CN106334210A - Multifunctional collagen nano-fibre repair film and preparation method thereof - Google Patents
Multifunctional collagen nano-fibre repair film and preparation method thereof Download PDFInfo
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- CN106334210A CN106334210A CN201610850674.XA CN201610850674A CN106334210A CN 106334210 A CN106334210 A CN 106334210A CN 201610850674 A CN201610850674 A CN 201610850674A CN 106334210 A CN106334210 A CN 106334210A
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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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
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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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
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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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
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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
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
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- 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
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
Abstract
The invention discloses a preparation method for a multifunctional collagen nano-fibre repair film. The preparation method comprises the following steps: (1) preparing a growth factor-containing collagen/polycaprolactone solution; (2) preparing a T4 bacteriophage-containing collagen/polycaprolactone solution; (3) quickly obtaining the nano-fibre repair film by adopting an electrospinning technology. The nano-fibre repair film integrates rapid hemostasis, long-acting bacteriostasis and cell repair, and is expected to provide full-function wound nursing for a patient.
Description
Technical field
The present invention relates to a kind of multi-functional collagen protein nano fiber repair membrane and preparation method thereof.
Background technology
After wound or operation, the gauze of wound healing and external wound is repaired and is mainly relied on two phenomenons: on
Skin is formed and granulation tissue is shunk, and the latter is most important for protective tissue seriality and reduction wound size.During contraction, phase
Adjacent myofibroblasts can be transitted very quickly into myofibroblast, and its feature is Tonofibrilses and smooth actin this two
Mark can exist.Especially, it is changed into this dissimilation meeting body of myofibroblast in wound contraction phase myofibroblasts
Reveal an important thing, that is, this process can signal to influence each other by mechanical stress and tgf β 1.Specifically,
Somatomedin tgf β 1 is considered as the direct inducement that myofibroblasts are changed into myofibroblast, this show smad2 and
The activation of smad3, the upper regulation of smad complex to nuclear migration and α sma expression.Lee et al. reports and only works as
The glue in class myofibroblast when myofibroblasts being carried out with the stimulation of short time with the tgf β 1 of appropriate concentration, in test tube
Former protein gel substrate can consumingly be shunk, and otherwise cell will occur permanent fibrosiss.Therefore, tgf β 1 is controlled by short-term
The mode of concentration, is helpful to for some wound healings.
The cell that the up-to-date direction of regenerative medicine is mainly concentrated in preparing similar cell epimatrix (ecm) at present props up
Frame, thus realizing in various dimensions space, the of short duration growth factor release speed controlling scalable cell function.Electrostatic spinning is
A kind of multi-functional polymer nanocomposite production technology, this allows have and is mixed with active growth factor and has micro nano structure cell
Support becomes to be easy to prepare, and this cytoskeleton as analog cell epimatrix, and can provide same yardstick and work(again simultaneously
Energy.The typical polymers that nanofiber can extensively be made include: synthetic polyester fibers, such as polylactic acid (pla), polyglycolic acid
(pga), PLGA (plga), poly- acetic acid lactone (pcl) etc., and natural polymer such as gelatin, silkworm
Silk, shitosan and purification i collagen type (hereinafter referred to as collagen protein, coll) etc..And collagen protein is as extracellular matrix
A kind of primary structure protein, be widely applied to medical apparatus and instruments aspect, such as wound dressing, skin regeneration, medicine
The aspects such as slow release, cornea replacement, tissue engineering bracket.
Content of the invention
It is an object of the present invention to provide a kind of multi-functional collagen protein nano fiber repair membrane and preparation method thereof, and directly quiet
Arteries and veins administration or oral medication are compared, can be long-acting as a certain specific part for the treatment of using the administration of organizational project mode specific aim
Medicine, this can reduce side effect and prevent medicine from decomposing, nanofiber repair membrane of the present invention is while strengthening medicinal effectiveness
Pcl/coll electrospun fibers containing tgf β 1 somatomedin, are used as a kind of special hemostatic material and artificial cell
Epimatrix (ecm), by adjusting its hemostatic bacteriostatic effect of INVESTIGATING THE MIXING PROPORTION of pcl/coll, controls the release of tgf β 1 simultaneously,
Thus adjusting myofibroblast rate of differentiation.This nanometer of repair membrane integrates quick-acting haemostatic powder, long-acting bacteriostatic, cytothesiss,
It is expected to provide global function wound care for patient.
