CN101072593B - Methods and apparatus for enhanced growth of peripheral nerves and nervous tissue - Google Patents
Methods and apparatus for enhanced growth of peripheral nerves and nervous tissue Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61B17/00—Surgical instruments, devices or methods
- A61B17/11—Surgical instruments, devices or methods for performing anastomosis; Buttons for anastomosis
- A61B17/1128—Surgical instruments, devices or methods for performing anastomosis; Buttons for anastomosis of nerves
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/043—Proteins; Polypeptides; Degradation products thereof
- A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
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Abstract
A medical device comprises a tubular body having a lumen and a long axis; and a plurality of silk elements laid substantially parallel along the long axis of the lumen of the tubular body. A method of manufacturing the medical device comprises forming the tubular body and introducing the silk elements into the lumen of the tubular body so as to lie substantially parallel along the long axis of the lumen of the tubular body. The device can be used in a method for the regeneration of nerve cells comprising the implantation of a medical device at a site for regeneration of nerves.
Description
Technical field
The present invention relates to be suitable for a kind of device that promotes peripheral nervous and central nervous tissue healing, and structure and using method.
Background technology
According to the position, the peripheral nerve injury that is caused by wound or surgical operation can cause the forfeiture feeling and move.Speed and the degree of rehabilitation are slow, and be normally incomplete and variable.Final afunction makes the patient very painful, and for example the damage of cavernous nerves of penis causes impotence.Spinal cord transection even have more serious consequence, and still recover the method that the nerve through Spinal Cord connects.The consequence of spinal cord injury comprise paralysis and the dermatotome of voluntary muscle consumption and transverse section afterbody supply feel completely lose.The forfeiture of urethra and Hyrtl's sphincter control ability causes dual incontinence.In addition, cross-section in the neck section tumor of spinal cord vertebra causes transeptate paralysis, this be because diaphragm be subjected to from the 3rd to the fifth cervical vertebra bone every innervation.The cross-section paralysis (arranged by thoracic nerves) that also causes Intercostal muscle in the neck section tumor of spinal cord vertebra.Therefore neck section tumor of spinal cord is cross-section stops respiratory movement, has potential lethal effect.Therefore, be necessary the patient with this type of damage is ventilated to keep the life of their remainder.In addition, degenerative disorders for example parkinson disease and multiple sclerosis causes the degeneration of central nervous system's nerve tract, and often causes weak and highly painful situation to occur, the dyskinesia for example, and sensory deprivation and awakening reduce.
To a certain degree recovery is common and by the regeneration of aixs cylinder with reconnect generation behind the peripheral nerve injury.Yet, after human spinal cord transection, do not observe heavily connection, and think and seldom reconnect generation in the nerve tract major injury hindbrain.
Various trials have correspondingly been made to promote the reparation of neural and nerve tract.
Three kinds of methods have been used for the peripheral nervous of surgical intervention damage: the directly again stitching of butt end; Autotransplantation substitutes; Various through being designed for the neural natural or synthetic materials that reconnect of guiding with use.First method has limitation.Make neural broken ends of fractured bone close enough with sew up can be described as impossible, even and may, the scar tissue that is caused by damage and surgical operation can stop the crosslinked of aixs cylinder and anastomotic position and sometimes cause being known as the entanglement of neuromatous nervous tissue.In the long situation of breach, autotransplantation is at present best selection, for example from the patient not damage location obtain one section sural nerve and sew up to replace the suture at nerve injury position.The shortcoming of the method comprises the sensory deprivation that donor tissue excision transplanting causes, pain increases, and the impracticability of excising sufficiently long graft for replacing long damage location strengthens the risk and extra cicatrix of transplanting the excision site infection.In addition, described restorative procedure is consuming time and a large amount of technical staff of needs.
Attempt multiple interchangeable neural transplantation material, comprised the empty perineurium that is expected to overcome neural autotransplantation shortcoming.Aim to provide the axon growth passage although attempt design, and the prevention fibroblast is invaded and neural neoplastic cuff, conduit, big envelope and pipe have very long history, these all do not obtain gratifying effect at present.
The collagen tube that is obtained by the decalcification skeleton is used in the earliest trial that is provided for the conduit of Neurotherapeutic.This causes the irreversible fiber adhesion of function usually.Attempt subsequently a large amount of other tissues and material, comprised vascular, binder, fat, muscle, fibrin, parchment paper, gelatin and various metal.The fibrosis that is caused by tissue injury and embedded material causes the failure of described device.The non-again absorbing material that uses needs further surgical operation to remove them usually.
Provide the early stage material of conduit to propose to improve to these peripheral nervouss for damage.For example, Ducker etc. are at Vol.28, and Journal ofNeurosurgery has reported the application of the silicone rubber sleeve that is used for the peripheral nervous reparation among the pp.582-587 (1968).Midgley etc. are at Vol.19, SurgicalForum, pp.519-528 (1968) Zhong He Lun Debao etc. at Vol.41, Journal ofNeuropathologyand Experimental Neurology has reported among the pp.412-422 (1982) to be used for the neural silicone rubber cover of repairing.Molander etc. are at Vol.5, Muscle﹠amp; Nerve, reported among the pp.54-58 (1982) can biological resorbent PLGA (polyglactin) webmaster application.Uzman etc. are at Vol.9, and Journal of Neuroscience Research discloses the application of semi-permeable acrylic acid copolymer property management in neuranagenesis among the pp.325-338 (1983).Empty perineurium pipe also is used as the passage of Bridging nerve breach, as at Y.Restrepo etc., (Microsurgery 4:105-112,1983) in " Fascicular nerve Graft Using An Empty perineurial Tube:An ExperimentalStudy in the Rabbit " and at Y.Restrepo etc., disclosed in " the Empty perineurial Tube Graft Used to repair A Digital Nerve:A First CaseReport " of (Microsurgery 6:73-77,1985).Nyilas etc. are at Vol.29, Transactions Am.Soc.Artif.InternalOrgans, and that has reported polyester and other polymer among the pp.307-313 (1983) can biological resorbent neural guiding channel.Joseph M.Rosen etc. disclose the application of polyglycolic acid as artificial neuron bundle film at Ann.Plast.Surg.11 among the pp 397-411.
United States Patent (USP) 6716225 has been instructed the application by biocompatibility and the longitudinal ridge hollow conduit can biological resorbent biopolymerization material made.United States Patent (USP) 5,026,381,4,963,146 and United States Patent (USP) 5,019,087 instructed the many walls hollow conduit with micro-pore wall by type i collagen preparation.United States Patent (USP) 6676675 discloses for stimulating neural regeneration, has sheet or the pipe of longitudinal ridge or contains the application of the pipe of poly-(vinyl alcohol).United States Patent (USP) 6,589,257 disclose by polyglycolic acid, polylactic acid, poly-(glycolic-lactic acid) copolymer or relevant synthetic again absorbing material preparation and are coated with gelatin or collagen and contain the application of the fibriilar again absorption tube of longitudinal register cross-linked rubber that is coated with laminin，LN.United States Patent (USP) 6,090,117 have instructed the application of similar pipe, and wherein the space-filling between the collagen fiber has the matrix gel that contains collagen, laminin，LN, Heparan sulfate mucin, nestin and somatomedin.United States Patent (USP) 5,834,029 has instructed the biocompatibility semipermeability conduit that contains any substrate of deriving in known three laminin，LN sequences important in Cell binding.
