CN100370958C - Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold - Google Patents
Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold Download PDFInfo
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- CN100370958C CN100370958C CNB2005100122014A CN200510012201A CN100370958C CN 100370958 C CN100370958 C CN 100370958C CN B2005100122014 A CNB2005100122014 A CN B2005100122014A CN 200510012201 A CN200510012201 A CN 200510012201A CN 100370958 C CN100370958 C CN 100370958C
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- 238000009413 insulation Methods 0.000 claims abstract description 12
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- 238000011049 filling Methods 0.000 claims abstract description 9
- 230000007935 neutral effect Effects 0.000 claims abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 22
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 14
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 13
- 229960002897 heparin Drugs 0.000 claims description 13
- 229920000669 heparin Polymers 0.000 claims description 13
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims description 12
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
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- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 6
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- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
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- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
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- 210000003437 trachea Anatomy 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
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- Materials For Medical Uses (AREA)
Abstract
The present invention relates to a multi-channel nerve rehabilitating guide tube, and a preparation method and a dedicated mold thereof. The guide tube is composed of a circular pipe made of chitosan and inner base materials with axial multiple channels; the mold is composed of a thermal insulation bottom seat made of foamed polystyrene and a stainless steel cover plate; the thermal insulation bottom seat is regularly distributed with a plurality of cylindrical holes of different internal diameters, and the bottom of each hole is provided with a thin pinhole communicated with the bottom of the bottom seat. The preparation method of the multi-channel nerve rehabilitating guide tube is characterized in that circular pipes with different diameters made of chitosan are inserted into the holes with corresponding diameters on the bottom seat of the mold, and then, various filling base materials are injected into the circular pipes made of chitosan according to the need; the precooled stainless steel cover plate rapidly covers the circular pipes, and then, the circular pipes are placed into a low-temperature refrigerator for 12 hours; next, the circular pipes are frozen, dried, deacidified and rinsed by distilled water until the PH value is neutral; then, at last, the circular pipes are naturally dried under room temperature so as to form the multi-channel nerve rehabilitating guide tube. The multi-channel nerve rehabilitating guide tube has the advantages of simple preparation process, stable result and suitable bulk production.
Description
Technical field
The present invention relates to a kind of preparation method and particular manufacturing craft thereof of multi-channel nerve rehabilitating tube, belong to bio-medical material and tissue engineering technique field.
Background technology
The long method apart from peripheral nerve defection of treatment is a nerve autograft clinically at present.But the autologous nerve source is limited, and is accompanied by for district's function impaired.Along with the development of medical science, biology and tissue engineering, be subjected to paying close attention to widely from disconnected neural research with the nerve rehabilitating tube bridge joint.With two severed nerve adventitias and tube wall sutured, the neural axon far-end of can growing into along tube chamber from near-end.Nerve trachea can prevent the intrusion of fibrous scar tissue in the process of neuranagenesis, improve the concentration of local nerve trophic factors, the guiding axon growth.But the hollow conduit that former studies is used as autogenous vein, silica gel tube and the artificial nerve catheter made of PLGA, collagen, chitosan etc., lacks enough internal surface areas, is unfavorable for the apposition growth of cell and aixs cylinder, and is easy to subside.Given this, people begin to consider filling substrate material in conduit, in order to growth that guides aixs cylinder and the slow-released carrier that becomes trophic factors.Chinese patent application 00810000.4 provides a kind of polyester material nerve trachea, fills the collagen hydrochloric acid solution obtains having microfibreization through lyophilization collastromin content in the tube chamber.This method weak point is that the matrix structure that makes is rambling, is unfavorable for that aixs cylinder passes through conduit with the shortest distance.Chinese patent application 02113103.1 is inserted chitosan conduit with chitin fiber and is prepared the pure chitosan nerve trachea; Chinese patent application 02105864.4 is placed the folding porous polyester film with convex-concave surface that curls in the hollow pipe that polyester material makes to increase inner cavity surface long-pending, makes the multi-channel type nerve regeneration conduit.Similarly method also has a lot, and subject matter is that fiber and the swelling of thin film in conduit are bigger, and the two all is free state in conduit, is not easy to keep stable structure.U.S. Pat 5925053 (1999) and US 6214021 (2001) provide the method for making multitube chamber (multi-lumen) nerve trachea, this method is placed some fine fibres in advance in mould, the macromolecular solution that will contain organic solvent again injects in the mould, solidified through freezing method afterwards, through the distillation mode solvent in this material is extracted out again, at last fine fibre is extracted out and the nerve trachea in formation multitube chamber.But this method gained nerve trachea does not have outer wall, be unfavorable for fixing with nerve suture, and fibrous tissue is easily invaded on every side.
