Anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells, and preparation method and application thereof

Abstract

The application belongs to the field of osteoarthritis drug research, and discloses an anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells and a preparation method and application thereof. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells provided by the application is composed of mesenchymal stem cells, piezoelectric nanotubes, temperature-sensitive hydrogel and glucocorticoids; wherein the mesenchymal stem cells are extracted from SD rat bone marrow mesenchymal stem cells and cultured to the third generation, the piezoelectric nanotubes are formed by self-assembly of phenylalanine dipeptide molecules, the temperature-sensitive hydrogel is prepared by compounding poloxamer 407 and methyl cellulose, and the glucocorticoids can be hydrocortisone sodium succinate, dexamethasone sodium phosphate, betamethasone sodium phosphate and the like. The application further provides a method for extracting and culturing the mesenchymal stem cells, a method for preparing the piezoelectric nanotubes and the temperature-sensitive hydrogel. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells is expected to become a new drug in the field of osteoarthritis and has great potential in clinical application.

Description

Technical Field

[0001] This invention belongs to the field of osteoarthritis drug research, and particularly relates to an anti-inflammatory piezoelectric hydrogel. Background Technology

[0002] Osteoarthritis (OA) is a chronic joint disease characterized by degenerative changes in articular cartilage, synovial inflammation, cartilage matrix degradation, and abnormal subchondral bone remodeling. OA commonly affects the knee joint, with a high prevalence in people over 40 years of age. Aging, obesity, and mechanical stress are the main risk factors. Current clinical treatment primarily involves stepwise interventions. Basic treatments and drug therapies can only relieve symptoms. Oral or intra-articular injections of nonsteroidal anti-inflammatory drugs (NSAIDs) have systemic or local adverse reactions. Severe OA often requires joint replacement. Overall, there is a lack of effective treatments to reverse or slow the degenerative process of cartilage. Articular cartilage lacks blood supply and nerves, resulting in extremely poor self-repair capabilities. Mesenchymal stem cells (MSCs) can achieve cartilage repair through multi-lineage differentiation and paracrine effects, becoming an important direction for OA treatment. However, simple intra-articular injection suffers from low cell retention and survival rates, susceptibility to the inflammatory microenvironment, and insufficient chondrogenic differentiation efficiency, making it difficult to achieve stable and lasting repair effects.

[0003] Biopiezoelectric materials can convert mechanical stimulation into local electrical signals through the piezoelectric effect, wirelessly inducing MSCs to differentiate into chondrocytes without an external power source, overcoming the limitations of traditional electrical stimulation devices in clinical applications. Natural biopiezoelectric materials have excellent biocompatibility but low piezoelectric coefficients. Synthetic piezoelectric materials, such as barium titanate, have better piezoelectric properties but suffer from poorer biocompatibility.

[0004] Hydrogels, with their three-dimensional structure similar to the extracellular matrix, can protect cells, improve retention and survival rates, and achieve precise enrichment at damaged sites when used as MSC delivery carriers. They can also load active factors to improve the local microenvironment. Thermosensitive hydrogels, in particular, can be injected at room temperature and gel in situ at body temperature, offering better minimally invasiveness and operability. Combining biopiezoelectric materials with thermosensitive hydrogels can integrate functions such as cell delivery protection, in situ piezoelectric stimulation-induced chondrogenic differentiation, and regulation of the inflammatory microenvironment, providing a feasible approach to overcome the current bottlenecks in MSC therapy and construct a highly efficient integrated OA cartilage repair delivery system.

[0005] The existing technology has the following insurmountable technical defects:

[0006] (1) MSCs have a low retention rate and are easily lost when injected directly into the joint cavity. The high inflammatory microenvironment of OA significantly inhibits their survival and chondrogenic differentiation.

[0007] (2) Traditional piezoelectric materials (such as PZT, PVDF, barium titanate) are non-degradable and have poor biocompatibility, and cannot be used for long-term in vivo repair.

[0008] (3) Single anti-inflammatory treatment can only relieve symptoms and cannot achieve cartilage regeneration;

[0009] (4) Conventional hydrogels do not have electrical signal response function and cannot provide physical signals for the directional differentiation of stem cells;

[0010] (5) Although existing piezoelectric hydrogel technology, represented by CN113507946A, has achieved a basic combination of piezoelectric stimulation and stem cell delivery, it lacks the function of regulating the inflammatory microenvironment and has not solved the problem of stem cell protection. Various single or combination therapies are difficult to achieve the synergistic repair of "anti-inflammation-electric stimulation induction-MSCs protection", and the therapeutic effect is limited.

[0011] In summary, there is an urgent need in this field for an integrated osteoarthritis repair material that is injectable, gels in situ, biodegradable, wirelessly piezoelectrically stimulated, synchronously anti-inflammatory, and protects MSCs. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention proposes an anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells, its preparation method, and its application.

[0013] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0014] An anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells (BMSCs) comprises BMSCs, piezoelectric nanotubes, a thermosensitive hydrogel, and glucocorticoids. This anti-inflammatory piezoelectric hydrogel is in a solution state at room temperature, rapidly transforms into a gel state at physiological temperatures, and generates a millivolt-level open-circuit potential under ultrasound.

[0015] Glucocorticoids are clinically approved intra-articular injection drugs with rapid onset of action, excellent anti-inflammatory effects, and high water solubility. Loading them into hydrogels can further delay their release, improve bioavailability, and enhance efficacy. Specifically, glucocorticoids may be at least one of hydrocortisone sodium succinate (HSS), dexamethasone sodium phosphate, and betamethasone sodium phosphate.

[0016] The aforementioned piezoelectric nanotubes are self-assembled piezoelectric nanotubes of phenylalanine dipeptide (FF) molecules.

[0017] The concentration of the aforementioned FF molecule self-assembled piezoelectric nanotubes in the anti-inflammatory piezoelectric hydrogel is 2-10 mg / mL. For example, the concentration of FF molecule self-assembled piezoelectric nanotubes in the anti-inflammatory piezoelectric hydrogel is 2-6 mg / mL; the concentration of FF molecule self-assembled piezoelectric nanotubes in the anti-inflammatory piezoelectric hydrogel is 6-10 mg / mL; the concentration of FF molecule self-assembled piezoelectric nanotubes in the anti-inflammatory piezoelectric hydrogel is 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL or 10 mg / mL, as long as it is within the above range.

[0018] The concentration of the aforementioned glucocorticoids in the anti-inflammatory piezoelectric hydrogel is 0.03 μg / mL to 0.25 mg / mL. For example, the concentration of glucocorticoids in the anti-inflammatory piezoelectric hydrogel is 0.03 μg / mL to 0.01 mg / mL; the concentration of glucocorticoids in the anti-inflammatory piezoelectric hydrogel is 0.011 mg / mL to 0.1 mg / mL; the concentration of glucocorticoids in the anti-inflammatory piezoelectric hydrogel is 0.11 mg / mL to 0.25 mg / mL; the concentration of glucocorticoids in the anti-inflammatory piezoelectric hydrogel is 0.03 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, or 0.25 mg / mL, as long as it is within the above range.

[0019] The aforementioned thermosensitive hydrogel comprises poloxamer 407 (P407) and methylcellulose (MC). Further, the mass ratio of P407 to MC is 7-10:1. For example, the mass ratio of P407 to MC is 7-8:1; 8-9:1; 9-10:1; or 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1, etc.

[0020] The aforementioned mesenchymal stem cells can be extracted from rat bone marrow cavities, etc. Furthermore, BMSCs were extracted from rat bone marrow and cultured to the third generation.

[0021] The preparation method of the above-mentioned anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells includes the following steps:

[0022] (1) Weigh MC powder and add it to physiological saline preheated to 90°C while stirring. Continue stirring in a 90°C water bath until the MC powder is completely wetted and evenly dispersed. Then add 10 mL of physiological saline precooled to 4°C and stir at room temperature for 3 min until the system gradually turns into a pale yellow transparent colloidal state, which is the 0.18 g / mL MC solution. Let it stand at 4°C for more than 12 h.

[0023] (2) Weigh P407 powder, add 7.109 mL of BMSCs complete culture medium and 1 mL of MC solution prepared in step (1) under sterile conditions, stir well, incubate on ice for 30 min, filter through a 0.1 μm filter membrane, and refrigerate at 4℃ for more than 12 h to obtain sterile P407-MC hydrogel matrix. The components of the complete culture medium include: 10% fetal bovine serum and 90% DMEM low glucose culture medium.

[0024] (3) Add FF lyophilized powder to sterile physiological saline, sonicate for 20 min, anneal at 80℃ for 30 min, and let stand at room temperature for more than 72 h. Centrifuge at 12000 rpm for 5 min, discard the supernatant, and the resulting precipitate is FF piezoelectric nanotube;

[0025] (4) Add FF piezoelectric nanotubes to the sterile P407-MC hydrogel matrix obtained in step (2) to obtain piezoelectric P407-MC hydrogel (gel+FF).

[0026] (5) Under light-protected conditions, the glucocorticoid is added to the piezoelectric hydrogel obtained in step (4) to obtain the anti-inflammatory piezoelectric hydrogel loaded with glucocorticoid. When the glucocorticoid is hydrocortisone sodium succinate, the anti-inflammatory piezoelectric hydrogel loaded with glucocorticoid is named HSS+gel+FF.

[0027] (6) Under sterile conditions, add mesenchymal stem cells cultured to the third generation to the anti-inflammatory piezoelectric hydrogel obtained in step (5), and mix thoroughly to obtain an anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells (BMSCs) (gel+FF+BMSCs+HSS). The density of BMSCs in the BMSC-loaded anti-inflammatory piezoelectric hydrogel is 0.5-3×10⁻⁶. 6 cells / mL.

[0028] The above-mentioned anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells is used in the recruitment of mesenchymal stem cells.

[0029] The above-mentioned anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells is used to induce the differentiation of mesenchymal stem cells into chondrocytes.

[0030] The above-mentioned anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells is used in the treatment of osteoarthritis.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention uses P407-MC injectable thermosensitive hydrogel as a carrier to achieve room temperature injection and in-situ gelation at body temperature, which has good tissue adhesion and biocompatibility, and can effectively solve the key problems of MSCs being easily lost and difficult to colonize at the site of injury.

[0033] (2) This invention combines biodegradable and highly biocompatible FF self-assembled piezoelectric nanotubes with thermosensitive hydrogels for the first time, overcoming the problems of non-degradability and insufficient biosafety of traditional inorganic piezoelectric materials and weak electrical signals of organic piezoelectric materials, and realizing mild and sustainable wireless in-situ electrical stimulation.

[0034] (3) The present invention constructs a three-in-one synergistic repair system of "anti-inflammatory + electrical stimulation induction + MSCs protection", which breaks through the limitations of a single strategy that cannot simultaneously improve inflammation, promote directional differentiation and increase cell retention rate, and effectively improves cartilage repair efficiency. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 TEM and optical microscope images of FF self-assembled piezoelectric nanotubes.

[0037] Figure 2 OCP of FF self-assembled nanotubes under different ultrasonic intensities.

[0038] Figure 3 To investigate the self-assembly of nanotubes with different concentrations of FF at 0.5 W / cm 2 OCP at 3 min of ultrasound treatment.

[0039] Figure 4 Photographs of the hydrogel sol-gel transition.

[0040] Figure 5 For different formulations at 0.5 W / cm 2 OCP under ultrasound for 3 minutes.

[0041] Figure 6 For morphological observation of BMSCs.

[0042] Figure 7 The effect of ultrasound conditions on the survival rate of BMSCs.

[0043] Figure 8 A study on the cell safety of co-culturing BMSCs with different formulations for 5 days (n=3).

[0044] Figure 9 The effect of anti-inflammatory piezoelectric hydrogel on IL-6 secretion levels in inflammatory BMSCs (n=3).

[0045] Figure 10 Alcian blue staining image of BMSCs undergoing chondrogenic differentiation under normal conditions (scale bar: 200 μm).

[0046] Figure 11 Alcian blue staining image of BMSCs undergoing chondrogenic differentiation under inflammatory conditions (scale bar: 200 μm).

[0047] Figure 12 The expression levels of three proteins in cells under different treatment groups under normal conditions.

[0048] Figure 13 The expression levels of three proteins in cells under different treatment groups during inflammation.

[0049] Figure 14 SF staining was performed on the knee joints of rats in each group (scale bar: 2 mm).

[0050] Figure 15 OARSI score for SF staining (n=3).

[0051] Figure 16 Immunohistochemical staining of IL-6 in the knee joints of rats in each group (scale bar: 200 μm). Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Extraction and culture of bone marrow mesenchymal stem cells (BMSCs): Two healthy male rats aged 8 weeks were selected and euthanized by cervical dislocation after anesthesia. The femur and tibia were separated under aseptic conditions, and the surface soft tissue was removed. After disinfection with 75% alcohol, the bones were washed 3-4 times with sterile PBS. The metaphysis at both ends of the bones was removed to expose the medullary cavity. The medullary cavity was repeatedly flushed with a sterile syringe containing basal culture medium. The bone marrow cell suspension was collected and filtered through a 70 μm cell sieve.

[0054] The filtered cell suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in PBS, and red blood cell lysis buffer was added. The cells were lysed on ice for 5 min, then centrifuged at 1000 rpm for 5 min, and washed twice with PBS. The cells were then resuspended in BMSCs complete medium and seeded in 60 mm culture dishes. The cells were cultured at 37°C in a 5% CO2 incubator. The medium was changed for the first time after 3 days to remove non-adherent cells. Thereafter, the medium was changed every 2 days. The cells were passaged when the confluence reached approximately 80%.

[0055] During passage, wash cells twice with PBS, add trypsin and digest at 37°C for 1 min, add complete culture medium to stop digestion, gently pipette to disperse cells, collect and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend cells in complete culture medium, and passage at a ratio of 1:3.

[0056] The results are as follows Figure 6 As shown, the cells exhibited a long spindle-shaped and fusiform fibrous cell morphology under standard BMSC culture conditions, with an oval nucleus in the center. The morphology was uniform and consistent with the characteristics of BMSCs, indicating that the BMSCs were successfully extracted.

[0057] BMSCs were extracted from the bone marrow of SD rats and cultured to the third generation.

[0058] Example 1

[0059] The preparation method of the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells in this embodiment includes the following steps:

[0060] (1) Weigh 3.6 g of methylcellulose (MC) powder and add it to 10 mL of physiological saline preheated to 90°C while stirring. Continue stirring in a 90°C water bath until the MC powder is completely wetted and evenly dispersed. Then add 10 mL of physiological saline precooled to 4°C and stir at room temperature for 3 min until the system gradually turns into a pale yellow transparent colloidal state, which is the 0.18 g / mL MC solution. Let it stand at 4°C for more than 12 h.

[0061] (2) Take 1.3499 g of poloxamer 407 (P407), add 7.181 mL of complete culture medium of BMSCs and 1 mL of MC solution prepared in step (1) under sterile conditions, stir evenly, incubate on ice for 30 min, filter through a 0.1 μm filter membrane, and stand at 4℃ for 12 h to obtain sterile P407-MC hydrogel (gel).

[0062] (3) Add 8.0 mg of lyophilized phenylalanine dipeptide (FF) powder to 1 mL of sterile physiological saline, sonicate for 20 min, anneal at 80℃ for 30 min, and let stand at room temperature for more than 72 h. Centrifuge at 12000 rpm for 5 min, discard the supernatant, and the resulting precipitate is the piezoelectric nanotube (gel+FF).

[0063] (4) Add the piezoelectric nanotubes (concentration of 8 mg / mL) prepared in step (3) to the sterile P407-MC hydrogel obtained in step (2) and mix evenly to obtain piezoelectric hydrogel; then add 0.05 mg of HSS to the above 2 mL piezoelectric hydrogel to obtain anti-inflammatory piezoelectric hydrogel (gel+FF+HSS).

[0064] (5) Prepare a cell suspension from SD rat BMSCs cultured to the third generation, count the cells, and adjust the cell concentration to 1×10⁻⁶ using complete culture medium. 6 Cells / mL, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and an equal volume of anti-inflammatory piezoelectric hydrogel was added. After thorough mixing, the anti-inflammatory piezoelectric hydrogel loaded with BMSCs (gel+FF+BMSCs+HSS) was obtained.

[0065] Implementation Results Example

[0066] 1. Properties of FF self-assembled piezoelectric nanotubes

[0067] The FF self-assembled structure prepared in Example 1 was observed under an optical microscope and a transmission electron microscope, respectively, and the results are as follows: Figure 1 As shown, FF molecules self-assemble through non-covalent interactions such as π-π stacking and hydrogen bonding to form ordered tubular structures with a diameter of approximately 500 nm and a length on the micrometer scale. This non-centrosymmetric structure allows the molecular dipoles to be oriented axially, and its rigid framework can effectively transfer mechanical stress and achieve a cooperative dipole response, providing a structural basis for its piezoelectric properties.

[0068] A three-electrode electrochemical testing system was constructed using an electrochemical workstation. Before each test, the system was allowed to stand for 10–20 min until the potential fluctuation was less than 5 mV / min before starting the test. No ultrasound was applied during the initial stage of the test (0–10 s), and the ultrasound device was turned on at the 10th second.

[0069] (1) Changes in OCP under different ultrasonic intensities: 8 mg of FF lyophilized powder was added to sterile physiological saline, and after sonication, annealing, and standing, the piezoelectric nanotubes were self-assembled. OCP was then applied at different intensities of 0.25, 0.5, 1, 1.5, 2, and 2.5 W / cm². 2 Ultrasonic intensity, measured by the OCP after 3 minutes of ultrasonic treatment.

[0070] The results are as follows Figure 2 As shown, the FF self-assembled nanotube solution exhibits a significant OCP under ultrasonic irradiation, which increases with increasing ultrasonic intensity, indicating that the FF self-assembled nanotubes possess a piezoelectric effect and can generate millivolt-level voltages under ultrasonic irradiation. (0–2.5 W / cm²) 2 Within the specified range, OCP is basically positively correlated with ultrasonic intensity, indicating that the material mainly undergoes elastic deformation and no irreversible structural damage occurs.

[0071] (2) OCP of FF piezoelectric nanotubes at different concentrations under the same ultrasonic conditions: 2, 4, and 8 mg of FF lyophilized powder were respectively placed in 1 mL of physiological saline, and after ultrasonication, annealing, and standing, FF piezoelectric nanotube solutions with concentrations of 2, 4, and 8 mg / mL were obtained; under 0.5 W / cm 2 The samples were treated under ultrasonic conditions for 3 minutes, and the OCP was measured. All experiments were performed in triplicate.

[0072] The results are as follows Figure 3 As shown, there is a significant correlation between OCP and FF self-assembled nanotube concentration. 8 mg / mL FF self-assembled nanotubes can generate 13-15 mV OCP, which meets the electrical stimulation intensity required for MSC chondrogenic differentiation and is beneficial to promoting the MSC chondrogenic differentiation process.

[0073] 2. Testing of the piezoelectric properties of anti-inflammatory piezoelectric hydrogels

[0074] The gelation temperature (T0) and gelation time at 37°C of the anti-inflammatory piezoelectric hydrogel prepared in Example 1 were determined using the tilted test tube method. 1 mL of sample was placed in a 5 mL EP tube, the thermometer probe was immersed below the sample surface, and the EP tube was placed in a constant temperature water bath. The water bath temperature was set not to exceed 15°C. After equilibration for 5 min, the temperature was increased at a rate of 0.5°C / min, with the test tube tilted approximately 45° every 1 min. The temperature at which the sample stopped flowing was recorded as the gelation temperature. Each group was tested in triplicate, and the average value was taken. The results are as follows: Figure 4 As shown, the gelation temperature of the anti-inflammatory piezoelectric hydrogel is 28.5℃, and the gelation time at physiological temperature (37℃) is about 1 min.

[0075] OCP testing was conducted following the same method used for testing the piezoelectric properties of FF self-assembled nanotubes. Four groups were set up: gel (P407-MC) group, FF (piezoelectric material) group, gel+FF (piezoelectric hydrogel) group, and gel+FF+HSS (anti-inflammatory piezoelectric hydrogel, Example 1) group, with FF concentration of 8 mg / mL and HSS concentration of 0.25 mg / mL. Each group was tested at 0.5 W / cm². 2 OCP was measured 3 minutes after ultrasound treatment, and each group of experiments was tested in triplicate. Results are as follows: Figure 5As shown, the gel group exhibits virtually no piezoelectric potential under ultrasound, indicating that P407-MC itself does not possess a piezoelectric effect. The piezoelectric effects of the FF group, gel+FF group, and gel+FF+HSS group all originate from FF self-assembled nanotubes. Under the same ultrasound conditions, the OCP of the three groups showed no significant difference, indicating that the addition of P407-MC gel and anti-inflammatory drugs does not affect the piezoelectric properties of FF self-assembled nanotubes, and they can be used for subsequent cell and animal studies.

[0076] 3. Evaluation of cell safety under ultrasound conditions

[0077] P3 generation BMSCs were used at 3×10 3 Cells / well were seeded at a density of 1,000 cells / well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. Different sonication conditions were set (0.5, 0.75, 1, 1.5, 2, 2.5 W / cm²). 2 Cells were sonicated once daily for 5 minutes, with the culture medium changed every other day. Cell viability was assessed using the CCK-8 assay after 3 and 7 days of sonication. The procedure was as follows: discard the old culture medium, add 100 μL of serum-free medium containing 5% (v / v) CCK-8 reagent to each well under light-protected conditions, incubate at 37°C for 1 h, and then measure the OD value at 450 nm using a microplate reader. Cell viability was calculated according to Formula 1. The experiment was repeated three times.

[0078]

[0079] In the formula, A sample A represents the absorbance of the experimental group. control The absorbance of the control group, A blank The absorbance is for the blank group.

[0080] The results are as follows Figure 7 As shown, 0.5 W / cm 2 After 5 minutes of daily ultrasound treatment for 7 consecutive days, the cell survival rate remained close to 100%, indicating that the ultrasound conditions had good biosafety for BMSCs.

[0081] 4. Safety evaluation of anti-inflammatory piezoelectric hydrogel cells

[0082] P3 generation BMSCs were used at 3×10 3 Cells / well were seeded at a density of 1:100 in 96-well plates and cultured for 24 h to allow for full cell adhesion. The experiment consisted of 8 groups: Control group, FF group, gel group, gel+FF group, HSS group, FF+US group, HSS+gel+FF group, and HSS+gel+FF+US group. The FF concentration was 8 mg / mL, the HSS concentration was 0.25 mg / mL, and the US treatment condition was 0.5 W / cm².2 Continue for 3 minutes, then 0.25 W / cm 2 Incubate for 2 minutes. Add 60 μL of the corresponding hydrogel to each well of the gel group and incubate at 37°C for 5 minutes to trigger gel formation, then cover with 60 µL of complete culture medium to prevent drying. Change the medium every other day for a total of 5 days, and perform corresponding sonication treatments daily according to the group. After culture, cell viability is determined using the CCK-8 assay and calculated according to Formula 1. The experiment is repeated 3 times. Results are as follows: Figure 8 As shown, the anti-inflammatory piezoelectric hydrogel and its components all exhibit excellent biocompatibility, which can meet the requirements of BMSCs loading and long-term co-culture, and is conducive to the smooth conduct of subsequent experiments.

[0083] 5. Evaluation of the anti-inflammatory effect of anti-inflammatory piezoelectric hydrogel

[0084] P3 generation BMSCs were used at 2×10 5 Cells / well were seeded at a density of 6-well plates and cultured for 24 h to allow for full cell adhesion. Six groups were set up for this experiment: Control group (no LPS stimulation), Model group (LPS stimulation), HSS group, gel+FF group, gel+FF+US+HSS group, and gel+FF+US group. The concentrations of FF and HSS were 8 mg / mL and 0.25 mg / mL, respectively, and the US stimulation was performed at 0.5 W / cm². 2 Continue for 3 minutes, then 0.25 W / cm 2 The stimulation lasted for 2 minutes. Except for the Control group, all other groups were stimulated with LPS (10 µg / mL) for 24 h to establish a BMSCs inflammation model. After successful model establishment, the culture medium was replaced with fresh complete medium containing the corresponding drugs, and the cells were sonicated according to their respective groups for another 72 h. Cell supernatant was collected, and IL-6 levels were detected using ELISA to evaluate the anti-inflammatory effect.

[0085] The results are as follows Figure 9 The IL-6 level in the Model group was significantly higher than that in the Control group, indicating that the inflammation model was successfully established. The IL-6 levels in the HSS group and the gel+FF+US+HSS group were not significantly different from those in the Control group, indicating that HSS can effectively inhibit the inflammatory response of BMSCs, and its anti-inflammatory activity was not affected by the gel or ultrasound after loading with piezoelectric hydrogel. Furthermore, although the IL-6 level in the gel+FF+US group was higher than that in the Control group, it was significantly lower than that in the Model group, indicating that the piezoelectric hydrogel and the electrical stimulation it generates also have a certain anti-inflammatory effect, possibly related to its activation of the TGF-β / Smad pathway.

[0086] 6. Alixin Blue staining of anti-inflammatory piezoelectric hydrogels promoting chondrogenic differentiation of BMSCs

[0087] P3 generation BMSCs were used at 1×10 4 Cells / well were seeded at a density of 1:1 ratio in 48-well plates and incubated at 37°C with 5% CO2 for 24 h to allow for full cell adhesion. The experiment was divided into normal and inflammatory states, with the specific groupings as follows:

[0088] (1) Under normal conditions, the experiment was set up with 8 groups: Control group, Differentiation Medium group (positive control group), Differentiation Medium+FF+US group, gel+FF group, US group, gel+US group, FF+US group, gel+FF+US group, where the concentration of FF was 8 mg / mL.

[0089] (2) Under inflammatory conditions, four groups were set up: Model group, HSS group, gel+FF+US group, and gel+FF+US+HSS group, with FF concentration of 8 mg / mL and HSS concentration of 0.25 mg / mL. Each group established a BMSCs inflammatory model in accordance with the evaluation of anti-inflammatory piezoelectric hydrogel anti-inflammatory effect.

[0090] After cell adhesion, the old culture medium was discarded, and different preparations were added according to the grouping. Specific experimental groups were then subjected to ultrasound treatment using an ultrasound therapy device with the ultrasound parameters set to 0.5 W / cm². 2 Continue for 3 minutes, then switch to 0.25 W / cm². 2 Continue for 2 minutes. Culture continuously for 7 days, sonicating once daily for a total of 7 times. After the experiment, GAG secretion levels were detected using Alixin Blue staining: After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes, stained according to the chondrogenic differentiation induction staining kit instructions, washed, and then observed under a microscope.

[0091] The results are as follows Figure 10 Under normal conditions, the US group, gel+US group, gel+FF group, and Control group showed almost no GAG secretion, indicating that they could not induce chondrogenic differentiation of BMSCs. The differentiation medium group, FF+US group, and gel+FF+US group showed a distinct blue color, indicating that the GAG ​​content was significantly higher than that of the Control group, suggesting that by day 7, both electrical stimulation and chemical induction had initiated chondrogenic differentiation of BMSCs. The induction medium + FF+US group, due to the dual regulatory effect of both electrical stimulation and chemical induction factors, showed a further increase in GAG content, indicating a higher degree of chondrogenic differentiation of BMSCs. Figure 11As shown, under inflammatory conditions, the GAG ​​content in the gel+FF+US group was lower than that in the normal state, indicating that the inflammatory microenvironment can inhibit the chondrogenic differentiation capacity of BMSCs; after the addition of HSS, the GAG ​​content significantly increased, suggesting that the chondrogenic differentiation capacity of BMSCs recovered to near-normal levels. Figure 9 It is known that HSS inhibits inflammation and can restore the differentiation function of BMSCs.

[0092] 7. Detection of expression levels of proteins related to the promotion of chondrogenic differentiation of BMSCs by anti-inflammatory piezoelectric hydrogels

[0093] BMSCs were extracted from 8-week-old male rats and cultured to passage P3. Experiments were conducted under normal and inflammatory conditions. Under normal conditions, four groups were set up: Control group, gel+FF group, US group, and gel+FF+US group, with FF concentration of 8 mg / mL. Under inflammatory conditions, four groups were set up: Model group, HSS group, gel+FF+US group, and gel+FF+US+HSS group, with FF concentration of 8 mg / mL and HSS concentration of 0.25 mg / mL. A BMSCs inflammatory model was established as described above. After cell adhesion, the corresponding preparations were added according to the groups, and the designated groups were subjected to sonication (0.5 W / cm²). 2 Duration 3 min; 0.25 W / cm 2 Process for 2 minutes). Repeat the treatment for 7 consecutive days, once a day.

[0094] After treatment, cells were collected, and total cellular protein was extracted using lysis buffer. Protein concentration was determined by the BCA method. Proteins were treated with loading buffer, separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder, and sequentially incubated with primary antibody, GAPDH primary antibody, and secondary antibody for the target protein. Finally, ECL chemiluminescence imaging was used to detect the expression levels of type II collagen (COL2), SOX9, and ACAN to evaluate the chondrogenic differentiation degree of BMSCs.

[0095] The results are as follows Figure 12 As shown, under normal conditions, there were no significant differences in the expression levels of type II collagen, SOX9, and ACAN proteins among the Control group, gel+FF group, and US group; however, under the action of piezoelectric hydrogel-based electrical stimulation (gel+FF+US), the expression levels of all three proteins in the cells were significantly increased. This result is consistent with the results of Alixin blue staining, further confirming that electrical stimulation can effectively promote the chondrogenic differentiation process of BMSCs. Figure 13As shown, under the in vitro simulated OA joint cavity inflammatory microenvironment, gel+FF+US still promoted the chondrogenic differentiation of BMSCs, but the effect was limited, further indicating that the inflammatory environment can inhibit the differentiation potential of BMSCs. After adding HSS (gel+FF+US+HSS) to alleviate the inflammatory state, the expression levels of all three proteins were significantly increased, indicating that the anti-inflammatory piezoelectric hydrogel loaded with HSS can effectively reverse the inhibitory effect of the inflammatory state on the chondrogenic differentiation process of BMSCs. This result is consistent with... Figure 11 Consistent.

[0096] 8. In vivo pharmacodynamic evaluation of anti-inflammatory piezoelectric hydrogels loaded with mesenchymal stem cells

[0097] An osteoarthritis (OA) rat model was established using sodium iodoacetate (MIA). Male rats were acclimatized for one week, and the hair around the knees was removed and the area disinfected. 50 μL of MIA solution (20 mg / mL) was injected into the joint cavity via a needle inserted medially or laterally into the patellar tendon. The control group received an equal volume of saline. After injection, the knee joint was gently moved 5-10 times. Three days after model establishment, the OA rats were randomly divided into six groups (n=6 per group): Model group, HSS group, BMSCs group, gel+FF+US group, gel+FF+US+BMSCs group, and gel+FF+US+BMSCs+HSS group. Six healthy rats served as the control group. The concentration of FF was 8 mg / mL, and the concentration of HSS was 0.25 mg / mL. The control and Model groups received saline injections into the right knee joint cavity, while the other groups received the corresponding preparations, 100 μL each time, administered once every 7 days for a total of 4 times. The ultrasound group received daily ultrasound therapy at a parameter of 0.5 W / cm². 2 Process for 3 min, then at 0.25 W / cm 2 Treatment lasted 2 minutes. During the treatment period, rat body weight was recorded on days 0, 3, 10, 17, 24, and 31, and bilateral knee joint width was measured to assess the degree of knee joint swelling.

[0098] Seven days after the last administration, rats were sacrificed, and the gross morphology of the knee joint was observed and photographed. Cartilage damage was evaluated according to the ICRS criteria. After fixation, decalcification, dehydration, clearing, paraffin embedding, and sectioning, the knee joints were stained with H&E and Safranin O-Fix Green (SF), and cartilage pathological damage was evaluated according to the OARSI criteria. In addition, the expression of type II collagen, MMP-13, ACAN, SOX9, and IL-6 was detected by immunohistochemistry.

[0099] The results are as follows Figure 14-15As shown, in the Control group, the articular cartilage showed uniform red staining and sufficient proteoglycan content; the cartilage surface was smooth and continuous, the joint structure was intact, the tide line was clear, and there were no signs of degeneration. In the Model group, the cartilage showed significantly lighter red staining, a large loss of proteoglycan, rough and peeling cartilage surface, Fast Green staining penetrated the surface layer, accompanied by tide line breakage and exposure of subchondral bone, indicating severe cartilage degeneration. The HSS group showed less structural damage than the Model group, but the cartilage still showed lighter red staining, a thinner cartilage layer, and visible ulcers and fibrosis, indicating limited repair capacity. In the BMSCs group and the gel+FF+US group, the red staining deepened, the cartilage thickness recovered, and the surface became smoother, with reduced ulcer depth and area, indicating that both electrical stimulation and BMSCs alone could delay proteoglycan loss, and there was no significant difference in OARSI scores between the two groups. The gel+FF+US+BMSCs group showed more significant recovery of cartilage thickness, a smoother surface, and fewer ulcers, with a better repair effect than the single-factor treatment group, indicating that electrical stimulation and BMSCs combined have a synergistic effect and a better OARSI score. The gel+FF+US+BMSCs+HSS group showed the most extensive red staining of cartilage, rich proteoglycan content, smooth and continuous cartilage surface, and thickness close to the normal group. The OARSI score showed that it had the best repair effect among all treatment groups.

[0100] Results of staining for inflammatory factor levels ( Figure 16The results showed that the control group exhibited very weak positive expression of IL-6, indicating that under healthy physiological conditions, the level of inflammation within the joint cavity was extremely low, IL-6 maintained a baseline expression level, and the joint microenvironment was stable. The model group showed strong positive expression, with high expression observed in synovial tissue, degenerated cartilage, and subchondral bone, indicating that IL-6 is a core driver of OA pathological progression and pain, and its abnormally high expression can accelerate cartilage damage and inflammation. The HSS group showed almost no IL-6 secretion, consistent with its pharmacological mechanism of action: HSS is hydrolyzed in vivo to hydrocortisone, which binds to glucocorticoid receptors, inhibiting the expression of inflammation-related genes, thereby exerting a direct and rapid chemo-anti-inflammatory effect. The gel+FF+US group showed a slight decrease in IL-6 positive signal, suggesting that it exerts a mild anti-inflammatory effect by regulating related cell signaling pathways, but its effect on combating the severe inflammation of OA when used alone is limited. The IL-6 secretion level in the BMSCs group was further reduced compared to the gel+FF+US group, indicating that BMSCs can secrete anti-inflammatory factors such as TSG-6, PGE2, and IL-1Ra through a strong paracrine effect, systematically inhibiting IL-6 production. Its anti-inflammatory effect is superior to simple electrical stimulation intervention. The gel+FF+US+BMSCs group exhibited a clear synergistic anti-inflammatory effect; electrical stimulation enhanced MSC homing, allowing them to exert their anti-inflammatory effects more fully and further reducing IL-6 expression levels. The gel+FF+US+BMSCs+HSS group combined the chemical anti-inflammatory effects of HSS with the paracrine anti-inflammatory effects of BMSCs. Electrical stimulation may enhance the synergistic effect of these two effects by regulating related signaling pathways, ultimately restoring the local inflammatory microenvironment of OA to near-normal levels and creating more favorable conditions for cartilage repair.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells, characterized in that, The anti-inflammatory piezoelectric hydrogel includes mesenchymal stem cells, piezoelectric nanotubes, thermosensitive hydrogels, and glucocorticoids.

2. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to claim 1, characterized in that, The piezoelectric nanotubes are self-assembled piezoelectric nanotubes of phenylalanine dipeptide molecules.

3. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to claim 2, characterized in that, The concentration of the phenylalanine dipeptide self-assembled piezoelectric nanotubes in the anti-inflammatory piezoelectric hydrogel is 2-10 mg / mL; the glucocorticoid is at least one of hydrocortisone sodium succinate, dexamethasone sodium phosphate and betamethasone sodium phosphate, and the concentration in the anti-inflammatory piezoelectric hydrogel is 0.03 μg / mL-0.25 mg / mL.

4. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to claim 1, characterized in that, The thermosensitive hydrogel comprises poloxamer 407 and methylcellulose; the mesenchymal stem cells are obtained from the bone marrow cavity of rats and cultured in vitro to the third generation.

5. The anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to claim 4, characterized in that, The mass ratio of poloxamer 407 to methylcellulose is 7-10:

1.

6. The method for preparing the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to any one of claims 1-5, characterized in that, The steps are as follows: (1) Dissolve methylcellulose in sterile physiological saline to prepare methylcellulose solution; add poloxamer 407 powder to mesenchymal stem cell complete culture medium and methylcellulose solution, stir evenly, then ice bath, filter to remove bacteria, and obtain sterile poloxamer 407-methylcellulose hydrogel matrix. (2) Phenylalanine dipeptide was added to sterile physiological saline, sonicated, and then annealed, allowed to stand at room temperature and centrifuged to obtain phenylalanine dipeptide piezoelectric nanotubes. (3) Add the phenylalanine dipeptide piezoelectric nanotubes obtained in step (2) to the sterile poloxamer 407-methylcellulose hydrogel matrix obtained in step (1) to obtain the piezoelectric poloxamer 407-methylcellulose hydrogel. (4) Under light-protected conditions, add glucocorticoids to the piezoelectric poloxamer 407-methylcellulose hydrogel obtained in step (3) to obtain an anti-inflammatory piezoelectric hydrogel loaded with glucocorticoids. (5) Under sterile conditions, the mesenchymal stem cells cultured to the third generation are added to the anti-inflammatory piezoelectric hydrogel obtained in step (4), and after thorough mixing, the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells is obtained.

7. The method for preparing the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to claim 6, characterized in that, The concentration of the methylcellulose solution in step (1) is 0.18 g / mL.

8. The use of the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells as described in any one of claims 1-5 in the recruitment of mesenchymal stem cells.

9. The use of the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to any one of claims 1-5 in inducing the differentiation of mesenchymal stem cells into chondrocytes.

10. The use of the anti-inflammatory piezoelectric hydrogel loaded with mesenchymal stem cells according to any one of claims 1-5 in the treatment of osteoarthritis.

Images