CN106110405A - A kind of functionalization guides muscular tissue regeneration membrane and its preparation method and application - Google Patents

A kind of functionalization guides muscular tissue regeneration membrane and its preparation method and application Download PDF

Info

Publication number
CN106110405A
CN106110405A CN201610461624.2A CN201610461624A CN106110405A CN 106110405 A CN106110405 A CN 106110405A CN 201610461624 A CN201610461624 A CN 201610461624A CN 106110405 A CN106110405 A CN 106110405A
Authority
CN
China
Prior art keywords
spinning
solution
polycaprolactone
dissolved
regeneration membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610461624.2A
Other languages
Chinese (zh)
Other versions
CN106110405B (en
Inventor
陈春英
刘颖
刘柱
郭梦雨
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Center for Nanosccience and Technology China
Original Assignee
National Center for Nanosccience and Technology China
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
Application filed by National Center for Nanosccience and Technology China filed Critical National Center for Nanosccience and Technology China
Priority to CN201610461624.2A priority Critical patent/CN106110405B/en
Publication of CN106110405A publication Critical patent/CN106110405A/en
Application granted granted Critical
Publication of CN106110405B publication Critical patent/CN106110405B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • 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
    • 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
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic 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
    • D04H1/4326—Condensation or reaction polymers
    • D04H1/435—Polyesters
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/30—Materials or treatment for tissue regeneration for muscle reconstruction

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Surgery (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Vascular Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Textile Engineering (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses a kind of functionalization and guide muscular tissue regeneration membrane and its preparation method and application.This regeneration membrane is with polycaprolactone as major matrix material, the film being prepared as by electrostatic spinning；Preferably on film, form fine and close poly-norepinephrine layer by polyreaction；In conjunction with the protein promoting cell growth.In the present invention feature of membrane material be diameter range be 1 20 μm fiber composition multilayer porous film material.This regeneration membrane preparation technology is simple, can effectively facilitate the Regeneration and Repair of muscular tissue, has good biocompatibility, mechanical performance excellence, can be widely used in the medical fields such as guiding skin damage reparation, muscle damage reparation, medical releasing film.

