CN100396340C - Composite nanometer hydroxy apatitel medical polymer material tissue engineering stent material and preparation method - Google Patents
Composite nanometer hydroxy apatitel medical polymer material tissue engineering stent material and preparation method Download PDFInfo
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- CN100396340C CN100396340C CNB200610021763XA CN200610021763A CN100396340C CN 100396340 C CN100396340 C CN 100396340C CN B200610021763X A CNB200610021763X A CN B200610021763XA CN 200610021763 A CN200610021763 A CN 200610021763A CN 100396340 C CN100396340 C CN 100396340C
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Abstract
A tissue-engineered scaffold material for medical purpose is composed of nano-class hydroxy apatite and the medical high-molecular material, and has the pores communicated with each other. It features that the dispersed microporous microballs are proportionally contained by it. Its preparing process includes such steps as proportionally mixing said microballs, nano-class hydroxy apatite, medical high-molecular slurry and the salt able to thermally generate gas, heating, and freeze-drying at -20 deg.C for at least 24 hr.
Description
Technical field
What the present invention relates to is a kind of compound nanometer hydroxy apatitel medical polymer material tissue engineering timbering material and preparation method thereof.
Background technology
Tissue engineering is application cell biology and engineering principle, with the biological substitution thing is purpose, studying, develop, repair and safeguard or improve a science of tissue and part organ dysfunction and form, is to be used to repair and the dysfunction of treated tissue or organ or a kind of important measures and the means of forfeiture.
About the nanometer hydroxyapatite/polymer composite that is prepared from by nanometer hydroxyapatite and various medical macromolecular materials is the organizational project repair materials and/or its preparation method on basis, and much more report is arranged at present.As, publication number is the porous bone prosthesis that the patent documentation of CN1460526A discloses a kind of polyamide-based composition of hydroxy apatitel medical by 1/1～1/0.3 composition form.Notification number is respectively CN1200043C, the Chinese patent literature of CN1225290C and CN1230210C discloses a kind of by weight ratio to be the composite bio-active porous material of 0.25/1～1.5/1 nanometer hydroxy apatitel medical polyamide 66 form respectively, a kind of is 0.25/1～1.5/1 nanometer hydroxy apatitel medical polyamide composition composite organization engineering stent material by using with mol ratio, with a kind of be the preparation method of 8/2～2/8 polyamide/nano hydroxyapatite series of biologic medical composite material by weight ratio.The Chinese patent of notification number CN1238062C discloses 5%～60% the apatitel medical polymer multi-element biologic composite that a kind of nano-apatite accounts for the composite gross weight.The content that has proposed a kind of apatite in the document of publication number CN1765423A is the preparation method of 0.1～9 times the nano-apatite/medical high polymer composition biology active porous stent material of macromolecular material weight.
The key that tissue engineering technique successfully uses is the selection and the preparation of porous timbering material.Ideal tissue engineering bracket removes should be possessed as excellent biological compatibility and good three-dimensional, the loose structure that network connects, porosity generally should and have suitable pore size between 75%～95%, and have certain biomechanical property and be easy to outside the molding, also proposed to carry requirement as osteoinductive protein, cell growth factor and antibiotic isoreactivity material and related drugs composition.
At present the preparation porosity reaches the method for the highly porous timbering material more than 90%, can have mainly that fiber is bonding, solution-cast/particle leaching, melt molding, gas foaming, be separated/emulsifying, lyophilization, thermic gelation etc.Wherein common and noticeable with particle leaching method, freeze-drying, gas foaming technology, wherein the gas foaming technology adopts gas as porogen, the porosity of gained support can be up to 93%, the advantage that bore dia can reach 100 these technology of μ m. is not need the filtering process, has also avoided with an organic solvent.Itself and phase transfer method coupling can be guaranteed into porosity can guarantee connection between the hole again.
The method of the medicine carrying or the relative growth factor in timbering material is that these active component are directly put into timbering material at present mostly.After in the implanted body of these materials,, can cause the prominent of medicine to release, be unfavorable for the tissue growth needs because a large amount of medicine isoreactivity compositions contacts body fluid simultaneously.For some medicine isoreactivity composition then is to tell on by its long-term release.Therefore preparation has excellent drug isoreactivity slowly released component effect, and the emphasis that pore-forming is good, voidage is high, connective good organization bracket has become research.
