WO2020252825A1 - 一种多层梯度生物膜及其制备方法 - Google Patents

一种多层梯度生物膜及其制备方法 Download PDF

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WO2020252825A1
WO2020252825A1 PCT/CN2019/095664 CN2019095664W WO2020252825A1 WO 2020252825 A1 WO2020252825 A1 WO 2020252825A1 CN 2019095664 W CN2019095664 W CN 2019095664W WO 2020252825 A1 WO2020252825 A1 WO 2020252825A1
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gelatin
biofilm
polycaprolactone
gradient
solution
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French (fr)
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李吉东
任欣
金蜀鄂
李玉宝
左奕
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四川大学
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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
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
    • D04H1/413—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties containing granules other than absorbent substances
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
    • D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
    • D04H1/4382—Stretched reticular film fibres; Composite fibres; Mixed fibres; Ultrafine fibres; Fibres for artificial leather
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces

Definitions

  • the invention belongs to the technical field of nanofiber membrane preparation, and particularly relates to a multilayer gradient biofilm and a preparation method thereof.
  • Electrospinning can simply and effectively prepare micro-nano fiber scaffolds.
  • This scaffold has a unique microstructure, appropriate mechanical properties, and a structure similar to natural extracellular matrix, so as to achieve bionic structural characteristics, so electrospinning becomes One of the most promising technologies in tissue engineering applications.
  • the nanofiber biofilm prepared by the electrospinning method can not only simulate the structure and environment of the extracellular matrix, but also adjust the composition, fiber diameter, porosity, etc. Therefore, the use of electrospinning to prepare nanofiber membrane materials has a wide range of applications in the field of biomedical materials.
  • the reported biomedical materials prepared by electrospinning include biofilms, wound coating materials, hemostatic materials, and artificial blood vessels. , Drug and gene delivery, tissue engineering scaffold, etc.
  • polymers that can prepare ultrafine nanofibers by electrospinning, including natural polymers and synthetic polymers, natural polymers such as gelatin, collagen, silk fibroin, chitosan, and hyaluronic acid. Acid, fibrin, etc., synthetic polymers such as polycaprolactone, polylactic acid, polyvinyl alcohol, polyurethane, polyamide, polylactic acid-glycolic acid copolymer, etc.
  • Natural polymers such as gelatin and collagen are common spinning materials, which have good biocompatibility, low antigenicity, biodegradability, and non-toxic side effects of degradation products in the body.
  • nanofiber membranes prepared from natural polymers such as gelatin and collagen have shortcomings such as low mechanical strength and excessive degradation speed, which limit their application. Therefore, it is necessary to combine synthetic polymers with better mechanical properties to strengthen and improve the properties of nanofiber membranes such as gelatin and collagen, so that they can be used in the field of medical biomaterials.
  • Polycaprolactone has good biocompatibility, good mechanical properties, etc., has been approved by the US Food and Drug Administration, and is widely used in the biomedical field. Therefore, blending polycaprolactone with gelatin, collagen, etc. for electrospinning can not only provide nanofiber membranes with good mechanical properties, but also make nanofiber membranes with good biocompatibility, thereby improving nanofiber membranes. The performance makes it more beneficial to be used in the biomedical field.
  • the current biofilms used to guide tissue regeneration can be divided into two categories: absorbable biofilms, mainly collagen films, chitosan films, polylactic acid films, polyglycolic acid films, and copolymers of polylactic acid and polyglycolic acid
  • absorbable biofilms mainly collagen films, chitosan films, polylactic acid films, polyglycolic acid films, and copolymers of polylactic acid and polyglycolic acid
  • These membranes have disadvantages such as poor mechanical properties, rapid degradation, and no isolation
  • non-absorbable biofilms mainly polytetrafluoroethylene membranes, expanded polytetrafluoroethylene and tetrafluoroethylene-hexafluoropropylene copolymer membranes , Titanium membranes, etc.
  • these membranes have stable physical and chemical properties and better isolation, they have disadvantages such as tearing of the tissue valve, exposure of the membrane, and secondary operations.
  • the purpose of the present invention is: the present invention provides a multi-layer gradient biofilm and a preparation method thereof.
  • natural polymers such as gelatin and collagen are blended with other synthetic polymers and a suitable crosslinking agent is used for controllable cross-linking.
  • Lianlai prepares nanofiber membranes with certain mechanical strength to solve the shortcomings of insufficient mechanical properties and excessive degradation of single natural polymers; on the other hand, by adding calcium phosphate salt to adjust the tissue regeneration performance of nanofiber membranes, In addition, the calcium phosphate added and the nanofiber membrane without calcium phosphate added are combined to form a multilayer nanofiber membrane, which not only provides better mechanical properties, but can simultaneously induce the regeneration of soft and hard tissues in both directions.
  • a multi-layer gradient biofilm comprising an upper, a middle, and a lower three layers of biofilm with a thickness of 0.1-2mm
  • the upper film is a gelatin/polycaprolactone nanofiber biofilm
  • the gelatin content is 40-90wt%
  • the middle layer film is a gelatin/polycaprolactone nanofiber biofilm, wherein the gelatin content is 1-50wt%
  • the lower layer film is a gelatin/polycaprolactone/apatite biofilm, wherein the gelatin content is 10-90wt% %, the apatite content is 1-40wt%.
