Preparation method of injectable piezoelectric hydrogel for resisting fibrosis, piezoelectric hydrogel and application
The piezoelectric hydrogel formed by cross-linking amino-containing degradable biomaterials with hyaluronic acid, combined with micron piezoelectric materials, solves the problems of existing materials being difficult to adapt to damaged tissue structures and difficult to administer, realizes the formation of a protective layer around the damaged tissue and endogenous repair, and has a good anti-fibrosis effect.
Patent Information
- Application Number
- CN202510960143.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-09-12
AI Technical Summary
Existing anti-fibrosis materials are difficult to adapt to the complex structure of damaged tissue sites and are difficult to administer, resulting in poor anti-fibrosis effects. Existing gel injection materials have relatively single effects and are difficult to achieve good anti-fibrosis effects.
Amino-containing biodegradable biomaterials are reacted with hyaluronic acid through Schiff base reaction to form a cross-linked structure, combined with micron piezoelectric materials, and hydrogen bonding forces are used to construct piezoelectric hydrogels, which are injectable and retentive, and provide endogenous repair through ultrasonic stimulation.
It forms a protective layer around the damaged tissue, provides a good physical barrier effect, and synergistically achieves anti-fibrosis. It is suitable for anti-fibrosis after injuries to multiple parts of the body, such as tendon rupture, endometrial injury, and peritoneal injury. It has good retention and injectability, and promotes endogenous repair.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method of an injectable piezoelectric hydrogel for anti-fibrosis, the piezoelectric hydrogel and applications. Background Art
[0002] Fibrosis is a repair response of the body to injury, but excessive fibrosis can cause serious damage to tissue structure, leading to various functional disorders. Existing treatments often lack sufficient specificity and are difficult to act accurately on the fibrosis process, which may lead to damage to normal tissues and systemic side effects. For example, many anti-fibrotic drugs have a short duration of efficacy and require frequent administration, which not only increases the treatment burden on patients, but may also cause side effects due to drug accumulation. At present, scaffolds and films based on macromolecular materials (such as polylactic acid, chitosan, cellulose, etc.) can be used as physical barriers to separate damaged tissue from mucosal tissue and avoid the formation of fibrosis. By adjusting the surface structure of biomaterials, constructing super-hydrophobic surfaces, hydrophilic materials (forming a hydration layer), zwitterionic materials, etc., the deposition of collagen matrix can be further reduced. However, these materials are usually prepared in the form of scaffolds or films, which are difficult to adapt to the complex anatomical structure of the damaged tissue site, and the scaffolds or films make drug administration difficult; injecting gel materials into the damaged tissue site by injection has the advantages of simple administration method, precise administration site, and can adapt to the complex structure of the damaged tissue site, but the existing gel injection materials have a relatively single effect on fibrosis caused by tissue muscle damage, and it is difficult to achieve a good anti-fibrosis effect. Summary of the Invention
[0003] In response to the shortcomings of the existing technology, the first purpose of the present invention is to provide a method for preparing injectable piezoelectric hydrogels for anti-fibrosis. This method can utilize piezoelectric materials, amino-containing degradable biomaterials, and aqueous dispersions of hyaluronic acid to construct piezoelectric hydrogels with piezoelectric properties, which can be injected and extruded and have good retention and anti-adhesion effects.
[0004] The second object of the present invention is to provide an injectable piezoelectric hydrogel for anti-fibrosis prepared by the above method, which has piezoelectric properties, can be injected and extruded, and has good retention and anti-adhesion effects.
[0005] The third object of the present invention is to provide the use of the above-mentioned piezoelectric hydrogel in the preparation of drugs or medical devices for preventing fibrosis after tissue damage, which shows a good effect in preventing fibrosis after tissue damage.
[0006] In order to achieve the above object, the technical solution provided by the present invention is:
[0007] According to the first aspect of the present invention, the present invention provides a method for preparing a piezoelectric hydrogel for preventing fibrosis after tissue damage, comprising the following steps:
[0008] Step (1): providing an aqueous dispersion comprising a micron piezoelectric material, an amino-containing degradable biomaterial, and hyaluronic acid;
[0009] Step (2): A cross-linking agent containing an aldehyde group or a ketone group is used to react the amino-containing biodegradable material in the aqueous dispersion with a Schiff base to obtain a cross-linking structure, and the micron piezoelectric material and hyaluronic acid are combined and distributed in the cross-linking structure by hydrogen bonding to form a piezoelectric hydrogel.
