Biological valve material of bionic endothelial glycocalyx-like hydrogel coating and preparation method of biological valve material
By constructing a bionic endothelial glycocalyx-like hydrogel coating on biological valve materials, the thrombosis and calcification problems of biological valve materials during blood contact are solved, better biocompatibility and anti-calcification performance are achieved, and the stability and safety of the valve are improved.
Patent Information
- Application Number
- CN202510759239.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-08-15
AI Technical Summary
Existing biological valve materials are prone to thrombosis and calcification during blood contact, and the aldehyde groups produced by the glutaraldehyde cross-linking process are cytotoxic, affecting the long-term stability and safety of the valve.
The preparation method of a bionic endothelial glycocalyx-like hydrogel coating was used to carry out amide condensation reaction between caffeic acid and biomaterials, graft the catechol groups, and oxidation coupling reaction was carried out using Alg-DA solution to construct a stable hydrogel coating.
It significantly improves the biocompatibility and hemocompatibility of biological valves, reduces platelet adhesion and calcification, and improves the stability and safety of the valve.
Smart Images
Figure CN120478731A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological valve materials, and in particular to a biological valve material with a biomimetic endothelial glycocalyx-like hydrogel coating and a preparation method thereof. Background Art
[0002] Valvular heart disease (VHD) is a leading cause of cardiovascular morbidity and mortality worldwide, affecting more than 2% of the general population. With an aging population, the prevalence of VHD is projected to double by 2050. As there is currently no effective medical treatment, the treatment of VHD relies primarily on heart valve replacement. There are two types of prosthetic valves: mechanical and bioprosthetic. Mechanical valves are robust and durable, but due to their unique hemodynamics, they carry significant risks of bleeding and thrombosis, necessitating lifelong anticoagulation. In contrast, bioprosthetic valves are increasingly becoming the preferred choice for patients due to their superior hemodynamic performance and the general absence of the need for long-term anticoagulation.
[0003] Bioprosthetic valves are collagen-based biomaterials that readily absorb blood components such as fibrin and platelets, which can activate and aggregate platelets, leading to thrombosis and accelerated degeneration. Currently, bioprosthetic valves used clinically are typically made from porcine or bovine pericardium cross-linked with glutaraldehyde. While glutaraldehyde cross-linking can improve the collagen stability and mechanical properties of bioprosthetic valves, the glutaraldehyde cross-linking process produces residual aldehyde groups, which are cytotoxic and can easily lead to valve calcification. Summary of the Invention
[0004] In view of the above problems, the present invention aims to provide a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating and a preparation method thereof.
[0005] The technical solutions of the present invention are as follows:
[0006] In one aspect, a method for preparing a bioprosthetic valve material having a biomimetic endothelial glycocalyx-like hydrogel coating is provided, comprising the following steps:
[0007] S1: obtaining biological materials and pre-treating the biological materials;
[0008] S2: preparing an activated caffeic acid solution, and soaking the pretreated biomaterial in the activated caffeic acid solution to graft caffeic acid onto the biomaterial;
[0009] S3: preparing an Alg-DA solution containing an initiator, and immersing the grafted biomaterial therein to perform an oxidative coupling reaction, and washing after the reaction to obtain the bioprosthetic valve material with the biomimetic endothelial glycocalyx-like hydrogel coating.
[0010] Preferably, in step S1, the biological material is any one of pericardium, valve, intestinal membrane, meninges, pulmonary membrane, blood vessel, skin or ligament.
[0011] Preferably, in step S1, the preprocessing includes the following sub-steps:
[0012] S11: Store the obtained biological material in a low-temperature and humidified state at 0-4°C;
[0013] S12: preparing a mixed solution comprising sodium deoxycholate and sodium lauryl sulfate, wherein the mass ratio of the sodium deoxycholate to the sodium lauryl sulfate is 0.1%-0.5%;
[0014] S13: Soak the biomaterial stored in step S11 in the mixed solution and shake it continuously at room temperature for 1-2 days, and then wash it with deionized water until there is no visible adherent non-pericardial or non-collagenous tissue.
[0015] Preferably, in step S2, the caffeic acid solution is activated using an amide condensation agent.
[0016] Preferably, the amide condensation agent is any one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide.
[0017] Preferably, in step S2, the solvent in the caffeic acid solution is any one or more of water, methanol, and ethanol, and the mass fraction of caffeic acid is 1%-5%.
[0018] Preferably, in step S2, after soaking, the mixture is shaken at 75-100 rpm for 1-3 days at 25-40°C, and then washed with ethanol to obtain the grafted biomaterial.
[0019] Preferably, in step S3, in the Alg-DA solution, the molar concentration of the initiator is 20-50 mM, and the mass fraction of Alg-DA is 2%-5%.
[0020] Preferably, in step S3, the oxidative coupling reaction is carried out at 25-40° C. for 12-24 hours.
[0021] On the other hand, a bio-valve material with a biomimetic endothelial glycocalyx-like hydrogel coating is also provided, which is prepared using any of the above-mentioned methods for preparing a bio-valve material with a biomimetic endothelial glycocalyx-like hydrogel coating, and includes heart valves and venous valves.
[0022] The beneficial effects of the present invention are:
[0023] The present invention activates the carboxyl groups of caffeic acid to enable them to undergo an amide condensation reaction with the amino groups of the biomaterial, thereby introducing catechol groups into the biomaterial, and further oxidatively polymerizing with the catechol groups on the sodium alginate grafted with dopamine, thereby simultaneously achieving cross-linking and hydrogel coating construction. The resulting bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating has excellent biocompatibility, blood compatibility and anti-calcification properties. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0025] Figure 1 Schematic diagram of the principle of the preparation method of the bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating of the present invention;
[0026] Figure 2 This is a comparison chart of Alcian blue staining of the bioprosthetic valve materials of Example 1 and Comparative Example 1;
[0027] Figure 3 Schematic diagram comparing the surface morphologies of the bioprosthetic valve materials of Example 1 and Comparative Example 1;
[0028] Figure 4 Schematic diagram of water contact angles of bioprosthetic valve materials of Example 1 and Comparative Example 1;
[0029] Figure 5 Schematic diagram comparing the water contact angles of the bioprosthetic valve materials of Example 1 and Comparative Example 1.
[0030] Figure 6 ABST· + Schematic diagram of clearance ability comparison;
[0031] Figure 7 ·OH of the bioprosthetic valve material of Example 1 and Comparative Example 1 - Schematic diagram of clearance ability comparison;
[0032] Figure 8 Schematic diagram comparing platelet adhesion of the bioprosthetic valve material of Example 1 and Comparative Example 1;
[0033] Figure 9 Schematic diagram of the comparison of lactate dehydrogenase activity detection of biological valve materials in Example 1 and Comparative Example 1;
[0034] Figure 10Schematic diagram of HE staining comparison of the bioprosthetic valve material of Example 1 and Comparative Example 1;
[0035] Figure 11 and Figure 12 Schematic diagram of immunohistochemical CD3 / CD68 comparison of the bioprosthetic valve material of Example 1 and Comparative Example 1;
[0036] Figure 13 and Figure 14 Schematic diagram of immunofluorescence staining comparison of the bioprosthetic valve material of Example 1 and Comparative Example 1;
[0037] Figure 15 This is a schematic diagram showing the comparison of Alizarin Red staining of the bioprosthetic valve material of Example 1 and Comparative Example 1;
[0038] Figure 16 Schematic diagram of the comparison of von Kossa staining of the biological valve materials of Example 1 and Comparative Example 1. DETAILED DESCRIPTION
[0039] The present invention is further described below with reference to the accompanying drawings and examples. It should be noted that, in the absence of conflict, the embodiments in this application and the technical features in the embodiments can be combined with each other. It should be noted that, unless otherwise specified, all technical and scientific terms used in this application have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The use of similar words such as "include" or "comprising" in the present invention means that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.
[0040] In one aspect, the present invention provides a method for preparing a bioprosthetic valve material having a biomimetic endothelial glycocalyx-like hydrogel coating, comprising the following steps:
[0041] S1: Obtaining biological materials and pre-treating the biological materials.
[0042] In a specific embodiment, the biological material is any one of pericardium, valve, intestinal membrane, meninges, lung membrane, blood vessel, skin or ligament.
[0043] In a specific embodiment, the pre-processing comprises the following sub-steps:
[0044] S11: Store the obtained biological material in a low-temperature and humidified state at 0-4°C;
[0045] S12: preparing a mixed solution comprising sodium deoxycholate and sodium lauryl sulfate, wherein the mass ratio of the sodium deoxycholate to the sodium lauryl sulfate is 0.1%-0.5%;
[0046] S13: Soak the biomaterial stored in step S11 in the mixed solution and shake it continuously at room temperature for 1-2 days, and then wash it with deionized water until there is no visible adherent non-pericardial or non-collagenous tissue.
[0047] It should be noted that the purpose of pretreatment of the biomaterial is to remove non-pericardial or non-collagenous tissue adhered to the surface of the biomaterial. The pretreatment method in the above embodiment is only one of the preferred methods to achieve this purpose. Other pretreatment methods in the prior art that can achieve this purpose can also be applied to the present invention.
[0048] S2: preparing an activated caffeic acid solution, and soaking the pretreated biomaterial in the activated caffeic acid solution to graft caffeic acid onto the biomaterial.
[0049] In a specific embodiment, the caffeic acid solution is activated using an amide condensation agent. Optionally, the amide condensation agent is any one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide.
[0050] In a specific embodiment, the solvent in the caffeic acid solution is any one or more of water, methanol, and ethanol, and the mass fraction of caffeic acid is 1%-5%.
[0051] In a specific embodiment, the pretreated biomaterial is immersed in the activated caffeic acid solution and then shaken at 100 rpm at 37° C. for 1-2 days, and then washed with ethanol to obtain the grafted biomaterial.
[0052] S3: preparing an Alg-DA solution containing an initiator, and immersing the grafted biomaterial therein to perform an oxidative coupling reaction, and washing after the reaction to obtain the bioprosthetic valve material with the biomimetic endothelial glycocalyx-like hydrogel coating.
[0053] In a specific embodiment, the Alg-DA solution has an initiator concentration of 20-50 mM and a mass fraction of Alg-DA of 2%-5%. Optionally, the initiator is ammonium persulfate or potassium persulfate. It should be noted that the initiator is intended to initiate the oxidative coupling reaction between the grafted biomaterial and the Alg-DA. In addition to the initiator used in this embodiment, other initiators known in the art that can achieve this purpose are also suitable for use in the present invention.
[0054] In a specific embodiment, the oxidative coupling reaction is carried out at 25-40° C. for 12-24 hours.
[0055] like Figure 1As shown, in the present invention, the carboxyl groups of caffeic acid are activated to enable an amide condensation reaction with the amino groups of the biomaterial, thereby introducing catechol groups into the biomaterial. Using an Alg-DA solution, the catechol groups in the dopamine-grafted sodium alginate polymerize with the catechol in the caffeic acid-grafted biomaterial, achieving simultaneous crosslinking and hydrogel coating construction, thereby enhancing the stability of the hydrogel coating.
[0056] On the other hand, the present invention also provides a bio-valve material with a biomimetic endothelial glycocalyx-like hydrogel coating, which is prepared using any of the above-mentioned methods for preparing a bio-valve material with a biomimetic endothelial glycocalyx-like hydrogel coating, including heart valves and venous valves.
[0057] Example 1
[0058] A bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating is prepared by the following steps: freshly collected porcine pericardium is placed in a solution of 0.5% (w / w) SD and 0.5% (w / w) SDS at 4°C and 100 rpm, and shaken continuously at room temperature for 1 day. The pericardium is then thoroughly washed with deionized water and then immersed in an ethanol solution of caffeic acid (5% by mass) activated by 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (0.3M) for 1-2 days to fully graft the caffeic acid. The ungrafted caffeic acid on the surface is then washed with 50% ethanol and then immersed in a 5% Alg-DA solution containing 20mM ammonium persulfate for oxidative coupling reaction at 37°C for 24 hours. Finally, the residue on the surface of the material is washed with distilled water to obtain the bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating.
[0059] Example 2
[0060] Different from Example 1, in this example, the pretreated porcine pericardium was immersed in an ethanol solution of caffeic acid (mass fraction 2%) activated by 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (0.1 M) for 2-3 days.
[0061] Example 3
[0062] Different from Example 1, in this example, the grafted porcine pericardium was immersed in a 15 mM Alg-DA solution containing 2.5% ammonium persulfate by mass, and the oxidative coupling reaction was carried out at 37° C. for 12 h.
[0063] Comparative Example 1
[0064] A biological valve material is prepared by the following steps: freshly collected porcine pericardium is washed with distilled water at 4°C and 100 rpm for 2 hours, then immersed in a 0.625% by mass glutaraldehyde solution for 24 hours, and after the reaction is completed, taken out and immersed in a 0.25% by mass glutaraldehyde solution for preservation to obtain the biological valve material.
[0065] Test Example 1: Alcian Blue Staining:
[0066] The bioprosthetic valve materials obtained in Comparative Example 1 and Example 1 were cut into 1*1 cm square pieces, and the surface coating morphology was observed by Alcian blue staining. The experimental results are shown in FIG. Figure 2 As shown. Figure 2 It can be seen that a distinct colored coating appeared in Example 1, indicating the successful construction of the hydrogel coating.
[0067] Test Example 2: Scanning electron microscope observation of surface morphology:
[0068] The bioprosthetic valve materials obtained in Comparative Example 1 and Example 1 were cut into 0.5*0.5 cm square pieces, placed between two glass slides and flattened, freeze-dried in vacuum and sprayed with gold, and then the surface morphology of the materials was observed using a scanning electron microscope. The experimental results are shown in FIG. Figure 3 As shown. Figure 3 It can be seen that the surface gap of the biological valve material of Example 1 is reduced compared with that of Comparative Example 1, and the surface is smoother.
[0069] Test Example 3: Water contact angle test:
[0070] The bioprosthetic valve materials obtained in Comparative Example 1 and Example 1 were cut into 1*1 cm square pieces, placed between two glass slides and flattened, and then freeze-dried under vacuum to perform water contact angle tests. The experimental results are shown in FIG. Figure 4 and Figure 5 As shown. Figure 4 and Figure 5 It can be seen that the water contact angle of the bioprosthetic valve material of Example 1 is significantly reduced, and the hydrophilicity of the material is enhanced.
[0071] Test Example 4: Antioxidant Performance Test:
[0072] Using ABST + , ·OH - The free radical scavenging experiment was used to evaluate the antioxidant capacity of the samples. Specifically, the bioprosthetic valve materials obtained in Comparative Example 1 and Example 1 were cut into circular shapes (Φ=10 mm), and 1 mL of diluted ABTS· +The free radical solution was incubated in the dark for 1 hour. The absorbance of the supernatant at 517 nm was detected and the clearance rate was calculated. The Fenton reaction between H2O2 and FeSO4 was used to test its ability to scavenge ·OH. After reacting with the bioprosthetic valve material prepared in Comparative Example 1 and Example 1 for 1 hour, the specific probe salicylic acid (SA) was added and reacted for 15 minutes. The absorbance of each group was measured at 510 nm and the clearance rate was calculated. The experimental results are shown in Figure 2. Figure 6 and 7 As shown. Figure 6 and Figure 7 It can be seen that the bioprosthetic valve material of Example 1 has a significant effect on ABST· + and OH - The cleaning ability is significantly improved.
[0073] Test Example 5: Platelet Adhesion and Lactate Dehydrogenase Activity Test:
[0074] Collect fresh rabbit blood, centrifuge at 1500rpm for 15min, and obtain platelet-rich plasma. Cut the biological valve material obtained in Comparative Example 1 and Example 1 into discs (Φ=10mm) and rinse with PBS 3 times, put into a 48-well plate, add 100μL platelet-rich plasma and incubate at 37°C for 1h. At the same time, select 100μL platelet-rich plasma as a positive control for quantitative detection. After incubation, rinse with PBS 3 times. Use a scanning electron microscope to observe the platelets adhered to the surface of the material. In addition, a lactate dehydrogenase assay kit was used to determine the relative number of platelet adhesion. Use an enzyme marker to record the absorbance of each group at 490nm, and calculate the relative activity of lactate dehydrogenase in each group. The experimental results are as follows: Figure 8 and 9 As shown. Figure 8 It can be seen that the construction of the hydrogel coating in the bioprosthetic valve material of Example 1 improves its anti-platelet adhesion ability, and the platelet adhesion is significantly reduced compared with that of Comparative Example 1. Figure 9 It can be seen that, when platelet adhesion is further quantitatively detected by lactate dehydrogenase activity, the platelet adhesion of Example 1 is significantly reduced compared with that of Comparative Example 1.
[0075] Test Example 6: Subcutaneous inflammation test:
[0076] The experimental animals and all protocols were approved by the Ethics Committee of Sichuan University. An incision was made on the back of 45-50g male SD rats, and the bioprosthetic valve materials prepared in Comparative Example 1 and Example 1 were implanted subcutaneously. The skin was then sutured. Samples were taken out after 7 days and HE staining was used to show the inflammation in the samples. Samples were taken out after 14 days and immunohistochemistry CD3 / CD68 and immunofluorescence staining were used to show the immune status in the samples. The experimental results are shown in Figure 2. Figure 10-14 As shown. Figure 10 It can be seen that the inflammatory cells near the bioprosthetic valve material in Example 1 were significantly reduced. Figure 11 and Figure 12 It can be seen that the CD3 / CD68 positivity of the bioprosthetic valve material in Example 1 is significantly lower than that in Comparative Example 1, indicating that subcutaneous inflammation in Example 1 is alleviated. Figure 13 and Figure 14 It can be seen that the inflammatory factors of the biological valve material in Example 1 are significantly reduced compared with those in Comparative Example 1, indicating that the subcutaneous inflammation in the example is alleviated.
[0077] Test Example 7: Calcification Test:
[0078] The bioprosthetic valve materials prepared in Comparative Example 1 and Example 1 were placed subcutaneously in rats, and the skin was sutured. After 90 days, the samples were taken out and stained with Alizarin Red and Von Kossa to observe the calcification of the sample surface. The experimental results are shown in FIG. Figure 15 and Figure 16 As shown. Figure 15 It can be seen that the alizarin red colored area of the bioprosthetic valve material in Example 1 is significantly smaller than that in Comparative Example 1, indicating that the degree of subcutaneous calcification in Example 1 is significantly smaller than that in Comparative Example 1. Figure 16 It can be seen that the vonkossa-stained area in the biological valve material of Example 1 is significantly reduced compared with that of Comparative Example 1, indicating that the degree of subcutaneous calcification in Example 1 is significantly reduced compared with that of Comparative Example 1.
[0079] It should be noted that the results of the above test examples are only the test results of some embodiments of the present invention. The biological valve material with the biomimetic endothelial glycocalyx-like hydrogel coating prepared by the preparation method of the present invention has similar properties, and both have excellent biocompatibility, blood compatibility and anti-calcification properties.
[0080] In summary, the present invention can produce a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating that has excellent biocompatibility, blood compatibility, and anti-calcification properties. Compared with the prior art, the present invention represents a significant improvement.
[0081] The above description is merely a representative embodiment of the present invention and does not constitute any form of limitation to the present invention. Any technical personnel familiar with the present invention who, without departing from the scope of the technical solution of the present invention, makes slight changes or modifications to the embodiments disclosed above using the technical contents disclosed above are equivalent embodiments of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. A method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating, characterized in that: The following steps are involved: S1: obtaining biological materials and pre-treating the biological materials; S2: preparing an activated caffeic acid solution, and soaking the pretreated biomaterial in the activated caffeic acid solution to graft caffeic acid onto the biomaterial; S3: preparing an Alg-DA solution containing an initiator, and immersing the grafted biomaterial therein to perform an oxidative coupling reaction, and washing after the reaction to obtain the bioprosthetic valve material with the biomimetic endothelial glycocalyx-like hydrogel coating.
2. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S1, the biological material is any one of pericardium, valve, intestinal membrane, meninges, pulmonary membrane, blood vessel, skin or ligament.
3. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S1, the preprocessing includes the following sub-steps: S11: Store the obtained biological material in a low-temperature and humidified state at 0-4°C; S12: preparing a mixed solution comprising sodium deoxycholate and sodium lauryl sulfate, wherein the mass fractions of the sodium deoxycholate and the sodium lauryl sulfate are both 0.1% to 0.5%; S13: Soak the biomaterial stored in step S11 in the mixed solution and shake it continuously at room temperature for 1-2 days, and then wash it with deionized water until there is no visible adherent non-pericardial or non-collagenous tissue.
4. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S2, the caffeic acid solution is activated using an amide condensation agent.
5. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 4, characterized in that: The amide condensation agent is any one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide.
6. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S2, the solvent in the caffeic acid solution is any one or more of water, methanol, and ethanol, and the mass fraction of caffeic acid is 1%-5%.
7. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S2, after soaking, the mixture is shaken at 75-100 rpm for 1-3 days at 25-40° C., and then washed with ethanol to obtain the grafted biomaterial.
8. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S3, in the Alg-DA solution, the molar concentration of the initiator is 20-50 mM, and the mass fraction of Alg-DA is 2%-5%.
9. The method for preparing a bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating according to claim 1, characterized in that: In step S3, the oxidative coupling reaction is carried out at 25-40° C. for 12-24 h.
10. A bioprosthetic valve material with a biomimetic endothelial glycocalyx-like hydrogel coating, characterized in that: The biovalve material is prepared by the preparation method of the biomimetic endothelial glycocalyx-like hydrogel coating according to any one of claims 1 to 9, and includes heart valves and venous valves.