CN110812531A

CN110812531A - Composite material, preparation method thereof and application thereof in decalcified bone matrix scaffold

Info

Publication number: CN110812531A
Application number: CN201911098346.9A
Authority: CN
Inventors: 王贤松; 郑志伟; 陈亚红
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2020-02-21
Anticipated expiration: 2039-11-12
Also published as: CN110812531B

Abstract

A composite material is a particle formed by oxidized graphene and baicalin through pi-pi bonds, the release speed of the baicalin is remarkably reduced, the long-term stable release of BAI is realized, osteogenic differentiation and formation of a bone formation microenvironment are facilitated, and acute inflammation caused by biological materials in a bone healing process is well controlled. The composite material provided by the invention is loaded on a decalcified bone matrix to form a decalcified bone matrix scaffold with bionic modification, proinflammatory macrophages are polarized into healing-promoting M2 type macrophages by releasing BAI, and the scaffold is endowed with dual regulation effects of osteoblasts and inflammatory cells, so that new bone regeneration is up-regulated, excessive fibrosis formation is inhibited, and remarkable curative effect is obtained in treatment of bone defect.

Description

Composite material, preparation method thereof and application thereof in decalcified bone matrix scaffold

Technical Field

The invention relates to a magnetic composite material, in particular to a nano particle formed by coating a polymer outside a magnetic particle and a preparation method thereof.

Background

Bone is one of the organs which are easy to be damaged in human body, and bone defect is usually caused by congenital disease, trauma or tumor excision and the like, and is usually repaired by means of operation with the help of bone substitute. The traditional principle for constructing bone substitutes is to regulate the physicochemical and biological properties of the material, providing a microenvironment conducive to osteoblast ingrowth and osteoclast differentiation. Using this principle, many bone substitutes have been invented and applied clinically, but their effectiveness varies widely. Acellular matrix bone is a foreign bone tissue that has been freed of inorganic minerals, retaining organic collagen. Because the acellular matrix has better biocompatibility and bioactivity, the acellular matrix is widely applied to clinical and basic researches on bone tissue repair. Because it is derived from fresh allogeneic or xenogeneic bone tissue, acellular matrix, it still causes graft rejection. However, elimination of its antigenicity inevitably destroys the bone formation. In order to solve the above problems, researchers have performed complex transplantation of acellular matrix and osteogenic active substance.

The Chinese patent application 201810344533.X discloses an integrated bone-cartilage repair scaffold and a preparation method thereof, which can prevent blood vessels from invading a cartilage layer and improve the connection tightness and stability between the bone layer and the cartilage layer. The cartilage repair layer is a formed porous calcium phosphate bioceramic, the middle layer is sulfhydryl-hyaluronic acid hydrogel, the cartilage repair layer is composed of I type collagen hydrogel and chondrocytes or mesenchymal stem cells, and the middle layer is positioned between the cartilage repair layer and in the porous structure of the formed porous calcium phosphate bioceramic to isolate the cartilage repair layer from the cartilage repair layer.

The Chinese patent application 201811443770.8 discloses a skull prosthesis for inducing bone tissue regeneration and a preparation method thereof, which can establish a digital model according to skull data of a patient, so that the skull prosthesis has higher matching degree with a skull defect part, and the operation difficulty during operation is reduced. The biological activity of the composite material is as follows, an inner inducing layer, a supporting layer and an outer inducing layer are sequentially arranged from inside to outside; the supporting layer is made of polyaryletherketone materials with the strength, hardness and weight equivalent to those of human bones, and plays a supporting role; the inner inducing layer and the outer inducing layer are made of bioactive materials and are respectively arranged on the inner surface and the outer surface of the supporting layer, so that the bone tissue regeneration inducing layer has good bioactivity and bone inductivity and can induce bone tissue regeneration; the supporting layer is provided with a plurality of grid holes, and bioactive materials are filled in each grid hole, so that the exchange of nutrient substances inside and outside the skull restoration can be promoted, and the tissue ingrowth is facilitated.