  • the above-mentioned preparation method of multilayer gradient biofilm includes the following steps:
  • Adding apatite to an organic solvent for ultrasonic dispersion to obtain an apatite-containing organic solvent wherein the mass of apatite accounts for 5 to 35% of the total mass of the apatite-containing organic solvent; wherein the mass of apatite
  • the total mass of the organic solvent containing apatite is preferably 5-15%;
  • step S2 Mix gelatin and polycaprolactone according to the gelatin content of 10-90wt%, and dissolve the gelatin/polycaprolactone mixture in the apatite-containing organic solvent obtained in step S1 under constant stirring, wherein the gelatin/polycaprolactone
  • the mass-volume ratio of caprolactone and apatite-containing organic solvent is 6-20g: 100mL, preferably 10-14g: 100mL;
  • step S3 Add 1-3vt% acetic acid solution dropwise to the mixture obtained in step S2 and stir well to obtain spinning solution A;
  • step S4 The spinning solution A obtained in step S3 is used to prepare the lower membrane by the electrostatic spinning method;
  • step S6 Add 1-3vt% acetic acid solution dropwise to the mixed solution obtained in step S5 and stir thoroughly to obtain spinning solution B;
  • step S7 The spinning solution B obtained in step S6 is prepared on the basis of the lower film by electrostatic spinning to prepare the middle film;
  • step S9 Add 1-3vt% acetic acid solution dropwise to the mixed solution obtained in step S8 and stir well to obtain spinning solution C;
  • step S10 The spinning solution C obtained in step S9 is prepared on the basis of the middle layer film by electrostatic spinning to prepare the upper layer film to obtain a three-layer biofilm;
  • step S11 After drying the three-layer biofilm obtained in step S10, place it in a cross-linking solution for cross-linking for 0.5-72 hours, and then wash and dry it.
  • the multi-layer gradient biofilm of the present invention has a three-layer film structure.
  • Each biofilm contains gelatin and polycaprolactone in different proportions. Its mechanical properties, porosity, degradation behavior, etc. have adjustable characteristics,
  • the asymmetric membrane structure material with a certain gradient is prepared. Gradient biofilms with different degradation rates and mechanical properties can be designed and prepared according to the changes in gelatin and polycaprolactone content. The higher the gelatin content, the faster the degradation rate and the lower the mechanical properties. The higher the polycaprolactone content, the slower the degradation. The better the mechanical properties.
  • the upper layer of gelatin has the highest content, good cell and tissue compatibility, faster degradation, and can effectively guide soft tissue regeneration;
  • the middle layer has the highest content of polycaprolactone, slower degradation, and the highest tensile strength, acting as a mechanical support and barrier, and can maintain It guides the integrity of the membrane structure in the process of tissue regeneration, and can be used as a transition layer to connect the upper and lower membranes;
  • the lower layer contains calcium and phosphorus components, which give the membrane better biological activity and promote bone tissue regeneration.
  • the gelatin is one of pig skin gelatin, fish skin gelatin, type I collagen, type II collagen, type III collagen, type IV collagen, type VI collagen, type X collagen, and type XI collagen.
  • the apatite is one of hydroxyapatite, tricalcium phosphate, calcium hydrogen phosphate, calcium pyrophosphate, and octacalcium phosphate, and the particle size of the apatite is 20-200 nm, preferably 30-100 nm.
  • the organic solvent is trifluoroethanol or hexafluoroisopropanol.
  • polycaprolactone can be replaced with one of polylactic acid, polylactic acid-glycolic acid and polyurethane.
  • the electrospinning voltage is 4-12kV
  • the receiving plate distance is 10-20cm
  • the bolus speed is 0.1-1mL/h; preferably, the electrospinning voltage is 6-10kV, and the receiving plate distance is 13- 17cm, the bolus rate is 0.3-0.7mL/h.
  • the ultrasonic power is 200-400W and the ultrasonic time is 30-120min; preferably, the ultrasonic power is 250-350W and the ultrasonic time is 30-60min.
  • the cross-linking temperature in step S11 is 0-5°C
  • the cross-linking solution is an ethanol mixed solution with a cross-linking agent concentration of 5-100 mM
  • the cross-linking solution can also be mixed with ethanol water with a cross-linking agent concentration of 5-100 mM Solution in which the water content is 5vt%.
  • crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, genipin, glutaraldehyde and vanillin. At least one of.
  • a three-layer gradient biofilm is prepared by electrospinning technology, each layer has a thickness of 0.1-2mm, and the fiber diameter is distributed between 300-500nm.
  • the composite film has good biocompatibility, biodegradability and The ability to guide the growth of soft and hard tissues;
  • the upper, middle and lower three-layer films of the present invention all contain gelatin and polycaprolactone in different ratios, and their mechanical properties, porosity, degradation behavior, etc. have the characteristics of controllable, and they can be prepared with a certain gradient.
  • Symmetrical membrane structure material
  • the asymmetric structure of the upper and lower layers of the biofilm of the present invention can respectively induce the growth of soft and hard tissues.