Description

A kind of functionalization guides muscular tissue regeneration membrane and its preparation method and application
Technical field
The invention belongs to technical field of biological material, be specifically related to a kind of using polycaprolactone as major matrix material and on surface Membrane material guiding muscular tissue regeneration coating medicine and its preparation method and application.
Background technology
Skeletal muscle is the vital tissue of human body, and the main reason of the impaired particularly fibro-muscular of skeletal muscle is impaired flesh Meat multiplication capacity is less than fibrosis, and all the time, skeletal muscle is impaired faces lot of challenges and difficulty in treatment.For organizational project Speech, the selection of embedded material is suitable important.Different from natural polymer, synthesis macromolecule molecule instrument physicochemical property can Control adjustable, quality stability is excellent, low price, obtains well application at biomedical sector.At present, lead in biological medicine What territory application had comparative advantage is biodegradable polyesters, and such as PLGA, PLA, PCL and PGA, these are all FDA approval of certification, Particularly polycaprolactone is a kind of to be relatively easily available with inexpensive Biodegradable polymer material PCL also than PGA and PLGA It is a kind of elastomeric material, its good mechanical performance, it is widely used in bone tissue engineer field.It is connected owing to there are 5 methylene Short carbon chain make it have the strongest hydrophobicity, need its surface is processed to strengthen hydrophilic.And norepinephrine Being a kind of clinical medicine, containing catechol and hydroxyl, poly-norepinephrine can be with substrate P C with covalent bond jail as tie It is solidly connected and also grappling can stick associated protein, change substrate hydrophilic, promote the absorption of albumen, thus promote the increasing of muscle cell Grow and unfold, be more beneficial for the impaired reparation of skeletal muscle.
Electrostatic spinning technique is a kind of simple general-purpose method preparing nanometer and micrometer fibers, due to its medicine load mode Simple, in electrostatic spinning or spinning last handling process, different medicines and biomacromolecule are easy to be loaded into fiber table Face, it addition, the medicine of muscle reparation will not occur performance to change after being coated onto fiber surface, remains to the property keeping its muscle to repair Can, can be used to the wound of the muscle such as prosthesis.Therefore, the nano-micrometre fiber functional membrane that prepared by Electrospun has good Potential applicability in clinical practice.
Summary of the invention
For the deficiencies in the prior art, present invention aim at providing a kind of functionalization to guide muscular tissue regeneration membrane and system thereof Preparation Method and application.
The technical scheme realizing the object of the invention is as follows:
A kind of functionalization guides muscular tissue regeneration membrane, and it is with polycaprolactone as major matrix material, passes through electrostatic spinning The film that technology is prepared as.
Further, above-mentioned functionsization guiding muscular tissue regeneration membrane forms fine and close noradrenaline by polyreaction Element layer；And combine the protein promoting cell growth.
Preferably, the protein of described promotion cell growth is basic fibroblast growth factor, insulin-like growth One or more in the somatomedin being correlated with in factor family (IGF), heparin, transforming growth factor (TGF), platelet source etc..
Wherein, basic fibroblast growth factor is widely distributed in Skeletal Muscle Cell, can regulate skeletal muscle satellite thin The proliferation and differentiation activity of born of the same parents, promotes its cultivation effect.Insulin like growth factor family (IGF), including insulin-like growth factor Sub-II and insulin-like growth factor I two kinds, after the damage of skeletal muscle satellite cell, IGF plays and significantly promotes that regeneration is made With.Heparin is an important cytokine, can promote that satellite cell is bred, it is also possible to activate the satellite cell of dormancy.Convert Somatomedin (TGF) family mainly includes TGF-α and TGF-β, and TGF-β is divided into again TGF-β 1, TGF-β 2 and TGF-β 3, and it is external The deposition of substrate promotes have facilitation.Additionally, the somatomedin being correlated with in platelet source is maintaining tissue growth and function to live Property aspect plays an important role.The mutual transmission by intercellular signal of the protein of above-mentioned promotion cell growth, damages at skeletal muscle The repair process of wound plays the part of important role.
Preferably, described regeneration membrane has the micron order fibre structure of random arrangement, and average bridging aperture is 1-16 μm.
Preferably, the multilayer porous film that described regeneration membrane is made up of the fiber that diameter range is 1-20 μm.
