BIOPROSTHETIC VALVE AND PREPARATION METHOD THEREOF

Abstract

A bioprosthetic valve and a preparation method thereof are provided. The bioprosthetic valve includes a stent and a functional biological tissue material attached to the stent. The functional biological tissue material is a biologicaltissue covalently bonded with an active group and a functional molecule or group. The method improves the anti-thrombosis and anti-calcification functions by covalently modifying the surface of a biological valve using an active group and a functional molecule or group with a substantial degree of grafting. The new bioprosthetic valve does not include aldehyde residues, exhibits excellent biocompatibility, optimal mechanical properties, high stability, and can meet the performance requirements of a biological valve delivered through a catheter.

Claims

1. A bioprosthetic valve, comprising a stent and a functional biological tissue material, wherein the functional biological tissue material is attached to the stent, and the functional biological tissue material is a biological tissue linked with an active group and a functional molecule or a functional group by covalent bonds; the functional molecule or the functional group is at least one selected from the group consisting of 15,000 Mw heparin, hyaluronic acid (HA), polyethylene glycol (PEG), argatroban, 4,500 Mw-5,000 Mw low-molecular-weight heparin (LMWH), hirudin, bivalirudin, fucoidan, peptide ACH.sub.11, antithrombin-III, sodium citrate, albumin, urokinase, hydroxyl, sulfonyl, sulfophenyl and zwitterion.

2. The bioprosthetic valve according to claim 1, wherein, a structural formula of the zwitterion is shown as follows: ##STR00002##

3. The bioprosthetic valve according to claim 1, wherein, the functional biological tissue material is prepared by soaking the biological tissue in deionized water to obtain a first mixture, and adding the active group into the first mixture to enable a volume concentration of the active group is 3-10% to obtain a second mixture; performing a reaction on the second mixture at room temperature for 12-36 h to obtain a reacted biological tissue, washing the reacted biological tissue to obtain a washed biological tissue; soaking the washed biological tissue in a functional molecule solution or a functional group solution overnight at 35° C.-40° C., wherein, a final concentration of the functional molecule or the functional group is 20 mM-500 mM; and adding an initiator for initiating a polymerization.

4. The bioprosthetic valve according to claim 3, wherein, the active group is added to enable the volume concentration of the active groups is 4%, followed by reacting at room temperature for 24 h.

5. The bioprosthetic valve according to claim 3, wherein, the washed biological tissue is soaked in the functional molecule solution or the functional group solution overnight at 37° C., wherein, the final concentration of the functional molecule or the functional group is 500 mM.

6. The bioprosthetic valve according to claim 1, wherein, the biological tissue is one selected from the group consisting of pericardium, aortic root, aortic valve, pulmonary root, pulmonary valve, tendon, ligament, skin, dura, peritoneum, blood vessel, pleura, diaphragm, mitral valve and tricuspid valve.

7. The bioprosthetic valve according to claim 3, wherein, the active group is one selected from the group consisting of acrylamide, methacrylamide, acrylate, methacrylate and allyl group.

8. The bioprosthetic valve according to claim 3, wherein, a weight of the functional molecule or the functional group is 1-30% of a dry weight of the functional biological tissue material.

9. The bioprosthetic valve according to claim 3, wherein, the polymerization initiated by the initiator is conducted at a temperature of no more than 50° C.

10. A preparation method of the bioprosthetic valve according to claim 1, comprising the following steps: preparing the functional biological tissue material, cutting the functional biological tissue material to form a tissue valve leaflet assembly, and attaching the tissue valve leaflet assembly to the stent to obtain the bioprosthetic valve.

11. The bioprosthetic valve according to claim 2, wherein, the functional biological tissue material is prepared by soaking the biological tissue in deionized water to obtain a first mixture, and adding the active group into the first mixture to enable a volume concentration of the active group is 3-10% to obtain a second mixture; performing a reaction on the second mixture at room temperature for 12-36 h to obtain a reacted biological tissue, washing the reacted biological tissue to obtain a washed biological tissue; soaking the washed biological tissue in a functional molecule solution or a functional group solution overnight at 35° C.-40° C., wherein, a final concentration of the functional molecule or the functional group is 20 mM-500 mM; and adding an initiator for initiating a polymerization.

12. The bioprosthetic valve according to claim 2, wherein, the biological tissue is one selected from the group consisting of pericardium, aortic root, aortic valve, pulmonary root, pulmonary valve, tendon, ligament, skin, dura, peritoneum, blood vessel, pleura, diaphragm, mitral valve and tricuspid valve.

13. The preparation method according to claim 10, wherein, a structural formula of the zwitterion is shown as follows: ##STR00003##

14. The preparation method according to claim 10, wherein, the functional biological tissue material is prepared by soaking the biological tissue in deionized water to obtain a first mixture, and adding the active group into the first mixture to enable a volume concentration of the active group is 3-10% to obtain a second mixture; performing a reaction on the second mixture at room temperature for 12-36 h to obtain a reacted biological tissue, washing the reacted biological tissue to obtain a washed biological tissue; soaking the washed biological tissue in a functional molecule solution or a functional group solution overnight at 35° C.-40° C., wherein, a final concentration of the functional molecule or the functional group is 20 mM-500 mM; and adding an initiator for initiating a polymerization.

15. The preparation method according to claim 14, wherein, the active group is added to enable the volume concentration of the active groups is 4%, followed by reacting at room temperature for 24 h.

16. The preparation method according to claim 14, wherein, the washed biological tissue is soaked in the functional molecule solution or the functional group solution overnight at 37° C., wherein, the final concentration of the functional molecule or the functional group is 500 mM.

17. The preparation method according to claim 10, wherein, the biological tissue is one selected from the group consisting of pericardium, aortic root, aortic valve, pulmonary root, pulmonary valve, tendon, ligament, skin, dura, peritoneum, blood vessel, pleura, diaphragm, mitral valve and tricuspid valve.

18. The preparation method according to claim 14, wherein, the active group is one selected from the group consisting of acrylamide, methacrylamide, acrylate, methacrylate and allyl group.

19. The preparation method according to claim 14, wherein, a weight of the functional molecule or the functional group is 1-30% of a dry weight of the functional biological tissue material.

20. The preparation method according to claim 14, wherein the polymerization initiated by the initiator is conducted at a temperature of no more than 50° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a flow chart of the preparation of a bioprosthetic valve.

[0046] FIG. 2 is a flow chart of the preparation of a biological tissue with anti-thrombosis and anti-calcification.

[0047] FIG. 3 is a schematic diagram illustrating the preparation mechanism of the functional biological tissue with anti-thrombosis and anti-calcification.

[0048] FIG. 4 is a scanning electron microscope (SEM) image illustrating the platelet adsorption of an MPC-20 biological tissue.

[0049] FIG. 5 is an SEM image illustrating the platelet adsorption of an MPC-500 biological tissue.

[0050] FIG. 6 is an SEM image illustrating the platelet adsorption of an SBMA-20 biological tissue.

[0051] FIG. 7 is an SEM image illustrating the platelet adsorption of an SBMA-500 biological tissue.

[0052] FIG. 8 is an SEM image illustrating the platelet adsorption of an unmodified biological tissue.

[0053] FIG. 9 is an SEM image illustrating the platelet adsorption of a glutaraldehyde-crosslinked biological tissue.

[0054] FIG. 10 is a diagram illustrating the growth of endothelial cells on the SBMA-500 biological tissue under a fluorescence microscope.

[0055] FIG. 11 is a diagram illustrating the growth of endothelial cells on the unmodified biological tissue under the fluorescence microscope.

[0056] FIG. 12 is a diagram illustrating the growth of endothelial cells on the glutaraldehyde-crosslinked biological tissue under the fluorescence microscope.

[0057] FIG. 13 is a diagram illustrating the pulsatile flow of a bioprosthetic valve made from the SBMA-500 (open state).

[0058] FIG. 14 is a diagram illustrating the pulsatile flow of a bioprosthetic valve made from the SBMA-500 (closed state).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiment 1 Preparation of a Functional Biological Tissue Material (a Biological Material with Anti-Coagulation and Anti-Calcification)

[0059] The preparation flow chart is shown in FIG. 2, and the preparation mechanism is shown in FIG. 3. The specific steps were as follows:

[0060] Fresh porcine pericardium was thoroughly washed with deionized water and then immersed in deionized water; methacrylic anhydride was added dropwise in a 4° C. ice bath under stirring to enable a final concentration of the anhydride is 4% (v/v), and the pH was adjusted to 7 with a sodium hydroxide solution; after the dropwise addition was completed, reaction was conducted at room temperature for 24 h; the porcine pericardium modified with methacrylic anhydride was thoroughly washed and then soaked overnight at 37° C. in a zwitterionic monomer solution with a final concentration shown in the table below; then an initiator was added for cross-linking, and reaction was conducted at 37° C. for 24 h to obtain the functional biological tissue material. The obtained porcine pericardium was thoroughly washed with deionized water and then stored in a 25% isopropanol (i-propanol) solution for later use.

[0061] Unmodified biological tissue: the preparation steps before the initiator was added for cross-linking were the same as above, but the porcine pericardium was directly added with the initiator for cross-linking without being soaked in the monomer solution. The obtained porcine pericardium was thoroughly washed with deionized water and then stored in a 25% i-propanol solution for later use.

[0062] The specific parameters are as follows:

TABLE-US-00001 Type and MPC/SBMA Sample concentration content name of monomer Initiator concentration (w/w) MPC-20 MPC: 20 mM 50 mM APS and 50 mM 1.6% sodium bisulfite MPC-500 MPC: 500 mM 50 mM APS and 50 mM 15.6%  sodium bisulfite SBMA-20 SBMA: 20 mM 50 mM APS and 50 mM 5.2% sodium bisulfite SBMA-500 SBMA: 500 mM 50 mM APS and 50 mM 24.6%  sodium bisulfite Unmodified 0 50 mM APS and 50 mM   0%* sodium bisulfite Notes: SBMA: [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide; MPC: 2-methacryloyloxyethyl phosphocholine; *Unmodified is used as a baseline, set as 0%; Initiator concentration refers to the concentration of an initiator immediately after being added to a reaction system; and MPC/SBMA content refers to the content of MPC/SBMA in a prepared biological material that is lyophilized.

[0063] Since the functional molecule MPC includes phosphorus which is almost not found in biological materials, and SBMA includes unique sulfur, inductively coupled plasma-optic emission spectroscopy (ICP-OES) can be used to analyze the content of sulfur or phosphorus in a biological material, which can be converted into the content of a functional molecule, so as to obtain the modification degree of the functional molecule on the biological tissue, facilitating the screening of experimental conditions.

[0064] The specific conversion method is as follows:

[00001]$\mathrm{Functional}\mathrm{molecule}\mathrm{content}\left(\%\right)=\frac{C_{S\left(P\right)}\times 10/M_{S\left(P\right)}\times M_{\mathrm{SBMA}\left(\mathrm{MPC}\right)}}{W}\times 100\%$

[0065] where, C.sub.S(P) (mg/L) is a concentration of sulfur or phosphorus in a solution determined by ICP-OES; M.sub.S(P) is a relative atomic mass of sulfur or phosphorus; M.sub.SBMA(MPC) is a relative molecular mass of a functional molecule; and W (mg) is a dry weight of a biological material.

[0066] It can be seen from the above table that, compared with the control group, the biological material prepared in the present invention shows that the biological tissue is well modified with the functional molecule, especially the SBMA-500 group, with the highest modification degree.

Embodiment 2 Anticoagulant Performance of the Biological Material with Anti-Coagulation and Anti-Calcification

[0067] The prepared biological material with anti-coagulation and anti-calcification was washed, cut into a suitable size, and incubated with platelet-rich plasma (PRP) for 1 h. The amount of lactate dehydrogenase (LDH) after platelets lysis adhered to the material was determined to indicate the number of adhered platelets, and the results are shown in the table below. The adhesion of platelets on the material was observed by SEM, and the specific results are shown in FIG. 4-FIG. 9.

TABLE-US-00002 Sample name Absorbance value (490 nm) MPC-20 0.30 MPC-500 0.29 SBMA-20 0.24 SBMA-500 0.110 Unmodified 0.32 Glutaraldehyde-crosslinked 0.35

[0068] It can be seen from the above table that the higher the absorbance value at 490 nm, the higher the content of LDH, that is, the more the platelets adhered to the material. Therefore, it can be seen from the above table that the biological material prepared in the present invention exhibits an anticoagulant effect significantly better than the anticoagulant effect of the unmodified biological tissue and the glutaraldehyde-crosslinked biological tissue (porcine pericardium was subjected to cross-linking for 24 h at room temperature and pH=7.4 in a glutaraldehyde aqueous solution with a volume concentration of 0.625%, and obtained porcine pericardium was dehydrated and dried). The SBMA-modified biological tissue exhibits a better anticoagulant effect than the MPC-modified biological tissue, and the SBMA-modified biological tissue with substantial grafting capability exhibits the best anticoagulant effect.

[0069] It can be seen from FIG. 4-FIG. 9 that the biological material prepared in the present invention exhibits an anticoagulant effect significantly better than the anticoagulant effect of the unmodified biological tissue and the glutaraldehyde-crosslinked biological tissue (porcine pericardium was subjected to cross-linking for 24 h at room temperature and pH=7.4 in a glutaraldehyde aqueous solution with a volume concentration of 0.625%, and obtained porcine pericardium was dehydrated and dried). The SBMA-modified biological tissue exhibits a better anticoagulant effect than the MPC-modified biological tissue, and the SBMA-modified biological tissue with substantial grafting capability exhibits the best anticoagulant effect.

[0070] The qualitative observation results of SEM in FIG. 4-FIG. 9 are consistent with the quantitative measurement results of LDH.

Embodiment 3 Endothelialization Performance of the Biological Material with Anti-Coagulation and Anti-Calcification

[0071] The prepared biological material with anti-coagulation and anti-calcification was inoculated with endothelial cells on the surface, then incubated for 3 days, and stained with DAPI- and FITC-labeled phalloidin. The results are shown in FIG. 10-FIG. 12.

[0072] It can be seen from FIG. 10-FIG. 12 that the growth of endothelial cells on the SBMA-500 modified biological tissue is better than the growth of endothelial cells on the unmodified and glutaraldehyde-crosslinked biological tissues.

Embodiment 4 Characterization of Anti-Calcification and Immune Response of the Biological Material with Anti-Coagulation and Anti-Calcification

[0073] The prepared biological material with anti-coagulation and anti-calcification was cut into 1 cm.sup.2 sheets, implanted subcutaneously into Sprague-Dawley (SD) rats, and taken out 90 days later. The calcium content in the sample was determined by ICP-OES. The results are as follows:

TABLE-US-00003 Sample name Calcium content (g/kg) SBMA-500 12.8 Unmodified 38.4 Glutaraldehyde-crosslinked 117.3

[0074] It can be seen from the above table that the calcification degree of the SBMA-500 modified biological tissue is significantly lower than the calcification degree of the unmodified and glutaraldehyde-crosslinked biological tissues.

Embodiment 5 Stability of the Biological Material with Anti-Coagulation and Anti-Calcification

[0075] The prepared biological material with anti-coagulation and anti-calcification was cut into small pieces of 3-8 mg, and the moisture on the surface was removed with filter paper. The sample was put in an aluminum crucible and sealed, and the crucible with the sample was placed in an instrument for conducting thermal denaturation temperature analysis by differential scanning calorimeter (DSC), to characterize the stability of the biological tissue. The results are as follows:

TABLE-US-00004 Sample name Thermal denaturation temperature (° C.) SBMA-500 80.15 Fresh tissue 68.88

[0076] It can be seen from the above table that the biological material with anti-coagulation and anti-calcification prepared in the present invention exhibits stronger high-temperature-resistance than fresh tissue, indicating that the biological tissue has superior stability.

Embodiment 6 Implantation of a Vascular Graft of the Biological Material with Anti-Coagulation and Anti-Calcification

[0077] The prepared biological material with anti-coagulation and anti-calcification was rolled into a tube with a diameter of about 2 mm, and a medical adhesive was used at the interface for bonding. The moisture on the surface was removed with filter paper. Then the tube was weighed, and recorded. The vascular anastomosis was used to replace a rabbit carotid artery with a length of about 2 cm. A period of time after the implantation, the material was taken out for observation. The moisture on the surface of the material was removed with filter paper, then the material was weighed again, and the weight gain was calculated, namely, the thrombus production. The results are as follows:

TABLE-US-00005 Initial Final Thrombus weight weight production Sample name (mg) (mg) (mg) SBMA-500 80.9 83.6 2.7 Unmodified 78.6 262.4 183.8 Glutaraldehyde-crosslinked 82.5 295.3 212.8

[0078] It can be seen from the above table that there is a thrombus production more than 2 times the tissue weight in the glutaraldehyde-crosslinked and unmodified biological tissues, and there is almost no thrombus production in the SBMA-500.

Embodiment 7 Preparation of a Bioprosthetic Valve (see FIG. 1 for the Preparation Process)

[0079] The biological tissue material with anti-coagulation and anti-calcification prepared in embodiment 1 that had an SBMA monomer concentration of 500 mM was laser-cut to form a tissue valve leaflet assembly, and the tissue valve leaflet assembly was attached to a nickel-titanium alloy stent by suturing to obtain an implantable bioprosthetic valve.

Embodiment 8 In vitro Hydrodynamic Performance Test of the Bioprosthetic Heart Valve

[0080] A optimal bioprosthetic heart valve with a diameter of 29 mm (modified with SBMA-500) was subjected to an in vitro hydrodynamic performance test under standard test conditions (heart rate: 70 beats/min, average cardiac output: 5 L/min, and average aortic pressure: 100 mmHg), and the following results were obtained: an effective opening area=2.855 cm.sup.2 and regurgitation ratio=9.998%, which met the requirements in ISO 5840-1:2015, indicating that the bioprosthetic valve met the hydrodynamic requirements. The specific results are shown in FIG. 13 and FIG. 14.

Embodiment 9 In Vitro Accelerated Fatigue Performance Test of the Bioprosthetic Heart Valve

[0081] The optimal bioprosthetic heart valve with a diameter of 29 mm (modified with SBMA-500) was subjected to an in vitro accelerated fatigue test on an in vitro accelerated fatigue tester. After 50 million heartbeats, the SBMA-500 modified valve still had excellent appearance, while leaflets of the unmodified biological valve were torn.

Embodiment 10 In Vivo Experiment on Implantation of a Bioprosthetic Venous Valve in Large Animals

[0082] A prepared bioprosthetic venous valve with a diameter of 12 mm (modified with SBMA-500) was sterilized and installed on a catheter delivery system before operation. The bioprosthetic valve was implanted into the iliac vein of a dog through the delivery system. The dog was followed up for 3 months, and then it was found through vascular ultrasound diagnosis that the vessel exhibited excellent patency, indicating that the SBMA-500 modified valve had a prominent anti-thrombosis function.