  • the middle layer acts as a barrier to prevent soft tissue and connective tissue from growing into the bone defect area; on the other hand, it acts as a transitional connection It is tightly connected with the upper and lower membranes, which can provide better mechanical properties for the biofilm while preventing the separation of the upper and lower membranes;
  • the upper layer of the biofilm of the present invention has the highest gelatin content, faster degradation, and can effectively induce soft tissue regeneration;
  • the middle layer has the highest content of polycaprolactone, slower degradation, the highest tensile strength, acts as a mechanical support, barrier, and can maintain guidance
  • the integrity of the membrane structure in the process of tissue regeneration and can be used as a transition layer to connect the upper and lower membranes;
  • the lower layer contains calcium and phosphorus components, which gives the biofilm better biological activity and can promote bone tissue regeneration;
  • the biofilm of the present invention solves the shortcomings of insufficient mechanical properties of a single natural polymer and excessive degradation speed, and not only provides better mechanical properties, but also can simultaneously induce the regeneration of soft and hard tissues in both directions, which is better, It is more widely used in the field of guiding tissue regeneration.
  • Figure 1 is a scanning electron micrograph of the surface of a multilayer gradient biofilm prepared in Example 1 of the present invention
  • Example 2 is a scanning electron micrograph of a cross-section of a multilayer gradient biofilm prepared in Example 1 of the present invention
  • Figure 3 shows the stress-strain curves of polycaprolactone/gelatin biofilms with different gelatin content
  • Figure 4 shows the morphology of polycaprolactone/gelatin biofilm after 1 day of degradation in vitro
  • Figure 5 shows the morphology of the polycaprolactone/gelatin biofilm after 7 days of degradation in vitro
  • Figure 6 shows the morphology of polycaprolactone/gelatin biofilm after 84 days of degradation in vitro
  • Figure 7 is a scanning electron micrograph of bone marrow mesenchymal stem cells cultured on a three-layer gradient biofilm for 7 days;
  • Figure 8 is a scanning electron microscope image of bone marrow mesenchymal stem cells cultured on polycaprolactone/gelatin/hydroxyapatite for 7 days;
  • Figure 9 is a scanning electron microscope image of bone marrow mesenchymal stem cells cultured on polycaprolactone/gelatin for 7 days;
  • Figure 10 is a scanning electron micrograph of bone marrow mesenchymal stem cells cultured on polycaprolactone fiber membrane for 7 days;
  • Figure 11 is a scanning electron microscope image of gingival fibroblasts cultured on a three-layer gradient biofilm for 7 days;
  • Figure 12 is a scanning electron micrograph of gingival fibroblasts cultured on polycaprolactone/gelatin/hydroxyapatite for 7 days;
  • Figure 13 is a scanning electron microscope image of gingival fibroblasts cultured on polycaprolactone/gelatin for 7 days;
  • Figure 14 is a scanning electron micrograph of gingival fibroblasts cultured on polycaprolactone fiber membrane for 7 days.
  • first and “second” and other relational terms are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply one of these entities or operations. There is any such actual relationship or order between.
  • the terms “include”, “include” or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device that includes a series of elements includes not only those elements, but also includes Other elements of, or also include elements inherent to this process, method, article or equipment. If there are no more restrictions, the element defined by the sentence “including a" does not exclude the existence of other same elements in the process, method, article, or equipment including the element.
  • a multilayer gradient biofilm is prepared, and the specific steps of the preparation method are as follows:
  • the stainless steel plate with tin foil is used as the receiving device, and the solution C is spun to form the lower layer.
  • Biomembrane on the lower membrane, with a receiving distance of 15cm, a spinning voltage of 6kV for the positive electrode, -2kV for the negative electrode, and a bolus speed of 0.5mL/h as the spinning parameters, spin solution B to produce an intermediate layer of biofilm; finally in the middle
  • the receiving distance is 15cm
  • the spinning voltage is 8kV for the positive electrode, -2kV for the negative electrode
  • the injection speed is 0.5mL/h as the spinning parameters to spin solution A to form the upper biofilm.
  • Mark the upper and lower layers of the obtained three-layer biofilm dry it under vacuum at 25°C for 24h, and then cross-link it in ethanol solution containing 50mM EDC/50mM NHS for 24h. After the cross-linking is completed, wash it with absolute ethanol several times. Drying, packaging, sterilization, etc., can obtain the multilayer gradient biofilm of the present invention.
  • the thickness of the prepared biofilm is 0.2-0.3mm. After gold-plating and S-450 scanning electron microscope observation, the prepared biofilm is composed of fibers with a diameter of 300-500nm, forming a porous structure.
  • a multilayer gradient biofilm is prepared, and the specific steps of the preparation method are as follows:
  • the solution C is spun to form the lower layer Biomembrane; on the lower membrane, with a receiving distance of 15cm, a spinning voltage of 6kV for the positive electrode, -2kV for the negative electrode, and a bolus speed of 0.5mL/h as the spinning parameters, spin solution B to produce an intermediate layer of biofilm; finally in the middle On the layer of film, the receiving distance is 15cm, the spinning voltage is 8kV for the positive electrode, -2kV for the negative electrode, and the injection speed is 0.5mL/h as the spinning parameters to spin solution A to form the upper biofilm.
  • the thickness of the prepared biofilm is 0.2-0.3mm. After gold-plating and S-450 scanning electron microscope observation, the prepared biofilm is composed of fibers with a diameter of 300-500nm, forming a porous structure.
  • a multilayer gradient biofilm is prepared, and the specific steps of the preparation method are as follows:
  • the solution C is spun to form the lower layer Biomembrane; on the lower membrane, with a receiving distance of 15cm, a spinning voltage of 6kV for the positive electrode, -2kV for the negative electrode, and a bolus speed of 0.5mL/h as the spinning parameters, spin solution B to produce an intermediate layer of biofilm; finally in the middle On the layer of film, the receiving distance is 15cm, the spinning voltage is 8kV for the positive electrode, -2kV for the negative electrode, and the injection speed is 0.5mL/h as the spinning parameters to spin solution A to form the upper biofilm.