Preferably, described regeneration membrane film thickness is 50-500 μm.
The present invention also provides for above-mentioned functionsization and guides the preparation method of muscular tissue regeneration membrane, comprises the following steps: will be poly-own Lactone is dissolved in organic solvent, prepares solution A；Then carry out electrostatic spinning, prepare electricity spinning fibre film, be drying to obtain functionalization Guide muscular tissue regeneration membrane.
Further, above-mentioned preparation method also includes norepinephrine and promotes that the protein of cell growth is dissolved in In Tris-HCl solution or PBS, prepare B solution；Gained electricity spinning fibre film is immersed in a timing in above-mentioned B solution Between, it is dried after taking-up, to obtain final product.
Described being dried can be air-dried, lyophilization or vacuum drying.
Preferably, described organic solvent is chloroform, or the mixture of chloroform and ethanol.Ethanol is 7 with the volume ratio of chloroform: 10-7:1, such as, can be 7:10,7:9,7:8,7:7,7:6,7:5,7:4,7:3,7:2 or 7:1.The mol ratio of the two is limited It is in order to avoid affecting spinning process because certain solution is too much or very few in above-mentioned scope.
Preferably, in described solution A, the mass fraction of polycaprolactone is 3%-45%, such as, can be 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.The height of polycaprolactone concentration directly affects the diameter of fiber And spinning process parameters.
Specifically, described electrostatic spinning comprises the following steps:
A, above-mentioned solution A is placed in syringe, utilizes propeller to promote, after droplets stable flows down, heighten electricity Pressure；
B, with rustless steel roller bearing for receive device, continue spinning obtain complete electricity spinning fibre film.
Preferably, angle of rake fltting speed described in step a (i.e. spinning liquid flow rate) is 1-15mL/h, the most permissible It is 1mL/h, 2mL/h, 3mL/h, 5mL/h, 6mL/h, 8mL/h, 10mL/h, 12mL/h or 15mL/h.Research finds, excessive velocities Spinning solution can be caused to ooze or fibre diameter is excessive, speed is crossed and then can be lengthened the spinning time slowly.
Preferably, voltage described in step a is 7-20kV, such as, can be 7kV, 8kV, 10kV, 12kV, 15kV, 18kV or 20kV.Voltage limits the smooth formation being to ensure that continuous print fiber in this range, and brownout spinning solution cannot Forming fiber, overtension then makes spinning process unstable, causes spinning discontinuous.
Preferably, receiving range described in step b is 8-30cm, such as, can be 8cm, 10cm, 12cm, 15cm, 18cm, 20cm, 22cm, 25cm or 30cm.
Preferably, the speed of rustless steel roller bearing described in step b is 100-2000rmp, such as, can be 100rmp, 200rmp, 400rmp, 500rmp, 800rmp, 1000rmp, 1200rmp, 1500rmp, 1800rmp or 2000rmp.
Preferably, the spinning time described in step b is 2-30h, such as, can be 2h, 4h, 8h, 10h, 15h, 20h, 25h or 30h。
Preferably, in above-mentioned B solution, solvent is Tris-HCl solution or PBS solution, and pH value is respectively 8-10, the most permissible It is 8,8.5,9,9.5 or 10.Wherein, Tris-HCl solution uses Tris configuration, and PBS uses Na2HPO4、KH2PO4、 NaCl and KCl configures, and regulates pH value with HCl or NaOH.
Preferably, in above-mentioned B solution, the concentration of norepinephrine and promotion cell growth cytokine is respectively 0.1- 10mg/ml, such as, can be 0.1mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 4mg/ml, 6mg/ml, 8mg/ Ml or 10mg/ml.
Specifically, above-mentioned functionsization guides the preparation method of muscular tissue regeneration membrane, comprises the following steps:
(1) fibre diameter be 1-8 micron functionalization guide muscular tissue regeneration membrane preparation
(1) being dissolved in chloroform by polycaprolactone, room temperature magnetic agitation 12-24h, obtaining mass fraction is the poly-own of 3-45% Lactone solution；
(2) above-mentioned polycaprolactone solution is carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate For 100-2000rpm, spinning liquid flow rate is 1-15mL/h, voltage 7-20kV, receiving range 8-30cm, spinning 2-30h, Electricity spinning fibre film to thickness 50-500 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes cell growth cytokine 12-24h in Tris-HCl solution, after taking-up, in fume hood, room temperature is placed 2-7 days, to obtain final product；
(2) fibre diameter be 8-20 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7 with the volume ratio of ethanol: 3), room temperature magnetic agitation 12-24h, obtain the polycaprolactone solution that mass fraction is 14-30%；
(2) above-mentioned polycaprolactone solution is carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate For 700-2600rpm, spinning liquid flow rate is 2-20mL/h, voltage 12-25kV, receiving range 10-35cm, spinning 1-30h, Obtain the electricity spinning fibre film of thickness 100-600 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes cell growth cytokine 12-24h in Tris-HCl solution, after taking-up, in fume hood, room temperature is placed 2-7 days, to obtain final product.
