CN101579247B - I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof - Google Patents
I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof Download PDFInfo
- Publication number
- CN101579247B CN101579247B CN2009101481385A CN200910148138A CN101579247B CN 101579247 B CN101579247 B CN 101579247B CN 2009101481385 A CN2009101481385 A CN 2009101481385A CN 200910148138 A CN200910148138 A CN 200910148138A CN 101579247 B CN101579247 B CN 101579247B
- Authority
- CN
- China
- Prior art keywords
- collagen
- sheath pipe
- peripheral nerve
- nerve sheath
- helix structure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to a peripheral nerve sheath, a preparation method and applications thereof. The peripheral nerve sheath is prepared by using I-type collagen keeping the peculiar triple helical structure and is characterized by (1) being the pure collagen sheath which has complete, compact and disordered pores or has no pore; (2) being a composite collagen sheath consisting of the pure collagen sheath and an internal cylindrical collastromin which has completely matched diameter and has pores with specific size and vertical axis orientation; and (3) being the pure collagen sheath which has compact and disordered pores with vertical axis openness or no pores. The peripheral nerve sheath has the function of enhancing repairing and regeneration of the damaged nerve, is applicable to repairing and regeneration of peripheral nerve which is damaged and defective, and has the characteristics of having stable structure and good biocompatibility, being capable of maintaining internal and external environments of damaged nerves and optimal and controllable degradation, absorption rate and reducing/preventing tissue adhesion and the like.
Description
Technical field
The present invention relates to medical science, medical biotechnology materialogy, medical tissue engineering and regeneration medicine technology field, be specifically related to the peripheral nerve sheath pipe that a kind of high-purity keeps the I type medical collagen material of collagen original specific triple helix structure.
Background technology
The twentieth century end is along with the high speed development of holistic medicine, and cytobiology, molecular biology, genetic, tissue engineering and regenerative medicine be constantly progress and development thereupon also.Biomaterial with superperformance is also promptly being found and is being utilized simultaneously.Tissue engineering and the regenerative medicine quick emergence in clinical medicine has in recent years just proved this point.Use type i collagen to obtain significant development and application as the organizational project of carrier and the product of regenerative medicine.
How to find and extract effective substances is served as the extracellular matrix material in tissue engineering and regenerative medicine material that making the histiocyte of damaged that the environment of good reparative regeneration can be arranged is a very important problem.For the damaged tissue cell provides approaching as far as possible normal internal and external environment, make it cell migration, differentiation and regeneration reach formation and organize identical tissue from the body local damage.
Type i collagen is most tissues in the human body, and the material that the extracellular matrix that the cell of organ is depended on for existence is required is damaged tissue organ indispensable material in the middle of repair process.Type i collagen is the structural protein of extracellular matrix, has three strands of α-peptide chains that covalent bond connects in the molecule, and three kinds of power between triple helix are to guarantee the stable core of collagen helix.Collagen is when extraction separation, and is different with condition with method, can produce collagen, three kinds of products of gelatin and collagen protein.Collagen, gelatin and collagen protein are different.What can be called as collagen must be the protein that its triple-helix structure does not have change, keeps its biological activity.Say that from structure collagen is the protein with three strands of α-peptide chains of covalent bond connection.Gelatin is that covalent bond is interrupted, and the protein of only surplus secondary key is the distortion product of collagen, and the triple helical body is totally released or part is unclamped loss of biological activity.Collagen protein is the segment of more free α-peptide chain or gelatin molecule, protein properties is arranged but does not have whole collagen characteristic.Say that from broad sense gelatin also belongs to collagen protein, its molecular weight ratio collagen protein is high.
Type i collagen exists in the intravital different tissues organ of the many animals of occurring in nature; Tail like muroid; The skin of animal etc.; Just content and quality are all different, and the type i collagen content of triple-helix structure is up to 98%, so the cattle heel string is the abundant source of extracting the type i collagen material in the heel string of cattle.But extract highly purified type i collagen with triple-helix structure is that very difficulty is with complicated, and how finding and collect the type i collagen with triple-helix structure is a very important problem.
Peripheral nerve is invaded in time in wound and tumor, can cause damaged and damage in various degree, thereby causes the dysfunction of peripheral nervous system, forms the histoorgan degradation degeneration that peripheral nerve is controlled then.With regard to neural, reparative regeneration is the problem of a difficult and complicated behind the peripheral nerve injury.Neural reparative regeneration after damaged is the individual problem of assaulting fortified position always.
At present common Therapeutic Method is direct end to end anastomosis or autotransplantation, because that autologous tissue obtains the source is limited, and has left over and supplies the district to feel to go down, and causes patient to supply to distinguish the painful of extra operation and bears extra problems such as financial burden.Therefore how solving nerve injury repairing as early as possible and good artificial bio-membrane's material is provided after damaged is a very important problem.Characteristics of the present invention are simple type i collagen sheath pipes of the maintenance collagen original specific triple helix structure that can sew up in impaired peripheral nerve outerwrap or use the simple sheath pipe of the type i collagen/compound sheath pipe that keeps the collagen original specific triple helix structure to carry out bridge type to connect damaged position, peripheral nerve end and coincide, make it reach the bridge interconnect function.The damaged of the special position, end end that is to use the compound sheath pipe of type i collagen that keeps the collagen original specific triple helix structure that proposes coincide, and grows fast through the collastromin of same trend in the hope of neural axis.Type i collagen with triple-helix structure is simple/and compound sheath pipe connects the peripheral nerve surface of damage, defect; Parcel covers; Not only make and mechanically protect organizing of perineural essential site not disturbed by the intrusion of its hetero-organization of periphery; Also protected the interior environment of damaged nerve tissue simultaneously; The somatomedin disorder that prevents to damage in the nervous tissue of back is lost, and keeps interior environment relatively stable of damaged tissues, for the reparation and the regeneration of repairing the nervous tissue after damaging provides suitable environment.
