CN103908698A - Superparamagnetic iron oxide nano-particle-collagen-chitosan magnetic degradable nerve conduit and preparation method thereof - Google Patents
Superparamagnetic iron oxide nano-particle-collagen-chitosan magnetic degradable nerve conduit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a superparamagnetic iron oxide nano-particle-collagen-chitosan magnetic degradable nerve conduit and a preparation method thereof. The preparation method comprises the steps of (1) preparing SPIONs (Superparamagnetic Iron Oxide Nano-particles); (2) preparing an SPIONs-collagen-chitosan magnetic degradable nerve conduit; and (3) carrying out disinfection treatment. With the adoption of the preparation method, in the process of preparing the superparamagnetic iron oxide nano-particle-collagen-chitosan magnetic degradable nerve conduit, as superparamagnetic iron oxide nano-particles with a certain concentration are adopted to form a magnetizable magnetic system, regenerated nerves can be guided to carry out orientated growth, the secretion of nerve nutrient substances can also be promoted, and damaged nerves can be more effectively promoted to regenerate. With the adoption of the prepared nerve conduit, the regenerated nerves can carry out orientated growth and accelerated repair to compound a myelin sheath by virtue of an internal magnetic field of the conduit and can be used for further improving the repair effect in nerve defect.
Description
Technical field
The present invention relates to long segment peripheral nerve injury recovery technique field, in particular Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit and preparation method thereof.
Background technology
Peripheral nerve defection is one of clinical modal wound.The nerve injury meeting of the type makes to get involved, and the neural distal limb appearance of arranging is felt completely and the dual disappearance of motor function, thereby causes handicapped generation, bring tremendous influence can to patient's work and quality of life.
At present, clinically for more short-range peripheral nerve defection, on the principle basis of sewing up without tension force, the method that adopts the neural broken ends of fractured bone directly to sew up is repaired more; And for the peripheral nerve defection of growing distance, often adopt the method for nerve autograft to repair.But, research shows: nerve autograft only can be realized the recovery of partial nerve function, and only have 35%～45% patient after accepting to transplant, can realize the recovery completely of motor function, and sensorineural functional rehabilitation effect is neural poorer than nervus motorius or mixed type; Meanwhile, still there are many restraining factors in the method application of this treatment, for example second operation,, ligand receptor neural the disable problem of not mating, easily form intractable neuroma and neural confession district limited for somatic nerves amount.
To be a kind of high molecular polymer take natural biologic material or synthetic be prepared from as raw material tissue engineering nerve support, there is special three dimensional structure, for near-end and the far-end of Bridging nerve defect, lay passage guiding regenerating nerve and grow into, and realize regenerating nerve and cross over a kind of tissue engineering bracket of damaged growth.At present, the emphasis that the relatively simple nerve trachea graft of structure is research, through multinomial animal or clinical research confirmation, it repairs long apart from neurologic defect definite effect.Mainly concentrate on this respect through FDA approval commercialization and the tissue engineering nerve support and the Related product that are applied to clinical bridge joint patient neurologic defect.But the tube wall of most of nerve tracheas is sealed design, both be unfavorable for regenerating nerve oriented growth, also be unfavorable for the transportation of nutrient substance, oxygen and metabolite, and need second operation to take out residual implant infrastructure, thereby limited the universal of multiple nerve trachea.Along with further going deep into of research, scholars also more and more pay attention to the research of peripheral nerve injury local microenvironment, thirst for promoting the regeneration of injured nerve and the recovery of function by improving damaged tissue local microenvironment.
Magnetic signal is the physical signalling that is prevalent in life, and the growth that magnetic field can directional guide various kinds of cell is pointed out in increasing research, thereby and can promote various kinds of cell secretion nutrient substance Promote cell's growth.Clinically, magnetic field is widely used as a kind of method of Noninvasive, as nuclear magnetic resonance, low-frequency magnetic therapy etc., but not yet has the report that can be repaired by the magnetized nerve trachea of external magnetic field neurologic defect.Therefore, invent and a kind ofly new can pass through the magnetized magnetic systems in external magnetic field, in nerve trachea, grow to promote injured nerve recovery just to seem particularly important by directional guide regenerating nerve.
In order to overcome this difficult problem, increasing scholar starts to pay close attention to a kind of new material-Superparamagnetic Iron Oxide nanoparticle (Superparamagneticironoxidenanopaticles, SPIONs).SPIONs is owing to having special magnetic conductance tropism, superparamagnetism, and surface can connect the characteristics such as biochemical activity functional group, make its application in fields such as foranalysis of nucleic acids, clinical diagnosis, targeted drug, enzyme and cell fixations obtain development widely, be characterized in: 1. superparamagnetism, SPIONs is FeO and Fe
2o
3mixed valence compound, its physical length is just in nanometer scale (below 50nm), do the used time being subject to externally-applied magnetic field, in magnetic nanometer particles there is align consistent with magnetization direction in the unordered magnetic moment of the heat of adjacent atom or ion to a certain extent, and after externally-applied magnetic field disappears, the magnetic of particle disappears.2. targeting, SPIONs, under externally-applied magnetic field guide effect, can targeting move, assemble, and produces local specificity and concentrates, thereby realize selective imaging.SPIONs is injected mouse vein by Chertok etc., in 0.4T magnetic field, can selective aggregation develop in mouse brain glioma.3. good biocompatibility, although SPIONs metabolic process in vivo is not also illustrated completely at present, a large amount of research shows that it has good biocompatibility.After Feng etc. inject in Mice Body by SPIONs, the metabolite in its urine and serum is analyzed, is found that the metabolite amounts such as α-phenyl succinimide-n-valeric acid only have slight change, on mice physiology without impact.Levy etc. imitate internal milieu, show that nanoparticle is decomposed into nanocrystal, and energy stable suspersion is in body fluid.The report SPIONs such as Stamopoulos have no significant effect human leucocyte, erythrocyte and platelet.Coated (dip-coating) method of infiltration of simple economy for N.Bock, by compound to SPIONs and hydroxyapatite-chitosan bone regeneration support, prepare novel magnetic bone tissue engineering scaffold, it is detected, find that its magnetic temperature stability is good, and on the activity of mesenchymal stem cells MSCs without impact.Find in addition, this magnetic bracket can pass through magnetic drive power, the somatomedin in attraction receptor, thus promote the damaged regeneration of bone.
Based on the above-mentioned advantage of Superparamagnetic Iron Oxide nanoparticle, perhaps, SPIONs can grow by directional guide regenerating nerve, promote nutrient substance secretion, thereby promote the survival of the seed cell of transplanting, thereby improve the repair ability of functionalized organization's engineering rack.But, not yet there is the bibliographical information for the neurocyte support in animal body by SPIONs at present.
Summary of the invention
The present invention is uncertain in order to solve the regenerating nerve direction of growth, make existing Nerve Scaffold repairing effect not good, slow down nerve growth speed, cause arranging the bad problem of district's functional rehabilitation, Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit and preparation method thereof is provided.
Technical scheme of the present invention is as follows:
The preparation method of Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit, its step is as follows:
(1) prepare SPIONs
Prepare respectively the Fe of 0.1mol/L
3+and Fe
2+solution, under logical condition of nitrogen gas, according to Fe
3+: Fe
2+mol ratio is that 2:1 mixes, and mechanical agitation 3～4h, fully mixes it; Then to the NaOH solution of mixed solution and dripping 1mol/L, until mixed solution pH=9～10 obtain black precipitate; After heating in water bath 2h, leave standstill 5h, remove the supernatant, by the particle decompress filter generating, repeatedly wash with distilled water and dehydrated alcohol, wash away the foreign ion of particle surface; By particle vacuum drying at 50 ℃, grinding is sieved, and obtains SPIONs;
(2) prepare SPIONs-collagen-chitin magnetic Biodegradable nerve conduit
Type i collagen albumen dirty solution and chitosan dirty solution are mixed, then add the SPIONs making in (1), at 2～6 ℃, 15000rpm, stir 80～120min, make mixing dirty solution, will mix dirty solution evacuation, leave standstill 10h～14h, make SPIONs-collagen-chitin gel dirty solution; Then be poured in nerve trachea shaping dies, with the two ends of the fixing nerve trachea shaping dies of little iron clamp, under miniature velometer, in the mode of fishing by nerve trachea shaping dies direct-axis to 2 × 10
-5the speed of m/s slowly immerses in liquid nitrogen, and the SPIONs-collagen-chitin gel dirty solution that injects nerve trachea shaping dies is carried out to the processing of stranguria of cold type solid, immerses completely after liquid nitrogen until nerve trachea shaping dies, continues to retain 10h～14h in liquid nitrogen; The SPIONs-collagen-chitin gel dirty solution that nerve trachea shaping dies is injected together with inside together proceeds to the refrigerator and cooled Tibetan of-80 ℃～-70 ℃, after cold preservation 24h, take out, remove rapidly iron clamp and the copper pipe at nerve trachea shaping dies two ends, be positioned over afterwards lyophilizing 48h～50h in the freezer dryer that pre-cooling is good, wherein the pre-cooling parameter of freezer dryer setting is :-60 ℃, 100mtorr; Nerve trachea shaping dies after frozen dried be positioned under vacuum state and be warming up to 0 ℃, keeping 5.5h～6.5h, being warming up to again afterwards 20 ℃～24 ℃, keeping 30min～60min, removing vacuum state, finally rising to room temperature; Finally nerve trachea is taken out from nerve trachea shaping dies, obtain SPIONs-collagen-chitin magnetic Biodegradable nerve conduit;
(3) disinfect
The SPIONs-collagen-chitin magnetic Biodegradable nerve conduit making in (2) is put into genipin and dehydrated alcohol mixed solution carries out crosslinking Treatment, crosslinking time is 46h～50h, and the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of every gram adds genipin and the dehydrated alcohol mixed solution of 20ml; After crosslinking Treatment, SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is alternately cleaned to 25min～35min with the ethanol that deionized water and mass percentage concentration are 95%; Then be placed under room temperature and be dried one week, finally use
60co irradiates SPIONs-collagen-chitin magnetic Biodegradable nerve conduit, disinfection, the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after being sterilized.
Described preparation method, in step (2), described type i collagen albumen dirty solution, its preparation method is that every 20mgI collagen type adds 1ml glacial acetic acid, through the dissolution process of 22h～26h, makes type i collagen albumen dirty solution; Described chitosan dirty solution, its preparation method is that every 20mg chitosan adds 1.2ml glacial acetic acid, through the dissolution process of 22h～26h, makes chitosan dirty solution.
Described preparation method, in step (2), type i collagen albumen dirty solution and chitosan dirty solution are that 0.25～4:1 mixes according to the mass ratio of type i collagen albumen and chitosan; The quality of the SPIONs adding is 5～20% of type i collagen albumen and chitosan gross mass.
Described preparation method, in step (3), in genipin and dehydrated alcohol mixed solution, genipin accounts for 1% of mixed solution gross mass.
Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit that described preparation method makes.
The present invention is in the process of preparation Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit, adopt certain density Superparamagnetic Iron Oxide nanoparticle, formation has magnetizable magnetic systems, not only can guide regenerating nerve oriented growth, can also promote the secretion of neurotrophy material, more effective promotion injured nerve regeneration.Nerve trachea prepared by the present invention can make regenerating nerve pass through the reparation of catheter interior field orientation Acceleration of growth and become myelin, further improves the repairing effect of neurologic defect.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit preparation method of the present invention; In figure, 1 collagen-chitin dirty solution, 2 Superparamagnetic Iron Oxide nanoparticles, 3 miniature velometers, nerve trachea shaping dies shown in 4 Fig. 2,5 liquid nitrogen containers.
Fig. 2 is the structure chart of the nerve trachea shaping dies that adopts in preparation method of the present invention; In figure, 6 solid stainless steel tubes, 7 Superparamagnetic Iron Oxide nanoparticle-collagen-chitin gel dirty solutions of filling, 8 hollow silica gel tubes, 9 copper pipes.
Fig. 3 is the transmission electron microscope picture of the Superparamagnetic Iron Oxide nanoparticle prepared of the present invention.
Fig. 4 is Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit magnetic detection figure that the present invention prepares.
Fig. 5 is that the present invention prepares Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit cross section scanning electron microscope (SEM) photograph.
Fig. 6 is Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit cross section scanning electron microscope enlarged drawing that the present invention prepares.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
(1) prepare respectively the Fe of 0.1mol/L
3+and Fe
2+solution, under logical condition of nitrogen gas, according to Fe
3+: Fe
2+mol ratio is that 2:1 mixes, and mechanical agitation 3h, fully mixes it.Continuing under logical condition of nitrogen gas, at the NaOH of mixed solution and dripping 1mol/L solution, until mixed solution pH=9 obtains black precipitate.Then heating in water bath 2h, then leaves standstill after 5h, removes the supernatant, by the particle decompress filter generating, repeatedly wash with distilled water and dehydrated alcohol, wash away the foreign ion of particle surface, by the particle vacuum drying at 50 ℃ obtaining, grinding is sieved, and obtains SPIONs.
(2) take respectively type i collagen albumen and chitosan, the quality of type i collagen albumen and chitosan is respectively 50mg, 50mg, takes the SPIONs5mg making in (1).
(3) add 1ml glacial acetic acid by every 20mgI collagen type, glacial acetic acid is mixed with type i collagen albumen, make type i collagen albumen dirty solution through the dissolution process of 22h; Add the glacial acetic acid of 1.2ml by the chitosan of every 20mg, the glacial acetic acid solution measuring is mixed with chitosan, through the dissolution process of 22h, make chitosan dirty solution; The type i collagen albumen dirty solution of preparation in (2) is mixed with chitosan dirty solution, then add 5mgSPIONs, under 2 ℃ of conditions, constant temperature stirs 100min, and mixing speed is 15000rpm, obtains mixing dirty solution; Mixing dirty solution is first carried out to evacuation and process afterwards standing 10h again, obtain SPIONs-collagen-chitin gel dirty solution.
(4) the SPIONs-collagen-chitin gel dirty solution in (3) is injected to nerve trachea shaping dies, with the two ends of the fixing nerve trachea shaping dies of little iron clamp; Under miniature velometer, in the mode of fishing by nerve trachea shaping dies direct-axis to 2 × 10
-5the speed of m/s slowly immerses in liquid nitrogen, and the SPIONs-collagen-chitin gel dirty solution that injects nerve trachea shaping dies is carried out to the processing of stranguria of cold type solid, immerses completely after liquid nitrogen until nerve trachea shaping dies, continues to retain 10h in liquid nitrogen; The SPIONs-collagen-chitin gel dirty solution that nerve trachea shaping dies is injected together with inside together proceeds to the refrigerator and cooled Tibetan of-80 ℃;
(5) after cold preservation 24h, nerve trachea shaping dies is taken out from the refrigerator of-80 ℃, remove rapidly iron clamp and the copper pipe at nerve trachea shaping dies two ends; Nerve trachea shaping dies is positioned over to lyophilizing 48h in the Alphal-2 type freezer dryer that pre-cooling is good; Wherein the parameter of freezer dryer setting is :-60 ℃, 100mtorr; Nerve trachea shaping dies after frozen dried be first positioned under vacuum state and be warming up to 0 ℃ of maintenance 5.5h, being warming up to again afterwards 20 ℃, keeping 30min, removing vacuum state, finally rising to room temperature; Nerve trachea is taken out from nerve trachea shaping dies, by different requirements, be cut into the nerve trachea of various different lengths, the external diameter of nerve trachea cross section is 2.5mm, internal diameter is 1.5mm, prepares SPIONs-collagen-chitin magnetic Biodegradable nerve conduit.
(6) genipin taking is mixed with dehydrated alcohol, the quality of genipin accounts for 1% of mixed solution gross mass; Genipin and the dehydrated alcohol mixed solution of the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit making in (5) being put into preparation carry out crosslinking Treatment, crosslinking time is 46h, and the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of every gram adds genipin and the dehydrated alcohol mixed solution of 20ml; After crosslinking Treatment, SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is cleaned to 25min repeatedly with the ethanol that deionized water and mass percentage concentration are 95%; SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after cleaning is placed under room temperature and is dried one week, finally use
60co irradiates the disinfection of SPIONs-collagen-chitin magnetic Biodegradable nerve conduit, the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after being sterilized.
Embodiment 2
(1) prepare respectively the Fe of 0.1mol/L
3+and Fe
2+solution, under logical condition of nitrogen gas, according to Fe
3+: Fe
2+mol ratio is that 2:1 mixes, and mechanical agitation 3.5h, fully mixes it.Continuing under logical condition of nitrogen gas, at the NaOH of mixed solution and dripping 1mol/L solution, until mixed solution pH=9.5 obtains black precipitate.Then heating in water bath 2h, then leaves standstill after 5h, removes the supernatant, by the particle decompress filter generating, repeatedly wash with distilled water and dehydrated alcohol, wash away the foreign ion of particle surface, by the particle vacuum drying at 50 ℃ obtaining, grinding is sieved, and obtains SPIONs.
(2) take respectively type i collagen albumen and chitosan, the quality of type i collagen albumen and chitosan is respectively 80mg, 20mg, takes the SPIONs10mg making in (1).
(3) add 1ml glacial acetic acid by every 20mgI collagen type, glacial acetic acid is mixed with type i collagen albumen, make type i collagen albumen dirty solution through the dissolution process of 24h; Add the glacial acetic acid of 1.2ml by the chitosan of every 20mg, the glacial acetic acid solution measuring is mixed with chitosan, through the dissolution process of 24h, make chitosan dirty solution; The type i collagen albumen dirty solution of preparation in (2) is mixed with chitosan dirty solution, then add 10mgSPIONs, under 4 ℃ of conditions, constant temperature stirs 80min, and mixing speed is 15000rpm, obtains mixing dirty solution; Mixing dirty solution is first carried out to evacuation and process afterwards standing 12h again, obtain SPIONs-collagen-chitin gel dirty solution.
(4) the SPIONs-collagen-chitin gel dirty solution in (3) is injected to nerve trachea shaping dies, with the two ends of the fixing nerve trachea shaping dies of little iron clamp; Under miniature velometer, in the mode of fishing by nerve trachea shaping dies direct-axis to 2 × 10
-5the speed of m/s slowly immerses in liquid nitrogen, and the SPIONs-collagen-chitin gel dirty solution that injects nerve trachea shaping dies is carried out to the processing of stranguria of cold type solid, immerses completely after liquid nitrogen until nerve trachea shaping dies, continues to retain 12h in liquid nitrogen; The SPIONs-collagen-chitin gel dirty solution that nerve trachea shaping dies is injected together with inside together proceeds to the refrigerator and cooled Tibetan of-75 ℃;
(5) after cold preservation 24h, nerve trachea shaping dies is taken out from the refrigerator of-75 ℃, remove rapidly iron clamp and the copper pipe at nerve trachea shaping dies two ends; Nerve trachea shaping dies is positioned over to lyophilizing 49h in the Alphal-2 type freezer dryer that pre-cooling is good; Wherein the parameter of freezer dryer setting is :-60 ℃, 100mtorr; Nerve trachea shaping dies after frozen dried be first positioned under vacuum state and be warming up to 0 ℃ of maintenance 6h, being warming up to again afterwards 20 ℃, keeping 45min, removing vacuum state, finally rising to room temperature; Nerve trachea is taken out from nerve trachea shaping dies, by different requirements, be cut into the nerve trachea of various different lengths, the external diameter of nerve trachea cross section is 2.5mm, internal diameter is 1.5mm, prepares SPIONs-collagen-chitin magnetic Biodegradable nerve conduit.
(6) genipin taking is mixed with dehydrated alcohol, the quality of genipin accounts for 1% of mixed solution gross mass; Genipin and the dehydrated alcohol mixed solution of the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit making in (5) being put into preparation carry out crosslinking Treatment, crosslinking time is 48h, and the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of every gram adds genipin and the dehydrated alcohol mixed solution of 20ml; After crosslinking Treatment, SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is cleaned to 35min repeatedly with the ethanol that deionized water and mass percentage concentration are 95%; SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after cleaning is placed under room temperature and is dried one week, finally use
60co irradiates the disinfection of SPIONs-collagen-chitin magnetic Biodegradable nerve conduit, the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after being sterilized.
Embodiment 3
(1) prepare respectively the Fe of 0.1mol/L
3+and Fe
2+solution, under logical condition of nitrogen gas, according to Fe
3+: Fe
2+mol ratio is that 2:1 mixes, and mechanical agitation 4h, fully mixes it.Continuing under logical condition of nitrogen gas, at the NaOH of mixed solution and dripping 1mol/L solution, until mixed solution pH=10 obtains black precipitate.Then heating in water bath 2h, then leaves standstill after 5h, removes the supernatant, by the particle decompress filter generating, repeatedly wash with distilled water and dehydrated alcohol, wash away the foreign ion of particle surface, by the particle vacuum drying at 50 ℃ obtaining, grinding is sieved, and obtains SPIONs.
(2) take respectively type i collagen albumen and chitosan, the quality of type i collagen albumen and chitosan is respectively 20mg, 80mg, takes the SPIONs20mg making in (1).
(3) add 1ml glacial acetic acid by every 20mgI collagen type, glacial acetic acid is mixed with type i collagen albumen, make type i collagen albumen dirty solution through the dissolution process of 26h; Add the glacial acetic acid of 1.2ml by the chitosan of every 20mg, the glacial acetic acid solution measuring is mixed with chitosan, through the dissolution process of 26h, make chitosan dirty solution; The type i collagen albumen dirty solution of preparation in (2) is mixed with chitosan dirty solution, then add 5mgSPIONs, under 6 ℃ of conditions, constant temperature stirs 120min, and mixing speed is 15000rpm, obtains mixing dirty solution; Mixing dirty solution is first carried out to evacuation and process afterwards standing 14h again, obtain SPIONs-collagen-chitin gel dirty solution.
(4) the SPIONs-collagen-chitin gel dirty solution in (3) is injected to nerve trachea shaping dies, with the two ends of the fixing nerve trachea shaping dies of little iron clamp; Under miniature velometer, in the mode of fishing by nerve trachea shaping dies direct-axis to 2 × 10
-5the speed of m/s slowly immerses in liquid nitrogen, and the SPIONs-collagen-chitin gel dirty solution that injects nerve trachea shaping dies is carried out to the processing of stranguria of cold type solid, immerses completely after liquid nitrogen until nerve trachea shaping dies, continues to retain 14h in liquid nitrogen; The SPIONs-collagen-chitin gel dirty solution that nerve trachea shaping dies is injected together with inside together proceeds to the refrigerator and cooled Tibetan of-70 ℃;
(5) after cold preservation 24h, nerve trachea shaping dies is taken out from the refrigerator of-70 ℃, remove rapidly iron clamp and the copper pipe at nerve trachea shaping dies two ends; Nerve trachea shaping dies is positioned over to lyophilizing 50h in the Alphal-2 type freezer dryer that pre-cooling is good; Wherein the parameter of freezer dryer setting is :-60 ℃, 100mtorr; Nerve trachea shaping dies after frozen dried be first positioned under vacuum state and be warming up to 0 ℃ of maintenance 6.5h, being warming up to again afterwards 24 ℃, keeping 60min, removing vacuum state, finally rising to room temperature; Nerve trachea is taken out from nerve trachea shaping dies, by different requirements, be cut into the nerve trachea of various different lengths, the external diameter of nerve trachea cross section is 2.5mm, internal diameter is 1.5mm, prepares SPIONs-collagen-chitin magnetic Biodegradable nerve conduit.
(6) genipin taking is mixed with dehydrated alcohol, the quality of genipin accounts for 1% of mixed solution gross mass; Genipin and the dehydrated alcohol mixed solution of the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit making in (5) being put into preparation carry out crosslinking Treatment, crosslinking time is 50h, and the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of every gram adds genipin and the dehydrated alcohol mixed solution of 20ml; After crosslinking Treatment, SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is cleaned to 35min repeatedly with the ethanol that deionized water and mass percentage concentration are 95%; SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after cleaning is placed under room temperature and is dried one week, finally use
60co irradiates the disinfection of SPIONs-collagen-chitin magnetic Biodegradable nerve conduit, the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after being sterilized.
In embodiment 1,2,3, the primary raw material that preparation SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is used is type i collagen albumen, chitosan and Superparamagnetic Iron Oxide nanoparticle.Wherein type i collagen albumen is mainly by fibroblast or as synthetic in: osteoblast, chondroblast with the similar cell in its source, in organism, collagen is synthetic, generally comprise a series of process, it forms precollagen by synthetic collagen molecules in born of the same parents, and then further aggregate into collagen fiber and collagen bundle outward born of the same parents, when type i collagen albumen is cultivated support as cell in vitro, having the effect that promotes cell adhesion and induced growth differentiation, is good cultivation binder.Between collagen and different cell, there is very strong affinity, and between the somatomedin playing a key effect, also there is special affinity in the agglutination of wound, after blood coagulation, can also prevent from again occurring hemorrhage by histio-irritative regeneration and reparation.
Chitosan is the product of chitin N-deacetylation; there is avirulence, non-immunogenicity, without heat source response, the characteristic such as haemolysis not; its biodegradable and good biocompatibility, film property, become a kind of desirable safe and reliable natural bioactive timbering material.
Superparamagnetic Iron Oxide nanoparticle, owing to thering is special magnetic conductance tropism, superparamagnetism, and surface can connect the characteristics such as biochemical activity functional group, make its application in fields such as foranalysis of nucleic acids, clinical diagnosis, targeted drug, enzyme and cell fixations obtain development widely, there is superparamagnetism, targeting and good biocompatibility.
The nerve trachea shaping dies that preparation SPIONs-collagen-chitin magnetic Biodegradable nerve conduit adopts, as shown in Figure 2, include hollow silica gel tube 8, the two ends of hollow silica gel tube 8 are respectively connected with a hollow copper pipe 9 that size is identical, cavity in hollow silica gel tube 8 is a solid stainless steel tube 6, between solid stainless steel tube 6 and hollow silica gel tube 8, be filled with SPIONs-collagen-chitin gel dirty solution 7, wherein, hollow copper pipe 9 is of a size of: long 2cm, cross section external diameter is 2.5mm, and cross sectional inner diameter is 1.5mm; Hollow silica gel tube 8 is of a size of: long 10cm, and cross section external diameter is 3.0mm, cross sectional inner diameter is 2.5mm; Solid stainless steel tube 6 is of a size of: long 10cm, cross-sectional diameter is 1.5mm.
Embodiment 4
Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit of preparing in application electrophysiological technique and the present embodiment 2 is in conjunction with external pulse electromagnetic field (2mT, 20Hz; PulsedElectromagneticField, PEMF) effect of adult SD rats 15mm Sciatic reparation is assessed, and compare in conjunction with the reparation of external pulse electromagnetic field with nerve autograft reparation, collagen-chitin nerve trachea, result is as follows:
SD rat was at the 4th week and the 8th week that implants after same material not, same time point use Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit of the present invention in conjunction with motor nerve conduction velocity (MNCV) recovery rate of external pulse electromagnetic field, wave amplitude recovery rate than collagen-chitin nerve trachea in conjunction with significantly the increasing of external pulse electromagnetic field (P<0.05), it does not have significant difference (P>0.05) compared with autologous nerve group motor nerve conduction velocity recovery rate, wave amplitude recovery rate.
Table 1MNCV and wave amplitude recovery rate
Embodiment 5
The degradation rate of the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of different proportionings
Table 2SPIONs-collagen-chitin nerve trachea degradation rate (%)
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (5)
1. the preparation method of Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit, is characterized in that, its step is as follows:
(1) prepare SPIONs
Prepare respectively the Fe of 0.1mol/L
3+and Fe
2+solution, under logical condition of nitrogen gas, according to Fe
3+: Fe
2+mol ratio is that 2:1 mixes, and mechanical agitation 3～4h, fully mixes it; Then to the NaOH solution of mixed solution and dripping 1mol/L, until mixed solution pH=9～10 obtain black precipitate; After heating in water bath 2h, leave standstill 5h, remove the supernatant, by the particle decompress filter generating, repeatedly wash with distilled water and dehydrated alcohol, wash away the foreign ion of particle surface; By particle vacuum drying at 50 ℃, grinding is sieved, and obtains SPIONs;
(2) prepare SPIONs-collagen-chitin magnetic Biodegradable nerve conduit
Type i collagen albumen dirty solution and chitosan dirty solution are mixed, then add the SPIONs making in (1), at 2～6 ℃, 15000rpm, stir 80～120min, make mixing dirty solution, will mix dirty solution evacuation, leave standstill 10h～14h, make SPIONs-collagen-chitin gel dirty solution; Then be poured in nerve trachea shaping dies, with the two ends of the fixing nerve trachea shaping dies of little iron clamp, under miniature velometer, in the mode of fishing by nerve trachea shaping dies direct-axis to 2 × 10
-5the speed of m/s slowly immerses in liquid nitrogen, and the SPIONs-collagen-chitin gel dirty solution that injects nerve trachea shaping dies is carried out to the processing of stranguria of cold type solid, immerses completely after liquid nitrogen until nerve trachea shaping dies, continues to retain 10h～14h in liquid nitrogen; The SPIONs-collagen-chitin gel dirty solution that nerve trachea shaping dies is injected together with inside together proceeds to the refrigerator and cooled Tibetan of-80 ℃～-70 ℃, after cold preservation 24h, take out, remove rapidly iron clamp and the copper pipe at nerve trachea shaping dies two ends, be positioned over afterwards lyophilizing 48h～50h in the freezer dryer that pre-cooling is good, wherein the pre-cooling parameter of freezer dryer setting is :-60 ℃, 100mtorr; Nerve trachea shaping dies after frozen dried be positioned under vacuum state and be warming up to 0 ℃, keeping 5.5h～6.5h, being warming up to again afterwards 20 ℃～24 ℃, keeping 30min～60min, removing vacuum state, finally rising to room temperature; Finally nerve trachea is taken out from nerve trachea shaping dies, obtain SPIONs-collagen-chitin magnetic Biodegradable nerve conduit;
(3) disinfect
The SPIONs-collagen-chitin magnetic Biodegradable nerve conduit making in (2) is put into genipin and dehydrated alcohol mixed solution carries out crosslinking Treatment, crosslinking time is 46h～50h, and the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit of every gram adds genipin and the dehydrated alcohol mixed solution of 20ml; After crosslinking Treatment, SPIONs-collagen-chitin magnetic Biodegradable nerve conduit is alternately cleaned to 25min～35min with the ethanol that deionized water and mass percentage concentration are 95%; Then be placed under room temperature and be dried one week, finally use
60co irradiates SPIONs-collagen-chitin magnetic Biodegradable nerve conduit, disinfection, the SPIONs-collagen-chitin magnetic Biodegradable nerve conduit after being sterilized.
2. preparation method according to claim 1, is characterized in that, in step (2), described type i collagen albumen dirty solution, its preparation method is that every 20mgI collagen type adds 1ml glacial acetic acid, through the dissolution process of 22h～26h, make type i collagen albumen dirty solution; Described chitosan dirty solution, its preparation method is that every 20mg chitosan adds 1.2ml glacial acetic acid, through the dissolution process of 22h～26h, makes chitosan dirty solution.
3. preparation method according to claim 1, is characterized in that, in step (2), type i collagen albumen dirty solution and chitosan dirty solution are that 0.25～4:1 mixes according to the mass ratio of type i collagen albumen and chitosan; The quality of the SPIONs adding is 5～20% of type i collagen albumen and chitosan gross mass.
4. preparation method according to claim 1, is characterized in that, in step (3), in genipin and dehydrated alcohol mixed solution, genipin accounts for 1% of mixed solution gross mass.
5. the Superparamagnetic Iron Oxide nanoparticle-collagen-chitin magnetic Biodegradable nerve conduit making according to the arbitrary described preparation method of claim 1～4.
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| CN104225680A (en) * | 2014-09-18 | 2014-12-24 | 中国人民解放军第四军医大学 | Compound seed cell repairing method adopting novel functional magnetic induction nerve scaffold |
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| CN104225680A (en) * | 2014-09-18 | 2014-12-24 | 中国人民解放军第四军医大学 | Compound seed cell repairing method adopting novel functional magnetic induction nerve scaffold |
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| CN107740171A (en) * | 2017-09-14 | 2018-02-27 | 浙江大学 | Magnetic response mineralized collagen coating of structure-controllable for medical metal implant surfaces and preparation method thereof |
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