CN114470326A

CN114470326A - Preparation method and application of biomimetic mineralized collagen-glycosaminoglycan material

Info

Publication number: CN114470326A
Application number: CN202111497665.4A
Authority: CN
Inventors: 焦凯; 牛丽娜; 万千千; 马雨轩; 万美辰; 王晨语; 许克惠
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-12-06
Filing date: 2021-12-09
Publication date: 2022-05-13
Anticipated expiration: 2041-12-09
Also published as: CN114470326B

Abstract

The invention discloses a preparation method and application of a biomimetic mineralized collagen-glycosaminoglycan material. The disclosed method comprises soaking the collagen-glycosaminoglycan material in a mineralization liquid, which is a solution containing cerium acetylacetonate, to obtain the biomimetic mineralized collagen-glycosaminoglycan material. The material prepared by the invention has the functions of promoting the growth of local nerve blood vessels and improving the bone defect repair effect when being used as a bionic periosteum.

Description

Preparation method and application of biomimetic mineralized collagen-glycosaminoglycan material

Technical Field

The invention relates to a bionic mineralized collagen-glycosaminoglycan material technology, in particular to a preparation method and application of a cerium oxide bionic mineralized collagen-glycosaminoglycan material.

Background

The biomimetic mineralization material is a material in which inorganic elements are deposited in an organic scaffold material in a graded and ordered manner. Currently, biomimetic calcification and biomimetic silicification in collagen fibers and biomimetic mineralization in collagen fibers of zirconia are realized by utilizing the biomimetic mineralization principle. The biomimetic mineralized material has shown good application prospect in the field of bone defect repair filling materials.

Disclosure of Invention

The invention provides a preparation method of a biomimetic mineralized collagen-glycosaminoglycan material.

To this end, a method is provided comprising: soaking the collagen-glycosaminoglycan material in a mineralization liquid to obtain the biomimetic mineralized collagen-glycosaminoglycan material, wherein the mineralization liquid is a solution containing cerium acetylacetonate.

Further, the soaking comprises soaking at normal temperature and then soaking at 55-65 ℃.

Optionally, the solvent of the mineralized liquid is methanol and water, or ethanol and water.

Further, the collagen-glycosaminoglycan material is pretreated before being soaked in a mineralization liquid, the pretreatment comprises the steps of pretreating and soaking in a pretreatment liquid I and a pretreatment liquid II in sequence, the pretreatment liquid I is prepared from collagenase or mercaptopropionic acid, and the pretreatment liquid II is prepared from polyacrylic acid or ammonium polyphosphate.

Further, the polyacrylic acid has a molecular weight of 450000 or less.

Further, the pretreatment soaking is performed at normal temperature.

Optionally, the collagen-glycosaminoglycan material is a membrane material.

Optionally, the collagen-glycosaminoglycan material is selected from livestock and poultry egg membranes.

The biomimetic mineralized collagen-glycosaminoglycan membrane material prepared by the invention is used for preparing a biomimetic periosteum. The biomimetic mineralized collagen-glycosaminoglycan material prepared by the invention is also used for preparing bone defect repair materials.

Drawings

FIG. 1 is a scanning electron microscope image of mineralized egg membrane and unmineralized egg membrane; (a) is a scanning electron microscope picture of a cerium oxide biomimetic mineralized egg membrane; (b) is a mouse skull periosteum scanning electron microscope picture.

FIG. 2 is an infrared spectrum of mineralized egg membrane and unmineralized egg membrane, wherein Ce-ESM represents the mineralized egg membrane; ESM means untreated egg membrane; P-ESM represents the egg membrane treated by the pretreatment liquids (I and II); AmideA represents the normalized reference amide A peak; amide I represents Amide I peak; amide II represents the Amide II peak.

FIG. 3 is a Raman spectrum of the mineralized egg membrane and the unmineralized egg membrane.

FIG. 4 is transmission electron micrographs of mineralized day 2 and day 7 egg membrane fibers.

FIG. 5 is a cross-sectional energy spectrum analysis of mineralized egg membrane fibers.

FIG. 6 is X-ray diffraction analysis spectrum of mineralized egg membrane and unmineralized egg membrane.

FIG. 7 is a thermogravimetric plot of mineralized egg membrane and unmineralized egg membrane.

