Oryzias latipes collagen piezoelectric hydrogel and application thereof in preparation of biomaterials for treating tissue injury

Abstract

The application discloses a tilapia collagen piezoelectric hydrogel and application of the tilapia collagen piezoelectric hydrogel in preparation of biomaterials for treating tissue injury. The piezoelectric hydrogel is collagen / oxidized gellan gum / barium titanate hydrogel, presents an irregular interconnected porous structure, the compressive strength of the piezoelectric hydrogel is 46.26-95.74 kPa, and the output voltage is 4.47-90.59 mV. The piezoelectric hydrogel based on tilapia collagen is introduced, amino titanium acid barium and oxidized gellan gum are introduced to improve the physical properties of the hydrogel, piezoelectric characteristics are integrated into the hydrogel, and therefore, the dependence of a traditional electric stimulation device on an external power supply is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a tilapia collagen piezoelectric hydrogel and application thereof in preparing a biomaterial for treating tissue damage. BACKGROUND

[0002] The healing of tissue damage is a complex biological process involving multiple types of tissues, including skin, bone, cartilage, muscle, etc. Different types of tissues have different healing mechanisms and time frames, but the overall process generally includes the following stages: inflammation, proliferation and remodeling, among which controlling the inflammatory response in damaged tissues, vascular regeneration, etc. is still a major clinical challenge. Growth factors are widely recognized as being able to promote cell proliferation, control the immune microenvironment and angiogenesis, and therefore their application in the regeneration of damaged tissues has received a high degree of attention. However, the use of growth factors has problems such as high cost, short application period and difficulty in controlling local concentration, which greatly limits their clinical application. In addition, the stability of growth factors and the duration of their effect at the site of injury are short, often requiring repeated administration, increasing the burden on patients and treatment costs.

[0003] Stimulation can promote biological processes such as cell proliferation, migration, macrophage polarization and vascular regeneration. However, the commonly used electrical stimulation technology currently requires an external power source and electrodes, which not only increases the cost of equipment, but also can cause inconvenience and discomfort, limiting its widespread application in actual treatment.

[0004] Gels are an ideal biomaterial and are widely used in tissue engineering and regenerative medicine. However, traditional hydrogel materials, especially those based on natural high molecular weight polymers such as collagen, often face problems such as uncontrollable degradation rate, poor mechanical properties and high biodegradability, which make them difficult to meet the requirements of long-term use in clinical practice. At the same time, the physical properties of traditional hydrogels are difficult to control, limiting their application in the treatment of different types of tissue damage. SUMMARY

[0005] The present application solves the problems existing in the prior art and provides a tilapia collagen piezoelectric hydrogel and application thereof in preparing a biomaterial for treating tissue damage. The piezoelectric hydrogel based on tilapia collagen (Col) (Col / OG / BTN hydrogel) proposed in the present application improves the physical properties of the hydrogel by introducing amino titanium barium (BTN) and oxidized gellan gum (OG), and at the same time incorporates piezoelectric properties, thereby avoiding the dependence on external power supply of traditional electrical stimulation devices.

[0006] The first object of the present application is to provide a tilapia collagen piezoelectric hydrogel, which is a collagen / oxidized gellan gum / barium titanate amino hydrogel (Col / OG / BTN hydrogel) and presents an irregular interconnected porous structure, and the compressive strength of the piezoelectric hydrogel is 46.26-95.74 kPa, and the output voltage is 4.47-90.59 mV.

[0007] The components of the hydrogel interact with each other, and the tilapia collagen, oxidized gellan gum and barium titanate amino synergistically form a gel within 2 min.

[0008] The present application improves the mechanical strength, stability, biodegradation resistance and piezoelectric properties of the hydrogel by introducing the composite material of barium titanate amino BTN and oxidized gellan gum OG, and has a longer service life, better mechanical properties and stronger output voltage compared with pure collagen hydrogel, which can better meet the requirements in the process of tissue damage treatment. The tilapia collagen piezoelectric hydrogel proposed in the present application is resistant to degradation, has high mechanical strength, can generate output voltage under mechanical stimulation, can regulate the immune environment and promote vascular regeneration, thereby promoting tissue healing.

[0009] The piezoelectric hydrogel generates a spontaneous electric field through the internal piezoelectric material (BTN), providing electrical stimulation, avoiding the use of external power supply and electrodes, and thus improving the convenience of treatment and the comfort of patients. This feature enables the hydrogel to generate output voltage through ultrasonic stimulation during the healing process of damaged tissue, promoting cell proliferation, migration and adhesion. The Col / OG / BTN hydrogel not only has good biocompatibility, but also can induce M1 macrophages to transform into M2 macrophages through ultrasonic stimulation, and play an anti-inflammatory and immune regulation role. This mechanism helps to reduce the inflammatory response and promote tissue repair. After ultrasonic stimulation, the Col / OG / BTN hydrogel can generate piezoelectricity, stimulate macrophages, and make the macrophages secrete growth factor VEGF to promote the formation of blood vessels, avoiding the problems of high cost, short application period and difficult local concentration control of direct use of growth factors.

[0010] The second object of the present application is to provide a preparation method of a tilapia collagen piezoelectric hydrogel, comprising the following steps:

[0011] S1, preparing tilapia collagen, dissolving the tilapia collagen in an acetic acid solution to obtain an acidic collagen solution, mixing the acidic collagen solution with a PBS solution, adjusting the pH to neutral to obtain a collagen solution Col;

[0012] S2, adding sodium periodate to the gellan gum GG solution to obtain oxidized gellan gum OG;

[0013] S3, stirring barium titanate BTO into H2O2, washing and drying, adding the dried material into ethanol solution, and adding (3-aminopropyl) trimethoxysilane dropwise to reflux reaction, after washing and drying treatment, obtaining aminated barium titanate BTN;

[0014] S4, dissolving oxidized gellan gum OG in water to prepare OG solution, mixing the collagen solution obtained in step S1 with the OG solution to obtain Col / OG solution, adding the BTN powder obtained in step S3 into the Col / OG solution to stir until completely mixed, the concentration of the OG solution is 0.05-0.2 g / mL, the volume ratio of the collagen solution to the OG solution is 8-12:1, and the mass-volume ratio of the BTN to the Col / OG solution is 0.005-0.02 g / mL, obtaining Col / OG / BTN piezoelectric hydrogel;

[0015] Or dissolving the collagen obtained in step S1, the oxidized gellan gum obtained in step S2, and the aminated barium titanate obtained in step S3 in a solvent to mix at room temperature for 1-3 days to obtain a mixed solution, the concentration of the fish skin collagen in the mixed solution is 2-20 wt%, the oxidized gellan gum is 1-5 wt%, and the aminated barium titanate is 1-5 wt%, and then electrospinning the mixed solution, soaking in PBS to swell, obtaining Col / OG / BTN piezoelectric hydrogel.

