CN116036375A - Vinyl dicyclo oxazolidine crosslinked biological valve and preparation method and application thereof - Google Patents
Vinyl dicyclo oxazolidine crosslinked biological valve and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a vinyl dicyclo oxazolidine crosslinked biological valve, and a preparation method and application thereof, and belongs to the field of biological valve materials. The invention combines the main framework of OX-OH with functional vinyl to develop a functional dicyclooxazolidine cross-linking agent: ethylene-bicyclo oxazolidines (OX-VI). Then utilizing the porcine pericardium treated by OX-VI to further modify to obtain the non-glutaraldehyde crosslinked multifunctional biological valve. The multifunctional biological valve has excellent mechanical property, biocompatibility, calcification resistance, anticoagulation, low immune rejection and endothelialization promoting performance, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of biological heart valve materials, and particularly relates to a vinyl dicyclooxazolidine crosslinked biological valve, and a preparation method and application thereof.
Background
With the growth of population and the development of aging, more and more people suffer from heart disease. Valvular heart disease is a heart disease that threatens the lives of over 1 million patients worldwide. However, due to the special physiological environment of the heart valve, no specific medicine for treating the heart valve disease exists at present. Replacing heart valves with prosthetic heart valves is currently the best method for treating severe heart valve disease. The heart valves used in clinical practice include mechanical valves and Biological Heart Valves (BHV). In recent years, with the development of minimally invasive interventional techniques, small trauma, rapid recovery transcatheter heart valve replacement (transcatheter heart valve replacement, THVR) has become the primary solution for heart valve therapy. BHV has superior hemodynamic properties, good anticoagulation properties and satisfactory compressibility compared to mechanical petals, and has become the first choice for THVR, and clinical applications have increasingly demanded BHV in recent years.
Commercial artificial BHV is typically made of Porcine Pericardium (PP) or Bovine Pericardium (BP) crosslinked with glutaraldehyde (Glut) to enhance the mechanical properties of the biomaterial and prevent degradation of the biomaterial after implantation. However, a fatal disadvantage of this BHV is a short lifetime, and BHV after THVR can only be used for 10-15 years. Therefore, BHV must be extended in service life to service a wider patient population. Calcification was found to be the first problem to be resolved in glutaraldehyde-treated BHV. Calcification can harden BHV, limit its movement, and eventually cause BHV to fail to function properly, reducing its useful life. Meanwhile, thrombus is another cause of short service life of glutaraldehyde-treated BHV in clinical applications. In addition, the high toxicity of glutaraldehyde-treated BHV surface residual aldehyde groups also limits endothelial cell adhesion and proliferation, further exacerbating calcification and thrombus formation with the absence of valve surface endothelial layers.
Over the past several decades, researchers have tried a number of approaches to address the problems with glutaraldehyde-treated prosthetic heart valves. Some non-glutaraldehyde compounds, such as epoxy compounds, gini and carbodiimides, were used to explore the possibility of being a BHV crosslinking agent. However, most of the non-glutaraldehyde compounds do not solve the basic problems of BHV mechanical properties and calcification, and no further clinical application of non-glutaraldehyde-crosslinked BHV is seen.
Document (T.Yu et al; nonglutaraldehyde treated porcine pericardium with good biocompatibility, reduced calcification and improved Anti-coagulation for bioprosthetic heart valve applications; chemical Engineering Journal 414 (2021) 128900) reports a non-glutaraldehyde heart valve cross-linker: hydroxymethyl dicyclooxazolidine (OX-OH), and preparing the OX-OH crosslinked BHV (OX-OH-PP) by taking OX-OH and pig pericardium as raw materials. Compared with BHV crosslinked by glutaraldehyde, the OX-OH-PP can not only maintain similar mechanical properties, but also has better calcification resistance and biocompatibility. However, in order to meet clinical demands, the anti-inflammatory and anticoagulant properties of OX-OH-PP are still to be further optimized to reduce the inflammatory response at early stage of implantation and to expand its application in prosthetic biological valves with higher anticoagulation requirements for pulmonary valves, venous valves, etc.
Disclosure of Invention
The invention aims to provide a vinyl dicyclooxazolidine crosslinked biological valve, and a preparation method and application thereof.
The invention provides a biological valve material, which is prepared from a pericardium and OX-VI as raw materials, wherein the structure of the OX-VI is shown as follows:
further, the pericardium is a decellularized pericardium, preferably a decellularized porcine pericardium.
The invention also provides a method for preparing the biological valve material, which comprises the following steps: immersing the pericardium in OX-VI solution, and reacting at 15-35deg.C for 6-8 days to obtain biological valve material; the concentration of the OX-VI solution is 9wt% to 11wt%.
