The specific embodiment
In order to understand better the advantage of this utility model embodiment; hereinafter in connection with preferred embodiment and embodiment the technical solution of the utility model is described, but protection domain of the present utility model is not subjected to the restriction of these specific embodiment and embodiment.
According to an embodiment of the present utility model, a kind of complex biological sleeve pipe is provided, it comprises the biological duct of hollow and the slow-releasing microcapsule layer that exists at the inwall of the biological duct of this hollow.
By having the slow-releasing microcapsule layer at the biological duct inwall according to the hollow of complex biological sleeve pipe of the present utility model, when this complex biological sleeve pipe being applied to Small gap bridging reparation peripheral nerve, slow-releasing microcapsule on the postoperative internal surface of sleeve pipe can provide short nerve growth material for a long time for injured nerve, is conducive to improve the repairing effect of peripheral nerve injury.
According to preferred implementation of the present utility model, the internal diameter of described complex biological sleeve pipe is 0.5mm~10mm.1mm~8m more preferably, even be preferably 1.5mm~7mm.For example, the internal diameter of described complex biological sleeve pipe can be 2mm, 3mm, 4mm, 5mm or 6mm.
To determine according to the needs of actual usefulness according to the length of stating the complex biological sleeve pipe of the present utility model.The length of general preferred described complex biological sleeve pipe is 3mm~20cm.For example, the length of described complex biological sleeve pipe can be chosen as 3.5mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 2cm, 5cm, 7cm, 10cm, 15cm, 18cm or 20cm.
According to another preferred implementation of the present utility model, the pipe thickness of described complex biological sleeve pipe is less than or equal to 2mm.Further preferred its pipe thickness is 0.1~2mm.Even preferably its pipe thickness is 0.15~1.5mm; More preferably its pipe thickness is 0.15~1.0mm; Most preferably its pipe thickness is 0.15~0.85mm.
According to preferred implementation of the present utility model, the slow-releasing microcapsule layer in the described complex biological sleeve pipe is to contain the slow-releasing microcapsule layer that promotes the nerve growth material.Preferably, the material of described promotion nerve growth is to be selected from least a in nerve growth factor, Brain Derived Neurotrophic Factor, ciliary neurotrophic factor, neurenergen 3, insulin like growth factor, the gluey cell source neurotrophic factor.
According to another preferred implementation of the present utility model, the biological duct of described hollow is the biological duct of the hollow made by biodegradation material.Preferably, described biodegradation material is to be selected from least a in chitosan, chitin, collagen, chitosan, polylactic acid, the polyglycolic acid.Even preferably, the biological duct of described hollow is the biological duct of the hollow for preparing of chitosan.Most preferably, be 27% by the deacetylation of the biological duct of the hollow of chitosan preparation, in 0.9% sodium-chloride water solution, without dissolving, under hygrometric state, thread a needle without embrittlement.
According to another embodiment of the present utility model, provide a kind of method by comprising the steps to prepare above-mentioned complex biological sleeve pipe:
Biodegradable material is dissolved in the solvent, forms the still spinning solution of Biodegradable material;
From the hollow spinneret orifice, extrude this spinning solution, obtain the biological duct of hollow;
The liquid that comprises the sustained-release micro-spheres of nerve growth factor by the multi-emulsion method preparation; And the liquid that will comprise above-mentioned sustained-release micro-spheres is injected into the inner chamber of the biological duct of described hollow, and dry under vacuum and low temperature, obtains to have at the inwall of the biological duct of described hollow the complex biological sleeve pipe of microcapsule layer.
According to preferred implementation of the present utility model, the material of described promotion nerve growth is to be selected from least a in nerve growth factor, Brain Derived Neurotrophic Factor, ciliary neurotrophic factor, neurenergen 3, insulin like growth factor, the gluey cell source neurotrophic factor etc.
According to this utility model further preferred embodiment, described biodegradation material is to be selected from least a in chitosan, chitin, collagen, chitosan, polylactic acid, the polyglycolic acid.More preferably, described Biodegradable material is chitosan.Preferred described solvent is dilute acetic acid aqueous solution, and for example the acetic acid weight content is 2%~10% dilute acetic acid aqueous solution, for example is preferably the dilute acetic acid aqueous solution of 2 % by weight, 4 % by weight and 5 % by weight.
According to this utility model further preferred embodiment, be 0.5mm~10mm according to the internal diameter of the complex biological sleeve pipe of this utility model method preparation, length is 3mm~20cm, and pipe thickness is 0.1~2mm.
According to this utility model further preferred embodiment, described drying steps is under 4~10 ℃ of conditions, lyophilization on the vacuum freeze drier.