Summary of the invention
One aspect of the present invention provides a kind of peripheral nerve sheath pipe, and its I type medical collagen material of serving as reasons maintenance collagen original specific triple helix structure is processed, and it is complete to have integral body, and tube wall has fine and close unordered hole/imporous simple collagen sheath pipe.
Another aspect of the present invention provides a kind of peripheral nerve sheath pipe; Its I type medical collagen material of serving as reasons maintenance collagen original specific triple helix structure is processed; Complete by integral body; Tube wall has fine and close unordered hole/imporous simple collagen sheath pipe, and the composite nerve sheath pipe formed of the cylindric collastromin with the specific big fine pore of longitudinal axis orientation that matees fully of built-in diameter.
Another aspect of the present invention provides a kind of peripheral nerve sheath pipe, and its I type medical collagen material of keeping the collagen original specific triple helix structure of serving as reasons is processed, and it is open fully to have a side Y-direction, and tube wall has fine and close unordered hole/imporous simple collagen sheath pipe.
Another aspect of the present invention, the pipe thickness of above-mentioned peripheral nerve sheath pipe are 0.5～1.5 millimeter, and the pipe diameter is 0.2～8 millimeter, said peripheral nerve sheath pipe can be in 3-12 month degraded and absorbed.
The I type medical collagen material of maintenance collagen original specific triple helix structure involved in the present invention is to be organized as raw material with the cattle heel string; Active through enzyme defat and chemical reaction Deproteinization, the purity that obtains through the extraction of chemical reaction program is up to the I type medical collagen material of the solid-state/cotton-shaped maintenance collagen original specific triple helix structure more than 98%.Its preparation method is: be organized as raw material with the cattle heel string, through freezing, cut into the thin slice that thickness is the 0.1-3 millimeter, through screening, under stirring condition, it is immersed in 0.1% sodium dihydrogen phosphate/sodium hydroxide solution and soaked 1-3 hour, make its PH keep neutral.The ficoin of 1: 5 (w/w) is added fully reaction in the substance solution of above-mentioned processing.With regularly stirring, handled 6 hours with 1% ammonium nitrate thereafter.Clean then.Temperature is handled and controlled to the sodium chloride that adds 1M more again, regulates PH, and 4 ℃ of following hold over night are thereafter with the normal sodium hydroxide solution reaction of 2M.Thereafter with 50% sulphuric acid neutralization buffer and regulate PH and make it be slant acidity, take out precipitate, washing was soaked 6 hours, removed moisture then, through the be maintained I type medical collagen material of collagen original specific triple helix structure of lyophilization.
Another aspect of the present invention provides the method for preparing of above-mentioned peripheral nerve sheath pipe.Particularly, the I type medical collagen that keeps the collagen original specific triple helix structure is dissolved in the glacial acetic acid of 0.01-0.5M, forms the solution of 0.5%-5%; Stir through 15,000～25,000 rev/min high-speed low temperature;, after 15～60 minutes the collagen suspension is injected in the mould, after the lyophilization program through 3000～4000 rev/mins of high speed centrifugations again; Strengthen chain attachment between tropocollagen molecule through physical method or chemical method, through the sterilization of low temperature oxirane disinfection, prepare the peripheral nerve sheath pipe again.
Wherein, described lyophilization program is freezing-distillation (temperature) drying, cryogenic temperature be set to (1)-10～-4 ℃/30 minutes/be persistent state;～-40 ℃ (2)-60/180-240 minute/curved state;～-40 ℃ (3)-60/60-120 minute/be persistent state; The temperature of freezer dryer refrigerator should keep-80 ℃ in the process, and the temperature of sublimation stage is set to (1) 0 ℃/maintenance 18-24 hour thereafter; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 30-120 minute, and above-mentioned all programs carry out that vacuum is arranged on the 200-350 millitorr in the process.
Keep the I type medical collagen diaphragm of collagen original specific triple helix structure to have biology/physical function of different nature for making; Can strengthen the interchain connection of tropocollagen molecule through physical method or chemical method and make its collagen protein physicochemical property change; Described physical method is vacuum high-temperature evaporation (DHT): will keep the type i collagen peripheral nerve sheath pipe of collagen original specific triple helix structure to put into vacuum oven; Its negative pressure is set to negative 1 atmospheric pressure; Temperature is 100～120 ℃, handles 24～72 hours; Described chemical method is a 1-ethyl ester-3,3-dimethyl propyl amine-carbonization DMA (EDAC) method: type i collagen peripheral nerve sheath pipe 1～5 hour is soaked to strengthen the tropocollagen molecule chain in EDAC, NHS and COOH liquid (1-ethyl ester-3-3-dimethyl propyl amine-carbonization DMA, HOSu NHS and carboxyl) 5: 2: 5 in molar ratio configuration back.
The sterilization of described low temperature oxirane disinfection is to use the eo sterilization sterilization, and the 30% pure ethylene oxide in addition carbon dioxide of 50%-70% is sterilized in disinfector, and sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 50%-70%RH; Forevacuum is-20～-40Kpa; Ventilation vacuum is-40～-60Kpa; Rate of ventilation is 8 times; Sterilization time is 3-4 hour.
In the preparation process; The suspension of variable concentrations; Different suspension intakes, different cryogenic temperatures and the different strong methods that adds are complementary, and can reach collagen nerve sheath pipe and 80～250 microns column collastromins of holding single Y-direction traveling hole of 5～180 microns big or small lack of alignment holes; Have different stress simultaneously, different degraded and absorbed rates.The preferred pore size of tube wall is 5～180 microns, more preferably 5～100 microns, most preferably is 50 microns.