The invention also provides a modified biological valve material, which is obtained by carrying out free radical polymerization reaction on the biological valve material and 4-vinylphenylboronic acid under the action of an initiator; the initiator is preferably ammonium persulfate and sodium cetyl sulfate.
The invention also provides a method for preparing the modified biological valve material, which comprises the following steps: immersing the biological valve material into a mixed solution containing 4-vinylphenylboronic acid, ammonium persulfate and sodium hexadecyl sulfate, and reacting for 20-28 hours at room temperature to obtain a modified biological valve material; in the mixed solution, the concentration of 4-vinylbenzene boric acid is 2.0-3.0 wt%, the concentration of ammonium persulfate is 45-55mM, and the concentration of sodium hexadecyl sulfate is 45-55mM.
The invention also provides a functional biological valve material, which is obtained by taking the modified biological valve material and polyvinyl alcohol as raw materials to react.
The invention also provides a method for preparing the functional biological valve material, which comprises the following steps: immersing the modified biological valve material into a polyvinyl alcohol solution, and reacting for 20-28 hours at room temperature to obtain a functional biological valve material; the concentration of the polyvinyl alcohol solution is 9-11 wt%.
The invention also provides a multifunctional biological valve material, which is obtained by taking the functional biological valve material and fucoidin as raw materials to react.
The invention also provides a method for preparing the multifunctional biological valve material, which comprises the following steps: immersing the functional biological valve material into fucoidin solution, and reacting for 20-28 hours at room temperature to obtain the multifunctional biological valve material; the concentration of the fucoidin solution is 4wt% to 6wt%.
The invention also provides the application of the biological valve material, the modified biological valve material, the functional biological valve material and the multifunctional biological valve material in preparing artificial biological valves; the prosthetic biological valve is preferably an artificial heart valve, a prosthetic lung valve or a prosthetic venous valve.
The invention combines the main framework of OX-OH with functional vinyl to develop a functional dicyclooxazolidine cross-linking agent: ethylene-bicyclo oxazolidines (OX-VI). And then polymerizing the porcine pericardium treated by the OX-VI with a 4-vinylphenylboronic acid monomer to prepare the biological heart valve PB@OX-PP. PB@OX-PP is further modified by polyvinyl alcohol, so that the biological heart valve PBA@OX-PP is obtained. The PBA@OX-PP further reacts with fucoidin to obtain the biological heart valve PBAF@OX-PP.
The biological heart valve material provided by the invention has the following beneficial effects:
1. the functional dicyclooxazolidine crosslinking of the invention avoids the problem of aldehyde residue in the traditional glutaraldehyde crosslinking process, and can fundamentally avoid the risks of poor biocompatibility, easy calcification and the like inherent in glutaraldehyde crosslinking agents.
2. The biological heart valve material provided by the invention has excellent mechanical property, biocompatibility, calcification resistance, anticoagulation, low immune rejection and endothelialization promoting performance, and has wide application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: schematic flow chart of the preparation of vinyl dicyclooxazolidine crosslinked bioprosthetic heart valves.
Fig. 2: synthetic route for OX-VI; OX-VI 1 H NMR spectroscopy; OX-VI 13 C NMR spectrum. * Represents CDCl 3 Is a characteristic peak of (2).
Fig. 3: results of studies on the resistance to enzymatic degradation.
Fig. 4: cytotoxicity study results.
Fig. 5: endothelial cell adhesion study results.
Fig. 6: blood experimental study results. A. Schematic diagram of half internal circulation experiment; B. half-in-vivo circulation experimental photographs; C. photographs of the sample after semi-in vivo circulation; D. scanning electron microscope images of the samples after semi-body circulation.
Fig. 7: the rats were subcutaneously implanted for inflammatory response. A. Immunofluorescence of interleukin 6 and interleukin 10; B. interleukin 6 marks the cell number; C. interleukin 10 marks cell number.
Fig. 8: the rats were subcutaneously implanted for calcification. A. Alizarin red-stained photographs; B. implanting for 30 days to obtain calcium element content; C. calcium element content was implanted for 90 days.
Detailed Description
Unless otherwise indicated, the materials and equipment used in the present invention are known products and are obtained by purchasing commercially available products.
The temperature of the operating procedure in the examples was room temperature (i.e., 25.+ -. 5 ℃ C.) unless otherwise indicated.
The solutions described in the examples are aqueous solutions unless otherwise specified.