By preparing complex biological sleeve pipe of the present utility model according to above-mentioned method.This preparation method obtains the biological duct of hollow by simple extruding spinning solution, and the perfusion liquid that comprises above-mentioned sustained-release micro-spheres can obtain complex biological sleeve pipe of the present utility model to the inner chamber of the biological duct of described hollow and the processing step such as dry.This preparation method technique is simple, with low cost, is easy to promote clinical practice.In addition, complex biological sleeve pipe according to the said method preparation can be applied to Small gap bridging reparation peripheral nerve, operation rear sleeve inwall slow-releasing microcapsule can also provide short nerve growth material for a long time for injured nerve, is conducive to improve the repairing effect of peripheral nerve injury.
Further specify the structure of complex biological sleeve pipe of the present utility model below in conjunction with accompanying drawing.
As shown in Figure 1, Fig. 1 is the schematic cross-section of a kind of complex biological sleeve pipe of the present utility model.Among the figure, 1 represents the complex biological sleeve pipe; 2 represent the biological duct of hollow; 3 represent the slow-releasing microcapsule layer.Complex biological sleeve pipe 1 comprises biological duct 2 and the slow release microcapsule layer 3 of hollow.Repair when perineural when this complex biological sleeve pipe 1 being applied to Small gap bridging, postoperative internal surface of sleeve pipe slow-releasing microcapsule can also provide short nerve growth material for a long time for injured nerve, is conducive to improve the peripheral nerve injury repairing effect.
Complex biological sleeve pipe 1 be shaped as tubulose, be preferably the cylindrical tubular of rule make because general complex biological sleeve pipe 1 is comparatively soft material, therefore, even some distortion of cylindrical tubular also is acceptable.
Below in conjunction with specific embodiment complex biological sleeve pipe of the present utility model and concrete preparation method thereof are described.
Embodiment 1: the preparation of complex biological sleeve pipe
1. the biological duct for preparing hollow
With commercially available deacetylation be 82%, weight average molecular weight is that 37.7 ten thousand chitosan (buying from Shanghai good and bio tech ltd) is by 4% concentration (W/W, weight ratio) is dissolved in the dilute acetic acid aqueous solution of 2% (weight ratio), obtains high viscosity solution.Vacuumizing and defoaming obtained still spinning solution in 12 hours.The air pressure of spinning solution with 0.6MPa (MPa) extruded from still, measure through spinning pump, extrude from the cortex of hollow spinneret orifice, meanwhile, with the air pressure of 0.2MPa 5%NaOH (W/W) aqueous solution (as coagulant) is extruded from the sandwich layer of hollow spinneret orifice.Spinning solution is extruded rear 5%NaOH (W/W) aqueous coagulation bath that directly enters, and chitosan is separated out and solidified under acting in extraneous and sandwich layer coagulant.Then, draw 1.25 times of first break draft through deflector roll, obtain continuous chitosan hollow pipe, bore 0.95mm, pipe thickness 0.15mm.The chitosan hollow pipe is washed with water to pH=7, is placed in 40 ℃, 5% acetic anhydride-methanol solution (V/V, volume ratio) with methanol dehydration, take out after 50 minutes, use methanol wash.Then place 1% dilute acetic acid aqueous solution to embathe, air-dry, again with 75% washing with alcohol, it is for subsequent use to be soaked in 75% ethanol sterilization after the washing.Wherein, the deacetylation of gained chitin hollow conduit is 27%, without dissolving, threads a needle without embrittlement under the hygrometric state in normal saline (mass fraction is 0.9% NaCl aqueous solution).
2. preparation sustained-release micro-spheres and performance test thereof
2.1 multi-emulsion method preparation contrast PLGA microsphere
Adopt W/O/W emulsion solvent evaporation method to prepare sustained-release micro-spheres.The surfactant polyethylene 400 of 50 μ L is dissolved in the deionized water of 100 μ L and obtains interior water (W1); (poly (lactic-co-glycolic acid, Poly(D,L-lactide-co-glycolide) is dissolved in and obtains oil phase (O) in the 3mL dichloromethane with the PLGA of 50mg; Outer water (W2) is 1.5% poly-vinyl alcohol solution 30ml; W1 is poured among the O, and supersound process obtained colostrum (W1/O) in 30 seconds, and colostrum is poured among the W2, stirred 6 minutes with 1000 rev/mins on cantilever type stirrer, obtained the W1/O/W2 emulsion.Emulsion is stirred 4 hours volatilization residual organic solvents in cantilever type stirrer with 500 rev/mins under room temperature (~25 ℃), by 13800 rev/mins of centrifugal collection microspheres, and with deionized water wash 3 times, sealing is placed in subzero 20 ℃ of refrigerators, lyophilization on the vacuum freeze drier, obtain blank sustained-release micro-spheres, place 4 ℃ of lower preservations.
