CN103263694B - Collagen-based dura and preparation method thereof - Google Patents
Collagen-based dura and preparation method thereof Download PDFInfo
- Publication number
- CN103263694B CN103263694B CN201310176893.0A CN201310176893A CN103263694B CN 103263694 B CN103263694 B CN 103263694B CN 201310176893 A CN201310176893 A CN 201310176893A CN 103263694 B CN103263694 B CN 103263694B
- Authority
- CN
- China
- Prior art keywords
- collagen
- type
- pig dermis
- preparation
- sodium
- 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
Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention provides a collagen-based dura on the basis of a process-friendly type ion liquid on-site spinning technology, and a preparation method thereof. The preparation method comprises the steps of: dissolving I type collagen by a novel process-friendly solvent system-ion liquid and sodium salt composite system, fully soaking by taking decellularized pig dermis as a substrate through an electrolyte solution, carrying out electrostatic spinning by taking air as a medium, and regulating the pore structure of collagen fibers by adjusting the electrospinning conditions; removing residual ion liquid with a proper eluent, and carrying out freeze-drying, sterilization and packaging to obtain the collagen-based dura. The collagen-based dura has two or more layers of structures: the aperture of collagen fibers at one side close to brain tissue is controlled to be 3mu m and below, and the aperture of collagen fibers at one side far away from brain tissue is controlled to be 20mu m and above. Compared with the traditional solvent spinning, the preparation method is process-friendly, the toxic and side effects are reduced, and the triple-helical structure of the collagen can be furthest retained.
Description
Technical field
The invention belongs to biomedical materials field, be specifically related to a kind of collagen-based cerebral dura mater and preparation method thereof.
Background technology
Cerebral dura mater consists of dense connective tissue, is attached at skull inner surface, is a kind of double-deck fibrous membrane, and Main Function is protection brain, participates in surrounding brain and is full of the effects such as lacuna of liquid around, most important to maintaining the normal physiological function of brain.Dural defect repair is comparatively common in neurosurgery, and open craniocerebral injury, inflammation, brain swelling, cerebroma are intruded into meninges, too the factors such as meninges of fulgerizing all may cause cerebral dura mater in art to close, and form damaged.And the dural damaged complication such as cerebrospinal leak, intracranial infection, otch protracted course of disease, epilepsy that cause.Therefore, look for and prepare a kind of suitable dural substitutes and become to delay anxious.
At present, commercially available cerebral dura mater succedaneum or applied for that the cerebral dura mater preparation method of Patents is quite a lot, in general, is divided into that degradable absorbs and non-degradable absorption two large classes.Degradable absorbs class, and especially to take such as pig source pericardium, cattle source pericardium, sheep source pericardium etc. in natural biological source be representative.Although this type of cerebral dura mater succedaneum can be simulated the histological structure of human dura mater substantially, has certain curative effect, because each animal only has a secondary pericardium, therefore its source is limited, should not produce in batch, cannot meet growing market capacity demand.It is collagen-based material that another kind of degradable absorbs class cerebral dura mater succedaneum, the product Duragen that the cattle heel string collagen of take as simple is raw material, Duramax etc.Although such cerebral dura mater succedaneum good biocompatibility, can obviously promote the growth of fibroblast in brain surrounding tissue etc., but mechanical property is not good enough, cannot bear the pressure that in larger brain, cerebrospinal fluid produces, cause bulging amount too high, usually cause cerebrospinal leak, cannot form environment in closed brain, in addition, because collagen sponge class succedaneum porosity is excessive, growing into of cerebral tissue peripheral cell be can cause, thereby glutinous gluing, the generation of epileptics formed.And non-degradable absorbs the dural succedaneum of class as the Neuro-Patch product of the U.S., its main component is polyester ethane, and while being expert at cerebral dura mater prosthesis, this product exists as permanent foreign body, nonabsorable, can cause comparatively serious chronic inflammatory reaction of later stage.
As can be seen here, a kind of desirable dural substitutes must have following features concurrently: (1) can prevent cerebrospinal leak; (2) after generating, class cerebral dura mater sample tissue can be absorbed by body; (3) there is enough ductility and intensity; (4) can not cause any known or potential infection risk; (5) material is easy to preserve and easily obtain.
Collagen is as a kind of special extracellular matrix, extensively be present in the tissues such as numerous mammiferous heel strings, skin, bone, growth and the propagation that can promote significantly cell, contribute to the quickly-healing of the open sore part, and be deeply subject to liking of vast medical science and technology worker.But the dissolubility of collagen in conventional solvent is limited, be unfavorable for the machine-shaping of product, product now or patent be mainly with sponge, gel, and the forms such as thin film occur, during as one-component, its mechanical property is not good enough, and embossability is poor, and surface topography is uncontrollable, the randomness of hole is large, and for it, the application on cerebral dura mater is totally unfavorable to the present situation that this hole can not regulate and control, mechanical property is not good enough.Existing employing nano electrostatic spining technology becomes collagen fiber by collagen electrospinning, by regulation and control concentration of dope, ejection speed, the conditions such as receiving screen and needle point distance or positive and negative high pressure can regulate and control final collagen product aperture and porosity within certain scope, the good way of the controlled utilization of collagen of can yet be regarded as.But, most of patents or bibliographical information adopt acetic acid, hexafluoroisopropanol, trifluoroacetic acid, water or its interworking thing as the solvent of electrospinning collagen, the solvent of this type has strong impulse abnormal smells from the patient, very big to operator's self-inflicted injury, eyes and skin are had to strong corrosiveness, respiratory tract is had to stimulation, especially fluorinated alcohols, after suction, make people drowsiness, limbs fatigue.What is more important, have been reported demonstration, its main component of the spun collagen fiber of fluorinated alcohols electricity has been degraded to the catabolite of gelatin-a kind of collagen, the distinctive triple helix structure of collagen is destroyed already, and this special triple helix structure of collagen is to maintain the architecture basics of its physiology characteristic.Well imagine, look for and develop a kind of process close friend, the electrospinning dicyandiamide solution that can at utmost retain collagen structure integrity is also extremely urgent.
About collagenolysis degree aspect, be different from conventional acid, alkali, and fluorinated alcohols above-mentioned, trifluoroacetic acids etc. have outside the solvent of larger harm to operator's health, fragmentary article and patent report adopt that the dicyandiamide solution that a class is new---ionic liquid dissolves biomass, relating to relevant biomass focuses mostly in cellulose, lignin, chitosan etc., but relate to, do not adopt ionic liquid to carry out any breakthrough or the report of collagen electrostatic spinning, also only rest on the dissolubility aspect to collagen, and, also equivocal to maintaining the triple helix structure of collagen special physiological function.Especially, the kind of ionic liquid at room temperature is thousands of, and its combining form is numerous and complicated also, and for one thing cited, ten thousand may have been left out unavoidably for the report of finding at present, therefore, explores and find suitable collagen solvent be really well worth doing in this field.
Acellular dermal matrix material is a kind of host material of taking as the leading factor with collagen composition of having removed antigenic substance completely, according to the source of material, is divided into again acellular matrix of the same race and xenogenesis acellular matrix material.One large main source of acellular matrix material of the same race is cadaver skin, and its source is limited, and development has been subject to certain limitation, and has the drawback of carrying HIV (human immunodeficiency virus).Application is at present more xenogenesis acellular matrix material, present situation in view of the livestock breeding industry of China, especially to take off cell pig dermis, take as the leading factor, its wide material sources, draw materials conveniently, it is worth mentioning that at present domestic existing some ecologic breeding bases of specially taking as the leading factor with breeding ecological pig, for the further deep processing of such material, process and established solid material base.More than 95% composition of de-cell pig dermis is collagen, but compare with pure collagen, its mechanical strength is a large advantage, can prepare as required intensity at 1～30N/mm2, the de-cell pig dermis of elongation at break between 5～40%, and its inner duct communicates with one another, and runs through whole skin corium, be applicable to very much cell, especially fibroblastic growing into.According to the literature and reports related patents, the use of de-cell pig dermis is more confined in the treatment of skin trauma and the use of body inner stuffing, its main cause is because Corii Sus domestica fat content is high, be difficult for except degrease, make most medical material workers be reluctant to develop this huge treasure-house, seriously restricted its application in biomedical materials field, if can overcome this defect, be applied to outside more wide field, prospect will be known no measure.
Consider the particularity of cerebral dura mater organizational structure, press close to corticocerebral material and partly need comparatively fine and close network structure, prevent brain tissue cell, especially fibroblastic growing into, need in addition to possess suitable degradation rate, can, in meningeal tissue growth, little by little decompose, or be absorbed by body.According to Patents, human body cell diameter is on average at 10～20 μ m, and general fibroblast diameter, at 20～30 μ m, organizes and press close to brain one side the collagen fiber that are mainly scattered in fibrocyte and secretion thereof, average pore size, can effectively prevent that brain from sticking sticky generation at 3 μ m once.The material part that leans against brain tissue is just the opposite, needs cell or tissue around to grow into, and promotes the healing of wound, also needs to bear especially in addition higher intracranial hypertension, and for brain is built the environment of a sealing, this just needs certain mechanical strength.
In sum, adopt at present collagen be cerebral dura mater succedaneum prepared by material of main part also exist or preparation process or properties of product on deficiency, while adopting electrostatic spinning technique, exist production process unfriendly, the most of forfeiture of biological activity of collagen is fallen, the defect of mechanical property deficiency, simple acellular matrix material, because pore structure runs through wherein, has the risk of seepage.As can be seen here, the cerebral dura mater succedaneum of single material is difficult to meet this application request that seems contradiction simultaneously, and it is inevitable that the road of walking composite seems to become.
Summary of the invention
The object of the invention is to overcome the deficiency of existing collagen-based cerebral dura mater production technology, the artificial dura mater of the friendly Collagen Type VI base of a kind of process based on ionic liquid electrostatic spinning technique nano composite structure is provided, the artificial dura mater of collagen fiber composite construction prepared by the method has the good mechanical property of excellent biocompatibility and the de-cell pig dermis of collagen, such as pliability, elasticity, hot strength etc. concurrently.And prepared material possesses suitable pore structure, can effectively prevent that brain is glutinous sticky, can bear higher intracranial hypertension, promote dura mater process, controlled degradation in the process of Ke brain tissue healing, and can cut randomly into required form, convenient storage, clinical practice is easy to operation.
Another object of the present invention is to provide a kind of above-mentioned dural method of preparing.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of collagen-based cerebral dura mater based on the friendly type ionic liquid of process original position spining technology, adopt original position electrostatic spinning technique, using different electrolyte solutions as wetting agent, utilize ionic liquid for solvent, take and take off cell pig dermis as substrate, by collagen electrospinning on it, form the controlled nano fibrous membrane in aperture, described cerebral dura mater can comprise taking off the two-layer or two-layer above structure that cell pig dermis is substrate, and collagen fiber part wherein has farthest retained architecture basics---the triple helix structure of its performance biotic induce effect.
(1) by collagenolysis in ion liquid solvent system, its temperature range is 0～37 ℃, and being mixed with concentration is the homogeneous phase spinning solution of 0.01%wt～20% wt, during standing or excusing from death ripple concussion deaeration, field of view uniformity under PLM with heating stage, without obvious phenomenon of phase separation;
(2) preparation of de-cell pig dermis: fresh ecological Corii Sus domestica is cutd open to the thin slice that layer is 0.5～1.0mm for thickness, supersound process 1h～3h under the peracetic acid ultrasound wave that is 3.0～5.0% by mass concentration, carry out inactivation of virus, and then adopting mass concentration is that 2～5% vigor is the trypsin of 50,000 units, mass concentration is that 5～10% peregal (AEO-25) is purchased from Chengdu Ke Long chemical reagent factory, take off cell and ungrease treatment, then lyophilizing is standby; Adopt the electrolyte solution of different proportionings to be sprayed at de-cell pig dermis surface, maybe will take off cell pig dermis and impregnated in this electrolyte solution, obtain the fully moistening de-cell pig dermis that is not polymerized to again water droplet, control its water content between 5% wt～100% wt;
(3) above-mentioned de-cell pig dermis is attached to electrostatic spinning machine touch screen metal roller one end, the air of take carries out electrostatic spinning as medium, control electrostatic field voltage at 5～50 kv, orifice diameter is at 0.05～3mm, microsyringe sample rate is 1～10 mL/h, the rotating speed of cylinder catcher is 5～1000r/min, spinning distance is between 10～50cm, ambient humidity is between 10～70%, making can be in the stable ejection of spinneret orifice through the collagen of ion liquid dissolving, and solidify in electrospinning process, can form the continuous collagen fiber rete of one deck on de-cell pig dermis surface,
(4) preparation eluate solvent, by de-cell pig dermis-collagen fiber composite film of above-mentioned gained, by the interworking thing of deionized water or deionized water and ethanol, wherein the interworking thing volume ratio of deionized water and ethanol is 1:9～9:1, washing by soaking 1～3 time, each 10～60 min, to remove residual ion liquid, again through lyophilization molding, obtain the collagen-based cerebral dura mater of two-layer composite, or further at de-cell pig dermis another side, repeat electrospinning step, in like manner can obtain the collagen-based cerebral dura mater of three-layer composite structure;
(5) the collagen-based cerebral dura mater of preparing by said method stores after sterilizing, packing;
Collagen in described step (1) is the type i collagen of being carried the commercially available skin from animal, heel string, tail tendon, bone, scale etc., its type comprises: cattle heel string type i collagen, Mus tail tendon type i collagen, Corii Sus domestica type i collagen, Corii Bovis seu Bubali type i collagen, fish skin type i collagen, donkey skin type i collagen, fish scale type i collagen, Os Bovis seu Bubali type i collagen, Os Sus domestica type i collagen or other commercially available type i collagen;
Ion liquid solvent system in described step (1) comprises the compound system that ionic liquid or ionic liquid and some sodium salts form; The cation of the ionic liquid in the ion liquid solvent system in described step (1) is 1-ethyl, 3-Methylimidazole. ([Emim]), 1-butyl, 3-Methylimidazole. ([Bmim]), 1-pi-allyl, a kind of in 3-Methylimidazole. ([Amim]), the anion of the ionic liquid in described step (1) is formate (COO-), acetate (CH3COO-), benzoate anion (Ba), glycine root (Gly), sulfonate radical (SO
3 2-), a kind of in lactate (La); Described sodium salt is sodium sulfate (Na
2sO
4), sodium bisulfate (NaHSO
4), sodium chloride (NaCl), sodium phosphate (Na
3pO
4), sodium hydrogen phosphate (Na
2hPO
4), sodium dihydrogen phosphate (NaH
2pO
4), Chile saltpeter (NaNO
3), sodium bicarbonate (NaHCO
3), sodium carbonate (Na
2cO
3) in a kind of, its percetage by weight content that accounts for ion liquid system is between 0.1～1%wt;
The electrolyte solution of the different proportionings that adopt in described step (2), the sodium chloride that its constituent is 0.9%wt (NaCl), the phosphate buffered solution of 0.01Mol/L (PBS), volume proportion scope NaCl:PBS is 1:9～9:1;
The pore size of the collagen fiber rete obtaining in described step (3) can be by regulating electrostatic field voltage, the orifice diameter of electrostatic spinning, the rotating speed of cylinder, spinning distance etc. effectively control, its pore-size distribution is than little 1～2 order of magnitude of fibroblastic dimension, average pore size is at 2 μ m and following, and fibre diameter is between 100～1000nm;
Deionized water in described step (4) or the interworking thing of deionized water and ethanol are for removing the de-residual ionic liquid of cell pig dermis-collagen fiber composite film, its deionized water: the volume proportion scope of ethanol is between 1:9 to 9:1; Lyophilization moulding process in described step (4) is programme-control freeze-dry process, and design parameter is as follows: first paragraph temperature-35 ℃, 2 ℃/min of rate of temperature fall, constant temperature 4h; Second segment temperature-25 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; The 3rd section of temperature-15 ℃, 1.5 ℃/min of heating rate, constant temperature time 10h; The 4th section of temperature-5 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; 21 ℃ of the 5th section of temperature, 1.5 ℃/min of heating rate, constant temperature time 6 h;
Sterilization method in described step (4) is low temperature ethylene oxide sterilizing, or adopts cobalt-60 to carry out irradiation sterilization, and sterilizing dose is 25～50kGy;
The collagen-based cerebral dura mater with composite construction that adopts said method to prepare, its key performance meets following requirement:
1. outward appearance: the membranaceous or tablet of white or milky, without the visible impurity of naked eyes;
2. moisture :≤10%(wt) (2010 editions-aquametry of Chinese Pharmacopoeia: first method (taking Xiu Shi method));
3. collagen fiber rete pore diameter range: be close to cerebral tissue face 3 μ m and following, away from cerebral tissue face 20 μ m and more than; Collagen fiber rete fiber diameter range: 100 nm～1000 nm; Collagen fiber thicknesses of layers: 0.05～0.1mm; De-cell pig dermis thickness: 0.3～0.7 mm;
4. cytotoxicity: 1 grade or above (ISO10993-5 that adopts international standards measures);
5. the collagen in collagen fiber rete has farthest retained the triple helix structure of natural collagen, can obviously see collagen peculiar three bands of a spectrum-α, β, γ chain (adopting SDS-PAGE electrophoresis to detect).
Compared to existing technology, the present invention has following advantage:
1. adopted the ion liquid system of the friendly type of process to substitute traditional high volatile volatile, the solvent of high toxic and side effects is prepared collagen electrostatic spinning liquid;
2. the new collagen dicyandiamide solution adopting has retained the distinctive triple helix structure of collagen, and this is that traditional fluorinated alcohols or three width acetic acid solvents can not match in excellence or beauty;
3. provide a kind of take take off cell pig dermis as substrate, carry out electrostatic spinning and prepare the dural simple and easy method of compound collagen-based, widened the application that the substrate of non-conductive property is applied to electrostatic spinning technique;
4. prepared compound collagen-based cerebral dura mater is compared traditional collagen cerebral dura mater, and its mechanical strength increases greatly, has met the demand of this series products applied technical field;
5. it is abundant that the artificial dura mater described in is prepared material source, cheap, fully excavated the potentiality of domestic ecological pig aquaculture, possesses and be easy to form scale industrial chain, obtains the good prospect of planningization, clustered benefit;
6. prepared collagen-based cerebral dura mater is simultaneously the collagen fiber membrane with control pore structure towards cerebral cortex, the glutinous sticky effect that can effectively prevent from growing into because cortex face is fibroblastic and cause, cerebral cortex is simultaneously de-cell pig dermis or highly porous collagem membrane dorsad simultaneously, there is flourishing porous network structure, be very suitable for growing into of fibroblast, vascular endothelial cell, make the vascularization rapidly of this product, integrated with autologous tissue, effectively prevent the seepage of cerebrospinal fluid.
Accompanying drawing explanation
Fig. 1 is the collagen-based cerebral dura mater structure scanning electron microscope sectional drawing that embodiment 1 makes, and upper strata is collagen fiber layer, and lower floor is de-cell pig dermis layer;
The surface topography SEM figure that Fig. 2 is de-cell pig dermis, its hole, between 20～100 μ m, as deviating from cerebral tissue one side, is suitable for entering of seed cell;
Fig. 3 is the SEM figure that is close to collagen fiber one side of cerebral tissue, can effectively organize growing into of cerebral tissue one side cell, avoids glutinous sticky generation;
Fig. 4 is that fibroblast is at the long SEM figure of collagen fiber one adnation.
The specific embodiment
Below by specific embodiment, the present invention is carried out to concrete elaboration; but it is important to point out; the present embodiment is only used to further illustrate the present invention; and can not be interpreted as the restriction to the present invention's protection domain, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of foregoing invention.
Embodiment 1:
(1) prepare collagen homogeneous phase electrospinning solution: 25 ℃ of NaH2PO4 room temperatures that is first 0.1%wt by mass fraction degree are dissolved in 1-pi-allyl-3-Methylimidazole. benzoate ([BMIM] [Ac]), obtain ionic liquid composite solvent system, Corii Bovis seu Bubali type i collagen is progressively dissolved at 4 ℃ to this ionic liquid composite solvent system, initially add 1% of ionic liquid composite solvent system quality percent, after fully dissolving, it adds successively again Corii Bovis seu Bubali type i collagen, until the mass percent content of Corii Bovis seu Bubali type i collagen in this ionic liquid composite solvent system is 5%, 25 ℃ of ultrasound wave deaerations are extremely completely without bubble,
(2) prepare the de-cell pig dermis substrate of electric conductivity: fresh ecological Corii Sus domestica is cutd open to the thin slice that layer is 0.5～1.0mm for thickness, supersound process 1h under the peracetic acid ultrasound wave that is 3.0% by mass concentration, carry out inactivation of virus, and then adopting vigor mass concentration is 2% 50,000 unit trypsin, mass concentration is that 5% peregal takes off cell and ungrease treatment, and then lyophilizing is standby.The sodium chloride that is 0.9%wt by constituent (NaCl), the phosphate buffered solution of 0.01Mol/L (PBS), the electrolyte buffer solution that volume proportion scope NaCl:PBS is 1:9 is evenly sprayed on de-cell pig dermis surface, controls the water content of de-cell pig dermis at 20 ± 5%wt;
(3) prepare collagen-based electrospinning rete: the de-cell pig dermis of the electric conductivity in (2) is fixed on the metal roller catcher of electrostatic spinning apparatus, connect negative high voltage, by in (1) join homogeneous phase collagen electrospinning solution and add in the syringe of electrostatic spinning apparatus, regulating the sample rate of micro-injection pump is 2 mL/h, electrostatic field voltage is 20 kv, orifice diameter is 0.5 mm, cylinder catcher rotating speed is 100 r/min, spinning distance is at 20 cm, electrospinning ambient humidity is 30%, the air of take carries out electrostatic spinning as medium, until collagen electrospinning layer thickness is 0.1 mm, the collagen fiber average diameter of preparation is 700 nm herein, fiber average pore size is 2 μ m, when this layer used as being close to cerebral tissue one side,
(4) resulting de-cell pig dermis-collagen fiber electrospinning layer composite membrane is soaked in eluate solvent to remove residual ion liquid solvent, eluent is the interworking thing of deionized water and ethanol, its volume ratio is deionized water: ethanol=20:80, repetitive operation 3 times, each 20 min;
(5) by having removed the de-cell pig dermis-collagen fiber electrospinning layer composite membrane after solvent, carry out lyophilization molding, lyophilizing molding technological condition is as follows: first paragraph temperature-35 ℃, 2 ℃/min of rate of temperature fall, constant temperature 4 h; Second segment temperature-25 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; The 3rd section of temperature-15 ℃, 1.5 ℃/min of heating rate, constant temperature time 10h; The 4th section of temperature-5 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; 21 ℃ of the 5th section of temperature, 1.5 ℃/min of heating rate, constant temperature time 6 h;
(6) by the collagen-based cerebral dura mater sterile packaged after lyophilization in packing bag for medical use, low temperature ethylene oxide sterilizing.
Embodiment 2:
(1) prepare collagen homogeneous phase electrospinning solution: 25 ℃ of NaHSO4 room temperatures that is first 0.5%wt by mass fraction degree are dissolved in 1-ethyl-3-methylimidazole glycinate ([EMIM] [Gly]), obtain ionic liquid composite solvent system, Corii Sus domestica type i collagen is progressively dissolved at 10 ℃ to this ionic liquid composite solvent system, initially add 3% of ionic liquid composite solvent system quality percent, after fully dissolving, it adds successively again Corii Sus domestica type i collagen, until the mass percent content of Corii Sus domestica type i collagen in this ionic liquid composite solvent system is 10%, 25 ℃ of ultrasound wave deaerations are extremely completely without bubble,
(2) prepare the de-cell pig dermis substrate of electric conductivity: de-cell pig dermis used is by being obtained in embodiment 1, the sodium chloride that is 0.9%wt by constituent (NaCl), the phosphate buffered solution of 0.01Mol/L (PBS), volume proportion NaCl:PBS is that the electrolyte buffer solution of 5:5 is evenly sprayed on de-cell pig dermis surface, controls the water content of de-cell pig dermis at 50 ± 5%wt;
(3) prepare collagen-based electrospinning rete: the de-cell pig dermis of the electric conductivity in (2) is fixed on the metal roller catcher of electrostatic spinning apparatus, connect negative high voltage, by in (1) join homogeneous phase collagen electrospinning solution and add in the syringe of electrostatic spinning apparatus, regulating the sample rate of micro-injection pump is 3 mL/h, electrostatic field voltage is 25kv, orifice diameter is 0.25 mm, cylinder catcher rotating speed is 250 r/min, spinning distance is at 15 cm, electrospinning ambient humidity is 35%, the air of take carries out electrostatic spinning as medium, until collagen electrospinning layer thickness is 0.1 mm, the collagen fiber average diameter of preparation is 1000 nm herein, fiber average pore size is 2 μ m, when this layer used as being close to cerebral tissue one side, again at de-cell pig dermis another side, carry out collagen solution Static Spinning, electrospinning parameter is: micro-injection pump sample rate is 5 mL/h, electrostatic field voltage is 15 kv, orifice diameter is 0.5 mm, cylinder catcher rotating speed is 100 r/min, spinning distance is at 30 cm, electrospinning ambient humidity is 40%, the air of take carries out electrospinning as medium, until collagen electrospinning layer thickness is 0.1 mm, the collagen fiber average diameter of preparation is 1000nm herein, fiber average pore size is 20 μ m, when this layer used as deviating from cerebral tissue one side,
(4) resulting de-cell pig dermis-collagen fiber electrospinning layer composite membrane is soaked in eluate solvent to remove residual ion liquid solvent, eluent is the interworking thing of deionized water and ethanol, its volume ratio is deionized water: ethanol=50:50, repetitive operation 3 times, each 30 min;
(5) by having removed the de-cell pig dermis-collagen fiber electrospinning layer composite membrane after solvent, carry out lyophilization molding, lyophilizing molding technological condition is as follows: first paragraph temperature-35 ℃, 2 ℃/min of rate of temperature fall, constant temperature 4 h; Second segment temperature-25 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; The 3rd section of temperature-15 ℃, 1.5 ℃/min of heating rate, constant temperature time 10h; The 4th section of temperature-5 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; 21 ℃ of the 5th section of temperature, 1.5 ℃/min of heating rate, constant temperature time 6 h;
(6) by the collagen-based cerebral dura mater sterile packaged after lyophilization in packing bag for medical use, cobalt-60 sterilizing, sterilizing dose 30kGy.
Embodiment 3
(1) prepare collagen homogeneous phase electrospinning solution: 25 ℃ of NaHCO3 room temperatures that is first 1%wt by mass fraction degree are dissolved in 1-pi-allyl-3-Methylimidazole. benzoate ([Amim] [Ba]), obtain ionic liquid composite solvent system, cattle heel string type i collagen is progressively dissolved in to this ionic liquid composite solvent system at 20 ℃, initially add 5% of ionic liquid composite solvent system quality percent, after fully dissolving, it adds successively again cattle heel string type i collagen, until the mass percent content of cattle heel string type i collagen in this ionic liquid composite solvent system is 15%, 25 ℃ of standing and defoaming are extremely completely without bubble,
(2) prepare the de-cell pig dermis substrate of electric conductivity: de-cell pig dermis used obtains in embodiment 1, the sodium chloride that is 0.9%wt by constituent (NaCl), the phosphate buffered solution of 0.01Mol/L (PBS), volume proportion NaCl:PBS is that the electrolyte buffer solution of 9:1 is evenly sprayed on de-cell pig dermis surface, controls the water content of de-cell pig dermis at 70 ± 5%wt;
(3) prepare collagen-based electrospinning rete: the de-cell pig dermis of the electric conductivity in (2) is fixed on the metal roller catcher of electrostatic spinning apparatus, connect negative high voltage, by in (1) join homogeneous phase collagen electrospinning solution and add in the syringe of electrostatic spinning apparatus, regulating the sample rate of micro-injection pump is 5 mL/h, electrostatic field voltage is 35kv, orifice diameter is 1.5 mm, cylinder catcher rotating speed is 500 r/min, spinning distance is at 40 cm, electrospinning ambient humidity is 60%, the air of take carries out electrostatic spinning as medium, until collagen electrospinning layer thickness is 0.1 mm, the collagen fiber average diameter of preparation is 800 nm herein, fiber average pore size is 2.5 μ m, when this layer used as being close to cerebral tissue one side, again at de-cell pig dermis another side, carry out collagen solution Static Spinning, electrospinning parameter is: micro-injection pump sample rate is 10 mL/h, electrostatic field voltage is 15 kv, orifice diameter is 2 mm, cylinder catcher rotating speed is 300 r/min, spinning distance is at 50 cm, electrospinning ambient humidity is 45%, the air of take carries out electrospinning as medium, until collagen electrospinning layer thickness is 0.1 mm, the collagen fiber average diameter of preparation is 1200 nm herein, fiber average pore size is 30 μ m, when this layer used as deviating from cerebral tissue one side,
(4) resulting de-cell pig dermis-collagen fiber electrospinning layer composite membrane is soaked in eluate solvent to remove residual ion liquid solvent, eluent is the interworking thing of deionized water and ethanol, its volume ratio is deionized water: ethanol=80:20, repetitive operation 3 times, each 40 min;
(5) by having removed the de-cell pig dermis-collagen fiber electrospinning layer composite membrane after solvent, carry out lyophilization molding, lyophilizing molding technological condition is as follows: first paragraph temperature-35 ℃, 2 ℃/min of rate of temperature fall, constant temperature 4 h; Second segment temperature-25 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; The 3rd section of temperature-15 ℃, 1.5 ℃/min of heating rate, constant temperature time 10h; The 4th section of temperature-5 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; 21 ℃ of the 5th section of temperature, 1.5 ℃/min of heating rate, constant temperature time 6 h;
(6) by the collagen-based cerebral dura mater sterile packaged after lyophilization in packing bag for medical use, cobalt-60 sterilizing, sterilizing dose 50kGy.
Claims (2)
1. the dural preparation method of collagen-based, described collagen-based cerebral dura mater be take the two-layer or two-layer above structure that to take off cell pig dermis be substrate, wherein being close to cerebral tissue one side is that average pore size is at 3 μ m and following collagen fiber, opposite side be average pore size at 20 μ m and above collagen fiber, the method comprises the following steps:
(1) by collagenolysis in ionic liquid/sodium salt compound system, its temperature range is 0～37 ℃, and being mixed with concentration is the homogeneous phase spinning solution of 0.01%wt～20% wt, during standing or excusing from death ripple concussion deaeration, field of view uniformity under PLM with heating stage, without obvious phenomenon of phase separation;
(2) preparation of de-cell pig dermis: fresh ecological Corii Sus domestica is cutd open to the thin slice that layer is 0.5～1.0mm for thickness, supersound process 1h～3h under the peracetic acid ultrasound wave that is 3.0～5.0% by mass concentration, carry out inactivation of virus, and then adopting mass concentration is that 2～5% vigor is the trypsin of 50,000 units, mass concentration is that 5～10% peregal AEO-25 takes off cell and ungrease treatment, and then lyophilizing is standby; Adopt the electrolyte solution of different proportionings to be sprayed at de-cell pig dermis surface, maybe will take off cell pig dermis and impregnated in this electrolyte solution, obtain the fully moistening de-cell pig dermis that is not polymerized to again water droplet, control its water content between 5% wt～100% wt;
(3) above-mentioned de-cell pig dermis is attached to electrostatic spinning machine touch screen metal roller one end, the air of take carries out electrostatic spinning as medium, control electrostatic field voltage at 5～50 kv, orifice diameter is at 0.05～3mm, microsyringe sample rate is 1～10 mL/h, the rotating speed of cylinder catcher is 5～1000r/min, spinning distance is between 10～50cm, ambient humidity is between 10～70%, making can be in the stable ejection of spinneret orifice through the collagen of ion liquid dissolving, and solidify in electrostatic spinning process, can form the continuous collagen fiber rete of one deck on de-cell pig dermis surface,
(4) preparation eluate solvent, by the de-cell pig dermis-collagen fiber composite film of above-mentioned gained, by the interworking thing of deionized water or deionized water and ethanol, wherein the interworking thing volume ratio of deionized water and ethanol is 1:9～9:1, washing by soaking 1～3 time, each 10～60 min, to remove residual ion liquid, again through lyophilization molding, obtain the collagen-based cerebral dura mater of two-layer composite, or further at de-cell pig dermis another side, repeat electrostatic spinning step, in like manner can obtain the collagen-based cerebral dura mater of three-layer composite structure.
2. the dural preparation method of collagen-based as claimed in claim 1, the cationic structural that it is characterized in that the ionic liquid in described ionic liquid/sodium salt compound system is selected from 1-ethyl 3-Methylimidazole. ([Emim]), 1-butyl, 3-Methylimidazole. ([Bmim]), 1-pi-allyl, one or more in 3-Methylimidazole. ([Amim]); The anion structure of ionic liquid is selected from formate (COO
-), acetate (CH
3cOO
-), benzoate anion (Ba), glycine root (Gly), sulfonate radical (SO
3 2-), one or more in lactate (La); Sodium salt is sodium sulfate (Na
2sO
4), sodium bisulfate (NaHSO
4), sodium chloride (NaCl), sodium phosphate (Na
3pO
4), sodium hydrogen phosphate (Na
2hPO
4), sodium dihydrogen phosphate (NaH
2pO
4), Chile saltpeter (NaNO
3), sodium bicarbonate (NaHCO
3), sodium carbonate (Na
2cO
3) in one or more, the percetage by weight content that sodium salt accounts for ion liquid system is between 0.1～1%wt.
3. the dural manufacture method of collagen-based as claimed in claim 1 or 2, it is characterized in that described collagen is the type i collagen being extracted the skin from animal, heel string, tail tendon, bone or scale, its type comprises: cattle heel string type i collagen, Mus tail tendon type i collagen, Corii Sus domestica type i collagen, Corii Bovis seu Bubali type i collagen, fish skin type i collagen, donkey skin type i collagen, fish scale type i collagen, Os Bovis seu Bubali type i collagen, Os Sus domestica type i collagen or other commercially available type i collagen.
4. the dural preparation method of collagen-based as claimed in claim 1 or 2, the sodium chloride (NaCl) that the constituent that it is characterized in that described electrolyte solution is 0.9%wt, the phosphate buffered solution of 0.01Mol/L (PBS), the volume ratio scope of phosphate buffered solution (PBS) solution of the sodium chloride of 0.9%wt (NaCl) solution and 0.01Mol/L is 1:9～9:1.
5. the dural preparation method of the collagen-based as described in claims 1 or 2, is characterized in that described lyophilization is stage control freeze drying process, and design parameter is as follows: first stage temperature-35 ℃, 2 ℃/min of rate of temperature fall, constant temperature 4 h; Second stage temperature-25 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; Phase III temperature-15 ℃, 1.5 ℃/min of heating rate, constant temperature time 10h; Fourth stage temperature-5 ℃, 1.5 ℃/min of heating rate, constant temperature time 8 h; 21 ℃ of five-stage temperature, 1.5 ℃/min of heating rate, constant temperature time 6 h.
6. the dural preparation method of the collagen-based as described in claims 1 or 2, characterized by further comprising sterilization treatment operation, adopts low temperature ethylene oxide sterilizing or cobalt-60 x ray irradiation x sterilizing, and irradiation dose is 30-60kGy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310176893.0A CN103263694B (en) | 2013-05-14 | 2013-05-14 | Collagen-based dura and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310176893.0A CN103263694B (en) | 2013-05-14 | 2013-05-14 | Collagen-based dura and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103263694A CN103263694A (en) | 2013-08-28 |
| CN103263694B true CN103263694B (en) | 2014-12-03 |
Family
ID=49007384
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310176893.0A Active CN103263694B (en) | 2013-05-14 | 2013-05-14 | Collagen-based dura and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103263694B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104189955B (en) * | 2014-08-08 | 2016-06-15 | 江西瑞济生物工程技术股份有限公司 | The duramater reparation support of degradable people's amnion and ox back tendon compound and preparation method |
| CN104888273B (en) * | 2015-05-14 | 2017-08-22 | 四川大学 | A kind of double-layer composite endocranium and preparation method thereof |
| CN106474548B (en) * | 2015-08-28 | 2020-01-21 | 北京华信佳音医疗科技发展有限责任公司 | Biological induction type artificial dura mater and preparation method thereof |
| CN106178133A (en) * | 2016-08-16 | 2016-12-07 | 烟台正海生物科技股份有限公司 | A kind of degradable meninges repair materials and preparation method thereof |
| CN107913435B (en) * | 2016-10-10 | 2022-09-09 | 北京邦塞科技有限公司 | Composite type dura mater (spinal) membrane implant, preparation method and use thereof |
| CN106310367A (en) * | 2016-10-13 | 2017-01-11 | 苏州希普生物科技有限公司 | Preparation method of absorbable cerebral dura mater patch |
| CN107551324B (en) * | 2017-10-27 | 2020-02-18 | 北京华信佳音医疗科技发展有限责任公司 | Preparation and application of suturable hard membrane repairing material |
| CN110240715B (en) * | 2019-06-26 | 2022-02-15 | 南京工业大学 | Method for quickly dissolving gelatin in Bronsted acidic ionic liquid at low temperature |
| CN111394891B (en) * | 2020-03-25 | 2021-04-16 | 武汉理工大学 | Preparation method of chitosan oligosaccharide and polyester composite material |
| CN112903146B (en) * | 2021-01-22 | 2022-07-15 | 中国科学院重庆绿色智能技术研究院 | Preparation method and application of dermal-based flexible pressure sensor |
| CN115089766B (en) * | 2022-03-22 | 2023-07-18 | 东华大学 | Method for preparing artificial skin by utilizing electrostatic spinning |
| CN118518439A (en) * | 2023-02-17 | 2024-08-20 | 清华大学 | Ionic liquid preparation, tissue treatment method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569245A (en) * | 2004-05-12 | 2005-01-26 | 四川大学 | Method for eliminating antigen from animal fur |
| CN1584150A (en) * | 2004-05-28 | 2005-02-23 | 四川大学 | Preparing method for collagen stroma composite medical fibre spinning stoste |
| CN1832773A (en) * | 2003-06-05 | 2006-09-13 | 巴克斯特国际有限公司 | Composition for repairing and regenerating human dura mater |
| CN101569765A (en) * | 2009-06-23 | 2009-11-04 | 许和平 | I-type medical collagen material keeping original specific triple helix structure of collagen, product and application thereof |
| CN101692986A (en) * | 2009-03-10 | 2010-04-14 | 广州迈普再生医学科技有限公司 | Artificial dura mater with bioactivity and preparation method thereof |
| CN102121133A (en) * | 2011-04-02 | 2011-07-13 | 四川大学 | Antigen-free porcine dermal collagen fibers |
| CN103007346A (en) * | 2012-12-26 | 2013-04-03 | 天津市赛宁生物工程技术有限公司 | Anti-adhesion endocranium prepared by physical sedimentation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2536923C (en) * | 2003-09-04 | 2012-10-09 | Cook Biotech Incorporated | Extracellular matrix composite materials, and manufacture and use thereof |
-
2013
- 2013-05-14 CN CN201310176893.0A patent/CN103263694B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1832773A (en) * | 2003-06-05 | 2006-09-13 | 巴克斯特国际有限公司 | Composition for repairing and regenerating human dura mater |
| CN1569245A (en) * | 2004-05-12 | 2005-01-26 | 四川大学 | Method for eliminating antigen from animal fur |
| CN1584150A (en) * | 2004-05-28 | 2005-02-23 | 四川大学 | Preparing method for collagen stroma composite medical fibre spinning stoste |
| CN101692986A (en) * | 2009-03-10 | 2010-04-14 | 广州迈普再生医学科技有限公司 | Artificial dura mater with bioactivity and preparation method thereof |
| CN101569765A (en) * | 2009-06-23 | 2009-11-04 | 许和平 | I-type medical collagen material keeping original specific triple helix structure of collagen, product and application thereof |
| CN102121133A (en) * | 2011-04-02 | 2011-07-13 | 四川大学 | Antigen-free porcine dermal collagen fibers |
| CN103007346A (en) * | 2012-12-26 | 2013-04-03 | 天津市赛宁生物工程技术有限公司 | Anti-adhesion endocranium prepared by physical sedimentation method |
Non-Patent Citations (6)
| Title |
|---|
| Evaluation of 1-ethyl-3-methylimidazolium acetate based ionic liquid systems as a suitable solvent for collagen;Yang Hu et al.;《Journal of Applied Polymer Science》;20130507;第130卷(第4期);摘要、第2247页左栏第2-4段 * |
| Yang Hu et al..Evaluation of 1-ethyl-3-methylimidazolium acetate based ionic liquid systems as a suitable solvent for collagen.《Journal of Applied Polymer Science》.2013,第130卷(第4期),摘要、第2247页左栏第2-4段. * |
| 京尼平交联脱细胞猪真皮基质材料的研究;但年华等;《功能材料》;20121231;第43卷(第21期);第2966-2969页 * |
| 但年华等.京尼平交联脱细胞猪真皮基质材料的研究.《功能材料》.2012,第43卷(第21期),第2966-2969页. * |
| 异体脱细胞真皮基质在硬脑膜修复中的应用;李德生等;《国外医学生物医学工程分册》;20051031;第28卷(第5期);第295页右栏第1段、第296页左栏第1-6段、右栏第2-3段 * |
| 李德生等.异体脱细胞真皮基质在硬脑膜修复中的应用.《国外医学生物医学工程分册》.2005,第28卷(第5期),第295页右栏第1段、第296页左栏第1-6段、右栏第2-3段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103263694A (en) | 2013-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103263694B (en) | Collagen-based dura and preparation method thereof | |
| Zhang et al. | Layered nanofiber sponge with an improved capacity for promoting blood coagulation and wound healing | |
| EP3015120B1 (en) | Tissue repair scaffold and preparation method and purpose thereof | |
| CN105194737B (en) | A kind of tissue recovery support and its preparation method and application | |
| Salehi et al. | Fabrication and characterization of electrospun PLLA/collagen nanofibrous scaffold coated with chitosan to sustain release of aloe vera gel for skin tissue engineering | |
| CN108478866B (en) | Tissue repair scaffold, preparation method and application thereof | |
| KR20070118730A (en) | Wound dressing materials with excellent ability to moisturized skin and method of manufacturing the same | |
| CN105031740A (en) | Waterproof and breathable bionic artificial skin and preparation method thereof | |
| CN103083720B (en) | Silk fibroin tube and preparation method thereof | |
| CN104414773A (en) | Anti-adhesion tissue repair membrane and preparation method thereof | |
| CN101716375A (en) | Artificial skin prepared from purely natural materials and having gradient hole structure and property | |
| CN103768650A (en) | Wound regenerating and repairing bandage and manufacture method thereof | |
| CN104013995A (en) | Oxidation chitosan graft modified porcine dermal collagen micro-nano fiber membrane and preparation method thereof | |
| CN103480042B (en) | Artificial dura mater spinalis, and preparation method and use method thereof | |
| CN106902389A (en) | Modified xenogenesis acellular nerve graft thing of a kind of nanofiber surface and preparation method thereof | |
| CN104027846A (en) | Non-woven material reinforced tissue engineering composite three-dimensional scaffold and preparation method thereof | |
| CN104383585B (en) | A kind of three-dimensional manometer composite adhesive bandage and preparation and application thereof | |
| CN110538006A (en) | Manufacturing method of fiber-reinforced three-dimensional printing cartilage acellular matrix scaffold | |
| CN104721873B (en) | The preparation of lamellar cross-linking hyaluronic acid sodium hydrogel | |
| El-Ghazali et al. | An overview of medical textile materials | |
| CN105457095A (en) | Keratin/silk fibroin composite dense membrane and preparing method thereof | |
| Kumar et al. | A review of wound dressing materials and its fabrication methods: emphasis on three-dimensional printed dressings | |
| CN101264337A (en) | Preparation of collagen base biological medical material | |
| Wang et al. | A sandwich-like silk fibroin/polysaccharide composite dressing with continual biofluid draining for wound exudate management | |
| CN113877001A (en) | Silk fibroin composite gel for injection |
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 |