阅读说明：本技术 包含金属-多酚网络的骨修复材料及其制备方法和用途 (Bone repair material containing metal-polyphenol network and preparation method and application thereof ) 是由 解慧琪 张卿义 谭杰 袁奇娟 黄锴 李千金 黄丽萍 于 2021-09-26 设计创作，主要内容包括：本发明属于骨缺损修复技术领域,具体涉及包含金属-多酚网络的骨修复材料及其制备方法和用途。本发明提供了金属-多酚网络用于制备促进骨组织修复的药物组合物或组织工程材料的用途。本发明还提供了一种骨修复材料,包括基材,所述基材表面覆盖有上述金属-多酚网络。该骨修复材料具有很好的亲水性、力学性能和生物相容性。且由于该骨修复材料能够有效降低氧化应激,具有骨诱导性和免疫调节功能,能够有效促进骨缺损的再生。因此,本发明在骨缺损的治疗和修复中具有很好的应用前景。(The invention belongs to the technical field of bone defect repair, and particularly relates to a bone repair material containing a metal-polyphenol network, and a preparation method and application thereof. The invention provides application of a metal-polyphenol network in preparing a pharmaceutical composition or a tissue engineering material for promoting bone tissue repair. The invention also provides a bone repair material which comprises a base material, wherein the surface of the base material is covered with the metal-polyphenol network. The bone repair material has good hydrophilicity, mechanical property and biocompatibility. And the bone repair material can effectively reduce oxidative stress, has osteoinductive and immunoregulatory functions, and can effectively promote regeneration of bone defect. Therefore, the invention has good application prospect in the treatment and repair of bone defects.)

1. Use of a metal-polyphenol network for the preparation of a material for promoting bone tissue repair, characterized in that: the polyphenol in the metal-polyphenol network is selected from at least one of protocatechuic aldehyde, procyanidin, tannic acid or epigallocatechin gallate; the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ、Mg^ⅡOr Cu^ⅡAt least one of (1).

2. Use according to claim 1, characterized in that: the material is a material that eliminates oxidative stress, inhibits inflammation, modulates the immune microenvironment, promotes osteoblast regeneration, and/or promotes mineralization.

3. Use according to claim 1, characterized in that: the polyphenols in the metal-polyphenol network are selected from protocatechualdehyde; and/or, the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ(ii) a And/or, the metal-polyphenol networkThe molar ratio of the metal ions to the polyphenol in the complex is 3: 1-6: 1; and/or the metal-polyphenol network is obtained by mixing, reacting and drying a polyphenol solution and a metal ion solution.

4. A bone repair material characterized by: comprises a substrate, wherein the surface of the substrate is covered with a metal-polyphenol network; the polyphenol in the metal-polyphenol network is selected from at least one of protocatechuic aldehyde, procyanidin, tannic acid or epigallocatechin gallate; the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ、Mg^ⅡOr Cu^ⅡAt least one of (1).

5. Bone repair material according to claim 4, characterized in that: the base material is a membrane made of at least one of the following materials: CaCO₃、Fe₃O₄、SiO₂Graphene oxide, hydroxyapatite, polystyrene, polycaprolactone, polyethylene glycol, polyetheretherketone, polylactic acid, silk fibroin, gelatin, collagen, chitosan, polyurethane or cellulose.

6. Bone repair material according to claim 4, characterized in that: the polyphenols in the metal-polyphenol network are selected from protocatechualdehyde; and/or, the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ(ii) a And/or the molar ratio of metal ions to polyphenol in the metal-polyphenol network is 3: 1-6: 1; and/or the thickness of a film formed by covering the metal-polyphenol network on the surface of the base material is 200 nm-800 nm; and/or the thickness of the substrate is 0.4-0.8 mm.

7. A method of preparing a bone repair material according to any of claims 4 to 6 comprising the steps of:

(1) preparing a mixed solution of polyphenol and metal ions;

(2) and (2) immersing the base material into the mixed solution obtained in the step (1) for reaction, thus obtaining the product.

8. The method of claim 7, wherein: in the step (1), the pH value of the mixed solution is 8.5-9.8; and/or in the step (2), the reaction is carried out at room temperature for 24-48 hours.

9. Use of the bone repair material according to any one of claims 4 to 6 for the preparation of periosteum.

10. A periosteum, comprising: it is made of the bone repair material according to any one of claims 4 to 6, the shape of the base material being a film, a block or a granule.

Technical Field

The invention belongs to the technical field of bone defect repair, and particularly relates to a bone repair material containing a metal-polyphenol network, and a preparation method and application thereof.

Background

Bone defects caused by various reasons are still the key points and difficulties in clinical diagnosis and treatment. Existing research has focused primarily on bone defects themselves, and various grafting strategies and tissue engineered bone materials have been developed for this purpose. However, delayed or even no healing of the bone still occurs in a significant proportion of patients. Indeed, the role of periosteum in bone healing has long been overlooked. Over the past decade, there has been increasing evidence that the periosteum is a vascularized bone-cartilage organ that plays an important role in the development, homeostasis, bone repair and remodeling of the skeletal system. Periosteum is reported to contribute more than 70% to bone and cartilage formation in the early stages of autograft-mediated repair. Consequently, periosteum has great potential in the treatment of bone defects.