Ideal nerve rehabilitating tube should be simulated normal neuromechanism, has the permeability of intensive axial passage, suitable aperture, porosity and outer wall.Timbering material should have excellent biological compatibility and biodegradable.U.S. Pat 4522753 (1990) and Chinese patent publication number CN1380115A inject silica gel tube with collagen and aminoglycan mixing suspension, and the two ends shutoff is slowly vertically inserted silica gel tube in the stranguria of cold type liquid again, can get the axially-aligned multiple channel duct after the lyophilizing.But operation is comparatively complicated, and efficient is lower, and silica gel tube separates easily with inner multichannel substrate after the lyophilizing, and silica gel tube can not degradation in vivo, needs second operation to take out.But the novel nerve rehabilitating tube that adopts simple method and/or mould efficient production to have axially-aligned micro-channel, Stability Analysis of Structures and vivo degradation is an important research direction.
Summary of the invention
The purpose of this invention is to provide a kind of multi-channel nerve rehabilitating tube and preparation method thereof and particular manufacturing craft, make the method therefor operating process simple, be easy to penetration and promotion and use; Make prepared multi-channel nerve rehabilitating tube result stable, can make the conduit of a large amount of different sizes simultaneously, be fit to standardization and produce in batches.
Technical scheme of the present invention is as follows:
But a kind of multi-channel nerve rehabilitating tube of the material preparation with mould and vivo degradation, this multi-channel nerve rehabilitating tube are by the chitosan pipe and have axial multichannel internal matrix and form; Described substrate is one or more the mixture in chitosan, collagen, gelatin, the heparin, and described axial multichannel internal diameter is 10～200 μ m; Described chitosan tube wall thickness is 0.2～1mm, and internal diameter is 1～5mm.
The invention provides a kind of particular manufacturing craft for preparing described multi-channel nerve rehabilitating tube, it is characterized in that: this mould is made up of foamed polystyrene heat insulation pedestal and rustless steel cover plate; Regular distribution the cylindrical cavity of a plurality of different inner diameters on heat insulation pedestal, and the hole degree of depth is 1～5cm; A fine needle hole that prevents that bubble from retaining is arranged at the hole bottom, and this fine needle hole and base bottom connect.
The present invention also provides a kind of method that adopts described particular manufacturing craft to prepare multi-channel nerve rehabilitating tube, it is characterized in that this method carries out as follows:
Step 1, at first prepare internal diameter 1～5mm, the chitosan pipe of wall thickness 0.2～1.0mm;
Step 2, with length is that the chitosan pipe of 1～5cm inserts in the hole of respective diameters on the foamed polystyrene heat insulation pedestal, in the chitosan pipe, inject filling substrate as required then, described filling substrate is 2～4% (w/v) chitosan acetic acid solution, or be 2～4% (w/v) chitosan acetic acid solution respectively with 1～3% (w/v) collagen hydrochloric acid solution, 1% (w/v) heparin solution, 1% (w/v) is in conjunction with bFGF, the heparin solution of VEGF, the mixed liquor of 10～20% (w/V) aqueous gelatin solution, rustless steel cover plate lid that rapidly will pre-cooling under-20 ℃～-80 ℃ temperature in the above, being placed on temperature then is that-20 ℃～-80 ℃ refrigerators are interior more than 12 hours, take out the back and remove the rustless steel cover plate, take out the pipe that contains the ice crystal post from heat insulation pedestal, lyophilization is at least 24 hours in freezer dryer;
Step 3, place the sodium hydrate aqueous solution of 2～4% (w/v) to soak 15～30 minutes the specimen after the step 2 lyophilization, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.2～7.4 at least 30 minutes, natural drying at room temperature, it is standby to sterilize, and obtains the needed multichannel nerve rehabilitating tube of axially-aligned that has.
