Anisotropic PVA (polyvinyl alcohol) hydrogel for tendon repair as well as preparation method and application of anisotropic PVA hydrogel

Abstract

The invention discloses anisotropic PVA hydrogel for tendon repair and a preparation method and application thereof, and belongs to the field of functional polymer materials, and the preparation method comprises the following steps: placing a PVA / DMSO solution in a glycerin / ethanol mixed solvent for solvent exchange to form PVA / glycerin / ethanol organic gel; the inferior solvent ethanol is volatilized through hot stretching treatment, and anisotropic PVA / glycerin organic gel is obtained; after the PVA hydrogel is recovered to room temperature, the PVA hydrogel is soaked in deionized water to remove glycerin, and the anisotropic PVA hydrogel is prepared. The PVA hydrogel prepared by the invention has excellent mechanical properties, fatigue resistance and biocompatibility, and also shows remarkable swelling resistance and capability of promoting directional growth of cells; experiments show that the hydrogel can effectively repair fractured tendons of rats and promote tendon regeneration, and has a wide application prospect in the field of biological materials.

Description

Technical Field

[0001] The present invention belongs to the field of functional polymer materials, and particularly relates to an anisotropic PVA hydrogel for tendon repair, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogels have high water content, flexibility, and biocompatibility, and are similar to the extracellular matrix (ECM) in terms of structure and composition. These properties make them widely applicable in many fields such as environmental engineering, flexible sensing, electrochemistry, especially in the biomedical field, including tissue repair, drug delivery systems, wound dressings, biosensors, contact lenses, artificial cells, etc., and have a wide range of applications. Due to the weak mechanical properties of traditional hydrogels, people are committed to developing high-strength and high-toughness hydrogels. Researchers have significantly improved the mechanical properties and anti-fatigue properties of hydrogels by introducing energy dissipation mechanisms, such as double-network hydrogels and multi-level structure hydrogels. However, isotropic hydrogels do not have an anisotropic structure similar to biological tissues, which limits the multifunctional uses of hydrogels.

[0003] Biological tissues, such as skeletal muscle, tendon, and cartilage, have excellent strength, toughness, and anti-fatigue properties. For example, skeletal muscle can withstand high stress (1 MPa) in millions of cycles without breaking. Although its water content is very high (≈80%), its fatigue threshold (i.e., the minimum fracture energy required for crack propagation under cyclic loading) exceeds 1000 J m −2 . The application of hydrogels is determined by their mechanical properties, including modulus, toughness, viscoelasticity, elasticity, and anti-fatigue characteristics. Tendons have very high strength, modulus, and toughness, and have long-term stability. Their anti-fatigue characteristics come from the micro / nano structure of hierarchically arranged collagen fibers. Biomimetic materials designed based on this structure are very promising to obtain excellent anti-fatigue properties.

[0004] In recent years, researchers have developed some strategies to induce the alignment of macromolecular chains to prepare anti-fatigue anisotropic hydrogels with stronger mechanical properties. Currently, several commonly used methods for inducing the ordered orientation of polymer molecular chains in hydrogels mainly include magnetic / electric field induction, directional freezing (ice templating), mechanical training, and pre-stretching methods, etc. Liu et al. successfully improved the mechanical properties such as anti-fatigue properties of water-soluble polymer hydrogels such as PVA, alginate, gelatin, and cellulose through directional freezing casting and annealing treatment (X. Liang, G. Chen, S. Lin, J. Zhang, L. Wang, P. Zhang, Z. Wang, Z. Wang, Y. Lan, Q. Ge, J. Liu, Anisotropically Fatigue-Resistant Hydrogels.Adv. Mater. 2021, 33, 2102011.). However, the improvement of the mechanical properties of hydrogels prepared by the ice-templating method is not satisfactory. Therefore, it is necessary to introduce ions into the hydrogels to further improve the mechanical properties through the "salting-out" effect. He et al. prepared anisotropic and hierarchically structured polyvinyl alcohol hydrogels with multiple length scales by directional freezing-assisted salting-out effect, which have excellent strength, toughness, and fatigue resistance. However, if used in vivo, these ions are easily migrated out of the hydrogels, which will not only lead to a significant decrease in mechanical properties but also may cause cell dehydration and rupture (Hua, M., Wu, S., Ma, Y. et al. Strong tough hydrogels via the synergy of freeze-casting and salting out. Nature 590, 594–599 (2021). Mechanical training usually needs to be completed by long-term cyclic stretching underwater, making the preparation more complex and increasing the cost. The tensile strength and fatigue resistance of the obtained hydrogels also need to be further improved (S. Lin, J. Liu, X. Liu, & X. Zhao, Muscle-like fatigue-resistant hydrogels by mechanical training, Proc. Natl. Acad. Sci. U.S.A. 116 (21) 10244-10249, (2019).). Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part, as well as in the abstract and title of the specification of this application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a preparation method for an anisotropic PVA hydrogel material for tendon repair.

