CN101874751B - Multi-layer porous scaffold and preparation method thereof - Google Patents
Multi-layer porous scaffold and preparation method thereof Download PDFInfo
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- CN101874751B CN101874751B CN 200910050499 CN200910050499A CN101874751B CN 101874751 B CN101874751 B CN 101874751B CN 200910050499 CN200910050499 CN 200910050499 CN 200910050499 A CN200910050499 A CN 200910050499A CN 101874751 B CN101874751 B CN 101874751B
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Abstract
The invention belongs to the technical field of biological materials and tissue repair and particularly relates to a multi-layer porous scaffold and a preparation method thereof. A suitable porous material is selected, after being clipped according to set size and requirements, the material is bonded by pore-forming adhesive which contains mixture of components of polymer/pore-forming particles/solvent, and pore-forming agent is removed after solidification and bonding, thus preparing the porous scaffold which has multi-layer structures communicated with each other. The scaffold has multi-layer structures, porous transition layers are among the layers of the scaffold, and the pores in an upper layer and a lower layer of the scaffold are communicated with each other; and the scaffold is suitable for transition of different cells and fusion of tissues in a process of tissue remodeling, and weakened separation between layers of the remolding tissue is avoided. The scaffold is suitable for a bionic three-dimensional cell scaffold which repairs a multi-layer tissue and other application fields.
Description
Technical field
The invention belongs to biomaterial and tissue repair technical field, be specifically related to porous support of a kind of multilamellar and preparation method thereof.
Background technology
Porous support can be widely used in fields such as tissue repair, cell culture vector, wound dressing, medicine controlled release carrier.Especially the polyalcohol stephanoporate three-dimensional rack that has high porosity has developed into the important component part of organizational project and original position tissue regeneration.
The ultimate principle that prior art discloses organizational project is: extract certain normal cell from tissue, after cultivating propagation, make up the cell material complex at external and degradable multiporous support, again with this damaged place of species complex implanting tissue, along with the continuous secretion of material degradation and extracellular matrix and the continuous formation of tissue, rebuild new tissue and the organ that adapts with self form and function, reach the purpose of tissue repair.Therefore porous support has considerable status in the tissue reconstruction process.
In addition, directly implant porous support, utilize in the body cell voluntarily the inductive technology (or claiming original position tissue regeneration) of organizing of repair tissue also depend on suitable porous support materials.
About tissue engineering bracket or organize making and the forming technique of induction rack, relatively abundanter approach and means have been arranged at present.For example, fibrous woven technology, particle remove technology, gas foaming technology, phase detachment technique, microsphere sintering technology etc.The tissue engineering bracket that different biomaterials and preparation means obtain, important indicators such as its pore morphology, porosity, specific surface area and degradation rate are all variant.For example, the nonwoven fabric construct that electrostatic spinning obtains is suitable for the structure of thin layer tissue such as skin heart; Particle is removed the three-dimensional rack that obtains high porosity, can be applicable to bone and cartilage tissue engineered; Utilize the method for microsphere sintering, can obtain high-intensity support, but voidage is not high, be suitable for the reparation of osseous tissue.Therefore and in the tissue reconstruction process, the construction features of support can have influence on the 26S Proteasome Structure and Function of cambium, the porous support that needs pore structure to adapt with it at the reconstruction of different tissues.
The human organ more complicated is not by single organizational composition, and needs to adopt the focus of organizational project reparation clinically, often includes the serious defect of multilayer tissue.The form of different tissues, structure, mechanical property have very big difference, and different classes of growth and proliferation of cell and tissue reconstruction speed are also variant.Therefore just need be according to the characteristics of these defective tissues, the timbering material that design different porosities, pore structure and degradation rate combine.In addition, in the organizational project practice process, even if to histioid reparation, also need to consider its with surrounding tissue between cooperate.For example, in the clinical example of repair of cartilage, except realizing the reparation of cartilaginous tissue, the problem that is connected to become key of the cartilage of reconstruction and lower floor's osseous tissue.Otherwise cartilage regeneration is connected the weak link that will become tissue repair with interface between the bone matrix.In addition, in the reparation of tissues such as skin, long dried bone, all need to make up the complicated bionical tissue of multiple form.And described single support pore moulding process before relying on generally can only prepare single support of planting material, single pore structure.In the face of the clinical demand of the structure of complex organization the time, it is bigger to have the independent the demand adjustable and support that still is interconnected on the whole of the multiple structure similar to defective tissue, each layer performance.
