Anisotropic cellulose piezoelectric hydrogel and application thereof in stem cell culture
Through a radio stimulation system that combines polydopamine-modified anisotropic cellulose piezoelectric hydrogel with ultrasound, the invasiveness and infection risks of traditional electrical stimulation methods are solved, and the safe transmission of radio signals and efficient neural differentiation of stem cells are achieved.
Patent Information
- Application Number
- CN202410175547.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-07
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art, neural network structures caused by nerve damage and neurodegenerative diseases are irreversible, and the induction of stem cells to neural differentiation is difficult to achieve, and traditional electrical stimulation methods require wire connections, resulting in secondary damage and infection risks.
Polydopamine-modified anisotropic cellulose piezoelectric hydrogels are used to provide radio signals through ultrasonic mechanical stimulation, and combined with chemical crosslinking and pre-stretching techniques, hydrogels with high voltage electrical output, antioxidant and biocompatible are prepared for radio stimulation and neural differentiation of stem cells.
It realizes the safe, controllable and continuous electrical signal transmission of the radio stimulation system, promotes the neural differentiation and directional arrangement of stem cells, reduces the risk of surgery, and improves the development efficiency of neurons.
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Figure CN120441915A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedical engineering, and in particular to anisotropic cellulose piezoelectric hydrogel and application thereof in stem cell culture. Background Art
[0002] Neurotrauma and neurodegenerative diseases, such as Alzheimer's and Parkinson's, can lead to irreversible damage to neural network architecture and cell death. Regeneration of damaged nervous systems remains a major challenge in neuroscience. Therefore, rapidly inducing stem cell differentiation to produce functional neurons to replace damaged cells has become a key challenge in treating neural injury.
[0003] In vivo electrical stimulation (ES) delivered via an external power source and percutaneous wires has been shown to significantly promote stem cell neural differentiation and neurogenesis. However, it still requires wires to connect implanted electrodes to stimulate cells. Furthermore, additional surgery is required to remove the electrodes or wires after stimulation. These surgeries may lead to secondary injury and infection. Therefore, there is an urgent need for a low-cost, safer, more durable, less invasive, and more controllable wireless local electrical stimulation system for assisted neuronal differentiation. Summary of the Invention
[0004] According to one aspect of the present application, a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, comprising: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.
[0005] In some embodiments, the modification amount of polydopamine is 0.4%-2% based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel. Optionally, the stretchability of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%.
[0006] According to another aspect of the present application, a method for preparing a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, comprising:
[0007] In an alkaline solution, dopamine is in situ polymerized on the anisotropic cellulose piezoelectric hydrogel to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.
[0008] In some embodiments, the preparation method comprises immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH value of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then washing with water to terminate the reaction to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel. Optionally, the alkaline solution is Tris-HCl buffer.
[0009] In some embodiments, the anisotropic cellulose piezoelectric hydrogel is prepared by the following steps:
[0010] 1) adding a crosslinking agent to the piezoelectric cellulose solution to chemically crosslink and gel to obtain a cellulose piezoelectric hydrogel; and
[0011] 2) Pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to align the cellulose in the cellulose piezoelectric hydrogel, and then immersing it in an aqueous sulfuric acid solution to permanently fix the oriented structure through physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.
[0012] In some embodiments, step 1) comprises dissolving the piezoelectric cellulose in a lithium hydroxide and urea solution to obtain the piezoelectric cellulose solution. Optionally, in the lithium hydroxide and urea solution, the concentration of lithium hydroxide is 3.6-5.4 wt %, and the concentration of urea is 12-18%. Preferably, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1 wt %-5 wt %. Optionally, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled at a temperature of -20°C to 0°C.
[0013] Optionally, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose and cotton.
[0014] Optionally, the cross-linking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin and polyethylene glycol diglycidyl ether,
[0015] Optionally, in step 1), the molar ratio of the cross-linking agent to the deoxyglucose unit in the piezoelectric cellulose solution is 1:1-3:1,
[0016] Optionally, step 1) includes dropwise adding the crosslinking agent to the cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring into a template, and gelling at 0-10° C. for 10 h to 100 h to obtain the cellulose piezoelectric hydrogel;
[0017] Optionally, the stretching ratio of the pre-stretching in step 2) is 80%-215%;
[0018] Optionally, step 2) comprises soaking the pre-stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution for physical crosslinking and terminating chemical crosslinking, and then washing with water to obtain the anisotropic cellulose piezoelectric hydrogel. Preferably, the concentration of the dilute sulfuric acid solution is 1 wt%-5 wt%.
[0019] According to another aspect of the present application, there is provided a polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to any of the aforementioned embodiments or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments for use as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.
[0020] According to another aspect of the present application, a wireless electric stimulation system is provided, comprising: a polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to any of the aforementioned embodiments, or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the aforementioned embodiments; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the action of ultrasound of the ultrasonic device.
[0021] Optionally, the ultrasonic frequency, ultrasonic intensity and ultrasonic time of the ultrasonic device can be programmably controlled.
[0022] Optionally, the ultrasound intensity of the ultrasound action is 0-5W / cm 2 .
[0023] According to another aspect of the present application, a method for culturing stem cells is provided, comprising contacting the stem cells with the aforementioned polydopamine-modified anisotropic cellulose piezoelectric hydrogel or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the aforementioned preparation method for culturing, and applying ultrasound so that the electrical stimulation generated by the polydopamine-modified anisotropic cellulose piezoelectric hydrogel under the ultrasound promotes the neural differentiation, development and / or directional arrangement of the stem cells.
[0024] In some embodiments, the stem cells are neural stem cells, mesenchymal stem cells or embryonic stem cells. Optionally, the ultrasound frequency, ultrasound intensity and ultrasound time of the ultrasound action can be programmably controlled. Optionally, the ultrasound intensity of the ultrasound action is 0-5 W / cm 2 .
[0025] Beneficial effects
[0026] The technical solution of this application can achieve at least one of the following effects:
[0027] (1) The polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in this application has the advantages of high piezoelectric output, antioxidant properties, flexibility, good biocompatibility, and degradability. The polydopamine-modified anisotropic cellulose hydrogel gives the cellulose hydrogel antioxidant ability and increases the piezoelectric output performance.
[0028] (2) The polydopamine-modified anisotropic cellulose piezoelectric hydrogel structure provided in this application can simulate the directional structure of regenerated natural tissues (such as nerves) and support and guide axonal projection.
[0029] (3) By using programmed ultrasound as a remote mechanical stimulus, the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of the present application can provide on-demand wireless electrical stimulation (ES) with adjustable timing, duration, and intensity.
[0030] (4) Programmable ultrasound combined with polydopamine-modified anisotropic cellulose piezoelectric hydrogel forms a radio stimulation system or radio neural regulation system, which provides on-demand radio stimulation, accelerates the process of stem cell differentiation and promotes neuronal development. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the embodiments of the present application 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. The drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0032] Figure 1 A schematic diagram of the process for preparing polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH) according to an embodiment of the present application is shown.
[0033] Figure 2 The stretchability of the chemically cross-linked cellulose hydrogel prepared in the examples of the present application is shown.
[0034] Figure 3 A physical picture of PDA / ACH-4 prepared in an example of the present application is shown.
[0035] Figure 4 Scanning electron microscope images of chemically cross-linked cellulose hydrogels ACH-1 (before stretching) and ACH-4 (after stretching, stretching rate 120%) prepared in the examples of the present application are shown, where the left image is ACH-1 and the right image is ACH-4.
[0036] Figure 5 The scanning electron microscopy observation and elemental analysis results of the polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH-4) prepared in the examples of the present application are shown, wherein the left figure is a scanning electron microscope image of PDA / ACH-4, and the right figure is an elemental analysis diagram of PDA / ACH-4.
[0037] Figure 6 The two-dimensional small-angle X-ray scattering (SAXS) (inset) and the azimuthal integrated intensity distribution curve of the SAXS pattern of different cellulose hydrogels prepared according to the examples of the present application are shown, where 0° represents the vertical direction.
[0038] Figure 7The piezoelectric characterization results of different cellulose hydrogels prepared in the examples of the present application are shown, where a: piezoelectric output of ACH-1, ACH-4, and PDA / ACH-4 under ultrasound, b: schematic diagram of ultrasonic piezoelectricity, c: butterfly amplitude ring of PDA / ACH-4, and d: phase potential hysteresis.
[0039] Figure 8 The antioxidant characterization results of different cellulose hydrogels prepared in the examples of this application are shown.
[0040] Figure 9 A schematic diagram of the PDA / ACH wireless stimulation system prepared based on an embodiment of the present application to promote neural differentiation and directional arrangement of neural stem cells is shown.
[0041] Figure 10 Real-time quantitative PCR is shown, in which the expression of neural-related target genes (Tuj1 and MAP2) was detected in neural stem cells cultured on glass slides (TCP) and PDA / ACH-4 on the 7th day without and with radiofrequency stimulation, respectively. The left figure shows the expression level of Tuj1, and the right figure shows the expression level of MAP2.
[0042] Figure 11 Representative immunostaining images of the mature neuron-specific marker MAP2 (red), the astrocyte marker GFAP (green), and the nuclear marker DAPI (blue) after 7 days of radio-frequency stimulation culture on TCP and PDA / ACH-4 are shown, where, from the left, Figure 1: TCP; Figure 2: TCP + ultrasound stimulation (US); Figure 3: PDA / ACH-4; Figure 4: PDA / ACH-4 + US.
[0043] Figure 12 Shown are the MAP2+ percentages for 7 days of TCP and PDA / ACH-4 radioelectric stimulation (n=5). DETAILED DESCRIPTION
[0044] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions of this application will be described in detail below. Obviously, the embodiments described are only some of the embodiments of this application, rather than all of them. Based on the embodiments in this application, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of this application.
[0045] According to one aspect of the present application, a polydopamine-modified anisotropic cellulose piezoelectric hydrogel is provided, which comprises: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.
[0046] The anisotropic cellulose piezoelectric hydrogel skeleton can also be called a multi-dimensionally oriented cellulose piezoelectric hydrogel skeleton. The anisotropy is generated from the directional distribution of cellulose chains in the hydrogel network.
[0047] Herein, "piezoelectric hydrogel" refers to a hydrogel having a piezoelectric effect, including but not limited to anisotropic cellulose hydrogel and bacterial cellulose hydrogel.
[0048] In some embodiments, the modification amount of polydopamine is 0.4%-2%, for example, 0.4%, 0.5%, 1%, 1.5%, or 2%, based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.
[0049] In some embodiments, the stretchability of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%, for example, 80%, 100%, 120%, 180%, 200%, 215%, or any value within a range thereof. As used herein, "stretchability" refers to the rate of change in the length of the stretched hydrogel (hydrogel skeleton) relative to the length of the hydrogel (hydrogel skeleton) before stretching in the stretching direction. This can improve the piezoelectric properties and oriented structure of the cellulose piezoelectric hydrogel.
[0050] According to another aspect of the present application, a method for preparing anisotropic cellulose piezoelectric hydrogel modified with polydopamine is provided, comprising: in an alkaline solution, in situ polymerizing dopamine on the anisotropic cellulose piezoelectric hydrogel to obtain the anisotropic cellulose piezoelectric hydrogel modified with polydopamine.
[0051] In some embodiments, a method for preparing an anisotropic cellulose piezoelectric hydrogel modified with polydopamine is provided, comprising immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then terminating the reaction by washing with water to obtain the anisotropic cellulose piezoelectric hydrogel modified with polydopamine. In one embodiment, the alkaline solution is a Tris-HCl buffer. This can improve the piezoelectric properties and biocompatibility of the cellulose piezoelectric hydrogel and impart antioxidant properties to the cellulose piezoelectric hydrogel.
[0052] In some embodiments, a method for preparing an anisotropic cellulose piezoelectric hydrogel is provided, comprising: 1) adding a cross-linking agent to a piezoelectric cellulose solution to chemically cross-link and gelate to obtain a cellulose piezoelectric hydrogel; and 2) pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to directional align the cellulose in the cellulose piezoelectric hydrogel, followed by immersing the pre-stretched cellulose in an aqueous sulfuric acid solution to permanently fix the directional aligned structure by physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.
[0053] In some embodiments, physical crosslinking is achieved through hydrogen bonding.
[0054] In some embodiments, step 1) includes dissolving a piezoelectric cellulose source in a lithium hydroxide and urea solution to obtain a piezoelectric cellulose solution. According to one embodiment, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1 wt%-5 wt%. In some embodiments, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 2 wt%. In some embodiments, the concentration of lithium hydroxide in the lithium hydroxide and urea solution is 3.6-5.4 wt%, and the concentration of urea is 12-18 wt%, for example, the concentration of lithium hydroxide is 4.5 wt%, and the concentration of urea is 15 wt%. This can produce a cellulose hydrogel with a low elastic modulus and a longer tensile length, providing the prerequisite for pre-stretching.
[0055] In some embodiments, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled to a temperature between -20°C and 0°C, for example, at -18°C, thereby obtaining a cellulose solution with higher solubility.
[0056] In some embodiments, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose, and cotton (e.g., cotton lint). In some embodiments, the piezoelectric cellulose is derived from filter paper, such as Advantec Toyo quantitative filter paper.
[0057] In some embodiments, the crosslinking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin, and polyethylene glycol diglycidyl ether. In some embodiments, the crosslinking agent is epichlorohydrin. Thus, a cellulose piezoelectric hydrogel with chemical crosslinking of hydroxyl groups is obtained.
[0058] In some embodiments, in step 1), the molar ratio of the crosslinking agent to the deoxyglucose units in the piezoelectric cellulose solution is 1:1-3:1, for example, 1.5:1. This can produce a cellulose hydrogel with a low elastic modulus and a longer tensile length, providing a prerequisite for pre-stretching.
[0059] In some embodiments, step 1) includes dropwise adding a crosslinking agent to the piezoelectric cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring the product into a template, and gelling at 0-10° C. for 10-100 hours to obtain the cellulose piezoelectric hydrogel. In some embodiments, gelling is performed at 4° C. for 30 hours.
[0060] In some embodiments, in step 2), the cellulose piezoelectric gel is stretched by 80% to 215% of its original length, thereby improving the piezoelectric properties and oriented structure of the cellulose piezoelectric hydrogel.
[0061] In some embodiments, step 2) includes immersing the stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution to physically crosslink and terminate chemical crosslinking, followed by washing with water to obtain an anisotropic cellulose piezoelectric hydrogel. According to one embodiment, the concentration of the dilute sulfuric acid solution is 1 wt% to 5 wt%, for example, 3 wt%. This results in an anisotropic cellulose piezoelectric hydrogel with a lower elastic modulus.
[0062] According to another aspect of the present application, there is provided the use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in any of the above embodiments or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any of the above embodiments as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.
[0063] According to another aspect of the present application, a radio stimulation system or a radio neural regulation system is provided, comprising: a polydopamine-modified anisotropic cellulose piezoelectric hydrogel provided in any of the above embodiments or a polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared by the preparation method according to any of the above embodiments; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the ultrasonic action of the ultrasonic device.
[0064] In some embodiments, the ultrasonic frequency, ultrasonic intensity, and ultrasonic time of the ultrasonic device in the wireless stimulation system or wireless neural regulation system provided by the present application can be programmably controlled. In some embodiments, the ultrasonic intensity of the ultrasonic action in the wireless stimulation system or wireless neural regulation system provided by the present application is 0-5W / cm 2 .
[0065] According to another aspect of the present application, a method for culturing stem cells is provided, comprising contacting the stem cells with the polydopamine-modified anisotropic cellulose piezoelectric hydrogel of the present application for culturing, and applying ultrasound so that the electrical signal generated by the polydopamine-modified anisotropic cellulose piezoelectric hydrogel under ultrasound stimulates and promotes the neural differentiation, development and / or directional arrangement of the stem cells.
[0066] In some embodiments, the stem cells are neural stem cells, mesenchymal stem cells, or embryonic stem cells. In particular, the embryonic stem cells are not obtained from human embryos, but are embryonic stem cell lines.
[0067] In some embodiments, the stem cells of the present application are selected from neural stem cells, adipose-derived, bone marrow-derived, dental pulp-derived, or umbilical cord-derived mesenchymal stem cells, hematopoietic stem cells, and embryonic stem cells.
[0068] In some embodiments, in the stem cell culture method provided herein, the ultrasound frequency, ultrasound intensity, and ultrasound time of the ultrasound action can be programmably controlled. In some embodiments, the ultrasound intensity of the ultrasound action is 0-5 W / cm 2 .
[0069] The present application prepares polydopamine-modified anisotropic cellulose piezoelectric hydrogels by chemical crosslinking, pre-stretching, physical crosslinking and in situ polymerization. By increasing the structural order and polydopamine modification, the piezoelectric properties of the hydrogel are improved. The hydrogel is used as an acoustic energy generator to convert ultrasonic energy into electrical energy, especially radio signals. The radio stimulation system or radio neural regulation system based on the polydopamine-modified anisotropic cellulose piezoelectric hydrogel combined with ultrasound can provide an antioxidant immune microenvironment and on-demand radio stimulation to accelerate neural stem cell-based neural regeneration and repair. Through programmed ultrasound as a remote mechanical stimulation, the anisotropic cellulose piezoelectric hydrogel can provide on-demand electrical stimulation with adjustable time, duration and intensity, activate the PI3K-AKT pathway to enhance the differentiation of nerve stems into neurons, promote the development of neurons, and promote the directional arrangement of differentiated cells.
[0070] Example
[0071] The test materials used in the examples of this application are all conventional test materials in the field and can be purchased through commercial channels.
[0072] Example 1
[0073] Preparation of polydopamine (PDA) modified anisotropic cellulose hydrogel (PDA / ACH), such as Figure 1 As shown:
[0074] (1) Preparation of chemically cross-linked cellulose hydrogel: Advantec Toyo quantitative filter paper was completely dried at 60°C and dissolved in a 4.5wt% lithium hydroxide / 15wt% urea solution pre-cooled at -18°C to obtain a transparent cellulose solution with a concentration of 2wt%. Epichlorohydrin was added dropwise to the dissolved cellulose solution, and the crude cellulose gel containing an epichlorohydrin (ECH) / anhydroglucose unit (AGI) molar ratio of 1.5 was centrifuged to remove bubbles, poured into the template, and reacted in a refrigerator at 4°C for 30 hours to obtain a chemically cross-linked cellulose hydrogel.
[0075] (2) Preparation of anisotropic cellulose hydrogel (ACH): Applying external force to the chemically cross-linked cellulose hydrogel obtained in step (1) to stretch the cellulose molecular chains inside the gel to cause the cellulose hydrogel to be oriented, such as Figure 2As shown in Figure 2, the stretchability of the chemically cross-linked hydrogel reached 215%. Using a hydrogel stretched to a specific length ratio of 120%, and soaked in a 3% H2SO4 solution, the oriented structure was permanently fixed by physical cross-linking (hydrogen bonding) between adjacent cellulose chains, and thoroughly washed with water, anisotropic cellulose hydrogel (ACH) was obtained.
[0076] (3) Preparation of polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH): The ACH obtained in step (2) was immersed in a 10 μM Tris-HCl buffer solution with a pH value of 8.8, and then dopamine at a concentration of 2 mg / mL was added for 2 hours. The reaction was terminated by repeated washing with water. Figure 3 As shown, a polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH) was obtained, wherein the modification amount of polydopamine was 0.96% based on the mass percentage of the N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.
[0077] Example 2 Investigation of the piezoelectric and antioxidant properties of anisotropic cellulose hydrogels
[0078] To facilitate subsequent stem cell differentiation into neurons, an anisotropic cellulose piezoelectric hydrogel with a smaller modulus was selected. The chemically cross-linked cellulose hydrogel was treated with a pre-stretch ratio of 120%. Other steps were followed as in Example 1 to prepare anisotropic cellulose hydrogel (ACH) and polydopamine-modified anisotropic cellulose hydrogel (PDA / ACH), yielding samples ACH-4 and PDA / ACH-4, respectively. The resulting anisotropic cellulose hydrogels were characterized by scanning electron microscopy.
[0079] like Figure 4 As shown, the left figure ACH-1 represents the chemically cross-linked cellulose hydrogel before stretching, which shows no directionality, while the right figure ACH-4 represents the chemically cross-linked cellulose hydrogel after stretching (stretching rate 120%), which has obvious directionality. Figure 5 The scanning electron microscopy observation and elemental analysis results of PDA / ACH-4 show that PDA / ACH-4 also has order, indicating that PDA modification does not affect the order of ACH. The nitrogen content in the elemental analysis is 0.96%, indicating the successful modification of PDA.
[0080] Two-dimensional small-angle X-ray scattering (SAXS) was used to characterize the effects of stretching and modification with different PDA contents on the anisotropy of the hydrogels. Figure 6, inset). For the ACH-1 hydrogel without pre-stretching treatment, a uniform intensity ring appears in the SAXS pattern, reflecting an isotropic structure. When the hydrogel is stretched to 120%, ACH-4 forms a sharp, elongated equatorial stripe, and the ring intensity is anisotropic. The results show that stress-induced submicrobeam orientation occurs along the stress direction, forming an anisotropic structure. At the same time, the azimuth-integrated intensity distribution curve of the pre-stretched hydrogel produces a sharp peak at 90° ( Figure 6 ), further demonstrating the anisotropy of the hydrogel. Furthermore, PDA modification did not significantly change the azimuthal integrated intensity distribution curve, indicating that PDA modification had little effect on the orientation of the cellulose hydrogel.
[0081] The piezoelectric properties of PDA / ACH-4 were tested by ultrasound and oscilloscope. The results are as follows: Figure 7 shown.
[0082] like Figure 7 As shown in Figure a, the voltage of ordered ACH-4 is approximately 1 V, significantly higher than that of disordered ACH-1 (approximately 0.5 V), indicating that the ordered structure improves the piezoelectric properties of the cellulose hydrogel. Furthermore, it was found that PDA modification significantly improved the piezoelectric properties of ACH, with the PDA / ACH-4 voltage reaching 1.5 V. This indicates that the ordered structure and the addition of PDA enhance the piezoelectric properties of the cellulose hydrogel.
[0083] The local piezoelectric activity of the hydrogel was studied using atomic force microscopy, and its amplitude image ( Figure 7 c) and phase image ( Figure 7 d). The hydrogel's piezoelectric response amplitude curve, also known as the butterfly curve, directly reflects the strain response of the area below the conductive probe under the action of the electric field.
[0084] like Figure 7 As shown in Figures 7c and 7d, when the bias voltage increases from -10V to +10V, the amplitude curve of PDA / ACH-4 exhibits significant hysteresis, sufficient to achieve polarity reversal. Regarding the piezoelectric properties of PDA / ACH-4, the phase curve of PDA / ACH-4 shows a wide hysteresis loop for the ±10V tip excursion, indicating that the polarity of PDA / ACH-4 exists and can be reversed in the presence of an applied electric field. This polarity reversal is primarily due to the presence of the polar β phase. These experimental results also demonstrate the excellent piezoelectric properties of PDA / ACH-4.
[0085] The antioxidant potential of various cellulose hydrogels was investigated using a 1-diphenyl-2-pyridylhydrazide (DPPH) free radical (DPPH) assay. DPPH free radical scavenging activity was determined by immersing 300 μL of different hydrogels (10 mg / mL hydrogel aqueous solution) or water (control) in 700 μL of a 0.1 mM DPPH solution (95 wt% ethanol) in the dark for 6 hours. The optical density (OD) of the mixture was measured at 517 nm using a microplate reader (BioTekCytation 3). The DPPH free radical scavenging activity was calculated using the following equation.
[0086]
[0087] like Figure 8 As shown, the scavenging capacity of the PDA-modified cellulose hydrogel PDA / ACH-4 was significantly higher than that of ACH-1 and ACH-4, reaching over 60%. The abundant antioxidant groups in PDA (such as phenolic hydroxyl groups) are responsible for scavenging ROS. Therefore, the PDA-modified hydrogel has excellent antioxidant activity.
[0088] Example 3
[0089] Polydopamine modified anisotropic cellulose piezoelectric hydrogel promotes neural differentiation and directional arrangement of neural stem cells, such as Figure 9 As shown:
[0090] The composition of neural stem cell proliferation medium is: DMEM / F12 + 2% B-27 supplement + 1% N-2 supplement + basic fibroblast growth factor (bFGF, 20ng mL -1 )+epidermal growth factor (EGF, 20ng mL -1 );
[0091] Neural stem cell differentiation medium: Neurobase 1 medium + 1% L-glutamine + 2% B-27 supplement + 1% double antibody + 1% fetal bovine serum.
[0092] (1) First, the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared in Examples 1 and 2 was attached to a cell culture plate. After the hydrogel was sterilized, a 2×10 5 A cell suspension of neural stem cells from SD rats was inoculated on polydopamine-modified anisotropic cellulose piezoelectric hydrogel. After resting for 20 minutes, an appropriate amount of neural stem cell proliferation culture medium was added to obtain a culture system. After culturing for 24 hours, when the neural stem cells were completely attached, the proliferation culture medium was replaced with differentiation culture medium.
[0093] (2) The above culture system was placed under an ultrasonic device (SCIENTZ-IID) and treated with 0W and 400W respectively under ultrasound to obtain a wireless radio stimulation system based on anisotropic cellulose piezoelectric hydrogel modified with polydopamine;
[0094] (3) The radio signals generated by the radio stimulation system are used to promote neural differentiation of neural stem cells. The treatment time is 7 days, 8 minutes each time, and twice a day.
[0095] At the same time, the glass slide (TCP) group was used as the control experimental group.
[0096] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to examine the effects of different materials on promoting neural differentiation under radiofrequency stimulation. Expression of neural target genes (Tuj1, MAP2, and GFAP) was detected in rNSCs cultured on TCP and PDA / ACH-4 cells on day 7 under radiofrequency stimulation. βIII tubulin (Tuj1) is an early marker of neuronal differentiation in stem cells. Microtubule-associated protein 2 (MAP2) is commonly used as a marker for mature neurons. Glial fibrillary acidic protein (GFAP) is a marker for astrocytes.
[0097] RT-qPCR results are as follows Figure 10 As shown in the data, under this radio stimulation, neural stem cells cultured on TCP and PDA / ACH-4 on the 7th day were detected to have neural-related target genes (Tuj1, MAP2 and GFAP) significantly upregulated on PDA / ACH-4, indicating that the radio neural regulation system based on polydopamine-modified anisotropic cellulose piezoelectric hydrogel promoted the neural differentiation of neural stem cells.
[0098] The detailed explanation is as follows: After 7 days of culture, radiofrequency stimulation did not significantly affect the expression of Tuj1 and MAP2 genes in rNSCs cultured on TCP, indicating that ultrasound stimulation has no effect on the neuronal differentiation of rNSCs in the short term. On PDA / ACH-4, the 0W group showed upregulation of Tuj1 and MAP2 gene expression. The 400W group showed significant upregulation of Tuj1 and MAP2 gene expression, which was significantly different from the other groups. These results indicate that radiofrequency signals triggered by ultrasound on the PDA / ACH-4 surface can significantly promote the neuronal differentiation of rNSCs.
[0099] In addition, after 7 days of culture, GFAP expression was significantly different in the other three groups compared with the 0W group, indicating that the electrical stimulation generated by the PDA / ACH-4 hydrogel-based wireless electrical stimulation system can accelerate the differentiation of astrocytes, thereby supporting the survival of neural stem cell-derived neurons.
[0100] Meanwhile, after 7 days of radio-induced stimulation culture on TCP and PDA / ACH-4, immunofluorescence staining of mature neurons, glial cells, and nuclei was performed, as shown in Figure 2 . Figure 11 As shown, red: mature neuron-specific marker MAP2, green: glial cell (astrocyte) marker GFAP; blue: cell nucleus marker DAPI. Figure 12 As shown, the percentage of MAP2+ after 7 days of TCP and PDA / ACH-4 radioelectric stimulation indicated that the radioelectric stimulation system based on PDA / ACH-4 piezoelectric hydrogel promoted the differentiation and directional arrangement of neural stem cells and increased the proportion of neural stem cells differentiating into neurons.
[0101] The above description is merely a specific embodiment of the invention covered by this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.
Claims
1. A polydopamine-modified anisotropic cellulose piezoelectric hydrogel, comprising: an anisotropic cellulose piezoelectric hydrogel skeleton; and polydopamine modified on the anisotropic cellulose piezoelectric hydrogel skeleton.
2. The polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1, wherein the modification amount of polydopamine is 0.4%-2% based on the mass percentage of N element in the polydopamine-modified anisotropic cellulose piezoelectric hydrogel, and optionally, the elongation rate of the anisotropic cellulose piezoelectric hydrogel skeleton is 80%-215%.
3. A method for preparing anisotropic cellulose piezoelectric hydrogel modified with polydopamine, comprising: In an alkaline solution, dopamine is in situ polymerized on the anisotropic cellulose piezoelectric hydrogel to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel.
4. The preparation method according to claim 3, comprising immersing the anisotropic cellulose piezoelectric hydrogel in an alkaline solution with a pH value of 8-12, adding dopamine at a concentration of 1-5 mg / mL, reacting for 1-12 hours, and then washing with water to terminate the reaction to obtain the polydopamine-modified anisotropic cellulose piezoelectric hydrogel, optionally, the alkaline solution is Tris-HCl buffer.
5. The preparation method according to claim 3, wherein The anisotropic cellulose piezoelectric hydrogel is prepared by the following steps: 1) adding a cross-linking agent to the piezoelectric cellulose solution to chemically cross-link and gelate to obtain a cellulose piezoelectric hydrogel; and 2) Pre-stretching the cellulose piezoelectric hydrogel obtained in step 1) to align the cellulose in the cellulose piezoelectric hydrogel, and then immersing it in an aqueous sulfuric acid solution to permanently fix the oriented structure through physical cross-linking between adjacent cellulose chains to obtain the anisotropic cellulose piezoelectric hydrogel.
6. The preparation method according to claim 5, wherein step 1) comprises dissolving the piezoelectric cellulose in a lithium hydroxide and urea solution to obtain the piezoelectric cellulose solution, optionally, in the lithium hydroxide and urea solution, the concentration of lithium hydroxide is 3.6-5.4wt%, and the concentration of urea is 12-18%. Preferably, the concentration of the piezoelectric cellulose in the piezoelectric cellulose solution is 1wt%-5wt%. Optionally, before adding the cross-linking agent, the piezoelectric cellulose solution is pre-cooled at a temperature of -20°C to 0°C. Optionally, the piezoelectric cellulose in the piezoelectric cellulose solution is derived from one or more of filter paper, wood, bacterial cellulose and cotton. Optionally, the cross-linking agent is selected from one or more of epichlorohydrin, glutaraldehyde, genipin and polyethylene glycol diglycidyl ether, Optionally, in step 1), the molar ratio of the cross-linking agent to the deoxyglucose unit in the piezoelectric cellulose solution is 1:1-3:1, Optionally, step 1) includes dropwise adding the crosslinking agent to the cellulose solution to obtain a crude cellulose piezoelectric hydrogel product, centrifuging to remove bubbles, pouring into a template, and gelling at 0-10° C. for 10 h to 100 h to obtain the cellulose piezoelectric hydrogel; Optionally, the stretching ratio of the pre-stretching in step 2) is 80%-215%; Optionally, step 2) comprises soaking the pre-stretched cellulose piezoelectric hydrogel in a dilute sulfuric acid solution for physical crosslinking and terminating chemical crosslinking, and then washing with water to obtain the anisotropic cellulose piezoelectric hydrogel. Preferably, the concentration of the dilute sulfuric acid solution is 1 wt%-5 wt%.
7. Use of the polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2 or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any one of claims 3 to 6 as an acoustic energy generator for converting ultrasonic energy into electrical energy, especially radio signals.
8. Radiofrequency stimulation system, including: The polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2, or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared according to the preparation method of any one of claims 3 to 6; and an ultrasonic device, wherein the polydopamine-modified anisotropic cellulose piezoelectric hydrogel is configured to generate electrical signal stimulation under the ultrasonic action of the ultrasonic device, Optionally, the ultrasonic frequency, ultrasonic intensity and ultrasonic time of the ultrasonic device can be programmably controlled. Optionally, the ultrasonic intensity of the ultrasonic action is 0-5W / cm 2 .
9. A method for culturing stem cells, comprising contacting the stem cells with the polydopamine-modified anisotropic cellulose piezoelectric hydrogel according to claim 1 or 2 or the polydopamine-modified anisotropic cellulose piezoelectric hydrogel prepared by the preparation method according to any one of claims 3 to 6 for culturing, and applying ultrasound so that the electrical stimulation generated by the polydopamine-modified anisotropic cellulose piezoelectric hydrogel under the ultrasound promotes the neural differentiation, development and / or directional alignment of the stem cells.
10. The stem cell culture method according to claim 9, wherein the stem cells are neural stem cells, mesenchymal stem cells or embryonic stem cells, and optionally, the ultrasound frequency, ultrasound intensity and ultrasound time of the ultrasound action are programmable. Optionally, the ultrasound intensity of the ultrasound action is 0-5 W / cm 2 .