The present invention compared with prior art has the following advantages and the salience effect: 1. the material of mould is foamed polystyrene and rustless steel, obtains easily and processes simple and easyly, and it is simple to prepare the conduit operating process with mould, is easy to penetration and promotion and uses.2. cryogenic temperature is controlled by refrigerator, and prepared multi-channel nerve rehabilitating tube stable performance can be made the conduit of a large amount of different sizes simultaneously, is fit to standardization and produces in batches.3. a series of nerve rehabilitating tubes with axial multichannel substrate are provided.This nerve rehabilitating tube is by the chitosan pipe and have the multichannel substrate of axially-aligned and form.Conduit outer wall and multichannel substrate are Biodegradable material and make, and therefore, can degrade in vivo after finishing neural repair function in good time, avoid second operation to take out conduit.4. heparin can with specific the mutually combining of many somatomedin (as bFGF, VEGF), and heparin also have the protection factor bonded with it effect.To mix as multichannel substrate with solution such as chitosans in conjunction with the heparin of the factors such as bFGF, VEGF, the support of axon elongation not only is provided, can also be as the slow-released carrier of somatomedin.
Description of drawings
Fig. 1 prepares the sketch map of multi-channel nerve rehabilitating tube for using mould.
Fig. 2 is the multi-channel nerve rehabilitating tube sketch map with the mould preparation.Form by chitosan pipe 2 and multichannel substrate 6; The position of corresponding A, B, C, D photo among rectangular box region representation Fig. 3 among the figure.
The scanning electron microscope picture of Fig. 3 multi-channel nerve rehabilitating tube different cross section.
The specific embodiment
According to above-mentioned preparation method and accompanying drawing, the present invention is described in further detail in conjunction with the embodiments:
Fig. 1 prepares the sketch map of multi-channel nerve rehabilitating tube for using mould.This mould is made up of foamed polystyrene heat insulation pedestal 4 and rustless steel cover plate 3; Regular distribution the cylindrical cavity 1 of a plurality of different inner diameters on heat insulation pedestal 4, and the hole degree of depth is 1～5cm; A fine needle hole 5 that prevents that bubble from retaining is arranged at the hole bottom, and this fine needle hole and base bottom connect.
Operating process is as follows: in the hole 1 on the chitosan pipe 2 insertion foamed polystyrene bases 4, inject various filling substrates as required in the chitosan pipe; Described filling substrate is 2～4% (w/v) chitosan acetic acid solution, or is 2～4% (w/v) chitosan acetic acid solution combines heparin solution, 10～20% (w/v) aqueous gelatin solution of bFGF, VEGF respectively with 1～3% (w/v) collagen hydrochloric acid solution, 1% (w/v) heparin solution, 1% (w/v) mixed liquor.Rustless steel cover plate 3 lids that rapidly will pre-cooling under-20 ℃～-80 ℃ temperature behind the filling substrate in the above, being placed on temperature be that-20 ℃～-80 ℃ refrigerators are interior more than 12 hours; Remove the rustless steel cover plate then, take out the pipe that contains the ice crystal post, place in the low-temperature (low temperature) vessel, lyophilization is at least 24 hours in freezer dryer.Place the sodium hydrate aqueous solution of 2～4% (w/v) to soak 15～30 minutes the specimen after the lyophilization, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.2～7.4 at least 30 minutes, natural drying at room temperature, it is standby to sterilize, and obtains the needed multichannel nerve rehabilitating tube of axially-aligned that has.
Because the foamed polystyrene base is wrapped in around the conduit, can reduce heat passage by tube wall, heat passage main rustless steel cover plate by the top is carried out, thereby cause thermograde from top to bottom in the pipe, ice crystal is generation suitable for reading in pipe at first, then to end opening growth, cause the directivity formation that is separated; The ice crystal diameter is by substrate concentration and cryogenic temperature decision.