[0010] Furthermore, the amino-containing degradable biomaterial in step (1) is selected from at least one of amino-containing chitosan, amino-containing chitosan derivatives, amino-containing cellulose, and amino-containing cellulose derivatives;
[0011] Preferably, the amino-containing biodegradable material in step (1) is selected from at least one of carboxymethyl chitosan, quaternary ammonium chitosan, carboxyethyl chitosan, cellulose methacrylamide, and cellulose acrylamide;
[0012] Preferably, the mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion in step (1) is 0.1-2%;
[0013] Preferably, the mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion in step (1) is 0.1-1%.
[0014] Furthermore, the micron piezoelectric material in step (1) is selected from at least one of polylactic acid, polyhydroxybutyrate valerate, phenylalanine dipeptide, collagen polypeptide and silk fibroin;
[0015] Preferably, the length of the micron piezoelectric material is 10-100 μm;
[0016] Preferably, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 1-10 mg / mL;
[0017] Preferably, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 4-6 mg / mL.
[0018] Furthermore, the concentration of the hyaluronic acid in the aqueous dispersion in step (1) is 0.1-2%;
[0019] Preferably, in step (1), the mass ratio of the amino-containing degradable biomaterial to the hyaluronic acid is 0.1-10:1.
[0020] Furthermore, the cross-linking agent containing an aldehyde group or a ketone group in step (2) is a biocompatible and degradable cross-linking agent;
[0021] Preferably, the cross-linking agent containing an aldehyde group or a ketone group is at least one of genipin and citric acid.
[0022] Furthermore, the mass ratio of the amino-containing biodegradable material in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 10-1000:1;
[0023] Preferably, the mass ratio of the amino-containing degradable biomaterial in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 50-1000:1.
[0024] Furthermore, the Schiff base reaction in step (2) is carried out under heating and stirring;
[0025] Preferably, the heating temperature is 40-70° C., the stirring speed is 400-600 rpm, and the reaction time is 18-96 h.
[0026] According to a second aspect of the present invention, the present invention provides a piezoelectric hydrogel for preventing fibrosis after tissue damage, wherein the piezoelectric hydrogel is prepared by the above method.
[0027] According to the third aspect of the present invention, the present invention provides the use of the above-mentioned piezoelectric hydrogel in the preparation of a drug or medical device for preventing fibrosis after tissue damage;
[0028] Preferably, the tissue damage is selected from at least one of tendon rupture, endometrial injury and peritoneal injury.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] The preparation method of the above-mentioned piezoelectric hydrogel provided in the present application is to obtain a cross-linked structure by Schiff base reaction of the amino-containing degradable biomaterial using a cross-linking agent containing an aldehyde group or a ketone group, which plays the role of a scaffold and a physical barrier; the micron piezoelectric material relies on the hydrogen bond force between it and hyaluronic acid to achieve the combination of the micron piezoelectric material in the hydrogel and uniform distribution, wherein the micron piezoelectric material generates electrical stimulation to the tissue cells under ultrasound, thereby providing an endogenous repair effect on the damaged tissue. The hydrogel obtained after the amino-containing degradable biomaterial is mixed with hyaluronic acid has the property of shear thinning, which makes it more convenient to deliver the material by injection to form a protective layer around the damaged tissue. The piezoelectric hydrogel provided in the present application has a long retention effect in the damaged tissue by virtue of its cross-linked structure and composition, providing a better physical barrier effect, and ultimately achieving anti-fibrosis through good retention, injectability, proliferation and repair of endogenous cells.
[0031] The piezoelectric hydrogel exhibits excellent efficacy in preventing fibrosis following tissue injury. The injectable piezoelectric hydrogel of the present invention is suitable for treating fibrosis following various injuries, such as tendon rupture, endometrial injury, and peritoneal injury, and has broad application prospects. Cutting the piezoelectric fibers into micron-sized short fibers allows for injectable piezoelectric materials, facilitating scalable production at a low cost, and facilitating clinical application. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art can derive other drawings based on these drawings without inventive effort.
