阅读说明：本技术 一种骨组织修复材料及其在眼眶骨缺损修复中的应用 (Bone tissue repair material and application thereof in orbital bone defect repair ) 是由 黄晓明 刘杨 孙丰源 吴桐 简天明 胡丹 于 2021-11-01 设计创作，主要内容包括：本发明公开了一种骨组织修复材料及其在眼眶骨缺损修复中的应用,涉及支架材料技术领域。该骨组织修复材料为生物可降解聚合物和羟基磷灰石的复合材料；上述复合材料基于硅烷偶联剂修饰后的羟基磷灰石,在其表面原位聚合形成可降解聚合物I,得到可降解聚合物I修饰的羟基磷灰石,再与可降解聚合物II共混；其中,可降解聚合物I反应单体至少包括苯乙烯；可降解聚合物II包括聚乙烯醇、接枝改性聚乙烯醇；接枝改性聚乙醇用接枝改性化合物结构中至少包含两个羧基。该骨组织修复材料具有更加优异的力学性能,其拉伸强度、抗弯强度和抗冲击强度显著提升,且耐磨性能提高；且亲水性良好,促成骨分化能力得到有效提升,可应用于眼眶骨缺损修复中。(The invention discloses a bone tissue repair material and application thereof in orbital bone defect repair, and relates to the technical field of scaffold materials. The bone tissue repair material is a composite material of a biodegradable polymer and hydroxyapatite; the composite material is based on hydroxyapatite modified by a silane coupling agent, a degradable polymer I is formed on the surface of the hydroxyapatite through in-situ polymerization to obtain hydroxyapatite modified by the degradable polymer I, and then the hydroxyapatite is blended with a degradable polymer II; wherein, the degradable polymer I reaction monomer at least comprises styrene; the degradable polymer II comprises polyvinyl alcohol and graft modified polyvinyl alcohol; the graft-modified polyvinyl alcohol contains at least two carboxyl groups in its structure. The bone tissue repair material has more excellent mechanical properties, the tensile strength, the bending strength and the impact strength of the bone tissue repair material are obviously improved, and the wear resistance is improved; and the hydrophilicity is good, the capacity of promoting bone differentiation is effectively improved, and the method can be applied to orbital bone defect repair.)

1. A bone tissue repair material which is a composite material of a biodegradable polymer and hydroxyapatite; the composite material is based on hydroxyapatite modified by a silane coupling agent, a degradable polymer I is formed on the surface of the composite material through in-situ polymerization, the hydroxyapatite modified by the degradable polymer I is obtained, and then the hydroxyapatite is blended with a degradable polymer II;

the silane coupling agent at least comprises a double bond structure;

the degradable polymer I reaction monomer at least comprises styrene;

the degradable polymer II comprises polyvinyl alcohol and graft modified polyvinyl alcohol; wherein, the graft modification compound structure for the graft modified polyethanol at least comprises two carboxyl groups.

2. A bone tissue repair material according to claim 1, wherein: the silane coupling agent includes vinyltriethoxysilane.

3. A bone tissue repair material according to claim 1, wherein: the graft modification compound comprises potassium dehydroandrographolide succinate.

4. A process for preparing graft-modified polyvinyl alcohol as claimed in claim 3, which comprises: the graft modified polyvinyl alcohol is prepared by the esterification reaction of potassium dehydroandrographolide succinate and polyvinyl alcohol.

5. The method for producing a graft-modified polyvinyl alcohol according to claim 4, wherein: the grafting rate of the graft modified polyvinyl alcohol is more than 67 percent.

6. Use of the bone tissue repair material according to any one of claims 1 to 3 for the preparation of scaffolds for bone tissue engineering.

7. Use according to claim 6, characterized in that: the bone tissue repair material has tensile yield strength of more than 26MPa, Young modulus of more than 7GPa and bending strength of more than 90 MPa.

Technical Field

The invention belongs to the technical field of scaffold materials, and particularly relates to a bone tissue repair material and application thereof in orbital bone defect repair.