Embodiment 1:
1) at first utilizes conventional method, as the method for Chinese patent application ZL 02149086.4, prepare internal diameter 1～5mm, the chitosan pipe of wall thickness 0.2～1.0mm;
2) as shown in Figure 1, in the hole 1 with respective diameters on the chitosan pipe 2 insertion mold bases 4 of the long different-diameter of 5cm, in chitosan pipe 2, inject 4% (w/v) chitosan solution (with the 0.2mol/L acetic acid solutions) then.Rustless steel cover plate 3 with-20 ℃ of pre-coolings covers in the above rapidly, be placed on-20 ℃ of refrigerators more than 12 hours, remove rustless steel cover plate 3 then, take out the pipe that contains the ice crystal post, place in the low-temperature (low temperature) vessel, lyophilization in freezer dryer (-40 ℃ approximately of trunk temperature) is more than 24 hours;
3) with step 2) to place concentration be that the sodium hydrate aqueous solution of 2% (w/v) soaked 30 minutes for specimen after the lyophilization, take out, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.4 30 minutes, natural drying at room temperature finally obtains needed nerve rehabilitating tube with axially-aligned multichannel (diameter range 30～200 μ m).
4) with gamma ray irradiation or the sterilization of 75% soak with ethanol.Use the physiological saline solution rinsing before using, soaked 30 minutes.
Be used for the reparation of nerve injury.
Embodiment 2
1) at first utilizes conventional method, as the method for Chinese patent application ZL 02149086.4, prepare internal diameter 1～5mm, the chitosan pipe of wall thickness 0.2～1.0mm;
2) as shown in Figure 1, the chitosan pipe 2 of the long different-diameter of 3cm is inserted in the hole 1 of respective diameters on the mold base 4, inject the mixture of 4% (w/v) chitosan solution (with the 0.2mol/L acetic acid solutions) and 10% aqueous gelatin solution then in the chitosan pipe, the two mass ratio is 1: 1.Rustless steel cover plate 3 with-80 ℃ of pre-coolings covers in the above rapidly, be placed on-80 ℃ of refrigerators more than 12 hours, remove rustless steel cover plate 3 then, take out the pipe that contains the ice crystal post, place in the low-temperature (low temperature) vessel, lyophilization in freezer dryer (-40 ℃ approximately of trunk temperature) is more than 24 hours;
3) with step 2) to place concentration be that the sodium hydroxide solution of 3% (w/v) soaked 20 minutes for specimen after the lyophilization, take out, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.2 30 minutes, natural drying at room temperature finally obtains needed nerve rehabilitating tube with axially-aligned multichannel (diameter range 10～100 μ m).
4) adopt the sterilization of 75% soak with ethanol.Use the physiological saline solution rinsing before using, soaked 30 minutes.Be used for the reparation of nerve injury.
Embodiment 3
1) at first utilizes conventional method, as the method for Chinese patent application ZL 02149086.4, prepare internal diameter 1～5mm, the chitosan pipe of wall thickness 0.2～1.0mm;
2) as shown in Figure 1, the chitosan pipe 2 of the long different-diameter of 1cm is inserted in the hole 1 of respective diameters on the mold base 4, in chitosan pipe 2, inject heparin and chitosan mixed solution glue then in conjunction with bFGF (20 μ g/L), VEGF (20 μ g/L), the heparin final concentration is 1%, and the chitosan final concentration is 3%.Rustless steel cover plate 3 with-80 ℃ of pre-coolings covers in the above rapidly, be placed on-80 ℃ of refrigerators more than 12 hours, remove rustless steel cover plate 3 then, take out the pipe that contains the ice crystal post, place in the low-temperature (low temperature) vessel, lyophilization in freezer dryer (-40 ℃ approximately of trunk temperature) is more than 24 hours;
3) with step 2) to place concentration be that the sodium hydroxide solution of 2% (w/v) soaked 15 minutes for specimen after the lyophilization, take out, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.4 30 minutes, natural drying at room temperature finally obtains needed nerve rehabilitating tube with axially-aligned multichannel (diameter range 10～150 μ m).
4) adopt the sterilization of 75% soak with ethanol.Use the physiological saline solution rinsing before using, soaked 30 minutes.Be used for the reparation of spinal cord injury.
Claims (2)
1. particular manufacturing craft of making multi-channel nerve rehabilitating tube, it is characterized in that: this mould is made up of foamed polystyrene heat insulation pedestal (4) and rustless steel cover plate (3); Regular distribution the cylindrical cavity (1) of a plurality of different inner diameters on heat insulation pedestal, and pore diameter range is 1.2mm～6mm, and the hole degree of depth is 1～5cm, and a fine needle hole (5) that prevents that bubble from retaining is arranged at the hole bottom, and this fine needle hole and base bottom connect.