  • the thickness of the prepared biofilm is 0.2-0.3mm. After gold-plating and S-450 scanning electron microscope observation, the prepared biofilm is composed of fibers with a diameter of 300-500nm, forming a porous structure.
  • a multilayer gradient biofilm is prepared, and the specific steps of the preparation method are as follows:
  • the solution C is spun to form the lower layer Biomembrane; on the lower membrane, with a receiving distance of 15cm, a spinning voltage of 6kV for the positive electrode, -2kV for the negative electrode, and a bolus speed of 0.5mL/h as the spinning parameters, spin solution B to produce an intermediate layer of biofilm; finally in the middle On the layer of film, the receiving distance is 15cm, the spinning voltage is 8kV for the positive electrode, -2kV for the negative electrode, and the injection speed is 0.5mL/h as the spinning parameters to spin solution A to form the upper biofilm.
  • the thickness of the prepared biofilm is 0.2-0.3mm. After gold plating and S-450 scanning electron microscope observation, the prepared biofilm is composed of fibers with a diameter of 300-500nm and has a porous structure.
  • a multilayer gradient biofilm is prepared, and the specific steps of the preparation method are as follows:
  • the solution C is spun to form the lower layer Biomembrane; on the lower membrane, with a receiving distance of 15cm, a spinning voltage of 6kV for the positive electrode, -2kV for the negative electrode, and a bolus speed of 0.5mL/h as the spinning parameters, spin solution B to produce an intermediate layer of biofilm; finally in the middle On the layer of film, the receiving distance is 15cm, the spinning voltage is 8kV for the positive electrode, -2kV for the negative electrode, and the injection speed is 0.5mL/h as the spinning parameters to spin solution A to form the upper biofilm.
  • the thickness of the prepared biofilm is 0.2-0.3mm. After gold-plating and S-450 scanning electron microscope observation, the prepared biofilm is composed of fibers with a diameter of 300-500nm, forming a porous structure.
  • Example 1 of the present invention The surface and cross-section of the multilayer gradient biofilm prepared in Example 1 of the present invention were respectively observed by scanning electron microscopy. The results are shown in Figs. 1 and 2, and it can be seen that the fiber morphology is good, showing a typical multilayer structure.
  • the polycaprolactone/gelatin biofilm prepared by the preparation method of the present invention was subjected to an in vitro degradation test, and the morphology was observed on 1 day, 7 days, and 84 days respectively. The results are shown in Figures 4-6. After the fiber pores become larger, the morphology is maintained well.

