CN111793225B

CN111793225B - Gelatin/gellan gum/hydroxyapatite composite hydrogel and preparation method thereof

Info

Publication number: CN111793225B
Application number: CN202010731727.2A
Authority: CN
Inventors: 汤克勇; 王绿阳; 李萌雅; 王芳; 刘捷; 郑学晶
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2020-06-19
Filing date: 2020-07-27
Publication date: 2022-05-27
Anticipated expiration: 2040-07-27
Also published as: CN111793225A

Abstract

The invention belongs to the technical field of tissue engineering, relates to a new material applied to a cartilage substitute, in particular to a gelatin/gellan gum/hydroxyapatite composite hydrogel and a preparation method thereof. The composite hydrogel is prepared from gelatin, low-acyl oxidized gellan gum and aminated hydroxyapatite, wherein the weight ratio of the gelatin to the low-acyl oxidized gellan gum is (10-30) to (1-3); the addition amount of the aminated hydroxyapatite is 0.5-2% of the total weight of the gelatin and the oxidized gellan gum. The particle size of the modified mHap is obviously reduced and is closer to a nanometer level, so that the mHap is distributed in the composite hydrogel more uniformly, a network of the hydrogel formed after the mHap is introduced into the Gel-OG is more compact, and the introduction of the mHap has a promoting effect on the proliferation of cells, so that the Gel-OG/mHap has good biocompatibility, degradability and self-healing property, and the application of the Gel-OG/mHap composite hydrogel in the field of cartilage tissue engineering is facilitated.

Description

Gelatin/gellan gum/hydroxyapatite composite hydrogel and preparation method thereof

Technical Field

The invention belongs to the technical field of tissue engineering, relates to a new material applied to a cartilage substitute, in particular to a gelatin/gellan gum/hydroxyapatite composite hydrogel and a preparation method thereof.

Background

Cartilage tissue is a highly ordered, fibrous, and cell-attached hydrogel-like tissue, which mainly comprises cartilage tissue cells and extracellular matrix (ECM), and is free of organs such as lymph and blood vessels. Cartilage tissue contains about 70% water, 20% collagen and 10% protease, and the tissue is gradually divided inwardly into four layers, i.e., surface, middle, deep and calcified cartilage regions, each having different physicochemical and biological properties. The cartilage tissue covers the end of the bone joint, has the functions of load decompression, joint protection from direct mechanical damage and the like, and is very important for the life activities of animals.

The damage of cartilage tissue can directly cause chronic diseases such as joint mechanical function degradation and osteoarthritis, and can lead to continuous pain and even disability. However, due to the lack of organs in human articular cartilage that can achieve self-repair, such as nerves, lymph, blood vessels, etc., the number of repair cells present is limited, and it is necessary to implant materials or cells that can help complete repair and healing from the outside. Currently, the traditional clinical approaches available for the treatment and repair of cartilage tissue mainly include abraded joint replacement surgery, Autologous Chondrocyte Implantation (ACI), and gene-induced autologous chondrocyte implantation (MACI), among others. These approaches often require long treatment times and may present immunological rejection, causing secondary harm to the patient.

In recent years, there has been an increasing search for materials and methods for reconstructing and developing cartilage tissue. Meanwhile, due to the progress and breakthrough of the cellular molecular biology field and the material chemistry field, the tissue engineering technology is used for repairing the damaged human tissue. The biological material is used as an important ring in the tissue engineering technology, can replace damaged tissues, and can also be used as a cell carrier to promote the regeneration of tissue cells. For cartilage tissue engineering, the replacement of damaged cartilage tissue by biological materials by implantation or local injection has been used as an effective way and means to repair damaged cartilage tissue.

At present, hydrogel and scaffold materials are mainly developed and utilized in tissue engineering to repair damaged cartilage tissues. The scaffold material has mechanical properties required by being used as an articular cartilage repair material, and the morphological structure of the scaffold material is similar to that of a natural cartilage tissue of a human body; however, the shape and mechanical properties of the stent material have certain fixity, which is not favorable for completely matching with the damaged tissue; moreover, the porous structure of the scaffold material is often compact and even non-porous, which will affect the fixation and growth of tissue cells on the scaffold, and limit its application in cartilage tissue engineering. As another new cartilage replacement and repair material, hydrogel has received great attention and research. The hydrogel is a material which contains a large amount of water and has a complete three-dimensional network structure, and can effectively inhibit hydrophilic substances from dissolving in an aqueous environment. Meanwhile, the physiological structures of the hydrogel and the extracellular matrix are similar, and good permeability is shown for metabolic substances and nutrient substances.

Currently, raw materials for preparing cartilage repair hydrogel mainly include natural polymers or synthetic polymers. The synthetic polymer itself generally has good mechanical properties, such as higher tensile strength, and stable physical and chemical properties. The synthetic polymers commonly used in surgical operations include polyurethane, polyethylene glycol, polypropylene glycol, and the like. However, synthetic polymers are usually accompanied by high cytotoxicity, are not favorable for tissue cell adhesion and proliferation, are not easy to be completely degraded, and pose challenges for research and application in tissue engineering.

Disclosure of Invention

The invention aims to provide gelatin/gellan gum/hydroxyapatite composite hydrogel and a preparation method thereof.

Based on the purpose, the invention adopts the following technical scheme: a gelatin/gellan gum/hydroxyapatite composite hydrogel is prepared from gelatin solution, oxidized gellan gum solution, and aminated hydroxyapatite.

Furthermore, the weight ratio of the gelatin to the oxidized gellan gum is (10-30) to (1-3); the addition amount of the aminated hydroxyapatite is 0.5-2% of the total weight of the gelatin and the oxidized gellan gum.

The preparation method of the composite hydrogel comprises the following steps:

(1) preparing a gelatin solution;

(2) preparing an oxidized gellan gum solution;

(3) dispersing the aminated hydroxyapatite powder into a gelatin solution to prepare a gelatin/hydroxyapatite mixed solution;

(4) dripping the oxidized gellan gum solution into the gelatin/hydroxyapatite mixed solution at the temperature of 30-50 ℃, uniformly mixing, pouring into a mold, and placing in a water bath at the temperature of 20-40 ℃ for reaction for 12-18 hours; and cooling to room temperature after the reaction is finished to obtain the composite hydrogel.

Further, the preparation method of the oxidized gellan gum comprises the following steps:

(1) dissolving and dispersing gellan gum in water at 90-100 ℃, and uniformly stirring to obtain a gellan gum solution;

(2) adding NaIO into gellan gum solution₄，NaIO₄The weight ratio of the oxide gellan gum to gellan gum is (0.5-1.3): 1, and the oxide gellan gum mixed solution is prepared by reacting for 4-12 hours at 35-55 ℃ in a dark place;

(3) placing the oxidized gellan gum mixed solution into a dialysis bag for dialysis, performing suction filtration on the mixed solution after dialysis, and performing freeze drying on the solution obtained by suction filtration to obtain oxidized gellan gum; wherein the molecular weight of the intercepted molecules of the dialysis bag is 8-14 kDa.

Further, the preparation method of the aminated hydroxyapatite comprises the following steps:

(1) dispersing hydroxyapatite powder into 70-90% absolute ethyl alcohol solution according to the proportion of 1g:100mL, and performing ultrasonic dispersion treatment for 10-15 min to prepare a mixed solution;

(2) adding ammonia water and ethyl orthosilicate into the mixed solution, stirring the mixture at the temperature of 60 ℃ for reaction, collecting precipitates, washing the precipitates with alcohol, washing the precipitates with water and drying the precipitates to obtain precipitates;

(3) and (3) dripping gamma-aminopropyltriethoxysilane into 70-90% absolute ethanol solution, adding the precipitate prepared in the step (2), stirring for 6-8 hours at 25-30 ℃, centrifuging, collecting the precipitate, and performing alcohol washing, water washing and drying to obtain the aminated hydroxyapatite.

Further, the preparation steps of the hydroxyapatite are as follows:

(1) respectively dissolving calcium nitrate and diammonium hydrogen phosphate in water to respectively prepare 1.2M Ca²⁺Solution, 0.72M HPO₄ ^2-A solution;

(2) at 40-50 deg.C, adding Ca²⁺The solution is dropped into HPO according to the volume ratio of 1:1₄ ^2-In the solution, the pH of the mixed solution is stabilized at 10.5 by ammonia water, and the reaction is continued for 2 hours;

(3) and standing the reaction solution at 50 ℃ for precipitation for 12h, collecting the precipitate, washing with alcohol, washing with water, drying, and calcining at 900 ℃ for 2h to obtain the hydroxyapatite.