[0016] Preferably, the tilapia collagen in step S1 is prepared by the following steps: washing the tilapia fish skin, then soaking in a mixed solution containing butanol and isopropyl alcohol, then transferring the tilapia fish skin to an acetic acid solution to stir overnight until the collagen is dissolved, then centrifuging the obtained crude collagen solution to remove particles, adding NaCl solution to the supernatant to precipitate collagen and stirring overnight, then centrifuging the supernatant to collect the collagen precipitate, dissolving the precipitated collagen in acetic acid again, and after dialysis and freeze-drying treatment, obtaining pure collagen.

[0017] Tilapia is a widely farmed fish, and a large amount of fish skin is discarded every year. The fish skin as a byproduct is usually low in cost, and by converting the fish skin into valuable products (collagen), the demand for fresh resources can be reduced, and the development of sustainable fisheries can be promoted by reasonably utilizing the fish skin, and the ecological balance can be promoted.

[0018] The tilapia collagen is directly extracted from the fish skin of tilapia by an acid dissolution method, and the gelatin is prepared by hydrolyzing and processing the animal collagen extracted by hot water. Compared with the gelatin, the tilapia collagen retains the characteristics of fish-specific collagen, and has a high proportion of type I collagen, which is very important in tissues such as skin, bone, tendon, ligament, blood vessel, cartilage and tooth. The hydrogel prepared from the tilapia collagen has good thermal stability and gelation capacity, can form a stable gel at a lower temperature, and remains stable at a physiological temperature, while the natural macromolecules such as gelatin lose the gelation capacity when heated after forming a gel, which limits the application thereof.

[0019] The acid extraction method of the tilapia collagen has higher extraction efficiency than other extraction methods (such as water extraction or enzyme extraction). A collagen solution with a higher concentration can be obtained in a shorter time. The acid extraction method is carried out under relatively mild conditions, and can better maintain the structure and functionality of the collagen, so as to ensure that the biological activity is retained. The acid extraction process is relatively simple, reduces the dependence on complex equipment and high technology, and thus reduces the production cost. Commonly used acids (such as acetic acid and citric acid) have less impact on the environment, are safer than some chemical solvents, and meet the concept of green extraction. The method is easy to standardize and scale up, is suitable for large-scale production, and meets the market demand.

[0020] Preferably, the acid collagen solution is mixed with the PBS solution in step S1, and after the pH is adjusted to neutral, the specific steps of obtaining the collagen solution Col are as follows: the acid collagen solution is mixed with 10×PBS solution at 4℃, the volume ratio of the acid collagen solution to the 10×PBS solution is 9:1, then the pH is adjusted to neutral by adding NaOH solution, and the collagen solution Col is obtained, and the concentration of the collagen solution is 3-6g / L.

[0021] The collagen extracted by the tilapia collagen extraction method has good biocompatibility, and the BTN nanoparticles can be well dispersed in the collagen hydrogel.

[0022] Further preferably, the volume ratio of the acid collagen solution to the 10×PBS solution is 8:1, and the concentration of the collagen solution is 5g / L (5mg / mL).

[0023] Preferably, the specific steps of step S2 are: dissolving the gelatin gum GG in deionized water, heating to 80-100°C until the gelatin gum GG solution is obtained, the concentration of the gelatin gum GG solution is 8-12 g / L, cooling the gelatin gum GG solution to 40°C, adding sodium periodate, stirring at room temperature in the dark for 7-10 hours, then transferring to dialysis membrane, dialysis with deionized water for 4-6 days, and freeze-drying to obtain the oxidized gelatin gum OG.

[0024] Further preferably, the molar ratio of gelatin gum GG to sodium periodate is 1:0.85-1:0.95, and the concentration of the gelatin gum GG solution is 10 g / L.

[0025] Further preferably, the specific steps of step S2 are: dissolving the gelatin gum GG in deionized water, heating to 80-100°C until the gelatin gum GG solution is obtained, the concentration of the gelatin gum GG solution is 10 g / L, cooling the gelatin gum GG solution to 40°C, adding sodium periodate, stirring at room temperature in the dark for 8 hours, then transferring to dialysis membrane, dialysis with deionized water for 5 days, and freeze-drying to obtain the oxidized gelatin gum OG.

[0026] Preferably, the specific steps of step S3 are: first adding barium titanate BTO powder to H2O2, stirring vigorously at 105°C for 2-6h, washing, vacuum drying, adding the dried material to an ethanol solution, and adding (3-aminopropyl)trimethoxysilane dropwise at 75-85°C for 16-24h of reflux reaction, washing, vacuum drying, to obtain the aminated barium titanate BTN.

[0027] The enhancement of the hydrogel piezoelectric performance by the BTN nanoparticles improves the thermal stability, degradability and mechanical properties of the cross-linking and physical structure; the degradation stability of the hydrogel is enhanced by the BTN, prolonging the use time of the hydrogel at the tissue damage site.

[0028] The modified aminated barium titanate has a coating layer of (3-aminopropyl)trimethoxysilane on the surface, with a thickness of 1-5 nm.

[0029] Further preferably, the specific steps of step S3 are: first adding barium titanate BTO powder to H2O2, stirring vigorously at 105°C for 4h, washing, vacuum drying, adding the dried material to an ethanol solution, and adding (3-aminopropyl)trimethoxysilane dropwise at 80°C for 20h of reflux reaction, washing, vacuum drying, to obtain the aminated barium titanate BTN.

[0030] Further preferably, the molar ratio of barium titanate BTO to H2O2 is 0.1-0.3:1, the mass-volume ratio of barium titanate BTO to ethanol solution is 1:3-5 g / mL, and the mass ratio of barium titanate BTO to (3-aminopropyl)trimethoxysilane is 1.5-2.5:1.

[0031] Further more preferably, the molar ratio of barium titanate BTO to H2O2 is 0.121:1, the mass-volume ratio of barium titanate BTO to ethanol solution is 1:4 g / mL, and the mass ratio of barium titanate BTO to (3-aminopropyl)trimethoxysilane is 2:1.

[0032] Preferably, the parameters of electrospinning in step S4 are as follows: the flow rate is 5-100 μL / min, the spinning distance is 2-30 cm, the voltage is 2-30 kV, and the drum rotation speed is 100-4000 rpm, and the solvent is hexafluoroisopropanol.

