the GMU particle gel in the ink is 5-50 mg / mL; The mass ratio of GMU particle gel and manganese dioxide-modified amyloid protein fibers was 5:(0.1~1).

4. The method for preparing the MnO2-protein fiber composite particle gel ink according to claim 1, characterized in that: The amyloid protein fiber solution is obtained by the following method: dissolving egg white lysozyme in an acid solution with a pH value of 2, and incubating at 85 to 95° C. for 8 to 12 hours to obtain the amyloid protein fiber solution.

5. The method for preparing the MnO2-protein fiber composite particle gel ink according to claim 4, characterized in that: The mass volume ratio of the hen's egg white lysozyme to the acid solution with pH=2 is 0.5 g: (4-5) mL.

6. A MnO2-protein fiber composite particle gel ink obtained by the preparation method according to any one of claims 1 to 5.

7. The use of the MnO2-protein fiber composite particle gel ink according to claim 6, characterized in that: The MnO2-protein fiber composite particle gel ink is used to prepare a gel product that can regulate the active oxygen microenvironment and cooperate with mesenchymal stem cells to promote osteogenesis.

8. The use of the MnO2-protein fiber composite particle gel ink according to claim 6, characterized in that: The gel ink is used for preparing bone repair materials or three-dimensional culture scaffold materials for stem cells.

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