Preparation method of degradable hydrogel based on dynamic crosslinking and application of degradable hydrogel to in-vitro amplification and recovery of mesenchymal stem cells

Abstract

The invention discloses a preparation method of degradable hydrogel based on dynamic crosslinking and application of the degradable hydrogel to in-vitro amplification and recovery of mesenchymal stem cells, and belongs to the field of biomedical engineering. The preparation method of the hydrogel comprises the following steps: reacting a cyclodextrin derivative with a zwitterionic monomer to form a copolymer, and uniformly mixing the copolymer with adamantane modified hyaluronic acid at room temperature to prepare the degradable hydrogel based on dynamic crosslinking. According to the degradable hydrogel based on dynamic crosslinking, zwitterions are introduced into a system, crosslinking is achieved through interaction of subjects and guests, the problems existing in existing stem cell in-vitro 3D multiplication culture can be solved, remarkable amplification of encapsulated stem cells can be promoted, and non-specific differentiation can be well inhibited; after the culture is finished, the hydrogel can be quickly degraded by adding competitive sodium adamantane carboxylate micromolecules, so that the stem cells can be released and recycled under mild conditions.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a method for preparing a degradable hydrogel based on dynamic cross-linking and its application in in vitro expansion and recovery of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells, as a cell population with self-renewal and multidirectional differentiation potential in organisms, are crucial in the fields of regenerative medicine and tissue engineering, and have significant therapeutic effects in repairing damaged tissues, treating blood diseases and treating immune metabolic diseases. However, the number of stem cells used in the clinical treatment of certain diseases is huge, and direct extraction from the body cannot meet the treatment needs at all. Therefore, the most ideal way to obtain a large number of stem cells is to carry out large-scale in vitro culture and expansion. The traditional two-dimensional (2D) culture model usually cannot fully simulate the in vivo cell microenvironment, resulting in a low self-proliferation ability of stem cells. Hydrogel, as a material with a three-dimensional (3D) network structure, has shown great advantages in the field of in vitro cell expansion in recent years. However, the existing 3D culture hydrogels also have many shortcomings. For example, during the long-term culture of stem cells in hydrogels, due to the influence of various factors, the cells undergo unwanted specific differentiation; in addition, there is still a lack of feasible methods to achieve efficient recovery of expanded cells from hydrogels. It is difficult for traditional methods to simultaneously meet high cell recovery rates and good activity of recovered cells. Therefore, it is of broad significance to develop a 3D culture hydrogel that can effectively proliferate stem cells and avoid nonspecific differentiation during the culture process, while also achieving rapid and efficient recovery in the subsequent process.

[0003] Hydrogels containing zwitterions exhibit superhydrophilic properties and can resist nonspecific protein adhesion, thereby providing a relatively "closed" undisturbed environment for the encapsulated stem cells, thereby achieving long-term maintenance of stem cell stemness. As a reversible binding, the host-guest interaction can be destroyed under the competitive action of the free host or guest, resulting in the degradation of the hydrogel. Studies have shown that within the appropriate (free molecule) concentration range, it does not affect the cell viability of cultured cells. Among the many host-guest pairs, β-CD has been approved by the FDA (U.S. Food and Drug Administration) as a food additive, and the corresponding guest adamantane will not be absorbed and digested by cells. However, the formation of hydrogels through the host-guest interaction of β-CD and adamantane cannot achieve efficient in vitro stem cell expansion and long-term maintenance of stem cell stemness, which needs to be solved urgently. Summary of the invention

[0004] The object of the present invention is to provide a preparation method of a degradable hydrogel based on dynamic crosslinking and its application in the in vitro expansion and recovery of mesenchymal stem cells, so as to solve the problems existing in the above-mentioned prior art. This hydrogel is prepared by introducing zwitterions into the system and achieving crosslinking through host-guest interaction. This hydrogel can overcome the problems existing in the existing in vitro 3D expansion culture of stem cells. It can not only promote the significant expansion of encapsulated stem cells, but also well inhibit the occurrence of non-specific differentiation. After the culture is completed, by adding a competitive small molecule of sodium adamantane carboxylate, the hydrogel will degrade rapidly, realizing the release and recovery of stem cells under mild conditions.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a preparation method of a degradable hydrogel based on dynamic crosslinking, including the steps of reacting a cyclodextrin derivative with a zwitterionic monomer to form a copolymer, and then uniformly mixing the copolymer with adamantane-modified hyaluronic acid at room temperature to obtain a degradable hydrogel based on dynamic crosslinking.

[0007] Preferably, the preparation method includes the following steps:

[0008] (1) Dissolve mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin in N,N-dimethylformamide, add ethyl isocyanate acrylate, react under a nitrogen atmosphere and ice bath conditions. After the reaction is completed, add acetone dropwise to precipitate, and vacuum dry to obtain β-cyclodextrin modified with ethyl isocyanate acrylate;

[0009] (2) Dissolve the β-cyclodextrin modified with ethyl isocyanate acrylate and the zwitterionic monomer in dimethyl sulfoxide, heat under a nitrogen atmosphere, and then add azobisisobutyronitrile and stir to react. After the reaction is completed, add methanol dropwise to precipitate, dialyze and freeze-dry to obtain a zwitterion-cyclodextrin copolymer;

[0010] (3) Dissolve the zwitterion-cyclodextrin copolymer and adamantane-modified hyaluronic acid in phosphate buffer solution respectively, and mix them evenly in equal volume to obtain a degradable hydrogel based on dynamic crosslinking.

[0011] Preferably, in step (1), the mass-volume ratio of the β-cyclodextrin to water is (1-10) g: 35 mL; and / or the mass ratio of the β-cyclodextrin to the p-toluenesulfonyl chloride is 10: (1-3); and / or the concentration of the p-toluenesulfonyl chloride acetonitrile solution is 15-50% w / v.