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Abstract

一种多层梯度生物膜及其制备方法。该纳米纤维生物膜组成和结构上呈梯度变化，通过明胶与聚己内酯比例的变化构建梯度结构生物膜，实现力学性能、降解行为可调控，赋予该生物膜的上下层具有同步引导软、硬组织再生的功能。该多层梯度生物膜通过静电纺丝法制备，再通过干燥、交联、灭菌等工艺过程形成生物膜。该梯度生物膜具有可控的力学性能、降解行为以及良好的生物相容性，且能同时诱导软硬组织再生。

Description

一种多层梯度生物膜及其制备方法 技术领域
本发明属于纳米纤维膜制备技术领域，尤其涉及一种多层梯度生物膜的及其制备方法。
背景技术
通过使用适当的材料和技术来模拟人体组织的结构和组成是仿生法在生物材料工程和组织工程领域的重要目标之一。静电纺丝法能够简单有效的制备微纳米纤维支架，这种支架具有独特的微观结构和适当的力学性能以及与天然细胞外基质相近的结构，从而能够达到仿生的结构特点，因此静电纺丝成为组织工程应用方面最具有潜力的技术之一。此外，通过静电纺丝法制备的纳米纤维生物膜不仅能够模拟细胞外基质的结构和环境，而且可调节组成成分、纤维直径、孔隙率等。因此，采用静电纺丝法制备纳米纤维膜材料在生物医用材料领域有着广泛的应用前景，已经报道的通过静电纺丝法制备的生物医用材料有生物膜、伤口包敷材料、止血材料、人造血管、药物及基因输送、组织工程支架等。
目前能通过静电纺丝法制备超细纳米纤维的高分子有上百种，其中包括天然的高分子和人工合成高分子，天然高分子如明胶、胶原、丝素蛋白、壳聚糖、透明质酸、纤维蛋白等，人工合成高分子如聚己内酯、聚乳酸、聚乙烯醇、聚氨酯、聚酰胺、聚乳酸-羟基乙酸共聚物等。
明胶、胶原等天然高分子是常见的纺丝材料，其具有良好的生物相容性，以及具有低抗原性、生物降解性以及体内降解产物无毒副作用等优点。但是明胶、胶原等天然高分子制备的纳米纤维膜具有机械强度低，降解速度过快等缺点，限制了其应用。因此，需要结合力学性能较好的合成高分子来加强并改善明胶、胶原等纳米纤维膜的特性，使其能够应用在医用生物材料领域。而聚己内酯具有良好的生物相容性、良好的力学性能等，已经获得美国食品和药物管理局批准，并广泛应用于生物医学领域。因此，将聚己内酯与明胶、胶原等进行共混静电纺丝，既可以为纳米纤维膜提供良好的力学性能，也可以使纳米纤维膜具有良好的生物相容性，从而改善纳米纤维膜的性能，使其更有利应用于生物医学领域。
另外，目前用于引导组织再生的生物膜可以分为两大类：可吸收性生物膜，主要有胶原膜、壳聚糖膜、聚乳酸膜、聚羟基乙酸膜以及聚乳酸和聚羟基乙酸共聚物膜等，这些膜存在力学性能较差、降解过快、无隔离作用等缺点；不可吸收生物膜，主要有聚四氟乙烯膜，膨体聚四氟乙烯和四氟乙烯六氟丙烯共聚膜、钛膜等，这些膜虽然具有稳定的物理化学性能，以及更好的隔离作用，但是存在易导致组织瓣裂开、膜暴露，二次手术等缺点。现有技术中有可以用于组织诱导的生物医用材料，具有较好的生物降解性，良好的生物相容性，低细胞 毒性。但是这些应用于引导组织再生的纳米纤维膜多是单一的引导硬组织或软组织再生膜，而同时兼具诱导软硬组织再生的生物膜鲜有报道。
由于单一的材料和结构难以满足理想的引导组织再生的要求，因此将几种材料结合制备出具有不同成分和结构的生物膜具有很大的研究前景和开发价值。
发明内容
本发明的目的在于：本发明提供一种多层梯度生物膜及其制备方法，一方面通过将明胶、胶原等天然高分子与其它合成高分子进行混纺并采用适当的交联剂进行可控交联来制备具有一定力学强度的纳米纤维膜，以此来解决单一天然高分子力学性能不足和降解速度过快的缺点；另一方面通过添加磷酸钙盐来调节纳米纤维膜的诱导组织再生性能，并使添加磷酸钙盐与未添加磷酸钙盐的纳米纤维膜相结合组成多层纳米纤维膜，既提供了更好的力学性能又可以同步双向诱导软硬组织组织再生。
本发明采用的技术方案如下：
一种多层梯度生物膜，包括上、中、下三层厚度为0.1-2mm的生物膜，所述上层膜为明胶/聚己内酯纳米纤维生物膜，其中明胶含量为40-90wt％，所述中层膜为明胶/聚己内酯纳米纤维生物膜，其中明胶含量为1-50wt％，所述下层膜为明胶/聚己内酯/磷灰石生物膜，其中明胶含量为10-90wt％，磷灰石含量为1-40wt％。
上述的多层梯度生物膜的制备方法，包括以下步骤：
S1.将磷灰石加入有机溶剂中进行超声分散，得到含磷灰石的有机溶剂，其中磷灰石的质量占含磷灰石的有机溶剂总质量的5-35％；其中磷灰石质量占含磷灰石的有机溶剂的总质量优选为5-15％；
S2.将明胶和聚己内酯按照明胶含量为10-90wt％混合，在不断搅拌作用下将明胶/聚己内酯混合物溶于S1步骤所得含磷灰石的有机溶剂中，其中明胶/聚己内酯与含磷灰石的有机溶剂的质量体积比为6-20g：100mL，优选为10-14g：100mL；
S3.在S2步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液A；
S4.将S3步骤所得纺丝液A通过静电纺丝法制备下层膜；
S5.将明胶和聚己内酯按照明胶含量为1-50wt％混合，在25-45℃，不断搅拌作用下将明胶/聚己内酯混合物溶于有机溶剂中，其中明胶/聚己内酯与有机溶剂的质量体积比为6-20g：100mL，优选为10-14g：100mL；
S6.在S5步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液B；
S7.将S6步骤所得纺丝液B在下层膜的基础上通过静电纺丝法制备中层膜；
S8.将明胶和聚己内酯按照明胶含量为40-90wt％混合，在25-45℃，不断搅拌作用下将明 胶/聚己内酯混合物溶于有机溶剂中，其中明胶/聚己内酯与有机溶剂的质量体积比为6-20g：100mL，优选为10-14g：100mL；
S9.在S8步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液C；
S10.将S9步骤所得纺丝液C在中层膜的基础上通过静电纺丝法制备上层膜，得到三层生物膜；
S11.将S10步骤所得三层生物膜干燥后置于交联溶液中交联0.5-72h，清洗干燥即得。
