Double-layer hydrogel stent capable of releasing medicine in time sequence as well as preparation method and application of double-layer hydrogel stent

Abstract

The invention discloses a double-layer hydrogel stent capable of releasing medicine in time sequence as well as a preparation method and application of the double-layer hydrogel stent. The scaffold comprises an inner hydrogel and an outer hydrogel coating the inner hydrogel, the outer hydrogel is loaded with cell factors, and the cell factors have the following effects of collecting neutrophils and promoting polarization of the neutrophils into subtypes with stem cell collection; the inner layer hydrogel is loaded with neurotrophic factors. According to the hydrogel scaffold, ordered slow release of cell factors (such as IL-8) and neurotrophic factors (such as NGF) is achieved through the structural design of the inner hydrogel layer and the outer hydrogel layer, neutrophils and sensory nerves can be activated in sequence in a bone defect area, the neutrophils are collected in the defect area, stem cell homing is promoted, and osteogenesis is started; then, local sensory nerves are induced to grow in through the lagged and continuously released NGF, the sensory nerves are stimulated to play an immune regulation role, inflammation dissipation is promoted, and finally repair and regeneration of refractory bone defects are promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a double-layer hydrogel scaffold capable of sequential drug release, a preparation method thereof, and an application thereof. Background Art

[0002] Bone is the main organ of the body that plays a role in support, movement, and protection. Bone tissue has a certain self-repair ability, and the completion of its repair is inseparable from local timely and appropriate inflammatory responses. Various cells in the inflammatory microenvironment of bone defects interact through a series of growth factors to promote bone defect repair.

[0003] Although inflammation is a necessary link in bone defect repair, continuous inflammatory infiltration in the defect area will seriously affect bone regeneration, thus forming intractable bone defects. With the acceleration of population aging, diabetes and aging have gradually become the main causes of continuous infiltration of low-intensity chronic inflammation in the defect area. The treatment of bone defects in such patients is the key and difficult point in clinical bone defect treatment. Different from the continuous inflammatory infiltration in the bone defect area caused by infection, the chronic inflammation caused by diabetes and aging presents the characteristics of aseptic and low-intensity, mainly macrophage infiltration, which is caused by changes in the body's endocrine, oxidative stress, release of inflammatory factors, etc., and cannot be controlled by using antibiotics. When traditional repair materials (usually combined with immunosuppressants, such as combined with hormones or non-steroidal anti-inflammatory drugs) are used for repair, it will instead lead to side effects such as osteoporosis, osteonecrosis, and metabolic disorders. At the same time, inhibiting inflammation will increase the probability of concurrent infection.

[0004] Therefore, how to efficiently repair intractable bone defects caused by chronic inflammation is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a double-layer hydrogel scaffold capable of sequential drug release, a preparation method thereof, and an application thereof, which are used to efficiently repair the problem of intractable bone defects caused by chronic inflammation.

[0006] To solve the above problems, the present invention first provides a double-layer hydrogel scaffold capable of sequential drug release, and the scaffold includes: an inner-layer hydrogel and an outer-layer hydrogel coating the inner-layer hydrogel, wherein, cytokines are loaded in the outer-layer hydrogel, and the cytokines have the following functions: recruiting neutrophils and promoting the polarization of neutrophils into a subtype with stem cell recruitment; a neurotrophic factor is loaded in the inner-layer hydrogel.

[0007] Preferably, the cytokine includes IL-8.

[0008] Preferably, the neurotrophic factor includes at least one of NGF, BDNF, NT-3, NT-4, and NT-5.

[0009] Preferably, the outer hydrogel comprises a first main material, and the inner hydrogel comprises a second main material. The first main material and the second main material include at least one of polyethylene glycol diacrylate (PEGDA), silk fibroin methacrylate (SilMA), hyaluronic acid methacrylate (HAMA), gelatin methacrylate (GeIMA), sodium alginate methacrylate (AlgMA), and dextran methacrylate (DexMA).

[0010] Preferably, the first main material and the second main material are the same or different.

[0011] Preferably, the thickness of the outer hydrogel is 0.2 mm - 0.6 mm, and the thickness of the inner hydrogel is 1.2 mm - 1.6 mm.

[0012] On the other hand, the present invention also provides a method for preparing the aforementioned bilayer hydrogel scaffold capable of sequential drug release, comprising the following steps:

[0013] Step S1, preparing an outer hydrogel mixture, including: a first main material, a cytokine, and a photoinitiator.