Multi-channel nerve rehabilitating tube with above-mentioned Mold Making has following feature: by the chitosan pipe with have axial multichannel internal matrix and form; Described substrate is one or more the mixture in chitosan, collagen, gelatin, the heparin, and described axial multichannel internal diameter is 10～200 μ m; Described chitosan tube wall thickness is 0.2～1mm, and internal diameter is 1～5mm.
2. a utilization prepares the method for multi-channel nerve rehabilitating tube as mould as described in the claim 2, it is characterized in that this method carries out as follows:
Step 1, at first prepare internal diameter 1～5mm, the chitosan pipe (2) of wall thickness 0.2～1.0mm;
Step 2, with length is that the chitosan pipe (2) of 1～5cm inserts in the hole (1) that foamed polystyrene heat insulation pedestal (4) goes up respective diameters, inject filling substrate then as required in chitosan pipe (2), described filling substrate is to be selected from a kind of in the following solution: 2～4% (w/v) chitosan acetic acid solution, 2～4% (w/v) chitosan acetic acid solution and the blended mixed liquor of 1～3% (w/v) collagen hydrochloric acid solution, 2～4% (w/v) chitosan acetic acid solution and the blended mixed liquor of 10～20% (w/v) aqueous gelatin solution, 2～4% (w/v) chitosan acetic acid solution and the blended mixed liquor of 1% (w/v) heparin solution or 2～4% (w/v) chitosan acetic acid solution and interpolation bFGF, the blended mixed liquor of 1% (w/v) heparin solution of VEGF; Rustless steel cover plate (3) lid that rapidly will pre-cooling under-20 ℃～-80 ℃ temperature in the above, being placed on temperature then is that-20 ℃～-80 ℃ refrigerators are interior more than 12 hours, take out the back and remove the rustless steel cover plate, from heat insulation pedestal, take out the pipe that contains the ice crystal post, lyophilization is after at least 24 hours in freezer dryer, becomes the specimen after the lyophilization;
Step 3, place the sodium hydrate aqueous solution of 2～4% (w/v) to soak 15～30 minutes the specimen after the step 2 lyophilization, extremely neutral with rinsed with deionized water, be dipped in again in the phosphate buffer of pH=7.2～7.4 at least 30 minutes, natural drying at room temperature, it is standby to sterilize, and obtains the needed multichannel nerve rehabilitating tube of axially-aligned that has.
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| CNB2005100122014A CN100370958C (en) | 2005-07-15 | 2005-07-15 | Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold |
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| CNB2005100122014A CN100370958C (en) | 2005-07-15 | 2005-07-15 | Multi-channel nerve rehabilitating tube, its preparation process and dedicated mold |
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| CN100370958C true CN100370958C (en) | 2008-02-27 |
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| CN102688110A (en) * | 2012-06-13 | 2012-09-26 | 北京天新福医疗器材有限公司 | Multi-aperture nerve repairing tube and preparation method and application thereof |
| CN103263308B (en) * | 2013-05-17 | 2015-07-29 | 中国人民解放军第四军医大学 | Many micropores degradable collagen-chitin nerve trachea and preparation method thereof |
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| CN1237914A (en) * | 1996-11-20 | 1999-12-08 | 清水庆彦 | Artificial neural canal |
| CN1568904A (en) * | 2004-05-14 | 2005-01-26 | 清华大学 | Preparation method of chitosan tubular bracket |
| CN1593354A (en) * | 2004-06-25 | 2005-03-16 | 清华大学 | Nerve tissue engineering tube type bracket and method for making same |
-
2005
- 2005-07-15 CN CNB2005100122014A patent/CN100370958C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1237914A (en) * | 1996-11-20 | 1999-12-08 | 清水庆彦 | Artificial neural canal |
| CN1568904A (en) * | 2004-05-14 | 2005-01-26 | 清华大学 | Preparation method of chitosan tubular bracket |
| CN1593354A (en) * | 2004-06-25 | 2005-03-16 | 清华大学 | Nerve tissue engineering tube type bracket and method for making same |
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