In order to obtain to make up the multiple structure that adapts with complex organization, consider from the angle of tissue repair, need to have good connectedness between the porous support of multilamellar sometimes.Be communicated with if two previously prepared porous supports connect to be easy to cause lack between the support simply.
Summary of the invention
The object of the present invention is to provide a kind of multi-layer porous scaffold and preparation method thereof.Be specifically related to a kind of by the multilamellar support bonding and layer with layer between have the multi-layer porous scaffold of good intercommunicating pore structure.
The invention provides a kind of porous support that possesses connective multilamellar.The present invention adopts a kind of binding agent that contains the pore particle, and it is independent adjustable to prepare between a kind of layer and the layer structure, and layer with layer between the timbering material that is communicated with of hole maintenance.Its core is the porous material that select to be fit to, after directly using or cutting out on request, but bonding by the binding agent of pore, remove porogen after the curing, make the porous support be interconnected multiple structure and complex appearance.
Particularly, multi-layer porous scaffold provided by the invention is characterized in that, has multiple structure, and each interlayer that constitutes rack body contain porous transition zone, make and be interconnected between the hole of levels.Each layer can be adopt particle stripping, fibrous woven, be separated, the porous support of any one or a few technology preparation in gas foaming or the microsphere sintering etc.One or more layers the aperture of forming this support is 10-900 μ m, porosity 20%-99%, the pore structure pattern of each layer and porosity are independent adjustable in the compound rest, the aperture of different layers and porosity can identical, also can be inequality, and the binding agent that contains porogen by use between each layer of support connects.
Among the present invention, adopt the mixture of being formed by polymer, solvent and pore particle as binding agent.Behind applying adhesive on the adhesive surface between layer and the layer, adhesive surface is harmonious, exert pressure.Solvent in the binding agent is partly dissolved fusion with support top layer hole wall by diffusion, treat solvent evaporates after, adhesive layer is fixed, the polymer of different layers just couples together by the adhesive layer of centre.The method that adopts particle to remove at last with the particle stripping of adhesive layer, namely obtains the porous compound support frame that layer and interlayer are interconnected.
The multi-layer porous scaffold that the present invention proposes it is characterized in that adopting the transition zone with loose structure that separate porous support layer and layer are connected into integral body, and the hole between layer and the layer has connectedness.Articulamentum thickness is generally about 0.5mm.
The host material of forming each layer support comprises macromolecular material, inorganic material, bio-derived material or the complex between them, and the host material between the different layers can identical, also can be inequality.
Described host material is to have from cementability, resolvability, plastic natural or synthesized polymer material, has both comprised degradable macromolecule, also comprises nondegradable macromolecular material.The host material of each layer support also comprises the complex between copolymer between the macromolecular material or blend and macromolecular material and bio-derived material, inorganic material etc.
Among the present invention, the macromolecular material of forming the porous support main body comprises degradable polymers such as polylactic acid, polyglycolic acid, poly butyric ester, polycaprolactone, Merlon, poe, poly-anhydride, poly-dioxane, chitosan, alginate, hyaluronate, collagen, gelatin, fibroin albumen, and any by two or more copolymer formed among them or blend; Also can be non-degradable polymer such as polystyrene, polrvinyl chloride, polyacrylate, polymethacrylates, Merlon, nylon, polyurethane, polyformaldehyde, polyvinyl alcohol, polyvinyl acetate, polysiloxanes, and any by two or more copolymer formed among them or blend.