Another aspect of the present invention provides the application of above-mentioned peripheral nerve sheath pipe in the material of the peripheral nerve tissue of preparation repair deficiency and/or damage.
Only if outside the certain illustrated, all percentage value units of the present invention are weight/volume.
Description of drawings
Fig. 1 is that the integral body that high-purity with 5～180 microns omnidirectional holes/0.1～2 millimeters thick tube wall has the medical simple collagen nerve sheath of an I type pipe of triple-helix structure is seen.
Fig. 2 is the integral body sight that association's 80～200 micrometer spindles have the medical composite collagen nerve sheath of the I type pipe of triple-helix structure in 5～180 microns omnidirectional holes/0.1～2 millimeters thick tube wall to the high-purity of the cylindric collastromin of orientation hole.
Fig. 3 is that the integral body of the I type medical axial open simple collagen nerve sheath pipe of the high-purity of 5～180 microns omnidirectional holes/0.1～2 millimeters thick tube wall with triple-helix structure is seen.
Fig. 4 is the operation instruction figure of simple collagen nerve sheath pipe to damaged nerve, the damaged peripheral nerve broken ends of fractured bone is placed in the two ends of collagen nerve sheath pipe respectively, with the capable epineurium of stitching thread of 10-0/9-0 and the stitching of sheath pipe.
Fig. 5 is the operation instruction figure of composite collagen nerve sheath pipe to damaged nerve; The damaged peripheral nerve broken ends of fractured bone is placed in the two ends of collagen nerve sheath pipe respectively; The section of injured nerve contacts with its interior collagen inner matter simultaneously, with the capable epineurium of stitching thread of 10-0/9-0 and the stitching of sheath pipe.
Fig. 6 is the operation instruction figure of axial open collagen nerve sheath pipe to injured nerve, and open nerve sheath pipe is wrapped in impaired neural periphery, and tube chamber is stitched and closed in the place of opening continuously with suture.
The specific embodiment
Below in conjunction with specific embodiment the present invention is described further.
Embodiment 1: extract the I type medical collagen material that keeps the collagen original specific triple helix structure
Select the cattle heel string tissue of 1kg for use, freezing, cut into the thin slice that thickness is the 0.1-3 millimeter, through screening, under stirring condition, it is immersed in 0.1% sodium dihydrogen phosphate/sodium hydroxide solution and soaked 1-3 hour, make its PH keep neutral.The ficoin of 1: 5 (w/w) is added fully reaction in the substance solution of above-mentioned processing.With regularly stirring, handled 6 hours with 1% ammonium nitrate thereafter.Clean then.Temperature is handled and controlled to the sodium chloride that adds 1M more again, regulates PH, and 4 ℃ of following hold over night are thereafter with the normal sodium hydroxide solution reaction of 2M.Thereafter with 50% sulphuric acid neutralization buffer and regulate PH and make it be slant acidity, take out precipitate, washing was soaked 6 hours, removed moisture content then, through the be maintained I type medical collagen material of collagen original specific triple helix structure of lyophilization.
Collagen, the difference of gelatin and collagen protein are the method and the controlled condition that in extracting manufacturing process, are adopted can keep the stable environment of collagen fiber helical structure for acid, and simultaneous temperature must be on the low side, so when making, must carry out at low temperatures.How to identify that the preceding collagen short fiber material of support can keep the characteristic of triple-helix structure, quantitatively learning is the comparison difficulty, and the microscopical sight of ultramicroscope and polarization optical is looked into its configuration and helped to confirm.The suspension of procollagen material after high-speed stirred obtains fibrous solid state collagen after treatment again; Behind acid leach solution, become emulsion again; Get of the inspection of its 1-2 drop of liquid through polariscope; It is thus clear that collagen short fiber and granule in it show bright bar institute's shape or shot-like particle under the polarisation background of dark, it is the refraction effects of collagen fiber under polarisation.Under high power lens more, present " band band or band fold " and the typical case's performance of type i collagen triple helical fiber just of this band band or band fold on visible its fiber form.This has explained that the collagen-based materials of embodiment 1 gained has kept the inherent collagen triple-helix structure of collagen fiber really.And gelatin and collagen protein get not so image.
Embodiment 2: prepare the unordered hole of complete densification/imporous simple collagen sheath pipe
The I type medical collagen of the maintenance collagen original specific triple helix structure of embodiment 1 preparation is dissolved 7.2 grams to be dissolved in the glacial acetic acid of 720 milliliters of 0.1M; Form 2% solution, stir through 18,000 rev/mins routines; Again after 4000 rev/mins of high speed centrifugation 15-30 minutes; The collagen suspension is injected in the mould, with the implantation of the die center property of the tissue adhesion of 1.5 millimeters of diameters wherein, put into the freezer dryer refrigerator and carry out lyophilization then.
Cryogenic temperature be set to (1)-7 ℃/30 minutes/be persistent state; ℃ (2)-40/200 minutes/curved state; ℃ (3)-40/70 minutes/persistent state be; The temperature of freezer dryer refrigerator keeps-80 ℃ in the process.The temperature of sublimation stage is set to (1) 0 ℃/maintenance 18 hours; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 40 minutes, and above-mentioned all programs carry out that vacuum is arranged on 200 millitorrs in the process.
The product that lyophilization obtains is put into vacuum oven, and it is provided with negative 1 atmospheric pressure, handles 24 hours down for 105 ℃ in temperature.