Background

With the rapid development of economy in China, the incidence of orbital wall fracture injury caused by various production activities, traffic accidents and the like is increasing year by year. The injury of the orbital wall bone can cause the problems of eyeball invagination or displacement, double vision, eyeball dyskinesia, infection and the like, thereby causing serious damage to visual function and appearance, seriously affecting the life and work of patients and greatly influencing the physiology and mind of the patients. The principle of orbital reconstruction is that the eyeball and the herniated orbital contents are surgically restored, and bone graft or artificial bone tissue repair materials are used to reconstruct orbital wall bone defects to restore the continuity of the orbital bone wall, correct the enlarged orbital volume, realize anatomical restoration, and restore the visual function and appearance of the patient.

Currently, autologous bone transplantation is still regarded as the gold standard for repairing orbital wall bone defects, but the orbital wall bone is special in shape and extremely difficult to obtain suitable materials, and has complications such as limited sources of transplanted bones, supply area infection, chronic pain and the like, so that finding an ideal orbital reconstruction implant material is one of the problems to be solved in orbital reconstruction treatment. Therefore, the choice of implant material is particularly important. The artificial material has the characteristics of low price and capability of being sterilized without causing change of chemical compositions; the shaping is easy to trim during the operation, the contour of the orbit is adapted, the fixation is easy, and the examination and the evaluation of the imaging are easy after the operation. At present, artificial materials are various in variety and can be divided into non-absorbable materials and absorbable materials, and can also be divided into single materials and composite materials. The ideal orbital reconstruction material needs to have reliable mechanical properties, a porous network structure for promoting vascularization and bone tissue in-situ regeneration, surface chemical properties and microstructures for supporting the growth and functional differentiation of osteoblast-related cells, a degradation rate matched with the generation of new bones at defect sites, and plasticity for adapting to the complex structures of the orbital and the individual customization requirements of patients.

Disclosure of Invention

The invention aims to provide a bone tissue repair material and application thereof in orbital bone defect repair, the bone tissue repair material has more excellent mechanical properties, the tensile strength, the bending strength and the impact strength of the bone tissue repair material are obviously improved, and the wear resistance of the bone tissue repair material is improved; and the hydrophilicity is good, and the capability of promoting bone differentiation is effectively improved.

The technical scheme adopted by the invention for realizing the purpose is as follows:

a bone tissue repair material which is a composite material of a biodegradable polymer and hydroxyapatite; the composite material is based on hydroxyapatite modified by a silane coupling agent, a degradable polymer I is formed on the surface of the hydroxyapatite through in-situ polymerization to obtain hydroxyapatite modified by the degradable polymer I, and then the hydroxyapatite is blended with a degradable polymer II;

the silane coupling agent at least comprises a double bond structure;

the degradable polymer I reaction monomer at least comprises styrene;

the degradable polymer II comprises polyvinyl alcohol and graft modified polyvinyl alcohol; wherein, the graft modification compound structure for the graft modified polyethanol at least comprises two carboxyl groups. The invention takes silane coupling agent as medium, adopts in-situ polymerization method to modify the surface of hydroxyapatite, and then mixes with degradable polymer to obtain the composite material, which can be applied to the preparation of bone tissue engineering scaffold or implant. The hydroxyapatite modified by in-situ polymerization and the degradable polymer II can be fully combined, so that the rheological property is better, the mechanical property is obviously improved, the tensile yield strength and Young modulus are obviously improved, and the impact strength and the bending strength are enhanced. Meanwhile, the existence of the potassium dehydroandrographolide succinate modified polyvinyl alcohol improves the hydrophilicity of the material, and further improves the adhesion, proliferation and differentiation capacities of cells on the surface of the material; obviously enhances the expression of the material to promote bone related genes, particularly promotes the expression of Col1, Opn and Runx2, improves the capacity of promoting cell osteogenic differentiation, and further effectively improves the bone repair effect of the material. The bone tissue repair material provided by the invention has excellent bone defect repair capacity and no toxicity to cells; the preparation method is simple, environment-friendly and has wide application prospect.

Preferably, the silane coupling agent comprises vinyltriethoxysilane.

Preferably, the graft modifying compound comprises potassium dehydroandrographolide succinate.