本发明的多层梯度生物膜为三层膜结构，每层生物膜中均含明胶与聚己内酯且两者比例不同，其力学性能、孔隙率、降解行为等具有可调控的特点，能制备出具有一定梯度的不对称膜结构材料。可根据明胶与聚己内酯含量的变化设计制备不同降解速度、力学性能的梯度生物膜，明胶含量越高，降解速度越快、力学性能降低，聚己内酯含量越高，降解越慢、力学性能越好。上层明胶含量最高，细胞和组织相容性好，降解较快，能有效引导软组织再生；中间层聚己内酯含量最高，降解较慢、拉伸强度最高，起力学支撑、屏障作用，能维持引导组织再生过程中膜结构的完整性，且能作为过渡层连接上下两层膜；下层则含有钙磷成分，赋予膜更好的生物活性，促进骨组织再生。
进一步地，明胶为猪皮明胶、鱼皮明胶、I型胶原、II型胶原、III型胶原、IV型胶原、VI型胶原、X型胶原、XI型胶原中的一种。
进一步地，磷灰石为羟基磷灰石、磷酸三钙、磷酸氢钙、焦磷酸钙和磷酸八钙中的一种，且磷灰石的粒径为20-200nm，优选为30-100nm。
进一步地，有机溶剂为三氟乙醇或六氟异丙醇。
进一步地，聚己内酯可替换为聚乳酸、聚乳酸-羟基乙酸和聚氨酯中的一种。
进一步地，静电纺丝的电压为4-12kV，接收板距离为10-20cm，推注速度为0.1-1mL/h；优选地，静电纺丝的电压为6-10kV，接收板距离为13-17cm，推注速度为0.3-0.7mL/h。
进一步地，S1步骤中超声功率为200-400W，超声时间30-120min；优选地，超声功率为250-350W，超声时间30-60min。
进一步地，S11步骤中交联温度为0-5℃，交联溶液为交联剂浓度为5-100mM的乙醇混合溶液；交联溶液还可采用交联剂浓度为5-100mM的乙醇水混合溶液，其中水含量为5vt％。
进一步地，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、京尼平、戊二醛和香草醛中的至少一种。
综上所述，由于采用了上述技术方案，本发明的有益效果是：
1、本发明通过静电纺丝技术制备了三层梯度生物膜，每层的厚度为0.1-2mm，纤维直径分布在300-500nm之间，复合膜具有良好的生物相容性、生物降解性以及引导软硬组织生长 的能力；
2、本发明的上中下三层膜中均含明胶与聚己内酯且两者比例不同，其力学性能、孔隙率、降解行为等具有可调控的特点，能制备出具有一定梯度的不对称膜结构材料；
3、本发明的生物膜的上、下层的不对称结构可以分别诱导软、硬组织生长，中间层一方面起到屏障作用，防止软组织和结缔组织长入骨缺损区；另一方面起到过渡衔接作用，与上下两层膜紧密连接，在防止上下两层膜出现分离情况的同时能够为生物膜提供更好的力学性能；
4、本发明的生物膜上层明胶含量最高，降解较快，能有效诱导软组织再生；中间层聚己内酯含量最高，降解较慢、拉伸强度最高，起力学支撑、屏障作用，能维持引导组织再生过程中膜结构的完整性，且能作为过渡层连接上下两层膜；下层则含有钙磷成分，赋予生物膜更好的生物活性，能促进骨组织再生；
5、本发明所述的生物膜解决了单一天然高分子力学性能不足和降解速度过快的缺点，且既提供了更好的力学性能又可以同步双向诱导软硬组织组织再生，能够更好、更广泛的应用于引导组织再生领域。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明实施例1制得的多层梯度生物膜表面的扫描电镜图；
图2为本发明实施例1制得的多层梯度生物膜断面的扫描电镜图；
图3为不同明胶含量的聚己内酯/明胶生物膜进行应力-应变曲线；
图4为聚己内酯/明胶生物膜体外降解1天后的形貌图；
图5为聚己内酯/明胶生物膜体外降解7天后的形貌图；
图6为聚己内酯/明胶生物膜体外降解84天后的形貌图；
图7为骨髓间充质干细胞在三层梯度生物膜上培养7天后的扫描电镜图；
图8为骨髓间充质干细胞在聚己内酯/明胶/羟基磷灰石上培养7天后的扫描电镜图；
图9为骨髓间充质干细胞在聚己内酯/明胶上培养7天后的扫描电镜图；
图10为骨髓间充质干细胞在聚己内酯纤维膜上培养7天后的扫描电镜图；
图11为牙龈成纤维细胞在三层梯度生物膜上培养7天后的扫描电镜图；
图12为牙龈成纤维细胞在聚己内酯/明胶/羟基磷灰石上培养7天后的扫描电镜图；
图13为牙龈成纤维细胞在聚己内酯/明胶上培养7天后的扫描电镜图；
图14为牙龈成纤维细胞在聚己内酯纤维膜上培养7天后的扫描电镜图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明，即所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件可以以各种不同的配置来布置和设计。
因此，以下对在附图中提供的本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，术语“第一”和“第二”等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本发明较佳实施例提供的一种多层梯度生物膜，其制备方法具体步骤如下：
在25℃，搅拌作用下将0.06g聚己内酯和0.54g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液A；在25℃，搅拌作用下将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液B；在25℃下，将0.0325g纳米羟基磷灰石加入到5mL三氟乙醇溶液中，在350W下超声振荡60min，然后在搅拌作用下将0.455g聚己内酯和0.195g明胶溶于羟基磷灰石的三氟乙醇溶液中，制得纳米羟基磷灰石/聚己内酯/明胶的纺丝溶液C。在接受距离15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h等纺丝参数下， 以贴有锡箔纸的不锈钢板为接收装置，先进行溶液C的纺丝，生成下层生物膜；再在下层膜上以接受距离15cm，纺丝电压正极6kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液B的纺丝，生成中间层生物膜；最后在中间层膜上以接受距离为15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液A的纺丝，形成上层生物膜。将所得三层生物膜标明上下层，再于真空、25℃下干燥24h，然后在含有50mM EDC/50mM NHS的乙醇溶液中交联24h，交联完成后用去无水乙醇清洗数次，再干燥，包装，灭菌等，即得到本发明所述的多层梯度生物膜。