The present invention also provides for above-mentioned functionsization and guides the another kind of preparation method of muscular tissue regeneration membrane, comprises the following steps:
(3) fibre diameter be 1-8 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7 with the volume ratio of ethanol: 3), in, room temperature magnetic agitation 12-24h, obtain the polycaprolactone solution that mass fraction is 5-19%；
(2) above-mentioned polycaprolactone solution is carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate For 100-2000rpm, spinning liquid flow rate is 0.5-10mL/h, voltage 7-20kV, receiving range 8-30cm, spinning 0.5- 30h, obtains the electricity spinning fibre film of thickness 25-250 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes cell growth cytokine 12-24h in PBS buffer solution, then, is vacuum dried it 4-12h, to obtain final product after freezing 6-12h at-60 DEG C～-20 DEG C；
(4) fibre diameter be 8-20 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7 with the volume ratio of ethanol: 3), room temperature magnetic agitation 12-24h, obtain the polycaprolactone solution that mass fraction is 13-29%；
(2) above-mentioned polycaprolactone solution is carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate For 500-2000rpm, spinning liquid flow rate is 0.5-10mL/h, voltage 14-30kV, receiving range 10-30cm, spinning 1.5- 30h, obtains the electricity spinning fibre film of thickness 70-400 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes cell growth cytokine 15-24h in PBS buffer solution, then, is vacuum dried it 4-12h, to obtain final product after freezing 6-12h at-60 DEG C～-20 DEG C.
Present invention additionally comprises above-mentioned functionsization guides muscular tissue regeneration membrane to promote the medicine of muscular tissue Regeneration and Repair in preparation In application.
Functionalization of the present invention guides muscular tissue regeneration membrane to be with polycaprolactone as main raw material(s) and coat promotion flesh The medicine that meat is repaired, is prepared by the method for electrostatic spinning and guides muscular tissue regeneration membrane；Inventive film material has the life of excellence The thing compatibility, mechanical performance and the Regeneration and Repair of muscular tissue can be effectively promoted.
Accompanying drawing explanation
Fig. 1 is scanning electron microscope (SEM) photo that embodiment 1-4 prepares film.2P, 2PP represent the PCL of 1-3 μ m diameter respectively The PCL fibrous membrane of the 1-3 μ m diameter of fibrous membrane and the poly-methylepinephrine of surface aggregate.10P, 10PP represent 8-12 μ m diameter respectively PCL fibrous membrane and the PCL fibrous membrane of 8-12 μ m diameter of the poly-methylepinephrine of surface aggregate.
Fig. 2 is that embodiment 1-4 prepares the x-ray photoelectron spectroscopy XPS of film to characterize (a) and poly-methylepinephrine fixing on film Rate (b).
Fig. 3 is poly-norepinephrine (PNE) release profiles on film that embodiment 3-4 prepares film.
Fig. 4 is that embodiment 1-4 prepares the film external degradation mass loss figure of 99 days.
Fig. 5 is that embodiment 1-4 prepares membrane degradation scanning electron microscope (SEM) figure after 33 days.
Fig. 6 is the cytotoxicity figure that embodiment 1-4 prepares film.
Fig. 7 is state diagram when growing 3d on film of experimental example 4 cell.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.In embodiment unreceipted specifically Technology or condition person, according to the technology described by the document in this area or condition, or carried out according to product description.Used Reagent or instrument unreceipted production firm person, be the conventional products can being commercially available by regular distributor.
Embodiment 1
1) 3g polycaprolactone (PCL) is dissolved in the mixed solvent of 20mL chloroform and ethanol (volume ratio 7:3), room temperature magnetic force Stirring 12h, obtains the spinning liquid that polycaprolactone concentration is 15% (w/v).
2) described spinning liquid being carried out electrostatic spinning, with stainless steel drum for receiving device, drum rotation speed is 200rpm, electricity Pressure 20KV, receiving range is 20cm, spinning liquid feed liquor speed 10mL/h, spinning 5h.Obtaining thickness is the Electrospun about 250 μm Fibrous membrane.Film after freezing 6-12h, is vacuum dried 4-12h, it is ensured that residual solvent fully volatilizees at-60 DEG C～-20 DEG C.This The named 2P of film that method obtains；Its scanning electron microscope result is as it is shown in figure 1, the fibre diameter of 2P is about 1-3 μm.
Embodiment 2
1) 3.5g polycaprolactone (PCL) is dissolved in 20mL chloroform solvent, room temperature magnetic agitation 12h, obtains polycaprolactone Concentration is the spinning liquid of 17.5% (w/v).
2) described spinning liquid being carried out electrostatic spinning, with stainless steel drum for receiving device, drum rotation speed is 700rpm, electricity Pressure 15KV, receiving range is 15cm, spinning liquid feed liquor speed 3.6mL/h, spinning 4h.Obtaining thickness is the electrospinning about 500 μm Silk fiber film.Film after freezing 6-12h, is vacuum dried 4-12h, it is ensured that residual solvent fully volatilizees at-60 DEG C～-20 DEG C. The named 10P of film that the method obtains；Its scanning electron microscope result is as it is shown in figure 1, the fibre diameter of 10P is about 8-12 μm.