[0033] The mass-volume ratio unit of the present application is g / mL.

[0034] The application also protects the use of the tilapia collagen piezoelectric hydrogel in the field of biological materials for treating tissue damage, including skin, bone, tendon, blood vessels, cartilage, teeth, etc.

[0035] The application also protects a biological material for treating wound healing, which uses the tilapia collagen piezoelectric hydrogel as an active ingredient and retains the triple helix structure of collagen.

[0036] The piezoelectric properties of the tilapia collagen hydrogel enable the hydrogel to generate an electric field spontaneously under external force, avoiding dependence on external power supply. This property helps to promote cell proliferation, migration, angiogenesis, and regulate immune response, accelerating the regeneration and healing of damaged tissues.

[0037] The Col / OG / BTN piezoelectric hydrogel can effectively regulate the polarization of macrophages and promote the transformation of M1-type macrophages to M2-type macrophages. M2-type macrophages have anti-inflammatory and immune-regulating effects, which can reduce the inflammatory response at the tissue damage site and create a favorable environment for tissue repair and regeneration. In addition, M2-type macrophages also promote angiogenesis, which is crucial for tissue regeneration.

[0038] The piezoelectric hydrogel can stimulate the secretion of growth factors VEGF, increase the expression of anti-inflammatory gene CD206, and reduce the expression of pro-inflammatory genes CD86 and iNOS in macrophages under ultrasound. At the same time, the released VEGF promotes the mRNA expression of b-FGF and Ang-1 in human umbilical vein endothelial cells (HUVEC), indicating the great potential of the piezoelectric hydrogel for tissue damage regeneration.

[0039] Compared with the prior art, the present application has the following advantages:

[0040] 1、The application utilizes tilapia collagen, a marine resource, as raw material, not only providing a new idea for the sustainable utilization of marine resources, but also making the hydrogel have good biocompatibility and safety. As a natural polymer material, collagen has excellent cell affinity and biodegradability, which helps to promote tissue regeneration and wound healing. Tilapia collagen not only has a rich source, but also has a similar amino acid sequence to human collagen, good biocompatibility, and can effectively promote cell adhesion and growth. This makes it an ideal tissue engineering material.

[0041] 2、The amino group on the surface of BTN can react with the aldehyde group on OG to increase the crosslinking density, in addition, the addition of nanoparticles itself has an enhancing effect, which increases the compressive strength of the hydrogel, and does not affect the piezoelectric properties of barium titanate itself.

[0042] 3、The application introduces amine titanium barium (BTN) and oxidized gellan gum (OG), so that the synthesized piezoelectric hydrogel has significantly improved physical properties. BTN can enhance the mechanical strength and piezoelectric properties of the hydrogel, reduce the biodegradation rate and swelling of the hydrogel, make it more stable under physiological conditions, and prolong its service life. Compared with pure tilapia collagen hydrogel, the Col / OG / BTN composite hydrogel shows obvious improvement in mechanical properties, which is particularly important for wound healing environment that needs to bear certain tensile force and shear force.

[0043] 4、The application introduces piezoelectric material (BTN), so that the hydrogel can spontaneously generate electric energy. This feature avoids the dependence on external power supply and electrodes of traditional electric stimulation devices, simplifies the treatment equipment, and reduces the discomfort and treatment cost of patients. The piezoelectric hydrogel promotes cell proliferation, migration and regulates immune microenvironment and angiogenesis through spontaneous electric field effect, not only reduces the complexity of the equipment, but also improves the treatment experience of the patients.

[0044] 5、The Col / OG / BTN piezoelectric hydrogel proposed in the application can effectively regulate the polarization of macrophages and promote the transformation of M1 type macrophages to M2 type macrophages. M2 type macrophages have anti-inflammatory and immune regulation effects, which can reduce the inflammatory response at the wound site and create a favorable environment for damaged tissue repair and regeneration. In addition, M2 type macrophages also promote angiogenesis, which is crucial for wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The FTIR spectrum of fish skin collagen extracted in Example 1 is shown in the figure;

[0046] Figure 2 The electrophoresis spectrum of fish skin collagen extracted in Example 1 is shown in the figure;

[0047] Figure 3 Amino acid content analysis chart for fish skin collagen extracted in Example 1;

[0048] Figure 4 FTIR spectra of oxidized gellan gum and aminated barium titanate in Example 1;

[0049] Figure 5 HNMR spectra of oxidized gellan gum in Example 1; 1 HNMR spectra;

[0050] Figure 6 TEM and TEM-EDS charts of aminated barium titanate in Example 1, 6a-b are TEM charts of aminated barium titanate, and 6c-f are TEM-EDS charts of aminated barium titanate;

[0051] Figure 7 SEM charts of piezoelectric hydrogels in Examples 1-3, 7a is a Col hydrogel SEM chart, 7b is a Col / OG hydrogel SEM chart, 7c is a Col / OG / 0.5% BTN hydrogel SEM chart, 7d is a Col / OG / 1% BTN hydrogel SEM chart, 7e is a Col / OG / 2% BTN hydrogel SEM chart, and 7f-j are Col / OG / 2% BTN hydrogel SEM-EDS charts;

[0052] Figure 8 Schematic diagram of piezoelectric hydrogel formation in Examples 1-3;

[0053] Figure 9 DSC chart of piezoelectric hydrogels in Examples 1-3;

[0054] Figure 10 Swelling property test chart of piezoelectric hydrogels in Examples 1-3;

[0055] Figure 11 Degradation property test chart of piezoelectric hydrogels in Examples 1-3;

[0056] Figure 12 Mechanical property test chart of piezoelectric hydrogels in Examples 1-3;

[0057] Figure 13 Piezoelectric property test chart of piezoelectric hydrogels in Examples 1-3;

[0058] Figure 14 Cell biocompatibility determination chart of piezoelectric hydrogels in Examples 1-3, 14a is a CCK-8 chart of cells on hydrogels, and 14b is a cell live and dead staining chart of cells on hydrogels;

[0059] Figure 15Figure 15a-15b. Effects of piezoelectric stimulation of piezoelectric hydrogels in Examples 1-3 on L929 cell proliferation. Figure 15a is immunofluorescence staining, and Figure 15b is quantitative analysis.

[0060] Figure 16 Figure 16a-16b. Cell migration of cells in piezoelectric hydrogels in Examples 1-3. Figure 16a is a cell scratch experiment, and Figure 16b is a cell Transwell experiment.

[0061] Figure 17 Figure 17a-17e. Macrophage immunomodulatory performance of piezoelectric hydrogels in Examples 1-3. Figure 17a is an immunofluorescence image of macrophage polarization, Figures 17b-d are relative mRNA expression of macrophages with respect to CD206, CD86 and iNOS, and Figure 17e is expression of VEGF protein in the supernatant of macrophages.

[0062] Figure 18 Figure 18a-18c. Angiogenic performance of HUVEC cells in piezoelectric hydrogels in Examples 1-3. Figure 18a is immunofluorescence staining of HUVEC cells with VEGF, and Figures 18b-c are mRNA expression of HUVEC cells with respect to b-FGF and Ang-1. DETAILED DESCRIPTION:

[0063] The following examples are further illustrations of the present application and are not intended to limit the present application in any way.

[0064] Unless otherwise defined, all terms used in connection with the present application, are to be interpreted according to their ordinary meaning to a person skilled in the art. The professional terms used in the present application are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present application. Unless otherwise specified, the experimental materials and reagents used in the present application are commercially available products commonly used in the art.

[0065] The detection instruments involved in the structural characterization in the following examples are as follows:

[0066] Infrared spectroscopy and Fourier transform spectroscopy (FTIR) (Thermo Fisher Scientific Nicolet iS20, USA) were used for molecular structure characterization.

[0067] SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used for molecular weight analysis.

[0068] An amino acid analyzer (Biochrom 30+, UK) was used for amino acid content analysis.

[0069] Morphological characterization was performed by transmission electron microscopy (TEM, Themis Z (3.2)). For TEM samples, BTN nanopowder was dissolved in absolute ethanol (5 mg / mL) for 10 min and then added dropwise to copper grids (accelerating voltage: 300 kV). The distribution of Ba, Ti, O, and N was analyzed using X-ray spectroscopy (EDS).

[0070] Field emission scanning electron microscopy (FE-SEM; Zeiss, Sigma, Germany) was used to characterize the morphology of the cross-section of the piezoelectric hydrogels. An energy dispersive spectrometer (Ultim Max 40, Oxford, UK) was used to scan the distribution of C, O, N, Ba, and Ti elements in the scaffolds.

[0071] Example 1

[0072] A method for preparing a tilapia collagen piezoelectric hydrogel, comprising the following steps:

[0073] S1, Fresh tilapia was scaled and the skin was washed with deionized water. The washed skin was cut into 1 x 1 cm 2 sized and washed in DPBS for 24 h, after which it was stirred in 0.1 M NaOH (800 mL) for 6 h, with the NaOH being replaced every 2 h. After 6 h, the skin was washed to neutrality with deionized water and soaked in a solution containing 10% butanol and 20% isopropanol (total 800 mL: 80 mL butanol, 160 mL isopropanol) for 24 h to remove attached fat. The skin was then transferred to a 0.5 M acetic acid solution with a volume of 3.4 L and stirred overnight until the collagen was dissolved. The resulting crude collagen solution was centrifuged at high speed (twice at 18,000 G for 30 min and once at 22,000 G for 30 min) to remove particles. The supernatant was added to a NaCl solution (the final NaCl solution concentration of the solution was 1 M) to precipitate the collagen, which was then centrifuged to collect the collagen precipitate (once at 12,000 G for 60 min and once at 18,000 G for 60 min). The precipitated collagen was redissolved in 0.5 M acetic acid (800 mL). The resulting solution was transferred to a dialysis membrane with a MWCO of 8-15 kDA and dialyzed in 0.1 M acetic acid for 24 h and then in distilled water for 48 h, after which the sample was freeze-dried to obtain pure collagen.

[0074] S2, 2 g of gellan gum GG (10 g / L) was dissolved in 200 mL of deionized water at a temperature of 90°C until a solution was formed. The solution was cooled to 40°C, and 0.359 g of sodium periodate was added (i.e., GG:NaIO4 = 1:0.9 mmol). It was stirred in the dark at room temperature for 8 hours, then transferred to a dialysis membrane and dialyzed with deionized water for 5 days, and freeze-dried to obtain oxidized gellan gum OG.

[0075] S3. Add 5g of barium titanate (BTO) powder to 20mL of H2O2, sonicate for 30min, stir vigorously at 105℃ for 4h, filter and wash three times with deionized water and ethanol, vacuum dry at 50℃ for 12h, add the dried product to 20mL of ethanol solution, and add 2.5g of (3-aminopropyl)trimethoxysilane. Reflux at 80℃ for 20h, filter and wash three times with deionized water and ethanol, and vacuum dry at 50℃ for 12h to obtain amino-modified barium titanate (BTN).

[0076] S4. Dissolve 50 mg of the pure collagen obtained in step S1 in 10 mL of 0.02 M acetic acid solution at 4 °C to obtain an acid-washed collagen solution of 5 mg / mL. Then, mix the acid-washed collagen solution with 10×PBS solution at a ratio of 8:1 (7.488:0.936 mL) at 4 °C. Adjust the pH to neutral by adding 0.126 mL of 1 N NaOH solution, then add 0.45 mL of deionized water to obtain 9 mL of collagen solution (Col). Pour this solution into a mold to form a collagen hydrogel (Col). Using the OG obtained in step S2, prepare 1 mL of 10% w / v OG solution. Mix the 9 mL collagen solution with the OG solution to obtain a Col / OG solution with an OG concentration of 1% w / v. Pour this solution into a mold to form a collagen hydrogel (Col / OG). BTN powder was stirred for 10 minutes at 0.5% w / v (0.005 g / mL) of the Col / OG volume until completely mixed. The mixture was then poured into a mold to form a hydrogel, resulting in Col / OG / 0.5% BTN hydrogel.

[0077] The pure collagen obtained in step S1 was analyzed for its molecular structure, molecular weight, and amino acid content, such as... Figures 1-3 As shown, Figure 1 The mid-FTIR spectrum showed five characteristic amide bands at wavenumbers of 3295, 2930, 1630, 1546, and 1236 cm⁻¹, corresponding to amide A, amide B, amide I, amide II, and amide III, respectively. At 1451 cm⁻¹... -1 At this point, the ratio of amide III to the amount absorbed is 1, indicating that the triple helix structure of collagen is intact. Figure 2 SDS-PAGE analysis revealed the presence of α, β, and γ subunits in the extracted fish skin collagen, which is characteristic of a typical type I collagen structure. Figure 3 Amino acid composition analysis of tilapia collagen showed that the collagen sample from tilapia skin contained a large amount of proline (114.602 mg / g) and hydroxyproline (200.949 mg / g), which increased the stability of the triple helix structure of collagen and were important contributors to the thermal stability of collagen.

[0078] The OG obtained in step S2 is structurally characterized, such as... Figures 4-5 As shown,Figure 4 The FTIR spectrum of OG at 1730 cm⁻¹ -1 A new absorption peak appeared at the point, indicating the C=O stretching vibration of the aldehyde group. Figure 5 middle 1 HNMR analysis further confirmed the success of the oxidation, with characteristic peaks of hemiacetal and aldehyde groups observed at approximately 4.86 ppm, 9.11 ppm, and 9.64 ppm.

[0079] The BTN obtained in step S3 is structurally characterized, such as... Figure 4 and 6 As shown, Figure 4 FTIR at 1536 cm⁻¹ indicates -1 A new peak value is found at NH bending vibration, 1130 and 997 cm. -1 The peak value at that point corresponds to the deformation vibration of Si-O-BT and Si-O28. Figure 6 TEM characterization revealed that the nanoparticles were approximately spherical with a diameter of less than 100 nm, and an amorphous layer was observed around the crystalline particles. TEM-EDS analysis of BTN showed a large amount of nitrogen on the surface of the nanoparticles, confirming the successful NH2 functionalization of BTO by APTMS.

[0080] Example 2

[0081] Same as in Example 1, except that: BTN powder was added to the Col / OG solution at 1% w / v (0.01 g / mL) of the Col / OG volume, and stirred for 10 minutes until completely mixed to obtain Col / OG / 1% BTN hydrogel.

[0082] Example 3

[0083] Same as in Example 1, except that: BTN powder was added to the Col / OG solution at 2% w / v (0.02 g / mL) of the Col / OG volume, and stirred for 10 minutes until completely mixed to obtain Col / OG / 2% BTN hydrogel.

[0084] The Col, Col / OG, Col / OG / 0.5% BTN, Col / OG / 1% BTN and Col / OG / 2% BTN hydrogels obtained in Examples 1-3 were characterized. Figure 7 SEM images showed that the Col, Col / OG, Col / OG / 0.5% BTN, Col / OG / 1% BTN, and Col / OG / 2% BTN hydrogels all exhibited irregular interconnected porous structures. Energy dispersive spectroscopy (EDS) was used to analyze the distribution of BTN in the composite hydrogels. Ba and Ti were uniformly distributed within the hydrogels, indicating that BTN was successfully incorporated into the hydrogels.

[0085] likeFigure 8 As shown, Col, Col / OG, Col / OG / 0.5% BTN, Col / OG / 1% BTN, and Col / OG / 2% BTN obtained from Examples 1-3 were all capable of rapid molding at 37°C within 2 min.

[0086] Experimental Example 1

[0087] The Col, Col / OG, Col / OG / 0.5% BTN, Col / OG / 1% BTN, and Col / OG / 2% BTN hydrogels obtained from Examples 1-3 were tested as follows:

[0088] The Col, Col / OG, Col / OG / 0.5% BTN, Col / OG / 1% BTN, and Col / OG / 2% BTN hydrogels were measured for heat distortion temperature by differential scanning calorimetry (DSC, NETZSCH DSC204F1).

[0089] Swelling performance test: The hydrogels were placed in PBS at pH 7.4, 37°C for 24 h. Excess water on the surface was wiped off, and the weight was recorded. The swelling ratio (SR) of the hydrogels in the PBS solution was calculated by Formula 1: SR (%) = (Wt-W0) / W0x100% (1); where: W0 is the initial weight of the dried hydrogel, and Wt is the mass of the hydrogel after swelling.

[0090] Degradation performance test: The hydrogels were soaked in 60 pg mL -1 Type I collagenase PBS at 37°C. The hydrogels were removed from the mixture at predetermined time intervals, freeze-dried, and then weighed again. The residual weight of the hydrogels was calculated by Formula 2.

[0091] Residual weight ratio (%) = Wt / W0x100% (2); where: W0 is the initial mass of the hydrogel, and Wt is the mass of the hydrogel after degradation.

[0092] Mechanical performance test: A texture analyzer (TA.HD Plus, UK) was used to test the compressibility of the hydrogels (h = 10 mm, d = 12 mm). The compression test was performed at a rate of 0.05 mm / s, with a maximum strain of 80%.

[0093] Piezoelectric performance test: The output voltage of the hydrogels was measured using a high-precision multimeter (Keithley DMM7510, USA). The sample was in the form of a cylinder (3 mm thick, 10 mm in diameter), and the hydrogel was layered with copper foil as the conductive material.

[0094] Biocompatibility test: The CCK-8 method and live / dead staining were used to evaluate the biocompatibility of the hydrogels. 3x10 3L929 cells were seeded onto different hydrogel surfaces in 96-well plates at a density of 1 x 10 2 Cells on hydrogels were stimulated with ultrasound (1 MHz, 2.5 W / cm2) for 5 min every 4 h, 3 times a day. After 1, 3, and 5 days of culture, CCK-8 solution (10 μL) was added to each well, followed by 1 h of incubation. Absorbance values were measured at a test wavelength of 450 nm using a plate reader in triplicate. In live / dead staining experiments, L929 cells were seeded onto different piezoelectric hydrogels in 24-well plates at a density of 1 x 10 4 Cells on hydrogels were stimulated with ultrasound (1 MHz, 2.5 W / cm2) for 5 min every 4 h, 3 times a day. After 1, 3, and 5 days of culture, CCK-8 solution (10 μL) was added to each well, followed by 1 h of incubation. Absorbance values were measured at a test wavelength of 450 nm using a plate reader in triplicate. In live / dead staining experiments, L929 cells were seeded onto different piezoelectric hydrogels in 24-well plates at a density of 1 x 10

[0095] Cell proliferation test: L929 cells were evaluated for proliferation on different hydrogels using the Edu Cell Proliferation Imaging Analysis Kit (KTA2030). Cells on hydrogels were stimulated with ultrasound (1 MHz, 2.5 W / cm2) for 5 min every 4 h, 3 times a day. Cells were then photographed using a laser confocal high-content screening system. At least 3 fields were selected for each sample, and the average proportion of EdU-positive cells was calculated using ImageJ.

[0096] Cell scratch and transwell experiments: 6-well plates were seeded with 6 x 105 L929 cells per well, and after the cells grew to a sufficient size, they were scratched with a straight blade using a 200-μL pipette tip. After scratching, the cells were washed with PBS 3 times and incubated with L929 and different hydrogels for 24 h. Cells on hydrogels were stimulated with ultrasound (1 MHz, 2.5 W / cm2) for 5 min every 4 h, 3 times a day. Photographs were taken under an inverted microscope at 0 and 24 h. At least 3 fields were selected for each sample, and the average cell area / initial area was analyzed using ImageJ. 200 μL (1 x 10 5 Cells on hydrogels were stimulated with ultrasound (1 MHz, 2.5 W / cm2) for 5 min every 4 h, 3 times a day. Photographs were taken under an inverted microscope at 0 and 24 h. At least 3 fields were selected for each sample, and the average cell area / initial area was analyzed using ImageJ. 200 μL (1 x 10

[0097] Modulation of macrophage phenotype in vitro by hydrogels: Raw 264.7 cells were seeded onto different piezoelectric hydrogels in 24-well plates at a density of 3 x 10 3Cell density was seeded on 96-well plates with different hydrogels for 24 hours. Subsequently, cells were treated with DMEM containing LPS (100 ng / mL) and IFN-γ (20 ng / mL) for 24 hours, and then treated with complete medium for 48 hours. Cells on hydrogels were stimulated with ultrasound for 5 minutes every 4 hours, 3 times a day. The morphological changes of co-cultured macrophages on different samples were observed by immunofluorescence. After PBS washing, cells were fixed with 4% paraformaldehyde for 30 minutes. After washing, 0.5% Triton X-100 permeation for 1 hour, 5% BSA blocking for 1 hour, DAPI staining the nucleus, immunofluorescence staining was used to detect macrophage polarization using iNOS (M1 marker) and CD206 (M2 marker) antibodies. Stained samples were imaged using a laser confocal high-content screening system, and at least 3 fields were randomly selected for each sample. Total RNA was extracted from cells in different samples using TRIzol reagent (Thermo Fisher, USA). According to the standard procedure, RT-qPCR (CFX96TOUCH, SG) was used to analyze iNOS, CD206, TNF-α, CD86 mRNA levels using Bio-Rad CFX Manager 3.1 software. The concentration of VEGF secreted by cells in the collected supernatant was detected using the corresponding ELISA kit. All enzyme-linked immunosorbent assays were performed according to the manufacturer's instructions.

[0098] In vitro evaluation of angiogenesis: The supernatant of macrophages cultured on different hydrogels was aspirated and mixed with complete culture medium at a ratio of 1:1 to configure a conditioned medium. Human umbilical vein endothelial cells (HUVECs) were co-cultured with the conditioned medium for 7 days, then the cells were fixed and stained for VEGF. Stained samples were imaged using a laser confocal high-content screening system, and at least 3 fields were randomly selected for each sample. RT-qPCR was used to analyze the mRNA expression of b-FGF, Ang-1.

[0099] Experimental results:

[0100] Regarding the thermal stability of the hydrogels: From Figure 9 As can be seen from the differential scanning calorimetry (DSC) curve, the Td value of Col / OG hydrogel is 2.3°C higher than that of Col, and the Td value moves to a higher temperature with the increase of BTN content, and the highest Col / OG / 2%BTN can reach 47.1°C. This indicates that the cross-linking bonds formed between the materials provide additional support and enhancement for the triple helix structure.

[0101] Regarding the swelling properties of the hydrogels: Uncontrolled swelling can cause a sharp decrease in its mechanical properties, which is not conducive to its sustained protection of the wound. With the incorporation of OG and BTN, the swelling rate is further reduced, as Figure 10As shown, the percentages are 379% (Col / OG), 289% (Col / OG / 0.5% BTN), 217% (Col / OG / 1% BTN), and 182% (Col / OG / 2% BTN), respectively. The main reason for the decrease in swelling ratio is the covalent cross-linking of the Schiff base and the increase in porosity.

[0102] Regarding the degradation properties of hydrogels: Due to the presence of collagenase in the human body, pure collagen hydrogels degrade too quickly, limiting their widespread application. Figure 11 This indicates that at 60 pg mL -1 In the presence of collagenase, pure collagen hydrogels were completely degraded within 200 h. The degradation rate of Col / OG hydrogel was 91.23%, while the degradation rate of Col / OG / 2% BTN (36.12%) was significantly lower than that of Col / OG / 0.5% BTN (65.48%) and Col / OG / 1% BTN (50.15%). This indicates that BTN has good interfacial interactions with the organic network, leading to an increase in cross-linking bonds and a decrease in cleavage sites for collagenase attachment.

[0103] Regarding the mechanical properties of hydrogels: Figure 12 The mechanical properties of the hydrogels were tested. At 80% deformation, the compressive strength of the Col hydrogel was 46.26 kPa, and the compressive strength of the composite hydrogels was higher than that of the Col hydrogel. The addition of BTN further improved the compressive strength of the hydrogels; the compressive strength of the Col / OG / 1%BTN hydrogel (95.74 kPa) was higher than that of the Col / OG / 0.5%BTN (75.50 kPa) and Col / OG / 2%BTN (77.74 kPa). The results indicate that appropriate crosslinking can improve the compressive strength of the material.

[0104] Regarding the piezoelectric properties of hydrogels: Figure 13 The output voltage of the hydrogels was evaluated. Two electrodes were placed at the top and bottom of the hydrogel, respectively, and the output voltage was measured under a cyclic compressive force of approximately 4 N. The output voltages of the Col and Col / OG hydrogels were 4.47 mV and 4.67 mV, respectively. When the BTN content increased from 0 w / v to 2% w / v, the output voltage of the piezoelectric hydrogel increased from 30.02 mV to 90.59 mV. The results indicate that an appropriate increase in BTN content can improve the piezoelectric properties of the material.

[0105] Regarding the biocompatibility of hydrogels: Mouse fibroblasts (L929 cells) are a typical cell type used in wound healing studies. Cells were cultured on different hydrogels and sonicated three times daily (2.5 W / cm²). 2 Cell biocompatibility was determined using the CCK-8 assay. Figure 14As shown, the number of fibroblasts gradually increased with the extension of co-culture time, and the number of cells in Col / OG / 2% BTN hydrogel was the largest, followed by Col / OG / 1% BTN hydrogel. This increase was attributed to the electric stimulation generated by the piezoelectric scaffold, which promoted the growth of fibroblasts. In addition, the piezoelectric composite hydrogel generated an electric field under ultrasonic stimulation, which affected cell behavior and promoted adhesion and diffusion. The effect of piezoelectric stimulation of Col / OG / BTN composite hydrogel on L929 cell proliferation was observed by 5-ethyl-2'-deoxyuridine (EdU) method as Figure 15 Col / OG / 2% BTN hydrogel had excellent proliferation rate (41.74%), and under ultrasonic stimulation, the proliferation rate of cells on piezoelectric composite hydrogel was significantly different from that on non-piezoelectric composite hydrogel. The results showed that the piezoelectric effect positively regulated cell proliferation. The effect of piezoelectric Col / OG / BTN hydrogel on cell migration was evaluated by in vitro Transwell migration experiment and scratch experiment. Figure 16 It was shown that after 24h incubation, a significant number of cells in piezoelectric hydrogel were stained with crystal violet compared with non-piezoelectric hydrogel. In addition, the wound site was reconstructed in the culture well to generate an acellular area, and the migration of cells to the scratched area was observed, and the results showed that Col / OG / 2% BTN hydrogel had the most significant promotion effect on fibroblast migration under ultrasonic stimulation.

[0106] Regarding the immune regulation performance of macrophages on hydrogel: The dynamic transformation of immune microenvironment is crucial for tissue repair and regeneration after injury. Inflammatory factors and cells, such as macrophages, show high sensitivity and plasticity to changes in microenvironment. When macrophage polarization is imbalanced, excessive accumulation of M1 macrophages leads to persistent inflammation, thereby inhibiting angiogenesis and interfering with fibroblast maturation. Therefore, in order to further understand the effect of piezoelectric stimulation on macrophage reprogramming, we polarized macrophages to M1 phenotype by treating macrophages with LPS and IFN-γ as a control group. Figure 17The process of macrophage polarization co-cultured with collagen-based piezoelectric hydrogel and the phenotype changes of macrophages under piezoelectric stimulation are shown. RAW264.7 macrophages were immunofluorescently stained for CD206 (M2 macrophage surface marker) and iNOS (M1 macrophage surface marker). After 2 days of co-culture, the control group had the highest iNOS fluorescence intensity. The iNOS fluorescence intensity in the collagen-based hydrogel showed a downward trend, while the CD206 fluorescence intensity in the Col / OG / 2%BTN piezoelectric hydrogel was significantly higher than that in the other groups. In addition, RT-qPCR was used to quantify pro-inflammatory genes (iNOS, CD86) and anti-inflammatory genes (CD206), and the results were consistent with the immunofluorescent staining. To further study whether the ultrasound-stimulated hydrogel could promote the secretion of the regenerative cytokine vascular endothelial growth factor (VEGF), enzyme-linked immunosorbent assay (ELISA) was used to detect the VEGF protein expression level. The study showed that the VEGF level was significantly increased. These results indicate that the incorporation of piezoelectric material BTN can significantly enhance the immune regulation function of macrophages, promote the reprogramming of M1 macrophages to M2 macrophages, change the immune microenvironment, and shorten the inflammation period. In addition, anti-inflammatory phenotype macrophages are proven to actively produce the pro-healing growth factor VEGF, which is considered necessary for angiogenesis in the proliferation phase.

[0107] Regarding the angiogenic performance of the hydrogel: After co-culturing different hydrogels with macrophages under ultrasound stimulation for 2 days, the supernatant was mixed with an equal amount of complete culture medium to culture huvec and study the angiogenic ability. Figure 18 The results show that the VEGF fluorescence intensity of HUVECs co-cultured with macrophage supernatant using Col / OG / 2%BTN hydrogel is significantly higher than that of other groups, indicating that the regulation of the immune microenvironment under piezoelectric stimulation effectively promotes angiogenesis. In addition, the RT-PCR results show that the expression of angiogenesis-related genes b-FGF and Ang-1 is also significantly up-regulated in HUVECs cultured in macrophage-conditioned medium using Col / OG / 2%BTN hydrogel.

[0108] Example 4

[0109] The same as Example 1, except that:

[0110] S4, 50 mg of collagen obtained in step S1, oxidized gellan gum obtained in step S2, and aminated barium titanate obtained in step S3 were dissolved in hexafluoroisopropanol solvent and mixed at room temperature for 2 days to obtain a mixed solution. The concentration of fish skin collagen in the mixed solution was 10 wt%, the concentration of oxidized gellan gum was 3 wt%, and the concentration of aminated barium titanate was 3 wt%. The mixed solution was then electrospun. The electrospinning parameters were as follows: flow rate was 20 μL / min, spinning distance was 10 cm, voltage was 15 kV, and drum rotation speed was 500 rpm. The electrospun product was soaked in PBS for swelling to obtain Col / OG / BTN piezoelectric hydrogel.

[0111] Example 5

[0112] The same as example 4, except that: collagen, oxidized gellan gum obtained in step S2 and aminated barium titanate obtained in step S3 are dissolved in hexafluoroisopropanol solvent, mixed at room temperature for 2 days to obtain a mixed solution, the concentration of fish skin collagen in the mixed solution is 2wt%, oxidized gellan gum 1wt%, aminated barium titanate 1wt%, and then the mixed solution is electrospun, the parameters of electrospinning are: flow rate is 5μL / min, spinning distance is 2cm, voltage is 2kV, drum speed is 100rmp, Col / OG / BTN hydrogel is obtained.

[0113] Example 6

[0114] The same as example 4, except that: collagen, oxidized gellan gum obtained in step S2 and aminated barium titanate obtained in step S3 are dissolved in hexafluoroisopropanol solvent, mixed at room temperature for 2 days to obtain a mixed solution, the concentration of fish skin collagen in the mixed solution is 20wt%, oxidized gellan gum 5wt%, aminated barium titanate 5wt%, and then the mixed solution is electrospun, the parameters of electrospinning are: flow rate is 100μL / min, spinning distance is 30cm, voltage is 30kV, drum speed is 4000rmp, Col / OG / BTN hydrogel is obtained.

[0115] Example 7

[0116] The same as example 3, except that:

[0117] Step S2: the concentration of gellan gum GG solution is 8g / L, the gellan gum GG solution is cooled to 40℃, sodium periodate is added, stirred at room temperature in the dark for 7 hours, then transferred to dialysis membrane, dialyzed with deionized water for 4 days, and freeze-dried to obtain oxidized gellan gum OG. The molar ratio of gellan gum GG to sodium periodate is 1:0.85.

[0118] Step S3: barium titanate BTO powder is added to H2O2 and stirred at 105℃ for 3h, (3-aminopropyl)trimethoxysilane is added dropwise and refluxed at 75℃ for 22h. The molar ratio of barium titanate BTO to H2O2 is 0.2:1, the mass-volume ratio of barium titanate BTO to ethanol solution is 1:3g / mL, and the mass ratio of barium titanate BTO to (3-aminopropyl)trimethoxysilane is 1.5:1.

[0119] Step S4: the concentration of OG solution is 0.05g / mL, and the volume ratio of collagen solution to OG solution is 8:1.

[0120] Example 8

[0121] The same as example 3, except that:

[0122] Step S2: The concentration of the gellan gum GG solution was 12 g / L, the gellan gum GG solution was cooled to 40℃, sodium periodate was added, and the mixture was stirred at room temperature in the dark for 10 hours, then transferred to a dialysis membrane and dialyzed with deionized water for 6 days, and then freeze-dried to obtain the oxidized gellan gum OG. The molar ratio of gellan gum GG to sodium periodate was 1:0.95.

[0123] Step S3: The barium titanate BTO powder was added to H2O2 and stirred at 105℃ for 5h, and (3-aminopropyl)trimethoxysilane was added dropwise and refluxed at 85℃ for 18h. The molar ratio of barium titanate BTO to H2O2 was 0.3:1, the mass-volume ratio of barium titanate BTO to ethanol solution was 1:5 g / mL, and the mass ratio of barium titanate BTO to (3-aminopropyl)trimethoxysilane was 2.5:1.

[0124] Step S4: The concentration of the OG solution was 0.2 g / mL, and the volume ratio of the collagen solution to the OG solution was 12:1.

[0125] The above examples are only used to help understand the technical solutions of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A tilapia collagen piezoelectric hydrogel, characterized in that, The piezoelectric hydrogel is a composite hydrogel of tilapia collagen / oxidized gellan gum / amino barium titanate. The amino barium titanate consists of approximately spherical nanoparticles with a diameter of less than 100 nm. The piezoelectric hydrogel exhibits an irregular interconnected porous structure. The compressive strength of the piezoelectric hydrogel at 80% deformation is 75.50~95.74 kPa, and the output voltage is 30.02~90.59 mV. The output voltage is tested by placing two electrodes at the top and bottom of the hydrogel and measuring the output voltage under a cyclic compressive force of 4 N.

2. The method for preparing the tilapia collagen piezoelectric hydrogel according to claim 1, characterized in that, Includes the following steps: S1. Preparation of tilapia collagen: Dissolve tilapia collagen in acetic acid solution to obtain acidic collagen solution. Mix acidic collagen solution with PBS solution, adjust pH to neutral, and obtain collagen solution Col. S2. Sodium periodate is added to the gellan gum GG solution for oxidation to obtain oxidized gellan gum OG; S3. Add barium titanate (BTO) to H2O2 and stir. Wash and dry the mixture. Add the dried substance to an ethanol solution and add (3-aminopropyl)trimethoxysilane dropwise. Reflux and react. After washing and drying, amino-modified barium titanate (BTN) is obtained. S4. Dissolve oxidized gellan gum (OG) in water to prepare an OG solution. Mix the collagen solution obtained in step S1 with the OG solution to obtain a Col / OG solution. Add the BTN powder obtained in step S3 to the Col / OG solution and stir until completely mixed. The concentration of the OG solution is 0.05-0.2 g / mL, the volume ratio of the collagen solution to the OG solution is 8-12:1, and the mass-volume ratio of BTN to the Col / OG solution is 0.005-0.02 g / mL to obtain a Col / OG / BTN piezoelectric hydrogel. Alternatively, the collagen obtained in step S1, the oxidized gellan gum obtained in step S2, and the barium titanate aminoide obtained in step S3 can be dissolved in a solvent and mixed at room temperature for 1-3 days to obtain a mixed solution. The concentration of fish skin collagen in the mixed solution is 2-20 wt%, oxidized gellan gum is 1-5 wt%, and barium titanate aminoide is 1-5 wt%. The mixed solution is then electrospun and soaked in PBS to swell, resulting in Col / OG / BTN piezoelectric hydrogel.

3. The preparation method according to claim 2, characterized in that, The tilapia collagen in step S1 is prepared by the following steps: tilapia skin is washed, then soaked in a mixed solution containing butanol and isopropanol, and then transferred to acetic acid solution and stirred overnight until the collagen is dissolved. The resulting crude collagen solution is centrifuged to remove particles, and the supernatant is added to NaCl solution to precipitate the collagen and stirred overnight. The collagen precipitate is then collected from the centrifuged liquid and dissolved in acetic acid. The resulting solution is dialyzed and freeze-dried to obtain pure collagen.

4. The preparation method according to claim 2 or 3, characterized in that, The specific steps for mixing the acidic collagen solution with PBS solution in step S1 and adjusting the pH to neutral to obtain collagen solution Col are as follows: Mix the acidic collagen solution with 10×PBS solution at 4℃, with a volume ratio of 9:

1. Then, add NaOH solution to adjust the pH to neutral to obtain collagen solution Col, with a concentration of 3-6 g / L.

5. The preparation method according to claim 2, characterized in that, The specific steps of step S2 are as follows: dissolve gellan gum GG in deionized water, heat to 80℃-100℃ until it becomes a gellan gum GG solution with a concentration of 8-12 g / L, cool the gellan gum GG solution to 40℃, add sodium periodate, stir at room temperature in the dark for 7-10 hours, then transfer to a dialysis membrane, dialyze with deionized water for 4-6 days, and freeze-dry to obtain oxidized gellan gum OG.

6. The preparation method according to claim 2, characterized in that, The specific steps of step S3 are as follows: First, add barium titanate BTO powder to H2O2, stir vigorously at 105℃ for 2-6 h, wash, and vacuum dry. Add the dried substance to an ethanol solution, and add (3-aminopropyl)trimethoxysilane dropwise. Reflux at 75℃-85℃ for 16-24 h, wash, and vacuum dry to obtain amino-modified barium titanate BTN.

7. The preparation method according to claim 6, characterized in that, The molar ratio of barium titanate (BTO) to H₂O₂ is 0.1-0.3:1, the mass-to-volume ratio of barium titanate (BTO) to ethanol solution is 1:3-5 g / mL, and the mass ratio of barium titanate (BTO) to (3-aminopropyl)trimethoxysilane is 1.5-2.5:

1.

8. The preparation method according to claim 2, characterized in that, The parameters for electrospinning in step S4 are: flow rate of 5-100 μL / min, spinning distance of 2-30 cm, voltage of 2-30 kV, and roller speed of 100-4000 rmp.

9. The application of the tilapia collagen piezoelectric hydrogel according to claim 1 in the preparation of biomaterials for treating tissue damage.

10. A biomaterial for treating tissue damage, characterized in that, The active ingredient is the tilapia collagen piezoelectric hydrogel as described in claim 1.

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