[0008] To solve the above technical problems, the present invention provides the following technical solutions: A preparation method for an anisotropic PVA hydrogel material, including, Adding PVA powder to a DMSO solution, heating and stirring to obtain a PVA / DMSO solution; After cooling the PVA / DMSO solution to room temperature, it is placed in a glycerol / ethanol mixed solvent for solvent exchange to obtain a PVA / glycerol / ethanol organic gel; The PVA / glycerol / ethanol organic gel is pre-stretched and then wet annealed to obtain an anisotropic PVA / glycerol organic gel; The anisotropic PVA / glycerol organic gel is immersed in deionized water. After the complete exchange of glycerol and water, an anisotropic PVA hydrogel is obtained.

[0009] As a preferred embodiment of the preparation method of the present invention, wherein: the mass fraction of PVA in the PVA / DMSO solution is 15%, the heating and stirring time is 3 h, and the heating and stirring temperature is 90 °C.

[0010] As a preferred embodiment of the preparation method of the present invention, wherein: in the glycerol / ethanol mixed solvent, the volume ratio of glycerol to ethanol is 1-4:1.

[0011] As a preferred embodiment of the preparation method of the present invention, wherein: the mixing solvent exchange time is 48 h.

[0012] As a preferred embodiment of the preparation method of the present invention, wherein: the pre-stretching ratio of the PVA / glycerol / ethanol organic gel is 0.1%-200%.

[0013] As a preferred embodiment of the preparation method of the present invention, wherein: the pre-stretching ratios of the PVA / glycerol / ethanol organic gel are 50%, 100%, 150% and 200%.

[0014] As a preferred embodiment of the preparation method of the present invention, wherein: the temperature of the wet annealing is 120 °C and the time is 30 min.

[0015] As a preferred embodiment of the preparation method of the present invention, wherein: the solvent exchange time between glycerol and water is 24-48 h.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide an anisotropic PVA hydrogel material for tendon repair.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of an anisotropic PVA hydrogel material in the preparation of tendon repair materials.

[0018] Advantages of the present invention: (1) The present invention cleverly selects a mixed poor solvent with different boiling points for solvent exchange to obtain an organic gel. The low-boiling solvent volatilizes during the thermal stretching process of the organic gel, and the high-boiling solvent ensures the thermal stability and structural stability of the organic gel during the annealing process.

[0019] (2) The present invention proposes a strategy of using a poor solvent-assisted thermal stretching. The poor solvent has a low boiling point and will volatilize during the thermal stretching process, enhancing the stress in the stretching direction, promoting the orientation and crystallization of macromolecular chains, facilitating the formation of neatly arranged nanofibrils, and greatly improving the mechanical properties of the material.

[0020] (3) The present invention proposes a technique of wet annealing during the stretching process. Different from the traditional dry annealing method (where the hydrogel loses water and becomes an aerogel for heat treatment), the macromolecular chains have sufficient mobility and can fully adjust their conformations. The prepared hydrogel has a tensile strength as high as 33.14 ± 2.05 MPa, a toughness as high as 44.1 ± 3.5 MJ m -3 , and a fracture energy as high as 106.18 ± 7.2 kJ m -2 .

[0021] (4) The anisotropic PVA hydrogel prepared by the present invention has excellent anti-fatigue performance. Cracks can only branch along the fiber orientation direction and cannot extend along the direction perpendicular to the fiber, showing a unique self-protection function.

[0022] (5) The anisotropic PVA hydrogel prepared by the present invention has excellent anti-swelling performance and biocompatibility, ensuring its application in vivo and can be used for the repair of transected tendons in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them: Figure 1 It is a schematic diagram for the preparation of the anisotropic PVA hydrogel of the present invention; where a is the preparation flow chart of the anisotropic PVA hydrogel, and b is the schematic diagram of ethanol volatilization during the thermal stretching process.