The Chinese patent application 201110094042.2 discloses a bone growth factor controlled release type bone repair material, a preparation method and application thereof, which have good mechanical properties and biocompatibility, realize the continuous slow combined release or sequential release of various bone growth factors, and improve the repair effect of bone defect tissues. The bone repair material is formed by wrapping two films on the pore surface of a porous bone repair inorganic material, wherein the first film mainly comprises bone growth factors and chitosan from inside to outside; the second layer of film is mainly prepared by mixing bone growth factors and biodegradable polyanion according to the mass ratio of (0-0.001) to 1.

Chinese patent application 201310723815.8 discloses a high-strength multi-level micro-nano structure silicon-based bone repair scaffold material, a preparation method and application thereof, wherein a mesoporous silicon-based xerogel is used as a matrix, and a mesoporous silica microsphere reinforcing agent is added to prepare the multi-level structure scaffold material with excellent mechanical properties. The silicon-based bone repair scaffold material prepared by the invention has highly communicated 200-micron macropores and 2-22nm nanometer mesopores, the porosity is up to 60-90%, and the mechanical property can be up to 10MPa, so that the silicon-based bone repair scaffold material can be used as a bone tissue engineering scaffold and a drug slow release carrier.

It has been found that the process of bone healing is biologically closely linked to the process of acute inflammation. Uncontrolled biomaterial-immune reactions lead to significant pathological changes in the local microenvironment, which is a bottleneck in the research of breakthrough solutions for regenerative medicine.

Disclosure of Invention

One object of the present invention is to provide a nanocomposite material having the effect of slowly releasing an anti-inflammatory active ingredient, which provides a good microenvironment for bone repair.

Another object of the present invention is to provide a nanocomposite material having the effect of slowly releasing an anti-inflammatory active ingredient to induce osteogenic differentiation of mesenchymal stem cells (BMSCs).

It is a further object of the present invention to provide a nanocomposite material having the effect of slowly releasing an anti-inflammatory active ingredient, which is advantageous for inducing macrophage polarization.

It is still another object of the present invention to provide a nanocomposite material having the effect of slowly releasing an anti-inflammatory active ingredient, which is advantageous for angiogenesis in tissue repair.

The fifth object of the present invention is to provide a method for preparing a nanocomposite, and the nanocomposite obtained by the method can be applied to bone repair.

A sixth object of the present invention is to provide a scaffold comprising a nanocomposite material, and a Demineralized Bone Matrix (DBM) forming a Demineralized Bone Matrix scaffold having a biomimetic modification for Bone regeneration.

A composite material which is a particle formed comprising Graphene Oxide (GO) and Baicalin (BAI).

Another composite material is a particle formed by a chemically weak interaction including Graphene Oxide (GO) and Baicalin (BAI).

Another composite material is a particle comprising Graphene Oxide (GO) and Baicalin (BAI) formed with pi-pi stacking weak interactions (i.e., weak interactions between aromatic rings).

The composite material is a nano-grade material, the particle size is 5-20 mu m, and the slow release of the baicalin is realized. Slow release is understood to mean that the amount of baicalin released in an in vitro solution environment (such as, but not limited to, water, aqueous solution or Phosphate Buffered Saline (PBS) at pH 7.4) is less than 40 w/w%, especially less than 30 w/w% within 24 hours. More preferably, the release amount of baicalin in the in vitro solution environment is less than 30 w/w% within 24 hours, and the release amount within 192 hours is less than 80 w/w%.

The composite material is a nano-scale material, and the graphene oxide contains carboxyl, carbonyl, hydroxyl, epoxy and other groups.

The composite material is a nano-grade material, and the ratio of the dosage of the graphene oxide to the dosage of the baicalin is 4-8: 1.

The composite material is a nano-scale material, and the dosage of graphene oxide is 40-80 and the dosage of baicalin is 10-20 according to weight.

