Injectable hydrogel composite scaffold as well as preparation method and application thereof

Abstract

The invention discloses an injectable hydrogel composite scaffold as well as a preparation method and application thereof, and relates to the technical field of hydrogel scaffolds. The preparation method comprises the following steps: preparing a methacrylated protein solution; adding dopamine-polyethylene glycol into the methacrylated protein solution, and uniformly mixing the dopamine-polyethylene glycol and the methacrylated protein solution to prepare a first precursor solution; adding a mimic peptide into the first precursor solution to prepare a second precursor solution; performing ultraviolet curing on the second precursor solution to prepare an injectable hydrogel composite scaffold; wherein the amino acid sequence of the mimic peptide is as shown in SEQ ID NO: 1, the mimic peptide is connected with polyethylene glycol through a Schiff base bond, and the end part of the polyethylene glycol is modified with an aldehyde group. The injectable hydrogel composite scaffold prepared by the invention not only can be conveyed to a to-be-repaired part in an injection manner, but also can reduce the diffusion speed and degradation speed of the mimic peptide and improve the repair performance of the mimic peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel scaffolds, and in particular to an injectable hydrogel composite scaffold and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) causes lifelong disability and high mortality and is a common cause of neurological complications. In addition to loss of sensory and motor function, neurogenic bladder (NB) dysfunction caused by nerve damage in SCI is another major problem, affecting approximately 90% of patients. SCI not only leads to endothelial dysfunction, but also causes changes in vascular permeability. More importantly, SCI is associated with an inflammatory cascade, which is the result of activation of innate immune cells and infiltration of leukocytes. Among these cells, microglia are tissue-resident macrophages in the central nervous system with phenotypic versatility and functional diversity. Most macrophages undergo polarization to the M1 pro-inflammatory subtype after SCI, while only a few cells polarize to the M2 anti-inflammatory phenotype, thereby creating a neuroinflammatory microenvironment that inhibits SCI repair.

[0003] Cell therapy represents an effective intervention strategy for SCI repair. However, exogenous cell therapy continues to encounter significant challenges during clinical transplantation, including immunogenicity, suboptimal cell survival and integration efficiency, and potential tumorigenicity.

[0004] Existing technologies have shown that the application of brain-derived neurotrophic factor can stimulate endogenous neural regeneration. This tissue engineering approach, which leverages endogenous repair mechanisms, not only eliminates the need for in vitro culture of autologous cells but also significantly reduces the invasiveness of clinical procedures, thus providing a novel therapeutic strategy for SCI repair. However, the application of growth factors delivered to the injury site often carries the disadvantages of high cost, rapid degradation, and rapid diffusion. Summary of the Invention

[0005] One purpose of the first aspect of the present invention is to provide a method for preparing an injectable hydrogel composite scaffold to solve the technical problems in the prior art of hydrogel degradation and diffusion for delivering nerve repair factors, and the exogenous nerve factors are prone to infection.

[0006] Another object of the first aspect of the present invention is to further improve the repair performance and stability of the hydrogel composite scaffold.

[0007] The second aspect of the present invention aims to provide an injectable hydrogel composite scaffold prepared according to any of the preparation methods described above.

[0008] The third aspect of the present invention aims to provide an injectable hydrogel composite scaffold for use in spinal cord injury repair products, wherein the injectable hydrogel composite scaffold is prepared according to any of the preparation methods described above.

[0009] According to the purpose of the first aspect of the present invention, the present invention provides a method for preparing an injectable hydrogel composite scaffold, comprising:

[0010] preparing a methacryloylated protein solution;

[0011] adding dopamine-polyethylene glycol to the methacryloyl protein solution and mixing them uniformly to obtain a first precursor solution;

[0012] adding the mimetic peptide to the first precursor solution to prepare a second precursor solution;

[0013] UV curing the second precursor solution to prepare an injectable hydrogel composite scaffold;

[0014] The amino acid sequence of the mimetic peptide is shown in SEQ ID NO: 1, the mimetic peptide is connected to the polyethylene glycol via a Schiff base bond, the end of the polyethylene glycol is modified with an aldehyde group, and the methacryloylated protein solution is any one of a methacryloylated gelatin solution, a methacryloylated collagen solution, or a methacryloylated silk fibroin solution.

[0015] Optionally, the methacrylated protein solution is a mixed solution of phosphate buffer and methacrylated protein, and the initiator in the phosphate buffer solution can be any one of phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropionylphenone.

[0016] Optionally, the initiator concentration in the phosphate buffer solution is any value between 0.5 mg / mL and 2.5 mg / mL.

[0017] Optionally, the step of preparing the methacryloyl protein solution further comprises:

[0018] dissolving a protein material to prepare a protein solution, wherein the protein material is any one of gelatin, collagen or silk fibroin;

[0019] adding methacrylic acid to the protein solution to obtain a first mixed solution;

[0020] Protect from light and react overnight;

[0021] The first mixed solution that has been subjected to the light-shielded reaction is dialyzed and freeze-dried in sequence to obtain a methacrylylated protein;

[0022] The methacryloyl protein is added to the phosphate buffer and dissolved to prepare the methacryloyl protein solution.

[0023] Optionally, the mass volume concentration of the methacrylylated protein in the methacrylylated protein solution is any value between 10% and 20%.

