CN100479785C - Producing method for double-layer artificial nerve catheter - Google Patents
Producing method for double-layer artificial nerve catheter Download PDFInfo
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- CN100479785C CN100479785C CN 200510063413 CN200510063413A CN100479785C CN 100479785 C CN100479785 C CN 100479785C CN 200510063413 CN200510063413 CN 200510063413 CN 200510063413 A CN200510063413 A CN 200510063413A CN 100479785 C CN100479785 C CN 100479785C
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Abstract
The invention discloses an artificial nerve conduit of double layer and the method for preparing same. The said nerve conduit comprises an inner conduit and an outer layer, which are made from degradable and adsorptive material in living body. The slit between said inner conduit and outer conduit is filled with fiber albumen glue (used for regeneration of nervous tissue and blood vessel). The artificial nerve conduit of double layer can be used for bypass surgery for damaged round nerve and spinal marrow, and it will be degraded and adsorbed in living body after nerve regeneration. The said artificial nerve conduit possesses good elasticity and strength, and the degradation time is adaptive to growing speed of nervous fiber (about 1-6 months).
Description
Technical field
The present invention relates to a kind of double-layer artificial nerve catheter and preparation method thereof.
Background technology
Peripheral nerve injury or when damaged, the method that people at first select for use is that the two nervus lateralis broken ends of fractured bone are directly coincide.If can not directly coincide, since around the obstructive action of connective tissue etc., then can cause newborn nerve fiber to arrive its position, target tissue place by defect, stump at regenerating nerve forms outgrowth neuroma sample tissue, consequently causes the dysfunction of wound place limbs.In addition, also there is the method that adopts nerve autograft to repair peripheral nerve injury.But adopt this method not only can cause the functional impairment at the position of drawing materials, and the position functional rehabilitation of transplanted nerve is also undesirable, and allogeneic nerve is transplanted the problems such as immunological rejection that can't overcome that exist.
Therefore, people begin attempting adopting two broken ends of fractured bone of the damaged nerve of artificial material conduit bridge joint, attempt to induce the nerve fiber regeneration of the broken ends of fractured bone to arrive far-end and rebulid with target tissue and get in touch.For example, chemical material silicone, polyethylene, polyglycolic acid, polylactic acid etc.; Biomaterial chitosan, gelatin, collagen protein etc.; Organism tubular bracket tremulous pulse, vein and musculomembranous tube etc. all are used for preparing conduit.But because the chemical material biocompatibility is relatively poor, the mechanical property of biomaterial is relatively poor, and the organism pipe also exists a series of problems such as mechanical property is relatively poor, up to now, induces length≤3 centimetre of neuranagenesis with conduit.
The research of repair of spinal cord injury has had the history of last 100 years, and tracing it to its cause mainly is that the partial microenvironment of damage is unfavorable for the regeneration of nerve fiber and extends to target site.In recent years, after people adopt methods such as sciatic nerve transplanting, artificial material conduit to prove Spinal Cord Injury in Rats,, be can be regenerated from disconnected nerve fiber as long as improve partial microenvironment.But, because problems such as the biocompatibility of conduit, mechanical property also do not have the comparatively ideal artificial nerve catheter that is used for spinal cord regeneration now.
Summary of the invention
The purpose of this invention is to provide a kind of double-layer artificial nerve catheter and preparation method thereof.
Double-layer artificial nerve catheter provided by the present invention, comprise by decomposing an inner conductor and the outer conductor that absorbent material is made in the organism, described inner conductor is set in described outer conductor inside, is filled with Fibrin Glue between the tube gap of described inner conductor and described outer conductor.
Fibrin Glue between these tube gaps not only can guarantee bonding mutually between the double-layer tube can also provide regenerated supporting structure for regenerated nervous tissue or blood vessel etc.For the nervous tissue in the pipe can be grown better, in Fibrin Glue, also be added with extracellular matrix and with nervous tissue's closely-related biomacromolecule material of regenerating, the consumption of these additives is collagen protein 50-100 μ g/ml, laminine 60-100ng/ml, Nogo antibody 30-60ng/ml, somatomedin 10-300ng/ml, L-poly-D-lysine 60-100 μ g/ml.Also can add in Schwann cell, astrocyte and the neural stem cell one or more.Import volume about these additional cells gets final product according to conventional method.
