CN1872354A - Duct filler material in use for bridge grafting nerves, and preparation method - Google Patents
Duct filler material in use for bridge grafting nerves, and preparation method Download PDFInfo
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- CN1872354A CN1872354A CN 200610066434 CN200610066434A CN1872354A CN 1872354 A CN1872354 A CN 1872354A CN 200610066434 CN200610066434 CN 200610066434 CN 200610066434 A CN200610066434 A CN 200610066434A CN 1872354 A CN1872354 A CN 1872354A
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Abstract
A filler of the guide tube used for bridging nerve is a high-purity transparent gel prepared from sodium hyaluronate and the physiologically balanced liquid through purifying by filter method. The cells or factors for promoting the repair and growth of nerve are added to the gel.
Description
Affiliated technical field
The present invention belongs to a kind of being used for to repair and regenerated novel organizational project packing material behind the neurologic defect, particularly is used in the bridge grafting nerves art conduit as the cell of short nerve growth, packing material of various factor carriers and preparation method thereof.
Background technology
At present, still take the method for nerve autograft in the clinical position mostly for the reparation of neurologic defect, get accessory relatively nerve and repair damaged nerve as donor.This method is except that existing the disadvantage of sacrificing for district function of nervous system, and also existing can't be to the wretched insufficiency of effectively treating than long section neurologic defect.Along with the fast development of tissue engineering, the appearance of conduit bridge joint art has solved this long-term puzzlement clinical position person's a difficult problem to a certain extent.Yet, though the bridge joint of neurologic defect has been used multiple biomaterials such as vein, silica gel tube and chitosan clinically, but still exist many deficiencies, for example bridge material only plays damaged broken ends of fractured bone interconnect function, neuranagenesis there is not facilitation, because the various short nerve growth composition that injects is in liquid condition, the part should not retain in conduit; Conduit subsides easily because of the self structure characteristics, influences the performance of its function, effect; And the formation of scar tissue.These problems all affect the reparation and the regeneration of damaged nerve, so restricted Clinical Application and popularization.
Ideal artificial neuron is to be carrier with the degradable high polymer with good biocompatibility, combine with the activated cell and the factor and form have specific three dimensional structure and a bioactive complex, allow the regenerating nerve fiber growth to the distally neural residul end, suppress neuromatous formation and fibrous scar tissue and grow into, can build characteristics such as favourable microenvironment with regeneration for the reparation of nerve.
Hyaluronic acid is by the linear polysaccharide class macromolecular substances of the disaccharidase formation of (1 → 4)-D-glucuronic acid-β (1 → 3)-N-acetylglucosamine, extensively is present in people and the various animal connective tissue.Because its intermolecular interaction forms three grades of complicated network structure, thereby has obtained many characteristics, as keeps tissue morphology, lubricated, mechanical function and important physical functions such as the gentle blow stress of diffusion barrier have significant viscoelasticity simultaneously again.Hyaluronic acid also is the main component that constitutes intercellular substance and extracellular matrix, and extracellular matrix has all been played the part of crucial role in the repair process of the Growth and Differentiation of keeping the normal physiological function of histoorgan, sustenticular cell and damaged tissue.In wound healing process, extracellular matrix has influenced the behavior performance of cell, and otherwise cell is also regulated by number of ways pair cell epimatrix, thereby has formed the microenvironment of a sustenticular cell growth promoter that runs through whole agglutination and constantly change.Reparation and the regenerative process of tissue is one and involves various kinds of cell and process that biomacromolecule carries out cell proliferation, differentiation and substrate reconstruction.Wound take place early stage, partial fibroblast, lift phagocyte etc. under the effect of somatomedin, synthetic hyaluronic quantity obviously increases.In contrast embryo and postnatal research, show that in the repair process of embryo skin wound, hyaluronic acid has kept very high concentration, thereby suppressed the contracture of collagenous tissue, obtain the reproducibility reparation and do not stay cicatrix until tissue.After the birth, hyaluronic acid has kept higher concentration in early days, but progressively is degraded under the effect of hyaluronidase subsequently, and collagen fiber is synthetic but in continuous increase simultaneously, has finally formed the cicatrix reparation.In addition, hyaluronic acid also has the effect that promotes angiogenic growth, improvement damage local microcirculation, promotes growth factor expression.Contain a large amount of carboxyls and hydroxyl in the hyaluronan molecule, in aqueous solution, form intramolecularly and intermolecular hydrogen bonding.This makes it have powerful water retention, can be in conjunction with more than 1000 times water itself.Existing in the prior art is main component with the hyaluronate sodium, be made into compound preparation with other adjuvants and pharmacological active substance such as chitosan, heparin, chondroitin sulfate, dermatan sulfate, epidermal growth factor, antibacterial etc. and be applied to clinically, be mainly used in the powder agent (patent No.: CN1335135) of treatment burn and scald; In addition, also brought into play the important function (patent No.: CN1295838) in the anti medical preparation of hyaluronate sodium in being applied to surgical operation.With the hyaluronate sodium is the duct filler material of main component as bridge grafting nerves, and retrieval is not at present seen as yet.