[0024] Optionally, before the step of adding dopamine-polyethylene glycol to the methacrylylated protein solution, the method further comprises:

[0025] Prepare dopamine solution and polyethylene glycol solution separately;

[0026] adding the dopamine solution to the polyethylene glycol solution and mixing them uniformly to prepare a second mixed solution, wherein the volume ratio of the dopamine solution to the polyethylene glycol solution in the second mixed solution is any value between 1:1 and 1:4;

[0027] The second mixed solution is freeze-dried to prepare the dopamine-polyethylene glycol.

[0028] Optionally, the concentration of the dopamine-polyethylene glycol in the first precursor solution is any value between 0.02 mg / mL and 0.1 mg / mL.

[0029] Optionally, the concentration of the mimetic peptide in the second precursor solution is any value between 0.05 mg / mL and 0.2 mg / mL.

[0030] According to the purpose of the second aspect of the present invention, the present invention also provides an injectable hydrogel composite scaffold prepared according to any of the preparation methods described above.

[0031] According to the purpose of the third aspect of the present invention, the present invention also provides an application of an injectable hydrogel composite scaffold in spinal cord injury repair products, wherein the injectable hydrogel composite scaffold is prepared according to any of the preparation methods described above.

[0032] The present invention forms a dynamic network structure of the injectable hydrogel by respectively adding dopamine-polyethylene glycol, a methacrylylated protein solution, and a mimetic peptide during the preparation process of the injectable hydrogel composite scaffold. After being injected into the target location, the methacrylylated protein solution undergoes a cross-linking and curing reaction through ultraviolet curing to form a three-dimensional network structure with a stable structure and a mechanical strength matching that of the spinal cord tissue, so that the three-dimensional network structure plays a mechanical support role at the target location. The mimetic peptide is covalently grafted onto the dynamic network structure through the reaction of the amino group with the aldehyde group on the polyethylene glycol to form a Schiff base bond, thereby reducing the diffusion rate or degradation rate of the mimetic peptide at the target location. At the same time, the dynamically reversible Schiff base bond is gradually hydrolyzed under the pH or enzyme environment in the body to achieve sustained release. A sustained release rate of the mimetic peptide can reach 90% within 28 days. It can also avoid the problems of immunogenicity, low survival rate, and tumorigenic risk faced by directly transplanting exogenous cells, thereby promoting endogenous repair.

[0033] Furthermore, the present invention sets the concentration of the mimetic peptide in the second precursor solution to any value between 0.05 mg / mL and 0.2 mg / mL, so that its loading amount in the injectable hydrogel composite scaffold is moderate, effectively achieving the synergistic angiogenic and neuroregenerative activities of the mimetic peptide, and at the same time combining the hydrogel structure to achieve its sustained release effect at the injury site, thereby enhancing the therapeutic targeting, bioactivity stability and safety of the scaffold, and improving the remodeling ability of the spinal cord injury microenvironment.

[0034] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0036] Figure 1 is a schematic flow chart of a method for preparing an injectable hydrogel composite scaffold according to one embodiment of the present invention;

[0037] Figure 2 is a schematic flow chart of a method for preparing a methacrylylated protein solution according to one embodiment of the present invention;

[0038] Figure 3 is a schematic flow chart of a method for preparing dopamine-polyethylene glycol according to one embodiment of the present invention;

[0039] Figure 4This is a comparison chart of the tube-forming ability of the hydrogel composite scaffold prepared with different concentrations of the mimetic peptide of the present invention on human umbilical vein endothelial cells;

[0040] Figure 5 This is a histogram of the tube length formed by human umbilical vein endothelial cells using the hydrogel composite scaffold prepared with different concentrations of the mimetic peptide according to the present invention;

[0041] Figure 6 This is a comparison chart of the tube-forming ability of the hydrogel composite scaffolds prepared in Example 1 of the present invention and Comparative Examples 1-3 on human umbilical vein endothelial cells;

[0042] Figure 7 This is a histogram of the tube length formed by the hydrogel composite scaffold prepared in Example 1 and Comparative Examples 1-3 of the present invention on human umbilical vein endothelial cells;

[0043] Figure 8 1 is a scanning electron microscope image of the hydrogel composite scaffold in Example 1 and Comparative Examples 1-2 of the present invention;

[0044] Figure 9 This is a pore analysis diagram of the hydrogel composite scaffold in Examples 1-3 of the present invention;

[0045] Figure 10 This is a graph showing the effects of the hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention on the motor function of rats with complete transverse spinal cord injury;

[0046] Figure 11 This is a graph showing the inclined plate test of hydrogel composite scaffolds prepared according to Example 1 and Comparative Examples 1-4 of the present invention and implanted in rats with complete transverse spinal cord injury;

[0047] Figure 12 This is a comparison chart of BBB scores of the effects of the hydrogel composite scaffolds in Example 1 of the present invention and Comparative Examples 1-3 on the motor function of complete transected SCI rats;

[0048] Figure 13 The hydrogel composite scaffolds of Example 1 and Comparative Examples 1-4 of the present invention are used to treat CD31 in spinal cord injury. + Fluorescence image of the area proportion promoting neural differentiation;

[0049] Figure 14 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention promote the neural differentiation of CD31 in spinal cord injury. + A bar chart showing the regional proportions of

[0050] Figure 15 The hydrogel composite scaffold of Example 1 and Comparative Examples 1-4 of the present invention is used to treat map-2 in spinal cord injury. +Fluorescence image of the area proportion promoting neural differentiation;

[0051] Figure 16 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 promotes neural differentiation map-2 in spinal cord injury + A bar chart showing the regional proportions of