Further in disinfector, use 30% pure ethylene oxide to add 70% carbon dioxide to sterilize with obtaining product, sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 60%RH; Forevacuum is-20Kpa; Ventilation vacuum is-40Kpa; Rate of ventilation is 8 times; Sterilized 3.5 hours, and prepared the peripheral nerve sheath pipe of 1.5 millimeters of internal diameters, 2.5 millimeters of external diameters.
Embodiment 3: prepare the unordered hole of complete densification/imporous simple collagen sheath pipe
The I type medical collagen of the maintenance collagen original specific triple helix structure of embodiment 1 preparation is dissolved 18 grams to be dissolved in the glacial acetic acid of 720 milliliters of 0.5M; Form 5% solution, stir through 24,000 rev/mins routines; Again after 4000 rev/mins of high speed centrifugation 30-45 minutes; The collagen suspension is injected in the mould, with the implantation of the die center property of the tissue adhesion of 2 millimeters of diameters wherein, put into the freezer dryer refrigerator and carry out lyophilization then.
Cryogenic temperature be set to (1)-7 ℃/30 minutes/be persistent state; ℃ (2)-40/200 minutes/curved state; ℃ (3)-40/70 minutes/persistent state be; The temperature of freezer dryer refrigerator keeps-80 ℃ in the process.The temperature of sublimation stage is set to (1) 0 ℃/maintenance 18 hours; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 40 minutes, and above-mentioned all programs carry out that vacuum is arranged on 200 millitorrs in the process.
The product that lyophilization obtains is put into vacuum oven, and it is provided with negative 1 atmospheric pressure, handles 24 hours down for 105 ℃ in temperature.
Further in disinfector, use 30% pure ethylene oxide to add 70% carbon dioxide to sterilize with obtaining product, sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 60%RH; Forevacuum is-20Kpa; Ventilation vacuum is-40Kpa; Rate of ventilation is 8 times; Sterilized 3.5 hours, and prepared the peripheral nerve sheath pipe of 1.5 millimeters of internal diameters, 2.5 millimeters of external diameters.
Embodiment 4: preparation composite collagen sheath pipe
Step 1: the unordered hole of the complete densification/imporous simple nerve sheath pipe for preparing 1.5 millimeters of internal diameters, 2.5 millimeters of external diameters through the method for embodiment 2;
Step 2: prepare cylindric collastromin core;
The I type medical collagen of the maintenance collagen original specific triple helix structure of embodiment 1 preparation is dissolved 3.6 grams to be dissolved in the glacial acetic acid of 720 milliliters of 0.1M; Form 0.5% solution, stir through 18,000 rev/mins routines; Again through 4000 rev/mins of high speed centrifugations after 30 minutes; The collagen suspension is injected in the columnar mould, and it is slowly freezing to put into-80 ℃ of chemical solutions with 1 millimeter/1 minute speed, carries the back and takes out and put into the freezer dryer refrigerator and carry out lyophilization.
Cryogenic temperature be set to (1)-7 ℃/30 minutes/be persistent state; ℃ (2)-40/200 minutes/curved state; ℃ (3)-40/70 minutes/persistent state be; The temperature of freezer dryer refrigerator keeps-80 ℃ in the process.The temperature of sublimation stage is set to (1) 0 ℃/maintenance 18 hours; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 40 minutes, and above-mentioned all programs carry out that vacuum is arranged on 200 millitorrs in the process.
The product that lyophilization obtains is put into vacuum oven, and it is provided with negative 1 atmospheric pressure, handles 24 hours down for 105 ℃ in temperature.
Further in disinfector, use 30% pure ethylene oxide to add 70% carbon dioxide to sterilize with obtaining product, sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 60%RH; Forevacuum is-20Kpa; Ventilation vacuum is-40Kpa; Rate of ventilation is 8 times; Sterilized 3.5 hours, and prepared the cylindric collastromin core of 1.5 millimeters of diameters.
Cylindric collastromin core is built in again prepares composite collagen sheath pipe in the simple nerve sheath pipe.
Embodiment 5: the open fine and close unordered hole/imporous simple collagen sheath pipe of preparation Y-direction
I type medical collagen 7.2 grams of the maintenance collagen original specific triple helix structure of embodiment 1 preparation are dissolved in the glacial acetic acid of 720 milliliters of 0.2M; Form 1% solution, stir through 25,000 rev/mins routines; Again through 3500 rev/mins of high speed centrifugations after 30 minutes; The collagen suspension is injected in 4 millimeters moulds with 2 millimeters openings of internal diameter, with the implantation of the die center property of the tissue adhesion of 2 millimeters of diameters wherein, put into the freezer dryer refrigerator and carry out lyophilization then.
Cryogenic temperature be set to (1)-4 ℃/30 minutes/be persistent state; ℃ (2)-50/200 minutes/curved state; ℃ (3)-50/100 minutes/persistent state be; The temperature of freezer dryer refrigerator keeps-80 ℃ in the process.The temperature of sublimation stage is set to (1) 0 ℃/maintenance 18 hours; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 70 minutes, and above-mentioned all programs carry out that vacuum is arranged on 200 millitorrs in the process.
The product that lyophilization is obtained soaked 3 hours in the mixing material of HOSu NHS and carboxyl in the 1-ethyl ester-3-3-dimethyl propyl amine-carbonization DMA according to 5: 2: 5 proportional arrangement.
Further in disinfector, use 30% pure ethylene oxide to add 70% carbon dioxide to sterilize with obtaining product, sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 65%RH; Forevacuum is-35Kpa; Ventilation vacuum is-55Kpa; Rate of ventilation is 8 times; Sterilized 4 hours, and prepared the peripheral nerve sheath pipe of 2 millimeters of internal diameters, 2.5 millimeters of external diameters.
