Preparation and application of dual-network hydrogel

Abstract

The invention discloses preparation and application of dual-network hydrogel. The dual-network hydrogel comprises the following components: (Z1) a spinal cord acellular matrix network DSM formed by self-assembly of a spinal cord acellular matrix dSECM; and (Z2) a metal-polyphenol network MPN formed by coordination of metal ions and polyphenols; wherein the DSM and the MPN are cross-linked through hydrogen bonds to jointly form a double-network structure. DSM provides a microenvironment suitable for cell survival and growth for the dual-network hydrogel and induces stem cells to differentiate into neurons, and MPN provides the dual-network hydrogel with biological effects of inhibiting inflammation and accumulation of reactive oxygen species (ROS). The dual-network hydrogel has the advantages of the two components, the active component neurotrophic factor and the inhibitor loaded on the dual-network hydrogel can regulate and control immune response after SCI, improve neuroinflammation microenvironment and promote nerve repair, and a new strategy is provided for in-situ tissue repair of SCI.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials and tissue engineering, in particular to preparation and application of a network hydrogel. BACKGROUND

[0002] Spinal cord injury (SCI) is a serious global disease, and its incidence is increasing year by year and often leads to loss of sensation and motor function below the injury plane. The pathogenesis of SCI includes primary injury and secondary injury. After primary injury, the local microenvironment homeostasis is unbalanced, a series of cascade biochemical reactions are activated, and tissue secondary injury is induced, including ischemia, ion imbalance, lipid peroxidation, glial scar formation and neural inflammation. Current clinical interventions can only delay the progression of secondary injury through surgical debridement and drug measures, but cannot reconstruct the neural pathway to promote tissue repair. In recent years, with the development of regenerative medicine technology, biocompatible material implantation strategies have provided a new direction for SCI repair. The core challenge is to develop a multi-dimensional repair interface that combines component structure simulation and construction of a pro-regenerative microenvironment.

[0003] Spinal cord decellularized matrix (dSECM) is a natural material prepared from spinal cord tissue by removing cellular components and retaining specific ECM active components of the spinal cord, which can provide a suitable microenvironment for cell proliferation, differentiation and growth. It has been widely used in tissue regeneration therapy. After processing, dSECM can be further assembled into a decellularized matrix network (DSM) hydrogel, which provides a suitable microenvironment for endogenous stem / progenitor cell recruitment and axon regeneration due to its high porosity. However, DSM has significant limitations such as weak mechanical strength, unstable gel formation, and lack of dynamic response capability, which makes it unsuitable for implantation alone to treat the complex injury microenvironment after SCI.

[0004] Metal-phenolic networks (MPN) is a supramolecular amorphous network formed by the mutual connection of polyphenols and metal ions, which has specific functions of metal ions and multiple advantages of polyphenols such as low cost, efficient free radical scavenging capacity, inflammation regulation function, and weak acid response to SCI pathology. MPN can exert the synergistic effect of metal and polyphenol and has a more stable structure. Previous studies have shown that MPN can play a neuroprotective role through good anti-inflammatory and antioxidant biological effects, and has been used in the fields of pharmaceuticals and tissue engineering in recent years. However, there are few studies on the double network structure of metal-phenolic network and decellularized matrix network.

[0005] Therefore, there is an urgent need in the art to provide a material with large mechanical strength, good stability, excellent dynamic response capability, and structure simulation and construction of a pro-regenerative microenvironment. SUMMARY

[0006] The present application aims to provide a material with structural bionics and promoting regenerative microenvironment construction, which has great mechanical strength, good stability and excellent dynamic response capability, and particularly relates to a preparation and application of a double-network hydrogel.

[0007] In a first aspect of the present application, a double-network hydrogel is provided, which comprises the following components: (Z1) a spinal cord decellularized matrix network DSM formed by self-assembly of a spinal cord decellularized matrix dSECM; and (Z2) a metal-polyphenol network MPN formed by coordination of metal ions and polyphenols, and (Z3) an optional loaded active ingredient; wherein the DSM and the MPN jointly constitute a double-network structure through hydrogen bond crosslinking.

[0008] In another preferred embodiment, the spinal cord decellularized matrix dSECM is derived from at least one of a pig spinal cord and a mouse spinal cord.

[0009] In another preferred embodiment, the concentration of the spinal cord decellularized matrix dSECM in the decellularized matrix network DSM is 10-30 mg / mL, preferably 10-25 mg / mL.

[0010] In another preferred embodiment, the metal ions are selected from trivalent or tetravalent metal ions, preferably the metal ions include at least one of Ce 3+ / Ce 4+ , Ti 4+ , Fe 3+ , Zr 4+ .

[0011] In another preferred embodiment, the polyphenols are selected from at least one of tannic acid TA, epigallocatechin gallate EGCG and dopamine DA.

[0012] In another preferred embodiment, the molar ratio of the metal ions to the polyphenols is 1:1-1:5.

[0013] In another preferred embodiment, the pore size of the double-network hydrogel is in the range of 30-100 μm, preferably 40-97 μm.

[0014] In another preferred embodiment, the compression modulus of the double-network hydrogel is 1.0-5.0 kPa, preferably 2.0-4.0 kPa.

[0015] In another preferred embodiment, the DNA content in the spinal cord decellularized matrix network DSM is <50 ng / mg.

[0016] In a second aspect of the present application, a preparation method of the double-network hydrogel of the first aspect is provided, which comprises the following steps:

[0017] (1) providing a spinal cord acellular matrix dSECM for pretreatment to prepare a foamed spinal cord acellular matrix dSECM;

[0018] (2) providing the foamed spinal cord acellular matrix dSECM of step (1) for treatment to prepare a spinal cord acellular matrix dSECM solution;

[0019] (3) providing a metal ion solution and a neutral polyphenol solution for mixing to prepare a metal-polyphenol network MPN solution;

[0020] (4) providing the prepared spinal cord acellular matrix dSECM solution and the metal-polyphenol network MPN solution for mixing to obtain a hydrogel pre-polymerization solution;

[0021] (5) heating the pre-polymerization solution obtained in step (4) for reaction to prepare a DSM-MPN double network hydrogel.