Summary of the invention
In order to replenish and to improve prior art and the bridge grafting nerves thing is only played the damaged broken ends of fractured bone be connected guiding, screen-wall effect, and neuranagenesis is lacked facilitation, and the various short nerve growth composition that injects in conduit is in liquid condition, the part should not retain in conduit, and conduit subsides easily because of the self structure characteristics, influence the problem of its function, effect performance, the invention provides a kind of packing material that is used for the bridge grafting nerves conduit and preparation method thereof.
The technical scheme that duct filler material adopted that the present invention is used for bridge grafting nerves is: said duct filler material includes the refining dry powder (A) of hyaluronate sodium, physiological balance liquid (B), the various cells (D) of short nerve growth cell growth factor (C) or short nerve growth.
The short nerve growth cell growth factor (C) of indication can be one or more of nerve growth factor, Brain Derived Neurotrophic Factor, acid fibroblast growth factor, alkaline sarcoplast somatomedin, ciliary nerves somatomedin, glial cell line-derived neurotrophic factor.
Said short nerve growth cell (D) can be one or more of Schwann cell, muscle stem cell, bone marrow stroma stem cell.
The said preparation method that is used for the duct filler material of bridge grafting nerves of the present invention comprises the steps:
(1) hyaluronate sodium dry powder (foreign biotech firm is contained in Qingdao) is added deionized water, be made into 0.01～0.2% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, add aseptic acetone or alcoholic solution that 1-5 doubly measures, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 1～10% hyaluronate sodium, be dissolved in 90%～99% physiological balance liquid (B), regulating its pH value is 7.0～7.4, fully stirs the hyaluronic acid sodium gel that is mixed with 1～10mg/ml concentration, discontinuous degassing, and 1～10 ℃ of cold preservation is standby;
(3) add 1%～10% short nerve growth factor (C) or the cell (D) of short nerve growth effect is arranged.
The packing material of this conduit is through above-mentioned preparation method, make a kind of gel, not only can supporting tube, solve the problem that conduit subsides, can also in conduit, build the microenvironment that helps nerve growth because of himself biological characteristics, promote, the guiding neuranagenesis.In addition can also be as the carrier that adds the various exogenous materials (as pharmaceutical agent, cell, gene etc.) in the conduit, the effect of performance pair cell and trophic factors controlled release or extracellular matrix carrier.
The invention has the beneficial effects as follows: in the bridge grafting nerves art, the nerve trachea wall is played effective supporting, can prevent that tube wall from subsiding, promote the effect of guiding and built to help neurologic defect reparation and regenerated microenvironment for performance in the reparation of neurologic defect and the regenerative process simultaneously.When repairing carrier with fertile absorber, various short neural in the conduit can also bring into play effect biological controlled-released or the extracellular matrix carrier as adding.The packing material that the present invention adopts is at the human body absorbable and degradable, and is nontoxic, need not second operation, and application operating is simple, can improve the curative effect that conduit is repaired neurologic defect greatly.
At present, in the rat body, test, and obtained remarkable result.Choose healthy adult cleaning level 30 of male rats of SD (Military Medical Science Institute's animal center provides), body weight 200～250g, average 223g.1% pentobarbital sodium (30mg/kg) intraperitoneal injection anesthesia.After the sterilization, get a strand back angular cut, expose right sciatic nerves, and about 8mm excises with sciatic nerve in the 5mm place below the piriformis outlet, let alone retraction, cause 10mm damaged, use not damaged micro suture line the nerve broken ends of fractured bone far away, near and bridge joint conduit sutured 2 pins, sewing method adopts two fixed point cover connections: respectively sew up 1 pin in 180 ° of two relative position, nerve trunk week footpath respectively, make the regenerating nerve broken ends of fractured bone be inserted in 1mm in the bridge pipe.Size of animal is equally divided into 5 groups:
A group: do not add any conduit implant.
B group: draw an amount of hyaluronic acid sodium gel with disposable sterilized injector (the special large aperture of socket syringe needle) and inject, make conduit fully full by conduit one side.
C group: draw the hyaluronic acid sodium gel that has added short nerve growth factor (ciliary nerves somatomedin) in right amount with disposable sterilized injector (the special large aperture of socket syringe needle) and inject, make conduit fully full by conduit one side.
D group: draw the hyaluronic acid sodium gel that has added short nerve growth cell (muscle-derived stem cell) in right amount with disposable sterilized injector (the special large aperture of socket syringe needle) and inject, make conduit fully full by conduit one side.
The E group:.Draw the hyaluronic acid sodium gel that has added short nerve growth factor (ciliary nerves somatomedin) and short nerve growth cell (muscle-derived stem cell) in right amount with disposable sterilized injector (the special large aperture of socket syringe needle) and inject, make conduit fully full by conduit one side.
Then muscle and skin incision behind the layer-by-layer suture thigh, standard is unified sub-cage rearing routinely.Treat to get respectively after 4,8 and 12 weeks and respectively organize laboratory animal and detect.
One, limbs are observed
Postoperative is promptly found rat right hind leg, toes bending, can't stretch, and the towing walking, acupuncture can not cause contraction; Postoperative finds respectively to organize heel heavy burden district, rat right side skin during 1 week all have redness in various degree, the slight ulcer in vola occurs in A, the group during 2 weeks, is tending towards subsequently healing.Local amyotrophy all appears in 3 weeks of postoperative; Postoperative 4 all amyotrophy have recovery in various degree, and muscular rigidity alleviates to some extent, and the towing degree reduces during walking, respectively have 1 and 3 (respectively organizing 6 of total number averages) that the acupuncture reaction is arranged in A, the B group, but not obvious; There are 5,5 and 6 (respectively organizing 6 of total number averages) that the acupuncture reaction is arranged respectively in C, D, each group of E; Have 25 rats during 8 weeks, wherein A, B group respectively has 2 and 3, and C, D, E group respectively have 5,4,5 (respectively organizing 5 of total number averages) right hind muscular rigidity degree obviously to reduce, external force can make it to stretch, toes have big grip, have obvious acupuncture reaction, and toes are observed the towing degree and obviously reduced.
Two, Anatomical Observation
12 weeks of postoperative, each group was got 1 rat, cut once more after 1% pentobarbital sodium (30mg/kg) the intraperitoneal injection anesthesia and expose sciatic nerve, as seen the nerve at conduit of Zhi Ruing and two ends is all holding the involucrum that is formed by fibrous connective tissue, does not have obvious adhesion with surrounding tissue.After cutting off and taking out conduit, except that A, all visible regenerating nerve of each group, far, the smooth non-cracking of near-end and normal neural junction, no scar tissue, neuroma form, the visible tiny blood vessels net in surface forms, outer likeness in form is normal neural, and the normal nerve trunk of diameter is thin, and C, D, E group slightly are coarser than the B group.Be full of flaxen liquid in the A group conduit, no regenerating nerve passes through.
Three, sciatic nerve index (SFI): the Bain formula calculates
SFI＝-38.3[(EPL-NPL)/NPL]+109.5[(ETS-NTS)/NTS]]+13.3[(EIT-NIT)/NIT]-8.8
SFI value 0%～± 11% expression function of nervous system is normal fully, and-100% expression function of nervous system completely loses, expression partial nerve functional rehabilitation between-11%～-100%.
See Table 1, each time point of A group postoperative is all not as good as other each groups, and difference has statistical significance (P＜0.01).Postoperative 4 all B, C, D group SFI compare no significance difference (p＞0.05); 8 weeks of postoperative, 12 weeks, C, D, E group SFI recovery situation obviously are better than the B group, and simultaneously, E group SFI is better than C, D group, and difference has statistical significance (p＜0.01).
Table 1 postoperative 4,8,12 is respectively organized sciatic nerve index (SFI) relatively (n=6, x ± s) during week
| Group | 4 weeks | 8 weeks | 12 weeks |
| A group B group C group D group E group | -92.732±4.07 -89.030±3.59 -88.928±3.31 -88.936±3.93 -86.832±3.15 | -91.394±5.67 -76.986±7.08 -70.961±6.12 -71.789±4.77 -67.407±3.44 | -89.891±5.35 -69.527±6.30 -61.540±4.22 -61.847±3.57 -56.532±3.02 |
Four, electrophysiologic study
12 weeks of postoperative are under the waking state, with the tricipital electromyogram of electromyograph(EMG detection of cancerous primary minimum lower limb.Under the narcotism, expose art side sciatic nerve, detect the wave amplitude of sciatic nerve conduction velocity, compound muscle action potential.
See Table 2, the A group is fibrillation potential, and visible positive sharp wave shows as fully inattentive through current potential; B, C, D, E group gastrocnemius electromyogram is the mixed phase current potential, is that part is inattentive to be showed through current potential; The motor nerve conduction velocity and the wave amplitude of E group are organized a little more than C, D, but apparently higher than B group (P＜0.01).
Table 2 postoperative 12 all neuroelectricity physiological detection (n=6, x ± s)
| Group | Motor nerve conduction velocity (m/s) | Wave amplitude (mv) | Electromyogram |
| A group B group C group D group E group | 32.811±3.12 42.973±2.84 42.208±2.79 45.812±2.64 | 2.98±3.25 3.70±3.21 3.55±3.27 4.08±3.10 | Fibrillation potential positive sharp wave mixed phase current potential mixed phase current potential mixed phase current potential mixed phase current potential |
Five, the complete gastrocnemius of taking off bilateral, ER-182A electronic balance (1/2000g) is measured its weight in wet base, calculates the wet weight of gastrocnemius muscle recovery rate.See the visible C of its table 3 result, D, E group wet weight of gastrocnemius muscle recovery rate and be better than A group and B group.Wherein E organizes index the best.
Table 3 postoperative 12 all wet weight of gastrocnemius muscle recovery rates (%) (n=6, x ± s)
| Group | Recovery rate (%) |
| A group B group C group D group E group | 50.312±4.33 67.544±3.71 71.231±3.25 70.057±3.47 75.042±2.96 |
Six, histological examination
1, observation by light microscope: the dyeing of HE longitudinal section shows, C, D group is good with E group bridge joint place's neuromechanism seriality, the regenerating nerve far-end of all growing into, and the regenerating nerve fiber alignment is neat, visible on every side a large amount of new vesselses are not seen situations such as degeneration necrosis and scarring are arranged.B group bridge joint place's neuromechanism seriality is bad, only has a small amount of nerve fiber to pass through, the far-end of growing into, and the disorder of regenerating nerve fiber alignment, new vessels is less.
The dyeing of HE transverse section shows that C, D group and all visible a large amount of regenerating nerve fiber of E group are arranged closely.And B group regenerating nerve fiber number is less, arranges loose.And the diameter of regenerating nerve fiber is more tiny.
The S-100 immunohistochemical staining shows that C, D and E organize the male Schwann cell of visible S-100 immunoreation and be scattered in ribbon along nerve fiber.And the male Schwann cell negligible amounts of B group S-100 immunoreation, and be not tangible ribbon distribution.
2, transmission electron microscope observing: E group regenerating nerve fiber aixs cylinder form has marrow and unmyelinated nerve fiber in a large number near normal, the shape comparison rule, and myelinated fiber myelin thickness is than normal thin.And the regenerating nerve fiber of B group is tiny, out-of-shape, and each bar nerve fiber is not of uniform size, arranges to loosen, the myelin thinner thickness, the aixs cylinder structure is imperfect.
3, the regenerating nerve fibre image is analyzed: graphical analysis the results are shown in Table 4, and in 12 weeks of postoperative, B, C, D, E group regeneration myelinated fiber density all are higher than normal group (P＜0.01); Relatively B, C, D, E organize simultaneously, and C, D, E group all are better than B group (P＜0.05 or P＜0.01) on the indexs such as myelinated nerve fiber density, nerve fiber effective area, myelinated nerve fiber quantity, diameter and myelin thickness.Wherein obvious with the E group.
Table 4 postoperative 12 all regenerating nerve aspect graphs are as analysis result (n=6, x ± s)
| Group | n | Nerve fiber effective area (n/104 μ m2) | Myelinated nerve fiber number (root) | Myelinated nerve fiber density (n/104 μ m2) | Myelinated fiber diameter (μ m) | Myelin thickness (μ m) |
| B group C group D group E group | 6 6 6 6 | 13.746±1.78 18.211±1.53 18.018±1.47 20.315±1.76 | 2603.400±39.95 3670.179±40.73 3640.400±38.44 4282.622±32.71 | 189.622±10.89 201.536±10.97 202.726±11.32 210.810±9.61 | 3.343±0.81 4.106±0.93 4.089±0.88 5.327±0.56 | 0.417± 0.07 0.521± 0.05 0.481± 0.05 0.598± 0.03 |
The specific embodiment
One, (1) adds deionized water with hyaluronate sodium dry powder, be made into 0.1% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, the aseptic acetone solution that adds 4 times of amounts, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 6mg hyaluronate sodium, be dissolved in 1ml physiological balance liquid (B) [77% DMEM (GIBCO company), 20%FBS (SIGMA company)], 2%Glutamine (SIGMA company) and 1% mycillin (Dalian Metro), it is 7.0～7.4 that caustic lye of soda is regulated its pH value, add an amount of (C) [nerve growth factor (20ng/ μ l)], make its concentration reach 5%, fully stir and be mixed with hyaluronic acid sodium gel.1～10 ℃ of cold preservation is standby.
The hyaluronic acid sodium gel that cold preservation is standby takes out the room temperature placement before the operation, in the art the capable end of neurologic defect place both sides broken ends of fractured bone finishing back application catheter-end adventitia is sewed up, reuse disposable sterilized injector (the special large aperture of socket syringe needle) is drawn an amount of hyaluronic acid sodium gel and is injected by conduit one side or puncture conduit, make its conduit fully full, thereby having formed one is outer wall with the conduit, hyaluronic acid sodium gel is a content, is rich in the favourable microenvironment of the short neural reparation and the regenerated factor.Simultaneously, because the physical behavior of hyaluronic acid sodium gel has also played the strong support effect to tube wall.
Two, (1) adds deionized water with hyaluronate sodium dry powder, be made into 0.1% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, the aseptic acetone solution that adds 4 times of amounts, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 6mg hyaluronate sodium, be dissolved in 1ml physiological balance liquid (B) [77%DMEM (GIBCO company), 20%FBS (SIGMA company)], 2%Glutamine (SIGMA company) and 1% mycillin (Dalian Metro), it is 7.0～7.4 that caustic lye of soda is regulated its pH value, add an amount of (C) [ciliary nerves somatomedin (60ng/ μ l)], make its concentration reach 5%, fully stir and be mixed with hyaluronic acid sodium gel.1～10 ℃ of cold preservation is standby.
The hyaluronic acid sodium gel that cold preservation is standby takes out the room temperature placement before the operation, in the art the capable end of neurologic defect place both sides broken ends of fractured bone finishing back application catheter-end adventitia is sewed up, reuse disposable sterilized injector (the special large aperture of socket syringe needle) is drawn an amount of hyaluronic acid sodium gel and is injected by conduit one side or puncture conduit, makes its conduit fully full.
Three, (1) adds deionized water with hyaluronate sodium dry powder, be made into 0.1% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, the aseptic acetone solution that adds 4 times of amounts, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 6mg hyaluronate sodium, be dissolved in 1ml physiological balance liquid (B) [77% DMEM (GIBCO company), 20%FBS (SIGMA company)], 2%Glutamine (SIGMA company) and 1% mycillin (Dalian Metro), it is 7.0～7.4 that caustic lye of soda is regulated its pH value, add an amount of (C) [ciliary nerves somatomedin (60ng/ μ l)], make its concentration reach 5%, fully stir and be mixed with hyaluronic acid sodium gel.1～10 ℃ of cold preservation is standby.
With getting muscle specimen (about 1cm * 1cm * 1cm or bigger) with the unparalleled anti-DMEM liquid of high sugared serum-free (GIBCO company) flushing 2 times, flush away blood and fat etc., reject fascia, the fragment (muddy flesh) that is cut into 1mm3 adds mixed enzyme (SIGMA company) and (1g/1.5ml) digests 45min after blowing and beating 5min with suction pipe under 37 ℃ of (getting final product in the incubator) conditions in the back, and every 15min blows and beats 1 time with suction pipe.After 45 minutes, muscular tissue should be in the pasty state, visual no obvious impurity, can add culture fluid (82%DMEM (GIBCO company) this moment, 15%FBS (SIGMA company), 2%Glutamine (SIGMA company) and 1% mycillin (Dalian Metro) stop digestion, cultivate liquid measure 2～3 times for the adding mixed enzyme.After 200 eye mesh screens filter, use the culture fluid re-suspended cell, per minute 1000 leaves heart 5min, abandons supernatant, behind the adding culture fluid re-suspended cell, is inoculated in 25cm
2In the culture bottle (CORNING company) and be labeled as P1.Put into 37 ℃, 5%CO
2Cultivate in the incubator, after 2 hours the cell of culture medium in the culture bottle and suspension is transferred to (P2) in the new culture bottle together.The supernatant that takes out P2 behind the 24h is displaced in the centrifuge tube, centrifugal 10 minutes of the speed of changeing with per minute 1000.Supernatant is removed in centrifugal end back, just can obtain the higher P3 of purity this moment.
The hyaluronic acid sodium gel that cold preservation is standby takes out the room temperature placement before the operation, and with micropipettor ready (D) evenly is stirred in the hyaluronic acid sodium gel.In the art the capable end of neurologic defect place both sides broken ends of fractured bone finishing back application catheter-end adventitia is sewed up, reuse disposable sterilized injector (the special large aperture of socket syringe needle) is drawn an amount of hyaluronic acid sodium gel and is injected by conduit one side or puncture conduit, makes its conduit fully full.Thereby having formed one is outer wall with the conduit, and hyaluronic acid sodium gel is a content, is rich in the favourable microenvironment of short neural reparation and regenerated cell and the factor.Tube wall is played when providing powerful support for effect, will better bring into play its curative effect.
Claims (10)
1. a duct filler material that is used for bridge grafting nerves includes the refining dry powder (A) of hyaluronate sodium, physiological balance liquid (B), it is characterized in that also containing various cells (D) composition of short nerve growth cell growth factor (C) or short nerve growth.
2. according to the said new catheter packing material that is used for bridge grafting nerves of claim 1, it is characterized in that said hyaluronate sodium makes with extra care the formation of dry powder (A), physiological balance liquid (B), short nerve growth cell growth factor (C) and be: (A) 1～10%; (B) 80%～99%; (C) 1～10%.
3. according to the said new catheter packing material that is used for bridge grafting nerves of claim 1, it is characterized in that said hyaluronate sodium makes with extra care the formation of the various cells (D) of dry powder (A), physiological balance liquid (B), short nerve growth and can be: (A) 1～10%; (B) 80%～99%; (D) trace.
4. according to the said duct filler material that is used for bridge grafting nerves of claim 1, it is characterized in that said hyaluronate sodium makes with extra care the formation of the various cells (D) of dry powder (A), physiological balance liquid (B), short nerve growth cell growth factor (C), short nerve growth and can be: (A) 1～10%; (B) 80%～99%; (C) 1～10%; (D) trace.
5. according to the said duct filler material that is used for bridge grafting nerves of claim 2, it is characterized in that said short nerve growth factor (C) can be one or more of fibroblast growth factor, ciliary nerves somatomedin, neurotrophic factor.
6. according to the said duct filler material that is used for bridge grafting nerves of claim 3, it is characterized in that the cell of said short nerve growth (D) can be one or more of Schwann cell, muscle stem cell, bone marrow stroma stem cell.
7. according to the said duct filler material that is used for bridge grafting nerves of claim 4, it is characterized in that said short nerve growth cell growth factor (C) can be one or more of nerve growth factor, Brain Derived Neurotrophic Factor, acid fibroblast growth factor, alkaline sarcoplast somatomedin, ciliary nerves somatomedin, glial cell line-derived neurotrophic factor, insulin like growth factor; Said short nerve growth cell (D) can be one or more of Schwann cell, bone marrow stroma stem cell, muscle stem cell.
8. one kind as claim 1 or the 2 said preparation methoies that are used for the duct filler material of bridge grafting nerves, it is characterized in that this method comprises the steps:
(1) hyaluronate sodium dry powder is added deionized water, be made into 0.01～0.2% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, add aseptic acetone or alcoholic solution that 1-5 doubly measures, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 1～10% hyaluronate sodium, be dissolved in 90%～99% physiological balance liquid (B), regulating its pH value is 7.0-7.4, fully stirs the hyaluronic acid sodium gel that is mixed with 1～10mg/ml concentration, discontinuous degassing, and 1-10 ℃ of cold preservation is standby;
(3) add the short nerve growth cell growth factor (C) of trace and be prepared into the duct filler material that is used for bridge grafting nerves.
9, a kind of as claim 1 or the 3 said preparation methoies that are used for the duct filler material of bridge grafting nerves, it is characterized in that this method comprises the steps:
(1) hyaluronate sodium dry powder is added deionized water, be made into 0.01～0.2% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, add aseptic acetone or alcoholic solution that 1-5 doubly measures, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 1～10% hyaluronate sodium, be dissolved in 90%～99% physiological balance liquid (B), regulating its pH value is 7.0-7.4, fully stirs the hyaluronic acid sodium gel that is mixed with 1～10mg/ml concentration, discontinuous degassing, and 1-10 ℃ of cold preservation is standby;
(3) add the micro-cell (D) of urging nerve growth and be prepared into the duct filler material that is used for bridge grafting nerves.
10, a kind of as claim 1 or the 4 said preparation methoies that are used for the duct filler material of bridge grafting nerves, it is characterized in that this method comprises the steps:
(1) hyaluronate sodium dry powder is added deionized water, be made into 0.01～0.2% sodium hyaluronate solution, carry out aseptic filtration by 0.22 μ m cellulose acetate membrane, collect degerming filtrate, add aseptic acetone or alcoholic solution that 1-5 doubly measures, collecting precipitation obtains the refining dry powder (A) of aseptic hyaluronate sodium behind the vacuum drying;
(2) get the refining dry powder (A) of 1～10% hyaluronate sodium, be dissolved in 90%～99% physiological balance liquid (B), regulating its pH value is 7.0-7.4, fully stirs the hyaluronic acid sodium gel that is mixed with 1～10mg/ml concentration, discontinuous degassing, and 1-10 ℃ of cold preservation is standby;
(3) add the micro-cell (D) of urging nerve growth cell growth factor (C) and short nerve growth and be prepared into the duct filler material that is used for bridge grafting nerves.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106237382A (en) * | 2016-08-26 | 2016-12-21 | 四川大学 | Promote CO2 laser weld pipe and preparation method thereof |
| CN110801534A (en) * | 2018-08-06 | 2020-02-18 | 华南协同创新研究院 | Biodegradable nerve conduit and preparation method thereof |
-
2006
- 2006-03-31 CN CN 200610066434 patent/CN1872354A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106237382A (en) * | 2016-08-26 | 2016-12-21 | 四川大学 | Promote CO2 laser weld pipe and preparation method thereof |
| CN110801534A (en) * | 2018-08-06 | 2020-02-18 | 华南协同创新研究院 | Biodegradable nerve conduit and preparation method thereof |
| CN110801534B (en) * | 2018-08-06 | 2022-01-14 | 华南协同创新研究院 | Biodegradable nerve conduit and preparation method thereof |
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