2.2 multi-emulsion method prepares NGF-β-PLGA sustained-release micro-spheres
Adopt W/O/W emulsion solvent evaporation method to prepare sustained-release micro-spheres.The surfactant polyethylene 400 of the bovine serum albumin of NGF-β, the 5mg of 10 μ g and 50 μ L is dissolved in the deionized water of 100 μ and obtains interior water (W1); The PLGA of 100mg is dissolved in and obtains oil phase (O) in the 4mL dichloromethane; Outer water (W2) is 1.5% poly-vinyl alcohol solution; W1 is poured among the O, supersound process obtained colostrum (W1/O) in 30 seconds, colostrum is poured among the W2, on cantilever type stirrer, stirred 6 minutes with 1000 rev/mins, obtain the W1/O/W2 emulsion, emulsion is poured in the 10% deionized water sodium chloride solution of 400mL, stir 4 hours volatilization residual organic solvents on the room temperature lower magnetic force blender, by 13800 rev/mins of centrifugal collection microspheres, and with deionized water wash 5 times, lyophilization is 48 hours on the vacuum freeze drier, obtains NGF-β-PLGA sustained-release micro-spheres, places under 4 ℃ the temperature to preserve.
2.3 the performance test of sustained-release micro-spheres
2.3.1 the form of sustained-release micro-spheres and particle diameter
The take a morsel NGF-β-PLGA sustained-release micro-spheres of preparation observes the sustained-release micro-spheres microstructure, specifically referring to Figure of description 2 in scanning electron microscope.
2.3.2 the drug loading of sustained-release micro-spheres and envelop rate
NGF-β-the drug loading of PLGA sustained-release micro-spheres and the measurement of envelop rate
Getting 10mg NGF-β-PLGA sustained-release micro-spheres is dissolved in the ethyl acetate of 0.5mL, then the deionized water that adds 2mL, abundant mixing leaves standstill on agitator, takes off clear liquid, NGF-β in the extraction ethyl acetate, repeat 3 times, under the 490nm wavelength, measure the absorbance of gained NGF-β solution with the ELISA method, with blank microsphere degradation solution as blank, the substitution standard curve, the content of calculating NGF-β.
Repeatedly repeated measuring results shows: the average content of NGF-β is 0.0047% in the sustained-release micro-spheres of this example preparation, and the envelop rate average is 18.2%.
2.3.3 the mensuration of the release in vitro of sustained-release micro-spheres
Accurately take by weighing the 20mg sustained-release micro-spheres and place bag filter, airtight sealing, then place 60mL as the PBS buffer (pH 7.4) of degraded medium, put into 37 ℃ constant temperature oscillation shaking bath, carry out jolting with 150 rev/mins speed, respectively when 2,4,8,12,24 hours and 2,4,7,10,14,21 and 28 days, take out respectively 2mL and put into-20 ℃ of refrigerator freezings and preserve, put back in the delayed release device behind the fresh PBS liquid of all the other additional equivalent.Adopt at last the ELISA method to measure NGF-β mass concentration, the NGF-β total amount that discharges when calculating every sub-sampling, and draw microsphere cumulative release curve.Microsphere cumulative release curve is referring to Figure of description 3.
This repaired the peripheral nerve regeneration rule with the Small gap bridging suturing skill and was consistent, and can provide good nutrient substance support for neuranagenesis for a long time about 2 weeks NGF-β deenergized period in this utility model.
2.3.4 the detection of NGF-'beta ' activity in the microsphere
Detect the activity of NGF by the counting to the PCI2 cell process.Well-grown PCI2 cell is inoculated in advance in 6 coated well culture plates of Mus tail collagen, and inoculum density is 2 * 10
4/ cm
2, every hole adds 2mL, and to contain volume fraction be that 10% horse serum and volume fraction are the DMEM culture medium of 5% hyclone, is 5%CO at 37 ℃, volume fraction
2And cultivate random packet behind the 3h under the saturated humidity condition, be divided into NGF group (positive controls), the blank microsphere group (negative control group) that does not contain NGF, contain NGF sustained-release micro-spheres group (experimental group), respectively with the NGF of 50pg/L, the sustained-release liquid of blank microsphere, each 2mL of sustained-release liquid (all microsphere sustained-release liquid are all used 0.22 μ m filter filtration sterilization) that contains NGF sustained-release micro-spheres group replaces the archeocyte culture fluid, continue to cultivate after 48 hours, on microscope, choose at random the visual field, count 100 cells, and calculate in every group the longest projection length of cell greater than the ratio of the cell (being positive cell) of cell space diameter, positive cell ratio (%)=positive cell number/counting cells number, every group of experiment repeats 3 times.