[0014] Step S2, preparing an inner hydrogel mixture, including: a second main material, a neurotrophic factor, and a photoinitiator.

[0015] Step S3, coaxial 3D printing the outer hydrogel mixture and the inner hydrogel mixture, and subjecting them to photocuring to obtain the bilayer hydrogel scaffold capable of sequential drug release.

[0016] Preferably, in the outer hydrogel mixture solution, the loading dose of the cytokine is 60 μg / mL - 100 μg / mL, and the concentration of the first main material is 10% - 20% w / v.

[0017] Preferably, in the inner hydrogel mixture, the loading dose of the neurotrophic factor is 80 μg / mL - 120 μg / mL, and the concentration of the second main material is 10% - 20% w / v.

[0018] On the other hand, the present invention also provides the aforementioned bilayer hydrogel scaffold, or the application of the bilayer hydrogel scaffold prepared by the aforementioned preparation method in the preparation of products for bone defect repair and / or bone regeneration.

[0019] Compared with the prior art, the beneficial effects of the present invention at least include:

[0020] 1. Different from traditional bone defect repair or bone regeneration materials that simply adopt an anti-inflammatory approach, the hydrogel scaffold provided by the present invention converts chronic persistent inflammation into transient acute inflammation through an immune regulation method of promoting inflammation first and then suppressing inflammation, ultimately promoting the repair and regeneration of refractory bone defects. Specifically, the present invention designs a double-layer hydrogel scaffold with a core-shell structure. The inner layer of the scaffold is loaded with neurotrophic factors (such as NGF), and the outer layer is loaded with cytokines (such as IL-8). Through the sequential degradation of the inner and outer layer hydrogels and the regulation of the thickness and concentration of the inner and outer layer hydrogels, an orderly slow release of the loaded factors is achieved, thereby sequentially activating neutrophils and sensory nerves in the bone defect area. First, an appropriate dose of IL-8 is released in the defect area to recruit neutrophils and induce their polarization into reparative subsets, thereby promoting stem cell homing and initiating osteogenesis. Subsequently, the NGF encapsulated in the inner layer hydrogel is released in a delayed manner to induce the ingrowth of local sensory nerves, stimulate the sensory nerves to play an immune regulation role to terminate the infiltration of neutrophils, promote the dissipation of inflammation, and reshape the local immune environment.

[0021] 2. Compared with the limited treatment time of traditional ion / drug sustained-release scaffolds, the present invention promotes the regeneration of local sensory nerves by delivering neurotrophic factors. Through the physiological regulation of inflammation by sensory nerves, a single implantation treatment can ensure a long-term inflammation regulation effect, avoiding the side effects brought by frequent and long-term use of anti-inflammatory drugs and reducing the inconvenience brought to patients by multiple visits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a preparation flow chart of a double-layer hydrogel scaffold capable of sequential drug release according to an embodiment of the present invention.

[0023] Figure 2 It is an external view of a double-layer hydrogel scaffold capable of sequential drug release prepared according to an embodiment of the present invention.

[0024] Figure 3 It is the protein release result under different preparation parameters of the outer layer hydrogel.

[0025] Figure 4 It is the protein release result under different preparation parameters of the inner layer hydrogel.

[0026] Figure 5 It is the result of the influence of different IL-8 loading amounts in the outer layer hydrogel on the stem cell recruitment efficiency and the polarization of neutrophils.

[0027] Figure 6 It is the result of the influence of different NGF loading amounts in the inner layer hydrogel on the efficiency of promoting the elongation of sensory neuron axons.

[0028] Figure 7 It is the in vivo anti-inflammatory effect of a double-layer hydrogel scaffold capable of sequential drug release prepared according to an embodiment of the present invention.

[0029] Figure 8 In vivo verification results of a bilayer hydrogel scaffold capable of sequential drug release prepared in an embodiment of the present invention for treating diabetic critical bone defects.

[0030] It should be noted that Figure 3 and Figure 4 in, the height shown by the column refers to the newly added release amount between two time points. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0032] The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.

[0033] Term description

[0034] Interleukin-8 (IL-8) is an important pro-inflammatory cytokine and belongs to the CXC chemokine family. IL-8 can attract and activate neutrophils, enabling them to migrate directionally to the inflammatory site and release active products, thereby triggering a local inflammatory response.

[0035] NGF (Nerve Growth Factor) is a neurotrophin that is crucial for the growth, differentiation, and survival of sensory afferent neurons during development and is involved in the synthesis of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) in sensory neurons.

[0036] As described in the background art, although inflammation is a necessary link in bone defect repair, continuous inflammatory infiltration in the defect area will seriously affect bone regeneration, thus forming refractory bone defects. However, different from the continuous inflammatory infiltration in the bone defect area caused by infection, the chronic inflammation caused by diabetes and aging is mainly macrophage infiltration, showing the characteristics of asepsis and low intensity, and cannot be controlled by using antibiotics. When traditional repair materials (usually combined with immunosuppressants, such as combined with hormones or non-steroidal anti-inflammatory drugs) are used for repair, it will instead lead to side effects such as osteoporosis, osteonecrosis, and metabolic disorders, and also increase the probability of concurrent infections. Therefore, how to develop an efficient method for repairing refractory bone defects caused by chronic inflammation is a technical problem that urgently needs to be solved in this field.

[0037] To solve the above problems, the present invention has conducted a large number of experiments and explorations: First, according to the bone self-repair mechanism, the present invention found that neutrophils are the first inflammatory cells infiltrating the defect site. Further research found that an appropriate dose of IL-8 can not only induce neutrophil recruitment, but also promote the anti-inflammatory "N2 type" polarization of neutrophils, releasing stromal cell-derived factor-1α (SDF-1α), thereby recruiting mesenchymal stem cells and promoting bone regeneration. Therefore, different from the "anti-inflammatory" repair method adopted by traditional repair materials, the present invention initially designed a repair material loaded with IL-8 to promote bone regeneration through "pro-inflammatory" means, with the expectation of ultimately completing the repair of refractory bone defects.

[0038] However, through experiments, the present invention found that persistent neutrophil infiltration would inhibit the repair of bone defects and ultimately lead to the failure of bone defect repair. To solve this problem, the present invention continued to explore and found that: some studies have shown that sensory nerves in various tissues such as the skin and intestinal mucosa have efficient immunomodulatory effects, which can control the inflammation level through cell contact or the release of neurotransmitters for dynamic interaction. However, after searching domestic and foreign literature, it was found that no researcher has applied the immunomodulatory effect of sensory nerves to the control of inflammation level.

[0039] Therefore, the present invention designed to incorporate NGF into the above-mentioned IL-8-loaded repair material, and through structural design, used coaxial 3D printing technology to prepare a hydrogel scaffold with a core-shell structure, with the shell layer loaded with IL-8 and the core layer loaded with NGF, for realizing the sequential slow release of IL-8 and NGF. It is expected that the hydrogel scaffold will first release IL-8 in the early stage of the bone defect area to induce neutrophil recruitment, further induce stem cell homing through neutrophils, and initiate osteogenesis; subsequently, the NGF encapsulated in the inner hydrogel will be released in a delayed manner to induce the ingrowth of local sensory nerves, thereby exerting an immunoregulatory effect to terminate neutrophil infiltration, promote the dissipation of inflammation, reshape the local immune environment, convert chronic persistent inflammation into transient acute inflammation, and thus be conducive to the regeneration and repair of refractory bone defects. Moreover, through subsequent experiments, the present invention has proved that the double-layer hydrogel scaffold with sequential drug release designed by the present invention can efficiently repair refractory bone defects caused by chronic inflammation, and has very good application prospects.

[0040] Based on the above, the present invention finally provides a bilayer hydrogel scaffold capable of sequential drug release, and the scaffold includes: an inner hydrogel and an outer hydrogel coating the inner hydrogel. Among them, cytokines are loaded in the outer hydrogel, and the cytokines have the following functions: recruiting neutrophils and promoting the polarization of neutrophils into a phenotype with stem cell recruitment; neurotrophic factors are loaded in the inner hydrogel, which is used to induce the ingrowth of local sensory neurons, and then stimulate the sensory nerves to play an immunomodulatory role to terminate the infiltration of neutrophils.

[0041] In some embodiments, the cytokine includes IL-8, and the neurotrophic factor includes at least one of NGF, BDNF, NT-3, NT-4, and NT-5.

[0042] The outer hydrogel includes a first main material, and the inner hydrogel includes a second main material. In some embodiments, the first main material and the second main material include at least one of polyethylene glycol diacrylate (PEGDA), silk fibroin methacrylate (SilMA), hyaluronic acid methacrylate (HAMA), gelatin methacrylate (GeIMA), sodium alginate methacrylate (AlgMA), and dextran methacrylate (DexMA).

[0043] Furthermore, the first main material and the second main material are the same or different.

[0044] In some embodiments, the thickness of the outer hydrogel is 0.2 mm - 0.6 mm, and the thickness of the inner hydrogel is 1.2 mm - 1.6 mm.

[0045] On the other hand, the present invention also provides a preparation method of the aforementioned bilayer hydrogel scaffold capable of sequential drug release, including the following steps:

[0046] Step S1, preparing an outer hydrogel mixture, including: a first main material, cytokines, and a photoinitiator.

[0047] Step S2, preparing an inner hydrogel mixture, including: a second main material, neurotrophic factors, and a photoinitiator;

[0048] Step S3, coaxial 3D printing the outer hydrogel mixture and the inner hydrogel mixture, and subjecting them to photocuring to obtain the bilayer hydrogel scaffold capable of sequential drug release.

[0049] In some embodiments, in the outer hydrogel mixed solution, the loading dose of cytokines is 60 μg / mL - 100 μg / mL, and the concentration of the first main material is 10% - 20% w / v.

[0050] In some embodiments, in the inner hydrogel mixture, the loading dose of the neurotrophic factor is 80 μg / mL - 120 μg / mL, and the concentration of the second matrix material is 10% - 20% w / v.

[0051] On the other hand, the present invention also provides the application of the aforementioned double-layer hydrogel scaffold, or the double-layer hydrogel scaffold prepared by the aforementioned preparation method, in the preparation of products for bone defect repair and / or bone regeneration.

[0052] The following combines specific embodiments and drawings to detail the experimental process of the present invention.

[0053] Figure 1 This is a flowchart for preparing a double-layer hydrogel scaffold capable of sequential drug release using 3D printing technology in this embodiment. It includes: incorporating the outer hydrogel mixture (pre-mixed cytokine) into the outer channel, and using a "projection type" photocuring 3D printing system. By adjusting parameters such as 3D printing parameters, the crosslinking degree of the outer hydrogel, and the loading dose of the cytokine, the outer hydrogel is prepared to form the outer layer of the scaffold, and the release curve of the cytokine in the outer hydrogel is designed as "moderate burst release in a short time"; injecting the inner hydrogel mixture (pre-mixed neurotrophic factor) into the inner channel, and then further curing it by photocrosslinking. By adjusting parameters such as 3D printing parameters, the crosslinking degree of the inner hydrogel, and the loading dose of the neurotrophic factor, the release curve of the neurotrophic factor in the inner hydrogel is designed as "delayed and sustained release". The injection schematic diagram is as Figure 1 shown in G.

[0054] In this embodiment, the cross-section of the formed double-layer hydrogel scaffold is as Figure 1 shown in I. The cross-section of the inner hydrogel is square, and the corresponding thickness of the inner hydrogel is the inner diameter dimension in the figure. The cross-section of the outer hydrogel is a square ring, and the corresponding thickness of the outer hydrogel is half of the difference between the outer diameter and the inner diameter in the figure. Adjusting the outer diameter and the inner diameter can change the release time of the two drugs in the inner and outer layers. The larger the inner diameter, the longer the release time of the inner drug. The larger the difference between the inner diameter and the outer diameter, the thicker the outer layer, the longer the release time of the outer drug, and the more delayed the release of the inner drug. In addition, different shapes of the scaffold can be designed through 3D modeling software to fit the defect area and adjust the contact area with the tissue.

[0055] In this embodiment, the preparation process of a scaffold with PEGDA as the matrix material of the outer hydrogel, IL-8 as the cytokine, SilMA as the matrix material of the inner hydrogel, and NGF as the neurotrophic factor is further described in detail.

[0056] (I) Screening of the shape parameters and crosslinking degree of the double-layer hydrogel scaffold

[0057] According to literature reports, when bones heal naturally, neutrophil infiltration mainly occurs three days after trauma. Subsequently, the inflammation is inhibited and the regeneration process is initiated. Therefore, in this embodiment, by incorporating bovine serum albumin BSA as a simulation factor release, the shape parameters and crosslinking degree of the double-layer hydrogel scaffold are screened to obtain a double-layer hydrogel scaffold structure that conforms to the rule of "appropriate burst release of cytokines within a short period (3 days in this embodiment) and delayed continuous release of neurotrophic factors".

[0058] 1. Experimental Materials and Instruments

[0059] The experimental materials include: polyethylene glycol diacrylate (PEGDA) (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-PEGDA-400), silk fibroin methacrylate (SilMA) (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-SilMA-001), bovine serum albumin BSA (Sigma, V900933), LAP photoinitiator (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-LAP), lemon yellow light blocker (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-UVAbsorber-001), etc.

[0060] The experimental instruments include: projection stereolithography three-dimensional bioprinter, ultraviolet light curing lamp, etc.

[0061] 2. Experimental Procedures and Results

[0062] Prepare the first mixture, containing: LAP photoinitiator (0.25% w / v), lemon yellow light blocker (0.05% w / v), PEGDA, and BSA (10 mg / mL). The three-dimensional printing light intensity is 8 mW / cm 2 , the exposure time is 7 seconds, the exposure layer thickness is 100 μm, the peeling distance is 6 mm, and the peeling speed is 25 mm / min. Through 3D printing technology, the outer hydrogel is formed; prepare the second mixture, containing: LAP photoinitiator (0.25% w / v), SilMA, and BSA (10 mg / mL). Inject the second mixture into the inner channels of the previously formed outer hydrogel and further cure it by light to form a double-layer hydrogel scaffold.

[0063] First, screen the preparation parameters of the outer hydrogel that conform to the rule of appropriate burst release of cytokines within a short period (3 days in this embodiment). In this embodiment, three different PEGDA concentrations of 10%, 15%, and 20% w / v and four printing thicknesses of 0.2 mm, 0.4 mm, 0.6 mm, and 0.8 mm are used for printing. Figure 3Figure of the outer shape of the partially prepared hydrogel scaffold by printing. The cross-section shows that the inner and outer hydrogels have uniform thickness. It was found in the experiment that since PEGDA with a concentration of 10% w / v could not be formed at a thickness of 0.2 mm, it was excluded. The remaining 11 parameter combinations correspond to 11 BSA drug release curves. The prepared scaffolds were immersed in PBS, and the extracts were collected at 1, 2, 3, 4, 5, 6, 24, 48, 72, 96, and 120 hours, respectively, and the protein concentration released was measured using a BCA kit.

[0064] The results are as Figure 3 shown. According to the results of two repeated tests of Repeat1 and 2, under the preparation parameters of the outer hydrogel with 20% w / v + 0.2 mm, 15% w / v + 0.4 mm, and 10% w / v + 0.6 mm, the BSA loaded in the outer hydrogel can complete an appropriate initial burst release within 72 hours. In the subsequent examples, 15% w / v + 0.4 mm was selected as the preparation parameter of the outer hydrogel.

[0065] After determining the crosslinking degree and thickness of the outer hydrogel, scaffolds with different preparation parameters of the inner hydrogel were further prepared to determine the drug release curve of the inner hydrogel. In the examples of the present invention, three different SilMA concentrations of 10%, 15%, and 20% w / v and three printing thicknesses of 0.4 mm, 0.8 mm, and 1.2 mm were used for printing, with a total of 9 parameter combinations, corresponding to the drug release results of 9 inner hydrogels.

[0066] According to the design idea, the factor release curve of the inner gel should meet the following: there is no obvious initial burst release in the first 24 hours, and it can still release continuously and gently after 72 hours. The results are as Figure 4 shown. According to the results of two repeated tests of Repeat1 and 2, 10% w / v + 1.6 mm, 15% w / v + 1.6 mm, 20% w / v + 1.2 mm, and 20% w / v + 1.6 mm were preferably selected as the preparation parameters of the inner hydrogel. In the subsequent examples, 20% w / v + 1.2 mm was selected as the preparation parameter of the inner hydrogel.

[0067] (2) Screening of the optimal loading dose of IL-8 / NGF

[0068] After screening and determining the crosslinking degree and thickness of the inner and outer hydrogels, the optimal drug loading dose of the inner and outer hydrogels was further screened.