OX-OH was prepared according to the method described in the literature (T.Yu et al; nonglutaraldehyde treated porcine pericardium with good biocompatibility, reduced calcification and improved Anti-coagulation for bioprosthetic heart valve applications; chemical Engineering Journal 414 (2021) 128900).
Example 1: preparation of vinyl dicyclooxazolidine crosslinked biological heart valve
A vinyldicyclooxazolidine crosslinked bioprosthetic heart valve was prepared according to the procedure shown in fig. 1. The method comprises the following specific steps:
synthesis of OX-VI
OX-VI was synthesized following the route shown in FIG. 2A. The specific operation is as follows:
OX-OH (14.50 g,0.10 mol) and triethylamine (13.15 g,0.13 mol) were added to the flask and dissolved in 200mL of anhydrous tetrahydrofuran; acryloyl chloride (9.96 g,0.11 mol) was diluted with 100mL anhydrous tetrahydrofuran and added slowly dropwise to the reaction solution. After 24 hours of reaction, the white solid was filtered off and the filtrate was concentrated by rotary evaporation. Then, the mixture was dissolved in ethyl acetate, and washed three times with ice-saturated brine. Finally, recrystallization using a low concentration of ethyl acetate/petroleum ether (wherein the volume ratio of ethyl acetate/petroleum ether is 1:10) gives OX-VI.
1 H NMR(400MHz,CDCl 3 )δ＝6.43(dd,J＝17.3Hz,1.3Hz,1H),6.14(dd,J＝17.3Hz,10.4Hz,1H),5.88(dd,J＝10.5Hz,1.4Hz,1H),4.59-4.41(m,4H),4.24(s,2H),3.88-3.69(m,4H). 13 C NMR(100MHz,CDCl 3 )δ＝165.8,131.6,127.8,88.3,73.8,71.4,66.6.HRMS(ESI + ):calcd for C9H13NO4[m/z] + 200.0878,found 200.0920.
2. Preparation of vinyl dicyclooxazolidine crosslinked biological heart valve
Collecting fresh pig pericardium, and cleaning with deionized water at 4deg.C and shaking at 100rpm for 2 hr; then, the animal biological tissue is subjected to decellularization treatment for 3 hours by adopting a mixed solution of 0.1 weight percent of sodium dodecyl sulfate and 0.1 weight percent of deoxycholic acid, and the decellularized pig pericardium (D-PP) is obtained. And immersing the D-PP into 10wt% of OX-VI solution, crosslinking and fixing for 7 days at 25 ℃, taking out, and ultrasonically cleaning for 5 times to obtain the OX-VI-PP.
Then, OX-VI-PP was immersed in a mixed solution containing 50mM Ammonium Persulfate (APS), 50mM Sodium Hexadecyl Sulfate (SHS) and 2.5wt% 4-vinylphenylboronic acid, reacted at room temperature for 24 hours, and 4-vinylphenylboronic acid was polymerized with a carbon-carbon double bond (c=c) on OX-VI-PP by a radical polymerization method, and after taking out, ultrasonic cleaning was performed for 5 times to obtain pb@ox-PP.
Then, the PB@OX-PP is soaked in a 10wt% polyvinyl alcohol solution for 24 hours, taken out and ultrasonically cleaned for 5 times to obtain PBA@OX-PP, then the PBA@OX-PP is soaked in a 5wt% fucoidan solution for 24 hours, taken out and ultrasonically cleaned for 5 times to obtain PBAF@OX-PP.
Comparative example 1: preparation of Glut-PP
Glutaraldehyde solution (w/v) at a concentration of 1.5% was prepared, and 6% NaCl (w/v) was added thereto, and pH was adjusted to 5 with hydrochloric acid at a concentration of 1M. And then vertically placing the fixed decellularized pig pericardium (D-PP) in the glutaraldehyde solution, and vibrating for 2 hours at room temperature to enable the glutaraldehyde solution to permeate into the pig pericardium as much as possible. Finally, slowly adjusting the pH value to be neutral by using a saturated sodium carbonate solution, oscillating for 24 hours at the room temperature at the speed of 120rpm to crosslink, taking out, and ultrasonically cleaning for 5 times to obtain the glutaraldehyde crosslinked biological heart valve: glut-PP.
The beneficial effects of the biological heart valve prepared by the invention are proved by experimental examples.
Experimental example 1: study of resistance to enzymatic degradation
1. Experimental method
The sample was lyophilized and then cut to 1cm x 1cm size (n=5), and its dry weight was measured and recorded as W 1 . A1 mg/mL (125U) collagenase solution was prepared with PBS buffer. After each sample was immersed in 5mL of collagenase solution, shaking was continued for 24 hours (120 rpm) at a constant temperature of 37 ℃. After the reaction was completed, the sample was centrifuged at 1X10 4 Centrifuging at rpm for 10min, washing with PBS buffer three times, freeze drying again, and weighing dry weight, denoted as W 2 . The weight loss ratio of the enzymatic degradation of the sample was calculated from the following formula.
2. Experimental results
The experimental results are shown in fig. 3, and it can be seen that the non-glutaraldehyde crosslinked multifunctional biological heart valve of the present invention has superior resistance to enzymatic degradation compared to glutaraldehyde crosslinked biological heart valves.
Experimental example 2: cytotoxicity study
1. Experimental method
The direct contact with blood of medical devices can be used to evaluate their cytotoxicity by leaching solutions, according to the relevant detection criteria for implantable medical devices. Samples were cut to 1cm x 1cm size (n=6), sterilized by 75% immersion for 24h and three treatments with sterile PBS buffer wash, placed in 12 well cell culture plates, respectively. The samples were submerged in 1mL PMI 1640 medium (containing 10% fetal bovine serum) in a sterile incubator (5% CO) at 37 ℃ 2 ) Medium without sample was incubated as a blank control for 72h in a thermostated incubator for 72h. Human vascular endothelial cells were selected as cytotoxic test cells, and 5x10 cells were seeded in each well of 96-well cell culture plate wells, respectively 3 Individual cells. After 24h of cell growth, the original medium was discarded, the medium after 72h of leaching sample was added, and the cells were again incubated for 24h and 48h, respectively. A10% solution (v/v) of CCK-8 was prepared in serum-free RPMI 1640 medium. After the end of the co-incubation time, 100. Mu.L of CCK-8 solution was added to each sample well and incubation was continued for 1.5h in a sterile thermostated incubator. Finally, the absorbance value of each sample at 450nm is measured by an enzyme-labeled instrument, and the corresponding cytotoxicity is calculated.
2. Experimental results
The experimental results are shown in fig. 4, and it can be seen that compared with glutaraldehyde-crosslinked biological heart valves, the non-glutaraldehyde-crosslinked multifunctional biological heart valves of the invention have significantly reduced cytotoxicity and better biocompatibility.
Experimental example 3: endothelial cell adhesion study
1. Experimental method
The samples are respectively cut into the pore sizes of 48 pore plates, and soaked for 24 hours by using 75% ethanol solution to sterilize the samples. After sterilization was completed, the samples were washed three times with sterile PBS solution to remove the surface residual ethanol solution. The treated samples were placed in 48-well plates and 1x10 were planted in each well 4 Density of individual endothelial cells human endothelial cells were seeded onto the samples. At 37℃and 5% CO 2 Is cultured in a sterile constant temperature incubator for 24 hours and 72 hours. Configuration with sterile PBSPI staining solution at a concentration of 10. Mu.g/mL. The original culture medium is discarded, and after the PI staining reagent is added and the sample is stained for 5min, the adhesion of endothelial cells on the surface of the sample is observed by using a fluorescence inverted microscope.
2. Experimental results
Experimental results as shown in fig. 5, it can be seen that the non-glutaraldehyde-crosslinked multifunctional bioprosthetic heart valve of the present invention has superior endothelial cell growth and adhesion promoting ability compared to glutaraldehyde-crosslinked bioprosthetic heart valves.
Experimental example 4: blood experiment research
1. Experimental method
Samples of different groups were cut into rectangles 15mm by 10mm size, sterilized and repeatedly rinsed with sterile PBS, after which all operations were performed under sterile conditions. And filling the sterilized sample into a PVC pipe, and assembling the PVC pipe into a pipeline capable of being connected with the artery and vein. Then, after the New Zealand white rabbits are anesthetized by pentobarbital sodium solution, the neck artery and vein are separated, and the neck artery and vein are connected with a pipeline after puncture, so as to establish a circulating loop. After 1 hour of circulation, the line was removed and the sample was washed three times with physiological saline. After the sample was weighed, it was fixed with a 2.5% glutaraldehyde solution. After dehydration, critical point drying, metal spraying and SEM observation of the sample surface.
2. Experimental results
The experimental results are shown in fig. 6, and it can be seen that the non-glutaraldehyde-crosslinked multifunctional bioprosthetic heart valve of the present invention has superior anticoagulation ability compared to glutaraldehyde-crosslinked bioprosthetic heart valves.
Experimental example 5: inflammatory response and calcification conditions in subcutaneous implantation in rats
1. Experimental method
Samples were cut to 1cm x 1cm size (n=6x3) and washed three times with PBS. Then soaking in 75% ethanol solution for 12 hr for sterilization, washing with PBS for 3 times after sterilization, and rinsing for 15min each time to ensure complete removal of residual ethanol. Male SD rats were selected as subjects, and after about 50g of body weight was fed for one week, the experiment was performed. After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, two 1cm mouthpieces were opened on both sides of the back of the rats and two pockets were formed by outward expansion without communication. The samples were placed in two pockets and the membrane was kept flat during implantation. After implantation, the wound was sutured with sutures and swabbed with iodophor to prevent bacterial infection. The material was removed at 30 and 60 days, respectively, and the removed material was divided into two parts, one part was paraffin-embedded after fixation with tissue fixative for immunohistochemistry and staining. And the other part of the material is directly subjected to calcification quantitative analysis.
2. Experimental results
Experimental results as shown in fig. 7 and 8, it can be seen that the non-glutaraldehyde-crosslinked multifunctional bioprosthetic heart valve of the present invention has a lower inflammatory response (fig. 7) than glutaraldehyde-crosslinked bioprosthetic heart valves, while being able to reduce calcification (fig. 8).
Claims (10)
1. A biological valve material characterized by: the preparation method is prepared from a pericardium and OX-VI as raw materials, wherein the structure of the OX-VI is shown as follows:
2. the biological valve material of claim 1, wherein: the pericardium is a decellularized pericardium, preferably a decellularized porcine pericardium.
3. A method of preparing the biological valve material of any one of claims 1-2, characterized by: it comprises the following steps: immersing the pericardium in OX-VI solution, and reacting at 15-35deg.C for 6-8 days to obtain biological valve material; the concentration of the OX-VI solution is 9wt% to 11wt%.
4. A modified biological valve material, characterized in that: the biological valve material is obtained by carrying out free radical polymerization reaction on the biological valve material according to any one of claims 1-2 and 4-vinylphenylboronic acid under the action of an initiator; the initiator is preferably ammonium persulfate and sodium cetyl sulfate.
5. A method of preparing the modified biological valve material of claim 4, characterized by: it comprises the following steps: immersing the biological valve material according to any one of claims 1-2 in a mixed solution containing 4-vinylphenylboronic acid, ammonium persulfate and sodium hexadecyl sulfate, and reacting for 20-28 hours at room temperature to obtain a modified biological valve material; in the mixed solution, the concentration of 4-vinylbenzene boric acid is 2.0-3.0 wt%, the concentration of ammonium persulfate is 45-55mM, and the concentration of sodium hexadecyl sulfate is 45-55mM.
6. A functional biological valve material characterized by: which is obtained by reacting the modified biological valve material of claim 4 with polyvinyl alcohol.
7. A method of preparing the functional biological valve material of claim 6, characterized by: it comprises the following steps: immersing the modified biological valve material in polyvinyl alcohol solution, and reacting for 20-28 hours at room temperature to obtain a functional biological valve material; the concentration of the polyvinyl alcohol solution is 9-11 wt%.
8. A multifunctional biological valve material, characterized in that: which is obtained by reacting the functional biological valve material of claim 6 with fucoidan.
9. A method of making the multi-functional biological valve material of claim 8, characterized by: it comprises the following steps: immersing the functional biological valve material in the fucoidin solution, and reacting for 20-28 hours at room temperature to obtain the multifunctional biological valve material; the concentration of the fucoidin solution is 4wt% to 6wt%.
10. Use of the biological valve material of any one of claims 1-2, the modified biological valve material of claim 4, the functional biological valve material of claim 6, the multifunctional biological valve material of claim 8 in the preparation of a prosthetic biological valve; the prosthetic biological valve is preferably an artificial heart valve, a prosthetic lung valve or a prosthetic venous valve.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211729662.3A CN116036375A (en) | 2022-12-30 | 2022-12-30 | Vinyl dicyclo oxazolidine crosslinked biological valve and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211729662.3A CN116036375A (en) | 2022-12-30 | 2022-12-30 | Vinyl dicyclo oxazolidine crosslinked biological valve and preparation method and application thereof |
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| CN116036375A true CN116036375A (en) | 2023-05-02 |
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| CN202211729662.3A Pending CN116036375A (en) | 2022-12-30 | 2022-12-30 | Vinyl dicyclo oxazolidine crosslinked biological valve and preparation method and application thereof |
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| CN (1) | CN116036375A (en) |
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- 2022-12-30 CN CN202211729662.3A patent/CN116036375A/en active Pending
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