Embodiment 3
1) 3g polycaprolactone (PCL) is dissolved in the mixed solvent of 20ml chloroform and ethanol (volume ratio 7:3), room temperature magnetic force Stirring 12h, obtains the spinning liquid that polycaprolactone concentration is 15% (w/v).
2) described spinning liquid being carried out electrostatic spinning, with stainless steel drum for receiving device, drum rotation speed is 200rpm, electricity Pressure 20KV, receiving range is 20cm, spinning liquid feed liquor speed 10mL/h, spinning 5h.Obtaining thickness is the Electrospun about 250 μm Fibrous membrane.
3) gained electricity spinning fibre film is immersed in dissolved with norepinephrine and the cytokine promoting cell growth In PBS buffer solution (pH=8.3,10mM), the cytokine concentrations of norepinephrine and promotion cell growth is respectively 0.1mg/mL.Film after freezing 6-12h, is vacuum dried 4-12h, it is ensured that residual solvent fully volatilizees at-60 DEG C～-20 DEG C. The named 2PP of film that the method obtains；Its scanning electron microscope result is as it is shown in figure 1, after the poly-methylepinephrine of 2PP surface aggregate, fine Dimension diameter is about 8-12 μm.
Embodiment 4
1) 3.5g polycaprolactone (PCL) is dissolved in the mixed solvent of 20ml chloroform and ethanol (volume ratio 7:3), room temperature magnetic Power stirring 12h, obtains the spinning liquid that polycaprolactone concentration is 17.5w/v%.
2) described spinning liquid being carried out electrostatic spinning, with stainless steel drum for receiving device, drum rotation speed is 200rpm, electricity Pressure 15KV, receiving range is 15cm, spinning liquid feed liquor speed 3.6mL/h, spinning 5h.Obtaining thickness is the electrospinning about 500 μm Silk fiber film.
3) gained electricity spinning fibre film is immersed in dissolved with norepinephrine and the cytokine promoting cell growth In PBS buffer solution (pH=8.3,10mM), the concentration of the cytokine of described norepinephrine and promotion cell growth is 0.1mg/mL.Film after freezing 6-12h, is vacuum dried 4-12h, it is ensured that residual solvent fully volatilizees at-60 DEG C～-20 DEG C. The named 10PP of film that the method obtains；Its scanning electron microscope result as it is shown in figure 1, after the poly-methylepinephrine of 10PP surface aggregate, Fibre diameter is about 8-12 μm.
Experimental example 1
4 kinds of films (2P, 2PP, 10P, 10PP) in embodiment 1-4 are cut into the size of 2mm × 2mm, with aluminum x-ray source (h υ=1486.6eV) as the x-ray photoelectron power spectrum (XPS) of emission source, operate voltage 15kV and electric current 15mA (225W).Adopt Electrostatic and the action of a magnetic field of area about 300 μ m 700 μm is being analyzed by mixing Electronic Speculum pattern.For each sample, relatively It it is 0 ° in sample surfaces angle of emergence, it is allowed to max survey depth is 10 nanometers.With the energy scan sample of 160eV, it is thus achieved that 0 The combination energy of 1200eV scope, has specific energy further according to every kind of element, so that it is determined that surface-element composition and content.On The x-ray photoelectron spectroscopy XPS sign stating 4 kinds of films (2P, 2PP, 10P, 10PP) is shown in Fig. 2 (a), and poly-methylepinephrine is fixing on film Rate is shown in Fig. 2 (b).
As shown in Figure 2, in nitrogen element spectrogram, the content of the nitrogen element of 2PP is 2 times of 10PP, and at the nitrogen carbon of 2PP Than be 5.87 than 10PP 2.94 closer to the nitrogen charcoal ratio of norepinephrine itself, also illustrate that the former is coated with the amount of PNE many, equally In high-resolution carbon is composed, the carbonnitrogen bond of 2PP is 2 times of 10PP, and this is mainly the diameter reason less than 10P of 2P.
Experimental example 2
4 kinds of films (2P, 10P, 2PP, 10PP) of preparation in embodiment 1-4 are broken into 48 orifice plate pore size disks, sterilizing (each 10min of ultra-vioket radiation positive and negative), is layered in 48 orifice plates, 75% soak with ethanol 30min, ultra-vioket radiation 10min, uses sealed membrane Seal, leave 4 DEG C in.When cell is in logarithmic (log) phase, digest L6 cell, be seeded in 48 orifice plates being covered with 2 kinds of films, cultivate 1d, 3d, 5d, 7d, every hole inoculates 3 × 10 respectively5、2×105、1×105、8×104Individual cell, every hole add 600 μ l (3d, 5d and 7d carries out and the most partly changes liquid), each film sets 6 parallel parallel blank groups only adding culture medium with 3.Film is taken out with tweezers It is individually placed in 48 new orifice plates, the Cell sap in sucking-off two boards, respectively adds the 10%CCK-8 of 220 μ L to it, cultivate 1- 4h, the 10%cck8 culture fluid of corresponding sucking-off 110 μ L joins in 96 orifice plates, surveys OD450Value.Above-mentioned 4 kinds of films (2P, 2PP, 10P, Cytotoxicity result 10PP) is shown in Fig. 6.
