WO2010102533A1 - 人工硬脑膜及其制备方法 - Google Patents

人工硬脑膜及其制备方法 Download PDF

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Publication number
WO2010102533A1
WO2010102533A1 PCT/CN2010/070566 CN2010070566W WO2010102533A1 WO 2010102533 A1 WO2010102533 A1 WO 2010102533A1 CN 2010070566 W CN2010070566 W CN 2010070566W WO 2010102533 A1 WO2010102533 A1 WO 2010102533A1
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WO
WIPO (PCT)
Prior art keywords
layer
hydrophobic
hydrophilic
electrospun layer
dura mater
Prior art date
Application number
PCT/CN2010/070566
Other languages
English (en)
French (fr)
Inventor
徐弢
袁玉宇
Original Assignee
广州迈普再生医学科技有限公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from CN2009100377365A external-priority patent/CN101507661B/zh
Priority claimed from CN200910039687.9A external-priority patent/CN101559242B/zh
Priority to KR1020147018543A priority Critical patent/KR20140090704A/ko
Priority to MX2011009282A priority patent/MX2011009282A/es
Priority to BRPI1006250 priority patent/BRPI1006250B1/pt
Priority to EP10750329.4A priority patent/EP2340785B1/en
Application filed by 广州迈普再生医学科技有限公司 filed Critical 广州迈普再生医学科技有限公司
Priority to RU2011140225/15A priority patent/RU2491961C2/ru
Priority to MX2015008387A priority patent/MX345863B/es
Priority to US13/255,356 priority patent/US8795708B2/en
Priority to MX2015008386A priority patent/MX345864B/es
Priority to JP2011553264A priority patent/JP5658175B2/ja
Publication of WO2010102533A1 publication Critical patent/WO2010102533A1/zh
Priority to US14/225,051 priority patent/US9211180B2/en
Priority to US14/225,136 priority patent/US9271822B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B5/00—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts
    • B32B5/22—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed
    • B32B5/24—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed one layer being a fibrous or filamentary layer
    • B32B5/26—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed one layer being a fibrous or filamentary layer another layer next to it also being fibrous or filamentary
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    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
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    • D01D5/0007—Electro-spinning
    • D01D5/0015—Electro-spinning characterised by the initial state of the material
    • D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
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Definitions

  • the invention relates to an artificial biofilm and a preparation thereof, in particular to an artificial dura mater and a preparation method thereof. Background technique
  • Dural defects are common in neurosurgical clinical work, and open brain injury (industry, transportation, war, etc.), tumor erosion, congenital meningeal defects, and other causes of craniocerebral diseases can cause dural defects.
  • Dural defects need to be repaired in time to prevent cerebrospinal fluid from spilling out, preventing brain bulging and atmospheric pressure, otherwise it will endanger human life.
  • the materials for repairing the dura mater can be divided into four types: autologous fascia, allogeneic materials, natural and synthetic materials, and dissimilar materials.
  • autologous fascia the material for repairing the dura mater
  • allogeneic materials the materials for repairing the dura mater
  • dissimilar materials when these materials are used in clinical practice, there are inevitably defects such as high clinical infection rate.
  • the infection rate of craniotomy is 4%
  • the dura mater prepared from pig small intestine mucosa has an infection rate of 3.4%
  • the meninges prepared with collagen have an infection rate of 3.8%.
  • Due to the presence of the blood-brain barrier intracranial infections are difficult to achieve at the required levels, causing obstacles to infection control.
  • the current artificial meninges generally do not mix drugs in the meninges, and thus have no control effect on postoperative infection.
  • meningeal graft repair surgery due to erosion of brain tumors is one of the common causes of meningeal repair. More than half of brain tumors cannot be completely removed, so chemotherapy is usually required after surgery. Many chemical drugs are highly toxic and cannot penetrate the blood-brain barrier, so they cannot achieve effective concentrations and affect the therapeutic effect.
  • the existing artificial dura mater has a high infection rate, poor biocompatibility, and cannot be completely
  • the human body absorbs and is difficult to add therapeutic drugs, and controls the defects of effective drug release.
  • the present invention provides an artificial dura mater which has the characteristics of good biocompatibility, prevention of adhesion, complete absorption, good mechanical and mechanical properties, low infection rate, and incorporation into other therapeutic substances.
  • the present invention also provides a method for preparing an artificial dura mater.
  • the artificial dura mater provided by the present invention is made of an electrospun layer produced by a method of electrospinning, and the electrospun layer is composed of at least one layer of a hydrophobic electrospun layer.
  • the hydrophobic electrospun layer may be prepared by electrospinning from one or more hydrophobic polymers selected from the group consisting of hydrophobic aliphatic polyesters, polyether esters. , polyorthoesters, polyurethanes, polyanhydrides, polyacrylonitriles, polyamino acids, or copolymers and mixtures of two or more of the above polymers.
  • the hydrophobic aliphatic polyester is selected from at least one of the group consisting of polylactic acid, polyglycolide, polycaprolactone, and polyhydroxybutyrate.
  • the polyether ester is selected from at least one of the group consisting of polydioxanone, ethylene glycol/lactic acid copolymer, and ethylene glycol/butylene terephthalate copolymer.
  • the polyanhydride is selected from at least one of the group consisting of polysebacic acid-hexadecanedioic acid anhydride, type I polyanhydride, type II polyanhydride, type III polyanhydride, and type IV polyanhydride.
  • the artificial dura mater made of hydrophobic electrospun layer is similar in strength to human dura mater. It can seal the brain, prevent cerebrospinal fluid leakage and protect the brain before the body's own dura mater regeneration. It is composed of hydrophobic material.
  • the electrospun layer is not conducive to the migration of cells, so that the purpose of anti-blocking can be achieved. In practice, multiple layers of hydrophobic electrospun layers can be provided as needed to meet different strength requirements.
  • At least one hydrophilic electrospun layer may be further disposed on the hydrophobic electrospun layer.
  • the hydrophilic electrospun layer is prepared by electrospinning from one or more hydrophilic polymeric materials selected from the group consisting of chondroitin sulfate, heparin, Agar, dextran, alginic acid, modified cellulose, alginic acid, starch, cellulose, gelatin, fibrin, silk protein, elastin mimetic peptide polymer, collagen, chitosan, modified chitosan , hydrophilic polyurethane, polyethylene glycol, decyl methacrylate, decyl methacrylate, polyhydroxybutyrate valerate, polyhydroxybutyrate hexanoate, polyvinyl alcohol or polylactide, or Two or more mixtures of the above materials.
  • the hydrophobic electrospun layer When the dura mater is implanted in the brain, the hydrophobic electrospun layer is placed adjacent to the surface of the brain to prevent adhesion; and the hydrophilic electrospun layer is placed away from the side of the brain for cell adhesion.
  • migration, proliferation, differentiation provide a good nano-cell scaffold, because this layer is made of a biocompatible hydrophilic material, which can effectively induce the migration of stem cells and fibroblasts, thereby promoting self-hardening. The growth of the meninges.
  • the hydrophilic electrospun layer can also be multi-layered as needed to meet different needs.
  • the artificial dura mater provided by the present invention may further be provided with a transition layer between the hydrophobic electrospun layer and the hydrophilic electrospun layer.
  • the transition layer is prepared by electrospinning from one or more polymeric materials, and the affinity of the transition layer for water is gradually increased from the hydrophobic electrospun layer to the hydrophilic electrospun layer.
  • the transition layer is arranged to enhance the affinity between the hydrophobic electrospun layer and the hydrophilic electrospun layer.
  • any one or more of the hydrophobic electrospun layer, the hydrophilic electrospun layer, and the transition layer may be blended with cytokines and/or drugs.
  • the blended electrospun layer is implanted in the brain tissue, with the absorption of the electrospun polymer, the blended cytokines and/or drugs are released to the local area to play a corresponding role to prevent local infection and anti-adhesion. , prevent brain tumor recurrence or promote autologous dura repair.
  • cytokines and/or drugs may be attached to the hydrophobic electrospun layer and/or the hydrophilic electrospun layer.
  • the cytokine of the present invention is a factor that acts on adhesion, migration, proliferation, and differentiation of fibroblasts, and is selected from the group consisting of: interleukin, colony stimulating factor, tumor necrosis factor, platelet-derived growth factor, Basic fibroblast factor, or a mixture of two or more of the above factors.
  • the role of cytokines can accelerate the process of the body's own dura mater.
  • the medicament of the present invention is selected from one or more of an antibiotic, a hemostatic agent, an anti-adhesion agent or an anti-tumor drug.
  • antibiotics are placed on the artificial dura mater provided by the present invention according to actual needs. After the implantation, the drug is gradually released to the local part with the absorption of the polymeric material and the process of self-dabilization of the body itself, thereby exerting therapeutic and preventive effects. Thereby overcoming the blocking effect of the blood-brain barrier on drugs.
  • cytokines and/or drugs can be coated in the hydrosol. Through the adhesion and fixation of the hydrosol, uniform or fixed-point release of the drug is achieved according to the local conditions of the treatment.
  • the hydrosol may be selected from one of a polysaccharide polymer, a polypeptide polymer, and a synthetic hydrophilic polymer. Several.
  • the hydrophobic electrospun layer of the artificial dura mater has a fiber diameter of 50 to 1000 nM. According to one embodiment of the present invention, the hydrophobic electrospun layer has a pore diameter of less than 3 ⁇ .
  • the hydrophilic electrospun layer has a fiber diameter of 5 to 200 ⁇ M and a pore diameter of 20 to 200 ⁇ M.
  • the pore size distribution obtained by electrospun fibers is largely dependent on the fiber diameter. As the fiber diameter decreases, the pore size also decreases at the same time. Therefore, by controlling the fiber diameter, the control of the electrospun layer pore size can be achieved.
  • the average diameter of human cells is 10 ⁇ 20 ⁇ .
  • the meninges mainly distribute fibroblasts and collagen fibers secreted by them. Generally, the diameter of fibroblasts is between 20 ⁇ 30 ⁇ .
  • the hydrophobic electrospun layer has a pore size of 3 ⁇ or less, which prevents cells from entering and further prevents brain adhesion.
  • the pore size of the hydrophilic electrospun layer is set to be equal to or greater than the average diameter of the cells in order to further promote cell migration.
  • the present invention also provides a method for preparing an artificial dura mater comprising the steps of: a) dissolving a hydrophobic polymer in a solvent to obtain a hydrophobic polymer electrospinning solution, said hydrophobic
  • the polymer is selected from the group consisting of hydrophobic aliphatic polyesters, polyether esters, polyorthoesters, polyurethanes, polyanhydrides, polyacrylonitriles, polyamino acids, or two or more of the above polymers. More than one type of copolymer and mixture; b)
  • the hydrophobic polymer electrospinning solution is electrospun into a film-like hydrophobic electrospun layer to obtain the artificial dura mater.
  • the hydrophobic aliphatic polyester is selected from at least one of the group consisting of polylactic acid, polyglycolide, polycaprolactone, and polyhydroxybutyrate.
  • the polyether ester is selected from at least one of the group consisting of polydioxanone, ethylene glycol/lactic acid copolymer, and ethylene glycol/butylene terephthalate copolymer.
  • the polyanhydride is selected from at least one of the group consisting of polysebacic acid-hexadecanedioic acid anhydride, type I polyanhydride, type II polyanhydride, type III polyanhydride, and type IV polyanhydride.
  • the method provided by the invention utilizes the principle of electrospinning to receive a specific type of high molecular polymer to form an artificial dura mater, which can effectively prevent the occurrence of brain adhesion after implantation.
  • the fiber diameter of the hydrophobic electrospun layer in the present invention can be controlled to be 50 to 1000 nM. According to one of the embodiments of the present invention, the hydrophobic electrospun layer has a pore diameter of less than 3 ⁇ .
  • the electrospinning process parameters in step b can be set as follows: the rate of the micro syringe pump is 0.1 to 5.0 ml/hr, the voltage of the high voltage generator is 5 to 40 KV, and the receiving distance is 5.0 30.0 cm.
  • the method provided by the present invention may further comprise the step of forming a hydrophilic electrospun layer by a similar electrospinning method on the hydrophobic electrospun layer: a')
  • the aqueous polymer is dissolved in a solvent to obtain a hydrophilic polymer electrospinning solution, the hydrophilic polymer being selected from the group consisting of chondroitin sulfate, heparin, agar, dextran, alginic acid, modified Cellulose, alginic acid, starch, cellulose, gelatin, fibrin, silk protein, elastin mimetic peptide polymer, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol , polydecyl methacrylate, polydecyl methacrylate, polyhydroxybutyrate valerate, polyhydroxybutyrate hexanoate, polyvinyl alcohol or polylactide; b'
  • the hydrophilic electrospun layer is placed on the side away from the surface of the brain in order to promote the migration of cells and form a self-forming neonatal dura mater.
  • the hydrophilic electrospun layer may have a fiber diameter of 5 to 200 ⁇ M and a pore diameter of 20 to 200 ⁇ M.
  • the electrospinning process parameters in step b' are: the rate of the microinjection pump is 0.1 ⁇ 20.0 ml/hr, and the voltage of the high voltage generator is 10 ⁇ 45 KV, and the receiving distance is 5.0 ⁇ 30.0 cm.
  • an electrospinning solution which is usually a volatile organic solvent, including but not limited to tannic acid, acetic acid, ethanol, Acetone, dimercaptoamide, dimercaptoacetamide, tetrahydrofuran, dimercaptosulfoxide, hexafluoroisopropanol, trifluoroethanol, dichlorodecane, trichlorodecane, decyl alcohol, ethanol, chloroform, two A mixture of two or more of oxane, trifluoroethane, trifluoroacetic acid, or a polymer solvent of the above.
  • a volatile organic solvent including but not limited to tannic acid, acetic acid, ethanol, Acetone, dimercaptoamide, dimercaptoacetamide, tetrahydrofuran, dimercaptosulfoxide, hexafluoroisopropanol, trifluoroethanol, dichlorodecane, trichlorodecane, de
  • the volatile organic solvent can be rapidly volatilized during the formation of the electrospun layer so that the final electrospun layer does not contain residual organic solvents.
  • the solvent may also be selected from water and removed by drying or drying in the form of an electrospun layer.
  • the present invention provides a method for preparing an artificial dura mater, which further comprises forming a transition layer by electrospinning between the hydrophobic electrospun layer and the hydrophilic electrospun layer before forming the hydrophilic electrospun layer.
  • the affinity of the transition layer for water is gradually increased from the hydrophilic electrospun layer to the hydrophobic electrospun layer.
  • the selection of the polymer material, the solvent and the electrospinning parameters of the electrospinning transition layer can be selected according to the actual conditions according to the actual situation, and the formation of the transition layer can increase the affinity between the hydrophilic layer and the hydrophobic layer.
  • cytokines and/or drugs may be mixed in the corresponding polymer electrospinning solution.
  • the electrospun layer formed becomes a blended electrospun layer of high molecular polymer and cytokine and/or drug composition, which can better meet the clinical needs and improve the therapeutic effect.
  • the method of preparing an artificial dura mater provided by the present invention may further comprise the step of forming a cytokine and/or drug distribution by cell printing on the hydrophobic electrospun layer and/or the hydrophilic electrospun layer.
  • Cell printing is the use of a hydrophobic electrospun layer and/or a hydrophilic electrospun layer as a scaffold ("bio-paper") on which cytokines and/or drugs are printed.
  • the cytokines and/or drugs may be coated in the hydrosol.
  • the cell printing of the cytokine and/or drug used in the present invention may take the following steps: a") mixing the hydrosol solution with the cytokine and/or drug to form a mixed solution; and b") utilizing the cell Printing techniques print the mixed solution onto the hydrophobic electrospun layer and/or the hydrophilic electrospun layer.
  • the hydrosol solution in the present invention may be an aqueous solution made of a polysaccharide polymer, a polypeptide polymer or a synthetic hydrophilic polymer.
  • the polysaccharide polymer includes, but is not limited to, starch, cellulose, alginic acid, hyaluronic acid or chitosan;
  • the polypeptide polymer includes, but not limited to, collagen, poly-L-lysine or poly-L ⁇ glutamic acid;
  • the synthetic hydrophilic high molecular polymer includes, but is not limited to, polyacrylic acid, polyacrylic acid acrylate, polyacrylamide or poly-N-polyacrylamide.
  • the hydrosol is usually in a liquid state under normal conditions, and at a suitable temperature or under certain conditions, it will become jelly in a short time, and thus has good adhesion.
  • some hydrosols require a crosslinking agent to participate in the reaction, and thus may further include pretreating the hydrophobic electrospun layer and/or the hydrophilic electrospun layer with a crosslinking agent solution before the printing step.
  • a step of. Pretreatment with a crosslinker solution causes the crosslinker to adhere to the electrospun layer; the hydrosol solution is mixed with appropriate cytokines and/or drugs and placed in a printhead.
  • the hydrosol solution in which the cytokine and/or drug is mixed in the head contacts the electrospun layer, it solidifies and adheres to the electrospun layer.
  • the choice of crosslinking agent is related to the type of hydrosol. For example, when the hydrosol is alginic acid, the crosslinking agent is calcium chloride; when the hydrosol is In the case of fibrin, the cross-linking agent is thrombin.
  • the present invention has the following beneficial effects:
  • the material used in the diaphragm is non-toxic and harmless to the human body, has good bio-tissue compatibility, can be completely absorbed after implantation, and avoids carcinogenesis of the membrane;
  • the membrane itself is not formed by biological components, which eliminates many risks such as immune rejection, viral transmission, and disease transmission;
  • the double-layer design promotes the growth of autologous cells while preventing adhesion, which can promote the early repair of autologous bodies;
  • the preparation method has simple process steps, low cost, and is easy to be industrialized.
  • the clinical application is simple and can be customized according to the patient's condition.
  • FIG. 1 is a schematic view showing an electrospinning process of an artificial dura mater according to the present invention
  • FIG. 2 is a schematic diagram showing the manufacturing process of the electrospinning bioprinting technology of the artificial dura mater of the present invention
  • Figure 4 is a schematic view of an artificial dura mater composed of a hydrophobic electrospun layer and a hydrophilic electrospun layer according to the present invention
  • Figure 5 is a schematic illustration of an artificial dura mater having a transition layer between the hydrophobic and hydrophilic electrospun layers in accordance with the present invention
  • Figure 6a is a schematic illustration of a blended artificial dura mater having a hydrophobic electrospun layer and a hydrophilic electrospun layer in accordance with the present invention
  • Figure 6b is an enlarged schematic view of the partial I region shown in Figure 6a;
  • Figure 7a is a schematic view of an artificial dura mater having a hydrophobic electrospun layer, a hydrophilic electrospun layer and a transition layer, which are blended according to the present invention
  • Figure 7b is an enlarged schematic view of a portion II region shown in Figure 7a;
  • Figure 8a is a schematic illustration of an artificial dura mater obtained in conjunction with a bioprinting technique in accordance with the present invention
  • Figure 8b is an enlarged schematic view of the partial ankle region shown in Figure 8a.
  • FIG. 1 it is a schematic diagram of an electrospinning process of the artificial dura mater of the present invention.
  • the electrospinning nozzle 1 is filled with a polymer solution, and the high voltage end of the high voltage power source 3 is connected to the electrospinning head 1.
  • the receiving device 4 has a cylindrical shape and can be moved left and right around the axis of the cylinder and the long axis of the cylinder according to actual operation.
  • the movement setting of the receiving device 4 can be realized by setting a corresponding program by the computer, so that the thickness of the electrospun layer formed is hooked.
  • the receiving device can also be arranged as a plane with a flat surface, and can be uniformly received by the left and right movements of the plane.
  • the receiving device 4 is in communication with the low voltage end of the high voltage power source 3 to form a higher voltage difference between the electrospinning head 1 and the receiving device 4.
  • a suitable electrospinning solution is first prepared.
  • Hydrophobic polymer electrospinning solutions can be used which are formulated by dissolving the hydrophobic polymer in a suitable solvent.
  • hydrophobic polymers include, but are not limited to, hydrophobic aliphatic polyesters (such as polylactic acid, polyglycolide, polycaprolactone, polyhydroxybutyrate), polyether esters (such as polydioxole) Ketones, polyorthoesters, polyurethanes, polyanhydrides (such as polysebacic acid-hexadecandioic acid anhydride), polyacetonitrile, polyamino acids, or copolymers and mixtures of two or more of the above polymers .
  • hydrophilic electrospun layer is further formed on the hydrophobic electrospun layer, it is necessary to simultaneously prepare a hydrophilic polymer electrospinning solution, which is a hydrophilic polymer. Formulated in a suitable solvent.
  • the hydrophilic polymer includes but is not limited to: chondroitin sulfate, heparin, agar, dextran, alginic acid, modified cellulose, alginic acid, starch, cellulose, gelatin, fibrin, silk protein, elastin Mimetic peptide polymer, collagen, chitosan, modified chitosan, hydrophilic polyurethane, polyethylene glycol, polydecyl methacrylate, polydecyl methacrylate, polyhydroxybutyrate valerate , polyhydroxybutyrate caproate, polyvinyl alcohol or polylactide.
  • a plurality of electrospinning nozzles 1 may be disposed as needed, and a hydrophobic or hydrophilic polymer electrospinning solution may be placed therein; or the electrospinning solution in the electrospinning nozzle 1 may be replaced after the formation of the hydrophobic electrospun layer. .
  • the solvent of the electrospinning solution may be water.
  • Volatile organic solvents may also be selected, including but not limited to citric acid, acetic acid, ethanol, acetone, dimercapto amide, dimercaptoacetamide, tetrahydrofuran, dimercaptosulfoxide, hexafluoroisopropanol, trifluoroethanol , dichlorodecane, trichlorodecane, decyl alcohol, ethanol, chloroform, dioxane, trifluoroethane, trifluoroacetic acid.
  • the electrospinning solution After the electrospinning solution is in place, set the parameters, turn on the power, and turn on the electrospinning device. As the electrospinning 2 is gradually pulled out of the electrospinning head 1, the receiving device 4 moves in accordance with the established procedure to ensure formation. Uniform electrospun membrane structure.
  • the process parameters for the formation of the hydrophobic electrospun layer are generally set as follows:
  • the rate of the microinjection pump is 0.1 to 5.0 ml/hr, the voltage of the high voltage generator is 5 to 40 KV, and the receiving distance is 5.0 to 30.0 cm.
  • the fiber diameter of the hydrophobic electrospun layer can be controlled to 50 to 1000 nM and the pore diameter is less than 3 ⁇ .
  • the process parameters for the formation of the hydrophilic electrospun layer are: the rate of the microinjection pump is 0.1 20.0 ml / hour, the voltage of the high voltage generator is 10 to 45 KV, and the receiving distance is 5.0 to 30.0 cm.
  • the hydrophilic electrospun layer has a fiber diameter of 5 to 200 ⁇ and a pore diameter of 20 to 200 ⁇ .
  • the above electrospinning step may be repeated a plurality of times according to actual needs to form a multilayer hydrophobic electrospun layer and/or a hydrophilic electrospun layer, as shown in Figs. 3 and 4.
  • Figure 3 is a three-layer hydrophobic electrospun layer of artificial dura mater, the strength is similar to the human dura mater, because the layer is composed of hydrophobic materials, which is not conducive to cell migration, and the materials used are safe and non-toxic, It can be absorbed by the body, so that it can achieve the purpose of anti-adhesion.
  • Fig. 4 is an artificial dura mater composed of a two-layer structure of a hydrophobic electrospun layer and a three-layer structure of a hydrophilic electrospun layer B.
  • the anti-adhesive hydrophobic electrospun layer A is placed adjacent to the surface of the brain, and the hydrophilic electrospun layer B is placed away from the side of the brain for cell adhesion, migration, Proliferation and differentiation provide a good nano-cell scaffold. Because this layer is made of a biocompatible hydrophilic material, and its pore size is large, stem cells and fibroblasts can be moved in, which promotes autologous Dural growth.
  • an appropriate drying or drying step can be selected depending on the composition of the electrospinning solution.
  • the solvent of the electrospinning solution is selected as a certain volatile organic solvent, such as hexafluoroisopropanol, the solvent is completely volatilized during the process in which the electrospinning 2 falls onto the receiving device 4 with a voltage difference.
  • the steps of drying or drying can be omitted.
  • a transition layer is formed between the two parts.
  • the affinity of the transition layer for water is gradually increased from the hydrophobic electrospun layer to the hydrophilic electrospun layer.
  • one or more suitable high molecular polymers can be selected according to the affinity for water.
  • Electrospinning process parameters of the transition layer The rate of the microinjection pump is 0.1 5.0 ml/hr, and the voltage of the high voltage generator is 5 ⁇ 40 KV. ⁇ Giant separation is 5.0 ⁇ 30.0 cm.
  • the artificial dura mater with a transition layer formed is shown in FIG.
  • the hydrophobic electrospun layer A is a two-layer structure
  • the hydrophilic electrospun layer B is a three-layer structure.
  • Layer C wherein one layer adjacent to the hydrophobic electrospun layer A is less hydrophilic than the layer adjacent to the hydrophilic electrospun layer B.
  • any one or more of the hydrophobic electrospun layer, the hydrophilic electrospun layer, and the transition layer may be blended with cytokines and/or drugs.
  • Cytokines and/or drugs may be added to the corresponding electrospinning solution as needed, and then electrospun according to the above method, and cytokines and/or drugs are mixed into the electrospinning 2 as the electrospinning 2 is formed. And a uniform film structure is formed on the receiving device 4.
  • the process can be repeated multiple times, and the cytokines and/or drugs mixed in each time can be the same or different.
  • the resulting artificial dura mater is shown in Figure 6.
  • the hydrophobic electrospun layer A has a 2-layer structure
  • the hydrophilic electrospun layer B has a 3-layer structure.
  • the hydrophobic electrospinning 7 is blended with a drug 8, a hydrophilic electrospinning 9
  • the medium blend has a cytokine 10.
  • Fig. 7a further provides a two-layer transition layer C structure on the structure of Fig. 6, and the layer adjacent to the hydrophobic electrospun layer A has a lower affinity for water than the layer adjacent to the hydrophilic electrospun layer B.
  • the hydrophobic electrospinning 7 is blended with the drug 8
  • the hydrophilic electrospinning 9 is blended with the cytokine 10
  • the two layers of the transition layer electrospinning 11 are respectively blended with the drug 8 and the cytokine. 9.
  • the present invention also provides a process for producing an artificial dura mater by electrospinning combined with bio-printing technology.
  • Bio-printing technology is a new technology that has emerged in recent years, using a special cell solution or a biologically active cytokine solution as a "bio-ink" to accurately print on a bio-degradable bio-paper in accordance with a predetermined schedule. After printing, stack the sheets in a certain order. Due to the use of printing technology, bio-ink composed of cells or/and cytokines can be precisely bonded to a predetermined site; and bio-sheets in a specific stacking manner form a three-dimensional structure.
  • a bioprinting head 5 is further provided on the basis of the apparatus shown in FIG.
  • the bio-printing head 5 can be modified using an existing ink jet printer, for example, the method disclosed in U.S. Patent No. 7,051,654.
  • the spray head contains cytokines and/or drugs, and the print mode and print position of the print head can be preset by a computer program.
  • the specific biological printing step can be carried out according to the prior art.
  • the cytokine and/or the drug may be coated in the hydrosol, and the hydrosol solution may be made of a polysaccharide polymer, a polypeptide polymer or a synthetic hydrophilic polymer. An aqueous solution.
  • the polysaccharide polymer includes, but is not limited to, starch, cellulose, alginic acid, hyaluronic acid or chitosan;
  • the polypeptide polymer includes, but not limited to, collagen, poly-L-lysine or poly-L ⁇ Glutamic acid;
  • the synthetic hydrophilic high molecular polymer includes, but is not limited to, polyacrylic acid, polyacrylic acid, polyacrylamide or poly-N-polyacrylamide.
  • the hydrosol is usually in a liquid state under normal conditions, and at a suitable temperature or under certain conditions, it will become jelly in a short time, and thus has good adhesion, thereby making the cytokines and/or drugs uniform or well-defined.
  • the ground is distributed on the electrospun layer.
  • the step of bioprinting with hydrosol is specifically as follows: a cytokine and/or a drug formulated with a corresponding liquid hydrosol is placed in the bioprinting head 5. After the electrospun layer is formed, the ink jet printer prints on the electrospun layer according to a preset procedure, and according to the selection of the hydrosol, the appropriate conditions are given, and the hydrosol rapidly becomes a jelly with good adhesion, The encapsulated cytokines and/or drugs adhere to the electrospun layer. When printing, it can be set by uniform printing method to form artificial dura mater. As shown in Figure 8a, a layer of drug can be printed on each layer of hydrophobic electrospun layer structure, in each layer of hydrophilic electrospinning.
  • a layer of cytokines can be printed on the layer structure.
  • the bioprinted film is different from the blend, and the cytokine and/or drug is applied to the electrospun layer composed of the hydrophobic electrospun 7 and/or the hydrophilic electrospun 9. Surface.
  • the cytokine and/or drug can be uniformly released.
  • local concentrated printing can be used to implant the human body, so that the cytokines and/or drugs are concentrated in the area of interest.
  • the curing of some hydrosols requires a crosslinking agent to participate in the reaction.
  • the step of bio-printing with hydrosol is as follows: A suitable crosslinking agent is preliminarily placed in the container 6, and after the electrospinning is started, the receiving device 4 is along the edge. During the rotation of the axis or the movement of the left and right, the crosslinking agent in the container 6 is brought into contact, so that the formed electrospun layer is adhered with the crosslinking agent molecule. Then, the biological printing is carried out according to the above method.
  • the liquid hydrosol in the biological printing head 5 When the liquid hydrosol in the biological printing head 5 is in contact with the crosslinking agent on the electrospun layer, it rapidly becomes a gel, and the cytokines and/or drugs encapsulated therein are adhered thereto. On the electrospinning layer.
  • the choice of crosslinking agent is related to the type of hydrosol. For example, when the hydrosol is alginic acid, the crosslinking agent is calcium chloride; when the hydrosol is fibrin, the crosslinking agent is thrombin.
  • the hydrophobic polymer electrospinning solution may be selected to be hydrophobic.
  • L-polylactic acid and ⁇ -caprolactone the weight ratio of the two is 50: 50 or 30: 70 or 70: 30, as a copolymerized polymer material, the number average molecular weight is 150,000 500,000, dissolved in hexafluoroisopropanol or two Chlorodecane.
  • a hydrophobic polymer electrospinning solution may be added: 0.01 to 3% antibiotic solution, or / and 0.001 to 3% hemostasis and anti-adhesion drug, mixed with L-polylactic acid and ⁇ -caprolactone solution, A homogeneous solution is obtained.
  • the hydrophilic polymer electrospinning solution may be selected from hydrophilic polyurethane and natural gelatin or chondroitin sulfate or polyethylene glycol, and the weight ratio is 20-80: 80-20, and the spinning solution is 3 to 15% of the total weight of the solution.
  • a basic fibroblast factor solution may be added to the hydrophilic polymer electrospinning solution to achieve a final concentration of cytokine of 0.001 to 0.5%.
  • the drugs added during the blending or bio-printing process can be selected according to the specific conditions of antibiotics or hemostasis or anti-adhesion drugs.
  • chemotherapy drugs for brain tumors can be used.
  • Antibiotics can be, but are not limited to, cephalosporins, benzathine, spiramycin, sulfonamides, quinolone antibiotics, and the like.
  • ceftriaxone sodium Because meningeal surgery usually requires craniotomy, most of the current intracranial infections after craniotomy are bacterial.
  • the main pathogens include: Staphylococcus aureus, Streptococcus, Pneumococcus, Escherichia coli, Salmonella and green. Pseudomonas and the like. Among them, Staphylococcus aureus is the most common. According to clinical reports, it is considered that the clinical treatment effect of ceftriaxone sodium is better.
  • Antineoplastic agents including but not limited to nimustine, semustine, liposomal doxorubicin, actinomycin. , vincristine, etc. The preferred choice is vincristine.
  • Hemostasis or anti-adhesion drugs can accelerate wound healing and prevent adhesions, including but not limited to hemostatic factors (which can also have hemostasis in the material), and inhibitors of collagen synthase (such as Trinister and Pisto, can inhibit Collagen synthase histamine and prostaglandin release, inhibit collagen regeneration), anticoagulants (such as dicoumarin, heparin, hirudin, etc.), anti-inflammatory drugs (such as promethazine, dexamethasone, hydrogenated Pine, prednisolone, ibuprofen, hydroxybutazone, etc.), calcium channel blockers (such as diltiazem hydrochloride, nifedipine, verapamil hydrochloride, etc.), cell growth inhibitors (such as fluorouracil), hydrolysis Enzymes (such as hyaluronidase, streptokinase, urokinase, pepsin, tPA), redox agents
  • the drug or factor in the blending process, is added to the electrospinning solution: 0.001 0.05% of the basic fibroblast growth factor, electrospinning solution containing the weight of the electrospinning solution 3% by weight of benzylamazine, a hemostatic factor of 0.001 to 0.05% by weight of the electrospinning solution, may be included in 0.01 to 5% of rimustine in meningeal repair surgery for brain tumors.
  • the prepared artificial dura mater is cleaned, sterilized, and stored after packaging.
  • L ⁇ polylactic acid and ⁇ -caprolactone were selected, the weight ratio of the two was 50:50, and the number average molecular weight was 260000.
  • the mixture was dissolved in hexafluoroisopropanol to form a hydrophobic electrospinning solution, which was placed in an electrospinning nozzle.
  • the output rate of the hydrophobic electrospinning solution was controlled by a micro-injection pump to be 5 ml/hr, the voltage of the high-voltage generator was adjusted to 30 kV, the receiving distance of the receiving device was adjusted to 20 cm, and the fiber was received into a film-like structure to form a fiber.
  • a hydrophobic electrospun layer having an average diameter of 300 nm. After receiving, turn off the electrospinning unit.
  • the prepared artificial meninges were rinsed 5 times with ethanol and distilled water, vacuum-packed, sterilized by 25 kGy cobalt-60, and stored at a low temperature of 20 °C.
  • the hydrophobic electrospun layer was prepared in the same manner as in Example 1.
  • the hydrophilic electrospinning solution has a mass ratio of polyethylene glycol to chondroitin of 70:30 and a spinning solution mass fraction of 9%.
  • the electrospinning apparatus was turned on, and the hydrophilic electrospun layer was continuously spun on the spun-stained electrospun layer of Example 1.
  • the receiving distance was 11 cm and the voltage was 20 KV, and the obtained hydrophilic electrospun layer had an average fiber diameter of 10 ⁇ m.
  • the cleaning and storage methods are the same as those in the first embodiment.
  • the preparation method of the hydrophobic electrospun layer is the same as the embodiment 1.
  • the polymer solution for the transition layer is: the mass ratio of polyurethane to hyaluronic acid is 70:30, and the mass fraction is 10%.
  • the electrospinning was turned on, and the receiving distance was 11 cm, the voltage was 20 KV, and the average fiber diameter was 5 ⁇ m, and the transition layer was continued on the spun hydrophobic electrospun layer.
  • the hydrophilic electrospun layer is then spun on the transition layer, and the embodiment of the hydrophilic electrospun layer is the same as in Example 4. Turn off electrospinning.
  • the cleaning and storage methods are the same as those in the first embodiment.
  • the above solution was added to an electrospinning head, the rate of the micro syringe pump was adjusted to 0.8 ml / hr, the voltage of the high voltage generator was adjusted to 12 KV, the receiving distance of the receiving device was adjusted to 15 cm, and the fiber was received as a film structure.
  • the hydrophobic electrospun layer was obtained to have a fiber diameter of 600 nm.
  • the hydrophilic electrospun layer continues to be spun on the spun layer.
  • the resulting hydrophilic electrospun layer has an average fiber diameter on the order of microns.
  • the cleaning and storage methods are the same as those in the first embodiment.
  • hydrophobic electrospun layer The hydrophobic polycaprolactone is selected, and the ratio of the mixed solvent of chloroform/nonanol is 1:1. Mix and vincristine at a concentration of 100 ng/ml. A homogeneous solution is obtained.
  • the above solution was added to an electrospinning head, the rate of the micro syringe pump was adjusted to 0.8 ml / hr, the voltage of the high voltage generator was adjusted to 12 KV, the receiving distance of the receiving device was adjusted to 15 cm, and the fiber was received as a film structure.
  • the hydrophobic electrospun layer was obtained to have a fiber diameter of 600 nm.
  • transition layer The mass ratio of polyurethane to hyaluronic acid is 70:30, and the mass fraction of spinning solution is 10%. Mixed ampicillin at a concentration of 3%. A homogeneous solution is obtained.
  • Electrospinning was initiated and the transition layer continued to be spun on the spun hydrophobic electrospun layer.
  • the receiving distance is from 11cm, the voltage is 20KV, and the average fiber diameter is 5 ⁇ ⁇ .
  • the electrospinning device was turned on, the receiving distance was adjusted to 10 cm, the voltage was 20 KV, and the hydrophilic electrospun layer was continuously spun on the spun transition layer.
  • the average fiber diameter is on the order of microns.
  • the cleaning and storage methods are the same as those in the first embodiment.
  • the hydrophobic electrospinning solution was selected from the hydrophobic L-polylactic acid and ⁇ -caprolactone, and the ratio of the two was 50:50.
  • the number average molecular weight was 260,000, which was dissolved in hexafluoroisopropanol.
  • the crosslinking agent solution was selected from a 0.1 M calcium chloride solution.
  • the cytokine-containing hydrosol solution was administered with a hemostatic factor alginate solution having a mass percentage concentration of hemostasis of 10 ppm in the cytokine alginate solution.
  • the prepared 0.1M calcium chloride solution is placed in a cell culture sub-diameter having a diameter of 150 mm, and the receiver common to the electrospinning device and the printer is placed in the culture sub-station, so that the receiving device can be cultured and jni when the electrospun layer is formed.
  • the containers in the container are in contact.
  • the cell printing nozzle is fixed directly under the electrospinning needle in the electrospinning device box, and is used as a hemostatic factor positioning printing; the prepared cytokine alginate solution is loaded into the inkjet printing cartridge; the ink cartridge model used in this embodiment For the HP51626A.
  • the hydrophobic electrospinning solution was added to the electrospinning nozzle, the rate of the micro-injection pump was adjusted to 5 ml/hr, the voltage of the high-voltage generator was adjusted to 30 KV, and the receiving distance of the receiving device was adjusted to 20 cm, and the fiber was received as Membrane structure. After electrospinning for 20 minutes, the electrospinning device was turned off.
  • the cytokine-containing hydrosol solution was printed on the nano-bionic scaffold by an inkjet printer, and the hydrosol was obtained after curing.
  • the mass ratio of polyethylene glycol to chondroitin sulfate is 70:30, the mass fraction of spinning solution is 9%, and the solution of crosslinker is 0.1M calcium chloride solution.
  • the mass percentage concentration of basic fibroblast factor in the cytokine alginate solution was 10,000 ppm.
  • the electrospinning parameters were adjusted to a microinjection pump at a rate of 0.8 ml/hr, the high voltage generator at a voltage of 20 KV, and the receiving device to a receiving distance of 11 cm.
  • the other preparation methods were the same as in the previous step.
  • the transition layer electrospinning solution was selected as follows: The mass ratio of polyurethane to hyaluronic acid was 70:30, and the spinning solution mass fraction was 10%. Mixed ampicillin at a concentration of 3%. A homogeneous solution is obtained. .
  • the crosslinking agent solution was selected from a 0.1 M calcium chloride solution.
  • the cytokine-containing hydrosol solution was administered with a hemostatic factor alginate solution having a mass percentage concentration of hemostasis of 10 ppm in the cytokine alginate solution.
  • the electrospinning device parameters were adjusted to a microinjection pump at a rate of 4 ml/hr, the high voltage generator at a voltage of 20 KV, and the receiving device to a receiving distance of 11 cm. Other preparation methods were the same as in the previous step.
  • the cytokine-containing hydrosol solution is printed on the transition layer by an ink jet printer, and the hydrosol is cured.
  • the preparation method was the same as in Example 6.
  • the cleaning and storage methods are the same as those in the first embodiment.
  • the canine animal experiment was carried out using the dura mater prepared in Example 1, and the control group used the animal-derived meningeal repair material which has been commercially used clinically.
  • the dogs were routinely fed and observed after surgery. At the end of each observation period, the specimens were collected at the repaired material site, and the gross specimens and the microscopic tissues were compared.
  • Each experimental animal was bred to an anesthetized animal after a set time, and the skull was exposed according to the above-mentioned craniotomy.
  • the outer surface of the repairing material was separated and exposed, and the animal was sacrificed by injecting air into the skull. After the skull was opened, the repairing material and surrounding tissues were cut open. The external surface, texture, relationship with the surrounding tissue, presence of edema, induration, and adhesion of the inner surface to the brain tissue were visually observed.
  • the specimen is bottled, soaked in formalin fixative, and the specimen bottle is labeled. After formalin fixation for 1 week at room temperature, the local tissue of the surgical site was taken, routinely paraffin-coated, and HE tissue sections were stained.
  • the canine animal experiment was carried out using the dura mater prepared in Example 2: the experimental dog weighed 15 to 20 KG, aged 1.5 to 2 years old, and male or female, 5 in total. After intramuscular injection of ketamine, after anesthesia shaving, the animals were placed on a dedicated operating table in the abdomen position. Disinfect with 2% iodine and 75% alcohol. The animal is in the middle of the head and cut longitudinally. The periosteum was separated with a stripper, the double-top skull plate was exposed, the skull was ground with a high-speed grinding drill, and a double-top portion formed a bone window. Use a small pair of scissors to cut off the 3 cm X 3 cm rectangular dura mater on both sides to create a dural defect at the top.
  • Electrocautery on the exposed brain surface resulting in six 1 mm x l mm lesions.
  • the artificial meninges prepared in Example 2 of the present invention were trimmed into repairing materials of corresponding shapes and sizes, and the hydrophobic electrospun layer was applied to the brain surface, and the 4/0 non-damaged silk thread was sutured intermittently, and the needle spacing was 4 mm, which was repaired on the top of the dog. Defect.
  • the muscles were sutured with a round needle 4 wire.
  • animal-derived meningeal repair materials that have been commercially used have been used. Animals were routinely fed and observed after surgery. After the operation, the animals recovered well, the incision healed well, no cerebrospinal fluid leakage, no epileptic seizures occurred. After the operation, the water intake was normal, the animal's outdoor activities were normal, no movement disorder was found, and the survival time was up to the predetermined time limit.
  • the animal was centered on the surgical site, and the specimen was taken at a distance of 1 cm larger than the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue. After cutting out the specimen, it can be seen that the joint between the artificial meninges and the dura mater is flat, without boundary, and has completely healed, only the sutured silk thread is seen. No obvious hyperemia, hemorrhage and other rejection reactions were found between the original dura mater. The implant site of the control group still showed undegraded implant material, and there was a little adhesion on the inner surface of the meninges at the implant site.
  • the experimental animals performed a top craniotomy, artificially creating a partial dural defect and brain tissue damage, and then Dural repair was performed with artificial meninges. Animals were routinely fed and observed after surgery. The animals recovered well after surgery. At 18 months after surgery, the animals were centered on the surgical site, and the specimens were taken at a distance of more than 1 cm from the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue.
  • the epithelial cells are covered on the inner surface, fibrous tissue proliferation can be seen under the epithelium, fibroblast proliferation, collagen fibers increase, resulting in the proliferation of new tissue with blood in the material, invasion of the host new tissue, material degradation, total Significantly reduced, capillaries visible inside.
  • fibrous tissue proliferation can be seen under the epithelium
  • fibroblast proliferation collagen fibers increase, resulting in the proliferation of new tissue with blood in the material, invasion of the host new tissue, material degradation, total Significantly reduced, capillaries visible inside.
  • There is no inflammatory cell reaction such as neutrophils and lymphocytes at the interface of new and old tissues, and no sacral wall is formed at the interface.
  • the arachnoid and brain tissues are normal.
  • the canine animal experiment was carried out using the dura mater prepared in Example 4: the specific animal experiment method was the same as in Experimental Example 2.
  • the animal was centered on the surgical site, and the specimen was taken at a distance of more than 1 cm from the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue.
  • the specimen was taken at a distance of more than 1 cm from the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue.
  • the joint between the artificial meninges and the dura mater is flat, without boundary, and has completely healed, only the sutured silk thread is seen. No obvious hyperemia, hemorrhage and other rejection reactions were found between the original dura mater.
  • the New Zealand rabbit animal experiment was carried out using the dura mater prepared in Example 5, and the control group was treated with an animal-derived meningeal repair material which had been commercially used clinically.
  • the specific experimental method is the same as in Example 3. 15 months after the operation, the animal was centered on the surgical site, and the specimen was cut out in the same manner as in Example 3. After excising the specimen, it can be seen that the epithelial cells are covered on the inner surface, fibrous tissue proliferation can be seen under the epithelium, fibroblast proliferation, collagen fibers increase, and the new tissue proliferation with blood in the material, host new tissue invasion, material degradation, total Significantly reduced, capillaries visible inside. There is no inflammatory cell reaction such as neutrophils and lymphocytes at the interface between the old and new tissues, and no sacral wall is formed at the interface. The arachnoid and brain tissues are normal. The implant site of the control group still showed undegraded implant material, and there was a little adhesion on the inner surface of the meninges at the implant site.
  • the animal was centered on the surgical site, and the specimen was taken at a distance of more than 1 cm from the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue. After cutting out the specimen, it can be seen that the joint between the artificial meninges and the dura mater is flat, without boundary, and has completely healed, only the sutured silk thread is seen. No obvious hyperemia, hemorrhage and other rejection reactions were found between the original dura mater.
  • the specific experimental method is the same as in Example 3.
  • the animals were centered on the surgical site, and specimens were taken at a depth of 1 cm larger than the surgical site to include the artificial meninges and the surrounding dura mater and the inner brain tissue.
  • the epithelial cells are covered on the inner surface, fibrous tissue hyperplasia can be seen under the epithelium, fibroblast proliferation, collagen fibers increase, and the new tissue proliferation with blood in the material, host new tissue invasion, material degradation, total Significantly reduced, capillaries visible inside.
  • the interface between the old and new tissues is free of neutrophils, lymphocytes and other inflammatory cells, and no sacral wall is formed at the interface.
  • the arachnoid and brain tissues are normal.

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Description

人工硬脑膜及其制备方法
技术领域
本发明涉及人工生物膜及其制备， 尤其是涉及人工硬脑膜及其制备方法。 背景技术
硬脑膜缺损在神经外科临床工作中常见， 开放性颅脑损伤 （工业、 交通、 战争等）、 肿瘤的侵蚀、 先天性脑膜缺损及其它颅脑疾患原因均可引发硬脑膜 缺损。硬脑膜缺损需及时修补以防脑脊液外溢，防止脑的膨出和大气压的压迫， 否则将危及人体生命。
目前硬脑膜的代用品虽有很多， 修复硬脑膜的材料可分为： 自体筋膜、 同 种异体材料、 天然及人工合成材料、 异种材料 4类。 但这些材料在应用于临床 时， 都不可避免有临床感染率较高等缺陷。据统计， 开颅手术的感染率为 4%; 以猪小肠黏膜为原料制备的硬脑膜， 其感染率在 3.4%; 而用胶原制备的脑膜， 感染率在 3.8%。 由于血脑屏障的存在， 颅内感染一旦发生， 颅内血药浓度艮 难达到所需水平， 给感染的控制带来障碍。 而目前的人工脑膜一般在脑膜内无 法混入药物， 因而对术后感染无控制效果。
此外，因脑肿瘤侵蚀破坏而进行脑膜移植修复手术是常见的脑膜修复原因 之一。 脑肿瘤有一半以上不能彻底切除， 因而手术之后通常还需要进行化疗。 许多化学药物毒性较大，且不能通透血脑屏障，因而同样不能达到有效的浓度， 影响治疗效果。
目前的人工脑膜一般不含有治疗性药物。根据人工脑膜的来源， 自体筋膜 根本不可能添加药物， 同种或异体材料难以实现药物加载， 而人工合成材料由 于良好可设计及加工性可对药物进行有效加载。此外， 受药物添加方式方法的 限制， 并非所有加工方式都可以顺利固定于人工脑膜上， 进而在脑膜移植后于 体内释放， 达到治疗的目的。 目前抗感染药物的添加方法一般是用抗感染药物 浸泡，但这种方法添加的药物绝大部分停留在人工脑膜基础材料的表面， 易于 流失， 也不能达到有效可控释放的目的。
综上所述， 现有的人工硬脑膜具有感染率高、 生物相容性差、 不可彻底为 人体吸收且难以添加治疗药物、 控制药物有效释放的缺陷。 发明内容
为了解决上述至少一个技术问题， 本发明提供一种人工硬脑膜， 具有生物 组织相容性佳、 防止粘连、 可完全吸收、 力学机械性能好、 感染率低、 可混入 其他治疗性物质的特点。
进一步地， 本发明还提供了一种人工硬脑膜的制备方法。
本发明提供的人工硬脑膜， 由通过静电纺丝的方法制成的电纺层制成， 所 述电纺层为至少一层疏水性电纺层构成。疏水性电纺层可以由一种或多种疏水 性聚合物通过静电纺制备而成的， 所述疏水性聚合物选自包括以下物质的组： 疏水性脂肪族聚酯类、 聚醚酯类、 聚原酸酯、 聚氨酯、 聚酸酐、 聚碑腈、 聚氨 基酸， 或以上聚合物的两种及两种以上的共聚物及混合物。
进一步地， 所述疏水性脂肪族聚酯类选自下述组中的至少一种： 聚乳酸、 聚乙交酯、聚己内酯、聚羟基丁酸酯。所述聚醚酯类选自下述组中的至少一种： 聚二氧杂环己酮、 乙二醇 /乳酸共聚物、 乙二醇 /对苯二曱酸丁二醇酯共聚物。 所述聚酸酐选自下述组中的至少一种：聚癸二酸 -十六烷二酸酸酐、 I型聚酸酐、 II型聚酸酐， III型聚酸酐、 IV型聚酸酐。
由疏水性电纺层制成的人工硬脑膜， 其强度类似于人硬脑膜， 可以在机体 自身硬膜再生之前起到密封脑、 防止脑脊液渗漏、保护脑的作用； 由疏水性材 料构成的电纺层， 不利于细胞的迁入， 从而可以达到防粘连的目的。 实际中根 据需要， 也可以设置多层疏水性电纺层， 以满足不同强度需要。
进一步地， 本发明所提供的人工硬脑膜上， 所述疏水性电纺层上还可以进 一步设置有至少一层亲水性电纺层。所述亲水性电纺层是由一种或多种亲水性 聚合材料通过静电纺制备而成的，所述亲水性聚合物材料选自包括以下物质的 组： 硫酸软骨素、 肝素、 琼脂、 葡聚糖、 褐藻酸、 改性纤维素、 海藻酸、 淀粉、 纤维素、 明胶、 纤维蛋白、 丝蛋白、 弹力蛋白拟态的肽聚合物、 胶原蛋白、 壳 聚糖、 改性壳聚糖、 亲水性聚氨酯、 聚乙二醇 、 聚曱基丙烯酸曱酯、 聚曱基 丙烯酸曱酯、 聚羟基丁酸戊酸酯、 聚羟基丁酸己酸酯、 聚乙烯醇或聚丙交酯， 或以上材料的两种及两种以上的混合物。 当该硬脑膜植入脑局部的时候， 疏水性电纺层被设置在临近大脑表面， 以 起到防粘连的作用； 而亲水性电纺层被设置在远离大脑一面， 为细胞的粘附、 迁移、 增殖、 分化提供了良好的纳米细胞支架， 由于该层釆用的是生物相容性 好的亲水性材料制成， 可有效诱导干细胞及成纤维细胞的迁入，从而促使自体 硬脑膜的生长。 实际中根据需要， 亲水性电纺层也可以为多层， 以满足不同需 要。
根据本发明的实施方式之一， 本发明所提供的人工硬脑膜， 所述疏水性电 纺层和亲水性电纺层之间还可以设有过渡层。所述过渡层是由一种或多种聚合 材料通过静电纺制备而成的，过渡层对水的亲和力按照从疏水性电纺层向亲水 性电纺层的方向逐步增加。过渡层的设置， 可以增强疏水性电纺层和亲水性电 纺层之间的亲和力。
才艮据本发明的另一实施方式， 在疏水性电纺层、 亲水性电纺层、 过渡层中 的任意一层或多层均可以混纺有细胞因子和 /或药物。 混纺电纺层在植入脑组 织局部后， 随着电纺层高分子聚合物的吸收， 混纺入的细胞因子和 /或药物被 释放至局部， 发挥相应的作用， 以预防局部感染、 防粘连、 防脑肿瘤复发或促 进自体硬膜修复。
根据本发明的再一实施方式， 在疏水性电纺层和 /或亲水性电纺层上还可 以附着有细胞因子和 /或药物。
本发明所述细胞因子是对成纤维细胞的粘附、 迁移、 增殖、 分化起作用的 因子， 选自包括以下物质的组： 白细胞介素、 集落刺激因子、 肿瘤坏死因子、 血小板源生长因子、碱性成纤维细胞因子， 或以上因子的两种及两种以上的混 合物。 细胞因子的作用， 可以加速机体自身硬膜化的过程。
本发明所述药物选自抗生素、止血剂、 防粘连剂或抗肿瘤药物的一种或几 种。这些药物被按照实际需要设置于本发明所提供的人工硬脑膜上，在植入后， 药物随着聚合材料的吸收和机体自身硬膜化的过程，逐步释放于局部，发挥治 疗和预防作用， 从而克服了血脑屏障对药物的阻挡作用。
进一步地， 细胞因子和 /或药物可被包覆于水溶胶之中。 通过水溶胶的粘 附和固定作用，按照处置局部的情况实现药物的均匀或定点释放。 所述水溶胶 可以选自多糖类聚合物， 多肽类聚合物，合成的亲水高分子聚合物中的一种或 几种。
根据本发明的实施方式之一，所述人工硬脑膜的疏水性电纺层的纤维直径 为 50~1000nM。 才艮据本发明的实施方式之一， 疏水性电纺层的孔径小于 3μΜ。 亲水性电纺层的纤维直径为 5~200μΜ, 孔径为 20~200μΜ。静电纺纤维得到的 孔隙大小分布很大程度上依赖于纤维直径， 当纤维直径减小时， 孔径也在同时 减小， 因此通过控制纤维直径， 可以实现对电纺层孔径的控制。 人体细胞直径 平均在 10~20 μ Μ, 脑膜主要分布成纤维细胞及其分泌的胶原纤维， 一般成纤 维细胞直径在 20~30 μ Μ之间。 疏水性电纺层的孔径设置为 3 μ Μ以下， 可以 阻止细胞进入， 进一步防止脑粘连的产生。 而亲水性电纺层的孔径设置为等于 或大于细胞的平均直径， 是为了进一步促进细胞的迁移长入。
根据本发明的另一方面， 本发明还提供了一种人工硬脑膜的制备方法， 包 括以下步骤： a) 将疏水性聚合物溶于溶剂中， 得到疏水性聚合物电纺溶液， 所述疏水性聚合物选自包括以下物质的组： 疏水性脂肪族聚酯类、 聚醚酯类、 聚原酸酯、 聚氨酯、 聚酸酐、 聚碑腈、 聚氨基酸， 或以上聚合物的两种及两种 以上的共聚物及混合物； b) 通过静电纺， 将所述疏水性聚合物电纺溶液制成 膜状的疏水性电纺层， 得到所述人工硬脑膜。
进一步地， 所述疏水性脂肪族聚酯类选自下述组中的至少一种： 聚乳酸、 聚乙交酯、聚己内酯、聚羟基丁酸酯。所述聚醚酯类选自下述组中的至少一种： 聚二氧杂环己酮、 乙二醇 /乳酸共聚物、 乙二醇 /对苯二曱酸丁二醇酯共聚物。 所述聚酸酐选自下述组中的至少一种：聚癸二酸 -十六烷二酸酸酐、 I型聚酸酐、 II型聚酸酐， III型聚酸酐、 IV型聚酸酐。
利用本发明提供的方法是釆用静电纺的原理 ,将特定种类的高分子聚合物 接收形成人工硬脑膜， 植入后可有效地防止脑粘连的发生。
为了进一步阻止脑粘连，可以通过控制纤维直径进而控制电纺层的孔径从 而阻止细胞迁入。 本发明中疏水性电纺层的纤维直径可被控制为 50~1000nM。 根据本发明的实施方式之一， 疏水性电纺层的孔径小于 3μΜ。
上述方法中， 步骤 b中所述静电纺的工艺参数可以设置为： 微量注射泵的 速率为 0.1~5.0 毫升 /小时， 高压发生器的电压为 5~40 KV, 接收距离为 5.0 30.0厘米。 为了达到临床上更好的治疗效果，本发明所提供的方法还可包括在所述疏 水性电纺层上通过类似的静电纺方法， 形成亲水性电纺层的步骤： a' )将亲水 性聚合物溶于溶剂中，得到亲水性聚合物电纺溶液， 所述亲水性聚合物选自包 括以下物质的组： 硫酸软骨素、 肝素、 琼脂、 葡聚糖、 褐藻酸、 改性纤维素、 海藻酸、 淀粉、 纤维素、 明胶、 纤维蛋白、 丝蛋白、 弹力蛋白拟态的肽聚合物、 胶原蛋白、 壳聚糖、 改性壳聚糖、 亲水性聚氨酯、 聚乙二醇 、 聚曱基丙烯酸 曱酯、 聚曱基丙烯酸曱酯、 聚羟基丁酸戊酸酯、 聚羟基丁酸己酸酯、 聚乙烯醇 或聚丙交酯； b' ) 通过静电纺， 将所述亲水性聚合物电纺溶液接收于所述疏 水性电纺层上， 形成亲水性电纺层。
设置亲水性电纺层于远离大脑表面的一面， 目的是为了促进细胞的迁入， 形成自体的新生硬膜。 为了进一步促进细胞进入， 可将亲水性电纺层的纤维直 径 5~200μΜ, 孔径为 20~200μΜ。
在形成亲水性电纺层的过程中，步骤 b'中静电纺的工艺参数为：微量注射 泵的速率为 0.1~20.0毫升 /小时， 高压发生器的电压为 10~45 KV, 接收距离 为 5.0~30.0厘米。
静电纺过程中 ,通常需要将疏水性聚合物或亲水性聚合物首先溶于适当的 溶剂中形成电纺溶液， 这些溶剂通常为易挥发有机溶剂， 包括但不限于曱酸、 乙酸、 乙醇、 丙酮、 二曱基曱酰胺、 二曱基乙酰胺、 四氢呋喃、 二曱基亚砜、 六氟异丙醇、 三氟乙醇、 二氯曱烷、 三氯曱烷、 曱醇、 乙醇、 氯仿、 二噁烷、 三氟乙烷、 三氟乙酸， 或以上聚合物溶剂的两种及两种以上的混合液。 易挥发 有机溶剂可以在形成电纺层的过程中快速挥发 ,使得最终的电纺层上不含有机 溶剂的残留。 在有些情况下， 溶剂也可以选用水， 通过烘干或阴干的方式在电 纺层形成后被去除。
进一步地， 本发明所提供制备人工硬脑膜的方法， 其在形成所述亲水性电 纺层之前，还包括在疏水性电纺层和亲水性电纺层之间通过静电纺形成过渡层 的步骤，所述过渡层对水的亲和力按照从亲水性电纺层向疏水性电纺层的方向 逐步增加。 静电纺形成过渡层的高分子材料、 溶剂、 静电纺参数的选取， 可以 根据实际情况按照通常的技术进行选取确定， 过渡层的形成， 可以增加亲水层 和疏水层之间的亲和力。 根据本发明的实施方式之一，在通过静电纺形成各电纺层的过程中， 可在 相应的聚合物电纺溶液中混有细胞因子和 /或药物。 混纺技术的引入， 形成的 电纺层成为高分子聚合物与细胞因子和 /或药物组成的混纺电纺层， 能更好地 适应临床需要， 提高治疗效果。
此外， 本发明所提供的制备人工硬脑膜的方法，还可进一步包括在所述疏 水性电纺层和 /或亲水性电纺层上通过细胞打印形成细胞因子和 /或药物分布的 步骤。 细胞打印就是以疏水性电纺层和 /或亲水性电纺层作为支架 （"生物纸 片"）， 在其上打印细胞因子和 /或药物。
为了使得细胞因子和 /或药物能够均匀地或定位明确地分布于电纺层上， 可以将所述细胞因子和 /或药物被包覆于水溶胶之中。
具体地， 本发明所釆用的细胞因子和 /或药物的细胞打印可以釆取如下步 骤： a" )将水溶胶溶液和细胞因子和 /或药物混合， 形成混合溶液； 和 b" )利 用细胞打印技术将所述混合溶液打印到所述疏水性电纺层和 /或亲水性电纺层 上。
本发明中水溶胶溶液可以为以多糖类聚合物、多肽类聚合物或合成亲水高 分子聚合物制成的水溶液。 其中， 所述多糖类聚合物包括但不限于淀粉、 纤维 素、 海藻酸、 透明质酸或壳聚糖； 所述多肽类聚合物包括但不限于胶原、 聚 L〜赖氨酸或聚 L〜谷胺酸；所述合成亲水高分子聚合物包括但不限于聚丙烯酸、 聚曱基丙烯酸、 聚丙烯酰胺或聚 N〜聚代丙烯酰胺。
水溶胶在常态下通常为液态，在适当温度或在特定条件下，会在短时间内 成为胶冻状， 因而具有较好的附着力。 根据本发明， 某些水溶胶需要交联剂参 与反应， 因此可以在所述打印步骤之前还包括将所述疏水性电纺层和 /或亲水 性电纺层用交联剂溶液进行预处理的步骤。用交联剂溶液进行预处理，使得交 联剂附着于电纺层上； 将水溶胶溶液混合适当的细胞因子和 /或药物， 置于打 印喷头内。 打印时， 当喷头内混有细胞因子和 /或药物的水溶胶溶液接触到电 纺层后， 凝固并粘附于电纺层上。 打印时， 可以釆用均匀打印的方法， 使得细 胞因子和 /或药物均勾释放， 也可以根据病人的个体情况， 釆用局部集中打印 的方法， 使得细胞因子和 /或药物集中释放于所关注的部位。 交联剂的选择与 水溶胶的种类有关， 例如当水溶胶为海藻酸时， 交联剂为氯化钙； 当水溶胶为 纤维蛋白时， 交联剂为凝血酶。
与现有技术相比， 本发明具有以下有益效果：
( 1 )机械特性能满足适应症对抗拉伸强度及柔韧性的要求， 并且不透水、 防粘连；
( 2 )膜片所使用的材料对人体均无毒无害， 具有良好的生物组织相容性， 植入后能够被完全吸收， 避免了膜片致癌；
( 3 )膜片本身并非由生物源成分形成， 免除了由此而带来的免疫排斥、 病毒传播、 疾病传染等诸多风险；
( 4 ) 双层的设计方案在防粘连的同时又促进了自体细胞的增长， 可促使 自体早日修复；
( 5 ) 结合生物打印技术， 在膜片中引入具有治疗作用的物质， 且植入后 可使这些物质可控释放， 增强了治疗效果；
( 6 )材料来源充分， 成本较低， 贮存运输简单；
( 7 )制备方法工艺步骤简单， 成本较低， 易于工业化推广。
( 8 ) 临床应用简单， 并且可以根据病人情况进行个性化定制。
本发明附加的方面和优点将在下面的描述中部分给出，部分将从下面的描 述中变得明显， 或通过本发明的实践了解到。 附图说明
本发明的上述和 /或附加的方面和优点从下面结合附图对实施例的描述中 将变得明显和容易理解， 其中：
图 1 为本发明所述人工硬脑膜的静电纺制作工艺示意图；
图 2 为本发明所述人工硬脑膜的静电纺结合生物打印技术制作工艺示意
Figure imgf000009_0001
图;
图 4 为根据本发明所述疏水性电纺层和亲水性电纺层组成的人工硬脑膜 的示意图；
图 5 为根据本发明所述疏水性和亲水性电纺层之间具有过渡层的人工硬 脑膜的示意图； 图 6a 为根据本发明所述混纺的具有疏水性电纺层和亲水性电纺层的人工 硬脑膜的示意图；
图 6b 为图 6a所示局部 I区域的放大示意图；
图 7a 为才艮据本发明所述混纺的具有疏水性电纺层、 亲水性电纺层和过渡 层的人工硬脑膜的示意图；
图 7b为图 7a所示局部 II区域的放大示意图；
图 8a为根据本发明所述结合生物打印技术得到的人工硬脑膜的示意图； 图 8b为图 8a所示局部 ΠΙ区域的放大示意图。
图中标号的含义如下：
1 静电纺喷头；
2 静电纺丝；
3 高压电源；
4 接收装置；
5 生物打印喷头；
6 谷 ；
7 疏水性电纺丝；
8 药物；
9 亲水性电纺丝；
10 细包因子；
11 过渡层电纺丝;
A 疏水性电纺层；
B 亲水性电纺层；
C 过渡层。 具体实施方式
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自 始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元 件。 下面通过参考附图描述的实施例是示例性的， 仅用于解释本发明， 而不能 解释为对本发明的限制。 下面结合图 1~8, 描述本发明所提供的人工硬脑膜及其制备方法。
如图 1所示， 为本发明所述人工硬脑膜的静电纺工艺示意图。静电纺喷头 1内装有高分子聚合物溶液， 高压电源 3的高压端接于静电纺喷头 1上。 接收 装置 4为圓柱体外形，可以按照实际操作需要绕圓柱体轴心以及圓柱体长轴方 向的左右移动。 接收装置 4 的移动设置可以通过计算机设定相应的程序来实 现， 使得形成的电纺层厚度均勾。 实际中， 接收装置还可以设置为一表面平整 的平面， 通过平面的左右、 前后移动实现均匀地接收。接收装置 4与高压电源 3的低压端相连通， 使得静电纺喷头 1和接收装置 4之间形成较高电压差。
静电纺步骤开始之前， 首先要配制适当的静电纺溶液。
可以选用疏水性聚合物电纺溶液，这些溶液是将疏水性聚合物溶于恰当溶 剂中而配成的。这些疏水性聚合物包括但不限于疏水性脂肪族聚酯类（如聚乳 酸、 聚乙交酯、 聚己内酯、 聚羟基丁酸酯）、 聚醚酯类 （如聚二氧杂环己酮）、 聚原酸酯、 聚氨酯、 聚酸酐（如聚癸二酸-十六烷二酸酸酐）、 聚碑腈、 聚氨基 酸， 或以上聚合物的两种及两种以上的共聚物及混合物。
根据设计需要，如果在形成疏水性电纺层后，还要在其上进一步形成亲水 性电纺层， 则需要同时配制亲水性聚合物电纺溶液， 这些溶液是将亲水性聚合 物溶于恰当溶剂中而配成的。 所述亲水性聚合物包括但不限于： 硫酸软骨素、 肝素、 琼脂、 葡聚糖、 褐藻酸、 改性纤维素、 海藻酸、 淀粉、 纤维素、 明胶、 纤维蛋白、 丝蛋白、 弹力蛋白拟态的肽聚合物、 胶原蛋白、 壳聚糖、 改性壳聚 糖、 亲水性聚氨酯、 聚乙二醇 、 聚曱基丙烯酸曱酯、 聚曱基丙烯酸曱酯、 聚 羟基丁酸戊酸酯、 聚羟基丁酸己酸酯、 聚乙烯醇或聚丙交酯。 这时可以根据需 要， 设置多个静电纺喷头 1 , 其内分别放置疏水性或亲水性聚合物电纺溶液； 也可以在疏水性电纺层形成后更换静电纺喷头 1内的电纺溶液。
电纺溶液的溶剂可以是水。也可以选择易挥发有机溶剂， 包括但不限于曱 酸、 乙酸、 乙醇、 丙酮、 二曱基曱酰胺、 二曱基乙酰胺、 四氢呋喃、 二曱基亚 砜、 六氟异丙醇、 三氟乙醇、 二氯曱烷、 三氯曱烷、 曱醇、 乙醇、 氯仿、 二噁 烷、 三氟乙烷、 三氟乙酸。
电纺溶液就位后， 设置好参数， 接通电源， 开启电纺装置。 随着静电纺丝 2逐渐从静电纺喷头 1中拉出， 接收装置 4会按照既定程序移动， 以保证形成 均一的电纺膜结构。
疏水性电纺层形成的工艺参数一般设置为： 微量注射泵的速率为 0.1~5.0 毫升 /小时， 高压发生器的电压为 5~40 KV, 接收距离为 5.0~30.0厘米。 疏水 性电纺层的纤维直径可被控制为 50~1000nM, 孔径小于 3μΜ。
亲水性电纺层形成的工艺参数为：微量注射泵的速率为 0.1 20.0毫升 /小 时， 高压发生器的电压为 10~45 KV, 接收距离为 5.0~30.0厘米。 亲水性电纺 层的纤维直径 5~200μΜ, 孔径为 20~200μΜ。
操作中可以根据实际需要，将上述静电纺步骤重复多次， 以形成多层疏水 性电纺层和 /或亲水性电纺层， 如图 3和图 4所示。
图 3为 3层疏水性电纺层构成的人工硬脑膜， 其强度类似于人硬脑膜， 由 于该层是由疏水性材料组成，因而不利于细胞的迁入，且所用材料均安全无毒， 可被人体吸收， 从而可以达到防粘连的目的。
图 4为由 2层结构的疏水性电纺层 Α和 3层结构的亲水性电纺层 B构成 的人工硬脑膜。 当该硬脑膜植入脑局部的时候， 防粘连的疏水性电纺层 A被 设置在临近大脑表面， 而亲水性电纺层 B被设置在远离大脑一面， 为细胞的 粘附、 迁移、 增殖、 分化提供了良好的纳米细胞支架， 由于该层釆用的是生物 相容性好的亲水性材料制成，且其孔径较大，可使得干细胞及成纤维细胞迁入， 从而促使自体硬脑膜的生长。
电纺层形成之后， 可以根据电纺溶液组成的不同， 选取适当的烘干或阴干 的步骤。当电纺溶液的溶剂被选择为某些易挥发的有机溶剂时，如六氟异丙醇， 在静电纺丝 2随着电压差下落到接收装置 4上的过程中， 溶剂已经完全挥发， 因而可以省略烘干或阴干的步骤。
由于疏水性电纺层和亲水性电纺层对水的亲和力差别较大，因此在使用时 可能会不易保持结构上的稳定， 为解决此问题、 增加两部分之间的亲和力， 还 可以在两部分之间形成过渡层。过渡层对水的亲和力按照从疏水性电纺层向亲 水性电纺层的方向逐步增加，实践中可以才艮据对水的亲和力的要求来选择一种 或多种适当的高分子聚合物和相应的溶剂，设置于相应的静电纺喷头内，按照 上述的方法， 在形成亲水性电纺层之前形成过渡层。 过渡层的电纺工艺参数： 微量注射泵的速率为 0.1 5.0毫升 /小时， 高压发生器的电压为 5~40 KV, 接 ^^巨离为 5.0~30.0厘米。
形成的具有过渡层的人工硬脑膜如图 5所示。 图中疏水性电纺层 A为 2 层结构， 亲水性电纺层 B为 3层结构， 在疏水性电纺层 A和亲水性电纺层 B 之间还设有 2层结构的过渡层 C, 其中靠近疏水性电纺层 A的一层亲水性弱 于靠近亲水性电纺层 B的一层。
进一步地， 为了实现在人工硬脑膜中加入细胞因子和 /或药物的目的， 可 以通过混纺的方法来实现。 具体地， 在疏水性电纺层、 亲水性电纺层、 过渡层 中的任意一层或多层均可以混纺有细胞因子和 /或药物。 细胞因子和 /或药物可 以根据需要添加到相应的电纺溶液之中， 然后按照上述方法进行静电纺， 细胞 因子和 /或药物随着静电纺丝 2的形成而混入到静电纺丝 2中，并在接收装置 4 上形成均一的膜结构。 同样的， 该过程也可重复进行多次， 每一次所混入的细 胞因子和 /或药物可以相同也可以不同。 得到的人工硬脑膜如图 6所示。 图 6a 中疏水性电纺层 A为 2层结构， 亲水性电纺层 B为 3层结构； 图 6b中可见， 疏水性电纺丝 7中混纺有药物 8, 亲水性电纺丝 9中混纺有细胞因子 10。
图 7a在图 6的结构上进一步设置了 2层的过渡层 C结构， 靠近疏水性电 纺层 A的一层对水的亲和力小于靠近亲水性电纺层 B的一层。从图 7b中可见， 疏水性电纺丝 7中混纺有药物 8, 亲水性电纺丝 9中混纺有细胞因子 10,在两 层过渡层电纺丝 11中分别混纺有药物 8和细胞因子 9。
此外，本发明还提供了一种静电纺结合生物打印技术的制作人工硬脑膜的 工艺。生物打印技术是近年来出现的新技术， 利用特制的细胞溶液或有生物活 性的细胞因子溶液作为 "生物墨水"， 按预定计划精确打印定位于在体内可降 解的生物纸片上。 打印后再将纸片按一定顺序的堆叠。 由于使用了打印技术， 可以将细胞或 /和细胞因子组成的生物墨水精确的结合到预定部位； 而按特定 的堆叠方式的生物纸片则会形成三维结构。
具体操作如图 2所示。在图 1所示的装置基础上进一步设置了生物打印喷 头 5。 该生物打印喷头 5可以是用现有的喷墨打印机进行改造而得到的， 例如 可以参照美国专利 US 7051654公开的方法。 喷头中含有细胞因子和 /或药物， 喷头的打印方式和打印位置可以通过计算机程序预先设定。具体生物打印步骤 可以根据现有技术进行。 根据本发明的实施方式之一， 细胞因子和 /或药物可以被包覆于水溶胶之 中， 水溶胶溶液可以为以多糖类聚合物、 多肽类聚合物或合成亲水高分子聚合 物制成的水溶液。 其中， 所述多糖类聚合物包括但不限于淀粉、 纤维素、 海藻 酸、 透明质酸或壳聚糖； 所述多肽类聚合物包括但不限于胶原、 聚 L〜赖氨酸 或聚 L〜谷胺酸； 所述合成亲水高分子聚合物包括但不限于聚丙烯酸、 聚曱基 丙烯酸、 聚丙烯酰胺或聚 N〜聚代丙烯酰胺。 水溶胶在常态下通常为液态， 在 适当温度下或在特定条件下，会在短时间内成为胶冻状， 因而具有较好的附着 力， 从而使得细胞因子和 /或药物均匀地或定位明确地分布于电纺层上。
利用水溶胶进行生物打印的步骤具体如下：在生物打印喷头 5中放入配制 好的、 混有相应液态水溶胶的细胞因子和 /或药物。 在电纺层形成之后， 喷墨 打印机按照预设程序在电纺层上进行打印，根据水溶胶的选择，给予适当条件， 水溶胶迅速地成为具有较好的附着力的胶冻状， 将其中所包裹的细胞因子和 / 或药物粘附于电纺层上。 打印时， 可以釆用均匀打印的方法进行设置， 形成人 工硬脑膜如图 8a所示，在每一层疏水性电纺层结构上均可以打印有一层药物， 在每一层亲水性电纺层结构上均可以打印有一层细胞因子。 如局部放大图 8b 所示， 生物打印形成的膜与混纺不同， 细胞因子和 /或药物是涂覆于有疏水性 电纺丝 7和 /或亲水性电纺丝 9构成的电纺层之表面的。 此类硬脑膜植入体内 后， 可以使得细胞因子和 /或药物均匀释放。 此外， 还可以根据病人的治疗需 要， 釆用局部集中打印的方法， 植入人体后， 使得细胞因子和 /或药物集中释 放于所关注的部位。
某些水溶胶的胶固化需要交联剂参与反应，这时利用水溶胶进行生物打印 的步骤具体如下： 在容器 6中预先放入适当的交联剂， 静电纺开始后， 接收装 置 4在沿轴线转动或左右移动的过程中会与容器 6中的交联剂相接触，使得形 成的电纺层上附着有交联剂分子。接着按照上述方法进行生物打印， 当生物打 印喷头 5中的液态水溶胶与电纺层上的交联剂接触时，迅速地成为胶状，将其 中所包裹的细胞因子和 /或药物粘附于电纺层上。 交联剂的选择与水溶胶的种 类有关，例如当水溶胶为海藻酸时，交联剂为氯化钙； 当水溶胶为纤维蛋白时， 交联剂为凝血酶。
根据本发明实施方式之一， 所述疏水性聚合物电纺溶液可选择疏水性的 L-聚乳酸和 ε -己内酯， 两者重量比为 50: 50或 30:70或 70:30, 作为共聚高分 子材料， 数均分子量是 150000 500000 , 溶于六氟异丙醇或二氯曱烷。 如果需 要混纺时， 疏水性聚合物电纺溶液中可以加入： 0.01~3%抗生素溶液， 或 /和 0.001~3%止血和防粘连药物， 与 L-聚乳酸和 ε -己内酯溶液混合， 得到均一溶 液。
根据本发明的另一实施方式，亲水性聚合物电纺溶液可以选择亲水性聚氨 酯与天然明胶或硫酸软骨素或聚乙二醇， 重量比 20~80： 80-20, 纺丝液为溶 液总重量的 3~15%。 混纺时， 亲水性聚合物电纺溶液中还可加入碱性成纤维 细胞因子溶液， 使细胞因子终浓度达为 0.001~0.5%。
混纺或生物打印过程中加入的药物可以依据具体情况选择抗生素或止血 或防粘连药物，在因脑肿瘤侵蚀而引起脑膜移植中， 可釆用针对脑肿瘤的化疗 药物。
抗生素可以为但不限于头孢类、 苄氨西林， 螺旋霉素， 磺胺， 喹诺酮类抗 生素等。 首选为头孢曲松钠。 因脑膜手术通常需要开颅， 目前绝大多数的开颅 术后颅内感染是细菌性的， 主要致病菌包括： 金黄色葡萄球菌、 链球菌、 肺炎 球菌、 大肠杆菌、 沙门菌属及绿脓杆菌等。 其中金黄色葡萄球菌最常见， 根据 临床报道， 多认为头孢曲松钠的临床治疗效果比较好。
抗肿瘤药物， 包括但不限于尼莫司汀、 司莫司丁、 脂质体阿霉素、 放线菌 素。、 长春新碱等。 首选为长春新碱。
止血或防粘连药物， 可加速创伤愈合， 防止粘连产生， 包括但不限于止血 因子 （可使材料同时具备止血功能）、 胶原合成酶的抑制剂 （如曲尼斯特和毗 嗜司特， 可以抑制胶原合成酶组胺和前列腺素的释放， 抑制胶原再生）、 抗凝 血药 （如双香豆素、 肝素钠、 水蛭素等）、 消炎药 （如异丙嗪、 地塞米松、 氢 化可的松、 泼尼松龙、 布洛芬、 羟基保泰松等）、 钙通道阻滞剂 （如盐酸地尔 硫卓、 硝苯地平、 盐酸维拉帕米等）、 细胞生长抑制剂 （如氟尿嘧啶）、 水解酶 (如玻璃酸酶、 链激酶、 尿激酶、 胃蛋白酶、 tPA )、 氧化还原剂 （如亚曱蓝） 等等。
根据本发明实施例之一， 混纺过程中， 电纺溶液中添加药物或因子的方案 是： 含有电纺溶液重量的 0.001 0.05%的碱性成纤维细胞成长因子， 电纺溶液 重量的 3%的苄氨西林， 电纺溶液重量的 0.001~0.05%的止血因子， 在用于因 脑肿瘤而导致的脑膜修复手术中， 可以包含有 0.01-5%的尼莫司汀。
制得的人工硬脑膜经清洗、 灭菌、 包装后贮存。
实施例 1
选择 L〜聚乳酸和 ε -己内酯，两者重量比为 50: 50,数均分子量是 260000, 溶于六氟异丙醇混匀形成疏水性电纺溶液， 置于静电纺喷头内。用调节微量注 射泵控制疏水性电纺溶液的输出速率为 5毫升 /小时，调节高压发生器的电压 为 30KV, 调节接收装置的接收距离为 20厘米， 并将纤维接收为膜状结构， 形成纤维直径平均为 300nm的疏水性电纺层。 接收完毕后， 关闭静电纺装置。
将制得的人工脑膜， 用乙醇及蒸馏水漂洗 5 遍， 经冻干后真空包装， 经 25kGy钴 -60灭菌后负 20摄氏度低温保存。
实施例 2
疏水性电纺层的制备方式同实施例 1。
亲水性电纺溶液选用聚乙二醇与硫酸软骨素质量比为 70:30,纺丝液质量 分数为 9%。
开启静电纺装置，在实施例 1已纺好的疏水性电纺层上继续纺亲水性电纺 层。 接收距离为 11cm, 电压 20KV, 所得亲水性电纺层的纤维平均直径在 10 μ m。
清洗、 保存方式同实施例 1。
实施例 3
疏水性电纺层的制备方式同实施例 1.
过渡层釆用的聚合物溶液为： 聚氨酯与透明质酸的质量比为 70:30,质量分 数为 10%。 开启静电纺丝， 设置接收距离为 11cm, 电压 20KV, 纤维平均直 径在 5 μ πι, 在已纺好的疏水性电纺层上继续纺过渡层。
然后在过渡层上继续纺亲水性电纺层，亲水性电纺层的实施方式同实施例 4。 关闭静电纺。
清洗、 保存方式同实施例 1。
实施例 4
( 1 )制备疏水性电纺层： 选择疏水性的聚已内酯， 氯仿 /曱醇混合溶剂比 例为 1 : 1。 混合头孢曲松钠， 浓度为 1%。 得到均一溶液。
将上述溶液加入静电纺喷头中，调节微量注射泵的速率为 0.8毫升 /小时， 调节高压发生器的电压为 12 KV, 调节接收装置的接收距离为 15厘米， 并将 纤维接收为膜状结构。 得到疏水性电纺层的纤维直径为 600纳米。
关闭静电装置。
( 2 )制备亲水性电纺层： 用亲水性丝素与天然明胶， 比例 20~80： 80-20 , 纺丝液质量分数为 9%。
配制碱性成纤维细胞因子溶液， 与上述电纺液混合均勾，使细胞因子终浓 度达为 0.001%, 接收距离 10 cm, 电压 20 KV, 开启静电纺丝， 在已纺好的疏 水性电纺层上继续纺亲水性电纺层。所得亲水性电纺层的纤维平均直径在微米 级。
清洗、 保存方式同实施例 1。
实施例 5
( 1 )制备疏水性电纺层： 选择疏水性的聚已内酯， 氯仿 /曱醇混合溶剂比 例为 1 : 1。 混合和长春新碱， 浓度为 100ng/ml。 得到均一溶液。
将上述溶液加入静电纺喷头中，调节微量注射泵的速率为 0.8毫升 /小时， 调节高压发生器的电压为 12 KV, 调节接收装置的接收距离为 15厘米， 并将 纤维接收为膜状结构。 得到疏水性电纺层的纤维直径为 600纳米。
关闭静电装置。
( 2 )制备过渡层： 选用聚氨酯与透明质酸的质量比为 70:30,纺丝液质量 分数为 10%。 混合氨苄西林， 浓度为 3%。 得到均一溶液。
开启静电纺丝， 在已纺好的疏水性电纺层上继续纺过渡层。
接收距离从 11cm, 电压 20KV, 纤维平均直径在 5 μ πι。
关闭静电纺。
( 3 )制备亲水性电纺层： 用亲水性丝素与天然明胶， 比例 20~80： 80-20 , 纺丝液质量分数为 9%。 配制氨苄西林溶液， 与上述电纺液混合均匀， 使抗生 素终浓度达为 3%。
开启静电纺装置， 调节接收距离 10 cm, 电压 20 KV, 在已纺好的过渡层 上继续纺亲水性电纺层。 纤维平均直径在微米级。 清洗、 保存方式同实施例 1。
实施例 6
( 1 )制备细胞打印的疏水性电纺层
疏水性电纺溶液选择疏水性的 L-聚乳酸和 ε -己内酯，两者比值为 50: 50, 作为共聚高分子材料， 数均分子量是 260000, 溶于六氟异丙醇。
交联剂溶液选用 0.1M氯化钙溶液。
含有细胞因子的水溶胶溶液釆用止血因子藻酸盐溶液，所述细胞因子藻酸 盐溶液中止血因子的质量百分比浓度为 10ppm。
将配置好的 0.1M氯化钙溶液放入直径 150mm的细胞培养亚中， 将静电 纺丝装置及打印机共用的接收器置于培养亚中 ,使得接收装置在形成电纺层时 可以和培养 jni中的容器相接触。细胞打印喷头固定在电纺丝装置箱内电纺针头 正下方， 用作止血因子定位打印； 将配好的细胞因子藻酸盐溶液装入喷墨打印 墨盒中； 本实施例釆用的墨盒型号为 HP51626A。
将疏水性电纺溶液加入静电纺喷头中，调节微量注射泵的速率为 5毫升 / 小时， 调节高压发生器的电压为 30 KV, 调节接收装置的接收距离为 20厘米， 用并将纤维接收为膜状结构。 静电纺持续 20分钟后， 关闭静电纺装置。
用喷墨打印机将含有细胞因子的水溶胶溶液打印到所述纳米仿生支架上， 水溶胶固化后即得。
( 2 )制备细胞打印的亲水性电纺层
制备电纺溶液、 含有药物的水溶胶溶液和交联剂溶液；
釆用亲水性材料， 聚乙二醇与硫酸软骨素质量比为 70:30,纺丝液质量分数 为 9%, 交联剂溶液选用 0.1M氯化钙溶液。 细胞因子藻酸盐溶液中碱性成纤维因子的质量百分比浓度为 1 OOppm。
将静电纺的参数调整为微量注射泵的速率为 0.8毫升 /小时， 高压发生器 的电压为 20 KV, 调节接收装置的接收距离为 11厘米， 其他制备方法同上一 步。将亲水性电纺层纤维接收为膜状结构后， 用喷墨打印机将含有碱性成纤维 细胞因子的水溶胶溶液打印到所述纳米仿生支架上， 水溶胶固化后即得。
清洗、 保存方式同实施例 1。 实施例 7
( 1 )制备细胞打印的疏水性电纺层
步骤同实施例 6。
( 2 )制备细胞打印的过渡层
过渡层电纺液选择以下方案： 聚氨酯与透明质酸的质量比为 70:30,纺丝液 质量分数为 10%。 混合氨苄西林， 浓度为 3%。 得到均一溶液。。
交联剂溶液选用 0.1M氯化钙溶液。
含有细胞因子的水溶胶溶液釆用止血因子藻酸盐溶液，所述细胞因子藻酸 盐溶液中止血因子的质量百分比浓度为 10ppm。
将静电纺装置参数调整为微量注射泵的速率为 4毫升 /小时，高压发生器 的电压为 20 KV, 调节接收装置的接收距离为 11厘米， 其他制备方法同上一 步骤。将纤维接收为膜状结构后， 用喷墨打印机将含有细胞因子的水溶胶溶液 打印到过渡层上， 水溶胶固化后即得。
( 3 )制备亲水性电纺层
制备方法同实施例 6。
清洗、 保存方式同实施例 1。
实验例 1
用实施例 1制得的硬脑膜进行犬动物实验，对照组釆用已商品化临床使用 的动物源性脑膜修补材料。 选择健康犬 3只， 雌雄不限， 体重 10-15kg之间， 观察期为 2-3个月。 进入实验的犬实行全麻， 双顶开颅， 人为制造双侧部分硬 脑膜缺损及脑组织损伤，然后以用实施例 1制得的硬脑膜以及对照组在同只实 验犬脑左右两侧分别实施硬脑膜修补术。 术后对犬进行常规的喂养及观察，每 个观察期满时在修补材料部位釆集标本，对比观察大体标本和镜下组织。各实 验动物饲养到设定的时间后麻醉动物，按前述开颅方式暴露颅骨， 分离暴露修 补材料外表面，静脉注入空气处死动物，锯开颅骨后掀开切取修补材料及周围 的组织。 肉眼观察修补材料外表面、 质地、 与周围组织的关系、 有无嚢肿、 硬 结及内表面与脑组织的粘连情况。 标本装瓶， 浸泡于福尔马林固定液， 把标本 瓶贴标签。室温下福尔马林固定 1周后，取手术部位局部组织，常规石蜡包理， HE组织切片染色。 手术后 3只犬恢复良好，切口愈合良好，无分泌物。术后进食，进水正常， 犬的户外活动正常， 没有发现运动障碍。 3个月后静脉内推入空气处死犬， 以 手术部位为中心，在大于手术部位 1 c m范围切取标本，使其包括修补材料及 周边硬脑膜及内面的脑组织。 切取出标本后， 逐层分离脑骨与脑膜， 可见实验 组植入部位的脑膜完整， 材料植入处已被纤维组织代替，植入材料与原生脑膜 融为一体，界限不分，且植入处脑膜内表面无粘连，与之对应脑组织表面光滑， 无粘连； 而对照组植入部位仍可见未降解的植入材料，且植入处脑膜内表面存 在少许祐连。
实验例 2
用实施例 2制得的硬脑膜进行犬动物实验： 实验犬体重 15 ~ 20 KG, 年龄 1.5~2岁， 雌雄不拘， 共 5只。 以氯胺酮肌肉注射全麻， 麻醉剃毛后， 将动物 置于专用手术台上， 腹卧位。 用 2%碘酒和 75%酒精消毒。 动物头顶正中， 纵 向切开。 用剥离器分离骨膜， 暴露双顶部颅骨板， 用高速磨钻磨开颅骨， 双顶 部形成骨窗。 用小剪刀剪掉双侧顶部 3 cm X 3cm 大小的矩形硬脑膜， 制造出 顶部的硬脑膜缺损。 在暴露的脑表面电灼， 造成 6个 1 mm x l mm大小的损 伤点。将用本发明实施例 2所制备的人工脑膜修剪成相应形状及尺寸的修补材 料， 疏水性电纺层向脑表面， 用 4 / 0无损伤丝线间断缝合， 针距 4 mm, 修补 于犬顶部的缺损。 用圓针 4号丝线缝合肌肉。对照组釆用已商品化临床使用的 动物源性脑膜修补材料。 术后对动物进行常规的喂养及观察。 术后动物恢复良 好， 切口愈合良好， 无脑脊液漏， 无癫痫发生。 术后进食进水正常， 动物的户 外活动正常， 没有发现运动障碍， 存活至预定期限。
术后 18个月， 动物以手术部位为中心， 在大于手术部位 1cm范围切取标 本， 使其包括人工脑膜及周边硬脑膜及内面的脑组织。 切取出标本后， 可见人 工脑膜与硬脑膜的连接处对合平整， 无分界， 已完全愈合， 仅见缝合的丝线。 原生硬脑膜之间未见明显充血， 出血等排异反应。 而对照组植入部位仍可见未 降解的植入材料， 且且植入处脑膜内表面存在少许粘连。
实验例 3
用实施例 3制得的硬脑膜进行新西兰兔动物实验：
实验的动物施行顶部开颅，人为制造部分硬脑膜缺损及脑组织损伤， 然后 以人工脑膜分别实施硬脑膜修补术。 术后对动物进行常规的喂养及观察。 术后 动物恢复良好。 术后 18个月， 动物以手术部位为中心， 在大于手术部位 lcm 范围切取标本，使其包括人工脑膜及周边硬脑膜及内面的脑组织。切取出标本 后， 可见内侧表面可见上皮细胞覆盖， 上皮下可见纤维组织增生， 纤维母细胞 增生，胶原纤维增多，致使材料内带血运的新生组织增生，宿主新生组织侵入， 材料降解， 总量明显减少， 内部可见毛细血管。 新老组织界面无中性粒细胞， 淋巴细胞等炎症细胞反应， 界面处无嚢壁形成。 蛛网膜及脑组织正常。
实验例 4
用实施例 4制备的硬脑膜进行犬动物实验：具体动物实验方法同实验例 2。 术后 15个月， 动物以手术部位为中心， 在大于手术部位 lcm范围切取标 本， 使其包括人工脑膜及周边硬脑膜及内面的脑组织。 切取出标本后， 可见人 工脑膜与硬脑膜的连接处对合平整， 无分界， 已完全愈合， 仅见缝合的丝线。 原生硬脑膜之间未见明显充血， 出血等排异反应。
实验例 5
用实施例 5制得的硬脑膜进行新西兰兔动物实验，对照组釆用已商品化临 床使用的动物源性脑膜修补材料。
具体实验方法同实施例 3。 术后 15个月， 动物以手术部位为中心， 切取 标本，具体方法同实施例 3。切取出标本后，可见内侧表面可见上皮细胞覆盖， 上皮下可见纤维组织增生， 纤维母细胞增生， 胶原纤维增多， 致使材料内带血 运的新生组织增生， 宿主新生组织侵入， 材料降解， 总量明显减少， 内部可见 毛细血管。 新老组织界面无中性粒细胞， 淋巴细胞等炎症细胞反应， 界面处无 嚢壁形成。蛛网膜及脑组织正常。而对照组植入部位仍可见未降解的植入材料， 且植入处脑膜内表面存在少许粘连。
实验例 6
用实施例 6制得的硬脑膜进行犬动物实验：
具体实验方法同实施例 2。
术后 12个月， 动物以手术部位为中心， 在大于手术部位 lcm范围切取标 本， 使其包括人工脑膜及周边硬脑膜及内面的脑组织。 切取出标本后， 可见人 工脑膜与硬脑膜的连接处对合平整， 无分界， 已完全愈合， 仅见缝合的丝线。 原生硬脑膜之间未见明显充血， 出血等排异反应。
实验例 7
用实施例 7制得的硬脑膜进行新西兰兔动物实验：
具体实验方法同实施例 3。 术后 12个月， 动物以手术部位为中心， 在大 于手术部位 1cm范围切取标本， 使其包括人工脑膜及周边硬脑膜及内面的脑 组织。 切取出标本后， 可见内侧表面可见上皮细胞覆盖， 上皮下可见纤维组织 增生， 纤维母细胞增生， 胶原纤维增多， 致使材料内带血运的新生组织增生， 宿主新生组织侵入， 材料降解， 总量明显减少， 内部可见毛细血管。 新老组织 界面无中性粒细胞， 淋巴细胞等炎症细胞反应， 界面处无嚢壁形成。蛛网膜及 脑组织正常。
尽管已经示出和描述了本发明的实施例， 对于本领域的普通技术人员而 言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多 种变化、 修改、 替换和变型， 本发明的范围由所附权利要求及其等同物限定。

Claims

权 利 要 求
1. 一种人工硬脑膜， 由通过静电纺丝的方法制成的电纺层制成， 所述电 纺层为至少一层疏水性电纺层构成。
2. 如权利要求 1 所述的人工硬脑膜， 其中所述电纺层是由一种或多种疏 水性聚合物通过静电纺制备而成的， 所述疏水性聚合物选自包括以下物质的 组： 疏水性脂肪族聚酯类、 聚醚酯类、 聚原酸酯、 聚氨酯、 聚酸酐、 聚碑腈、 聚氨基酸， 或以上聚合物的两种及两种以上的共聚物及混合物。
3. 如权利要求 2所述的人工硬脑膜， 其中所述疏水性脂肪族聚酯类选自 下述组中的至少一种： 聚乳酸、 聚乙交酯、 聚己内酯、 聚羟基丁酸酯。
4. 如权利要求 2所述的人工硬脑膜， 其中所述聚醚酯类选自下述组中的 至少一种： 聚二氧杂环己酮、 乙二醇 /乳酸共聚物、 乙二醇 /对苯二曱酸丁二醇 酯共聚物。
5. 如权利要求 2所述的人工硬脑膜， 其中所述聚酸酐选自下述组中的至 少一种： 聚癸二酸 -十六烷二酸酸酐、 I型聚酸酐、 II型聚酸酐， III型聚酸酐、 IV型聚酸酐。
6. 如权利要求 1 所述的人工硬脑膜， 所述疏水性电纺层上进一步设置有 至少一层亲水性电纺层。
7. 如权利要求 6所述的人工硬脑膜， 其中所述亲水性电纺层是由一种或 多种亲水性聚合物通过静电纺制备而成的，所述亲水性聚合物选自包括以下物 质的组： 硫酸软骨素、 肝素、 琼脂、 葡聚糖、 褐藻酸、 改性纤维素、 海藻酸、 淀粉、 纤维素、 明胶、 纤维蛋白、 丝蛋白、 弹力蛋白拟态的肽聚合物、 胶原蛋 白、 壳聚糖、 改性壳聚糖、 亲水性聚氨酯、 聚乙二醇 、 聚曱基丙烯酸曱酯、 聚曱基丙烯酸曱酯、 聚羟基丁酸戊酸酯、 聚羟基丁酸己酸酯、 聚乙烯醇、 聚丙 交酯， 或以上材料的两种及两种以上的混合物。
8. 如权利要求 6所述的人工硬脑膜， 其中所述疏水性电纺层和亲水性电 纺层之间还设有过渡层。
9.如权利要求 8所述的人工硬脑膜， 其中所述过渡层是由一种或多种聚合 物通过静电纺制备而成的，所述过渡层对水的亲和力按照从亲水性电纺层向疏 水性电纺层的方向逐步增加。
10.如权利要求 2或 7或 9所述的人工硬脑膜， 其中所述聚合物中还混有 细胞因子和 /或药物。
11. 如权利要求 1或 6所述的人工硬脑膜，其中所述疏水性电纺层和 /或亲 水性电纺层上还附着有细胞因子和 /或药物。
12. 如权利要求 11 所述的人工硬脑膜， 其特征在于所述细胞因子选自包 括以下物质的组： 白细胞介素、 集落刺激因子、 肿瘤坏死因子、 血小板源生长 因子、 碱性成纤维细胞因子、 或以上因子的两种及两种以上的混合物。
13. 如权利要求 11所述的人工硬脑膜， 其特征在于所述药物选自抗生素、 止血剂、 防粘连剂或抗肿瘤药物的一种或几种。
14. 如权利要求 11所述的人工硬脑膜， 其中所述细胞因子和 /或药物被包 覆于水溶胶之中。
15. 如权利要求 14所述的人工硬脑膜， 其中所述水溶胶选自多糖类聚合 物， 多肽类聚合物， 合成的亲水高分子聚合物中的一种或几种。
16. 如权利要求 1或 6或 8中任意一项所述的人工硬脑膜， 其中所述疏水 性电纺层的纤维直径为 50~1000nM。
17. 如权利要求 16所述的人工硬脑膜， 其中所述疏水性电纺层的孔径小 于 3μΜ。
18. 如权利要求 6或 8中任意一项所述的人工硬脑膜， 其中所述亲水性电 纺层的纤维直径为 5~200μΜ, 孔径为 20~200μΜ。
19. 一种人工硬脑膜的制备方法， 包括以下步骤：
a) 将疏水性聚合物溶于溶剂中， 得到疏水性聚合物电纺溶液， 所述疏水 性聚合物选自包括以下物质的组：疏水性脂肪族聚酯类、聚醚酯类、聚原酸酯、 聚氨酯、 聚酸酐、 聚碑腈、 聚氨基酸， 或以上聚合物的两种及两种以上的共聚 物及混合物；
b) 通过静电纺，将所述疏水性聚合物电纺溶液制成膜状的疏水性电纺层， 得到所述人工硬脑膜。
20. 如权利要求 19所述的制备方法， 其中所述疏水性脂肪族聚酯类选自 下述组中的至少一种： 聚乳酸、 聚乙交酯、 聚己内酯、 聚羟基丁酸酯。
21. 如权利要求 19所述的制备方法， 其中所述聚醚酯类选自下述组中的 至少一种： 聚二氧杂环己酮、 乙二醇 /乳酸共聚物、 乙二醇 /对苯二曱酸丁二醇 酯共聚物。
22. 如权利要求 19所述的制备方法， 其中所述聚酸酐选自下述组中的至 少一种： 聚癸二酸 -十六烷二酸酸酐、 I型聚酸酐、 II型聚酸酐， III型聚酸酐、 IV型聚酸酐。
23. 如权利要求 19所述制备方法， 其中所述疏水性电纺层的纤维直径为 50~1000nM。
24. 如权利要求 23 所述制备方法， 其中所述疏水性电纺层的孔径小于 3μΜ。
25. 如权利要求 19所述制备方法， 其中步骤 b中所述静电纺的工艺参数 为： 微量注射泵的速率为 0.1 5.0毫升 /小时， 高压发生器的电压为 5 40 KV, 接收巨离为 5.0~30.0厘米。
26. 如权利要求 19所述制备方法， 其进一步包括在所述疏水性电纺层上 形成亲水性电纺层的步骤：
a' )将亲水性聚合物溶于溶剂中， 得到亲水性聚合物电纺溶液， 所述亲水 性聚合物选自包括以下物质的组： 硫酸软骨素、肝素、琼脂、 葡聚糖、褐藻酸、 改性纤维素、 海藻酸、 淀粉、 纤维素、 明胶、 纤维蛋白、 丝蛋白、 弹力蛋白拟 态的肽聚合物、 胶原蛋白、 壳聚糖、 改性壳聚糖、 亲水性聚氨酯、 聚乙二醇 、 聚曱基丙烯酸曱酯、 聚曱基丙烯酸曱酯、 聚羟基丁酸戊酸酯、 聚羟基丁酸己酸 酯、 聚乙烯醇或聚丙交酯、 或以上材料的两种及两种以上的混合物；
b' ) 通过静电纺， 将所述亲水性聚合物电纺溶液接收于所述疏水性电纺 层上， 形成亲水性电纺层。
27. 如权利要求 26 所述制备方法， 其中所述亲水性电纺层的纤维直径 5~200μΜ, 孔径为 20~200μΜ。
28. 如权利要求 26所述的制备方法， 其中步骤 b'中所述静电纺的工艺参 数为： 微量注射泵的速率为 0.1 20.0毫升 /小时， 高压发生器的电压为 10~45 KV, 接收距离为 5.0~30.0厘米。
29. 如权利要求 19或 26所述的制备方法， 其中所述溶剂选自包括以下物 质的组： 曱酸、 乙酸、 乙醇、 丙酮、 二曱基曱酰胺、 二曱基乙酰胺、 四氢呋喃、 二曱基亚砜、 六氟异丙醇、 三氟乙醇、 二氯曱烷、 三氯曱烷、 曱醇、 乙醇、 氯 仿、 二噁烷、 三氟乙烷、 三氟乙酸、 水、 或以上聚合物溶剂的两种及两种以上 的混合液。
30. 如权利要求 26所述制备方法， 其在形成所述亲水性电纺层之前， 还 包括在疏水性电纺层和亲水性电纺层之间将一种或多种聚合物电纺溶液通过 静电纺形成过渡层的步骤，所述过渡层对水的亲和力按照从亲水性电纺层向疏 水性电纺层的方向逐步增加。
31. 如权利要求 19或 26或 30所述的制备方法， 其中所述聚合物电纺溶 液中还混有细胞因子和 /或药物。
32. 如权利要求 19或 26所述的制备方法，还进一步包括在所述疏水性电 纺层和 /或亲水性电纺层上通过细胞打印形成细胞因子和 /或药物分布的步骤。
33. 如权利要求 32所述的制备方法， 其中所述细胞因子和 /或药物的细胞 打印步骤如下：
a" )将水溶胶溶液和细胞因子和 /或药物混合， 形成混合溶液； 和
b" ) 利用细胞打印技术将所述混合溶液打印到所述疏水性电纺层和 /或亲 水性电纺层上。
34. 如权利要求 33 所述的制备方法， 其中在所述打印步骤之前可包括将
35. 如权利要求 33 所述的制备方法， 其中所述水溶胶溶液是以多糖类聚 合物、 多肽类聚合物、合成亲水高分子聚合物制、 或以上材料的两种及两种以 上的混合物成的水溶液。
PCT/CN2010/070566 2009-03-10 2010-02-08 人工硬脑膜及其制备方法 WO2010102533A1 (zh)

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MX2011009282A MX2011009282A (es) 2009-03-10 2010-02-08 Duramadre artificial y su metodo de fabricacion.
BRPI1006250 BRPI1006250B1 (pt) 2009-03-10 2010-02-08 dura - máter artificial e método de produção da mesma
EP10750329.4A EP2340785B1 (en) 2009-03-10 2010-02-08 Artificial dura mater and manufacturing method thereof
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MX2015008387A MX345863B (es) 2009-03-10 2010-02-08 Uso de duramadre artificial para tratar y reparar tejido dural defectuos en un paciente.
US13/255,356 US8795708B2 (en) 2009-03-10 2010-02-08 Treating defective dura with synthetic artificial dura substitute
US14/225,051 US9211180B2 (en) 2009-03-10 2014-03-25 Method for treating defective dura mater
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US8795708B2 (en) 2014-08-05
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