A hydrogel with controllable enhanced mechanical properties, its preparation and application

Abstract

The present invention provides a hydrogel with controllable enhanced mechanical properties and its preparation and application. The main components of the hydrogel are glycine-polyvinyl alcohol and methacrylated silk fibroin. The glycine-polyvinyl alcohol crystal film powder and methacrylated silk fibroin are pre-crosslinked by ultraviolet light to obtain a piezoelectric silk fibroin hydrogel, and the hydrogel is ultrasonically treated to obtain a piezoelectric silk fibroin hydrogel with enhanced mechanical strength. The glycine-polyvinyl alcohol piezoelectric material used in the present invention has a higher piezoelectric coefficient compared with traditional organic piezoelectric materials; the methacrylated silk fibroin and glycine-polyvinyl alcohol components have good biocompatibility; it can be used for the treatment of cartilage defects, and the change of the mechanical strength of the hydrogel material can be regulated by the time, sound intensity, frequency, etc. of applying external ultrasonic intervention, so that the mechanical strength of the hydrogel can dynamically adapt to different stages of the repair of locomotor system tissues including cartilage.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and hydrogel manufacturing technology, and particularly relates to a silk fibroin hydrogel with controllable enhanced mechanical properties and its preparation and application. Background Art

[0002] Osteoarthritis, as a degenerative disease, can lead to loss of motor ability, cause severe pain, and is prone to induce other chronic diseases. The onset of osteoarthritis usually results from the defect of cartilage tissue. And due to frequent stimulation during movement, it is difficult to recruit chondrocytes and easy to trigger inflammatory reactions; currently, the defect of cartilage is difficult to be repaired. Natural osteochondral tissue has the effect of generating bioelectric current in response to external stimuli. Currently, some studies have shown that externally applied electrical stimulation can promote the regeneration and differentiation of osteochondral cells. The piezoelectric effect is a physical and chemical effect of materials that can sense external mechanical stimuli, such as pressing, stretching, electromagnetic waves, ultrasonic wave stimulation, etc., so that internal molecules generate charge polarization and then form a transient current. Materials with piezoelectric effect can make up for the lack of the function of generating bioelectric signals in response to external stimuli caused by cartilage defects. Therefore, current studies have shown that piezoelectric materials can promote the regeneration of animal osteochondral defects.

[0003] In current research, the mechanical properties of the materials for promoting cartilage defect regeneration will remain stable after material preparation and will not change further. However, the mechanical properties of the materials have an impact on different stages of cartilage tissue repair. The development of osteochondral is a process from loose connective tissue to dense connective tissue. During this process, the mechanical properties of osteochondral gradually increase. Existing materials are difficult to continuously maintain a dynamic increase in mechanical properties after implantation in the body, so it is difficult to adapt to the enhanced mechanical properties of osteochondral development in terms of time.

[0004] Meanwhile, the inflammatory response and cell recruitment during osteochondral regeneration are also closely related to the mechanical properties of the material. Some studies have shown that with the increase in the mechanical strength of the implanted scaffold material, the polymerization of cellular actin can be promoted, thereby activating the Piezo 1 pathway and leading to an exacerbation of the inflammatory response. In addition, some studies have also shown that the increase in the mechanical strength of the implanted material will also promote the acetylation of intracellular tubulin, thereby promoting the migration and recruitment of cells at the defect site. It can be seen that in the inflammatory response period at the initial stage of osteochondral repair, the repair of the defect prefers scaffold materials with lower stiffness to reduce the local inflammatory response, while in the tissue repair period in the middle and late stages of osteochondral repair, the repair of the defect prefers scaffold materials with higher stiffness to promote cell recruitment and osteochondral regeneration. In the current research on the effects of material mechanics on inflammatory response and cartilage regeneration, it can be found that materials with a mechanical strength in the kPa range are beneficial to reducing the occurrence of inflammatory response in the initial stage of osteochondral regeneration; while when the mechanical strength reaches the 10 kPa range, the material is more beneficial to the expression of YAP protein and cell recruitment in the middle and late stages of osteochondral regeneration, thereby promoting cartilage tissue regeneration.

[0005] In the existing technology, a study has developed an inorganic piezoelectric material that responds to external force loading and promotes enhanced mechanical properties incorporated into a hydrogel. In this study, a phenomenon of internal charge polarization within molecules was proposed based on zinc oxide particles of piezoelectric materials during the process of external mechanical force loading, which was applied to initiate the generation of free radicals inside the hydrogel and then promote the free radical chain reaction of molecular monomers. The piezoelectric effect was used to trigger the increase in the mechanical strength of the hydrogel with external mechanical loading. However, in this study, a high-frequency and long-time loading was required to trigger the piezoelectric effect, and this mechanical loading method was difficult to be applied to the in vivo of actual living animals. At the same time, the inorganic piezoelectric material incorporated in this study was also difficult to be degraded in vivo. In addition, the range of change in the mechanical strength of the materials studied in this article was also difficult to adapt to different stages of cartilage repair.

[0006] Generally speaking, the existing technologies mainly have the following major problems: (1) The mechanical properties of existing hydrogel materials cannot dynamically adapt to the mechanical requirements of different repair stages after being implanted into osteochondral tissue defects, and there is a lack of hydrogel materials with dynamically enhanced mechanical properties; (2) Existing materials with enhanced mechanical properties have poor biocompatibility and the adjustable range of mechanical changes does not match the repair of osteochondral tissue, and there is a lack of materials with high biocompatibility and suitable for the mechanics of osteochondral repair; (3) The mechanical strength of existing materials with enhanced mechanical properties cannot be accurately regulated by controlling the time of external intervention, and there is a lack of research on the specific corresponding relationship between the mechanical strength of the material and the external loading time; (4) The external intervention methods and intervention conditions for triggering changes in the mechanical strength of existing materials with dynamically enhanced mechanical properties are not suitable for in vivo tissue repair, and there is a lack of minimally invasive and painless intervention methods for triggering enhanced mechanical properties of materials.