Binding agent of the present invention is characterized in that being made up of polymer, solvent and pore particle.Polymers compositions wherein can be degradable polylactic acid, polyglycolic acid, poly butyric ester, polycaprolactone, Merlon, poe, poly-anhydride, poly-dioxane, chitosan, alginate, hyaluronate, collagen, gelatin, fibroin albumen and the copolymer of two or more composition or blend any among them; Also can be nondegradable polystyrene, polrvinyl chloride, polyacrylate, polymethacrylates, Merlon, nylon, polyurethane, polyformaldehyde, polyvinyl alcohol, polyvinyl acetate, polysiloxanes, and any by two or more copolymer formed among them or blend.
Solvent composition in the binding agent (being solvent A), can be acetone, butanone, chloroform, dichloromethane, oxolane, benzene,toluene,xylene, ethylene glycol, Ketohexamethylene, dioxane, N, any or several mixture in dinethylformamide, formic acid, benzyl alcohol, the cyclohexane extraction.Described solvent orange 2 A has fine solubility to binder polymer component and rack body material, but can not change its structure and performance; The pore particle is not dissolved and do not change its structure and character; Have suitable volatility, under vacuum condition, can remove fully.
Among the present invention, the polymer of solvent B is insoluble or insoluble substantially, the pore particle there is good dissolving ability, and has suitable volatility, under vacuum condition, can be removed fully, adopted any or several mixture in pentane, hexane, heptane, octane, cyclohexane extraction or the water usually.
Among the present invention, the pore particle in the binding agent is dissolved in solvent B, but is insoluble to solvent orange 2 A.Described pore particle comprises inorganic salt, polysaccharide, organic micromolecule compound or the mixture of being made up of them, and particle can be that the ball-type of rule also can be irregular polyhedron, and particle diameter is in the 10-900 mu m range.The shared volume fraction of solvent orange 2 A is generally 20%-70% in the binding agent, and preferable scope is 30%-50%; The amount ranges of pore particle is the 20wt%-99wt% of mixture, and preferable scope is 70wt%-95wt%.
The present invention adopts the method for adhesives to prepare described multi-layer porous scaffold, by following step,
1, the solvent (solvent orange 2 A) of polymer raw material, polymer and pore particle are mixed after, obtain the binding agent of multilamellar support;
2, each ingredient of the porous support that will make is in advance directly used or is suitably cut out to obtain neat clean adhesive surface according to the tissue defect form;
3, the support binding agent is coated on the adhesive surface of each layer support, forms articulamentum, will treat that then bonding another layer support is placed on above, and exert pressure, treat to finalize the design after the solvent evaporates;
4, after typing is finished after a while, remove pressure, add new shelf layer as required, can repeat previous action;
5, interconnective multilamellar support is implemented the particle removing process, with the particle stripping of solvent B with articulamentum; Behind the dry removal residual solvent, make the MULTILAYER COMPOSITE porous support with the pore structure of being interconnected.
Among the present invention, can earlier polymeric material be dissolved in the solvent orange 2 A when obtaining binding agent, form polymer solution, then the pore particle be joined in the macromolecular solution, mix.
Among the present invention, the moulding process of each layer support can adopt particle pore, non-woven fabrics braiding, foaming, is separated, in 3 D-printing and the particles sintering technology any one or a few.
When support of the present invention is bonding, need give certain pressure and the time chien shih adhesive layer solidify.Can implement at small-sized molding apparatus, prerequisite is to destroy the structure of each layer support, is generally a few minutes to a few hours hardening time not wait, and is decided by the volatilization ability of use solvent orange 2 A.
The present invention need make solvent orange 2 A remove after obtaining multilamellar support after bonding, can under the air at room temperature environment solvent orange 2 A partly be volatilized earlier, removes fully under the vacuum condition room temperature then, generally at 12-48 hour.Solvent B should be excessive greatly, is generally the 50-1000 of binding agent use amount doubly.
Among the present invention, the above-mentioned compound rest that has removed particle is taken out, make most of solvent evaporates under the room temperature in the air, change over to then and remove residual solvent B under the vacuum condition.This process generally continues 12-48 hour, and vacuum drying oven is no more than the glass transition temperature of polymer, and general room temperature state gets final product.