Further, the concentration of the gelatin aqueous solution is 10-30 wt%.

Furthermore, the concentration of the oxidized gellan gum solution is 1-3 wt%.

Further, a gelatin solution and an oxidized gellan gum solution are added according to a mass ratio of 1:1.

The application of the composite hydrogel in cartilage tissue substitute materials.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, through optimizing the oxidation condition of the gellan gum, the aldehyde group content of the finally prepared oxidized gellan gum is higher, the particle size is reduced, the particle size distribution is uniform, and the crosslinking effect of the oxidized gellan gum as a chemical crosslinking agent is improved.

(2) The invention utilizes a precipitation method to prepare hydroxyapatite (Hap), and utilizes Tetraethoxysilane (TEOS) and gamma-Aminopropyltriethoxysilane (APTES) to modify the Hap to obtain aminated hydroxyapatite (mHap), and particle size tests and FESEM results show that after silanization modification, the particle size of the Hap is obviously reduced, is close to nanometer level, and is more uniformly distributed. FTIR and XRD results show that Si-O-Si and-NH 2 are successfully introduced into the Hap, the silane coupling agent forms a monolayer structure mainly on the surface of the Hap, and the crystal structure of the Hap is not changed.

(3) According to the invention, mHap is doped into Gel-OG to prepare the Gel-OG/mHap composite hydrogel, and a strong bonding effect exists between mHap and a Gel-OG network, so that the network formed by the Gel-OG/mHap composite hydrogel is more compact, and Ca, P and Si elements are uniformly distributed in the hydrogel.

(4) The results of mechanical, swelling and degradation tests show that the compression mechanical strength of the Gel-OG/mHap composite hydrogel is remarkably improved by the incorporation of mHap, the equilibrium swelling rate and the degradation rate are reduced, and the compression mechanical strength is doubled compared with that of the hydrogel without the incorporation of mHap. In addition, the Gel-OG/mHap composite hydrogel containing 1wt% of mHap has the highest compressive mechanical strength of 2.03MPa at 90% deformation, and can meet the requirement of the human articular cartilage on the lowest compressive mechanical strength of 0.78 MPa. In addition, the Gel-OG/mHap composite hydrogel has an equilibrium swelling ratio of 776% and the degradation rate is lowest; after 50 times of cyclic compression, the composite hydrogel can still retain 91 percent of the mechanical strength of the initial compression, and shows good elasticity and compressibility. In addition, it has excellent tensile mechanical strength and certain self-healing property. MTT test results show that the Gel-OG/mHap composite hydrogel has good cell compatibility to L929 cells, and the introduction of mHap has a promoting effect on the proliferation of cells, thereby being beneficial to the application of the Gel-OG/mHap composite hydrogel in human articular cartilage tissue engineering.

In conclusion, mHap is prepared by modifying Hap with TEOS and APTES, and is doped into Gel-OG to prepare the Gel-OG/mHap composite hydrogel. The particle size of the modified mHap is obviously reduced and is closer to the nanometer level, so that the mHap is more uniformly distributed in the composite hydrogel, a network of the hydrogel formed by introducing the mHap into the Gel-OG is more compact, and the Ca, P and Si elements are uniformly distributed in the hydrogel. In addition, the mHap is doped, so that the Gel-OG/mHap composite hydrogel has higher compressive mechanical strength and lower equilibrium swelling ratio and degradation rate, and can meet the basic requirements of articular cartilage on compressive stress. In addition, the introduction of mHap has the promotion effect on the proliferation of cells, shows good biocompatibility, has the properties of injectability and self-healing, and is favorable for the application of Gel-OG/mHap composite hydrogel in the field of cartilage tissue engineering.

Drawings

FIG. 1 is a line graph showing the aldehyde group content in OG;

FIG. 2 is a particle size distribution image of GG solution and OG solution;

FIG. 3 is an FTIR image of GG and OG;

FIG. 4 is a graph of the particle size distribution of Hap and mHap;

FIG. 5 is an FTIR image of Hap and mHap;

FIG. 6 is an XRD pattern of Hap and mHap;

FIG. 7 is a FESEM image of a Hap sample, an mHap sample;

FIG. 8 is a cross-sectional profile of a Gel-OG hydrogel with 10wt% Gel and different OG contents;

FIG. 9 is a graph of compressive stress-strain curves and compressive mechanical strength at 90% deformation for Gel-OG hydrogels of different OG content;

FIG. 10 is a graph of swelling curves for Gel-OG hydrogels with different OG contents;

FIG. 11 is a FTIR characterization of Gel-OG/mHap composite hydrogels with 10wt% gelatin, varying mHap incorporation;

FIG. 12 is a diagram of the reaction mechanism that may exist between Gel, OG and mHap;

FIG. 13 is an XRD image of Gel-OG/mHap composite hydrogels with different mHap incorporation levels;

FIG. 14 is a sectional profile of Gel-OG/mHap composite hydrogel with 10wt% gelatin and different mHap incorporation levels;

FIG. 15 is a low power FESEM image of Gel-OG hydrogel and 1wt% mHap composite hydrogel;

FIG. 16 is a graph of the compressive stress-strain curve of a hydrogel and the compressive mechanical strength at 90% deformation;

FIG. 17 is a graph of swelling curves of composite hydrogels of different mHap contents in PBS solution;

FIG. 18 is a graph of the degradation curves of composite hydrogels with different mHap contents in PBS solution;

FIG. 19 is a graph showing the relative proliferation rate (RGR/%) of L929 cells obtained by MTT assay on different days of culture.

Detailed Description

Example 1 preparation and characterization of oxidized gellan gum

Preparation method of oxidized gellan gum

The preparation process of the oxidized gellan gum (OG) is as follows:

(1) weighing a certain amount of low acyl Gellan Gum (GG), dispersing in 200mL of distilled water at 90 ℃, stirring for 30min to obtain a uniform GG solution, and gradually cooling to 40 ℃.

(2) Adding a certain amount of NaIO into GG solution₄And under the condition of keeping out of the sun, controlling the oxidation temperature and the oxidation time, and violently and uniformly stirring to obtain the oxidized gellan gum mixed solution.

(3) Pouring the mixture into dialysis bag with molecular weight of 8-14kDa, and dialyzing with distilled water at room temperature for 5 days to remove residual NaIO₄And carrying out suction filtration on the mixed solution after dialysis and the reaction by-products, prefreezing the solution obtained by suction filtration at-20 ℃ for 12h, then carrying out freeze drying by a freeze dryer to prepare the oxidized gellan gum, and storing at-4 ℃ for later use.

Secondly, influence of different reaction conditions on aldehyde group content in OG

Based on single-factor experimental analysis method, different NaIO are explored₄The influence of the dosage, the oxidation temperature and the oxidation time on the content of aldehyde groups in OG. Optimizing OG oxidation condition according to aldehyde group content test resultAnd provides raw materials for preparing the composite hydrogel.

Method for measuring aldehyde group content

And (3) measuring the content of the aldehyde group of the OG by adopting a potentiometric titration method, and optimizing the oxidation condition by comparing the content of the aldehyde group of the OG obtained under different oxidation conditions.

The principle of aldehyde group content determination is as follows: the aldehyde group can react with an amino group in hydroxylamine hydrochloride to produce oxime and HCl; and titrating the reaction system by using a NaOH solution with a certain concentration, thereby calculating the content of aldehyde groups in the OG.

The detailed operation process for determining the aldehyde group content is as follows: 0.5g OG is weighed and dissolved in 25 mL of distilled water at 40 ℃, and the pH value is adjusted to 5 by using NaOH; then, 20mL of hydroxylamine hydrochloride solution with the concentration of 0.72 mol/L, pH being 5 is added into the mixture, and the mixture is placed in a water bath environment at the temperature of 40 ℃ to be stirred and reacted for 4 hours; subsequently, the pH of the mixed solution after the reaction was titrated to 5 using 1mol/L NaOH solution, and the volume of the NaOH solution consumed was recorded as V₁(mL); at the same time, the same concentration of GG solution was measured by the above method, and this group was a blank control group, and the consumption of NaOH solution was designated as V₀(mL); the aldehyde group content AC (mmol/g) of OG is calculated by the following formula: AC = (V)₁-V₀）×C_NaoH/m_{Sample (A)}Wherein, C_NaoHRepresents the concentration (mol/L) of the NaOH solution, m_{Sample (A)}Representing the mass (g) of the samples, i.e. GG or OG samples, each set of samples was run in triplicate and averaged.