Embodiment 5: the zoopery of relevant peripheral nerve injury reparative regeneration
All animal models are selected Spraque Daley (SD) or Lewis, heavy 250-350 gram, the 6-8 rat in age in week for use.Expose the sciatic nerve of right lower extremity behind the general anesthesia, select its mid point to do the nerves transected art.Two broken ends of fractured bone are done the nerves transected art respectively, do not use/use different types of nerve sheath pipe to connect the nerve of the broken ends of fractured bone of both sides thereafter.Implanting length is 3 millimeters, uses the stitching thread that do not absorb of 10-0 to carry out epineurial neurorrhaphy.12 weeks of postoperative put to death animal and check substantially and histologic analysis.
Animal model is divided into:
1, the nerves transected postoperative keeps 5 millimeters, 10 millimeters and 15 mm clearance between the neural broken ends of fractured bone, does not have any nerve sheath pipe and gets involved connection: 18 of rats.
2, select for use silica gel tube as the nerve sheath pipe, every side is in the neural broken ends of fractured bone implant 3 millimeters, row epineural suture: 30 of rats.
(A) select 11 centimeter length silica gel tubes for use, keep 5 millimeters damaged;
(B) select 16 centimeter length silica gel tubes for use, keep 10 millimeters damaged;
(C) select 21 centimeter length silica gel tubes for use, keep 15 millimeters damaged.
3, select 1.5 millimeters of the internal diameters that the type i collagen that keeps the collagen original specific triple helix structure processes for use, in the simple collagen sheath pipe that external diameter is 2.5 millimeters, every nervus lateralis broken ends of fractured bone implant 3 millimeters, row epineural suture: 30 of rats.
(A) select 11 lis of above-mentioned nerve sheath pipes for use, keep 5 millimeters damaged in it;
(B) select 16 centimetres of above-mentioned nerve sheath pipes for use, keep 10 millimeters damaged in it;
(C) select the above-mentioned nerve sheath pipe of 21 centimeter length for use, keep 15 millimeters damaged in it.
4, select 1.5 millimeters of the internal diameters that the type i collagen that keeps the collagen original specific triple helix structure processes for use; The composite collagen sheath pipe that external diameter is 2.5 millimeters; Pipe is built-in with the collagen tubing string that the type i collagen with triple-helix structure of certain-length is made into; 3 millimeters also contact with the collagen tubing string in every nervus lateralis broken ends of fractured bone implant, row epineural suture: 30 of rats.
(A) select 11 lis of above-mentioned nerve sheath pipes for use, keep the collagen tubing string of 5 millimeters long in it;
(B) select 16 centimetres of above-mentioned nerve sheath pipes for use, keep 10 millimeters collagen tubing string in it;
(C) select the above-mentioned nerve sheath pipe of 21 centimeter length for use, keep 15 millimeters collagen tubing string in it.
Perusal:
All laboratory animal postoperative general health are all right, do not have death, do not have and infect, primary wound healing.
1 group: this group shows the leg muscle of all animals than the obvious atrophy of offside, and neural have serious tissue adhesion on every side, and cicatrization does not have any tissue and connects the neural broken ends of fractured bone.
2 groups: leg muscle is than the obvious atrophy of offside, and all models are the visible tissue adhesion that slight or moderate are arranged between sebific duct sheath pipe and muscular tissue.
(A) select 11 centimeter length silica gel tubes for use, keep 5 millimeters damaged: 100% presents tissue connection in the silica gel tube, but conjunctive tissue is tiny.
(B) select 16 centimeter length silica gel tubes for use, keep 10 millimeters damaged: 70% presents tissue connection in the silica gel tube, but organizing of middle interconnecting piece position is tiny especially.
(C) select 21 centimeter length silica gel tubes for use, keep 15 millimeters damaged: 10-20% presents tissue connection in the silica gel tube, but the middle interconnecting piece position organize tiny especially one-tenth wire.
3 groups: leg muscle is than the offside atrophy, and all models discoveries are visible between sebific duct sheath pipe and muscular tissue to have slight tissue adhesion, and the type i collagen pipe 70%-100% of triple-helix structure degraded forms a type epineurium structure.
(A) select 11 lis of above-mentioned nerve sheath pipes for use, keep 5 millimeters damaged in it: 100% presents tissue connection in the type i collagen pipe of triple-helix structure, and the diameter of regenerating tissues 90% is equal to the diameter of severed nerve.
(B) select 16 centimetres of above-mentioned nerve sheath pipes for use, keep 10 millimeters damaged in it: 100% presents tissue connection in the type i collagen pipe of triple-helix structure, and the near-end of regenerating tissues and former disconnected diameter are near the diameter nerve of the broken ends of fractured bone.
(C) select the above-mentioned nerve sheath pipe of 21 centimeter length for use, keep 15 millimeters damaged in it: 90% presents tissue connection in the type i collagen pipe of triple-helix structure, wherein is broken into comparatively significantly in 30% the connection to attenuate.
4 groups: leg muscle has slight atrophy than offside, and all models discoveries are visible between sebific duct sheath pipe and muscular tissue to have slight tissue adhesion, and the type i collagen pipe 70%-100% of triple-helix structure degraded forms a type epineurium structure.
(A) select 11 lis of above-mentioned nerve sheath pipes for use, keep 5 millimeters collagen column inner matter in it: type i collagen column 90% degraded of triple-helix structure forms the cambium of neuroid tissue's structure.The diameter of cambium 95% is equal to the diameter of severed nerve.
(B) select 16 centimetres of above-mentioned nerve sheath pipes for use, keep 10 millimeters collagen column inner matter in it: type i collagen column 90% degraded of triple-helix structure forms the cambium of neuroid tissue's structure.The diameter 80%-90% of cambium is equal to the diameter of severed nerve.