用游标卡尺测定，制备的生物膜的厚度为0.2-0.3mm，经镀金，S-450型扫描电镜观察，制备的生物膜由直径为300-500nm的纤维组成，成多孔结构。
实施例2
本发明较佳实施例提供的一种多层梯度生物膜，其制备方法具体步骤如下：
在25℃，搅拌作用下将0.18g聚己内酯和0.42g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液A；在25℃，搅拌作用下将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液B；在25℃下，将0.065g纳米羟基磷灰石加入到5mL三氟乙醇溶液中，在350W下超声振荡60min，然后在搅拌作用下将0.455g聚己内酯和0.195g明胶溶于羟基磷灰石的三氟乙醇溶液中，制得纳米羟基磷灰石/聚己内酯/明胶的纺丝溶液C。在接受距离15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h等纺丝参数下，以贴有锡箔纸的不锈钢板为接收装置，先进行溶液C的纺丝，生成下层生物膜；再在下层膜上以接受距离15cm，纺丝电压正极6kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液B的纺丝，生成中间层生物膜；最后在中间层膜上以接受距离为15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液A的纺丝，形成上层生物膜。将所得三层生物膜标明上下层，再于真空、25℃下干燥24h，然后在含有50mM EDC/50mM NHS的乙醇溶液中交联24h，交联完成后用去无水乙醇清洗数次，再干燥，包装，灭菌等，即得到本发明所述的多层梯度生物膜。
用游标卡尺测定，制备的生物膜的厚度为0.2-0.3mm，经镀金，S-450型扫描电镜观察，制备的生物膜由直径为300-500nm的纤维组成，成多孔结构。
实施例3
本发明较佳实施例提供的一种多层梯度生物膜，其制备方法具体步骤如下：
在25℃，搅拌作用下将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液A；在25℃，搅拌作用下 将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液B；在25℃下，将0.0975g纳米羟基磷灰石加入到5mL三氟乙醇溶液中，在350W下超声振荡60min，然后在搅拌作用下将0.455g聚己内酯和0.195g明胶溶于羟基磷灰石的三氟乙醇溶液中，制得纳米羟基磷灰石/聚己内酯/明胶的纺丝溶液C。在接受距离15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h等纺丝参数下，以贴有锡箔纸的不锈钢板为接收装置，先进行溶液C的纺丝，生成下层生物膜；再在下层膜上以接受距离15cm，纺丝电压正极6kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液B的纺丝，生成中间层生物膜；最后在中间层膜上以接受距离为15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液A的纺丝，形成上层生物膜。将所得三层生物膜标明上下层，再于真空、25℃下干燥24h，然后在含有50mM EDC/50mM NHS的乙醇溶液中交联24h，交联完成后用去无水乙醇清洗数次，再干燥，包装，灭菌等，即得到本发明所述的多层梯度生物膜。
用游标卡尺测定，制备的生物膜的厚度为0.2-0.3mm，经镀金，S-450型扫描电镜观察，制备的生物膜由直径为300-500nm的纤维组成，成多孔结构。
实施例4
本发明较佳实施例提供的一种多层梯度生物膜，其制备方法具体步骤如下：
在25℃，搅拌作用下将0.42g聚己内酯和0.18g明胶溶于5ml三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液A；在25℃，搅拌作用下将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液B；在25℃下，将0.0325g纳米羟基磷灰石加入到5mL三氟乙醇溶液中，在350W下超声振荡60min，然后在搅拌作用下将0.455g聚己内酯和0.195g明胶溶于羟基磷灰石的三氟乙醇溶液中，制得纳米羟基磷灰石/聚己内酯/明胶的纺丝溶液C。在接受距离15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h等纺丝参数下，以贴有锡箔纸的不锈钢板为接收装置，先进行溶液C的纺丝，生成下层生物膜；再在下层膜上以接受距离15cm，纺丝电压正极6kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液B的纺丝，生成中间层生物膜；最后在中间层膜上以接受距离为15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液A的纺丝，形成上层生物膜。将所得三层生物膜标明上下层，再于真空、25℃下干燥24h，然后在含有50mM EDC/50mM NHS的乙醇溶液中交联24h，交联完成后用去无水乙醇清洗数次，再干燥，包装，灭菌等，即得到本发明所述的多层梯度生物膜。
用游标卡尺测定，制备的生物膜的厚度为0.2-0.3mm，经镀金，S-450型扫描电镜观察， 制备的生物膜由直径为300-500nm的纤维组成，成多孔结构。
实施例5
本发明较佳实施例提供的一种多层梯度生物膜，其制备方法具体步骤如下：