As shown in Figure 6, sarcoplast is different at the proliferative conditions of four kinds of films, 2P and 2PP more promotes carefully than 10P and 10PP The propagation of born of the same parents, 2PP and 10PP more promotes the propagation of cell than 2P and 10P, this with relevant report be consistent, i.e. the propagation of cell and Adherent mainly by sticking speckle, the size sticking speckle is mainly affected by adhesion protein, and diameter is the least, and specific surface area is the biggest, absorption The amount of PNE the most, the amount of its fixing adhesion protein is the most, and cell proliferation is the strongest.But 2PP Yu 2P increment 5 days when Amplitude is little less than 10P with 10PP amplitude of rising in value, mainly due to 2PP fiber due to the cell cell than 10PP many, and cell Existence environment and nutrient substance is limited and the contact inhibition of cell so that the cell proliferation speed on 2PP fibrous membrane compares 10PP Slowly.
The degradation experiment of experimental example 3 film:
1) matter loss rate: 4 kinds of films (2P, 2PP, 10P and 10PP) of preparation in embodiment 1-4 of known quality are respectively put into In bottle equipped with 20ml normal saline, tighten in the constant incubator covering and being placed on 37 degree, within every 11 days, take out and be dried and difference Claim its quality, according to below equation calculating matter loss rate: matter loss rate %=(mt-m0)/m0, wherein mtIt it is every kind of each time point of film Take out dried quality, m0It it is the initial mass of every kind of film.The matter loss rate of above-mentioned 4 kinds of films (2P, 2PP, 10P, 10PP) is shown in Fig. 4.
2) the SEM picture of membrane degradation: by four kinds of films (2P, 2PP, 10P and 10PP) of preparation in a certain amount of embodiment 1-4 It is respectively put in the bottle equipped with 20ml normal saline, tightens in the constant incubator covering and being placed on 37 degree, within every 11 days, take out, dry Dry it is SEM with cold field emission scanning electron microscope S4800 two days later.The SEM figure of the degraded of above-mentioned 4 kinds of films (2P, 2PP, 10P, 10PP) Sheet characterizes sees Fig. 5.
3) poly-norepinephrine (PNE) residual volume measures: by the embodiment 3 of known quality, 2 kinds of films of preparation in 4 (2PP and 10PP) is respectively put in the bottle equipped with 20ml normal saline, tightens in the constant incubator covering and being placed on 37 degree, every Within 2 days, take out 1ml normal saline, supplement the fresh normal saline of 1ml simultaneously, utilize the 1ml that fluorescent spectrophotometer assay takes out PNE concentration in normal saline, exciting light and wavelength of transmitted light are respectively 410nm and 505nm.20ml is obtained raw according to standard curve The gross mass of PNE in reason saline.According to below equation calculating PNE residual volume: PNE residual volume=(m1-m2)/m1, wherein m2It is every Plant the quality of the PNE that each time point of film is degraded, m1It it is PNE original quality on film.Above-mentioned 2 kinds of films (2PP and 10PP) PNE residual volume characterizes sees Fig. 3.
For understanding this 4 kinds of films degraded situation in animal body, the interior environment in analogue body, four kinds of films are placed in equivalent and In the normal saline of charge, weighed every 11 days and be SEM and observe the change of pattern.As shown in Figure 4,4 kinds of films are in 99 days Quality is not changed in substantially, but in the SEM figure of 280 days, 4 kinds of fibre diameters diminish, and what this also met organizational project can The requirement of degraded support.
Experimental example 4
The process that cellular morphology observation experiment is cultivated with experimental example 2 cell is consistent, each time point, and sterile working takes out two Planting film (2PP and 10PP) and put into 48 new orifice plates, with PBS three times (serum affects the fixing of paraformaldehyde), every hole adds The 4% paraformaldehyde room temperature entering 500 microlitres fixes 30min, and exhaust liquid, washes 3 times with PBS, washes 5min every time.By rhodamine mark The methanol solution of the PI (phallodin) of note dilutes with 1:200 ratio 0.2%Triton/PBS, and every hole adds 400 μ L, room temperature Lucifuge 0.5h, washes 3 times with PBS, washes 5min every time.Blotting liquid, every hole adds the DAPI solution of 400 μ L, room temperature lucifuge 15min, washes 3 times with PBS, washes 5min every time, then film is put on coverslip, and that having a cell faces up, and drips upper one and prevents Quencher sealing agent, then covers microscope slide, then is inverted, made cell face down, and room temperature keeps 8min.Use confocal Fluorescence microscope takes the nucleus of blueness and the photo of the tubulin of redness under 403nm and 543nm passage.Above-mentioned 4 kinds The impact of cellular morphology is shown in Fig. 7 by film (2P, 2PP, 10P, 10PP).
Result as it is shown in fig. 7, under conditions of all conditions is identical, 2PP is more than 10PP cell number and cytochrome oxidase isozymes more Good.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Claims (10)

