CN117771428A

CN117771428A - Bletilla polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof

Info

Publication number: CN117771428A
Application number: CN202311744323.7A
Authority: CN
Inventors: 顾勇; 陈亮; 郗焜; 唐锦程; 王嘉豪
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2023-12-18
Filing date: 2023-12-18
Publication date: 2024-03-29

Abstract

The invention relates to the technical field of biological medicine, in particular to bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof. The Schiff base reaction of the invention respectively bridges the hydroformylation bletilla polysaccharide (oBSP) and gelatin by the amination mesoporous bioactive glass aMBGN to construct the inorganic-organic double-crosslinking excited bone immune-stem cell endogenous self-healing hydrogel (GBM) ^gel ). Experimental results show that the hydrogel stimulates endogenous stem cells to homing repair bone defects, enhances bone matrix deposition, mineralization and vascular maturation, and self-assembles in-situ gel at the bone defects by injection, thus being a defectThe lesion provides a good cell microenvironment and has the effect of promoting bone defect regeneration through immunoregulation, and has wide application potential in a treatment strategy for stimulating bone immune-stem cell endogenous self-healing program to promote bone healing.

Description

Bletilla polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof.

Background

Hydrogels, which are commonly used as biomaterials, are widely used in the field of tissue repair due to their properties similar to the extracellular matrix (ECM). In consideration of the specificity of critical bone defects, compared with other biological materials such as microspheres and electrospinning, the hydrogel formed by the Schiff base reaction has the characteristic of being capable of being injected into gel in situ, and is suitable for filling bone defects of various shapes. In addition, gelatin-based hydrogels have excellent biocompatibility and degradability, and can avoid toxic effects on the body. In previous studies, it was found that the aldehyde-modified glucan (Dex) structure was able to bind to amino groups in Gelatin (Gelatin) to form hydrogels, which were able to efficiently adhere to crushed bone pieces, indirectly leading to bone healing. However, dextran hydrogels have certain limitations in immunomodulation and promotion of bone growth and neglect the role of stimulating endogenous self-healing processes in bone immune-stem cells. Therefore, development of hydrogels that can exert both immunomodulating and self-healing properties is of paramount importance.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof.

The invention provides a preparation method of bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel, which comprises the following steps:

step 1, performing an oxidation reaction on an aqueous solution of bletilla striata polysaccharide in the presence of sodium periodate to prepare and obtain aldehyde bletilla striata polysaccharide;

step 2, performing an amination reaction on MBGN in the presence of APTES to prepare and obtain amino MBGN;

and 3, reacting the aldehyde group bletilla polysaccharide with amino MBGN and gelatin in sequence to obtain the bletilla polysaccharide-mesoporous bioactive glass-gelatin hydrogel.

Further, the mass ratio of the aldehyde group bletilla polysaccharide to the amino MBGN to the gelatin is 10:3:10.

In a specific embodiment of the invention, the aldehyde bletilla striata polysaccharide is dissolved in PBS according to a solubility of 20% (w/v), and then 6% (w/v) of amino MBGN and 20% (w/v) of gelatin are added in sequence. Experimental results show that the gelling time can be effectively shortened when MBGN is added, and the gelling time is shortest when the mass ratio of the aldehyde group bletilla polysaccharide to the amino MBGN to the gelatin is 10:3:10.

The mass fraction of the bletilla polysaccharide in the bletilla polysaccharide aqueous solution is 0.5-1.25%, and the pH value is 7.5-8.5; preferably, the mass fraction of the bletilla polysaccharide in the bletilla polysaccharide aqueous solution is 1g, and the pH value is 8.5. Experimental results show that compared with other conditions, the conditions can obviously reduce the generation of viscous liquid and facilitate hydroformylation.

In step 2 of the preparation method of the invention,

the mass ratio of the bletilla polysaccharide to the sodium periodate in the bletilla polysaccharide aqueous solution is 2:1, a step of;

the conditions of the oxidation reaction are argon protection, and the mixture is uniformly mixed for 8 hours in a dark place;

the oxidation reaction is followed by a dialysis step;

the dialyzed solution is deionized water;

the molecular cut-off of the dialysis is 3500;

the dialysis time was 48 hours.

In step 2 of the preparation method of the invention,

the mass ratio of MBGN to APTES is 5:1, a step of;

the amination reaction is carried out under the condition of 60 ℃ and evenly mixed for 24 hours.

The dialysis further comprises a drying step;

the method further comprises the steps of cleaning and drying after the amination reaction;

the washing includes washing with ethanol three times followed by washing with deionized water three times.

In the step 3, the reaction condition is 45 ℃ for 10min.

The invention provides the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method.

The hydrogel is prepared by modifying chemical bonds of MBGN and bletilla polysaccharide BSP, and then the aminated MBGN can be bonded together through the Schiff base reaction of amino and aldehyde groups, and the bonding of the aminated MBGN is superior to the bonding mainly by hydrogen bonds. Based on the above, gelatin base is introduced, the main body is a hydrogel structure formed by bletilla polysaccharide BSP and gelatin, and MBGN is uniformly embedded in the hydrogel structure, so that the stability is further improved and the cell growth microenvironment is provided. The repair of bone tissue requires sufficient mechanical strength and an osteogenic effect. Compared with gelatin and bletilla polysaccharide systems, the introduction of MBGN can provide high enough mechanical strength for repairing bone tissues on one hand, and can supplement the effect of bletilla polysaccharide on the aspect of bone formation on the other hand, and promote bone formation and differentiation in time. The macrophage targeting function proved by the bletilla polysaccharide can be used for filling critical bone defects, targeting surrounding macrophages and promoting stem cell homing.

The invention provides an application of bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method or the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel in targeted capture of bone marrow mesenchymal stem cells, polarization of the bone marrow mesenchymal stem cells towards the direction of macrophages, promotion of migration of the bone marrow mesenchymal stem cells, promotion of homing of the stem cells, promotion of osteogenic differentiation of the bone marrow mesenchymal stem cells, promotion of angiogenesis-related cytokine expression, promotion of bone repair, promotion of bone regeneration, promotion of macrophage secretion of anti-inflammatory cytokines (CSF-3 and Cxcl 3), and inhibition of inflammatory related factors (Mpo and cd5 l) of macrophages.

The test results of the present invention show that the gel speed is significantly faster compared to hydrogels with and without the addition of aminated MBGN; the elasticity is larger, the compression resistance is stronger, and the mechanical stress is obviously improved; the swelling rate is reduced, and the gel structure is more stable; significantly promoting macrophage polarization; significantly promoting osteogenic differentiation and bone repair; the in vitro blood vessel has higher branch and total length, remarkably promotes the generation of blood vessels, and has wide application potential in the treatment strategy of stimulating the endogenous self-healing procedure of bone immune-stem cells to promote bone healing.

MBGN has high biological safety to cells, and experimental results show that after the hydrogel is co-cultured with BMSC (mesenchymal stem cells), the survival rate of the cells is not obviously statistically different from that of a control group.

The invention provides an application of bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method or the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel in preparation of products for promoting bone repair, bone regeneration and/or stem cell homing.

The invention provides a product for promoting bone repair, promoting bone regeneration and/or promoting stem cell homing, which comprises at least one of a carrier or an auxiliary material and the bletilla polysaccharide-mesoporous bioactive glass-gelatin hydrogel.

Further, the carrier comprises gauze, non-woven fabrics and a syringe; the auxiliary materials comprise medicines, growth factors and the like which assist the hydrogel.

The Schiff base reaction of the invention respectively bridges the hydroformylation bletilla polysaccharide (oBSP) and gelatin by the amination mesoporous bioactive glass aMBGN to construct the inorganic-organic double-crosslinking excited bone immune-stem cell endogenous self-healing hydrogel (GBM) ^gel ). Experimental results show that the hydrogel stimulates endogenous stem cells to homing and repair bone defects, enhances bone matrix deposition, mineralization and vascular maturation, self-assembles and forms gel in situ at the bone defects by injection, provides good cell microenvironment for the defects, has the effect of promoting bone defect regeneration through immunoregulation, and has wide application potential in a treatment strategy of stimulating bone immune-stem cell endogenous self-healing procedures to promote bone healing.