Currently, artificial periosteum, which is partially synthesized based on natural or high molecular materials, is used for clinical and preclinical research, such as polycaprolactone, (nano) hydroxyapatite, polyethylene glycol, polyether ether ketone, polylactic acid, silk fibroin, gelatin, collagen, chitosan and the like. The existing artificial periosteum is mainly used for soft tissue ingrowth, and is used as a scaffold for assisting cell migration to a defect area so as to promote bone regeneration or provide antibacterial performance and an osteoinductive signal. However, during bone repair, oxidative stress also inhibits stem/progenitor cell proliferation and differentiation, causing cell damage, apoptosis and necrosis, affecting the quality of bone regeneration repair. Meanwhile, oxidative stress can also induce osteoclast generation, so that osteoclastolysis is caused, and the dilemma of bone regeneration and repair is further aggravated. The existing artificial periosteum material can not eliminate the adverse effect of oxidative stress on the bone repair process, so that the performance of the existing artificial periosteum in the aspect of promoting bone tissue repair is limited.

Metal-polyphenol networks (MPNs) are network structures formed by self-assembly of natural polyphenols and Metal ions through coordination driving action. At present, the metal-polyphenol network is mainly used for medicine carrying in the field of medicine and is prepared into various targeted preparations. For example: the Chinese patent application CN202011371775.1 provides a medicinal preparation for treating lung diseases and a preparation method thereof, which comprises cells, a metal-polyphenol network and a medicament wrapped between the cells and the metal-polyphenol network. The Chinese patent application CN202011337685.0 provides a targeted nano vaccine and a product thereof based on a metal-polyphenol network structure, which comprises mesoporous silica nanoparticles loaded with ovalbumin OVA, wherein the surface of the mesoporous silica nanoparticles is coated with a metal-polyphenol network coating.

At present, no influence of the metal-polyphenol network on bone growth is found, and no report on the application of the metal-polyphenol network in treating or repairing bone defects is found.

Disclosure of Invention

In view of the defects of the prior art, the invention provides the application of a metal-polyphenol network, a bone repair material, a preparation method and application thereof. Aims to provide an artificial bone repair material which can eliminate oxidative stress in the bone repair process.

Use of a metal-polyphenol network, wherein the polyphenol in the metal-polyphenol network is selected from at least one of protocatechuic aldehyde, procyanidins, tannic acid, or epigallocatechin gallate, for the preparation of a material for promoting bone tissue repair; the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ、Mg^ⅡOr Cu^ⅡAt least one of (1).

Preferably, the material has the functions of eliminating oxidative stress, inhibiting inflammation, regulating immune microenvironment and promoting osteoblast regeneration and mineralization in the bone tissue repair process.

Preferably, theThe polyphenol in the metal-polyphenol network is selected from protocatechualdehyde; and/or, the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ(ii) a And/or the molar ratio of metal ions to polyphenol in the metal-polyphenol network is 3: 1-6: 1; and/or the metal-polyphenol network is obtained by mixing, reacting and drying a polyphenol solution and a metal ion solution.

The invention also provides a bone repair material, which comprises a substrate, wherein the surface of the substrate is covered with a metal-polyphenol network; the polyphenol in the metal-polyphenol network is selected from at least one of protocatechuic aldehyde, procyanidin, tannic acid or epigallocatechin gallate; the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ、Mg^ⅡOr Cu^ⅡAt least one of (1).

Preferably, the substrate is a membrane made of at least one of the following materials: CaCO₃、Fe₃O₄、SiO₂Graphene oxide, polystyrene, (nano) hydroxyapatite, polycaprolactone, polyethylene glycol, polyetheretherketone, polylactic acid, silk fibroin, gelatin, collagen, chitosan, polyurethane or cellulose. The polyurethane can be polyurethane with molecular weight of 80K-120K; the cellulose may be a cellulose having a molecular weight of 60K to 80K.

Preferably, the polyphenol in the metal-polyphenol network is selected from the group consisting of protocatechualdehyde; and/or, the metal ion in the metal-polyphenol network is selected from Zn^Ⅱ(ii) a And/or the molar ratio of metal ions to polyphenol in the metal-polyphenol network is 3: 1-6: 1; and/or the thickness of a film formed by covering the metal-polyphenol network on the surface of the base material is 200 nm-800 nm; and/or the thickness of the substrate is 0.4-0.8 mm.

Experiments prove that the composite material with bone repair performance can be obtained by modifying different base materials by adopting the metal-polyphenol network, for example, the metal-polyphenol network modified polyurethane or cellulose formed by protocatechuic aldehyde and Zn, and can promote osteoblast regeneration, biomimetic mineralization and bone defect regeneration repair. Therefore, the bone repair material can be used for bone repair of any scene of a human body, such as periosteum repair.

