CN108478862B

CN108478862B - Hydroxyapatite ceramic/graphene composite bone tissue replacement material and preparation thereof

Info

Publication number: CN108478862B
Application number: CN201810543759.2A
Authority: CN
Inventors: 杨伟; 喻鹏; 石小军; 周凯; 杨鸣波
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2020-02-18
Anticipated expiration: 2038-05-31
Also published as: CN108478862A

Abstract

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

Description

Hydroxyapatite ceramic/graphene composite bone tissue replacement material and preparation thereof

Technical Field

The invention belongs to the field of bone tissue replacement materials, and particularly relates to a porous hydroxyapatite ceramic/graphene composite 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, and has the advantages of large specific surface area, strong drug-loading capacity, capability 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. Although the special surface structure of graphene is beneficial to the attachment of cells, thereby promoting the proliferation and differentiation of osteoblasts, the mechanical strength of the hydrogel prepared by using graphene alone is poor. Therefore, the graphene material is often used as a composite material by being compounded with other materials.

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 nano hydroxyapatite, chitosan and graphene as raw materials, adopts a specific preparation method, has a porous structure and higher mechanical strength and porosity, and can meet the requirements of the bone tissue replacement material.

The technical scheme of the invention is as follows:

the first technical problem to be solved by the invention is to provide a preparation method of a hydroxyapatite ceramic/graphene composite bone tissue replacement material, which sequentially comprises the following steps: preparing nano hydroxyapatite/chitosan/graphene slurry, preparing a porous hydroxyapatite/graphene composite scaffold, and sintering and molding; the method for preparing the nano-porous hydroxyapatite/graphene composite scaffold comprises the following steps: immersing polyurethane sponge in hydroxyapatite/chitosan/graphene slurry, filling the pores in the polyurethane sponge with the slurry by extruding the polyurethane sponge, and then taking out and carrying out vacuum drying on the obtained product to obtain a porous hydroxyapatite/graphene composite scaffold; in the vacuum drying process, the vacuum temperature is 40-80 ℃ (preferably 60 ℃), and the vacuum degree is 60-90 KPa, preferably 80-85 kPa.

Further, the method for preparing the nano hydroxyapatite/chitosan/graphene slurry comprises the following steps: dispersing nano hydroxyapatite powder in graphene dispersion liquid, performing ultrasonic treatment to uniformly disperse hydroxyapatite (ultrasonic treatment is performed for 1 hour), adding acetic acid, adding chitosan powder while stirring, and uniformly stirring (generally stirring for 2 hours) to obtain hydroxyapatite/chitosan/graphene slurry; wherein the mass of the nano hydroxyapatite is 20-80% of that of the graphene dispersion liquid, and preferably 60%; the volume of acetic acid is 0.5-3%, preferably 2% of the volume of the graphene dispersion liquid; the mass of the chitosan is 0.5-4%, preferably 3% of the mass of the graphene dispersion liquid.

Further, in the method for preparing the nano hydroxyapatite/chitosan/graphene slurry, the concentration of the graphene dispersion liquid is 0.05 wt% -1 wt%. In the invention, the graphene dispersion liquid is prepared by the following method: and (3) carrying out ultrasonic treatment on the graphene and the aqueous solution at normal temperature and uniformly mixing to obtain the graphene dispersion liquid (generally requiring two hours).

Further, the sintering and forming method comprises the following steps: sintering the porous nano hydroxyapatite/graphene composite scaffold under the protection of inert gas to obtain the porous nano hydroxyapatite ceramic/graphene composite bone tissue replacement material, wherein the sintering 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 2-4h, and naturally cooling to normal temperature.

Further, polyurethane sponge gap size is 100 ~ 300um to be the through-hole structure.

The second technical problem to be solved by the invention is to provide a hydroxyapatite ceramic/graphene composite bone tissue replacement material, which is prepared by the 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 graphene material is added, so that the bone formation induction capability is better than that of pure hydroxyapatite ceramic.

4) The main components of the final material are hydroxyapatite and a small amount of carbon, so that the material has good biocompatibility and is basically a completely degradable material.

In addition, the PU sponge is green, environment-friendly and safe light-density foam, has the characteristics of high aperture ratio, water absorption, uniform cavities, through holes, low cost and the like, and can be carbonized at high temperature, so that the PU sponge can be used as a template to prepare a hydroxyapatite porous scaffold, and finally, a complete carbon skeleton can be formed in the carbonization process to increase the strength of the scaffold.

Drawings

FIGS. 1a and 1b are SEM and magnified views of the material obtained in example two, respectively. As can be seen from figure 1, more through hole structures of 200-400um exist in the material, such a large-aperture through hole structure is very suitable for the growth of osteoblasts, meanwhile, the material is further amplified, graphene is well loaded in a matrix of porous hydroxyapatite ceramic, the sheet structure of the graphene greatly increases the specific surface area of the material, more places can be provided for cell adhesion, and the graphene has certain capacity of inducing cell osteogenesis, a hydroxyapatite skeleton loaded with the graphene in the material not only provides a suitable living environment for osteoblasts, but also can play a role in inducing osteoblast differentiation.

FIG. 2 shows the compressive strength results of examples I, II, III and comparative examples I, II, III. As can be seen from fig. 2, as the amount of added hydroxyapatite increases, the compressive strength of the material also increases, but the viscosity of the slurry prepared after too much hydroxyapatite increases, increasing the difficulty of impregnating into PU resin, and decreasing the porosity, so that the second embodiment has higher strength while ensuring the porosity. The compressive strength of the first comparative example is slightly reduced compared with that of the second example, probably because the second example undergoes two sintering processes, so that the hydroxyapatite ceramic is more compact, and the introduction of the graphene increases the strength of the porous ceramic.

FIG. 3 shows the results of cell proliferation activity assay (CCK-8) of example two, comparative example one of example two, and blank without sample (blank for short). The quantitative detection through a CCK-8 experiment shows that: after 7 days after the co-culture of the first comparative example, the second comparative example and the first comparative example and the mBMSCs cells, compared with a blank group, each group can promote the cell proliferation (P is less than 0.01), wherein the second example has the most remarkable promotion effect, the quality ratio of rGO is increased, and the promotion effect is reduced; the content of the graphene in the hydroxyapatite composite scaffold is in an optimal range in the aspect of promoting cell proliferation, and the optimal promoting effect cannot be achieved when the content is too small.

Fig. 4A-4C show alizarin red staining after 21 days of bmscs cells grown on material example two (fig. 4A), comparative example one (fig. 4B), and blank without material (fig. 4C), respectively. As can be seen from FIGS. 4A to 4C, after the osteogenesis inducing solution was cultured, brick red nodules were formed over a large area in the material group of example two, and almost the entire visual field was occupied; the material group of comparative example has brick red nodules in part and is relatively dispersedly arranged; the blank without material was uniformly reddish colored and no brick-red deposition occurred in the field. The extracellular matrixes of the experimental group and the control group are proved to have generation and precipitation of inorganic calcium; meanwhile, the composite material of the second embodiment can promote osteogenic differentiation of mBMSCs and increase matrix mineralization.

Fig. 5 is a graph of porosity and compressive modulus for examples one, two, three and comparative examples two, three, and it can be seen that the compressive modulus increases with increasing hydroxyapatite content, and the increase is greatest when 6g of HA is added, i.e., example two, but the porosity decreases 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., comparative example two, and the third sample did not have high porosity and strength because uniform through-hole formation was not caused in the interior of the material dried under normal pressure and without addition of CS.

Detailed Description

The first technical problem to be solved by the invention is to provide a preparation method of a hydroxyapatite ceramic/graphene composite bone tissue replacement material, which comprises the following steps:

(1) preparing hydroxyapatite/chitosan/graphene slurry: dispersing nano hydroxyapatite powder in graphene dispersion liquid, performing ultrasonic treatment to uniformly disperse the nano hydroxyapatite (ultrasonic treatment is performed for 1h), adding acetic acid, adding chitosan powder while stirring, and uniformly stirring (generally stirring for 2h) to obtain hydroxyapatite/chitosan/graphene slurry; wherein the mass ratio of the nano hydroxyapatite to the chitosan to the graphene is as follows: 20-80 parts of nano hydroxyapatite, 0.5-4 parts of chitosan and 0.05-1 part of graphene;

(2) preparing a porous hydroxyapatite composite scaffold: immersing polyurethane sponge in the hydroxyapatite/chitosan/graphene slurry obtained in the step (1), filling the pores in the polyurethane sponge with the slurry by extruding the polyurethane sponge, taking out and carrying out vacuum drying on the slurry to obtain a porous hydroxyapatite/graphene composite scaffold, wherein the vacuum temperature is 40-80 ℃ (preferably 60 ℃), the vacuum degree is 60-90 KPa, and preferably 80-85 kPa;

(3) sintering and forming: and (3) sintering the dried composite support obtained in the step (2) under the protection of inert gas to obtain the porous hydroxyapatite ceramic/graphene composite bone tissue replacement material.

The invention mainly discloses a preparation method of a porous hydroxyapatite ceramic/graphene composite material, which comprises the steps of adopting Polyurethane (PU) foam as a template for forming the porous hydroxyapatite ceramic, utilizing strong hydrogen bond interaction between Chitosan (CS) and Graphene Oxide (GO) and the polyurethane foam, dipping slurry prepared from GO, CS and nano Hydroxyapatite (HA) into the polyurethane foam, obtaining a porous hydroxyapatite composite scaffold by utilizing vacuum drying, and preparing a three-dimensional bone tissue replacement material which takes HA as a matrix and is loaded with graphene (G) in a sintering mode. The bone tissue replacement material prepared by the method has the advantages of high strength, high porosity, good biocompatibility and bone induction capability, and is expected to be applied to bone tissue repair. The method has the advantages of simple preparation process, low cost, short production period and the like in the implementation process.

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 invention obtains the porous hydroxyapatite ceramics with high strength, high porosity and suitable cell growth aperture by introducing CS which is a traditional biological friendly natural polymer and adopting methods such as vacuum drying and the like.

The forming mechanism of the porous scaffold of the invention is as follows: in the vacuum drying process of the polyurethane resin adsorbed with the slurry formed by HA, CS and GO, along with solvent volatilization, air holes appear in the support, the HA nano particles and the graphene oxide sheet layer are adhered together by chitosan molecular chain shrinkage, and the uniform through hole structure shown in figure 1 is finally formed due to the relationship of vacuum suction and through holes in the polyurethane resin.

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/GO/HA slurry: dispersing 3g of HA nano powder into 10ml of GO solution with the mass concentration of 0.1 wt%, carrying out ultrasonic treatment for 1h, adding 0.2ml of acetic acid (the acetic acid is added to dissolve chitosan), carrying out magnetic stirring for 10min, adding 0.3gCS powder, and stirring for 2h to obtain CS/GO/HA slurry.

(2) Preparation of PU of different shapes: commercially available melamine sponges were cut with a razor blade into 3mm by 5mm cuboids.

(3) Preparing a porous hydroxyapatite composite scaffold: immersing the blocky PU sponge obtained in the step (2) in the slurry obtained in the step (1), sucking the slurry into the sponge by extruding the melamine sponge, and then drying the blocky PU sponge in a vacuum oven with the temperature of 60 ℃ under the condition that the vacuum degree is 80kPa to obtain the porous hydroxyapatite composite scaffold.

(4) Preparing porous hydroxyapatite composite ceramic: and (3) placing the dried porous hydroxyapatite composite scaffold obtained in the step (3) into a crucible and sintering under the protection of nitrogen, wherein the sintering condition is that the temperature is increased to 330 ℃ at the rate of 5 ℃/min, the temperature is maintained for 1h, then the temperature is increased to 1350 ℃ at the rate of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally reduced to the normal temperature in a tube furnace to obtain a final product.

Example two

(1) Preparing CS/GO/HA slurry: dispersing 6g of HA nano powder into 10ml of GO solution with the mass concentration of 0.1 wt%, carrying out ultrasonic treatment for 1h, adding 0.2ml of acetic acid, carrying out magnetic stirring for 10min, adding 0.3gCS powder, and carrying out stirring for 2h to obtain CS/GO/HA slurry.

(4) Preparing porous hydroxyapatite composite ceramic: and (3) placing the dried porous hydroxyapatite composite scaffold obtained in the step (3) into a crucible and sintering under the protection of nitrogen, wherein the sintering condition is that the temperature is firstly raised to 330 ℃ at a rate of 5 ℃/min, the temperature is maintained for 1h, then the temperature is raised to 1350 ℃ at a rate of 10 ℃/min, the temperature is maintained for 2h, and then the temperature is naturally lowered to the normal temperature in a tube furnace to obtain a final product.

Comparative example one of example two

(1) Preparing CS/GO/HA slurry: dispersing 6g of HA nano powder into 10ml of GO solution with the mass concentration of 0.2 wt%, carrying out ultrasonic treatment for 1h, adding 0.2ml of acetic acid, carrying out magnetic stirring for 10min, adding 0.3gCS powder, and carrying out stirring for 2h to obtain CS/GO/HA slurry.

EXAMPLE III

(1) Preparing CS/GO/HA slurry: dispersing 8g of HA nano powder into 10ml of GO solution with the mass concentration of 0.1 wt%, carrying out ultrasonic treatment for 1h, adding 0.2ml of acetic acid, carrying out magnetic stirring for 10min, adding 0.3gCS powder, and carrying out stirring for 2h to obtain CS/GO/HA slurry.

Comparative example No GO

(1) Preparing CS/HA slurry: dispersing 6g of HA nano powder into 10ml of aqueous solution, carrying out ultrasonic treatment for 1 hour, adding 0.2ml of acetic acid, carrying out magnetic stirring for 10min, adding 0.3gCS powder, and stirring for 2 hours to obtain CS/HA slurry.

Comparative example two drying

(1) Preparing CS/GO/HA slurry: dispersing 6g of HA nano powder into 10ml of 0.1 wt% GO solution, adding 0.2ml of acetic acid after ultrasonic treatment for 1h, adding 0.3gCS powder after magnetic stirring for 10min, and stirring for 2h to obtain CS/GO/HA slurry.

(3) Preparing a porous hydroxyapatite composite scaffold: immersing the blocky PU sponge obtained in the step (2) in the slurry obtained in the step (1), sucking the slurry into the sponge by extruding the melamine sponge, and then drying the sponge in an oven with the temperature of 60 ℃ (the vacuum degree is 0) to obtain the porous hydroxyapatite composite scaffold.

Comparative example No. C

(1) Preparing GO/HA slurry: dispersing 6g of HA nano powder in 10ml of 0.1 wt% GO solution, and magnetically stirring for 2 hours to obtain GO/HA slurry.

And (3) performance testing:

as shown in fig. 1, it can be seen that more through hole structures of 200-400um exist in the material, such a large-aperture through hole structure is very suitable for the growth of osteocytes, and at the same time, through further amplification of the material (fig. 1b), it can be seen that graphene is well loaded in the matrix of the porous hydroxyapatite ceramic, the sheet structure of graphene greatly increases the specific surface area of the material, and can provide more places for cell adhesion, and graphene has a certain capacity of inducing cell osteogenesis, and the hydroxyapatite skeleton loaded with graphene in the material not only provides a suitable living environment for osteoblasts, but also can play a role in inducing osteoblast differentiation.

As shown in fig. 2, as the amount of the hydroxyapatite is increased, the compressive strength of the material is increased, but the viscosity of the slurry prepared after the amount of the hydroxyapatite is too large is increased, the difficulty of impregnation into the PU resin is increased, and the porosity is reduced, so that the second embodiment has higher strength while ensuring the porosity. The compression strength of the comparative example one is slightly reduced compared to that of the example two, probably because the example two undergoes the process of two times of sintering, so that the hydroxyapatite ceramic is more compact.

Quantitative detection by CCK-8 assay found (FIG. 3): compared with a blank group without a sample (called a blank group for short), the cell proliferation (P is less than 0.01) of each group can be promoted by the first example, the second example and the first comparative example, wherein the promotion effect of the second example is most remarkable, the quality ratio of rGO is increased, and the promotion effect is reduced; the content of the graphene in the hydroxyapatite composite scaffold is in an optimal range in the aspect of promoting cell proliferation, and the optimal promoting effect cannot be achieved when the content is too small.

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 hydroxyapatite ceramic/graphene composite bone tissue replacement material is characterized by sequentially comprising the following steps: preparing nano hydroxyapatite/chitosan/graphene slurry, preparing a nano porous hydroxyapatite/graphene composite scaffold, and sintering and molding; the method for preparing the nano-porous hydroxyapatite/graphene composite scaffold comprises the following steps: immersing polyurethane sponge in the nano hydroxyapatite/chitosan/graphene slurry, filling the pores in the polyurethane sponge with the slurry by extruding the polyurethane sponge, taking out the polyurethane sponge, and performing vacuum drying to obtain a nano porous hydroxyapatite/graphene composite scaffold; in the vacuum drying process, the vacuum temperature is 40-80 ℃, and the vacuum degree is 60-90 KPa.

2. The method for preparing a hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 1, wherein the vacuum degree is 80-85 kPa.

3. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 1 or 2, wherein the method for preparing the nano hydroxyapatite/chitosan/graphene slurry comprises the following steps: dispersing nano hydroxyapatite powder in graphene dispersion liquid, performing ultrasonic treatment to uniformly disperse hydroxyapatite, adding acetic acid, adding chitosan powder while stirring, and stirring and uniformly mixing to obtain nano hydroxyapatite/chitosan/graphene slurry; wherein the mass of the nano hydroxyapatite is 20-80% of that of the graphene dispersion liquid; the volume of acetic acid is 0.5-3% of the volume of the graphene dispersion liquid; the mass of the chitosan is 0.5-4% of that of the graphene dispersion liquid.

4. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 3, wherein the mass of the nano hydroxyapatite is 60% of the mass of the graphene dispersion liquid; the volume of acetic acid is 2% of the volume of the graphene dispersion liquid; the mass of the chitosan is 3% of that of the graphene dispersion liquid.

5. The method for preparing a hydroxyapatite/graphene composite bone tissue replacement material according to claim 3, wherein in the method for preparing the nano hydroxyapatite/chitosan/graphene slurry, the concentration of the graphene dispersion liquid is 0.05 wt% to 1 wt%.

6. The method for preparing a hydroxyapatite/graphene composite bone tissue replacement material according to claim 4, wherein in the method for preparing the nano hydroxyapatite/chitosan/graphene slurry, the concentration of the graphene dispersion liquid is 0.05 wt% to 1 wt%.

7. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 3, wherein the graphene dispersion liquid is prepared by adopting the following method: and carrying out ultrasonic treatment on the graphene and the aqueous solution at normal temperature and uniformly mixing to obtain the graphene dispersion liquid.

8. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 1 or 2, wherein the sintering molding method comprises the following steps: sintering the nano-porous hydroxyapatite/graphene composite scaffold under the protection of inert gas to obtain the nano-porous hydroxyapatite ceramic/graphene composite bone tissue replacement material, wherein the sintering 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 2-4h, and naturally cooling to normal temperature.

9. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 3, wherein the sintering molding method comprises the following steps: sintering the nano-porous hydroxyapatite/graphene composite scaffold under the protection of inert gas to obtain the nano-porous hydroxyapatite ceramic/graphene composite bone tissue replacement material, wherein the sintering 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 2-4h, and naturally cooling to normal temperature.

10. The preparation method of the hydroxyapatite ceramic/graphene composite bone tissue replacement material according to claim 1 or 2, wherein the polyurethane sponge has a pore size of 100-300 um and a through hole structure.

11. A hydroxyapatite ceramic/graphene composite bone tissue replacement material, which is characterized by being prepared by the method of any one of claims 1 to 10.