(C) select the above-mentioned nerve sheath pipe of 21 centimeter length for use, keep 15 millimeters collagen column inner matter in it: type i collagen column 90% degraded of triple-helix structure forms the cambium of neuroid tissue's structure.The diameter of cambium 80% is equal to the diameter of severed nerve.
Histological examination:
Sciatic nerve excision with integral body becomes two parts in middle point cut, after cured/JB-4 embedding, processes one one of 5-6 micron slab and does HE dyeing, and another part is done Epon dyeing, and the regeneration situation of neural axis is dyed and observed to the tissues observed form thereafter.
HE dyeing:
For the sciatic nerve of no any nerve sheath pipe connection injured nerve broken ends of fractured bone, mirror has a large amount of scar tissues to form around showing injured nerve down.A large amount of fibroblasts are wrapped in the injured nerve broken ends of fractured bone and form neurofibroma.No any tissue forms-0% between the nearly neural broken ends of fractured bone far away.
Show that at the sciatic nerve BIAO and BEN that connects with silica gel tube the damaged interneural connection rate of long distance is almost 0%.The damaged connection rate of short distance is auspicious dye can reach 80% but do not have and have the sheath neural axis to pass through.
Use the animal model of the type i collagen sheath pipe that has triple-helix structure merely, the damaged sciatic connection rate of short distance is almost 100%, and the connection rate of the damaged nerve of long distance can reach 60%-70%.But the density of the conjunctive tissue that the stage casing is newborn reduces, and diameter diminishes, the visible neuroma property parcel of distal nerve stump.A little cicatrization is arranged between collagen sheath pipe and peripheral tissues.
Use compound animal model with type i collagen sheath pipe of triple-helix structure, be connected to 100% between the neural broken ends of fractured bone, the density of cambium increases, near normal morphology.Collagen sheath pipe and peripheral tissues do not have clear scar and form.
Epon dyeing:
This kind specific stain can show the quantity that the sheath neural axis is arranged in the nervous tissue, and size and form are a kind of special effective methods of identifying neurocyte.
Through to quantitatively the finding of 4 treated animal models with analyzing contrast qualitatively, do not have that any junctional complex uses first group show neurocyte-have sheath neural axis be regenerated as 0%.
For the 3rd group of result's demonstration of using simple type i collagen sheath pipe, there is the quantity of sheath neural axis to be higher than the quantity in the normal nerve in newborn the connection in the neural tissue of each root with triple-helix structure.60% of the diameter that new life has the diameter of sheath neural axis to reach the sheath neural axis is normally arranged can only reach 25% of normal nerve but diameter surpasses 6 microns the quantity that the sheath neural axis is arranged.
Use the result of compound type i collagen sheath pipe to show for the 4th group with triple-helix structure; Be higher than 90% of diameter that quantity new life in the normal nerve has the diameter of sheath neural axis to reach the sheath neural axis is normally arranged in the newborn quantity that the sheath neural axis is arranged in the neural tissue that connects of each root; And diameter surpasses 6 microns the quantity that the sheath neural axis is arranged and can reach more than 50% of normal nerve, is ideal neural reparative regeneration material.
Characteristics of the present invention are damaged coincideing in position, end end of the type i collagen sheath pipe with triple-helix structure that can sew up in impaired peripheral nerve outerwrap and the damaged peripheral nerve simple sheath pipe of type i collagen that carries out triple-helix structure; Damaged the coincideing in position, end end that makes it reach the bridge interconnect function or use the compound sheath pipe of type i collagen of triple-helix structure grown through collastromin in the hope of neural axis fast.Type i collagen with triple-helix structure is simple/and compound sheath pipe connects parcel to the peripheral nerve surface of damaging damaged damage position and covers; Be not only and mechanically protect organizing of perineural essential site not disturbed by the intrusion of its hetero-organization of periphery; Also protected the interior environment of damaged nerve tissue simultaneously; Somatomedin disorder after preventing to damage in the nervous tissue is lost; Interior environment relatively stable that keeps damaged tissues is for the reparation and the regeneration of repairing the nervous tissue after the damage provides suitable environment.
Claims (8)
1. method for preparing the peripheral nerve sheath pipe, said peripheral nerve sheath pipe are to be processed by the I type medical collagen material that keeps the collagen original specific triple helix structure, have whole complete tube wall and have fine and close unordered hole/imporous simple collagen sheath pipe; It is characterized in that to keep the I type medical collagen of collagen original specific triple helix structure to be dissolved in the glacial acetic acid of 0.01-0.5M, form the solution of 0.5%-5%, through 15; 0000～25; 000 rev/min high-speed low temperature stirs, and, after 15～60 minutes the collagen suspension is injected in the mould through 3000～4000 rev/mins of high speed centrifugations again; Through lyophilization program aftershaping; Strengthen chain attachment between tropocollagen molecule through physical method or chemical method, through the sterilization of low temperature oxirane disinfection, prepare described peripheral nerve sheath pipe again.
2. method for preparing the peripheral nerve sheath pipe, said peripheral nerve sheath pipe are to be processed by the I type medical collagen material that keeps the collagen original specific triple helix structure, have fine and close unordered hole/imporous simple collagen sheath pipe by the complete tube wall of integral body; And the composite nerve sheath pipe formed of the cylindric collastromin that matees fully of built-in diameter with the specific big fine pore of longitudinal axis orientation; It is characterized in that to keep the I type medical collagen of collagen original specific triple helix structure to be dissolved in the glacial acetic acid of 0.01-0.5M, form the solution of 0.5%-5%, through 15; 0000～25; 000 rev/min high-speed low temperature stirs, and, after 15～60 minutes the collagen suspension is injected in the mould through 3000～4000 rev/mins of high speed centrifugations again; Through lyophilization program aftershaping; Strengthen chain attachment between tropocollagen molecule through physical method or chemical method, through the sterilization of low temperature oxirane disinfection, prepare described peripheral nerve sheath pipe again.
