A composite scaffold material with functions of promoting bone sensory nerve remodeling, blood vessel regeneration and bone rapid repair and a preparation method thereof

Abstract

The application discloses a composite scaffold material with the functions of promoting bone sensory nerve remodeling, blood vessel regeneration and bone rapid repair and a preparation method thereof. The preparation process comprises the following steps: ①preparing a core particle with a porous core-shell structure hydroxyapatite (HA) coating through biomimetic mineralization and in-situ pore forming technology; ②placing the particle into a buffer solution containing a first growth factor, electrostatically adsorbing the growth factor in the pores through ultra-high specific surface area, and further coating the pores with in-situ self-polymerization dopamine (DA) on the surface to obtain a composite particle; ③obtaining a decellularized matrix by treating animal soft tissues, sequentially adding a second growth factor and the composite particle, and preparing the composite scaffold material through low-speed centrifugal defoaming and low-temperature freeze-drying technology. The composite scaffold material has excellent degradation performance, biocompatibility, factor controlled release performance, nerve promoting performance, vascularization performance and bone induction performance, and can realize the treatment of refractory bone defects of different sizes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composite scaffold material that promotes bone sensory nerve remodeling, angiogenesis and rapid bone repair, as well as its preparation method and application. Background Technology

[0002] Bone defects of critical size caused by trauma and other diseases pose a challenging clinical problem. This challenge stems from the fact that bone repair is a complex, multi-stage process involving inflammation, neurovascular network remodeling, rapid bone mineralization, and bone remodeling. Bone healing strategies targeting one or more stages of this process have been developed to accelerate bone healing. For example, dual VEGF / BMP-2 delivery systems are designed to first promote angiogenesis to accelerate bone healing, thereby achieving pro-angiogenic osteogenesis. However, the role of the nervous system in bone regeneration has been largely underestimated. In fact, bone is innervated by sensory and autonomic nerve fibers distributed in the periosteum, bone marrow, growth plate, and mineralized trabecular / cortical bone. Increasing evidence suggests that the nervous system plays an irreplaceable role in bone development and metabolism by directly or indirectly regulating the activity of osteoblasts and osteoclasts, thus allowing for the development of new bone healing strategies that utilize the regulatory functions of the nervous system during bone healing. Clinical and animal studies have shown that loss of sensory nerves easily leads to increased bone loss, resulting in delayed fracture healing. Qin et al. reported that calcitonin gene-related peptide (CGRP) secreted by sensory neurons has a positive effect on regulating vascular network remodeling. Furthermore, sensory nerve secretions such as substance P (SP) and CGRP in human serum can regulate the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, sensory nerves play an important biological role in regulating bone formation. Due to the multiple roles of sensory nerves in bone metabolism and homeostasis, reconstructing sensory neural networks at bone defect sites is considered a feasible strategy for regulating new bone formation.

[0003] The germination and branching of sensory nerves in the new callus are important upstream features of bone repair. Current data indicate that NGF-TrkA signaling is crucial for sensory nerve remodeling. While direct in situ release of NGF can enhance sensory nerve regeneration and subsequent osteogenic and bone remodeling, these methods are limited by the tendency of these factors to inactivate and release explosively. Natural extracellular matrix (ECM) has been widely used in bone repair due to its inherent ability to locally bind and control the release of soluble bioactive factors to regulate tissue regeneration. The decellularized extracellular matrix possesses cell adhesion and extensibility, and also enhances neurite elongation and controls neurite growth orientation. Considering these advantages, ECM can serve as an ideal carrier for NGF release, achieving rapid sensory nerve innervation.

[0004] In summary, this invention addresses the shortcomings of existing bone repair materials and meets market demands by developing a bone repair material and its preparation method. The bone repair material of this invention exhibits independently controlled release of multiple proteins, including growth factors, with release time adjustable from several days to several months; excellent pro-neurogenic osteogenic properties, angiogenic properties, and bone remodeling function; and excellent biocompatibility and biodegradability, with a degradation rate matching the osteogenic process. Summary of the Invention

[0005] To address the issues of existing bone repair materials neglecting the role of nerve innervation and lacking bioactivity in the scaffold, this invention provides a composite scaffold material and its preparation method that promotes bone sensory nerve remodeling, angiogenesis, and rapid bone repair.

[0006] The present invention adopts the following technical solution:

[0007] A method for preparing a composite scaffold material that promotes bone sensory nerve remodeling, angiogenesis, and rapid bone repair includes the following steps:

[0008] 1) First, inorganic salts and pore-forming agents are added to deionized water to prepare a modified biomimetic mineralization solution. Then, degradable core particles are incubated in the modified biomimetic mineralization solution. The biomimetic mineralization solution is changed every day and incubated for 7 days to form a mineral coating layer with a porous structure. The mineralized particles are washed with deionized water and dried for later use.

[0009] 2) Disperse the mineralized particles obtained in step 1) in a 0.2% BSA solution (pH=7.4) containing the first growth factor, and incubate at 37°C for 4.0 hours by rotation to obtain mineralized particles loaded with the first growth factor;

[0010] 3) Place the mineralized particles obtained in step 2) in a 10 mM Tris buffer solution (pH=8.5) containing dopamine (DA), and react for 2-6 hours to obtain composite particles containing the first growth factor.

[0011] 4) Process animal soft tissue to obtain decellularized matrix;

[0012] 5) Wash the decellularized matrix prepared in step 4) multiple times with PBS buffer, adjust the pH of the decellularized matrix to 7.20, add the second growth factor and mix thoroughly, and obtain the decellularized matrix containing the second growth factor through electrostatic adsorption.

[0013] 6) Add the composite particles obtained in step 3) to the decellularized matrix obtained in step 5), stir and electrostatically adsorb, disperse evenly, centrifuge at low speed to degas to obtain a mixture of semi-fluid composite materials, transfer the mixture to a mold, and let it stand at a temperature of -20℃ to -80℃ for 24 hours to obtain a frozen mixture.

[0014] 7) The frozen mixture prepared in step 6) is subjected to vacuum freeze-drying below 0°C to obtain the composite scaffold material.

[0015] In the above technical solution, further, in step 1), the inorganic salts in the biomimetic mineralization solution are improved by sequentially adding the following reagents to deionized water to form corresponding concentrations: 141mM NaCl, 4mM KCl, 0.5mM MgSO4, 1.0mM MgCl2, 25mM NaHCO3, 20.0mM HEPES, 5mM CaCl2, and 2mM KH2PO4; then a pore-forming agent is added, and the pH value of the biomimetic mineralization solution should be adjusted to 6.80.

[0016] Further, in step 1), the pore-forming agent used in the biomimetic mineralization process includes one or more of polymethyl methacrylate, methyl methacrylate, polyethylene glycol, polyvinyl alcohol, and polyvinyl butyral, with a concentration of 1-10 mg / mL.

[0017] Furthermore, the degradable core microparticles are one or more of β-TCP particles, magnesium phosphate particles, polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), and polyurethane (PU), and the ratio of the degradable core microparticles to the modified biomimetic mineralization solution is 25 mg: 50 mL.

[0018] Furthermore, in the rotation incubation process in step 2), the rotation speed is 100 rpm and the temperature is 37°C; the first growth factor is BMP-2, insulin-like growth factor, or fibroblast growth factor, dispersed in BSA solution at a concentration of 1-10 mg / mL.

[0019] Furthermore, in step 3), the concentration of DA in the Tris solution is 2 mg / ml, which needs to be dissolved in 10 mM Tris buffer solution under sonication. The solution pH is 8.5, and the incubation process needs to be kept in the dark throughout.

[0020] Further, in step 4), the decellularized matrix is ​​derived from soft tissues of pigs, cattle, or humans; the soft tissues include one or more of skin, blood vessels, ligaments, tendons, diaphragms, and mesentery.

[0021] Further, step 4) specifically involves cleaning the animal soft tissue and cutting it into tissue raw materials of the required size. Then, the raw materials are placed in a 1.0% sodium deoxycholate solution and placed on a shaker at 37°C and 120 rpm for 24 hours to remove cells. After that, the raw materials are soaked in a solution of 40 U / mL DNase I and 10 mM MgCl for 90 minutes to completely remove cell residues. Finally, the raw materials are sterilized with a 0.1% peracetic acid / 4% ethanol solution for 2 hours. After centrifugation to remove the sterilization solution, the raw materials are washed with a 0.9% physiological saline shaking bath for 30 minutes. After three cycles, the decellularized matrix is ​​cryopreserved at low temperature.

[0022] Further, in step 5), the second growth factor is one or more of nerve growth factor, transforming growth factor β, basic fibroblast growth factor, insulin-like growth factor, vascular endothelial cell growth factor, fibroblast growth factor, acidic and basic fibroblast growth factor, with a concentration of 1-10 mg / mL.

[0023] Furthermore, the bone repair material can be cut to a suitable size according to the size of the bone defect, or a mold can be customized using 3D printing technology based on imaging data to construct a bone repair material that conforms to the size of the bone defect.

[0024] Compared with the prior art, the present invention has the following technical features:

[0025] 1) The mineralized particles with long-lasting sustained-release properties used in this composite scaffold material are self-designed functional fillers. They are prepared by combining components such as biodegradable core particles (e.g., β-TCP particles), modified biomimetic mineralization solutions, pore-forming agents, growth factors, and dopamine as raw materials, and by combining multi-step mineralization coating construction, in-situ loading of functional proteins, and in-situ protein encapsulation processes. Biodegradable β-TCP particles are used as the core, and a porous hydroxyapatite mineralization coating is constructed on the surface of β-TCP using a self-designed modified biomimetic mineralization solution. The introduction of pore-forming agents forms a large number of radial pore structures on the mineralization coating, giving it a larger specific surface area to achieve efficient loading of growth factors. Meanwhile, dopamine polymerizes on the surface of mineralized particles loaded with functional proteins such as BMP-2 to form a polydopamine coating, thereby achieving in-situ encapsulation of pores loaded with growth factors, resulting in composite particles such as β-TCP / BMP-2 / polydopamine composite particles, which can further improve the preservation and independent controlled release performance of proteins, and the release time can be regulated from several days to several months.

[0026] 2) The decellularized material used in this composite scaffold is obtained by decellularizing the raw materials through a series of pretreatment steps involving animal tissues and a biochemical solution with multiple modified formulations. The decellularized matrix prepared through the aforementioned modified decellularization process retains its natural nanostructure, thereby protecting the numerous growth factor affinity sites on its surface to achieve specific adsorption and controlled release of various growth factors; it protects the original three-dimensional scaffold structure of the decellularized matrix and effectively removes antigens from the raw materials that could trigger an immune response in the human body, exhibiting certain mechanical properties and excellent biocompatibility; it retains the main components of the extracellular matrix, which can better promote cell adhesion, migration, and growth.

[0027] 3) This composite scaffold material is prepared by combining the aforementioned mineralized particles and decellularized matrix through a combination of molding, low-temperature freeze-drying, and other processes. By controlling the release rates of various proteins independently through different components, the growth rates of nerves, blood vessels, and bone at different stages of bone tissue repair can be regulated. The decellularized material slowly releases neuroinducible proteins to promote early bone sensory nerve regeneration. The polydopamine-encapsulated mineralized particles release bone-inducing proteins and amino acids and calcium and phosphorus ions required for bone repair over a long period, achieving rapid healing of bone defects, more accurately mimicking the normal bone repair process, and regulating bone sensory nerve and blood vessel regeneration, bone mineralization, and bone remodeling processes. The composite scaffold material prepared by this invention has excellent pro-neurogenic osteogenic properties, releasing neuropeptides to increase bone tissue growth rate by promoting sensory neuronization at the defect site; it also has excellent bone remodeling function, with sensory nerves in the new bone optimizing the osteogenic process through nerve innervation and avoiding ectopic osteogenic formation. Attached Figure Description

[0028] Figure 1 A schematic diagram of the preparation of the composite scaffold material (referred to as ECM@S / BMP-2) in Example 1;

[0029] Figure 2 Representative scanning electron microscope images of composite scaffold materials.

[0030] Figure 3 Example 2: Factor release rate diagram of bone repair materials: release rate diagram of materials BMP-2 and NGF;

[0031] Figure 4 Example 5: In vivo adaptive osteogenic properties of bone repair materials: A) Micro CT reconstructed images of the skull at 4 or 8 weeks post-surgery in each group; B and D) Quantitative data on bone volume (BV), bone volume fraction (BV / TV), and trabecular bone number (Tb.N) for different groups.

[0032] Figure 5Example 5: In vivo nerve regeneration performance of bone repair material: 28 days after surgery, immunofluorescence staining of sensory nerve-specific target calcitonin gene-related peptide (CGRP) was performed.

[0033] Figure 6 Example 5: In vivo vascular regeneration performance of bone repair material: Immunohistochemical staining of vascular-specific target CD31 on tissue 28 days after surgery in each group. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0035] Unless otherwise defined, the technical or scientific terms used in this disclosure shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0036] The modified biomimetic mineralization solution (i.e., modified simulated body fluid) of this invention is formulated as follows: the following reagents are added sequentially to deionized water to form the corresponding concentrations: 141mM NaCl, 4mM KCl, 0.5mM MgSO4, 1.0 mM MgCl2, 25mM NaHCO3, 20.0 mM HEPES, 5mM CaCl2, and 2mM KH2PO4; then 1-10 mg / mL pore-forming agent is added, and the pH value of the biomimetic mineralization solution should be adjusted to 6.80.

[0037] The pore-forming agent includes one or more of polymethyl methacrylate, methyl methacrylate, polyethylene glycol, polyvinyl alcohol, and polyvinyl butyral.

[0038] According to a specific embodiment of the present invention, the degradable core microparticles may be β-TCP particles with an average diameter of about 10 μm; in addition, they may be any one of magnesium phosphate particles, polylactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), and polyurethane (PU).

[0039] Example 1

[0040] β-TCP, after sieving, was placed in a modified simulated body fluid for biomimetic mineralization. After mineralization and loading with BMP-2, it was immersed in Tris buffer containing DA to form a DA coating on the surface. It was then washed with deionized water and freeze-dried to obtain β-TCP / BMP-2 / polydopamine composite particles. Fresh pigskin was washed, cut, and homogenized. It was then decellularized and sterilized using decellularization fluid and peracetic acid, and NGF affinity loading was added to obtain an NGF-containing decellularized matrix. The mineralized composite particles and the dry weight of the decellularized matrix were weighed at a ratio of 1:2. 10g of ECM (6.8g dry weight) and 3.4g of composite particles were weighed. Both were placed in a homogenizer, and an equal volume of physiological saline was gradually added and stirred until homogeneous. The resulting semi-fluid composite material was then freeze-dried in a container to obtain the composite scaffold material.

[0041] See the flowchart for composite scaffold material preparation. Figure 1 The resulting composite scaffold material, such as... Figure 2 As shown in the electron micrograph, the three-dimensional spatial structure of the decellularized matrix remains intact, and the pore size is uniform, exhibiting a good porous structure.

[0042] Example 2

[0043] The material from Example 1 was cut into small pieces of equal mass and placed in EP tubes. 4 mL of SBF (similar body fluid) was added, and the tubes were placed in a 37°C, 100 rpm air bath shaker to simulate in vitro release for 4 weeks. At specific time points, all released fluid was collected, frozen, and stored in the dark. Simultaneously, an equal volume of SBF was added to the release system. The concentrations of each factor in the released fluid at each time point were detected using NGF and BMP-2 corresponding ELISA kits, and the cumulative release amount of the factors was calculated.

[0044] See bone repair material factor release rate. Figure 3 .from Figure 3 As can be seen, this material has a sustained-release function for both NGF and BMP-2, and can continuously release both for more than 30 days. Moreover, the release rate of NGF bound to ECM is relatively fast in the first 15 days, reaching its peak at around 20 days, which can more quickly promote the sensory nerveization of surrounding tissues, thereby promoting bone growth.

[0045] Example 3

[0046] Eight-week-old rats were intraperitoneally injected with 4% sodium pentobarbital (40 mg / kg). −1After anesthesia, the rats were cut open along the sagittal midline to expose the skull. Using a bone-harvesting drill, two circular defects, each 6 mm in diameter, were drilled along the central line of the skull. The scaffold material from Example 1 was cut into circular slices with a radius of 3 mm and a thickness of approximately 0.2 mm and inserted into the bone defects. The control group underwent the same treatment but without any implantation of material. Six weeks later, the rats were euthanized, and the skulls were removed. A large amount of soft tissue was removed, and the skulls were fixed in 10% formalin solution for 48 hours. Micro-CT scans were then performed to analyze the defect sites.

[0047] Adaptive modulation of BMP-2-mediated bone formation capacity in bone repair materials (see [link to relevant documentation]). Figure 4 .like Figure 4 As shown, compared with the control group, the experimental group showed large areas of new bone growth at 4 weeks and almost completely covered the defect area at 8 weeks, demonstrating excellent osteogenic performance.

[0048] Example 4

[0049] The tissue specimens obtained in Example 3 were decalcified with 10% EDTA solution for 20 days. The prepared tissues were then dehydrated and embedded in paraffin. After sectioning, the slides were dewaxed and dehydrated with graded alcohols, and surface antigens were repaired using trypsin. The slides were washed three times with PBS for 5 min each time. The washed slides were infiltrated with 0.5% (v / v) Triton X-100 for 20 min and blocked with 1% (w / v) goat serum albumin at 37°C for 1 hour. They were then incubated overnight at 4°C with anti-TUBB3 and CD31 antibodies, and then incubated with secondary antibodies at 37°C for 1 hour. Finally, the cell nuclei were stained with 0.1 g / mL 4',6-diamidinyl-2-phenylindole (DAPI) for 5 min, and the samples were imaged and observed using a Leica fluorescence microscope.

[0050] See also: Neuroremodeling function and angiogenesis properties of bone repair materials Figure 5 , 6 .like Figure 5 As shown, compared with the blank group, the experimental group showed a large number of CGRP positive stainings and a large amount of sensory nerve remodeling in immunofluorescence. Figure 6 The experimental group showed numerous ring-shaped CD31 stainings at the site of new bone formation, along with extensive capillary regeneration.

[0051] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a composite scaffold material that promotes bone sensory nerve remodeling, angiogenesis, and rapid bone repair, characterized in that, Includes the following steps: 1) First, inorganic salts and a pore-forming agent are added to deionized water to prepare a modified biomimetic mineralization solution. Then, biodegradable core particles are incubated in the modified biomimetic mineralization solution, with the solution being changed daily for 7 days to form a mineral coating layer with a porous structure. The mineralized particles are washed with deionized water and dried for later use. The modified biomimetic mineralization solution contains: 141 mM NaCl, 4 mM KCl, 0.5 mM MgSO4, 1.0 mM MgCl2, 25 mM NaHCO3, 20.0 mM HEPES, 5 mM CaCl2, and 2 mM KH2PO4. Then, a pore-forming agent is added, and the pH value is adjusted to 6.

80. 2) Take the mineralized particles obtained in step 1) and disperse them in a 0.2% BSA solution containing the first growth factor, pH=7.4, and incubate them at 37℃ for 4.0 hours by rotation. This results in mineralized particles loaded with the first growth factor. 3) Place the mineralized particles obtained in step 2) in a 10 mM Tris buffer solution containing dopamine (DA), pH=8.5, and react for 2-6 hours to obtain composite particles loaded with the first growth factor. 4) Process animal soft tissue to obtain decellularized matrix; 5) Wash the decellularized matrix prepared in step 4) multiple times with PBS buffer, adjust the pH of the decellularized matrix to 7.20, add the second growth factor and mix thoroughly, and obtain the decellularized matrix containing the second growth factor through electrostatic adsorption. 6) Add the composite particles obtained in step 3) to the decellularized matrix obtained in step 5), stir and electrostatically adsorb, disperse evenly, centrifuge at low speed to degas to obtain a mixture of semi-fluid composite materials, transfer the mixture to a mold, and let it stand at a temperature of -20℃ to -80℃ for 24 hours to obtain a frozen mixture. 7) The frozen mixture prepared in step 6) is subjected to vacuum freeze-drying below 0°C to obtain the composite scaffold material.

2. The preparation method according to claim 1, characterized in that, In step 1), the pore-forming agent used in the biomimetic mineralization process includes one or more of polyethylene glycol and polyvinyl alcohol, with a concentration of 1-10 mg / mL.

3. The preparation method according to claim 1, characterized in that, The degradable core microparticles are one or more of β-TCP particles and magnesium phosphate particles, and the ratio of the degradable core microparticles to the modified biomimetic mineralization solution is 25mg:50mL.

4. The preparation method according to claim 1, characterized in that, In step 2), the rotational incubation process is carried out at a rotational speed of 100 rpm and a temperature of 37°C. The first growth factor is BMP-2, insulin-like growth factor, or fibroblast growth factor, which is dispersed in BSA solution at a concentration of 1-10 mg / mL.

5. The preparation method according to claim 1, characterized in that, In step 3), the concentration of DA in the Tris solution is 2 mg / ml. It needs to be dissolved in 10 mM Tris buffer solution under sonication. The solution pH is 8.5, and the incubation process needs to be kept in the dark throughout.

6. The preparation method according to claim 1, characterized in that, In step 4), the decellularized matrix is ​​derived from soft tissues of pigs, cattle, or humans; the soft tissues include one or more of skin, blood vessels, ligaments, tendons, diaphragms, and mesentery.

7. The preparation method according to claim 1, characterized in that, Step 4) Specifically, after cleaning the animal soft tissue, cut it into tissue raw materials of the required size, then place it in a 1.0% sodium deoxycholate solution and place it on a shaker at 37°C and 120 rpm for 24 hours to remove cells. After that, soak it in a solution of 40 U / mL DNase I and 10 mM MgCl2 for 90 minutes to completely remove cell residues. Finally, sterilize it with a 0.1% peracetic acid / 4% ethanol solution for 2 hours, centrifuge it to remove the sterilization solution, wash it with a 0.9% physiological saline shaking bath for 30 minutes, cycle it 3 times, and then freeze it at low temperature.

8. The preparation method according to claim 1, characterized in that, In step 5), the second growth factor is one or more of nerve growth factor, transforming growth factor β, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor, with a concentration of 1-10 mg / mL.

9. A composite scaffold material that combines the functions of promoting bone sensory nerve remodeling, angiogenesis, and rapid bone repair, characterized in that, It is prepared by the method described in any one of claims 1-8.

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