The prepared multi-layer porous scaffold of the present invention is applicable to as organizational projects such as skin, bone/cartilage, blood vessel, esophagus, tracheas repairs employed cytoskeleton, is used for the artificial constructed of tissues such as skin, bone/cartilage, blood vessel, esophagus, trachea.
The present invention compares with existing compound rest technology of preparing, has following advantage and characteristics:
1, prepared multi-layer porous scaffold layer with the layer between be connected by the adhesive layer transition, different with common chemical adhesive, the adhesive layer that the present invention finally obtains also has loose structure, and and realize between each layer of support being interconnected, be suitable for the fusion between transition of different cells in the tissue reconstruction process and tissue, avoided rebuilding organized layer with layer between weakened separation.
2, prepared support, different layers can be different materials, different pore technology, also can be same moulding process different porosities support, and above combination arbitrarily, this flexible and changeable system all has practical significance on bionical two general orientation of 26S Proteasome Structure and Function.
3, the polymers compositions of the binding agent of the present invention's use is the material with excellent biological compatibility, can select and the rack body identical materials, also can select other suitable tissue engineering material,
4, the present invention can also pass through bonding technology, by the method that each several part splices and combines, makes the large-scale porous support of complex contour.
5, can avoid residues of harmful substances to greatest extent in the preparation method involved in the present invention, be fit to the manufacturing of medical embedded device.
For the ease of understanding, below will describe in detail of the present invention by concrete drawings and Examples.It needs to be noted, instantiation and accompanying drawing only are in order to illustrate, obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.
Description of drawings
Fig. 1 is the application principle of multilamellar support on organizational project (be example with bone/repair of cartilage) sketch map, wherein the 1, the 3rd, and the polylactic acid of two-layer different degradation cycles (PLA) porous support, 2 is porous tack coat part.
Fig. 2 is aperture 350-455 μ m, the polylactic acid of porosity 90% and copolymer (PLGA) the porous support scanning electron microscope picture of polyglycolic acid by adopting the paraffin ball to obtain as the pore particle.
Fig. 3 adopts adhesive bonding method the bonding back of the PLGA porous support of two spherical pore structures to be formed the adhesive interface stereoscan photograph of compound rest, binding agent is made up of PLGA/ salt particle/dichloromethane, and the irregular porous zone about middle wide 400 μ m is adhesive layer.Aperture 350-455 μ m, porosity 90%.
Fig. 4 is the outward appearance photo that bonding front and back porosity reaches 90% PLGA support, and a is two independent racks before bonding, and b is the two-layer compound support that bonding back forms.
The specific embodiment
Embodiment 1
At first prepare salt grain and spherical paraffin pore particle.The salt grain uses machinery to smash the back by the standard screen screening, collects the particle of different-grain diameter scope.The paraffin microsphere uses 1.0g gelatin, 20.0g paraffin, joins in the 400ml deionized water, is heated to 80 ℃ under the 400rpm mechanical agitation, adds 300ml frozen water quenching then, and the wax ball is solidified.Filter, with deionized water rinsing twice, screening is shone with standard in dry back, collects the particle of different-grain diameter scope.
Then prepare binding agent.Be that 300,000 PLGA85/15 is dissolved in the 8.0ml dichloromethane with the 0.5g molecular weight, the sodium chloride salt particle of 9.5g particle diameter 350-455 μ m is joined in the macromolecular solution mixing and stirring.
Get two column PLGA85/15 porous supports with the pore of paraffin microsphere (high 5mm, diameter 10mm, porosity 90%, aperture 350-455 μ m), the end equating is whole.Binding agent is coated in equably on the bonding plane of a support wherein, the bonding plane with another piece support is buckled on the rack surface that has applied binding agent then.Put in the mold pressing utensil, apply the pressure of 40N, kept 6 hours.Pressure relief is taken out support, continues solvent flashing after 12 hours in air, changes vacuum under the room temperature over to greater than dry 24 hours of the vacuum drying oven of 755mmHg; Compound rest is put into the pore particle that the beaker that contains the 500ml deionized water leaches adhesive layer, changed water one time in per 4 hours, detect with silver nitrate solution after 48 hours, white precipitate does not appear in leachate, illustrates that particle is except clean.Compound rest is taken out from water, the filter paper suction, air at room temperature changes vacuum over to greater than the vacuum drying oven drying of 755mmHg after dry 24 hours.Make by two PLGA porous supports and form the complex stephanoporate bracket that communicates with each other.Porosity 90%, aperture 350-455 μ m.