Drawings

FIG. 1 is a schematic diagram of hydrogel composition and action;

FIG. 2 is SEM and TEM observations of the synthesized MBGN, where A and B are SEM images of MBGN and aMBGN; c and D are TEM images of MBGN and aMBGN;

FIG. 3 is GB ^gel And GBM ^gel SEM images of (a);

FIG. 4 is a graph showing physicochemical properties of a gel, wherein A is a particle size distribution diagram of aMBGN; b is the XRD pattern of aMBGN; c is GB ^gel EDS analysis; d is GBM ^gel EDS analysis of (C);

FIG. 5 is a plot of Gel formation versus Gel effect of different ratios of Gel and oBSP, where A is a plot of 10% Gel and 20% oBSP; b is a Gel forming diagram of 20% Gel and 20% oBSP; c is a Gel forming diagram of 20% Gel and 10% oBSP; d is a Gel-forming diagram of 10% Gel, 20% oBSP and 6% aMBGN; e is a Gel forming diagram of 20% Gel, 20% oBSP and 6% aMBGN; f is a Gel plot of 20% Gel, 20% oBSP and 6% aMBGN;

FIG. 6 is GB ^gel And GBM ^gel Forming a glue pattern;

FIG. 7 is a gel spectrum and rheological analysis, wherein A is FT-IR analysis of different materials; b is GB ^gel And GBM ^gel Is a rheological analysis of (2);

FIG. 8 is GBM ^gel Injection schematic;

FIG. 9 shows the mechanical, swelling and degradation of the gel and the release of Si ions, wherein A is GB ^gel And GBM ^gel Is a mechanical graph of (2); b is GB ^gel And GBM ^gel Swelling curve and degradation curve of (a); c is GBM ^gel Si ion release profile of (2); d is Si ⁴⁺ Is a release rate profile of (2);

FIG. 10 is a cell viability study of a composite hydrogel, wherein A is a schematic of co-culturing a material with cells; b is a live-dead fluorescent staining chart after 7 days of co-culture with BMSCs; c is the semi-quantitative analysis of the fluorescent staining of liveness after 7 days of co-culture with BMSCs; d is cck-8 analysis co-cultured with BMSCs; e is the effect of M-CSF and a hydrogel containing the bletilla polysaccharide BSP on BMDM;

FIG. 11 is a flow chart of the composite hydrogel after 3 days of co-culture with BMDMs, wherein A is a flow chart of the composite hydrogel after 3 days of co-culture with BMDMs; b is CD11B after 3 days of co-culture with BMDMs ⁺ /CD86 ⁺ Semi-quantitative analysis of the duty cycle; c is CD11b after 3 days of co-culture with BMDMs ⁺ /CD206 ⁺ Semi-quantitative analysis of the duty cycle;

FIG. 12 is a flow chart of the composite hydrogel after 3 days of co-culture with BMDMs, wherein A is a flow chart of the composite hydrogel after 7 days of co-culture with BMDMs; b is co-culture with BMDMs CD11b after 7 days ⁺ /CD86 ⁺ Semi-quantitative analysis of the duty cycle; c is CD11b after 7 days of co-culture with BMDMs ⁺ /CD206 ⁺ Semi-quantitative analysis of the duty cycle;

FIG. 13 is an image of iNOS immunofluorescence staining after 3 days and 7 days of co-culture of the composite hydrogel with BMDMs;

FIG. 14 is a graph of CD206 immunofluorescence staining of composite hydrogels after 3 days and 7 days of co-culture with BMDMs;

FIG. 15 is a schematic of co-culture of composite hydrogels with BMDMs, wherein A is an INOS staining semi-quantitative analysis after 3 days of co-culture with BMDMs; b is a semi-quantitative analysis of CD206 staining after 7 days co-culture with BMDMs; c is an INOS staining semi-quantitative analysis after 7 days of co-culture with BMDMs; d is a semi-quantitative analysis of CD206 staining after 7 days of co-culture with BMDMs;

FIG. 16 is a graph of the effect of co-culture of composite hydrogels with BMDMs on BMSCs, wherein A is a cell scratch experiment for each group of BMSCs; b is a cell transwell migration experimental diagram of each group of BMSCcs; c is the semi-quantitative analysis of cell scratch healing of each group of BMSCs; semi-quantitative analysis of cell transwell migration cell number for each group of BMSCc;

FIG. 17 is an ALP staining chart of each group of BMSCs after co-culturing the composite hydrogel with BMDMs;

FIG. 18 is an OCN immunofluorescence staining chart of each group of BMSCs after co-culturing the composite hydrogel with BMDMs;

FIG. 19 is a graph of calcium nodule staining of various groups of BMSCs after co-culturing the composite hydrogel with BMDMs;

FIG. 20 shows ALP staining semi-quantitative analysis and OCN immunofluorescence semi-quantitative analysis of each group of BMSCs after co-culturing of the composite hydrogel with BMDMs, wherein A is ALP staining semi-quantitative analysis; b is OCN immunofluorescence semi-quantitative analysis;

FIG. 21 is a graph of quantitative analysis of each group of BMSCs after co-culturing the composite hydrogel with BMDMs, wherein A is a semi-quantitative analysis of calcium nodule staining of each group of BMSCs after co-culturing the composite hydrogel with BMDMs; b is the PCR analysis of RUNX-2; c is the PCR analysis of ALP; PCR analysis with D being OCN;

FIG. 22 is a angiogram of each group of HUVECs after co-culturing the composite hydrogel with BMDMs; influence of HUVECs on vascularization

FIG. 23 is the effect of composite hydrogels on vascularization of HUVECs after co-culture with BMDMs, wherein A is a semi-quantitative analysis of the vascularization number of each group of HUVECs; b is the semi-quantitative analysis of the number of blood vessel segments of each group of HUVECs; c is the semi-quantitative analysis of the length of the assembly tube of each group of HUVECs; ELISA assay for VEGF; ELISA assay with E being PDGF-BB;

FIG. 24 is a time axis of in vivo experiments in rats;

FIG. 25 is a schematic representation of a rat skull defect model;

FIG. 26 is a schematic of micro-CT after each of the groups 4W and 8W, macrophage-indicating depletion of macrophages in vivo; macrophage+ indicates that macrophages in the body are not depleted;

FIG. 27 is a plot of HE staining for each group;

FIG. 28 is a map of the masson staining of each group;

FIG. 29 is a quantitative graph of imaging metrics for each group, wherein A) each group is quantitatively analyzed by BV/TV; b) Semi-quantitative analysis of BMD for each group; quantitative analysis of new bone tissue when C is 4W of each group; d is quantitative analysis of new bone tissue at 8W for each group; e is quantitative analysis of red-stained new bone tissue when each group is 4W; f is quantitative analysis of red-stained new bone tissue when each group is 8W;

FIG. 30 is an experimental immunostaining and corresponding quantitative analysis in rats, wherein A is an OCN staining pattern after each of groups 4W and 8W; b is a CD31 staining chart after 4W and 8W of each group; c is an OPN staining chart after 4W and 8W of each group; d is VWF staining after each group of 4W and 8W;

FIG. 31 is a quantitative graph of the bone and vascularization index for each component, wherein A is the OCN semi-quantitative analysis for each group of 4W; b is 8W of each group of OCN semi-quantitative analysis; semi-quantitative analysis of CD31 in each group with C being 4W; d is 8W of semi-quantitative analysis of each group of CD 31; e is the semi-quantitative analysis of OPN of each group of 4W; f is 8W of semi-quantitative analysis of OPN of each group; semi-quantitative analysis of VWF for each group with G being 4W; semi-quantitative analysis of VWF for each group with H being 4W;

FIG. 32 is an immunofluorescent staining of experimental macrophages in rats, wherein A is a plot of immunofluorescent staining of each of the groups CD90 and F4/80; b is RUNX-2 immunofluorescence staining patterns of each group; c is an alpha-SMA immunofluorescence staining chart of each group;

FIG. 33 is a graph of experimental macrophage quantification in rats, wherein F4/80+ cell fraction semi-quantitative analysis for each group of 3D; CD90+ cells when B is 3DSemi-quantitative analysis of the duty ratio; f4/80+ cell ratio semi-quantitative analysis when C is 7D of each group; half-quantitative analysis of cd90+ cell fraction for each group 7D; e is quantitative analysis of RUNX-2 fluorescence areas of each group; f is a group alpha-SMA ⁺ Quantitative analysis of cell ratio;

FIG. 34 is a PCR analysis of inflammation-associated protein in rats, wherein A is CSF; b is OSM; c is TNF-alpha; d is iL-1 beta; e is iL-12; f is iL-4;

FIG. 35 proteomic analysis;

FIG. 36 is a schematic diagram of differential gene numbers;

FIG. 37 is GO analysis;

FIG. 38 is a KEGG pathway analysis;

FIG. 39 is a WB assay;

FIG. 40 is a quantitative graph of WB assay.

Detailed Description

The invention provides bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel and application thereof, and a person skilled in the art can properly improve process parameters by referring to the content of the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

In the research, in order to establish a bone tissue engineering treatment strategy for regulating and controlling the quantity of macrophages and triggering a bone immune-stem cell endogenous bone repair program by a space-time phenotype, an inorganic-organic double-crosslinking excited bone immune-stem cell endogenous self-healing hydrogel (GBM) is constructed by bridging an amino mesoporous bioactive glass aMBGN with an aldehyde group bletilla polysaccharide (oBSP) and gelatin respectively through Schiff base reaction ^gel )。GBM ^gel Capturing and polarizing M1 type macrophage in early inflammation stage in a targeting way, rapidly starting endogenous stem cell recruitment and vascular sprouting repair mechanism of organism, and accompanying stable release of Si by aMBGN ⁴⁺ Stem cell osteogenic differentiation programmed to induce homing of M2 macrophages andand (5) maturing blood vessels. The structure and the function of the composite scaffold are researched by a physicochemical method, and on the basis of a macrophage and bone marrow mesenchymal stem cells (BMSCs) co-culture system, the biological mechanism of regulating and controlling bone immunoosteogenesis by the composite scaffold is evaluated by adopting protein transcriptome analysis and related signal path research. GBM was further validated in a bone critical bone defect rat model of chlorophosphonate liposome-depleted macrophages ^gel The therapeutic strategy of selectively targeting captured macrophages to stimulate bone immune-stem cell endogenous self-healing processes to promote bone healing is a very potential and attractive solution.

The reagent consumable adopted by the invention is a common commercial product and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 hydrogel preparation and physicochemical Property characterization

1. Hydrogel preparation

In the research, the bone immune-stem cell-stimulating composite hydrogel GBM is constructed by self-assembling the bletilla polysaccharide structure with macrophage affinity and the nano-scale mesoporous bioactive glass through Schiff base reaction ^gel Chemotactic macrophages are targeted through Mannose Receptors (MR), and the phenotype macrophages are repaired through space-time phase regulation, so that endogenous stem cells are stimulated to homing to repair bone defects. The hydrogel can be injected into the bone defect in situ to form gel, and provides good mechanical stress support. More importantly, we found GBM ^gel The method is characterized in that seed stem cells and vascular sprouts required by bone regeneration are captured and driven by macrophages at high efficiency in early stage of bone injury, continuous release of inorganic silicon ions and recognition and phagocytosis by the macrophages can play a role in coordinating polarized anti-inflammatory phenotype macrophages at later stage, bone matrix deposition, mineralization and vascular maturation are further enhanced, the mixed biological material is constructed by injecting self-assembled in-situ gel at a bone defect site, a good cell microenvironment is provided for the defect site, and the bone defect regeneration is promoted by immunoregulation, and the gel composition and the action are shown in a schematic diagram in figure 1.

The technical scheme comprises the following steps:

1. preparation of aldehyde-based bletilla striata polysaccharide (oBSP)

Bletilla polysaccharide powder BSP from bletilla root was subjected to ultraviolet sterilization and then dissolved in deionized water to prepare a 1% (w/v) solution. Adding oxidant sodium periodate (NaIO 4), stirring under the protection of argon and light-shielding condition for reaction for 8 hours, and adding glycol to terminate the reaction. Then transferring into a dialysis bag with molecular weight of 3500, dialyzing in deionized water for 48h, filtering after dialysis, preserving at-20 deg. after filtration, and freeze drying for 48h to obtain aldehyde group bletilla striata polysaccharide (oBSP). The percentage of oxidation of the oBSP was determined by the previously reported method, with the oBSP reacted with an excess of t-butylhydrazinoformate, the unreacted t-BC was quantified by adding trinitrobenzenesulfonic acid and used to quantify the colored derivative resulting from the reaction of the excess t-BC with TNBS by a spectro-luminance meter at 334nm, yielding a percentage of oxidation of 70.5%.

2. Preparation of aminated mesoporous bioactive glass (aMBGN)

Synthetic reference related studies of MBGN. Through preparing TRIS-HCL buffer solution with pH=8, taking a certain amount, placing the buffer solution into a flask, stirring at 60 ℃, sequentially adding CTAB (cetyltrimethylammonium bromide), calcium nitrate tetrahydrate (CN), tetraethoxysilane (TEOS) and triethyl phosphate (TEP), reacting for 24 hours, and centrifuging at a high speed after the reaction is finished. And (3) sequentially cleaning the centrifugal product with absolute ethyl alcohol and deionized water, and finally drying and calcining in a muffle furnace at 650 ℃ for three hours to obtain the MBGN.

0.4g of MGBN was placed in n-hexane and shaken while 5ml of APTES (3-aminopropyl triethoxysilane) was added and stirred at 60℃for 24h. Washing with absolute ethanol and deionized water for three times in sequence, and drying the obtained product to obtain aminated MBGN (aMBGN).

3、GB ^gel And GBM ^gel Is prepared from

Dissolving oBSP and gelatin GEL in PBS respectively with 20% (w/v) solubility, stirring, mixing the two liquids, injecting, allowing the residual amino bond on gelatin to react with aldehyde group on oBSP at room temperature to form GB ^gel A hydrogel.

In addition, oBSP is set to 2Dissolving 0% (w/v) of the solution in PBS, stirring uniformly, dissolving aMBGN in an oBSP solution at a solubility of 6% (w/v), stirring uniformly by magnetic force, reacting aldehyde groups of oBSP with amino groups on aMGBN at normal temperature to form covalent bonding by Schiff base, injecting gelatin solution with the same solubility, mixing uniformly, allowing redundant hydroxyl groups on MBGN to form hydrogen bonds with carboxyl groups on gelatin, and standing for a certain time to form GBM ^gel A hydrogel.

2. Characterization of physicochemical Properties

1. Sub-microscopes (sem) and transmission electron microscopes (tem)

The surface morphology of MBGN and AMBGN was observed by scanning electron microscopy SEM (Hitachi, S-4800, japan) and transmission electron microscopy. Before testing, MBGN and AMBGN are respectively dispersed in absolute ethyl alcohol, then are dripped on a silicon wafer for air drying, and finally are stuck on a sample table, and gold is sprayed for 60 seconds by using gold sputtering coating equipment (QuorumTechnologies, SC 7620). Before the test, the hydrogel sample needs to be frozen and crisp in liquid nitrogen after being frozen and dried in a freezing machine for 3 days, and then the sample is buried at a metal spraying position, so that the conductivity of the sample is improved. Meanwhile, EDS elemental analysis was performed on carbon (C), oxygen (O), silicon (Si), phosphorus (P), calcium (Ca) and nitrogen (N).

2. Particle size analysis

MBGN and AMBGN were sonicated in absolute ethanol and particle size analysis was performed by a research grade light scattering system.

3、XRD

The synthesized MBGN was measured by an X-ray diffractometer (XDR, ultimaIII, japan) at a scanning speed of 2 °/min in the range of 10 to 80 ° of diffraction angle (20). And adopting a Cu target K alpha ray, and scanning the sample under the condition that the tube current is 40mA and the tube voltage is 40 KV.

4. Fourier transform infrared spectroscopy (FTIR)

At 4000cm using FTIR spectrometer ^-1 -500 ^-1 In-range scan MBGN, AMBGN, BSP, OBSP, GB ^gel And GBM ^gel FT-IR spectrum of hydrogel dried samples.

5. Rheometry measurement

The hydrogel samples were rheologically measured using a rheometer (thermo scientific, HAAKE RheoStress 6000, usa) under an oscillation frequency sweep test. The storage modulus (G ') and loss modulus (G') of viscoelasticity are measured at 28.+ -. 1 ℃ and 5% shear strain, oscillation frequency ranging from 0.1 to 15 HZ. The self-healing properties of the hydrogels are scanned by oscillating strains of the hydrogels on a rheometer of 1% to 104%. After allowing the hydrogel to heal for 15 minutes, a shaking time scan was again performed to characterize the healing effect.

6. Measurement of mechanical Properties

The hydrogel samples were measured by a universal mechanical tester (Hengfei, HY-1080, shanghai, china). The hydrogel formed into a column was pretreated in PBS for 12 hours and subjected to compression test. The compressive modulus of the hydrogel was obtained by calculating the slope of the stress-strain curve through the origin.

7. Silicon ion Release experiment

The same volume of material was placed in 1mL of human Simulated Body Fluid (SBF), respectively, periodically replaced and the supernatant collected. The silicon ion content of the supernatants collected at each time point was measured by inductively coupled plasma emission spectroscopy (ICP) (PerkinElmer, optima 7300, usa).

8. Measurement of swelling Rate and degradation Rate of hydrogels

The swelling ratio of the hydrogels was measured in PBS (pH 7.4) to evaluate swelling in vitro. Briefly, approximately 1mL of freshly prepared hydrogel was added to 3mL fbs at 37 ℃. PBS was removed and the quality of the hydrogel was measured periodically. Calculation using equation (1): (W1-W0)/W0X 100%. Wherein W1 and W0 are the mass of the hydrogel after and before soaking in PBS, respectively.

The degradation rate of the hydrogels was measured in PBS (pH 7.4) to evaluate in vitro degradation. Briefly, approximately 1mL of freshly prepared hydrogel was added to 3mL fbs at 37 ℃. PBS was removed and the quality of the hydrogel was measured periodically. Calculation using equation (2): (W0-W2)/W0X 100%. Wherein W2 and W0 are the mass of the hydrogel after and before soaking in PBS, respectively.

3. Intervention of hydrogels on in vitro cells

1. Cell viability study

Mouse bone marrow mesenchymal cells (BMSCs) were extracted from 8 week old male C57 mice following the protocol approved by the ethical committee of the first affiliated hospital at the university of su. Mouse bone marrow mesenchymal stem cells (BMSCs) were used for in vitro assays. BMSCs in 5% CO in alpha-DMEM (Gibico, USA) supplemented with 10% FBS (fetal bovine serum, gibico, USA) and 1% penicillin-streptomycin (Gibico, USA) ₂ Culturing in an incubator at 37 ℃. BMSCs were run at 1X 10 per well ⁴ The density of individual cells was seeded on the surface of sterilized hydrogel (Φ8mm×1mm,75% ethanol sterilization) in 48-well plates. Following 1 day, 3 days, 5 days and 7 days of culture, cell proliferation was assessed on a hydrogel using the CCK-8 assay, as per manufacturer's instructions. Optical Density (OD) values (n=3) at a wavelength of 450nm were measured periodically by means of an enzyme-labeling instrument. After 7 days of incubation, the cell viability on the hydrogels was assessed using a calcein-AM/propidium iodide-PI live/dead affinity assay kit and using a laser inverted microscope. Semi-quantitative measurements of cell activity were performed using ImageJ software (national institutes of health, bescens, maryland, usa).

2. Cell culture and collection of conditioned Medium

Mouse bone marrow mesenchymal cells (BMSCs) and mouse primary macrophages (BMDMs) were extracted from 8 week old male C57 mice according to the protocol approved by the ethical committee of the first affiliated hospital at the university of su. Bone Marrow Derived Macrophages (BMDM) were harvested from mice and purified in the presence of macrophage colony stimulating factor (M-CSF, 20ng mL ^-1 ) In a culture medium of 1×10 ⁶ The cells were cultured at a density of cells/mL for 6 days. And simultaneously selecting bone marrow mesenchymal stem cells (BMSCs) obtained from the bone marrow of the mice for 3-5 generations for experiments.

BMDMs cells were plated at 3X 10 per well ⁶ Is inoculated into 6-well plates and cultured with DMEM containing FBS and P/S. BMDMs cells were cultured with fresh DMEM supplemented with FBS and P/S for 48 hours, and then the medium was collected. The collected media was centrifuged at 1000rpm for 3 minutes, and the supernatant was collected, stored and labeled as Control conditioned media (Control). At the same time, the sterilized hydrogel was placed in a 6-well plate at the bottom, BMSCs cells were plated at 3X 10 per well ⁶ Is inoculated in 6 holes containing hydrogelIn the plates, experiments were performed after a certain time of co-culture with DME containing FBS and P/S, while medium was collected, centrifuged at 1000rpm for 3min, and supernatant was collected, stored and labeled as a specific medium.

3. Morphology and behavior analysis of macrophage co-culture

Macrophages co-cultured with the hydrogel for a certain period of time were fixed with 4% paraformaldehyde for 30 minutes at room temperature. Samples were prepared using an alcohol gradient dehydration method, and gold was sprayed for 60 seconds using a gold sputter coating apparatus for scanning electron microscope SEM and transmission electron microscope TEM observations.

4. Flow analysis

Expression of macrophage markers such as CD86, CD206, CD11b, and CD45 in co-cultured BMSC cells was examined by flow cytometry (BD, canto II, USA). Washed twice with cold PBS and stained with FITC-labeled anti-CD 86 antibody (BD Pharmingen, USA), PE-labeled anti-CD 206 antibody (BD Pharmingen, USA), APC-labeled anti-CD 11b antibody (BD Pharmingen, USA) and PE-CY 7-labeled anti-CD 45 antibody (BD Pharmingen, USA). To assess the percentage of macrophage polarization.

5. Immunofluorescence analysis

Macrophage markers such as iNOS (Abcam, USA), CD206 (Abcam, USA) and F4/80 (Abcam, USA) were tested for expression in co-cultured BMSC cells by laser confocal microscopy (CLSM, ZEISS, LSM, germany). Cells were fixed with 4% paraformaldehyde (Biosharp, shanghai, china), blocked with a quick blocking solution, and incubated sequentially with primary/secondary antibodies and DAPI (Yesen, shanghai, china). Immunofluorescent cells were observed under a mirror and photographed.

6. Proteomic analysis

The co-cultured hydrogels were analyzed by proteomic analysis. Samples (n=3/group) were collected after 7 days of co-culture to extract proteins for reverse phase high performance liquid chromatography (RP-HPLC). Mass spectral results were analyzed qualitatively and quantitatively on the MaxQuant/Andromeda software (version 1.3.0.5). By DAVID, string, cytoscape and omictstudio analysis, the differentially expressed proteins were defined as P <0.05.

7. Western blot experiment (WB)

BMSCs were collected periodically for a certain period of co-culture, total protein was extracted using RIPA lysis buffer (Beyotidme, shanghai, china), and protein lysates were quantified using the bicinchoninic acid (BCA) kit (Solarbio, beijing, china). Subsequently, 20. Mu.g of protein was loaded onto a 10% SDS PAGE gel (New Cell & Molecular, suzhou, china), followed by electrophoresis, membrane transfer and blocking. The membrane is then incubated with the relevant primary antibody. Antibodies used include anti-TLR 4 (Abcam, uk), anti-P65 (Abcam, uk), anti-STAT 3 (Abcam, uk), anti-P-STAT 3 (Abcam, uk), anti-ERK (Abcam, uk), anti-P-ERK (Abcam, uk), gapdh (Abcam, uk). After washing in Tris buffered saline-tween (TBST), color was developed using HRP conjugated secondary antibodies and visualized using an imaging system (Bio-rad). Quantitative analysis of proteins was performed using ImageJ.

8. qRT-PCR experiments

BMSCs were collected for 7 days of co-culture, and total RNA was extracted from cells cultured on each sample using Trizol reagent. The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Takara, japan).

qRT-PCR was performed using MaximaTM SYBR Green/ROX qPCR Master Mix (Thermo). All reactions were performed in triplicate.

9. Cell scratch assay

BMSC cells were plated at 1X 10 per well ⁶ The density of individual cells was seeded in 6-well plates (corning, usa) and cultured for 24 hours, respectively. After that, the medium was removed, and then a scratch was made at the bottom of each well with a 200. Mu.L pipette tip. Then, after washing the cells twice with PBS, different BMDM conditioned media were added for culturing. Cells were observed periodically with an inverted microscope (Lecia, DMI 3000B, germany) and images were taken at different time points to monitor migration of cells to the scratched areas.

10. transwell migration experiments

BMSC cells were plated at 2X 10 per well ⁵ Is inoculated in the upper chamber of a 24-well Transwell plate (corning, usa) and cultured with fresh DMEM without FBS and P/S. Different BMDM conditioned media were addedTo the lower chamber of a 24 well Transwell plate for 12 hours. The upper chamber was then removed from the 24-well Transwell plate, cells were fixed with 4% paraformaldehyde (Biosharp, shanghai, china), and then stained with gentian violet. Cells were observed with an inverted microscope (Lecia, DMI 3000B, germany) and images were taken.

11. Osteogenic differentiation

BMDM and BMSC were co-cultured using 24 well transwell cell culture plates (Corning, USA). BMDM is cultured on the hydrogel in the upper chamber, while BMSC is cultured in the lower chamber. After co-cultivation for 7d, different groups of BMSCs were stained and quantified using an ALP chromogenic solution kit (bi yun, shanghai, china) and an alkaline phosphatase activity quantification kit (established, south kyo, china). After co-incubation for 14d, different groups of BMSCs were immunofluorescent stained with OCN primary antibody (SAB, USA) following the immunofluorescent staining procedure described above. After 21 days of co-culture, BMSCs were stained using the cytosine red calcium staining kit (Cyagen, guangzhou, china). The calcium nodules were detected by light microscopy and photographed. Then, calcium nodules were lysed using perchloric acid and the absorbance at 420nm wavelength was measured using an enzyme-labeled instrument. Total RNA of osteoblasts was extracted for a certain period of time and subjected to qRT-PCR experiments, as described above.

12. Angiogenic differentiation

HUVEC at 1X 10 ⁵ mL ^-1 Is plated in 24-well plates (corning, usa) coated with low growth factor matrix gel and BMDM specific medium is added. After 3 days of incubation, the cells were fixed with 4% paraformaldehyde. The cells were then observed under an inverted fluorescence microscope and photographed. Semi-quantitative analysis of data was performed using imagej. Cell culture supernatant was collected. The content of VEGF, PDGF-BB in the supernatant was quantified by the corresponding ELISA kit (Elabscience, wuhan, china) according to the instructions.

4. In vivo evaluation of hydrogels

1. Preparation of animal models

Animal experiments were performed using SD rats. All SD rats were purchased from megaly research new drug research center limited (su zhou, china). All rats were male with an average body weight of 200-220 g. All animals were treated during the experimentSurgical procedures were all approved by the first hospital ethics committee affiliated with the university of su. First, a rat model of macrophage depletion was established to study macrophage presence in GBM ^gel Hydrogels recruit key roles in repairing cells. The chlorophosphonate liposomes (Yeasen, shanghai, china) were intraperitoneally injected one week before and once a week after the operation to deplete macrophages in SD rats. SD rats were then anesthetized with 2% pentobarbital at a dose of 2.5mLkg ^-1 . After complete anesthesia, the rat skull was shaved and skin disinfection with anions was prepared. A median incision of approximately 2.5cm length was made along the longitudinal axis of the skull. The skin and fascia are separated layer by layer until the skull is exposed. Two circular defects were made on either side of the skull using a 5 mm diameter dental trephine. After the hemostasis was rinsed, the wound was covered with the ungelatinized hydrogel by in situ injection, waiting for 10 minutes for adequate gelling, and the control group was injected with the same volume of PBS. The incision was then sutured layer by layer and sterilized with iodophor. Penicillin is injected into muscle for 3 days after the operation day, so as to prevent infection.

2. Animal specimen sampling

SD rats were sacrificed 3 days, 7 days, 14 days, 4 weeks, and 8 weeks post-surgery. Different sets of skull specimens were collected and fixed with 4% paraformaldehyde (Biosharp, shanghai, china) for 24h.

3. Microscopic CT analysis

The samples of the skull at 4 and 8 weeks post-surgery were scanned and processed using micro CT (SkyScan, skyScan1176 belgium). The scan voltage was 65kV, the current was 385mA, and the resolution was 7. Mu.m. Transverse, sagittal and coronal sections of the radius were reconstructed by CTAnalyzer software (Skyscan, belgium) and cylindrical ROIs were defined for morphological and histological analysis of Bone Mineral Density (BMD) of fracture site bone volume fraction (bone volume (BV)/Total Volume (TV)). The sample model obtained from the software was reconstructed at the surface by means of the mic software (belgium).

4. Histological analysis

The collected skull samples were decalcified with ethylenediamine tetraacetic acid (EDTA) solution for 4 weeks, and then gradient dehydrated with ethanol solutions of different concentrations. The dehydrated sample was immersed in a pure xylene solution and then embedded in paraffin.

Histological analysis is divided into two parts. Specimens at 3 and 7 days post-surgery were decalcified, embedded and sectioned as described above. Samples were immunofluorescent stained with F4/80 (Abcam, UK), CD90 (Abcam, UK), iNOS (Abcam, UK) and ARG-1 (Abcam, UK), respectively, to examine macrophage and stem cell distribution in vivo. Samples taken at 14 days after surgery were immunofluorescent stained with RUNX-2 (Abcam, uk), samples taken at 4 and 8 weeks after surgery were stained with hematoxylin and eosin (H & E) and Masson for bone regeneration, and then immunohistochemical staining was performed on OCN (Abcam, uk), CD31 (Abcam, uk), and immunofluorescent staining was performed on OPN (Abcam, uk), VWF (Abcam, uk). The above indicators were used to evaluate bone tissue, periosteum regeneration and vascularization of bone defects, respectively.

5. qRT-PCR experiments

Samples were collected 3 days and 7 days after surgery, ground after liquid nitrogen treatment, and total RNA was extracted from each sample using Trizol reagent (Invitrogen, USA). The RNA was then reverse transcribed into cDNA using a reverse transcription kit (Takara, japan).

5. Results

1. Physical and chemical Properties of hydrogels

We have designed a gelatin-based (gelatin) polymeric hydrogel (GBM) loaded with a complex of bletilla polysaccharide BSP and biological mesoporous active glass MBGN ^gel) For treating critical bone defects. The mixed biological material is constructed by injecting the mixed biological material into a bone defect site to form gel in situ, so that a good cell microenvironment is provided for the defect site, and the regeneration of the bone defect is promoted through immunoregulation, and the gel action is shown in a schematic diagram in figure 1. The physicochemical properties of the prepared hydrogel are as follows:

BSP is used as polysaccharide with high biocompatibility and macrophage affinity, and is further subjected to hydroformylation modification to obtain the aldehyde bletilla polysaccharide oBSP. The invention optimizes the step of the BSP hydroformylation of the bletilla polysaccharide, and dissolves the bletilla polysaccharide in water. The rhizoma bletilla polysaccharide is prepared from glucose and mannoseThe beta glycosidic bond is polymerized to form the glucomannan which can be dissolved in water to form viscous hydrophilic glue solution and is easy to lose viscosity when meeting alkali. The pH of the solution is adjusted to 8.5, and the mass volume percentage concentration of the bletilla polysaccharide to the water is 0.5-1.25%, preferably 1%, namely 0.5-1.25 g, preferably 1g of the bletilla polysaccharide is added into 100mL of water, taking the characteristic that the bletilla polysaccharide is soluble in water but is easy to be sticky into consideration. The dissolution was smooth by slow addition under magnetic stirring for 2 minutes. Can obviously reduce the generation of viscous liquid and facilitate the hydroformylation. Adding sodium periodate (NaIO) as oxidant into rhizoma bletilla polysaccharide solution dissolved in water ₄ ) Stirring and reacting for 8 hours under the protection of argon and light shielding, and adding ethylene glycol to terminate the reaction. Then transferring into a dialysis bag with molecular weight of 3500, dialyzing in deionized water for 48h, filtering after dialysis, preserving at-20 deg. after filtration, and freeze drying for 48h to obtain aldehyde group bletilla striata polysaccharide (oBSP). The percentage of oxidation of oBSP was determined by the previously reported method, with oBSP reacted with excess t-butylhydrazinoformate, the unreacted t-BC was quantified by adding trinitrobenzenesulfonic acid and used at 334nm by spectro-luminance to quantify the colored derivative resulting from the reaction of excess t-BC with TNBS, yielding an optimized percentage of oxidation of 70.5%

MBGN is mesoporous bioactive glass, which is prepared according to a previous procedure, and is further subjected to amination modification to obtain aMBGN. Successful synthesis of the aldehyde-modified bletilla polysaccharide and amination of MBGN were first successfully demonstrated by FTIR. After the bletilla polysaccharide is subjected to oxidation reaction, FTIR is positioned at 1730cm ^-1 The peak of the reaction aldehyde group appears, which proves that the aldehyde group generated on the molecular chain of the bletilla striata polysaccharide can be used for the subsequent experiment. In addition, the APTES-modified MBGN was 1535cm ^-1 The peaks of the reaction amino groups appear, and the modified amino groups of MBGN are proved to be available for subsequent experiments.

SEM and TEM observations of the synthesized MBGN revealed that MBGN had a smooth spherical shape and a uniform size distribution, while aMBGN and MBGN were not significantly different in morphology and distribution. Dynamic Light Scattering (DLS) results showed that the average diameter of aMBGN was 221.3±246nm (a in fig. 4) and the polydispersity index (PDI) was 0.0532. The XRD pattern showed a broad diffuse diffraction peak at 15-30 °, which is a typical characteristic peak of amorphous bioactive glass (B in fig. 4).

SEM results showed that with the addition of aMBGN, the pore size of the hydrogel did not change significantly, averaging about 115 μm by measurement, but the pore structure was gradually dense, while the hydrogel was seen to show asperities with obvious spherical protrusions (fig. 3). It is believed that a pore size of about 100 μm helps promote cell migration and tissue ingrowth while the rugged surface helps cell attachment. EDS spectrum of hydrogel shows GBM ^gel Compared with GB ^gel The presence of calcium (Ca), silicon (Si) and phosphorus (P) in the structure confirmed that MBGN was incorporated in the hydrogel (C and D in FIG. 4, where C is GB ^gel D is GBM ^gel )。

To investigate the effect of different ratios of Gel and oBSP on Gel formation, two liquid Gel formations of different concentrations and different ratios were selected, and the results are shown in FIG. 5, wherein the Gel formation time for Ser. No. 1 (10% Gel and 20% oBSP) and the Gel formation time for Ser. No. 3 (20% Gel and 10% oBSP) were greater than 30 minutes, with Ser. No. 1 being about 37 minutes, and Ser. No. 3 being about 34 minutes; the Gel time for sequence number 4 (10% Gel, 20% oBSP and 6% aMBGN) and sequence number 6 (20% Gel, 20% oBSP and 6% aMBGN) were approximately 19 minutes, while the Gel time for sequence number 2 (20% Gel and 20% oBSP) was approximately 10 minutes, and the Gel time for sequence number 5 (20% Gel, 20% oBSP and 6% aMBGN) was only 3 minutes. And the glue forming strength of the serial numbers 5 and 2 is obviously superior to that of other groups. This suggests that Gel and oBSP can significantly shorten the Gel time at approximately the same ratio, which may be related to the corresponding number of two groups of amino and aldehyde groups, while the addition of aMBGN can also significantly shorten the Gel time. So we choose the ratio of sequence number 2 to sequence number 5 for the next experiment.