Can decompose the copolymer etc. that absorbent material comprises chitosan and derivant, collagen protein, gelatin, L-poly-D-lysine, Polyethylene Glycol acid, polylactic acid or lactic acid and ε-caprolactone in the organism commonly used, its netted screen distance with about 10-500 μ m.
Preferred, outer conductor can be made by chitosan, and inner conductor can be made by collagen protein.
The preparation method of above-mentioned double-layer artificial nerve catheter comprises the steps:
1) preparation chitosan catheter: chitosan-acetic acid solution is poured in the core film die, is warmed to 80-160 ℃, take out behind the distilled water immersion, then in the acetum of chitosan repeatedly the dipping, air-dry, obtain chitosan catheter;
2) preparation collagen protein conduit: pour the collagen protein hydrochloric acid solution in core film die molding,, carry out the mechanical presses molding again, obtain the collagen protein conduit through after the lyophilization;
3) fiberfill fibers albumin glue: gained collagen protein conduit is inserted in the chitosan catheter, Fibrin Glue is injected between the tube gap of two-layer conduit then, obtain described double-layer artificial nerve catheter.
The described chitosan of step 1) is that deacetylation is 80-85% chitosan; Described chitosan-acetic acid solution is dissolved in 100-200ml, 2% acetum with the 200mg chitosan and obtains.After having prepared chitosan catheter, also can pass through the NaOH solution soaking.
Step 2) the collagen protein hydrochloric acid solution described in is dissolved in 100 milligrams of collagen protein in 30-100 milliliters of 1mol/L hydrochloric acid and obtains.
Layer artificial nerve catheter of the present invention can be by the position of operation transplantation to nerve injury.As when being used for peripheral nerve injury and repairing, can will be inserted in the conduit about 2-5 millimeter from disconnected nerve two broken ends of fractured bone and sew up processing with operation suture thread; When being used for repair of spinal cord injury, damaged position can be transplanted and be fixed to conduit.Its length and diameter can be according to the different of neureotomy length and thickness and difference.For example, damaged with 10 centimetres of sheep sciatic nerves is model, and the internal diameter of employed artificial nerve catheter is the 6-10 millimeter, length 11-12 centimetre.In addition, when being used for repair of spinal cord injury, the length of this pipe and diameter still need to determine according to the position of operation and the segments of spinal cord of operation.The catheter diameter that is adopted when for example, the rat thoracic spinal cord damages is preferably about 1.5-2.5 millimeter; Then should elect the 2-15 millimeter as at large animal and man-hour, particularly about 10 millimeters.
Clinically, can adopt double-layer artificial nerve catheter of the present invention to carry out bypass surgery after peripheral nerve and the spinal cord injury, and after neuranagenesis is finished, in animal body degraded and absorbed progressively.Compare with nerve trachea in the past, the reproduction speed that cuts off nerve is very fast, and the quality of regenerating nerve is also better.Double-layer artificial nerve catheter of the present invention is owing to adopt double-decker, has better elastic and intensity, the speed of the degradation time of institute's materials used and nerve fiber growth adapts (individual month of about 1-6), can be applied to the reparation behind the peripheral nerve injury and spinal cord injury clinically.
Description of drawings
Fig. 1 is chitosan catheter and collagen protein conduit photo;
Fig. 2 is a double-layer artificial nerve catheter;
Fig. 3 is newborn sheep sciatic nerve;
Fig. 4 is newborn rat spinal cord tissue.
The specific embodiment
Embodiment 1, preparation double-layer artificial nerve catheter
Present embodiment ectomesoderm conduit Preparation of Chitosan, inner conductor prepares with collagen protein, then inner conductor is inserted in the outer conductor, and is last, fibrin gel is injected among the gap of double-layer tube, promptly makes double-layer artificial nerve catheter.
