CN108578766A - A kind of bone tissue alternate material and preparation method thereof - Google Patents
A kind of bone tissue alternate material and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to bone tissue alternate material fields, and in particular to a kind of preparation method of chitosan graphene hydroxyapatite composite material as bone tissue alternate material.The present invention provides a kind of preparation method of bone tissue alternate material, and the method includes successively：Prepare hydroxyapatite/chitosan slurry, the melamine sponge for preparing load hydroxyapatite/chitosan slurry, sinter molding, the reduction for loading graphene oxide and graphene oxide；The method of melamine sponge for preparing load hydroxyapatite/chitosan slurry is：Melamine sponge is submerged in hydroxyapatite/chitosan slurry and is filled up the hole in melamine sponge by slurry by squeezing melamine sponge, then takes out the melamine sponge for be dried in vacuo loading nano-hydroxyapatite/chitosan；True temp is 40~80 DEG C, and vacuum degree is 60~90KPa.Gained bone tissue alternate material has porous structure, has higher mechanical strength and porosity, disclosure satisfy that the requirement of bone tissue alternate material.
Description
Technical field
The invention belongs to bone tissue alternate material fields, and in particular to a kind of chitosan (CS)-graphene (G)-hydroxyl phosphorus
Lime stone (HA) trielement composite material is as bone tissue alternate material and preparation method thereof.
Background technology
Hydroxyapatite (HA) is the main component of inorganic matter in people's bone, and there is good biocompatibility, biology can drop
Xie Xing.Currently, commercial artificial bone nearly all contains this ingredient of hydroxyapatite, it can be obtained by high temperature sintering
The high hydroxylapatite ceramic material of intensity, common are hydroxyl apatite bioceramic, coral hydroxyapatite, but they are more
The shortcomings of few all exist difficult moulding, and porosity is low, and pore size is inadequate and self-bone grafting energy force difference.
Chitosan (CS) is widely present in nature, it has good biocompatibility, can be degraded into ammonia in vivo
Base glucose, Glucosamine is in neutrality or alkalescent, will not cause local inflammation, and can be fully absorbed by human body.And CS
It can also promote sticking for osteocyte and fibroblast, break up and be proliferated.CS and HA has good biocompatibility, is suitble to
It is used to prepare bone renovating material.In addition, chitosan also has certain antiinflammation, also has to the inflammation in affected part and inhibit to make well
With.
Graphene receives significant attention in recent years, with great specific surface area, has good adhesive attraction to cell,
Drug loading capacity is strong, and has the advantages that capable of promoting bone cell proliferation, differentiation.These advantages also make researchers be applied to successively
In bone tissue material.
There are many kinds of the technology and methods for being presently available for preparing bone tissue engineering scaffold, such as：Method of electrostatic spinning, phase point
From/freeze-drying, solvent cast/particle leaching technique, rapid shaping technique and gas foaming techniques prepare timbering material, but
It is that these technologies all have the features such as complex process prepared, production cost is high, and porosity is low, intensity difference.
Invention content
The present invention provides a kind of bone tissue alternate material, the bone tissue alternate material with hydroxyapatite, chitosan and
Graphene is raw material, and using specific preparation method, gained bone tissue alternate material has porous structure, has higher machinery
Intensity and porosity disclosure satisfy that the requirement of bone tissue alternate material.
Technical scheme of the present invention：
A kind of preparation method of bone tissue alternate material, the preparation method include successively：Preparation of nano hydroxyapatite/
Sinter molding obtains porous in chitosan slurry, the melamine sponge for preparing load nano-hydroxyapatite/chitosan slurry, air
Hydroxylapatite ceramic, the reduction for loading graphene oxide and graphene oxide；Wherein, preparation load hydroxyapatite/
The method of the melamine sponge of chitosan slurry is：Melamine sponge is submerged close by squeezing in hydroxyapatite/chitosan slurry
Amine sponge is filled up the hole in melamine sponge by slurry, then take out be dried in vacuo load nanometer hydroxyapatite/
The melamine sponge of chitosan；Wherein, true temp is 40~80 DEG C, and vacuum degree is 60~90KPa.
Further, the method for preparing hydroxyapatite/chitosan slurry is：By nanometer hydroxyapatite powder in water
It is uniformly dispersed in solution, acetic acid is added afterwards, Chitosan powder is then added while stirring, be stirred for mixing and obtain nano-hydroxy-apatite
Stone/chitosan slurry；Wherein, the mass ratio of nanometer hydroxyapatite and chitosan is：20~80 parts by weight of hydroxyapatite, shell
0.5~4 parts by weight of glycan.
Preferably, in the method for preparing hydroxyapatite/chitosan slurry, nanometer hydroxyapatite and chitosan
Mass ratio is：60~80 parts by weight of hydroxyapatite, 0.5~4 parts by weight of chitosan；More preferably：60 weight of hydroxyapatite
Part, 0.3 parts by weight of chitosan
Further, in the method for preparing hydroxyapatite/chitosan slurry, hydroxyapatite powder is aqueous solution matter
The 20%-80% of amount, preferably 60%；Acetic acid volume be aqueous solution volume 0.5%-3%, preferably 2%；Chitosan mass is water
The 0.5%-4% of solution quality, preferably 3%.
Further, the method for the load graphene oxide is：Porous Hydroxyapatite Ceramic is immersed in graphite oxide
The alcoholic solution of alkene keeps 5~15min, then takes out the hydroxyapatite porous ceramics for drying to load graphene oxide.
Further, in the method for the load graphene oxide, the mass concentration of the graphene oxide alcoholic solution is
0.2~0.5% (i.e. the quality accounting of graphene oxide in graphene oxide alcoholic solution).The concentration of graphene alcoholic solution is such as
Fruit is too low, and the effect of graphene modification is not achieved, and the lamella of the too high graphene of concentration can block the porous structure of porous material, presses down
Cell processed is grown into.
Further, the restoring method of the graphene oxide is：The porous pottery of hydroxyapatite that graphene oxide will be loaded
Porcelain carries out thermal reduction reaction in atmosphere of inert gases in 900~1100 DEG C, and bone tissue alternate material is obtained after cooling.
Further, the melamine sponge pore size is 100~300um, and is through-hole.
Further, the sintering forming process is：330 DEG C first are warming up to 5 DEG C/min~15 DEG C/min, after keeping 1h,
1200~1350 DEG C are warming up to 5 DEG C/min~15 DEG C/min again, (keeps structure more stable) after keeping 2~4h, Temperature fall
To room temperature.
Further, in the restoring method of the graphene oxide, the thermal reduction technique is：First with 5 DEG C/min-15 DEG C/
Min is warming up to 300~1000 DEG C, then Temperature fall to room temperature.
The invention solves second technical problem be to provide a kind of bone tissue alternate material, the bone tissue replaces material
Material is prepared by the above method.
Beneficial effects of the present invention：
It is had the following advantages using bone tissue alternate material made from the method for the present invention：
1) there is higher mechanical strength, disclosure satisfy that the requirement of bone tissue alternate material.
2) there is very high porosity, hole is uniform and is through-hole, can meet osteoblast and be wanted to growing space and environment
It asks.
3) grapheme material added, there is certain self-bone grafting ability.
4) main component of final material is hydroxyapatite and a small amount of carbon, there is good biocompatibility.
In addition, melamine sponge is that a kind of environmentally protective safe light density bubble is continuous, there is high opening rate, water imbibition, cavity is
One and be through-hole, it is cheap the features such as, and it can distil when temperature reaches and spent 300 more, and present invention choosing uses it as
Template can be removed easily in subsequent sintering process.
Description of the drawings
Fig. 1 a and Fig. 1 b are respectively the electron microscope and its partial enlarged view of two gained porous support materials of embodiment.
Fig. 2 is the electromicroscopic photograph of the comparative example two of embodiment two, it can be seen that due to aoxidizing stone in the alcoholic solution of immersion
The concentration of black alkene is higher, and graphene oxide layer can block the hole in porous hydroxyapatite holder so that osteoblast without
Method is grown into, so needing graphene additive amount appropriate, has not only ensured that cell can be adhered to preferably, but also can not block in holder
Through-hole structure.
Fig. 3 a, Fig. 3 b are respectively the electron microscope of comparative example two and comparative example three, it can be seen that are drying or be not added
Uniform porous structure as shown in Figure 1 cannot be formed under conditions of CS.
Fig. 4 is result of the nourishing the bone bone marrow-drived mesenchymal stem after three weeks in graphene membrane surface, it can be seen that in graphite
Alkene surface, cell can be very good to carry out adherency growth.
Fig. 5 is the testing result figure of culture solution alkaline phosphatase (ALP) content, as shown in Figure 5, in no introducing graphite
The osteocyte broken up in the comparative example one of alkene is minimum, and one material of comparative example of osteogenic induction liquid is added and introduces the reality of graphene
Applying example two has relatively high differentiation rate；This just illustrates that the introducing of graphene improves the Osteoblast Differentiation of Osteogenic Stem really
Ability.
Fig. 6 be embodiment one, two, three, comparative example two, three porosity and compression modulus figure.
Specific implementation mode
The present invention is intended to provide a kind of prepare with suitable pore size, higher porosity, high mechanical strength, and have very well
The compound bone substitution material of self-bone grafting ability；Nano HA, GO are dispersed in CS solution, since CS and melamine sponge are good
Compatibility so that HA, GO are loaded in sponge matrix well, excellent to obtain uniform performance in subsequent sintering process
Good bone tissue alternate material.
The present invention is used as soft template by melamine resin, will wherein impregnate the slurry containing hydroxyapatite and chitosan,
Then by vacuum drying method, porous hydroxyapatite scaffold has been obtained；Porous hydroxyapatite can be obtained by sintering again
Apatite Ceramic is further steeped molten into the alcohol containing GO using a large amount of oxygen-containing group in hydroxylapatite ceramic surface
In liquid, GO is allowed to load in Porous Hydroxyapatite Ceramic, is heat-treated again after drying and (be equivalent to double sintering), second
Sintering, which can not only restore GO and obtain rGO, reduces its dispersibility in aqueous solution, it is made not to be lost in vivo, can be with
Make hydroxyapatite recrystallize again to be combined with each other with rGO, due to good compatible between hydroxylapatite ceramic and graphene
Property, so graphene has been loaded to well in the holder of porous hydroxyapatite；Uniform load graphene is finally obtained
Porous Hydroxyapatite Ceramic holder.Compared with traditional bone tissue alternate material, the porous knot of present invention gained composite material
Structure is more advantageous to the increment of osteocyte and grows into, and due to graphene this drawing with certain induced osteogenesis ability material
Enter, be then easier that osteoblast is allowed on holder to adhere to and promote proliferation and the differentiation of osteocyte；Therefore the material is compared with existing
Commercial material there are preferably potentiality as bone tissue alternate material.
It is found in experimentation of the present invention, prepares during hydroxyapatite porous ceramics that there are one using sinter molding
A major issue, i.e., there are holes to burn well between nano particle during nanometer hydroxyapatite sinter molding
Knot cannot improve the mechanical strength of material in turn together；But it if is unable to get again by the method being sintered after compacting porous
Structure；Therefore the present invention uses vacuum drying etc. by the natural polymer of introducing this traditional biological friendly of CS
Method has obtained having high intensity, high porosity, and with the Porous Hydroxyapatite Ceramic for being suitble to cell growth aperture, is based on
Our porous material, the later stage, graphene was also introduced into our system by we, and discussed graphene in terms of skeletonization
Influence.
The mechanism that the formation mechenism and graphene of porous support of the present invention are embedded on hydroxyapatite porous support is：It inhales
The melamine resin of slurry with HA and CS is during vacuum drying, and as solvent volatilizees, stomata, shell occurs in internal stent
HA nano-particles are stained with by the contraction of glycan molecule chain to stick together, due to there is the relationship of through-hole inside pull of vacuum and melamine resin,
Ultimately form the through-hole structure of figure one；Upper GO is then carried on it, and it is heat-treated, during reduction, due to
The recrystallization of nanometer hydroxyapatite, the graphene in attachment are just embedded in hydroxylapatite ceramic surface.
The present invention wants the preparation method of bone tissue alternate material that following steps can be used：
(1) hydroxyapatite/chitosan slurry is prepared：Hydroxyapatite powder is uniformly dispersed in aqueous solution and (such as may be used
Pass through ultrasonic 1h), acetic acid is then added, Chitosan powder is added while stirring, is stirred for mixing (need to generally stir 2h) and obtains hydroxyl
Base apatite/chitosan slurry；Wherein, the mass ratio of hydroxyapatite and chitosan is：Hydroxyapatite 20-80 parts by weight,
Chitosan 0.5-4 parts by weight；
(2) the melamine sponge of load hydroxyapatite/chitosan slurry is prepared：Melamine sponge is immersed in step (1) to obtain
To hydroxyapatite/chitosan slurry in by squeeze melamine sponge the hole in melamine sponge is filled up by slurry, then
Take out the melamine sponge for be dried in vacuo loading nano-hydroxyapatite/chitosan, wherein true temp is 40-80 DEG C
(preferably 60 DEG C), vacuum degree 60-90KPa, preferably 80-85kPa；
(3) sinter molding：The melamine sponge of the load hydroxyapatite/chitosan for the drying that step (2) is obtained is in sky
It is sintered to obtain Porous Hydroxyapatite Ceramic in gas；
(4) graphene oxide is loaded：The Porous Hydroxyapatite Ceramic that step (3) obtains is immersed in graphene oxide
Alcoholic solution keeps 5~15min (preferably 10min), then takes out drying；
(5) reduction of graphene oxide：The hydroxyapatite porous ceramics for the load graphene oxide that step (4) is obtained
Thermal reduction reaction is carried out in 900~1100 DEG C (preferably 1000 DEG C) in atmosphere of inert gases, obtaining bone tissue after cooling replaces
Conversion materials.
Following embodiment is several typical embodiments, can not play the effect of the limitation present invention, this field
Technical staff is referred to embodiment and is reasonably designed technical solution, can equally obtain the result of the present invention.
Embodiment one
(1) CS/HA slurries are prepared：3gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in slurry
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4), which is immersed in mass concentration, is
(the quality accounting for referring to graphene oxide in graphene oxide/alcoholic solution is the alcoholic solution of the graphene oxide of 0.3wt%
0.3%) drying is taken out in after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
Embodiment two
(1) CS/HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in slurry
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.3% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
The comparative example one of embodiment two
(1) CS/HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in slurry
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.1% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
The comparative example two of embodiment two
(1) CS/HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in slurry
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.6% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
Embodiment three
(1) CS/HA slurries are prepared：8gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
Slurry is sucked in sponge by squeezing melamine sponge in slurry, is in vacuum degree as in 60 degree of vacuum drying ovens after being drawn off
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.3% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
Comparative example one is without graphene
(1) CS/HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in slurry
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall obtains product to room temperature in tube furnace.
Comparative example two is dried
(1) CS/HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, after ultrasonic 1h
200ul acetic acid is added, CS powder 0.3g, which is added, after magnetic agitation 10min is stirred for 2h.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load C S/HA slurries is prepared：The blocky melamine sponge obtained in (2) is immersed in (1)
Slurry is sucked in sponge by squeezing melamine sponge in slurry, as dry (vacuum in 60 degree of vacuum drying ovens after being drawn off
0) degree is.
(4) sinter molding：By the melamine sponge of the load C S/HA of the drying obtained in (3) as in crucible and in air
Under conditions of under be sintered, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then is warming up to 10 DEG C/min
1200 DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.3% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
Comparative example three is without CS
(1) HA slurries are prepared：6gHA nanometer hydroxyapatite powder is scattered in 10ml aqueous solutions, ultrasonic 1h obtains hydroxyl
Base apatite slurry.
(2) preparation of melamine sponge of different shapes：The melamine sponge bought is cut into 0.5cm*0.5cm* with blade
The small cuboid of 1cm.
(3) the melamine sponge of load HA slurries is prepared：The slurry blocky melamine sponge obtained in (2) being immersed in (1)
It is in vacuum degree by squeezing melamine sponge and in slurry sucking sponge, will be drawn off being placed in 60 degree of vacuum drying ovens in material
It is dry under conditions of 80kPa.
(4) sinter molding：By the melamine sponge of the load HA of the drying obtained in (3) as in crucible and in the item of air
It is sintered under under part, temperature program is to be warming up to 330 DEG C with 5 DEG C/min, after keeping 1h, then with 10 DEG C/min is warming up to 1200
DEG C, after keeping 2h, Temperature fall is to room temperature in tube furnace.
(5) introducing of graphene oxide：The hydroxyapatite porous ceramics obtained in (4) is immersed in 0.3% oxidation
Drying is taken out in the alcoholic solution of graphene after 10min.
(6) reduction of graphene oxide：The load obtained in (5) is had to the hydroxyapatite porous ceramics of graphene oxide
It is placed in tube furnace under high pure nitrogen protection and carries out thermal reduction reaction, thermal reduction temperature program is that 10 DEG C/min is warming up to 1000
DEG C, final sample is obtained after furnace cooling.
Microstructure and performance test：
One a of figure and one b of figure is respectively the electron microscope and its partial enlarged view (figure one of two gained porous support materials of embodiment
B), it can be seen that material internal has the formation of many holes, these holes provide for the attachment of Osteogenic Stem, Proliferation, Differentiation
Suitable environment, and due to the presence of through-hole, cell can be grown into material.Finally, in vivo with hydroxyapatite
It decomposes, the growth of osteocyte, area of new bone finally replaces the material.From the partial enlarged view on the right, it is seen that, graphene
It is fitted in hydroxyapatite surface, there is good compatibility, graphene to have the ratio of bigger compared with hydroxyapatite between the two
Surface area, it is easier to the attachment of cell.And studies have reported that graphene has the ability that induced osteogenesis breaks up, so being attached in this way
The porous hydroxyapatite holder of graphene can be better than pure ha porous support to bone tissue reparation effect.
Figure two is the electromicroscopic photograph of the comparative example two of embodiment two, it can be seen that due to aoxidizing stone in the alcoholic solution of immersion
The concentration of black alkene is higher, and graphene oxide layer can block the hole in porous hydroxyapatite holder so that osteoblast without
Method is grown into, so needing graphene additive amount appropriate, has not only ensured that cell can be adhered to preferably, but also can not block in holder
Through-hole structure.
Figure three a, b are respectively the electron microscope of comparative example two and comparative example three, it can be seen that in drying or that CS is not added
Under conditions of cannot form the uniform porous structure such as figure one, the formation mechenism and graphene of one porous support of figure are embedded in
Mechanism on hydroxyapatite porous support is analyzed：The melamine resin of the slurry of HA and CS is adsorbed with vacuum drying
In the process, as solvent volatilizees, there is stomata in internal stent, and HA nano-particles are stained with by chitosan molecule chain contraction to stick together,
Due to there is the relationship of through-hole inside pull of vacuum and melamine resin, the through-hole structure of figure one is ultimately formed；With GO on back loading, and
It is heat-treated；During reduction, due to the recrystallization of nanometer hydroxyapatite, the graphene in attachment is just inlayed
On hydroxylapatite ceramic surface.
To understand the bio-toxicity and cell adherence ability of graphene, pure graphene film is immersed in analogue body by us
In liquid SBF solution, and mesenchymal stem cell is put into SBF liquid and is cultivated, graphene membrane surface cell is viscous after three weeks
Attached situation is as shown in Figure IV, it can be seen that on the surface of graphene, cell can be very good to carry out adherency growth；To illustrate graphite
Alkene does not have bio-toxicity to cell and can promote its Proliferation, Differentiation well.
In order to further appreciate that the differentiation situation of cell, the present invention carry out culture solution alkaline phosphatase (ALP) content
(ALP is the secretory product of osteoblast, and the differentiation feelings of osteocyte are generally judged by ALP contents in culture solution for detection
Condition)；As a result as shown in Figure 5, the osteocyte broken up in no comparative example one for being introduced into graphene is minimum, and skeletonization is added and lures
One material of comparative example of drain and the embodiment two for introducing graphene have relatively high differentiation rate；This just illustrates graphene
Introduce the Osteoblast Differentiation ability for improving Osteogenic Stem really.
Figure six is embodiment one, embodiment two, embodiment three, comparative example two, the porosity and compression mould of comparative example three
Spirogram, it can be seen that with the increase of hydroxyapatite content, compression modulus is being continuously increased, and when 6gHA is added, is increased
Add amplitude maximum, but with the increase of HA, porosity is constantly declining.The sample of embodiment two is before ensureing high porosity
It putting, intensity is also maintained at a higher level, the dry under normal pressure and sample i.e. comparative example two that CS is not added, and three
Then there is no high porosity and intensity, this is because it is drying under normal pressure and that CS is not added in the case of material internal simultaneously
There is no the reason of uniform through-hole formation.
Although describing the present invention above in conjunction with embodiment, it would be clear to those skilled in the art that not departing from power
In the case of the spirit and scope that profit requires, above-described embodiment can be carry out various modifications.
Claims (10)
1. a kind of preparation method of bone tissue alternate material, which is characterized in that the preparation method includes successively：Preparation of nano hydroxyl
It is sintered into base apatite/chitosan slurry, the melamine sponge for preparing load nano-hydroxyapatite/chitosan slurry, air
The reduction that type obtains Porous Hydroxyapatite Ceramic, loads graphene oxide and graphene oxide；Wherein, the preparation loads nanometer
The method of the melamine sponge of hydroxyapatite/chitosan slurry is：Melamine sponge is immersed in nano-hydroxyapatite/chitosan
The hole in melamine sponge is filled up by squeezing melamine sponge by slurry in slurry, then takes out and be dried in vacuo obtaining load
The melamine sponge of nano-hydroxyapatite/chitosan；Wherein, true temp is 40~80 DEG C, and vacuum degree is 60~90KPa.
2. a kind of preparation method of bone tissue alternate material according to claim 1, which is characterized in that the preparation of nano
The method of hydroxyapatite/chitosan slurry is：Nanometer hydroxyapatite powder is uniformly dispersed in aqueous solution, vinegar is added afterwards
Then Chitosan powder is added in acid while stirring, be stirred for mixing and obtain nano-hydroxyapatite/chitosan slurry；Wherein, nanometer
The mass ratio of hydroxyapatite and chitosan is：20~80 parts by weight of nanometer hydroxyapatite, 0.5~4 parts by weight of chitosan；It is excellent
Choosing, the mass ratio of nanometer hydroxyapatite and chitosan is：60~80 parts by weight of nanometer hydroxyapatite, chitosan 0.5~4
Parts by weight.
3. a kind of preparation method of bone tissue alternate material according to claim 1 or 2, which is characterized in that the load
The method of graphene oxide is：By Porous Hydroxyapatite Ceramic be immersed in graphene oxide alcoholic solution keep 5~
15min then takes out the hydroxyapatite porous ceramics for drying to load graphene oxide.
4. according to a kind of preparation method of bone tissue alternate material of claims 1 to 3 any one of them, which is characterized in that institute
In the method for stating load graphene oxide, a concentration of 0.2wt% of graphene oxide in the graphene oxide alcoholic solution~
0.5wt%.
5. a kind of preparation method of bone tissue alternate material according to claim 2, which is characterized in that the preparation hydroxyl
In the method for apatite/chitosan slurry, nanometer hydroxyapatite powder is the 20%~80% of aqueous solution quality, preferably
60%；Acetic acid volume is the 0.5%~3% of aqueous solution volume, preferably 2%；Chitosan mass be aqueous solution quality 0.5%~
4%, preferably 3%.
6. according to a kind of preparation method of bone tissue alternate material of Claims 1 to 5 any one of them, which is characterized in that institute
It is 100um~300um to state melamine sponge pore size, and is through-hole.
7. according to a kind of preparation method of bone tissue alternate material of claim 1~6 any one of them, which is characterized in that institute
Stating sintering forming process is：First be warming up to 330 DEG C with 5 DEG C/min~15 DEG C/min, after keeping 1h, then with 5 DEG C/min~15 DEG C/
Min is warming up to 1200~1350 DEG C, after keeping 2~4h, Temperature fall to room temperature.
8. according to a kind of preparation method of bone tissue alternate material of claim 1~7 any one of them, which is characterized in that institute
The restoring method for stating graphene oxide is：The hydroxyapatite porous ceramics of graphene oxide will be loaded in atmosphere of inert gases
Thermal reduction reaction is carried out in 900~1100 DEG C, and bone tissue alternate material is obtained after cooling.
9. a kind of preparation method of bone tissue alternate material according to claim 8, which is characterized in that the graphite oxide
In the restoring method of alkene, the thermal reduction technique is：300~1000 DEG C first are warming up to 5 DEG C/min~15 DEG C/min, then certainly
So cool to room temperature.
10. a kind of bone tissue alternate material, which is characterized in that the bone tissue alternate material is by any one of claim 1~9 institute
The method stated is prepared.
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| KR20120119891A (en) * | 2012-09-20 | 2012-10-31 | 순천향대학교 산학협력단 | Method of producing artificial bone and artificial bone made thereby |
| CN105536069A (en) * | 2016-01-06 | 2016-05-04 | 四川大学 | Hydroxyapatite-graphene-chitosan tri-crosslinking reduction compound material and preparing method thereof |
| JP2016158680A (en) * | 2015-02-27 | 2016-09-05 | 国立大学法人北海道大学 | Scaffold for osteoanagenesis or for skin regeneration using graphene oxide |
| CN105999421A (en) * | 2016-05-21 | 2016-10-12 | 南昌航空大学 | Method for in-situ preparation of wollastonite porous biological ceramic bone repair materials by adoption of shells |
| CN106075590A (en) * | 2016-07-29 | 2016-11-09 | 福州大学 | A kind of composite of organic/inorganic heterogeneous induction nanometer hydroxyapatite |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120119891A (en) * | 2012-09-20 | 2012-10-31 | 순천향대학교 산학협력단 | Method of producing artificial bone and artificial bone made thereby |
| JP2016158680A (en) * | 2015-02-27 | 2016-09-05 | 国立大学法人北海道大学 | Scaffold for osteoanagenesis or for skin regeneration using graphene oxide |
| CN105536069A (en) * | 2016-01-06 | 2016-05-04 | 四川大学 | Hydroxyapatite-graphene-chitosan tri-crosslinking reduction compound material and preparing method thereof |
| CN105999421A (en) * | 2016-05-21 | 2016-10-12 | 南昌航空大学 | Method for in-situ preparation of wollastonite porous biological ceramic bone repair materials by adoption of shells |
| CN106075590A (en) * | 2016-07-29 | 2016-11-09 | 福州大学 | A kind of composite of organic/inorganic heterogeneous induction nanometer hydroxyapatite |
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