FIG. 8 is an electron energy spectrum of X-ray diffraction of mineralized and unmineralized egg membranes; (a) the figure is a complete spectrum; (b) the figure is a cerium element fine spectrum; the spectrum of Ce3d can be described by different parameters, which are respectively related to Ce³⁺(u0, v0, u 'and v') and Ce⁴ ⁺(u, v, u ", v", u '"and v'"), and Ce is obtained from the ratio of the peak intensities it occupies³⁺And Ce⁴⁺And (4) proportion.

FIG. 9 is an atomic force microscope representation of the surface morphology and elastic properties of an unmineralized egg membrane and a mineralized egg membrane; (a) an atomic force microscope three-dimensional reconstruction picture of the unmineralized egg membrane is obtained; (b) is a mineralized egg membrane atomic force microscope three-dimensional reconstruction picture; (c) is a histogram of the surface elasticity of the un-mineralized egg membrane and the mineralized egg membrane.

FIG. 10 is the release curve of cerium from 0-14 days after mineralization of egg membrane.

FIG. 11 shows the CD4/CD8 ratio of blood lymphocytes at day 7 and day 14 after flow cytometry analysis, with Ctrl representing the control.

FIG. 12 shows the hematoxylin-eosin (HE) staining and quantitative analysis of the number of blood vessels in the operation area at day 14 after the operation, i represents the sham operation control group and the partial enlarged view; ii represents the mineralized egg membrane implantation group and a partial enlarged view.

FIG. 13 shows the analysis of blood inflammatory factor content by enzyme-linked immunosorbent assay (Elisa) on days 1, 7 and 14 after the operation.

FIG. 14 is a CCK8 analysis of the effect of different cerium stimulation concentrations and different incubation times on cell viability.

FIG. 15 is a flow cytometry analysis of the effect of cerium on apoptosis.

FIG. 16 is a graph showing the physical barrier effect of the egg membrane after the in vitro cell culture and the green fluorescence in FIG. 16 shows the cytoskeleton, which indicates the existence of the cells.

FIG. 17 is HE staining and van Giesen staining of neogenetic periosteal area and quantitative analysis of neogenetic periosteal thickness at 4 weeks post-surgery.

FIG. 18 shows the immunofluorescence staining and quantitative analysis of Periostin in the neoperiosteal region at 4 weeks after surgery.

FIG. 19 is a MicroCT three-dimensional reconstruction and quantitative analysis for evaluation of post-operative 4-and 8-week bone defect repair.

FIG. 20 is von Kossa staining and quantitative analysis to assess post-operative 8-week bone defect repair.

FIG. 21 shows immunofluorescence staining and quantitative analysis of post-operative 2 and 4 weeks neogenetic periosteal area and neogenetic bone area neurovascular ingrowth.

Detailed Description

Unless otherwise specified, the terms or methods herein are understood or implemented using established methods of correlation, as recognized by one of ordinary skill in the relevant art.

The collagen-glycosaminoglycan material is a material mainly containing collagen and glycosaminoglycan, such as egg membranes of livestock and poultry (egg membranes, duck egg membranes, goose egg membranes and the like), and mainly containing collagen and glycosaminoglycan (namely the collagen-glycosaminoglycan material), has good biocompatibility and certain bioactivity, and has been applied in the aspects of wound dressings and the like.

According to the invention, cerium oxide is loaded in the collagen-glycosaminoglycan material by adopting a biomimetic mineralization principle, an initial membrane material is selected, and the mineralized material can be used as a biomimetic bone membrane material, so that the material has a physical barrier function, promotes the growth of nerve vessels in a defect area, and is beneficial to improving the bone defect repair effect.

The egg membrane is selected as an initial membrane material in the following embodiments, is a daily agricultural byproduct, is green and environment-friendly, and is easy to obtain. The reagents and materials used in the following examples are all commercially available products.

Example 1:

the embodiment is to soak an egg membrane in a mineralized liquid to prepare the mineralized collagen material, and the specific scheme is as follows:

(1) stripping egg membrane from the inner side of a commercially available egg membrane shell, and fully washing with deionized water for three times for use;

(2) soaking the egg membrane treated in the step (1) in a pretreatment liquid I (for 2 hours) and a pretreatment liquid II (for 8 hours) in sequence; wherein the pretreatment solution I is collagenase type I (# C0130; Millipore Sigma, Burlington, MA, USA) dissolved in Hank's balanced salt solution at a concentration of 100 mU/. mu.L, and the pretreatment solution II is 6.67X 10^-4M high molecular polyacrylic acid solution (HPAA, CAS # 9003-01-4; molecular weight: 450000; Millipore Sigma);

(3) soaking the membrane material treated in the step (2) in a mineralization solution, wherein the mineralization solution is prepared from 3mL of 24mg/mL cerium acetylacetonate (CAS # 206996-61-4; Millipore Sigma) methanol solution and 0.03mL of deionized water, and the mineralization solution is replaced once a day for 7 days; then, the egg membrane soaked in the mineralized liquid is processed for 48 hours at 60 ℃ under the sealed state;

(4) after washing, drying and ethylene oxide disinfection, the cerium oxide biomimetic mineralized egg membrane can be directly applied.

Example 2: EXAMPLE 1 characterization of the materials prepared

1. Scanning electron microscope to observe the surface structure of mineralized egg membrane and natural periosteum:

obtaining periosteum from the skull of a C57BL/6J mouse approved by the medical ethics committee, and then fixing in 2% glutaraldehyde buffer for 12 hours; the cerium oxide biomimetic mineralization of the egg membrane and the periosteum is dehydrated by gradient ethanol (50-100%) and incubated overnight in hexamethyldisilazane; then, the sample was sputtered with gold/palladium and observed with a scanning electron microscope (S-4800, Hitachi, Tokyo, Japan), and as a result, as shown in FIG. 1, it was found that the fibers of the mineralized egg membrane surface traveled and the fiber diameter distribution was similar to that of the periosteum.

2. Fourier infrared spectrum analysis of the mineralized egg membrane components:

drying the cerium oxide biomimetic mineralized egg membrane, the untreated egg membrane and the pretreated egg membrane with anhydrous calcium sulfate for 24 hours; fourier Infrared Spectroscopy (Shimadzu 8400S, Shimadzu Corp., Kyoto, Japan) was used at 4 cm^-1Has a resolution (number of scans: 32) of from 4000 cm^-1To 400 cm^-1Collecting and scanning; at the amide A peak (. about.3300 cm)^-1) The spectrum is normalized; the results of the spectroscopic analysis using IR solution software (Shimadzu) are shown in FIG. 2, and the sum of the peaks of amide I in the IR spectrum and 1440cm after pretreatment as compared with untreated egg membrane^-1The peak at (b) is higher and corresponds to the C ═ O stretching vibration peak in the carboxyl group. Amide I Peak (1630 cm) when comparing mineralized egg membrane to pretreated egg membrane^-1) And original 1440cm^-1The peaks at left and right drift to 1520cm^-1And 1450cm^-1This is probably due to the chelation of the carboxyl groups with cerium. Meanwhile, the mineralized egg membrane is 1390cm^-1Left and right (1382 cm)^-1Peak shift) increase in absorbance at 500-650 cm^-1The absorbance increases in the range corresponding to O-Ce-O stretching vibration. These results indicate that pretreatment of the egg membrane may bring more carboxyl groups to the egg membrane, which may then chelate with cerium, thereby facilitating sufficient penetration and mineralization of cerium oxide.

3. Analyzing the ingredients of the mineralized egg membrane by Raman spectroscopy:

anhydrous calcium sulfate for cerium oxide bionic mineralized egg membrane and unmineralized egg membraneDrying for 24 hours; raman spectroscopy (LabRAM HR Evolution, Horiba Scientific, Horiba ltd., Kyoto, Japan) was used, with a 785nm continuous semiconductor laser (Toptica Photonics,

germany) as an excitation source and 462cm after mineralization treatment^-1The peak is significant, suggesting symmetric vibration of oxygen atoms around the cerium atom.

4. Observation by a transmission electron microscope:

on days 2 and 7 of mineralization, the samples were fixed in 2% glutaraldehyde, dehydrated with ascending gradient ethanol series, immersed in propylene oxide as the transition medium, and embedded in blocks with epoxy resin; ultrathin transmission electron microscope sections (90 nm thick) are prepared on the polyvinyl acetate and carbon-coated nickel grids and examined by a transmission electron microscope, and as a result, as shown in fig. 4, uniform high-density development in the egg membrane fibers is found, which indicates that mineralization exists in the collagen core and glycosaminoglycan shell part, and sufficient mineralization permeability and uniform mineralization distribution are realized.