(II) analysis of different NaIO by one-factor test₄Influence of dosage, oxidation temperature and oxidation time on aldehyde group content in OG

1. Controlling the oxidation temperature to be 40 ℃ and the oxidation time to be 8h, and adjusting GG and NaIO₄The aldehyde group content of the obtained OG is shown in fig. 1 (a) when the mass ratio is 1:0.5, 1:0.7, 1:0.9, 1:1.1 and 1:1.3 in this order. With NaIO₄The using amount is increased, and the aldehyde group content of the obtained OG is greatly increased and then tends to be balanced. This is probably due to the limited number of hydroxyl groups that can be modified on the GG molecular chain, and the excess NaIO when oxidation reaches saturation₄The content of aldehyde groups is not obviously increased. When GG and NaIO₄When the mass ratio is 1:0.9, the aldehyde group content of the obtained OG is 3.70 mmol/g, the use amount of GG is kept inconvenient, and NaIO is continuously added₄The dosage and the increasing rate of the aldehyde group content in the obtained OG are obviously reduced, so GG and NaIO are selected₄The mass ratio is preferably 1: 0.9.

2. Control of GG and NaIO₄The mass ratio is 1:0.9, the reaction time is 8h, and when the oxidation temperature is adjusted to 35 ℃, 40 ℃, 45, 50 and 55 ℃ in sequence, the aldehyde group content of the obtained OG is shown in figure 1 (B), and the aldehyde group content of the obtained OG shows a trend that the aldehyde group content is increased firstly and then decreased along with the gradual increase of the oxidation temperature. When the oxidation temperature is 40 ℃, the highest aldehyde group content of the obtained OG reaches 3.75 mmol/g. This is probably due to the lower temperature, which is not conducive to the oxidation reaction, and the slower reaction rate; when the temperature is too high, part of NaIO₄Decomposition occurs, affecting the oxidation reaction with GG. In addition, the aldehyde group may undergo a condensation reaction at a high temperature, which may eventually result in a decrease in the aldehyde group content, and thus the oxidation temperature is preferably 40 ℃.

3. Control of GG and NaIO₄The mass ratio is 1:0.9, the oxidation temperature is 40 ℃, the oxidation time is adjusted to be 4, 6, 8, 10 and 12 hours in sequence, the aldehyde group content of the obtained OG is shown in figure 1 (C), the aldehyde group content of the OG shows a trend that the aldehyde group content is increased firstly and then reduced along with the increase of the oxidation time, and the aldehyde group content reaches the maximum value at 8 hours and is 3.51 mmol/g. The oxidation time is short, and the oxidation degree is small; when the oxidation time reaches about 8h, the oxidation process may be substantially complete, and if the oxidation time is continuously increased, the aldehyde group content begins to decrease, which may be due to the fact that part of aldehyde groups continue to react to generate carboxyl groups. In addition, since part of aldehyde groups can perform condensation with hydroxyl groups on the molecular chain of the polysaccharide, the obtained hemiacetal structure can also cause the reduction of the content of aldehyde groups. Therefore, the oxidation time is preferably 8 hours.

In conclusion, according to the test result of the aldehyde group content in OG, by combining with the factors such as economic benefit, energy consumption and oxidation reaction degree, the oxidation condition that GG is more suitable is obtained as follows: GG and NaIO₄The mass ratio is 1:0.9, the oxidation temperature is 40 ℃, the oxidation time is 8h, and the aldehyde group content of the obtained OG is about 3.75 mmol/g.

Third, particle size analysis

The diffusion speed is determined through the scattering of dynamic light scattering laser in GG solution before and after oxidation, and the particle size of a sample is detected so as to explore the influence of the oxidation process on the particle size of GG. Small, equal amounts of GG and OG, respectively, were dissolved in a quantity of deionized water. And (3) detecting the particle sizes of GG and OG in the solution by using a Dynamic Light Scattering (DLS) particle size analyzer, drawing an intensity autocorrelation curve, and showing the distribution information of the particle sizes in the solution.

The particle size distribution images of the GG solution and the OG solution are shown in FIG. 2, in which FIG. 2 (A) is a GG particle size distribution diagram and FIG. 2 (B) is an OG particle size distribution diagram. In FIG. 2 (A), the GG particle size distribution is mainly at 518.4 nm, and a portion of the GG particle size distribution is at about 11.2 μm, which may be caused by the existence of GG particles of this size, or by the formation of physical cross-links between GG molecular chains in solution, which are entangled with each other. In FIG. 2 (B), the OG particles are mainly distributed at 269.0 nm, and the overall particle size is reduced relative to GG. On the one hand, this is probably due to the degradation of GG by the oxidation process, resulting in the shortening of the molecular chain; on the other hand, this is probably due to the fact that after oxidation, the bonding between GG molecular chains is weakened, the OG solution has relatively enhanced fluidity, and it is not easy to form a larger particle size. These results indicate that the OG obtained after oxidation has a smaller particle size, a narrower particle size distribution in the solution, and a more uniform appearance, compared to GG, which is advantageous for its crosslinking effect as a chemical crosslinking agent.

Four, Fourier Infrared Spectroscopy (FTIR) characterization

The GG and OG are characterized by FTIR to analyze the change of chemical groups during the reaction process and detect whether the oxidation and the reaction process are successful. Cutting the freeze-dried GG and OG samples into pieces, respectively, uniformly mixing with KBr in a mass ratio of 1:100, grinding into powder, and detecting by using an FTIR instrument (SpectrumGX, USA) with a scanning range of 400cm^-1～4000 cm^-1。

FTIR images of GG and OG are shown in FIG. 3, 1069cm^-1、1638 cm^-1And 2930cm^-1The vibration peaks at (A) correspond to the C-O-C group and the C = O group in GG and OG, respectivelyAnd C-H groups. FTIR curves for OG at 1725 cm compared to those for GG^-1A new peak appears corresponding to the stretching vibration peak of the aldehyde group, and the result shows that the aldehyde group is introduced into GG and NaIO is used₄GG was successfully oxidized. In addition, no other new peaks appear in FTIR curves of GG before and after oxidation, which indicates that other groups on GG molecular chains and the overall structure of GG are not changed in the oxidation modification process.

Example 2 preparation and characterization of aminated hydroxyapatite

Preparation of aminated hydroxyapatite

The preparation process of the aminated hydroxyapatite comprises the synthesis of hydroxyapatite (Hap) and the preparation process of aminated hydroxyapatite (mHap) from hydroxyapatite, and comprises the following specific steps:

synthesis of hydroxyapatite

(1) Respectively dissolving calcium nitrate and diammonium hydrogen phosphate in distilled water to respectively obtain 1.2M Ca²⁺Solution, 0.72M HPO₄ ^2-And (3) solution.

(2) At 40 deg.C, adding Ca²⁺The solution is slowly dropped into HPO according to the volume ratio of 1:1₄ ^2-In the solution, the pH of the mixture was stabilized at 10.5 with ammonia water and the reaction was continued for 2 hours.

(3) Standing the reaction solution at 50 ℃ for precipitation for 12h, centrifuging to collect white precipitate, alternately washing the precipitate with absolute ethyl alcohol and distilled water for at least three times to remove residual ammonia water and reaction reagents, drying the collected white precipitate in an electric air blowing drying box at 60 ℃ for 2h, then transferring the white precipitate to a muffle furnace, and calcining at 900 ℃ for 2h to obtain the hydroxyapatite.

Synthesis of (di) aminated hydroxyapatite

(1) 2g of hydroxyapatite powder is weighed and added into 200mL of distilled water containing 90% of absolute ethyl alcohol, and ultrasonic dispersion treatment is carried out for 15min.

(2) Adding 1mL of ammonia water and 1.5mL of Tetraethoxysilane (TEOS) into the mixed solution, vigorously stirring for 2h at 60 ℃, centrifuging to collect precipitate, washing the precipitate by using absolute ethyl alcohol and distilled water alternately for at least three times to remove residual TEOS and ammonia water, transferring the precipitate into an electric drum air drying box, and drying for 2h at 60 ℃ to obtain a white solid.

(3) 0.44g of gamma-Aminopropyltriethoxysilane (APTES) was weighed out and dropped into 200mL of distilled water containing 90% absolute ethanol, and 1.83g of a white solid was added thereto, and vigorously stirred at 25 ℃ for 6 hours, and then the mixed solution was separated by centrifugation, and the precipitate was collected, and the collected precipitate was washed with distilled water and absolute ethanol alternately to remove residual APTES and aqueous ammonia, followed by drying at 60 ℃ to obtain aminated hydroxyapatite (mHap).