A method of making the composite material of the present invention comprises the steps of:

an aqueous solution of 0.2 + -0.1 mg/mL BAI is added to an aqueous suspension of 0.5 + -0.1 mg/mL GO and stirred for 24 hours at 4 deg.C + -0.2 deg.C (e.g., ice water bath). The resulting mixture is then collected (e.g., centrifuged) and washed twice to remove unbound BAI.

The composite material prepared by the invention can be prepared into a composition (such as a preparation or a scaffold material) as an inducer for inducing macrophage polarization, or as an accelerant for promoting BMSCs to realize osteogenic differentiation and promote angiogenesis, and can be used for up-regulating new bone regeneration and inhibiting excessive fibrosis formation.

The composite material prepared by the invention is loaded on a bracket, such as: DBM scaffold, and preparing the decalcified bone matrix scaffold (GO-BAI/DBM scaffold) with bionic modification. The specific method comprises the following steps:

the DBM scaffold was immersed in a solution containing 1. + -. 0.2mg/mL of the composite material (GO-BAI) for 15. + -.5 minutes at 37. + -. 0.5 ℃ under degassing conditions, and then dehydrated (e.g., overnight under vacuum).

The GO-BAI/DBM scaffold is implanted into a bone tissue defect part, can remarkably promote calcium precipitation (P <0.05) of the defect tissue, and remarkably improves the rate of new bone formation and the rate of angiogenesis.

The technical scheme of the invention has the following beneficial effects:

the composite material provided by the invention is a particle mainly formed by oxidized Graphene (GO) and Baicalin (BAI) through pi-pi bonds, has biocompatibility, low toxicity to cells and high safety. The graphene oxide realizes the sustained and controlled release of the baicalin, remarkably reduces the release speed of the baicalin, realizes the long-term stable release of the BAI, is beneficial to osteogenic differentiation and formation of a bone formation microenvironment, and well controls the acute inflammation caused by biological materials in the bone healing process.

Graphene Oxide (GO) with bioactivity is used as an inorganic component, and the excellent physical, mechanical and biological properties of the graphene oxide can endow DBM with a better osteogenic differentiation and bone formation microenvironment. GO, which is rich in oxygen-containing functional group (e.g., carboxyl, carbonyl, hydroxyl and epoxy) ions, provides nucleation sites for the deposition of hydroxyapatite layers, a prerequisite for bone formation.

And use of such things as: compared with the components for regulating the immune system on cytokines such as IL4, IL13 and the like, baicalin avoids the condition that the biological activity is easy to lose due to the complex tertiary structure of protein and the defect of short biological half-life, reduces the using amount of active molecules (the biological active factors usually need to use high dose), and is more beneficial to the clinical application of the stent in bone repair.

The GO-BAI/DBM can provide an ideal microenvironment for inducing the differentiation of the BMSCs; at the same time, pro-inflammatory macrophages are polarized by the release of BAI to healing-promoting M2-type macrophages. By dual regulation of osteoblasts and inflammatory cells, thereby up-regulating new bone regeneration and inhibiting excessive fibrosis formation, a dual functional GO-BAI/DBM scaffold can be used as an effective solution to ensure a significant therapeutic effect in the treatment of bone defects.

Drawings

FIG. 1 is a UV-Vis spectrum of BAI, GO and the composite material of the present invention releasing BAI in vitro;

FIG. 2 is a graph of the in vitro release of BAI from a composite material of the present invention;

FIG. 3a is a drawing; an immunofluorescence staining pattern of M1 macrophage iNOS contained in RAW264.7 cells under the action of GO and the composite material;

FIG. 3b shows the M2 macrophage CD206 contained by RAW264.7 cells treated with GO and the composite material of the present invention⁺Immunofluorescent staining patterns;

FIG. 3c is an SEM electron micrograph of the morphology of RAW264.7 cells upon which GO and the composite of the present invention act;

FIG. 4a is a graph showing the co-incubation of GO and RAW264.7 cells acting as a composite material of the present invention and the ALP and ARS coloration results thereof;

FIG. 4b is a data graph of GO co-incubation with RAW264.7 cells and ALP staining thereof;

FIG. 4c is a graph of GO and composite effect RAW264.7 cell co-incubation and ARS staining data thereof;

FIG. 4d is a graph showing mRNA expression data of ALP, OCN, OPN and OSX when GO and RAW264.7 cells of the composite material of the present invention are incubated for 7 days;

FIG. 4e is a graph showing the results of electrophoresis of ALP, OCN, OPN and OSX proteins after GO and RAW264.7 cells of the composite material of the present invention are incubated for 7 days;

FIG. 4f is a graph of the in vitro co-incubation results of GO and human umbilical vein endothelial cells affected by the composite material of the present invention;

FIG. 4g is a graph showing the results of quantitative analysis of the total length of the resulting tissue, the length of the segments, and the total bifurcation length;

FIG. 5a is a graph of the results of 3D reconstruction at 6 and 12 weeks after implantation of the scaffold into a skull-deficient animal;

fig. 5b is a BV/TV result at 6 and 12 weeks after implantation of the scaffold into the skull-deficient animal (P <0.05, P < 0.01);

fig. 5c is a graph of BMD

morphometric analysis results

6 and 12 weeks after implantation of the scaffold into the skull-deficient animal (P <0.05, P < 0.01).

Detailed Description

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

In the following examples of the present invention, all data presented are expressed as mean ± standard deviation. Each experiment was repeated at least three times. The sstistical analysis is data obtained using the t-test (between groups) or one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. P <0.05, indicating statistically significant differences.

In the following embodiments of the present invention, the detection items and corresponding methods used are as follows:

and (3) detecting the activity of the cells: to assess cell viability after seeding cells onto scaffolds, live/dead cell assays were performed according to the manufacturer's instructions (Life Technologies, Calif., USA). Samples were imaged using confocal laser scanning microscopy (CLSM, Leica, Wetzlar, germany) on days 1 and 3 post-cell seeding.

Alkaline phosphatase (ALP) activity assay: according to the method described in the previous study (Inflammation,38,2015, 1493-1501). Briefly, BMSCs were incubated with 10, 20 and 40 μ g/mL GO or GO-BAI in culture for 3 days, then fixed and stained with an ALP kit (Beyotime, Shanghai, China).

Example 1

An aqueous solution of BAI was gradually added to an aqueous suspension of GO and stirred in an ice-water bath for 24 hours. Subsequently, the resulting mixture was collected by centrifugation and unbound BAI was removed by washing twice in ultrapure water. Characterization of the prepared nanocomposite particles was determined by UV-visible spectroscopy, and pure water was used as a reference sample (see fig. 1). The UV-Vis spectrum absorption spectrum shifts from 280nm to 316nm, from which it can be determined that BAI is anchored to GO,

to detect the BAI released from GO-BAI, 2mg GO-BAI solids and an equal amount of BAI were added to 10mL of ultra pure water. After they were mixed into a homogeneous solution, it was transferred to a dialysis tube (Spectra/Por Biotech; cellulose ester; MWCO 100,000) and immersed in 100mL of ultrapure water at 37 ℃ with slow stirring. Supernatants were collected at set time points and the concentration of BAI was determined by UV-visible spectroscopy (see FIG. 2). The BAI bound to GO released more slowly with a total release of 81.7% on day 14. The slow release of BAI favors osteoinduction.

Example 2

The DBM scaffold was immersed in a solution containing 1mg/mL GO or GO-BAI for 15 minutes at 37 ℃ under degassed conditions, then dehydrated overnight under vacuum. SEM characterizes the surface morphology of the composite scaffold. The water absorption, water retention and mechanical properties of the scaffolds were evaluated using the protocol described in the literature (J Control Release, 254,2017, 65-74; Biomaterials,33,2012, 72-79). The dried scaffolds were weighed, immersed in distilled water for 24 hours, blotted dry with filter paper, and weighed again to determine water absorption, while their water retention was determined by centrifugation (500rpm, 3 minutes) of the wet scaffolds. Compression testing was performed using an Instron 5542 universal tester (Instron, MA, USA) with a 500N load cell to evaluate mechanical properties. A stress-strain (σ - ∈) curve was determined, and the compressive modulus was calculated from the stress-strain curve based on the slope of the initial linear portion of the stress-strain curve (n ═ 5).

Example 3GO-BAI nanocomposites induce macrophage polarization

The toxicity of GO on RAW264.7 cells was first determined. GO at concentrations of 1, 5, 10, 20, 50 and 100 μ g/mL or RAW264.7 cells were incubated in culture for 24 hours. Cell viability was measured using the apoptosis detection kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions. The percentage of viable cells was calculated by flow cytometry.

Expression of M1 (inducible nitric oxide synthase, iNOS) and M2 macrophage (CD206) surface marker (CD206) was determined by immunofluorescence. After 2 days of culture, the conditioned media were filter sterilized and frozen at-80 ℃ until analysis or use. Cells were then fixed with 4% PFA and permeabilized with 0.1% Triton X-100. After blocking with 1% BSA, cells were incubated with anti-mouse iNOS or CD206 antibody (Abcam, MA, USA) overnight at 4 ℃ and then with Alexa Fluor 488-conjugated secondary antibody (Jackson Immuno Research Laboratories, Inc.). Then, cytoskeletal structure and nucleus of cells were counterstained with phalloidin (azo Life Science, Exeter, UK) and DAPI, respectively. RAW264.7 cells cultured in normal complete medium were used as a control. In cells co-incubated with GO, the proportion of M1 macrophages was higher than the control (Ctrl) and conversely the proportion of M2 macrophages was lower, whereas the proportion of M2 macrophages was significantly increased after co-incubation with BAI or GO-BAI composites (see FIGS. 3a and 3b)

The morphology of RAW264.7 cells when incubated for 2 days was further evaluated by SEM. Macrophages were sequentially fixed with 2% glutaraldehyde and dehydrated using a series of increasing concentrations of alcohol. After critical point drying and gold palladium coating, cell morphology was observed under SEM. Under electron microscopy, untreated control cells were round and no cytoplasmic spreading was seen. The cells of GO co-incubated group were surrounded by M1 macrophages, and the cells of GO-BAI co-incubated group were surrounded by M2 macrophages, but the outward expansion was also less (see FIG. 3c)

The gene expression of RAW264.7 cells was determined by RT-PCR using SYBR Premix Ex Taq (Takara, Japan) assay kit after 2 days of culture, total RNA was harvested and the mRNA expression levels of IL-1 β - α, arginase 1(Arg1) and resistin- α (Retn1- α) were quantified, normalized by Δ Δ Ct method using housekeeping gene GAPDH expression, the expression of inflammatory factors in RAW264.7 cells was determined by enzyme linked immunosorbent assay (ELISA), according to the manufacturer's instructions, the production of TNF- α and IL-10 in the culture medium was measured using ELISA kit (ExCellBio, shanghai china), M1 macrophage marker IL-1 β - α was highest in the co-GO group, the co-GO group was found to inhibit efficiently the genes of inflammatory cytokines and the modified marker of M2 resulted in a significant increase in the secretion of TNF-3 by incubation of macrophage secretion marker in the co-GO group of GO-BAI-96, TNF-3 secretion was found to be higher after incubation with TNF-3, TNF-9, TNF-7 was found to be able to inhibit the increased by TNF-7 secretion of TNF-7.

Example 4 osteogenic differentiation of BMSCs in RAW cell conditioned Medium

Osteogenic differentiation performance assay of BMSCs: differentiation of BMSCs on GO-BAI/DBM scaffolds was measured by immunofluorescence staining 14 days after cell inoculation. Briefly, the scaffolds were fixed with 4% Paraformaldehyde (PFA) for 20 minutes. The specimens were incubated with a secondary antibody targeting osteocalcin antibody (OCN, Abcam, MA, usa) and conjugated fluorescent dye and counterstained with DAPI.

To examine the osteogenic differentiation ability of BMSCs, BMSCs were administered at 1X 10⁴Cells were plated at a density per square centimeter. After 24 hours of incubation, the medium was replaced with RAW conditioned mediumTo which was added osteogenic supplement [5mM β -glycerophosphate (Millipore Sigma, Mo., USA), 10nM dexamethasone (Millipore Sigma) and 50 μ M L-ascorbic acid (Millipore Sigma)]. During the experiment, the medium was changed every three days. After 14 days of culture in osteogenic medium, BMSCs were fixed using 4% PFA, then ALP staining and Alizarin Red Staining (ARS) were performed according to the manufacturer's instructions (Beyotime, shanghai, china). Semi-quantitative analysis of ALP and ARS activity was performed as described above (Inflammation,38,2015,1493-1501; Acta bionatrielia, 3,2007,597-605), and the results were divided by the total protein content to obtain the semi-quantitative ALP analysis results.

After 7 days of induction culture, total RNA was extracted and gene expression levels of ALP, Osteopontin (OPN), OCN and Ostrix (OSX) were analyzed for Western blot analysis, 30mg of total protein was loaded on SDS/PAGE gel, which was incubated with anti-rat OPN or OCN antibody (Abcam) overnight at 4 ℃ and anti-rat β -actin monoclonal antibody (Invitrogen, CA, USA) was used as a control.

ALP staining showed a significant increase in BMSC cells after co-incubation of RAW264.7 cells with GO-BAI (GO-BAI + RAW) compared to control (Ctrl + RAW) or GO (GO + RAW), and ARS staining showed significant calcium deposition seen in GO-BAI co-incubated group (P < 0.05). While GO-BAI improved osteogenic differentiation of BMSC cells, ALP, OCN, OPN, OSX, etc. were significantly increased at 7 days of co-incubation (see fig. 4 a-4 e).

Example 5 evaluation of angiogenesis in Human Umbilical Vein Endothelial Cells (HUVEC) in RAW cell conditioned Medium

Angiogenesis at the site of injury is critical for rapid tissue regeneration. Here, in vitro angiogenesis experiments (EMBO journal,28,2009,2114-2127) were carried out according to the methods reported previously. Total capillary length, segment length and branch length were counted for each image using ImageJ 1.45 software (national institute of health, maryland).

No tubular body formation occurred 3 hours after macrophage-only medium was used, but in GO-or GO-BAI-containing medium, a net-like body could be clearly seen (see FIG. 4 f). Further quantitative analysis showed that the culture medium in which GO-BAI gave a mesh with a significantly larger bifurcation length and total length than the mesh obtained in GO medium (see fig. 4 g).

Example 6 in vivo evaluation of GO-BAI Implantation

PBS solutions containing GO or GO-BAI were prepared under sterile conditions. 100 μ L of GO-BAI (5mg/kg) in PBS (equivalent to about 1mg GO per rat) was injected subcutaneously using an insulin syringe. To limit variability between groups, each rat was injected subcutaneously with PBS, GO and GO-BAI simultaneously at three different sites. At the indicated time points required, mice were euthanized and the PBS, GO and GO-BAI administration areas and surrounding subcutaneous tissues were dissected. Tissues were immediately transferred to 4% PFA fixative followed by trichrome staining with hematoxylin-eosin (HE) and Masson.

Paraffin embedded tissue sections were deparaffinized and hydrated. M2 macrophages were stained with CD206 (1: 1000) and CD68 (1: 200) primary anti-body (Abcam) (overnight at 4 ℃) and then with anti-mouse Alexa Fluor 594 (1: 200) and anti-rabbit Alexa Fluor 488 (1: 200) secondary antibodies. DAPI was used to stain nuclei. M1 macrophages were stained with iNOS (1: 200) and CD68 (1: 200) primary antibody (Abcam).

NF- κ B signaling pathway activity was detected using Electrophoretic Mobility Shift Assay (EMSA). Nuclear and cytoplasmic extraction reagents (Thermo Scientific) were used to extract nuclear extracts from tissue surrounding the implantation site according to the manufacturer's instructions.

Female SD rats of 8 weeks of age were anesthetized, prepared for skin, and sterilized with 70% ethanol. Rat craniums were perforated using 5mm diameter trephines (Nouvag AG, Goldach, Switzerland). After the construct was implanted into the defect, the incision was sutured. Rats were randomized into four groups: (1) GO-BAI/DBM, (2) GO/DBM, (3) DBM and (4) NC (negative control).

Micro CT and histological observations: animals were euthanized under

general anesthesia

6 and 12 weeks post-surgery, and the skull was harvested and fixed in 4% PFA. The morphology of the reconstructed skull was assessed using an animal micro CT scanner (μ CT-80, Scanco Medical, Switzerland). After Micro CT scanning, Bone visualization was performed using 3D iso-surface rendering, and the ratio of Bone mass (Bone Volume) to Tissue mass (Tissue Volume) (BV/TV) and Bone Mineral Density (BMD) were calculated. The GO-BAI/DBM implanted mice had higher BV/TV values and BMD values than GO/DBM implanted mice and DBM implanted mice (see FIG. 5).

After detection by micro-CT, the samples were decalcified in 10% EDTA and then embedded in paraffin. The portion near the central region of the implant was stained with HE and Masson trichrome and observed using an optical microscope. It was found that there was capsule formation near the aggregates of GO and GO-BAI, with GO-BAI mediated capsules being smaller in area and thinner, but with more thick capsules around GO.

Fluorescent double labeling of calcein and alizarin red: to assess the rate of new bone formation, 3 mice were randomly injected with alizarin red (30mg/kg, millipore sigma) and calcein (20mg/kg, millipore sigma) from each group at week 6 and week 9, respectively. Calvarial bones of mice were taken at week 12, fixed overnight in 4% PFA, embedded in methyl methacrylate, and sectioned in the sagittal plane to 50mm thick undecalcified sections. Unstained sections were examined using CLSM. New bone formation was analyzed using Image-Pro Plus software (Media Cybernetics, MD, USA). For each sample, images were taken randomly for integrated densitometry (n-5).

The results show that the area of the new bone in the GO-BAI/DBM group is 47%, which is much higher than the area of the new bone in the GO/DBM group of 36.9% and the area of the new bone in the DBM group of 26.4%, and the obvious curative effect is obtained in the treatment of the bone defect.

Claims

1. A composite material, characterized by comprising particles formed by weak interaction of graphene oxide and baicalin in pi-pi stacking.

2. The composite material according to claim 1, characterized in that the particles have a size of between 5 μm and 20 μm.

3. The composite material of claim 1, wherein the release of baicalin from said particles in an in vitro solution environment is less than 40 w/w% within 24 hours.

4. The composite material of claim 1, wherein said particles release less than 30w/w baicalin within 24 hours and less than 80w/w baicalin within 192 hours of an in vitro solution environment.

5. The composite material according to claim 1, wherein the graphene oxide contains one or more groups selected from carboxyl, carbonyl, hydroxyl and epoxy.

6. The composite material according to claim 1, wherein the graphene oxide is used in an amount of 40 to 80% by weight, and the baicalin is used in an amount of 10 to 20% by weight.

7. Use of a composite material according to any one of claims 1 to 6 as a carrier in the preparation of a composition for inducing macrophage polarization, or promoting osteogenic differentiation and angiogenesis of BMSCs, up-regulating new bone regeneration and inhibiting excessive fibrosis formation.

8. A contrast agent characterized by comprising the magnetic nanoparticles according to any one of claims 1 to 5 as a carrier for MRI imaging, X-ray detection or fluorescence detection.

9. A scaffold comprising the composite material of any one of claims 1 to 6 and a demineralized bone matrix, said composite material being supported on said demineralized bone matrix.

10. A method for preparing a composite material according to any one of claims 1 to 6, characterized in that it comprises the following steps:

an aqueous solution of BAI was added to an aqueous suspension of GO and stirred at 4 ℃. + -. 0.2 ℃ for 24 hours. Subsequently, the resulting mixture was collected and washed twice to remove unbound BAI.