[0024] Figure 2 It is the scanning electron microscope image of the hydrogel of the present invention; where a is the cross-sectional electron microscope image of Comparative Example 2, b is the cross-sectional electron microscope image perpendicular to the orientation direction of Example 2, and c is the cross-sectional electron microscope image parallel to the orientation direction of Example 2.

[0025] Figure 3 It is the tensile stress-strain curve graph of the gels prepared in Comparative Examples 1 - 5 of the present invention.

[0026] Figure 4 It is the tensile stress-strain curve graph of the gels prepared in Comparative Example 6 and Examples 1, 5, 6, 7 of the present invention.

[0027] Figure 5 Tensile stress-strain curve graphs of the gels prepared in Comparative Example 2 and Examples 1, 2, 3, and 4 of the present invention.

[0028] Figure 6 Summary graphs of the modulus and toughness of the gels prepared in Comparative Example 2 and Examples 1, 2, 3, and 4 of the present invention.

[0029] Figure 7 Summary graph of the fracture energy of the gels prepared in Comparative Example 2, Examples 1, 2, 3, and 4 of the present invention.

[0030] Figure 8 Cyclic tensile-recovery stress-strain curve graphs of the gels prepared in Comparative Example 2, Examples 1, 2, 3, and 4 of the present invention; wherein, a is Comparative Example 2, b is Example 4, c is Example 3, d is Example 1, and e is Example 2.

[0031] Figure 9 Fatigue test result graphs of the gels prepared in Example 1 and Comparative Example 2 of the present invention; wherein, a is Comparative Example 2 and b is Example 1.

[0032] Figure 10 For the gel prepared in Example 1 of the present invention at energy release rates of 495.5 J / m 2 and 1011.5 J / m 2 Notch photos at the 1st, 1000th, and 10,000th times during 10,000 tensile cycle tests.

[0033] Figure 11 Schematic diagram of the anti-fatigue mechanism of the anisotropic PVA hydrogel prepared in Example 1 of the present invention.

[0034] Figure 12 Swelling rate graphs of the anisotropic PVA hydrogel prepared in Example 1 of the present invention after swelling in deionized water, PBS buffer solution, and physiological saline for 7 days.

[0035] Figure 13 Mechanical property graphs of the anisotropic PVA hydrogel prepared in Example 1 of the present invention before and after swelling in deionized water for 7 days, wherein, a is the stress-strain curve graph and b is the mechanical property retention coefficient graph.

[0036] Figure 14 Biocompatibility test of the anisotropic PVA hydrogel prepared in Example 1 of the present invention, wherein, a is the cell dead / alive staining image and b is the cell survival rate graph.

[0037] Figure 15The process of implanting the anisotropic PVA hydrogel prepared in Example 1 of the present invention into the tendon of a rat. Among them, a is a schematic diagram, b is the surgical process, and c is the appearance diagram of the tendon injury area at the 4th and 8th weeks.

[0038] Figure 16 The tendon regeneration conditions at different times after implanting the anisotropic PVA hydrogel prepared in Example 1 of the present invention into the rat body. Among them, a is the HE section image and Masson staining image of the regenerated tendon at different times, b is the statistical chart of the thickness of the regenerated tendon at different times, c is the statistical chart of the width of the regenerated tendon at different times, d is the statistical chart of the macroscopic score of the regenerated tendon at different times, and e is the statistical chart of the histological score of the regenerated tendon at different times.

[0039] Figure 17 The collagen regeneration conditions at different times after implanting the anisotropic PVA hydrogel prepared in Example 1 of the present invention into the rat body. Among them, a is the immunofluorescence staining image of the regenerated tendon at different times, b is the statistical chart of collagen regeneration at the 4th week, and c is the statistical chart of collagen regeneration at the 8th week. Detailed implementation manners

[0040] To make the above objects, features and advantages of the present invention more obvious and understandable, the following will describe the detailed implementation manners of the present invention in conjunction with the embodiments of the specification.

[0041] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0042] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that can be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not all refer to the same embodiment, nor is it a separate or selectively exclusive embodiment from other embodiments.

[0043] The materials in the embodiments of the present invention: PVA-1799 (Aladdin), dimethyl sulfoxide (DMSO, Aladdin), glycerol (Aladdin), absolute ethanol (analytical pure) were all used directly without further purification; Fetal bovine serum (FBS), medium (DMEM), penicillin / streptomycin (PS), calcein-AM and propidium iodide (PI) were all purchased from Thermo Fisher Scientific; Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories; Paraformaldehyde (PFA; 4.0%, mass / volume ratio) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); all aqueous solutions were prepared using ultrapure deionized water.

[0044] Example 1 (1) Preparation of anisotropic PVA hydrogel: Dissolve 4.5 g of PVA in 25.5 g of DMSO solution, stir at 90 °C for 3 h, pour the well-stirred solution into a mold, and place it at room temperature. Then put it into a mixed solvent of glycerol / ethanol with a ratio of 4:1 for solvent exchange for 48 h to obtain a PVA / glycerol / ethanol organogel. Pre-stretch the PVA / glycerol / ethanol organogel to 150%, fix both ends of the pre-stretched gel, put it into an oven at 120 °C for wet annealing for 30 min, and then place it at room temperature. At this time, the ethanol has completely evaporated to obtain an anisotropic PVA / glycerol organogel. Put the anisotropic PVA / glycerol organogel into deionized water for solvent exchange for 48 h to obtain an anisotropic PVA hydrogel.

[0045] The preparation process of the anisotropic PVA hydrogel is as Figure 1 shown.

[0046] (2) Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine. The anisotropic PVA hydrogel was subjected to 1000 cycles of tensile-recovery performance test and fatigue test using a fatigue tensile machine.

[0047] The test results of the tensile stress-strain curve are as Figure 4 , 5 shown, and the tensile strength of the anisotropic PVA hydrogel is as high as 19.54 ± 1.12 MPa.

[0048] The Young's modulus and toughness are as Figure 6 shown, and the toughness reaches 44.1 ± 3.5 MJ m -3 . The test results of the fracture energy are as Figure 7 shown, and the fracture energy reaches 79.4 ± 7.9 kJ m -2 .

[0049] The test results of the cyclic tensile-recovery stress-strain curve are as Figure 8As shown in d. 1000 tensile loading-unloading tests were carried out, and the hydrogel showed obvious mechanical hysteresis in the first cycle, namely the so-called Mullins effect. As the pre-stretching ratio increases, the hysteresis loop becomes more obvious. A higher pre-stretching ratio enhances hydrogen bond interaction and increases crystallinity, so more energy is required to break hydrogen bonds and orient crystalline domains during stretching. After 50 cycles, the loading and unloading curves almost coincide and there is no hysteresis loop, that is, the dissipated energy is close to zero.

[0050] The fatigue test results by the single-notch method are as Figure 9 shown, as Figure 9 shown in a, the fatigue threshold of the isotropic hydrogel is 187 J m -2 . As Figure 9 shown in b, when the initial crack is perpendicular to the alignment direction of the nanofibers, when λ is less than 1.3, the crack does not propagate under 10,000 loading cycles, and the energy release rate is 495.5 J m -2 . When λ increases to 1.4, 1.5 and 1.6, crack branching is observed under 10,000 cyclic loadings, and the energy release rates are 834.9 J m -2 , 1011.6 J m -2 and 1386.3 J m -2 respectively.

[0051] The notch photos at the 1st, 1000th and 10,000th times in the 10,000 tensile cycle tests of the gel prepared in Example 1 at energy release rates of 495.5 J m -2 and 1011.5 J m -2 are as Figure 10 shown. As the tensile deformation increases, the crack does not propagate along the initial direction, but branches and propagates along the loading direction. This defect insensitivity can effectively protect the notched hydrogel without causing catastrophic damage. Although most tough hydrogels have a large fatigue threshold, cracks can still propagate under long-term cyclic loading; however, the anisotropic PVA hydrogel can deflect the crack along the load direction until the notched part tears, leaving the remaining hydrogel intact. It should be noted that crack branching also consumes a large amount of energy, indicating that the hydrogel is insensitive to initial defects.

[0052] The anti-fatigue mechanism of the anisotropic PVA hydrogel prepared in Example 1 is as Figure 11As shown. The anisotropic PVA hydrogel has a unique hierarchical structure, namely a dense polymer network composed of fiber bundles, nanofibers composed of neatly arranged macromolecular chains, crystalline domains, and intermolecular hydrogen bonds. On the micron scale, when the crack is perpendicular to the direction of the neatly arranged fiber bundles, due to the neatly arranged thick fibers in the parallel direction, the crack bends, and the crack propagation path deviates from the maximum stress surface at the initial point, reducing the stress concentration at the crack tip. As the tensile strain increases, the crack tends to propagate along the notch, the nanofibers slip, are pulled out and torn in the bridging area, further dispersing the stress and dissipating a large amount of energy. On the molecular scale, the crystalline domains, as rigid and highly functional crosslinking agents, can fix the cracks, thus blunting the cracks. In addition, a large number of hydrogen bonds are also the reason for energy dissipation. Finally, the hydrogel achieves multi-level strengthening and anti-fatigue characteristics, and the crack can only propagate along the tensile direction rather than the notch direction. Therefore, the hydrogel is only partially damaged and exhibits a self-protection function. On the other hand, when the crack is parallel to the direction of the neatly arranged fiber bundles, the strength of the gap area between these fiber bundles is insufficient to withstand the stress concentration at the crack tip, so the crack will rapidly propagate along these weak areas, resulting in the rapid failure of the hydrogel.

[0053] (3)Anti-swelling property test: The mechanical properties of the hydrogel before and after swelling were tested using a universal testing machine.

[0054] To evaluate the anti-swelling property of the hydrogel, the hydrogel was immersed in water, normal saline, and PBS buffer solution for 3 days and 7 days, respectively.

[0055] As Figure 12 shown, after 7 days, the swelling rate of the gel in water, normal saline, and PBS solution was less than 5%. The hydrogel was prepared by solvent exchange of glycerol and water, and the macromolecular network almost reached an equilibrium state in water. Therefore, the obtained hydrogel hardly swells when used in an underwater environment. In addition, the excellent anti-swelling property also comes from the dense crystalline domains, which can act as crosslinking points to prevent the swelling of macromolecules.

[0056] Figure 13 Figure a shows the stress-strain curves of the gel before and after swelling in water for 7 days. The tensile strength and fracture strain of the gel decreased slightly after swelling, and the retention coefficients were 96% and 99% respectively ( Figure 13 Figure b).

[0057] (4)Bioactivity test: The CCK-8 assay and LIVE / DEAD staining method of mouse embryonic fibroblasts (NIH-3T3) were used to verify the cytocompatibility of the material.

[0058] The specific steps are as follows: Cultivate NIH / 3T3 mouse embryonic fibroblasts in DMEM medium containing 10.0% (volume fraction) fetal bovine serum (FBS) and 1.0% (volume fraction) penicillin / streptomycin, and place them in a cell incubator at 37 °C with a CO2 concentration of 5% for routine cultivation.

[0059] Qualitatively evaluate the cytotoxicity of the hydrogel by the CCK-8 method, and quantitatively evaluate the proliferation rate of NIH / 3T3 cells by adding the CCK-8 solution at regular intervals; The specific steps are as follows: Add 300 μL of cell suspension and 1 cm² of hydrogel material to a 48-well plate, and replace it with the CCK-8 solution for continued incubation for 45 minutes after co-incubation for 1, 3, and 7 days respectively; Further evaluate the cytotoxicity of the hydrogel by the live / dead cell double staining method (LIVE / DEAD): Inoculate 1.0 mL of NIH / 3T3 cell suspension in a 6-well plate, co-culture it with the hydrogel material for 1, 3, and 7 days, stain it with a mixed staining agent containing 1.0 μM calcein-AM and 1.0 μM propidium iodide (PI), and finally observe and collect cell morphology images through a fluorescence microscope.

[0060] As Figure 14 shown in b, the cells growing on the empty tissue culture plate were used as the control group. The cell survival rate was greater than 85% after the gel was co-cultured with NIH-3T3 for 1, 3, and 7 days. On the seventh day, the cell survival rate of the gel group reached more than 97.92% ± 2.71%, showing no significant difference from the blank control group. That is, the hydrogel has excellent biocompatibility, and the dead / live staining images can also prove this trend ( Figure 14 a).

[0061] (5) Rat tendon repair experiment: The surgical procedure is as Figure 15 shown in a, b. Dissect and cut the bilateral patellar tendons of 6-8-week-old female SD rats (purchased from Shanghai Slake Experimental Animal Co., Ltd. (Shanghai, China), raised in a constant temperature environment at 25 °C) to form a full-thickness tendon defect. Use surgical sutures to suture the two ends of the 5 mm 2 gel to the two ends of the severed tendon to replace the tendon tissue, and directly suture the severed tendon as the blank group.

[0062] As Figure 15 shown in c, at the 4th week and the 8th week, the tendon injury area in the gel group was fully filled with regenerated tissue, and its surface was smooth. The surface of the regenerated tendon showed a milky white tissue appearance consistent with that of natural tendons. In the blank group, tendon tissue edema and thickening were generally observed.

[0063] As Figure 16As shown in a, there are more mature tenocytes and tendon cells in the gel group, with a high density and close distribution of collagen fibers. Moreover, the cell morphology and distribution are mainly near-circular and regionally arranged at week 4; and mainly spindle-shaped and parallel arranged at week 8. Through the analysis of the Masson staining images at week 8, it can be seen that there are more myofibers formed in the regenerated tendon of the gel group. Myofibers not only have the ability to contract to improve the tensile strength of the tendon, but also can provide blood for the tendon tissue. As Figure 16 As shown in b and c, the thickness and width of the regenerated tendon in the gel group are smaller than those in the blank group, indicating that the gel group produces less scar tissue during tendon repair. As Figure 16 As shown in d and e, through the macroscopic scoring and histological scoring of tendon repair conditions from multiple factors such as inflammation, tendon adhesion, defect area filling, shape and color, the score of the gel group is significantly lower than that of the blank group, indicating that the anisotropic PVA hydrogel prepared in Example 1 is beneficial to the remodeling and regeneration of tendon tissue.

[0064] Use a microtome to prepare tissue sections, and perform hematoxylin-eosin (H&E) staining and Masson staining to evaluate the morphological structure of tendons at different stages; fix the treated tissue with 4% paraformaldehyde for 48 h, and then perform paraffin embedding; conventional paraffin-embedded tissue sections with a thickness of about 5.0 μm need to be dewaxed, hydrated, stained and dehydrated, and images are collected using an upright optical microscope (Nikon, Eclipse E100); Subsequently, immunohistochemical staining is performed on the sections using type I collagen antibody (Wuhan Sevier Biotechnology Co., Ltd.), type III collagen antibody (Wuhan Sevier Biotechnology Co., Ltd.) and nuclear antibody (Wuhan Sevier Biotechnology Co., Ltd.) to evaluate the regeneration of collagen. Briefly, type I collagen is stained red, type III collagen is stained green, and the nucleus is stained blue; images are collected using an upright optical microscope (Nikon, Eclipse E100); finally, the histological evaluation of hematoxylin-eosin (H&E) staining images is performed using the modified Mankin scoring method.

[0065] During tendon repair, the expression level of COL1 / COL3 is crucial for the physiological healing of tendons. As Figure 17 As shown, the staining area of COL1 in the gel group is larger and the fluorescence intensity is stronger. Combining the quantitative data, it can be seen that the regeneration rate of COL1 in the gel group is significantly faster. Thus, it can be seen that the implantation of the anisotropic PVA hydrogel prepared in Example 1 plays a promoting role in the regeneration of collagen.

[0066] Example 2 (1) Preparation of anisotropic PVA hydrogel: This example is basically the same as Example 1, and the only difference is that the pre-stretching ratio is 200%.

[0067] (2)Morphology observation: The anisotropic PVA hydrogel was observed for its morphology using a scanning electron microscope.

[0068] The SEM image of the cross-section of the anisotropic PVA hydrogel is as shown in Figure 2 c. It can be seen that the hydrogel has an oriented structure. The SEM image of the longitudinal section is as shown in Figure 2 b, which has a uniform and dense porous structure.

[0069] (3)Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine, and the 1000-cycle tensile-recovery performance test of the anisotropic PVA hydrogel was carried out using a fatigue tensile machine.

[0070] The test results of the tensile stress-strain curve are as shown in Figure 5 . The tensile strength of the anisotropic PVA hydrogel reaches 33.2 ± 2.1 MPa, and the fracture energy reaches 106.18 ± 7.2 kJ m -2 ( Figure 7 ).

[0071] The test results of the 1000-cycle tensile-recovery performance are as shown in Figure 8 e. It shows obvious mechanical hysteresis in the first cycle and dissipates a large amount of energy.

[0072] Example 3 (1)Preparation of anisotropic PVA hydrogel: This example is basically the same as Example 1, and the only difference is that the pre-stretching ratio is 100%.

[0073] (2)Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine, and the 1000-cycle tensile-recovery performance test of the anisotropic PVA hydrogel was carried out using a fatigue tensile machine.

[0074] The test results of the tensile stress-strain curve are as shown in Figure 5 . The tensile strength of the anisotropic PVA hydrogel reaches 12 ± 0.8 MPa, and the fracture energy reaches 46.53 ± 1.04 kJ m -2 ( Figure 7 ).

[0075] The test results of the 1000-cycle tensile-recovery performance are as shown in Figure 8As shown in c, significant mechanical hysteresis was exhibited in the first cycle, dissipating a large amount of energy.

[0076] Example 4 (1) Preparation of anisotropic PVA hydrogel: This example is basically the same as Example 1, and the only difference is that the pre-stretching ratio is 50%.

[0077] (2) Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine, and the 1000-cycle tensile-recovery performance test of the anisotropic PVA hydrogel was carried out using a fatigue tensile machine.

[0078] The test results of the tensile stress-strain curve are as Figure 5 shown. The tensile strength of the anisotropic PVA hydrogel reached 7.35 ± 0.15 MPa, and the fracture energy reached 14.7 ± 2.2 kJm -2 ( Figure 7 )

[0079] The test results of the 1000-cycle tensile-recovery performance are as Figure 8 shown in b. Significant mechanical hysteresis was exhibited in the first cycle, dissipating a large amount of energy.

[0080] Example 5 (1) Preparation of anisotropic hydrogel: This example is basically the same as Example 1, and the only difference is that the ratio of glycerol / ethanol is 2:1.

[0081] (2) Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine. The test results of the tensile stress-strain curve are as Figure 4 shown.

[0082] The tensile strength of the anisotropic PVA hydrogel reached 15.07 MPa.

[0083] Example 6 (1) Preparation of anisotropic hydrogel: This example is basically the same as Example 1, and the only difference is that the ratio of glycerol / ethanol is 1:1.

[0084] (2) Mechanical property test: All mechanical property tests of the anisotropic PVA hydrogel were carried out using a universal testing machine. The test results of the tensile stress-strain curve are as Figure 4 shown. The tensile strength of the anisotropic PVA hydrogel reached 16.56 MPa.

[0085] Example 7 (1) Preparation of anisotropic hydrogel: This example is basically the same as Example 1, and the only difference is that the ratio of glycerol / ethanol is 0:1.

[0086] (2) Mechanical property test: All mechanical property tests were carried out on the anisotropic PVA hydrogel using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 4 shown. The tensile strength of the anisotropic PVA hydrogel reached 13.58 MPa.

[0087] Comparative Example 1 (1) Preparation of isotropic hydrogel: 4.5 g of PVA was dissolved in 25.5 g of DMSO solution, stirred at 90 °C for 3 h, and the well-stirred solution was poured into a mold. After standing at room temperature, it was placed in a mixed solvent of glycerol / ethanol with a ratio of 1:0 for solvent exchange for 48 h to obtain a PVA / glycerol / ethanol organogel.

[0088] The PVA / glycerol / ethanol organogel was placed in an oven at 120 °C for wet annealing for 30 min, and then placed at room temperature. At this time, the ethanol had completely evaporated to obtain an isotropic PVA / glycerol organogel.

[0089] The isotropic PVA / glycerol organogel was placed in deionized water for solvent exchange for 48 h to obtain an isotropic PVA hydrogel.

[0090] (2) Mechanical property test: All mechanical property tests were carried out on the isotropic PVA hydrogel using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 3 shown.

[0091] The tensile strength of the isotropic PVA hydrogel reached 4.32 MPa.

[0092] Comparative Example 2 (1) Preparation of isotropic PVA hydrogel: This comparative example is basically the same as Comparative Example 1, and the only difference is that the ratio of glycerol / ethanol is 4:1.

[0093] Morphology observation: The morphology of the isotropic PVA hydrogel was observed using a scanning electron microscope.

[0094] The scanning electron micrograph of the cross-section of the isotropic PVA hydrogel is as Figure 2As shown in a, it can be seen that it has a uniform porous structure with an average pore size of about 256 nm, which is larger than that of the anisotropic PVA hydrogel and is closely related to the water content.

[0095] (2) Mechanical property test: All mechanical property tests of the isotropic PVA hydrogel were carried out using a universal testing machine, and the 1000-cycle tensile-recovery performance test and single-notch fatigue test of the anisotropic PVA hydrogel were carried out using a fatigue tensile machine.

[0096] The test results of the tensile stress-strain curve are as Figure 5 shown. The tensile strength of the isotropic PVA hydrogel reaches 3.65 MPa, and the fracture energy reaches 4.9 ± 0.8 kJ m -2 ( Figure 7 )

[0097] The test results of the 1000-cycle tensile-recovery performance are as Figure 7 shown in a. It shows obvious mechanical hysteresis in the first cycle and dissipates a large amount of energy.

[0098] The test results of the single-notch fatigue test are as Figure 9 shown in a. The fatigue threshold of the isotropic PVA hydrogel reaches 187 Jm -2 .

[0099] Comparative Example 3 (1) Preparation of isotropic hydrogel: This comparative example is basically the same as Comparative Example 1, and the only difference is that the ratio of glycerol / ethanol is 2:1.

[0100] (2) Mechanical property test: All mechanical property tests of the isotropic PVA hydrogel were carried out using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 3 shown. The tensile strength of the isotropic PVA hydrogel reaches 3.14 MPa.

[0101] Comparative Example 4 (1) Preparation of isotropic hydrogel: This comparative example is basically the same as Comparative Example 1, and the only difference is that the ratio of glycerol / ethanol is 1:1.

[0102] (2) Mechanical property test: All mechanical property tests of the isotropic PVA hydrogel were carried out using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 3 shown. The tensile strength of the isotropic PVA hydrogel reaches 2.71 MPa.

[0103] Comparative Example 5 (1)Preparation of isotropic hydrogel: This comparative example is basically the same as Comparative Example 1, and the only difference is that the ratio of glycerol / ethanol is 0:1.

[0104] (2)Mechanical property test: All mechanical property tests were performed on the isotropic PVA hydrogel using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 3 shown. The tensile strength of the isotropic PVA hydrogel reached 2.36 MPa.

[0105] Comparative Example 6 (1)Preparation of anisotropic hydrogel: This comparative example is basically the same as Example 1, and the only difference is that the ratio of glycerol / ethanol is 1:0.

[0106] (2)Mechanical property test: All mechanical property tests were performed on the anisotropic PVA hydrogel using a universal testing machine, and the test results of the tensile stress-strain curve are as Figure 4 shown. The tensile strength of the anisotropic PVA hydrogel reached 8.39 MPa.

[0107] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered within the scope of the present invention.

Claims

1. A preparation method of an anisotropic PVA hydrogel material for tendon repair, characterized in that: including Adding PVA powder into DMSO solution, heating and stirring to obtain PVA / DMSO solution; After cooling the PVA / DMSO solution to room temperature, placing it into a glycerol / ethanol mixed solvent for solvent exchange to obtain PVA / glycerol / ethanol organogel; Pre-stretching the PVA / glycerol / ethanol organogel, and then performing wet annealing to obtain anisotropic PVA / glycerol organogel; Soaking the anisotropic PVA / glycerol organogel in deionized water, and after the complete exchange of glycerol and water, obtaining anisotropic PVA hydrogel.

2. The preparation method according to claim 1, characterized in that: The mass fraction of PVA in the PVA / DMSO solution is 15%, the heating and stirring time is 3 h, and the heating and stirring temperature is 90 °C.

3. The preparation method according to claim 1 or 2, characterized in that: The glycerol / ethanol mixed solvent, wherein the volume ratio of glycerol to ethanol is 1-4:

1.

4. The preparation method according to claim 3, characterized in that: The mixing solvent exchange time is 48 h.

5. The preparation method according to claim 1 or 4, characterized in that: The pre-stretching ratio of the PVA / glycerol / ethanol organogel is 0.1%-200%.

6. The preparation method according to claim 5, characterized in that: The pre-stretching ratios of the PVA / glycerol / ethanol organogel are 50%, 100%, 150% and 200%.

7. The preparation method according to claim 1 or 6, characterized in that: The temperature of the wet annealing is 120 °C and the time is 30 min.

8. The preparation method according to claim 7, characterized in that: The solvent exchange time between glycerol and water is 24-48 h.

9. An anisotropic PVA hydrogel material for tendon repair prepared by the preparation method according to any one of claims 1-8.

10. Use of the anisotropic PVA hydrogel material according to claim 9 in the preparation of a tendon repair material.

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