On the other hand, microsphere has obtained generally acknowledging as a kind of slow releasing carrier of medication at present.The method of making microsphere at present has emulsion process, film emulsion process, microchannel emulsion process, spray drying method etc.Be written into medicine or other active component in microsphere, can have tangible slow release effect, the drug level of both can having remained valid can keep secular pharmaceutical release time again, can effectively lower because medicine is dashed forward simultaneously and release the malicious attached effect that brings.Brought Gospel for the patient of long-term prescription, sustained-release micro-spheres also is major fields of drug release studies at present.
Summary of the invention
At above-mentioned situation, the present invention's purpose at first provides and a kind ofly is loaded with the composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material of microsphere composition at the nanometer hydroxy apatitel medical polymer material tissue engineering timbering material, to satisfy at present to tissue engineering bracket material augmented performance requirement day by day.On this basis, another object of the present invention provides the preparation method of the composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material of this year microsphere composition.
Composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material of the present invention, constitute framing structure by nanometer hydroxyapatite composition and medical macromolecular materials composition with mutual perforation hole, in the framing structure material, be scattered with the microsphere composition of band microcellular structure, the weight ratio of microsphere and nanometer hydroxy apatitel medical polymer material composition＜2: 9 wherein, and the amount of microsphere composition is not 0.
Wherein, saidly constitute nanometer hydroxy apatitel medical polymer material framing structure by nanometer hydroxyapatite composition and medical macromolecular materials composition with mutual perforation hole, can be the nanometer hydroxy apatitel medical polymer material of currently reported various corresponding forms such as above-mentioned document, preparation method, and by its tissue engineering bracket material of making, nanometer hydroxyapatite and multiple medical polyamide-based composition composite as the existing different proportion composition form of reporting, the composite material of nanometer hydroxyapatite and chitosan form, and the respective holder structure that further is prepared into.
The method of making microsphere at present has emulsion process, film emulsion process etc.Chang Yong emulsion process for example, it is the inconsistent principle of utilization profit, in oil phase, add surfactant (as Tween 80, sorbester p37, Polyethylene Glycol, sodium stearate or the like), (as being generally～4000 rev/mins of 200 commentaries on classics) adds the aqueous solution that is prepared into by the water soluble polymer composition under the condition of certain mixing speed, water-soluble polymer solution is dispersed in uniformly forms microemulsion in the oil phase, drop is glomeration under surface tension condition.Can form gel after microemulsion is stable, gel and washing after the collection fixed in shape can obtain microsphere.Energy commonly used polymer substance water-soluble and the formation thick liquid has: polyvinyl alcohol water solution, chitosan aqueous solution, polyvinyl alcohol dimethyl sulfoxide solution, sodium alginate aqueous solution, aqueous gelatin solution etc.Water soluble polymer has thickening power, its mechanism and hydrogen bond, and hydrophobic interaction and electrostatic interaction are relevant, and the ion-type water soluble polymer generally has stronger thickening capabilities than non-ionic water-soluble macromolecule.Other also has solvent evaporated method, spray drying method, and film emulsion processes etc. also can prepare microsphere.The said microsphere of the present invention with adopt that the emulsion process mode prepares for good.Wherein, under the stirring of～4000 rev/mins of 200 commentaries on classics, the macromolecule aqueous solution disperses to form microemulsion in oil phase.Wherein the big I of mixing speed influences the size that emulsion droplet forms in the emulsion, and mixing speed is big more, and the microsphere particle of preparing is more little, therefore by changing and the control mixing speed, can adjust and control the microsphere composition that obtains required suitable size.
In above-mentioned tissue engineering bracket material of the present invention, said microsphere composition is currently reported by the different preparation methods microsphere that obtain and/or multi-form except that using, preferably adopt emulsion process/inner gel method by chitosan and two kinds of compositions of sodium alginate, prepare that to become microsphere and chitosan with sodium alginate be the chitosan/sodium alginate complex microsphere of coating.Its concrete preparation method can be that to become microsphere and chitosan be the chitosan/sodium alginate complex microsphere of coating for the sodium alginate of gel preparation with the calcium ion at EuropeanJournal of Pharmaceutical Sciences report referring to Antonio J.Ribeiro etc.The complex microsphere that this is formed by two kinds of compositions, not only can overcome need introduce when compositions such as present single chitosan prepare microsphere to the disadvantageous organic compound of health, and chitosan can also form poly-electric matter with sodium alginate, helps improving the microsphere mechanical property.For example, the chitosan microball with the glutaraldehyde as cross linker preparation of existing wide coverage though glutaraldehyde cross-linking effect wherein is good, because of carcinogenecity is arranged, can cause harmful effect to organism; With sodium sulfate, sodium citrate, polyphosphoric acids etc. is chitosan crosslinked dose, has limited its application again owing to its cross-linking effect is not good.And be the microsphere of raw material with the sodium alginate, it can form the bigger gel of intensity with different kinds of ions such as calcium ion existing reported in literature, and therefore in the sodium alginate emulsion behind these gel components of adding, sodium alginate micro ball can be fixed.And used calcium ion, zinc ion, the agent of magnesium ion isogel then has no adverse effect to human body is several.Add chitosan on this basis again, form poly-electric matter and improve mechanical strength each other with sodium alginate by its interaction, thereby improved the mechanical strength of microsphere.
