SURFACE MODIFICATION METHOD AND STRUCTURE FOR IMPROVING HEMOCOMPATIBILITY OF BIOMEDICAL METALLIC SUBSTRATES

Abstract

The present invention relates to a surface modification method for improving the hemocompatibility of biomedical metallic substrate, comprising: fixing a sulfur-containing monomolecular film on the surface of oxide layer of a biomedical metallic substrate by molecular self-assembly. The surface modification will improve the hydrophilicity and hemocompatibility of the biomedical metallic substrate in contact with the blood, and ensure that the biomedical metallic substrate is non-toxic to the endothelial cells.

Claims

1. A surface modified structure of biomedical metallic substrate for improving hemocompatibility, comprising: a silanol-oxide layer formed on a surface of a biomedical metallic substrate, and a sulfur-containing monomolecular film with exposed sulfur-containing functional groups formed on the surface of the silanol-oxide layer.

2. The surface modification structure of claim 1, further comprising a nitric oxide (NO) layer formed on the surface of the sulfur-containing monomolecular film.

3. A method for preparing the surface modified structure of claim 1, comprising: contacting a biomedical metallic substrate having an oxide layer with a solution of a silanol chemical derivative containing mercapto group or sulfur atom for a predetermined period of time to form a silanol-oxide layer on the biomedical metallic substrate, and a sulfur-containing monomolecular film exposing sulfur-containing functional groups on outermost surface of the silanol-oxide layer by means of molecular self-assembly.

4. The method of claim 3, wherein the predetermined period of time is 10 minutes to 24 hours.

5. The method of claim 3, wherein the silanol chemical derivative is 3-mercaptopropyltrimethoxysilane (MPTMS).

6. The method of claim 3, wherein the solution of silanol chemical derivative has a volume concentration of 0.1%20%.

7. The method of claim 3, further comprising a step of forming a nitric oxide (NO) layer on the surface of the sulfur-containing monomolecular film.

8. A surface modification method for improving hemocompatibility of biomedical metallic substrate, comprising: forming a nitric oxide (NO) layer on the surface of an oxide layer of a biomedical metallic substrate, and forming a sulfur-containing monomolecular film on the surface of the nitric oxide (NO) layer by means of molecular self-assembly.

9. The surface modification method of claim 8, wherein the molecular self-assembly comprises: contacting the biomedical metallic substrate having the nitric oxide (NO) layer with a solution of a silanol chemical derivatives containing mercapto group or sulfur atom for a predetermined period of time to form a sulfur-containing monomolecular film exposing functional mercapto group or sulfur atom on the surface of the nitric oxide (NO) layer by self-assembly.

10. The surface modification method of claim 9, wherein the predetermined period of time is 10 minutes to 24 hours.

11. The surface modification method of claim 9, wherein the silanol chemical derivative is 3-mercaptopropyltrimethoxysilane (MPTMS).

12. The method of claim 9, wherein the solution of silanol chemical derivative has a volume concentration of 0.1%20%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1A illustrates the chemical structure of a titanium dioxide substrate surface modified with a monomolecular film consisting of sulfur-containing silanol functional group. FIG. 1B illustrates the chemical structure of a titanium dioxide substrate surface modified with a nitric oxide (NO) layer and a monomolecular film consisting of sulfur-containing silanol functional group.

[0025] FIG. 2 shows the ESCA analyses of S.sub.2p3/2 scan for the four samples described in the Example.

[0026] FIG. 3 shows the results of hydrophilicity evaluation of the four samples described in the Example by droplet angle goniometry.

[0027] FIG. 4 shows the Field-emission scanning electron microscope (FESEM) graph of the platelet-adsorbed surface of the four samples described in the Example.

[0028] FIG. 5 shows the FESEM graph of the erythrocyte-adsorbed surface of the four samples described in the Example.

[0029] FIG. 6 shows the fluorescent staining of endothelial cells cultured on the surface of the four samples described in the Example.

[0030] FIG. 7 shows the quantitative analyses of endothelial cell numbers cultured on the surface of the four samples described in the Example.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The characteristics and advantages of the present invention will be further illustrated and described in the following examples. The examples described herein are used for illustrations, not for limitations of the invention.

[0032] The surface modification of the present invention for improving the hemocompatibility of titanium- or titanium alloy-based biomedical metallic substrate comprises: forming a sulfur-containing monomolecular film on the surface of oxide layer of a biomedical metallic substrate by molecular self-assembly.

[0033] The said oxide layer may be a native oxide or an oxide layer created by a surface modification technique. In the present invention, the oxide layer (TiO) is created on the surface of a titanium or titanium alloy substrate (referred to as a titanium dioxide substrate) by an anode oxidation process or gas plasma surface treatment. The oxide layer provides the chemical bonding required in the subsequent molecular self-assembly.

[0034] In certain embodiments of the present invention, the steps of said molecular self-assembly comprise: immersing the titanium dioxide substrate in a 0.1%20% solution of a silanol chemical derivative containg mercapto group for a period of 10 minutes to 24 hours. During the immersion, the molecular self-assembly is performed on said oxide layer through silanol group, and the sulfur-containing functional groups are then exposed on the outermost surface of the metals.

[0035] As shown in FIG. 1, the sulfur-containing functional group (SH) was immobilized on the surface of titanium dioxide substrate by the silanol group binding with titanium (TiOSi).

[0036] Additionally, a further nitric oxide (NO) layer is formed on the surface of the sulfur-containing monomolecular film by plasma treatment. Alternately, the nitric oxide (NO) layer is formed on the surface of titanium dioxide substrate, and a sulfur-containing monomolecular film is formed on the surface of the nitric oxide (NO) layer by molecular self-assembly.

[0037] To confirm the hemocompatibility of the surface modified substrate as described, four samples were prepared to carry out the related measurement and analysis. In the following, MPTMS stands for 3-mercaptopropyltrimethoxysilane. The four samples include: A, titanium substrate (Ti); B, titanium dioxide substrate modified with MPTMS (MPTMS-ATN), with a chemical structure as shown in FIG. 1A; C, titanium dioxide substrate modified with NO-coated MPTMS (NO-MPTMS-ATN); and D, NO-coated titanium dioxide substrate modified with MPTMS (MPTMS-NO-ATN), with a chemical structure as shown in FIG. 1B. The substrate A is a control, and substrates B, C and D are exemplary substrates of present invention.

[0038] To make sure if the sulfur-containing functional group (S) was successfully immobilized on the surface of titanium dioxide substrate, the chemical elements contained in the substrate surface were analyzed by using electron spectroscopy for chemical analysis (ESCA). As shown in FIG. 2, after the scanning of ESCA, S.sub.2p3/2 scan shows the substrates B, C and D possessed the signals of (1) SH and SN of 163.6 eV; (2) SH of 164.6 eV; (3) SO.sub.4.sup.2 of 169 eV; (4) SO.sub.4.sup.2 of 169 eV. The substrate A showed no S signal. It is proven that sulfur-containing functional group has successfully modified the surface of substrates B, C and D.

[0039] The hydrophilicity of the four samples was evaluated by droplet angle goniometry. Results are shown in FIG. 3. The contact angle of substrate A (Ti) was almost 90, indicating it was hydrophobic. The contact angles of substrates B (MPTMS-ATN), C (NO-MPTMS-ATN) and D (MPTMS-NO-ATN) were less than 60, indicating they were relatively hydrophilic.

[0040] Blood testing. The four substrates were incubated with fresh blood respectively to investigate the coagulation and anticoagulation actions of the substrates. Platelet-poor plasma (PPP), platelet-rich plasma (PRP) and red blood cells (RBCs) were isolated from fresh blood by centrifugation. The PPP was contacted with the four substrates and incubated at 37 C. in CO.sub.2 incubator for one hour, then subjected to the tests of PT and aPTT, and the measurement of fibrinogen concentration. Furthermore, the four substrates were contacted with PRP and RBC and incubated at 37 C. in CO.sub.2 incubator for one hour, then the adhesion of platelet or blood cell on the surface of four substrates were observed by Field-emmision scanning electron microscope (FESEM).

[0041] The results of PT, aPTT and fibrinogen concentration analyses are listed in Table 1. It is shown that the PT and aPTT values of the four substrates are in the normal range, indicating that no negative effects on the exogenous coagulation pathway and endogenous coagulation pathway of blood were produced, and the dynamic equilibrium between blood coagulation and anti-coagulation was maintained. The fibrinogen concentrations on the surface of the four substrate groups were all lower than the normal value, and no blood clotting was observed on the surface of substrates.

TABLE-US-00001 TABLE 1 fibrinogen concentration PT(sec) aPTT(sec) (mg/dL) Normal range 8.0~12.0 23.9~35.5 200.0~400.0 Substrate A (Ti) 11.08 0.08 29.36 0.51 195.64 3.28 Substrate B 11.27 0.4 29.23 0.59 191.27 5.93 (MPTMS-ATN) Substrate C 11.3 0.2 29.8 0.7 189.23 5.03 (NO-MPTMS-ATN) Substrate D 11.17 0.31 29.33 0.31 188.67 2.04 (MPTMS-NO-ATN)

[0042] The observed results of platelet adhesion on the surface of the four substrates by FESEM are shown in FIG. 4. Platelets were adsorbed on the substrate A (Ti), while no platelets were adsorbed on the substrates B (MPTMS-ATN), C (NO-MPTMS-ATN) and D (MPTMS-NO-ATN). Platelet activation occurred on the substrate A, but no platelet activation occurred on the substrates B, C and D of the present invention. The platelet activation will prime blood coagulation. Platelet activation did not occur on the surface of substrates B, C and D of the present invention, which ensures that no blood clots are formed on the substrate surface.

[0043] The observed results of red blood cells (RBCs) adhesion on the surface of the four substrates by FESEM are shown in FIG. 5. There was no red blood cell adsorbing on the surface of the four substrates. The expected physiological effect of no RBC adhesion on the surface of present substrates is that no thrombus formation is induced.

[0044] For the vascular endothelium test, vascular endothelial cells were attached to the surface of the four substrates. The nucleus of endothelial cell was stained with the fluorescein dye DAPI, and the staining result was observed using a fluorescence microscope. The growth of endothelial cell on the surface of the four substrates at Day 1, Day 3 and Day 5 are shown in FIG. 6. FIG. 7 shows the statistic analyses of the number of growing endothelial cells. As shown in the FIGS. 6 and 7, the number of endothelial cells increased with the progressive days, especially the cell numbers were greater in the substrate B, C and D groups than in the substrate A. The results indicate that the substrates B, C and D of the present invention were non-toxic to endothelial cells, promising the normal growth and proliferation of endothelial cells on the substrate surface. The expected physiological effect is no activation of the coagulation and thrombosis.