CN107875452A - Porous compound support frame for bone tissue engineer and preparation method thereof - Google Patents
Porous compound support frame for bone tissue engineer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of porous compound support frame for bone tissue engineer, its preparing raw material includes the component of following parts by weight：100 parts of glucan, 1~7 part of nano micro crystal cellulose and 11~33 parts of sodium trimetaphosphate.Porous compound support frame of the present invention is made by glucan and nano micro crystal cellulose, nano micro crystal cellulose is uniformly distributed in support, so that support good mechanical performance, and the support has good biocompatibility, it would be preferable to support and promote the cartilage differentiation of bone marrow-drived mesenchymal stem；(2) complex stephanoporate bracket has the advantages of porosity high, uniform pore diameter, and can be degradable in time in human body, will not extended residual in human body；(3) method that glucan, nano micro crystal cellulose are crosslinked and are freeze-dried by polysaccharide by the present invention, glucan/nano micro crystal cellulose porous support is formed, preparation technology is simple, material source is extensive, and production efficiency is high, cost is low, can be applied to industrialized production.
Description
Technical field
The present invention relates to technical field of polymer materials, especially a kind of porous compound support frame for bone tissue engineer and
Its preparation method.
Background technology
Bone is the support of human body, and protection and supporting function are played to human body, and plays leverage during exercise.Periosteum, bone
Matter and marrow are the three parts for forming bone.But because cartilage does not have the characteristic of blood vessel, so it is difficult to having the energy that itself heals
Power, but Cranial defect is common disease clinically, according to statistics, Cranial defect is sick caused by disease, wound, tumour etc. every year in China
For people more than 1,000,000, the reparation of a wide range of Cranial defect is still at present one of problem of orthopaedics therapy, at present, treats the side of Cranial defect
Method has bone collection, artificial bone graft's alternative materials and tissue engineering technique etc., in addition, being lacked using bone tissue engineer technology treatment bone
Damage can overcome the shortcomings of autologous bone transplanting donor deficiency and allogenic bone transplantation immunological rejection.Therefore, bone tissue engineer is utilized
To carry out the focus that Bone Defect Repari is treatment Cranial defect.
Glucan (Dextran, Dex), also known as dextran.For a kind of polysaccharide.It is present in certain micro-organisms growing
In the mucus secreted in journey.Glucan has higher molecular weight, mainly by D- glucopyanosyls with α, 1 → 6 key connection, side chain
Point has 1 → 2,1 → 3,1 → 4 connection.With the difference of microbe species and growth conditions, its structure also has difference.Except this it
There is beta glucan outside, there is immune activation function, protect body health, while in the research of many bone tissue engineers regeneration,
Glucan is frequently as a kind of pharmaceutical carrier, because it has good biocompatibility, can stimulate the growth of bone matrix, but due to
Its poor mechanical performance, so limiting again the application of bone tissue engineer.
Nano micro crystal cellulose (Nanocrystal Cellulose, CNCs), is by native cellulose or microcrystalline cellulose
By chemistry, the method for physics or biochemistry is prepared, and nano micro crystal cellulose not only has the relevant nature of nanoparticle
Such as：Larger ratio surface, high intensity and high-crystallinity；Therefore there is very strong mechanical performance, there is obvious enhancing effect,
Taste and be applied to biological field as a kind of reinforcing agent, be in addition also equipped with the basic structure and characteristic of cellulose, such as：
The features such as non-toxic, biocompatibility and degradability；In addition, the role that nano-cellulose typically serves as among compound is
Reinforced filling, to improve the heat endurance of compound and mechanical performance.Moreover, contain abundant hydroxyl in nano micro crystal cellulose
Base, add in compound, the hydrophilicity of compound can be improved.
In recent years, although the poor mechanical performance of glucan limits its application in bone tissue engineer, due to it
Unique biological property, it is always the problem of people are of interest to study his potential value, but nano-cellulose is normal in the recent period
A kind of apparent reinforcing agent is often studied as, improves the hydrophily of matrix, heat endurance and mechanical performance, but due to tool
There is poor degradation property, so individually nanofiber also limit the application of its own.
The content of the invention
Based on this, it is excellent to provide a kind of mechanical property it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art
Porous compound support frame good, that there is good biocompatibility, can be degraded in time in human body.
To achieve the above object, the technical scheme taken of the present invention is：A kind of porous composite support for bone tissue engineer
Frame, the preparing raw material of the porous compound support frame include the component of following parts by weight：100 parts of glucan, 1~7 part of nanometer
Microcrystalline cellulose and 11~33 parts of sodium trimetaphosphate.It should be noted that when in compound rest nano micro crystal cellulose contain
Measure for 3~7% when, the compressive property of compound rest is preferable, and cytotoxicity is smaller, and biocompatibility is preferable, and degradability is higher；
When the content of nano micro crystal cellulose in compound rest is 5%, the cytotoxicity of compound rest is minimum, and biocompatibility is most
It is good, while crushing resistance and degradability are preferable.
Preferably, in the component of the preparing raw material, the mass ratio of the glucan and nano micro crystal cellulose is 1：
0.03~0.07.It is highly preferred that the mass ratio of the glucan and nano micro crystal cellulose is 1：0.05.When glucan and nanometer
The mass ratio of microcrystalline cellulose is 1：When 0.05, the good mechanical properties of support, one-tenth porosity is high, and the size in hole approaches, meanwhile, branch
The consistency and elasticity modulus of frame is suitable.
Preferably, the porosity of the porous compound support frame is 91~96%, and aperture is 80~223 μm.
Preferably, the nano micro crystal cellulose is club shaped structure, and the length of the club shaped structure is 140~360nm, wide
Spend for 20~40nm.
As another aspect of the present invention, present invention also offers the preparation method of above-mentioned porous compound support frame, including
Following steps：
(1) glucan is dissolved in deionized water and dissolved by heating；
(2) nano micro crystal cellulose is added in dextran solution obtained by step (1) and be well mixed, successively add aqueous slkali
With sodium trimetaphosphate solution, quick freeze after reacting completely, then freezed at -80 DEG C, obtain described porous compound support frame.
It should be noted that the cross-linking process for preparing support betides supercooling link, quick freeze can terminate the ring opening process of crosslinking agent,
It is lyophilized to can guarantee that support obtains more homogeneous pore.
Preferably, heating-up temperature is 40~60 DEG C in the step (1).
Preferably, the temperature of quick freeze is -80 DEG C in the step (2).
Preferably, the preparation method of the nano micro crystal cellulose comprises the following steps：Cellulose is added into mass concentration
In 35~70% concentrated sulfuric acid, the magnetic agitation under 30~65 DEG C of waters bath with thermostatic control, to react 1~6h, then adding deionized water
With terminating reaction, through ultrasonic disperse after cooling, dialysed afterwards using deionized water stable to pH, after -80 DEG C of freeze-dryings i.e.
Obtain described nano micro crystal cellulose.By nano micro crystal cellulose made from the above method into bar-shaped, size is in nanoscale
In other scope, while surface carries sulfonic group, in negative electricity effect, can prevent the aggregation between nano micro crystal cellulose, easily
In being scattered in good solvent.
Preferably, the cellulose is plant cellulose.
In summary, beneficial effects of the present invention are：
(1) porous compound support frame of the present invention is made by glucan and nano micro crystal cellulose, and nano micro crystal cellulose is uniform
It is distributed in support so that support good mechanical performance, and also the support has good biocompatibility, it would be preferable to support and promote
Enter the cartilage differentiation of bone marrow-drived mesenchymal stem；
(2) complex stephanoporate bracket has the advantages of porosity high (91~96%), uniform pore diameter (165-223um), and
And can in human body in time it is degradable, will not extended residual in human body；
(3) method that glucan, nano micro crystal cellulose are crosslinked and are freeze-dried by polysaccharide by the present invention, forms Portugal
Glycan/nano micro crystal cellulose porous support, preparation technology is simple, material source is extensive, and production efficiency is high, and cost is low, can answer
For industrialized production.
Brief description of the drawings
Fig. 1 is the transmission electron microscope figure of nano micro crystal cellulose；
A, B, C, D are that CNCs weight/mass percentage composition is 1%, 3%, 5%, 7% porous compound support frame respectively in Fig. 2
200 times of scanning electron microscope (SEM) photographs；
Fig. 3 is 500 times of scanning electron microscope (SEM) photographs that CNCs weight/mass percentage composition is 5% porous compound support frame；
Fig. 4 is the modulus of compressibility figure of different porous compound support frames；
Fig. 5 is the light absorption value of the cell cultivated on porous compound support frame in 450nm；
Fig. 6 is scanning electron microscope (SEM) photograph of the cell when being grown 3 days on porous compound support frame material.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
Embodiment 1
A kind of embodiment of the porous compound support frame for bone tissue engineer of the present invention, its preparing raw material include following heavy
Measure the component of part：100 parts of glucan, 7 parts of nano micro crystal cellulose and 11 parts of sodium trimetaphosphate.Porous compound support frame
Porosity is 91%, and aperture is 80~223 μm.Wherein, nano micro crystal cellulose is club shaped structure, the length of the club shaped structure
For 140~360nm, width is 20~40nm.
The preparation method of above-mentioned porous compound support frame includes step in detail below：
Weigh 5g cotton celluloses to be added in the concentrated sulfuric acid of 42ml 70% (mass concentration), in 30 DEG C of water bath with thermostatic control
In, mechanical agitation, react 6 hours, add a large amount of distilled water terminating reactions, after deionized water dialysis being added after ultrasonic disperse 3 days,
The freeze overnight at -20 DEG C, is then placed in freeze dryer, and -80 DEG C are made nano micro crystal cellulose in lyophilized 48 hours；
2.7g glucans are dissolved in 27ml deionized waters again, 40 DEG C of heating water bath magnetic agitation dissolvings is placed in, claims 189mg
Nano micro crystal cellulose is added to mixing in dextran solution and stirred evenly, and 1ml 10mol/L NaOH solution is added dropwise, adds 1ml
0.3g/ml sodium trimetaphosphate solution, reaction are immediately placed at -80 DEG C after 20 minutes and freezed, and are freezed at subsequent -80 DEG C, 7% receives
Glucan/nano micro crystal cellulose porous compound support frame of rice microcrystalline cellulose enhancing.
Embodiment 2
A kind of embodiment of the porous compound support frame for bone tissue engineer of the present invention, its preparing raw material include following heavy
Measure the component of part：100 parts of glucan, 5 parts of nano micro crystal cellulose and 22 parts of sodium trimetaphosphate.Porous compound support frame
Porosity is 93%, and aperture is 80~223 μm.Wherein, nano micro crystal cellulose is club shaped structure, the length of the club shaped structure
For 140~360nm, width is 20~40nm.
The preparation method of above-mentioned porous compound support frame includes step in detail below：
Weigh 5g cotton celluloses to be added in the concentrated sulfuric acid of 45ml 65% (mass concentration), in 42 DEG C of water bath with thermostatic control
In, mechanical agitation, react 3 hours, add a large amount of distilled water terminating reactions, after deionized water dialysis being added after ultrasonic disperse 3 days,
The freeze overnight at -20 DEG C, is then placed in freeze dryer, and -80 DEG C are made nano micro crystal cellulose in lyophilized 48 hours；
2.7g glucans are dissolved in 27ml deionized waters again, 50 DEG C of heating water bath magnetic agitation dissolvings is placed in, claims 135mg
Nano micro crystal cellulose is added to mixing in dextran solution and stirred evenly, and 1ml 10mol/L NaOH solution is added dropwise, adds 2ml
0.3g/ml sodium trimetaphosphate solution, reaction are immediately placed at -80 DEG C after 20 minutes and freezed, and freeze, obtain at subsequent -80 DEG C
Glucan/nano micro crystal cellulose porous compound support frame of wt5% nano micro crystal celluloses enhancing.
Embodiment 3
A kind of embodiment of the porous compound support frame for bone tissue engineer of the present invention, its preparing raw material include following heavy
Measure the component of part：100 parts of glucan, 3 parts of nano micro crystal cellulose and 33 parts of sodium trimetaphosphate.Porous compound support frame
Porosity is 95%, and aperture is 80~223 μm.Wherein, nano micro crystal cellulose is club shaped structure, the length of the club shaped structure
For 140~360nm, width is 20~40nm.
The preparation method of above-mentioned porous compound support frame includes step in detail below：
The preparation process of glucan/nano micro crystal cellulose porous compound support frame of 3% nano micro crystal cellulose enhancing：Claim
5g cotton celluloses are taken to be added in the concentrated sulfuric acid of 53ml 55% (mass concentration), in 50 DEG C of water bath with thermostatic control, machinery stirs
Mix, react 5 hours, a large amount of distilled water terminating reactions are added, after deionized water dialysis being added after ultrasonic disperse 3 days, at -20 DEG C
Freeze overnight, it is then placed in freeze dryer, -80 DEG C are made nano micro crystal cellulose in lyophilized 48 hours；
2.7g glucans are dissolved in 27ml deionized waters again, 50 DEG C of heating water bath magnetic agitation dissolvings is placed in, claims 81mg
Nano micro crystal cellulose is added to mixing in dextran solution and stirred evenly, and 1ml 10mol/L NaOH solution is added dropwise, adds 3ml
0.3g/ml sodium trimetaphosphate solution, reaction are immediately placed at -80 DEG C after 20 minutes and freezed, and freeze, obtain at subsequent -80 DEG C
Glucan/nano micro crystal cellulose porous compound support frame of 3% nano micro crystal cellulose enhancing.
Embodiment 4
A kind of embodiment of the porous compound support frame for bone tissue engineer of the present invention, its preparing raw material include following heavy
Measure the component of part：100 parts of glucan, 1 part of nano micro crystal cellulose and 11 parts of sodium trimetaphosphate.Porous compound support frame
Porosity is 96%, and aperture is 80~223 μm.Wherein, nano micro crystal cellulose is club shaped structure, the length of the club shaped structure
For 140~360nm, width is 20~40nm.
The preparation method of above-mentioned porous compound support frame includes step in detail below：
The preparation process of glucan/nano micro crystal cellulose porous compound support frame of 1% nano micro crystal cellulose enhancing：Claim
5g cotton celluloses are taken to be added in the concentrated sulfuric acid of 84ml35% (mass concentration), in 65 DEG C of water bath with thermostatic control, mechanical agitation,
Reaction 1 hour, a large amount of distilled water terminating reactions are added, it is cold at -20 DEG C after deionized water dialysis being added after ultrasonic disperse 3 days
Freeze overnight, be then placed in freeze dryer, -80 DEG C are made nano micro crystal cellulose in lyophilized 48 hours；
2.7g glucans are dissolved in 27ml deionized waters again, 60 DEG C of heating water bath magnetic agitation dissolvings is placed in, claims 27mg
Nano micro crystal cellulose is added to mixing in dextran solution and stirred evenly, and 1ml 10mol/L NaOH solution is added dropwise, adds 1ml
0.3g/ml sodium trimetaphosphate solution, reaction are immediately placed at -80 DEG C after 20 minutes and freezed, and freeze, obtain at subsequent -80 DEG C
Glucan/nano micro crystal cellulose porous compound support frame of 1% nano micro crystal cellulose enhancing.
The performance test of the porous compound support frame of the present invention of embodiment 5
(1) the compressive property test of porous compound support frame of the invention
Test object：The wt1% of embodiment 1~4,3%, 5%, glucan/nano micro crystal cellulose of 7%CNCs enhancings
Support；
Method of testing：Certain surface accumulates dry state support at room temperature under pressure test instrument Instron 4505 with 2mm/
Min pushes the compressive property parameter that speed obtains different samples；
Test result：
As shown in figure 4, prepare 1% respectively by the method in above-described embodiment, 3%, 5%, the Portugal of 7%CNCs enhancings
Glycan/nano micro crystal cellulose support, can significantly find out from figure, with CNCs content increase, the resistance to compression mould of support
Amount enhancing, illustrates that CNCs significantly improves the shortcomings that glucan mechanical property is weak.
(2) absorbance of porous compound support frame of the invention
Test object：The wt1% of embodiment 1~4,3%, 5%, glucan/nano micro crystal cellulose of 7%CNCs enhancings
Support；
Method of testing：After porous support carries out ultraviolet sterilization, it is put into 96 well culture plates.With 0.25% pancreatin/PBS solution
It is from culture plate digestion, centrifugal condition by ATDC5 cells：5min, 1500rpm, after removing supernatant, concentration is added as 10%
Fresh α-MEM the culture mediums of hyclone, 2.5 × 10 are adjusted to by concentration of cell suspension6/mL.Then by 10 μ L cell suspensions
Add in porous support, after hatching 2h, 100 μ L culture mediums are added per hole, are cultivated in 37 DEG C of incubators to required time.Every other day
Liquid is changed, to keep the nutrition supply of cell.
In 96 orifice plates, 100 μ L MTT solution are added per hole, is placed in 37oC incubators and continues to be incubated 4h.Taken with tweezers
Go out above resulting compound rest to be put into 2mL centrifuge tubes, add 1mL DMSO, blow and beat to crystal and be completely dissolved.Draw
200 μ L are added in ELISA Plate, and 570nm absorbance is determined on ELIASA (Bio-Rad 550).
Test result：
In cytotoxicity experiment, as shown in figure 5, the growth rate of cell can be reflected by the height of absorbance, 1%,
3%th, 5%, 7%CNCs compound rest all has preferable cell compatibility, but cell increases in the support of 5%CNCs contents
Grow that speed is more obvious, show that too high CNCs contents (7%CNCs) are unsuitable for the growth of cell.
(3) biocompatibility of porous compound support frame of the invention
Test object：The wt1% of embodiment 1~4,3%, 5%, glucan/nano micro crystal cellulose of 7%CNCs enhancings
Support；
Method of testing：It is planted in after above-mentioned ATDC5 passages in different component CNCs support, 37 DEG C.5%CO2Ring
Cultivated 3 days in the incubator of border, after rinsed 3 times with PBS, then with 2.5% glutaraldehyde fixer fix 2h, then use successively
30%, 50%, 70%, 80%, 90%and 100% ethanol dehydration are handled, 10 minutes every time, after supercritical CO again2Dry, use
SEM is observed.
Test result：
As shown in fig. 6, clearly it can be seen that growth of the cell on support, adhesion, propagation, Yi Jixi under ESEM
Interaction between born of the same parents, cell survival is less in figure A (support of 1%CNCs contents), and possible mechanical strength is insufficient, culture medium
Original shape can not be kept long afterwards by contacting, and porosity declines, and permeability reduces, thin in figure D (support of 7%CNCs contents)
Born of the same parents retain less, and CNCs nano-particles that may be higher both reduce one-tenth porosity, expand CNCs toxicity cooperative effect resistance again
Hindered the propagation of cell, in figure B (support of 3%CNCs contents), C (support of 5%CNCs contents) cell of cell survival compared with
More, C becomes apparent, and the result of this reaction is consistent with Fig. 5.
(4) degradation property of porous compound support frame of the invention
Test object：The wt1% of embodiment 1~4,3%, 5%, glucan/nano micro crystal cellulose of 7%CNCs enhancings
Compound rest；
Method of testing：The compound rest for weighing certain mass is placed in (sample in the buffer solution with trypsase：PBS：Pancreas
Enzyme=10mg:2ml:100 μ L, pH=7.4), 37 degrees Celsius of lower dynamic cultivations 1,3,5,7,10,14,21 days, every other day half measure
More renew liquid, every group is done 3 parallel samples.
Test result：Several supports generally can slowly degrade, but with the increase (such as 7%) of CNCs contents, it is residual
Stay support quality more, so CNCs amount needs to control within the scope of one (such as 1~7%), can preferably reduce its band
The negative effect come.
By summary embodiment and comparative example analysis shows, wt5% concentration Cs NCs compound rest shows
Go out optimal biocompatibility, its mechanical performance is also preferable, and can be degraded in time in human body, will not extended residual patient
In vivo, it is suitable as sclerotin timbering material.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (10)
- A kind of 1. porous compound support frame for bone tissue engineer, it is characterised in that the preparing raw material of the porous compound support frame Include the component of following parts by weight：100 parts of glucan, 1~7 part of nano micro crystal cellulose and 11~33 parts of three metaphosphoric acids Sodium.
- 2. porous compound support frame according to claim 1, it is characterised in that in the component of the preparing raw material, the Portugal The mass ratio of glycan and nano micro crystal cellulose is 1：0.03~0.07.
- 3. porous compound support frame according to claim 2, it is characterised in that the glucan and nano micro crystal cellulose Mass ratio is 1：0.05.
- 4. porous compound support frame according to claim 1, it is characterised in that the porosity of the porous compound support frame is 91 ~96%, aperture is 80~223 μm.
- 5. porous compound support frame according to claim 1, it is characterised in that the nano micro crystal cellulose is bar-shaped knot Structure, the length of the club shaped structure is 140~360nm, and width is 20~40nm.
- 6. the preparation method of the porous compound support frame as described in any one of Claims 1 to 5, it is characterised in that including following step Suddenly：(1) glucan is dissolved in deionized water and dissolved by heating；(2) nano micro crystal cellulose is added in dextran solution obtained by step (1) and be well mixed, successively add aqueous slkali and three Sodium metaphosphate solution, quick freeze after reacting completely, then freezed at -80 DEG C, obtain described porous compound support frame.
- 7. preparation method according to claim 6, it is characterised in that heating-up temperature is 40~60 DEG C in the step (1).
- 8. preparation method according to claim 6, it is characterised in that the temperature of quick freeze is -80 in the step (2) ℃。
- 9. preparation method according to claim 6, it is characterised in that the preparation method of the nano micro crystal cellulose includes Following steps：Cellulose is added in the concentrated sulfuric acid that mass concentration is 35~70%, magnetic force stirs under 30~65 DEG C of waters bath with thermostatic control Mix, react 1~6h, then add deionized water with terminating reaction, through ultrasonic disperse after cooling, dialysed afterwards using deionized water It is stable to pH, described nano micro crystal cellulose is obtained after -80 DEG C of freeze-dryings.
- 10. preparation method according to claim 9, it is characterised in that the cellulose is plant cellulose.
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