[0012] Preferably, in step (1), the mass-volume ratio of mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin to N,N-dimethylformamide is 1 g:(5-10) mL; and / or the mass-volume ratio of mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin to ethyl isocyanate acrylate is 3 g:(250-600) μL; and / or the time of the ice bath reaction is 10-60 min.

[0013] Preferably, in step (2), the mass ratio of the ethyl isocyanate acrylate-modified β-cyclodextrin, zwitterionic monomer and azobisisobutyronitrile is (1-3) g:(1-2.5) g:(50-100) mg; and / or heat to 60-80 °C, add the azobisisobutyronitrile and stir for reaction for 12-48 h; and / or the zwitterionic monomer includes sulfobetaine zwitterionic monomer, carboxybetaine zwitterionic monomer or phosphorylcholine zwitterionic monomer.

[0014] Preferably, in step (3), the concentration of the zwitterionic-cyclodextrin copolymer dissolved in phosphate buffer solution is 20-30% w / v, and the concentration of the adamantane-modified hyaluronic acid dissolved in phosphate buffer solution is 5-8% w / v.

[0015] Preferably, in step (3), the preparation method of the adamantane-modified hyaluronic acid includes the following steps:

[0016] Add tetrabutylammonium hyaluronic acid, carboxyl-containing adamantane and 4-dimethylaminopyridine to DMSO, stir under nitrogen atmosphere until completely dissolved, add di-tert-butyl dicarbonate, react at room temperature for 12-36 h, after the reaction is completed, dialyze and freeze-dry to obtain adamantane-modified hyaluronic acid; wherein, the mass-volume ratio of the tetrabutylammonium hyaluronic acid, carboxyl-containing adamantane, 4-dimethylaminopyridine and di-tert-butyl dicarbonate is (0.5-2) g:(0.5-1) g:(0.05-0.25) g:(100-300) μL, and the carboxyl-containing adamantane includes adamantane formic acid, adamantane acetic acid or adamantane propionic acid.

[0017] The present invention also provides an application of the degradable hydrogel prepared by the above preparation method in the in vitro expansion and recovery of mesenchymal stem cells.

[0018] Preferably, the method for in vitro expansion of mesenchymal stem cells includes:

[0019] Resuspend the zwitterionic-cyclodextrin copolymer in α-MEM basal medium, then add 5-10 mg / mL of collagen, filter to obtain a zwitterionic-cyclodextrin copolymer precursor solution;

[0020] Resuspend the adamantane-modified hyaluronic acid in α-MEM basal medium to obtain an adamantane-modified hyaluronic acid precursor solution;

[0021] Resuspend the mesenchymal stem cells in the zwitterionic-cyclodextrin copolymer precursor solution, and mix it with the adamantane-modified hyaluronic acid precursor solution to form a hydrogel encapsulating the mesenchymal stem cells. Then, immerse the hydrogel encapsulating the mesenchymal stem cells in α-MEM basal medium and culture it;

[0022] The method for in vitro recovery of the mesenchymal stem cells includes: transferring the hydrogel encapsulating the mesenchymal stem cells to a 2-10 mM sodium adamantane carboxylate solution for degradation for 30-60 min. After completion, filter the suspension through a cell strainer to remove undegraded hydrogel blocks, and then collect the cells by centrifugation using a centrifuge.

[0023] The above-mentioned sodium adamantane carboxylate includes sodium adamantane carboxylate Ad-COONa, sodium adamantane acetate Ad-CH2COONa, or sodium adamantane propionate Ad-CH2CH2COONa.

[0024] The above-mentioned mesenchymal stem cells include adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, or umbilical cord mesenchymal stem cells.

[0025] The present invention discloses the following technical effects:

[0026] The present invention first prepares a copolymer based on a cyclodextrin derivative and a zwitterionic monomer, and adamantane-modified hyaluronic acid through free radical polymerization. The two solutions can form a dynamic cross-linked hydrogel based on host-guest recognition under physiological conditions. By introducing collagen, the hydrogel can simulate the composition of the extracellular matrix and achieve continuous amplification of mesenchymal stem cells in vitro. The hydrogel has excellent anti-fouling performance and cell compatibility, and can effectively maintain the stem cell phenotype during cell proliferation. More importantly, the hydrogel realizes specific degradation under physiological conditions and efficient recovery of mesenchymal stem cells by introducing a competitive small molecule sodium adamantane carboxylate. The present invention realizes continuous proliferation of mesenchymal stem cells in vitro and recovery under physiological conditions through a dynamically cross-linked degradable hydrogel, greatly reducing the stimulation to cells during the recovery process, and providing an effective method for stem cell amplification for stem cell clinical treatment. Description of the Drawings

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 (A) FTIR and (B) HNMR spectra of P(SBMA-co-CD) prepared in Example 5 of the present invention; 1 HNMR spectrum;

[0029] Figure 2 Schematic diagrams and optical photographs of the formation of HSC hydrogel in Experimental Example 2 of the present invention (A) and schematic diagrams and optical photographs of the inability to form hydrogel in the presence of competing molecules (B) (the red solution is caused by adding a small amount of rhodamine B);

[0030] Figure 3 Frequency sweep (A) and time sweep (B) rheological behaviors of HSC 20 HSC 25 HSC 30 and HC hydrogels measured in Experimental Example 3 of the present invention;

[0031] Figure 4 Degradation conditions of HSC 20 HSC 25 HSC 30 and HC hydrogels; (A) Degradation curves, (B) Equilibrium swelling ratio (ESR);

[0032] Figure 5 SEM images (A) and pore sizes (B) of HSC 20 HSC 25 HSC 30 and HC hydrogels taken in Experimental Example 3 of the present invention;

[0033] Figure 6 Optical photographs of the degradation of HSC 30 hydrogel (rhodamine stained) in different solutions; (A) PBS solution, (B) 2 mM Ad-COONa solution, (C) 5 mM Ad-COONa solution, (D) 10 mM Ad-COONa solution;

[0034] Figure 7 Degradation curves (A) and degradation times (B) of HSC 30 hydrogel in different concentrations of Ad-COONa solutions measured in Experimental Example 4 of the present invention;

[0035] Figure 8 Anti-protein adhesion behaviors of the hydrogels measured in Experimental Example 5 of the present invention; (A) Fluorescence images, (B) Data statistical charts;

[0036] Figure 9Proliferation activities of ADSCs cultured in different hydrogels for 1, 4, 7, and 10 days as measured in Experimental Example 6 of the present invention; (A) AO / PI fluorescence staining images, (B) Alamar Blue reduction rate, (C) amplification fold;

[0037] Figure 10 For the recovery of ADSCs from HSC hydrogels under different conditions as measured in Experimental Example 7 of the present invention 30 ; (A) Calcein-AM / PI fluorescence staining images, (B) flow cytometry results, (C) cell viability statistics, (D) harvest rate statistical results;

[0038] Figure 11 For the expression of surface protein markers of primary ADSCs and ADSCs recovered from HSC and HC hydrogels respectively as measured in Experimental Example 8 of the present invention; (A) immunofluorescence staining images, (B) flow cytometry results, (C) statistical analysis of flow cytometry results. Detailed implementation manners

[0039] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0042] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0043] Terms such as "comprising", "including", "having", "containing", etc. used in this article are all open-ended terms, meaning including but not limited to.

[0044] The single-(6-ethylenediamino-6-deoxy)-β-cyclodextrin used in the following examples was purchased from Shanghai Aladdin Biochemical Reagent Co., Ltd.

[0045] Example 1: Preparation of isocyanate ethyl acrylate-modified β-cyclodextrin (β-CD-AOI)

[0046] 0.5 g of single-(6-ethylenediamino-6-deoxy)-β-cyclodextrin (β-CD-EDA) reagent was dissolved in 5 mL of N,N-dimethylformamide (DMF). After stirring to dissolve, 100 μL of isocyanate ethyl acrylate (AOI) was added and the reaction was carried out for 10 min under nitrogen protection and ice bath conditions. After the reaction was completed, precipitation was carried out by dripping acetone, and vacuum drying was carried out to obtain a white solid product (β-CD-AOI).

[0047] Example 2: Preparation of isocyanate ethyl acrylate-modified β-cyclodextrin (β-CD-AOI)

[0048] 1.5 g of β-CD-EDA was dissolved in 10 mL of N,N-dimethylformamide (DMF). After stirring to dissolve, 170 μL of isocyanate ethyl acrylate (AOI) was added and the reaction was carried out for 30 min under nitrogen protection and ice bath conditions. After the reaction was completed, precipitation was carried out by dripping acetone, and vacuum drying was carried out to obtain a white solid product (β-CD-AOI).

[0049] Example 3: Preparation of isocyanate ethyl acrylate-modified β-cyclodextrin (β-CD-AOI)

[0050] 3 g of β-CD-EDA was dissolved in 15 mL of N,N-dimethylformamide (DMF). After stirring to dissolve, 250 μL of isocyanate ethyl acrylate (AOI) was added and the reaction was carried out for 60 min under nitrogen protection and ice bath conditions. After the reaction was completed, precipitation was carried out by dripping acetone, and vacuum drying was carried out to obtain a white solid product (β-CD-AOI).

[0051] Example 4: Preparation of zwitterionic-cyclodextrin copolymer

[0052] Weigh 1.0 g of sulfobetaine zwitterionic monomer (SBMA) and 1 g of β-CD-AOI, dissolve them in 20 mL of DMSO, and heat them in a water bath to 60 °C under nitrogen protection. After a period of time, add 50 mg of azobisisobutyronitrile (AIBN), and stir the reaction for 12 h. After the reaction is completed, use methanol dropwise precipitation to obtain a large amount of white product. Filter and collect the product, dissolve the product in NaCl solution, perform dialysis with deionized water, and freeze-dry to obtain the product P(SBMA-co-CD). The above zwitterionic monomer can also be carboxybetaine zwitterionic monomer (CBMA) or phosphorylcholine zwitterionic monomer (MPC).

[0053] Example 5: Preparation of Zwitterionic-Cyclodextrin Copolymer

[0054] Weigh 1.7 g of SBMA and 2 g of β-CD-AOI, dissolve them in 50 mL of DMSO, and heat them in a water bath to 70 °C under nitrogen protection. After a period of time, add 74 mg of AIBN, and stir the reaction for 24 h. After the reaction is completed, use methanol dropwise precipitation to obtain a large amount of white product. Filter and collect the product, dissolve the product in NaCl solution, perform dialysis with deionized water, and freeze-dry to obtain the product P(SBMA-co-CD). The above zwitterionic monomer can also be carboxybetaine zwitterionic monomer (CBMA) or phosphorylcholine zwitterionic monomer (MPC).

[0055] Example 6: Preparation of Zwitterionic-Cyclodextrin Copolymer

[0056] Weigh 2.5 g of SBMA and 3 g of β-CD-AOI, dissolve them in 100 mL of DMSO, and heat them in a water bath to 80 °C under nitrogen protection. After a period of time, add 100 mg of AIBN, and stir the reaction for 48 h. After the reaction is completed, use methanol dropwise precipitation to obtain a large amount of white product. Filter and collect the product, dissolve the product in NaCl solution, perform dialysis with deionized water, and freeze-dry to obtain the product P(SBMA-co-CD). The above zwitterionic monomer can also be carboxybetaine zwitterionic monomer (CBMA) or phosphorylcholine zwitterionic monomer (MPC).

[0057] Example 7: Synthesis Method of Adamantane-Modified Hyaluronic Acid

[0058] Weigh 0.5 g of tetrabutylammonium hyaluronic acid (HA-TBA), 0.5 g of carboxyl-containing adamantane (any one of adamantane formic acid, adamantane acetic acid, and adamantane propionic acid, and adamantane formic acid is selected in this example), and 0.05 g of 4-dimethylaminopyridine (DMAP), and dissolve them in 30 mL of dimethyl sulfoxide (DMSO). Continuously stir under nitrogen protection until HA-TBA is completely dissolved. Add 100 μL of di-tert-butyl dicarbonate (BOC2O) and react at room temperature for 24 h. After the reaction is completed, dialyze with deionized water and perform freeze-drying treatment to obtain the product HA-Ada.

[0059] Example 8: Synthesis method of adamantane-modified hyaluronic acid

[0060] Weigh 1 g of HA-TBA, 0.8 g of carboxyl-containing adamantane (any one of adamantane formic acid, adamantane acetic acid, and adamantane propionic acid, and adamantane acetic acid is selected in this example), and 0.13 g of DMAP, and dissolve them in 50 mL of DMSO. Continuously stir under nitrogen protection until HA-TBA is completely dissolved. Add 200 μL of BOC2O and react at room temperature for 12 h. After the reaction is completed, dialyze with deionized water and perform freeze-drying treatment to obtain the product HA-Ada.

[0061] Example 9: Synthesis method of adamantane-modified hyaluronic acid

[0062] Weigh 2 g of HA-TBA, 1 g of carboxyl-containing adamantane (any one of adamantane formic acid, adamantane acetic acid, and adamantane propionic acid, and adamantane propionic acid is selected in this example), and 0.25 g of DMAP, and dissolve them in 100 mL of DMSO. Continuously stir under nitrogen protection until HA-TBA is completely dissolved. Add 300 μL of BOC2O and react at room temperature for 36 h. After the reaction is completed, dialyze with deionized water and perform freeze-drying treatment to obtain the product HA-Ada.

[0063] Example 10: Preparation method of a dynamically crosslinked degradable hydrogel

[0064] Add 0.20 g of P(SBMA-co-CD) synthesized in Example 5 to 1 mL of phosphate buffer solution and stir until dissolved; add 0.16 g of HA-Ada synthesized in Example 9 to 2 mL of phosphate buffer solution and stir until completely dissolved. Aspirate equal volumes of the P(SBMA-co-CD) solution and the HA-Ada solution, and mix them evenly at room temperature to form a dynamically crosslinked degradable hydrogel (denoted as HSC 20 ).

[0065] Example 11: Preparation method of a dynamically crosslinked degradable hydrogel

[0066] Add 0.25 g of P(SBMA-co-CD) synthesized in Example 5 to 1 mL of phosphate buffer and stir until dissolved; add 0.16 g of HA-Ada synthesized in Example 9 to 2 mL of phosphate buffer and stir until completely dissolved. Pipette equal volumes of the P(SBMA-co-CD) solution and the HA-Ada solution and mix well at room temperature to form a dynamically crosslinked degradable hydrogel (denoted as HSC 25 ).

[0067] Example 12: Preparation method of a dynamically crosslinked degradable hydrogel

[0068] Add 0.3 g of P(SBMA-co-CD) synthesized in Example 5 to 1 mL of phosphate buffer and stir until dissolved; add 0.16 g of HA-Ada synthesized in Example 9 to 2 mL of phosphate buffer and stir until completely dissolved. Pipette equal volumes of the P(SBMA-co-CD) solution and the HA-Ada solution and mix well at room temperature to form a dynamically crosslinked degradable hydrogel (denoted as HSC 30 ).

[0069] Example 13: Detection of the amplification and proliferation activities of mesenchymal stem cells in the hydrogel

[0070] Prepare the P(SBMA-co-CD) solution and the HA-Ada solution according to the steps of Example 11, but use α-MEM basal medium instead of phosphate buffer, and add a small amount of collagen (5 - 10 mg / mL, 5 mg / mL is selected in this example) to the P(SBMA-co-CD) precursor solution, and filter through a syringe filter. Resuspend white adipose mesenchymal stem cells (ADSCs) at a density of 2×10 6 cells / mL in the P(SBMA-co-CD) precursor solution and quickly mix well with the HA-Ada precursor solution in TCPS (standard 48-well polystyrene tissue culture plate) to form a hydrogel encapsulating ADSCs. Then, add α-MEM complete medium to each well to submerge the hydrogel, and culture routinely at 37 °C and 5% CO2. Replace the fresh medium every 24 h and culture for a total of 10 d. Stain the cells in the hydrogel with AO / PI at 1, 4, 7, and 10 d respectively to observe the growth state and the number of live and dead cells of ADSCs in the hydrogel, and detect the proliferation of ADSCs in the hydrogel using an Alamar Blue kit.

[0071] Example 14: Detection of the amplification and proliferation activities of mesenchymal stem cells in the hydrogel

[0072] Prepare the P(SBMA-co-CD) solution and the HA-Ada solution according to the steps of Example 11, but replace the phosphate buffer solution with α-MEM basal medium, and add a small amount of collagen (5 - 10 mg / mL, 5 mg / mL is selected in this example) to the P(SBMA-co-CD) precursor solution, and filter it through a syringe filter. Resuspend the bone marrow mesenchymal stem cells (BMSCs) at a density of 2×10 6 cells / mL in the P(SBMA-co-CD) precursor solution, and quickly mix it with the HA-Ada precursor solution in TCPS to form a hydrogel encapsulating BMSCs. Then, add α-MEM complete medium to each well to submerge the hydrogel, and culture it routinely at 37 °C and 5% CO2. Replace the fresh medium every 24 h, and culture for a total of 10 d. Perform AO / PI staining on the cells in the hydrogel at 1, 4, 7, and 10 d respectively, observe the growth state and the number of live and dead cells of BMSCs in the hydrogel, and detect the proliferation of BMSCs in the hydrogel by the AlamarBlue kit.

[0073] Example 15: Detection of the amplification and proliferation activity of mesenchymal stem cells in the hydrogel

[0074] Prepare the P(SBMA-co-CD) solution and the HA-Ada solution according to the steps of Example 11, but replace the phosphate buffer solution with α-MEM basal medium, and add a small amount of collagen (5 - 10 mg / mL, 5 mg / mL is selected in this example) to the P(SBMA-co-CD) precursor solution, and filter it through a syringe filter. Resuspend the umbilical cord mesenchymal stem cells (UCMSCs) at a density of 2×10 6 cells / mL in the P(SBMA-co-CD) precursor solution, and quickly mix it with the HA-Ada precursor solution in TCPS to form a hydrogel encapsulating UCMSCs. Then, add α-MEM complete medium to each well to submerge the hydrogel, and culture it routinely at 37 °C and 5% CO2. Replace the fresh medium every 24 h, and culture for a total of 10 d. Perform AO / PI staining on the cells in the hydrogel at 1, 4, 7, and 10 d respectively, observe the growth state and the number of live and dead cells of UCMSCs in the hydrogel, and detect the proliferation of UCMSCs in the hydrogel by the AlamarBlue kit.

[0075] Example 16: Release and recovery of stem cells in the hydrogel

[0076] After culturing ADSCs in the hydrogel for 10 days according to the method in Example 13, the hydrogel was taken out of the well plate and placed into a pre-prepared 2 mM sodium adamantane carboxylate (sodium adamantane methanoate) solution. The hydrogel was gently blown with a pipette to float in the solution and left standing for 30 min. After that, the suspension was filtered through a cell strainer. The cells were recovered by centrifugation and the recovered cells were resuspended in complete medium. The activity of the recovered ADSCs was observed and evaluated by AM / PI staining and flow cytometry experiments.

[0077] Example 17: Release and recovery of stem cells in hydrogel

[0078] After culturing ADSCs in the hydrogel for 10 days according to the method in Example 13, the hydrogel was taken out of the well plate and placed into a pre-prepared 5 mM sodium adamantane carboxylate (sodium adamantane acetate) solution. The hydrogel was gently blown with a pipette to float in the solution and left standing for 45 min. After that, the suspension was filtered through a cell strainer. The cells were recovered by centrifugation and the recovered cells were resuspended in complete medium. The activity of the recovered ADSCs was observed and evaluated by AM / PI staining and flow cytometry experiments.

[0079] Example 18: Release and recovery of stem cells in hydrogel

[0080] After culturing ADSCs in the hydrogel for 10 days according to the method in Example 13, the hydrogel was taken out of the well plate and placed into a pre-prepared 10 mM sodium adamantane carboxylate (sodium adamantane propionate) solution. The hydrogel was gently blown with a pipette to float in the solution and left standing for 60 min. After that, the suspension was filtered through a cell strainer. The cells were recovered by centrifugation and the recovered cells were resuspended in complete medium. The activity of the recovered ADSCs was observed and evaluated by AM / PI staining and flow cytometry experiments.

[0081] Example 19: Detection of surface markers of ADSCs after amplification in hydrogel

[0082] After culturing ADSCs in the hydrogel for 14 days according to the method in Example 13, the ADSCs were recovered according to the method in Example 17, and the surface protein markers (CD29, CD31, CD44, CD45 and CD90) of ADSCs were detected by immunofluorescence staining and flow cytometry.

[0083] Comparative Example 1: Preparation of a hydrogel not containing zwitterionic moiety

[0084] Cyclodextrin-grafted hyaluronic acid (HA-CD) was prepared with reference to the existing synthesis method (DOI: 10.1038 / nprot.2017.053). 0.10 g of HA-CD was added to 2 mL of phosphate buffer solution, and at the same time, 0.10 g of HA-Ada synthesized in Example 8 was added to 2 mL of phosphate buffer solution. After the two solutions were stirred until dissolved respectively, equal volumes were taken and mixed evenly at room temperature to form a zwitterion-free hydrogel (denoted as HC). Subsequent cell experiments were all operated according to Examples 13 - 19.

[0085] Experimental Example 1: Structural Characterization of P(SBMA-co-CD) Prepared in Example 5

[0086] As Figure 1 shown in A and B, in the FTIR spectrum of the synthesized P(SBMA-co-CD), the stretching vibration characteristic peak of the C-OH group is at 1079 cm -1 ; the in-plane bending vibration peak of the methylene group is at 1558 cm -1 , both belonging to the glycoside structure of cyclodextrin. Combining with the FTIR spectrum of pure PSBMA, it can be shown that the synthesized P(SBMA-co-CD) indeed contains β-CD units. At the same time, the characteristic peak a (4.85 ppm, H-1) belonging to cyclodextrin also appears in the 1 H NMR spectrum of P(SBMA-co-CD). By calculating the integral area of characteristic peak a and the integral area of characteristic peak 5 (3.32 ppm) belonging to SBMA, it can be known that the proportion of β-CD units on the synthesized P(SBMA-co-CD) is about 10%.

[0087] Experimental Example 2: Preparation and Formation of HSC Hydrogel

[0088] As Figure 2 shown in A, according to what was described in Example 12, P(SBMA-co-CD) and HA-Ada were respectively dissolved in PBS, and after equal-volume mixing and continuous stirring for a while, a zwitterionic hydrogel based on host-guest interaction could be formed. The reason for its formation is that the guest unit Ada on HA-Ada enters the inner cavity of the host unit β-CD on P(SBMA-co-CD) and binds under hydrophobic interaction, thus forming cross-linking sites. As Figure 2 shown in B, it was also found that when P(SBMA-co-CD) was fully mixed with a certain concentration of sodium adamantane carboxylate in advance and then mixed with the HA-Ada precursor solution, no hydrogel could be formed. This shows that the presence of competitive molecules will hinder the formation of host-guest interaction and thus prevent cross-linking.

[0089] Experimental Example 3: The hydrogels prepared in Examples 10 - 12 and Comparative Example 1 were subjected to rheological testing, swelling behavior testing, and observation of the surface morphology after freeze-drying.

[0090] (a) 600 μL of the prepared hydrogel was placed on the sample stage, maintaining a strain of 1% and a temperature of 37 °C, and an oscillatory frequency sweep was performed in the scanning frequency range of 0.1 - 100 Hz. The results are as Figure 3 shown in A. The hydrogel showed a certain frequency-dependent enhancement. The storage modulus (G') and loss modulus (G”) increased with the increase of the scanning frequency and showed an intersection at about 10 Hz. After the intersection, G' was greater than G”. Maintaining a strain of 1% and a temperature of 37 °C, a time sweep was performed on the hydrogel at a frequency of 10 Hz. The results are as Figure 3 shown in B. The HSC hydrogel could maintain a stable gel state, and with the increase of the content of P(SBMA-co-CD) in the hydrogel, the G' of the HSC hydrogel increased from 344 Pa to 683 Pa. This is because the increase in the content of P(SBMA-co-CD) brought more cross-linking sites to the system.

[0091] (b) The swelling performance of the hydrogel was tested by the weighing method for a total of 14 days, and the equilibrium swelling ratio was taken as the swelling ratio value when the hydrogel reached swelling equilibrium. The results are as Figure 4 shown. The HSC 20 hydrogel and the HSC 25 hydrogel would undergo a certain degree of degradation after about 7 - 9 days, while the HSC 30 hydrogel and the HC hydrogel showed excellent stability within 14 days. The HSC 30 hydrogel also showed the lowest ESR, only 169%, indicating that with the increase of the polymer solid content, the swelling degree of the hydrogel gradually decreased.

[0092] (c) The pore structure of the hydrogel in the dry state was observed by scanning electron microscopy. As Figure 5 shown, the increase in the content of P(SBMA-co-CD) led to a decrease in the pore size structure of the hydrogel. The pore size of the HSC 20 hydrogel was about 94 μm, while the pore size of the HSC 30 hydrogel was about 66 μm. The existence of the pore size can ensure that the hydrogel exhibits better water molecule transport and diffusion ability.

[0093] Experimental Example 4: Specific Degradation of Hydrogel

[0094] For the HSC prepared in Example 12 30The degradation process was tested according to the hydrogel degradation method in Examples 16 - 18. The prepared hydrogel was placed in a circular petri dish with a diameter of 60 mm, and PBS solution or Ada - COONa solutions with different concentrations, which were 10 times the volume of the hydrogel, were added. The degradation of the hydrogel was observed, and it was weighed and photographed. As Figure 6 shown, the HSC 30 hydrogel did not degrade significantly in the PBS solution and could exist stably after 80 min. In the Ada - COONa solution with a lower concentration, the hydrogel would first swell to a certain extent and then gradually show a significant decrease in volume; in the Ada - COONa solution with a concentration of 10 mM, there was no obvious swelling phenomenon. This is because at a lower concentration, the destruction process of the hydrogel network occurs relatively slowly, and at first, some external solution would re - enter the hydrogel interior, resulting in a slight increase in volume; but at a higher concentration, the destruction of the network occurs rapidly, and the hydrogel degrades quickly, so the swelling phenomenon is not obvious. As Figure 7 shown, in the Ada - COONa solution with a concentration of 2 mM, the mass of the hydrogel was still 16.2% after 80 min, and it took 92 min for the hydrogel to degrade completely. As the solution concentration increased, the time required for the complete degradation of the hydrogel was shortened to 63 min (5 mM) and 51 min (10 mM). This shows that under the action of the competitive molecule Ada - COONa, the host - guest interaction in the hydrogel was indeed destroyed, leading to the degradation of the gel.

[0095] Experimental Example 5: Protein anti - adhesion experiment of hydrogel

[0096] The hydrogels prepared in Examples 10 - 12 and Comparative Example 1 were tested for protein anti - adhesion. Fluorescein isothiocyanate - labeled bovine serum albumin (FITC - BSA) was used for qualitative testing. 0.5 mL of the hydrogel was prepared in a standard 48 - well polystyrene tissue culture plate (TCPS). After swelling to equilibrium, it was immersed in a 1 mg / mL FITC - BSA solution. After incubating in a constant - temperature shaker at 37 °C for 2 h, the hydrogel was washed 3 times with PBS buffer. Then, the fluorescence intensity on the surface of the gel was observed. As Figure 8 shown in A, it was observed that there was obvious green fluorescence on the surface of the TCPS, indicating that a large amount of protein adhered to the hydrophobic TCPS surface; while no protein adhesion was observed on the surface of the HSC 30 surface.

[0097] The anti - protein adhesion performance of the hydrogel was quantitatively characterized using a BCA kit. Similarly, 0.5 mL of the hydrogel was immersed in 1 mg / mL solutions of BSA (bovine serum albumin), Ly (lysozyme), Fib (fibrinogen), and CollⅠ(type I collagen). After incubation in a shaker at 37 °C for 2 h, the protein solution was aspirated, and the hydrogel was washed 3 times with PBS. 500 μL of 1 wt% sodium dodecyl sulfate (SDS) solution was added, and after soaking for 1 h, 100 μL was aspirated and placed into a new well plate, and 50 μL of BCA reagent solution was added. Incubate in the dark in a shaker at 37 °C for 30 min, and measure the absorbance at 562 nm using an enzyme - linked immunosorbent assay (ELISA) reader and compare. As Figure 8 shown in B, different hydrogels all reduced the amount of protein adhesion to a certain extent. The adhesion performance of the HSC hydrogel to the four model proteins was basically less than 20%, and as the content of zwitterions increased, the anti - adhesion performance of the hydrogel to proteins gradually increased. HSC 30 The relative adhesion amounts of the HSC hydrogel to the four proteins BSA, Ly, Fib, and Coll I were only 8.52%, 12.58%, 16.42%, and 15.52% respectively.

[0098] Experimental Example 6: Proliferation activity test of ADSCs in the hydrogel

[0099] The proliferation activity of ADSCs in the hydrogel was tested by immunofluorescence staining according to the method in Example 13. As Figure 9 shown in A, ADSCs could proliferate continuously in the hydrogel, and no cell death was observed. And as the number of culture days increased, ADSCs maintained good proliferation activity, and it could be clearly seen that the cells existed in a spherical state and proliferated in the hydrogel.

[0100] Quantitative detection was carried out using the Alamar Blue reagent. The reduction rate of each group was calculated according to the following formula, and the proliferation multiple was converted to illustrate the proliferation behavior of ADSCs in the hydrogel:

[0101]

[0102] E570 and E600 are the extinction coefficients of oxidized Alamar blue at wavelengths of 570 nm and 600 nm respectively (E570 = 80568 and E600 = 117612); E570' and E600' are the extinction coefficients of reduced Alamar blue at wavelengths of 570 nm and 600 nm respectively (E570' = 155677 and E600' = 14652); A570 and A600 are the absorbance values of the sample group at wavelengths of 570 nm and 600 nm, and C570 and C600 are the absorbance values of the control group at wavelengths of 570 nm and 600 nm.

[0103] As shown Figure 9 in B and C, after 10 days of culture, the HSC 30 alarm blue reduction rate in the hydrogel can reach 73%, and the amplification multiple of ADSCs also reaches 8.8 times that of the initial state. In the first 4 days, there was no significant difference in the proliferation activity of ADSCs in the hydrogels of each group. But after 4 days, the HSC 20 proliferation activity of ADSCs in the hydrogel was significantly lower than that of the other hydrogels. After 10 days, the alarm blue reduction rate was only 61%, and the amplification multiple was only 5.9 times. This may be due to the HSC 20 network of the hydrogel being unable to maintain a stable structure during a long culture time. As the culture time increased, part of the hydrogel degraded, resulting in a certain number of cell losses. Due to the HSC 30 hydrogel having better stability, ADSCs showed better amplification effects than in the other two groups of HSC hydrogels.

[0104] Experimental Example 7: Release and recovery of ADSCs in hydrogel

[0105] Cell release and recovery experiments were carried out according to Examples 16 - 18, and the activity of the recovered ADSCs was observed and evaluated by AM / PI staining and flow cytometry experiments. The results are as Figure 10 shown in A. Using the competitive monomer Ad - COOH for the stimulating degradation of the hydrogel, the recovered cells showed good cell activity, and only part of the cells died. And it showed that with the increase of the degradation solution concentration and the extension of the degradation time, the cell survival rate of the recovered ADSCs gradually decreased. As Figure 10 shown in B and D, the experimental results showed that under most conditions, the survival rate of ADSCs remained above 75%. When using a 2 mM sodium adamantane carboxylate solution for degradation, the cell survival rate of the recovered ADSCs could reach about 90%. At 30 min of degradation, the cell survival rate was 93.4%. Even when the degradation time was extended to 60 min, the cell survival rate was as high as 86.7%. This shows that a low - concentration sodium adamantane carboxylate solution does not have obvious cytotoxicity to cells. When using a 5 mM sodium adamantane carboxylate solution for the experiment, when the degradation time was extended to 60 min, the survival rate of the recovered cells decreased to 77.9%. This shows that when using a 5 mM sodium adamantane carboxylate solution for degradation, too long a degradation time will cause obvious cell damage. When the salt solution concentration was increased to 10 mM, the number of dead cells in the recovered ADSCs became more, and the cell survival rate was relatively low at this time, with a minimum of only 57.3%.

[0106] With the prolongation of the degradation time, the cell recovery rate gradually increases, which is accompanied by the improvement of the degradation degree of the hydrogel. When the concentration of the Ad-COOH solution increases, the cell recovery rate of ADSCs also shows an increasing trend ( Figure 10 in C). Especially when the concentration of the degradation solution increases from 2 mM to 5 mM, the cell recovery rate will increase significantly. For example, when degraded for 45 min, the cell recovery rate will increase from 28.7% to 53.2%, almost doubling. This is because at 2 mM, the HSC 30 hydrogel degrades slowly, the cell release ability is not strong, and the cell recovery rate after 60 min of degradation is only 36.9%. At a concentration of 10 mM of sodium adamantane carboxylate solution, the cell recovery rate reaches 48.3% at 30 min and as high as 68.3% at 60 min.

[0107] In summary, using the HSC 30 hydrogel in Example 12 of the present invention to recover the encapsulated ADSCs can ensure a high cell recovery rate while maintaining good cell viability of the recovered cells.

[0108] Experimental Example 8: Detection of surface markers of ADSCs after amplification in hydrogel

[0109] The surface markers of ADSCs in the hydrogel were detected by immunofluorescence staining and flow cytometry according to the method in Example 19. The results are as shown in Figure 11 A and B. The cell membranes of ADSCs recovered from the HSC 30 hydrogel still highly express three marker proteins, CD29 (92.3%), CD44 (99.6%) and CD90 (94.4%). The expression levels are basically the same as those on primary ADSCs, all maintaining at a very high level, and there is no statistical difference; CD31 (0.40%) and CD45 (0.53%) are basically not expressed, which is also consistent with primary ADSCs. For ADSCs recovered from the HC hydrogel, the expression levels of CD29, CD44 and CD90 are significantly lower, while the expression levels of CD31 and CD45 are slightly increased. Statistical analysis also shows that the expression levels of the markers are significantly different from those of primary stem cells and ADSCs recovered from the HSC 30 hydrogel. This shows that the zwitterionic hydrogel HSC 30 in Example 12 of the present invention can well maintain the cell phenotype expression of ADSCs during the culture process without obvious loss of stemness.

[0110] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a degradable hydrogel based on dynamic cross-linking, characterized in that: The method comprises the steps of reacting a cyclodextrin derivative with a zwitterionic monomer to form a copolymer, and then uniformly mixing the copolymer with adamantane-modified hyaluronic acid at room temperature to prepare a degradable hydrogel based on dynamic cross-linking.

2. The preparation method according to claim 1, characterized in that The preparation method comprises the following steps: (1) Mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin is dissolved in N,N-dimethylformamide, ethyl isocyanate acrylate is added, and the mixture is reacted under a nitrogen atmosphere and an ice bath. After the reaction is completed, acetone is added dropwise to precipitate the mixture, and the mixture is dried in a vacuum to obtain ethyl isocyanate acrylate-modified β-cyclodextrin; (2) dissolving the isocyanate ethyl acrylate modified β-cyclodextrin and zwitterion monomer in dimethyl sulfoxide, heating in a nitrogen atmosphere, then adding azobisisobutyronitrile and stirring for reaction, adding methanol dropwise for precipitation after the reaction, dialyzing, and freeze-drying to obtain a zwitterion-cyclodextrin copolymer; (3) The zwitterion-cyclodextrin copolymer and the adamantane-modified hyaluronic acid are respectively dissolved in a phosphate buffer solution, and then mixed evenly in equal volumes to prepare a degradable hydrogel based on dynamic cross-linking.

3. The preparation method according to claim 2, characterized in that: In step (1), the mass volume ratio of mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin to N,N-dimethylformamide is 1 g:(5-10) mL; and / or the mass volume ratio of mono-(6-ethylenediamino-6-deoxy)-β-cyclodextrin to isocyanate ethyl acrylate is 3 g:(250-600) μL; and / or the ice bath reaction time is 10-60 min.

4. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of the isocyanate ethyl acrylate modified β-cyclodextrin, the zwitterionic monomer and azobisisobutyronitrile is (1-3) g: (1-2.5) g: (50-100) mg; and / or the mixture is heated to 60-80° C., and the azobisisobutyronitrile is added and stirred for reaction for 12-48 hours; and / or the zwitterionic monomer comprises a sulfonic acid betaine zwitterionic monomer, a carboxylic acid betaine zwitterionic monomer or a phosphorylcholine zwitterionic monomer.

5. The preparation method according to claim 2, characterized in that: In step (3), the concentration of the zwitterion-cyclodextrin copolymer dissolved in the phosphate buffer solution is 20-30% w / v, and the concentration of the adamantane-modified hyaluronic acid dissolved in the phosphate buffer solution is 5-8% w / v.

6. The preparation method according to claim 2, characterized in that: In step (3), the method for preparing adamantane-modified hyaluronic acid comprises the following steps: Tetrabutylammonium hyaluronic acid, carboxyl-containing adamantane and 4-dimethylaminopyridine are added to DMSO, stirred under a nitrogen atmosphere until completely dissolved, and di-tert-butyl dicarbonate is added. The mixture is reacted at room temperature for 12-36 hours. After the reaction is completed, the mixture is dialyzed and freeze-dried to obtain adamantane-modified hyaluronic acid; wherein the mass volume ratio of the tetrabutylammonium hyaluronic acid, carboxyl-containing adamantane, 4-dimethylaminopyridine and di-tert-butyl dicarbonate is (0.5-2) g:(0.5-1) g:(0.05-0.25) g:(100-300) μL, and the carboxyl-containing adamantane includes adamantane carboxylic acid, adamantane acetic acid or adamantane propionic acid.

7. Use of the degradable hydrogel prepared by the preparation method according to any one of claims 1 to 6 in the in vitro expansion and recovery of mesenchymal stem cells.

8. The use according to claim 7, characterized in that The method for in vitro expansion of mesenchymal stem cells comprises: resuspending the zwitterion-cyclodextrin copolymer in α-MEM basal medium, then adding 5-10 mg / mL collagen, filtering, and obtaining a zwitterion-cyclodextrin copolymer precursor solution; resuspending the adamantane-modified hyaluronic acid in α-MEM basal culture medium to obtain an adamantane-modified hyaluronic acid precursor solution; resuspending mesenchymal stem cells in the zwitterion-cyclodextrin copolymer precursor solution and mixing with the adamantane-modified hyaluronic acid precursor solution to form a hydrogel encapsulating the mesenchymal stem cells, and then immersing the hydrogel encapsulating the mesenchymal stem cells in α-MEM basal culture medium for culturing; The in vitro recovery method of mesenchymal stem cells comprises: transferring the hydrogel encapsulating the mesenchymal stem cells into a 2-10 mM sodium adamantane carboxylate solution for degradation for 30-60 minutes, and after the degradation, filtering and centrifuging to collect the cells.

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