Wherein, the preparation method of chitosan catheter is:
1, chitosan is dissolved in the acetum;
2, chitosan solution is poured into the core die for molding, and carried out hot dehydrated crosslinking and handle;
3, the gained conduit is flooded in chitosan solution and air-dry operation several repeatedly.
The preparation method of collagen protein conduit is:
1, collagen protein is dissolved in the hydrochloric acid solution;
2, collagen solution is poured into the core die for molding, and carried out lyophilization and handle;
3, the gained conduit is carried out mechanical compress, obtain highdensity microfibre collagen protein conduit.
In Fibrin Glue, can also contain some supplementary elements, as collagen protein, laminine, Nogo antibody, somatomedin, L-poly-D-lysine, Schwann cell, astrocyte and neural stem cell etc., the introduction method of these additives gets final product according to a conventional method.
The concrete operations of this preparation process are as follows:
1, chitosan is made tubulose: prepare about 11 centimetres of length, 9 millimeters of internal diameters, the conduit that thickness is about 1 millimeter.With deacetylation is that the chitosan 200mg of 80-85% is dissolved among the 2% acetum 100ml, and to 9 millimeters of diameter of mandrel, telescopic internal diameter is in 10 millimeters the mould, to heat to 120 ℃, with distilled water immersion, takes out conduit with above-mentioned injection of solution.Again with conduit dipping and air-dry operation 10 times repeatedly in above-mentioned chitosan solution, and soak in the NaOH of 1N solution, reach pH7 up to the pH value of conduit leachate, this conduit is shown in A among Fig. 1.
2, preparation collagen protein pipe: type i collagen protein 10 0mg is dissolved in the 60ml 1N hydrochloric acid solution, then this solution is injected 8 millimeters of diameter of mandrel, telescopic internal diameter is 8.5 millimeters a die for molding, and adopts freezer dryer to carry out vacuum drying (drying is 24 hours under 70 ℃); Then, the conduit that makes is carried out the mechanical presses molding, obtain highdensity microfibre collagen protein pipe, this conduit is shown in B among Fig. 1.
3, the collagen protein pipe is inserted in the chitosan catheter, and between tube gap, injects the fiberfill fibers albumin glue, promptly make double-layer artificial nerve catheter, as shown in Figure 2.
Wherein, used Fibrin Glue prescription is the medical protein glue that sell in market, and by specification uses and gets final product.
Embodiment 2, double-layer artificial conduit are used for the sciatic reparation of sheep
Method according to embodiment 1 prepares double-layer artificial nerve catheter, wherein is added with 100ng nerve growth factor NGF in every milliliter of Fibrin Glue.
10 centimetres of the common peroneal nerves of excision sheep (body weight 20kg) insert the neural broken ends of fractured bone of both sides in the above-mentioned conduit, with this conduit and neural eclipsed part 10-0 nylon line suture, dynamic observe 1 year, and carry out morphology, electrophysiology and ethological evaluation.
Result of study shows, carries out after the transplant operation 3rd month, and the behavior disorder of animal begins to recover, and do not have obvious action obstacle on the 6th month, recovers normal not having under the situation of bearing a heavy burden on the 12nd month.Employing WGA-HRP carries out neural spike, Neurfilament (NF) immunohistochemical staining proves the reconstruction that morphosis is arranged; Auric chloride dyeing proof has the recovery of motor end plate.In electrophysiological research, also proved the recovery of somatosensory evoked potential (SEP) and Motion Evoked Potential (MEP).Newborn sheep sciatic nerve as shown in Figure 3.
Embodiment 3, double-layer artificial conduit are used for the reparation of rat spinal cord tissue
According to the method for embodiment 1, long 5 millimeters of preparation, the conduit that external diameter is 2.5 millimeters.
With the wistar rat spinal cord of growing up complete cross-section is model: promptly carry out the conventional surgical operation, open vertebral plate, rat spinal cord breast 9-10 sections is excised 5 millimeters fully, above-mentioned conduit is transplanted to the part of damage, stitching spinal dura mater.Dynamic observed 1 year, and carry out the ethological evaluation of morphology, electrophysiology and BBB21.