The invention also provides a preparation method of the bone repair material, which is characterized by comprising the following steps:

(1) preparing a mixed solution of polyphenol and metal ions;

(2) and (2) immersing the base material into the mixed solution obtained in the step (1) for reaction, thus obtaining the product.

Preferably, in the step (1), the pH of the mixed solution is 8.5-9.8; and/or in the step (2), the reaction is carried out at room temperature for 24-48 hours.

The invention also provides application of the bone repair material in preparing periosteum.

The invention also provides a periosteum which is prepared from the bone repair material, and the shape of the base material is membranous, massive or granular.

In the present invention, the "Zn" is^Ⅱ”“Mg^Ⅱ'OR' Cu^Ⅱ"refers to divalent Zn, Mg or Cu ions, respectively.

The invention applies a metal-polyphenol network to a medicament or tissue engineering material for bone repair for the first time, and provides a bionic bone membrane material based on the metal-polyphenol network.

Experiments prove that after different base materials are modified by the metal-polyphenol network, oxidative stress can be eliminated, inflammation can be inhibited, immune microenvironment can be regulated and controlled, osteoblast regeneration can be promoted, and biomimetic mineralization can be realized in the bone tissue repair process. Therefore, the metal-polyphenol network can greatly promote the regeneration and repair of bone defects. It is to be noted that Zn is known in the prior art^ⅡThe metal ions have the effect of promoting bone defect repair, but the effect of directly using the metal ions in a free state on bone defect repair is very limited, and the metal-polyphenol network can generate very excellent bone defect repair effect compared with the metal ions in the free state.

The bone repair material has good hydrophilicity, mechanical property and biocompatibility, can effectively reduce oxidative stress, has osteoinductivity, inflammation inhibition and immunosuppression, can effectively promote the regeneration of bone defects, and has good application prospect in the treatment and repair of bone defects.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows Control group, PCA group and PCA + Zn in Experimental example 1^ⅡA photograph of the group;

FIG. 2 shows Control group, PCA + Zn in Experimental example 1^ⅡGroup, PCA + Mg^ⅡGroup and PCA + Cu^ⅡThe results of the hydrophilic performance test of the group;

FIG. 3 shows Control group, PCA + Zn in Experimental example 1^ⅡGroup, PCA + Mg^ⅡGroup and PCA + Cu^ⅡThe scanning electron microscope and EELS test results of the group;

FIG. 4 shows Control group, PCA + Zn in Experimental example 1^ⅡGroup, PCA + Mg^ⅡGroup and PCA + Cu^ⅡXPS analysis of the panel;

FIG. 5 shows Control group, PCA group and PCA + Zn in Experimental example 1^Ⅱ(ii) raman spectroscopy test results for the panel;

FIG. 6 shows Control group, PCA group and PCA + Zn in Experimental example 1^ⅡThe mechanical property test result of the group;

FIG. 7 shows the results of Alamar Blue experiment in Experimental example 2;

FIG. 8 shows the results of live and dead staining of cells in Experimental example 2;

FIG. 9 shows the results of conventional and biochemical tests of 1/2 months post-operation blood in rats in Experimental example 2;

FIG. 10 shows the results of pathological examination of the organ tissue structures of heart, liver, spleen, lung, kidney and small intestine of the rat in Experimental example 2 after 1 month of surgery;

FIG. 11 shows the results of the DCFH-DA probe assay for detecting intracellular ROS levels in Experimental example 3;

FIG. 12 shows MDA level, SOD level and H concentration in each experimental group based on polyurethane in Experimental example 3₂O₂Results of cellular LDH release and apoptosis experiments under stimulation;

FIG. 13 shows the levels of SOD, ROS, MDA and H concentration in experiment example 3 for each experiment group using cellulose as a base₂O₂Results of LDH release experiments under stimulation;

FIG. 14 shows the results of detection of apoptosis in each experimental group based on cellulose in Experimental example 3;

FIG. 15 shows the results of alkaline phosphatase staining and intracellular ALP activity assay at 1/3 weeks and alizarin red staining and quantitative analysis at 2/3 weeks after differentiation induction in Experimental example 4;

FIG. 16 shows the results of three-dimensional reconstruction and quantitative analysis of bone density and bone volume in mcrio-CT scan in Experimental example 5;

FIG. 17 shows the results of H & E staining in Experimental example 5;

FIG. 18 shows immunohistochemical results for ALP, OCN and BMP-2 in Experimental example 5;

FIG. 19 shows immunohistochemical results of inflammation-related markers in Experimental example 5.

Detailed Description

Reagents and materials of which the sources are not specifically described in examples and experimental examples of the present invention are commercially available.

Example 1 Artificial bionic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the base material is polyurethane, and the metal-polyphenol network is formed from protocatechualdehyde and Zn^ⅡAnd (4) forming.