1. a functionalization guides muscular tissue regeneration membrane, it is characterised in that it is with polycaprolactone as major matrix material, passes through The film that electrostatic spinning is prepared as.
Regeneration membrane the most according to claim 1, it is characterised in that the fine and close norepinephrine of polymerization on described regeneration membrane Layer, and combine the protein promoting cell growth；Preferably, the protein of described promotion cell growth is basic fibroblast The growth being correlated with in somatomedin, insulin like growth factor family (IGF), heparin, transforming growth factor (TGF), platelet source One or more in the factor.
Regeneration membrane the most according to claim 1 and 2, it is characterised in that it has the micron order fibre structure of random arrangement, Average bridging aperture is 1-16 μm；Preferably, described fibre diameter is 1-20 μm.
Regeneration membrane the most according to claim 1 and 2, it is characterised in that its thickness is 50-500 μm.
5. the preparation method of regeneration membrane described in any one of claim 1-4, it is characterised in that comprise the following steps: will be poly-own interior Ester is dissolved in organic solvent, prepares solution A；Then carry out electrostatic spinning, prepare electricity spinning fibre film, be dried.
Preparation method the most according to claim 5, it is characterised in that also include norepinephrine and promote that cell is raw Long protein is dissolved in Tris-HCl solution or PBS, prepares B solution；Gained electricity spinning fibre film is immersed in institute State certain time in B solution, be dried after taking-up, to obtain final product.
7. according to the preparation method described in claim 5 or 6, it is characterised in that described organic solvent is chloroform, or chloroform and second The mixture of alcohol；Being preferably ethanol with chloroform volume ratio is the mixture of 7:10-7:1；
And/or, in described solution A, the mass fraction of polycaprolactone is 3%-45%；
And/or, described Tris-HCl solution, the pH value of PBS solution are respectively 8-10；
And/or, in described B solution, the concentration of norepinephrine and promotion cell growth cytokine is respectively 0.1-10mg/ ml。
8. according to the preparation method described in claim 5 or 6, it is characterised in that described electrostatic spinning comprises the following steps:
A, described solution A is placed in syringe, utilizes propeller to promote, after droplets stable flows down, heighten voltage；
B, with rustless steel roller bearing for receive device, continue spinning obtain complete electricity spinning fibre film；
Preferably, angle of rake fltting speed described in step a is 1-15mL/h；And/or,
Voltage described in step a is 7-20kV；And/or,
Receiving range described in step b is 8-30cm；And/or,
The speed of rustless steel roller bearing described in step b is 100-2000rmp；And/or,
The spinning time described in step b is 2-30h.
9. according to the preparation method described in any one of claim 5-8, it is characterised in that include following (one), (two), (three), (4) arbitrary described step:
(1) fibre diameter be 1-8 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in chloroform, room temperature magnetic agitation 12-24h, obtains the polycaprolactone that mass fraction is 3-45% Solution；
(2) above-mentioned polycaprolactone solution being carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate is 100-2000rpm, spinning liquid flow rate is 1-15mL/h, and voltage 7-20kV, receiving range 8-30cm, spinning 2-30h obtain The electricity spinning fibre film of thickness 50-500 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes that cell grows the Tris-of cytokine 12-24h in HCl solution, after taking-up, in fume hood, room temperature is placed 2-7 days, to obtain final product；
(2) fibre diameter be 8-20 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7:3 with the volume ratio of ethanol), Room temperature magnetic agitation 12-24h, obtains the polycaprolactone solution that mass fraction is 14-30%；
(2) above-mentioned polycaprolactone solution being carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate is 700-2600rpm, spinning liquid flow rate is 2-20mL/h, voltage 12-25kV, receiving range 10-35cm, spinning 1-30h, Electricity spinning fibre film to thickness 100-600 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes that cell grows the Tris-of cytokine 12-24h in HCl solution, after taking-up, in fume hood, room temperature is placed 2-7 days, to obtain final product；
(3) fibre diameter be 1-8 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7:3 with the volume ratio of ethanol), In, room temperature magnetic agitation 12-24h, obtain the polycaprolactone solution that mass fraction is 5-19%；
(2) above-mentioned polycaprolactone solution being carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate is 100-2000rpm, spinning liquid flow rate is 0.5-10mL/h, voltage 7-20kV, receiving range 8-30cm, spinning 0.5-30h, Obtain the electricity spinning fibre film of thickness 25-250 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes that the PBS of cell growth cytokine delays 12-24h in dissolved liquid, then, is vacuum dried it 4-12h, to obtain final product after freezing 6-12h at-60 DEG C～-20 DEG C；
(4) fibre diameter be 8-20 micron functionalization guide muscular tissue regeneration membrane preparation
(1) polycaprolactone is dissolved in the mixed solvent of chloroform and ethanol (preferably, chloroform is 7:3 with the volume ratio of ethanol), Room temperature magnetic agitation 12-24h, obtains the polycaprolactone solution that mass fraction is 13-29%；
(2) above-mentioned polycaprolactone solution being carried out electrostatic spinning, with stainless steel drum for receiving device, cylinder slewing rate is 500-2000rpm, spinning liquid flow rate is 0.5-10mL/h, voltage 14-30kV, receiving range 10-30cm, spinning 1.5- 30h, obtains the electricity spinning fibre film of thickness 70-400 μm；
(3) gained electricity spinning fibre film it is immersed in dissolved with norepinephrine and promotes that the PBS of cell growth cytokine delays 15-24h in dissolved liquid, then, is vacuum dried it 4-12h, to obtain final product after freezing 6-12h at-60 DEG C～-20 DEG C.
10. the regeneration membrane that prepared by method described in regeneration membrane described in any one of claim 1-4 or any one of claim 5-9 is in system Application in the standby medicine promoting muscular tissue Regeneration and Repair.
CN201610461624.2A 2016-06-23 2016-06-23 A kind of functionalization guidance muscular tissue regeneration membrane and its preparation method and application Active CN106110405B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610461624.2A CN106110405B (en) 2016-06-23 2016-06-23 A kind of functionalization guidance muscular tissue regeneration membrane and its preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610461624.2A CN106110405B (en) 2016-06-23 2016-06-23 A kind of functionalization guidance muscular tissue regeneration membrane and its preparation method and application

