阅读说明：本技术 一种明胶/结冷胶/羟基磷灰石复合水凝胶及其制备方法 (Gelatin/gellan gum/hydroxyapatite composite hydrogel and preparation method thereof ) 是由 汤克勇 王绿阳 李萌雅 王芳 刘捷 郑学晶 于 2020-07-27 设计创作，主要内容包括：本发明属于组织工程技术领域,涉及一种应用于软骨替代物的新材料,具体涉及一种明胶/结冷胶/羟基磷灰石复合水凝胶,同时还涉及其制备方法。复合水凝胶由明胶、低酰基氧化结冷胶和氨基化羟基磷灰石制成,其中,明胶与低酰基氧化结冷胶的重量比为(10～30):(1～3)；氨基化羟基磷灰石的添加量为明胶和氧化结冷胶总重量的0.5%～2%。经过改性后的mHap的粒径明显降低,更接近纳米级,使得mHap在复合水凝胶中分布更为均匀,并且,在Gel-OG中引入mHap后所形成的水凝胶的网络更为致密,且mHap的引入对细胞的增殖有促进作用,使得Gel-OG/mHap具有良好的生物相容性、可降解性和自愈合性,有利于Gel-OG/mHap复合水凝胶在软骨组织工程领域的应用。(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.)

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.

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

(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.

4. The method for preparing the composite hydrogel according to claim 3, wherein the step of preparing the oxidized gellan gum comprises:

(1) dissolving and dispersing gellan gum in water, 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; the molecular weight of the intercepted molecules of the dialysis bag is 8-14 kDa.

5. The method for preparing the composite hydrogel according to claim 3, wherein the preparation 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) 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.

6. The method for preparing the composite hydrogel according to claim 5, wherein the step of preparing the hydroxyapatite comprises:

(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 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;

(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.

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

8. 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%.

9. The method for preparing the composite hydrogel according to claim 7 or 8, wherein the gelatin aqueous solution and the oxidized gellan gum solution are added in a mass ratio of 1:1.

10. 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 8 in cartilage tissue replacement materials.

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 tissue engineering technology, can replace damaged tissues, and can also be used as a cell carrier to promote tissue cell regeneration. 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 mainly forms a monolayer structure 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 graph of particle size distribution 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 different mHap incorporation of 10wt% gelatin;

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