Second, particle size analysis

Since the particle size of apatite has a large influence on the stability of the composite hydrogel, the particle size of apatite is measured and analyzed by a dynamic light scattering method.

The specific determination method comprises the following steps: respectively adding equal amount of Hap and mHap into deionized water to obtain mixed solution of Hap and mHap with the same concentration, and performing ultrasonic treatment for 10 min. The particle sizes of Hap and mHap in the mixed solution were measured using a dynamic light scattering particle size analyzer.

The particle size distribution images of Hap and mHap are shown in fig. 4, in which fig. 4 (a) is a Hap particle size distribution image and fig. 4 (B) is an mHap particle size distribution image. In FIG. 4 (A), the particle size distribution of the Hap is broad, mainly around 750.2 nm, and still distributed at 1.6 μm. In FIG. 4 (B), the particle size distribution of mHap is narrow, and the particle size of mHap is reduced from that of Hap, and is mainly distributed around 187.8 nm. This shows that after the silanization modification, the particle size of mHap is obviously reduced, which is close to nanometer level, and the distribution in the mixed solution is more uniform. It is reasonable to speculate that mHap has reduced surface binding energy compared to Hap, is less likely to form aggregated forms, and is beneficial to forming good dispersion in organic matrices.

Characterization by FTIR

1. Detection method

Respectively mixing the prepared Hap, mHap and KBr in a ratio of 1:100, grinding into powder, tabletting, detecting by an FTIR instrument, and setting a scanning rangeIs 400cm^-1～4000cm^-1. FTIR characterization can analyze the change of chemical groups in the reaction process and verify whether the experiment successfully prepares mHap, i.e. whether Si-O-Si and-NH are successfully introduced into the Hap₂A group.

2. The result of the detection

FTIR images of Hap and mHap are shown in FIG. 5, in which 3573 cm^-1The peak corresponding to the-OH group is the oscillation peak. To a certain extent, the smaller the number of residual hydroxyl groups on the modified nanoparticles, the more complete the monolayer structure formed on the particle surface by the silane coupling agent. The intensity of the-OH peak in the FTIR curve of mHap is significantly reduced compared to Hap, probably due to the fact that TEOS and APTES form mainly a monolayer structure on the Hap surface; in the FTIR profile of mHap, 1382 cm^-1And 3130 cm^-1The vicinity of the peak corresponds to the stretching vibration peak of the C-N group and the N-H group, 807 cm^-1The bending vibration peak corresponding to the siloxane group (Si-O-Si) confirms the presence of mHap surface silicon element. As can be seen from the above analysis, the method of the present invention successfully converts Si-O-Si and-NH₂Introducing Hap to prepare mHap.

Four, X-ray diffraction (XRD) characterization

1. Characterization method

XRD characterization can be used to analyze the crystal structure of a sample as well as the crystallographic changes of crystalline compounds. Hap and mHap samples were ground to powders, respectively, and then examined using an XRD instrument. The scanning speed is 20 degrees/min, and 5 degrees to 80 degrees are selected to characterize the sample.

2. Characterization results

The XRD patterns of Hap and mHap are shown in fig. 6, and the corresponding peaks at 5.8 °, 31.8 °, 32.9 ° and 34.1 ° for 2 θ are due to the reflections of (002), (211), (300) and (202) of Hap, respectively, which demonstrates that Hap was successfully synthesized. The synthesized Hap is treated by TEOS and APTES to obtain mHap, and the XRD diffraction pattern of the mHap is not obviously changed compared with that of the Hap, so that the silanization modification does not change the crystal structure of a Hap sample, and the bioactivity of the Hap is favorably kept.

Fifth, FESEM and EDS characterization

1. Characterization method

The detection of the cross-sectional morphology and the elemental composition of the sample can be realized by the combination of FESEM and an EDS instrument. Respectively weighing a certain mass of the Hap and mHap powder, adding the powder into an ethanol water solution to obtain a mixed solution of the Hap and the mHap with the same mass fraction, and carrying out ultrasonic treatment for 10 min. Then, a small amount of the uniform mixed solution is respectively taken and dripped on the surface of the silicon chip, and the silicon chip is naturally dried. And performing platinum sputtering coating on the sample to be detected by using a Hitachi E1010 type ion sputtering coating machine, and observing the cross section appearance of the sample in an FESEM and EDS instrument.

2. Characterization results

The FESEM images of the Hap and mHap samples are shown in FIG. 7, in which FIG. 7 (A) is the FESEM image of the Hap sample, and FIG. 7 (B) is the FESEM image of the mHap sample. As shown in fig. 7 (a), the Hap particles tend to aggregate and even to undergo sheeting, probably due to the higher surface binding energy of Hap. In FIG. 7 (B), it can be seen that the mHap particles are spherical or nearly spherical, they are uniformly distributed and have an average particle diameter of around 100 nm. Materials with or containing nanoscale levels have been shown to provide regulatory effects on osteoblasts and bone tissue cells in a variety of ways. Furthermore, the Hap surface becomes rough after silanization modification, probably due to the fact that the Hap surface is covered with a silicon coating. Compared with the Hap particles, the mHap particles have lower aggregation property and good dispersibility, can provide more opportunities for combining with an organic matrix, ensure that the compounded hydrogel material is more uniform, and are favorable for showing better mechanical properties.

Example 3 preparation and characterization of gelatin/oxidized gellan hydrogel

Preparation method of gelatin/oxidized gellan gum hydrogel

The gelatin/oxidized gellan gum (Gel-OG) hydrogel was prepared as follows:

(1) 1g of Gel was weighed and dissolved in distilled water at 50 ℃ to prepare a 20wt% Gel solution.

(2) Weighing a certain amount of OG and dissolving the OG in distilled water at 40 ℃ to respectively prepare OG solutions with the weight percentages of 1wt%, 1.5wt%, 2wt%, 2.5wt% and 3 wt%; wherein the oxidized gellan gum (OG) is prepared by the conditions optimized in example 1, namely, the method for preparing the oxidized gellan gum (OG)GG and NaIO in preparation process₄The mass ratio is 1:0.9, the oxidation temperature is 40 ℃, and the oxidation time is 8 h.

(3) And respectively mixing OG solutions with different concentrations and Gel solutions according to the mass ratio of 1:1 at 40 ℃.

(4) Pouring the mixed solution into five glass test tubes respectively, wherein the inner diameter of each glass test tube is 13mm, the depth of each glass test tube is 100mm, sealing the glass test tubes, and placing the glass test tubes in a 30 ℃ water bath for 12 h. And finally, removing the test tube, cooling to room temperature, peeling off the test tube to obtain Gel-OG hydrogel samples 3-1-3-5 respectively, wherein the samples 3-1-3-5 are prepared by reacting 1wt%, 1.5wt%, 2wt%, 2.5wt% and 3wt% of OG with GG respectively.

Second, characterization by Field Emission Scanning Electron Microscope (FESEM)

1. Characterization method

The 10wt% Gel hydrogel and the Gel-OG hydrogel of samples 3-1 to 3-5 were prefrozen in liquid nitrogen, and then lyophilized in a lyophilizer (GT 2-Type-8, LYOTECH, Germany) for 48 h. The cross-section of the freeze-dried Gel hydrogel and Gel-OG hydrogel samples was brittle in a liquid nitrogen environment, and a platinum layer was sprayed on the surface of the samples using a small ion sputtering apparatus (Hitachi E1010, Japan), followed by observation of the cross-sectional morphology of the samples in a FESEM apparatus (S4800, Hitachi, Japan).

2. Characterization results and analysis

2.3.4 FESEM characterization

The sectional topography of the 10wt% Gel hydrogel and the Gel-OG hydrogels of samples 3-1 to 3-5 are shown in FIG. 8, wherein FIG. 8 (A) is a sectional topography of 10wt% Gel; FIGS. 8 (B), (C), (D), (E) and (F) are sectional profiles of Gel-OG hydrogels of samples 3-1 to 3-5, respectively.

As can be seen from FIG. 8, the Gel hydrogel and the Gel-OG hydrogel of the samples 3-1 to 3-5 both show good three-dimensional pore structures, the pore size of the Gel hydrogel is about 25 mu m, and the pore size of the Gel-OG hydrogel is smaller than 10 mu m, so that the network structure formed by the Gel-OG hydrogel is more compact and regular relative to 10wt% of the Gel hydrogel, and the generated crosslinking points are more due to Schiff base chemical crosslinking in the preparation process of the Gel-OG hydrogel.

Furthermore, as the concentration of the OG solution increases from 1wt% to 2wt%, the pore diameter of the resulting Gel-OG hydrogel decreases from about 10 [ mu ] m to about 3 [ mu ] m as shown in FIGS. 8 (B) - (D). This is probably because the number of chemical cross-linking points generated by Schiff base reaction with Gel increases after the addition of OG increases, and a more compact network structure is easily formed. When the OG solution concentration is increased from 2wt% to 3wt%, the network formed by the obtained Gel-OG hydrogel gradually becomes loose and the pore structure becomes irregular as shown in FIGS. 8 (D) to (F). The reason for this is probably that the cross-linking agent OG solution has a large concentration and is locally easy to rapidly cross-link with Gel molecular chains, which hinders the OG from contacting with other Gel molecular chains, and causes the chemical cross-linking network formed by the OG and the Gel to be irregular and the size of the pores to be uneven. In summary, when the OG solution concentration is 2wt%, the network structure of the obtained Gel-OG hydrogel is the most regular and compact, the pores are the smallest, and the higher mechanical strength is probably corresponded to.

Third, compression mechanical property test

1. Compression mechanical property testing method

The Gel-OG hydrogel of samples 3-1 to 3-5 was prepared into a cylindrical sample with a diameter of 13mm and a height of 15mm, and placed on a physical property tester (TA. XTPlus, UK) test bed at room temperature, and the compression test element was compressed downward at a speed of 0.5mm/s, with a constant compression set at 90%. Each set of samples was run in triplicate and averaged.

2. Compression mechanical property test results and analysis

The compressive stress-strain curves of Gel-OG hydrogels (samples 3-1 to 3-5) with different OG contents are shown in FIG. 9 (A), and the compressive mechanical strength of the Gel-OG hydrogels with different OG contents at 90% deformation is shown in FIG. 9 (B).

As can be seen in FIG. 9 (A), none of the five Gel-OG hydrogels tested exhibited fracture at 90% deformation, showing good toughness. As the OG solution concentration increases from 1wt% to 3wt%, the compressive mechanical strength of the hydrogel at 90% deformation shows a tendency to increase and then decrease, as shown in FIG. 9 (B). Wherein the maximum mechanical strength at 2wt% is 1.81 MPa. This is probably because the OG solution as a cross-linking agent is more uniform and cross-linked with Gel when the concentration of the OG solution is not more than 2 wt%; when the OG solution concentration is higher than 2wt%, quick crosslinking can be formed due to excessive local crosslinking agent concentration by dropping the OG solution into the Gel, so that an uncrosslinked Gel part exists in the Gel-OG hydrogel, and the overall mechanical strength of the Gel-OG hydrogel is reduced. These results indicate that the use of 2wt% OG solution to react with Gel facilitates the formation of Gel-OG hydrogels with a more uniform chemical cross-linked network and higher compressive mechanical strength.

Fourthly, swelling behavior test analysis

1. Test method

PBS solution at pH 7.4 was prepared as in Table 1. The Gel-OG hydrogels of samples 3-1 to 3-5 were cut into slices of 13mm in diameter and 2mm in thickness and lyophilized. The lyophilized samples were then weighed individually and recorded as W₀(g) Then immersed in a PBS solution and transferred to an electric heating incubator at 37 ℃. After a certain time, the sample was removed and the solution on its surface was gently wiped with filter paper, and the sample was weighed again and recorded as W_s(g) In that respect Swelling ratio SR (%) of hydrogel sample according to the formula SR (%) = [ (W)_s-W₀)/W₀]X 100 was calculated.

2. The result of the detection

The swelling curves of the Gel-OG hydrogels (namely samples 3-1-3-5) obtained by different OG solution concentrations are shown in FIG. 10, the five measured Gel-OG hydrogels reach swelling equilibrium within about 28 hours, and the equilibrium swelling ratio of the hydrogel tends to decrease first and then increase along with the increase of the OG solution concentration from 1wt% to 3 wt%. The equilibrium swelling ratio of the hydrogel prepared from 2wt% of OG solution is the lowest, namely 920%, because the pore structure of the hydrogel is more compact and regular than other hydrogels, and the formed pores are smaller, which is consistent with the detection result of the section morphology of the Gel-OG hydrogel. When the concentration of the OG solution is 3wt%, the swelling rate of the obtained hydrogel in 0-5h at the initial swelling stage is lower than that of other hydrogels, which is probably because the hydrogel locally contains a denser network structure; after 10h of swelling, the swelling rate of the hydrogel increases faster, since more chemically uncrosslinked moieties may be present in the hydrogel, increasing the swelling rate of the overall hydrogel. Dense pore structures tend to result in lower equilibrium swelling ratios. On the one hand, this is because dense pores limit the entry of the swelling solution; on the other hand, the hydrogel with the compact pore structure has higher stability and is not easy to expand and break. The hydrogel sample is lyophilized, so that the sample absorbs a large amount of swelling liquid, and the measured equilibrium swelling ratio is larger. The hydrogel material applied to the in vivo environment needs to have a low equilibrium swelling ratio, so that the OG solution concentration is set to be 2wt% rather.

In conclusion, when the concentration of the Gel solution is 20wt% and the concentration of the OG solution is 2wt%, the Gel-OG hydrogel prepared from the Gel and the OG has the advantages of the most regular and compact network structure, the smallest hole and lower swelling rate, and is suitable for the articular cartilage tissue of the human body; when the hydrogel is in 90% deformation, the compressive mechanical strength reaches 1.81MPa, according to the reports of the prior art (Sun W, Xue B, Li Y, et al, Polymer-porous Polymer double-network hydrogel [ J ]. Advanced Functional Materials, 2016, 26(48): 9044-9052.), the compressive stress of the human articular cartilage is 0.78-10 MPa, and the compressive mechanical strength of the Gel-OG hydrogel prepared from Gel and OG can meet the minimum requirement of the human articular cartilage on the compressive mechanical strength, but still needs to be further improved.

Example 4 Effect of different amounts of aminated hydroxyapatite on the Properties of composite hydrogels

Preparation of gelatin/oxidized gellan gum/aminated hydroxyapatite composite hydrogel

A preparation method of gelatin/oxidized gellan gum/aminated hydroxyapatite composite hydrogel comprises the following steps:

(1) preparing a gelatin solution: 1g of gelatin is weighed into distilled water at 50 ℃ to prepare a gelatin solution with the concentration of 20wt%, and the gelatin solution is stored at 40 ℃ for standby.

(2) Preparing an oxidized gellan gum solution: weighing 0.1g of oxidized gellan gum, and dissolving in distilled water at 40 deg.C to obtain 2wt% oxidized gellan gum solutionExample 1 optimized preparation of conditions, i.e. GG and NaIO in OG preparation₄The mass ratio is 1:0.9, the oxidation temperature is 40 ℃, and the oxidation time is 8 h.

(3) A gelatin/aminated hydroxyapatite solution was prepared by adding a certain amount of aminated hydroxyapatite prepared by the method described in example 2 to a gelatin solution under stirring.

(4) Dropping the oxidized gellan gum solution into the gelatin/aminated hydroxyapatite solution at 40 ℃, uniformly stirring, transferring into a transparent glass test tube, placing the glass test tube into water at 30 ℃, reacting in a water bath for 12 hours, taking out, and cooling to room temperature to obtain the gelatin/oxidized gellan gum/aminated hydroxyapatite composite hydrogel.

Referring to the above method, 0wt%, 0.5wt%, 1.0wt%, 1.5wt%, 2.0wt% of aminated hydroxyapatite was added to gelatin in step (3), and the finally prepared composite hydrogel was sequentially designated as sample 4-1, sample 4-2, sample 4-3, sample 4-4, and sample 4-55, and samples 4-1 to 4-5 were all freeze-dried for use. Wherein, the addition amount of the aminated hydroxyapatite is calculated by the total weight percentage of the gelatin and the oxidized gellan gum; the preparation process of the sample 4-1 is not added with the aminated hydroxyapatite, namely the sample 1 is gelatin/oxidized gellan gum hydrogel, and the samples 4-2-4-5 are gelatin/oxidized gellan gum/aminated hydroxyapatite composite hydrogel.

Second, FTIR characterization

1. Inspection method

Taking 10wt% of gelatin as a control, respectively uniformly mixing and grinding freeze-dried samples 4-1-4-5, the control gelatin and KBr in a ratio of 1:100 into powder, tabletting, detecting by using an FTIR instrument, and setting the scanning range to be 400cm^-1～4000 cm^-1。

2. The result of the detection

FTIR characterization results of 10wt% gelatin and samples 4-1 to 4-5 are shown in FIG. 11, in which 3439 cm^-1The wide absorption peak is caused by the stretching vibration of the bonding of N-H and O-H, and moves to the direction of high wave number along with the rise of mHap content,this indicates that the hydrogen bonding between Gel, OG and mHap in the composite hydrogel is enhanced. An FTIR profile of the composite hydrogel (Gel-OG/mHap) at 566cm could be observed compared to that of sample 4-1 (Gel-OG)^-1、602cm^-1、631cm^-1And 961 cm^-1All have vibration peaks corresponding to the phosphate group (-PO) of Hap₄ ^3-) Indicating that mHap was successfully incorporated into the Gel-OG network. Similarly, 900 cm in FTIR curves of composite hydrogels of different mHap content^-1～1200 cm^-1The relative intensities in the wavenumber range are very different, 1031 cm^-1The peak of stretching vibration corresponding to C-O bond in OG is shifted to low wavenumber direction in FTIR curve of Gel-OG compared with FTIR curve of Gel due to Schiff base chemical cross-linking between Gel and OG; as mHap content increases, the peak gradually shifts to a high wavenumber, probably because the increased chemical cross-linking between mHap and OG affects the chemical reaction between OG and Gel. The results show that the mHap and the organic matrix have stronger interaction, and the Gel-OG/mHap composite hydrogel is successfully prepared.

Based on the above analysis, the possible reaction mechanism between Gel, OG and mHap is shown in fig. 12. the-CHO on the OG molecular chain can directly crosslink the-NH on the Gel molecular chain₂Forming a Gel-OG network; at the same time, -NH on mHap₂Can react with-CHO on OG molecular chains and enhance the interaction with an organic matrix by forming hydrogen bonds with a Gel-OG network.

Characterization by X-ray diffraction (XRD)

1. Characterization method

Respectively grinding 10wt% of a gelatin freeze-dried sample and 4-1-4-5 freeze-dried samples into powder, detecting the gelatin and the samples 4-1-4-5 by using an XRD instrument at a scanning speed of 20 DEG/min, and selecting 5-80 DEG to characterize the samples to be detected.

2. Characterization results

XRD patterns of samples 4-1 to 4-5 are shown in FIG. 13, and the peak at 20 ℃ is generated by Gel-OG hydrogel, and the strength of the crystallization peak gradually weakens with the increase of mHap incorporation, and becomes blunted, which shows that hydrogen bonding among OG, Gel and mHap is enhanced. After mHap is doped into Gel-OG, a distinct crystallization peak appears at 31.8 degrees, the peak corresponds to a typical crystallization peak of the Hap, the intensity of the peak is increased along with the increase of the mHap content, and the position of the crystallization peak is not obviously moved. Furthermore, the XRD profile of the composite hydrogel appears predominantly as an organic phase, similar to that of Gel-OG hydrogels, due to the lower level of incorporation of mHap. From XRD images of composite hydrogels with different mHap contents, the crystal structure of mHap was found to remain good in all hydrogels, indicating good blending of mHap with organic matrix.

Fourth, FESEM and EDS characterization

1. Characterization method

The gelatin and the freeze-dried samples 4-1-4-5 are brittle-broken in a liquid nitrogen environment, platinum sputtering coating is carried out on all the samples 4-1-4-5 by using a Hitachi E1010 type ion sputtering coating machine, and the samples are observed in an FESEM and an EDS instrument.

2. Characterization results

The profile plots of 10wt% Gel and samples 4-1 to 4-5 are shown in FIG. 14, in which FIG. 14 (A) is the profile plot of 10wt% Gel hydrogel, and FIGS. 14 (B), (C), (D), (E), (F) are the profile plots of samples 4-1 to 4-5 hydrogel, respectively.

10wt% of Gel and samples 4-1 to 4-5 of hydrogel all showed good three-dimensional pore structures. Among them, 10wt% Gel hydrogel formed larger pores than other hydrogels because other hydrogels all underwent chemical crosslinking reaction. As the mHap content increases from 0 to 1wt%, the hydrogel-forming network becomes progressively denser and has some orientation. The form change is probably because mHap and Gel are connected through the action of hydrogen bonds in the composite hydrogel, and the formation of the hydrogen bonds and covalent bonds between OG and mHap further increases the contact chance between Gel and OG molecular chains, thereby being beneficial to forming a more compact hydrogel network. The composite hydrogel containing 1wt% mHap shows a large number of small pores in the porous structure, and the network is denser than other hydrogels. As the mHap content continues to increase, the composite hydrogel exhibits a relatively loose network structure, possibly due to the excess mHap occupying the Gel and OG reaction sites, impeding relative motion and contact between the Gel and OG molecular chains. However, the network formed by the composite hydrogel was denser than the Gel-OG hydrogel (sample 4-1), probably due to increased hydrogen bonding in the hydrogel. From fig. 14 (C) - (F), it can be concluded that mHap particles are likely to be uniformly distributed in the composite hydrogel network rather than aggregated on the surface, indicating that the good interaction between mHap and the organic matrix is beneficial to improve the mechanical properties of the hydrogel.

Low power FESEM images of Gel-OG hydrogel (sample 4-1) and 1wt% mHap composite hydrogel (sample 4-3) are shown in FIG. 15, where FIG. 15 (A) is a topographic cross-sectional view of the Gel of sample 4-1 and the corresponding EDS map; FIG. 15 (B) is a cross-sectional profile of the composite hydrogel of sample 4-3 and the corresponding EDS map. As can be seen from FIG. 15, both Gel-OG and 1wt% mHap composite hydrogels formed three-dimensional ordered pore structures, with the latter network structure being denser than the former. Compared with Gel-OG hydrogel, the composite hydrogel obviously contains C, Ca, P and Si elements, and the Si element is shown to be contained in mHap and successfully incorporated into blank hydrogel. In addition, Ca, P and Si elements are distributed uniformly, which shows that mHap particles are distributed uniformly in the composite hydrogel and do not generate obvious aggregation phenomenon, and further shows that the inorganic particles and the organic matrix have good combination effect. Because the mass of the added mHap is lower than that of the Gel-OG hydrogel, the element points of Ca, P and Si are not dense as C element, and the brightness is lower.

Fifth, testing mechanical properties

1. Test method

The compressive mechanical strength of the hydrogel can represent the pressure bearing capacity of the material, and is one of important parameters for designing the loaded hydrogel, and the method for measuring the compressive mechanical strength of the hydrogel comprises the following steps: a sample 4-1 to 4-5 of hydrogel having a diameter of 13mm and a height of 15mm was cut into a cylindrical sample, and placed on a test bed of a physical property tester at room temperature, and a compression test element was compressed downward at a speed of 0.5mm/s with a constant compression set at 90%. The composite hydrogel containing 1wt% mHap (i.e., sample 4-3) was selected for cyclic compression testing, the number of cycles was set to 50, and the constant deformation was 85%. Each set of samples was run in triplicate and averaged.

2. Test results

The compressive stress-strain curve and the compressive mechanical strength at 90% deformation of the hydrogel are shown in FIG. 16. FIG. 16 (A) is a photograph of hydrogels of samples 4-1 to 4-5, which gradually transition from brown-yellow to white in color as the mHap content increases. Furthermore, it can be observed that mHap is distributed uniformly without obvious aggregation. From fig. 16 (B) and (C) which are the compression property test pictures and knotting and tensile property test pictures of the samples 4-3, it can be seen that the composite hydrogel shows better toughness, tensile property and mechanical strength.

FIG. 16 (E) is a result of the compressive mechanical strength of samples 4-1 to 4-5, the compressive mechanical strength of the Gel-OG hydrogel at 90% deformation is 1.18MPa, and the compressive mechanical strength of the composite hydrogel (samples 4-2 to 4-5) detected is higher than that of the Gel-OG hydrogel (sample 4-1). On the one hand, this is due to the fact that the network formed by the composite hydrogel is denser than the Gel-OG hydrogel, which results in energy dissipation over a larger area; on the other hand, the tight connection between the mHap and the organic matrix can inhibit the growth of micro-cracks in the composite hydrogel during compression. Wherein, when the mHap content is 1wt%, the compressive mechanical strength of the obtained composite hydrogel at 90% deformation is as high as 2.03MPa, and the composite hydrogel is not cracked, and compared with the Gel-OG hydrogel not doped with mHap, the compressive strength of the Gel-OG/mHap composite hydrogel doped with 1% mHap is doubled. According to the published documents (W, Sun, B, Xue, Y, Li, M, Qin, J, Wu, K, Lu, J, Wu, Y, Cao, Q, Jiang, W, Wang, Advanced Functional Materials 2016, 26, 9044), the compressive stress of the human articular cartilage reaches 0.780-10 MPa, and can meet the requirement of the human articular cartilage tissue on the compressive mechanical strength, and compared with the Gel-OG hydrogel without mHap, the Gel-OG/mHap composite hydrogel expands the application range in the human articular cartilage tissue.

When the mHap content is increased from 1 to 2wt%, the compressive mechanical strength of the resulting composite hydrogel is reduced, probably because too much mHap interferes with the formation of Gel-OG organic networks, resulting in a loose network structure, reducing the mechanical strength of the hydrogel. These results indicate that good integration between mHap and the organic matrix increases the compressive strength of the hydrogel.

The cyclic compressive stress-strain curve of the composite hydrogel containing 1wt% mHap (samples 4-3) is shown in FIG. 16 (F), and after 50 cyclic compressions (constant compression set 85%), the compressive mechanical strength of the composite hydrogel still has the first 91%, indicating that the composite hydrogel has good deformability and elasticity. This is due to the presence of reversible stored energy in the hydrogel, and the high elasticity also demonstrates the reusability of the hydrogel. In conclusion, the silane coupling agent method is utilized to promote the interaction between mHap and the organic matrix, so that the flexibility of the hydrogel is improved, and the application of the hydrogel in biological soft tissue engineering is facilitated.

Sixth, self-healing behavior

1. Detection method

A1 wt% mHap-containing composite hydrogel (sample 4-3) having a diameter of 13mm and a thickness of 2mm was selected for a macroscopic self-healing performance test. First, a hydrogel sheet was cut into two parts, stained with a methylene blue solution, spliced together and left to stand at 37 ℃ for 12 hours, and the hydrogel was observed for morphological change.

2. The result of the detection

The photograph of the healing performance test containing 1wt% mHap composite hydrogel (sample 4-2) is shown in FIG. 16 (D). The intact composite hydrogel was cut into two portions, then stained with methylene blue solution, and after splicing the two portions together and allowed to stand at 37 ℃ for 12h, the boundary between the two portions of the composite hydrogel was found to have almost disappeared, indicating that the two portions were bonded together as a unit. Then, the composite hydrogel is stretched by applying an external force, and the two parts of the healing hydrogel are not separated. This repairable hydrogel may be based on the reversibility of imine bonds at 37 ℃ in the absence of any external stimulus. The test shows that the Gel-OG/mHap composite hydrogel with good self-healing capability is beneficial to prolonging the service life of the hydrogel in the human body environment.

Seventh, swelling behavior

1. Detection method

First, a PBS solution having a pH of 7.4 was prepared as shown in Table 1. Hydrogels with different mHap contents (samples 4-1 to 4-5) were cut into discs with a diameter of 13mm and a thickness of 2mm, and lyophilized. The lyophilized samples were then weighed individually and recorded as W₀(g) Then immersed in a PBS solution and transferred to an electric heating incubator at 37 ℃. After a certain time, the sample was removed and the solution on its surface was gently wiped with filter paper, and the sample was weighed again and recorded as W_s(g) In that respect Swelling ratio SR (%) of hydrogel sample according to the formula SR (%) = [ (W)_s-W₀)/W₀]X 100 was calculated.

2. The result of the detection

The swelling behavior, which is one of the important considerations for hydrogels for tissue engineering, has the function of transferring nutrients and waste products in the human body. The swelling curves of the composite hydrogels (samples 4-1 to 4-5) with different mHap contents in PBS solution at 37 ℃ are shown in FIG. 17. The hydrogel samples tested all reached swelling equilibrium within 30h, and this equilibrium state can be maintained for 20h due to their highly interconnected porous structure. Furthermore, the equilibrium swelling ratio of the composite hydrogel was lower than that of the Gel-OG hydrogel (sample 4-1). This is due to the fact that the composite hydrogel forms a more dense network, resulting in a reduced permeability of the swelling solution. When the mHap content is increased from 0wt% to 1wt%, the equilibrium swelling ratio of the hydrogel is reduced from 1010% to 769%, which may be that the network compactness influences the swelling properties of the hydrogel. When the mHap content was increased from 1 to 2wt%, the equilibrium swelling ratio of the hydrogel increased from 769 to 937%. On the one hand, this may be due to the presence of a certain number of free amino and hydroxyl groups on mHap, which are hydrophilic; on the other hand, excessive mHap can affect the formation of Gel-OG cross-linked network in the composite hydrogel, so that a macroporous structure is formed, the hydrogel can absorb more swelling liquid, and higher swelling rate is shown. Based on the above results, the strong interaction between mHap and the organic matrix will result in a lower swelling ratio, which is beneficial for the hydrogel to maintain the structural and performance stability of the in vivo environment.

Collagenase degradation behaviour

1. Measurement method

In vitro environment, using collagenase-containing compositionsThe degradation rate of the lyophilized hydrogel was determined with the PBS solution of (a). First, the hydrogels (n = 5) with different mHap contents after lyophilization were weighed individually and recorded as W₀(g) Then, the cells were immersed in a PBS solution containing 2. mu.g/mL type I collagenase at 37 ℃. After a certain time the samples were taken out, rinsed with 3 times of distilled water and freeze-dried, weighed again and the mass recorded as W_t(g) The Degradation Rate (DR) of the hydrogel sample was calculated using the following formula: DR (%) = [ (W)₀-W_t)/W₀]×100。

2. Measurement results

The structural stability and the corresponding degradation behavior of the hydrogel are particularly important for the application of the hydrogel in cartilage tissue engineering. Composite hydrogels with different mHap contents were obtained at 37^o2 microgram g mL of C^-1The degradation profile of collagenase type I in PBS solution is shown in figure 18. All hydrogels tested were completely degraded within 6 days due to the presence of collagenase type I and showed varying degrees of mass loss. With the addition of mHap, the composite hydrogel has a lower degradation rate than the Gel-OG hydrogel, which is similar to the swelling behavior study of the hydrogel. The degradation rate of the composite hydrogel containing 1wt% of mHap is the lowest, which is related to its network compactness and the strong interaction of mHap with the organic matrix. The rate of hydrogel degradation increased slightly as the mHap content increased from 1wt% to 2 wt%. In addition, the mHap particles are firmly embedded in the composite hydrogel and have good interfacial interaction with the organic network, and the Hap is released at the degradation rate of the organic matrix, which is beneficial to the formation of inorganic mineral phase crystals and wound healing. In conclusion, the strong interaction between mHap and the organic network can reduce the cracking of imine bonds between Gel and OG, and further inhibit the degradation of the composite hydrogel.

Nine, cell compatibility

1. Detection method

The cell compatibility of the hydrogel with different mHap contents is detected by adopting a thiazole blue (MTT) colorimetric method. In the experiment, mouse fibroblast (L929) cells are selected as test cell objects. And (3) sterilizing the hydrogel sample by using ultraviolet light, sterilizing the hydrogel sample by using absolute ethyl alcohol, soaking the hydrogel sample into DMEM to prepare a leaching solution, and culturing the L929 cell by using the leaching solution as a cell culture solution of a sample group. The absorbance of different groups is measured by an MTT method, and the relative cell proliferation rate (RGR/%) of the sample group is calculated by a certain formula, so that the biocompatibility and cytotoxicity of the hydrogel are evaluated.

Cell recovery and culture: first, the cryopreservation tube in which the L929 cells were encapsulated was taken out of the liquid nitrogen, transferred to a 37 ℃ water bath and slowly shaken to melt it as much as possible within 3 min. Then, the mixture was sterilized by spraying 75% ethanol, transferred to a centrifuge and centrifuged at 1200rpm/min for 4min, and the supernatant was discarded. Sterilizing, transferring to a clean bench, adding 1mL 10% bovine serum culture medium, subpackaging the culture medium containing cells into culture bottles, transferring to 37 deg.C, and transferring to 5% CO₂The cells were incubated in the incubator, and the culture medium was changed every two days. The 10% serum-containing medium was prepared by mixing DMEM and FBS at a ratio of 9:1, adding a small amount of diabody thereto, and mixing with a vortex apparatus.

Cell passage and plating: the culture flask containing the L929 cells was observed under a microscope, and when the density of the cells in the culture flask exceeded 80% and most of them exhibited fibrous growth, the passaging operation was performed. First, the old culture medium was aspirated, and the wall of the flask on the cell side was gently rinsed with PBS solution, taking care to prevent loss of cells. Adding appropriate amount of trypsin solution, transferring to 37 deg.C, and adding 5% CO₂Digesting in a constant temperature incubator to separate the cells from the wall of the culture flask. After 3min the flask was removed and placed under a microscope for observation, and if most cells were spherical and the flask was gently shaken to show wall detachment, the trypsin solution was carefully aspirated, an appropriate amount of culture medium was added and the cells were carefully blown off the wall. Subsequently, the cell suspension was dispensed into a centrifuge tube and centrifuged at 1200rpm/min for 4min, and the supernatant was discarded. An appropriate amount of culture medium was added and the cells were carefully blown to obtain a suspension in which the cells were uniformly dispersed.

The number of cells in the suspension was counted using a hemacytometer cytometry. First, the cell suspension was diluted to some extent and then seeded into 3 96-well plates at a rate of about 5000 cells per well. Each well plate comprises a positive control group, a negative control group and a sample group, wherein a DMSO solution is added into the wells of the positive control group; adding a DMEM medium solution into the wells of the negative control group; the wells of the sample set were loaded with a leaching solution of the hydrogel samples in DMEM.

Experiment: first, 3 well plates seeded with cells were removed and transferred to 37 ℃ in 5% CO₂The culture is carried out for 48 hours in a constant temperature incubator, and the culture solution is changed at regular time. Then, the old culture medium in 3 96-well plates was aspirated, and each well was replaced with the leaching solution of the hydrogel material, the culture medium of the negative control group and the culture medium of the positive control group, and then transferred to 37 ℃ with 5% CO₂The culture is carried out in a constant temperature incubator, and the culture solution is replaced at regular time. On

days

1, 3 and 5, 1 well plate was removed, followed by addition of 0.5% MTT solution at a concentration of 10. mu.L/well, and transferred to 37 ℃ with 5% CO₂Reacting for 4 hours in the constant temperature incubator. Then, the 96-well plate where the crystallization occurred was removed, the supernatant was carefully aspirated (without touching the crystals), and after adding an appropriate amount of DMSO to each well, the plate was transferred to a shaker and slowly shaken for 10min to ensure complete dissolution of the crystals. The absorbance of each well solution was measured at 570 nm with a microplate reader and was recorded as OD. Wherein, the larger the OD value, the larger the number of L929 cells indicating a viable form in the well. The relative cell proliferation rate (RGR/%) was calculated using the formula, RGR (%) = (OD)_sample/OD_control) X 100, wherein OD_sampleAnd OD_controlThe OD values of the tested sample group and the negative control group are respectively corresponded.

The obtained RGR was calculated according to the formula, and the cytotoxicity of the corresponding hydrogel material was judged in combination with Table 2 (ISO 10, 993-5 standard). Wherein, 0 grade and 1 grade represent that the material is non-toxic to cells; level 2 represents the toxicity of the material to cells, and the comprehensive analysis should be carried out according to the specific existing forms of the cells; grades 3-5 indicate that the material has significant cytotoxicity and is not suitable for use as biomedical materials.

2. The result of the detection

Cell compatibility is an important consideration in determining whether materials, particularly modified materials, can be used in tissue engineering. The relative proliferation rate (RGR/%) of L929 cells obtained by MTT assay on different days of culture is shown in FIG. 19, i.e., cytotoxicity of hydrogel material was evaluated after culturing cells for 1, 3 and 5 days. If the material is not cytotoxic, its RGR should be higher than 75%, corresponding to a cytotoxicity rating of 0 or 1 (see Table 2). As shown in FIG. 19, the RGR of the cells in the sample group ranged from 86% to 124%, indicating that the Gel-OG hydrogel and the mHap composite hydrogel at various levels were not significantly cytotoxic. In addition, at

days

1, 3 and 5, the RGR of the hydrogels of the sample groups showed a tendency to increase and then decrease as the mHap content increased, with the 1wt% of the mHap content being the highest RGR. There may be two reasons for this: on one hand, the composite hydrogel containing 1wt% of mHap has smaller pores than other hydrogels, which may limit the release of self-degradation products when the hydrogel swells in DMEM, resulting in less degradation products in the sample leaching solution; on the other hand, the high or low cell viability of the sample group may be related to the content of Hap. Furthermore, it has been shown that the use of silane coupling agents to modify Hap does not increase its toxicity and is more beneficial to cell proliferation than Hap, since mHap presents many efficient cell binding sites and therefore some mHap particles may be released from the hydrogel scaffold into the sample leachate, which aids in cell proliferation. The increase in RGR value of the composite hydrogel may be mainly due to the dissolution of the non-crosslinked Gel and polysaccharide having good biocompatibility in the culture solution. The RGR of the hydrogel decreased on day 3 and increased on day 5 compared to the negative control group. This may be due to cell loss due to media exchange and other manipulations in the MTT experiment. In conclusion, the composite hydrogel has better cell compatibility, and the silylated and modified Hap is favorable for the proliferation of the L929 type cells.

In conclusion, mHap is prepared by modifying Hap with TEOS and APTES, and is doped into Gel-OG to prepare the Gel-OG/mHap composite hydrogel. The particle size of the modified mHap is obviously reduced and is closer to the nanometer level, so that the mHap is more uniformly distributed in the composite hydrogel, a network of the hydrogel formed by introducing the mHap into the Gel-OG is more compact, and the Ca, P and Si elements are uniformly distributed in the hydrogel. In addition, compared with Gel-OG hydrogel, the Gel-OG/mHap composite hydrogel has higher compressive mechanical strength, lower equilibrium swelling ratio and lower degradation rate. In addition, the introduction of mHap has the promotion effect on the proliferation of cells, shows good biocompatibility, has the properties of injectability and self-healing, and is favorable for the application of Gel-OG/mHap composite hydrogel in the field of cartilage tissue engineering.

Claims

1. The gelatin/gellan gum/hydroxyapatite composite hydrogel is characterized by being prepared from a gelatin solution, an oxidized gellan gum solution and aminated hydroxyapatite, wherein the oxidized gellan gum is prepared by the following steps:

(1) dissolving and dispersing gellan gum in water, and uniformly stirring to obtain a gellan gum solution;

(3) placing the oxidized gellan gum mixed solution into a dialysis bag for dialysis, performing suction filtration on the mixed solution after dialysis, and performing freeze drying on the solution obtained by suction filtration to obtain oxidized gellan gum; the molecular weight of the intercepted molecules of the dialysis bag is 8-14 kDa;

the preparation method of the aminated hydroxyapatite comprises the following steps:

(1) dispersing hydroxyapatite into 70-90% absolute ethyl alcohol solution according to the proportion of 1g:100mL, and performing ultrasonic dispersion treatment for 10-15 min to prepare a mixed solution;

2. The composite hydrogel according to claim 1, wherein the weight ratio of the gelatin to the oxidized gellan gum is (10-30) to (1-3); the addition amount of the aminated hydroxyapatite is 0.5-2% of the total weight of the gelatin and the oxidized gellan gum.

3. A method for preparing a composite hydrogel according to claim 1 or 2, comprising the steps of:

(1) preparing a gelatin solution;

(2) preparing an oxidized gellan gum solution;

(4) dripping the oxidized gellan gum solution into the gelatin/hydroxyapatite mixed solution at the temperature of 30-50 ℃, uniformly mixing, pouring into a mold, and placing in a water bath at the temperature of 20-40 ℃ for reaction for 12-18 hours; after the reaction is finished, cooling to room temperature to obtain the composite hydrogel;

the preparation method of the oxidized gellan gum comprises the following steps:

4. The method for preparing the composite hydrogel according to claim 3, wherein the hydroxyapatite is prepared by the following steps:

(2) at 40-50 deg.C, adding the same volume of Ca²⁺Solution and HPO₄ ^2-Mixing the solutions, stabilizing the pH of the mixed solution at 10.5 by using ammonia water, and continuously reacting for 2 hours;

5. The method for preparing the composite hydrogel according to claim 3, wherein the concentration of the gelatin aqueous solution is 10 to 30 wt%.

6. The method for preparing the composite hydrogel according to claim 3, wherein the concentration of the oxidized gellan gum solution is 1 to 3 wt%.

7. The preparation method of the composite hydrogel according to claim 5 or 6, wherein the gelatin aqueous solution and the oxidized gellan gum solution are added in a mass ratio of 1:1.

8. Use of the composite hydrogel according to claim 1 or 2 or the composite hydrogel prepared by the method for preparing the composite hydrogel according to any one of claims 3 to 6 in cartilage tissue replacement materials.