Above-mentioned said chitosan/sodium alginate complex microsphere, special recommendation is used is to prepare the microsphere that become by following mode: be that 1%～2.5% sodium alginate aqueous solution is under 200～4000 rev/mins mixing speed with by weight/volume, dropwise join and be mixed with the water-insoluble organic facies liquid that volume content is 1%～5% surface active ingredient, as liquid paraffin, Oleum Terebinthinae, phenol, vegetable oil, Oleum Glycines, chloroform, methane, dichloromethane, monochloro methane, petroleum ether etc. and the immiscible organic facies solvent of water, abundant mix homogeneously, sodium alginate is added behind balling-up and the homodisperse in oil phase as calcium ion, zinc ion, the agent of magnesium ion isogel, mix homogeneously.To use the calcium carbonate gel to be example, this moment can be by adding acetic acid in emulsion, divided calcium carbonate is separated discharged calcium ion, and emit carbon dioxide simultaneously, calcium ion can make sodium alginate form gel, sodium alginate micro ball is fixed and had certain intensity, the carbon dioxide of emitting then can form micropore simultaneously in microsphere.Then, to be mixed with by weight/volume by the chitosan of said proportional quantities and be 2%～3% under fully stirring, also to be mixed with volume content be that the chitosan aqueous solution of 1%～5% surface active ingredient adds wherein, after stirring abundant reaction (for example generally can react at least 1 hour) down, obtain said chitosan/sodium alginate complex microsphere after filtration washing and the lyophilization.
Surfactant is to select low toxicity or nontoxic being advisable for use, as the type emulsifying agent of carboxylate, sulfate and sulfonate form such as soap, stearic acid sodium salt, sodium lauryl sulphate salt and calcium salt of dodecylbenzene sulfonate commonly used; Cation type emulsifying agents such as N-domiphen; Paraoctyl phenol polyoxyethylene ether etc. are commonly used polyethenoxy ether class and polyoxypropylene ethers nonionic emulsifier etc.The consumption of surfactant generally can be 1%～10%, does not reach ideal emulsifying effectiveness when very few, crosses unnecessary at most.
In case of necessity, can also use the gel of appropriate format in the usual way, more help the formation of gel.For example in above-mentioned sodium alginate microemulsion, can also add calcium ion in advance, or as other gels such as zinc ion, copper ion, magnesium ions, itself and sodium alginate are fully reacted after, two kinds of microemulsions are mixed obtain complex microsphere.
Microsphere has been proved to be and can have had excellent drug load and sustained release performance, can be as the excellent drug carrier.Therefore; in tissue engineering bracket material, be written into microsphere; the microsphere that in material, scatters particularly with homogeneous form roughly; can be medicine or other active component; as bone morphogenetic protein (BMPs) commonly used; transforming growth factor-beta (TGF-β); insulin like growth factor (IGF); fibroblast growth factor (FGFs); basic fibroblast growth factor (bFGF); vascular endothelial cell growth factor (VRGF); platelet-derived growth factor (PDGF); neoplasm necrosis somatomedin tissue growth factor compositions such as (TNF); and/or penicillin; gentamycin; tobramycin, vancomycin, antibiotic compositions such as berberine; and the Radix Astragali; astragaloside, Radix Notoginseng, Radix Notoginseng total glycosides; Radix Salviae Miltiorrhizae; ingredients such as Herba Epimedii, and/or comprise copper; zinc; silver; lanthanum; cerium; neodymium metal ion and corresponding copper sulfate thereof; copper chloride; zinc sulfate; zinc chloride; silver nitrate; Lanthanum (III) nitrate; cerous nitrate; inorganic antibacterial compositions such as neodymium nitrate salt compounds, and the good carrier of needed other active substance composition; both reach these material compositions of protection, and can give play to the effect of slow release again.
The basic preparation method of the above-mentioned composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material of the present invention, be to adopt blending method with suitable microsphere composition, for example above-mentioned chitosan/sodium alginate complex microsphere, even with the slurry mixed together of the nanometer hydroxy apatitel medical polymer material of aforementioned existing bibliographical information, use gas foaming method and phase transfer method subsequently, can obtain of the present invention year microsphere composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material.
Complex microsphere composition with above-mentioned chitosan/sodium alginate, with the composite parts slurry with nanometer hydroxyapatite/chitosan form be example, its year, the basic preparation process of compound tissue engineering bracket material of microsphere was: with the slurry of microsphere composition, nanometer hydroxy apatitel medical polymer composite parts and under≤85 ℃ of heating conditions, can decompose the abundant mix homogeneously of salt constituents (as sodium bicarbonate commonly used or ammonium bicarbonate etc.) that produces gas, be warming up to≤85 ℃ said salt constituents is decomposed produce gas.Optimal way to the heating rate of mixed material is to be 0.5～3 ℃/minute.Under≤-20 ℃ of conditions freezing at least 24 hours, remove wherein water constituent through lyophilization again, obtain the said year compound tissue engineering bracket material of microsphere.Wherein, the weight ratio of said microsphere composition and nanometer hydroxy apatitel medical polymer material composition＜2: 9, and the amount of microsphere composition is not 0, and the part by weight of water content is 50%～60% in the said nanometer hydroxy apatitel medical polymer composite parts slurry.
Wherein, said nanometer hydroxy apatitel medical polymer composite parts slurry is the slurry of nanometer hydroxyapatite and chitosan composite parts, and the part by weight scope of nanometer hydroxyapatite and chitosan composite parts is 1/9～8/2; Said microsphere composition can to become microsphere and chitosan with sodium alginate be the chitosan/sodium alginate complex microsphere of coating for above-mentioned.
Test shows that the microsphere amount that adds is excessive, can reduce the mechanical property of gained tissue engineering bracket material, also can reduce the connectivity of material mesopore in the nanometer hydroxy apatitel medical polymer composite parts.
Adding can be reacted the salt of aerogenesis, decomposes the gas that produces by it under heating condition, can form bubble in the mixed slurry raw material.The consumption of these salt constituents generally can be about 4%～6% of composite material compositions quality.Then it is freezing, thereby except that can make in the material by the aerogenesis salt decompose formed micropore in refrigerating chamber because of freezing hard being fixed that become, can also make the moisture in the slurry mix form ice crystal therein, in freezing dry process subsequently, promptly stay aperture after the ice crystal drying, thereby also guaranteed interpore connection, and hard solid-state ice crystal also there is the effect of further reinforcing material mechanical property.
By test shows, in the porous nano hydroxyapatite/chitosan tissue engineering bracket for preparing in the above described manner, microsphere does not have agglomeration, does not deform yet, and microsphere combines closely with support, is evenly distributed.Brace aperture rate height, hole connects.It is a kind of tissue engineering rack material with function admirable of sustained drug release effect.
Except that the above-mentioned composite nanometer hydroxy apatitel that is loaded with chitosan/sodium alginate complex microsphere/chitosan tissue engineering bracket material, nanometer hydroxy apatitel medical polymer material slurry with aforementioned documents record form, it is raw material that other composition/method of reaching obtains the microsphere composition, the corresponding method of carrying the compound tissue engineering bracket material of microsphere of preparation can be carried out with reference to above-mentioned basic skills.
The above-mentioned utilization gas foaming of the present invention method, gas expansion takes up space and forms micropore, under freezing, pass through phase transition behavior, be water in the support forms the space occupy-place earlier under the lyophilization state ice, direct gasification is removed and is stayed the micropore that occupy-place forms under vacuum state then, thereby the internal stent structure is being carried out the fixed while, can in composite material bracket, form the perforation between micropore and retaining hole again effectively.Microsphere composition by uniformly dispersing load in timbering material, can make the cell growth factor, Chinese herbal medicine effective ingredients or the antibiotics medicine that add valid density in the tissue engineering bracket material effectively, can keep secular releasing effect again, from provide for reparation and treated tissue or organ a kind of can have simultaneously repair or replace the New-support material of damaged organ and two kinds of functions of medicament slow release.
Below in conjunction with the specific embodiment of accompanying drawing illustrated embodiment, foregoing of the present invention is described in further detail.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements or change according to ordinary skill knowledge and customary means are made all should comprise within the scope of the invention.
Description of drawings
Fig. 1 is the microscopic pattern with the porous support materials of the not load microsphere of the method preparation of gas foaming and phase transfer.
Fig. 2 is the stereoscan photograph that the present invention adopts the microsphere of inner gel/emulsion process preparation.
Fig. 3 is the scanning electron microscope picture that carries microsphere porous tissue engineering bracket of the inventive method preparation.
The specific embodiment
Carry the preparation of the nanometer hydroxyapatite/chitosan tissue engineering bracket of alginate microsphere
1 ': the preparation of alginate microsphere
The chitosan raw material joined contain in 1% (w/v) aqueous acetic acid, stir and make dissolving, the chitosan aqueous acetic acid that is prepared into 3% (w/v) is standby.In addition the sodium alginate raw material is dissolved in to heat and remain in 70 ℃ deionized water for stirring and make dissolving, the sodium alginate soln for preparing 2% (w/v) is standby.
In beaker, pour liquid paraffin 150ml and Oleum Terebinthinae 50ml and 4ml Tween 80 into, stir, obtain containing 2% (v/v) Tween 80 oil mixed phase liquid.Though the consumption of used liquid paraffin and Lignum Pini Nodi wet goods oil phase material need not too much qualification, not helping microsphere can have enough dispersions and unlikely reunion in oil phase, generally think add 2 times of liquor capacity or more mostly be suitable.
Get 2% standby sodium alginate soln 80ml, the amount of pressing 0.01g/ml adds CaCO
3And after mixing evenly, at rotating speed is under the stirring condition of 3000～3500 commentaries on classics/min, be added drop-wise in the above-mentioned mixing oil phase liquid with 1～2 droplet/minute speed, minim by 0.01ml/ml is added in mixed solution and the mix homogeneously that is mixed with the 0.8ml glacial acetic acid in the 2ml liquid paraffin that contains 1% (v/v) Tween 80 subsequently, reacted 1 hour, and made calcium carbonate and acetic acid reaction, discharge calcium ion as gel, and have carbon dioxide to generate simultaneously, in microsphere, form micropore.
Other gets 3% standby chitosan solution 80ml, and (how many consumptions does not make significant difference with the liquid paraffin uniform mixing dilution formation emulsion that contains 1% (v/v) Tween 80, for making it that better mix homogeneously degree can be arranged in sodium alginate soln, measuring big effect, generally to be often better than consumption little), join subsequently in the above-mentioned sodium alginate micro ball emulsion, keeping rotating speed is 3000～3500 rev/mins stir about 1 hour, after chitosan and sodium alginate micro ball are fully acted on, filtering and washing is lyophilization again, obtains the alginate complex microsphere.Its state as shown in Figure 2, wherein Fig. 2 (b) is the local magnifying state among Fig. 2 (a).
2 ': the preparation of nanometer hydroxyapatite/chitosan composite material sizing agent
According to chitosan and 70: 30 ratio of nanometer hydroxyapatite weight ratio of constituents, the aqueous solution and nanometer hydroxyapatite slurry (moisture content the is 80%) mix homogeneously of 3% (w/v) chitosan of acetic acid will do not contained, promptly, get chitosan 500ml in the 600ml beaker, take by weighing slurry 32.15 grams that contain the 6.43g nanometer hydroxyapatite again, under rotating speed is 300～500 rev/mins lasting stirring, join in the chitosan solution and mix, and maintain the temperature at 60 ℃～70 ℃, reach at 50%～60% o'clock to the moisture content of mixed slurry and stop heating, obtain the slurry of nanometer hydroxyapatite/chitosan composite.
3 ': the preparation of carrying the nanometer hydroxyapatite/chitosan tissue engineering bracket of microsphere
Get microsphere 5 grams of above-mentioned preparation, with the slurry 50ml mix homogeneously of nanometer hydroxyapatite/chitosan composite, the ratio with 0.03g/ml under stirring condition adds 1.5 gram NaHCO
3With the abundant mix homogeneously of slurry, put in the vacuum drying oven afterwards and heat, at 40 ℃～85 ℃, in the time of 85 ℃, be incubated 5 minutes with 1 ℃/minute heating rate control temperature at last.This moment NaHCO in microsphere and compound slurry mixed material
3React, produce partial CO 2 and a small amount of water vapor and discharge.Taking-up is subsequently put into-20 ℃ of refrigerating chamber quick-freezings and was kept 24 hours, the hole that has formed is fixed, and made the water of not removing form ice crystal drilling once more.Compound material after freezing is put into the freezer dryer lyophilization, remove the wherein water of ice crystal state, promptly obtain said year microsphere tissue engineering scaffold material.Its state as shown in Figure 3, wherein Fig. 3 (b) is the local magnifying state of Fig. 3 (a).By clearly visible among the figure, be substantially and evenly be dispersed in the microsphere that is distributed in the timbering material surface, there is not agglomeration, do not deform yet, microsphere combines closely with support.The not load that shown in Figure 1 is contrasts with it has the micro state of the single nanometer hydroxyapatite/chitosan tissue engineering bracket material of microsphere composition, and wherein Fig. 1 (b) is the local magnifying state of Fig. 1 (a).
Embodiment 2
Carry the preparation of the nanometer hydroxyapatite/chitosan tissue engineering bracket of chitosan microball
1 ': the preparation of chitosan microball
The chitosan raw material joined contain in 2% (w/v) aqueous acetic acid, stir and make dissolving, the chitosan aqueous acetic acid that is prepared into 3% (w/v) is standby.
In beaker, pour liquid paraffin 100ml and Oleum Terebinthinae 60ml and 2.5ml Tween 80 and 2.5ml span (span) into, stir, obtain containing 3.12% (v/v) Tween 80/span oil mixed phase liquid.
Get 50% glutaraldehyde solution 2ml, be mixed with out the solution 10ml that contains glutaraldehyde 10% with distilled water.It is mixed the back with the 10ml liquid paraffin standby again.
Get 3% standby chitosan solution 60ml, at rotating speed is under the stirring condition of 3300～3500 commentaries on classics/min, be added drop-wise in the above-mentioned mixing oil phase liquid with 1～2 droplet/minute speed, after half an hour, in chitosan emulsion, splash into the mixed liquor of glutaraldehyde and liquid paraffin with 1～2 droplet/second speed with separatory funnel.Stir after 3 hours, take out the washing lyophilization, obtain chitosan microball.
2 ': the preparation of nanometer hydroxyapatite/chitosan composite material sizing agent
According to chitosan and 80: 20 ratio of nanometer hydroxyapatite weight ratio of constituents, with 3% chitosan solution and nanometer hydroxyapatite slurry (its moisture content is 80%) mix homogeneously under agitation, promptly get chitosan solution 300ml in the 600ml beaker, take by weighing slurry 11.25 grams that contain the 2.25g nanometer hydroxyapatite again, under rotating speed is 300～500 rev/mins lasting stirring, join in the chitosan solution and mix, and maintain the temperature at 60 ℃～70 ℃, reach at 50%～60% o'clock to the moisture content of mixed slurry and stop heating, obtain the slurry of nanometer hydroxyapatite/chitosan composite.
3 ': the preparation of carrying the nanometer hydroxyapatite/chitosan tissue engineering bracket of microsphere.
Get chitosan microball 3 grams of above-mentioned preparation, with the slurry 60ml mix homogeneously of nanometer hydroxyapatite/chitosan composite, the ratio with 0.01g/ml under stirring condition adds 0.6 gram NH
4HCO
3With the abundant mix homogeneously of slurry, put in the vacuum drying oven afterwards and heat, at 40 ℃～45 ℃, be incubated 5 minutes with 1 ℃/minute heating rate control temperature.This moment NH in microsphere and compound slurry mixed material
4HCO
3React, emit carbon dioxide and ammonia and part water vapour and discharge.Taking-up is subsequently put into-20 ℃ of refrigerating chamber quick-freezings and is spent the night, and the hole that has formed is fixed, and made the water of not removing form ice crystal drilling once more.Compound material after freezing is put into the freezer dryer lyophilization, remove the wherein water of ice crystal state, promptly obtain said year microsphere tissue engineering scaffold material.
Embodiment 3
Carry the preparation of the nanometer hydroxyapatite/chitosan tissue engineering bracket of sodium alginate micro ball
1 ': the preparation of sodium alginate micro ball
Sodium alginate is mixed with 2% solution for standby.
In beaker, pour liquid paraffin 100ml and 2.5ml Tween 80 into, stir, obtain containing 2.5% (v/v) Tween 80 mixing oil phase liquid.
Get CaCl
210g and ZnCl
25g adds in the 10ml distilled water, is mixed with solution for standby.
Get 2% standby sodium alginate soln 60ml, at rotating speed is under the stirring condition of 3000～3400 commentaries on classics/min, be added drop-wise in the above-mentioned mixing oil phase liquid with 1～2 droplet/minute speed, after half an hour, in sodium alginate soln emulsion, splash into CaCl with 1～2 droplet/second speed with separatory funnel
2And ZnCl
2Mixing oil phase liquid.Stir after 3 hours, treat that sodium alginate fully forms gel after, take out the washing lyophilization, obtain chitosan microball.
2 ': the preparation of nanometer hydroxyapatite/chitosan composite material sizing agent
According to chitosan and 80: 20 ratio of nanometer hydroxyapatite weight ratio of constituents, with 3% chitosan solution for preparing and nanometer hydroxyapatite slurry (its moisture content is 80%) mix homogeneously under agitation, promptly get chitosan solution 200ml in the 600ml beaker, take by weighing slurry 30 grams that contain the 6.0g nanometer hydroxyapatite again, under rotating speed is 300～500 rev/mins lasting stirring, join and mix and keep 60 ℃～70 ℃ of temperature in the chitosan solution, reach at 50%～60% o'clock to the moisture content of mixed slurry and stop heating, obtain the slurry of nanometer hydroxyapatite/chitosan composite.
3 ': the preparation of carrying the nanometer hydroxyapatite/chitosan tissue engineering bracket of microsphere.
Get sodium alginate micro ball 5 grams of above-mentioned preparation, slurry 100ml mix homogeneously with nanometer hydroxyapatite/chitosan composite, ratio with 0.01ml/ml under stirring condition adds hydrogen peroxide 1ml and the abundant mix homogeneously of slurry, put in the vacuum drying oven afterwards and heat, be warming up to about 60 ℃ of temperature and be incubated 20 minutes with the speed of 0.5 degrees celsius/minute.This moment, the hydrogen peroxide decomposes in microsphere and compound slurry mixed material discharged oxygen and part steam.Taking-up is subsequently put into-20 ℃ of refrigerating chamber quick-freezings and is spent the night, and the hole that has formed is fixed, and made the water of not removing form ice crystal drilling once more.Compound material after freezing is put into the freezer dryer lyophilization, remove the wherein water of ice crystal state, promptly obtain said year microsphere tissue engineering scaffold material
Embodiment 4
1 ': the preparation of alginate microsphere
The chitosan raw material joined contain in 1% (w/v) aqueous acetic acid, stir and make dissolving, the chitosan aqueous acetic acid that is prepared into 3% (w/v) is standby.In addition the sodium alginate raw material is dissolved in to heat and remain in 70 ℃ deionized water for stirring and make dissolving, the sodium alginate soln for preparing 2% (w/v) is standby.
In beaker, pour liquid paraffin 150ml and Oleum Terebinthinae 50ml and 4ml Tween 80 into, stir, obtain containing 2% (v/v) Tween 80 oil mixed phase liquid.Though the consumption of used liquid paraffin and Lignum Pini Nodi wet goods oil phase material need not too much qualification, not helping microsphere can have enough dispersions and unlikely reunion in oil phase, generally think add 2 times of liquor capacity or more mostly be suitable.
Get 2% standby sodium alginate soln 80ml, the amount of pressing 0.01g/ml adds CaCO
3And after mixing evenly, at rotating speed is under the stirring condition of 3000～3500 commentaries on classics/min, be added drop-wise in the above-mentioned mixing oil phase liquid with 1～2 droplet/minute speed, minim by 0.01ml/ml is added in mixed solution and the mix homogeneously that is mixed with the 0.8ml glacial acetic acid in the 2ml liquid paraffin that contains 1% (v/v) Tween 80 subsequently, reacted 1 hour, and made calcium carbonate and acetic acid reaction, discharge calcium ion as gel, and have carbon dioxide to generate simultaneously, in microsphere, form micropore.
Other gets 3% standby chitosan solution 80ml, and (how many consumptions does not make significant difference with the liquid paraffin uniform mixing dilution formation emulsion that contains 1% (v/v) Tween 80, for making it that better mix homogeneously degree can be arranged in sodium alginate soln, measuring big effect, generally to be often better than consumption little), join subsequently in the above-mentioned sodium alginate micro ball emulsion, keeping rotating speed is 3000～3500 rev/mins stir about 1 hour, after chitosan and sodium alginate micro ball are fully acted on, filtering and washing is lyophilization again, obtains the alginate complex microsphere.
2 ': the preparation of nanometer hydroxy apatitel medical polyamide 66 (PA66) composite material sizing agent
The ratio of nanometer hydroxyapatite and PA66 is 50: 50, getting 10 gram PA66 is dissolved in ethanol/calcium chloride mixed solution, solution is 300ml altogether, take by weighing 10 gram hydroxyapatite dry powder again, put into drying baker in advance, remove contained moisture, with PA66 solution mix homogeneously, can obtain nanometer hydroxyapatite/PA66 composite material sizing agent then.
3 ': the preparation of carrying the nanometer hydroxy apatitel medical polyamide 66 tissue engineering bracket of microsphere.
Get alginate complex microsphere 3 grams of above-mentioned preparation, slurry 100ml mix homogeneously with nanometer hydroxyapatite/PA66 composite, in heating jacket, keep 60 degrees centigrade heated at constant temperature to discharge fully, promptly obtain said year microsphere tissue engineering scaffold material up to ethanol.
Claims (10)
1. composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material, constitute framing structure by nanometer hydroxyapatite composition and medical macromolecular materials composition with mutual perforation hole, it is characterized in that in the framing structure material, being scattered with the microsphere composition of band microcellular structure, the weight ratio of microsphere composition and nanometer hydroxy apatitel medical polymer material composition＜2: 9 wherein, and the amount of microsphere composition is not 0.
2. composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material as claimed in claim 1 is characterized in that said microsphere composition is the chitosan/sodium alginate complex microsphere of coating for becoming microsphere and chitosan with sodium alginate.
3. composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material as claimed in claim 1 is characterized in that said medical macromolecular materials are chitosan, and nanometer hydroxyapatite is 1/9～8/2 with the quality of chitosan than scope.
4. as the described composite nanometer hydroxy apatitel medical polymer material tissue engineering bracket material of one of claim 1 to 3, it is characterized in that load has the active component that comprises medicine or Biological somatomedin in the said microsphere.
5. the method for preparing described year microsphere composite nanometer hydroxy apatitel medical polymer material of claim 1 tissue engineering bracket material, it is characterized in that slurry with microsphere composition and nanometer hydroxy apatitel medical polymer composite parts, and under≤85 ℃ of heating conditions, can decompose the abundant mix homogeneously of salt constituents that produces gas, be warming up to≤85 ℃ said salt constituents is decomposed produce gas after, under≤-20 ℃ of conditions freezing at least 24 hours, remove wherein water constituent through lyophilization again, obtain said tissue engineering bracket material, the weight ratio of wherein said microsphere composition and nanometer hydroxy apatitel medical polymer material composition＜2: 9, and the amount of microsphere composition is not 0, and the percentage by weight of water content is 50%～60% in the said nanometer hydroxy apatitel medical polymer composite parts slurry.
6. preparation method as claimed in claim 5, it is characterized in that said nanometer hydroxy apatitel medical polymer composite parts slurry is the slurry of nanometer hydroxyapatite and chitosan composite parts, the part by weight scope of nanometer hydroxyapatite and chitosan composite parts is 1/9～8/2.
7. as claim 5 or 6 described preparation methoies, it is characterized in that said microsphere composition is for formed to become microsphere and chitosan with sodium alginate be the chitosan/sodium alginate complex microsphere of coating by chitosan and sodium alginate.
8. preparation method as claimed in claim 7, it is characterized in that said chitosan/sodium alginate complex microsphere is the microsphere that becomes by following mode: be that 1%～2.5% sodium alginate aqueous solution is under 200～4000 rev/mins mixing speed with weight/volume percent, dropwise join and be mixed with the abundant mix homogeneously of water-insoluble organic facies liquid that volume content is 1%～5% surface active ingredient, and adding gel, to be mixed with weight/volume percent then by the chitosan of said proportional quantities and be 2%～3% under fully stirring, also to be mixed with volume content be that the chitosan aqueous solution of 1%～5% surface active ingredient adds wherein, after stirring abundant down reaction, obtain said chitosan/sodium alginate complex microsphere after filtration washing and the lyophilization.
9. preparation method as claimed in claim 5 is characterized in that said heating rate to mixed material is 0.5～3 ℃/minute.
10. preparation method as claimed in claim 5 is characterized in that the said salt constituents that can decompose generation gas is sodium bicarbonate or ammonium bicarbonate.
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| CN102977385A (en) * | 2012-12-19 | 2013-03-20 | 江南大学 | Preparation method of nano-hydroxyapatite/chitosan |
| CN105013005B (en) * | 2015-08-20 | 2018-03-02 | 重庆科技学院 | A kind of complex microsphere |
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| CN111346263A (en) * | 2020-04-22 | 2020-06-30 | 吉林大学 | Aseptic bone scaffold material with anti-infection and bone formation dual functions and preparation method thereof |
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