[0022] In another preferred embodiment, the preparation method of the spinal cord acellular matrix dSECM in step (1) comprises the following steps:

[0023] a) freeze-thaw cycle: freeze the spinal cord tissue in a -80℃ refrigerator and then melt to room temperature, repeat the freezing and melting for 3-5 times;

[0024] b) chemical enzymatic hydrolysis under 37℃ oscillation for three cycles to obtain the acellular matrix:

[0025] First cycle: ultrapure water treatment for 5-10h, 3v / v% Triton X-100 treatment for 5-7h, ultrapure water treatment for 20min / time×3 times, 4w / v% sodium deoxycholate SDC treatment for 9-12h, ultrapure water treatment for 20min / time×3 times, DNA enzyme treatment for 4-6h;

[0026] Second cycle: ultrapure water treatment for 20min / time×3 times, 3v / v% Triton X-100 treatment for 5-7h, ultrapure water treatment for 20min / time×3 times, 4w / v% sodium deoxycholate SDC treatment for 9-12h, ultrapure water treatment for 20min / time×3 times, DNA enzyme treatment for 4-6h;

[0027] Third cycle: ultrapure water treatment for 20min / time×3 times, 3v / v% Triton X-100 treatment for 5-7h, ultrapure water treatment for 20min / time×3 times, 4w / v% sodium deoxycholate SDC treatment for 9-12h, ultrapure water treatment for 20min / time×3 times, DNA enzyme treatment for 4-6h, ultrapure water treatment for 20min / time×3 times, 4v / v% ethanol treatment for 1-4h, ultrapure water treatment for 20min / time×3 times; to obtain the acellular matrix dSECM;

[0028] c) the resulting decellularized matrix is pre-treated comprising the steps of:

[0029] i. the resulting decellularized matrix is ground in an acidic solution having a pH in the range of 1.5-2.5;

[0030] ii. 0.1-1 mg / mL pepsin is added to the ground solution of the decellularized matrix and incubated at room temperature for 12-16 h;

[0031] iii. the digested decellularized matrix solution is snap frozen in liquid nitrogen and dried to obtain a foamy dSECM.

[0032] In another preferred embodiment, the DNase treatment comprises the step of dissolving DNase in 1 M sodium chloride solution (containing 0.015 M magnesium chloride, 0.001 M calcium chloride) to obtain a DNase solution of 40-50 kU / mL for treating the spinal cord tissue.

[0033] In another preferred embodiment, the pre-treatment in step (1) comprises the steps of:

[0034] i. the resulting decellularized matrix dSECM is ground in an acidic solution having a pH in the range of 1.5-2.5 to obtain a ground solution containing dSECM;

[0035] ii. 0.1-1 mg / mL pepsin is added to the ground solution obtained in step (i) and incubated at room temperature for 12-16 h to obtain a digested decellularized matrix solution;

[0036] iii. the digested decellularized matrix solution obtained in step (ii) is snap frozen in liquid nitrogen and dried to obtain a foamy dSECM.

[0037] In another preferred embodiment, the pre-treatment in step (1) comprises the steps of:

[0038] i. the resulting decellularized matrix dSECM is ground in 0.5 M glacial acetic acid;

[0039] ii. 0.1-1 mg / mL pepsin is added to the ground solution of the decellularized matrix and incubated at room temperature for 12-16 h;

[0040] iii. the digested decellularized matrix solution is snap frozen in liquid nitrogen and dried to obtain a foamy dSECM.

[0041] In another preferred embodiment, the treatment in step (2) comprises the steps of:

[0042] the foamy dSECM is ground in 0.5 M glacial acetic acid at 0-4 °C to obtain a dSECM solution.

[0043] In another preferred embodiment, the concentration of the dSECM solution is 10-30 mg / mL, preferably 10-25 mg / mL.

[0044] In another preferred embodiment, the concentration of the polyphenol solution is 1-5 w / v%; the molar ratio of the metal ion to the polyphenol is 1:1-1:5.

[0045] In another preferred embodiment, the content of the MPN solution in the hydrogel pre-polymerization solution is 1-20 v / v%, preferably 2-15 v / v%, most preferably 2-10 v / v%.

[0046] In another preferred embodiment, the content of the dSECM solution in the hydrogel pre-polymerization solution is 70-95 v / v%, preferably 80-90 v / v%.

[0047] In another preferred embodiment, the method for preparing the double network hydrogel comprises the steps of:

[0048] i. dissolving the foamy spinal cord acellular matrix dSECM in an acidic solution with a pH in the range of 1.5-2.5 at 0-4°C to obtain a dSECM solution;

[0049] ii. adjusting the pH of the polyphenol solution to neutral, mixing the metal ion solution and the neutral polyphenol solution to obtain an MPN solution;

[0050] iii. mixing the MPN solution and the dSECM solution at 0-4°C to obtain a hydrogel pre-polymerization solution;

[0051] iv. heating the pre-polymerization solution obtained in step (iii) at 35-40°C for 5-10 min to obtain the DSM-MPN double network hydrogel.

[0052] In another preferred embodiment, the dSECM solution in step i) is prepared by the following steps:

[0053] dissolving the foamy dSECM in 0.01 M hydrochloric acid at 0-4°C to obtain a dSECM solution.

[0054] In another preferred embodiment, the dSECM solution in step i) is prepared by the following steps:

[0055] grinding the foamy dSECM in 0.5 M glacial acetic acid at 0-4°C to obtain a dSECM solution.

[0056] In another preferred embodiment, the MPN solution in step ii) is prepared by the following steps:

[0057] Dissolving tannic acid TA in ultrapure water to obtain a TA solution of 1-5 w / v%, adjusting the pH of the TA solution to neutral with 1M sodium hydroxide solution, dissolving ceric sulfate Ce(S04)2*4H20 in ultrapure water to obtain a Ce ion solution, mixing the Ce ion solution and the neutral TA solution to obtain an MPN solution, wherein the molar ratio of the Ce ion to the TA is 1:1-1:5, preferably 1:2.

[0058] In another preferred embodiment, the MPN solution in step ii) is prepared by the following steps:

[0059] Dissolving tannic acid TA in ultrapure water to obtain a TA solution of 1-5 w / v%, adjusting the pH of the TA solution to neutral with 1M sodium hydroxide solution, dissolving ceric sulfate Ce(S04)2*4H20 in ultrapure water to obtain a Ce ion solution, mixing the Ce ion solution and the neutral TA solution to obtain an MPN solution, wherein the molar ratio of the Ce ion to the TA is 1:1-1:5, preferably 1:2. 4+ adding 20 v / v% dimethyl sulfoxide DMSO solution to the solution to obtain a Ti 4+ precursor solution, mixing the Ti 4+ precursor solution and the neutral TA solution to obtain an MPN solution, wherein the molar ratio of the Ti 4+ to the TA is 1:1-1:5.

[0060] In another preferred embodiment, the hydrogel pre-polymer solution in step iii) is prepared by the following steps:

[0061] mixing the MPN solution and the dSECM solution at 0-4°C, adjusting the pH to the range of 7.2-7.8 with 10M sodium hydroxide solution, adding 10 v / v% 10x PBS solution to the mixed solution to obtain a hydrogel pre-polymer solution; preferably, the pH value is 7.4.

[0062] In a third aspect of the present application, a double network hydrogel composite is provided, the double network hydrogel composite comprising:

[0063] (Y1) the double network hydrogel of the first aspect and the second aspect; and

[0064] (Y2) an active component;

[0065] wherein the active component comprises at least one of a neurotrophic factor, an inhibitor.

[0066] In another preferred embodiment, the neurotrophic factor is selected from the group consisting of NGF, BDNF, GDNF, NT-3, NT-4, CNTF, or a combination thereof.

[0067] In another preferred embodiment, the inhibitor is selected from the group consisting of MMP408, edaravone, fasudil mesylate, or a combination thereof.

[0068] In another preferred embodiment, the double network hydrogel composite has one or more features selected from the group consisting of:

[0069] (a) the pore size of the composite ranges from 30 to 100 μm, preferably from 40 to 95 μm;

[0070] (b) the compression modulus of the composite ranges from 1.0 to 5.0 kPa, preferably from 2.0 to 4.0 kPa;

[0071] (c) the composite reduces the DPPH concentration by 80% to 90% within 48 h, having good antioxidant properties;

[0072] (d) the composite increases the relative cell viability by 10% to 20%;

[0073] (e) the composite reduces the content of pro-inflammatory factor IL-6 and increases the content of anti-inflammatory factor IL-4, and has an inhibitory effect on the inflammatory response after SCI.

[0074] In another preferred embodiment, the composite reduces the pro-inflammatory factor IL-6 by 50% to 60% compared to the SCI group.

[0075] In another preferred embodiment, the composite increases the anti-inflammatory factor IL-4 by 30% to 40% compared to the SCI group.

[0076] In another preferred embodiment, the preparation method of the double network hydrogel composite comprises the following steps:

[0077] (1) providing a spinal cord decellularized matrix dSECM solution;

[0078] (2) providing a metal-polyphenol network MPN solution;

[0079] (3) providing an active ingredient solution;

[0080] (4) mixing the dSECM solution, the MPN solution, and the active ingredient to obtain a hydrogel pre-polymerization solution;

[0081] (5) heating the pre-polymerization solution obtained in step (4) at 35-40°C for 5-15 min to obtain the double network hydrogel composite.

[0082] In another preferred embodiment, the preparation method of the double network hydrogel composite comprises the following steps:

[0083] i. dissolving the foamed dSECM in an acidic solution with a pH ranging from 1.5 to 2.5 at 0-4°C to obtain a dSECM solution;

[0084] ii. Adjusting the pH of the polyphenol solution to neutral, mixing the metal ion solution and the neutral polyphenol solution to obtain an MPN solution;

[0085] iii. Mixing the MPN solution, the dSECM solution and the active component at 0-4℃ to obtain a hydrogel pre-polymerization solution;

[0086] iv. Heating the pre-polymerization solution at 35-40℃ for 5-10 min to obtain the double network hydrogel loaded with the active component.

[0087] In another preferred embodiment, the preparation method of the dSECM solution in step i) comprises the following steps:

[0088] At 0-4℃, the foamy dSECM is ground in 0.5M glacial acetic acid to obtain a dSECM solution.

[0089] In another preferred embodiment, the preparation method of the hydrogel pre-polymerization solution in step iii) comprises the following steps:

[0090] (1) Dissolving the MMP408 inhibitor in a DMSO solution to obtain a 1w / v% MMP408 solution; and

[0091] (2) Mixing the MPN solution, the dSECM solution and the MMP408 solution at 0-4℃ to obtain a hydrogel pre-polymerization solution; wherein the content of the MMP408 solution is 1-3v / v%.

[0092] In the fourth aspect of the present application, the double network hydrogel of the first aspect or the double network hydrogel complex of the third aspect is used for preparing a preparation for treating spinal cord injury.

[0093] In another preferred embodiment, the DSM in the double network hydrogel is used for providing a microenvironment suitable for cell survival and growth and inducing stem cells to differentiate into neurons, and the MPN is used for inhibiting inflammation and ROS accumulation.

[0094] In another preferred embodiment, the double network hydrogel is used for loading active components neurotrophic factors and inhibitors to regulate immune response after SCI, improve the inflammatory microenvironment of nerves and promote nerve repair.

[0095] Compared with the prior art, the present application has the following beneficial effects:

[0096] (1) The present application constructs a double network hydrogel crosslinked by DSM and MPN, which has cell adhesion, temperature sensitivity, biocompatibility and a natural extracellular matrix microenvironment, and the pore size and mechanical strength of the material are controllable, so that it is a good tissue engineering biomaterial.

[0097] (2) The double network hydrogel for SCI nerve repair of the present application has the biocompatibility of natural materials, the mechanical properties of synthetic materials and the porous network structure suitable for cell growth, can induce stem cells to differentiate into neurons, and has obvious repair effect on tissue repair after loading active components neurotrophic factors and inhibitors.

[0098] (3) The double network hydrogel of the present application regulates the immune response in the microenvironment of the spinal cord after SCI, inhibits inflammation and active oxygen ROS accumulation, and provides a favorable microenvironment for nerve regeneration and tissue repair. The present application provides a new strategy for in situ tissue repair of SCI.

[0099] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described below (such as the examples) can be combined with each other to form a new or preferred technical scheme. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Due to the limited space, they will not be listed one by one. BRIEF DESCRIPTION OF DRAWINGS

[0100] Figure 1 The figure shows the DNA content of normal spinal cord tissue and tissue under dSECM dry conditions. **** in the figure means t test between two experimental groups, p value less than 0.0001.

[0101] Figure 2 The figure shows the micro-morphology and pore size of the double network hydrogel DSM-MPN of the present application and the double network hydrogel MMP408@DSM-MPN loaded with inhibitor MMP408 after freeze-drying, wherein a is the scanning electron microscope graph of DSM-MPN hydrogel, b is the scanning electron microscope graph of MMP408@DSM-MPN hydrogel, c is the pore size of DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel, and ns in the figure means t test between two experimental groups, p value greater than 0.05, no significant statistical difference.

[0102] Figure 3 The figure shows the compression mechanical property characterization of the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel of the present application, wherein a is the stress-strain curve graph of the two experimental groups (strain range: 0-50%), b is the compression modulus comparison calculated based on the linear region of stress-strain (strain 20%) of the two experimental groups. In the figure, ns means t test between two experimental groups, p value greater than 0.05, no significant statistical difference.

[0103] Figure 4 The figure shows the hydrogel mass degradation graph of the DSM hydrogel and the DSM-MPN hydrogel of the present application within 11 days. The figure shows the hydrogel mass degradation graph of the DSM hydrogel and the DSM-MPN hydrogel of the present application within 11 days.

[0104] Figure 5 The figure shows the DPPH free radical scavenging activity of the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel within 48h. * means t-test between two experimental groups, p value less than 0.05; ** means t-test between two experimental groups, p value less than 0.01; **** means t-test between two experimental groups, p value less than 0.0001. All of the above are considered to have statistical difference between the two groups compared, the smaller the p value, the more significant the statistical difference.

[0105] Figure 6 The figure shows the relative cell viability of the DSM-MPN hydrogel extract and the MMP408@DSM-MPN hydrogel extract cultured cells for 5 days detected by CCK-8, and *** means t-test between two experimental groups, p value less than 0.001; **** means t-test between two experimental groups, p value less than 0.0001, all of the above are considered to have statistical difference between the two groups compared, the smaller the p value, the more significant the statistical difference.

[0106] Figure 7 The figure shows the spinal cord tissue morphology of SCI mice in different experimental groups after 10 weeks of treatment.

[0107] Figure 8 The figure shows the content of inflammatory factors in the spinal cord tissue of SCI mice in different experimental groups after 1 week of treatment, wherein a is the content of pro-inflammatory factor interleukin-6 (IL-6) in the spinal cord tissue of different experimental groups, and b is the content of anti-inflammatory factor interleukin-4 (IL-4) in the spinal cord tissue of different experimental groups. ** means t-test between two experimental groups, p value less than 0.01; *** means t-test between two experimental groups, p value less than 0.001; **** means t-test between two experimental groups, p value less than 0.0001. All of the above are considered to have statistical difference between the two groups compared, the smaller the p value, the more significant the statistical difference. DETAILED DESCRIPTION

[0108] The inventors of the present application have carried out extensive and in-depth research, and through a large number of experimental screening, for the first time developed a double network hydrogel constructed by using a spinal cord acellular matrix network DSM and a metal-polyphenol network MPN, the double network hydrogel has temperature sensitivity, biocompatibility and important components in a natural extracellular matrix, and the pore size and mechanical strength of the double network hydrogel obtained through specific component proportioning are controllable, and the double network hydrogel is a good tissue engineering biomaterial. In addition, after loading active components (such as neurotrophic factors, inhibitors) in the double network hydrogel, on the one hand, the composite has the advantages of good biocompatibility, excellent mechanical properties, a porous network structure suitable for cell growth and the like, can induce stem cells to differentiate into neurons, and is beneficial to tissue repair after SCI; on the other hand, the immune response of the composite in the microenvironment of the spinal cord after SCI can inhibit inflammation and accumulation of reactive oxygen species ROS, and provides a favorable microenvironment for nerve regeneration and tissue repair; therefore, the present application provides a new strategy for in situ tissue repair of SCI. On this basis, the inventors of the present application have completed the present application.

[0109] Terms

[0110] Abbreviation explanation

[0111] SCI: spinal cord injury

[0112] dSECM: spinal cord acellular matrix

[0113] DSM: acellular matrix network

[0114] MPN: metal-polyphenol network

[0115] Triton X-100: Triton X-100

[0116] SDC: sodium deoxycholate

[0117] NGF: nerve growth factor

[0118] GDNF: glial cell-derived neurotrophic factor

[0119] NT-3: neurotrophic factor-3

[0120] TA: tannic acid

[0121] DPPH: 2,2-diphenyl-1-picrylhydrazyl

[0122] IL-4: interleukin-4

[0123] IL-6: interleukin-6

[0124] Double network hydrogel of the present application

[0125] The application provides a kind of acellular matrix and metal polyphenol double network hydrogel, comprising the acellular matrix network DSM formed by spinal cord acellular matrix dSECM self-assembly, and the metal-polyphenol network MPN formed by metal ion and polyphenol coordination, and DSM and MPN are jointly constituted double network structure by hydrogen bond crosslinking.

[0126] The application also provides a preparation method of the double network hydrogel

[0127] In the application, the preparation method of the double network hydrogel comprises the following steps:

[0128] i. Dissolving the foamy dSECM in an acidic solution with pH in the range of 1.5-2.5 to obtain a dSECM solution at 0-4℃;

[0129] ii. Adjusting the pH of the polyphenol solution to neutral, mixing the metal ion solution and the neutral polyphenol solution to obtain an MPN solution;

[0130] iii. Mixing the MPN solution and the dSECM solution at 0-4℃ to obtain a hydrogel pre-polymerization solution;

[0131] iv. Heating the above pre-polymerization solution at 35-40℃ for 5-10 min to obtain the double network hydrogel.

[0132] The spinal cord acellular matrix dSECM is prepared by the following steps:

[0133] a) Freeze-thaw cycle: freezing the spinal cord tissue in a refrigerator at -80℃ and then thawing to room temperature, repeating the freezing and thawing for 3-5 times;

[0134] b) Chemical enzymatic hydrolysis under 37℃ oscillation for three cycles to obtain the acellular matrix:

[0135] First cycle: ultrapure water treatment for 5-10 h, 3v / v% Triton X-100 treatment for 5-7 h, ultrapure water treatment for 20 min / time×3 times, 4w / v% sodium deoxycholate SDC treatment for 9-12 h, ultrapure water treatment for 20 min / time×3 times, DNA enzyme treatment for 4-6 h;

[0136] Second cycle: ultrapure water treatment for 20 min / time×3 times, 3v / v% Triton X-100 treatment for 5-7 h, ultrapure water treatment for 20 min / time×3 times, 4w / v% sodium deoxycholate SDC treatment for 9-12 h, ultrapure water treatment for 20 min / time×3 times, DNA enzyme treatment for 4-6 h;

[0137] Third cycle: ultrapure water treatment 20 min / time x 3 times, 3v / v% Triton X-100 treatment 5-7h, ultrapure water treatment 20 min / time x 3 times, 4w / v% deoxycholate sodium SDC treatment 9-12h, ultrapure water treatment 20 min / time x 3 times, DNA enzyme treatment 4-6h, ultrapure water treatment 20 min / time x 3 times, 4v / v% ethanol treatment 1-4h, ultrapure water treatment 20 min / time x 3 times;

[0138] c) the obtained decellularized matrix pre-treatment comprises the following steps:

[0139] i. the obtained decellularized matrix is dissolved in an acidic solution with pH in the range of 1.5-2.5 and ground;

[0140] ii. 0.1-1mg / mL pepsin is added to the ground solution of the decellularized matrix and digested at room temperature for 12-16h;

[0141] iii. the digested decellularized matrix solution is quickly frozen in liquid nitrogen and dried to obtain a foamy dSECM.

[0142] The application also provides a dual-network hydrogel loaded with active components, which comprises the dual-network hydrogel and is loaded with active components, and the active components include at least one of neurotrophic factors (NGF, GDNF, NT-3) and inhibitors (MMP408, edaravone, fasudil mesylate).

[0143] Dual-network hydrogel loaded with active components

[0144] The application also provides application of the dual-network hydrogel loaded with active components, neurotrophic factors and inhibitors, in SCI treatment.

[0145] The application realizes the collection of advantages such as biological activity, biocompatibility, structure adjustability and controllable mechanical strength by constructing a dual-network hydrogel. The prepared dual-network hydrogel is used for SCI treatment, which can not only deliver active components, neurotrophic factors or inhibitors in the damaged area, but also induce stem cells to differentiate into neural lineage cells. The dual-network hydrogel of the application cooperates with active components to play an immunoregulatory role in the damaged environment after SCI, reduces nerve inflammation and promotes tissue repair.

[0146] The application will be further described in conjunction with specific examples. It should be understood that these examples are used only for illustrating the application and are not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions (such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts. In the present application, w / v refers to g / mL.

[0147] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are illustrative only and not intended to be limiting.

[0148] Example 1 Preparation of double network hydrogel

[0149] In this example, a decellularized matrix and metal polyphenol double network hydrogel was constructed from DSM and MPN, and the preparation steps were as follows:

[0150] (1) Preparation of porcine spinal cord decellularized matrix dSECM

[0151] The obtained porcine spinal cord tissue was frozen in a -80℃ refrigerator for more than 1h, then thawed to room temperature, and the freezing and thawing process was repeated for 3 times.

[0152] Chemical enzymatic digestion was carried out under 37℃ oscillation for three cycles, and the reagents used were: ultrapure water, 3v / v% Triton X-100 (Sigma, T8787) solution, 0.04g / mL sodium deoxycholate (SDC, Sigma, 30970), 40kU / mL DNAase (DNase I, MCE, HY-108882), magnesium chloride (MgCl2·6H2O, Makeline, M813913), calcium chloride (CaCl2·2H2O, Makeline, C804986), sodium chloride (NaCl, Makeline, S805275), 4v / v% ethanol (Titan, G73537F).

[0153] The DNAase was dissolved in 1M sodium chloride solution (containing 0.015M magnesium chloride and 0.001M calcium chloride) to obtain a 40kU / mL DNAase solution.

[0154] The treatment reagents and treatment time are listed in order:

[0155] First cycle: ultrapure water (6h), 3v / v% Triton X-100 (6h), ultrapure water (20min x 3), 0.04g / mL SDC (10h), ultrapure water (20min x 3), DNase (4h);

[0156] Second cycle: ultrapure water (20min x 3), 3v / v% Triton X-100 (6h), ultrapure water (20min x 3), 0.04g / mL SDC (10h), ultrapure water (20min x 3), DNase (4h);

[0157] Third cycle: ultrapure water (20min x 3), 3v / v% Triton X-100 (6h), ultrapure water (20min x 3), 0.04g / mL SDC (10h), ultrapure water (20min x 3), DNase (4h), ultrapure water (20min x 3), 4v / v% ethanol (4h), ultrapure water (20min x 3);

[0158] After the above treatment, the decellularized matrix dSECM is obtained.

[0159] (2) Pretreatment of decellularized matrix dSECM

[0160] The obtained decellularized matrix is dissolved in 0.5M glacial acetic acid (Macklin, A801295) and ground, 1mg / mL pepsin (MCE, HY-P1635) is added and the solution is digested at room temperature for 12h, and the digested decellularized matrix solution is quickly frozen in liquid nitrogen and dried to obtain a foamed dSECM.

[0161] (3) Construction of DSM-MPN double network hydrogel

[0162] The foamed dSECM is dissolved in 0.5M glacial acetic acid at 0℃ to obtain a 20mg / mL dSECM solution (the dSECM solution contains 20mg / mL foamed dSECM).

[0163] 10mg / mL tannic acid TA (Macklin, T818845) is dissolved in ultrapure water to obtain a 5.88mM TA solution, the pH of the TA solution is adjusted to neutral with 1M sodium hydroxide (Macklin, S835850) solution, 1.19mg / mL ceric sulfate Ce(SO4)2·4H2O (Qisong, QS8878) is dissolved in ultrapure water to obtain a 2.94mM Ce ion solution, and the Ce ion solution and the neutral TA solution are mixed to obtain a metal-polyphenol network MPN solution, wherein the molar ratio of Ce ion to TA is 1:2.

[0164] The MPN solution and the dSECM solution were mixed at 0°C, and the pH of the mixed solution was adjusted to 7.4 with a 10M sodium hydroxide solution. A 10v / v% 10×PBS solution (phosphate buffer solution) was added to the mixed solution to obtain a hydrogel pre-polymerization solution, wherein the content of the MPN solution in the hydrogel pre-polymerization solution was 5v / v%, and the content of the dSECM solution in the hydrogel pre-polymerization solution was 82v / v%.

[0165] The pre-polymerization solution was heated at 37°C for 10min to obtain the DSM-MPN double network hydrogel.

[0166] Example 2 Preparation of a double network hydrogel

[0167] In this example, a decellularized matrix and metal polyphenol double network hydrogel was constructed from DSM and MPN, and the preparation method was the same as that in Example 1, except that the prepared DSM contained 25mg / mL dSECM, the content of the MPN solution in the hydrogel pre-polymerization solution was 10v / v%, the content of the dSECM solution in the hydrogel pre-polymerization solution was 78.7v / v%, the concentration of the dSECM solution in step (3) was 25mg / mL, and the content of the MPN solution in the hydrogel pre-polymerization solution was 10v / v%, and the remaining steps were the same as those in Example 1.

[0168] Example 3 Preparation of a double network hydrogel loaded with active components

[0169] In this example, a decellularized matrix and metal polyphenol double network hydrogel loaded with active components was constructed from DSM, MPN, and MMP408 (sigma, 444291), wherein the DSM contained 20mg / mL dSECM, the content of the MPN was 5v / v%, and the content of MMP408 was 2v / v%. The preparation method referred to Example 1, except that step (3) was different from that in Example 1, wherein step (3) was as follows:

[0170] (3) Construction of a double network hydrogel loaded with active components MMP408@DSM-MPN

[0171] The 20mg / mL foamed dSECM was dissolved in 0.5M ice acetic acid at 0°C to obtain a dSECM solution.

[0172] The 10mg / mL tannic acid TA was dissolved in ultrapure water to obtain a 5.88mM TA solution. The pH of the TA solution was adjusted to neutral with a 1M sodium hydroxide solution. The 1.19mg / mL ceric sulfate Ce(SO4)2·4H2O was dissolved in ultrapure water to obtain a 2.94mM Ce ion solution. The Ce ion solution and the neutral TA solution were mixed to obtain an MPN solution, wherein the molar ratio of Ce ion to TA was 1:2.

[0173] The MMP408 solution was prepared by dissolving 10 mg / mL of MMP408 inhibitor in DMSO solution. The MPN solution, the dSECM solution and the MMP408 solution were mixed at 0°C. The pH of the mixed solution was adjusted to 7.4 by using 10 M sodium hydroxide solution. The 10 v / v% 10x PBS solution was added to the mixed solution to obtain a hydrogel pre-polymerization solution, wherein the content of the MPN solution in the hydrogel pre-polymerization solution was 5 v / v%, the content of the MMP408 solution in the hydrogel pre-polymerization solution was 2 v / v%, and the content of the dSECM solution in the hydrogel pre-polymerization solution was 80.6 v / v%.

[0174] The pre-polymerization solution was heated at 37°C for 10 min to obtain the active substance-loaded double network hydrogel MMP408@DSM-MPN double network hydrogel loaded with the inhibitor.

[0175] Example 4 Characterization of spinal cord decellularized matrix

[0176] The dSECM obtained in Example 1 was evaluated for the effect of cell nucleus removal. DNA content detection was used for quantitative evaluation. The specific method is as follows:

[0177] DNA was purified by using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and then the dsDNA content was quantitatively detected by using a fluorescence quantifier (Thermo Fisher Scientific, Waltham, USA). The detection results are shown in Figure 1 As shown in the table, the ratio of the DNA content of the freeze-dried spinal cord tissue after decellularization to that before decellularization was 2.8%, and the DNA content in the dSECM was less than 50 ng / mg, which is considered to be a safe level for clinical application.

[0178] Example 5 Characterization of DSM hydrogel, DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel

[0179] (1) Morphological characterization

[0180] The DSM-MPN hydrogel obtained in Example 1 and the MMP408@DSM-MPN hydrogel obtained in Example 3 were characterized for micro-morphology by using a scanning electron microscope (SEM, Regulus 8100, Hitachi, Japan). The detailed steps are that the hydrogel was quickly frozen in liquid nitrogen and then vacuum freeze-dried for 3 days. The freeze-dried hydrogel was cut into thin slices, sprayed with platinum, and then characterized by SEM at an acceleration voltage of 5 kV. The scanning electron microscope images are shown in Figure 2 As shown in the pictures, the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel present a porous network structure in the dry state, which is a structure suitable for cell survival and growth.

[0181] Based on the scanning electron microscope images of hydrogels, the pore size was calculated using ImageJ-win64 software. The measurement results show that the pore sizes of the DSM-MPN hydrogel in Example 1 and the MMP408@DSM-MPN hydrogel in Example 3 are 66.75 ± 26.16 μm and 70.16 ± 26.19 μm, respectively, which is conducive to the exchange of substances between cells and microenvironments.

[0182] (2) Compression modulus test

[0183] In order to ensure that the hydrogel has sufficient mechanical strength to support cell growth, the DSM-MPN hydrogel obtained in Example 1 and the MMP408@DSM-MPN hydrogel obtained in Example 3 were subjected to compression test, as shown in Figure 3 The stress-strain curve of the hydrogel, and the compression modulus of the hydrogel was determined by calculating the slope of the linear region of the stress-strain curve (limited to the first 20% strain). The specific method is as follows:

[0184] First, a cylindrical hydrogel sample with a diameter of 10 mm and a height of 5 mm was prepared using a special mold, and then a dynamic biomaterial mechanics test system (450N / Electroforce 3230, USA) was used to compress the sample at a constant strain rate of 0.02 mm / s. The compression modulus of the hydrogel of Example 1 was 2.79 ± 0.37 kPa, and the compression modulus of the hydrogel of Example 3 was 3.11 ± 0.12 kPa, close to the compression modulus of the natural mouse spinal cord tissue.

[0185] (3) Stability test of DSM and DSM-MPN hydrogel gelation

[0186] In order to explore the effect of the addition of MPN on the stability of DSM gelation, the degradation rate of DSM hydrogel and DSM-MPN hydrogel was tested to compare the stability of the structure, as shown in Figure 4 The degradation mass curve of the hydrogel. The specific method is as follows:

[0187] Cylindrical hydrogels with a diameter of 10 mm and a height of 5 mm were prepared using a special mold, and the initial weight of the hydrogels was recorded. 4 mL of PBS solution with a pH of 6.0 was added to a 5 mL centrifuge tube, and the hydrogels were soaked in the acidic PBS solution and reacted in a 37°C shaking bed at 120 rpm. The hydrogels were taken out on the 1st, 2nd, 3rd, 4th, 5th, 7th, 9th, and 11th days, weighed, and replaced with fresh acidic PBS solution. The remaining mass percentage of the hydrogels was calculated to obtain the degradation curve of the hydrogels. On the 11th day, the remaining mass of the DSM hydrogel was 40.92%, and the remaining mass of the DSM-MPN hydrogel was 47.78%, indicating that the double network structure formed by the addition of MPN to DSM was more stable than the single DSM hydrogel.

[0188] Example 6 Antioxidant detection of DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel

[0189] In this example, the free radical scavenging ability of the DSM-MPN hydrogel of Example 1 and the MMP408@DSM-MPN hydrogel of Example 3 was detected using 2,2-diphenyl-1-picrylhydrazyl (DPPH, Mcllhinney, D807297), and the antioxidant performance of the hydrogels was detected.

[0190] A 0.1 mM DPPH working solution was prepared using anhydrous ethanol, and its absorbance at a wavelength of 517 nm was detected to draw a standard curve. 10 mg of hydrogel was weighed and added to 2 mL of working solution, and the control group was 2 mL of working solution without hydrogel. The absorbance was detected at a specific time point using a 10 mm cuvette, and the lower the absorbance, the higher the DPPH clearance rate. As shown in Figure 5 DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel have good and sustained free radical scavenging ability, reducing the concentration of DPPH by 87.62% ± 3.62% (DSM-MPN) and 85.87% ± 2.18% (MMP408@DSM-MPN) within 48 h, indicating that DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel have good antioxidant performance.

[0191] Example 7 Biocompatibility detection of DSM-MPN hydrogel and MMP408@DSM-MPN hydrogel

[0192] To detect the biocompatibility of the hydrogels, the extract of the DSM-MPN hydrogel of Example 1 and the MMP408@DSM-MPN hydrogel of Example 3 was used to culture mouse hippocampal neuron cell line HT22.

[0193] The DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel were respectively soaked in the complete culture medium DMEM added with 10% serum and 1% double antibody at 37°C for 24 h, the hydrogel concentration was 0.1 g / mL, and the bacteria were removed by filtering with a 0.22 μm needle filter to obtain the extraction liquid of the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel.

[0194] The HT22 interface plate was cultured in a 96-well plate, and the DMEM added with 10% serum and 1% double antibody was cultured at 37°C in a 5% CO2 incubator for 1 day, the medium was replaced with the DSM-MPN hydrogel extraction liquid and the MMP408@DSM-MPN hydrogel extraction liquid for 5 days, and the relative cell viability was detected and calculated using CCK-8. The results are shown in Figure 6 The relative cell viability of the cells cultured with the DSM-MPN hydrogel extraction liquid and the MMP408@DSM-MPN hydrogel extraction liquid was 119.86% and 115.78% respectively compared with the control group, indicating that the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel had good biocompatibility.

[0195] Example 8 Evaluation of the tissue repair effect of the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel on SCI mice

[0196] In this embodiment, a spinal cord injury (SCI) hemisection model was established in 7w female C57BL / 6 mice. First, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and the back hair of the mice was shaved. Then, a 1.5 cm incision was made on the back skin, the muscle tissue was removed, the vertebrae were exposed, the cone plate at T9-T10 was knocked out, and a 1.5 mm long spinal cord tissue was cut off on the right side at the position of the spinal cord T9-T10 using a surgical knife to establish a spinal cord injury hemisection model.

[0197] Two experimental groups were set up, one SCI group and one control group, and the experimental groups were respectively implanted with the DSM-MPN hydrogel obtained in Example 1 and the MMP408@DSM-MPN hydrogel obtained in Example 3 into the injury area to treat the mice with the above established spinal cord injury hemisection model. Subsequently, the skin incision was sutured, and the mice were recovered, and were respectively marked as the “DSM-MPN” group and the “MMP408@DSM-MPN” group. The mice which were not treated after modeling and directly sutured the skin incision were the “SCI” group. The skin was only cut and sutured, and the mice which were not modeled were the control group.

[0198] The mice were sacrificed at 10 weeks after treatment, and the spinal cord tissue was taken out for observation. The spinal cord tissue diagram is as shown in Figure 7As shown, it can be seen that the spinal cord tissue defect area of the mice in the "MMP408@DSM-MPN" group is obviously smaller than that of the untreated SCI mice, and the spinal cord tissue defect area of the mice in the "DSM-MPN" group is between that of the SCI group and the "MMP408@DSM-MPN" group. It indicates that both the "DSM-MPN" hydrogel and the "MMP408@DSM-MPN" hydrogel have beneficial effects on tissue repair after SCI, and the effect of the latter is more obvious.

[0199] Example 9 Evaluation of the anti-inflammatory effect of the DSM-MPN hydrogel and the MMP408@DSM-MPN hydrogel on SCI mice

[0200] The SCI mouse model was established as in Example 8, and the mice were sacrificed at 1 week after treatment, the spinal cord tissue was taken out, diluted with PBS solution and ground thoroughly, then centrifuged to take the supernatant for testing. The ELISA kit of interleukin IL-6 (Xinboseng, EMC004.96) and interleukin IL-4 (Xinboseng, EMC003.96) was used to detect the content of inflammatory factors in the supernatant, and all the operation steps were carried out according to the instructions. Figure 8 As shown, the content of pro-inflammatory factor IL-6 in the "DSM-MPN" group and the "MMP408@DSM-MPN" group was significantly reduced, and the content of anti-inflammatory factor IL-4 was significantly increased. Specifically, the content of IL-6 in the "DSM-MPN" group and the "MMP408@DSM-MPN" group was reduced by 32.13% and 55.64% respectively compared with the SCI group, and the content of IL-4 was increased by 33.44% and 95.88% respectively compared with the SCI group. It indicates that both the "DSM-MPN" hydrogel and the "MMP408@DSM-MPN" hydrogel have inhibitory effect on the inflammatory response after SCI, and the effect of the latter is more significant.

[0201] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or amendments to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A dual-network hydrogel, characterized in that, The dual-network hydrogel comprises the following components: (Z1) The decellularized matrix network (DSM) of the spinal cord, which is formed by the self-assembly of the decellularized matrix dSECM of the spinal cord; (Z2) A metal-polyphenol network (MPN) formed by coordination of metal ions and polyphenols; and (Z3) Optional loaded active ingredient; The DSM and MPN are cross-linked by hydrogen bonds to form a dual network structure.

2. The dual-network hydrogel as described in claim 1, characterized in that, The decellularized spinal cord matrix dSECM is derived from at least one of porcine spinal cord and mouse spinal cord.

3. The dual-network hydrogel as described in claim 1, characterized in that, The metal ion is selected from trivalent or tetravalent metal ions, preferably including Ce. 3+ / Ce 4+ Ti 4+ Fe 3+ Zr 4+ At least one of them.

4. The dual-network hydrogel as described in claim 1, characterized in that, The polyphenols are selected from at least one of tannic acid (TA), epigallocatechin gallate (EGCG), and dopamine (DA).

5. A method for preparing a dual-network hydrogel as described in any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Provide spinal cord decellularized matrix dSECM for pretreatment to prepare foam-like spinal cord decellularized matrix dSECM; (2) The foamy spinal cord decellularized matrix dSECM from step (1) is processed to prepare a spinal cord decellularized matrix dSECM solution; (3) Mix metal ion solution and neutral polyphenol solution to prepare metal-polyphenol network (MPN) solution; (4) Provide the prepared spinal cord decellularized matrix dSECM solution and metal-polyphenol network MPN solution, and mix them to obtain a hydrogel prepolymer solution; (5) The prepolymer solution obtained in step (4) is heated to prepare DSM-MPN dual-network hydrogel.

6. The preparation method according to claim 5, characterized in that, The concentration of the dSECM solution is 10-30 mg / mL, preferably 10-25 mg / mL.

7. The preparation method according to claim 5, characterized in that, The MPN solution has a content of 1-20 v / v in the hydrogel prepolymer solution, preferably 2-15 v / v, and most preferably 2-10 v / v.

8. The preparation method according to claim 5, characterized in that, The content of the dSECM solution in the hydrogel prepolymer solution is 70-95 v / v, preferably 80-90 v / v.

9. A dual-network hydrogel composite, characterized in that, The dual-network hydrogel complex comprises: (Y1) The dual-network hydrogel according to any one of claims 1-8; and (Y2) Active component; The active component includes at least one of neurotrophic factors and inhibitors.

10. The application of a dual-network hydrogel as described in any one of claims 1-8 or the dual-network hydrogel composite as described in claim 9, characterized in that, Used to prepare formulations for treating spinal cord injuries.

Images