Polyurethane: the molecular weight is 80K-120K, PDI is less than or equal to 5, and the synthesis method comprises the following steps: 0.02mol of polycaprolactone (2000g/mol), 0.03mol of 4,4' -methylenebis (phenylisocyanate) and 20ppm of dibutyltin dilaurate were mixed and stirred at 85 ℃ for 2 hours to obtain an isocyanate-terminated prepolymer. Slowly adding 0.01mol of ethylenediamine for full chain extension, and stopping reaction by using diethylamine. Precipitating the solution in a large amount of water, drying for 24 hours at 80 ℃ to obtain a dry polymer, and carrying out electrostatic spinning on the obtained polymer to prepare the base material membrane.

The preparation method of the bionic periosteum comprises the following steps:

1g of Protocatechualdehyde (PCA) was dissolved in 50ml of deionized water, and the pH was adjusted to 9.8 by dropwise addition of aqueous ammonia. Adding zinc chloride (ZnCl)₂) Adding the mixture into a solution system according to the molar ratio of 6:1 to PCA, and fully and uniformly mixing by adopting an ultrasonic dispersion method. The substrate (polyurethane/cellulose film) was immersed in the above solution system and reacted at room temperature for 48 hours. After the reaction is finished, fully washing with deionized water, and drying to obtain PCA + Zn^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Zn^ⅡGroups).

Example 2 Artificial bionic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the substrate is a cellulose membrane, and the metal-polyphenol network is composed of protocatechuic aldehyde and Zn^ⅡAnd (4) forming.

The preparation method was the same as in example 1 except that the substrate was replaced with a cellulose film.

The cellulose molecular weight is 60K-80K, PDA is less than or equal to 3, and the preparation method of the cellulose membrane is as follows:

30g of bleached filter paper are first placed in 3000ml of distilled water and stirred for 12h until completely dispersed in the water. Then, 0.468g of tetramethylpiperidine nitroxide (TEMPO) (0.1mmol/g of cellulose) and 3.086g of sodium bromide (1.0mmol/g of cellulose) were added, respectively. When TEMPO and sodium bromide were completely dissolved, a solution of 193.6g,11 wt.% sodium hypochlorite (10mmol/g cellulose) was added. The pH of the system was adjusted to 10-10.5 at room temperature with continuous stirring with 10.1M hydrochloric acid. During the reaction, the pH gradually decreases. To maintain the pH at 10, 0.5M sodium hydroxide solution was added and monitored with a pH meter. The reaction was terminated when the pH of the system did not change. The rapidly oxidized cellulose is washed to neutrality. The obtained cellulose pulp was then diluted to 0.5 wt.% and sonicated for 2 hours at 900W using an ultrasound cell disruptor (JY99-IIDN, scientific biotechnology limited, ningbo, china). Finally, centrifugation was carried out at 10000rpm for 30min to remove undispersed impurities, resulting in CNF completely dispersed in water. Then, the cellulose membrane is prepared by adopting the processes of suction filtration, vacuum drying and the like.

This example gives PCA + Zn^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Zn^ⅡGroups).

Example 3 Artificial bionic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the substrate is polyurethane film, and the metal-polyphenol network is composed of protocatechuic aldehyde and Mg^ⅡAnd (4) forming.

The preparation is identical to example 1, except that zinc chloride is replaced by magnesium chloride. Obtaining PCA + Mg^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Mg^ⅡGroups).

Example 4 Artificial biomimetic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the substrate is a cellulose membrane, and the metal-polyphenol network is composed of protocatechuic aldehyde and Mg^ⅡAnd (4) forming.

The preparation is identical to example 2, except that zinc chloride is replaced by magnesium chloride. Obtaining PCA + Mg^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Mg^ⅡGroups).

Example 5 Artificial biomimetic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the substrate is a polyurethane film, and the metal-polyphenol network is composed of protocatechuic aldehyde and Cu^ⅡAnd (4) forming.

The preparation is identical to example 1, except that zinc chloride is replaced by magnesium chloride. Obtaining PCA + Cu^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Cu^ⅡGroups).

Example 6 Artificial bionic periosteum

The embodiment provides a bionic periosteum, which comprises a substrate, wherein a metal-polyphenol network is covered on the surface of the substrate. Wherein the substrate is a cellulose membrane, and the metal-polyphenol network is composed of protocatechuic aldehyde and Cu^ⅡAnd (4) forming.

The preparation is identical to example 2, except that zinc chloride is replaced by magnesium chloride. Obtaining PCA + Cu^ⅡMPN surface modification material (in the following experimental examples, the corresponding experimental group of samples is PCA + Cu^ⅡGroups).

Comparative example 1 base Material

The pure substrate polyurethane film was prepared in the same manner as in example 1, and the sample in the following experimental examples was designated as Control group.

Comparative example 2 base Material

A pure substrate cellulose membrane was prepared in the same manner as in example 2, and the sample-corresponding experimental group in the following experimental examples was designated as Control group.