The technical solution used in the present invention is:
A kind of preparation method of multi-functional collagen protein nano fiber repair membrane, comprises the steps:
1) configuration of collagen protein containing somatomedin/polycaprolactone solution
Because purification i collagen type (hereinafter referred to as collagen protein, coll) spinnability is poor, therefore add polycaprolactone
(polycaprolactone, pcl) carries out blending.Polycaprolactone pcl is dissolved in hexafluoro according to the mass ratio of 5～15:100 different
Obtain solution a in propanol, collagen protein coll is dissolved in hexafluoroisopropanol according to the mass ratio of 5～15:100 and obtains solution b；
Again solution a and solution b is mixed and utilize magnetic stirring apparatuses continuous stirring 4-8h, until obtaining transparent and homogeneous
Macromolecule pcl/coll spinning solution.Again by transforming growth factor tgf β 1 with 10-3%～10-4The proportioning of %, w/w is uniformly added
To in pcl/coll spinning solution, obtain collagen protein containing somatomedin/polycaprolactone solution.
2) configuration of the collagen protein of phage containing t4/polycaprolactone solution
Polycaprolactone pcl is dissolved in hexafluoroisopropanol according to the mass ratio of 0.5～15:100 and obtains solution c, by collagen
Albumen coll is dissolved in hexafluoroisopropanol according to the mass ratio of 0.5～15:100 and obtains solution d, then solution c and solution d is mixed
And utilize magnetic stirring apparatuses continuous stirring 1-8h, until obtain the macromolecule pcl/coll spinning solution of transparent and homogeneous.To train
The t4 phage supported concentrates the pcl/coll spinning that liquid is uniformly added to rapidly with the proportioning of 0.1～1.5/100, v/v molten
In liquid, obtain the collagen protein of phage containing t4/polycaprolactone solution.
3) setting of electrospinning parameters
Homogeneous nano fibrous membrane can be obtained rapidly using electrostatic spinning technique, electrostatic spinning as shown in Figure 1 shows
It is intended to.Specific process parameter is as follows: voltage, 5～20kv；Feed rate, 0.1～1ml/h；Sedimentation time, 3～6h；Spinning away from
From 8～20cm；Relative humidity, 20-35%；Room temperature.All of fiber is placed in 24～36h in freezer dryer after spinning, complete
It is stored in -20 DEG C after white drying.
Brief description
Fig. 1 is electrostatic spinning schematic diagram.
Fig. 2-1 is the surface topography map of the pcl/coll fiber loading tgf β 1.
Fig. 2-2 is the pcl/coll fiber diameter distribution profile loading tgf β 1.
Fig. 3 is pcl/coll blend fibre and pure pcl, the miscibility comparative study of pure coll.
Fig. 4 is combined the contrast of blend fibre tensile strength for pcl/coll.
Fig. 5 is the contact angle change contrast of pcl/coll blend fibre and pcl and coll
Fig. 6-1 is loaded with the impact of the tgf β 1 cell proliferation rate of variable concentrations for pure collagen fiber.
Fig. 6-2 loads the impact of 1ng/mg tgf β 1 cell proliferation rate for pcl/coll blend fibre.
Fig. 7 is the amount of bleeding of different haemostatic membranes in rabbit arteria auricularises and liver injury model.
Fig. 8 is the micro-structure diagram of different samples: (a) phage tem figure；B pcl/collb blending that () contains phage is fine
Dimension sem figure；C (), (d) contain the pcl/collb blend fibre microcosmic tem figure of phage.
Fig. 9 is rt-pcr gene expression detection situation.Wherein, (a) mice myofibroblast marker gene α sma；(b)
Mouse muscle fiber cell sign gene fsp1；(c-h) it is cell differentiation situation on the 3rd day different materials；(i n) is the 5th
Cell differentiation situation on its different materials；(o t) is the cell differentiation situation on the 7th day different materials.
Figure 10 is to be 1 week, 2 weeks, 4 weeks, coll the and pcl/coll b histopathology photo of 8 weeks the implantation phase.
Specific embodiment
A kind of preparation method of multi-functional collagen protein nano fiber repair membrane of embodiment 1
Comprise the steps:
1) configuration of collagen protein containing somatomedin/polycaprolactone solution
By polycaprolactone pcl according to 10% mass ratio be dissolved in hexafluoroisopropanol polycaprolactone hexafluoroisopropanol is molten
Liquid, collagen protein coll is dissolved in hexafluoroisopropanol according to 10% mass ratio and obtains collagen protein hexafluoroisopropanol solution,
By polycaprolactone hexafluoroisopropanol solution and collagen protein hexafluoroisopropanol solution according to w/w, 40/60 proportioning mixes and utilizes
Magnetic stirring apparatuses continuous stirring 6h, until obtain pcl/coll b spinning liquid (40/60, w/w) of transparent and homogeneous.
Again by transforming growth factor tgf β 1 according to high concentration (10-3%, w/w) uniformly it is added to pcl/coll b spinning liquid
(40/60, w/w), obtains collagen protein containing somatomedin/polycaprolactone solution.
2) configuration of the collagen protein of phage containing t4/polycaprolactone solution
Polycaprolactone is dissolved in hexafluoroisopropanol according to 10% mass ratio and obtains polycaprolactone hexafluoroisopropanol solution,
Collagen protein coll is dissolved in hexafluoroisopropanol according to 10% mass ratio and obtains collagen protein hexafluoroisopropanol solution, will gather
Caprolactone hexafluoroisopropanol solution and collagen protein hexafluoroisopropanol solution mix according to the proportioning of (40/60, w/w) and utilize magnetic
Power agitating device continuous stirring 6h, until obtain pcl/coll b spinning liquid (40/60, w/w) of transparent and homogeneous.Will be cultured
T4 phage concentrates liquid to be uniformly added to rapidly pcl/coll b spinning liquid (40/60, w/w) according to the proportioning of 1/100, v/v,
Obtain the collagen protein of phage containing t4/polycaprolactone solution.
3) adopt the homogeneous nano fibrous membrane of the rapid acquisition of electrostatic spinning technique.Specific process parameter is as follows: voltage,
20kv；Feed rate, 1ml/h；Sedimentation time, 3h；Spinning distance, 10cm；Relative humidity, 20-35%；Room temperature.All of fibre
Dimension is placed in 24h in freezer dryer after spinning, is stored in -20 DEG C after being completely dried.
Embodiment 2 contrast test
First group: only change embodiment 1 step 1) in by transforming growth factor tgf β 1 according to high concentration 10-3%, w/w are equal
Even it is added in pure coll spinning liquid；Step 2) in will cultured t4 phage concentrate liquid fast according to the proportioning of 1/100, v/v
Speed is uniformly added in pure coll spinning liquid；Other processes are constant.
Second group: only change embodiment 1 step 1) in by transforming growth factor tgf β 1 according to high concentration 10-3%, w/w are equal
Even it is added in pcl/coll a spinning liquid (33.3/66.7, w/w)；Step 2) in by cultured t4 phage concentration liquid press
Proportioning according to 1/100, v/v is uniformly added to rapidly in pcl/coll a spinning liquid (33.3/66.7, w/w)；Other processes are not
Become.
3rd group: embodiment 1.
4th group: only change embodiment 1 step 1) in by transforming growth factor tgf β 1 according to high concentration 10-3%, w/w are equal
Even it is added in pcl/coll c spinning liquid (50/50, w/w)；Step 2) in cultured t4 phage is concentrated liquid according to 1/
100, v/v proportioning is uniformly added to rapidly in pcl/coll c spinning liquid (50/50, w/w)；Other processes are constant.
5th group: only change embodiment 1 step 1) in by transforming growth factor tgf β 1 according to high concentration 10-3%, w/w are equal
Even it is added in pcl/coll d spinning liquid (66.7/33.3, w/w)；Step 2) in by cultured t4 phage concentration liquid press
Proportioning according to 1/100, v/v is uniformly added to rapidly in pcl/coll d spinning liquid (66.7/33.3, w/w)；Other processes are not
Become.
6th group: only change embodiment 1 step 1) in by transforming growth factor tgf β 1 according to high concentration 10-3%, w/w are equal
Even it is added in pure pcl spinning liquid；Step 2) in will cultured t4 phage concentrate liquid rapid according to the proportioning of 1/100, v/v
Uniformly it is added in pure pcl spinning liquid；Other processes are constant.
Experiment and analysis:
1st, as shown in Fig. 2-1 and 2-2, above-mentioned made multiple static spinning membranes are analyzed its surface topography using sem, on
The multiple static spinning membranes stating preparation have special porous pattern.The wherein spinnability of 100%coll is very poor, electrostatic spinning institute
The collagen fiber size of system is extremely uneven, rough, seriality extreme difference, 7.45 ± 2.21 μm of fibre diameter.100%pcl
Good in fibre, distribution of fiber diameters is at 0.78 ± 0.54 μm.During therefore by pcl with coll blending, with the ratio increasing pcl
Example, gained fiber stock solution spinnability is obviously improved, and fiber surface slickness lifting fibre diameter is gradually lowered: pcl/coll a is straight
Footpath is 1.67 ± 0.72 μm；A diameter of 1.49 ± 0.87 μm of pcl/coll b；A diameter of 1.19 ± 0.38 μm of pcl/coll c；
A diameter of 1.08 ± 0.44 μm of pcl/coll d.All of distribution of fiber diameters is as shown in the rectangular histogram in Fig. 2-2.From Fig. 2-1
Sem in figure understand, micro tgf β 1 and t4 phage have no too big impact to fiber macro morphology.
2nd, by pcl and the miscibility of collagen protein bi-material be have detected using the analysis method of dsc.In Fig. 3
Shown, the heat absorption denaturation temperature of collagen protein at 214.88 DEG C, this with document in report freeze-dried pure collagen protein
Heat absorption deformation temperature is consistent, and the heat absorption denaturation temperature of pure pcl is then at 56 DEG C, and this is consistent with its melting temperature (tm).Institute
The pcl/coll blend fibre of four kinds of different proportions of preparation is all using hexafluoroisopropanol as solvent, the dsc spectrogram being obtained
In, also the fusing point with pure pcl is consistent, near 56 DEG C for heat absorption denaturation temperature.Therefore, pcl and collagen protein are in hexafluoroisopropanol
In miscibilty fabulous, during electrostatic spinning sprays solidification, do not go out in composite fibre between two kinds of different materials
Existing split-phase.
3rd, tensile strength tests the performance for test material under axial tension load.Fig. 4 shows, pcl mechanical property
Most preferably, in the composite, with the increase of the mass fraction of collagen protein, the tensile property of material is gradually lowered.Wherein, due to
This material is crosslinked without firming agent, and the tensile strength of 100%coll is worst to be 304 ± 102kpa；100%pcl has splendid
Pliability, its tensile strength be 4754 ± 100kpa.The tensile strength of pcl/coll a is 711 ± 98kpa；pcl/coll b
Tensile strength be 1398 ± 76kpa；The tensile strength of pcl/coll c is 1904 ± 97kpa；The stretching of pcl/coll d is strong
Spend for 2808 ± 150kpa.
4th, macroscopical wettability of material, is checked by detecting the contact angle between liquid and material matrix.In Fig. 5 in detail
Describe contact angle and its contact angle final state figure of different materials.Greatly, liquid is almost complete at the material angle of 100%pcl
Nonwetting, and the contact angle of 100%coll is minimum, wettability is fabulous, drop almost at once on base material drawout come.Doping glue
From the point of view of pcl/coll blend fibre after former albumen is with respect to two kinds of original materials, it is provided with more gentle contact angle, material
Wettability be in moderate state.This has carboxyl and amino mainly due to collagen protein is a kind of splendid material of hydrophilic
Material, its addition enhances the wettability of material.
5th, the impact of different materials cell proliferation rate: (a) pure collagen fiber are loaded with the tgf β 1 of variable concentrations, (b)
Pcl/coll blend fibre loads 1ng/mg tgf β 1.As in Figure 6-1, all there is cell in all of cell after cultivating 7 days
Propagation, the matched group tcpt that cell obtains under the conditional stimulus of tgf β 1 simultaneously is than normal control group tcp being not added with tgf β 1
Group has shown higher rate of increase.Extremely micro tgf β 1 (1ng/mg) is loaded on coll l fiber to nih3t3 mice
Fibroblast proliferation has obvious stimulation.However, after the loading capacity of tgf β 1 expands ten times greater, the tgf β 1 in coll h fiber
Content reaches 10ng/mg, but the propagation of cell is but inhibited by, and it is sizable that this illustrates that the tgf β 1 of high concentration has to cell
Cytotoxicity.Therefore, the loading concentration of tgf β 1 is most widely suited for 1ng/mg for pcl/coll blend fibre, such
Concentration both ensure that its differentiation function, is unlikely to produce toxic and side effects to cell again.Just as shown in Fig. 6-2, pcl/coll a
Fiber had the effect of significant proliferative induction before the 5th day to cell, and subsequent cell quantity fails rapidly, this mainly due to
First week latter stage, tgf β 1 had a large amount of releases of an explosion type, and to cell, instantaneous toxicity increases.In all of group, bag
Include pcl/coll blending group and tcpt group, cell has best propagation and differentiation performance in pcl/coll b group.
6th, anthemorrhagic performance test
By the Hemorrhage Model in Hepar Leporis seu Oryctolagi portion and ear artery, using pure collagen and pure pcl as reference, to different ratio
Pcl/coll film has carried out anthemorrhagic performance assessment.Shown in table 1, when the liver of pure pcl and pcl/coll d and ear's hemostasis
Between be all higher than 500s, almost there is no haemostatic effect.
When the blending haemostatic membrane high using collagen content covers on liver's wound surface, average bleeding stopping period is fast
Prompt drop is low, and wherein, the average bleeding stopping period of pcl/coll a, pcl/coll b, pcl/coll c is respectively 95.34 ± 10.05*
(ear), 67.05 ± 7.15* (liver)；106.66 ± 12.31* (ear), 75.56 ± 3.89* (liver)；320.63±
41.01* (ear), 298.67 ± 31.58* (liver).By data analysiss, compare with pure collagem membrane, exist substantially between its group
Difference (p < 0.05), has statistical significance.Subsequently, by the weighing (Fig. 7) of amount of bleeding, can more preferable clear and definite different materials
Haemostatic effect.The surface wettability of pure pcl is worst, and blood is not absorbed by material, and the amount of bleeding that therefore it obtains is minimum.Pure
The amount of bleeding of collagen group is larger, and this is that all of blood is all absorbed by material because collagem membrane has good surface wettability.
In pcl/coll blending film, due to the addition of the pure collagen of different proportion, great changes have taken place for its surface wettability and bleeding stopping period.
Pcl/coll a, pcl/coll b amount of bleeding quite, though and in pcl/coll c the content of pcl more absorb more
Amount of bleeding, illustrate that the haemostatic effect of this material is general.By data analysiss, all exist between all groups notable difference (p <
0.05), there is statistical significance.
Therefore, when for the haemostatic effect of material, pcl/coll a, pcl/coll b haemostatic membrane have shorter hemostasis
Between and less amount of bleeding.Because in both materials, collagen content is higher, and collagen protein is in hemostasis
Play Main Function, therefore, pcl/coll a, pcl/coll b hemostasis experiment effect optimal.
The average bleeding stopping period of different haemostatic membranes in 1 two kinds of trauma models of table
table 1the mean hemostatic time of different film in two rabbit
injury models
statistical analysis was performed by t-text
(*p<0.05compared to the coll group).
7th, bacteriophage activity and fiber morphology observation
The great feature of microscopic pattern of phage, is to be made up of head and afterbody, as shown in figure 8 a, wherein all of something lost
Pass material to be present among head.After t4phages is added to pcl/coll blend fibre, sem in figure is not seen any
Change, basically identical with when being not added with, but more accurately in tem picture it can be seen that being uniformly distributed on single fiber
T4phages (Fig. 8 b-d).
8th, confocal microscope is observed
In order to further illustrate the process of cell differentiation, continue using fluorescence microscope cell in cell differentiation procedure
In morphology change, and marker protein expression situation of change.Wherein, nucleus adopt hoechst to dye (blue cell core),
Actin filament in cytoskeleton adopts phalloidin to dye (green cell skeleton), the mark α of myofibroblast
Sma albumen adopts first antibody (mice α sma) and second antibody (orchil) to be demarcated.
Fig. 9 is rt-pcr gene expression detection situation: (a) mice myofibroblast marker gene α sma (b) Mouse Muscle
Fibrocyte marker gene fsp1., respectively at tcp (no tgf β 1), tcpt is (containing 10ng/ for the fluorescence immunoassay (red) of α sma
Ml tgf β 1) and different proportion pcl/coll blend fibre (β of tgf containing 1ng/mg 1) differentiation state.Wherein, (c-h) is
Cell differentiation situation on 3rd day different materials, (i n) is the cell differentiation situation on the 5th day different materials, and (o t) is
Cell differentiation situation on 7th day different materials.It is that myofibroblasts are glimmering to myofibroblast differentiation process in Fig. 9 c-t
Light immunity microgram.In initial three days, there is no obvious α sma protein expression (Fig. 9 c-h).When the 5th day, with feminine gender
Comparison tcp group is compared (Fig. 9 i), and only a small amount of α sma albumen can be in positive control tcpt group (Fig. 9 j) and pcl/coll b
Fiber group (Fig. 9 l) can detect.After the culture of seven days, substantial amounts of α sma albumen is in positive controls (Fig. 9 p), pcl/coll
Express in a (Fig. 9 q) and pcl/coll b (Fig. 9 r), but only in negative control group tcp (Fig. 9 o) and pcl/coll b
In fiber group (Fig. 9 r), the normal pattern of cell ability unsoundness, in other groups, cell morphology is bad, and activity is poor.
Such result explanation, the microcosmic quantitative analysiss in conjunction with rt-pcr and confocal microscope macroscopic observation,
Cell on pcl/coll b fiber has optimal cellular morphology, and major part has been divided into myofibroblast simultaneously.
9th, according to the haemostatic effect of material, fungistatic effect and other physicochemical property, biological safety, cell differentiation row
For research, pcl/coll b compound hemostatic film has the resultant effect of optimum, therefore, only with pure coll film and pcl/coll b
Film carries out degradation experiment in animal body.
As can be seen from Figure 10, after coll film implantation in rabbit leg muscle or dorsal sc, surrounding materials inflammatory reaction
Inconspicuous, when implanting one week, material is broken down into fine debris；When implanting two weeks, the fragment of material is bitten carefully by huge further
Born of the same parents surround；After implanting surrounding, coll film almost disappears, but still there is substantial amounts of inflammatory cell in implanted region, sends out
Give birth to inflammatory infiltration reaction；After implanting eight weeks, rabbit leg muscle restores completely, and subcutaneous tissue of back also grows hair follicle again
Recover original cellular morphology and function of organization.
Compare the vivo degradation situation of coll film, the degradation rate of pcl/coll b film is longer due to the addition of degradation cycle
Pcl after be greatly reduced.When implanting one week, pcl/coll b film is still larger fragment, and surrounding materials exist tighter
The inflammatory cell of weight, simultaneously because material is larger, implant site tissue is seriously damaged, and there are a large amount of non-viable non-apoptotic cells；Work as plant
When entering two weeks, pcl/coll b film is broken down into the less fragment of size in complicated internal milieu, and necrosis about is thin
Born of the same parents' quantity has reduced；When implanting three weeks, the pcl/coll b film in rabbit leg muscle is absorbed substantially, and rabbit back is subcutaneous
Still with the presence of minimum material fragment, this enriches mainly due to leg muscle part blood capillary, leg blood during rabbit motion
Flow velocity is very fast, and cell metabolic rates are very fast, and dorsal sc is relatively slow due to blood circulation, and therefore metabolic rate is also relatively
Slowly；And when implant eight weeks after, in two kinds of models, pcl/coll b film does not all detect, but compare purer coll implantation experiment and
Speech, the pathology in figure of pcl/coll b film still can see a small amount of inflammatory cell, but cellular morphology is substantially good, tissue
Function substantially recovers it can be seen that the hair follicle tissue of morphologically normal myofibroblasts and dorsal sc.
Claims (8)
1. a kind of preparation method of multi-functional collagen protein nano fiber repair membrane is it is characterised in that comprise the steps:
1) prepare collagen protein containing somatomedin/polycaprolactone solution, polycaprolactone is dissolved in hexafluoroisopropanol and obtains solution
A, collagen protein is dissolved in hexafluoroisopropanol and obtains solution b；Again solution a and solution b is mixed and utilizes magnetic stirring apparatuses
Continuous stirring, obtains the collagen protein/polycaprolactone spinning solution of transparent and homogeneous, then transforming growth factor tgf β 1 is uniformly added
It is added in collagen protein/polycaprolactone spinning solution, obtain collagen protein containing somatomedin/polycaprolactone solution；
2) configure the collagen protein of phage containing t4/polycaprolactone solution, polycaprolactone is dissolved in hexafluoroisopropanol and obtains solution
C, collagen protein is dissolved in hexafluoroisopropanol and obtains solution d, then solution c and solution d is mixed and utilizes magnetic stirring apparatuses
Continuous stirring, obtains the collagen protein/polycaprolactone spinning solution of transparent and homogeneous, and t4 phage concentration liquid is uniformly added rapidly
In the collagen protein being added to/polycaprolactone spinning solution, obtain the collagen protein of phage containing t4/polycaprolactone solution；
3) nanofiber repair membrane is obtained rapidly using electrostatic spinning technique.
2. preparation method as claimed in claim 1 is it is characterised in that step 1) and step 2) collagen protein/polycaprolactone
In spinning solution, collagen protein and the mass ratio of polycaprolactone are 66.7-33.3:33.3-66.7.
3. preparation method as claimed in claim 1 is it is characterised in that step 1) and step 2) polycaprolactone be according to 5～
The mass ratio of 15:100 is dissolved in hexafluoroisopropanol and is configured；Collagen protein is to be dissolved according to the mass ratio of 5～15:100
Hexafluoroisopropanol is configured.
4. preparation method as claimed in claim 1 is it is characterised in that step 1) transforming growth factor tgf β 1 is with 10-3%～
10-4The proportioning of %, w/w is uniformly added in collagen protein/polycaprolactone spinning solution.
5. preparation method as claimed in claim 1 is it is characterised in that step 2) t4 phage concentrates liquid with 0.1～1.5/
In the collagen protein that 100, v/v proportioning is uniformly added to rapidly/polycaprolactone spinning solution.
6. preparation method as claimed in claim 1 is it is characterised in that step 1) utilize magnetic stirring apparatuses continuous stirring 4-
8h.
7. preparation method as claimed in claim 1 is it is characterised in that step 2) utilize magnetic stirring apparatuses continuous stirring 1-
8h.
8. preparation method as claimed in claim 1 is it is characterised in that step 3) electrostatic spinning specific process parameter is as follows: electricity
Pressure, 5～20kv；Feed rate, 0.1～1ml/h；Sedimentation time, 3～6h；Spinning distance, 8～20cm；Relative humidity, 20-
35%；Room temperature, all of fiber is placed in 24～36h in freezer dryer after spinning, is completely dried storage at -20 DEG C.
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| CN107519524A (en) * | 2017-09-21 | 2017-12-29 | 滨州医学院 | A kind of polycaprolactone/collagen/quaternary ammonium salt electrospun composite fibers film and preparation method thereof |
| CN107630292A (en) * | 2017-09-18 | 2018-01-26 | 吉林大学 | A kind of bioactive film and its electrostatic spinning preparation method for loading spermidine |
| 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 |
| CN111560709A (en) * | 2019-07-26 | 2020-08-21 | 上海交通大学医学院附属上海儿童医学中心 | Nanofiber electrospun membrane containing axitinib and preparation method and application thereof |
| CN114685822A (en) * | 2020-12-31 | 2022-07-01 | 财团法人工业技术研究院 | Non-fibrous membrane and cell layer sheet |
| CN114949322A (en) * | 2022-04-13 | 2022-08-30 | 齐齐哈尔医学院 | Preparation method of tissue-induced nanofiber biomaterial capable of trapping host cells by graphene quantum dot tweezers and homing |
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| CN107630292A (en) * | 2017-09-18 | 2018-01-26 | 吉林大学 | A kind of bioactive film and its electrostatic spinning preparation method for loading spermidine |
| CN107630292B (en) * | 2017-09-18 | 2019-04-30 | 吉林大学 | A kind of bioactive film and its electrostatic spinning preparation method loading spermidine |
| CN107519524A (en) * | 2017-09-21 | 2017-12-29 | 滨州医学院 | A kind of polycaprolactone/collagen/quaternary ammonium salt electrospun composite fibers film and preparation method thereof |
| 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 |
| CN111560709A (en) * | 2019-07-26 | 2020-08-21 | 上海交通大学医学院附属上海儿童医学中心 | Nanofiber electrospun membrane containing axitinib and preparation method and application thereof |
| CN114685822A (en) * | 2020-12-31 | 2022-07-01 | 财团法人工业技术研究院 | Non-fibrous membrane and cell layer sheet |
| CN114949322A (en) * | 2022-04-13 | 2022-08-30 | 齐齐哈尔医学院 | Preparation method of tissue-induced nanofiber biomaterial capable of trapping host cells by graphene quantum dot tweezers and homing |
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