Up to now, following three kinds of peripheral nervous regeneration conduits have obtained the FDA approval and have been used for the neural sleeve pipe (Salubria Nerve Cuff) of clinical experiment: Salubria, and Integra Neurosciences can absorb collagen tube and Neurogen neurocele (Neurogen Neurotube) again.These devices promote the ability of peripheral nervous healing to leave very large room for improvement.None is entirely satisfactory for repairing peripheral nervous for these devices or above-mentioned material or method, and none proof has the purposes of promotion central nervous system (CNC) axon regeneration up to now.
Summary of the invention
The present invention relates to a kind of implantable device of eliminating or significantly reducing the most shortcomings relevant with the prior art of be intended to regenerate peripheral nervous and maincenter white matter.
One aspect of the present invention provides a kind of medical treatment device, and it comprises:
Body with inner chamber and major axis; With
A plurality of thread composition (silk elements) along the substantially parallel placement of major axis of tube cavity.
Described body can contain can resorbent material.For example, protein or based on the material of protein, it can be natural or synthetic.The synthetic material that relates to comprises by chemical method and the synthetic material of recombinant DNA technology method.The composite construction that preferably contains the fiber that places substrate.The tube wall of described device can contain silk fiber and suitable protein material.For example, Antherea pernyii (Antherea pernyi Guerin-Meneville) silk and Bombyx mori (silkworm) albumen substrate of regenerating.
The redissolution fibroin that described substrate can for example be obtained by Bombyxmori Linnaeus or non-Bombyxmori Linnaeus by fibroin, or the natural silk heart protein that is obtained by Bombyxmori Linnaeus or non-Bombyxmori Linnaeus forms.For example, Antherea pernyii (Antherea pernyi Guerin-Meneville) silk.Described substrate can by crosslinked stable, for example be used formaldehyde gas, glutaraldehyde, citrate ion, ribose, Biformyl or genipin (genipin).
The fiber that forms body can comprise that spiral type is placed or the silk fiber of braiding.
Thread composition in the inner chamber preferably is separated from each other with the distance between about 1 μ m and the about 100 μ m.
Device of the present invention can suitably have from per 10,000 μ m
2About 1 to about 30, preferred per 10,000 μ m
2About 1 to about 10, or per 10,000 μ m
2The about 5 thread composition packed densities to about 10 scopes.
According to this aspect of the invention, described device can comprise having from about 1.0 to about 2.5mm, preferably from about 1.5mm to about 2.0mm, or from about 1.0mm to about 1.5mm, and the about body of the external diameter of 1.4mm or 1.5mm most preferably.
The wall of described body can have from about 250 μ m to about 750 μ m, suitably from about 300 μ m to about 600 μ m, and the thickness of the value about can preferred 300 to 350 μ m.
The length of described device can be from about 0.5mm to about 150mm.Can select to be suitable for to have the described device length of the nerve that described device to be used repairs.For example, described device can be used for repairing than nervelet, and described device can be suitably from about 1.0mm to 5.0mm, or 1.5mm to 2.5mm, or 1.0mm to 2.0mm.For the reparation of large scale nerve, described device can correspondingly strengthen, for example from about 10mm to 20mm.It can be identical size that the length of 20mm to 130mm and device of the present invention are successfully used in the autotransplantation of people's nerve.
Described thread composition can have from about 5 μ m to about 50 μ m, suitably from the diameter of about 10 to 20 μ m.
In some preferred embodiment of the present invention, described device can be 2.0mm length and the diameter with 0.5mm.
Thread composition or fiber that described device uses can comprise mulberry silk, non-mulberry silk, spider dragline silk and the filament (filament) that is spun into by recombinant fibroin or albumen analog.Non-mulberry silk particularly preferably.Suitable example is Antherea pernyii silk.
Described thread composition is the form of bar silk (sliver silk) or filature (reeled silk) or twisted silk (twisted silk) typically.Thread composition relatively device wall is substantially longitudinally arranged easily.
In order to promote cell migration, described thread composition preferably has main fibroin (principal silk protein), it comprises the triplet RGD of at least eight repetitions, and wherein the triplet RGD of at least some repetitions preferably is positioned to be close to the corner of main fibroin structure or the corner of expectation.Main fibroin preferably has such site: be blocked to regulate cytoadherence from one or more arginine groups of these described major protein in site.Described blocking-up can be by one or many deaminizating, sulphation, one-tenth amide and use the blocking-up of cyclohexanedione to reach.
Also can use easily blocker to generate from the density gradient of the distal-to-proximal free arginine group of described device.This can reach by slowly and little by little the near-end of described device at first being reduced in the blocker solution.Alternatively, before introducing the inner chamber of body, the gradient of free arginine group can be introduced in the described thread composition thread composition.This type of gradient can be linear or nonlinear.Described gradient can promote the process that neurocyte and silk fiber separate at described device near-end.
In order to promote neurocyte to enter and to leave the process of described device, the thread composition of the basic longitudinal register of preferred arrangement so that they than one or two of described device inner chamber body terminal outstanding 0.1 to 10mm.
Particularly preferably be with described thread one-tenth be placed in contain can the inner chamber substrate of resorbent biocompatible polymer in, the example of described biocompatible polymer such as hydrogel, for example alginate or hyaluronic acid or casein, described alginate or hyaluronic acid have or do not have polylysine.The component that also can have other, extracellular matrix (ECM) for example is such as fibronectin splicing variants (fibronectin) and/or laminin，LN (laminin).These materials can add in the inner chamber substrate in the conduit or be coated on the filament (silk filament) of the silk in the inner chamber substrate.
A second aspect of the present invention comprises the method for preparing a kind of medical treatment device, and described method comprises in the inner chamber that forms body and thread composition is introduced body so that the substantially parallel placement of its major axis along tube cavity.
The formation of described body can further comprise the steps:
Preparation forms the former of body;
Fiber is put into described former;
Substrate is applied to fiber to form complex; With
Remove described former.
The formation of body also can comprise crosslinked substrate.
Also introduce the inner chamber matrix components between preferred thread composition in tube cavity.
Described thread composition can use enveloping agent solution to wash to remove possible pollutant, such as transition metal ions that may be poisonous; The example of chelating agent such as ethylenediaminetetraacetic acid (EDTA) sodium salt.Other chelating agent also can use.Preferably, with described degumming of silk.This can be by reaching with the Protease Treatment silk, the example of protease such as subtilisin, but other gentle proteolytic enzyme also can use.Then after handling with described enzyme flush away.
A third aspect of the present invention provides a kind of method for nervous cell regenerating, and it comprises a kind of medical treatment device of implanting according to first aspect present invention.
The present invention relates to a kind of implantable device, it can be eliminated or significantly reduce the many shortcomings relevant with the prior art of be intended to regenerate peripheral nervous and maincenter white matter.
More specifically, this device can by biocompatibility, can resorbent material construction, can be neural axon to described material, execute ten thousand Schwann Cells (Schwann cell) and the neurogliocyte growth provides the ability of binding site to regulate.
Preferred structure according to device of the present invention comprises a multiple tube, described silk multiple tube both ends open and within it the chamber contain the filament (silk filaments) of directed silk.The wall of described multiple tube generally has the thickness of basic homogeneous, and according to position to be implanted, and has from 0.1 to 25mm, preferably the diameter of from 250 to 750 mu m ranges.
The silk multiple tube contains the bar filament (non-mulberry silk sliver filament) of meticulous non-mulberry silk usually, regeneration substrate is placed and placed to its helicon mode with about 55 ° intersection angle, described regeneration substrate is the redissolution fibroin that is obtained by Bombyxmori Linnaeus or non-Bombyxmori Linnaeus, but should be appreciated that other can resorbent biocompatibility filament (filaments) and can replace use by resorbent biocompatible matrix.In further embodiment, described substrate contains in fact the natural silk heart protein by the sericterium extraction of Bombyxmori Linnaeus or non-Bombyxmori Linnaeus.Described substrate is stable by covalent cross-linking.In one embodiment, this is to reach by using formaldehyde gas to process, but other cross-linking agent also can use.In further embodiment, the silk multiple tube can be by using braider directly from 1 or the braiding fiber tube of the 7-13 silk non-mulberry silk preparation of coming unstuck and making.Described braiding fiber tube processes to form substrate between braiding fiber tube silk thread with the solution that one or more can resorbent biocompatible polymer, and the example of described biocompatible polymer is such as Bombyxmori Linnaeus or the non-mulberry silk of regeneration.
Described silk multiple tube contains the filament (filaments) of non-mulberry silk, and it places and contains hyaluronic inner chamber substrate (other inner chamber host material comprises that hydrogel is such as the hyaluronic acid with polylysine, the alginate that has or do not have polylysine and casein).Described filament is substantially longitudinally to locate and trimmed by the end with described pipe with respect to the major axis of silk multiple tube.In further embodiment, filament (silk filamens) and the inner chamber substrate of described silk or extend beyond the terminal short distance of described pipe or short distance termination before described pipe end.Described filament generally is filled in together in described tube cavity, and its density is per 10,000 μ m
21 to 10 filament is arranged, and average headway is about 30 to 100 μ m between filament, but the filling of less dense also can be used.
In further embodiment, described device can comprise one or more bioactive materials in addition.Described material is optional from growing the factor, cytokine, antibiotic, immunosuppressant, steroid, nonsteroidal antiinflammatory drug (NSAIDs).Described somatomedin can be nerve growth factor.For example, nerve growth factor can be added in the filament inner chamber substrate on every side.The nerve growth factor that can be used for this purpose is included in the peripheral nervous somatomedin (NGF) that situation that described device is used for promoting peripheral nervous or nervus centralis nutrient protein-3 (NT3) to restore uses and the Brain Derived Neurotrophic Factor (BDNF) of using in the situation that described device is used for brain or spinal cord.It should be understood that and other can be promoted medicine or the factor that neuranagenesis or inhibition glioma or fibrosis form to add in the filament inner chamber substrate on every side.Should also be noted that also and medicine and other factor that improves described apparatus function can be added in the substrate of a multiple tube.For example, antibiotic, immunosuppressant, steroid or nonsteroidal antiinflammatory drug (NSAIDs).Other bioactive substance comprises, but be not limited to, promote the cAMP promoter (such as rolipram or db-cAMP) of regeneration, reduce molecule such as TFG β antiserum and/or the chondroitinase of cicatrization, or reduce the molecule of myelin inhibitory action (such as anti-Nogo treatment).
Also can imagine and cell can be added in the device of the present invention, as help neuranagenesis long and/or the neural stem cell myelin forms executes ten thousand Schwann Cells or Olfactory essheathing cell (olfactory ensheathing cells, OECs).The cell type that also can add on request other.Described cell can be the endogenous cell from the patient who remains to be implanted described device, or described cell can be the foreign cell from external source, such as the cell of growing in the culture.In other words, described cell can be from body or non-from body with respect to patient's immune system.
Nerve trachea section prepared in accordance with the present invention also can be inserted in brain or the spinal cord to promote the reparation of damage or degeneration white matter.They can use with guidance and the promotion in conjunction with cell culture technology and are connected with central nervous system's suitable part by the implantable neural cell that the neuroblast stem cell of implanting forms.
Bioactive substance or cell are being added in the situation of apparatus of the present invention, can use at an end (for example near-end) of described device with material or the cell of another end (for example far-end) high concentration of comparing and set up Concentraton gradient (linear or non-linear).Alternatively, the bank of material or cell can only add an end of described device.
In further embodiment, can omit described silk multiple tube, and directly implant the directed filament (silk filaments) that places again absorption base.
Select the device of suitable diameter to implant according to the diameter of nerve to be repaired or white matter bundle.Use sharp blade or other instrument that described device is downcut suitable length.In one embodiment, by the one or many stitching described device is kept in position.In another embodiment, can use Fibrin Glue that described device is kept in position.Described device can dryly be implanted or soak five minutes to five hours in suitable normal saline solution before use.
Therefore, according to the present invention, provide a kind of device that is used for as mentioned above nervous cell regenerating.Described device can have concrete application in spinal nerves cell or peripheroneural regeneration.
Therefore, device of the present invention has practicality in nerve of animal body or many traumatic nerve injuries or injury in treating.Therefore the present invention can be applicable to people's medical science and veterinary.The nerve of human maximum is sciatic nerve, and the diameter of its maximum is only less than 20mm.The length of the appropriate device of using in people's medical science can change, but according to the nerve injury that the needs that clinical observation is arrived are treated, is generally from about 10mm to about 20mm.
Therefore, device of the present invention is used in to rebuild between the nerve of the damage of central nervous system or peripheral nervous system or infringement and connects.The invention provides cell/extracellular environment of existing before a kind of means utilization and the nerve injury roughly similar environment rebuild nerve or spinal cord.In the situation of peripheral nerve injury, this comprises that myelin executes ten thousand Schwann Cells, and it also requires the appropriate electrical conductance of electric pulse in aixs cylinder and the extracellular matrix molecule except other, the example of extracellular matrix molecule such as laminin，LN.Therefore, device of the present invention can comprise extracellular matrix components (ECMs) in addition, and for example fibronectin splicing variants and/or laminin，LN, and foreign cell are for example executed ten thousand Schwann Cells (Schwann cells).
Medicable peripheral damage type is those wherein neural types of having damaged according to the present invention, wherein nerves transected may occur.Described damage can be described as neurotmesis.The clinical definition of this type of damage is also mentioned with fourth or the 5th degree neurotmesis under " Sunderland System ".In fourth neurotmesis, all nerves and supporting element interrupt, and epineurium can be complete and described neural expansion.In the 5th degree neurotmesis, have and follow the fully cross-section of neural asynechia.
The preferred feature that is used for second aspect present invention and aspect subsequently is the change (mutatis mutandis) about first aspect.
Description of drawings
The present invention will further specify by the mode of reference following examples and accompanying drawing now, and described embodiment and accompanying drawing only provide for the purpose of explanation, but are not considered as limiting the present invention.With reference to a plurality of accompanying drawings, wherein:
What Fig. 1 showed Hoechst (nihexyn) dyeing executes the nuclear dorsal root ganglion of ten thousand Schwann Cells (DRG) graft, shows manyly to execute ten thousand Schwann Cells and move out and adhere on the silk fiber from graft.
Fig. 2 also shows the DRG graft of Hoechst dyeing, shows manyly to execute ten thousand Schwann Cells and move out and adhere on the silk fiber from described graft.In addition, observe the immunoreactive aixs cylinder of GAP-43 (arrow) along each silk fiber extension and go back in some cases each fiber of bridge joint.
Fig. 3 shows the sciatic nerve graft of Hoechst labelling and observes the GAP-43 immunoreactivity and execute ten thousand Schwann Cells and move out and adhere on the silk fiber from described graft.
Fig. 4 shows executing the nuclear adult DRG of ten thousand Schwann Cells culture and adhering to the GAP-43 immunoreactivity aixs cylinder of each silk fiber that is coated with laminin，LN of Hoechst labelling.
Fig. 5 shows executing the nuclear adult DRG of ten thousand Schwann Cells culture and adhering to the GAP-43 immunoreactivity aixs cylinder of each silk fiber that is coated with laminin，LN of Hoechst labelling.
Fig. 6 shows the cell marking of using neuroglia specific marker thing S100, and it is relevant with silk fiber to show that most S100 immunoreactivities is executed ten thousand Schwann Cells.
Fig. 7 shows the cell marking of using GAP-43 and Neuron-specific label β III tubulin, shows that the nucleus (arrow) of some Hoechst labelling and meticulous GAP-43 immunoreactivity process are the neurocyte origins.
Fig. 8 shows silk fiber and spinal cord (white fiber, Fig. 8, a left side).Use the labelling of astrocyte label GFAP to show common described silk fiber closely near adjacent complete spinal cord, between host's spinal cord and implant, have seldom or without slough (Fig. 8, the right side).
The macrophage that Fig. 9 is presented in the silk fiber bundle of implanting spinal cord and surrounding tissue is invaded.
Figure 10 shows that the growth of use aixs cylinder label PGP9.5 labelling enters a graft and the aixs cylinder (arrow) parallel with the silk fiber direction.
Figure 11 shows the aixs cylinder confocal microscopy photo along the PGP9.5 labelling of growing between each silk fiber and each silk fiber.The left side picture disply uses the aixs cylinder of arrow labelling, and the right side picture disply is by the silk fiber of arrow labelling.
Figure 12 shows the double labeling that uses aixs cylinder label PGP9.5 and Shi Wan Schwann Cells label p75.Left side picture disply aixs cylinder, the right side picture disply is executed ten thousand Schwann Cells.
Figure 13 is presented at the silk fiber conduit of arranging in the catheter core.
Figure 14 shows the guide-tube structure of implanting spinal cord, and each epitheca wall looks like the slice (arrowhead) that little silk fragment bar (arrow) and internal core are portrait orientation.
Figure 15 shows the silk fiber similar (referring to Fig. 9) that macrophage is pricked with bundle on apparent and degree the intrusion of silk conduit.In addition, in 8 weeks after implanting, can be observed macrophage and be gathered in around each silk fiber.
Figure 16 is presented at the aixs cylinder of the PGP9.5 dyeing of growing between silk fiber.
Figure 17 shows the double labeling that uses aixs cylinder label PGP9.5 and Shi Wan Schwann Cells label p75, the aixs cylinder that shows growth and maturity with execute between ten thousand Schwann Cells substantially corresponding.Left side picture disply aixs cylinder, the right side picture disply is executed ten thousand Schwann Cells.
Figure 18 shows the stereoscan photograph of nerve trachea according to an embodiment of the invention.
The preparation of apparatus of the present invention
Some or all step below the preparation of described nerve regeneration conduit needs: preparation former; Fiber is put into former; The applying waterborne protein solution forms multiple tube; Shift out from former; Wax removing; Crosslinked described multiple tube; In the filament inlet tube with directed silk; Between intraluminal filament, add matrix components; Introduce extracellular matrix components for example fibronectin splicing variants and/or laminin，LN; Introduce nerve growth factor, depyrogenation and sterilization; Add nerve growth factor; Dry described device is also cut to Len req.Although above operating sequence has obtained good effect, the order of some step is not crucial.For example crosslinked step can add at the filament with silk described Guan Zhonghou execution; And the step of depyrogenation can be carried out before or after adding NGF; If sterilization is when implementing by radiation gamma then can adds NGF before sterilizing.
The preparation of cylinder former
Former is prepared as follows.The simplest method of preparation former is to use stainless steel tube or the stainless steel strip of suitable diameter.Clean and polish for these before use.Described pipe can skid off from described former after employed host material drying easily.For the former of minor diameter, at first will be relatively hard straight steel wire coating is with the paraffin that melts when the above relative low temperature of room temperature or the thin layer of some other material.Coating even can be by obtaining in the wax that steel wire is dipped vertically into fusing.The external diameter of wax coating defines the internal diameter (diameter of inner chamber) of the pipe that forms thereon on the former.The larger diameter former that diameter reaches 30mm can prepare by casting or machine-building wax bar or with the cylinder that wax is coated with suitable diameter.Other method that the preparation former is arranged wherein can be from around shifting out described former the described former fiber tube inner chamber that form, that this area workman can obtain easily.
Fiber is put on the former
The filament of the silk of following three types is preferably used for the fiber strengthened in the tube wall that forms the nerve regeneration conduit outer wall: strand (silk sliver) (combing out the filament that comes unstuck with combing by piques); The sub-thread silk that comes unstuck by the silk preparation of from a cocoon, extracting out at every turn; The 7-13 silk 20-37 danier silk that comes unstuck by the silk preparation of from 7-13 cocoon, extracting out at every turn.Used the tussah silk from Antherea pernyi Guerin-Meneville (antheraea pernyi), but the fibroin of filament, restructuring or the regeneration of natural any mulberry silk of extruding or non-mulberry silk or silk can replace using.
The 7-13 silk 20-37 danier silk that comes unstuck has provided good effect.At first use the described silk of dilute solution washing of ethylenediaminetetraacetic acid (EDTA) sodium salt to remove possible pollutant, transition metal ions that for example may be poisonous.Also can use other chelating agent.Preferably, described silk is come unstuck.This can pass through to use for example described silk acquisition of subtilisin processing of protease, but also can use other gentle proteolytic enzyme.After handling with described enzyme flush away.
The Antherea pernyi Guerin-Meneville strand contains a large amount of meticulous parallel filaments, and it also can provide good result.Described parallel filament can grip and be wound in the spiral layers that described former obtains having intersection angle between 40 and 50 degree on every side between thumb and finger.A kind of wind can be used for this operating process of mechanization.Alternatively, the mode that sub-thread or 7-13 silk line can spirals is wound on the described former.Filament for continuous silk can use a kind of simple device that spiral layers is wound on the former.This has used the small size motor that drives cylinder former slow circumvolve and its cam follower that silk is dispensed to centrifugal cam on the former.The device that the filament of silk is continued to be wound on the pliable and tough cylinder former is to make up easily.
Alternatively, can use braider from 1 or the direct a kind of braided tube of preparation of 7-13 cocoon degumed silk.Described braided tube can be used for forming silk multiple tube as described below.
The applying waterborne protein solution forms multiple tube
Most protein can be used for supplying the substrate of described silk multiple tube.The solution of the fresh regenerated silkworm that use prepares in the 6.3M lithium bromide water solution by the fibroin powder dissolution that will be purchased (bombyx mori) fibroin 10-40%w/v concentration has obtained good effect.Remove described lithium bromide by thoroughly dialysing in distilled water at 4 ℃.In Dialysis tubing, concentrate described dialysis solution by evaporation or anti-dialysis.Regenerated silk heart protein solution coat with gained when silk thread is still on former obtains non-perforated pipe in silk thread.Dry fibroin solution.Now prepare the regenerated silk of gained/tussah silk multiple tube is shifted out from described former.Also can form described multiple tube in the same solution by immersing with the fibroin solution spraying of regeneration or with described former.The concentrated fibroin solution that is directly obtained by mulberry silk or non-mulberry silk can replace the regenerated silk heart protein to use.Multiple proteins also can replace the regenerated silk heart protein to use.These comprise fibroin white glues, gelatin dilute solution or serum albumin.Other water soluble protein, hyaluronic acid or other biocompatible polymer can replace using.Replace alternatively using the silk layer at former, can use stromatin or other polymer solution by spraying or immerse coating braiding fiber tube.
Shift out from former
In the situation of using the rustless steel former, can easily it be shifted out by skid off described silk multiple tube from described former.For narrow multiple tube, this can use fine forceps to realize.In the situation of the former that uses coating wax, by being melted gradually, the wax on the former or other low melting point coating shift out described silk multiple tube.Perhaps, the former that can use diameter to reduce for example before the silk multiple tube that it is skidded off around it, is removed centronucleus.
Wax removing
In the situation of using wax, can be residual by in benzene, dimethylbenzene or other wax solvent, soaking the wax of removing on described silk multiple tube.
Crosslinked described multiple tube
Following operational approach can be used for the stromatin of crosslinked silk multiple tube.Excessive anhydrous paraformaldehyde is put into the container bottom of sealing and the 0.2ml distilled water is added to the gram of 2 in 0.5 liter of container paraformaldehyde.Put thereon with filter paper covering paraformaldehyde and with described silk multiple tube.Behind sealed container, be heated one hour to 80 ℃.Shift out fiber tube and use warm water thoroughly to wash from described container after the cooling.
In the filament inlet tube with directed silk
The filament of strand is introduced in the silk multiple tube of following drying.At first use the strand threading in pin or the awl of appropriate size.Sack needle is used for larger-diameter silk multiple tube.Then typically the filament of described silk is coated with the hyaluronic acid solution of suitable viscosity.Push through described silk multiple tube and make it fill up the filament of directed silk putting on the pin of line or awl, excessive in the case hyaluronic acid oozes out from the cut ends of pipe.Other the hyaluronic acid that can resorbent gel can replace uses.In one embodiment, omit the use that hyaluronic acid or other can resorbent gels.Such as needs, the pin of putting on line is repeated to push through described silk multiple tube until obtain the suitable packed density of strand filament at tube cavity.Rule of thumb, this can judge by eyes.Alternatively, the accurate measurement of density that the filament of silk is packed in the pipe can followingly obtain: the silk multiple tube that will measure length is weighed, and again weighs after trimming at the filament of introducing silk with them and pipe end.The filament of silk can add or shift out from pipe until the filament weight in the tube cavity meets the requirements.The stereoscopic microscope that the right angle eyepiece graticule is installed is used for measuring the filament number of the every square of mm that crosses tube section.What the scanning electron microscope inspection before experiment is implanted showed the best is that packed density is per 10,000 μ m
210 to 1 filaments, the average headway between filament are about 30 to 100 μ m.
Between intraluminal filament, add matrix components
Inner chamber matrix components between described silk filament be used in the device preparation and all follow-up phases of inserting in the filament of silk is remained on its position, keep appropriate intervals between filament by forming hydrogel simultaneously.They also promote nerve growth to enter in the described device.Multiple biopolymer can be used for providing the substrate of the inner chamber between filament.These hydrogels comprise the hyaluronic acid that has or do not have polylysine, the alginate that has or do not have polylysine, casein, Fibrin Glue, serum albumin and gelatin.Use in case of necessity warm water to prepare these high molecular aqueous solutions.Other solvent can replace water to use.The chamber silk multiple tube that contains the filament of directed silk is dipped in the solution that contains one or more these polymer within it.Can use vacuum to help to infiltrate.In the situation of Fibrin Glue, can at first use the infiltration of fibrin solution to contain the silk multiple tube of the filament of directed silk, then use thrombin solution to infiltrate to start the formation of Fibrin Glue.
Depyrogenation and sterilization
Depyrogenation is implemented before being preferably in and adding nerve growth factor.Preferably contain final concentration 0.1%v/v Tween 20 by use
TM1%v/v dimethyl sulphoxide aqueous solution washing implement.Usually use this solution to implement two to five times washing.Behind the depyrogenation, can in aseptic and pyrogen-free normal saline, wash described device.All contact the glass that is used for depyrogenation or plastic ware or other experimental facilities with solution should be 240 ℃ of bakings at least 2 hours to remove pyrogen.
Introduce bioactive substance such as nerve growth factor (NGF)
A series of bioactive substances for example nerve growth factor can be introduced in the described device.These are included in the peripheral nervous NGF that the situation that be used for to promote peripheral nervous or nervus centralis neurenergen 3 (NT3) to restore in described device uses and the Brain Derived Neurotrophic Factor (BDNF) of using in the situation that described device is used for brain or spinal cord.Nerve growth factor is preferably in when forming between filament the inner chamber matrix components and adds.They can be mixed with the inner chamber matrix solution, then add the silk multiple tube that inner chamber contains the filament of directed silk.
Drying device is also cut to Len req
Described device is removed first unnecessary solution before drying.Multiple drying means be can use, air drying or lyophilization comprised.Preferably downcut the conduit of suitable length from the device of drying.These conduits can directly implant or before implantation in aseptic and pyrogen-free 0.9%w/v saline rehydration.
Implant described device
Cross-section for treating spinal cord transection or part, downcut the device for disc of 2 to 10mm thickness from the filament that contains silk, the prepared silk multiple tube sections of diameter 2-20mm.With the horizontal insertion spinal cord injury of described device position.Tearing under the degree of impairment, can insert conduit to connect spinal cord and the root of tearing.
For promoting peripheral nervous regeneration, conduit is standby by the narrow control of diameter 1-15mm, and described diameter depends on size and the position of nerve to be repaired.Described device should be sewn into the position lightly.
Also the nerve trachea section can be inserted in the brain, be intended to promote to damage or the reparation of the white matter of degenerating.They can use in conjunction with cell culture technology, and suitable part is connected to be intended to implantable neural cell that guidance and the promotion forms by the neuroblast stem cell of implanting and central nervous system.Cell culture technology also can use with spinal cord or peripheral nervous implant.
Device described above provides four advantages that are better than prior art.
The first, described device is owing to following reason has higher tensile property.This unusual tough and tensile non-mulberry silk by the composite attribute that can be used for a multiple tube and described pipe and inner chamber content thereof produces.In addition, the placement of the fiber spiral in the pipe is designed to offer pipe vertically and radially intensity and toughness.Described device body is also further reinforced by its compound structure.
The second, the design of described device so that the migration of aixs cylinder can optimize by device.This is because the packed density of the filament of silk and channel size therebetween are to regulate easily.In addition, the non-mulberry silk that uses in the described device carries the cell adhesion sequence RGD (preferably at least eight) of a plurality of repetitions naturally, comprises that the Cell binding of aixs cylinder is in this.At least the cell adhesion sequence RGD of some repetition is positioned near the corner of the corner of protein or expectation.In addition, be understood that the migration for aixs cylinder, the density of binding site needs carefully control.If can be excessive near RGD position density, then the external surplus of aixs cylinder be combined with silk fiber too firmly and it can not occur from the opposite end of device.On the other hand, if can be too small near RGD position density, then the adhesion of the external surplus of aixs cylinder and the filament of silk not and their abilities of migrating into described device reduce.Therefore, the external surplus of aixs cylinder can be regulated by the RGD position density on the filament that changes silk with the combination of the filament of silk.This can realize in two ways.The natural density at RGD position along with the difference of kind one of every molecule to the range changing of per molecule more than 12.Therefore, can select to have the silk at suitable density RGD position.The RGD group of natural density has obtained good effect in the tussah silk.In addition, as the skilled artisan will appreciate, also might replace the amino density of further regulating the RGD group of arginine ε by come part with gentle retardance.Method including, but not limited to sulphation with become the amide effect.Described group also can block by cyclohexanedione.
The 3rd, described device is stiff when dry and plastifies bending and the tensile property that obtains being similar to natural nerve when moistening.Hardness when drying or part aquation is conducive to insert described device and is sewn into the position, and the pliability when moistening is similar to the pliability of natural nerve.
The 4th, hyaluronic acid derivatives when described device is dry in the inner chamber substrate helps the filament with silk to remain on the appropriate location in the inner chamber, makes the filament in the fine filaments in the described device inner chamber easily described device be downcut and process to required length in free of losses or in without the situation of confusion.In addition, hyaluronic acid has stimulated the inside growth of neurocyte when aquation.
Embodiment 1: the DRG/ silk that separates in the culture
Silk tussah fibre in the initial experiment in vitro proof culture is supported the growth of aixs cylinder by peripheral nervous system (PNS) neurocyte (Dorsal Root Ganglion Neurons) and is also supported adhesion and the migration of PNS sustenticular cell (executing ten thousand Schwann Cells).The DRG cell that the dorsal root ganglion (DRG) of use neonate rat (P3) separates with sciatic nerve implant and rat adult is implemented experiment.
Method
By sucking the CO of high concentration
2To grow up or newborn (P3) rat execution, and use the method (Huang etc., Neuroreport 16:89-93 (2005)) of announcing according to british animal (scientific procedure) bill to cultivate the DRG neurocyte.Dorsal root ganglion is shifted out and cleans, then, in Bottenstein and SatoShi serum-free medium (BSF-2; The streptomycin that in HamShi F-12 basal medium, contains the penicillin of 0.3% bovine serum albumin (BSA), 1%N-2 fill-in and 100 units/ml/100 μ g/ml, all reagent are all from U.S. Life Technologies company) middle chemical (0.125% collagenase, 2 hours; Sigma, Britain) and mechanical separation.Then with cell suspension with 600rpm centrifugal 5 minutes, suspend by 15% BSA subsequently and with 900rpm centrifugal 10 minutes for the second time.With the sedimentation cell Eddy diffusion in BSF-2, then with the density of 900-1000 neurocyte/coverslip be seeded to the coverslip that is attached with silk fiber on.At 37 ℃, 95% air and 5%CO
2Humidification atmosphere under, the maintain thing is 7 days in the BSF-2 that is added with 100ng/ml nerve growth factor (NGF).The preparation of coverslip is by following method: use first the coating of poly-L-Lysine (100 μ g/ml) and rat tails collagen, silk fiber is adhered on the collagen.In some cases, before using the inoculation of DRG cell, will be stained with the coverslip of silk fiber with the laminin，LN coating of 10 μ g/ml.In addition, DRG uses with the DRG cell that the sciatic nerve implant replaces separating in some experiments.In this type of situation, by sucking the CO of high concentration
2Put to death newborn (P3) rat, cut off lumbar nerves dorsal root ganglion and sciatic nerve section, it is sticked on the microscope slide of the silk fiber that is stained with the laminin，LN coating of poly-L-Lysine and collagen coating, and in being added with the BSF-2 of NGF, cultivated 7-10 days.
When culture period finishes, culture mixed in the paraformaldehyde of 100% methanol or 4% and use following reagent labelling: the mice β III tubulin (1: 1000) of announcement DRG cell space and process, disclose regeneration DRG process and non-myelin and execute the rabbit GAP-43 antibody (1: 1000) of ten thousand Schwann Cells processes, the rabbit S100 antibody (1: 1000) of ten thousand Schwann Cells is executed in announcement, and is used as the Hoechst 3342 (2 μ g/ml) of general kernel counterstaining.Use anti-rabbit TRITC (tetramethyl isothiocyanic acid Luo Daming) and anti-mice FITC (Fluorescein isothiocyanate) the second antiserum to carry out visual to the first antiserum.Then observe specimen at Zeiss LSM-510 confocal microscope.
The result
Newborn DRG and sciatic nerve implant
In the DRG implant (Fig. 1,2), Hoechst dyeing shows that most ten thousand Schwann Cells of executing move out and adhere on the silk fiber from implant.In addition, can clearly observe GAP-43 immunoreactivity aixs cylinder extends and also each fiber of bridge joint in some cases along each silk fiber.
In the sciatic nerve implant (Fig. 3), most Hoechst labellings and GAP-43 immunoreactivity are executed ten thousand Schwann Cells and are moved out and adhere on the silk fiber from described implant, confirm that described fiber provides good substrate for executing the connection of ten thousand Schwann Cells.
The adult DRG cell that separates
In adult DRG culture (Fig. 4), observe most Hoechst labellings execute ten thousand Schwann Cells nuclear and GAP-43 immunoreactivity aixs cylinder adheres on each silk fiber, confirm that described fiber also supports growth and the support neurogliocyte of adult PNS neurocyte.Be coated with in the culture of laminin，LN at silk that (Fig. 5) all observes widely growth among (Fig. 4) and the culture without laminin，LN, confirm that silk itself is the good substrate of nerve growth and does not need other extracellular matrix coating.
In order further to characterize the growth that observation in vitro arrives, use the label β III tubulin of the specific label S100 of neuroglia and Neuron-specific to implement labelling.This has confirmed that most S100 immunoreactivities execute ten thousand Schwann Cells relevant with silk fiber (Fig. 6), and meticulous GAP-43 immunoreaction process is neurocyte source property (Fig. 7).Most Hoechst nuclear is oval, the tubulin feminine gender, and belong to and execute ten thousand Schwann Cells.Yet some is circular and tubulin immunoreactive (arrow among Fig. 7), and extends tubulin and GAP-43 immunoreactivity neurocyte process along silk.These be adhered to silk and along silk extend process, by the relevant DRG neurocyte that ten thousand Schwann Cells are supported of executing.
In vitro study shows by the fiber of silk preparation to be supported the growth of aixs cylinder and also supports to execute connection and the migration of ten thousand Schwann Cells by the newborn PNS neurocyte (DRG cell) of being connected with adult.This is important feature, because knownly execute ten thousand Schwann Cells and help axon growth.
Embodiment 2: silk fiber is implanted spinal cord of adult rats
The effect that silk fiber is implanted spinal cord is investigated in initial in vivo test.Interested especially is the degree of axon growth and orientation (with respect to silk fiber) and implant on the impact of complete nervous tissue on every side (i.e. the degree of downright bad and inflammatory reaction).
Method:Carry out at the fibre bundle of not restrainting bundle (namely not being included in the conduit) about the in vivo test of silk being implanted spinal cord at first.Guideline and testing program according to the approval of the Britain Ministry of Internal Affairs are implemented animal treatment and operation.Use halothane (4% derivant, 2% keep agent) the young Thirty male rats of anesthesia.Cover at epispinal skin and muscle is cut open and implement laminectomy between T7 and T9 position with horizontal.Open pachymeninx and use iris scissors shift out from center line extend toward the side nearly 0.5mm and from spinal cord surface veutro extend 1mm near one section long spinal cord of 2mm.Then the silk fiber bundle is put into the pathological changes chamber, make silk fiber have the orientation parallel with longitudinal spinal cord axis.Then use gelfoam to cover muscle and the skin of diseased region and stitching covering.Animal after implanting the time-to-live between 1 and 8 weeks.After the suitable time-to-live, use pentobarbital sodium (Sagatal, RMB, 60mg/kg) the deep anaesthesia animal, and use the phosphate buffer (PBS) of 50ml 0.01M to pour into by ascending aorta succeeded by the paraformaldehyde that is dissolved in pH7.40.01M phosphate buffer 4%.Spinal cord is cut open, in 4% paraformaldehyde, fix afterwards (postfixed) 1-2 hour, and low-temperature protection is spent the night in being dissolved in 15% sucrose of PBS.Take out the longitudinal section of 10-12mm thickness by implant site.
The immunohistochemistry processing is carried out in the section that then will contain implant site.Use protein gene product antibody 9.5 (PGP9.5) that the inside growth of aixs cylinder is characterized, and low-affinity p75 receptor antibody is used for ten thousand Schwann Cells of executing that labelling is invaded implant site.In addition, macrophage antibody (ED1) at first is used for characterizing the inflammatory reaction to implant, and astrocyte label glial fibrillary acidic protein (GFAP) is used for estimating the implant neuroglia reaction of complete tissue on every side.Immunohistochemical general step is as follows: cultivated 48 hours in primary antibodie, with the phosphate buffer washed twice, each ten minutes, during resisting with two of tetramethyl isothiocyanic acid Luo Daming (TRITC) or Fluorescein isothiocyanate (FITC) (both all obtain from Jackson Immunoresearch Laboratories company) conjugation, cultivated 2 hours.Washing is more than three times, each 10 minutes, containing 2.5%1,4-diazabicyclo-(2 subsequently, 2,2)-give the microscope slide covered in the PBS glycerol (1: 3) of octane or carry out two anti-immunohistochemistries and process to be equal to mode described above.
Silk fiber self produces fluorescence and can clearly observe (white fiber, Fig. 8, a left side) in spinal cord.Use the labelling of astrocyte label GFAP to show: common described silk fiber is near adjacent complete spinal cord (Fig. 8, the right side), has seldom between host's spinal cord and implant or without slough.In addition, observing the astrocyte reaction is typical in spinal cord injury.These two kinds of features show that spinal cord has good toleration to silk fiber.
In implanting the silk fiber bundle of spinal cord and the intrusion (Fig. 9) of having observed macrophage in the surrounding tissue.The degree of this kind intrusion reduces gradually and usually seldom observe macrophage outside distance implant 2mm.Other implant (for example fibronectin splicing variants) of the degree of described inflammatory reaction and use and the inflammatory reaction after spinal cord injury is not treated are compared more favourable, further show the excellent compatibility of silk fiber and host's spinal cord.
A large amount of aixs cylinder (representing with the arrow) growth of labelling (Figure 10) demonstration of use aixs cylinder label PGP9.5 enters in the described silk implant and usually shows the orientation that is parallel to silk fiber.4 weeks after implantation (the nearest time point of investigation) are observed the growth of maximum.
Confocal microscope is analyzed (Figure 11) and is shown that further the aixs cylinder of PGP9.5 labelling grows along each silk fiber and between each silk fiber.In addition, at this moment between point (4 week) without any the sign of silk fiber degraded.
Figure 12 shows the double labeling that uses aixs cylinder label PGP9.5 and Shi Wan Schwann Cells label p75 and has disclosed that to execute ten thousand Schwann Cells substantially corresponding with the aixs cylinder of growth and maturity.This show the major part growth of in implant, observing may be outer perigene and/or stimulate by setting up the good neurotrophy support relevant with executing ten thousand Schwann Cells.
Embodiment 3: the implantation of silk fiber (repeating first) in the silk conduit
At first in the spinal cord based on the study tour of the conduit purposes of silk a kind of conduit, described conduit contains a conduit (Figure 13) of closely filling silk fiber in nuclear.
Method:Middle describe identical in method for implantation and (2) is except the slightly wide pathological changes chamber of diameter (near 1mm) requirement preparation in spinal cord of implant.
The result:The result shows that these implants are not integrated into spinal cord and come off from spinal cord in the cutting tissue process.To such an extent as to this may be since in the conduit packed density of fiber do not allow any endogenous component to infiltrate through in the implant too greatly, so so that all impossible with the integration of any kind of host's spinal cord.Yet, importantly be noted that the size in pathological changes chamber and implant in all animals must be consistent and implant site spinal cord on every side without downright bad sign, show that host's spinal cord has good toleration to these implants.
Embodiment 4: contain the implantation of silk fiber in the hyaluronic silk conduit
Do not observe the integration (referring to more than) of a conduit, show that silk fiber in the catheter core needs fiber to be suspended in as aixs cylinder and other endogenous component and invades in the biodegradable matrices that the space is provided and allows for axon growth.In the conduit implant core that therefore, will be formed by the silk epitheca with silk fiber of being suspended in the hyaluronic acid.
Method:It is identical that the method for implantation and dyeing and above (embodiment 3) are described, except the slightly wide pathological changes chamber of diameter (near 1mm) requirement preparation in spinal cord of implant.
The result:The structure of conduit clearly visible (Figure 14), each epitheca wall look like little silk slip (arrow) and internal core is the slice (arrowhead) of portrait orientation.For the silk (referring to section 2) that uses bundle to prick, the GFAP labelling shows that a silk implant is integrated in host's spinal cord well, has seldom between astrocyte cicatrix and implant or without slough.
With respect to Fig. 9, it is similar that extremely the macrophage of a conduit is invaded (Figure 15) of observing with the silk fiber of restrainting bundle on apparent and degree.In addition, 8 weeks after implanting, can be observed macrophage be gathered in each silk fiber around, but still the sign that begins to decompose without silk fiber.
For the silk (referring to embodiment 3) that uses bundle to prick, the aixs cylinder that can be observed a large amount of PGP9.5 dyeing between silk fiber, grow (referring to Figure 16).Differ widely with the silk fiber of not restrainting bundle, can be observed the growth of many ingrown axon fasciculations ground.
In addition, for the silk (referring to embodiment 2) that uses bundle to prick, use aixs cylinder that the double labeling of aixs cylinder label PGP9.5 and Shi Wan Schwann Cells label p75 shows growth and maturity and to execute ten thousand Schwann Cells substantially corresponding.
Claims (35)
1. medical treatment device that is used for nervous cell regenerating, it comprises:
Body with inner chamber and major axis; With
Along a plurality of thread composition of the substantially parallel placement of major axis of the inner chamber of described body,
Wherein said body comprises albumen or based on the material of albumen, and described thread composition is selected from silkworm silk that the silkworm of mulberry silk, non-Bombyxmori Linnaeus produces, spider dragline silk and the filament that is spun into by recombinant fibroin at least a.
2. device as claimed in claim 1, wherein said body contains can resorbent material.
3. device as claimed in claim 2, wherein said body has the composite construction that comprises the fiber that places substrate.
4. device as claimed in claim 3, wherein said substrate is fibroin.
5. device as claimed in claim 4, wherein said fibroin are the redissolution fibroins that is obtained by Bombyxmori Linnaeus or non-Bombyxmori Linnaeus, or the natural silk heart protein that is obtained by Bombyxmori Linnaeus or non-Bombyxmori Linnaeus.
6. device as claimed in claim 4, wherein said substrate is by the crosslinked stabilizing treatment of carrying out.
7. device as claimed in claim 6, wherein said crosslinked be to use formaldehyde gas, citrate ion, ribose, Biformyl or genipin and realize.
8. device as claimed in claim 3, the described fiber that wherein forms body is placed or is woven by spiral.
9. device as claimed in claim 3, the described fiber that wherein forms body is silk fiber.
10. device as claimed in claim 1, wherein said thread composition is separated from each other with the distance between 1 μ m and the 100 μ m.
11. device as claimed in claim 1, the packed density of wherein said thread composition is from per 10,000 μ m
2In 1 to 30 the scope.
12. device as claimed in claim 1, wherein said body has from 1.0 to 2.5mm external diameter.
13. device as claimed in claim 1, the wall of wherein said body have from the thickness of 250 μ m to 750 μ m.
14. device as claimed in claim 1, the length of wherein said device are from 0.5mm to 20.0mm.
15. device as claimed in claim 1, wherein said thread composition have from the diameter of 5 μ m to 50 μ m.
16. device as claimed in claim 1, wherein said thread composition comprise silkworm silk, the spider dragline silk that the silkworm of mulberry silk, non-Bombyxmori Linnaeus produces and the filament that is spun into by recombinant fibroin.
17. device as claimed in claim 16, wherein said thread composition is by the silk preparation from Antherea pernyi Guerin-Meneville.
18. device as claimed in claim 17, wherein said thread composition are the forms of bar silk or filature or twisted silk.
19. device as claimed in claim 1, wherein said thread composition has main fibroin, and described main fibroin contains the triplet RGD of at least eight repetitions.
20. device as claimed in claim 19, wherein the triplet RGD of at least some described repetitions is positioned at corner or the expectation corner of the structure of the described main fibroin of next-door neighbour.
21. device as claimed in claim 19, wherein said main fibroin has following site: the one or more arginine groups from the described main fibroin in described site have been blocked to regulate cytoadherence.
22. device as claimed in claim 21, wherein said blocking-up realizes by the blocking-up of deaminizating, sulphation, one-tenth amide and the use cyclohexanedione of one or many.
23. device as claimed in claim 1, wherein said a plurality of thread compositions be placed in comprise can the inner chamber substrate of resorbent biocompatible polymer in.
24. device as claimed in claim 23 wherein saidly can comprise hydrogel by resorbent biocompatible polymer.
25. device as claimed in claim 24, wherein said hydrogel are hyaluronic acid or alginate or casein, described hyaluronic acid or alginate have or do not have polylysine.
26. device as claimed in claim 1, wherein said inner chamber or thread composition also contain extracellular matrix in addition.
27. device as claimed in claim 26, wherein said extracellular matrix comprises fibronectin splicing variants and/or laminin，LN.
28. device as claimed in claim 1, wherein said device also contain one or more bioactive substances in addition.
29. device as claimed in claim 28, wherein said bioactive substance are selected from somatomedin, cytokine, antibiotic, immunosuppressant, steroid and nonsteroidal antiinflammatory drug NSAIDs.
30. device as claimed in claim 1, wherein said device also comprise cell mass in addition.
31. device as claimed in claim 30, wherein said cell are to execute ten thousand Schwann Cells or Olfactory essheathing cell OECs.
32. method for the preparation of the medical treatment device of nervous cell regenerating, it comprise in the inner chamber that forms body and thread composition is introduced described body so that described thread composition along the substantially parallel placement of the major axis of tube cavity, wherein said body comprises albumen or based on the material of albumen, and described thread composition is selected from silkworm silk that the silkworm of mulberry silk, non-Bombyxmori Linnaeus produces, spider dragline silk and the filament that is spun into by recombinant fibroin at least a.
33. method as claimed in claim 32, the formation of wherein said body comprises the steps:
-preparation forms the former of described body;
-fiber is placed on the described former;
-substrate is applied to described fiber to form complex; And
-remove described former.
34. method as claimed in claim 33, it further comprises crosslinked described substrate.
35. method as claimed in claim 32, it further is included in and introduces the inner chamber matrix components between described thread composition in the described tube cavity.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0420411A GB2417904A (en) | 2004-09-14 | 2004-09-14 | Tubular prosthesis for nerve regeneration |
| GB0420411.1 | 2004-09-14 | ||
| GB0422019.0 | 2004-10-05 | ||
| GB0422019A GB0422019D0 (en) | 2004-10-05 | 2004-10-05 | Medical device |
| PCT/GB2005/003456 WO2006030182A2 (en) | 2004-09-14 | 2005-09-08 | Methods and apparatus for enhanced growth of peripheral nerves and nervous tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101072593A CN101072593A (en) | 2007-11-14 |
| CN101072593B true CN101072593B (en) | 2013-01-02 |
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| CN200580036978.2A Active CN101072593B (en) | 2004-09-14 | 2005-09-08 | Methods and apparatus for enhanced growth of peripheral nerves and nervous tissue |
Country Status (2)
| Country | Link |
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| CN (1) | CN101072593B (en) |
| GB (1) | GB2417904A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103405289B (en) * | 2013-07-26 | 2015-04-15 | 清华大学 | Device based on liquid metal and used for repairing defective peripheral nerve function |
| CN104721567A (en) * | 2013-08-19 | 2015-06-24 | 陈霞 | Externally applied nursing dressing medicament after nerve injuries |
| CN103877623B (en) * | 2014-02-19 | 2016-03-02 | 西南大学 | A kind of engineered artificial neuron and preparation method thereof |
| WO2017031167A1 (en) * | 2015-08-17 | 2017-02-23 | The Johns Hopkins University | Fiber-hydrogel composite surgical meshes for tissue repair |
| CN109939257B (en) * | 2019-02-28 | 2021-04-09 | 李琳 | Antibacterial medical bandage and preparation method thereof |
| CN110101908B (en) * | 2019-02-28 | 2021-07-06 | 李琳 | Degradable tissue repair material and preparation method thereof |
| CN111603608B (en) * | 2020-05-15 | 2021-12-10 | 南通大学 | Pullulan-hyaluronic acid-microfiber spinal cord scaffold and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1411873A (en) * | 2001-10-09 | 2003-04-23 | 中国纺织科学研究院 | Partial chitosan bio-canula capable of inducing and promoting effective regeneration of nerve and its preparation method |
| GB2386841A (en) * | 2002-03-11 | 2003-10-01 | Ind Tech Res Inst | Multi-channel bioresorbable nerve regeneration conduit and process for preparing the same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5656605A (en) * | 1994-01-26 | 1997-08-12 | Institute Of Molecular Biology, Inc. | Device to promote drug-induced nerve regeneration |
| SE9601243D0 (en) * | 1996-03-29 | 1996-03-29 | Hans Arne Hansson | Promotion of regeneration of organized tissues |
| AU2003249366A1 (en) * | 2002-06-24 | 2004-01-06 | Tufts University | Silk biomaterials and methods of use thereof |
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2004
- 2004-09-14 GB GB0420411A patent/GB2417904A/en not_active Withdrawn
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1411873A (en) * | 2001-10-09 | 2003-04-23 | 中国纺织科学研究院 | Partial chitosan bio-canula capable of inducing and promoting effective regeneration of nerve and its preparation method |
| GB2386841A (en) * | 2002-03-11 | 2003-10-01 | Ind Tech Res Inst | Multi-channel bioresorbable nerve regeneration conduit and process for preparing the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101072593A (en) | 2007-11-14 |
| GB2417904A (en) | 2006-03-15 |
| GB0420411D0 (en) | 2004-10-20 |
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