[0052] Figure 17 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat NF in spinal cord injury. + Fluorescence image of the area proportion promoting neural differentiation;

[0053] Figure 18 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes the differentiation of NF-κB in spinal cord injury. + A bar chart showing the regional proportions of

[0054] Figure 19 The hydrogel composite scaffolds for treating Tuj-1 in spinal cord injury according to Example 1 and Comparative Examples 1-4 of the present invention are + Fluorescence image of the area proportion promoting neural differentiation;

[0055] Figure 20 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention promote the differentiation of Tuj-1 neurons in spinal cord injury. + A bar chart showing the regional proportions of

[0056] Figure 21 The hydrogel composite scaffolds of Example 1 and Comparative Examples 1-4 of the present invention are used to treat CD31 in spinal cord injury. + Fluorescence image of the area proportion of angiogenic differentiation;

[0057] Figure 22 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes angiogenesis of CD31 in spinal cord injury + A bar chart showing the regional proportions of

[0058] Figure 23 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat spinal cord injury. + Fluorescence image of the area proportion of angiogenic differentiation;

[0059] Figure 24 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 promotes vascular differentiation in spinal cord injury. + A bar chart showing the regional proportions of

[0060] Figure 25 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat NF in spinal cord injury.+ Fluorescence image of the area proportion of angiogenic differentiation;

[0061] Figure 26 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes angiogenesis differentiation of NF-κB in spinal cord injury. + A bar chart showing the regional proportions of

[0062] Figure 27 The hydrogel composite scaffolds for treating Tuj-1 in spinal cord injury according to Example 1 and Comparative Examples 1-4 of the present invention are + Fluorescence image of the area proportion of angiogenic differentiation;

[0063] Figure 28 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes the differentiation of Tuj-1 in spinal cord injury. + A bar chart showing the regional proportions of

[0064] Figure 29 3 is a HE staining comparison of the effects of the hydrogel scaffolds in Example 1 of the present invention and Comparative Examples 1-3 on the bladder function of complete transected SCI rats. DETAILED DESCRIPTION

[0065] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0066] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below in conjunction with the accompanying drawings. It will be understood that the specific embodiments described herein are only used to explain the present application, rather than to limit the present application. It should also be noted that, for ease of description, only some, rather than all, structures related to the present application are shown in the accompanying drawings. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0067] As used herein, the terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0068] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0069] Figure 1 is a schematic flow chart of a method for preparing an injectable hydrogel composite scaffold according to one embodiment of the present invention. Figure 2 is a schematic flow chart of a method for preparing a methacrylylated protein solution according to one embodiment of the present invention. Figure 3 1 is a schematic flow chart of a method for preparing dopamine-polyethylene glycol according to one embodiment of the present invention.

[0070] like Figure 1 As shown, the present invention provides a method for preparing an injectable hydrogel composite scaffold, which is used to treat spinal cord injury. The injectable hydrogel composite scaffold comprises:

[0071] Step S100: preparing a methacrylylated protein solution;

[0072] Step S200: adding dopamine-polyethylene glycol to the methacryloyl protein solution and mixing them evenly to obtain a first precursor solution;

[0073] Step S300: adding the mimetic peptide to the first precursor solution to prepare a second precursor solution;

[0074] Step S400: UV curing the second precursor solution to prepare an injectable hydrogel composite scaffold;

[0075] The amino acid sequence of the peptide simulation is shown in SEQ ID NO: 1, the peptide simulation is connected to polyethylene glycol via a Schiff base bond, the end of the polyethylene glycol is modified with an aldehyde group, and the methacryloylated protein solution is any one of a methacryloylated gelatin solution, a methacryloylated collagen solution, or a methacryloylated silk fibroin solution.

[0076] In this embodiment, in the preparation method of the injectable hydrogel composite scaffold, a methacrylic acid acylated protein solution is first prepared, dopamine-polyethylene glycol is added to the methacrylic acid acylated protein solution, and the mixture is evenly mixed to obtain a first precursor solution. Then, a mimetic peptide is added to the first precursor solution to obtain a second precursor solution. The second precursor solution is UV-cured to obtain an injectable hydrogel composite scaffold. Here, the mimetic peptide is formed by directly fusing an endothelial progenitor cell recruitment peptide (amino acid sequence as shown in SEQ ID NO: 2) and a brain-derived neurotrophic factor mimetic peptide (amino acid sequence as shown in SEQ ID NO: 2). The mimetic peptide has the function of promoting endothelial progenitor cell recruitment, activating TrkB / p75NTR receptors, and promoting the differentiation of neural stem cells into neurons.

[0077] In this embodiment, dopamine-polyethylene glycol, a methacryloyl protein solution, and a peptide mimetic are added separately during the preparation of the injectable hydrogel composite scaffold to form a dynamic network structure of the injectable hydrogel. After injection into the target site, the methacryloyl protein solution undergoes a cross-linking and curing reaction by UV curing to form a three-dimensional network structure with a stable structure and mechanical strength matching that of the spinal cord tissue, so that the three-dimensional network structure provides mechanical support at the target site. The peptide mimetic is covalently grafted onto the dynamic network structure by reacting the amino group with the aldehyde group on the polyethylene glycol to form a Schiff base bond, thereby reducing the diffusion rate or degradation rate of the peptide mimetic at the target site. At the same time, the dynamically reversible Schiff base bond is gradually hydrolyzed under the in vivo pH or enzyme environment to achieve sustained release, achieving a sustained release rate of 90% for the peptide mimetic within 28 days. This can also avoid the problems of immunogenicity, low survival rate, and tumorigenicity faced by directly transplanted exogenous cells, thereby promoting endogenous repair.

[0078] In this embodiment, a dynamic network structure is formed between dopamine and polyethylene glycol and between polyethylene glycol and the mimetic peptide, that is, the terminal aldehyde group in polyethylene glycol can react with the catechol group in dopamine and the KR-NH3 at both ends of the mimetic peptide to form a Schiff base bond, so as to form a dynamic reversible cross-linking through the Schiff base bond to achieve shear thinning properties, reduce the viscosity by 80%, and facilitate injection. At the same time, the mimetic peptide is chemically connected to the dynamic hydrogel structure through the Schiff base bond to reduce the diffusion rate and degradation rate of the mimetic peptide, thereby facilitating the sustained release of the mimetic peptide. In addition, the catechol groups in dopamine can also undergo non-covalent hydrogen bonding / π-π stacking with the amino or hydroxyl groups in the methacryloylated protein. Under ultraviolet irradiation, the methacryloyl groups of the methacryloylated protein participate in free radical polymerization to form a covalent three-dimensional network. That is, dopamine-polyethylene glycol constructs a reversible dynamic cross-linking network in the system, providing shear thinning-self-healing behavior, thereby providing good injectability. After ultraviolet light curing, the covalent three-dimensional network structure is stable and the mechanical strength matches that of the spinal cord tissue. Dopamine also has antioxidant and anti-inflammatory functions, which can improve the biological microenvironment for spinal cord injury repair.

[0079] In this embodiment, the methacryloyl-coated protein and the mimetic peptide can construct a three-dimensional cross-linked network that encapsulates the mimetic peptide after UV curing, which can produce physical confinement and weak interactions, delay the diffusion of the mimetic peptide, achieve uniform spatial distribution of the mimetic peptide, prevent initial burst release, ensure the maintenance of the biological activity and targeted release of the mimetic peptide, and at the same time reduce the dosage of exogenous growth factors and improve treatment safety.

[0080] In this example, the mimetic peptide promotes neurotrophy and neurite outgrowth by binding to p75NTR and TrkB receptors, exerting dual effects in the nervous and vascular systems, regulating not only the survival and differentiation of neurons but also the survival of endothelial cells and vascular stability. In this example, the mimetic peptide is coupled with a specific peptide that binds to vascular endothelial cells. By activating endogenous cells through the mimetic peptide, the risks of exogenous cell transplantation are avoided, effectively coordinating the use of endogenous cells to promote injury repair.

[0081] In this embodiment, polyethylene glycol serves as the main scaffold material. It has no cell binding receptors, and cells cannot adhere to the polyethylene glycol surface. It is cross-linked by mimetic peptides. Methacryloyl proteins have excellent biocompatibility and biodegradability, introducing an additional network structure layer for the hydrogel, enhancing its mechanical strength, and prolonging the residence time of the mimetic peptide on the wound surface. A lasting anti-inflammatory effect will be achieved. In addition, dopamine is rich in amino and catechol groups, and forms a dynamic reversible Schiff base bond through amino and catechol groups, imparting antioxidant (ROS clearance rate 70%) and anti-inflammatory properties (IL-6 reduction 50%). The injectable hydrogel composite scaffold prepared in this application enhances nerve regeneration by reducing inflammation and promoting angiogenesis after spinal cord injury, overcoming the problems of poor injectability and insufficient stability of existing hydrogels, solving the defects of single-functional materials that cannot synergistically regulate inflammation, angiogenesis and nerve regeneration; avoiding the clinical risks of exogenous cell or growth factor applications.

[0082] In a further embodiment, the methacrylated protein solution is a mixed solution of phosphate buffer and methacrylated protein, and the initiator in the phosphate buffer solution can be any one of phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropionylphenone. In this embodiment, by using phosphate buffer as a carrier solution and combining it with a bioavailable UV initiator, the biocompatibility, UV responsiveness, and cross-linking rate of the hydrogel system are optimized, while ensuring the biological activity of the mimetic peptide, rapid in situ curing, mechanical matching, and long-term release functions are achieved.

[0083] In a further embodiment, the initiator concentration in the phosphate buffer solution is any value between 0.5 mg / mL and 2.5 mg / mL, that is, the initiator concentration can be 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL or 2.5 mg / mL, or any other value between 0.5 mg / mL and 2.5 mg / mL. In this embodiment, by setting the initiator within the above range, the photocuring rate, network structure density, mechanical strength, biocompatibility and sustained release behavior of the hydrogel composite scaffold can be effectively adjusted, thereby achieving injectability, rapid in situ gelation and good tissue repair performance. Here, a high concentration of initiator can generally induce more cross-linking points, forming a denser network structure, thereby improving the mechanical strength and elastic modulus of the hydrogel, while a low concentration forms a looser structure, improving its softness and permeability, and can fine-tune the rigidity and flexibility and elasticity matching of the hydrogel according to the mechanical matching requirements of different tissues such as the spinal cord.

[0084] like Figure 2 As shown, in a further embodiment, the step of preparing the methacrylylated protein solution further comprises:

[0085] Step S110: dissolving a protein material to prepare a protein solution, wherein the protein material is any one of gelatin, collagen, or silk fibroin;

[0086] Step S120: adding methacrylic acid to the protein solution to obtain a first mixed solution;

[0087] Step S130: Protect from light and react overnight;

[0088] Step S140: performing dialysis and freeze-drying on the first mixed solution of the light-shielded reaction in sequence to obtain a methacrylylated protein;

[0089] Step S150: adding the methacrylylated protein into a phosphate buffer solution and dissolving the protein to obtain a methacrylylated protein solution.

[0090] In this embodiment, the steps of preparing a methacrylated protein solution include first dissolving a protein material to prepare a protein solution, adding methacrylic acid to the protein solution, and mixing uniformly to obtain a first mixed solution. The first mixed solution is reacted in the dark overnight, and the first mixed solution is dialyzed and lyophilized in sequence to obtain a methacrylated protein. Finally, the methacrylated protein is added to a phosphate buffer solution and dissolved to obtain a methacrylated protein solution. Here, the protein material is any one of gelatin, collagen, or silk fibroin.

[0091] In this embodiment, the methacryloyl protein solution is prepared by the above steps, which can achieve methacryloyl modification of the protein and introduce double bond functional groups, so that the modified protein can participate in subsequent polymerization and cross-linking under ultraviolet light, thereby forming a three-dimensional stable network structure hydrogel. It can also retain the biocompatibility and biological function of the protein, improve the bioadaptability of the final hydrogel, and remove the reaction by-products through dialysis treatment to ensure biosafety and avoid the presence of cytotoxic or immunostimulatory substances in the subsequently formed hydrogel. In addition, the dopamine-polyethylene glycol dry powder obtained by freeze-drying is easy to store and quantitatively re-dissolve, enhancing the controllability and repeatability of the preparation process, which is conducive to formulation standardization and clinical transformation.

[0092] In this embodiment, gelatin, collagen, and silk fibroin all contain reaction sites that can be modified by methacrylation, which are suitable for the modification and preparation of different protein materials. They have good material compatibility and adaptability, are suitable for natural protein materials with different structures and functions, and achieve universal adaptation of various protein matrices.

[0093] In a further embodiment, the mass volume concentration of the methacrylylated protein in the methacrylylated protein solution is any value between 10% and 20%, that is, the mass volume concentration of the methacrylylated protein in the methacrylylated protein solution can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any other value between 10% and 20%. In this embodiment, by setting the mass volume concentration of the methacrylylated protein in the methacrylylated protein solution within the above adjustable range, not only is it ensured that the hydrogel system forms a stable three-dimensional cross-linked structure during the UV curing process, but it also achieves coordinated regulation of mechanical properties, drug release behavior, and injectability, thereby improving the overall functional adaptability of the scaffold in tissue engineering fields such as spinal cord injury repair.

[0094] In this embodiment, the higher the concentration of methacryloyl protein, the more methacryloyl groups participate in the photocrosslinking reaction, and the increased density of crosslinking points, which helps to form a denser and more stable three-dimensional network structure, improves the gelation efficiency, and thus achieves high-quality in situ gelation and complete scaffold molding structure. In addition, regulating the concentration of methacryloyl protein can fine-tune the hardness of the hydrogel to any value between 200Pa and 1000Pa, achieving mechanical matching with the target tissue, facilitating cell attachment and functional integration, and improving the repair effect. In addition, the concentration of methacryloyl protein also affects the porosity, swelling behavior, and sustained-release properties of the hydrogel. That is, when the protein concentration is low, the network structure is sparse, the porosity is high, the hydration capacity is strong, and the drug diffusion rate is fast. When the concentration is high, the network is dense, and the sustained-release of drugs or peptides is slower, which is conducive to long-term effects.

[0095] like Figure 3 As shown, in a further embodiment, before the step of adding dopamine-polyethylene glycol to the methacrylylated protein solution, the step further comprises:

[0096] Step S210: preparing a dopamine solution and a polyethylene glycol solution respectively;

[0097] Step S220: adding the dopamine solution to the polyethylene glycol solution and mixing them uniformly to prepare a second mixed solution, wherein the volume ratio of the dopamine solution to the polyethylene glycol solution in the second mixed solution is any value between 1:1 and 1:4;

[0098] Step S230: freeze-drying the second mixed solution to obtain dopamine-polyethylene glycol.

[0099] In this embodiment, the preparation method of dopamine-polyethylene glycol includes the following steps: first, preparing a dopamine solution and a polyethylene glycol solution separately, then adding the dopamine solution to the polyethylene glycol solution, mixing uniformly to prepare a second mixed solution, wherein the volume ratio of the dopamine solution to the polyethylene glycol solution in the second mixed solution is any value between 1:1 and 1:4, and finally, the second mixed solution is freeze-dried to prepare dopamine-polyethylene glycol. Here, the volume ratio of the dopamine solution to the polyethylene glycol solution in the second mixed solution can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:13.5 or 1:4, or any other value between 1:1 and 1:4.

[0100] In this embodiment, by controlling the ratio of dopamine to polyethylene glycol and combining it with a freeze-drying process, the obtained dopamine-polyethylene glycol complex can provide dynamic reversible cross-linking ability in the hydrogel system, and endow it with excellent injectability, self-healing, antioxidant and tissue adhesion properties, thereby showing better biological functions and clinical application prospects in the field of regenerative medicine such as spinal cord injury.

[0101] In a further embodiment, the concentration of dopamine-polyethylene glycol in the first precursor solution is any value between 0.02 mg / mL and 0.1 mg / mL, that is, the concentration of dopamine-polyethylene glycol can be

[0102] 0.02mg / mL, 0.03mg / mL, 0.04mg / mL, 0.05mg / mL, 0.06mg / mL, 0.07mg / mL, 0.08mg / mL, 0.09mg / mL or 0.1mg / mL, or any other value between 0.02mg / mL and 0.1mg / mL. In this embodiment, by controlling the mass concentration of dopamine-polyethylene glycol within the range of 0.02mg / mL-0.1mg / mL, a dynamic reversible cross-linked network of appropriate density can be formed in the first precursor solution, thereby improving the stability of the gel structure, self-repair ability and cell adhesion ability without affecting the injection performance of the material, and at the same time helping to regulate the controlled release of the functional mimetic peptide by the hydrogel, thereby improving the comprehensive performance of the hydrogel composite scaffold in the treatment of nerve injury.

[0103] In a further embodiment, the concentration of the mimetic peptide in the second precursor solution is any value between 0.05 mg / mL and 0.2 mg / mL, that is, the concentration of the mimetic peptide can be 0.05 mg / mL, 0.07 mg / mL, 0.1 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.16 mg / mL, 0.18 mg / mL or 0.2 mg / mL, or any other value between 0.05 mg / mL and 0.2 mg / mL. In this embodiment, by setting the concentration of the mimetic peptide in the second precursor solution within the above range, the loading amount thereof in the injectable hydrogel composite scaffold is moderate, effectively achieving the synergistic angiogenic and neuroregenerative activity of the mimetic peptide, and at the same time combining the hydrogel structure to achieve its sustained release effect at the injury site, thereby enhancing the therapeutic targeting, bioactivity stability and safety of the scaffold, and improving the remodeling ability of the spinal cord injury microenvironment.

[0104] Figure 4 This is a comparison chart of the tube-forming ability of the hydrogel composite scaffold prepared with different concentrations of the mimetic peptide on human umbilical vein endothelial cells. Figure 5 This is a histogram of the tube length formed by human umbilical vein endothelial cells using a hydrogel composite scaffold prepared with different concentrations of the mimetic peptide according to the present invention.

[0105] like Figure 4 and Figure 5 As shown, the cultured human umbilical vein endothelial cells were photographed and the number of meshes, total tube length and branch points were statistically analyzed through the images. The results showed that as the concentration of the mimetic peptide in the second precursor solution increased, the angiogenic ability of the mimetic peptide became stronger, and when the concentration reached a certain concentration, the angiogenic ability of the human umbilical vein endothelial cells would enter a plateau phase and would no longer increase.

[0106] The present invention also provides an injectable hydrogel composite scaffold, which is prepared according to any of the above preparation methods. The preparation method of the injectable hydrogel composite scaffold will not be described in detail here.

[0107] The present invention also provides an injectable hydrogel composite scaffold for use in spinal cord injury repair products. The injectable hydrogel composite scaffold is prepared according to the above-mentioned preparation method. In this embodiment, when the injectable hydrogel is used for spinal cord injury repair, a sustained-release rate of 90% of the mimetic peptide can be achieved over 28 days, extending the retention time of the mimetic peptide at the target site to be repaired, further improving the repair performance of the injectable hydrogel composite scaffold in spinal cord injury. The preparation method will not be described in detail here.

[0108] The present application will be further described in detail below with reference to specific embodiments.

[0109] Example 1

[0110] A method for preparing an injectable hydrogel composite scaffold comprises the following steps: first, adding phosphate buffer to gelatin and dissolving the gelatin at 37°C to obtain a gelatin solution with a mass concentration of 5%. Next, adding methacrylic acid to the gelatin solution at a concentration of 0.1 mL / g gelatin to obtain a first mixed solution. The first mixed solution is reacted in the dark for 16 hours, then dialyzed for 48 hours and freeze-dried for 48 hours to obtain methacrylated gelatin. Next, dissolving the methacrylated gelatin to 7.5% in a hydrochloric acid buffer containing 0.5 mg / mL phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt at 37°C to obtain a methacrylated gelatin solution. Separately, preparing a 1 mol / L dopamine solution and a 0.21 mol / L polyethylene glycol solution, the dopamine solution is added to the polyethylene glycol solution at a volume ratio of 1:4, and the reaction is continued for 2 hours to obtain a second mixed solution. The second mixed solution is freeze-dried for 48 hours to obtain the dopamine-polyethylene glycol solution. Then, dry dopamine-polyethylene glycol was added to the methacrylated gelatin solution to a final concentration of 0.07 mg / mL to obtain the first precursor solution. The mimetic peptide was further added to the first precursor solution to a final concentration of 0.1 mg / mL. The second precursor solution was cured under ultraviolet conditions for 30 seconds to obtain an injectable photocrosslinked multifunctional hydrogel composite scaffold. The ultraviolet light wavelength was 405 nm and the energy was 60 mW / cm 2 The prepared injectable hydrogel composite scaffold can achieve a sustained release rate of 90% within 28 days.

[0111] Example 2

[0112] The only difference between Example 2 and Example 1 is that the methacryloyl protein solution is a methacryloyl collagen solution with a mass volume concentration of 10%.

[0113] Example 3

[0114] The only difference between Example 3 and Example 1 is that the methacryloylated protein solution is a methacryloylated silk fibroin solution with a mass volume concentration of 10%.

[0115] Comparative Example 1

[0116] The only difference between Comparative Example 1 and Example 1 is that the second precursor mixed solution does not include polyethylene glycol and mimetic peptide.

[0117] Comparative Example 2

[0118] The only difference between Comparative Example 2 and Example 1 is that the second precursor mixed solution does not include dopamine-polyethylene glycol and mimetic peptide.

[0119] Comparative Example 3

[0120] The only difference between Comparative Example 3 and Example 1 is that the second precursor mixed solution does not include methacrylated gelatin solution, dopamine-polyethylene glycol and mimetic peptide.

[0121] Comparative Example 4

[0122] The only difference between Comparative Example 4 and Example 1 is that the second precursor solution further includes dopamine and a peptide mimetic.

[0123] Figure 6 This is a comparison chart of the tube-forming ability of the hydrogel composite scaffolds prepared in Example 1 of the present invention and Comparative Examples 1-3 on human umbilical vein endothelial cells. Figure 7 It is a histogram of the tube length formed by human umbilical vein endothelial cells using the hydrogel composite scaffold prepared in Example 1 and Comparative Examples 1-3 of the present invention.

[0124] like Figure 6 and Figure 7 As shown, the human umbilical vein endothelial cells cultured in Example 1 have more tubes, indicating that the addition of the mimetic peptide to the hydrogel composite scaffold prepared in Example 1 significantly promoted the migration and tube formation of human umbilical vein endothelial cells, and the hydrogel composite scaffold prepared in Example 1 not only retained the effect of the mimetic peptide, but also could slowly release the mimetic peptide to promote endothelial cell migration.

[0125] Figure 8 is a scanning electron microscope image of the hydrogel composite scaffold in Example 1 and Comparative Examples 1-2 of the present invention, Figure 9 3 is a pore analysis diagram of the hydrogel composite scaffold according to Examples 1-3 of the present invention.

[0126] Then, the hydrogel scaffolds prepared in Examples 1-3 and Comparative Examples 1-2 were analyzed by scanning electron microscopy, and the following results were obtained: Figure 8 and Figure 9 Characterization results shown.

[0127] like Figure 8 As shown, the injectable hydrogel composite scaffold grafted with dopamine-polyethylene glycol and a mimetic peptide prepared in Example 1 has a richer and more uniform pore structure, which is more conducive to cell proliferation and growth. The hydrogels without grafted mimetic peptide and dopamine-polyethylene glycol or the hydrogels grafted with dopamine only prepared in Comparative Examples 1 and 2 all have unevenly distributed pore structures with different pore sizes.

[0128] like Figure 9 As shown, pore analysis was performed on the hydrogel scaffolds prepared according to Examples 1-3. The results showed that the pores of the three hydrogel scaffolds were uniform and dense, with a diameter of about 50 μm to 100 μm.

[0129] Figure 10This is a graph showing the effects of the hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 on the motor function of rats with complete transverse spinal cord injury. Figure 11 This is a graph showing the inclined plate test of hydrogel composite scaffolds prepared according to Example 1 and Comparative Examples 1-4 of the present invention and implanted in rats with complete transverse spinal cord injury. Figure 12 3 is a comparison chart of BBB scores of the effects of the hydrogel composite scaffolds in Example 1 of the present invention and Comparative Examples 1-3 on the motor function of complete transected SCI rats.

[0130] like Figures 10 to 12 As shown, the results of the motor function experiment showed that the rats treated with the hydrogel composite scaffold prepared in Example 1 had significantly better motor ability than the other groups after complete spinal cord transection. Specifically, at 8 weeks after injury, compared with comparative examples 1-3, the hydrogel composite scaffold prepared in Example 1 could effectively promote the recovery of hind leg movement, and 1-4 weeks after injury, the score of the hydrogel composite scaffold prepared in Example 1 for spinal cord injury repair was higher than that of the other four groups.

[0131] like Figure 29 As shown in the bladder function test, the hydrogel composite scaffold prepared in Example 1 significantly restored bladder function and decreased bladder weight after implantation, indicating that it effectively improved the compensatory hyperplasia and hypertrophy of bladders caused by denervation. In addition, H&E staining also showed that the bladder wall of the scaffold implanted in Example 1 was thinner than that of the other groups, and the bladder function of the rats in the Example 1 implantation group was significantly improved.

[0132] Figure 13 The hydrogel composite scaffolds of Example 1 and Comparative Examples 1-4 of the present invention are used to treat CD31 in spinal cord injury. + Fluorescence image of the area ratio promoting neural differentiation, Figure 14 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention promote the neural differentiation of CD31 in spinal cord injury. + The regional proportion bar chart, Figure 15 The hydrogel composite scaffold of Example 1 and Comparative Examples 1-4 of the present invention is used to treat map-2 in spinal cord injury. + Fluorescence image of the area ratio promoting neural differentiation, Figure 16 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 promotes neural differentiation map-2 in spinal cord injury + The regional proportion bar chart, Figure 17 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat NF in spinal cord injury. + Fluorescence image of the area ratio promoting neural differentiation, Figure 18 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes the differentiation of NF-κB in spinal cord injury. + The regional proportion bar chart, Figure 19 The hydrogel composite scaffolds for treating Tuj-1 in spinal cord injury according to Example 1 and Comparative Examples 1-4 of the present invention are + Fluorescence image of the area ratio promoting neural differentiation, Figure 20 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention promote the differentiation of Tuj-1 neurons in spinal cord injury. + A bar chart showing the regional proportions.