3. method for preparing the peripheral nerve sheath pipe, said peripheral nerve sheath pipe are to be processed by the I type medical collagen material that keeps the collagen original specific triple helix structure, and it is open fully to have a side Y-direction; Tube wall has fine and close unordered hole/imporous simple collagen sheath pipe, it is characterized in that the I type medical collagen that keeps the collagen original specific triple helix structure is dissolved in the glacial acetic acid of 0.01-0.5M, forms the solution of 0.5%-5%; Stir through 15,0000～25,000 rev/min high-speed low temperature; Again through 3000～4000 rev/mins of high speed centrifugations after 15～60 minutes; The collagen suspension is injected in the mould,, strengthen chain attachment between tropocollagen molecule through physical method or chemical method through lyophilization program aftershaping; Through the sterilization of low temperature oxirane disinfection, prepare described peripheral nerve sheath pipe again.
4. like the arbitrary described method for preparing the peripheral nerve sheath pipe of claim 1-3, the diameter that it is characterized in that said simple collagen sheath pipe is 0.2～8 millimeter, and pipe thickness is 0.5～1.5 millimeter, and the aperture of hole is 5～180 microns.
5. the method for preparing the peripheral nerve sheath pipe as claimed in claim 2, the aperture that is characterised in that the axial orientation property hole of said cylindric collastromin is 80～250 microns.
6. like the arbitrary described method for preparing the peripheral nerve sheath pipe of claim 1-3, it is characterized in that described lyophilization program be freezing-distillation and heat up dry, cryogenic temperature be set to (1)-10～-4 ℃/30 minutes/be persistent state;～-40 ℃ (2)-60/180-240 minute/curved state;～-40 ℃ (3)-60/60-120 minute/be persistent state; The temperature of freezer dryer refrigerator should keep-80 ℃ in the process, and the temperature of sublimation stage is set to (1) 0 ℃/maintenance 18-24 hour thereafter; (2) temperature of secondary temperature elevation is set to 20 ℃, and the time is 30-120 minute, and above-mentioned all programs carry out that vacuum is arranged on the 200-350 millitorr in the process.
7. like the arbitrary described method for preparing the peripheral nerve sheath pipe of claim 1-3; It is characterized in that described physical method is vacuum high-temperature evaporation (DHT): will keep the type i collagen peripheral nerve sheath pipe of collagen original specific triple helix structure to put into vacuum oven; Its negative pressure is set to negative 1 atmospheric pressure; Temperature is 100～120 ℃, handles 24～72 hours; Described chemical method is a 1-ethyl ester-3; 3-dimethyl propyl amine-carbonization DMA (EDAC) method: with EDAC, NHS and COOH liquid is 1-ethyl ester-3, and type i collagen peripheral nerve sheath pipe 1～5 hour is soaked to strengthen the tropocollagen molecule chain in 5: 2: 5 in molar ratio configuration backs of 3-dimethyl propyl amine-carbonization DMA, HOSu NHS and carboxyl.
8. like the arbitrary described method for preparing the peripheral nerve sheath pipe of claim 1-3; It is characterized in that described low temperature oxirane disinfection sterilization is to use the eo sterilization sterilization; The 30% pure ethylene oxide carbon dioxide of 50%-70% is in addition sterilized in disinfector, and sterilising temp is 54 ℃ ± 2 ℃; Sterilization humidity is 50%-70%RH; Forevacuum is-20～-40Kpa; Ventilation vacuum is-40～-60Kpa; Rate of ventilation is 8 times; Sterilization time is 3-4 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101481385A CN101579247B (en) | 2009-06-23 | 2009-06-23 | I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101481385A CN101579247B (en) | 2009-06-23 | 2009-06-23 | I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101579247A CN101579247A (en) | 2009-11-18 |
| CN101579247B true CN101579247B (en) | 2012-02-29 |
Family
ID=41361662
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101481385A Active CN101579247B (en) | 2009-06-23 | 2009-06-23 | I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101579247B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014130419A1 (en) * | 2013-02-19 | 2014-08-28 | Board Of Regents, The University Of Texas System | Devices and methods for the prevention and treatment of neuromas |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104213238B (en) * | 2014-08-11 | 2016-04-27 | 武汉轻工大学 | Improve the method for collagenous fiber material tensile strength |
| CN107812244B (en) * | 2017-10-25 | 2020-09-29 | 北京华信佳音医疗科技发展有限责任公司 | Preparation of liquid collagen filler |
| CN108383904A (en) * | 2018-02-26 | 2018-08-10 | 王由 | Application of the extracting method, collagenous fibres of collagenous fibres on manufacture tissue filling agent |
| CN112870451B (en) * | 2021-03-02 | 2022-09-06 | 山东奥精生物科技有限公司 | Nerve sheath tube and preparation method and application thereof |
| CN114917406B (en) * | 2022-04-14 | 2023-08-08 | 北京大学第三医院（北京大学第三临床医学院） | Nanotube capable of promoting self-selective nerve repair |
| CN115634317B (en) * | 2022-10-20 | 2023-09-08 | 上海市第六人民医院 | Collagen fiber composite membrane for nerve injury repair |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298746A (en) * | 1999-12-09 | 2001-06-13 | 浙江大学 | Process for preparing polycarbonate-type nerve catheter |
| CN1380115A (en) * | 2002-03-25 | 2002-11-20 | 中国人民解放军第四军医大学 | Preparation process of spinal cord and peripheral nerve repairing material |