Comparative example 3PCA surface modified substrate

A PCA surface modified substrate prepared as follows:

1g of PCA was dissolved in 50ml of deionized water, and the pH was adjusted to 9.8 by dropwise addition of aqueous ammonia. The substrate (the polyurethane film prepared in example 1) was immersed in the above solution system and reacted at room temperature for 48 hours. After the reaction is finished, fully washing with deionized water, and drying to obtain the PCA surface modification material (the sample corresponding experimental group in the experimental example is denoted as PCA group).

Comparative example 4PCA surface modified substrate

A PCA surface modified substrate prepared as follows:

1g of PCA was dissolved in 50ml of deionized water, and the pH was adjusted to 9.8 by dropwise addition of aqueous ammonia. The substrate (cellulose film prepared in example 1) was immersed in the above solution system and reacted at room temperature for 48 hours. After the reaction is finished, fully washing with deionized water, and drying to obtain the PCA surface modification material (the sample corresponding experimental group in the experimental example is denoted as PCA group).

In order to further explain the technical scheme of the invention, the beneficial effects of the invention are further explained by experimental examples.

Experimental example 1 Material characterization

The experimental example characterizes the materials prepared in the examples and comparative examples.

1. Experimental methods

The hydrophilicity of the material was evaluated by a water drop contact angle test, 8. mu.L of deionized water was dropped on the surface of the material, and Data-Physics OCA25 was used to measure the contact angle. The scanning electron microscope is used for evaluating the surface appearance of each group of materials, and an Electron Energy Loss Spectrum (EELS), an energy dispersion X-ray element spectrum (EDX) and an X-ray photoelectron spectrum (XPS) are used for analyzing the surface element composition, content and valence state of each group of materials. Uniaxial tensile experiments were used to characterize the mechanical properties of the materials of each group. All the above experiments measured 4 samples for statistical analysis.

2. Results of the experiment

FIG. 1 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡPhotograph of the group (sample of example 2), from which it can be seen that the pure substrate of the Control group is white; after PCA surface modification, the material is dark brown; PCA + Zn^ⅡAfter the MPN surface is modified, the color of the material is further deepened to be black.

FIG. 2 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), PCA + Zn^ⅡGroup (sample of example 1), PCA + Mg^ⅡGroup (sample of example 3) and PCA + Cu^ⅡIn the hydrophilic performance test of the group (sample of example 5), the surface of the material of the Control group is hydrophobic, the contact angle of a water drop reaches 112 degrees, and after the PCA surface is modified, the contact angle is reduced to 48 degrees. And PCA + Zn after MPN surface modification^ⅡGroup, PCA + Mg^ⅡGroup, PCA + Cu^ⅡThe group contact angles are respectively 18 degrees, 2 degrees and 21 degrees. This shows that the PCA surface modification can improve the hydrophilicity of the substrate, and the MPN surface modification can further improve the hydrophilicity of the substrate, which provides a basis for the good biocompatibility of the material.

FIG. 3 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), PCA + Zn^ⅡGroup (sample of example 1), PCA + Mg^ⅡGroup (sample of example 3) and PCA + Cu^ⅡScanning electron microscopy and EELS test results for the group (sample of example 5). The scanning electron microscope results indicate that after PCA and MPN modification, the surface of the material is not modifiedSignificant aggregation was found and the microporous structure was retained. The EELS test result shows that various metal ions are uniformly distributed on the surface of the material. This indicates that MPN can be uniformly modified on the substrate.

The results of EDX elemental spectroscopy are shown in the following table:

Wt％	Control	PCA	PCA+ZnⅡ	PCA+MgⅡ
					C	71.4	71	61.2	59.2
O	28.6	29	28	27.8
					Zn	-	-	1.3	-
Mg	-	-	-	1.2

EELS test and EDX element spectrum analysis after the modified material is cleaned show that the metal elements can stably exist on the surface of the material. This shows that the MPN surface modification method of the present invention has good metal ion loading performance and stability.

FIG. 4 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), PCA + Zn^ⅡGroup (sample of example 1), PCA + Mg^ⅡGroup (sample of example 3) and PCA + Cu^ⅡXPS analysis of the set (samples of example 5), PCA + Zn^ⅡGroup, PCA + Mg^ⅡGroup and PCA + Cu^ⅡThe XPS spectral line of the group obviously forms a characteristic spectral peak of Zn, Mg or Cu, which indicates that the MPN surface modification method has good metal ion loading performance and stability.

FIG. 5 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡRaman spectrum characterization of the set (sample of example 2) from which PCA + Zn can be seen^ⅡSet up in 499 and 667cm^-1A new Raman peak appears, and the Raman peak is attributed to Zn^ⅡCoordination bonds with PCA catechol. And 1200 + 1500cm^-1The peak at (a) corresponds to the catechol structure of the PCA. It can thus be shown that the method according to the invention does succeed in the production of MPN networks on the surface of a substrate.

FIG. 6 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), and PCA + Zn^ⅡAnalysis of the mechanical properties of the set (samples from example 1) the stress-strain curves suggest that the mechanical properties after surface modification are substantially similar. PCA and PCA + Zn^ⅡThe elastic modulus of the group is reduced from (38.9 +/-7.8) MPa of the Control group to (20.5 +/-5.1) MPa and (22.8 +/-2.5) MPa respectively. Control group, PCA group and PCA + Zn^ⅡThe tensile strengths of the groups were (32.7. + -. 1.0)(38.3 +/-6.6) and (38.2 +/-4.7) MPa, and the elongation at break is (73.5 +/-7.1)%, (122.1 +/-12.4)% and (121.2 +/-9.3)%, respectively. The above results show that PCA and PCA + Zn^ⅡThe MPN surface modification can moderately improve the strength of the composite material.

To sum up, this experiment demonstrates that examples 1-3 successfully modify the MPN network on the surface of the substrate, and the hydrophilicity and mechanical properties of the substrate are improved after the MPN network is modified.

Experimental example 2 evaluation of biocompatibility

1. Experimental methods

1) In vitro experiments

The materials of each experimental group are cut to be proper in size and adhered to the bottoms of the cell culture plates with different specifications. After uv sterilization, the medium was soaked overnight for subsequent experiments.

Cell survival on each set of materials was assessed using live-dead-cell staining: urine-derived stem cells (USCs) were seeded at a density of 10,000cells/well in 48-well plates and tested at 1/3 days post-seeding. Cell proliferation was assessed using Alamar Blue assay, cells were seeded at 3,000cells/well density in 96-well plates, Alamar Blue working solution was added at 1/4/7 days after seeding to incubate, and fluorescence intensity (excitation wavelength 550nm, emission wavelength 590nm) was recorded for each group. Cell morphology was assessed by cytoskeletal staining, cells were seeded at 10,000cells/well density in 48-well plates and examined at 1/3 days post-seeding. And meanwhile, measuring the cell stretching area, and further evaluating the influence of different surface modifications on the cell adhesion condition.

2) Evaluation of in vivo safety:

animal molding: six-week-old male Sprague-dawley (SD) rats were used for in vivo functional verification of material. 3% sodium pentobarbital is used for intraperitoneal injection for anesthesia. After sufficient shaving and sterilization, a longitudinal incision is made in the middle of the surgical area, the soft tissue is carefully separated, and the skull is exposed. Two symmetrical defects with a diameter of 5mm were made. Each group of materials is used for covering the defect area, and the Blank group is only used for drilling and molding and is not covered with any material. The soft tissue is closed layer by layer. Penicillin was injected 1 time a day for 3 consecutive days after surgery.

Each group of materials covered the surface of the SD rat cranial defect. Routine and biochemical tests of blood circulation were performed at post-operative month 1/3. And the materials are taken in the 1 st month after operation, and the important visceral organs of the SD rat are subjected to pathological examination.

2. Results of the experiment

Control group (sample of comparative example 1) PCA + Zn^ⅡGroup (sample of example 1), PCA + Mg^ⅡGroup (sample of example 3) and PCA + Cu^ⅡThe results of the Alamar Blue experiments for the groups (samples from example 5) are shown in the left panel of FIG. 7, and show that the cells show similar proliferation tendency on each group of materials, and the MPN surface modification does not affect the proliferation tendency of the cells. As shown in the right of FIG. 7, the live-dead-cell staining indicates that USCs are in the Control group (sample of comparative example 1), the PCA group (sample of comparative example 3), and PCA + Zn^ⅡThe surface of the material of group (sample of example 1) survived well and no significant dead cells were observed. Meanwhile, the proliferation tendency of the three groups of cells is similar.

Control group (sample of comparative example 1), PCA group (sample of comparative example 3), and PCA + Zn^ⅡThe cytoskeleton staining results of the group (sample of example 1) are shown in FIG. 8, and PCA group and PCA + Zn of cells^ⅡThe surface adhesion condition of the material is better than that of the Control group, and the cell extension area is obviously improved.

Control group (sample of comparative example 2), PCA group (sample of comparative example 4), and PCA + Zn^ⅡThe results of the evaluation of the in vivo biocompatibility of the group (sample of example 2) are shown in fig. 9 and 10, and no significant difference was observed between the groups of the conventional blood and the biochemical blood (red blood cell count, platelet count, white blood cell count, lymphocyte percentage, monocyte percentage, neutrophil percentage, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, blood urea nitrogen, albumin, and blood creatinine) at 1/2 months after the operation of the rat. After the operation for 1 month, the tissue structures of important organs such as heart, liver, spleen, lung, kidney, small intestine and the like of the rat are not abnormal. The above results suggest that the MPN surface-modified material has good biocompatibility.

The experimental results show that the bionic bone membrane material provided by the invention has good biocompatibility.

Experimental example 3 evaluation of antioxidant stress ability

1. Experimental methods

The materials of each experimental group are cut to be proper in size and adhered to the bottoms of the cell culture plates with different specifications. After uv sterilization, the medium was soaked overnight for subsequent experiments.

Wherein the culture medium comprises the following components: 50% v/v K-SFM + 33.75% v/v DMEM-HG + 11.25% v/v Ham's F12+ 5% v/v FBS, supplemented with 5ng/ml epidermal growth factor +50ng/ml bovine pituitary extract + 0.4. mu.g/ml hydrocortisone + 5. mu.g/ml transferrin +5ng/ml insulin +0.18mmol/L adenine +2nmol 3,3, 5-Triiodo-L-thyronine +100U/ml penicillin + 100. mu.g/ml streptomycin.

USCs were seeded at 10,000cells/well density in 48-well plates, 100. mu.M hydrogen peroxide (H)₂O₂) After 24 hours of stimulation, the DCFH-DA probe was used to detect intracellular Reactive Oxygen Species (ROS) levels. In addition, USCs were seeded at 100,000cells/well density in 6-well plates at 100uM H₂O₂After stimulation, the levels of intracellular Superoxide dismutase (SOD) and glutaraldehyde (MDA), a lipid oxidation end product, are detected, and the oxidative stress state of the cells is evaluated.

Evaluation of the respective groups of materials at high concentrations H₂O₂Protective effect on cells under stimulation. Cells were seeded at 10,000cells/well density in 96-well plates at 500-₂O₂(500. mu. M H for the experimental group with polyurethane as substrate₂O₂(ii) a For the experimental group with cellulose as substrate 1000. mu. M H was used₂O₂) After 24 hours of stimulation, culture supernatants were assayed for Lactate Dehydrogenase (LDH) activity to reflect cell damage. Similarly, cells were seeded at 200,000cells/well density in 96-well plates at 500. mu. M H₂O₂After 24 hours of stimulation, flow cytometry was performed to evaluate apoptosis and necrosis after stimulation.

2. Results of the experiment

FIG. 11 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), and PCA + Zn^ⅡGroup (sample of example 1), PCA + Mg^ⅡGroup (sample of example 3) and PCA + Cu^ⅡDCFH of groups (samples of example 5)Results of ROS detection by DA Probe, FIG. 12 shows Control group (sample of comparative example 1), PCA group (sample of comparative example 3), and PCA + Zn^ⅡMDA level, SOD level and high concentration H of group (sample of example 1)₂O₂Results of experiments on LDH activity and apoptosis under stimulation. FIG. 13 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡMDA levels and high concentrations H for the group (sample of example 2)₂O₂Results of experiments on LDH activity under stimulation. FIG. 14 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡApoptotic results of group (sample of example 2).

DCFH-DA probes show that PCA and MPN surface modification can effectively reduce H₂O₂Intracellular ROS levels after stimulation. Further experiments showed that PCA and PCA + Zn^ⅡThe SOD level of the group is obviously higher than that of the Control group, and the MDA level is obviously reduced. At the same time, under high concentration stimulation, PCA and PCA + Zn^ⅡThe group can significantly reduce cell damage. Furthermore, PCA + Zn^ⅡDegree of MDA level reduction and PCA + Zn in the group^ⅡThe reduction degree of the cell damage of the group is obviously better than that of the PCA group. The invention shows that the bionic periosteum material obtained by modifying the surface of polyurethane or cellulose by MPN has good protection effect on the damage of ROS to cells, and the protection effect of the bionic periosteum material is better than that of the bionic periosteum material obtained by simply modifying polyphenol compounds on the surface of a base material.

Experimental example 4 evaluation of osteoinductive ability

1. Experimental methods

Periodontal ligament stem cells (PDLSCs) were obtained by culturing in the following medium: alpha-MEM + 10% v/v FBS +100U/ml penicillin + 100. mu.g/ml streptomycin. And (3) inoculating the PDLSCs on the surfaces of the materials, and starting osteogenesis induction when the cell fusion rate reaches 70-80%. The osteogenic induction medium is: alpha-MEM + 10% v/v FBS + 50. mu.g/ml sodium ascorbate +100nmol/L dexamethasone +10mmol/L sodium beta-glycerophosphate +100U/ml penicillin + 100. mu.g/ml streptomycin. Alkaline phosphatase staining (ALP staining) and intracellular ALP activity were measured at 1/3 weeks after induction of differentiation, and Alizarin red staining (ARS staining) and quantitative analysis were performed at 2/3 weeks.

2. Results of the experiment

Control group (sample of comparative example 1), PCA group (sample of comparative example 3), and PCA + Zn^ⅡThe results of the group (sample of example 1) are shown in FIG. 15, and the ALP staining and intracellular ALP activity measurement results (on FIG. 15) at 1/3 weeks indicate that PCA + Zn^ⅡThe composition can effectively promote the expression of cell ALP. Similarly, ARS staining at 2/3 weeks and quantitative analysis (FIG. 15 lower) indicated PCA + Zn^ⅡThe composition can promote calcium salt deposition. The results show that the bionic periosteum material obtained by modifying the surface of polyurethane by MPN can effectively promote cell osteogenic differentiation and calcium salt deposition.