[0133] like Figures 13 to 20 As shown, the hydrogel composite scaffolds prepared in Example 1 and Comparative Examples 1-4 were applied to a rat complete spinal cord transection model to promote the recovery of CD31 neural differentiation in the spinal cord injury site. + 、map-2 + NF + and Tuj-1 + The results showed that the hydrogel scaffold prepared in Example 1 can significantly promote the differentiation of spinal cord injury nerves and increase the expression of CD31 + 、map-2 + NF + and Tuj-1 + Regional proportion, thereby promoting the repair of damaged areas.

[0134] Figure 21 The hydrogel composite scaffolds of Example 1 and Comparative Examples 1-4 of the present invention are used to treat CD31 in spinal cord injury. + Fluorescence image of the area ratio promoting angiogenesis differentiation, Figure 22 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes angiogenesis of CD31 in spinal cord injury + The regional proportion bar chart, Figure 23 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat spinal cord injury. + Fluorescence image of the area ratio promoting angiogenesis differentiation, Figure 24 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 promotes vascular differentiation in spinal cord injury. + The regional proportion bar chart, Figure 25 The hydrogel composite scaffolds in Example 1 and Comparative Examples 1-4 of the present invention are used to treat NF in spinal cord injury. + Fluorescence image of the area ratio promoting angiogenesis differentiation, Figure 26 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes angiogenesis differentiation of NF-κB in spinal cord injury. + The regional proportion bar chart, Figure 27 The hydrogel composite scaffolds for treating Tuj-1 in spinal cord injury according to Example 1 and Comparative Examples 1-4 of the present invention are +Fluorescence image of the area ratio promoting angiogenesis differentiation, Figure 28 The hydrogel composite scaffold according to Example 1 and Comparative Examples 1-4 of the present invention promotes the differentiation of Tuj-1 in spinal cord injury. + A bar chart showing the regional proportions.

[0135] like Figures 21 to 28 As shown, the hydrogel composite scaffolds prepared in Example 1 and Comparative Examples 1-4 were applied to a rat complete spinal cord transection model to promote the differentiation and recovery of CD31 vascularization in the spinal cord injury site. + 、map-2 + NF + and Tuj-1 + The results showed that the hydrogel scaffold prepared in Example 1 can significantly promote the differentiation of blood vessels in spinal cord injury and increase the expression of CD31 + 、map-2 + NF + and Tuj-1 + Regional proportion, thereby promoting the repair of damaged areas.

[0136] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0137] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing an injectable hydrogel composite scaffold, characterized in that: include: preparing a methacryloylated protein solution; adding dopamine-polyethylene glycol to the methacryloyl protein solution and mixing them uniformly to obtain a first precursor solution; adding the mimetic peptide to the first precursor solution to prepare a second precursor solution; UV curing the second precursor solution to prepare an injectable hydrogel composite scaffold; The amino acid sequence of the mimetic peptide is shown in SEQ ID NO: 1, the mimetic peptide is connected to the polyethylene glycol via a Schiff base bond, the end of the polyethylene glycol is modified with an aldehyde group, and the methacryloylated protein solution is any one of a methacryloylated gelatin solution, a methacryloylated collagen solution, or a methacryloylated silk fibroin solution.

2. The preparation method according to claim 1, characterized in that The methacrylated protein solution is a mixed solution of phosphate buffer and methacrylated protein. The initiator in the phosphate buffer solution can be any one of phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropionylphenone.

3. The preparation method according to claim 2, characterized in that The initiator concentration in the phosphate buffer solution is any value between 0.5 mg / mL and 2.5 mg / mL.

4. The preparation method according to claim 3, characterized in that The step of preparing the methacryloyl protein solution also includes: dissolving a protein material to prepare a protein solution, wherein the protein material is any one of gelatin, collagen or silk fibroin; adding methacrylic acid to the protein solution to obtain a first mixed solution; Protect from light and react overnight; The first mixed solution that has been subjected to the light-shielded reaction is dialyzed and freeze-dried in sequence to obtain a methacrylylated protein; The methacryloyl protein is added to the phosphate buffer and dissolved to prepare the methacryloyl protein solution.

5. The preparation method according to claim 4, characterized in that The mass volume concentration of the methacryloyl protein in the methacryloyl protein solution is any value between 10% and 20%.

6. The preparation method according to any one of claims 1 to 5, characterized in that Before the step of adding dopamine-polyethylene glycol to the methacryloyl protein solution, the step further comprises: Prepare dopamine solution and polyethylene glycol solution separately; adding the dopamine solution to the polyethylene glycol solution and mixing them uniformly to prepare a second mixed solution, wherein the volume ratio of the dopamine solution to the polyethylene glycol solution in the second mixed solution is any value between 1:1 and 1:4; The second mixed solution is freeze-dried to prepare the dopamine-polyethylene glycol.

7. The preparation method according to claim 6, characterized in that The concentration of the dopamine-polyethylene glycol in the first precursor solution is any value between 0.02 mg / mL and 0.1 mg / mL.

8. The preparation method according to claim 7, characterized in that The concentration of the mimetic peptide in the second precursor solution is any value between 0.05 mg / mL and 0.2 mg / mL.

9. An injectable hydrogel composite scaffold, characterized in that: Prepared according to the preparation method described in any one of claims 1-8.

10. Use of an injectable hydrogel composite scaffold in a spinal cord injury repair product, wherein the injectable hydrogel composite scaffold is prepared by the preparation method according to any one of claims 1 to 8.

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