在25℃，搅拌作用下将0.54g聚己内酯和0.06g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液A；在25℃，搅拌作用下将0.30g聚己内酯和0.30g明胶溶于5mL三氟乙醇中，待其完全溶解后加入0.015mL乙酸并充分搅拌，制得聚己内酯/明胶的纺丝溶液B；在25℃下，将0.065g纳米羟基磷灰石加入到5mL三氟乙醇溶液中，在350W下超声振荡60min，然后在搅拌作用下将0.455g聚己内酯和0.195g明胶溶于羟基磷灰石的三氟乙醇溶液中，制得纳米羟基磷灰石/聚己内酯/明胶的纺丝溶液C。在接受距离15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h等纺丝参数下，以贴有锡箔纸的不锈钢板为接收装置，先进行溶液C的纺丝，生成下层生物膜；再在下层膜上以接受距离15cm，纺丝电压正极6kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液B的纺丝，生成中间层生物膜；最后在中间层膜上以接受距离为15cm，纺丝电压正极8kV、负极-2kV，推注速度0.5mL/h为纺丝参数进行溶液A的纺丝，形成上层生物膜。将所得三层生物膜标明上下层，再于真空、25℃下干燥24h，然后在含有50mM EDC/50mM NHS的乙醇溶液中交联24h，交联完成后用去无水乙醇清洗数次，再干燥，包装，灭菌等，即得到本发明所述的多层梯度生物膜。
用游标卡尺测定，制备的生物膜的厚度为0.2-0.3mm，经镀金，S-450型扫描电镜观察，制备的生物膜由直径为300-500nm的纤维组成，成多孔结构。
实验例1
对本发明实施例1制得的多层梯度生物膜的表面和断面分别进行扫描电镜观察，结果如图1和图2所示，可见纤维形貌良好，呈典型的多层结构。
实验例2
对采用本发明的制备方法制备的不同明胶含量的聚己内酯/明胶生物膜进行应力-应变曲线测定，结果如图3所示，可知当聚己内酯含量为90％、明胶含量为10％时纤维膜拉伸强度最大。
实验例3
对采用本发明的制备方法制备的聚己内酯/明胶生物膜进行体外降解试验，分别在1天、7天、84天观察其形貌，结果如图4-6所示，可知较长时间后纤维孔隙变大，形貌保持良好。
实验例4
分别用骨髓间充质干细胞在三层梯度生物膜(A)、聚己内酯/明胶/羟基磷灰石(B)、聚己内酯/明胶(C)及聚己内酯纤维膜(D)上的培养，培养7天后进行扫描电镜观察，结果如图7-10 所示，可知骨髓间充质干细胞在梯度生物膜上生长良好。
实验例5
分别用牙龈成纤维细胞在三层梯度生物膜(A)、聚己内酯/明胶/羟基磷灰石(B)、聚己内酯/明胶(C)及聚己内酯纤维膜(D)上的培养，培养7天后进行扫描电镜观察，结果如图11-14所示，可知牙龈成纤维细胞在梯度生物膜上生长良好。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种多层梯度生物膜，其特征在于：包括上、中、下三层厚度为0.1-2mm的生物膜，所述上层膜为明胶/聚己内酯纳米纤维生物膜，其中明胶含量为40-90wt％，所述中层膜为明胶/聚己内酯纳米纤维生物膜，其中明胶含量为1-50wt％，所述下层膜为明胶/聚己内酯/磷灰石生物膜，其中明胶含量为10-90wt％，磷灰石含量为1-40wt％。
  2. 权利要求1所述的多层梯度生物膜的制备方法，其特征在于，包括以下步骤：
    S1.将磷灰石加入有机溶剂中进行超声分散，得到含磷灰石的有机溶剂，其中磷灰石的质量占含磷灰石的有机溶剂总质量的5-35％；
    S2.将明胶和聚己内酯按照明胶含量为10-90wt％混合，在不断搅拌作用下将明胶/聚己内酯混合物溶于S1步骤所得含磷灰石的有机溶剂中，其中明胶/聚己内酯与含磷灰石的有机溶剂的质量体积比为6-20g：100mL；
    S3.在S2步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液A；
    S4.将S3步骤所得纺丝液A通过静电纺丝法制备下层膜；
    S5.将明胶和聚己内酯按照明胶含量为1-50wt％混合，在25-45℃，不断搅拌作用下将明胶/聚己内酯混合物溶于有机溶剂中，其中明胶/聚己内酯与有机溶剂的质量体积比为6-20g：100mL；
    S6.在S5步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液B；
    S7.将S6步骤所得纺丝液B在下层膜的基础上通过静电纺丝法制备中层膜；
    S8.将明胶和聚己内酯按照明胶含量为40-90wt％混合，在25-45℃，不断搅拌作用下将明胶/聚己内酯混合物溶于有机溶剂中，其中明胶/聚己内酯与有机溶剂的质量体积比为6-20g：100mL；
    S9.在S8步骤所得混合液中滴加1-3vt％的乙酸溶液并充分搅拌，得到纺丝液C；
    S10.将S9步骤所得纺丝液C在中层膜的基础上通过静电纺丝法制备上层膜，得到三层生物膜；
    S11.将S10步骤所得三层生物膜干燥后置于交联溶液中交联0.5-72h，清洗干燥即得。
  3. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述明胶为猪皮明胶、鱼皮明胶、I型胶原、II型胶原、III型胶原、IV型胶原、VI型胶原、X型胶原和XI型胶原中的一种。
  4. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述磷灰石为羟基磷灰石、磷酸三钙、磷酸氢钙、焦磷酸钙和磷酸八钙中的一种，且所述磷灰石的粒径为20-200nm。
  5. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述有机溶剂为三 氟乙醇或六氟异丙醇。
  6. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述聚己内酯可替换为聚乳酸、聚乳酸-羟基乙酸和聚氨酯中的一种。
  7. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述静电纺丝的电压为4-12kV，接收板距离为10-20cm，推注速度为0.1-1mL/h。
  8. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述S1步骤中超声功率为200-400W，超声时间30-120min。
  9. 根据权利要求2所述的多层梯度生物膜的制备方法，其特征在于：所述S11步骤中交联温度为0-5℃，交联溶液为交联剂浓度为5-100mM的乙醇混合溶液。
    根据权利要求9所述的多层梯度生物膜的制备方法，其特征在于：所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、京尼平、戊二醛和香草醛中的至少一种。
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