An amount of aMBGN was mixed in the oBSP solution while mixing the gelatin solution of the same solubility, which formed hydrogels by schiff base reaction and hydrogen bonding. When the formation of the hydrogel was observed, it was found that the color of the hydrogel group of the composite aMBGN was significantly cloudy (FIG. 6), while the gel time was significantly shortened, compared with GB without aMBGN ^gel The group required 10 minutesGel time, GBM loaded with aMBGN ^gel The group only needs 3 minutes of gel forming time, so that the hydrogel liquid loss caused by overlong gel forming time when the hydrogel is injected in situ can be avoided. FTIR results for hydrogels of different compositions showed 1630cm on both sets of hydrogels ^-1 The c=n stretching vibration absorption peak of the schiff base bond appears, indicating that the schiff base reaction of-NH 2 on the gelatin backbone and aMBGN with c=o on the aldehyde-formed bletilla polysaccharide oBSP forms a c=n bond. With GB ^gel Compared with the spectrum of the hydrogel, the MBGN-loaded hydrogel appeared to be positioned at 799 and 1100cm ^-1 Two new bands at this point, respectively attributable to Si-O-Si bending and stretching vibrations of silicate glass, indicate GBM ^gel MBGN is present in the hydrogel (a in fig. 7).

The viscoelasticity of the hydrogels was evaluated by rheology experiments, and the results of comparing the oscillation frequencies of the two sets of hydrogels at 0-12.5 hz, indicate that both sets can stably maintain the storage modulus (G ') higher than the loss modulus (G'), indicating the construction of the elastic network of hydrogels. Simultaneous GBM ^gel The storage modulus (G') of the group is higher than GB ^gel Group, indicating GBM ^gel Group possession is superior to GB ^gel The applicability of the group may be due to the loading of MBGN (B in fig. 7). To further evaluate the effect of MBGN loading on hydrogel mechanical properties, compression testing was performed on the samples. The compression mechanical behavior is an important parameter for hybrid hydrogels, as this type of hydrogel is suitable for bone regeneration applications where the biomaterial is mainly subjected to compressive stress. The strain-stress curve of the hydrogel was obtained by mechanical compression experiments. The results show that compression analysis of the two sets of hydrogels showed typical "J-shaped" stress strain curves, similar to biological tissue. Simultaneous GBM ^gel The group showed greater elasticity (a in fig. 9), GBM ^gel The compressive strength of the group was within the reported range of human cancellous bone compressive strength values (-0.15 to 13.7 MPa). With GB ^gel Compared with hydrogel, GBM ^gel The compressive modulus of the hydrogel was increased by about 10 times (a in fig. 9). It was demonstrated that the loading of MBGN did contribute to the improvement of mechanical properties, which may be related to the relatively high surface reactivity of MBGN, which leads to the release of ions by MBGNThese ions may interact (e.g., ionically crosslink) with the hydrogel, thereby altering the mechanical properties of the hydrogel matrix. Self-healing also allows injectability of hydrogels, such as GB that will fully set ^gel Hydrogel injection into bottles containing PBS solution was demonstrated. (FIG. 8) the relative stability of the hydrogel in its morphology in the physiological environment of the body helps the hydrogel to exert a cell guiding effect while avoiding deviation of the hydrogel from its original position, while ensuring early support for tissue regeneration, whereas slow degradation of the hydrogel may provide room for the growth of new tissue. MBGN-loaded GBM by measuring the swelling rate and degradation rate of hydrogels ^gel The swelling ratio of the group is significantly lower than GB ^gel Group (B in fig. 9), probably due to the crosslinking of MBGN with the hydrogel, limits the swelling of the hydrogel, contributing to the shape stabilization of the hydrogel. Simultaneous GBM ^gel The degradation rate of the group is obviously lower than GB ^gel Group (C in fig. 9), the loading of surface MBGN helped the structural stabilization of the hydrogel. SI in MBGN to load ⁴⁺ The detection was carried out, and the results were shown to be within the first 12h, si ⁴⁺ Is released at a faster rate and then slowed down, and is shelf stable and sustained release over a period of time (D in fig. 9). This change may be related to the ionic solubility in solution, si at the early stage of hydrogel placement ⁴⁺ Is accelerated to achieve ion balance in the solution, and then continuously and slowly releases the active ingredients to help MBGN to play the roles of promoting the immunity and the osteogenesis of macrophage-stem cell axes and simultaneously reduce Si ⁴⁺ Too fast release results in toxicity to the body. )

2. Intervention of hydrogels on in vitro cells

Biocompatibility is an important factor for the application of materials in vivo, and the co-culture of materials with cells is schematically shown as a in fig. 10. In vitro experiments were used to assess the biocompatibility of the composite hydrogels. Live/dead staining results after 7 days of cell co-culture with hydrogel showed that BMSC cells adhered and spread well on different sets of hydrogels (as B in fig. 10). There was no significant difference in the number of cell survival between the groups compared to the blank group, indicating that the composite hydrogel had no significant effect on cell viability (e.g., C in FIG. 10). In addition, the cytotoxicity of the hydrogels was evaluated in vitro by CCK-8 assay (as in D in FIG. 10). In CCK-8 experiments, BMSCs (bone marrow mesenchymal stem cells) proliferate after co-culture with different hydrogels, there was no significant statistical difference in survival rate between groups compared to control groups, while there was no significant difference in survival rate between MBGN-loaded hydrogel groups and MBGN-unloaded hydrogel groups, in the relevant literature regarding MBGN's potential cytotoxicity to cells, while after amine modification, the acmbgn partially circumvented MBGN's potential cytotoxicity. In this study we used the natural bletilla polysaccharide BSP with good biocompatibility to target mannose receptor MR on macrophages, which proved to be effective in past studies. We selected in vitro experiments with primary BMDM extracted from mouse bone marrow, which could be stimulated by M-CSF to adhere. Comparing the effect of M-CSF and the hydrogel containing the polysaccharide BSP on BMDM, it was found by cell counting that BMDM adhered to the hydrogel containing the polysaccharide BSP in large amounts, without significant difference from the amount of M-CSF stimulated adherence (as in E in FIG. 10).

We further explored the changes that were made after hydrogel targeted capture of BMDM by flow analysis. The flow results indicated (FIGS. 11 and 12, where FIG. 11 is co-cultured with BMDMs for 3 days; FIG. 12 is co-cultured with BMDMs for 7 days), that the inflammatory environment was simulated with intervention of 100ng/mL of co-LPS, and that CD86 at 3 days in comparison of the groups co-cultured for 3 days and co-cultured for 7 days ⁺ the/CD 11b cell fraction exhibited a high fraction, CD206 ⁺ the/CD 11b cells were low duty cycle. Whereas over time, co-culture was carried out for 7 days of CD86 ⁺ /CD11b ⁺ Cells showed a tendency to decrease in duty cycle, CD206 ⁺ the/CD 11b cell ratio shows an upward trend. This represents a dynamic regulation of BMDM in inflammatory environments. On co-cultivation for 3 days, GB group and GBM compared to untreated cells ^gel CD68 in group treatment group ⁺ Cell (representing M1 macrophage) occupancy increased compared to control and gel alone, and CD206 ⁺ Cells (representing M2 macrophages) were not significantly induced, which may be associated with involvement of bletilla polysaccharide in the hydrogel. Mannose on bletilla polysaccharide binds to mannose receptor of BMDM, affecting BMDM to M1 type giantThe phagocytes are polarized in the direction. Interestingly, GBM after 7 days of co-cultivation ^gel Group CD206 ⁺ The cell fraction was at the highest level, up to 25.9%, among several groups, which may be related to MBGN involvement. Over time, MBGN loaded in hydrogels was gradually exposed, enriched in SI ⁴⁺ It has been demonstrated that there is a tendency to promote polarization of BMDM toward the M2 type.

Similar results were also observed with cell immunofluorescent staining, and isolated BMDM cells were stained with green fluorescent light of macrophage surface marker F4/80 (FIGS. 13 and 14). After 3d co-culture, fluorescence of the surface marker INOS of M1 type macrophages exhibited a high intensity fluorescent appearance in each group of cells. Meanwhile, fluorescence of the surface marker CD206 of M2 type macrophages exhibits weak expression. After 3d co-cultivation, GB ^gel Group and GBM ^gel The INOS fluorescence was significantly stronger in the group than in the other two groups (fig. 13 and 15), whereas GBM after 7d co-culture ^gel The fluorescence of CD206 of group was significantly stronger than that of the other groups (FIGS. 14 and 15), and GBM ^gel Cells on one group have a more elongated morphology and more pseudopodia than cells in the other group. Semi-quantitative analysis of fluorescence corresponding to INOS and CD206 showed the same trend after 3 and 7 days of co-culture. In combination with the above phenotypic analysis, we can conclude that in GBM ^gel In the co-culture process of the group and the BMDM, the duty ratio of the BMDM in the M1 type is obviously improved at 3 days, and the duty ratio of the BMDM in the M2 type is obviously improved at 7 days, so that the trend of time sequence conversion is presented. Is capable of binding to the receptor on BMDM at an early stage with sustained release of SI ⁴⁺ GBM of ions ^gel Hydrogels are expected to achieve ligand selective targeting to capture macrophages, transforming the phenotype of macrophages at different times.

Cell scratch experiments and Transwell chemotactic migration experiments were used to evaluate the effect on BMSCs after hydrogel targeting affected BMDM. GB compared with the group without the bletilla polysaccharide BSP ^gel Group and GBM ^gel The mobility of the BMSCs of the group was higher (a and B in fig. 16). After 24 hours, GB ^gel The mobility of the group was 30.36% at maximum, whereas the mobility of the control group was only 10.19%; after 48 hours, GB ^gel Of groups ofThe mobility was up to 67.13% and significantly higher than in the control and gelatin gel alone groups. Transwell migration experiments also presented similar results (C and D in fig. 16). GBM co-cultured with BMDM for 7 days ^gel After 24 hours of incubation of BMSCs in the special culture medium of the group, more BMSC cells migrate through the Transwell membrane. The results indicate that BSP can affect homing ability of stem cells early by targeting BMDM.

To further investigate the effect of hydrogels on bone differentiation of BMSCs following intervention of BMDM in the local microenvironment formed by secreted bone-related cytokines, BMDM and BMSCs were co-cultured with different hydrogels using transwell plates. Alkaline phosphatase (ALP) secreted by osteoblasts can directly reflect the activity and function of osteoblasts, and is a marker of early osteoblast differentiation and bone mineralization. After 7 days of co-culture, BMSCs were collected for ALP staining (FIG. 17) and activity measurement (A in FIG. 20). GB (GB) ^gel Group and GBM ^gel More stained osteoblasts in the group, while GBM ^gel Groups had significantly deeper ALP staining than the other groups. Osteocalcin (OCN), a calbindin synthesized and secreted by mature osteoblasts, is the major component of bone non-collagens and is considered as a marker of osteoblast differentiation towards the mineralization stage. BMSCs were collected after 14d of co-culture and subjected to OCN immunofluorescent staining (fig. 18). GBM (GBM) ^gel BMSCs of the group exhibited the most intense red fluorescence, representing OCN, and cells spread better in all groups. This was confirmed by semi-quantitative analysis of OCN immunofluorescence staining (B in FIG. 20). Calcium nodules are mineralized extracellular matrix formed by osteoblasts, indicating the final stage of osteogenic differentiation. BMSCs were collected for alizarin red staining 21 days after co-culture (fig. 19). GBM (GBM) ^gel The calcium nodules in the group were the most abundant and dense in all groups. Quantitative analysis of calcium nodules produced results consistent with staining results (a in fig. 21). qRT-PCR detection of RUNX2, ALP and OCN, GBM ^gel The groups all exhibited high expression results, which were identical to those of the above experiment (B, C, D in fig. 21).

The stimulatory effect of the particular medium on angiogenesis after co-culture of hydrogels with BMDM was evaluated by an in vitro angiogenesis assay. In use special After 4 hours of culture in the medium, the control group did not form a distinct capillary network in the conditioned medium of untreated macrophages. In contrast, GB ^gel Group and GBM ^gel Group of media showed rich capillary tube formation, and GBM ^gel Group co-cultures had higher branches and overall lengths. Investigation of cytokines associated with cell migration and angiogenesis by ELISA, GB ^gel Group and GBM ^gel The expression levels of both VEGF and PDGF-BB are significantly increased in the group ^gel Group B is also compared with GB ^gel The group is lifted more (fig. 22 and 23).

3. In vivo evaluation of hydrogels

The experimental imaging performance and histological analysis of the rat in vivo are shown in fig. 24, and the skull defect model of the rat is shown in fig. 25. Bone defect repair was analyzed by injecting hydrogel in situ over the rat skull defect and gelling it, and collecting skull specimens 4 and 8 weeks after surgery. Bone tissue repair was studied using micro-CT and histological examination. The collected specimens were reconstructed by micro-CT scanning to evaluate bone repair. Reconstructed images of bone defects showed normal GBM at 4 and 8 weeks post-operative ^gel Is significantly better than the other groups (fig. 26). After 4 weeks of hydrogel implantation, normal GBM ^gel New autologous bone tissue appeared around the critical dimension defect edge of the group, whereas macrophage depleted GBM ^gel The formation of new bones in the group is limited. After 8 weeks of hydrogel implantation, normal GBM ^gel The critical size defects of the group have a large area of new bone formation, and the bone defects are almost entirely covered by closely packed new bone tissue. Macrophage depleted GBM ^gel The group also had a portion of the new osteogenesis demonstrated better than the macrophage depleted control group, which may be associated with GBM ^gel Group-provided scaffolds, cellular microenvironments and supported MBGN are themselves capable of dissolving to form calcium silicon layers contributing to bone-related, but macrophage-depleted GBM ^gel The group performed worse than the normal control group. As expected, quantitative micro-CT analysis showed normal GBM ^gel The group significantly increased the BV/TV and BMD values over the other groups, indicating GBM ^gel The number of newly formed bone trabeculae in the group is high,the packing is more dense. The BV/TV and BMD values of the second normal control group were higher than those of the macrophage depleted GBM ^gel Group, again higher than the control group depleted of macrophages (a and B in fig. 29). The results of the micro-CT and quantitative analysis show that GBM in the presence of macrophages ^gel The bone defect regeneration capacity of the group was significantly better than the other groups, which might suggest GBM ^gel Can promote the endogenous self-healing process of bone immune-stem cells in vivo to help the regeneration of bone defects. Meanwhile in the group of depleted macrophages, GBM ^gel The group had a certain degree of ability to promote regeneration of bone defects compared to the control group, but the condition of promoting regeneration of bone defects was worse than the control group without depleting macrophages, suggesting the importance of macrophages in the endogenous self-healing process of bone immune-stem cells for regeneration of bone defects, further suggesting GBM ^gel The targeted bone defect regeneration is mainly acted by targeted capture macrophages. Histological analysis of the skull specimens was performed. H&E staining results show (C and D in FIG. 27, FIG. 29), in normal GBM ^gel Bone repair was superior to the other groups at different time points in the group, while exhibiting the highest bone tissue continuity. Masson staining was used to assess the maturity of new bone tissue, especially from crust to mature remodeling during repair of bone defects, and red-blue cross staining showed that new bone tissue was formed at all groups of bone defects. Normal GBM ^gel The group had a larger, more continuous sheet-like red-stained area consistent with the quantitative analysis of the red-stained area (fig. 28, E and F in fig. 29). Shows normal GBM ^gel The bone formation and remodeling activities of the group are enhanced. In contrast, in macrophage depleted GBM ^gel The group contained thicker collagen fibers than the normal control group, suggesting that the lack of macrophages involved in the repair process resulted in an inability to precisely regulate stem cell differentiation, resulting in scar hyperplasia.

Immunohistochemical staining of late cell osteogenic differentiation index OCN, OPN showed (A and C in FIG. 30), normal GBM ^gel Positive expression of OCN in the group was significantly higher at various time points than in the other groups (A and B in FIG. 31), indicating good ability of bone repair. The result shows that it is normalGBM of (c) ^gel The group had better ability to promote bone tissue maturation (E and F in fig. 31), and changes in osteogenic index at different times suggested GBM ^gel Has a positive effect throughout the osteogenesis process.

To assess the ability to regenerate blood vessels at the defect. Angiogenesis at the defects of the different treatments was studied by IH staining of CD31 and IF staining of VWF (B and D in fig. 30). The results indicate that at 4 weeks, there was a greater amount of CD31 positive staining structure in normal mice than in macrophage depleted rats; in normal rats, GBM is used ^gel The number of CD31 positive stained capillaries in hydrogel-treated wounds was large compared to sham-operated groups. In contrast, in macrophage depleted rats, GBM was used ^gel The number of capillaries positively stained for CD31 in hydrogel-treated wounds was similar to that of the control group. After 8W, the CD31 stained area then showed a decreasing trend (C and D in fig. 31). VWF is considered an indicator of neovasculature, GBM in normal rats at 8 weeks ^gel Group VWF expressed significantly less than macrophage depleted GBM ^gel This reduction may be due to the fact that less nutrition and oxygen are required after defect repair and that the vessels formed during regeneration subsequently degenerate. The above results indicate GBM ^gel Bone defects can heal faster and more effectively without depleting macrophages (G and H in fig. 31).

To verify the hypothesis, we reselected samples of 3, 7, 14, 4W and 8W post-surgery on the macrophage depleted skull defect model for the experiment.

We labeled macrophages with F4/80 and stem cells with CD 90. F4/80 observed at defective sites in groups of macrophage depleted rats on day 3 and day 7 ⁺ Far fewer cells than normal rat groups indicate that injection of chlorophosphonate liposomes is effective for macrophage depletion. CD90 is also visible at the defect site in macrophage depleted rats ⁺ The cells are unevenly distributed and accumulate at the periphery of the defect. In GBM ^gel F4/80 at the third day is visible in the inserted group ⁺ The number of cells is greater than that of the control group, while CD90 ⁺ Obviously present with F4/80 ⁺ Cell-like distribution. F4/80 was clearly visible on day seven ⁺ Cell infiltration into defect with CD90 ⁺ And also distributed therewith. Whereas GBM is depleted in macrophages ^gel CD90 placed in group ⁺ Cells were also distributed at the periphery of the defect, with no significant statistical difference from the macrophage depleted control group (a in fig. 32, A, B, C and D in fig. 33). The results indicate that the distribution of stem cells is affected by macrophages while GBM ^gel Can affect the distribution of macrophages but not directly the distribution of stem cells.

We performed a PCR analysis of secreted related factors for each group at two time points (A, B, C, D, E and F in FIG. 34), indicating GBM of non-depleted macrophages ^gel The expression of factors iL-1, iL-4, iL-12, TNF- α was significantly increased in the group at 3 days compared to the control group, while the expression was significantly decreased at 7 days compared to the control group, and these factors were highly correlated with inflammation. Whereas M-CSF and OSM factor are expressed significantly higher than the control group on both 3 and 7 days, M-CSF is a cytokine highly correlated with macrophage recruitment and OSM is a cytokine highly correlated with stem cell homing, which may suggest that BSP regulates macrophage characteristics after bone defect occurs. In the literature, macrophages are analyzed for their similar behavior in cytokine expression in different interventions of BSP and LPS. RUNX-2 immunofluorescent staining in the 14 day post-operative group revealed GBM of non-depleted macrophages ^gel The RUNX-2+ region of the group was significantly more than that of the control group, whereas the GBM of macrophages was depleted ^gel There was no significant difference between the RUNX-2+ region of the group and the control group, indicating GBM ^gel Has the effect of promoting bones in early stages. (B in FIG. 32, E in FIG. 33) immunofluorescent staining of alpha-SMA at the site of bone defect at 8W, the high expression region of alpha-SMA probably marks the pathological package of a large number of macrophages, and GBM of unconsumed macrophages can be found ^gel No significant aggregation of α -sma+ cells was seen in group w, in contrast to the aggregated α -sma+ cells seen in the control group. This may prompt GBM ^gel The regulation and control of macrophages are controllable, and the formation of pathological packages can be avoided. (C in FIG. 32, F in FIG. 33))

To further investigate GBM ^gel Mechanisms to promote macrophage transformation to the immune activation phenotype at various time points we performed proteomic analysis of co-cultured BMDM (fig. 35). From the control group and GBM, respectively, by mass spectrometry ^gel 1877 and 1169 proteins were identified. In the control group and GBM ^gel Showing obvious change>1.5-fold) protein of CSF-3, cxcl3 and ARG-1 in GBM ^gel Up-regulation of Mpo, cd5l and other proteins in GBM ^gel Down-regulation (fig. 36). CSF-3, cxcl3, aid in recruitment and adhesion of macrophages, ARG-1 aids in secretion of anti-inflammatory cytokines by macrophages, while Mpo, cd5l are inflammatory related factors by macrophages.

Gene Ontology (GO) enrichment analysis showed GBM ^gel The groups showed significant differences in the proteomes with respect to immune system processes, cell migration and cell adhesion compared to the control group (FIG. 37), which suggested GBM ^gel The group has obvious capacity of regulating macrophage behavior and polarization. KEGG enrichment analysis showed GBM ^gel The group was significantly different from the TNF signaling pathway, NF-. Kappa.B signaling pathway, PI3K-AKT signaling pathway and MAPK signaling pathway in the control group (FIG. 38).

To further verify the effect of hydrogels on cytokines of BMDM analysis, cells were subjected to qRT-PCR for genomic analysis after 3 and 7 days of co-culture. The results indicate GBM ^gel The group exhibited high expression of OSM and m-CSF at both 3 and 7 days, m-CSF proved to be associated with recruitment and adhesion of macrophages, whereas OSM proved to be associated with homing of stem cells of the surrounding tissues, a key cytokine directly inducing osteogenesis, a member of the pro-inflammatory interleukin 6 (IL-6) family, the elevation of which was a beneficial cue for the subsequent function of bone cells. GBM (GBM) ^gel The ability to promote macrophage and stem cell homing is sustained throughout the process of interfering with macrophages. GBM for the expression of inflammatory-related iNOS and TNF-alpha ^gel The group had a significantly high expression at 3 days, while the expression of the relevant inflammatory factor was significantly down-regulated at 7 days, while GBM was expressed for Arg-1, il-10 of the anti-inflammatory factor ^gel There was no significant difference between the control group at 3 days, whereas Arg-1, il-10 expression was significantly up-regulated at 7 days compared to the control group. GBM is shown for the opposite trend of inflammatory and anti-inflammatory factors at different time points ^gel The macrophage is stimulated to secrete inflammatory factors in early stage, and has various beneficial effects in mobilizing osteoprogenitor cells, inducing osteogenic differentiation and improving angiogenesis. And after 7 days with GBM ^gel MBGN in (b) is recognized and endocytosed by macrophages, stimulates the macrophages to reduce the expression of inflammatory factors, secretes anti-inflammatory factors to timely control inflammation, and interestingly, GBM ^gel At 3 days, it exhibited an up-regulated expression of CD206 (mannose receptor MR) compared to the control group, suggesting GBM ^gel Is bound by activating CD206 on macrophages.

To further understand GBM ^gel The differences in time relationship between the groups compared to the control groups were shown, and we performed relevant WB experimental analyses for different time points (fig. 39 and 40). The JAK/STAT-3 and TLR4/NF- κb and MAPK pathways were selected selectively for the results of the proteomic analysis. Recent studies have shown that natural polysaccharides induce immune cell activation through MAPK and NF- κB phosphorylation. WB results show that GBM at 3 days ^gel Expression of the groups p-NF-. Kappa.Bp 65/NF-. Kappa.Bp 65 and p-ERK/ERK was increased compared to the control group. At 7 days there was no significant difference in the expression of p-NF-. Kappa.Bp 65/NF-. Kappa.Bp 65 and p-ERK/ERK compared to the control, whereas p-STAT3/STAT3 expression was significantly up-regulated. Results hint GBM ^gel The group activated NF- κB signaling pathway through binding to TLR4 receptor at early stage to promote polarization of BMDM to M1 type, while activating ERK signaling pathway reduces inflammatory factor release, and at late stage significantly up-regulates JAK/STAT3 signaling pathway to promote polarization of BMDM to M2 type. The results further demonstrate that GBM ^gel Compared with GB not being taken by MBGN ^gel The polarization of BMDM and the release of relevant cytokines are accurately regulated and improved obviously.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The preparation method of the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel is characterized by comprising the following steps of:

2. The preparation method according to claim 1, wherein the mass ratio of the aldehyde group bletilla polysaccharide, the amino MBGN and the gelatin is 10:3:10.

3. The preparation method according to claim 1, wherein the mass fraction of the bletilla polysaccharide in the bletilla polysaccharide aqueous solution is 1%, and the pH is 8.5.

4. The method according to claim 1, wherein in the step 1,

the mass ratio of the bletilla polysaccharide to the sodium periodate in the bletilla polysaccharide aqueous solution is 2:1;

the oxidation reaction is followed by a dialysis step;

the dialyzed solution is deionized water;

the molecular cut-off of the dialysis is 3500;

the dialysis time was 48 hours.

5. The method according to claim 1, wherein in the step 2,

the mass ratio of the MBGN to the APTES is 1:5;

6. The method according to claim 6, wherein,

in the step 3, the reaction condition is 45 ℃ for 10min.

7. The bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method of any one of claims 1-6.

8. Use of the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method according to any one of claims 1-6 or the bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel according to claim 7 for targeted capture of bone marrow mesenchymal stem cells, polarization of bone marrow mesenchymal stem cells towards macrophages, promotion of migration of bone marrow mesenchymal stem cells, promotion of stem cell homing, promotion of bone marrow mesenchymal stem cell osteogenic differentiation, promotion of angiogenesis-related cytokine expression, promotion of bone repair, promotion of bone regeneration, promotion of macrophage secretion of anti-inflammatory cytokines (CSF-3, cxcl 3), inhibition of macrophage inflammatory-related factors (Mpo, cd5 l).

9. Use of a bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel prepared by the preparation method according to any one of claims 1-6 or a bletilla striata polysaccharide-mesoporous bioactive glass-gelatin hydrogel according to claim 7 for preparing a product for promoting bone repair, promoting bone regeneration and/or promoting stem cell homing.

10. A product for promoting bone repair, promoting bone regeneration and/or promoting stem cell homing, comprising at least one of a carrier or an adjuvant and the bletilla polysaccharide-mesoporous bioactive glass-gelatin hydrogel of claim 7.