Experimental example 5 in vivo functional verification

1. Experimental methods

Six-week-old male Sprague-dawley (SD) rats were used for in vivo functional verification of material. 3% sodium pentobarbital is used for intraperitoneal injection for anesthesia. After sufficient shaving and sterilization, a longitudinal incision is made in the middle of the surgical area, the soft tissue is carefully separated, and the skull is exposed. Two symmetrical defects with a diameter of 5mm were made. Each group of materials is used for covering the defect area, and the Blank group is only used for drilling and molding and is not covered with any material. The soft tissue is closed layer by layer. Penicillin was injected 1 time a day for 3 consecutive days after surgery.

Taking materials 1 month after operation, performing three-dimensional reconstruction after mcrio-CT scanning, and quantitatively analyzing bone density and bone volume. After fixation and decalcification, the cells were embedded in paraffin and sliced at 3 μm. Conventional Hematoxylin & Eosin (H & E) staining, Masson staining, osteogenesis related indicators [ ALP, Osteocalcin (OCN), Bone morphogenetic protein-2 (Bone morphogenetic protein-2, BMP-2) ] and inflammation related indicators [ (Tumor necrosis factor-alpha (TNF-alpha), Interleukin-6 (Interleukin, IL-6), Macrophage mannose receptor protein (Macrophage mannose receptor, MMR, alpha known as CD206) ] immunohistochemical staining were performed.

2. Results of the experiment

FIG. 16 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡMcrio-CT scanning of the panels (samples of example 2) three-dimensional gravimetricAnd establishing a bone density and bone volume quantitative analysis result. After 4 weeks of operation, no obvious bone regeneration is seen in the defects of Blank group and Control group in micro-CT three-dimensional reconstruction, the defect repair degree of PCA group is superior to the two groups, and PCA + Zn is added^ⅡThe group repair effect is optimal. Bone density and bone volume analysis showed the same trend.

FIG. 17 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡGroup H (sample of example 2)&E staining result shows that PCA and PCA + Zn^ⅡThere was a large amount of new bone formation in the group, and cubic osteoblasts were visible at the edges of the new bone, suggesting the formation of a functional layer with osteogenic activity. Furthermore, PCA + Zn^ⅡThe osteogenic effect of the group was superior to that of the PCA group, consistent with in vitro studies. In contrast, in the Blank and Control groups, the defect area was filled with a large amount of soft tissue, impeding the bone defect repair process. In Masson color staining, PCA + Zn^ⅡThe group presented the most abundant red-blue transition and red-stained areas, suggesting a significant mineralization process and bone tissue reconstruction for the group, second to the PCA group. The defect area of Blank and Control group is full of blue-stained loose connective tissue, and no obvious new bone formation occurs.

FIG. 18 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡALP, OCN and BMP-2 immunohistochemistry results for group (sample of example 2) showed PCA + Zn^ⅡThe expression level of the constitutive bone-related protein is highest, especially in osteoblasts surrounding new bones, and meanwhile, bone cells which are not fully mature in the bone pit also stain positively. The PCA group expressed the next level. While OCN, ALP and BMP-2 were expressed at the lowest level in Blank and Control groups.

FIG. 19 shows Control group (sample of comparative example 2), PCA group (sample of comparative example 4) and PCA + Zn^ⅡResults of immunohistochemistry for inflammation-related indices in group (sample of example 2) revealed that TNF-. alpha.and IL-6 were highly expressed in the matrix outside the bone tissue in Blank and Control groups, while in PCA group and PCA + Zn^ⅡThe group expression level is obviously reduced. The markers of M2-type macrophages positive for CD206 expression showed a reverse trend, in PCA and PCA + Zn^ⅡExpression was clearly up-regulated in the group.

The results show that in an in vivo experiment, the bionic periosteum material obtained by surface modification of cellulose by MPN has excellent osteoinductivity, inflammation inhibition and immunosuppression, and can effectively promote regeneration of bone defect.

According to the embodiments and experimental examples, the metal-polyphenol network is applied to the field of bone repair and used for preparing a bone repair material, so that the bionic bone membrane material based on the metal-polyphenol network is obtained. The bionic bone membrane material has good hydrophilicity, mechanical property and biocompatibility. And the bionic periosteum material can effectively reduce oxidative stress, has osteoinductive, inflammation inhibition and immunosuppression effects, and can effectively promote the regeneration of bone defect. In addition, tests are carried out on two different base materials of polyurethane and cellulose in experimental examples, and the metal-polyphenol network modified on different base materials can obtain the composite material with bone repair performance. Therefore, the invention has good application prospect in the treatment and repair of bone defects.