CN113174082B

CN113174082B - Preparation method and application of bionic silicification collagen material

Info

Publication number: CN113174082B
Application number: CN202110345013.2A
Authority: CN
Inventors: 焦凯; 牛丽娜; 马雨轩; 万美辰; 万千千
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2022-12-02
Anticipated expiration: 2041-03-30
Also published as: CN113174082A

Abstract

The invention discloses a preparation method and application of a bionic silicification collagen material. The preparation method comprises the step of soaking ACE collagen in a pretreatment solution to obtain the bionic silicification collagen material, wherein the pretreatment solution is composed of choline chloride and a silicic acid solution. The bionic silicified collagen material constructed by the invention has good physical and chemical properties, and can promote the growth of peripheral sensory nerve axons and activate a sensory nerve mTOR signal pathway to secrete Semaphorins 3A.

Description

Preparation method and application of bionic silicification collagen material

Technical Field

The invention relates to a bionic collagen material technology, in particular to a preparation method and application of a bionic silicification collagen material.

Background

The existing bionic silicification collagen material is prepared by the previous method that polypropylene ammonium chloride is used as a collagen pretreating agent, and the polypropylene ammonium chloride has dose-dependent toxicity, so that the industrial application of the bionic silicification collagen material is limited.

Disclosure of Invention

Aiming at the defects or shortcomings of the prior art, the invention provides a preparation method of a bionic silicification collagen material.

Therefore, the preparation method provided by the invention comprises the step of soaking ACE collagen in a pretreatment solution to obtain the bionic silicification collagen material, wherein the pretreatment solution consists of choline chloride and a silicic acid solution.

Further, the pH value of the pretreatment liquid is 5-6.

Further, the silicic acid solution is prepared from Silbond40, ethanol and water.

Choline chloride is a hydrochloride of choline, is a high-efficiency nutritional supplement, is used as a vitamin product, and is widely applied to medicines, health products and food nutritional additives. The invention creatively uses choline chloride as a pretreating agent and a stabilizing agent for biomimetic silicification of collagen fibers, and synthesizes novel biomimetic silicified collagen scaffold materials (SCSs) by using only choline chloride. The novel bionic silicification material with stable pure choline has the advantages of simple synthesis process, stable performance of reaction products and potential clinical application prospect.

Semaphorin 3A (semaphorin 3A, sema 3A) is a diffusible protein with chemochemotaxis and growth-guiding effects on nerve axons, and plays an important role in various physiological processes such as embryonic and nervous system development, angiogenesis, tumorigenesis, immunoregulation and the like. Sema3A is a secreted protein belonging to the Semaphorin family, which is a neurite-directing factor and is secreted by sensory neurons such as the dorsal root ganglion (Semaphorin III can repulse and inhibit adapt sensing activities in vivo). In recent years, increasing research shows that Sema3a is widely expressed in the process of embryonic development and influences the growth direction and aggregation of nerve fibers, so Sema3a is considered to play an important role in the processes of nerve growth, development and damage repair. The bionic silicification collagen material constructed by the invention has good physical and chemical properties, and the novel bionic silicification material promotes the growth of peripheral sensory nerve axons and activates a sensory nerve mTOR signal path to secrete Semaphorins 3A.

Based on the method, the invention also provides the application of the bionic silicified collagen material prepared by the method in preparing the Sema3A secretion promoter in dorsal root ganglion cells.

Further provides the application of the leaching liquor of the bionic silicification collagen material prepared by the method in preparing the Sema3A secretion promoter in dorsal root ganglion cells.

Meanwhile, the invention provides application of silicic acid in preparing a promoter for Sema3A secretion in dorsal root ganglion cells.

Drawings

FIG. 1 is a recombinant type I collagen scaffold before and after siliconization according to example 1, wherein the left side is before siliconization; the right side is after silicification;

FIG. 2 is the morphological change and elemental analysis of the biomimetic siliconized collagen scaffold and the simple collagen scaffold of example 1; wherein: a: observing the collagen scaffold (ruler =500 nm) B under a scanning electron microscope: elemental analysis; CS is a pure collagen scaffold; SCS: siliconizing the collagen scaffold;

fig. 3 is a transmission electron micrograph of the biomimetic siliconized collagen scaffold of example 1 (ruler =200 nm);

FIG. 4 is an infrared spectroscopic analysis of the biomimetic siliconized collagen scaffolds and simple collagen scaffolds of example 1;

FIG. 5 is a silicic acid release profile of the biomimetic siliconized collagen scaffold prepared in example 1;

FIG. 6 is the porosity of the biomimetic siliconized collagen scaffold prepared in example 1;

FIG. 7 is the tensile strength of the biomimetic siliconized collagen scaffold prepared in example 1;

FIG. 8 is an immunofluorescence staining of the morphological effects of dorsal root ganglion cells of the leaching solution of biomimetic silication collagen scaffold in example 2; (a) representative immunofluorescence images of dorsal root ganglion cells (scale: 200 μm), (B) quantitative analysis of neurite length, (C) quantitative analysis of neuronal viability;

FIG. 9 is a graph showing the effect of real-time quantitative PCR detection of the leaching solution of the silicated collagen scaffold on the mRNA expression of each neuropeptide of dorsal root ganglion in example 2;

FIG. 10 is a graph of the effect of immunofluorescence assays of the siliconized collagen scaffold leachate on the expression of dorsal root ganglion Sema3a and Sema4d proteins in example 2 (scale =50 μm);

FIG. 11 is an immunofluorescent staining of the morphological effect of different concentrations of silicic acid on dorsal root ganglion cells of example 3; (a) representative immunofluorescence images of dorsal root ganglion cells cultured for 3 days at different concentrations of silicic acid, (ruler =200 μm), (B) quantification of neurite length, (C) quantification of neuronal survival;

FIG. 12 is the real-time quantitative PCR assay of mRNA expression of each neuropeptide of dorsal root ganglia in 10. Mu.M silicic acid medium in example 3;

figure 13 is an immunofluorescence assay of example 3 for dorsal root ganglion neuropeptide expression in 10 μ M silicic acid medium (scale =50 μ M);

FIG. 14 shows the activation of the PI3K-Akt-mTOR signaling pathway in example 3;

fig. 15 is a Sema3A immunofluorescent stain in dorsal root ganglion tissue in example 4 (scale =200 nm).

Detailed Description

Unless otherwise indicated, the terminology herein is to be understood in light of the knowledge of one of ordinary skill in the relevant art.

Unless otherwise specified, reagents and equipment used in the following examples are commercially available products. In addition, the statistical analyses involved in the following examples are: the data obtained by detection are subjected to normal distribution test and variance homogeneity test, and the two groups are compared by using two-tail T test (alpha = 0.05).

The orthosilicic acid solution used in the present invention can be prepared by using an existing method. The components of the pretreatment solution are choline chloride and an orthosilicic acid solution, the concentration of the choline chloride and the orthosilicic acid solution and the final pH value of the pretreatment solution can be optimized according to actual conditions, and the goal is to obtain the bionic silicified collagen material with better physicochemical characteristics and corresponding activity.

The leaching liquor of the bionic silicification collagen material is obtained by soaking the bionic silicification collagen material by adopting organism body fluid simulation liquid, and for example, buffer solution or double distilled water are common organism body fluid simulation liquid.

Example 1: preparation of bionic silicified collagen material

Mixing Silbond40, absolute ethanol, deionized water and 37% hydrochloric acid (pH adjuster) at room temperature in a molar ratio of 1.875 396.79;

uniformly mixing the 3% ortho-silicic acid solution with 36mM (Choline chloride, molecular weight: 139.62, sigma-Aldrich, st.Louis, MO, USA) in equal volume to prepare a pretreatment solution, and adjusting the pH value of the pretreatment solution to 5.5 and the concentration of the ortho-silicic acid to 1.5% in volume;

trimming three-dimensional recombinant type I collagen sponge (ACE collagen, ACE Surgical Supply Co., inc, MA, USA) into collagen blocks with the diameter of 0.3 cm, and washing with Milli-Q deionized water for three times for later use;

biomimetic silicification was performed on collagen sponges using four protocols shown in table 1, respectively:

TABLE 1

In each protocol, collagen pieces were soaked in 1ml of choline chloride stabilized silicic acid solution, incubated at 37 ℃ for 7 days, and the silicic acid precursor solution was changed daily.

1.1 Observation of biomimetic siliconized collagen scaffold Material

After the ACE collagen is placed in a choline-stabilized silicic acid precursor solution for 7 days, anhydrous calcium sulfate is dried overnight, and general observation is carried out; wherein the stent material prepared according to the third embodiment is shown in fig. 1 (the stent material prepared according to the third embodiment is tested and described in the following tests and subsequent examples);

rinsing the bionic silicified collagen scaffold and the ACE collagen scaffold which is not treated by any treatment for three times by using deionized water, and then sequentially carrying out gradient dehydration on the bionic silicified collagen scaffold and the ACE collagen scaffold for 30 minutes by using 50%, 70%, 80% and 95% ethanol; dehydrating with 100% ethanol for 3 times, each for 15 min; HMDS was dehydrated for 30 minutes; the fixed samples were air dried overnight;

fixing the sample on an aluminum base by using conductive double-sided carbon adhesive, and drying the sample by using anhydrous calcium sulfate overnight; after the sample was sprayed with gold/palladium using an ion sprayer (Humme, technologies inc., alexandria, VA, USA), the ultrastructure of the collagen scaffold was observed under a scanning electron microscope (Hitachi, tokyo, japan) at a voltage of 10 to 15kV, and a selective elemental analysis was performed on the mineralized surface of the collagen fiber.

Under a scanning electron microscope, the surface morphologies of biomimetic silicified collagen and ACE collagen without any treatment are shown in the figure and 2: the outline of single collagen before and after silicification is clear without collapse; comparing the forms of the collagen in a scanning electron microscope, the untreated collagen scaffold has a specific periodic strip pattern of the collagen; the biomimetic siliconized collagen scaffold showed clear signs of extrafibrous mineral deposition; elemental analysis determined that the content of Si element in biomimetic siliconized collagen was significantly higher than that of the control group.

1.2 scanning Electron microscopy of biomimetic siliconized collagen scaffold Material

Washing the bionic silication collagen scaffold for three times by using deionized water, and then performing gradient dehydration on the bionic silication collagen scaffold for 30 minutes by using 50%, 70%, 80% and 95% ethanol in sequence; dehydrating with 100% ethanol for 3 times, each for 15 min; 100% propylene oxide soak 3 times for about 30 minutes each; the propylene oxide and embedding resin are soaked for 2 times according to the volume ratio of 1; impregnating the epoxypropane and embedding resin for 12 hours according to the volume ratio of 1; 100% embedding resin is soaked for 12 hours; then placing the bionic silicification collagen scaffold in a silica gel mold, completely embedding the bionic silicification collagen scaffold by embedding resin, placing the bionic silicification collagen scaffold in a 48 ℃ drying oven for 4 hours and a 80 ℃ drying oven for 24 hours, and curing to form an embedding block; a transmission electron microscope workpiece with the thickness of 60-90nm is cut by a Leica EM UC7 ultrathin slicer (Leica, wetzlar, germany); the ultrastructure of the sample was observed by a JEM-1230 Transmission Electron microscope (JEOL, tokyo, japan) with a voltage parameter of 110kV.

Transmission electron microscopy images without staining showed that the collagen scaffold after siliconization achieved good intrafibrous mineralization (fig. 3); mineral deposits within the fibers display the microstructure of collagen fibers, and the ordered ribbon-like mineralized structure mimics the structural features of the internal mineralization of collagen fibers in natural bone tissue.

1.3 scanning Electron microscopy of biomimetic siliconized collagen scaffold Material

The bionic silicified collagen scaffold and the ACE collagen scaffold without any treatment are washed for three times by deionized water and then are placed in anhydrous calcium sulfate for drying for 48 hours. Collecting infrared spectrum of sample by FTIR8400S attenuated total reflection-Fourier transform infrared spectrometer (Shimadzu, tokyo, japan), setting spectral wavelength range at 400-4000cm ^-1 The number of scanning times is 32, and the resolution is 4cm ^-1 (ii) a The obtained infrared spectrogram is showed at amide A bond (-3300 cm) ^-1 ) A normalization process is performed to facilitate the comparison.

Analysis of the infrared spectrogram shows that the collagen scaffold subjected to the siliconization treatment simultaneously has characteristic peaks of collagen and silicon dioxide (figure 4); the characteristic peaks of collagen fibers, including 3300cm, were observed for the collagen scaffold without siliconization ^-1 The NH stretching peak corresponding to amide A; at 2900cm ^-1 The asymmetric stretching peak of CH2 corresponding to the amide B; at 1650cm ^-1 C = O peak corresponding to amide i; at 1550cm ^-1 The NH bend peak at position corresponds to amide II. The spectrum of the collagen scaffold after siliconization was 1650cm ^-1 The characteristic collagen peaks described above are still visible after standardized transformation of the C = O contractile peaks in collagen type I. In addition, silicidation introduces the properties of hydrated silicaCharacteristic peak, wherein 460cm ^-1 、800cm ^-1 、1070cm ^-1 The TO1, TO2 and TO3 modes corresponding TO the Si-O-Si are processed; 940-960cm ^-1 Corresponds to the Si-OH vibration peak.

1.4 silicic acid sustained release determination of biomimetic silication collagen scaffold

The dried biomimetic siliconized collagen scaffold prepared in protocol three above (100 mg) was soaked in 10ml Tris hcl buffer (PH = 7.4) at 37 ℃; at a specified time period: 400 μ Ι _ of soak solution was recovered on days 0.125, 0.25, 0.5, 1, 2, 4, and 8 to assess silicic acid release; measuring absorbance (λ =700 nm) by silicomolybdic acid spectrophotometry; measuring silicic acid standard substance by the same method, drawing standard curve, calculating silicic acid release concentration in solution by using the standard curve, and drawing corresponding curve, wherein the silicic acid release curve is shown in FIG. 5, SCS soaked in Tris-HCl has sustained release of silicic acid, has a rapid release stage within the first 4 days, and is maintained at a stable release stage.

1.5 porosity measurement

The porosity of the material was measured using absolute ethanol as a displacement fluid.

A known volume (V) of a pre-weighed (Wdry) sample was immersed in ethanol in vacuo for 1 hour to saturate the pores and then reweighed (Wsat); porosity is determined by the following formula: (Wsat-Wdry)/(ρ V). Times.100, where ρ is the density of the alcohol.

The porous scaffold with high porosity is beneficial to exchange of gas and nutrient substances, thereby promoting the adhesion and proliferation of cells. The scaffold material should have sufficient porosity to meet the requirements of tissue engineering. As shown in fig. 6, the porosity of the biomimetic siliconized collagen scaffold was 86.7 ± 1.5%, which is significantly higher than that of the unmineralized collagen scaffold (76.8 ± 2.4%). It is demonstrated that although the deposition of silica inside and outside the fibers improves the physical properties of the collagen scaffold material, it has a higher porosity.

1.6 determination of tensile Strength

The cutting size is 40 multiplied by 8 multiplied by 2mm ³ (length x width x height) collagen scaffolds were siliconized as described in protocol three. Average thickness of the samples was measured before and after siliconization and the samples were dipped in PBS before tensile testingSimulating an in vivo environment; the specimen was fixed between two opposing pinch points of a tensile tester (Shimadzu, kyoto, japan), the specimen was stressed to failure using a speed of 1mm/min, and the tensile modulus was determined as the slope of the linear region of the stress-strain curve.

The mechanical strength comparison graph of the collagen scaffold before and after biomimetic silicification shows that the durability of the material in vivo is influenced by too low tensile strength of the material, and the tensile modulus of the biomimetic silicification material is 5.96 +/-0.73 MPa and is obviously higher than that of the unmineralized collagen scaffold (2.88 +/-0.51 MPa) as shown in figure 7.

The results of the above studies indicate that choline chloride is used to stabilize the siliconized media as a first step in the siliconization within the fibers. The bionic silicified collagen scaffold has a highly mineralized surface morphology, and can generate micro-scale surface roughness to enhance cell attachment; the high porosity of the bionic silicified collagen scaffold can promote infiltration and proliferation of cells, and the increase of the tensile modulus creates better mechanical stability; the bionic silication collagen bracket stably and continuously carries out the slow release of the silicic acid in a liquid environment.

The above examples confirm that the biomimetic silication collagen scaffold material can stably and continuously release silicate ions, has good porosity and mechanical strength, and the inventor further explores the effect of the biomimetic silication collagen scaffold leaching solution and silicic acid on peripheral sensory nerve cells (primary isolated dorsal root ganglion cells).

Example 2: effect of silicated collagen scaffold leach solutions on dorsal root ganglion cell morphology and neuropeptide expression

2.1 preparation of Bionically silicated collagen scaffold extract

The dried biomimetic siliconized collagen scaffold (100 mg) was soaked in 10ml Tris-hcl buffer (PH = 7.4) at 37 ℃ for 24 hours and the material was removed and the buffer collected, as per 1.4 in example 1;

the above buffer was mixed with Neurobasal complete medium containing 20ng/mL of nerve growth factor (R & D Systems, minneapolis, MN, USA) at a ratio of 1. Neurobasal-A complete medium was prepared by adding 0.5mM/L glutamine (Gibco, gaithersburg, md., USA), 1% penicillin-streptomycin (Invitrogen, carlsbad, calif., USA) and 2% B-27 (Gibco, gaithersburg, md., USA) to Neurobasal-A medium (Gibco, gaithersburg, md., USA).

2.2 isolation and culture of dorsal root ganglion cells

Taking a 5-day-old newborn SPF clean-grade Sprague-Dawley rat suckling mouse (purchased from the center of laboratory animals of the air force military medical university, the animal experiment meets the standard of the application guidance of laboratory animals of the national health institution and is approved by the committee of animal management and use of the air force military medical university, the animal experiment ethical examination number is IACUC-20190110), cutting the head of the rat, soaking the rat in 75% alcohol for 10 minutes for disinfection, and shearing the skin along the center of the back of the rat by an ophthalmic scissors to expose the spine;

carefully separating soft tissues by using an ophthalmologic scissors, completely taking out a spinal column, longitudinally cutting a back vertebral plate along the spinal cord by using a microscopic orthopedic scissors, taking sterile gauze to wipe off the spinal cord and redundant bleeding, and making each section of the spinal cord and dorsal root ganglia in a hidden pit visible under a body type magnifier to be in a transparent spherical shape and be connected with peripheral sensory nerve fibers; taking dorsal root ganglia on two sides of a newborn mouse one by using micro-tweezers, and placing the dorsal root ganglia in a serum-free DMEM culture medium precooled in advance; removing nerve fibers and surface capsule connected with ganglion with micro forceps and micro scissors, and cutting the extracted ganglion into pieces with micro scissors;

after washing 3 times with DMEM medium (Gibco, gaithersburg, MD, USA), the dorsal root ganglion tissue fragment was transferred together with DMEM medium into a 15ml centrifuge tube, centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, 0.1% collagenase (Gibco, gaithersburg, MD, USA) and 0.25% trypsin (Corning, lowell, MA, USA) were added to the centrifuge tube to digest the dorsal root ganglion tissue fragment, which was placed in a 37 ° constant temperature water bath after shaking uniformly and digested for 40 minutes, during which time it was shaken 1 time every 10 minutes; after completion of digestion, DMEM medium containing 10% serum (Gibco, gaithersburg, md., USA) was added to stop digestion, centrifuged again at 1000 rpm for 3min, and the supernatant was discarded;

resuspending the isolated cells in Neurobasal complete medium at a density of 10000 cells/mL; 24-well plates (Corning, lowell, MA, USA), cell slides (Corning, lowell, MA, USA) or Transwell chambers (Corning, lowell, MA, USA) were coated overnight with 0.01% polylysine (Gibco, gaithersburg, MD, USA) in advance, washed 3 times with pre-PBS buffer (Corning, lowell, MA, USA) for 5 minutes each, after which dorsal root ganglion cells were inoculated uniformly, transferred to a cell culture chamber of 5 Fisher CO2 (Thermo Scientific, USA) at 37 ℃ after 4h with Neurobasal complete medium (control) containing 20ng/mL nerve growth factor or leach liquor medium (leach group) formulated at a ratio of 1; then, the solution was changed every 2 to3 days, and the cell state was observed under an inverted phase contrast microscope every day.

2.3 immunofluorescent staining experiment

In order to discuss the influence of the bionic silicified collagen scaffold leaching solution on the dorsal root ganglion cells, the separated dorsal root ganglion cells are inoculated on a cell climbing sheet of a 24-well plate, a leaching solution group is cultured for 3 days by using a leaching solution culture medium obtained by the experiment, and a control group is cultured for 3 days by using a Neurobasal complete culture medium containing 20ng/mL nerve growth factors. The culture medium in the petri dish was removed, washed 3 times with PBS buffer for 5 minutes each, and the cells were fixed with 4% paraformaldehyde (Macklin, shanghai, china) for 30 minutes and then aspirated, washed 3 times with PBS buffer for 5 minutes each;

the specimens were then punched with 0.1% Triton-100 (Sigma-Aldrich, st.Louis, mo, USA) for 15 minutes, washed with PBS buffer for 5 minutes each, rinsed 3 times, blotted for excess liquid on the sections, then blocked with 5% serum-containing blocking solution for 1 hour, and after completion of blocking, blotted for excess liquid, the sections were incubated with primary antibody at 4 deg.C: immunofluorescence incubation of neuro-specific anti- β -III tubulin antibodies (ab 78078, abcam, cambridge, MA, USA) and anti-NeuN antibodies (ab 177487, abcam, cambridge, MA, USA) overnight;

the following day, sections were removed, washed 3 times with PBS buffer to remove primary antibody for 10 minutes each, and incubated with fluorescent secondary antibody of the corresponding species (Jackson Immuno Research, west Grove, PA, USA) for 1 hour at room temperature;

after incubation, washing the fluorescent secondary antibody with PBS buffer solution in dark condition for 10 minutes and 3 times, counterstaining the cell nucleus with DAPI-containing long-acting anti-quencher (Invitrogen, san Diego, CA, USA), and mounting with cover glass, fixing the edge of the cover glass with a small amount of nail polish to prevent the cover glass from sliding;

finally, the staining results were observed under a confocal laser microscope (BX-60, olympus, japan) and images were collected, and the fluorescence intensity was analyzed by ImageJ software.

As shown in fig. 8, after 3 days of culturing dorsal root ganglion cells in the control and the culture medium containing the silicified collagen scaffold leaching solution, respectively, the silicified collagen scaffold leaching solution had no significant inhibitory effect on neuron survival (P > 0.05) but significantly promoted the increase in neuron axon length (P < 0.01) compared to the control group. 2.4 real-time quantitative polymerase chain reaction (qRT-PCR) detection

The real-time quantitative polymerase chain reaction was used to detect the effect of the bionic silication collagen scaffold leaching solution on dorsal root ganglion cell neuropeptide gene expression, and primers (Bao bioengineering, dalian, china) were designed based on the published cDNA sequence, as shown in Table 2.

TABLE 2 real-time quantitative PCR primer sequences

After 3 days of culture of dorsal root ganglion cells in the culture medium of the siliconized collagen scaffold extract obtained in the above experiment, the following operations were performed:

extracting total RNA, namely operating according to Trizol (Beyotime, shanghai, china) instructions, adding Trizol into a culture plate, repeatedly blowing and beating until cells fall off, sucking the cells into a 1.5mL enzyme-free EP tube by using an enzyme-free gun head, respectively adding trichloromethane with the volume of 1/5 of that of the Trizol, violently shaking for about 30 seconds until mixed liquid presents pink color, standing for about 5 minutes at 4 ℃, layering the liquid, placing the EP tube at 4 ℃, centrifuging at 12000rpm for 15 minutes, separating the mixed liquid into three layers, and carefully collecting an upper colorless aqueous phase into a new enzyme-free EP tube; then, an equal volume of isopropanol was added, mixed well at room temperature, left to stand for 10 minutes, the EP tube was again placed in a high-speed centrifuge, separated at 4 ℃ and 12000rpm for 15 minutes, and a white gel-like RNA precipitate at the bottom of the tube was observed, the supernatant was carefully decanted, and the RNA precipitate was washed by adding 1ml of 75% ethanol in DEPC water (Beyotime, shanghai, china) and centrifugation at 12000rpm for 5 minutes at 4 ℃. The supernatant was discarded, dried at room temperature for 5-10 minutes (over-drying should not be done), and the RNA was re-solubilized with 10. Mu.L of DEPC water to determine the concentration.

RNA quality detection: the enzyme-linked immunosorbent assay is used for determining the ratio of the 10 mu L solution A260/A280, namely the RNA purity; DEPC water is added to adjust the RNA concentration so that the total amount of RNA meets the requirements of the reagent specification (1000 ng), and the ratio of the sample A260/A280 is adjusted to be between 300 and 500.

Genome DNA removal: the reaction was carried out according to the kit instructions of TaKaRa (Takara, shiga, japan), and the whole reaction was carried out on ice; adding the reagent or the sample into an enzyme-free EP tube according to the following sequence and dosage to obtain a 10 mu L system, and centrifuging after uniformly mixing; the enzyme-free EP tube was placed into a reverse transcriptase (Berkeley, california, USA) with parameters set: temperature 42 ℃, time: 2 minutes; temperature: 4 ℃, time: the reaction system was maintained as shown in Table 3.

TABLE 3 degenomic DNA System

cDNA synthesis was carried out according to the kit instructions of TaKaRa, and the whole reaction was carried out on ice. Adding the reagent or the sample into an enzyme-free EP tube according to the following sequence and dosage to obtain a 20 mu L system, and centrifuging after uniformly mixing; and (3) placing the enzyme-free EP tube into a reverse transcription instrument for reverse transcription, and setting parameters: temperature: 37 ℃, time: 15 minutes, temperature: 85 ℃, time: 5 minutes; temperature: 4 ℃, time: maintaining; the reverse transcription system is shown in Table 4.

TABLE 4RNA reverse transcription System

And (3) real-time quantitative PCR detection: the reaction is carried out on ice according to the instruction of a kit of TaKaRa company; after the grouping layout and the repeated sample pore volume are designed, adding a reagent or a sample into an enzyme-free EP tube according to the following sequence and dosage to obtain a 25 mu L system, and centrifuging after uniformly mixing; PCR amplification was performed on a fluorescence quantitative instrument (Berkeley, california, USA) with parameter settings: temperature: 95 ℃, time: 10 minutes; temperature: at 95 ℃ for 2 seconds; temperature: 60 ℃ for 20 seconds; temperature: 70 ℃, time: 10 seconds (40 cycles), the PCR system is shown in Table 5.

TABLE 5 real-time quantitative PCR System

Using glyceraldehyde 3-phosphate dehydrogenase (GADPH) as housekeeping gene, 2 ^-ΔΔCt The method calculates the results obtained after correcting the GADPH expression level and expresses them as fold increase relative to the control;

as shown in fig. 9, the siliconized collagen scaffold leachate significantly promoted the expression of Sema3A and Sema4D genes in dorsal root ganglion cells compared to the control group, however, the expression of CGRP, SP, sema3E and Sema7D was not significantly different from the control group.

2.5 Immunofluorescent staining for Sema3A and Sema4D

The procedure was as in 2.2, the primary antibodies selected were anti-Sema 3A antibody (sc-74555, santa Cruz Biotechnology, inc., dallas, TX, USA) and anti-Sema 4D antibody (sc-136250, santa Cruz Biotechnology, inc., dallas, TX, USA).

As shown in fig. 10, in the laser confocal microscope, the expression intensities of the silicified collagen scaffold leaching solution stimulated group and the neuron axons Sema3A and Sema4D were significantly increased compared with the control group, and an obvious axon morphology was shown; neuN is used as a neuron nucleus specific antibody to mark peripheral sensory neurons, and immunofluorescence staining is used for detecting that siliconized collagen scaffold leaching liquor promotes the growth of sensory nerve axons in vitro and the expression of Sema3A and Sema 4D.

Example 3: effect of silicic acid on dorsal root ganglion cell morphology and neuropeptide expression

3.1 isolation and culture of dorsal root ganglion cells

The same as in example 2.

3.2 immunofluorescence staining experiments

To investigate the effect of silicic acid stimulation on dorsal root ganglion cells, fresh siliceous media were prepared by diluting appropriate amounts of sodium orthosilicate in complete media with a final silicon concentration between 5 μ M and 40 μ M; inoculating the separated dorsal root ganglion cells on a cell climbing sheet of a 24-pore plate, culturing for 3 days by using silicon-containing culture media with different concentrations, and performing an immunofluorescence staining experiment; the concrete method is the same as the embodiment 2;

3.3 assay of the Effect of silicic acid on the morphology of dorsal root ganglion cells

After 3 days of culture of dorsal root ganglion cells in 5, 10, 20 and 40 mu M silicic acid culture media respectively, compared with a control group, the cell number in the 40 mu M silicic acid culture medium is obviously reduced, but silicic acid has no obvious inhibition effect on neuron survival along with the reduction of the concentration of silicic acid, compared with the control group, the 5, 10 and 20 mu M silicic acid culture media have no obvious inhibition effect on neurite growth, wherein, when the concentration is 10 mu M, the length of neuron axons is obviously increased (figure 11), the primary culture of the dorsal root ganglion cells is a good model for in-vitro culture of the neuron cells, and the growth of the neuron cell axons can be promoted while the survival of the neuron cells is not influenced by the silicic acid condition culture medium with proper concentration.

3.4 determination of the Effect of silicic acid on Gene expression in dorsal root ganglion cells

After 3 days in 10 μ M silicic acid-cultured dorsal root ganglion cells, the expression of Sema3A and Sema4d was significantly increased in the dorsal root ganglion cells compared to the control group; in contrast, the expression of CGRP, SP, sema3E and Sema7D was not significantly different from that of the blank control (fig. 12), CGRP and SP secreted from sensory nerves could simultaneously act on osteoblasts and osteoclasts to regulate bone metabolism, however, in the silicic acid conditioned medium, the expression of CGRP and SP was not significantly changed, demonstrating that CGRP and SP may not be the key factors for regulating the bone regeneration process promoted by the sensory nerves regulated by the biomimetic siliconized material. 3.5 Effect of silicic acid on the expression of Sema3A and Sema4d proteins by dorsal root ganglion cells

Under confocal laser microscopy, as shown in fig. 13, the neuron axons Sema3A and Sema4D were expressed with increased intensity in 10 μ M of silicic acid medium, and showed a distinct axon morphology. NeuN is used as a neuron nucleus specific antibody to mark peripheral sensory neurons; sema4D exists mainly as a membrane protein on neuronal cell bodies, with only a small amount in a secreted form; sema3A exists primarily as a secreted protein; immunofluorescent staining detected that silicic acid promoted DRG axonal growth and Sema3A and Sema4D expression in vitro.

3.6 Signal pathway Western blot detection

Detecting the protein expression conditions in the dorsal root ganglion cells of the control group and the silicic acid stimulation group by a western blot method, which comprises the following specific steps:

extracting total protein: mu.L of lysis solution containing PMSF and RIPA (phosphatase inhibitor) was added to each well of cells, lysed on ice for 30 minutes and transferred to a pre-cooled centrifuge tube.

Protein concentration determination: protein quantification kits were used according to the instructions to prepare corresponding protein standard solution gradients and working solutions. Covering the mixed 96-well plate with tinfoil, and incubating at 37 ℃ for 30min; and (3) measuring the absorbance of each group of samples by using a microplate reader, setting the wavelength to be 562nm, drawing a standard protein curve according to the concentration and the absorbance value of the standard substance, and calculating the protein concentration of the samples according to the standard curve and the absorbance value of the samples.

Polyacrylamide gel electrophoresis: SDS-PAGE polyacrylamide gel was prepared using a kit from Biyunnan. Adding 5 x loading buffer according to the protein volume of each sample to prepare a protein mixed solution with the protein concentration of 3 mug/muL, matching the volume shortage with PBS, carrying out electrophoresis at a concentration gel voltage of 80V for about 20min, at a separation gel voltage of 120V for about 90min, and stopping electrophoresis when bromophenol blue runs to the bottom of a glass plate and the separation distance of Marker strips is enough. The PVDF membrane is cut by wearing clean PE gloves, is put into methanol for activation for 5 minutes, and is put into a membrane transferring liquid.

After electrophoresis is finished, cutting off gel in the whole plate area according to Marker instructions, immersing the gel in a membrane transferring liquid, then putting the gel into a membrane transferring template according to the sequence of sponge-filter paper-gel-PVDF membrane-filter paper-sponge, putting the membrane transferring template into an electric rotating tank after the clamps are stabilized, keeping the ice bath constant current for 200mA for 90 minutes, and transferringAfter completion of the membrane incubation, the membrane was removed and placed in a pre-formulated blocking solution (5% fetal bovine serum), blocked for 1 hour at room temperature, the blocking solution was aspirated off, the membrane was added with primary antibody diluted with TBST, placed in a refrigerator at 4 ℃ overnight for incubation, the membrane washed with TBST 3 times (10 min/time), the membrane was added with diluted secondary antibody ((Santa Cruz Biotechnology, inc., dallas, TX, USA)), incubated for 60min at room temperature, the membrane washed with TBST 5 times (5 min/time), illuminated with freshly prepared chemiluminescence solution (GeneTex, irvine, CA, USA), and then placed in a chemiluminescence detector (ChemiDoc) ^TM Imaging system) and taking pictures. The data was analyzed using ImageJ software to scan and quantify the bands. Protein expression levels were normalized to β -actin and plotted.

Primary antibodies used in Western blot detection include: active-beta-catenin (Millipore Corp., billerica, MA, USA), beta-catenin (Cell Signaling Technology, inc. Danvers, MA, USA), P38 MAPK ((Cell Signaling Technology, inc. Danvers, MA, USA), phosphor-P38 MAPK (Cell Signaling Technology, inc. Danvers, MA, USA), phosphor 3-kinase (PI 3K) (Cell Signaling Technology, danvers, MA, USA), phosphor-Akt (Cell Signaling Technology, inc. Danvers, MA, USA), AKT antipody (GeneTex, irvine, CA, USA), mTOR antipody (GeneTex, irvine, CA, USA), phosphor-mTOR (Cell Signaling Technology, inc. Danvers, MA, USA), beta-actin (GeneTex, irvine, CA, USA)

The Wnt/beta-catenin and MAPK/p38 pathways have no obvious change in the two groups. The expression of phosphorylated PI3K, akt, and rapamycin (mTOR) was significantly elevated, while the total protein content of PI3K, akt, and mTOR was not significantly altered; as shown in FIG. 14, the PI3K/Akt/mTOR signaling pathway is activated during the process of biomimetic silicified collagen stimulation of sensory nerve growth and Sema3A secretion.

Example 4: influence of implantation of bionic silicification material into mouse femoral condyle defect region on dorsal root ganglion sensory neuropeptide expression

4.1 establishment of rat femur distal defect model

And establishing a femur distal defect model to verify the capability of SCS implantation for promoting the expression of dorsal root ganglion cell Sema 3A.

Male 2-month-old Sprague-Dawley rats were purchased from the animal testing center of the air force military medical university, fed on a regular diet, in an SPF-grade environment. 1% sodium pentobarbital is administered by intraperitoneal injection (dosage: 20 mg/kg), fixed on an operating table in supine position, shaved around the knee of the right hind limb, and spread with a towel after being sterilized by 75% ethanol. A linear incision of about 1.5cm is made on the epiphyseal surface of the distal femur on the right side of a rat, the skin, subcutaneous tissues and muscle membranes are incised layer by layer, then the muscles of the condyle of the femur are stripped in a blunt manner, and the condyle of the femur is exposed. Under the continuous washing of normal saline, a trephine is used for forming a hole with the diameter of 3mm and the depth of 4mm at a position perpendicular to the distal end of the femur, the normal saline is continuously washed to remove residual bone fragments, the bionic silicification collagen scaffold group rat implantation material can absorb suture lines to sew up an incision layer by layer, the rat is raised conventionally after reviving, the animal activity is not limited, and the health state is observed every day. The blank group was not implanted with filler.

4.2 Heart perfusion fixation and sampling

After the rat femur distal end defect model is established for six weeks, the SD rat is weighed and is provided with 1% sodium pentobarbital for intraperitoneal injection anesthesia (the dosage is 20 mg/kg);

fixing a rat in a supine position, slightly clamping the skin on the upper part of the sternal xiphoid process by holding a pair of forceps with a left hand, cutting a V-shaped incision with an upward opening from the sternal xiphoid process by holding a pair of scissors with a right hand, respectively enlarging the incision at the upper left and the upper right along the V-shaped incision, and cutting the skin, subcutaneous tissues and abdominal muscles to the lower edge of the costal arch;

clamping the skin and muscle tissue at the tail end of a sternum handle by hemostatic forceps, slightly pulling upwards to expose the septal muscle, carefully cutting the skin and muscle tissue obliquely upwards along the septal muscle by using an ophthalmic scissors to expose the heart and great vessels, carefully opening the pericardium by using forceps at the left hand, slightly clamping the lower end of the heart by using forceps at the left hand, obliquely inserting the right hand-held perfusion needle into the left ventricle from the apex of the heart to the upper left of the heart, and delivering the perfusion needle to the aorta. At the moment, the right hand straightening forceps firmly clamp cardiac muscle at the cardiac apex and the perfusion needle to prevent the perfusion needle from falling off;

the normal saline is injected rapidly by the transfusion device, and the right auricle is cut off at the same time, so that the blood is discharged. Along with the perfusion of the normal saline, the gradual whitening of the liver, the swelling of viscera and the clear liquid flowing out of the right auricle are observed, at the moment, the perfusate is changed into 4 percent paraformaldehyde stationary liquid, the muscle twitching of four limbs of a rat is observed, and then the material can be obtained after the organ tissues of the whole body are stiff;

cutting out the femoral condyle and the part of the femur connected with the upper end of the femoral condyle as a specimen, and soaking the specimen in 4% paraformaldehyde for preservation at 4 ℃;

the dorsal root ganglion is positioned at the inner side edge of the intervertebral foramen and is tightly attached to the vertebral canal wall, and the L3-L5 dorsal root ganglion is carefully taken out and then is placed into a refrigerator with the temperature of 70 ℃ below zero for storage for frozen sectioning.

4.3 frozen tissue sections and immunofluorescence experiments

OCT embedded dorsal root ganglion tissue was sectioned at 15 μm thickness using a cryomicrotome. Airing at room temperature for 20 minutes to ensure that the cut slide is firmly and flatly stuck on the glass slide;

immersing the section in PBS buffer for 10 minutes, and then subjecting the specimen to a punch treatment with 0.1% Triton-100 solution for 15 minutes;

the punch was washed with PBS buffer for 5 minutes each and 3 times. After sucking off the redundant liquid on the section, sealing the specimen for 1 hour by using a sealing liquid containing 5% serum;

after the blocking was completed and excess liquid was blotted, the sections were incubated with primary antibody overnight at 4 ℃. The next day, sections were removed and primary antibody washed with PBS buffer for 10 minutes each, 3 times. Incubating for 1 hour at room temperature with fluorescent secondary antibody of corresponding species;

after the incubation is finished, washing the fluorescent secondary antibody by using a PBS buffer solution under the condition of keeping out of the sun, wherein the washing is carried out for 3 times after 10 minutes each time;

counterstaining the cell nucleus with a long-acting anti-quencher containing DAPI, and mounting the cell nucleus with a cover slip; a small amount of nail polish secured the coverslip edges to prevent the coverslip from slipping. And finally observing a dyeing result under a confocal microscope, collecting an image, and analyzing the fluorescence intensity by ImageJ software.

The results show that: the expression of the sensory neuron Sema3A protein of the bionic silicified collagen scaffold implanted group dorsal root ganglion is remarkably increased compared with that of a blank control group (fig. 15), which is consistent with an in vitro experiment.

Claims

1. The application of the bionic silicified collagen material in preparing a Sema3A secretion promoter in dorsal root ganglion cells; the bionic silicification collagen material is obtained by soaking ACE collagen in a pretreatment solution, and the pretreatment solution consists of choline chloride and a silicic acid solution.

2. The use of claim 1, wherein the pretreatment fluid has a pH of 5 to 6.

3. The use according to claim 1, wherein the solution of orthosilicic acid is formulated from Silbond40, ethanol and water.

4. The leaching liquor of the bionic silicified collagen material is used for preparing a Sema3A secretion promoter in dorsal root ganglion cells; the bionic silication collagen material is obtained by soaking ACE collagen in pretreatment liquid, and the pretreatment liquid consists of choline chloride and silicic acid solution.