A multi-level pore hydrogel bone scaffold and a preparation method and application thereof

Abstract

The present application belongs to the technical field of biological scaffolds, and particularly relates to a preparation method of a porous biomimetic bone scaffold and the biomimetic bone scaffold. The preparation method of the porous biomimetic bone scaffold comprises the following steps: mixing a hydrogel main material, water, a crosslinking agent, an alkyl amine compound and an optional complex high polymer material; and performing directional freezing treatment on the obtained mixed product, wherein the hydrogel main material is selected from one or more than two of silk fibroin, collagen and gelatin, and the complex high polymer material is selected from one or more than two of hyaluronic acid, chitosan and other biological source natural polymer and synthetic organic high polymer compound. The porous biomimetic bone scaffold with certain pore size directional micropore channels and nanochannels formed along the side walls of the directional micropore channels can be prepared by the method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological scaffolds, in particular to a multi-level pore hydrogel bone scaffold and a preparation method and application thereof, and especially to a vascularized multi-level pore biomimetic directional bone scaffold and a preparation method and application thereof. BACKGROUND

[0002] Bone defects caused by severe trauma, tumors and other diseases have always been the focus of orthopedic treatment. Although the human skeleton has a certain self-repairing regeneration ability, it is difficult to rely on pure bone healing for larger defect repair (diameter > 5mm). At present, autologous, allogeneic or xenogeneic transplantation methods are mainly used to fill and repair the defect site, but autologous bone is scarce and has many postoperative complications, and allogeneic bone has weak combination ability with the host and has rejection problems. Therefore, large area bone injury is still a problem that has not been completely solved in clinical treatment. With the development of bone tissue engineering technology, artificial implanted scaffolds can fill the injury site and induce bone tissue regeneration, which is expected to replace traditional human or animal bone, avoiding the problems of source difficulty and immune rejection.

[0003] Patents CN109498846B and CN108478877A are both based on directional freezing strategy, using chitosan and collagen as biological materials, and doping zinc, strontium and calcium ions or graphene oxide and other functional materials into the scaffold body, so as to have functional ion slow release after implantation and promote bone cell differentiation. However, the above method only realizes the formation of one-way (one-dimensional) pore of the hydrogel scaffold through directional freezing technology, and the bone tissue structure formed by the pores is mainly one-way (axial) structure, and the structure between the bone tissues formed by the pores is sparse, resulting in poor radial mechanical properties.

[0004] Patent CN109010926B uses sodium alginate as the main material, constructs a three-dimensional porous scaffold through freeze-drying and calcium chloride cross-linking process, which can promote tissue regeneration, and the porous structure can efficiently promote stem cell adhesion and expansion. Patent CN115920133A proposes a porous combined scaffold, which combines a large pore (200-250μm) scaffold with a mesoporous (2-50nm) scaffold, absorbs cell-free fat extract (Ceffe) into the large pores through the mesopores and releases it into the large pores, induces cells to adhere to the large pores to grow and differentiate, and realizes bone tissue regeneration. However, the above strategy only allows cells to grow in disorder, forming homogeneous and disordered bone tissue without anisotropic structure and performance of natural bone tissue. To accelerate bone tissue regeneration, the implanted scaffold serves as a place for cells to improve residence, metabolism and material exchange, and it not only has the ability to recruit and induce bone cells to adhere, but also provides sufficient nutrition for adhered bone cells, which is the basis for bone cells to differentiate into bone and construct bone morphology, structure and physiological function. Referring to the natural bone, the biomimetic construction of directional bone structure and the realization of tissue vascularization are the key prerequisites to ensure cell metabolism, nutrition and waste exchange.

[0005] Patent CN110227178A proposes to combine ceramic slurry with three-dimensional printed polymer porous template to form precursor, to build three-dimensional porous structure with polymer template, to freeze the precursor in a certain direction, to freeze dry in turn, and to remove the polymer template by high temperature sintering, so as to obtain three-dimensional structure bioceramic scaffold, the porous structure formed by the communication of the directional pores and the three-dimensional pores is beneficial to the multidirectional growth of cells, and can form a biomimetic structure similar to bone tissue, but the process flow is long, the polymer porous sacrificial template is difficult to completely remove by high temperature sintering, the organic residue has biological toxicity, and the biological activity is difficult to guarantee. Patent CN112137768A proposes a layered biomimetic pore composite scaffold, which directly mixes inorganic bioceramics and organic biopolymers to form slurry, builds three-dimensional porous structure by 3D printing technology and sintering melting, and fuses with bioceramics to form a stable multi-dimensional composite material. Since the actual blood vessels of the bone tissue are mostly bifurcated reticular structures similar to branches, and the pore size is between 100-300 μm, the actual pore of the porous structure constructed by traditional three-dimensional printing is difficult to accurately control, and the pore size is mostly in the range of 500-1000 μm. SUMMARY

[0006] The purpose of the present application is to solve the problem that the existing technology is difficult to meet the requirements of making the bone scaffold have the anisotropic structure and performance of natural bone tissue while accurately controlling the size of the porous structure of the bone scaffold between the size of the blood vessels of the bone tissue when preparing a bone defect vascular repair scaffold. Therefore, a multi-level pore hydrogel bone scaffold and a preparation method and application thereof are proposed. In the present application, a specific type of natural organic polymer compound in animals is used as the main body of the hydrogel, a crosslinking agent and an alkyl amine compound are added for treatment, and finally a multi-level pore hydrogel bone scaffold with directional micropores and nanochannels formed along the sidewalls of the directional micropores is obtained by directional freezing technology. The multi-level pore hydrogel bone scaffold prepared by the present application has similar structural characteristics to natural fibrous cartilage tissue and has a biomimetic structure mechanics. The size of the directional micropores is 10-250 μm.

[0007] The first aspect of the present application proposes a multi-level pore hydrogel bone scaffold, which has directional micropores and nanochannels formed along the sidewalls of the micropores. The diameter of the micropores is 10-250 μm.

[0008] Preferably, the diameter of the nanochannels is 30-400 nm.

[0009] The second aspect of the present application proposes a preparation method of a multi-level pore hydrogel bone scaffold, which comprises the following steps:

[0010] (1) Preparation of hydrogel preform: mixing hydrogel main material, water, crosslinking agent, alkyl amine compound and optional complex polymer material;

[0011] (2) Construction of multi-level pore scaffold: directional freezing treatment of the mixed product obtained in step (1),

[0012] The hydrogel main material is selected from one or more than two of silk fibroin, collagen and gelatin, and the complex polymer material is a natural organic polymer compound and / or a synthetic organic polymer compound.

[0013] Preferably, the alkyl amine compound is selected from one or more than two of tetramethyl ethylenediamine, diethylene triamine and triethylene tetramine.

[0014] Preferably, the natural organic polymer compound is selected from one or more than two of sodium alginate, hyaluronic acid, chitosan and cellulose.

[0015] Preferably, the synthetic organic polymer compound is selected from one or more than two of polyvinyl alcohol, polylactic acid and polyvinyl pyrrolidone.

[0016] Preferably, in step (1), the hydrogel main material, water and optional complex polymer material are first mixed to obtain a mixed solution, and then the crosslinking agent and alkyl amine compound are added to the mixed solution for second mixing. More preferably, the conditions for the first mixing include a temperature of 40-80℃, a stirring speed of 100-300 rpm and a time of 30-90 min. Further preferably, the conditions for the second mixing include a temperature of 40-60℃, a stirring speed of 150-400 rpm and a time of 60-120 min.

[0017] Preferably, in step (2), the conditions for the directional freezing treatment include a temperature of -80 to -60℃ and a time of 5-30 min.

[0018] Preferably, when no complex polymer material is added in step (1), the mass ratio of the amount of the hydrogel main material to water is 1:5-20.

[0019] Preferably, when no complex polymer material is added in step (1), the volume ratio of the amount of the alkyl amine compound to the mixed solution is 0.1-1:100.

[0020] Preferably, when no complex polymer material is added in step (1), the amount ratio of the hydrogel main material to the crosslinking agent is 1g:1-20mmol.

[0021] Preferably, when no complexing high molecular material is added in step (1), the mass fraction of the mixed solution is 5-20% by weight.

[0022] Preferably, when the complexing high molecular material is added in step (1), the mass ratio of the amount of the complexing high molecular material to the amount of the hydrogel base material is 1-3:10.

[0023] Preferably, when the complexing high molecular material is added in step (1), the mass ratio of the total amount of the hydrogel base material and the complexing high molecular material to the amount of water is 1:10-20.

[0024] Preferably, when the complexing high molecular material is added in step (1), the volume ratio of the amount of the alkyl amine compound to the amount of the mixed solution is 0.1-1:100.

[0025] Preferably, when the complexing high molecular material is added in step (1), the ratio of the total amount of the hydrogel base material and the complexing high molecular material to the amount of the crosslinking agent is 1g:1-20mmol.

[0026] Preferably, when the complexing high molecular material is added in step (1), the mass fraction of the mixed solution is 5-10% by weight.

[0027] Preferably, the crosslinking agent is butanediol diglycidyl ether and / or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide; more preferably, the mass ratio of the N-hydroxysulfosuccinimide to the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:2-5.

[0028] Preferably, when no complexing high molecular material is added in step (1), the mass ratio of the amount of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide to the amount of the hydrogel base material is 1-5:100.

[0029] Preferably, when the complexing high molecular material is added in step (1), the mass ratio of the amount of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide to the total amount of the hydrogel base material and the complexing high molecular material is 1-5:100.

[0030] The third aspect of the present application provides a multi-level porous hydrogel bone scaffold prepared by the above method.

[0031] The fourth aspect of the present application provides an application of the above multi-level porous hydrogel bone scaffold in vascularized repair of bone defects.

[0032] The multi-level pore hydrogel bone scaffold and the preparation method thereof have at least the following beneficial effects:

[0033] (1) In the present application, water molecules in the hydrogel form a guide crystallization structure in the directional freezing process, which promotes the high molecules in the hydrogel to form unidirectional and uniformly distributed micropore channels around the ice crystals of water molecules, which is beneficial to the cell aggregation and growth in the channels, and provides conditions for blood vessel preforming. Meanwhile, after the completion of the catalytic polymer crosslinking reaction of the alkyl amine compound between the molecular chains of the hydrogel, the alkyl amine compound molecules are separated from the side walls of the polymer and form nanochannels along with the phase separation process of the polymer and water molecules. The nanochannels can further induce cell adhesion and form capillary blood vessels along the nanochannels, and form a biomimetic reticular structure between the channels, forming a three-dimensional oriented channel scaffold with a three-dimensional oriented channel scaffold similar to the blood vessels in the natural bone tissue.

[0034] (2) In the present application, a specific type of hydrogel main polymer material is selected, which forms micropore channels with a pore size of 10-250 μm around the ice crystals of water molecules under the action of directional freezing. The channel size is beneficial to cell-induced adhesion and vascularization, and provides an adequate nutrient environment for bone cell differentiation into bone.

[0035] (3) In the present application, under the preferred condition, by adjusting the key parameters such as the concentration of the hydrogel polymer, the content of the crosslinking agent and the content of the alkyl amine compound, the size of the nanochannels formed on the side walls can be controlled and adjusted between 30-400 nm.

[0036] (4) The method of the present application is based on the principle of hydrogel molecular crosslinking and directional freezing technology. A porous biomimetic bone scaffold with directional micropore channels and nanochannels formed along the side walls of the directional micropore channels can be prepared by a simple and efficient operation method. Meanwhile, the directional and lateral compressive strength of the biomimetic bone scaffold is relatively good. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a scanning electron microscope image of the porous channel in the multi-level pore hydrogel bone scaffold prepared in Example 1 of the present application;

[0038] Figure 2 is a scanning electron microscope image of the channel in the bone scaffold prepared in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0039] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0040] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Each integer value within the range is also specifically disclosed. Any numerical value, however, can only be approximate. The endpoints of the ranges and any value are provided as a separate value from but within the range. Any numerical value, however, can only be approximate although exact values are provided in some instances. The endpoints of the ranges and any value are provided as a separate value from but within the range. Numerical ranges include all values from and including the lower and the upper values, in increments of one unit per interval. For ranges generated using the "from about" and "up to about" or "from about and / or "to about" phrases, the approximate values appropriate are disclosed in the specification. For a numerical range that includes only one value, that value is the exact value used in the value list preceding the "and / or" phrase. For a numerical range that includes only one number, that number is the exact value used in the value list preceding the "and / or" phrase. Numerical ranges include endpoints.

[0041] The first aspect of the present application provides a multi-porous hydrogel bone scaffold, which has oriented micropores and nanopores formed along the side walls of the micropores, wherein the diameter of the micropores is 10-250 μm, specifically, for example, 10 μm, 20 μm, 40 μm, 50 μm, 80 μm, 110 μm, 120 μm, 150 μm, 180 μm, 200 μm, 230 μm or 250 μm.

[0042] In the bone scaffold of the present application, the diameter of the nanopores can be 30-400 nm, specifically, for example, 30 nm, 40 nm, 80 nm, 100 nm, 120 nm, 150 nm, 200 nm, 260 nm, 300 nm, 340 nm, 380 nm or 400 nm.

[0043] The second aspect of the present application provides a method for preparing a multi-porous hydrogel bone scaffold, which comprises the following steps:

[0044] (1) Preparation of a hydrogel preform: mixing a hydrogel main material, water, a crosslinking agent, an alkylamine compound and an optional complex polymer material;

[0045] (2) Construction of a multi-porous scaffold: subjecting the mixed product obtained in step (1) to directional freezing treatment,

[0046] wherein the hydrogel main material is selected from one or more than two of silk fibroin, collagen and gelatin, and the complex polymer material is a natural organic polymer compound and / or a synthetic organic polymer compound.

[0047] The hydrogel preform prepared in step (1) is subjected to directional freezing, in which the high molecular (hydrogel main material and optional matching high molecular material) and water molecules are phase separated, the water molecules form a guide crystallization structure in the process of directional freezing, which promotes the high molecular to form a micro-porous channel in the freezing direction around the ice crystal of water molecules, with a pore size of 10-250 μm, at the same time, the alkyl amine compound in the hydrogel preform plays a catalytic function, which promotes the cross-linking reaction of the molecular chain of the high molecular in the hydrogel, with the phase separation of the high molecular and water molecules, the alkyl amine compound is precipitated from the side wall of the high molecular and forms a nano-porous channel, with a pore size of 30-400 nm, thereby forming a multi-level porous channel scaffold structure.

[0048] In the method of the present application, the cross-linking agent is butanediol diglycidyl ether (BDDE) and / or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / N-hydroxysulfosuccinimide (NHS), which is referred to as EDC / NHS in the present application, and specifically, the EDC / NHS can be prepared by conventional technical means in the art, i.e. EDC and NHS are sequentially added to the buffer solution, and the mass ratio of NHS to EDC is 1:2-5.

[0049] In the method of the present application, in a preferred case, the cross-linking agent is butanediol diglycidyl ether (BDDE).

[0050] In the method of the present application, the alkyl amine compound is selected from one or more than two of tetramethyl ethylenediamine, diethylene triamine and triethylene tetramine. In a preferred case, the alkyl amine compound is tetramethyl ethylenediamine.

[0051] In the method of the present application, the natural organic high molecular compound is selected from one or more than two of sodium alginate, hyaluronic acid, chitosan and cellulose.

[0052] In the method of the present application, the synthetic organic high molecular compound is selected from one or more than two of polyvinyl alcohol, polylactic acid and polyvinyl pyrrolidone.

[0053] In the method of the present application, in step (1), the hydrogel main material, water and optional matching high molecular material are first mixed to obtain a uniform mixed solution, and then the cross-linking agent and the alkyl amine compound are added to the mixed solution for second mixing.

[0054] In the method of the present application, the first mixing and the second mixing are both carried out under stirring. The conditions of the first mixing include: temperature of 40-80℃, stirring speed of 100-300rpm, and time of 30-90min. The conditions of the second mixing include: temperature of 40-60℃, stirring speed of 150-400rpm, and time of 60-120min.

[0055] In the method of the present application, the hydrogel host material can be directly used to construct the multi-dimensional pore channel scaffold, or can be used to form a multi-phase composite hydrogel with a matching polymer material to construct the multi-dimensional pore channel scaffold. In the specific operation process, when no matching polymer material is added in step (1), the hydrogel host material is directly mixed with water for the first time to obtain a uniform mixed solution, and then the crosslinking agent is slowly dropped into the mixed solution under stirring for the second mixing with the alkyl amine compound.

[0056] In the method of the present application, when no matching polymer material is added in step (1), the mass ratio of the use amount of the hydrogel host material to water can be 1:5-20, and specifically, for example, can be 1:5, 1:10, 1:15 or 1:20.

[0057] In the method of the present application, when no matching polymer material is added in step (1), in order to make the size of the nano-pore channel formed by the micropore side wall controllable and adjustable between 30-400nm, the volume ratio of the use amount of the alkyl amine compound to the mixed solution can be 0.1-1:100, and specifically, for example, can be 0.1:100, 0.2:100, 0.5:100, 0.8:100 or 1:100. In a preferred case, the volume ratio of the use amount of the alkyl amine compound to the mixed solution is 0.2-0.5:100.

[0058] In the method of the present application, when no matching polymer material is added in step (1), in order to make the size of the micropore channel of the hydrogel scaffold controllable and adjustable between 10-250μm during the directional freezing construction, the mass fraction of the mixed solution should be controlled to be 5-20wt%, and specifically, for example, can be 5wt%, 10wt%, 15wt% or 20wt%.

[0059] In the method of the present application, when no matching polymer material is added in step (1), the use amount ratio of the hydrogel host material to the crosslinking agent can be 1g:1-20mmoL, and specifically, for example, can be 1g:1mmoL, 1g:5mmoL, 1g:10mmoL, 1g:15mmoL or 1g:20mmoL.

[0060] In the method of the present application, when no complexing polymer material is added in step (1), and when the crosslinking agent uses EDC / NHS, the mass ratio of the amount of use of the EDC / NHS to the hydrogel base material can be 1-5:100, and specifically, for example, can be 1:100, 2:100, 3:100, 4:100, or 5:100.

[0061] In the method of the present application, in the specific operation process, when the complexing polymer material is added in step (1), the hydrogel base material is first mixed with the complexing polymer material to form a composite polymer material, then the composite polymer material is mixed with water for the first time to obtain a uniform mixed solution, and then the mixed solution is slowly dripped with the crosslinking agent and the alkyl amine compound under stirring for the second time.

[0062] In the method of the present application, when the complexing polymer material is added in step (1), the mass ratio of the amount of use of the complexing polymer material to the hydrogel base material can be 1-3:10, and specifically, for example, can be 1:10, 2:10, or 3:10.

[0063] In the method of the present application, when the complexing polymer material is added in step (1), the mass ratio of the total content of the composite polymer material (hydrogel base material and complexing polymer material) to the amount of use of water can be 1:10-20, and specifically, for example, can be 1:10, 1:15, or 1:20.

[0064] In the method of the present application, when the complexing polymer material is added in step (1), in order to control and adjust the size of the nanopore channel formed by the micropore side wall to be between 30-400 nm, the volume ratio of the amount of use of the alkyl amine compound to the mixed solution can be 0.1-1:100, and specifically, for example, can be 0.1:100, 0.2:100, 0.5:100, 0.8:100, or 1:100. In a preferred case, the volume ratio of the amount of use of the alkyl amine compound to the mixed solution is 0.8-1:100.

[0065] In the method of the present application, when the complexing polymer material is added in step (1), in order to control and adjust the size of the micropore channel of the hydrogel scaffold directional freezing construction to be between 10-250 μm, the mass fraction of the mixed solution should be controlled to be 5-10 wt%, for example, can be 5 wt% or 10 wt%.

[0066] In the method of the present application, when the complex polymer material is added in step (1), the ratio of the use amount of the complex polymer material (hydrogel base material and complex polymer material) to the crosslinking agent can be 1 g: 1-20 mmol, specifically, for example, 1 g: 1 mmol, 1 g: 5 mmol, 1 g: 10 mmol, 1 g: 15 mmol or 1 g: 20 mmol.

[0067] In the method of the present application, when the complex polymer material is added in step (1), and when the crosslinking agent uses EDC / NHS, the mass ratio of the use amount of the EDC / NHS to the complex polymer material (hydrogel base material and complex polymer material) can be 1-5: 100, specifically, for example, 1: 100, 2: 100, 3: 100, 4: 100 or 5: 100.

[0068] In the method of the present application, in step (2), the hydrogel solution is directionally and gradually frozen from the bottom to the top by a unidirectional cold source until the whole hydrogel is completely frozen. Since the thermal conductivity of copper is 1600 times that of plastic, the freezing of the hydrogel (SF) solution occurs in the direction from bottom to top. In a specific embodiment, the mixed product obtained in step (1) is injected into a mold with copper at the bottom, and then the lower half of the mold is immersed in a liquid nitrogen bath to reduce the temperature of the copper to below -70°C, and the directional freezing treatment is carried out until the hydrogel in the mold is completely frozen, and the directional freezing treatment is completed. The conditions of the directional freezing treatment include a temperature of -80 to -60°C and a time of 5-30 min. In a preferred case, the conditions of the directional freezing treatment include a temperature of -80 to -70°C and a time of 10-25 min.

[0069] In the method of the present application, in step (2), the method further comprises sequentially subjecting the product after the directional freezing treatment to thawing soaking and freeze-drying.

[0070] In the method of the present application, the thawing soaking can be a conventional operation in the art, and the specific implementation process is as follows: the product after the directional freezing treatment is placed in a refrigerator at -20°C for a freezing reaction for 24-72 h, then thawed at room temperature for 12 h, and then soaked in deionized water for one week, and the water is changed every 24-48 h during the soaking to remove soluble impurities.

[0071] In the method of the present application, the freeze-drying can be a conventional operation in the art, and the specific implementation process is as follows: the hydrogel scaffold sample after the thawing soaking is taken out and re-placed in a refrigerator at -20°C for freezing for 24 h, and then freeze-dried to a constant weight.

[0072] The third aspect of the present application provides a multi-level porous hydrogel bone scaffold prepared by the above-mentioned method.

[0073] The fourth aspect of the present application provides an application of the above-mentioned multi-porous hydrogel bone scaffold in the vascularized repair of bone defects.

[0074] The multi-porous hydrogel bone scaffold and the preparation method thereof described in the present application are further illustrated by the following examples. The examples are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0075] In the following examples, the experimental methods are all conventional methods in the art unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0076] Example 1

[0077] (1) Hydrogel preform preparation: first, the silk fibroin powder and deionized water are mixed at a mass ratio of 1:20 for the first time, and mixed at 50°C and 100 rpm for 30 min to form a uniform mixed solution with a mass fraction of 5 wt%, and then BDDE and TEMED are slowly dropped into the uniform mixed solution for the second time at 50°C and 150 rpm, wherein the amount ratio of BDDE to silk fibroin is 10 mmol:1 g, and the volume ratio of TEMED to the uniform mixed solution is 0.5:100;

[0078] (2) Multi-porous scaffold construction: the mixed product obtained in step (1) is poured into a square mold with an inner side length of 10 mm, the bottom of the mold is a copper plate, the lower half of which is immersed in a liquid nitrogen bath, and the temperature of the copper plate is reduced to-70°C. After complete freezing, the reactant is placed in a refrigerator at-20°C for 24 h of freezing reaction, and then thawed at room temperature for 12 h, and then immersed in deionized water for one week, and the water is changed every 24 h during the immersion period to remove soluble impurities. The immersed hydrogel scaffold sample is taken out and re-placed in a refrigerator at-20°C for 24 h of freezing, and then freeze-dried to constant weight to obtain a multi-porous biomimetic bone scaffold.

[0079] Example 2

[0080] According to the manner of example 1, the difference is that the silk fibroin powder and deionized water are mixed at a mass ratio of 1:10 to obtain a multi-porous biomimetic bone scaffold.

[0081] Example 3

[0082] (1) Hydrogel preform preparation: First, the silk fibroin powder and gelatin particles are mixed in a mass ratio of 10:1, then the resulting product is mixed with deionized water in a mass ratio of 1:20, and mixed at 70°C, 100 rpm for 60 min to form a uniform mixed solution with a mass fraction of 5 wt%, and finally slowly drop into the uniform mixed solution at 60°C, 250 rpm, mixed with EDC / NHS (NHS and EDC in a mass ratio of 1:2) and tetramethyl ethylene diamine (TEMED), wherein the amount ratio of EDC / NHS to composite polymer material (hydrogel main material and complex polymer material) is 20 mmol:1 g, and the volume ratio of TEMED to uniform mixed solution is 1:100;

[0083] (2) Multistage pore scaffold construction: The mixed product obtained in step (1) is poured into a square mold with an inner side length of 10 mm, the bottom of the mold is a copper plate, the lower half of which is immersed in a liquid nitrogen bath, and the copper plate temperature is reduced to -70°C. After complete freezing, the reactant is placed in a refrigerator at -20°C for 24 h of freezing reaction, then thawed at room temperature for 12 h, then immersed in deionized water for a week, and the water is changed every 48 h during the immersion period to remove soluble impurities. The water-soaked hydrogel scaffold sample is taken out and re-placed in a refrigerator at -20°C for 24 h of freezing, and then freeze-dried to constant weight to obtain a porous biomimetic bone scaffold.

[0084] Example 4

[0085] (1) Hydrogel preform preparation: First, the silk fibroin powder and gelatin particles are mixed in a mass ratio of 10:1, then the resulting product is mixed with deionized water in a mass ratio of 1:20, and mixed at 70°C, 100 rpm for 60 min to form a uniform mixed solution with a mass fraction of 5 wt%, and finally slowly drop into the uniform mixed solution at 60°C, 250 rpm, mixed with EDC / NHS (NHS and EDC in a mass ratio of 1:2) and tetramethyl ethylene diamine (TEMED), wherein the amount ratio of EDC / NHS to composite polymer material (hydrogel main material and complex polymer material) is 20 mmol:1 g, and the volume ratio of TEMED to uniform mixed solution is 1:100;

[0086] (2) Multistage pore scaffold construction: The mixed product obtained in step (1) is cast into a square mold with an inner edge length of 10 mm, the bottom of the mold is a copper plate, the lower half of which is immersed in a liquid nitrogen bath, and the temperature of the copper plate is reduced to -70°C. After complete freezing, the reactants are placed in a refrigerator at -20°C for 24 h of freezing reaction, then thawed at room temperature for 12 h, then immersed in deionized water for a week, and the water is changed every 24 h during the immersion period to remove soluble impurities. The soaked hydrogel scaffold sample is taken out and re-placed in a refrigerator at -20°C for 24 h of freezing, and then freeze-dried to constant weight to obtain a porous biomimetic bone scaffold.

[0087] Example 5

[0088] (1) Hydrogel preform preparation: First, collagen powder and hyaluronic acid are mixed according to a mass ratio of 10:5 to obtain a composite polymer material (hydrogel main material and complex polymer material), then the composite polymer material (hydrogel main material and complex polymer material) is mixed with deionized water at a mass ratio of 1:10, and mixed at 60°C, 100 rpm for 60 min to form a uniform mixed solution with a mass fraction of 10 wt%, and finally EDC / NHS (NHS and EDC in a mass ratio of 1:3) and diethylene triamine are slowly added dropwise into the uniform mixed solution at 60°C, 300 rpm, wherein the amount ratio of EDC / NHS to composite polymer material (hydrogel main material and complex polymer material) is 20 mmol:1 g, and the volume ratio of diethylene triamine to uniform mixed solution is 1:100;

[0089] (2) According to the manner of the embodiment, a porous biomimetic bone scaffold is obtained.

[0090] Comparative Example 1

[0091] According to the manner of Example 1, except that in step (1), no tetramethyl ethylenediamine (TEMED) is added, a bone scaffold is obtained.

[0092] Comparative Example 2

[0093] According to the manner of Example 3, except that in step (1), tetramethyl ethylenediamine (TEMED) is replaced by glutaraldehyde, a bone scaffold is obtained.

[0094] Comparative Example 3

[0095] According to the manner of Example 1, except that in step (1), tetramethyl ethylenediamine (TEMED) is replaced by ethanolamine, a bone scaffold is obtained.

[0096] Comparative Example 4

[0097] The procedure was carried out in accordance with Example 1, except that in step (1), silk fibroin powder was replaced with hyaluronic acid to obtain a bone scaffold.

[0098] Comparative Example 5

[0099] The procedure was carried out in accordance with Example 1, except that in step (1), silk fibroin powder was replaced with chitosan to obtain a bone scaffold.

[0100] Comparative Example 6

[0101] The procedure was carried out in accordance with Example 5, except that in step (1), collagen powder was replaced with sodium alginate to obtain a bone scaffold.

[0102] Comparative Example 7

[0103] The procedure was carried out in accordance with Example 5, except that in step (1), collagen powder was replaced with polyvinyl alcohol to obtain a bone scaffold.

[0104] Comparative Example 8

[0105] The procedure was carried out in accordance with Example 1, except that in step (1), butanediol diglycidyl ether (BDDE) was replaced with calcium chloride to obtain a bone scaffold.

[0106] Test case

[0107] Scanning electron microscope (SEM) images of the bone scaffolds prepared in Example 1 and Comparative Example 1: A neat cross-section A was cut from the freeze-dried scaffold using a sharp blade perpendicular to the freezing direction. Simultaneously, a neat cross-section B was cut from the freeze-dried scaffold using a blade parallel to the freezing direction. Cross-sections A and B were placed on a stage coated with conductive adhesive and sputtered with gold. The structure of the scaffold was observed under an accelerating voltage of 5 kV. Analysis of cross-section A revealed the micron-scale pore morphology and size formed by directional freezing, while analysis of cross-section B revealed the nanopore morphology of the sidewalls of the directional freezing channels.

[0108] The directional compressive strength of the bone scaffolds prepared in Examples 1-5 and Comparative Examples 1-8: The freeze-dried scaffold material was cut into cubes of regular size, and a universal testing machine was used to perform a compression test parallel to the directional freezing channel direction of the scaffold. The compression speed was 3 mm / min, and the sample was compressed until it ruptured or the strain range reached 70%.

[0109] Lateral compressive strength of bone scaffolds prepared in Examples 1-5 and Comparative Examples 1-8: As above, compression tests were performed using a universal testing machine in the direction of the directional freezing channels perpendicular to the scaffold.

[0110] The results of directional compressive strength and lateral compressive strength of the bone scaffolds prepared in Examples 1-5 and Comparative Examples 1-8 are shown in Table 1.

[0111] Table 1

[0112]

[0113]

[0114] It can be seen from the scanning electron microscope images of the bone scaffold prepared according to Example 1 and Comparative Example 1 that the biomimetic bone scaffold prepared by the method of the present application has directional micron channels and nanochannels formed along the sidewalls of the micron channels. Meanwhile, it can be seen from the data in Table 1 that the sidewall nanochannel diameter of the biomimetic bone scaffold prepared by the method of the present application is 10-250 μm, and the nanochannel diameter formed by the sidewalls is controllable and adjustable between 30-400 nm. The channel size is conducive to cell-induced adhesion and vascularization, provides sufficient nutritional environment for osteocyte differentiation into bone, and the biomimetic bone scaffold has excellent directional and lateral compressive strength.

[0115] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A method for preparing a multi-porous hydrogel bone scaffold, characterized in that, The method comprises the following steps: (1) Preparation of hydrogel preform: mixing hydrogel main material, water, crosslinking agent, alkyl amine compound and optional coordinated polymer material; (2) Construction of multi-level pore scaffold: directional freezing treatment is performed on the mixed product obtained in step (1), Wherein, the hydrogel main material is selected from one or more than two of silk fibroin, collagen and gelatin, and the coordinated polymer material is a natural organic polymer compound and / or a synthetic organic polymer compound; The crosslinking agent is butanediol diglycidyl ether and / or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide; The mass ratio of N-hydroxysulfosuccinimide to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:2-5; When no coordinated polymer material is added in step (1), the mass ratio of the use amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide to the hydrogel main material is 1-5:100; When no coordinated polymer material is added in step (1), the mass ratio of the use amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysulfosuccinimide to the total content of the hydrogel main material and the coordinated polymer material is 1-5:

100.

2. The method of claim 1, wherein, The alkyl amine compound is selected from one or more than two of tetramethylethylenediamine, diethylenetriamine and triethylenetetramine; The natural organic polymer compound is selected from one or more than two of sodium alginate, hyaluronic acid, chitosan and cellulose; The synthetic organic polymer compound is selected from one or more than two of polyvinyl alcohol, polylactic acid and polyvinylpyrrolidone.

3. The method according to claim 1 or 2, characterized in that, In step (1), the hydrogel main material, water and optional coordinated polymer material are first mixed to obtain a mixed solution, and then the crosslinking agent and alkyl amine compound are added to the mixed solution for second mixing; The conditions of the first mixing include: temperature of 40-80℃, stirring speed of 100-300rpm, and time of 30-90min; The conditions of the second mixing include: temperature of 40-60℃, stirring speed of 150-400rpm, and time of 60-120min; In step (2), the conditions of the directional freezing treatment include: temperature of-80--60℃, and time of 5-30min.

4. The method of claim 3, wherein, When no coordinated polymer material is added in step (1), the mass ratio of the use amount of the hydrogel main material to water is 1:5-20; When no coordinated polymer material is added in step (1), the volume ratio of the use amount of the alkyl amine compound to the mixed solution is 0.1-1:100; When no coordinated polymer material is added in step (1), the use amount ratio of the hydrogel main material to the crosslinking agent is 1g:1-20mmol; When no coordinated polymer material is added in step (1), the mass fraction of the mixed solution is 5-20wt%.

5. The method of claim 3, wherein, When the complex high molecular material is added in step (1), the mass ratio of the use amount of the complex high molecular material to the use amount of the hydrogel main material is 1-3:10; When the complex high molecular material is added in step (1), the mass ratio of the total content of the hydrogel main material and the complex high molecular material to the use amount of water is 1:10-20; When the complex high molecular material is added in step (1), the volume ratio of the use amount of the alkyl amine compound to the mixed solution is 0.1-1:100; When the complex high molecular material is added in step (1), the ratio of the total content of the hydrogel main material and the complex high molecular material to the use amount of the crosslinking agent is 1g:1-20mmol. When the complex high molecular material is added in step (1), the mass fraction of the mixed solution is 5-10wt%.

6. A multi-level pore hydrogel bone scaffold prepared by the method according to any one of claims 1-5.

7. Application of the multi-level pore hydrogel bone scaffold according to claim 6 in preparation of a bone defect vascularization repair product.

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