[0069] 1. Experimental materials and instruments

[0070] The experimental materials included: Alkaline Phosphatase Staining Kit (Beyotime Institute of Biotechnology, C3206), absolute ethanol (Maclean, E809056), RNA extraction kit (TAKARA, 9796), reverse transcription kit (TAKARA, RR036A), crystal violet staining solution (Beyotime Institute of Biotechnology, C0121), Wright-Giemsa staining solution (Beyotime Institute of Biotechnology, C0060), Transwell chamber (Falcon, 353097), etc.

[0071] Experimental cells: Mouse bone marrow stem cells (BMSCs), mouse neutrophils (NEUs), and mouse dorsal root ganglion neurons (DRGs), all isolated from 4-week-old C57 mice.

[0072] The experimental equipment included: fluorescence quantitative PCR instrument, incubator, microscope, centrifuge, pipette, etc.

[0073] 2. Experimental procedures and results

[0074] (1) Optimization of the concentration of IL-8 enhancing the effect of neutrophil-mediated stem cell recruitment

[0075] BMSCs (bone marrow mesenchymal stem cells) in good growth state were seeded on the upper chamber of the Transwell chamber. A double-layer hydrogel scaffold was placed in the lower chamber of the Transwell chamber for IL-8 release, and the doses of IL-8 loaded in the outer hydrogel were 0 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL, respectively. In the NEUs group, neutrophils were simultaneously seeded in the lower chamber of the Transwell for co-culture, and in the control group (CON group), no NEUs cells (neutrophils) were seeded in the lower chamber of the Transwell. After 72 hours of incubation, the non-migrated BMSCs in the upper chamber of the Transwell were wiped off with a cotton swab, and the migrated BMSCs in the lower chamber were stained with crystal violet dye to determine the recruitment efficiency of stem cells at different IL-8 loading doses. Meanwhile, the NEUs cells after co-culture were collected, and the nuclear morphology was observed by Wright-Giemsa staining to determine the effect of different IL-8 loading doses on neutrophil polarization.

[0076] The results were as Figure 5 shown. The scaffold did not show a significant direct effect on stem cell recruitment (i.e., the Con group), but under the condition of co-culture of neutrophils with stem cells / scaffolds (NEUs group), when the dose of IL-8 loaded in the outer hydrogel was 40 μg / mL - 120 μg / mL, it could promote the recruitment of stem cells. Among them, when the loading dose was 60 μg / mL - 100 μg / mL, the recruitment efficiency of stem cells was better. Figure 5A). In addition, different IL-8 loading doses lead to different polarizations of neutrophils. When the IL-8 loading dose is around 80 μg / mL, the nuclear morphology of neutrophils is "doughnut-like", which is consistent with the characteristics of N2-type polarization of neutrophils reported in previous studies; while the nuclear morphology of neutrophils in the control group (CON group) or the high-concentration group is "kidney-shaped" or "segmented lobed", indicating lower or higher maturity ( Figure 5 B).

[0077] (2) Optimization of the concentration of NGF enhancing the axonal extension effect of sensory neurons

[0078] DRG neurons in good growth state were seeded in cell culture plates. The hydrogel scaffolds were placed on the upper chamber of the Transwell chamber for NGF release. The doses of NGF loaded in the inner hydrogel were 0 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL respectively. After incubation for 72 hours, immunofluorescence staining was used to judge the promotion efficiency of different NGF doses on neuronal axonal extension.

[0079] The results are as Figure 6 shown. When the dose of NGF loaded in the inner hydrogel is 40 μg / mL - 120 μg / mL, it can promote the axonal elongation of sensory neurons, and the promotion efficiency of axonal elongation is the highest when it is 80 μg / mL - 120 μg / mL.

[0080] (3) Preparation and in vivo performance evaluation of the double-layer hydrogel scaffold

[0081] 1. Experimental materials and instruments

[0082] Experimental materials: Polyethylene glycol diacrylate (PEGDA) (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-PEGDA-400), Silk fibroin methacrylate (SilMA) (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-SilMA-001), Cytokine IL-8 (Novoprotein, C035), Neurotrophic factor NGF (Novoprotein, C060), LAP photoinitiator (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-LAP), Tartrazine light blocker (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-UV Absorber-001), etc.

[0083] Experimental instruments: Projection stereolithography 3D bioprinter, UV curing lamp, etc.

[0084] Experimental animals: 8-week-old type II diabetic C57 mice, 12-week-old type II diabetic New Zealand white rabbits.

[0085] 2. Experimental procedures and results

[0086] (1) Preparation of a bilayer hydrogel scaffold for sequential release of IL-8 / NGF

[0087] Use 3D modeling software to construct a scaffold model file that conforms to the shape of the defect. The scaffold consists of a pipeline with a bilayer structure. The specific steps are as follows:

[0088] Prepare the outer layer hydrogel mixture, including: LAP photoinitiator (0.25% w / v), tartrazine light-blocking agent (0.05% w / v), PEGDA (15% w / v), and IL-8. The printing thickness of the outer layer hydrogel is 0.4 mm, the 3D printing light intensity is 8 mW / cm 2 , the exposure time is 7 seconds, the exposure layer thickness is 100 μm, the peeling distance is 6 mm, and the peeling speed is 25 mm / min. Through 3D printing technology, the outer layer hydrogel is formed.

[0089] Prepare the inner layer hydrogel mixture, including: LAP photoinitiator (0.25% w / v), SilMA (20% w / v), and NGF. Inject the second mixture into the inner channel of the previously formed outer layer hydrogel. The diameter of the inner layer hydrogel is 1.2 mm, and after photocuring, a bilayer hydrogel scaffold is formed.

[0090] Among them, in the bilayer hydrogel scaffolds of different groups, the concentrations of the inner and outer loading factors are shown in Table 1 and Table 2. Table 1 and Table 2 are shown later.

[0091] (2) In vivo verification of the anti-inflammatory effect of the bilayer hydrogel scaffold for sequential release of IL-8 / NGF

[0092] Take 8-week-old diabetic C57 mice, weigh them, and anesthetize the mice by injecting 3% pentobarbital solution at a dose of 100 - 200 μL / 100 g body weight. After the righting reflex and corneal reflex of the mice disappear, shave the hair, disinfect the anterior surgical area of the hind limb with 75% alcohol, make an incision along the body surface projection of the long axis of the femur to the bone surface, separate the muscle, use a pioneer drill of the implanting machine to prepare a circular bone defect with a diameter of 2 mm in the middle of the femur, and use PBS to rinse the drill bit to cool down.

[0093] Implant the following groups of hydrogel scaffolds with the concentration combinations shown in Table 1 at the defect site for in vivo experiments. Use the scaffold loaded only with IL-8 implanted at the defect site as the control group (CON group), and the sample size of each group is n = 3. Suture the muscle and skin in sequence, disinfect the surgical area again, confirm that the breathing and mental state are stable, and after making good marks, put the mice back into the cage to wake up. Five days after the operation, euthanize the mice and collect the scaffolds and surrounding tissues, prepare a single cell suspension using an enzyme mixture, and perform flow cytometry to detect the mature neutrophils (CD45 + LY6G + CD11B +) to reflect the role of the reaction scaffold in inhibiting inflammation in diabetic bone regeneration.

[0094] Table 1 Factor loading doses of different groups in the in vivo anti-inflammatory efficiency experiment

[0095]

[0096] The results are as Figure 7 shown. Compared with the control group (CON group), the number of mature neutrophils in the bones of diabetic mice in the NGF group decreased significantly, indicating the remission of acute inflammation.

[0097] (3) In vivo verification of a bilayer hydrogel scaffold with sequential release of IL-8 / NGF for treating diabetic critical bone defects

[0098] Twelve-week-old New Zealand white rabbits with type II diabetes were weighed and anesthetized with 3% pentobarbital solution at a dose of 100 - 200 μL / 100 g body weight. After the righting reflex and corneal reflex of the rabbits disappeared, the hair was shaved, the surgical area on the top of the skull was disinfected with 75% alcohol, a sagittal incision was made along the midcranial suture to the periosteum, the periosteum was separated, and a bone drill was used with a planting machine to prepare circular bone defects with a diameter of 7 mm symmetrically on both sides of the skull. The drill bit was cooled with PBS flushing.

[0099] To compare the effects of simultaneous mixed release and sequential release on bone healing, the following groups of scaffolds with the concentration combinations shown in Table 2 were implanted at the defect sites for in vivo experiments, with the blank defect group as the control group (Blank group), and the sample size of each group was n = 6. The periosteum and skin were sutured in sequence, the surgical area was disinfected again, and after confirming that the respiration and mental state were stable and making good marks, the rabbits were put back into the cage to wake up. At 4 weeks and 8 weeks after surgery, the rabbits were euthanized, the specimens were fixed with 10% formalin solution for 12 - 24 hours, the specimens were rinsed with water for 30 minutes, and micro-CT imaging was used to qualitatively and quantitatively detect the bone formation of each group.

[0100] Table 2 Factor loading doses of different groups in the in vivo experiment for treating critical bone defects

[0101]

[0102]

[0103] The results are as Figure 8 shown. The Micro-CT results indicate that compared with the control group and the mixed delivery of the IL-8 + NGF group, the sequential drug delivery scheme of the IL-8 / NGF group can effectively promote the healing of the skull, effectively increase the bone tissue ratio (BV / TV), increase the trabecular number (Tb.N), and decrease the trabecular separation (Tb.sp).

[0104] The above results indicate that the bilayer hydrogel scaffold with sequential release of IL-8 / NGF has an efficient bone regeneration function under inflammatory conditions and can efficiently repair the refractory bone defects caused by chronic inflammation.

[0105] In summary, through the structural design of the inner and outer layer hydrogels, the hydrogel scaffold provided by the present invention realizes the sequential slow release of cytokines (such as IL-8) and neurotrophic factors (such as NGF), thereby being able to sequentially activate neutrophils and sensory nerves in the bone defect area. By first recruiting neutrophils in the defect area, it promotes stem cell homing and initiates osteogenesis; then, the locally sensory nerve ingrowth is induced by the lagging and continuous release of NGF, stimulating the sensory nerves to play an immune regulation role and promoting the dissipation of inflammation, ultimately being beneficial to promoting the repair and regeneration of refractory bone defects.

[0106] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A double-layer hydrogel scaffold capable of timed drug release, characterized in that: The scaffold comprises: an inner layer hydrogel and an outer layer hydrogel covering the inner layer hydrogel, wherein the outer layer hydrogel is loaded with cytokines, and the cytokines have the following functions: recruiting neutrophils and promoting polarization of neutrophils into a type with stem cell recruitment; the inner layer hydrogel is loaded with neurotrophic factors.

2. The double-layer hydrogel scaffold capable of timed drug release according to claim 1, characterized in that: The cytokines include IL-8.

3. The double-layer hydrogel scaffold capable of timed drug release according to claim 1, characterized in that: The neurotrophic factor includes at least one of NGF, BDNF, NT-3, NT-4, and NT-5.

4. The double-layer hydrogel scaffold capable of timed drug release according to claim 1, characterized in that: The outer layer hydrogel includes a first main material, and the inner layer hydrogel includes a second main material. The first main material and the second main material include at least one of polyethylene glycol diacrylate (PEGDA), methacrylated silk protein (SilMA), methacrylated hyaluronic acid (HAMA), methacrylated gelatin (GeIMA), methacrylated sodium alginate (AlgMA), and methacrylated dextran (DexMA).

5. The double-layer hydrogel scaffold capable of timed drug release according to claim 1, characterized in that: The first host material and the second host material are the same or different.

6. The double-layer hydrogel scaffold capable of timed drug release according to claim 1, characterized in that: The thickness of the outer layer hydrogel is 0.2 mm-0.6 mm, and the thickness of the inner layer hydrogel is 1.2 mm-1.6 mm.

7. The method for preparing a double-layer hydrogel scaffold capable of timed drug release according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step S1, preparing an outer layer hydrogel mixture, including: a first main body material, a cytokine and a photoinitiator; Step S2, preparing an inner layer hydrogel mixture, including: a second main body material, a neurotrophic factor and a photoinitiator; Step S3, the outer layer hydrogel mixture and the inner layer hydrogel mixture are coaxially 3D printed and photocured to obtain the double-layer hydrogel scaffold capable of timed drug release.

8. The preparation method according to claim 7, characterized in that: In the outer layer hydrogel mixed solution, the loading dose of cytokines is 60 μg / mL-100 μg / mL, and the concentration of the first main material is 10%-20% w / v.

9. The preparation method according to claim 7, characterized in that: In the inner layer hydrogel mixture, the loading dosage of the neurotrophic factor is 80 μg / mL-120 μg / mL, and the concentration of the second main body material is 10%-20% w / v.

10. Use of the double-layer hydrogel scaffold according to any one of claims 1 to 6, or the double-layer hydrogel scaffold prepared according to the preparation method according to any one of claims 7 to 9 in preparing products for bone defect repair and / or bone regeneration.

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