Support adopts two different porosities to get the column PLGA85/15 porous support of paraffin microsphere pore, porosity 95% wherein, another piece 85% (high 5mm, diameter 10mm, aperture 350-455 μ m), remaining condition is identical with embodiment 1, makes a double-layer porous support of being made up of the different porosities material.
Support adopts two column PLGA85/15 porous support (high 5mm that different pore technologies obtain, diameter 10mm, porosity 90%, aperture 350-455 μ m), wherein one by the pore of sodium chloride salt grain, and one by the pore of paraffin microsphere, and all the other conditions are identical with embodiment 1, make one deck regular spherical hole, the complex stephanoporate bracket in the irregular hole of another layer.
Embodiment 4
Prepare three with column PLGA85/15 porous support (the high 5mm of paraffin microsphere pore, diameter 10mm, porosity 90%, aperture 350-455 μ m), after first and second bonding, again at second upper surface application of adhesive, the 3rd is bondingly got on, all the other methods are identical with embodiment 1, make three layers of complex stephanoporate bracket.
Embodiment 5
Binder particles is changed the paraffin microsphere of making 350-455 μ m, column PLGA85/15 porous support (high 5mm with the pore of two paraffin microspheres, diameter 10mm, porosity 90%, aperture 350-455 μ m) press embodiment 1 mode bonding after, place in the apparatus,Soxhlet's, be solvent extraction 48 hours with pentane, took out the support air at room temperature dry 8 hours, vacuum drying oven vacuum was greater than 755mmHg drying at room temperature 48 hours.Obtaining adhesive layer is the two-layer compound porous support of spherical pore.
Embodiment 6
Use 0.3g molecular weight is 50,000 polycaprolactone (PCL), is dissolved in the 10ml acetone, and the salt particle 2.7g of 350-455 μ m particle diameter, mix homogeneously is made binding agent, and all the other methods are identical with embodiment 1, make the PLGA85/15 complex stephanoporate bracket that is connected by PCL.
Claims (17)
1. a multi-layer porous scaffold is characterized in that, described support has multiple structure, and each interlayer of support contains the transition zone of porous, is interconnected between the hole of support levels; Described transition zone makes by following method: adopt the mixture of being made up of polymer, solvent and pore particle as binding agent, remove particle after the curing, form the transition layer structure that is interconnected; Described multi-layer porous scaffold prepares by following method:
(1) will make the solvent of polymer raw material, polymer of support and pore particle and stir into the binding agent that obtains the multilamellar support behind the mixture, described solvent is solvent orange 2 A;
(2) porous support that will make in advance respectively forms the direct use of shelf layer or cuts out the acquisition adhesive surface by required form;
(3) binding agent of step 1) is coated on the adhesive surface, behind the formation articulamentum, will treat placed on it of bonding another layer support, and exerts pressure, and treats to finalize the design after the solvent evaporates;
(4) after typing is finished, remove pressure, if also need add shelf layer, repeat previous action;
(5) above-mentioned interconnective multilamellar support is implemented particle and remove, use the solvent of particle with the particle stripping of articulamentum, behind the dry removal residual solvent, the MULTILAYER COMPOSITE porous support that must have the pore structure of being interconnected, the solvent of described particle are solvent B.
2. multi-layer porous scaffold according to claim 1 is characterized in that, one or more layers in the described multi-layer porous scaffold includes the loose structure of aperture 10-900 μ m, porosity 20%-99%, and the aperture of different layers is identical with porosity or inequality.
3. multi-layer porous scaffold according to claim 1 is characterized in that, the host material of forming each layer support comprises macromolecular material, inorganic material, bio-derived material or their complex; The host material of forming different layers is identical or inequality.
4. multi-layer porous scaffold according to claim 3 is characterized in that described host material is to have from cementability, resolvability or plastic natural or synthesized polymer material.
5. multi-layer porous scaffold according to claim 3 is characterized in that described macromolecular material is selected from one of the complex of complex, macromolecular material and the inorganic material of degradable macromolecular material, nondegradable macromolecular material, macromolecular material and bio-derived material.
6. multi-layer porous scaffold according to claim 5 is characterized in that described degradable macromolecular material is selected from copolymer or the blend of polylactic acid, polyglycolic acid, poly butyric ester, polycaprolactone, Merlon, poe, poly-anhydride, poly-dioxane, chitosan, alginate, hyaluronate, collagen, gelatin or fibroin albumen or the two or more compositions in them.
7. multi-layer porous scaffold according to claim 5 is characterized in that described nondegradable macromolecular material is selected from polystyrene, polrvinyl chloride, polyacrylate, polymethacrylates, Merlon, nylon, polyurethane, polyformaldehyde, polyvinyl alcohol, polyvinyl acetate or polysiloxanes or by the two or more copolymers of forming or blend in them.
8. according to the described arbitrary multi-layer porous scaffold of claim 1-7, any one or a few preparation in particle hole forming technology, non-woven fabrics technology, foaming technology, phase detachment technique or the 3 D-printing technology is adopted in the molding that it is characterized in that described each layer of support, and wherein the thickness of each layer support is 0.1-100mm.
9. according to the multi-layer porous scaffold of claim 1, it is characterized in that the amount ranges of pore particle accounts for the 20wt%-99wt% of mixture in the described binding agent, the volume fraction of solvent is 20%-70%, and the thickness of adhesive layer is 0.01-5mm.
10. according to the multi-layer porous scaffold of claim 1, it is characterized in that the polymer in the described binding agent is selected from a kind of of copolymers a kind of or two or more compositions among them in degradable polylactic acid, polyglycolic acid, poly butyric ester, polycaprolactone, Merlon, poe, poly-anhydride, poly-dioxane, chitosan, alginate, hyaluronate, collagen, gelatin, the fibroin albumen or blend; Or be selected from a kind of of copolymers a kind of or two or more compositions among them in nondegradable polystyrene, polrvinyl chloride, polyacrylate, polymethacrylates, Merlon, nylon, polyurethane, polyformaldehyde, polyvinyl alcohol, polyvinyl acetate, the polysiloxanes or blend.
11. the multi-layer porous scaffold according to claim 1, it is characterized in that, solvent orange 2 A in the described binding agent is selected from acetone, butanone, chloroform, dichloromethane, oxolane, benzene,toluene,xylene, ethylene glycol, Ketohexamethylene, dioxane, N, the mixture of one or more in dinethylformamide, formic acid, benzyl alcohol, the cyclohexane extraction.
12. the multi-layer porous scaffold according to claim 1 is characterized in that, the mixture that the pore particle in the described binding agent is selected from inorganic salt, polysaccharide, organic micromolecule compound or is made up of them; Described particle is ball-type or the irregular polyhedron of rule, and particle size range is 10-900 μ m.
13. the multi-layer porous scaffold according to claim 1 is characterized in that, described solvent B is selected from one or more the mixture in pentane, hexane, heptane, octane, cyclohexane extraction or the water.
14. the multi-layer porous scaffold according to claim 1 is characterized in that, the amount ranges of pore particle accounts for the 70wt%-95wt% of mixture in the described binding agent.
15. the multi-layer porous scaffold according to claim 1 is characterized in that, the shared volume fraction of solvent is 30%-50% in the described binding agent.
16. the multi-layer porous scaffold according to claim 1 is characterized in that, solvent has dissolubility to binder polymer component or backing substrate material in the described binding agent, does not change its structure and performance; The pore particle is not dissolved and do not change its structure and character; Under vacuum condition, remove fully.
17. the purposes of the multi-layer porous scaffold of claim 1 in the cytoskeleton of preparation reparation skin, bone or cartilage, blood vessel, esophagus or tracheal tissue.
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