The preparation method of the graft modified polyvinyl alcohol comprises the following steps: is prepared from potassium dehydroandrographolide succinate and polyvinyl alcohol through esterification.

Further, the preparation method of the graft modified polyvinyl alcohol specifically comprises the following steps:

adding polyvinyl alcohol into deionized water, wherein the concentration is 0.08-0.14 g/mL; heating to 90-94 ℃ to completely dissolve the polyvinyl alcohol; and then cooling to 50-60 ℃, adding a potassium dehydroandrographolide succinate solution (the solvent is 0.8-1.5 wt% of NaCl solution, and the concentration is 2-6 mg/mL) at the same temperature into a constant-pressure dropping funnel, stirring for reaction for 3-5 hours, adding ethanol for settling, performing suction filtration, extracting in a Soxhlet extractor with ethanol for 10-12 hours, and performing vacuum drying to obtain the graft modified polyvinyl alcohol.

Preferably, the mass ratio of the potassium dehydroandrographolide succinate to the polyvinyl alcohol is 3-4.5: 1.

preferably, the graft ratio of the graft-modified polyvinyl alcohol is > 67%.

The preparation method of the bone tissue repair material comprises the following steps:

carrying out in-situ copolymerization treatment, mixing deionized water, hydroxyapatite and vinyl triethoxysilane, and stirring in ultrasonic waves at normal temperature for 2-3 h; adding SDS, heating the system to 70-75 ℃, slowly adding APS and styrene, reacting for 4-6 h, heating to 80-85 ℃, curing for 1-2 h, cooling to room temperature, demulsifying, and vacuum drying to obtain hydroxyapatite modified by degradable polymer I;

preparing a composite repairing material, namely melting and blending hydroxyapatite modified by a degradable polymer I and graft modified polyvinyl alcohol (the filling amount of the hydroxyapatite modified by the degradable polymer I is 20-26 wt%), compounding and granulating, setting the temperature of a charging barrel to be 180-190 ℃, 200-220 ℃ and 200-220 ℃, respectively, forming by using a flat plate vulcanizing machine model, controlling the temperature of an upper template and a lower template to be (170 +/-2) DEG C and (160 +/-2) DEG C, and controlling the pressure to be 13-16 MPa, so as to prepare a composite repairing material plate, and cutting to obtain the bone tissue repairing material.

Preferably, the mass ratio of the hydroxyapatite to the vinyltriethoxysilane is 1: 0.2 to 0.3; the solid-to-liquid ratio of the hydroxyapatite to the deionized water is 0.01-0.02 g: 1 mL; the mass ratio of the styrene to the hydroxyapatite is 1: 18-24; the adding amount of SDS is 1-3 wt% of the total amount of the monomers; the addition amount of the APS is 0.2-0.6 wt% of the total amount of the monomers.

More preferably, during the in-situ copolymerization treatment, the polymerized monomers further comprise betulonic acid; wherein the molar ratio of betulonic acid to styrene is 0.2-0.4: 1. the betulonic acid is used as one of crosslinking monomers, is compounded with styrene and is crosslinked on the surface of hydroxyapatite modified by a silane coupling agent to form a polymer in situ, and the polymer is applied to the preparation of bone tissue repair materials, so that the tensile yield strength, Young modulus, impact strength and bending strength of the materials are obviously enhanced, and the mechanical property is further improved; the bonding property of the hydroxyapatite and the polymer II interface is effectively improved, the defects of the material body and the surface are reduced, and the wear resistance is greatly improved; and the cell osteogenic differentiation promoting capacity of the material is improved to a certain extent, and particularly, the expression of Opn and Bsp genes is promoted. When the betulonic acid and the modified polyvinyl alcohol are compounded for use, the effect of enhancing the cell osteogenic differentiation promoting capability of the bone tissue repair material is better, and the addition of the betulonic acid plays a role in synergism.

The invention also aims to provide application of the bone tissue repair material in preparing a bone tissue engineering scaffold.

Preferably, the bone tissue repair material has a tensile yield strength > 26 MPa; young's modulus > 7GPa, more preferably, Young's modulus > 9.8 GPa; the bending strength is more than 90 MPa; more preferably, the flexural strength is ≥ 100 MPa.

Compared with the prior art, the invention has the following beneficial effects:

the invention takes silane coupling agent as medium, adopts in-situ polymerization method to modify the surface of hydroxyapatite, and then mixes the hydroxyapatite with potassium dehydroandrographolide succinate modified polyvinyl alcohol to obtain the bone tissue repair material, the mechanical property is obviously improved, the tensile yield strength and Young modulus are obviously improved, and the impact strength and the bending strength are enhanced. Meanwhile, the existence of the potassium dehydroandrographolide succinate modified polyvinyl alcohol improves the hydrophilicity of the material, and further improves the adhesion, proliferation and differentiation capacities of cells on the surface of the material; obviously enhances the expression of the material to promote bone related genes, improves the capacity of promoting cell osteogenic differentiation, and further effectively improves the bone repair effect of the material. In addition, the betulonic acid is used as one of crosslinking monomers, is compounded with styrene and is crosslinked on the surface of hydroxyapatite modified by a silane coupling agent to form a polymer in situ, and the polymer is applied to the preparation of bone tissue repair materials, so that the tensile yield strength, Young modulus, impact strength and bending strength of the materials are obviously enhanced, and the mechanical property is further improved; the wear resistance is greatly improved; and the cell osteogenic differentiation promoting capacity of the material is improved to a certain extent. The bone tissue repair material provided by the invention has excellent bone defect repair capacity, no toxicity to cells and excellent degradation performance; the preparation method is simple, environment-friendly and has wide application prospect, for example, the method can be suitable for repairing orbital bone defects.

Therefore, the invention provides a bone tissue repair material and application thereof in orbital bone defect repair, the bone tissue repair material has more excellent mechanical properties, the tensile strength, the bending strength and the impact strength of the bone tissue repair material are obviously improved, and the wear resistance of the bone tissue repair material is improved; and the hydrophilicity is good, and the capability of promoting bone differentiation is effectively improved.

Drawings

FIG. 1 shows the results of IR spectroscopy (A-polyvinyl alcohol, B-graft-modified polyvinyl alcohol from example 1) according to the invention;

FIG. 2 is a SEM scan of a bone tissue repair material in example 1 of the present invention;

FIG. 3 shows SEM scanning results of a bone tissue repair material in example 4 of the present invention;

FIG. 4 shows the results of the cytotoxicity test according to the present invention;

FIG. 5 shows the results of the level test of the expression of the osteogenesis-related gene in the present invention.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:

the hydroxyapatite used in the embodiment of the invention is in a nano level and is all commercially available.

Example 1:

preparation of graft-modified polyvinyl alcohol:

adding polyvinyl alcohol (with the hydroxyl content of 0.0455 mol) into deionized water, wherein the concentration is 0.1 g/mL; heating to 90 ℃ to completely dissolve the polyvinyl alcohol; then, the temperature is reduced to 56 ℃, and a potassium dehydroandrograpolide succinate solution (a 1.2wt% NaCl solution as a solvent and the concentration of 3.6 mg/mL) with the same temperature is added by using a constant pressure dropping funnel, wherein the mass ratio of potassium dehydroandrograpolide succinate to polyvinyl alcohol is 3.7: 1; stirring for reaction for 4h, adding ethanol for settling, performing suction filtration, extracting with ethanol in a Soxhlet extractor for 11h, and vacuum drying to obtain the graft modified polyvinyl alcohol with the grafting rate of 72.3%.

Preparing a bone tissue repair material:

in-situ copolymerization treatment, mixing deionized water, hydroxyapatite and vinyl triethoxysilane, and stirring in ultrasonic wave at normal temperature for 2 h; adding SDS, heating the system to 72 ℃, slowly adding APS and styrene, reacting for 4.5 hours, heating to 83 ℃, curing for 1 hour, cooling to room temperature, demulsifying, and vacuum drying to obtain hydroxyapatite modified by degradable polymer I; in the preparation process, the mass ratio of the hydroxyapatite to the vinyltriethoxysilane is 1: 0.25; the solid-to-liquid ratio of the hydroxyapatite to the deionized water is 0.015 g: 1 mL; the mass ratio of the styrene to the hydroxyapatite is 1: 21; the amount of SDS added was 2.2wt% of the total amount of monomers; the addition amount of APS is 0.41wt% of the total amount of the monomers;

preparing a composite repairing material, namely melting and blending hydroxyapatite modified by degradable polymer I and the graft modified polyvinyl alcohol (the filling amount of the hydroxyapatite modified by the degradable polymer I is 23.8 wt%), compounding and granulating, setting the temperature of a charging barrel to be 185 ℃, 210 ℃ and 210 ℃, respectively, forming by using a flat vulcanizing machine model, controlling the temperature of an upper template and a lower template to be (170 +/-2) DEG C and (160 +/-2) DEG C, and controlling the pressure to be 14.5MPa, so as to prepare a composite repairing material plate, and cutting to obtain the bone tissue repairing material.

Example 2:

the graft-modified polyvinyl alcohol was prepared differently from example 1: the mass ratio of the potassium dehydroandrographolide succinate to the polyvinyl alcohol is 3: 1; the graft ratio of the graft-modified polyvinyl alcohol was 67.8%.

The bone tissue repair material was prepared differently from example 1: the mass ratio of the hydroxyapatite to the vinyltriethoxysilane is 1: 0.2; the mass ratio of the styrene to the hydroxyapatite is 1: 18.2; the amount of SDS added was 1.4% by weight based on the total amount of monomers; the addition amount of APS is 0.28wt% of the total amount of the monomers; the filling amount of the hydroxyapatite modified by the degradable polymer I is 20.6 wt%.

Example 3:

the graft-modified polyvinyl alcohol was prepared differently from example 1: the mass ratio of the potassium dehydroandrographolide succinate to the polyvinyl alcohol is 4.2: 1; the grafting ratio of the graft-modified polyvinyl alcohol was 70.6%.

The bone tissue repair material was prepared differently from example 1: the mass ratio of the hydroxyapatite to the vinyltriethoxysilane is 1: 0.27; the mass ratio of the styrene to the hydroxyapatite is 1: 23; the amount of SDS added was 2.5% by weight of the total amount of monomers; the addition amount of APS is 0.52wt% of the total amount of the monomers; the filling amount of the hydroxyapatite modified by the degradable polymer I is 25.4 wt%.

Example 4:

the graft-modified polyvinyl alcohol was prepared in the same manner as in example 1.

The bone tissue repair material was prepared differently from example 1: in the in-situ copolymerization treatment process, the polymerized monomer also comprises betulonic acid; wherein the molar ratio of betulonic acid to styrene is 0.33: 1.

example 5:

the graft-modified polyvinyl alcohol was prepared in the same manner as in example 4.

The preparation of the bone tissue repair material differs from that of example 4: the molar ratio of betulonic acid to styrene was 0.28: 1.

example 6:

the graft-modified polyvinyl alcohol was prepared in the same manner as in example 4.

The preparation of the bone tissue repair material differs from that of example 4: the molar ratio of betulonic acid to styrene is 0.39: 1.

example 7:

the preparation of the bone tissue repair material differs from that of example 4: polyvinyl alcohol is adopted to replace graft modified polyvinyl alcohol.

Comparative example 1:

the bone tissue repair material was prepared differently from example 1: polyvinyl alcohol is adopted to replace graft modified polyvinyl alcohol.

Characterization of the Infrared Spectrum

The test method is a potassium bromide tabletting method, and a Fourier transform infrared spectrometer is adopted to characterize the chemical structure. Wherein the test wave number is 4000-500 cm < -1 >.

The above tests were carried out on polyvinyl alcohol and graft-modified polyvinyl alcohol obtained in example 1, and the results are shown in FIG. 1. As can be seen from the analysis of the figure, in the infrared spectrum of the modified polymethylsiloxane obtained in example 1, a characteristic absorption peak of C = O appears in the vicinity of 1750cm-1, a characteristic absorption peak of C = C bond appears in the vicinity of 1690cm-1, and a characteristic absorption peak of C-O-C bond appears in the vicinity of 1215 cm-1, as compared with the infrared test result of polyvinyl alcohol; the above results show that the graft-modified polyvinyl alcohol of example 1 was successfully prepared.

The graft ratio was calculated according to the following formula:

percent grafting (% = (m 2-m 1)/m 1X 100%

Wherein m1 is the mass of unmodified polyvinyl alcohol; m2 represents the mass of the graft-modified polyvinyl alcohol.

SEM characterization

The prepared bone tissue repair material is quenched and brittle-broken in liquid nitrogen, and the morphological structure of the bone tissue repair material is observed under a scanning electron microscope after the surface is sprayed with gold.

The bone tissue repair materials prepared in examples 1 and 4 were subjected to the above-described tests, and the results are shown in fig. 2 and 3. As can be seen from the analysis of the figure, the bone tissue repair material section prepared in example 4 has substantially no apparent voids, compared to the bone tissue repair material section prepared in example 1, indicating that hydroxyapatite is dispersed in the polymer matrix in a more uniform size and the interface bonding state is further improved. The results show that the betulonic acid is used as one of the crosslinking monomers, and the polymer is formed on the surface of the hydroxyapatite modified by the silane coupling agent through in-situ polymerization, so that the prepared bone tissue repair material has a better compatibilization effect by forming the polymer through in-situ polymerization, the dispersibility of the hydroxyapatite is promoted, and the interface bonding strength is further improved.

Mechanical Property test

The test refers to GB/T1040-; and (4) testing the notch impact strength of the test sample on an impact testing machine according to the GB/T1043-2018 standard.

The results of the above tests on bone tissue repair materials prepared in comparative example 1 and examples 1 to 7 are shown in table 1:

as can be seen from the analysis in Table 1, the tensile yield strength and Young's modulus of the bone tissue repair material prepared in example 1 are significantly higher than those of comparative example 1, which indicates that the tensile yield strength and Young's modulus of the material can be significantly enhanced and the mechanical properties thereof can be effectively improved by applying the dehydroandrographolide succinate modified polyvinyl alcohol to the preparation of the bone tissue repair material. The effect of example 4 is better than that of example 1, and the effect of example 7 is better than that of comparative example 1, which shows that betulonic acid is used as one of crosslinking monomers, and the polymer is formed by in-situ polymerization on the surface of hydroxyapatite modified by a silane coupling agent, so that the tensile yield strength and Young modulus of the prepared bone tissue repair material are further improved, and the mechanical property is further improved.

Meanwhile, the notch impact strength of the bone tissue repair material prepared in the example 1 is obviously higher than that of the comparative example 1, which shows that the impact strength of the material can be obviously enhanced by applying the potassium dehydroandrographolide succinate modified polyvinyl alcohol to the preparation of the bone tissue repair material. The effect of example 4 is better than that of example 1, and the effect of example 7 is better than that of comparative example 1, which shows that betulonic acid is adopted as one of crosslinking monomers, and the polymer is formed by in-situ polymerization on the surface of hydroxyapatite modified by silane coupling agent, so that the impact strength of the prepared bone tissue repair material is further enhanced.

Bending strength test

The test adopts a three-point bending resistance method, the bending resistance of the bone tissue repair material is measured by a universal testing machine, the span of the test point is 25mm, and the loading rate is 5.00 mm/min.

The results of the above tests on bone tissue repair materials prepared in comparative example 1 and examples 1 to 7 are shown in table 2:

from the analysis in table 2, it can be seen that the bending strength of the bone tissue repair material prepared in example 1 is significantly higher than that of comparative example 1, indicating that the bending strength of the material can be significantly enhanced by applying the dehydroandrographolide succinate modified polyvinyl alcohol to the preparation of the bone tissue repair material. The effect of example 4 is better than that of example 1, and the effect of example 7 is better than that of comparative example 1, which shows that betulonic acid is adopted as one of crosslinking monomers, and the betulonic acid is polymerized in situ on the surface of hydroxyapatite modified by silane coupling agent to form polymer, so that the bending strength of the prepared bone tissue repair material is further enhanced.

Abrasion Performance test

The test was performed using a computer controlled pin-disc abrasion tester. Before testing, the test pieces were polished with silicone paper and then polished with ultra-fine diamond paste. During testing, a layer of fetal bovine serum is coated on the surface of a sample, and then the sample is placed on a sample rotating disc and is placed under a fixed tungsten carbide abrasion ball. The environment temperature of the device is controlled at 37 ℃, the abrasion diameter of each sample is 4mm, the load is 30N, and the rotating speed is 100 rpm; the wear volume was calculated using a Form talsrf PG surface texture tester to measure the profilometry of the abrasive traces.

The results of the above tests on bone tissue repair materials prepared in comparative example 1 and examples 1 to 7 are shown in table 3:

from the analysis in table 3, it can be seen that the wear volume of the bone tissue repair material prepared in example 1 is not significantly different from that of comparative example 1, which indicates that the wear resistance of the bone tissue repair material is not negatively affected by the use of the dehydroandrographolide succinate modified polyvinyl alcohol in the preparation of the bone tissue repair material. The wear volume of the bone tissue repair material prepared in the embodiment 4 is obviously less than that of the embodiment 1, the effect of the embodiment 7 is better than that of the comparative example 1, and the bone tissue repair material is prepared by adopting betulonic acid as one of the crosslinking monomers, polymerizing the betulonic acid on the surface of the hydroxyapatite modified by the silane coupling agent in situ to form a polymer, and then compounding the polymer with the polymer II, so that the interface bonding property of the hydroxyapatite and the polymer II is further improved, the defects of the material body and the surface are reduced, and the wear resistance is greatly improved.

Hydrophilicity test

Test hydrophilicity of a sample was evaluated by measuring the contact angle of water with the sample using a contact angle tester.

The results of the above tests on bone tissue repair materials prepared in comparative example 1 and examples 1 to 7 are shown in table 4:

from the analysis in table 4, it can be seen that the contact angle of the bone tissue repair material prepared in example 1 is significantly lower than that of comparative example 1, which indicates that the use of the potassium dehydroandrographolide succinate modified polyvinyl alcohol in the preparation of the bone tissue repair material can effectively improve the hydrophilicity of the bone tissue repair material, and further improve the adhesion, proliferation and differentiation capabilities of cells on the surface of the material. The contact angle of the bone tissue repair material prepared in example 4 is equivalent to that of example 1, and the effect of example 7 is equivalent to that of comparative example 1, which shows that the bone tissue repair material prepared by using betulonic acid as one of the crosslinking monomers, performing in-situ polymerization on the surface of hydroxyapatite modified by a silane coupling agent to form a polymer, and then compounding the polymer with the polymer II does not have negative influence on the hydrophilicity of the material.

Characterization of bone repair Performance

Cell culture

Test objects: human adipose mesenchymal stem cells (hydroxyapatite DSCs). Suspending hydroxyapatite DSCs cells in a DMEM/F12 culture medium containing 10% fetal calf serum, culturing for 7d at 37 ℃ under 5% CO2, discarding original culture solution, washing for 3 times by PBS, adding 0.25% trypsin (containing 0.02% EDTA), digesting at 37 ℃ for 5min at constant temperature, observing that the adherent cells are converted into floating spheres under a light microscope, adding a complete culture medium with the same volume as the EDTA to stop digestion, blowing and mixing the cell suspension uniformly, transferring the cell suspension into a centrifuge tube, centrifuging for 5min at the rotation speed of 800rpm/min, and discarding supernatant; then carrying out resuspension passage on the cells to obtain 1 st generation hydroxyapatite DSCs; after the cell proliferation reaches about 80%, carrying out passage to the 2 nd generation, after reaching the passage density, carrying out pancreatin digestion and centrifugation, and removing the supernatant; then adding 1mL of complete culture solution, uniformly mixing and resuspending, and carrying out passage plating, wherein when the proliferation of 3 rd generation hydroxyapatite DSCs reaches 80%, the cells are reserved.

Toxicity detection

Taking 3 rd generation hydroxyapatite DSCs cells, digesting, centrifuging and resuspending the 3 rd generation hydroxyapatite DSCs cells by pancreatin, and planting the cells when the spreading area of the cells accounts for 80% of the total area; before planting cells, placing the sterilized bone tissue repair material into a 48-hole culture plate, soaking for 12 hours by using a PBS solution, absorbing the PBS solution, then injecting diluted cell suspension (the number of the planted cells in each hole is 5 multiplied by 104), culturing under the conditions of 37 ℃ and 5% CO2, changing the solution every other day, sucking out the culture medium after culturing for 3 days, and transferring the sample into a new 48-hole culture plate; then adding 1mL of CCK-8 solution into 9mL of complete culture medium under the condition of keeping out of the light, uniformly mixing, adding 300 mu L of CCK-8 solution into each hole, and placing in a constant-temperature water bath kettle at 37 ℃ to incubate for 1h in the absence of the light. And sucking 100 mu L of solution from each hole in dark, transferring the solution to a 96-hole culture plate, and testing the absorbance OD value at 450nm by using a multifunctional microplate reader.

The results of the above tests on bone tissue repair materials prepared in comparative example 1 and examples 1 to 7 are shown in FIG. 4. Analysis in the figure shows that compared with a blank control group, the absorbance OD value at 450nm of the bone tissue repair material prepared by the invention is not reduced after treatment, namely, the proliferation of hydroxyapatite DSCs is not obviously influenced, and the bone tissue repair material prepared by the invention basically does not produce toxic effect on cells and has good use safety.

RNA extraction and RT-PCR assay

Culturing cells on a material by adopting a 24-hole culture plate, wherein the number of the inoculated cells in each hole is 6 multiplied by 104, culturing by using osteogenic induction liquid, changing the liquid every other day, sucking out the culture liquid after culturing for 14 days, and washing by using PBS (phosphate buffer solution) for 3 times. Collecting the total RNA of the purified hydroxyapatite DSCs by using an RNA purification kit. The specific steps are carried out according to the instruction of the specification.

After obtaining purified RNA, determining the purity and concentration of the extracted cell RNA by using an enzyme-labeling instrument; then reverse transcription is carried out by using a Prime Script RT kit; and (3) performing optimization analysis on quantitative RT-PCR on a PCR detection system by adopting POWERSBR PCR mix enzyme. The sequences of the relevant primers are shown in Table 5.

The experimentally detected osteogenesis related genes comprise human type I collagen (Col 1), osteopontin (Opn), Lanta related transcription factor 2 (Runx 2) and bone sialoprotein (Bsp), and the test results are shown in FIG. 5. From the analysis in the figure, it can be seen that after the bone tissue repair material prepared in example 1 is treated, the relative expression amounts of mRNA of Col1, Opn and Runx2 are obviously higher than those of comparative example 1, which indicates that the use of the dehydroandrographolide succinate modified polyvinyl alcohol in the preparation of the bone tissue repair material can significantly enhance the expression of the bone-related gene of the material, improve the osteogenic differentiation capacity of the cell and improve the bone repair effect of the material. After the bone tissue repair material prepared in the embodiment 7 is treated, the relative expression quantity of mRNA of Opn and Bsp is obviously higher than that of the mRNA of the Opn and Bsp in the comparative example 1, which shows that the betulonic acid is adopted as one of the crosslinking monomers, and the polymer is formed by in-situ polymerization on the surface of hydroxyapatite modified by the silane coupling agent, so that the cell osteogenic differentiation promoting capability of the prepared bone tissue repair material is improved to a certain extent. After the bone tissue repairing material prepared in the embodiment 4 is treated, the relative expression amounts of mRNA of Col1, Opn, Runx2 and Bsp are obviously higher than those of the mRNA of the Col1, the Opn, the Runx2 and the Bsp in the embodiment 1, which shows that the betulonic acid is adopted as one of crosslinking monomers, the polymer is formed by in-situ polymerization on the surface of hydroxyapatite modified by a silane coupling agent, and the polymer is compounded with modified polyvinyl alcohol for use, so that the effect of enhancing the cell osteogenesis and differentiation promoting capability of the bone tissue repairing material is better, and the addition of the betulonic acid has a synergistic effect on the dehydroandrographolide modified polyvinyl alcohol.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.