[0033] Figure 1 The hydrogel morphology diagrams with different cross-linker ratios;
[0034] Figure 2 The morphology of hydrogels with different carboxymethyl chitosan contents;
[0035] Figure 3 The injectability of hydrogels with different cross-linker ratios;
[0036] Figure 4 The rheological test results of hydrogel;
[0037] Figure 5 is the hydrogel compatibility test result;
[0038] Figure 6 is the piezoelectric output of the hydrogel under ultrasound driving;
[0039] Figure 7 The results of hydrogel anti-fibroblast adhesion;
[0040] Figure 8 The results of hydrogel anti-macrophage adhesion;
[0041] Figure 9 The hydrogel promoted the polarization of M2 macrophages;
[0042] Figure 10 Masson staining results of hydrogel used for tendon adhesion (fibrosis);
[0043] Figure 11 The results of immunofluorescence staining of hydrogel tendon stem cell proliferation are shown;
[0044] Figure 12 Gait and Achilles tendon function index of rats after 7 days of hydrogel treatment;
[0045] Figure 13 is the retention of the hydrogel in the uterus;
[0046] Figure 14 This is the morphology of the uterine cavity after hydrogel treatment;
[0047] Figure 15 The results show the effect of the culture supernatant of M1 macrophages after hydrogel intervention on the differentiation of endometrial stromal cells into myofibroblasts;
[0048] Figure 16 The results show the effect of piezoelectric stimulation generated by piezoelectric short fibers in hydrogel on the proliferation of vascular endothelial cells;
[0049] Figure 17 Results of fertility assessment of female mice after hydrogel treatment. DETAILED DESCRIPTION
[0050] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0051] An embodiment of the present invention provides a method for preparing a piezoelectric hydrogel for preventing fibrosis after tissue damage, comprising the following steps:
[0052] Step (1): providing an aqueous dispersion comprising a micron piezoelectric material, an amino-containing degradable biomaterial, and hyaluronic acid;
[0053] Step (2): A cross-linking agent containing an aldehyde group or a ketone group is used to react the amino-containing biodegradable material in the aqueous dispersion with a Schiff base to obtain a cross-linking structure, and the micron piezoelectric material and hyaluronic acid are combined and distributed in the cross-linking structure by hydrogen bonding to form a piezoelectric hydrogel.
[0054] The construction principle and anti-fibrosis effect of the above-mentioned piezoelectric hydrogel provided in the present application are that the degradable biomaterial containing amino groups is reacted with a cross-linking agent containing aldehyde or ketone groups to obtain a cross-linked structure by Schiff base reaction, which plays a role as a support and physical barrier; the micron piezoelectric material relies on the hydrogen bond force between it and hyaluronic acid to achieve the uniform distribution of the micron piezoelectric material in the hydrogel, and the micron piezoelectric material therein generates electrical stimulation to the tissue cells under ultrasound, thereby providing an endogenous repair effect on the damaged tissue. The hydrogel obtained by mixing the degradable biomaterial containing amino groups with hyaluronic acid has the property of shear thinning, and it is more convenient to deliver the material by injection to form a protective layer around the damaged tissue, and has a longer retention effect in the damaged tissue to provide a better physical barrier effect, and finally achieves anti-fibrosis in a coordinated manner through good retention, injectability, proliferation and repair of endogenous cells.
[0055] The amino group in the amino-containing degradable biomaterial serves as a cross-linking functional group, and the degradability of the material ensures that the material can be degraded and absorbed in the tissue to avoid residue. As a preferred embodiment, the amino-containing degradable biomaterial in step (1) is selected from at least one of amino-containing chitosan, amino-containing chitosan derivatives, amino-containing cellulose, and amino-containing cellulose derivatives; as some specific embodiments, the amino-containing degradable biomaterial in step (1) is at least one of carboxymethyl chitosan, quaternary ammonium salt chitosan, carboxyethyl chitosan, cellulose methacrylamide, and cellulose acrylamide; according to a preferred embodiment, the amino-containing degradable biomaterial is carboxymethyl chitosan, which has excellent tissue compatibility. The mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion has a significant effect on the appearance of the hydrogel. The mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion of step (1) of the present application is 0.1-2%. Within the above range, it can be ensured that the prepared hydrogel has a good injection and a good retention effect in the tissue. In the actual preparation process, the mass concentration of the amino-containing degradable biomaterial can be adjusted based on the specific purpose of use. As a specific embodiment, its mass concentration can be any value within the above range, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%. Further, the mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion of step (1) is 0.1-1%.
[0056] The micron-sized piezoelectric material mixed in the hydrogel can generate electrical stimulation under the action of ultrasound, promoting endogenous repair of tissue damage sites. Faster tissue repair reduces the occurrence of fibrosis. As a preferred embodiment, the micron piezoelectric material in step (1) is selected from at least one of polylactic acid, polyhydroxybutyrate valerate, phenylalanine dipeptide, collagen polypeptide and silk fibroin; the length of the micron piezoelectric material is 10-100 μm, and the micron piezoelectric material within the above length range can have a better dispersion effect under the action of hyaluronic acid to avoid agglomeration; as a preferred embodiment, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 1-10 mg / mL. In a specific embodiment, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) can be any value in the range of 1-10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL; further, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 4-6 mg / mL.
[0057] As a preferred embodiment, the concentration of the hyaluronic acid in the aqueous dispersion in step (1) is 0.1-2%; further, the mass ratio of the amino-containing degradable biomaterial to the hyaluronic acid in step (1) is 0.1-10:1.
[0058] As a preferred embodiment, the cross-linking agent containing an aldehyde group or a ketone group in step (2) is a biocompatible degradable cross-linking agent; further, the cross-linking agent containing an aldehyde group or a ketone group is at least one of genipin and citric acid.
[0059] The amount of cross-linking agent used can play a role in regulating the cross-linking degree of the hydrogel. The amount of cross-linking agent used has a significant effect on the morphology of the hydrogel. As a preferred embodiment, the mass ratio of the amino-containing degradable biomaterial in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 10-1000:1. The mass ratio of the amino-containing degradable biomaterial in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) can be any value within the range of 10-1000:1, for example, 10:1, 50:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1. Furthermore, the mass ratio of the amino-containing degradable biomaterial in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 50-1000:1. Within the above range, it can be ensured that the prepared hydrogel has a good injectable agent and a good retention effect in the tissue. In the actual preparation process, the mass ratio of the amino-containing degradable biomaterial to the aldehyde- or ketone-containing cross-linking agent in step (2) can be adjusted based on the specific purpose of use.
[0060] As a preferred embodiment, the Schiff base reaction in step (2) of the present application is carried out under heating and stirring; further, the heating temperature is 40-70°C, the stirring speed is 400-600 rpm, and the reaction time is 18-96h.
[0061] In order to better illustrate the construction principle and the anti-fibrosis effect of the present application's piezoelectric hydrogel, it is described below by the present application's preferred embodiment, according to the present application's preferred embodiment, the present application obtains a cross-linked structure by utilizing genipin and carboxymethyl chitosan to produce Schiff base reaction, due to the high surface energy of PLLA, short fibers are difficult to be evenly dispersed in aqueous solution. In this system, hyaluronic acid (carboxyl) and PLLA (hydroxyl) are adsorbed by hydrogen bonding, so that the PLLA surface hydrophilicity increases, reducing surface energy; The hydrogen bonding between hyaluronic acid (carboxyl) and cross-linked network (amino and carboxyl) and water (hydroxyl) prompts PLLA to be evenly dispersed in the cross-linked network under mechanical stirring, and finally constructs uniform piezoelectric hydrogel. The present invention utilizes hyaluronic acid to replace medical sodium hyaluronate to promote the retention and physical barrier duration of hydrogel, while introducing the carboxymethyl chitosan that can produce a negative charge and the negative charge on the surface of cells to form a repulsive force to enhance the ability to prevent cell adhesion. The anti-inflammatory and anti-adhesion effects of the hydrogel itself reduce the formation of fibrosis (i.e., inhibit exogenous healing that causes fibrosis). The coated piezoelectric short fibers can promote endogenous healing under the action of ultrasound to avoid the occurrence of exogenous healing. The two pathways synergistically inhibit the fibrosis of damaged tissue and promote functional recovery of the injured area.
[0062] Repeated inflammatory reactions and fibroblasts in mucosal tissues are another major cause of fibrosis in damaged tissues. The materials used in this application are all natural products with excellent biocompatibility. They can adjust the pro-inflammatory microenvironment formed after tissue damage to an anti-inflammatory microenvironment that is conducive to tissue repair. In addition, inflammatory cells and fibroblasts recruited by damage signals cannot attach to the surface of the hydrogel. The hydrogel's physical barrier, anti-inflammatory and endogenous repair effects prevent fibrosis in damaged tissues.
[0063] Another embodiment of the present invention further provides a piezoelectric hydrogel for preventing fibrosis after tissue damage, wherein the piezoelectric hydrogel is prepared by the above method. The piezoelectric hydrogel provided in this application has piezoelectric properties and shear thinning properties, and can be conveniently delivered by injection to form a protective layer around damaged tissue. It has a long retention effect in the damaged tissue and provides a good physical barrier effect. Ultimately, it synergistically achieves anti-fibrosis through good retention, injectability, and the proliferation and repair of endogenous cells.
[0064] Preferably, the piezoelectric hydrogel is an injectable.
[0065] Another embodiment of the present invention also provides the use of the piezoelectric hydrogel in the preparation of a drug or medical device for preventing fibrosis after tissue damage;
[0066] Preferably, the tissue damage is selected from at least one of tendon rupture, endometrial damage and peritoneal damage, and exhibits a better effect in preventing fibrosis after tissue damage.
[0067] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.
[0068] Example 1
[0069] Carboxymethyl chitosan, hyaluronic acid, and PLLA short fibers (30 μm in length) were added to physiological saline and blended, wherein the concentration of carboxymethyl chitosan was 0.5%, the concentration of hyaluronic acid was 0.5%, and the concentration of PLLA short fibers was 5 mg / mL. Carboxymethyl chitosan was cross-linked with genipin at 1% w / w of the carboxymethyl chitosan mass. The hydrogel (called cCMCS-HA / PLLA hydrogel) was obtained by reacting at a temperature of 65°C and a rotation speed of 550 rpm for 24 hours.
[0070] The preparation method of PLLA short fibers is to prepare an oriented PLLA material by electrospinning. 1g of PLLA (intrinsic viscosity: 1.2-1.5dL / g) is dissolved in 5mL of hexafluoroisopropanol solution and stirred at room temperature for 2 hours until the PLLA is completely dissolved to obtain a uniform spinning solution. The above solution is added to a syringe, and the syringe is installed on a syringe pump. The polymer solution is set to a flow rate of 1mL / h and a time of 40min. It flows through a G22 needle, a positive voltage of 12kV is applied, and it is collected on a roller covered with aluminum foil at a speed of 2500rpm / min to obtain an oriented nanofiber membrane. A negative voltage of -2kV is applied to the roller, and the relative humidity is controlled at 30±10%. The PLLA nanofiber membrane is annealed at 120°C for 8h and then slowly cooled to room temperature to obtain annealed PLLA. The prepared oriented PLLA film is embedded in a cryoembedding medium and cut into short fibers of ~30μm using a cryostat.
[0071] Example 2
[0072] Carboxymethyl chitosan, hyaluronic acid and PLLA short fibers (30 μm in length) were added to physiological saline and blended, wherein the carboxymethyl chitosan concentration was 1%, the hyaluronic acid concentration was 1%, and the PLLA short fiber concentration was 5 mg / mL. Carboxymethyl chitosan was cross-linked with 1% w / w genipin by weight of carboxymethyl chitosan, and the hydrogel (called cCMCS-HA / PLLA hydrogel) was obtained by reacting at a temperature of 65 ° C and a rotation speed of 550 rpm for 24 h. The PLLA short fibers were the same as in Example 1.
[0073] Example 3
[0074] The method of Example 1 was followed, except that 0.1% w / w of carboxymethyl chitosan was used as cross-linked carboxymethyl chitosan with genipin.
[0075] Example 4
[0076] The method of Example 1 was followed, except that 0.5% w / w of carboxymethyl chitosan was cross-linked with genipin.
[0077] Example 5
[0078] The method of Example 1 was followed, except that 2% w / w of carboxymethyl chitosan was used to cross-link the carboxymethyl chitosan with genipin.
[0079] Comparative Example 1
[0080] Carboxymethyl chitosan and hyaluronic acid were added to water and blended, wherein the carboxymethyl chitosan concentration was 0.5%, the hyaluronic acid concentration was 0.5%, and the carboxymethyl chitosan was cross-linked with genipin at 1% w / w of the carboxymethyl chitosan mass. The reaction was carried out at a temperature of 65°C and a rotation speed of 550 rpm for 24 hours to obtain a hydrogel (called cCMCS-HA hydrogel);
[0081] Test example
[0082] (1) Appearance, injectability and rheological properties test
[0083] The morphology of the hydrogels prepared in Examples 1, 3 to 5 using different amounts of crosslinking agents is as follows: Figure 1 As shown, from Figure 1 It can be seen that the fluidity of hydrogels prepared with different amounts of cross-linking agent is quite different.
[0084] The morphology of the hydrogels prepared in Example 1 and Example 2 is as follows Figure 2 As shown, the results show that the fluidity of hydrogels prepared with different carboxymethyl cellulose chitosan contents is quite different.
[0085] The hydrogels prepared in Examples 1 and 3 to 5 were placed in a 1 mL syringe and injected. The test results are shown in FIG. Figure 3 As shown, the results show that the hydrogels in Examples 1 and 3 to 5 can be extruded, indicating that the hydrogels prepared in Examples 1 and 3 to 5 of the present application can be administered by injection.
[0086] The piezoelectric hydrogel prepared in Example 1 was further subjected to rheological test using a rheometer. The time scan was set at 25°C, a fixed frequency of 1 Hz, and a scan time of 600 s. The frequency scan was set at a fixed amplitude of 0.1% and a frequency of 0.01-100 Hz. The rheological analysis results are shown in FIG. Figure 4 shown.
[0087] The results show that at a shear frequency of 1 Hz, the storage modulus (G') of the hydrogel is always higher than the loss modulus (G"), which indicates that the hydrogel is in a gel state at low frequencies. However, when the shear frequency of the hydrogel is higher than 1 Hz, G' suddenly drops below G", indicating that the cCMCS-HA material transitions from a gel state to a sol state.
[0088] (2) Biocompatibility
[0089] The cCMCS-HA-PLLA material and PLLA material prepared in Example 1 of the present application, as well as the cCMCS-HA material of Comparative Example 1, were subjected to biocompatibility testing. The biocompatibility testing method was as follows: 10 mg of the material was co-cultured with NIH3T3 cells in a 96-well plate for 24 hours, 48 hours, and 72 hours. CCK-8 was used to react with the living cells for 45 minutes to 1 hour. The absorbance of the supernatant after the CCK-8 reaction with the cells was measured at a wavelength of 450 nm using a microplate reader.
[0090] Cell viability (%) = (absorbance of material group - absorbance of apoptosis well) / (absorbance of blank group - absorbance of apoptosis well) * 100
[0091] Compatibility test results are as follows Figure 5 As shown, the results showed that the materials used in cCMCS-HA-PLLA, PLLA materials and cCMCS-HA were non-cytotoxic, that is, biocompatible.
[0092] (3) Piezoelectricity test
[0093] 500 μL of cCMCS-HA-PLLA hydrogel prepared in Example 1 was dropped onto a glass plate and dried for 4 h (drying temperature was 60° C.) to form a film. The film was then ultrasonically treated (frequency was 1 MHz and intensity was 0.5 W / cm 2 , duty cycle is 50%), use an oscilloscope to collect the piezoelectric output, the results are as follows Figure 6 As shown, it is shown that PLLA can generate electrical output when ultrasound is present.
[0094] (4) Anti-macrophage and fibroblast adhesion
[0095] The cCMCS-HA-PLLA hydrogel prepared in Example 1 was coated on one side of a 24-well plate and sterilized under ultraviolet light. Raw 264.7 (macrophages) and NIH3T3 cells (fibroblasts) were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / mL and cultured for 48 h. A blank control group (cells were inoculated in a well plate without any material) and a cCMCS-HA group (cCMCS-HA hydrogel was coated in one well of a 24-well plate) were set up at the same time. Images were taken using a bright field microscope. The results are shown in Figure 2. Figure 7 and Figure 8 shown.
[0096] As can be seen from the figure, cells do not adhere to the locations of cCMCS-HA hydrogel and cCMCS-HA-PLLA hydrogel, which indicates that the anti-adhesion properties of cCMCS-HA hydrogel will not be invalidated by the introduction of PLLA.
[0097] (5) Promote M2 macrophage polarization (anti-inflammatory)
[0098] The cCMCS-HA-PLLA hydrogel prepared in Example 1 was incubated with M1 macrophages (1×10 5 After 2 days of co-culture, a blank control group (no material was added to the cell culture medium) and a cCMCS-HA group (cells co-cultured with cCMCS-HA hydrogels) were set up at the same time. Immunofluorescence staining was then performed, using CD206 to label M2 macrophages and DAPI to label cell nuclei. Laser confocal microscopy was used to take pictures. The results are shown in the figure. Figure 9 .
[0099] Since CD206 is a marker for M2 macrophages, it can be seen from the figure that cCMCS-HA-PLLA hydrogel and cCMCS-HA hydrogel can promote the polarization of macrophages into repair-type M2 macrophages, thereby reducing the inflammatory response.
[0100] (6) Anti-fibrosis
[0101] The Achilles tendon of the rat was cut and sutured using the Kessler suture method. The cCMCS-HA-PLLA hydrogel prepared in Example 1 was injected into the injured tendon with an injection volume of 200 μL. Ultrasound (frequency of 1 MHz and intensity of 0.5 W / cm 2 The blank control group received no treatment after Achilles tendon suture, while the cCMCS-HA group received 200 μL of cCMCS-HA hydrogel injected into the Achilles tendon. Masson staining was performed 14 days after Achilles tendon modeling. Figure 10 shown.
[0102] Primary tendon stem cells were obtained from rat Achilles tendons, and 0.1 mg / mL PLLA and cCMCS-HA (i.e., cCMCS-HA-PLLA hydrogel) were added to the culture medium. The cells were then subjected to ultrasound (frequency 1 MHz, intensity 0.5 W / cm 2 , duty cycle of 50%, twice a day, 1 min each time). A blank control group (no material was added to the cell culture medium), a PLLA+US group (0.1 mg / mL PLLA was added to the culture medium and the same dose of ultrasound as the cCMCS-HA-PLLA group) and a cCMCS-HA group (cells were co-cultured with cCMCS-HA hydrogel) were also set up. Immunofluorescence staining was performed 2 days after the intervention of tendon stem cells, using Ki67 as a marker to evaluate cell proliferation. The results are shown in Figure 11 7 days after treatment, the rats' footprints were collected and the results are shown in Figure 12 .
[0103] Figure 10The blank control group had more collagen (blue) (dark color), indicating obvious adhesion and fibrosis. The cCMCS-HA-PLLA and cCMCS-HA groups had almost no fibrosis and were normal loose connective tissue. Figure 11 The results showed that the piezoelectric stimulation of PLLA in the cCMCS-HA-PLLA group could promote the proliferation of tendon stem cells (high expression of Ki67), thereby promoting endogenous healing after tendon injury. Figure 12 It can be seen that the right footprint of the ruptured Achilles tendon in the cCMCS-HA-PLLA hydrogel group was more complete than that in the blank control group and the cCMCS-HA group, and the Achilles tendon function index was higher.
[0104] (7) Hydrogel retention and therapeutic effect on intrauterine adhesions
[0105] A uterine injury model was established by the following steps: After conventional curettage of the uterus of SD rats, 95% alcohol was injected into the bilateral uterine cavity for 90 seconds, followed by washing away residual alcohol with saline. The cCMCS-HA-PLLA hydrogel prepared in Example 2 was used for the following experiments:
[0106] cCMCS-HA-PLLA hydrogel was injected into the injured uterus and opened on the ninth day. Figure 13 As shown, it was found that a small amount of hydrogel (blue) was still retained, which indicated that the hydrogel could be retained for more than 9 days;
[0107] 500 μL cCMCS-HA-PLLA hydrogel was injected into the injured uterine cavity of rats, and ultrasound (frequency of 1 MHz, intensity of 0.5 W / cm 2 , duty cycle is 50%, once a day, 20 minutes each time) After 9 days of continuous treatment, the results are shown Figure 14 As shown in the figure, the uterine cavity did not become closed and glands regenerated. The uterine cavity of the cCMCS-HA group (500 μL cCMCS-HA hydrogel was injected into the damaged uterine cavity) did not become closed, but there was no gland regeneration, while the uterine cavity of the blank control group (only the uterine injury model was constructed and no hydrogel was injected) was almost closed and complete fibrosis occurred. The culture supernatant of the culture medium after the cCMCS-HA-PLLA hydrogel and cCMCS-HA hydrogel intervention in M1 macrophages was extracted and used to intervene in endometrial stromal cells for 5 days. Immunofluorescence staining was performed using α-SMA as an indicator of differentiation into myofibroblasts. The results are shown in Figure 2. Figure 15As shown, the results showed that the endometrial stromal cells in the control group could differentiate into myofibroblasts, while the cells in the hydrogel group were almost undifferentiated (low expression of α-SMA, red fluorescence), indicating that cCMCS-HA-PLLA hydrogel and cCMCS-HA hydrogel can inhibit the occurrence of fibrosis that causes uterine adhesions by reducing inflammation and its induced myofibroblast differentiation.
[0108] 0.1 mg / mL PLLA and cCMCS-HA (i.e., cCMCS-HA-PLLA hydrogel) were added to the culture medium of vascular endothelial cells, and ultrasound (frequency of 1 MHz, intensity of 0.5 W / cm 2 , duty cycle of 50%, twice a day, 1 min each time), and set up a blank control group (no material was added to the cell culture medium), a PLLA+US group (0.1 mg / mL PLLA was added to the culture medium, and the same dose of ultrasound as the cCMCS-HA-PLLA group was applied) and a cCMCS-HA group (cells were co-cultured with cCMCS-HA hydrogel). Immunofluorescence staining was performed using ki67 as a marker to evaluate cell proliferation. The piezoelectric stimulation generated by PLLA in the PLLA+US group and the cCMCS-HA-PLLA group can promote the proliferation of vascular endothelial cells, thereby promoting the repair of damaged uterus. The results are shown in Figure 16 As shown, Ki67 was highly expressed (red fluorescence) in immunofluorescence staining.
[0109] After 9 days of treatment, the female mice were allowed to rest for a week and then placed in the same cage with the male mice. After 2 weeks, the female mice's uterus was removed for observation. The results are shown in the figure. Figure 17 , and found that the female mice in the cCMCS-HA-PLLA and cCMCS-HA groups became pregnant normally, while the control group failed to become pregnant due to uterine atresia.
[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solutions claimed for protection by the present invention.
Claims
1. A method for preparing a piezoelectric hydrogel for preventing fibrosis after tissue damage, characterized in that: The following steps are involved: Step (1): providing an aqueous dispersion comprising a micron piezoelectric material, an amino-containing degradable biomaterial, and hyaluronic acid; Step (2): A cross-linking agent containing an aldehyde group or a ketone group is used to react the amino-containing biodegradable material in the aqueous dispersion with a Schiff base to obtain a cross-linking structure, and the micron piezoelectric material and hyaluronic acid are combined and distributed in the cross-linking structure by hydrogen bonding to form a piezoelectric hydrogel.
2. The preparation method according to claim 1, characterized in that The amino-containing degradable biomaterial in step (1) is selected from at least one of amino-containing chitosan, amino-containing chitosan derivatives, amino-containing cellulose, and amino-containing cellulose derivatives; Preferably, the amino-containing biodegradable material in step (1) is selected from at least one of carboxymethyl chitosan, quaternary ammonium chitosan, carboxyethyl chitosan, cellulose methacrylamide, and cellulose acrylamide; Preferably, the mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion in step (1) is 0.1-2%; Preferably, the mass concentration of the amino-containing degradable biomaterial in the aqueous dispersion in step (1) is 0.1-1%.
3. The preparation method according to claim 1, characterized in that The micron piezoelectric material in step (1) is selected from at least one of polylactic acid, polyhydroxybutyrate valerate, phenylalanine dipeptide, collagen polypeptide and silk fibroin; Preferably, the length of the micron piezoelectric material is 10-100 μm; Preferably, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 1-10 mg / mL; Preferably, the concentration of the micron piezoelectric material in the aqueous dispersion in step (1) is 4-6 mg / mL.
4. The preparation method according to any one of claims 1 to 3, characterized in that The concentration of the hyaluronic acid in the aqueous dispersion of step (1) is 0.1-2%; Preferably, in step (1), the mass ratio of the amino-containing degradable biomaterial to the hyaluronic acid is 0.1-10:
1.
5. The preparation method according to claim 1, characterized in that The cross-linking agent containing an aldehyde group or a ketone group in step (2) is a biocompatible and degradable cross-linking agent; Preferably, the cross-linking agent containing an aldehyde group or a ketone group is at least one of genipin and citric acid.
6. The preparation method according to claim 1 or 5, characterized in that The mass ratio of the amino-containing biodegradable material in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 10-1000:1; Preferably, the mass ratio of the amino-containing degradable biomaterial in step (1) to the aldehyde- or ketone-containing cross-linking agent in step (2) is 50-1000:
1.
7. The preparation method according to claim 1, characterized in that The Schiff base reaction in step (2) is carried out under heating and stirring; Preferably, the heating temperature is 40-70° C., the stirring speed is 400-600 rpm, and the reaction time is 18-96 h.
8. A piezoelectric hydrogel for preventing fibrosis after tissue damage, characterized in that: The piezoelectric hydrogel is prepared by the method according to any one of claims 1 to 7.
9. The piezoelectric hydrogel according to claim 8, characterized in that: The piezoelectric hydrogel is an injection.
10. Use of the piezoelectric hydrogel according to claim 8 or 9 in the preparation of a drug or medical device for preventing fibrosis after tissue damage; Preferably, the tissue damage is selected from at least one of tendon rupture, endometrial injury and peritoneal injury.