| CN1463984A (en) * | 2002-06-10 | 2003-12-31 | 于海鹰 | Medicinal collagen material and its making process |
| CN1589913A (en) * | 2003-09-02 | 2005-03-09 | 中国人民解放军第四军医大学口腔医学院 | Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method |
| CN1593354A (en) * | 2004-06-25 | 2005-03-16 | 清华大学 | Nerve tissue engineering tube type bracket and method for making same |
| CN1795932A (en) * | 2004-12-29 | 2006-07-05 | 东华大学 | Composite collagen nerve ductus for promoting neural regeneration, and method for forming filature from hollow wet process |
| CN1843307A (en) * | 2005-04-07 | 2006-10-11 | 首都医科大学北京神经科学研究所 | Double-layer artificial nerve catheter and preparation method thereof |
| CN101204336A (en) * | 2007-12-06 | 2008-06-25 | 崔树森 | Autologous function-amplifying artificial nerve |
| CN101312756A (en) * | 2005-11-17 | 2008-11-26 | 格利达股份公司 | Nerve guide |
-
2009
- 2009-06-23 CN CN2009101481385A patent/CN101579247B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298746A (en) * | 1999-12-09 | 2001-06-13 | 浙江大学 | Process for preparing polycarbonate-type nerve catheter |
| CN1380115A (en) * | 2002-03-25 | 2002-11-20 | 中国人民解放军第四军医大学 | Preparation process of spinal cord and peripheral nerve repairing material |
| CN1463984A (en) * | 2002-06-10 | 2003-12-31 | 于海鹰 | Medicinal collagen material and its making process |
| CN1589913A (en) * | 2003-09-02 | 2005-03-09 | 中国人民解放军第四军医大学口腔医学院 | Tissue engineering peripheral nerve used for repairing peripheral nerve defect and its preparation method |
| CN1593354A (en) * | 2004-06-25 | 2005-03-16 | 清华大学 | Nerve tissue engineering tube type bracket and method for making same |
| CN1795932A (en) * | 2004-12-29 | 2006-07-05 | 东华大学 | Composite collagen nerve ductus for promoting neural regeneration, and method for forming filature from hollow wet process |
| CN1843307A (en) * | 2005-04-07 | 2006-10-11 | 首都医科大学北京神经科学研究所 | Double-layer artificial nerve catheter and preparation method thereof |
| CN101312756A (en) * | 2005-11-17 | 2008-11-26 | 格利达股份公司 | Nerve guide |
| CN101204336A (en) * | 2007-12-06 | 2008-06-25 | 崔树森 | Autologous function-amplifying artificial nerve |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014130419A1 (en) * | 2013-02-19 | 2014-08-28 | Board Of Regents, The University Of Texas System | Devices and methods for the prevention and treatment of neuromas |
| US9950099B2 (en) | 2013-02-19 | 2018-04-24 | Board Of Regents, The University Of Texas System | Devices and methods for the prevention and treatment of neuromas |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101579247A (en) | 2009-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101569765B (en) | I-type medical collagen material keeping original specific triple helix structure of collagen, product and application thereof | |
| CN101579247B (en) | I-type collagen peripheral nerve sheath keeping the peculiar triple helical structure of collagen, preparation method and applications thereof | |
| Entekhabi et al. | Fabrication and in vitro evaluation of 3D composite scaffold based on collagen/hyaluronic acid sponge and electrospun polycaprolactone nanofibers for peripheral nerve regeneration | |
| Bian et al. | Evaluation of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) conduits for peripheral nerve regeneration | |
| Santos et al. | PEOT/PBT guides enhance nerve regeneration in long gap defects | |
| US9585984B2 (en) | Antibacterial cornea repair material and preparation method thereof | |
| JP6677813B2 (en) | Novel tissue-engineered neural construction via microRNA genes and its use in repairing neural defects | |
| CN104001212B (en) | Peripheral nerve injury containing double factor repairs collagen-based materials and preparation method thereof | |
| Yan et al. | Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration | |
| CN103920194B (en) | Nerve conduit and preparation method thereof | |
| CN105233336B (en) | Sericin nerve trachea and the preparation method and application thereof | |
| JP2010537761A (en) | Biomaterial scaffolds for controlled tissue growth | |
| CN106039416A (en) | Chitosan-sericin composite biological scaffold as well as preparation method and application thereof | |
| Nune et al. | Self-assembling peptide nanostructures on aligned poly (lactide-co-glycolide) nanofibers for the functional regeneration of sciatic nerve | |
| Yang et al. | Characteristics and biocompatibility of a biodegradable genipin‐cross‐linked gelatin/β‐tricalcium phosphate reinforced nerve guide conduit | |
| CN102813961A (en) | Injection gel containing submicron hyaluronic acid microspheres and preparation method thereof | |
| Hsiang et al. | Novel use of biodegradable casein conduits for guided peripheral nerve regeneration | |
| Yang et al. | Sciatic nerve repair by reinforced nerve conduits made of gelatin–tricalcium phosphate composites | |
| Jin et al. | Microtubes with gradient decellularized porcine sciatic nerve matrix from microfluidics for sciatic nerve regeneration | |
| CN111317867A (en) | Nerve conduit and preparation method thereof | |
| CN101072593A (en) | Methods and apparatus for enhanced growth of peripheral nerves and nervous tissue | |
| Liu et al. | bFGF-chitosan scaffolds effectively repair 20 mm sciatic nerve defects in adult rats | |
| CN101106955A (en) | Hyaluronic acid derivative and neural stem cells for SCI and PNT regeneration | |
| WO2023143337A1 (en) | Rotator cuff patch and preparation method therefor | |
| CN1580255A (en) | Tissue engineered peripheral nerve graft |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |