CN105326581B - A kind of method for preparing polyethylene glycol protein fiber composite heart valve prosthesis - Google Patents
A kind of method for preparing polyethylene glycol protein fiber composite heart valve prosthesis Download PDFInfo
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- CN105326581B CN105326581B CN201510633718.9A CN201510633718A CN105326581B CN 105326581 B CN105326581 B CN 105326581B CN 201510633718 A CN201510633718 A CN 201510633718A CN 105326581 B CN105326581 B CN 105326581B
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- polyethylene glycol
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- eggshell membrane
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 32
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- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 29
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims 8
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- Prostheses (AREA)
Abstract
The invention discloses a kind of method for preparing polyethylene glycol protein fiber composite heart valve prosthesis, belong to biomedical materials field.Composite is made by the coated egg membrane protein matter fiber of photo-crosslinking polyethylene glycol hydrogel, is further crosslinked using molecule cross-link agent (such as glutaraldehyde) and prepares complex layered heart valve prosthesis.The present invention is made polyethylene glycol hydrogel by optical cross-linking method and not only ensure that the integraty of artificial valve material, and protects coated protein fibre to prevent it from being degraded in vivo by enzymolysis, and prevents the calcification of artificial valve.Artificial heart valve membrane material prepared by this method has imitated the layer structure of human aortic valve, realize and its similar mechanical property, good biocompatibility is ensure that simultaneously, can meet that displacement acceptor is strong to valve durability, histocompatbility is good and need not lifelong anticoagulant demand.
Description
Technical field
The present invention relates to biology medical material technical field, and in particular to it is multiple that one kind prepares polyethylene glycol-protein fibre
The method for closing heart valve prosthesis.
Background technology
Heart valve disease is to jeopardize a kind of important diseases of human health, and valvular calcification and fibrosis cause a left side
Ventricular outflow tract blocks and aorta petal severe stenosis, does not have any surgical operation and medicine to cure calcified valves lesion so far
Etc. disease, the method for unique feasible is valve replacement, and being applied to clinical artificial valve now includes mechanical valve prosthesis and bioprosthetic valves
Film.But need to take anticoagulation medicine throughout one's life after Mechanical prosthetic valve replacement, the complication such as thrombus otherwise occur.In view of machinery
Valve, which implants, can cause adverse reaction, and biovalve is produced the 1970s and gradually grown up.Now, it is feasible
Biological cardiac valves have following several types：Pig xenogenesis valve, bovine pericardium valve, allogeneic valve (also cry homograft,
From donor) and autograft (tissue for coming from autologous patient).Allograft and heterograft can induce
The growth of fibr tissue, the incidence of disease of obstructive thrombus can be controlled between 0.2-1.8%, by valvular function sexual dysfunction disease
Sick incidence of disease control is between 2~4%.It is average but with the increase of transplant time, biovalve is easily decayed and calcification
Life-span is only 7-20, for teenager transplant recipient crowd, will be faced with the pain of Repeated Operation.Therefore,
Artificial valve of the development with more preferable structure and biological property is needed, to meet that displacement acceptor is strong to valve durability, organizes
Compatibility is good and without lifelong anticoagulant demand.
The content of the invention
The purpose of the present invention is need to take anticoagulation and biology throughout one's life for the artificial mechanical valve prosthesis commonly used on Present clinical
The problems such as calcification of valve and immunological rejection, there is provided it is valvular that one kind prepares polyethylene glycol-protein fibre combined artificial
Method, prepared using the good polyethylene glycol of the egg membrane protein matter fiber and biocompatibility of good mechanical performance for raw material
Complex layered heart valve prosthesis, and can be controlled by adjusting the number of plies of egg film, the volume of polyglycol solution and concentration multiple
The thickness and intensity of stratiform heart valve prosthesis are closed, there is important clinical value.
To achieve the above object, technical scheme is as follows：
One kind prepares polyethylene glycol-valvular method of protein fibre combined artificial, this method be with eggshell membrane and
Polyethylene glycol is raw material, prepares the lamellar composite of the coated protein fibre of polyethylene glycol hydrogel by photo-crosslinking method first
Material, then it is chemically crosslinked using molecule cross-link agent, obtains the polyethylene glycol-protein fibre combined artificial heart valve
Film.This method specifically comprises the following steps：
(1) PEGDA preparation：Passed through by polyethylene glycol (poly (ethylene glycol), write a Chinese character in simplified form PEG) and acryloyl chloride
Chemical reactive synthesis and purifying prepare the polyethyleneglycol diacrylate (poly (ethylene glycol) of Photocrosslinkable
Diacrylate, write a Chinese character in simplified form PEGDA)；
(2) pretreatment of eggshell membrane：Eggshell membrane after cleaning is immersed in PBS solution and is placed in storing in 4 DEG C of refrigerators,
Eggshell membrane is cut into the sample of required shape and size before photo-crosslinking, is placed in soaking 30 minutes in the pre- gelling solutions of PEGDA
More than；
(3) dimethyl silicone polymer of size and thickness (PDMS) template needed for preparing, one is hollowed out in PDMS templates center
It is individual with egg membrane sample same shape and size white space, the PDMS templates, which are placed on, a piece of to be hadCoating
Slide on, then white space in PDMS templates places pretreated egg membrane sample, is supplemented on sample
The pre- gelling solutions of PEGDA fill up PDMS template central clears region, and another has in PDMS template upper coversThe slide sealing of coating；
(4) cool white light or ultraviolet light cross-linking are utilized, the pre- gelling solutions of PEGDA on eggshell membrane surface are cross-linked to form PEG water-settings
Glue, ultimately form the complex layered materials of the coated eggshell membrane of PEG-hydrogel；According to cool white photo-crosslinking, crosslinking time 1-2
Minute；According to ultraviolet light cross-linking, crosslinking time is 10-30 minutes.
(5) complex layered materials obtained by step (4) are immersed in molecule cross-link agent (such as glutaraldehyde) solution, be chemically crosslinked
Polyethylene glycol-protein fibre combined artificial cardiac valves is obtained afterwards.
In above-mentioned steps (2), the cleaning process of eggshell membrane is：The yolk and egg white in raw egg are removed, from egg air chamber
After eggshell membrane and eggshell are peeled off by place, cleaned using deionized water；Then removed using acid soak method and remained on eggshell membrane
Calcium carbonate (CaCO3) particle, then to spend Ion Cleaning clean.The CaCO on eggshell membrane is removed using acid soak method3Particle
When, used acid is concentration 1-8mol/L acetum, and processing time is 2-12 hours, and treatment temperature is 4 DEG C；Using this
Egg membrane protein matter fiber intact can preserve after technique ensures acid treatment, and mechanical property is unaffected.
In above-mentioned steps (2), the PEGDA that step (1) obtains first is prepared into the pre- gelling solutions of PEGDA, it is prepared
In the pre- gelling solutions of PEGDA, the pre- gelling solutions of PEGDA for cool white photo-crosslinking are 10-30%PEGDA containing mass percent
(molecular weight 1000-3400g/mol), percent by volume 1-2% triethanolamines (triethanolamine, writing a Chinese character in simplified form TEOA), 10 μ
Mol/L eosins (Eosin Y) and percent by volume 0.375%N- vinyl pyrrolidones (N-vinylpyrrolidone, are write a Chinese character in simplified form
NVP PBS solution)；The pre- gelling solutions of PEGDA for ultraviolet light cross-linking are the (2-hydroxy- of Irgacure2959 containing 3g/L
40- (2-hydroxyethoxy) -2-methylpropiophe-none) PBS solution.
In above-mentioned steps (3), the thickness for making PDMS templates determines (individual layer eggshell membrane by the number of plies of used eggshell membrane
Thickness is 60-100 μm), PDMS template thickness 0.3-1mm.
In above-mentioned steps (5), the molecule cross-link agent is glutaraldehyde solution, and glutaraldehyde mass percent concentration is 0.25-
2%；The chemical crosslinking time is 2-12 hours.
Design principle of the present invention is as follows：
Composite is made by the coated egg membrane protein matter fiber of photo-crosslinking polyethylene glycol hydrogel in the present invention, further
It is crosslinked using molecule cross-link agent (such as glutaraldehyde) and prepares complex layered heart valve prosthesis.The present invention is with eggshell membrane and poly- second two
Alcohol is raw material, wherein eggshell membrane abundance, is made up of protein fibre；And polyethylene glycol be it is more conventional have it is good raw
The high polymer material of thing compatibility and biologically inert, polyethylene glycol hydrogel is made additionally by optical cross-linking method and not only ensure that people
The integraty of work valve material, and protect coated protein fibre to prevent it from being degraded in vivo by enzymolysis, and prevent artificial
The calcification of valve.In addition, it can be controlled by changing the eggshell membrane quantity of cladding and the concentration of polyethylene glycol hydrogel and volume
The thickness and intensity of valve material processed, to meet the individual demand of different patients.Heart valve prosthesis prepared by this kind of method
Material has imitated the layer structure of human aortic valve, realize with its similar mechanical property, while ensure that good life
Thing compatibility, it can meet that displacement acceptor is strong to valve durability, histocompatbility is good and need not lifelong anticoagulant demand.
Compared with existing cardiac valves technology of preparing, beneficial effects of the present invention are embodied in：
1st, the present invention realizes the mechanical performance similar to human body valve using eggshell membrane as inner layer material；Outer layer is coated
PEGDA hydrogels provide good biocompatibility and biologically inert, and protect the protein fibre of internal layer not to be degraded by enzymes.
2nd, polyethylene glycol proposed by the present invention-protein fibre combined artificial cardiac valves, can overcome existing machinery valve
The problems such as needing to use anticoagulation and the interior degraded of biovalve body and calcification throughout one's life.
3rd, the present invention can realize according to added by the egg film number of plies and polyglycol solution volume to prepare different-thickness and strong
The complex layered heart valve prosthesis of degree.It is 0.3-1mm that thickness can be made according to demand for the thickness of prepared valve material.Institute
The mechanical strength for preparing valve material is determined by the number of plies of the eggshell membrane added, the volume of PEGDA hydrogels and concentration.It is made
Modulus of elasticity >=3.3MPa of standby valve material, elongation percentage are more than 35%.
Brief description of the drawings
Fig. 1 is the stereoscan photograph of the protein fibre of original eggshell membrane；Wherein：(a) it is eggshell membrane outer layer；(b) it is
Egg film inner layer.
Fig. 2 is that PDMS masterplate methods prepare polyethylene glycol-protein fibre straticulate structure schematic diagram.
Fig. 3 is the poly- second two for using 4 layers of eggshell membrane to prepare 600 μ m-thicks for 20%PEGDA solution with mass percent concentration
Alcohol-protein fibre complex layered materials (molecule cross-link agent before processing).
Fig. 4 is the artificial heart for using 4 layers of eggshell membrane to prepare 600 μ m-thicks for 20%PEGDA solution with mass percent concentration
Dirty valve (after molecule cross-link agent processing).
The artificial heart valve that Fig. 5 is 4 layers of eggshell membrane and mass percent concentration is 20%PEGDA solution 600 μ m-thicks of preparation
Film after glutaraldehyde cross-linking after mouse subcutaneously embeds 2 weeks around histotomy HE stained photographs；Wherein：(a) it is 4 times of amplifications
Figure；(b) it is 10 times of enlarged drawings.
The artificial heart valve that Fig. 6 is 4 layers of eggshell membrane and mass percent concentration is 20%PEGDA solution 400 μ m-thicks of preparation
Film soaks the modulus of elasticity after 2 weeks after glutaraldehyde cross-linking and in type i collagen enzyme.
Embodiment
Technical scheme is described further with reference to embodiment and accompanying drawing.
The present invention is using eggshell membrane and polyethylene glycol as raw material, and egg film is by the fiber rich in collagen and keratin
The netted membrane structure of composition.By by the protein fibre of egg film with polyethylene glycol hydrogel is compound prepares heart valve prosthesis.
Technical process of the present invention is：Egg film raw material → removal CaCO3Particle → PDMS stamp fabrications → egg membrane fiber with
PEGDA hydrogels photo-crosslinking → molecule cross-link agent chemical crosslinking → heart valve prosthesis.Detailed process is as follows：By polyethylene glycol
(PEG, mean molecule quantity 3350g/mol) and acryloyl chloride reaction prepare polyethyleneglycol diacrylate (PEGDA) [Zhang X,
Xu B,Puperi DS,Yonezawa AL,Wu Y,Tseng H,Cuchiara ML,West JL,Grande-Allen
KJ.Integrating Valve-Inspired Design Features Into Poly(ethylene glycol)
Hydrogel Scaffolds for Heart Valve Tissue Engineering.Acta Biomaterialia
2015；14:11-21.].The yolk and egg white in raw egg are removed, is carefully peeled off eggshell membrane at egg air chamber, deionization
Water is cleaned 3 times, and egg film is immersed in into the CaCO in acetum removal egg film3Particle (saves eggshell membrane urporotein fibre
Tie up structure, Fig. 1), it is stored in PBS solution at 4 DEG C.Above-mentioned eggshell membrane is soaked in PEGDA solution 30 minutes before photo-crosslinking
More than.Using conventional method prepare different-thickness (0.3-1mm) dimethyl silicone polymer (PDMS) template [Mata A,
Fleischman AJ,Roy S.Characterization of Polydimethylsiloxane(PDMS)Properties
for Biomedical Micro/Nanosystems.Biomedical Microdevices 2005；7:281-293.],
PDMS templates center hollows out a semicircular area, and PDMS templates, which are placed on, a piece of to be hadThe glass slide of coating
On, the eggshell membrane handled well is put into hollow region, and the pre- gelling solutions of PEGDA are added dropwise, it is then another in capping above
OneCoating [Diaz ME, Cerro RL.Transition fromsplit streamlines to dip-
coating during Langmuir-Blodgett film deposition.Thin Solid Films 2004；460:
274-278.] load thin slice and seal (Fig. 2), be crosslinked to obtain polyethylene glycol-protein with cool white light source or ultraviolet source irradiation
Fiber composite stratiform material (Fig. 3), finally obtained complex layered materials are chemically crosslinked at 4 DEG C with glutaraldehyde solution,
Go PBS solution to clean 3 times, obtain heart valve prosthesis (Fig. 4).
Embodiment 1
Raw material and requirement：Fresh Egg film, polyethylene glycol, acryloyl chloride, triethanolamine (TEOA), eosin (Eosin
Y), (chemical reagent is for PBS solution, ethanol, NVP (NVP), 40% solution of sodium bisulfite, ultra-pure water
Analyze pure).
PEGDA is synthesized：Polyethyleneglycol diacrylate (PEGDA) is prepared by polyethylene glycol (PEG) and acryloyl chloride reaction；
Egg film processing：The yolk and egg white in raw egg are removed, is carefully peeled off eggshell membrane at egg air chamber, deionization
Water is cleaned 3 times, egg film is immersed in the acetic acid that concentration is 8mol/L, and 4 DEG C are handled 12 hours, and deionized water is cleaned 3 times.Afterwards
Clip diameter 12mm semicircle samples, soak at room temperature is in the pre- gelling solution of PEGDA white lights that mass percent concentration is 20%
30 minutes；
PDMS template constructs：Conventional method prepares thick dimethyl silicone polymer (PDMS) templates of 0.6mm, in PDMS templates
Center hollows out diameter 12mm semicircle shape model；
Photo-crosslinking：PDMS templates are placed on into one hasIn the glass slide of coating, in hollow region
The 8 layers of eggshell membrane handled well are inside put into, a small amount of pre- gelling solution of PEGDA white lights to template hollow region is instilled and all fills
Full, being capped another above hasThe slide sealing of coating, is irradiated 90 seconds with cool white light source；
Separation cleaning：Complex layered artificial heart valve membrane material is carefully separated from template, goes PBS solution soaking and washing 3
It is secondary；
Molecule cross-link agent is chemically crosslinked：It is in 4 DEG C, mass percent concentration by complex layered artificial heart valve membrane material
0.5% glutaraldehyde solution (utilizes 1mol/L NaHCO3Solution adjust pH value of solution=8) in crosslinking 6 hours, utilize quality hundred
Divide the solution of sodium bisulfite than 40% to clean 3 times, then clean 3 times with PBS solution and remove superfluous molecule cross-link agent, obtain people
Work cardiac valves.
Embodiment 2
This example will prepare the semicircle shape model that size is 12mm (diameter) × 0.6mm (thickness), choosing in PDMS templates
The 6 layers of eggshell membrane handled well filling PDMS templates are taken, remaining is the same as embodiment 1.Static tensile test measures heart valve prosthesis
Modulus of elasticity 3.43MPa, elongation percentage reach 43%.
Embodiment 3
This example will prepare the semicircle shape model that size is 12mm (diameter) × 0.6mm (thickness), choosing in PDMS templates
The 4 layers of eggshell membrane handled well filling PDMS templates are taken, remaining is the same as embodiment 1.Static tensile test measures heart valve prosthesis
Modulus of elasticity 3.30MPa, elongation percentage reach 35%.
Embodiment 4
This example will choose 4 layers of eggshell membrane handled well and fill PDMS templates, and remaining is the same as embodiment 1.By the sample of above-mentioned preparation
Product are cut into after 5mm x 5mm x 0.6mm samples that to be implanted to mouse back subcutaneous, rejection phenomenon are not observed, to planting after 2 weeks
Enter some of tissue slice HE dyeing (Fig. 5) and also do not observe inflammatory cell, these results illustrate above-mentioned artificial heart valve membrane material
With good biocompatibility.
Embodiment 5
This example will prepare the semicircle shape model that size is 12mm (diameter) × 0.4mm (thickness), choosing in PDMS templates
The 4 layers of eggshell membrane handled well filling PDMS templates are taken, remaining is the same as embodiment 1.By by above-mentioned heart valve prosthesis in 1mg/mL
Type i collagen enzyme (vigor 201u/mg) solution in soak 2 weeks, experimental result (Fig. 6) display enzymolysis before and after sample elastic modulus simultaneously
There was no significant difference (modulus of elasticity 3.71MPa before enzymolysis, enzymolysis after modulus of elasticity 3.56MPa), it was demonstrated that prepared artificial valve
Film has the ability to resistance to enzymic degradation.Gained heart valve prosthesis elongation percentage reaches 45%.
Embodiment 6
The pre- gelling solution mass percent concentrations of PEGDA that this example uses are 30%, and remaining is the same as embodiment 1.
Embodiment 7
The pre- gelling solution mass percent concentrations of PEGDA that this example uses are 30%, and remaining is the same as embodiment 3.
Claims (9)
1. one kind prepares polyethylene glycol-valvular method of protein fibre combined artificial, it is characterised in that：This method is first
The laminar composite of the coated protein fibre of polyethylene glycol hydrogel is prepared by photo-crosslinking method, then utilizes molecule cross-link
Agent is chemically crosslinked, and obtains the polyethylene glycol-protein fibre combined artificial cardiac valves；This method specifically includes as follows
Step：
(1) PEGDA preparation：By polyethylene glycol and acryloyl chloride the double propylene of polyethylene glycol are prepared through chemical reactive synthesis and purifying
Acid esters, i.e. PEGDA；
(2) pretreatment of eggshell membrane：Eggshell membrane after cleaning is immersed in PBS solution and is placed in storing in 4 DEG C of refrigerators, light is handed over
Eggshell membrane is cut into the sample of required shape and size before connection, is placed in soaking more than 30 minutes in the pre- gelling solutions of PEGDA；
(3) size and the PDMS templates of thickness needed for preparing, a shape identical with egg membrane sample is hollowed out in PDMS templates center
The white space of shape and size, the PDMS templates, which are placed on, a piece of to be hadOn the slide of coating, then in PDMS
Pretreated egg membrane sample is placed in white space in template, the pre- gelling solutions of PEGDA are supplemented by PDMS on sample
Template central clear region is filled up, then in PDMS template upper covers another haveThe slide sealing of coating；
(4) using cool white light or ultraviolet light cross-linking until forming the complex layered materials of the coated eggshell membrane of polyethylene glycol hydrogel；
(5) complex layered materials obtained by step (4) are immersed in molecule cross-link agent solution, poly- second two is obtained after chemical crosslinking
Alcohol-protein fibre combined artificial cardiac valves.
2. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：In step (2), the cleaning process of eggshell membrane is：The yolk and egg white in raw egg are removed, by egg at egg air chamber
After film is peeled off with eggshell, cleaned using deionized water；Then the calcium carbonate remained on eggshell membrane is removed using acid soak method
Particle, then cleaned up with deionized water.
3. according to claim 2 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：The CaCO on eggshell membrane is removed using acid soak method3During particle, used acid solution is concentration 1-8mol/L vinegar
Acid solution, processing time are 2-12 hours, and treatment temperature is 4 DEG C.
4. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：In step (2), in the pre- gelling solutions of PEGDA, the pre- gelling solutions of PEGDA for cool white photo-crosslinking are to contain quality
Percentage 10-30%PEGDA, percent by volume 1-2% triethanolamines, 10 μm of ol/L eosins and percent by volume 0.375%N-
The PBS solution of vinyl pyrrolidone；The pre- gelling solutions of PEGDA for ultraviolet light cross-linking are Irgacure2959 containing 3g/L
PBS solution.
5. polyethylene glycol-valvular method of protein fibre combined artificial is prepared according to claim 1 or 4, its
It is characterised by：In step (4), according to cool white photo-crosslinking, crosslinking time is 1-2 minutes；According to ultraviolet light cross-linking, during crosslinking
Between be 10-30 minutes.
6. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：In step (3), the thickness of PDMS template thickness 0.3-1mm, PDMS template is determined by the number of plies of used eggshell membrane.
7. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：In step (5), the molecule cross-link agent solution is glutaraldehyde solution, and glutaraldehyde solution mass percent concentration is 0.25-
2%, utilize 1mol/L NaHCO3Solution adjusts pH value of solution=8；The chemical crosslinking time is 2-12 hours.
8. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：The prepared valvular thickness of combined artificial determines by the number of plies of eggshell membrane used and the volume of PEGDA hydrogels, root
It is 0.3-1mm that thickness, which is made, according to demand；The prepared valvular mechanical strength of combined artificial by eggshell membrane used the number of plies,
The volume and concentration of PEGDA hydrogels determine.
9. according to claim 1 prepare polyethylene glycol-valvular method of protein fibre combined artificial, its feature
It is：The prepared valvular modulus of elasticity >=3.3MPa of combined artificial, elongation percentage are more than 35%.
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| CN108904877A (en) * | 2018-08-10 | 2018-11-30 | 中国科学院金属研究所 | Insertion type artificial cardiac valve and preparation method thereof based on macromolecular fibre composite material |
| CN110841110B (en) * | 2018-08-20 | 2021-03-12 | 四川大学 | Method for treating biological valve by combining enzyme crosslinking and tea polyphenol |
| CN109260517B (en) * | 2018-09-19 | 2020-10-30 | 杭州启明医疗器械股份有限公司 | Prefillable dry biological heart valve and preparation method thereof |
| CN109820624B (en) * | 2018-09-29 | 2019-12-06 | 四川大学 | Method for treating biological valve by adopting photo-crosslinking |
| CN109833519B (en) * | 2018-10-19 | 2021-03-12 | 四川大学 | Method for artificial biological valve |
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| CN102160899B (en) * | 2010-02-13 | 2013-10-30 | 华中科技大学同济医学院附属协和医院 | Polyethylene glycol crosslinked decellularized valve multi-signal composite scaffold material and preparation method thereof |
| US9351829B2 (en) * | 2010-11-17 | 2016-05-31 | Edwards Lifesciences Corporation | Double cross-linkage process to enhance post-implantation bioprosthetic tissue durability |
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