Biological Material And Preparation Method Therefor

Abstract

Provided are an anticoagulation and anticalcification biological material and a preparation method therefor. The preparation method includes the following steps: introducing, on a biological tissue, a polymerizable reactive group, and undergoing free radical copolymerization with a zwitterion. In the present disclosure, by introducing a reactive group capable of free radical polymerization to a biological tissue and undergoing free radical copolymerization with a zwitterionic monomer, collagen in the biological tissue is crosslinked at multiple sites by means of a polymer, thereby achieving sufficient crosslinking within and between collagen fibers, improving the stability of the biological tissue, and prolonging the service life of the biological tissue. Moreover, a zwitterion is introduced to the surface of the biological tissue, to improve the anticoagulation performance, promote the in-situ endothelialization of a biological valve, and prevent the calcium element deposition.

Claims

1. A method for preparing a biological material, comprising following steps: introducing, on a biological tissue, a polymerizable reactive group, and undergoing free radical copolymerization with a zwitterion, wherein the zwitterion has a specific structure as below: ##STR00002##

2. The method for preparing a biological material according to claim 1, wherein the biological tissue introduced with the reactive group is added to a zwitterion solution having a final concentration of 20 to 500 mM, and an initiator is added to initiate the polymerization, to obtain the biological material.

3. The method for preparing a biological material according to claim 1, wherein the biological tissue introduced with the reactive group is added to a zwitterion solution and soaked at 35 to 40° C., and an initiator is added to initiate the polymerization, to obtain the biological material.

4. The method for preparing a biological material according to claim 1, wherein the biological tissue is soaked in deionized water, and then the reactive group is added, to provide a concentration of the reactive group of 3-10% by volume.

5. The method for preparing a biological material according to claim 4, wherein the biological tissue is reacted with the reactive group at room temperature for 12 to 36 hrs, and then the biological tissue is washed.

6. The method for preparing a biological material according to claim 4, wherein the biological tissue is soaked in deionized water, and then the reactive group is added to provide a concentration of the reactive group of 4% by volume. and then reacted at room temperature for 24 hrs.

7. The method for preparing a biological material according to claim 1, wherein the zwitterion solution is added to the washed biological tissue, to provide a final concentration of the zwitterion of 500 mM, and the biological tissue is soaked at 37° C.

8. The method for preparing a biological material according to claim 1, wherein the biological tissue is pericardium, aortic root, aortic valve, pulmonary artery root, pulmonary artery valve, tendon, ligament, skin, dura mater, peritoneum, blood vessels, pleura, septum, mitral or tricuspid valve.

9. The method for preparing a biological material according to claim 1, wherein the reactive group is methacrylic anhydride, acrylamide, methacrylamide, acrylate, methacrylate or allyl.

10. The method for preparing a biological material according to claim 1, wherein the zwitterion accounts for 1 to 30% by weight based on a total weight of the biological material.

11. The method for preparing a biological material according to claim 2, wherein when the initiator initiates the polymerization, a reaction temperature is not higher than 50° C.

12. The method for preparing a biological material according to claim 2, wherein the initiator is a thermal initiator or a photoinitiator.

13. A biological material prepared by the preparation method according to claim 1.

14. A biological material prepared by the preparation method according to claim 2.

15. A biological material prepared by the preparation method according to claim 3.

16. A biological material prepared by the preparation method according to claim 4.

17. A biological valve prepared by the preparation method according to claim 1.

18. A biological valve prepared by the preparation method according to claim 2.

19. A biological valve prepared by the preparation method according to claim 3.

20. A biological valve prepared by the preparation method according to claim 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 is a flow chart illustrating a preparation of a biological tissue.

[0040] FIG. 2 is a schematic diagram showing the mechanism of preparation of a functionalized biological tissue.

[0041] FIG. 3 is an SEM image showing the platelet adsorption on MPC-20 biological tissue.

[0042] FIG. 4 is an SEM image showing the platelet adsorption on MPC-500 biological tissue.

[0043] FIG. 5 is an SEM image showing the platelet adsorption on SBMA-20 biological tissue.

[0044] FIG. 6 is an SEM image showing the platelet adsorption on SBMA-500 biological tissue.

[0045] FIG. 7 is an SEM image showing the platelet adsorption on an unmodified biological tissue.

[0046] FIG. 8 is an SEM image showing the platelet adsorption on a glutaraldehyde-crosslinked biological tissue.

[0047] FIG. 9 is a fluorescent micrograph showing endothelial cells grown on SBMA-500 biological tissue.

[0048] FIG. 10 is a fluorescent micrograph showing endothelial cells grown on an unmodified biological tissue.

[0049] FIG. 11 is a fluorescent micrograph showing endothelial cells grown on a glutaraldehyde-crosslinked biological tissue.

DETAILED DESCRIPTION

Example 1

[0050] A method for preparing an anticoagulation and anticalcification biological material is provided. The preparation process is shown in FIG. 1, and the mechanism of preparation is shown in FIG. 2, which specifically includes the steps as follows:

[0051] washing a fresh porcine pericardium thoroughly with deionized water, and then adding deionized water to completely submerge the tissue; adding methacrylic anhydride dropwise with stirring in an ice bath at 4° C., to obtain a final concentration of the acid anhydride of 4% (v/v), with the solution maintained at pH 7 with a sodium hydroxide solution; and after that, the reaction was continued at room temperature for 24 hrs; thoroughly washing the porcine pericardium modified with methacrylic anhydride, adding the porcine pericardium modified with methacrylic anhydride to a zwitterion monomer solution having a final concentration as shown in a table below, and soaking overnight at 37° C.; then adding an initiator for crosslinking reaction at 37° C. for 24 hrs; obtaining the prepared porcine pericardium. The prepared porcine pericardium was thoroughly washed with deionized water and stored in a 25% isopropanol solution for later use.

[0052] Unmodified biological tissue: the unmodified biological tissue was prepared by the steps described above before the initiator is added for crosslinking, and the porcine pericardium is directly cross-linked by adding an initiator without being soaked in a monomer solution. The unmodified porcine pericardium was prepared, and the prepared porcine pericardium was thoroughly washed with deionized water and stored in a 25% isopropanol solution for later use.

[0053] Specific formulas are shown below:

TABLE-US-00001 Functional Type and molecule Sample concentration (MPC/SBMA) name of monomer Initiator concentration content (w/w) MPC-20 MPC: 20 mM 50 mM ammonium  1.6% persulfate and 50 mM sodium hydrogen sulfite MPC-500 MPC: 500 mM 50 mM ammonium 15.6% persulfate and 50 mM sodium hydrogen sulfite SBMA-20 SBMA: 20 mM 50 mM ammonium  5.2% persulfate and 50 mM sodium hydrogen sulfite SBMA-500 SBMA: 500 mM 50 mM ammonium 24.6% persulfate and 50 mM sodium hydrogen sulfite Unmodified 0 50 mM ammonium    0%* persulfate and 50 mM sodium hydrogen sulfite Note: SBMA: [2-(methylacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide; MPC: 2-methylacryloyloxyethyl phosphorylcholine; *the unmodified is used as a reference line, set as 0%; and the initiator concentration is the concentration initially added to the reaction system.

[0054] The above-mentioned functional molecule content is derived from analyzing by ICP-OES the content of sulfur or phosphorus in the biological material after freeze-dried since phosphorus element are contained in the functional molecule MPC and sulfur element are contained in SBMA, both of which are hardly found in the biological material. Thus, the degree of modification with the functional molecule on the biological tissue can be obtained, which facilitates the screening of experimental conditions.

[0055] The specific conversion formula is as follows:

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

wherein C.sub.S(P) (mg/L) is the concentration of sulfur or phosphorus in solution measured by ICP-OES, M.sub.S(P) is the relative atomic mass of sulfur or phosphorus, M.sub.SBMA(MPC) is the relative molecular mass of the functional molecule, and W (mg) is the dry weight of the biological material.

[0056] As can be seen from the above table, compared with the control group, the biological material prepared by the present disclosure shows that the biological tissue is well modified with the functional molecule, particularly, the SBMA-500 group has the highest degree of modification.

Test Example 1: Stability of Biological Tissue

[0057] The obtained biological material was cut into small pieces of 3-8 mg, aspirated with filter paper to remove water on the surface, and then placed in an aluminum crucible and sealed. The crucible with the sample was placed in the instrument and the thermal denaturation temperature was analyzed by differential scanning calorimetry, to characterize the stability of the biological tissue. The results are shown below:

TABLE-US-00002 Thermal denaturation Sample name temperature (° C.) SBMA-500 80.15 Fresh tissue 68.88

[0058] As can be seen from the above table, the biological material prepared by the present disclosure has a higher temperature resistance compared with fresh tissue, indicating that the biological tissue has excellent stability.

Test Example 2: Anticoagulation Performance of Biological Tissue

[0059] The prepared biological material was washed, cut to have an appropriate size, and incubated with platelet-rich plasma for one hr. The amount of lactate dehydrogenase (LDH) after lysis of platelets adhering to the material was measured to express the amount of platelet adhered. The results are shown in a table below. The adhesion of platelets on the material was observed under SEM. The results are specifically shown in FIGS. 3-8.

TABLE-US-00003 Absorbency value Sample name (490 nm) MPC-20 0.30 MPC-500 0.29 SBMA-20 0.24 SBMA-500 0.110 Unmodified 0.32 Crosslinked with 0.35 glutaraldehyde

[0060] As can be seen from the above table, a higher absorbency value at 490 nm indicates a higher lactate dehydrogenase content, that is, the amount of platelets adhered to the material is higher. Therefore, as can be seen from the above table, the anticoagulation effect of the biological material prepared by the present disclosure is apparently better than those of unmodified biological tissue and glutaraldehyde-crosslinked biological tissue (prepared by immersing porcine pericardium in 0.625 vol % of glutaraldehyde aqueous solution, crosslinking for 24 hrs at pH 7.4 and room temperature, and then dehydrating and drying). The anticoagulation effect of the biological tissue modified with SBMA is better than that of the biological tissue modified with MPC, and the anticoagulation effect of the biological tissue with a high grafting degree of SBMA is the best.

[0061] The qualitative observation results under SEM in FIGS. 3-8 are consistent with the quantitative measurement results of LDH.

Test Example 3. Characterization of Anti-Calcification Performance and Immune Reaction of Biological Tissue

[0062] The prepared biological material was cut into pieces of 1 square centimeter, subcutaneously implanted in SD rats, and removed after 90 days. The calcium content in the samples was measured by ICP-OES. The results are shown below:

TABLE-US-00004 Sample name Calcium content (g/kg) SBMA-500 12.8 Unmodified 38.4 Crosslinked with 117.3 glutaraldehyde

[0063] As can be seen from the above table, the calcification of the biological tissue modified with SBMA-500 was significantly lower than those of unmodified biological tissue and glutaraldehyde-crosslinked biological tissue.

Test Example 4: Recellularization Performance of Biological Tissue

[0064] The surface of the prepared biological material was seeded with endothelial cells, incubated for 3 days, and then stained with DAPI and FITC labeled phalloidin. The results are shown in FIGS. 9-11.

[0065] As can be seen from FIGS. 9-11, the growth of endothelial cells on the biological tissue modified with SBMA-500 was better than those on unmodified biological tissue and glutaraldehyde-crosslinked biological tissue.

Test Example 5. Implantation of Artificial Blood Vessels

[0066] The prepared biological material was rolled into a tube with a diameter of about 2 mm and the junction was bonded with a medical adhesive, aspirated with filter paper to remove water on the surface, weighed and recorded. A rabbit carotid artery having a length of approximately 2 cm was replaced by the tube using vascular anastomosis. After implantation for a period of time, it was retracted, observed, aspirated with filter paper to remove water on the material, and weighed again. The weight gain was calculated, which is the amount of thrombus produced. The result is shown below.

TABLE-US-00005 Initial weight Final Amount of thrombus Sample name (mg) weight (mg) produced (mg) SBMA-500 80.9 83.6 2.7 Unmodified 78.6 262.4 183.8 Crosslinked with 82.5 295.3 212.8 glutaraldehyde

[0067] From the above table, it can be seen that for glutaraldehyde-crosslinked biological tissue and unmodified biological tissue, thrombus of more than 2 times of the tissue weight is produced, but almost no thrombus is produced for SBMA-500.