Result of study shows, adopts BDA to carry out the neural spike of direct motion, Neurfilament (NF) immunohistochemical staining, Gless silver staining etc. and all proves the reconstruction that morphosis is arranged; Adopt electron microscope observation to form to a large amount of have marrow and unmyelinated nerve fiber; In electrophysiological research, also proved the recovery of somatosensory evoked potential (SEP) and Motion Evoked Potential (MEP); The BBB21 scoring can reach 13 fens; Newborn rat spinal cord is organized as shown in Figure 4.
Claims (6)
1, prepares the method for double-layer artificial nerve catheter, comprise the steps:
1) preparation chitosan catheter: chitosan-acetic acid solution is poured in the core film die, is warmed to 80-160 ℃, take out behind the distilled water immersion, then in chitosan-acetic acid solution repeatedly the dipping, air-dry, obtain chitosan catheter;
2) preparation collagen protein conduit: pour the collagen protein hydrochloric acid solution in core film die molding,, carry out the mechanical presses molding again, obtain the collagen protein conduit through after the lyophilization;
3) fiberfill fibers albumin glue: gained collagen protein conduit is inserted in the chitosan catheter, Fibrin Glue is injected between the tube gap of two-layer conduit then, obtain double-layer artificial nerve catheter; This double-layer artificial nerve catheter, comprise by decomposing a collagen protein inner conductor and the chitosan outer conductor that absorbent material is made in the organism, described inner conductor is set in described outer conductor inside, is filled with Fibrin Glue between the tube gap of described inner conductor and described outer conductor.
2, method according to claim 1 is characterized in that: the described chitosan of step 1) is that deacetylation is 80-85% chitosan; Described chitosan-acetic acid solution is dissolved in 100-200ml2% acetum with the 200mg chitosan and obtains.
3, preparation method according to claim 2 is characterized in that: also need pass through the NaOH solution soaking through dipping, chitosan catheter after air-dry in the described step 1).
4, according to claim 1 or 2 or 3 described preparation methoies, it is characterized in that: step 2) described in the collagen protein hydrochloric acid solution 100 milligrams of collagen protein be dissolved in 30-100 milliliters of 1mol/L hydrochloric acid obtain.
5, according to claim 1 or 2 or 3 described preparation methoies, it is characterized in that: also contain additive in the described Fibrin Glue, described additive is selected from one or more in collagen protein, laminine, Nogo antibody, somatomedin, L-poly-D-lysine, Schwann cell, astrocyte and the neural stem cell.
6, preparation method according to claim 5 is characterized in that: the consumption of described additive is collagen protein 50-100 μ g/ml, laminine 60-100ng/ml, Nogo antibody 30-60ng/ml, somatomedin 10-300ng/ml, L-poly-D-lysine 60-100 μ g/ml.
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| US8758374B2 (en) | 2010-09-15 | 2014-06-24 | University Of Utah Research Foundation | Method for connecting nerves via a side-to-side epineurial window using artificial conduits |
| US9931121B2 (en) | 2011-10-17 | 2018-04-03 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| US10842494B2 (en) | 2011-10-17 | 2020-11-24 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| WO2024044026A1 (en) * | 2022-08-26 | 2024-02-29 | Checkpoint Surgical, Inc. | Chitosan tubular member and method of producing |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US8758374B2 (en) | 2010-09-15 | 2014-06-24 | University Of Utah Research Foundation | Method for connecting nerves via a side-to-side epineurial window using artificial conduits |
| US9931121B2 (en) | 2011-10-17 | 2018-04-03 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| US10772633B2 (en) | 2011-10-17 | 2020-09-15 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| US10842494B2 (en) | 2011-10-17 | 2020-11-24 | University Of Utah Research Foundation | Methods and devices for connecting nerves |
| WO2024044026A1 (en) * | 2022-08-26 | 2024-02-29 | Checkpoint Surgical, Inc. | Chitosan tubular member and method of producing |
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