CN108578766B

CN108578766B - Bone tissue replacement material and preparation method thereof

Info

Publication number: CN108578766B
Application number: CN201810543807.8A
Authority: CN
Inventors: 石小军; 周凯; 喻鹏; 杨伟; 杨鸣波
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2019-12-27
Anticipated expiration: 2038-05-31
Also published as: CN108578766A

Abstract

The invention belongs to the field of bone tissue replacement materials, and particularly relates to a preparation method of a chitosan-graphene-hydroxyapatite composite material as a bone tissue replacement material. The invention provides a preparation method of a bone tissue replacement material, which sequentially comprises the following steps: preparing hydroxyapatite/chitosan slurry, preparing melamine sponge loaded with the hydroxyapatite/chitosan slurry, sintering and molding, and reducing the loaded graphene oxide and the graphene oxide; the method for preparing the melamine sponge loaded with the hydroxyapatite/chitosan slurry comprises the following steps: immersing melamine sponge into hydroxyapatite/chitosan slurry, extruding the melamine sponge to fill the pores in the melamine sponge with the slurry, taking out the melamine sponge and carrying out vacuum drying to obtain nano hydroxyapatite/chitosan loaded melamine sponge; the vacuum temperature is 40-80 ℃, and the vacuum degree is 60-90 KPa. The obtained bone tissue replacement material has a porous structure, high mechanical strength and porosity, and can meet the requirements of the bone tissue replacement material.

Description

Bone tissue replacement material and preparation method thereof

Technical Field

The invention belongs to the field of bone tissue replacement materials, and particularly relates to a Chitosan (CS) -graphene (G) -Hydroxyapatite (HA) ternary composite material serving as a bone tissue replacement material and a preparation method thereof.

Background

Hydroxyapatite (HA) is the main component of inorganic substances in human bones, and HAs good biocompatibility and biodegradability. At present, commercial artificial bone materials almost contain hydroxyapatite which is a component, and hydroxyapatite ceramic materials with high strength can be obtained through high-temperature sintering, and hydroxyapatite biological ceramics and coral hydroxyapatite are common, but the hydroxyapatite ceramic materials have the defects of difficult shaping, low porosity, insufficient void size, poor bone induction capability and the like.

Chitosan (CS) is widely present in nature, has good biocompatibility, can be degraded into glucosamine in vivo, is neutral or weakly alkaline, does not cause local inflammation, and can be completely absorbed by human body. And CS can promote the adhesion, differentiation and proliferation of osteocytes and fibroblasts. Both CS and HA have good biocompatibility and are suitable for preparing bone repair materials. In addition, chitosan has certain anti-inflammatory effect and also has good inhibition effect on the inflammation of the affected part.

Graphene has attracted much attention in recent years, has a very large specific surface area, has a good adhesion effect on cells, has a strong drug-loading capacity, and has the advantages of promoting proliferation and differentiation of bone cells and the like. These advantages also allow researchers to apply them to bone tissue materials in succession.

There are many techniques and methods currently available for preparing scaffolds for bone tissue engineering, such as: the stent material is prepared by an electrostatic spinning method, a phase separation/freeze drying method, a solvent casting/particle leaching technology, a rapid prototyping technology and a gas foaming technology, but the technologies have the characteristics of complex preparation process, high production cost, low porosity, poor strength and the like.

Disclosure of Invention

The invention provides a bone tissue replacement material, which takes hydroxyapatite, chitosan and graphene as raw materials, adopts a specific preparation method, has a porous structure and high mechanical strength and porosity, and can meet the requirements of the bone tissue replacement material.

The technical scheme of the invention is as follows:

a preparation method of a bone tissue replacement material sequentially comprises the following steps: preparing nano hydroxyapatite/chitosan slurry, preparing melamine sponge loaded with the nano hydroxyapatite/chitosan slurry, sintering and molding in air to obtain porous hydroxyapatite ceramic, and reducing the loaded graphene oxide and the graphene oxide; the method for preparing the melamine sponge loaded with the hydroxyapatite/chitosan slurry comprises the following steps: immersing melamine sponge into hydroxyapatite/chitosan slurry, extruding the melamine sponge to fill the pores in the melamine sponge with the slurry, taking out the melamine sponge and carrying out vacuum drying to obtain nano hydroxyapatite/chitosan loaded melamine sponge; wherein the vacuum temperature is 40-80 ℃, and the vacuum degree is 60-90 KPa.

Further, the method for preparing the hydroxyapatite/chitosan slurry comprises the following steps: dispersing nano hydroxyapatite powder in water solution uniformly, adding acetic acid, adding chitosan powder while stirring, and stirring and mixing uniformly to obtain nano hydroxyapatite/chitosan slurry; wherein the mass ratio of the nano hydroxyapatite to the chitosan is as follows: 20-80 parts of hydroxyapatite and 0.5-4 parts of chitosan.

Preferably, in the method for preparing hydroxyapatite/chitosan slurry, the mass ratio of the nano hydroxyapatite to the chitosan is as follows: 60-80 parts of hydroxyapatite and 0.5-4 parts of chitosan; more preferably: 60 parts of hydroxyapatite and 0.3 part of chitosan.

Further, in the method for preparing the hydroxyapatite/chitosan slurry, the mass of the hydroxyapatite powder accounts for 20-80 percent of that of the aqueous solution, and preferably 60 percent; the volume of acetic acid is 0.5-3%, preferably 2% of the volume of the aqueous solution; the mass of the chitosan is 0.5-4%, preferably 3% of the mass of the aqueous solution.

Further, the method for loading graphene oxide comprises the following steps: and immersing the porous hydroxyapatite ceramic in an alcohol solution of graphene oxide for 5-15 min, and taking out and drying to obtain the graphene oxide-loaded hydroxyapatite porous ceramic.

Further, in the method for loading graphene oxide, the mass concentration of the graphene oxide alcohol solution is 0.2-0.5% (namely the mass ratio of graphene oxide in the graphene oxide alcohol solution). If the concentration of the graphene alcohol solution is too low, the graphene modification effect cannot be achieved, and when the concentration is too high, the sheet layer of the graphene blocks the porous structure of the porous material, so that the growth of cells is inhibited.

Further, the reduction method of the graphene oxide comprises the following steps: carrying out thermal reduction reaction on the hydroxyapatite porous ceramic loaded with the graphene oxide in an inert gas atmosphere at 900-1100 ℃, and cooling to obtain the bone tissue replacement material.

Further, the size of the melamine sponge gap is 100-300 um and is a through hole.

Further, the sintering and molding process comprises the following steps: heating to 330 ℃ at a speed of 5-15 ℃/min, keeping the temperature for 1h, heating to 1200-1350 ℃ at a speed of 5-15 ℃/min, keeping the temperature for 2-4 h (so that the structure is more stable), and naturally cooling to the normal temperature.

Further, in the graphene oxide reduction method, the thermal reduction process is as follows: firstly heating to 900-1100 ℃ at the speed of 5-15 ℃/min, and then naturally cooling to the normal temperature.

A second technical problem to be solved by the present invention is to provide a bone tissue replacement material prepared by the above method.

The invention has the beneficial effects that:

the bone tissue replacement material prepared by the method has the following advantages:

1) has higher mechanical strength and can meet the requirement of bone tissue replacement materials.

2) Has high porosity, uniform pores and through holes, and can meet the requirements of osteoblasts on growth space and environment.

3) The added graphene material has certain osteoinductive capacity.

4) The main components of the final material are hydroxyapatite and a small amount of carbon, and the final material has good biocompatibility.

In addition, the melamine sponge is green, environment-friendly and safe light-density foam, has the characteristics of high aperture ratio, water absorbability, uniform cavities, through holes, low price and the like, can be sublimated when the temperature reaches more than three hundred degrees, and can be easily removed in the subsequent sintering process by using the melamine sponge as a template.

Drawings

FIG. a and FIG. b are electron micrographs and partial enlargements of the porous scaffold material obtained in example two, respectively.

Fig. two is an electron microscope photograph of the comparative example of the second example, and it can be seen that since the concentration of graphene oxide in the immersed alcohol solution is high, the graphene oxide lamella blocks the holes in the porous hydroxyapatite scaffold, so that osteoblasts cannot grow into the porous hydroxyapatite scaffold, and therefore, a proper amount of graphene addition is required, which not only ensures better adhesion of cells, but also does not block the through hole structure in the scaffold.

In FIGS. III a and III b, which are electron micrographs of comparative example II and comparative example III, respectively, it can be seen that a uniform porous structure as shown in FIG. I could not be formed under the conditions of drying or without addition of CS.

The fourth graph is the result after three weeks of culturing the mesenchymal stem cells on the surface of the graphene membrane, and it can be seen that the cells can be well adhered and grown on the surface of the graphene membrane.

FIG. V is a graph showing the result of measurement of the content of alkaline phosphatase (ALP) in the culture solution, and it can be seen from the graph V that the differentiated bone cells were the least in the comparative example one in which graphene was not introduced, while the comparative example one material to which the osteogenesis inducing liquid was added and the example two in which graphene was introduced had relatively high differentiation rates; this indicates that the introduction of graphene actually improves osteogenic differentiation capacity of osteogenic stem cells.

Figure six is a plot of porosity and compression modulus for examples one, two, three, and comparative examples two, three.

Detailed Description

The invention aims to provide a composite bone replacement material which has proper pore size, higher porosity, high mechanical strength and good bone induction capability; the nano HA and GO are uniformly dispersed in the CS solution, and due to the good compatibility of CS and melamine sponge, the HA and GO are well loaded on the sponge matrix, so that a bone tissue replacement material with excellent uniformity and performance can be obtained in the subsequent sintering process.

The invention takes melamine resin as a soft template, slurry containing hydroxyapatite and chitosan is dipped in the soft template, and then a porous hydroxyapatite bracket is obtained by a vacuum drying method; porous hydroxyapatite ceramics can be obtained by sintering, a large amount of oxygen-containing groups on the surface of the hydroxyapatite ceramics are further soaked in alcohol solution containing GO, the GO is loaded in the porous hydroxyapatite ceramics, the porous hydroxyapatite ceramics are dried and then thermally reduced (equivalent to secondary sintering), the second sintering can reduce the GO to obtain rGO, reduce the dispersing capacity of the rGO in water solution, enable the rGO not to run off in vivo, and enable the hydroxyapatite to be recrystallized again and combined with the rGO mutually, and due to good compatibility between the hydroxyapatite ceramics and graphene, graphene is well loaded in a support of the porous hydroxyapatite; finally obtaining the porous hydroxyapatite ceramic bracket uniformly loading the graphene. Compared with the traditional bone tissue replacement material, the porous structure of the composite material obtained by the invention is more beneficial to the proliferation and the growth of bone cells, and due to the introduction of the graphene material with certain osteogenesis inducing capacity, the osteoblasts are more easily attached to the scaffold and the proliferation and the differentiation of the osteoblasts are promoted; therefore, the material has better potential as a bone tissue replacement material than the existing commercial material.

In the experimental process, the invention discovers that an important problem exists in the process of preparing the hydroxyapatite porous ceramic by sintering and forming, namely that pores exist among nano-particles in the process of sintering and forming the nano-hydroxyapatite, and the pores can not be well sintered together, so that the mechanical strength of the material can not be improved; but if the method of sintering after compaction is adopted, a porous structure cannot be obtained; therefore, the traditional biological friendly natural polymer CS is introduced, the porous hydroxyapatite ceramic with high strength and high porosity and suitable for cell growth pore size is obtained by adopting methods such as vacuum drying, and based on the porous material, graphene is introduced into the system in the later stage, and the influence of the graphene on osteogenesis is discussed.

The forming mechanism of the porous scaffold and the mechanism of embedding graphene on the hydroxyapatite porous scaffold are as follows: in the vacuum drying process of the melamine resin adsorbed with the slurry of HA and CS, along with solvent volatilization, pores appear in the support, chitosan molecular chains shrink to stick HA nano particles together, and due to the relationship of vacuum suction and through holes in the melamine resin, a through hole structure shown in the figure I is finally formed; then, GO is loaded on the nano-hydroxyapatite ceramic, and is subjected to thermal reduction, and in the reduction process, the attached graphene is inlaid on the surface of the hydroxyapatite ceramic due to recrystallization of the nano-hydroxyapatite.

The preparation method of the bone tissue replacement material can adopt the following steps:

(1) preparing hydroxyapatite/chitosan slurry: dispersing hydroxyapatite powder in water solution uniformly (for example, ultrasonic treatment can be carried out for 1h), then adding acetic acid, adding chitosan powder while stirring, and then stirring and uniformly mixing (generally stirring for 2h) to obtain hydroxyapatite/chitosan slurry; wherein the mass ratio of the hydroxyapatite to the chitosan is as follows: 20-80 parts of hydroxyapatite and 0.5-4 parts of chitosan;

(2) preparing melamine sponge loaded with hydroxyapatite/chitosan slurry: immersing melamine sponge in the hydroxyapatite/chitosan slurry obtained in the step (1), filling pores in the melamine sponge with the slurry by extruding the melamine sponge, taking out the melamine sponge, and performing vacuum drying to obtain the nano hydroxyapatite/chitosan-loaded melamine sponge, wherein the vacuum temperature is 40-80 ℃ (preferably 60 ℃), the vacuum degree is 60-90KPa, preferably 80-85 kPa;

(3) sintering and forming: sintering the dried melamine sponge loaded with hydroxyapatite/chitosan obtained in the step (2) in air to obtain porous hydroxyapatite ceramic;

(4) loading graphene oxide: immersing the porous hydroxyapatite ceramic obtained in the step (3) in an alcohol solution of graphene oxide for 5-15 min (preferably 10min), and then taking out and drying;

(5) and (3) reduction of graphene oxide: and (3) carrying out thermal reduction reaction on the graphene oxide-loaded hydroxyapatite porous ceramic obtained in the step (4) in an inert gas atmosphere at 900-1100 ℃ (preferably 1000 ℃), and cooling to obtain the bone tissue replacement material.

The following examples are only exemplary embodiments and are not intended to limit the present invention, and those skilled in the art can reasonably design the technical solutions with reference to the examples and can also obtain the results of the present invention.

Example one

(1) Preparing CS/HA slurry: dispersing 3g of HA nano hydroxyapatite powder in 10ml of water solution, carrying out ultrasonic treatment for 1h, adding 200ul of acetic acid, carrying out magnetic stirring for 10min, adding 0.3g of CS powder, and stirring for 2 h.

(2) Preparation of melamine sponges of different shapes: commercially available melamine sponges were cut into small rectangular cubes of 0.5cm by 1cm with a razor blade.

(3) Preparation of melamine sponge loaded with CS/HA slurry: immersing the blocky melamine sponge obtained in the step (2) in the slurry obtained in the step (1), sucking the slurry into the sponge by pressing the melamine sponge, taking out the sponge, and drying the sponge in a 60-degree vacuum oven under the condition that the vacuum degree is 80 kPa.

(4) Sintering and forming: and (4) placing the dried CS/HA-loaded melamine sponge obtained in the step (3) into a crucible and sintering the melamine sponge in the air, wherein the temperature rise procedure is that the temperature is raised to 330 ℃ at a speed of 5 ℃/min, the temperature is maintained for 1h, then the temperature is raised to 1200 ℃ at a speed of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally reduced to the normal temperature in a tube furnace.

(5) Introducing graphene oxide: immersing the hydroxyapatite porous ceramic obtained in the step (4) in an alcohol solution of graphene oxide with the mass concentration of 0.3 wt% (the mass ratio of the graphene oxide in the graphene oxide/alcohol solution is 0.3%) for 10min, and taking out and drying.

(6) And (3) reduction of graphene oxide: and (3) placing the hydroxyapatite porous ceramic loaded with the graphene oxide obtained in the step (5) into a tubular furnace to perform thermal reduction reaction under the protection of high-purity nitrogen, wherein the temperature rise procedure of thermal reduction is that the temperature rises to 1000 ℃ at a speed of 10 ℃/min, and the final sample is obtained after furnace cooling.

Example two

(1) Preparing CS/HA slurry: dispersing 6g of HA nano hydroxyapatite powder in 10ml of water solution, carrying out ultrasonic treatment for 1h, adding 200ul of acetic acid, carrying out magnetic stirring for 10min, adding 0.3g of CS powder, and stirring for 2 h.

(4) Sintering and forming: and (4) placing the dried CS/HA-loaded melamine sponge obtained in the step (3) into a crucible and sintering under the condition of air, wherein the temperature rise procedure is that the temperature is raised to 330 ℃ at the speed of 5 ℃/min, the temperature is maintained for 1h, then the temperature is raised to 1200 ℃ at the speed of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally lowered to the normal temperature in a tubular furnace.

(5) Introducing graphene oxide: immersing the hydroxyapatite porous ceramic obtained in the step (4) in 0.3% graphene oxide alcohol solution for 10min, and taking out and drying.

Comparative example one of example two

(5) Introducing graphene oxide: immersing the hydroxyapatite porous ceramic obtained in the step (4) in 0.1% graphene oxide alcohol solution for 10min, and taking out and drying.

Comparative example two to example two

(5) Introducing graphene oxide: immersing the hydroxyapatite porous ceramic obtained in the step (4) in 0.6% graphene oxide alcohol solution for 10min, and taking out and drying.

EXAMPLE III

(1) Preparing CS/HA slurry: dispersing 8g of HA nano hydroxyapatite powder in 10ml of water solution, carrying out ultrasonic treatment for 1h, adding 200ul of acetic acid, carrying out magnetic stirring for 10min, adding 0.3g of CS powder, and stirring for 2 h.

Comparative example graphene-free

(4) Sintering and forming: and (4) placing the dried CS/HA-loaded melamine sponge obtained in the step (3) into a crucible and sintering under the condition of air, wherein the temperature rise procedure is that the temperature is raised to 330 ℃ at a speed of 5 ℃/min, the temperature is maintained for 1h, then the temperature is raised to 1200 ℃ at a speed of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally lowered to the normal temperature in a tubular furnace to obtain the product.

Comparative example two drying

(3) Preparation of melamine sponge loaded with CS/HA slurry: immersing the massive melamine sponge obtained in the step (2) in the slurry obtained in the step (1), sucking the slurry into the sponge by squeezing the melamine sponge, taking out the slurry, and drying the slurry in a vacuum oven with the vacuum degree of 60 degrees (the vacuum degree is 0).

Comparative example No. C

(1) Preparing HA slurry: dispersing 6g of HA nano hydroxyapatite powder in 10ml of water solution, and carrying out ultrasonic treatment for 1h to obtain hydroxyapatite slurry.

(3) Preparation of HA slurry loaded melamine sponge: immersing the blocky melamine sponge obtained in the step (2) in the slurry obtained in the step (1), sucking the slurry into the sponge by pressing the melamine sponge, taking out the sponge, and drying the sponge in a 60-degree vacuum oven under the condition that the vacuum degree is 80 kPa.

(4) Sintering and forming: and (3) placing the dried HA-loaded melamine sponge obtained in the step (3) into a crucible and sintering under the condition of air, wherein the temperature rise procedure is that the temperature is raised to 330 ℃ at a speed of 5 ℃/min, the temperature is maintained for 1h, then the temperature is raised to 1200 ℃ at a speed of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally lowered to the normal temperature in a tube furnace.

And (3) microstructure and performance testing:

first and second figures are electron micrographs and partial enlargements (first figure) of the porous scaffold material obtained in the second example, respectively, and it can be seen that the inside of the material has many pores, which provide suitable environments for the attachment, proliferation and differentiation of osteoblasts, and the cells can grow into the material due to the existence of the through holes. Finally, as the hydroxyapatite is broken down in vivo, the bone cells grow and new bone eventually replaces the material. From the enlarged view on the right side, we see that the graphene is attached to the surface of the hydroxyapatite, the graphene and the hydroxyapatite have good compatibility, and the graphene has a larger specific surface area than the hydroxyapatite and is easier to attach to cells. And researches report that the graphene has the capacity of inducing osteogenic differentiation, so that the effect of the porous hydroxyapatite scaffold attached with the graphene on bone tissue repair is superior to that of a pure hydroxyapatite porous scaffold.

Fig. three a and b are electron microscope images of comparative example two and comparative example three, respectively, and it can be seen that a uniform porous structure as in fig. one cannot be formed under the condition of drying or without adding CS, and the formation mechanism of the porous scaffold in fig. one and the mechanism of embedding graphene on the hydroxyapatite porous scaffold are analyzed: in the vacuum drying process of the melamine resin adsorbed with the slurry of HA and CS, along with solvent volatilization, pores appear in the support, chitosan molecular chains shrink to stick HA nano particles together, and due to the relationship of vacuum suction and through holes in the melamine resin, a through hole structure shown in the figure I is finally formed; then loading GO, and carrying out thermal reduction on the GO; in the reduction process, due to the recrystallization of the nano hydroxyapatite, the attached graphene is embedded on the surface of the hydroxyapatite ceramic.

In order to understand the biotoxicity and cell adhesion capability of graphene, a pure graphene membrane is immersed in a simulated body fluid SBF solution, bone marrow mesenchymal stem cells are placed in the SBF solution for culture, after three weeks, the cell adhesion condition on the surface of the graphene membrane is shown in the fourth drawing, and the cells can be seen to be well adhered and grown on the surface of the graphene; therefore, the graphene has no biological toxicity to cells and can well promote the proliferation and differentiation of the cells.

In order to further understand the differentiation condition of the cells, the invention detects the content of alkaline phosphatase (ALP) in the culture solution (ALP is a secretion product of osteoblasts, and the differentiation condition of the osteoblasts is generally judged by the content of ALP in the culture solution); as a result, as shown in fig. five, the differentiated bone cells were the least in comparative example one in which no graphene was introduced, while the comparative example one material to which the osteogenesis inducing liquid was added and the example two in which graphene was introduced had relatively high differentiation rates; this indicates that the introduction of graphene actually improves osteogenic differentiation capacity of osteogenic stem cells.

Fig. six is a graph of porosity and compressive modulus for example one, example two, example three, comparative example two, and comparative example three, and it can be seen that the compressive modulus is increasing with increasing hydroxyapatite content, and when 6g of HA is added, the increase is greatest, but the porosity is decreasing with increasing HA. The strength of the sample of example two was maintained at a high level while ensuring high porosity, and the sample dried under normal pressure and without addition of CS, i.e., the sample of comparative example two, and the third sample did not have high porosity and strength because there was no uniform through-hole formation inside the material dried under normal pressure and without addition of CS.

While the invention has been described in conjunction with the embodiments above, it will be apparent to those skilled in the art that various modifications may be made to the embodiments described above without departing from the spirit and scope of the claims.

Claims

1. A preparation method of a bone tissue replacement material is characterized by sequentially comprising the following steps: preparing nano hydroxyapatite/chitosan slurry, preparing melamine sponge loaded with the nano hydroxyapatite/chitosan slurry, sintering and forming in air to obtain porous hydroxyapatite ceramic, and reducing the loaded graphene oxide and the graphene oxide; the method for preparing the melamine sponge loaded with the nano hydroxyapatite/chitosan slurry comprises the following steps: immersing melamine sponge in the nano hydroxyapatite/chitosan slurry, extruding the melamine sponge to fill the pores in the melamine sponge with the slurry, taking out the melamine sponge and carrying out vacuum drying to obtain the nano hydroxyapatite/chitosan loaded melamine sponge; wherein the vacuum temperature is 40-80 ℃, and the vacuum degree is 60-90 KPa.

2. The method for preparing bone tissue replacement material according to claim 1, wherein the method for preparing nano hydroxyapatite/chitosan slurry comprises: dispersing nano hydroxyapatite powder in water solution uniformly, adding acetic acid, adding chitosan powder while stirring, and stirring and mixing uniformly to obtain nano hydroxyapatite/chitosan slurry; wherein the mass ratio of the nano hydroxyapatite to the chitosan is as follows: nano hydroxyapatite: 20-80% of chitosan: 0.5 to 4.

3. The method for preparing bone tissue replacement material according to claim 2, wherein the mass ratio of the nano-hydroxyapatite to the chitosan is as follows: nano hydroxyapatite: 60-80 parts of chitosan: 0.5 to 4.

4. The method for preparing the bone tissue replacement material as claimed in claim 1 or 2, wherein the method for loading graphene oxide is as follows: and immersing the porous hydroxyapatite ceramic in an alcohol solution of graphene oxide for 5-15 min, and taking out and drying to obtain the graphene oxide-loaded hydroxyapatite porous ceramic.

5. The method for preparing a bone tissue replacement material according to claim 4, wherein in the method for loading graphene oxide, the concentration of graphene oxide in the graphene oxide alcohol solution is 0.2 wt% to 0.5 wt%.

6. The method for preparing bone tissue replacement material according to claim 2, wherein in the method for preparing nano hydroxyapatite/chitosan slurry, the nano hydroxyapatite powder accounts for 20-80% of the mass of the aqueous solution; the volume of the acetic acid is 0.5 to 3 percent of the volume of the aqueous solution; the mass of the chitosan is 0.5-4% of the mass of the aqueous solution.

7. The method for preparing bone tissue replacement material according to claim 6, wherein in the method for preparing nano hydroxyapatite/chitosan slurry, the nano hydroxyapatite powder accounts for 60% of the mass of the aqueous solution; the volume of acetic acid is 2% of the volume of the aqueous solution; the mass of the chitosan is 3 percent of the mass of the aqueous solution.

8. The method for preparing a bone tissue substitute material according to claim 1 or 2, wherein the melamine sponge has a void size of 100-300 um and is a through hole.

9. The method for preparing a bone tissue substitute material as claimed in claim 4, wherein the melamine sponge has a gap size of 100-300 um and is a through hole.

10. The method for preparing a bone tissue substitute material as claimed in claim 6, wherein the melamine sponge has a gap size of 100-300 um and is a through hole.

11. The method for preparing bone tissue substitute material according to claim 1, wherein the sintering and molding process comprises: heating to 330 ℃ at a speed of 5-15 ℃/min, keeping the temperature for 1h, heating to 1200-1350 ℃ at a speed of 5-15 ℃/min, keeping the temperature for 2-4 h, and naturally cooling to normal temperature.

12. The method for preparing bone tissue replacement material according to claim 1, wherein the graphene oxide is reduced by: carrying out thermal reduction reaction on the hydroxyapatite porous ceramic loaded with the graphene oxide in an inert gas atmosphere at 900-1100 ℃, and cooling to obtain the bone tissue replacement material.

13. The method for preparing bone tissue replacement material according to claim 12, wherein in the method for reducing graphene oxide, the thermal reduction process comprises: firstly heating to 900-1100 ℃ at the speed of 5-15 ℃/min, and then naturally cooling to the normal temperature.

14. A bone tissue replacement material prepared by the method of any one of claims 1 to 13.