CN109350766A - A kind of preparation method of agarose acetate nano fibrous membrane - Google Patents
A kind of preparation method of agarose acetate nano fibrous membrane Download PDFInfo
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Abstract
The invention belongs to bone material technical fields, more particularly to a kind of preparation method of the agarose acetate nano fibrous membrane of English bone tissue engineering scaffold, it is first that sepharose hydrophobic is modified, agarose acetate is mixed with bioactive substance bata-tricalcium phosphate (β-TCP), AGA/ β-TCP nano fibrous membrane is prepared by method of electrostatic spinning.The present invention solves the disadvantage that single bone renovating material in clinical application, through agarose acetate in conjunction with bioactive substance bata-tricalcium phosphate, it obtains that biocompatibility is excellent, configuration of surface is excellent, wetability and good mechanical stability and the apparent nanofiber membrane support of osteogenic action, improves the application value in terms of Bone Defect Repari.
Description
Technical field
The invention belongs to bone material technical fields, and in particular to a kind of agarose acetic acid of English bone tissue engineering scaffold
The preparation method of ester nano fibrous membrane.
Background technique
In recent years, polysaccharide has degradability outstanding, excellent biocompatibility, and has and promote cell adherence, proliferation
With the optical bio medicine capabilities of regeneration.Many different polysaccharide tissue engineering bracket, such as algae have been used at present
Hydrochlorate, starch-based material, cellulose, glucan and chitosan.
Agarose, a kind of by D- galactolipin and 3, the polysaccharide of 6- dehydration-L- galactopyranose composition comes from red algae cell
It wall and has been widely studied for cultured chondrocytes and regenerating bone or cartilage.Bone is very close with cartilage structure.Agarose these
Feature may be suitble to osteanagenesis.Gel based on agarose is also proved to adhere to osteoblast and be proliferated have good biology
Compatibility.The biologically inert of agarose, nontoxic and low inflammatory reaction play very important effect in these researchs.However,
Great amount of hydroxy group in agarose leads to the strongly hydrophilic of agarose, this leads to poor processability, and the bracket based on agarose
Mechanical weakness and porosity, this is unfavorable to bone defect healing.It therefore, is to widen it in organizational project by modification
The critical issue of middle application.Agarose acetate (AGA) is hydrophobicity agarose derivative, further to improve agarose biology
The machinability of medical application.Agarose acetate is more hydrophobic than agarose, is conducive to protein absorption or cell adherence.Agar
Sweet and sour acid esters is soluble in many organic solvents, and can be manufactured by a variety of forming methods.Such as agarose acetate
Can be with spinning, and its fiber is kept stablizing in water without expanding, and this is conducive to construct porous tissue engineering scaffold.
Bone is the mixed system of hydroxyapatite (HA) and Type I collagen fiber, with complicated and organized porous structure group
Dress.In bone tissue engineer research, calcium phosphate material has been proven that osteoinductive, and is usually added in bracket to change
Kind osteoinductive.It has been found that β-TCP is for having enough biological degradabilities in bone tissue engineer, good biocompatibility and
Osteoinductive.This mechanical performance that will also improve bracket with mixed with polymers inorganic particle.It is made of AGA and β-TCP
The advantages of compound rest can combine them in osteanagenesis.
Various processing technologies have been used for for different polymer (PLA, PCL, chitosan, collagen etc.) being processed into suitable
Bracket is mutually separated with meeting, and is freeze-dried, the basic demands such as self assembly and salt particle leaching.Electrostatic spinning is recognized extensively
To be the promising polymer processing techniques for manufacturing fibrous framework.Since natural extracellular matrix is by fibrous collagen
It is formed with proteoglycan, and collagenous fibres, with 3D porous structure tissue, some other forming processes cannot be imitated ideally carefully
Extracellular matrix architecture.Electrospinning fibre bracket is usually made of the fiber of nanometer diameter, has high surface-to-volume ratio and high hole
Gap rate, therefore it is very suitable to the physical structure of replicating cell epimatrix (ECM).Furthermore, it is possible to pass through incorporation bioactivity chemical combination
Object carrys out functionalization by bracket that electrospinning fibre forms to enhance cell activity.Therefore, electro spinning nano fiber is widely used in human body
Tissue, such as skin, bone, the regeneration of nerve and blood vessel.
Therefore, this field needs to develop that a kind of biocompatibility is excellent, configuration of surface is excellent, wetability and mechanical stability
The good and apparent nanofiber membrane support of osteogenic action, so that the shortcomings that overcoming single bone renovating material in clinical application, mentions
Rise the application value of Bone Defect Repari.
Summary of the invention
For the problems of the prior art, the present invention provides a kind of preparation method of agarose acetate nano fibrous membrane,
It solves the disadvantage that single bone renovating material in clinical application, passes through agarose acetate and bioactive substance bata-tricalcium phosphate
In conjunction with obtaining that biocompatibility is excellent, configuration of surface is excellent, wetability and good mechanical stability and osteogenic action are significantly received
Rice fiber membrane support, improves the application value in terms of Bone Defect Repari.
To realize the above technical purpose, the technical scheme is that
A kind of preparation method of agarose acetate nano fibrous membrane, preparation, electrospinning liquid including agarose acetate
Prepare the preparation with nano fibrous membrane；
The preparation of the agarose acetate includes:
Step 1, magnetic agitation 1-3h in glacial acetic acid is added in agarose, acetic anhydride and sulfuric acid reaction 0.5- is then added
5h obtains agarose acetate suspension；
Step 2, agarose acetate suspension is subjected to repeatedly distillation water washing, until filtered filtrate is in center,
Then the agarose acetate being obtained by filtration is freezed, it is dry；
The preparation of the electrospinning liquid includes:
Step 3, agarose acetate dry in step 2 is added into organic solvent, and 5-10h is stirred at room temperature, obtained
To homogeneous agarose acetic ester solution；
Step 4, bata-tricalcium phosphate powder is added and stirs 10-20h into agarose acetic ester solution, be then placed in 500W
It is ultrasonically treated 10-30min under 40% intensity in Ultrasound Instrument, obtains agarose acetate/bata-tricalcium phosphate composite solution, it is as electric
Spin liquid；
The preparation of the nano fibrous membrane includes:
Step 5, in the plastic injector for electrospinning liquid being passed through 10mL, and connect with syringe pump, then carry out high-pressure electrostatic
Spinning reaction, obtains nanofiber, and be dried under vacuum.
The weight average molecular weight of agarose in the step 1 is 100-400 ten thousand.
Concentration of the agarose in glacial acetic acid in the step 1 is 5-20% (g/mL).
The volume ratio of glacial acetic acid, acetic anhydride and sulfuric acid in the step 1 is 60-100:40-80:1.
The temperature of reaction in the step 1 is 45-55 DEG C.
The concentration of agar vinegar triacetate in the step 3 is 5-10% (g/mL).
Organic solvent in the step 3 is using one of dimethyl acetamide, acetone or methylene chloride.
It is 0-1:1 that bata-tricalcium phosphate powder in the step 4, which is added to the mass ratio of agarose acetate,.
High-voltage electrostatic spinning parameter in the step 5 is as follows: spinning voltage 10-30KV, fltting speed 0.5-
2.0mL/h, receiving distance is 10-30cm.
The present invention is first modified by sepharose hydrophobic, by agarose acetate and bioactive substance bata-tricalcium phosphate (β-
TCP it) mixes, AGA/ β-TCP nano fibrous membrane is prepared by method of electrostatic spinning.The avarage fiber diameter of nano fibrous membrane is with β-
The increase of TCP granule content and increase；After being blended with β-TCP, the hydrophily and mechanical performance of agarose acetate are improved.
Rat bone marrow mesenchymal stem cells (rBMSCs) is cultivated on the nano fibrous membrane based on AGA to contain compared with single AGA film
More rBMSCs are divided into osteoblast-like cells on the nano fibrous membrane of β-TCP, when rBMSCs is in the nanometer containing β-TCP
When cultivating on tunica fibrosa, more alkaline phosphatases (ALP) and mineralized dentin matrix can detecte.Biological evaluation result shows to contain
There is the nano fibrous membrane of β-TCP to promote the proliferation and Osteoblast Differentiation of rat bone marrow mesenchymal stem cells (rBMSCs).In addition, receiving
Rice tunica fibrosa is during surrounding implantation in vivo without apparent inflammatory reaction.Therefore, with biocompatibility based on
The nano fibrous membrane of AGA/ β-TCP potential can be used for bone tissue reparation.
From the above, it can be seen that the present invention has following advantages:
1. the present invention solves the disadvantage that single bone renovating material in clinical application, living by agarose acetate and biology
Property substance bata-tricalcium phosphate combine, obtain biocompatibility is excellent, configuration of surface is excellent, wetability and good mechanical stability and
The apparent nanofiber membrane support of osteogenic action, improves the application value in terms of Bone Defect Repari.
2. the avarage fiber diameter of nano fibrous membrane prepared by the present invention increases with the increase of β-TCP granule content, can
Control property is strong, and controls simple.
3. the agarose acetate that the present invention uses has good biocompatibility, degradable, it is capable of providing continuous
Structure, it is easy to form, it can be achieved that high porosity and high surface area, meet the requirement of Bone Defect Repari.
4. the present invention, as bioactive materials, utilizes the inorganic constituents phase of its ingredient and bone matrix using bata-tricalcium phosphate
Seemingly, the feature good with synosteosis, it is ensured that human body cell can normal growth, differentiation and breeding on bata-tricalcium phosphate material.
Detailed description of the invention
Fig. 1 is the fiber SEM figure of the nano fibrous membrane of the embodiment of the present invention 1, and amplification factor is 5000 times.
Specific embodiment
In conjunction with Fig. 1, the specific embodiment that the present invention will be described in detail, but any limit is not done to claim of the invention
It is fixed.
A kind of preparation method of agarose acetate nano fibrous membrane, preparation, electrospinning liquid including agarose acetate
Prepare the preparation with nano fibrous membrane；
The preparation of the agarose acetate includes:
Step 1, magnetic agitation 1-3h in glacial acetic acid is added in agarose, acetic anhydride and sulfuric acid reaction 0.5- is then added
5h obtains agarose acetate suspension；
Step 2, agarose acetate suspension is subjected to repeatedly distillation water washing, until filtered filtrate is in center,
Then the agarose acetate being obtained by filtration is freezed, it is dry；
The preparation of the electrospinning liquid includes:
Step 3, agarose acetate dry in step 2 is added into organic solvent, and 5-10h is stirred at room temperature, obtained
To homogeneous agarose acetic ester solution；
Step 4, bata-tricalcium phosphate powder is added and stirs 10-20h into agarose acetic ester solution, be then placed in 500W
It is ultrasonically treated 10-30min under 40% intensity in Ultrasound Instrument, obtains agarose acetate/bata-tricalcium phosphate composite solution, it is as electric
Spin liquid；
The preparation of the nano fibrous membrane includes:
Step 5, in the plastic injector for electrospinning liquid being passed through 10mL, and connect with syringe pump, then carry out high-pressure electrostatic
Spinning reaction, obtains nanofiber, and be dried under vacuum.
The weight average molecular weight of agarose in the step 1 is 100-400 ten thousand.
Concentration of the agarose in glacial acetic acid in the step 1 is 5-20% (g/mL).
The volume ratio of glacial acetic acid, acetic anhydride and sulfuric acid in the step 1 is 60-100:40-80:1.
The temperature of reaction in the step 1 is 45-55 DEG C.
The concentration of agar vinegar triacetate in the step 3 is 5-10% (g/mL).
Organic solvent in the step 3 is using one of dimethyl acetamide, acetone or methylene chloride.
It is 0-1:1 that bata-tricalcium phosphate powder in the step 4, which is added to the mass ratio of agarose acetate,.
High-voltage electrostatic spinning parameter in the step 5 is as follows: spinning voltage 10-30KV, fltting speed 0.5-
2.0mL/h, receiving distance is 10-30cm.
Embodiment 1
A kind of preparation method of agarose acetate nano fibrous membrane, includes the following steps:
(1) preparation of agarose acetate:
Dried 3g agarose (Mw=300 ten thousand) is weighed, is put into the flask for filling 30mL glacial acetic acid, magnetic agitation fine jade
Lipolysaccharide is swollen 2h, adds 24mL acetic anhydride and the sulfuric acid of 0.4mL, reacts 2.0h at 50 °C.After reaction, with steaming
Repeatedly, suction filtration is in neutrality to filtrate for distilled water washing, is finally freezed agarose acetate, is drying to obtain product.
(2) preparation of electrospinning liquid:
In order to prepare polymer solution, agarose acetate 6g is dissolved in 100mL dimethyl acetamide and acetone first
Mixed organic solvents in (V:V=1:1), be stirred at room temperature 6h dissolution, obtain agarose acetic acid ester concentration be 6% (g/mL)
Homogeneous phase solution.Then 3g bata-tricalcium phosphate powder is then added in above-mentioned solution and continues to stir 15h, keep powder evenly dispersed
And obtain the solution of stable uniform.Finally mixed liquor is placed under 40% intensity of 500W Ultrasound Instrument and is ultrasonically treated 20min, is prepared
Agarose acetate/bata-tricalcium phosphate composite solution (electrospinning liquid).
(3) preparation of tunica fibrosa:
The solution for preparing composite material is injected into the plastic injector of 10mL, syringe needle is that internal diameter is the stainless of 0.5mm
The flat needle of steel, and be connected on syringe pump.Spinning voltage 20KV, fltting speed 1.0mL/h, receiving distance is 15cm, is covered with aluminium foil
It covers on electrostatic drum collector and collects, obtain thin nanofiber.The nano fibrous membrane of deposition is carefully divided from collector
It separates out and 1h is dried under vacuum.
Through detecting, the scanning SEM figure of the nano fibrous membrane of embodiment preparation is as shown in Figure 1.
Embodiment 2
A kind of preparation method of agarose acetate nano fibrous membrane, includes the following steps:
(1) preparation of agarose acetate:
Dried 6g agarose (Mw=200 ten thousand) is weighed, is put into the flask for filling 40mL glacial acetic acid, magnetic agitation fine jade
Lipolysaccharide is swollen 3h, adds 35mL acetic anhydride and the sulfuric acid of 0.5mL, reacts 2.0h at 50 °C.After reaction, with steaming
Repeatedly, suction filtration is in neutrality to filtrate for distilled water washing, is finally freezed agarose acetate, is drying to obtain product.
(2) preparation of electrospinning liquid:
In order to prepare polymer solution, agarose acetate 8g is dissolved in 100mL dimethyl acetamide and acetone first
Mixed organic solvents in (V:V=1:1), be stirred at room temperature 10h dissolution, obtain agarose acetic acid ester concentration be 8% (g/
ML) homogeneous phase solution.Then 2.4g bata-tricalcium phosphate powder is then added in above-mentioned solution and continues to stir 10h, keep powder equal
Even dispersion and the solution for obtaining stable uniform.Finally mixed liquor is placed under 40% intensity of 500W Ultrasound Instrument and is ultrasonically treated
25min prepares agarose acetate/bata-tricalcium phosphate composite solution (electrospinning liquid).
(3) preparation of tunica fibrosa:
The solution for preparing composite material is injected into the plastic injector of 10mL, syringe needle is that internal diameter is the stainless of 0.5mm
The flat needle of steel, and be connected on syringe pump.Spinning voltage 30KV, fltting speed 1.2mL/h, receiving distance is 15cm, is covered with aluminium foil
It covers on electrostatic drum collector and collects, obtain thin nanofiber.The nano fibrous membrane of deposition is carefully divided from collector
It separates out and 0.5h is dried under vacuum.
Embodiment 3
A kind of preparation method of agarose acetate nano fibrous membrane, includes the following steps:
(1) preparation of agarose acetate:
Dried 8g agarose (Mw=400 ten thousand) is weighed, is put into the flask for filling 40mL glacial acetic acid, magnetic agitation fine jade
Lipolysaccharide is swollen 3h, adds 32mL acetic anhydride and the sulfuric acid of 0.4mL, reacts 5h at a temperature of 55 DEG C.After reaction, with distillation
Water washing is multiple, and suction filtration is in neutrality to filtrate, finally freezes agarose acetate, is drying to obtain product.
(2) preparation of electrospinning liquid:
In order to prepare polymer solution, agarose acetate 10g is dissolved in 100mL acetone first, is stirred at room temperature
10h dissolution is mixed, obtaining agarose acetic acid ester concentration is 10% (g/mL) homogeneous phase solution.Then again by 10g bata-tricalcium phosphate powder
It is added in above-mentioned solution and continues to stir 20h, keep powder evenly dispersed and obtain the solution of stable uniform.Finally by mixed liquor
It places under 40% intensity of 500W Ultrasound Instrument and is ultrasonically treated 30min, prepare agarose acetate/bata-tricalcium phosphate composite solution
(electrospinning liquid).
(3) preparation of tunica fibrosa:
The solution for preparing composite material is injected into the plastic injector of 10mL, syringe needle is that internal diameter is the stainless of 0.5mm
The flat needle of steel, and be connected on syringe pump.Spinning voltage 30KV, fltting speed 2.0mL/h, receiving distance is 30cm, is covered with aluminium foil
It covers on electrostatic drum collector and collects, obtain thin nanofiber.The nano fibrous membrane of deposition is carefully divided from collector
It separates out and 2h is dried under vacuum.
Embodiment 4
A kind of preparation method of agarose acetate nano fibrous membrane, includes the following steps:
(1) preparation of agarose acetate:
Dried 2g agarose (Mw=100 ten thousand) is weighed, is put into the flask for filling 40mL glacial acetic acid, magnetic agitation fine jade
Lipolysaccharide is swollen 1h, adds 26.7mL acetic anhydride and the sulfuric acid of 0.7mL, reacts 0.5h at a temperature of 45 DEG C.After reaction, it uses
It is multiple to distill water washing, suction filtration is in neutrality to filtrate, is finally freezed agarose acetate, is drying to obtain product.
(2) preparation of electrospinning liquid:
In order to prepare polymer solution, agarose acetate 5g is dissolved in 100mL methylene chloride first, at room temperature
5h dissolution is stirred, obtaining agarose acetic acid ester concentration is 5% (g/mL) homogeneous phase solution.Then again by 0.1g bata-tricalcium phosphate powder
It is added in above-mentioned solution and continues to stir 10h, keep powder evenly dispersed and obtain the solution of stable uniform.Finally by mixed liquor
It places under 40% intensity of 500W Ultrasound Instrument and is ultrasonically treated 10min, prepare agarose acetate/bata-tricalcium phosphate composite solution
(electrospinning liquid).
(3) preparation of tunica fibrosa:
The solution for preparing composite material is injected into the plastic injector of 10mL, syringe needle is that internal diameter is the stainless of 0.5mm
The flat needle of steel, and be connected on syringe pump.Spinning voltage 10KV, fltting speed 0.5mL/h, receiving distance is 10cm, is covered with aluminium foil
It covers on electrostatic drum collector and collects, obtain thin nanofiber.The nano fibrous membrane of deposition is carefully divided from collector
It separates out and 0.5h is dried under vacuum.
Performance detection
Comparative example uses commercially available agarose acetate nano fibrous membrane.
In conclusion the invention has the following advantages that
1. the present invention solves the disadvantage that single bone renovating material in clinical application, living by agarose acetate and biology
Property substance bata-tricalcium phosphate combine, obtain biocompatibility is excellent, configuration of surface is excellent, wetability and good mechanical stability and
The apparent nanofiber membrane support of osteogenic action, improves the application value in terms of Bone Defect Repari.
2. the avarage fiber diameter of nano fibrous membrane prepared by the present invention increases with the increase of β-TCP granule content, can
Control property is strong, and controls simple.
3. the agarose acetate that the present invention uses has good biocompatibility, degradable, it is capable of providing continuous
Structure, it is easy to form, it can be achieved that high porosity and high surface area, meet the requirement of Bone Defect Repari.
4. the present invention, as bioactive materials, utilizes the inorganic constituents phase of its ingredient and bone matrix using bata-tricalcium phosphate
Seemingly, the feature good with synosteosis, it is ensured that human body cell can normal growth, differentiation and breeding on bata-tricalcium phosphate material.
It is understood that being merely to illustrate the present invention above with respect to specific descriptions of the invention and being not limited to this
Technical solution described in inventive embodiments.Those skilled in the art should understand that still can be carried out to the present invention
Modification or equivalent replacement, to reach identical technical effect；As long as meet use needs, all protection scope of the present invention it
It is interior.
Claims (9)
1. a kind of preparation method of agarose acetate nano fibrous membrane, it is characterised in that: preparation including agarose acetate,
The preparation of electrospinning liquid and the preparation of nano fibrous membrane；
The preparation of the agarose acetate includes:
Step 1, magnetic agitation 1-3h in glacial acetic acid is added in agarose, acetic anhydride and sulfuric acid reaction 0.5-5h is then added, obtains
To agarose acetate suspension；
Step 2, agarose acetate suspension is subjected to repeatedly distillation water washing, until filtered filtrate is in center, then
The agarose acetate being obtained by filtration is freezed, it is dry；
The preparation of the electrospinning liquid includes:
Step 3, agarose acetate dry in step 2 is added into organic solvent, and 5-10h is stirred at room temperature, obtained
The agarose acetic ester solution of phase；
Step 4, bata-tricalcium phosphate powder is added and stirs 10-20h into agarose acetic ester solution, be then placed in 500W ultrasound
It is ultrasonically treated 10-30min under 40% intensity in instrument, obtains agarose acetate/bata-tricalcium phosphate composite solution, as electrospinning
Liquid；
The preparation of the nano fibrous membrane includes:
Step 5, in the plastic injector for electrospinning liquid being passed through 10mL, and connect with syringe pump, then carry out high-voltage electrostatic spinning
Reaction, obtains nanofiber, and be dried under vacuum.
2. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
The weight average molecular weight of agarose in step 1 is 100-400 ten thousand.
3. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
Concentration of the agarose in glacial acetic acid in step 1 is 5-20% (g/mL).
4. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
The volume ratio of glacial acetic acid, acetic anhydride and sulfuric acid in step 1 is 60-100:40-80:1.
5. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
The temperature of reaction in step 1 is 45-55 DEG C.
6. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
The concentration of agar vinegar triacetate in step 3 is 5-10% (g/mL).
7. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
Organic solvent in step 3 is using one of dimethyl acetamide, acetone or methylene chloride.
8. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
It is 0-1:1 that bata-tricalcium phosphate powder in step 4, which is added to the mass ratio of agarose acetate,.
9. a kind of preparation method of agarose acetate nano fibrous membrane according to claim 1, it is characterised in that: described
High-voltage electrostatic spinning parameter in step 5 is as follows: spinning voltage 10-30KV, fltting speed 0.5-2.0mL/h, receive away from
From for 10-30cm.
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| CN106540329A (en) * | 2016-12-10 | 2017-03-29 | 深圳清华大学研究院 | Nanofiber complex calcium phosphate bone support and preparation method thereof |
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