Cultivation results shows: NGF organizes (positive controls) and contains NGF sustained-release micro-spheres group (experimental group) Growth of Cells situation similar, the blank microsphere group (negative control group) that does not more contain NGF, cultured cells density is large, quantity is more, and cell process length is longer.NGF group (positive controls), do not contain NGF blank microsphere group (negative control group), contain NGF sustained-release micro-spheres group (experimental group) positive cell ratio and be respectively: 57.3%, 29.6%, 54.2%.The result shows that sustained-release micro-spheres can reach abundant release function, and can guarantee the biologic activity of NGF.
3. the preparation of complex biological sleeve pipe
The sustained-release micro-spheres of above-mentioned fresh preparation is placed deionized water (sustained-release micro-spheres and deionized water weight ratio are 1: 100), make the sustained-release micro-spheres suspension, the suspension for preparing is injected into the inner chamber of the biological duct of described hollow, then this conduit was placed on the vacuum freeze drier lyophilization 24 hours, so that microsphere is affixed on the hollow pipe inwall, thereby acquisition has the complex biological sleeve pipe of microcapsule layer at the inwall of the biological duct of described hollow.
Embodiment 2: complex biological sleeve pipe Small gap bridging is repaired the animal peripheral nerve defection
Select 30 of the healthy male SD rats of SPF level (220~250g), be divided at random A, B, C, D, E group, adopt pentobarbital sodium (2ml/kg (ml/kg), lumbar injection) anesthesia, expose right sciatic nerves under the aseptic condition.
The A group: the 10mm place cuts off tibial nerve under the sciatic nerve crotch, sews up with 10-0 microsutures film;
The B group: the 10mm place cuts off tibial nerve under the sciatic nerve crotch, sews up with 10-0 nylon wire 2 pin adventitias.The stitch points localized pulverization contains nerve growth factor, neurotrophic factor mixed liquor 2ml after sewing up;
The C group: the 10mm place cuts off tibial nerve under the sciatic nerve crotch, sews up with the simple sleeve pipe Small gap bridging of 10-0 nylon wire 2 pins;
The D group: the 10mm place cuts off tibial nerve under the sciatic nerve crotch, sews up with the simple sleeve pipe Small gap bridging of 10-0 nylon wire 2 pins, contains nerve growth factor, neurotrophic factor mixed liquor 50ul (microlitre) with microsyringe to being socketed to inject in the gap;
The E group: the 10mm place cuts off tibial nerve under the sciatic nerve crotch, sews up with 10-0 nylon wire 2 pin composite bushing Small gap bridgings.
Observation item and detection method:
1) ordinary circumstance is observed: rat ordinary circumstance, operation side are suffered from limb active situation and ulcer situation.
2) 12 weeks after operation carries out respectively:
A. rat tibial nerve function score: use the ink marks method and measure rat tibial nerve function score.
Evaluate formula is:
Tibial nerve function index=-37.2 * footmark length factor+104.4 footmark width factors+middle toes width factor.
B. electrophysiologicalmeasurements measurements; The result adopts spss11.0 software to carry out one factor analysis of variance, compares between organizing.
C. the bilateral nerve is drawn materials, osmic acid dyeing, myelinated nerve fiber histological observation and counting.As a result statistical disposition: unit visual field myelinated nerve fiber count results adopts SPSS11.0 to carry out one factor analysis of variance, compares between organizing.
D. tibial nerve domination muscle histology is observed.
The experimental result of observing and detecting is as follows:
1) ordinary circumstance: all experimental rats are all performed the operation smoothly, and none is only dead, and all rats suffer from limb all without foot ulcers occurring.
2) rat tibial nerve function score: meansigma methods E group>C group>D group>B group>A group (the higher representative functional rehabilitation of value is better) does not wherein have significant difference between A, B, C, the D group, and the E group all has significant difference with other four groups.
3) electric physiology result: E group nerve conduction velocity is apparently higher than 4 groups of other A, B, C, D, and difference has statistical significance, does not have significant difference between other four groups.
4) osmic acid dyeing: E group myelinated nerve fiber is than other four groups of homogeneous, and myelin thickness is also thicker, no significant difference between other four groups.Myelinated nerve fiber counting E group is apparently higher than other four groups, and difference has statistical significance.
5) muscle histology is observed, each group hinder pleural muscle meat all strong pleural muscle meat to a certain degree atrophy is arranged, diameter of muscle fiber is also calibration constant once all, but no significant difference between each group.
Utilize complex biological sleeve pipe of the present utility model to be applied to Small gap bridging and repair when perineural, ulcer can not occur, electric physiological structure is excellent, and myelinated nerve fiber is than homogeneous, and myelin thickness is also thicker, and the myelinated nerve fiber counting is high.