[0007] Since the repair process of osteochondral defects is a process that gradually increases the requirements for the mechanical properties of materials, and there are corresponding requirements for the biocompatibility of the materials themselves, the range of mechanical enhancement, and the external intervention methods that trigger mechanical enhancement. Therefore, the tissue engineering repair of osteochondral materials poses a new demand for a piezoelectric scaffold material with controllable mechanical property enhancement, and it is necessary to develop a hydrogel material with good biocompatibility, in-situ dynamic enhancement in vivo, controllable mechanical strength enhancement, and a method of triggering mechanical enhancement and applying external intervention that is beneficial to the repair of tissues such as osteochondral to effectively address the above deficiencies. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the present invention provides a hydrogel with controllable mechanical property enhancement and a preparation method thereof. The main components of the hydrogel are glycine-polyvinyl alcohol and methacrylated silk fibroin. The glycine-polyvinyl alcohol crystal film powder and methacrylated silk fibroin are pre-crosslinked by ultraviolet light to obtain a piezoelectric silk fibroin hydrogel, and the hydrogel is ultrasonically treated to obtain a piezoelectric silk fibroin hydrogel with enhanced mechanical strength. The glycine-polyvinyl alcohol piezoelectric thin film and powder used in the present invention have a higher piezoelectric coefficient compared with traditional organic piezoelectric materials; the methacrylated silk fibroin and glycine-polyvinyl alcohol components have good biocompatibility; it can be used for the treatment of cartilage defects, and the change of the mechanical strength of the hydrogel material can be regulated by the time of applying external ultrasonic intervention, so that the mechanical strength of the hydrogel can dynamically adapt to different cartilage tissue repair stages.

[0009] On the one hand, the present invention provides a hydrogel with dynamically enhanced mechanical properties triggered by the piezoelectric effect. The hydrogel includes a piezoelectric component and a colloidal component and is prepared by crosslinking; the piezoelectric component includes any one or more of barium titanate (BaTiO3), glycine-polyvinyl alcohol crystals, and the colloidal component includes any one or more of methacrylated silk fibroin (SilMA), methacrylated gelatin (GelMA), and acrylamide (AM).

[0010] The piezoelectric effect refers to the phenomenon that pressure, tension, or waves applied to certain crystal or ceramic materials cause changes in the electric potential of the materials, which in turn leads to the rearrangement of internal charges and uneven charge distribution, and then generates a potential difference and electric field strength at both ends of the materials. Materials with piezoelectric effects can compensate for the loss of the function of generating bioelectric signals in response to external stimuli caused by cartilage defects. In current research, the mechanical properties of piezoelectric materials that promote cartilage defect regeneration remain stable after material preparation and will not change further. The hydrogel material provided by the present invention can trigger the dynamic enhancement of the mechanical properties of the hydrogel through the piezoelectric effect, with controllable enhanced mechanical strength, and can adapt to different stages of cartilage repair.

[0011] Further, the piezoelectric component includes glycine - polyvinyl alcohol crystal, and the colloid component includes methacrylated silk fibroin.

[0012] Currently, most of the piezoelectric materials used in the prior art have low piezoelectric coefficients and high costs. For example, the polyvinylidene fluoride material has a relatively high cost, may be affected by strong oxidants under extreme conditions, and has great processing difficulty. The zinc oxide material has a low piezoelectric coefficient due to its inherent potential shielding effect, which affects its actual application effect as a piezoelectric material. The glycine - polyvinyl alcohol piezoelectric thin film and powder used in the piezoelectric component of the hydrogel in the present invention have a higher piezoelectric coefficient compared with traditional organic piezoelectric materials, providing a higher piezoelectric voltage to initiate the generation of free radicals inside the molecules and carry out chain reactions, and its preparation method and conditions are mild, and the raw materials are easily available.

[0013] Meanwhile, the methacrylated silk fibroin and glycine - polyvinyl alcohol components used in the colloid component of the hydrogel in the present invention have good biocompatibility, have no obvious adverse effects on the growth and proliferation of in - vivo cells, and are beneficial to the migration and spreading of cells during tissue repair, solving the problems such as insufficient upper limit of the material mechanical strength and inability to perform in - situ mechanical enhancement in animals caused by the colloid materials such as polyvinyl alcohol and chitosan used in the prior art.

[0014] Further, the mass ratio of the methacrylated silk fibroin to glycine - polyvinyl alcohol is (10 - 20):1.

[0015] On the other hand, the present invention provides a preparation method of a hydrogel with dynamically enhanced mechanical properties induced by piezoelectric effect, which is characterized by including the following steps:

[0016] (1) Dissolve the colloid component in a solvent to prepare a hydrogel pre - solution; the colloid component includes any one or more of methacrylated silk fibroin (SilMA), methacrylated gelatin (GelMA), and acrylamide (AM).

[0017] (2) Add the piezoelectric component to the hydrogel pre - solution and mix evenly to prepare a hydrogel solution; the piezoelectric component includes any one or more of barium titanate (BaTiO3) and glycine - polyvinyl alcohol crystal;

[0018] (3) Cross - link the hydrogel solution to obtain it.

[0019] Further, the colloid component in the step (1) includes methacrylated silk fibroin; the piezoelectric component in the step (2) includes glycine - polyvinyl alcohol powder.

[0020] In some ways, the pre-solution in step (1) is obtained by dissolving methacrylated silk fibroin (SilMA) in PBS solution to obtain a SilMA solution; then, PEG-thiol (PEG-SH) as a radical initiator is dissolved in the photo-crosslinking agent lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) under light-shielding conditions to obtain a PEG-SH-LAP solution; finally, the above PEG-SH-LAP solution is added to the SilMA solution and mixed evenly to obtain a SilMA solution containing a photo-crosslinking agent and a radical initiator, that is, the pre-solution.

[0021] Preferably, the final concentration of PEG-thiol in the SilMA solution is 2% (m / v), and the final concentration of LAP in the SilMA solution is 0.2% (m / v).

[0022] In some ways, the piezoelectric component glycine-polyvinyl alcohol crystal powder in step (2) is formed by crushing a glycine (γ-Gly) thin film assisted by polyvinyl alcohol (PVA) for γ-crystallization through a ball mill to form PVA-γ-Gly powder, and then the pre-solution in step (1) is added to the PVA-γ-Gly powder and mixed evenly quickly.

[0023] Preferably, during the preparation of the glycine thin film assisted by polyvinyl alcohol for γ-crystallization, the concentrations of glycine and polyvinyl alcohol before mixing are both 10% (m / v). After mixing evenly, the homogeneous glycine-polyvinyl alcohol solution is spread in a petri dish to form a liquid film and then dried and crystallized in an oven at 65 °C for no less than 35 min. The glycine-polyvinyl alcohol crystal film needs to be treated with liquid nitrogen for 10 min before being crushed by a ball mill. The frequency of the ball mill is set to 50 Hz, and the crushing time is set to 1 min.

[0024] Furthermore, the final concentration of methacrylated silk fibroin in the hydrogel solution is 10% - 30% (m / v), and the final concentration of glycine-polyvinyl alcohol powder is 1% - 3% (m / v).

[0025] Preferably, the final concentration of methacrylated silk fibroin in the hydrogel solution is 30% (m / v), and the final concentration of glycine-polyvinyl alcohol powder is 2% (m / v).

[0026] In some ways, the hydrogel solution in step (3) is pre-crosslinked by ultraviolet light for 30 s to obtain a piezoelectric silk fibroin hydrogel with relatively low initial mechanical strength; the piezoelectric silk fibroin hydrogel with low mechanical strength is placed on the probe of an ultrasonic therapy instrument for ultrasonic treatment to obtain a piezoelectric silk fibroin hydrogel with enhanced mechanical strength.

[0027] On the other hand, the present invention provides a method for using a piezoelectric effect-induced hydrogel with dynamically enhanced mechanical properties. By subjecting the hydrogel to ultrasonic waves, its mechanical properties are enhanced, and the hydrogel is as described above.

[0028] In some ways, the generation of the piezoelectric effect inside the hydrogel and the enhancement of the mechanical strength of the hydrogel by the piezoelectric effect can be achieved by ultrasonic loading through various ultrasonic wave generating devices. The portability of the ultrasonic device is beneficial for the development of treatment. The high penetrability of high-frequency ultrasonic waves is beneficial for loading the hydrogel scaffold on the defect layer of cartilage tissue. At the same time, the damage and pain in the animal body caused by ultrasonic loading are relatively low. The enhancement of the mechanical properties of the hydrogel induced by the ultrasonic device for physical therapy has a change range of kPa - 10 kPa. The lower limit of the change in mechanical strength in the kPa order is beneficial for avoiding the occurrence of inflammatory reactions in the initial stage of tissue repair, and the upper limit of the change in mechanical strength of 10 kPa order is beneficial for promoting the regeneration of osteochondral tissue. Using a high-frequency focused ultrasonic device for non-physical therapy may enable the mechanical strength of the material to reach a higher level.

[0029] Furthermore, the frequency of the ultrasonic waves for physical therapy is 1 - 5 MHz, and the sound intensity is 1 - 3 W / cm 2 , and the time of ultrasonic treatment is not less than 2 h. The frequency of the high-frequency focused ultrasonic waves for non-physical therapy is generally 2.5 - 5 MHz, and the sound intensity can reach 100 W / cm 2 or above.

[0030] Preferably, the frequency of the ultrasonic waves for physical therapy is 1 MHz, and the sound intensity is 2.5 W / cm 2 , and the time of ultrasonic treatment is not less than 2 h.

[0031] In some ways, during the process of enhancing the mechanical strength of the hydrogel by the piezoelectric effect, the change in the mechanical strength of the hydrogel material can be regulated by the time, sound intensity, and frequency of applying external ultrasonic intervention. The storage modulus of the hydrogel material is in a process of dynamically controllable increase during ultrasonic loading. By adjusting the time of ultrasonic loading, the specific mechanical strength that the hydrogel finally reaches can be controlled.

[0032] In some embodiments, other methods such as magnetic fields can be applied to enhance the hydrogel by the piezoelectric effect, and the method of inducing mechanical enhancement can be selected according to the convenience of specific users.

[0033] On the other hand, the present invention provides a use of a mixture for preparing a hydrogel that can be ultrasonically induced to have a piezoelectric effect, thereby dynamically enhancing the mechanical properties. The mixture is characterized in that it is prepared by crosslinking a piezoelectric component and a colloid component; the piezoelectric component includes any one or more of barium titanate and glycine-polyvinyl alcohol crystals, and the colloid component includes any one or more of methacrylated silk fibroin, methacrylated gelatin, and acrylamide.

[0034] Further, the piezoelectric component includes glycine-polyvinyl alcohol crystals, and the colloid component includes methacrylated silk fibroin.

[0035] On the other hand, the present invention provides a use of a mixture for preparing a hydrogel with dynamically enhanced mechanical properties for improving the treatment effect of cartilage defects. The mixture is prepared by crosslinking a piezoelectric component and a colloid component; the piezoelectric component includes any one or more of barium titanate and glycine-polyvinyl alcohol crystals, and the colloid component includes any one or more of methacrylated silk fibroin, methacrylated gelatin, and acrylamide.

[0036] Further, the hydrogel is ultrasonically induced to have a piezoelectric effect, thereby dynamically enhancing the mechanical properties.

[0037] It can be understood that, in addition to the ultrasound used in the preferred embodiments of the present invention, other methods that can induce the mechanical enhancement effect of the hydrogel using the principle of piezoelectric effect are applicable to the present invention.

[0038] On the other hand, the present invention provides a use of glycine-polyvinyl alcohol crystals for preparing a hydrogel that can be ultrasonically induced to have a piezoelectric effect, thereby dynamically enhancing the mechanical properties.

[0039] Glycine-polyvinyl alcohol piezoelectric films and powders have higher piezoelectric coefficients compared to traditional organic piezoelectric materials, providing higher piezoelectric voltages to initiate free radical generation and chain reactions inside molecules.

[0040] On the other hand, the present invention provides a use of methacrylated silk fibroin for preparing a hydrogel that can be ultrasonically induced to have a piezoelectric effect, thereby dynamically enhancing the mechanical properties.

[0041] Methacrylated silk fibroin has good biocompatibility, which is beneficial for the migration and spreading of cells during tissue repair.

[0042] Further, the hydrogel with dynamically enhanced mechanical properties is beneficial for the regeneration and repair of the osteochondral tissue described in the present invention.

[0043] In some embodiments, the hydrogel with dynamically enhanced mechanical properties is also applicable to tissues with dynamic mechanical requirements during the regeneration and repair processes of bone tissue and the like through formulation adjustment and selection of methods for inducing mechanical enhancement.

[0044] Compared with the prior art, the present invention has the following advantages and effects:

[0045] 1. The glycine-polyvinyl alcohol piezoelectric thin film and powder have a higher piezoelectric coefficient compared with traditional organic piezoelectric materials, providing a higher piezoelectric voltage to initiate the generation of free radicals inside molecules and carry out chain reactions, and their preparation methods and conditions are mild, and the raw materials are easily available;

[0046] 2. Methacrylated silk fibroin and glycine-polyvinyl alcohol components have good biocompatibility, have no obvious adverse effects on the growth and proliferation of cells in vivo, and are beneficial to the migration and spreading of cells during tissue repair;

[0047] 3. The dynamic enhancement range of the mechanical properties of the piezoelectric hydrogel induced by a physical therapy ultrasound device is from kPa to 10 kPa. The lower limit of the change in mechanical strength in the kPa order of magnitude is beneficial to avoiding the occurrence of inflammatory reactions in the initial stage of tissue repair, and the upper limit of the change in mechanical strength in the 10 kPa order of magnitude is beneficial to promoting the regeneration of osteochondral tissue; when the ultrasound device has a higher ultrasound frequency and ultrasound intensity, the mechanical properties may reach a higher level.

[0048] 4. During the process of enhancing the mechanical strength of the hydrogel by the piezoelectric effect, the change in the mechanical strength of the hydrogel material can be regulated by the time of applying external ultrasound intervention, and the storage modulus of the hydrogel material is in a process of dynamically controllable increase during the ultrasound loading process. By adjusting the time of ultrasound loading, the specific mechanical strength that the hydrogel finally reaches can be controlled;

[0049] 5. The generation of the piezoelectric effect inside the hydrogel and the method for enhancing the mechanical strength of the hydrogel are ultrasonic wave loading by a handheld ultrasound device. The portability of the ultrasound device is beneficial to the development of treatment, the high penetrability of high-frequency ultrasonic waves is beneficial to loading the hydrogel scaffold on the cartilage tissue defect layer, and at the same time, the damage and pain caused by ultrasonic wave loading in the animal body are relatively low. Description of the Drawings

[0050] Figure 1 It is the preparation flow chart of the silk fibroin hydrogel provided by the present invention in Example 1;

[0051] Figure 2 It is the piezoelectric coefficient of the PVA-glycine piezoelectric crystal film and the cross-sectional structure of the piezoelectric hydrogel in Example 6;

[0052] Figure 3Transient voltage generated by the piezoelectric hydrogel in Example 6 and piezoelectric coefficient D of the material at different γ-Gly concentrations 33 ;

[0053] Figure 4 Macroscopic mechanical characteristics of the piezoelectric hydrogel in Example 6 under different conditions;

[0054] Figure 5 Effect of different pre-crosslinking times on the mechanical strength of the piezoelectric hydrogel in Example 7;

[0055] Figure 6 Specific mechanical strength of the piezoelectric hydrogel in Example 8 under different ultrasonic times;

[0056] Figure 7 Mechanical strength of silk fibroin hydrogels with different concentration gradients under 30 s of photocrosslinking in Example 9;

[0057] Figure 8 Mechanical strength of the 10% silk fibroin piezoelectric hydrogel in Example 10 under different ultrasonic times;

[0058] Figure 9 Mechanical strength of the 20% silk fibroin piezoelectric hydrogel in Example 10 under different ultrasonic times;

[0059] Figure 10 Mechanical strength of hydrogels with different PVA-γ-Gly concentrations in Example 11 under different ultrasonic times;

[0060] Figure 11 Mechanical strength of the GelMA piezoelectric hydrogel in Example 12 under different ultrasonic times;

[0061] Figure 12 Mechanical strength of the BaTiO3-silk fibroin hydrogel in Example 13 under different ultrasonic times;

[0062] Figure 13 Biocompatibility of the piezoelectric hydrogel and its effect on cell morphology in Example 15;

[0063] Figure 14 Effect of the mechanically controllable enhanced piezoelectric hydrogel on chondrogenic gene expression of cells in Example 16;

[0064] Figure 15 Effect of the mechanically controllable enhanced piezoelectric hydrogel on the repair of rabbit osteochondral tissue in Example 17. Detailed implementation manners

[0065] The present invention will be further described in detail below in conjunction with embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way. Unless otherwise specified, the raw materials used in the embodiments are commercially purchased.

[0066] Example 1. Preparation of the hydrogel provided by the present invention (30% silk fibroin, 2% PVA-γ-Gly)

[0067] (1) Degum and air-dry mulberry silk. Weigh the degummed silk and methacrylamide according to a mass ratio of (1:4). Dissolve the degummed silk in the methacrylamide solution by stirring to obtain a methacryloylated silk fibroin solution (SilMA). Freeze-dry the solution to form SilMA powder.

[0068] (2) Weigh the SilMA powder and PBS solution according to a mass ratio of (3:10) to prepare a SilMA mixed solution. Then, weigh polyethylene glycol-thiol (PEG-SH) as a radical initiator and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photo-crosslinking agent according to a mass ratio of (1:1). Dissolve them under light-shielded conditions to obtain a PEG-SH-LAP solution. Then, mix the SilMA mixed solution and the PEG-SH-LAP solution evenly according to a volume ratio of (20:1) to obtain a pre-solution.

[0069] (3) Weigh glycine (Gly) powder and double-distilled water according to a mass-to-volume ratio of (1:10) and dissolve it. Weigh polyvinyl alcohol (PVA) and double-distilled water according to a mass-to-volume ratio of (1:10) and dissolve it. Then, mix the glycine solution and the PVA solution according to a volume ratio of (1:1) to obtain a solution.

[0070] (4) Spread the mixed solution obtained in step (3) in a petri dish to form a liquid film, and then dry it in an oven at 65 °C for no less than 35 min to obtain a crystal film. Treat the crystal film with liquid nitrogen for 10 min and then crush it with a ball mill to obtain PVA-γ-Gly powder. The frequency of the ball mill is 50 Hz, and the crushing time is 1 min.

[0071] (5) Weigh the pre-solution obtained in step (2) and the PVA-γ-Gly powder according to a concentration ratio of 30% (m / v): 2% (m / v), quickly mix them evenly, and perform ultraviolet pre-crosslinking for 30 s to obtain a piezoelectric silk fibroin hydrogel with a relatively low initial mechanical strength.

[0072] (6) Place the hydrogel obtained in step (5) on the probe of an ultrasonic therapeutic instrument for ultrasonic treatment. The ultrasonic frequency is 1 MHz, and the sound intensity is 2.5 W / cm 2 , and the required time is not less than 2 h to obtain a piezoelectric silk fibroin hydrogel with enhanced mechanical strength. The preparation process is asFigure 1 as shown

[0073] Example 2: Preparation of 20% silk fibroin hydrogel

[0074] According to the preparation steps in Example 1, in step (5), the pre-solution and PVA-γ-Gly powder are weighed and mixed at a concentration ratio of 20% (m / v): 2% (m / v), and ultrasonic treatment is carried out to obtain 20% silk fibroin hydrogel.

[0075] Example 3: Preparation of 10% silk fibroin hydrogel

[0076] According to the preparation steps in Example 1, in step (5), the pre-solution and PVA-γ-Gly powder are weighed and mixed at a concentration ratio of 10% (m / v): 2% (m / v), and ultrasonic treatment is carried out to obtain 10% silk fibroin hydrogel.

[0077] Example 4: Preparation of 1% PVA-γ-Gly hydrogel

[0078] According to the preparation steps in Example 1, in step (5), the pre-solution and PVA-γ-Gly powder are weighed and mixed at a concentration ratio of 30% (m / v): 1% (m / v), and ultrasonic treatment is carried out to obtain 1% PVA-γ-Gly hydrogel.

[0079] Example 5: Preparation of 3% PVA-γ-Gly hydrogel

[0080] According to the preparation steps in Example 1, in step (5), the pre-solution and PVA-γ-Gly powder are weighed and mixed at a concentration ratio of 30% (m / v): 3% (m / v), and ultrasonic treatment is carried out to obtain 3% PVA-γ-Gly hydrogel.

[0081] Example 6: Performance test of the hydrogel provided by the present invention

[0082] The hydrogel prepared in Example 1 is subjected to physicochemical property analysis by energy dispersive spectroscopy, scanning electron microscopy observation and compression elastic modulus testing machine, macroscopic electrical property analysis by an electrochemical workstation, and macroscopic mechanical property analysis by a rheometer testing method.

[0083] (I) Physicochemical properties

[0084] It can be seen from Figure 2 that the synthesized glycine-polyvinyl alcohol is γ-crystalline glycine with strong piezoelectric properties, and the crosslinking degree of the silk fibroin hydrogel with piezoelectric effect increases after ultrasonic loading.

[0085] (II) Macroscopic electrical properties

[0086] It can be seen from Figure 3It can be seen that the piezoelectric performance of the hydrogel doped with γ-crystalline glycine is significantly improved, and the transient voltage generated under ultrasonic conditions reaches 0.16 V, indicating that the piezoelectric material γ-Gly can significantly improve the piezoelectric performance of the hydrogel. After further testing the piezoelectric coefficient D of the material under different γ-Gly concentration conditions 33 as Figure 3 shown, we found that the piezoelectric coefficient of γ-Gly at a concentration of 0.5% is only 0.62 pm / V. At glycine concentrations of 1% and 2%, the piezoelectric coefficients of the material reach 1.68 pm / V and 3.52 pm / V respectively, and reach 3.84 pm / V at a higher γ-Gly concentration of 4%. This further verifies that γ-glycine can significantly improve the piezoelectric performance of the hydrogel. Considering that the increase in the piezoelectric coefficient is slow when the concentration of γ-Gly is above 2%, γ-crystalline glycine assisted by 2% concentration of polyvinyl alcohol, namely glycine-polyvinyl alcohol, is finally selected for subsequent experiments.

[0087] (III) Macroscopic mechanical properties

[0088] It is Figure 4 known that the compressive modulus of the piezoelectric silk fibroin hydrogel increases with the prolongation of ultrasonic time. After ultrasonic treatment, the anti-compressive performance is stable, the swelling rate decreases, and the macroscopic mechanical strength shows an upward trend. The range of mechanical property changes is from kPa to 10 kPa. The lower limit of the mechanical strength change in the kPa order is beneficial to avoiding the occurrence of inflammatory reactions in the initial stage of tissue repair, and the upper limit of the mechanical strength change in the 10 kPa order is beneficial to promoting the regeneration of osteochondral tissue.

[0089] Example 7. Changes in the mechanical strength of the hydrogel at different pre-crosslinking times

[0090] According to the hydrogel preparation steps provided in Example 1, where the pre-crosslinking time is set to 15 s, 30 s, 45 s, and 60 s, hydrogels under different pre-crosslinking times are prepared. The mechanical properties of the hydrogels under different pre-crosslinking conditions are analyzed, and the test results are shown in Table 1 and Figure 5 , where the storage modulus G' and the loss modulus G" represent the stiffness and state of the material respectively.

[0091] Table 1. Influence of different pre-crosslinking times on the mechanical strength of the hydrogel

[0092]

[0093] According to the results, it can be seen that the storage modulus increases with the prolongation of the pre-crosslinking time. In order to keep the initial mechanical strength of the hydrogel at a relatively low level after pre-crosslinking, which is beneficial to adapting to the inflammatory reaction period in the initial stage of cartilage tissue defect, the pre-crosslinking time used in the present invention is 30 s.

[0094] Example 8. Changes in the mechanical properties of the hydrogel at different ultrasonic times

[0095] The hydrogels prepared in Example 1 were subjected to ultrasonic treatment for different lengths of time, wherein the controls were the lack of piezoelectric material and no ultrasonic loading, no ultrasonic loading, and the lack of piezoelectric material and only ultrasonic loading (2 h). The mechanical properties of the hydrogels under different ultrasonic conditions were then analyzed. The test results are shown in Tables 2 and Figure 6 .

[0096] Table 2 Effects of different ultrasound times on the mechanical properties of hydrogels

[0097]

[0098] According to the above results, there is no significant change in the mechanical properties of hydrogels without ultrasonic loading or with ultrasonic loading only in the absence of piezoelectric materials; the mechanical properties of silk hydrogels significantly improve with the extension of ultrasonic loading time, and finally reach a storage modulus of more than 10kPa.

[0099] Example 9 Mechanical strength of hydrogels with different SilMA concentrations at a pre-crosslinking time of 30 s

[0100] According to the hydrogel preparation steps provided in Examples 2 and 3, hydrogels with different SilMA concentrations were prepared, wherein the PVA-γ-Gly concentration was 2%. The mechanical properties were then tested, and the test results are shown in Figure 7 .

[0101] By comparing Examples 7 and 9, it can be seen that when the silk fibroin concentration of the hydrogel is low (10%, 20%), its mechanical properties are less affected by the photocrosslinking time, and the mechanical properties are less than the kPa level under short-time pre-crosslinking; when the silk fibroin concentration of the hydrogel is 30%, the storage modulus can reach 2 kPa when the crosslinking time reaches 30 s, so the preferred silk fibroin concentration of the present invention is 30%.

[0102] Example 10 Mechanical strength of hydrogels with different SilMA concentrations at different ultrasound times

[0103] According to the method of Example 2 and Example 3, hydrogels with different SilMA concentrations were prepared, wherein the ultrasound time was set to 0 h, 1 h, and 2 h, and then mechanical properties were tested respectively. The test results are shown in Table 3. Figure 8 and Figure 9 .

[0104] Table 3 Effect of different ultrasound times on the mechanical strength of hydrogels with different SilMA concentrations

[0105]

[0106] Comparing Comparative Example 8 and Example 10, it can be seen that when the silk fibroin concentration of the hydrogel is relatively low (10%), the effect of improving the mechanical strength under ultrasonic influence is not obvious; when the silk fibroin concentration of the hydrogel is 20%, the mechanical strength is improved under ultrasonic influence, but the value that the mechanical strength can reach is limited (1500 Pa); when the silk fibroin concentration of the hydrogel is 30%, a storage modulus of more than 10 kPa can be achieved. Therefore, the present invention preferably uses a combination of silk fibroin with a concentration of 30% and ultrasonic treatment for 2 h to prepare the hydrogel.

[0107] Mechanical Strength of Hydrogels with Different Concentrations of PVA-γ-Gly in Example 11 at Different Ultrasonic Treatment Times

[0108] Prepare hydrogels with different concentrations of PVA-γ-Gly according to the methods of Examples 4 and 5, where the ultrasonic treatment time is set to 0 h, 1 h, and 2 h, and then mechanical property tests are carried out respectively. The test results are shown in Table 4 and Figure 10 .

[0109] Table 4 Effect of Different Ultrasonic Treatment Times on the Mechanical Strength of Hydrogels with Different Concentrations of PVA-γ-Gly

[0110]

[0111] Comparing Comparative Example 8 and Example 11, it can be seen that when the concentration of glycine-polyvinyl alcohol in the hydrogel is 1%, with the extension of the ultrasonic loading time, the mechanical strength of the hydrogel gradually increases, and the storage modulus finally reaches 8 kPa; when the concentration of glycine-polyvinyl alcohol in the hydrogel is 2%, the mechanical properties of the hydrogel are significantly improved with the extension of the ultrasonic loading time, and the storage modulus finally reaches more than 10 kPa; when the concentration of glycine-polyvinyl alcohol in the hydrogel is 3%, the increase in the storage modulus compared with the hydrogel with a glycine-polyvinyl alcohol concentration of 2% under the same ultrasonic conditions is not obvious. Therefore, the present invention preferably uses a glycine-polyvinyl alcohol concentration of 2% and an ultrasonic treatment time of 2 h.

[0112] Example 12, Influence of Different Colloidal Constituent Components on the Preparation of Piezoelectric Hydrogels with Dynamically Enhanced Mechanical Properties

[0113] According to the hydrogel preparation steps provided in Example 1, after replacing the colloidal constituent components with acrylamide gel (AM) and methacrylated gelatin (GelMA), the physical and chemical properties and mechanical properties of the hydrogel were tested and analyzed. The test results showed that the mechanical strength of the 20% (m / v) piezoelectric-acrylamide hydrogel increased from 2 kPa to 4 kPa as the ultrasonic time (0 - 2 h) extended. For the piezoelectric-methacrylated gelatin (GelMA) hydrogel, considering its dissolution properties, the mechanical properties were tested with a 10% (m / v) concentration of GelMA. The results showed that the mechanical properties of the piezoelectric-methacrylated gelatin hydrogel increased from 2 kPa to 3 kPa as the ultrasonic time extended, and the mechanical enhancement effect was obvious at 1 h (as Figure 11 shown).

[0114] Comparing with the results of Example 6, after replacing the methacrylated silk fibroin in the hydrogel colloidal constituent components with acrylamide gel (AM) and methacrylated gelatin (GelMA), the cross-linking degree and mechanical strength of the hydrogel both increased, but the storage modulus of the two hydrogels could only reach 3 - 4 kPa, and the effect was not as good as that of the hydrogel provided by the present invention. The hydrogel provided by the present invention can finally reach a mechanical strength of more than 10 kPa after ultrasonic loading, which is more conducive to the regeneration of cartilage tissue. In addition, the colloidal constituent components provided by the present invention have a better effect in promoting the repair of osteochondral tissue. It may be that the principle of the enhanced mechanical properties by the piezoelectric effect is to initiate free radicals to attack the carbon-carbon double bonds of the hydrogel molecules. Therefore, the hydrogel monomers that can be enhanced by the piezoelectric effect can be other biocompatible molecules modified with carbon-carbon double bonds.

[0115] Example 13. Influence of Different Piezoelectric Components on the Preparation of Piezoelectric Hydrogels with Dynamically Enhanced Mechanical Properties

[0116] According to the hydrogel preparation steps provided in Example 1, after replacing the piezoelectric constituent components with barium titanate (BaTiO3), the physical and chemical properties and mechanical properties of the hydrogel were tested and analyzed, and the test results are shown in Figure 12 .

[0117] Comparing Comparative Example 6 and 13, it can be seen that when the piezoelectric component is barium titanate, the crosslinking degree and mechanical strength of the hydrogel are also improved under ultrasonic loading. Since the vector sum of the electric dipole moments of amino acid crystals tends to cancel each other out under natural crystallization conditions, while a strong polarization intensity can be generated under specific crystallization conditions, glycine with the γ crystal form in the composition can be replaced by other amino acid crystals or piezoelectric materials with piezoelectric effect crystal forms. However, the mechanical strength of the hydrogel prepared from barium titanate can only reach a storage modulus of 6 kPa, which cannot meet the 10 kPa storage modulus beneficial for promoting the regeneration of osteochondral tissue, and the effect is not as good as the hydrogel provided by the present invention. Therefore, the piezoelectric component provided by the present invention has a better effect in promoting the repair of osteochondral tissue.

[0118] Example 14. Influence of Different Loading Modes Inducing Piezoelectric Effect on the Preparation of Piezoelectric Hydrogels with Dynamically Enhanced Mechanical Properties

[0119] According to the hydrogel preparation steps provided in Example 1, where the ultrasonic loading mode is replaced by external mechanical loading, and then the physicochemical properties and mechanical properties of the hydrogel are tested and analyzed. When a stress of 50 N is applied for loading, the mechanical properties of the hydrogel gradually increase from 1 kPa to 4 kPa. Comparing Comparative Example 6 and 14, it can be seen that the crosslinking degree and mechanical strength of the hydrogel can also be improved by using the external mechanical loading method, because the loading mode that induces the change of the dipole moment of the piezoelectric material molecules inside the hydrogel molecules can also trigger the free radical chain reaction inside the hydrogel, thereby increasing the mechanical strength of the hydrogel. However, using the external mechanical loading method can only make the storage modulus of the hydrogel reach 4 kPa, which cannot meet the 10 kPa storage modulus beneficial for promoting the regeneration of osteochondral tissue. Therefore, the loading mode for inducing piezoelectric effect provided by the present invention has a better effect in promoting the repair of osteochondral tissue and in the actual medical process.

[0120] Example 15. Biocompatibility Study of Hydrogels

[0121] The hydrogel prepared in Example 1 was simultaneously treated by adding piezoelectric materials (SFG) and adding piezoelectric materials with ultrasonic treatment (SFGP), and the control was the treatment of culturing in a material-free well plate. After 1 D, 3 D, and 5 D of treatment, the absorbance of the hydrogel at a wavelength of 450 nm was measured using an enzyme-labeled instrument by the CCK-8 kit detection method, and the change in the cell adhesion state on hydrogel materials with different hardnesses was analyzed by scanning electron microscopy observation.

[0122] As Figure 13As shown, compared with the control group, there was no significant difference in the biocompatibility results between the SFG group and the SFGP group, and the absorbance increased significantly at 3D, indicating that the piezoelectric material has no significant harm to cell proliferation. It can be seen that the hydrogel material provided by the present invention has good biocompatibility and has no obvious effect on cell growth and proliferation. At the same time, the mechanically enhanced piezoelectric hydrogel is beneficial to cell spreading and adhesion.

[0123] Example 16. Upregulation of chondrogenic gene expression by enhanced mechanical properties of hydrogel

[0124] To further illustrate the specific effect of the hydrogel in cartilage repair applications, qPCR technology was used to measure the gene expression of cells cultured on the surface of the hydrogel without ultrasonic loading and the hydrogel after ultrasonic loading under chondrogenic induction experimental conditions at 7 days and 14 days, respectively. The control group was cells cultured in a material-free well plate. The measurement results are shown in Figure 14 .

[0125] According to the results, under the experimental conditions at 7 days, the expression levels of the chondrogenic genes COL-2A1 and ACAN in the cells cultured in the ultrasonic loading treatment group were significantly upregulated, and there was no obvious difference in the expression of the SOX9 gene. Under the experimental conditions at 14 days, the expression levels of COL-2A1, SOX9, and ACAN in the cells cultured in the ultrasonic loading treatment group were all significantly increased, indicating that the hydrogel material after ultrasonic loading treatment can promote the expression of cartilage genes and contribute to the repair of osteochondral tissue.

[0126] Example 17. Repair of rabbit cartilage tissue by enhanced mechanical properties of hydrogel

[0127] To illustrate the specific effect of the mechanically enhanced hydrogel in in vivo chondral repair applications, a rabbit femoral cartilage defect model was used to verify the repair effects of rabbit cartilage at 4 weeks and 8 weeks of repair time points under the conditions of whether there was ultrasonic-induced mechanical enhancement of the material. The control group only underwent cartilage defect modeling; the experimental groups were piezoelectric hydrogel without ultrasonic enhancement treatment and piezoelectric hydrogel with ultrasonic enhancement treatment, respectively.

[0128] As Figure 15 shown, at 4 weeks and 8 weeks time points, the repair effect of the ultrasonic-loaded mechanically enhanced hydrogel on rabbit femoral cartilage defects was significantly better than that of the experimental group without ultrasonic enhancement and the defect control group, indicating that ultrasonic-induced mechanical enhancement of the piezoelectric hydrogel is beneficial to the regeneration of cartilage tissue.

[0129] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A hydrogel with dynamically enhanced mechanical properties induced by piezoelectric effect, characterized in that: The invention comprises glycine-polyvinyl alcohol crystals and methacrylylated silk fibroin, and a hydrogel is prepared by cross-linking.

2. The hydrogel according to claim 1, characterized in that The mass ratio of the methacrylylated silk fibroin to glycine-polyvinyl alcohol is (10-20):

1.

3. A method for preparing a hydrogel with dynamically enhanced mechanical properties induced by piezoelectric effect, characterized in that: The following steps are involved: (1) dissolving methacrylylated silk fibroin in a solvent to prepare a hydrogel pre-solution; (2) adding glycine-polyvinyl alcohol crystals to the hydrogel pre-solution and mixing to prepare a hydrogel solution; (3) The hydrogel solution is prepared by cross-linking.

4. The preparation method according to claim 3, characterized in that In the step (3), the concentration of the methacrylylated silk in the hydrogel solution is 10% to 30% m / v, and the concentration of the glycine-polyvinyl alcohol powder is 1% to 3% m / v.

5. A method for using a hydrogel with dynamic mechanical property enhancement induced by piezoelectric effect, characterized in that: By subjecting the hydrogel according to any one of claims 1 to 2 to ultrasound, its mechanical properties are enhanced.

6. The method of use according to claim 5, characterized in that: The frequency of the ultrasound is 1-3 MHz, and the sound intensity is 1-2.5 W / cm 2 The ultrasonic treatment time is not less than 2 h.

7. Use of a mixture for preparing a hydrogel whose mechanical properties are dynamically enhanced by piezoelectric effect induced by ultrasound, characterized in that: The mixture includes glycine-polyvinyl alcohol crystals and methacrylylated silk fibroin.

8. Use of a mixture for preparing a hydrogel with dynamic mechanical properties for improving the treatment effect of cartilage defects, characterized in that: The mixture includes glycine-polyvinyl alcohol crystals and methacrylylated silk fibroin.

9. The use according to claim 8, characterized in that The hydrogel dynamically enhances its mechanical properties by inducing piezoelectric effect through ultrasound.

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