5. The energy spectrum analysis further defines the element composition and distribution:

the above ultra-thin transmission electron microscope section (90 nm thick) was subjected to elemental energy spectrum analysis (Talos-F200X, Thermo Fisher Scientific) by scanning transmission electron microscope, and the results are shown in fig. 5, which further confirmed the sufficient penetration and distribution of cerium oxide in collagen fibers.

X-ray diffraction analysis:

drying the cerium oxide biomimetic mineralized egg membrane and the unmineralized egg membrane, grinding the dried egg membrane and the unmineralized egg membrane into powder, and checking the powder by using an X-ray diffractometer (Rigaku America, Woodlands, TX, USA); the detection parameters are as follows: nickel filtering copper target radiation: 30kv, 20ma, 2 θ angle: 5-90 degrees, the scanning speed is 4 degrees/min, and the scanning step length is 0.02 degrees; as a result, as shown in FIG. 6, no sharp crystal peak was observed, indicating that cerium oxide did not crystallize sufficiently in the egg membrane, and was in a unique amorphous state.

7. Thermogravimetric analysis:

thermogravimetric analysis of cerium oxide biomimetic mineralized and unmineralized egg membranes was performed using a Thermo Hake TG/DTA 320 instrument (Seiko Instruments USA inc., Torrence, CA, USA):

about 10mg of membrane material was placed in a platinum pan and heated to 1000 ℃ in air at a rate of 5 ℃/min; the data are expressed as the relationship between weight loss and temperature, and as shown in fig. 7, the residual weight of the mineralized egg membrane after combustion is 27.31%, which is higher than the residual weight of the unmineralized egg membrane of 8.49%, indicating that the degree of mineralization of the egg membrane is conservative.

X-ray photoelectron spectroscopy:

firstly, drying a cerium oxide biomimetic mineralized egg membrane and an egg membrane for 24 hours by using anhydrous calcium sulfate, and detecting the cerium oxide biomimetic mineralized egg membrane and the egg membrane by using an X-ray photoelectron spectroscopy (K-Alpha, Thermo Fisher Scientific) (n-3); the spectral peak was calibrated at 284.8ev, the C-C/C-H energy position where C1s adsorbs carbon; the chemical state of ce3d was analyzed according to XPS data handbook and NIST X-ray photoelectron spectroscopy database (https://srdata.nist.gov/xps/) (ii) a As a result, as shown in fig. 8, a characteristic peak of cerium was observed, and further fine spectrum peak fitting analysis resulted in that cerium was composed of trivalent cerium and tetravalent cerium, which was associated with a unique self-reduction function due to oxygen vacancy in the structure of cerium oxide.

9. Atomic force microscopy analysis:

surface topography imaging of cerium oxide biomimetic mineralized egg membranes and unmineralized egg membranes atomic force microscopy (Keysight Technologies, Santa Rosa, Calif., USA) was performed in tapping mode using the NSG01 probe (typical force constant 5.1N m) from NT-MDT^-1) And a classical resonance frequency of 150 kHz; the mechanical performance is monitored in real time by using nano-indentation based on an atomic force microscope, the atomic force microscope nano-indentation is carried out on the surfaces of the cerium oxide biomimetic mineralized egg membrane and the egg membrane, and a silicon atomic force microscope probe with 13kHz resonance frequency and 0.2N/m nominal spring constant (PPP-NCL-11; Nanosensors) is used for the nano-indentation in a tapping mode; the elastic modulus at each position was quantified at an indentation depth of 300nm, and the elastic modulus at 5 separate positions for each sample was measured and averaged. The results are shown in FIG. 9, which shows the mineralization front and back surfaces of the egg membraneSurface morphology, observed as an increase in diameter of the mineralized fibers; the elasticity of the mineralized egg membrane is obviously improved when the egg membrane is measured in a mechanical analysis mode.

10. Cerium release profile:

immersing a cerium oxide biomimetic mineralized egg membrane (100mg) in 10mL of cell culture solution (10mg/mL) and placing the immersed egg membrane at 37 ℃ (n-3); the cumulative release profile of cerium in the cerium oxide biomimetic mineralized egg membranes over 14 days was obtained by measuring the cerium concentration in the leachate daily using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7700; Agilent Technologies, Santa Clara, Calif., USA). As shown in fig. 10.

Example 3: EXAMPLE 1 evaluation of biocompatibility of the prepared Material

In this example, a series of biosafety evaluations were performed on the cerium oxide biomimetic mineralized egg membrane prepared in example 1, and it was found that the biosafety was good. The stimulation of the materials on the mouse immune system is preliminarily explored by adopting a subcutaneous implantation experiment and found as follows: compared with an untreated group, the content of inflammatory factors in the blood of the implanted mice is not obviously changed; the tissue morphology has no obvious inflammatory manifestation; the lymphocyte inflammatory response in blood is mild. In vitro cell experiments also prove that the material has good biological safety performance within a certain concentration range.

1. Subcutaneous implantation of mice:

(1) male C57BL/6J mice at 8 weeks of age were randomly divided into two groups (number n-24 mice per group) for subcutaneous implantation; after sterilization and anesthesia, an incision was made in the back of the mouse; inert separation under skin to form cavity, implanting 10mm × 10mm cerium oxide biomimetic mineralized egg membrane, and carefully suturing wound, wherein all operations are performed under aseptic condition; sham operated groups served as controls.

(2) Serum lymphocytes were tested for CD3, CD4, and CD8 expression (n-5) using flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) on days 7 and 14 post-surgery; specifically, lymphocytes obtained by centrifugal separation using a lymphocyte separation medium (purchased from Beijing Solebao scientific Co., Ltd.) were diluted to 1X 10⁶Density of individual cells/mL, then incubated at 4 ℃ with fluorescently labeled CD3-cy5 antibody, CD4-PE antibody and CD8-FITC antibody (BioLeg)end inc., San Diego, CA, USA) for 30 minutes; cells were washed twice with Phosphate Buffered Saline (PBS) to remove excess antibody and resuspended in 300. mu.L PBS for detection; non-specific isotype-matched controls were used to determine background fluorescence; CD4 was determined using CellQuest Pro software (BD Biosciences)⁺/CD8⁺The ratio, results are shown in FIG. 11, and it was found that the CD4/CD8 ratio was increased on day 7, indicating a mild inflammatory response in the early phase, and that the CD4/CD8 ratio was recovered at day 14 without statistical difference from the control group (sham) indicating the regression of the inflammatory response.

(3) On day 14 after surgery, after mice were anesthetized to death, surgical field tissue (n ═ 5) was collected for histological analysis: after the specimens were fixed in 10% neutral buffered formalin, they were embedded in paraffin and cut into 5 μm thick sections, which were then stained with hematoxylin and eosin (H & E) (wuhan Servicebio, hubei, china) and examined by BX53 microscope (Olympus Corp., japan); the number of capillaries was measured in three randomly selected non-overlapping fields, and then averaged for each sample (n ═ 5) using ImageJ software (http:// ImageJ. net/ImageJ). The results are shown in fig. 12, which shows that the cerium oxide biomimetic mineralized egg membrane subcutaneous implantation does not cause obvious local inflammatory reaction and can promote local blood vessel formation.

(4) Blood was collected from the orbital venous plexus (n-3) of mice on days 1, 7 and 14 after surgery; the concentration of plasma inflammatory factor in peripheral blood of mice was measured using a mouse interleukin-2 (IL-2) quantitative factor ELISA kit, a mouse interleukin-4 (IL-4) quantitative factor ELISA kit, and a mouse interferon gamma (IFN-. gamma.) quantitative factor ELISA kit (R & D systems, USA). The results are shown in fig. 13, and it is found that the content of the inflammatory-related factors in the blood of the implanted mineralized egg membrane group does not change obviously, which indicates that the implanted mineralized egg membrane group does not cause obvious inflammatory reaction of the body.

2. In vitro cell experiments:

(1) respectively diluting a material leaching liquor (10mg/mL) which is released and tends to be stable by cerium and is soaked for 7 days by a DMEM cell culture solution by 10 times (1000 mug/mL), 100 times (100 mug/mL) and 1000 times (10 mug/mL) to obtain culture media with different cerium concentrations; then, 200. mu.L of the solution was usedDMEM cell culture fluid, 10 μ g/mL extract, 100 μ g/mL extract or 1000 μ g/mL extract at 37 deg.C (n-3)³Human oral mucosal fibroblasts (ATCC cell bank) were seeded in 96-well plates for 4, 12, 24 and 48 hours (5% CO2 and 100% humidity); add 20uL CCK-8 reagent to the wells and incubate at 37 ℃ for 1 hour; the absorbance values at 450nm were recorded by a microplate reader (BioTek, Winooski, VT, USA). As shown in FIG. 14, no statistical difference was found in cell activity over a range of concentrations (100. mu.g/mL of extract).

(2) Culturing human oral mucosa fibroblast with a concentration of about 1 × 10⁵Human oral mucosal fibroblasts (Ce-ESM group: human oral mucosal fibroblasts cultured in 100. mu.g/mL of leachate for 12 and 48 hours; Ctrl group: human oral mucosal fibroblasts cultured in general Cell complete medium for 12 and 48 hours; n ═ 3) were cultured at 4 ℃ for 20 minutes together with FITC-labeled annexin V and propidium iodide (Cell Signaling Technology, USA); after three washes, the cells were examined by flow cytometry (BD Biosciences), and as a result, as shown in fig. 15, no statistical difference was found in the apoptosis ratio.

Example 4: the material prepared in example 1 is used as the physical barrier function research of the bionic periosteum

This example places a cerium oxide biomimetic mineralized eggshell membrane sample at the bottom of each well of a 24-well plate with the inner surface facing upward and approximately 5 x 10 eggs planted on the inner surface of the membrane (n-5)³Individual oral mucosal fibroblasts; after 3 days, cells were stained for F-actin with FITC Phalloidin (Abcam; ab 235137); observing the inner surface and the outer surface of the film by using a confocal laser scanning microscope; using ImageJ software, the number of cells on the inner and outer surface was measured in three randomly selected non-overlapping fields per section. The results are shown in FIG. 16, where it was observed that the cells could not cross the membrane from one side to the other, indicating that the membrane had a good physical barrier.

Example 5: the material prepared in the embodiment 1 is used as a bionic periosteum for treating bone defect, so that the vascular nerve growth is promoted, and the bone defect repair effect is improved:

this example applied cerium oxide composite egg membrane for bone defect repair achieved good bone repair with adequate neurovascularization. Micro-CT detects the osteogenesis condition, and histomorphometric analysis and immunofluorescence staining experiments all prove that the cerized egg membrane and bone powder collagen implantation group can realize obviously more nerve ingrowth and blood vessel ingrowth and realize good bone regeneration.

(1) After ethical approval, 8-week male C57BL/6J mice were randomized into four groups (n-20); after sterilization and anesthesia, a 6mm incision was made using a scalpel, and a full-thickness defect of 3.5mm in diameter was made in the central area of the skull using a sterile trephine (precision tools, inc., shanghai, china); will be provided with

Collagen (swiss geiger) was implanted in the defect area, and the defect surface was covered with different membranes: (1) Ce-ESM group, the surface is covered with cerium oxide biomimetic mineralized egg membrane; (2) ESM group, surface covering egg membrane (collagenase I pretreatment); (3) no film is coated on the surface of the control group; (4) surface covering

Group BG (Switzerland); finally, the scalp was sutured with 4-0 absorbable suture and penicillin was injected continuously for 2 days after surgery.

(2) After 4 weeks in the mouse skull defect model, histological analysis was performed to assess periosteal regeneration: skull specimens were collected 4 weeks (n ═ 5) post-operatively and fixed and decalcified (24 hours in 4% paraformaldehyde and 7 days decalcified with 25% (w/v) EDTA-2Na in an ultrasonic rapid decalcifier (Pro-Cure medical science, ltd., hong, china); then dehydrating the specimen, embedding in paraffin, and cutting into 7 μm sections; h & E staining and van Gieson staining (Servicebio) were performed and all slides were observed under a BX53 microscope; analyzing the digitized image of the stained section using ImageJ software; the newly formed periosteal area (when the periosteal area can be distinguished) is divided into four equal-width parts with dashed lines, and the length of the dashed lines is measured and averaged to represent the periosteal thickness of one visual field; periosteum thickness was measured in three randomly selected non-overlapping fields per section and averaged for each sample, which was then normalized to the periosteum thickness of the control group. As shown in FIG. 17, it was found that the mineralized egg membrane group had ordered periosteal tissue ingrowth and had a bilayer structure similar to that of the natural periosteum, with a denser upper layer and a loose vessel-rich structure in the lower layer.

(3) 4 weeks after surgery, skull specimens (n ═ 5) were collected, fixed in 4% paraformaldehyde for 24 hours, and decalcified with 25% (w/v) EDTA-2Na in an ultrasonic rapid decalcifier (hong kong Pro-Cure medical science and technology limited, china) for 7 days; the samples were then embedded in frozen section embedding medium (OCT) (beijing, Solarbio, china) and cut into sections of 7 μm thickness using a cryomicrotome (CM1950, wilhelmy, germany); after 0.3% Triton X-100 treatment and 10% serum blocking, sections were incubated in anti-periostin antibody (1: 500; ab 215199; Abcam) at 4 ℃; the corresponding secondary antibody (1: 400; ab150080, ab150113, ab 150077; Abcam) was then added to the sections for specific binding and the nuclei were stained with DAPI (ab 228549; Abcam) for 15 min at room temperature; finally, the stained sections were photographed using a confocal laser scanning microscope (nikon A1R, nikon corporation, midon, tokyo, japan); mean fluorescence intensity of periostin was measured by ImageJ in three randomly selected non-overlapping fields per section and averaged for each sample, which was then normalized to a control group, and as a result, as shown in fig. 18, it was observed that the mineralized egg membrane group had high expression of periostin.

(4) At 4 and 8 weeks post-surgery, 5 mice per group were examined using the Inveon micro CT system (Siemens Munich, Germany) (n-5); each mouse was anesthetized with 1% pentobarbital (5mL/kg body weight) and scanned at 80keV, 500mA, and 19 micron accuracy, generating isotropic resolution two-dimensional images and used for three-dimensional reconstruction; further, the area (ROI) of 3 × 3 × 0.5mm around the defect was calculated and analyzed for trabecular bone thickness (tb.th.), new bone volume fraction (bone volume/total volume, BV/TV) and bone density (BMD) at the defect site, and as a result, as shown in fig. 19, it was found that the bone defect of the mineralized egg membrane group was significantly superior to the early repair and regeneration effect of other groups at 4 weeks and 8 weeks.

(5) Mouse skull defect model analysis by VK staining after 8 weeks: skull specimens were collected 8 weeks after surgery (n-5) and then chemically dehydrated and infiltrated with purified methyl methacrylate. The embedded specimens were sectioned at a thickness of about 50 μm (Leica SP1600, Mannheim, Germany) and stained with von Kossa stain (Servicebio), the black particle region in the ROI was considered positive; calculating the ratio of positive area to total field of view area using ImageJ software; VK staining analysis is carried out on the mouse skull defect model after 8 weeks, and the result is shown in figure 20, and the mineralized omentum group is found to realize a significantly better bone defect repairing effect, and quantitative analysis is further verified.

(6) The mouse skull defect model was subjected to immunofluorescence staining analysis for neurovascularization level 2 weeks and 4 weeks later and subjected to quantitative analysis, and the results are shown in fig. 21, and it was found that the mineralized omentum group promotes the growth of neurovessels into neogenetic periosteum region and neogenetic bone region.

Claims

1. A preparation method of a biomimetic mineralized collagen-glycosaminoglycan material is characterized by comprising the following steps: soaking the collagen-glycosaminoglycan material in a mineralization liquid to obtain the biomimetic mineralized collagen-glycosaminoglycan material, wherein the mineralization liquid is a solution containing cerium acetylacetonate.

2. The method of claim 1, wherein the soaking comprises soaking at room temperature, and then soaking at 55-65 ℃.

3. The method of claim 1, wherein the mineralized liquid is methanol and water or ethanol and water.

4. The method for preparing a biomimetic mineralized collagen-glycosaminoglycan material according to claim 1, wherein the collagen-glycosaminoglycan material is pretreated before being soaked in the mineralization solution, the pretreatment comprises sequentially pretreating and soaking in a pretreatment I solution and a pretreatment II solution, the pretreatment I solution is prepared from collagenase or mercaptopropionic acid, and the pretreatment II solution is prepared from polyacrylic acid or ammonium polyphosphate.

5. The method of claim 4, wherein the polyacrylic acid has a molecular weight of 450000 or less.

6. The method of claim 4, wherein the pre-soaking is performed at room temperature.

7. The method of claim 1, wherein the collagen-glycosaminoglycan material is a membrane material.

8. The method of claim 1, wherein the collagen-glycosaminoglycan material is selected from the group consisting of poultry egg membrane.

9. Use of a biomimetic mineralized collagen-glycosaminoglycan material produced according to the method of claim 7 or 8 for the production of biomimetic periosteum.

10. Use of the biomimetic mineralized collagen-glycosaminoglycan material produced according to the method of claim 1 for the production of bone defect repair materials.