Publications (2)

Publication Number Publication Date
CN106110405A true CN106110405A (en) 2016-11-16
CN106110405B CN106110405B (en) 2019-10-11

Family

ID=57268635

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610461624.2A Active CN106110405B (en) 2016-06-23 2016-06-23 A kind of functionalization guidance muscular tissue regeneration membrane and its preparation method and application

Country Status (1)

Country Link
CN (1) CN106110405B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331579A (en) * 2019-06-21 2019-10-15 广东工业大学 A kind of surface-functionalized Oligoanilines nanofiber of antibacterial and its preparation method and application
CN110404117A (en) * 2018-04-28 2019-11-05 国家纳米科学中心 A kind of functionalization guidance muscular tissue repair membrane and its preparation method and application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103006359A (en) * 2012-12-24 2013-04-03 汪泱 Bionic three-dimensional tissue engineering scaffold and preparation method thereof
CN204618942U (en) * 2015-04-01 2015-09-09 上海交通大学医学院附属第九人民医院 Medical polycaprolactone diaphragm
KR20160047634A (en) * 2014-10-22 2016-05-03 한남대학교 산학협력단 Porous matrix for trachea regeneration and method for preparing thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103006359A (en) * 2012-12-24 2013-04-03 汪泱 Bionic three-dimensional tissue engineering scaffold and preparation method thereof
KR20160047634A (en) * 2014-10-22 2016-05-03 한남대학교 산학협력단 Porous matrix for trachea regeneration and method for preparing thereof
CN204618942U (en) * 2015-04-01 2015-09-09 上海交通大学医学院附属第九人民医院 Medical polycaprolactone diaphragm

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110404117A (en) * 2018-04-28 2019-11-05 国家纳米科学中心 A kind of functionalization guidance muscular tissue repair membrane and its preparation method and application
CN110404117B (en) * 2018-04-28 2022-03-15 国家纳米科学中心 Functional guided muscle tissue repair membrane and preparation method and application thereof
CN110331579A (en) * 2019-06-21 2019-10-15 广东工业大学 A kind of surface-functionalized Oligoanilines nanofiber of antibacterial and its preparation method and application
CN110331579B (en) * 2019-06-21 2021-12-17 广东工业大学 Antibacterial surface functionalized aniline oligomer nanofiber and preparation method and application thereof

Also Published As

Publication number Publication date
CN106110405B (en) 2019-10-11

Similar Documents

Publication Publication Date Title
Wu et al. In vitro and in vivo biocompatibility evaluation of a 3D bioprinted gelatin-sodium alginate/rat Schwann-cell scaffold
Zhu et al. 3D bioprinting mesenchymal stem cell-laden construct with core–shell nanospheres for cartilage tissue engineering
Abdollahiyan et al. The triad of nanotechnology, cell signalling, and scaffold implantation for the successful repair of damaged organs: An overview on soft-tissue engineering
Keskar et al. In vitro evaluation of macroporous hydrogels to facilitate stem cell infiltration, growth, and mineralization
Ardeshirylajimi et al. Enhanced reconstruction of rat calvarial defects achieved by plasma-treated electrospun scaffolds and induced pluripotent stem cells
Zou et al. Electrospun fibrous scaffolds with continuous gradations in mineral contents and biological cues for manipulating cellular behaviors
Li et al. From 2D to 3D: The morphology, proliferation and differentiation of MC3T3-E1 on silk fibroin/chitosan matrices
CN110408058B (en) Halloysite composite hydrogel for promoting bone defect repair and preparation method and application thereof
CN103751848B (en) A kind of preparation method of antibacterial repairing type electrostatic spinning collagen protein-Bacterial cellulose composite nano fiber scaffold and application thereof
Gugerell et al. Adipose-derived stem cells cultivated on electrospun l-lactide/glycolide copolymer fleece and gelatin hydrogels under flow conditions–aiming physiological reality in hypodermis tissue engineering
Kim et al. Eggshell membrane as a bioactive agent in polymeric nanotopographic scaffolds for enhanced bone regeneration
US10508264B2 (en) Cell culture method using bone marrow-like structure, and porous polyimide film for healing bone injury site
CN106110405A (en) A kind of functionalization guides muscular tissue regeneration membrane and its preparation method and application
EP3021880A1 (en) Three-dimensional scaffold functionalized with micro-tissues for tissue regeneration
Kaufman et al. Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration
Wu et al. Bacterial cellulose nanofiber distribution on gelatin and silk fibroin scaffolds and the cell behavior
CN110404117A (en) A kind of functionalization guidance muscular tissue repair membrane and its preparation method and application
US20160136330A1 (en) Three-Dimensional Scaffold Functionalized with Micro-Tissues for Tissue Regeneration
Das et al. Bone tissue engineering construct fabricated using a cell electrospinning technique with polyglutamic acid biopolymer
Gharravi et al. Status of tissue engineering and regenerative medicine in Iran and related advanced tools: Bioreactors and scaffolds
US20090186408A1 (en) Biocompatible bilayer porous matrix and preparation thereof
CN107674409B (en) Polyurethane/liquid crystal composite biological material and preparation method and application thereof
Jia et al. 3D Bioprinting-Based Dopamine-Coupled Flexible Material for Nasal Cartilage Repair
WO2019232736A1 (en) Hydrogel system, preparation method therefor and use thereof
Zhuang 3D bioprinting of biomimetic skeletal muscle tissue model

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant