A flexible conductive hydrogel scaffold with functions of skull repair and nerve signal monitoring

Abstract

The application discloses a flexible conductive hydrogel scaffold with functions of cranium repair and nerve signal monitoring, which is prepared from polyvinyl alcohol-collagen-polylysine as a matrix, doping magnesium oxide nanoparticles and black phosphorus nanosheets, and finally through freeze-induced phase separation and freeze drying. The application can be used as a cranium defect repair scaffold to promote bone tissue regeneration, and can also be used as a flexible ECoG electrode to realize high-fidelity brain electrical signal monitoring.

Description

Technical Field

[0001] This invention relates to the field of brain electrode manufacturing technology, and in particular to a flexible conductive hydrogel scaffold that combines cranial bone repair and neural signal monitoring functions. Background Technology

[0002] Traumatic brain injury (TBI) is a serious neurological disorder affecting more than 60 million people worldwide each year, with approximately 30% of patients also experiencing skull defects. During postoperative rehabilitation, these patients often face secondary neurological dysfunctions such as post-traumatic epilepsy (PTE) and cognitive decline due to insufficient brain tissue exposure and mechanical support caused by skull defects. Therefore, achieving structural reconstruction and real-time monitoring of neurological function in the skull defect area is crucial for patient rehabilitation.

[0003] Currently, commonly used cranioplasty materials include titanium mesh and polymethyl methacrylate (PMMA) bone cement. Their advantage lies in providing basic mechanical support, but their limitations are obvious: on the one hand, the materials are non-degradable and lack the ability to integrate with bone tissue, resulting in postoperative rejection and the risk of secondary surgery; on the other hand, existing materials lack electrical functions and cannot achieve real-time monitoring of postoperative nerve activity.

[0004] Traditional scalp electroencephalography (EEG) suffers from low signal-to-noise ratio and numerous artifacts due to skull attenuation and poor contact. Flexible ECoG electrode arrays (such as Neurogrid and PtNRGrids) have shown excellent performance in high-density monitoring of EEG signals, but they often use substrates such as polystyrene, polyimide, and silicon wafers, which have problems such as high rigidity, hydrophobicity, and non-degradability. They are prone to inducing glial scarring and interface instability, lack osseointegration capacity, and pose a risk of implantation damage. Furthermore, hydrophobic materials, due to their biological inertness, hinder the bone repair process, completely ignoring the need for skull repair.

[0005] Therefore, cranioplasty and neural signal monitoring are currently in a "disjointed" state, and there is an urgent need for a new integrated material platform that can both promote bone tissue regeneration and collect EEG signals stably over a long period of time, thereby achieving synergistic reconstruction of postoperative structure and function. Summary of the Invention

[0006] The purpose of this invention is to provide a flexible conductive hydrogel scaffold that combines cranial repair and neural signal monitoring functions. This integrated interface not only enables signal reading but also has an active repair function, laying the material foundation for a closed-loop brain-computer interface that integrates repair, compensation, and regulation.

[0007] The hydrogel scaffold of this invention possesses excellent mechanical strength, biocompatibility, and ion-release capabilities. The highly interconnected porous structure formed through unidirectional cryo-induced phase separation not only promotes cell migration and bone regeneration but also ensures low impedance and signal stability at the electrode-tissue interface, providing a new strategy for postoperative neurological rehabilitation assessment and closed-loop intervention.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A flexible conductive hydrogel scaffold that combines cranial bone repair and nerve signal monitoring functions is made by using polyvinyl alcohol-collagen-polylysine as a matrix, doping with magnesium oxide nanoparticles and black phosphorus nanosheets, and finally forming it through freeze-induced phase separation and freeze-drying.

[0010] In this invention, polyvinyl alcohol serves as the substrate for the hydrogel material, providing gelation and mechanical support. Collagen enhances cell adhesion and osteogenic induction. MgO is used to slowly release magnesium ions to promote osteogenic formation, while black phosphorus (BP) releases phosphate ions to form a conductive network with magnesium ions, reducing interfacial impedance. Simultaneously, magnesium ions and phosphate ions promote calcium deposition, thereby accelerating osteogenic repair. However, because black phosphorus creates a strongly acidic environment, the release of magnesium ions from MgO is too rapid, affecting osteogenic repair. This invention encapsulates MgO with polylysine to control the slow release of magnesium oxide, thus preventing excessively rapid magnesium ion release. Furthermore, magnesium oxide alone tends to deposit at the bottom of the composite precursor solution, failing to effectively disperse in the hydrogel system and exhibiting poor biocompatibility. This invention encapsulates MgO with polylysine, which has good water solubility, solving the MgO deposition problem and enabling uniform dispersion. Polylysine also has better biocompatibility, improving overall biocompatibility after encapsulation. Polylysine carries a positive charge and can assemble with black phosphorus nanosheets through electrostatic interactions.

[0011] This invention utilizes unidirectional freezing technology to form an interpenetrating porous structure. This scaffold possesses a suitable compressive modulus (0.1-1 MPa) and low interfacial resistance (<1 kΩ@1 Hz), and can controllably degrade and release Mg. 2+ In vitro experiments confirmed that it can significantly enhance osteoblast activity, alkaline phosphatase (ALP) expression, and calcium nodule formation. In a rat skull defect model, after 8 weeks of implantation, the bone volume fraction (BV / TV) reached approximately 65.2%, the bone mineral density (BMD) reached approximately 373.8 mgHA / cm³, and stable acquisition of high-fidelity cortical electroencephalography (ECoG) signals (amplitude >60 μV) was achieved simultaneously.

[0012] The porous structure of this invention has a pore size of 100-250 μm and a porosity of approximately 65%; electrical properties include a conductivity ≥1.5 S / m and an impedance ≤1 kΩ @ 1 kHz; and sustained-release properties, enabling the continuous release of Mg in simulated body fluid (SBF). 2+ The concentration remained stable within the range of 100-300 μg / mL for more than 28 days. The acquired EEG signals had amplitudes exceeding 60 μV, covered the δ-β band, and were clear and stable. The gel matrix exhibited good biocompatibility and biodegradability, avoiding the rigidity mismatch, immune rejection, and long-term implantation complications associated with traditional metal / polymer electrodes.

[0013] As a preferred option, the specific preparation method is as follows:

[0014] (1) Preparation of composite precursor solution: Polyvinyl alcohol was dissolved in deionized water, and then collagen, magnesium oxide nanocomposite coated with polylysine and black phosphorus nano dispersion were added and mixed to obtain composite precursor solution.

[0015] (2) Preparation of flexible conductive hydrogel scaffold: The composite precursor solution is injected into the mold and placed on a cold source for freeze-thaw cycles 1-5 times. Then, the flexible conductive hydrogel scaffold is obtained by vacuum freeze-drying.

[0016] Preferably, the components in a 100mL composite precursor solution are: 5-15g polyvinyl alcohol, 0.1-1g collagen, 0.2-6g magnesium oxide nanocomposite encapsulated with polylysine, and 10-40mg black phosphorus.

[0017] Preferably, the black phosphorus nano-dispersion is obtained by dispersing black phosphorus nanosheets in N-methylpyrrolidone, with a black phosphorus mass concentration of 0.1-1%. This invention disperses black phosphorus nanosheets using N-methylpyrrolidone, resulting in uniform dispersion of the black phosphorus nanosheets in the gel system, better compatibility, and a better and more uniform construction of a conductive network.

[0018] Preferably, the polylysine-encapsulated magnesium oxide nanocomposite is prepared by in-situ co-precipitation, specifically: MgCl2 solution and polylysine are mixed under alkaline conditions, NaOH solution is slowly added dropwise until no more precipitate forms, the reaction is continued for 2-3 hours, and then the product is obtained by centrifugation and washing. The concentration of NaOH solution is preferably 1M. The concentration of MgCl2 solution is preferably 50mM. Polylysine is added at a concentration of 1 mg / mL.

[0019] Preferably, the amount of MgCl2 solution and polylysine is measured and added in a mass ratio of magnesium oxide to polylysine of 1:1.

[0020] Preferably, the freeze-thaw cycle is set as follows: freezing is unidirectional freezing for 4-24 hours, and thawing is thawing at room temperature for 4-12 hours.

[0021] Preferably, the cold energy in the unidirectional freezing is transferred longitudinally from the bottom to the top of the mold, and the temperature of the bottom cold source is -10 to -80°C. The unidirectional freezing is carried out at room temperature, with the cold source located at the bottom of the mold.

[0022] Preferably, the vacuum freeze-drying temperature is -50~-60℃ and the time is 36-60 hours.

[0023] The beneficial effects of this invention are:

[0024] 1. Dual-function integrated design: This invention can serve as a scaffold for repairing skull defects and promoting bone tissue regeneration, and can also serve as a flexible ECoG electrode to achieve high-fidelity EEG signal monitoring.

[0025] 2. Ion-releasing mechanism: MgO particles achieve controllable Mg release within the hydrogel network. 2+ Release and 28-day simulated body fluid (SBF) immersion experiments verified its tunable degradation and sustained osteogenic induction effect;

[0026] 3. Conductive network construction: BP nanosheets form stable ion-electron coupling channels in the hydrogel, combined with a flexible matrix, to achieve mechanical matching with brain tissue, thereby reducing interfacial impedance (≤1 kΩ) and maintaining signal fidelity in the 0.1-10 kHz frequency band;

[0027] 4. Multi-scale porous structure: Highly interconnected pores are obtained through unidirectional cryogenic phase separation technology, which takes into account the synergistic performance of cell migration, nutrient transport and electrical signal conduction. Attached Figure Description

[0028] Figure 1 Transmission electron microscope image of polylysine-modified magnesium oxide nanoparticles;

[0029] Figure 2 Elemental analysis of polylysine-modified magnesium oxide nanoparticles;

[0030] Figure 3 Transmission electron microscope images of polylysine-modified magnesium oxide nanoparticles and black phosphorus nanosheets;

[0031] Figure 4 Elemental analysis of polylysine-modified magnesium oxide nanoparticles and black phosphorus nanosheets;

[0032] Figure 5 Electron microscopy images and elemental analysis of PCPL-MgO-BP composite hydrogels formed by dispersing polylysine-modified magnesium oxide nanoparticles and black phosphorus nanosheets in polyvinyl alcohol and collagen solutions.

[0033] Figure 6 Optical images of the PCPL-MgO-BP composite hydrogel;

[0034] Figure 7 Micro-CT structural image of PCPL-MgO-BP composite hydrogel;

[0035] Figure 8 Infrared (A) and Raman (B) spectra of the PCPL-MgO-BP composite hydrogel and its constituent materials.

[0036] Figure 9 The zeta potential of the PCPL-MgO-BP composite hydrogel and its constituent materials;

[0037] Figure 10 The pore size distribution (A) and porosity distribution (B) of the PCPL-MgO-BP composite hydrogel are shown.

[0038] Figure 11 Optical images and Micro-CT structural images of PCPL-MgO-BP composite hydrogel after 7 days of immersion and degradation in SBF solution;

[0039] Figure 12 Electron microscopy images and elemental analysis of PCPL-MgO-BP composite hydrogel after immersion in SBF solution for 7 days for degradation;

[0040] Figure 13 Optical images and Micro-CT structural images of PCPL-MgO-BP composite hydrogel after 28 days of immersion in SBF solution for degradation.

[0041] Figure 14 Electron microscopy images and elemental analysis of PCPL-MgO-BP composite hydrogel after 28 days of immersion and degradation in SBF solution;

[0042] Figure 15 The degradation rate of the PCPL-MgO-BP composite hydrogel;

[0043] Figure 16 The magnesium ion release rate during the degradation process of PCPL-MgO-BP composite hydrogel;

[0044] Figure 17 Compressive stress (A), compressive modulus, and compressive strength (B) of PCPL-MgO-BP composite hydrogel.

[0045] Figure 18Compressive stress (A), compressive modulus, and compressive strength (B) of PCPL-MgO-BP composite hydrogel after immersion in SBF solution for 14 days for degradation.

[0046] Figure 19 Storage modulus and loss modulus (A), viscosity (B), loss factor (C), and mechanical properties of PCPL-MgO-BP composite hydrogel after immersion in SBF solution for 14 days before degradation, and 10 cycles of compression mechanical properties (DE).

[0047] Figure 20 The graphs show the electrochemical impedance (A), resistance as a function of frequency (B), phase angle of electrochemical impedance as a function of frequency (C), cyclic voltammetry (D), and stability after 100 cycles of cyclic voltammetry (E) of the PCPL-MgO-BP composite hydrogel.

[0048] Figure 21 The graph shows the changes in the fidelity of the PCPL-MgO-BP composite hydrogel before and after immersion in SBF solution for degradation at different frequency signals.

[0049] Figure 22 A schematic diagram of the cell compatibility experiment design (A) and CCK-8 results are shown. The CCK-8 assay was used to detect the cell proliferation activity on days 1 (B), 2 (C), and 3 (D). All data are expressed as mean ± standard deviation (n=4).

[0050] Figure 23 Representative fluorescence images of live / dead cells stained (4× objective lens);

[0051] Figure 24 Semi-quantitative analysis of live cell fluorescence intensity on days 1 (A), 2 (B), and 3 (C); all data are expressed as mean ± standard deviation (n=4), *P<0.05, **P<0.01;

[0052] Figure 25 Representative images from the scratch test (4× objective lens);

[0053] Figure 26 Quantitative analysis of cell migration at 12h (A) and 24h (B); all data are expressed as mean ± standard deviation (n=4), *P<0.05;

[0054] Figure 27 Representative images of alkaline phosphatase (ALP) staining (days 7 and 14, 10× objective) (A) and semi-quantitative analysis of ALP activity (day 7 (B) and day 14 (C)); all data are expressed as mean ± standard deviation (n=4), *P<0.05, **P<0.01, ***P<0.001;

[0055] Figure 28 Representative images of alizarin red (ARS) staining (days 10 and 21, 10× objective) (A) and semi-quantitative analysis of calcium nodule deposition (days 10 (B) and 21 (C)); all data are expressed as mean ± standard deviation (n=4), *P<0.05, **P<0.01, ***P<0.001;

[0056] Figure 29 Representative images of type I collagen (Col1) immunofluorescence staining (scale bar: 50 μm) (A) and semi-quantitative analysis of fluorescence intensity (day 7 (B) and day 14 (C)); all data are expressed as mean ± standard deviation (n=4), *P<0.05, **P<0.01, ***P<0.001;

[0057] Figure 30 Representative images of osteopontin (OPN) immunofluorescence staining (scale bar: 50 μm) (A) and semi-quantitative analysis of OPN fluorescence intensity (day 7 (B) and day 14 (C)); all data are expressed as mean ± standard deviation (n=4), *P<0.05, **P<0.01, ***P<0.001;

[0058] Figure 31 The image shows the histological analysis of the skull defect repair area at 4 and 8 weeks post-surgery, with hematoxylin and eosin (HE) staining results. The upper half of the image is a low-power panoramic view (scale bar: 1 mm), and the lower half is a high-power magnification of the corresponding blue-framed area (scale bar: 200 μm).

[0059] Figure 32 The images show the results of Masson's trichrome staining for histological analysis of the skull defect repair area at 4 and 8 weeks post-surgery; the upper half of the image is a low-power panoramic view (scale bar: 1 mm), and the lower half is a high-power magnification of the corresponding blue-framed area (scale bar: 200 μm).

[0060] Figure 33 Micro-CT three-dimensional reconstruction images of the skull defect area in each group 4 weeks after surgery (A) and quantitative analysis of bone mineral density (B);

[0061] Figure 34 This is a representative ECoG signal waveform acquired 4 weeks post-surgery.

[0062] Figure 35 Micro-CT three-dimensional reconstruction images of the skull defect area in each group at 8 weeks postoperatively (A) and quantitative analysis of bone mineral density (B);

[0063] Figure 36 This is a representative ECoG signal waveform acquired 8 weeks post-surgery. Detailed Implementation

[0064] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0065] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0066] In this embodiment of the invention, the cold source can be a cold table (Polystat CC1, Huber, Germany) or other refrigeration equipment.

[0067] Example 1:

[0068] A flexible conductive hydrogel scaffold that combines cranial bone repair and nerve signal monitoring functions is made by using polyvinyl alcohol-collagen-polylysine as a matrix, doping with magnesium oxide nanoparticles and black phosphorus nanosheets, and finally forming it through freeze-induced phase separation and freeze-drying.

[0069] The specific preparation method is as follows:

[0070] (1) Preparation of composite precursor solution: Dissolve 5g of polyvinyl alcohol in deionized water, add 0.1g of collagen, 0.2g of magnesium oxide nanocomposite coated with polylysine and black phosphorus nanodispersion, mix well and make up to 100mL to obtain composite precursor solution with a final concentration of black phosphorus of 0.1mg / mL.

[0071] (2) Preparation of flexible conductive hydrogel scaffold: The composite precursor solution was injected into a mold and placed on a cold source for one freeze-thaw cycle. The freeze-thaw cycle was set as follows: freezing at -10°C for 24 hours at room temperature and thawing at room temperature for 4 hours; then vacuum freeze-drying (vacuum freeze dryer, model FreeZone 2.5L, manufacturer: Labconco, USA) was performed to obtain a flexible conductive hydrogel scaffold with an interpenetrating porous structure. The cold energy of the unidirectional freezing was transferred longitudinally from the bottom to the top of the mold. The vacuum freeze-drying temperature was -50°C, the time was 60 hours, and the vacuum degree was below 50 Pa.

[0072] Preparation of black phosphorus nano-dispersion: Block black phosphorus crystals were ultrasonically treated in N-methylpyrrolidone using an ultrasonic cell disruptor (Scientz-IID, China) at 600 W in an ice bath for 6 hours. The supernatant was then centrifuged to obtain the dispersion. The mass concentration of black phosphorus in the black phosphorus nano-dispersion was 0.1%.

[0073] The magnesium oxide nanocomposite encapsulated with polylysine was prepared by in-situ coprecipitation. Specifically, the magnesium oxide and polylysine were measured in a mass ratio of 1:1. A 50 mM MgCl2 solution and a 1 mg / mL polylysine solution were mixed under alkaline conditions at pH 10.5. A 1M NaOH solution was slowly added dropwise until no more precipitate was formed. After reacting for another 2 hours, the product was obtained by centrifugation and washing.

[0074] Example 2:

[0075] A flexible conductive hydrogel scaffold that combines cranial bone repair and nerve signal monitoring functions is made by using polyvinyl alcohol-collagen-polylysine as a matrix, doping with magnesium oxide nanoparticles and black phosphorus nanosheets, and finally forming it through freeze-induced phase separation and freeze-drying.

[0076] The specific preparation method is as follows:

[0077] (1) Preparation of composite precursor solution: Dissolve 15g of polyvinyl alcohol in deionized water, add 1g of collagen, 6g of magnesium oxide nanocomposite coated with polylysine and black phosphorus nanodispersion, mix well and make up to 100mL to obtain composite precursor solution with a final concentration of black phosphorus of 0.4mg / mL.

[0078] (2) Preparation of flexible conductive hydrogel scaffold: The composite precursor solution was injected into a mold and subjected to 5 freeze-thaw cycles on a cold source. The freeze-thaw cycle was set as follows: freezing at -80°C for 4 hours at room temperature and thawing at room temperature for 12 hours; then vacuum freeze-drying (vacuum freeze dryer, model FreeZone 2.5L, manufacturer: Labconco, USA) was performed to obtain a flexible conductive hydrogel scaffold with an interpenetrating porous structure. The cold energy of the unidirectional freezing was transferred longitudinally from the bottom to the top of the mold. The vacuum freeze-drying temperature was -60°C, the time was 36 hours, and the vacuum degree was below 50 Pa.

[0079] Preparation of black phosphorus nano-dispersion: Block black phosphorus crystals were ultrasonically treated in N-methylpyrrolidone using an ultrasonic cell disruptor (Scientz-IID, China) at 600 W in an ice bath for 6 hours. The supernatant was then centrifuged to obtain the dispersion. The mass concentration of black phosphorus in the black phosphorus nano-dispersion was 1%.

[0080] The magnesium oxide nanocomposite encapsulated with polylysine was prepared by in-situ coprecipitation. Specifically, the magnesium oxide and polylysine were measured in a mass ratio of 1:1. A 50 mM MgCl2 solution and a 1 mg / mL polylysine solution were mixed under alkaline conditions at pH 10.5. A 1M NaOH solution was slowly added dropwise until no more precipitate was formed. After reacting for another 3 hours, the product was obtained by centrifugation and washing.

[0081] Example 3:

[0082] A flexible conductive hydrogel scaffold that combines cranial bone repair and nerve signal monitoring functions is made by using polyvinyl alcohol-collagen-polylysine as a matrix, doping with magnesium oxide nanoparticles and black phosphorus nanosheets, and finally forming it through freeze-induced phase separation and freeze-drying.

[0083] The specific preparation method is as follows:

[0084] (1) Preparation of composite precursor solution: Dissolve 10g of polyvinyl alcohol in deionized water, add 0.5g of collagen, 3g of magnesium oxide nanocomposite coated with polylysine and black phosphorus nanodispersion, mix well and make up to 100mL to obtain composite precursor solution with a final concentration of black phosphorus of 0.2mg / mL.

[0085] (2) Preparation of flexible conductive hydrogel scaffold: The composite precursor solution was injected into a mold and placed on a cold source for three freeze-thaw cycles. The freeze-thaw cycle was set as follows: freezing at room temperature for 12 hours at -20°C and thawing at room temperature for 8 hours; then vacuum freeze-drying (vacuum freeze dryer, model FreeZone 2.5L, manufacturer: Labconco, USA) was performed to obtain a flexible conductive hydrogel scaffold with an interpenetrating porous structure, named PCPL-MgO-BP. The cold energy of the unidirectional freezing was transferred longitudinally from the bottom to the top of the mold. The vacuum freeze-drying temperature was -55°C, the time was 48 hours, and the vacuum degree was below 50 Pa.

[0086] Preparation of black phosphorus nano-dispersion: Block black phosphorus crystals were ultrasonically treated in N-methylpyrrolidone using an ultrasonic cell disruptor (Scientz-IID, China) at 600 W in an ice bath for 6 hours. The supernatant was then centrifuged to obtain the dispersion. The mass concentration of black phosphorus in the black phosphorus nano-dispersion was 0.5%.

[0087] The magnesium oxide nanocomposite encapsulated with polylysine was prepared by in-situ coprecipitation. Specifically, the magnesium oxide and polylysine were measured in a mass ratio of 1:1. A 50 mM MgCl2 solution and a 1 mg / mL polylysine solution were mixed under alkaline conditions at pH 10.5. A 1M NaOH solution was slowly added dropwise until no more precipitate was formed. After reacting for another 2 hours, the product was obtained by centrifugation and washing.

[0088] Example 4:

[0089] The difference between this embodiment and Example 3 is that the final concentration of black phosphorus is 0.1 mg / mL.

[0090] Example 5:

[0091] The difference between this embodiment and Example 3 is that the final concentration of black phosphorus is 0.4 mg / mL.

[0092] Example 6:

[0093] The difference between this embodiment and embodiment 3 is that this embodiment is placed on a cold source and subjected to a freeze-thaw cycle once.

[0094] Comparative Example 1:

[0095] The difference between this embodiment and Example 3 is that no black phosphorus nanodispersion was added. The resulting hydrogel was named PCPL-MgO.

[0096] Experimental Section:

[0097] 1. Materials Characterization and Testing

[0098] The morphology and size of PL-MgO nanoclusters (i.e., polylysine-encapsulated magnesium oxide nanocomposite) and BP nanosheets were observed using transmission electron microscopy (TEM, JEM-2100, JEOL, Japan). The microporous structure of the hydrogel scaffold was characterized using scanning electron microscopy (SEM, SU8010, Hitachi, Japan). Elemental surface scanning analysis was performed using TEM and SEM equipped with energy dispersive spectroscopy (EDS, X-MaxN, Oxford Instruments, UK). Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher, USA) and Raman spectroscopy (inVia, Renishaw, UK) were used to analyze the chemical composition and intermolecular interactions of the material. Three-dimensional scanning and reconstruction of the scaffold were performed using a Micro-CT system (Venus® micro-CT VNC-102, Ping Sheng Medical Technology Co., Ltd.). The internal pore connectivity was quantitatively assessed and pore size distribution and porosity were calculated using CT-Analyzer software. A Zeta potentiometer (Zetasizer Nano) was used. The surface charge properties of the composite material were determined using the ZS90 (Malvern Panalytical, UK) under different pH conditions.

[0099] 2. Evaluation of in vitro degradation and ion release behavior

[0100] Hydrogel samples were immersed in simulated body fluid (SBF, Sigma-Aldrich, USA) and continuously shaken (60 rpm) in a 37°C constant-temperature shaker (THZ-98A, Shanghai Yiheng). Samples were removed at preset time points (7 days and 28 days), gently rinsed with deionized water, and directly characterized. Macroscopic morphological changes were recorded using a digital camera (EOS 90D, Canon, Japan). Microstructural evolution and surface elemental distribution were observed using a scanning electron microscope (SU8010, Hitachi, Japan) and its accompanying energy dispersive spectroscopy (EDS). Three-dimensional reconstruction was performed using a Micro-CT system (Skyscan 1272, Bruker, Belgium) to assess the dynamic changes in the internal pore structure.

[0101] To accurately quantify the degradation rate of the material, precisely weighed cylindrical hydrogel samples (initial weight denoted as W0) were immersed in phosphate-buffered saline (PBS, HyClone, USA) and shaken in a 37°C constant-temperature shaker. Samples were removed at specific time points (days 1, 3, 7, 14, 28, and 56), rinsed with deionized water, thoroughly dried using a freeze dryer, and weighed again precisely (W1). The mass loss of the sample at different time points was calculated using the formula: mass loss rate (%) = (W0 – W1) / W0 × 100%, and a mass loss curve was plotted. To simultaneously assess the release behavior of magnesium ions and avoid interference from background ions in SBF, all soaking liquid was collected at each time point in the same PBS soaking system used for the above mass loss determination, and an equal volume of fresh PBS was added. The collected liquid was filtered and diluted using a 0.22 μm filter membrane, and the Mg content was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES, iCAP 7400, Thermo Fisher, USA). 2+ Concentration. Based on the measured concentration, solution volume, and initial sample mass, calculate the cumulative release amount (μg / mL / mg) and plot the release kinetic curve.

[0102] 3. Mechanical property testing

[0103] Quasi-static compression tests were conducted using a universal testing machine (Instron 5943, USA). Cylindrical samples (Φ10 mm × 5 mm) were compressed to 80% strain at a fixed rate, and stress-strain curves were recorded. The compressive modulus and maximum strength were calculated. To investigate the effect of degradation on mechanical properties, the samples were immersed in simulated body fluid (SBF, Sigma-Aldrich, USA) for 14 days, and the above tests were repeated. Dynamic rheological properties were characterized using a rotational rheometer (DHR-2, TA Instruments, USA). A parallel plate fixture was used to perform frequency scanning (0.1–10 Hz) in the linear viscoelastic region to obtain the storage modulus (G′), loss modulus (G″), and complex viscosity (η*), and the loss factor tanδ (= G″ / G′) was calculated. Cyclic compression performance was also evaluated using a universal testing machine. Samples immersed in SBF for 14 days were subjected to 9 consecutive compression-unloading cycles (50% strain). The fatigue resistance and structural recovery ability were evaluated by the changes in peak stress and modulus.

[0104] 4. Electrochemical performance testing

[0105] A three-electrode system was used to systematically electrochemically characterize the PCPL-MgO-BP hydrogel in phosphate-buffered saline (PBS, HyClone, USA, pH 7.4). An electrochemical workstation (CHI760E, Shanghai Chenhua) was used, with a flexible conductive hydrogel scaffold as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. Tests included: electrochemical impedance spectroscopy (EIS) measurements in the frequency range of 0.001 Hz–100 kHz (amplitude 10 mV); cyclic voltammetry at a scan rate of 50 mV / s within a potential window of -0.6 V to 0.6 V, with 100 consecutive scans to assess stability; and inputting sinusoidal signals of different frequencies (0.01 Hz, 1 Hz, 1000 Hz) using a function generator (AFG1022, Tektronix, USA), simultaneously recording the output waveform and amplitude to analyze signal fidelity. Some tests were repeated after soaking in PBS for 2 weeks to examine long-term electrochemical stability.

[0106] 5. Cell compatibility evaluation

[0107] 10 mg of hydrogel was immersed in 5 mL of α-MEM basal medium (Cyber-Tech (Shanghai) Biotechnology Co., Ltd.) at 37°C for 48 hours. The extract was then filtered through a 0.22 μm filter to obtain a sterile extract (concentration 0.2 mg / mL). MC3T3-E1 cells (Chinese Academy of Sciences Cell Bank) were cultured at 1×10⁻⁶... 4 1 / well (96-well plate) and 5×10 5Cells were seeded at a density of 10 cells / well (6-well plate) using α-MEM complete medium (penicillin-streptomycin antibiotics, Wuhan Pronosei Life Sciences Co., Ltd.) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin, and pre-cultured at 37°C in a 5% CO2 incubator for 24 hours. The experiment included a blank control group, a PCPL-MgO group (Comparative Example 1), and PCPL-MgO-BP1 (Example 4), PCPL-MgO-BP2 (Example 3), and PCPL-MgO-BP3 (Example 5) groups. Cell proliferation was detected using a CCK-8 assay kit (Beyotime). 10 μL of CCK-8 solution was added on days 1, 2, and 3 of co-culture, and after incubation for 1.5 hours, the absorbance at 450 nm was measured using a microplate reader. Cell viability was assessed using a Calcein-AM / PI double staining kit (Beyotime), observed using a fluorescence microscope, and semi-quantitatively analyzed using ImageJ. Cell migration was evaluated using a scratch assay. Scratches were created using a 200 μL pipette tip, and data were recorded under a microscope at 0, 12, and 24 hours. The scratched area was quantified using ImageJ software, and the migration rate was calculated using the following formula:

[0108] Cell migration % = (A0-A r Ar = ) / A0 × 100%, where A0 represents the initial scratch area at 0 hours and Ar represents the remaining scratch area at a specific time point.

[0109] 6. Evaluation of in vitro osteogenic differentiation performance

[0110] MC3T3-E1 cells were loaded at 5×10 4The cells were seeded at a density of cells / well in 12-well plates and pre-cultured for 24 hours in α-MEM complete medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-dextrin antibiotics. Then, the medium was replaced with osteogenic induction medium (containing 10 mM sodium β-glycerophosphate, Beijing Solarbio Science & Technology Co., Ltd.) containing extracts of each material (blank control group, PCPL-MgO group, PCPL-MgO-BP1 / 2 / 3 group), 50 μM ascorbic acid, and 100 nM dexamethasone for further culture. Early differentiation was assessed by alkaline phosphatase staining on days 7 and 14. Cells were fixed with 4% paraformaldehyde (Beyotime) and then developed using the BCIP / NBT kit (Beyotime). Observation was performed under an inverted microscope and semi-quantitative analysis was performed using ImageJ. Late mineralization was assessed by Alizarin Red S (Beyotime) staining on days 10 and 21. Calcium nodules were observed and stained areas were quantified. Osteogenesis-related proteins were detected by immunofluorescence staining. After permeabilization blocking, cells were incubated overnight at 4°C with Collagen I antibody (1:200, Affinity Biosciences, Jiangsu) and osteopontin antibody (1:200, Affinity Biosciences, Jiangsu). The corresponding fluorescent secondary antibody (1:500, Affinity Biosciences, Jiangsu) was incubated at room temperature in the dark for 2 hours. After DAPI counterstaining, fluorescence intensity was observed and quantified using a laser confocal microscope.

[0111] 7. In vivo cranial bone repair and electrophysiological monitoring

[0112] All animal experimental procedures in this study were approved by the animal ethics committee and strictly followed international guidelines for the husbandry and use of laboratory animals. The in vivo performance of the material was evaluated using a critical-sized skull defect model (8 mm in diameter) in SD rats. Sixty rats were randomly divided into four groups (Control group, PCPL-MgO group, PCPL-MgO-BP1 group, and PCPL-MgO-BP2 group), with 13 rats in each group used for evaluation at two time points (week 4, n=6; week 8, n=7), and the remaining 2 rats were reserved for surgery. After surgical implantation of the corresponding scaffold, the following evaluations were performed at weeks 4 and 8 postoperatively. Bone mineral density (BMD) and bone volume fraction (BV / TV) were calculated using an in vivo Micro-CT system. Cortical EEG signals were acquired in the conscious state using an implanted EEG recording system (CerePlex Direct, Blackrock Microsystems, USA) (sampling rate 1000 Hz, bandpass 0.5-100 Hz). In addition, after euthanizing animals at various time points, in vitro scanning was performed using a high-resolution Micro-CT system (80 kV, 125 μA, 10 μm resolution). Three-dimensional reconstruction was then performed using CTAn software to further analyze the microstructure and thickness distribution of newly formed bone. Simultaneously, tissue samples from the defect area were fixed in 4% paraformaldehyde, decalcified with EDTA, embedded in paraffin, and sectioned for hematoxylin-eosin (H&E) staining and Masson's trichrome staining to observe the morphology, cell distribution, and collagen deposition of the newly formed bone tissue.

[0113] 8. Statistical Analysis

[0114] All data are expressed as mean ± standard deviation, and each experiment was independently repeated at least four times. Statistical analysis was performed using GraphPad Prism 8.0 and SPSS 26.0 software. Intergroup comparisons were performed using t-tests or one-way ANOVA, combined with Tukey's post-hoc test; P < 0.05 was considered statistically significant. Intergroup correlations were analyzed using Pearson correlation coefficient analysis; P < 0.05 was considered a significant correlation.

[0115] 9. Results and Discussion

[0116] 9.1 Construction and hierarchical characterization of PCPL-MgO-BP hydrogel scaffold

[0117] To achieve the dual functions of cranial repair and neural signal monitoring, this study constructed a flexible conductive hydrogel scaffold (PCPL-MgO-BP) using polyvinyl alcohol-collagen-polylysine (PVA-Col-PL) as the matrix and doped with magnesium oxide (MgO) nanoparticles and black phosphorus (BP) nanosheets. The multi-level structure and compositional characteristics of the scaffold in Examples 3-5 are as follows. Figures 1-10 As shown.

[0118] The morphology and composite mechanism of nano-functional components are fundamental to material functionalization. Transmission electron microscopy images (…) Figure 1 The results showed that polylysine (PL)-encapsulated magnesium oxide formed uniform nanoclusters with a particle size of 600-800 nm. The organic coating on the surface indicated that PL was successfully modified onto the MgO surface, effectively improving its dispersion stability. Scanning transmission electron microscopy and corresponding elemental surface scanning analysis (STEM) were performed. Figure 2 This further reveals a typical "core-shell" structure: Mg and O elements are concentrated in the cluster core, while N and C elements contained in PL are uniformly coated on the periphery. When PL-MgO is combined with black phosphorus nanosheets ( Figure 3 The lamellar BP and granular PL-MgO form a tight "lamellar-granular" heterostructure. The corresponding elemental distribution diagram is shown below. Figure 4 The results show that P and Mg elements are spatially complementary, indicating that they achieve stable nanoscale composite through electrostatic interaction, laying the foundation for the subsequent construction of uniform functional regions in three-dimensional hydrogel networks.

[0119] The macroscopic morphology and three-dimensional porous structure of hydrogels are crucial to their biological functions. Scanning electron microscopy images ( Figure 5 The results showed that the PCPL-MgO-BP hydrogel possesses a highly interconnected honeycomb porous structure with pore sizes concentrated between 100-250 μm. This structure originates from the template effect of ice crystals during unidirectional freeze-induced phase separation, which is beneficial for cell migration and nutrient transport. Energy dispersive spectroscopy (EDS) elemental surface area scanning confirmed the uniform distribution of C, N, O, Mg, and P elements in the three-dimensional network, indicating good integration of functional components. Macroscopic photographs (…) Figure 6 The display scaffold possesses a complete disc-shaped structure and molding stability. Micro-CT 3D reconstructed images ( Figure 7 This further confirms that the internal channels have good connectivity, meeting the structural requirements of an ideal bone tissue engineering scaffold.

[0120] The chemical structures and interactions of each component were analyzed using spectroscopic methods. Fourier transform infrared spectroscopy (FTIR) Figure 8 A) indicates that the composite hydrogel retains PVA (3200-3600 cm⁻¹). -1 (-OH stretching vibration), Col (1650 cm) -1 amide I band), PL (1550 cm) -1Characteristic peaks of each component, including the amide II band, were observed, and no new covalent bond signals were found. However, the absorption peaks of the amide I band (C=O) and the amide II band (NH) were significantly broadened, indicating strong hydrogen bonding and electrostatic interactions between the components. This suggests that the composite material mainly relies on non-covalent mechanisms, which is beneficial for maintaining its biocompatibility and dynamic properties. Raman spectroscopy (… Figure 8 The characteristic vibrational peak of BP in (B) (~362 cm) -1 With ~467 cm -1 The fact that the crystal structure remains clear after composite formation indicates that its lattice structure was maintained during the preparation process, which is a necessary prerequisite for ensuring the high conductivity of the hydrogel.

[0121] The surface potential and porosity of a material are key physical parameters affecting its biological properties. Zeta potential testing ( Figure 9 The results showed that after the positively charged PL-MgO (+14.5 mV) recombines with the strongly negatively charged BP (-26.7 mV), the overall potential tends to be neutral (+1.2 mV). This charge neutralization phenomenon is not only key to driving the orderly assembly of nanocomponents, but also helps reduce non-specific protein adsorption caused by surface charge after implantation, thereby improving biocompatibility. Pore size and porosity statistics ( Figure 10 The results showed that the optimized group PCPL-MgO-BP2 had the most concentrated pore size distribution (120-180 μm) and the highest porosity (66.4% ± 1.6%). These parameters are close to the standard of natural cancellous bone, which can provide sufficient space for cell ingrowth and bone regeneration while providing a stable signal transduction environment for the neuroelectrode-tissue interface.

[0122] Through component design and process control, a PCPL-MgO-BP flexible conductive hydrogel scaffold with suitable chemical composition, an ideal three-dimensional porous structure, and a uniformly distributed functional network was successfully constructed. Its unique structural characteristics lay the material foundation for realizing the dual biological functions of bone repair and nerve signal monitoring.

[0123] 9.2 In vitro degradation behavior and functional ion release characteristics of hydrogel scaffolds

[0124] The degradation behavior and functional ion release characteristics of the PCPL-MgO-BP flexible conductive hydrogel scaffold in simulated physiological environments are core performance indicators for its use as a bone repair scaffold. Therefore, this study systematically evaluated its long-term stability, degradation, and ion release kinetics through structural evolution studies in simulated body fluid (SBF) and quantitative mass loss and ion release experiments in phosphate-buffered saline (PBS).

[0125] like Figure 11As shown, after immersion in SBF for 7 days, the macroscopic structure of the hydrogel remained intact, without structural disintegration. Micro-CT 3D reconstructed images ( Figure 11 The internal porous network remains highly interconnected, with pore sizes maintained within the ideal range of 100-250 μm for cell permeability and substance exchange. Scanning electron microscopy observations (…) Figure 12 ) revealed slight thinning of the pore walls and wrinkling at the edges, while elemental surface scanning analysis ( Figure 12 The uniform distribution of Mg and P elements was confirmed, indicating that the material maintained both structural integrity and the stability of functional components in the early stages of degradation. With an extended soaking time of 28 days, the material entered the active degradation and biomineralization stage. Macroscopic morphology ( Figure 13 The image shows moderate collapse at the edge of the stent and increased surface roughness. Micro-CT images ( Figure 13 This confirmed that the three-dimensional channel structure remained connected, but some of the pore walls underwent reconstruction. It is noteworthy that scanning electron microscopy (SEM)... Figure 14 ) Scale-like and granular deposits were observed to form on the pore wall surface, and energy dispersive spectroscopy analysis (EDS) Figure 14 The study revealed the co-location of Ca, P, and Mg elements in the region, indicating that the degradation process successfully induced the formation of a bone-like apatite layer, creating a biomineralized microenvironment conducive to bone regeneration.

[0126] The quantitative degradation behavior and ion release characteristics of the material show a significant component dependence. For example... Figure 15 Monitoring of mass loss in PBS revealed that on day 56, the PCPL-MgO-BP3 group had the highest mass loss rate (73.8% ± 2.5%), the PCPL-MgO-BP2 group had the lowest mass loss rate (70.2% ± 2.5%), and the PCPL-MgO group had the slowest mass loss rate (64.1% ± 3.5%). This indicates that the introduction of BP nanosheets effectively regulated the degradation rate through the oxidation of their edge sites and the promotion of hydrolysis of the polymer backbone.

[0127] To investigate the bioactive ion release performance of the hydrogel during degradation, the soaking solution was collected in the same PBS system, and magnesium ions (Mg) were determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+ Release concentration, results as follows Figure 16 As shown, all samples exhibited an initial release peak at 1-3 days, followed by a slow plateau, with Mg still detectable at 56 days. 2+ Specifically, the higher the BP content, the greater the early and overall release: the peak release in the BP3 group on day 1 was approximately 350 ± 50 mg / L. -1 The BP2 group received approximately 190 ± 15 mg / L. -1 The BP1 group received approximately 165 ± 20 mg / L.-1 The control group without BP had a BP level of approximately 150 ± 20 mg / L. -1 From day 4 onwards, all groups entered the sustained-release phase and tended to plateau. By day 56, the Mg levels in the BP3, BP2, and BP1 groups compared to the control group were... 2+ The amounts were approximately 80, 60, 40, and 38 mg·L, respectively. -1 The above data indicate that BP content affects Mg. 2+ Release showed a clear positive correlation, but excessively high BP (BP3 group) could trigger an overly strong early release peak, posing a potential biocompatibility risk; BP2 group maintained 100-200 mg / L on days 1-28. -1 The concentration range is within the ideal range for promoting angiogenesis and osteogenic differentiation, and the release lasts for more than 8 weeks. Therefore, it has a good balance in bone repair applications, providing a more stable release curve within the bioactive window, which is an optimal ratio between bone repair and safety.

[0128] 9.3 Mechanical properties and long-term stability of hydrogel scaffolds

[0129] Skull repair materials need to provide sufficient mechanical support while maintaining mechanical compatibility with brain tissue to ensure a stable electrode-tissue interface. The mechanical performance evaluation results of the PCPL-MgO-BP flexible conductive hydrogel scaffold are as follows: Figures 17-19 As shown.

[0130] With the increase of black phosphorus nanosheet content, the compressive modulus and strength of the hydrogel show a regular decrease. Figure 17 The PCPL-MgO group without BP exhibited the highest compressive modulus (1.24 ± 0.08 MPa) and strength (152 ± 9 kPa), while the PCPL-MgO-BP3 group showed the lowest (modulus 0.61 ± 0.05 MPa, strength 89 ± 7 kPa). This trend can be attributed to the interfacial effect of two-dimensional BP nanosheets in a three-dimensional network: although BP itself has excellent mechanical properties, its dispersion in the polymer matrix may, to some extent, disrupt the original physical cross-linking density between PVA / Col / PL-MgO, forming local stress concentration points. Notably, the PCPL-MgO-BP2 group, while maintaining functionality, has a compressive modulus (0.84 ± 0.06 MPa) that falls within the modulus range of human brain tissue (0.1–1 MPa) and cancellous bone of the skull (0.1–2 MPa). This characteristic helps alleviate the stress shielding effect caused by mechanical mismatch.

[0131] After 14 days of SBF immersion, the mechanical properties of all groups of hydrogels showed a moderate decrease. Figure 18However, the overall structure remains intact. The compressive modulus of the PCPL-MgO-BP2 group decreased from the initial 0.84 MPa to 0.58 MPa, and the strength decreased from 126 kPa to 82 kPa. This controlled decline in mechanical properties matches the expected bone repair process. During degradation, the material gradually transfers the mechanical load to the newly formed bone tissue, avoiding premature loss of mechanical properties that could lead to repair failure.

[0132] Dynamic rheological testing shows ( Figure 19 The PCPL-MgO-BP2 hydrogel (in the AC range) exhibited typical elastic solid characteristics (G'>G'') across the entire tested frequency range. Its storage modulus (G') remained in the range of 2.8–3.4 kPa, and its loss factor (tanδ) was between 0.28 and 0.35, indicating that the material possesses sufficient structural stiffness while also having a moderate energy dissipation capacity. This viscoelastic characteristic is crucial for adapting to the physiological pulsations of brain tissue. Furthermore, the shear-thinning behavior, where the complex viscosity significantly decreases with increasing frequency, indicates that the material can undergo moderate deformation under external force, which is beneficial for manipulating and shaping during surgical implantation.

[0133] Cyclic compression testing further verified the mechanical stability of the material under degradation conditions. Figure 19 (DE in the text). After 10 loading-unloading cycles at 50% strain, the stress-strain curves of the PCPL-MgO-BP2 hydrogel remained highly consistent, with a peak stress decay rate of less than 5%, demonstrating excellent fatigue resistance and structural recovery ability. This good cyclic stability mainly stems from the elastic characteristics of the polymer network and the synergistic effect between the nanocomposition components, ensuring that it can withstand the periodic mechanical stress under physiological conditions during long-term implantation.

[0134] The PCPL-MgO-BP flexible conductive hydrogel scaffold, especially the PCPL-MgO-BP2 group, exhibits good mechanical compatibility with brain tissue, controllable degradation-dependent mechanical attenuation, and excellent fatigue resistance. These mechanical properties enable it to provide temporary support for bone repair without causing significant stress shielding, while maintaining the long-term stability of the electrode-tissue interface, thus meeting the dual mechanical performance requirements of the integrated implantation platform.

[0135] 9.4 Electrochemical performance and signal transduction characteristics

[0136] As an implantable material that combines cranial repair and neurological monitoring functions, the electrochemical properties of PCPL-MgO-BP2 composite hydrogel directly affect its signal acquisition quality. Figure 20The Nyquist plot of A shows that the hydrogel exhibits a distinct semi-circular shape in the high-frequency region, with a fitted charge transfer resistance (Rct) of 730 Ω. After soaking in PBS for 2 weeks, the resistance only slightly increases to 812 Ω, indicating that its internal conductive network has good stability. Figure 20 The impedance spectrum shown in B indicates that the hydrogel impedance decreases with increasing frequency in the frequency range of 0.01-100 kHz, and the impedance is less than 1 kΩ at the characteristic frequency of 1 kHz. This value is significantly lower than that of traditional metal electrodes, which helps to reduce thermal noise and interference during signal acquisition.

[0137] Phase angle analysis ( Figure 20 The C-axis further reveals the material's capacitive properties, with a peak phase angle close to -75°, indicating that the electrode-electrolyte interface primarily exhibits capacitive behavior. This characteristic helps maintain the integrity of neural signal waveforms. Cyclic voltammetry results ( Figure 20 D) shows a symmetrical rectangular curve, indicating that the material has good electrochemical reversibility and charge storage capacity. After 100 consecutive cycles of testing ( Figure 20 The E and CV curves remain stable, and the current density decay is negligible, demonstrating that the material has long-term electrochemical stability.

[0138] Signal fidelity is a key indicator for evaluating the performance of neural electrodes. For example... Figure 21 As shown, at different input frequencies (0.01 Hz to 1000 Hz), the output signal remained highly consistent with the input signal, with no significant waveform distortion or phase delay. Particularly at the 1000 Hz high-frequency condition, the signal attenuation rate was less than 5%, indicating that the material has the ability to acquire high-frequency neural signals. This performance was well maintained after two weeks of PBS immersion, proving that this hydrogel electrode is suitable for long-term implantable neural signal monitoring applications.

[0139] The PCPL-MgO-BP2 composite hydrogel exhibits low interfacial impedance, good capacitance characteristics, excellent electrochemical stability, and high signal fidelity, meeting the requirements for high-quality neural electrical signal acquisition and providing electrical performance assurance for its application in integrated repair and monitoring after traumatic brain injury.

[0140] 9.5 Cell compatibility evaluation

[0141] Mouse embryonic osteoblast precursor cells (MC3T3-E1) were selected as the model cells. This cell line is derived from the mouse cranial parietal bone and exhibits typical osteogenic differentiation characteristics, making it an ideal model for evaluating the biocompatibility of bone repair materials. Figure 22 As shown in A, the biosafety of the PCPL-MgO-BP series materials and their potential mechanisms for promoting cell function were investigated through an in vitro evaluation experiment design with multiple time points and multiple indicators.

[0142] Regarding cell proliferation, the results of the CCK-8 experiment showed ( Figure 22 During co-culture with the material extract, cells in all groups maintained normal proliferation kinetics. During the 1-3 day culture period, there were no significant differences in absorbance values ​​among the experimental groups compared to the control group (P > 0.05), indicating that the material extract had no adverse effect on initial cell adhesion and early growth.

[0143] Cell viability staining analysis further verified the biosafety of the material. Figures 23-24 Fluorescence microscopy revealed that throughout the culture period, the experimental cells maintained typical osteoblast morphology, with intact cell membranes and fully extended pseudopodia. Semi-quantitative fluorescence intensity analysis was performed using ImageJ. Notably, compared to the CCK-8 assay, there was a significant difference in fluorescence intensity between the PCPL-MgO group and the control group on days 1 and 3 of culture (P < 0.05). This result may be attributed to errors in the semi-quantitative technique or cell plating technique, but overall, it aligns with the CCK-8 experimental data. Cell viability in all experimental groups was >80%, indicating that the material exhibited minimal cytotoxicity.

[0144] At the cellular function level, scratch assay and ImageJ semi-quantitative analysis revealed significant differences in cell migration ability among the groups. Figures 25-26 The results showed that the PCPL-MgO-BP1 and PCPL-MgO-BP2 groups exhibited higher migration rates after 12 hours of culture, which were significantly different from the control group (P < 0.05). After 24 hours of culture, all groups showed excellent migration ability compared with the control group. This significant migration-promoting effect can be attributed to the synergistic effect of multiple factors: firstly, the continuous release of Mg from the material... 2+ It has been shown to activate the integrin-FAK signaling pathway, promoting cytoskeleton rearrangement and pseudopodia formation; secondly, the phosphate ions generated by the degradation of BP nanosheets may participate in cellular energy metabolism, providing the necessary ATP support for the migration process; in addition, the presence of collagen in the material matrix provides adhesion sites required for cell migration, while the positive charge properties of polylysine may enhance the initial adhesion between cells and the material through electrostatic interactions.

[0145] The above results demonstrate that the PCPL-MgO-BP hydrogel material exhibits significant advantages in terms of cell compatibility. This advantage stems from the careful design and synergistic effect of the material's components: the PVA-Col-PL matrix provides the basic framework for biocompatibility, the MgO nanoparticles contribute bioactive ions, and the BP nanosheets optimize the material's surface properties and electrical performance.

[0146] 9.6 Evaluation of osteogenic differentiation performance

[0147] To systematically evaluate the osteogenic differentiation-promoting ability of PCPL-MgO-BP flexible conductive hydrogel scaffold material, an experimental design with multiple time points and multiple indicators was used to explore the effects of the material extract on the osteogenic differentiation process of MC3T3-E1 cells and its potential mechanisms.

[0148] In early osteogenic differentiation assessment, alkaline phosphatase (ALP) staining results showed ( Figure 27 After 7 days of induction with the extracts from each experimental group, MC3T3-E1 cells all showed significant ALP positivity, with the PCPL-MgO-BP2 group exhibiting significantly higher staining intensity than the control group. This phenomenon suggests that Mg released from the material... 2+ It may promote the expression of key enzymes in early osteogenic differentiation by activating the BMP / Smad signaling pathway. As the induction time was extended to 14 days, the ALP activity in all groups showed a time-dependent increase. The PCPL-MgO-BP2 group showed the strongest staining depth and distribution uniformity, and its semi-quantitative value reached about 2.5 times that of the control group, indicating that the material can continuously and effectively promote the early osteogenic differentiation process.

[0149] In assessing late-stage mineralization capacity, alizarin red S staining results ( Figure 28 The results showed that significant calcium nodule formation was observed as early as day 10 of induction culture, and the degree of mineralization further deepened by day 21. Semi-quantitative analysis indicated that calcium nodule production in the PCPL-MgO-BP2 group was significantly higher than in other experimental groups. Notably, the calcium nodules formed in the PCPL-MgO-BP2 group exhibited a typical clump-like distribution and a dense structure. This excellent mineralization induction ability may be attributed to the reaction of phosphate ions generated by the degradation of BP nanosheets with Mg. 2+ The synergistic effect of these factors provides an ideal microenvironment for the deposition of hydroxyapatite.

[0150] Immunofluorescence staining results of osteogenic-related proteins further confirmed the osteogenic differentiation-promoting effect of the material. Figure 29 Regarding the expression of the early marker type I collagen (Col I), the PCPL-MgO-BP2 group showed the strongest fluorescence signal on both days 7 and 14. This result echoes the ALP staining data, jointly indicating that the material can effectively promote the expression of proteins related to early osteogenic differentiation. Regarding the expression of the late marker osteopontin (OPN), all experimental groups showed a time-dependent increasing trend, with the PCPL-MgO-BP2 group reaching its highest fluorescence intensity on day 14, significantly higher than the control group. Figure 30 OPN, a key protein in bone matrix maturation and mineralization, demonstrates the material's role in promoting the entire osteogenic differentiation process through its high expression level.

[0151] The above results indicate that the PCPL-MgO-BP flexible conductive hydrogel scaffold material, especially the PCPL-MgO-BP2 group, effectively promotes osteoblast differentiation, maturation, and mineralization through multiple mechanisms. This excellent osteogenic performance is mainly due to the synergistic effect of the following factors: First, the continuous release of Mg from the material... 2+ It has been shown to activate multiple osteogenic signaling pathways, including the BMP / Smad and Wnt / β-catenin pathways. Secondly, the degradation of BP nanosheets not only provides phosphate ions required for bone mineralization, but their unique two-dimensional structure also provides a favorable interface for cell attachment and proliferation. Furthermore, the collagen component in the material matrix mimics the microenvironment of the natural bone matrix, providing essential biological clues for osteogenic differentiation. Of particular note is that the PCPL-MgO-BP2 group showed the best performance across all tested indicators, indicating that the precise ratio of each component in the material is crucial for achieving optimal osteogenic induction.

[0152] Thus, the PCPL-MgO-BP flexible conductive hydrogel scaffold material shows promising application prospects in bone repair. Through ingenious material design and component optimization, it successfully constructs a microenvironment conducive to osteogenic differentiation, providing important experimental evidence and theoretical support for the development of novel bone tissue engineering materials.

[0153] 9.7 Simultaneous Monitoring and Analysis of In Vivo Cranial Repair and Neurological Function

[0154] Although previous in vitro experiments have confirmed that PCPL-MgO-BP2 material possesses good osteogenic activity and electrochemical properties, its osteointegration effect and long-term signal stability in a real physiological environment still need to be verified through in vivo experiments. Therefore, this study established a rat skull defect model to systematically evaluate the repair and monitoring performance of this material under complex in vivo conditions. Given that the PCPL-MgO-BP3 group exhibited excessively rapid degradation characteristics in vitro (degradation rate of approximately 87.4% after 56 days), it was not included in the in vivo study to ensure the long-term stability of the scaffold during in vivo repair. Ultimately, based on comprehensive in vitro evaluation, the PCPL-MgO-BP1 and PCPL-MgO-BP2 groups were selected for in vivo functional verification.

[0155] The in vivo tissue repair performance of different composite materials was evaluated using hematoxylin-eosin (H&E) and Masson's trichrome staining. H&E staining results showed ( Figure 31At week 4 post-surgery, the control group showed poor defect repair with disordered cell arrangement; the PCPL-MgO group showed initial signs of cell infiltration and repair; while the PCPL-MgO-BP1 and PCPL-MgO-BP2 groups exhibited a greater number of cells and better tissue repair. By week 8, the control group still had significant defects, the PCPL-MgO group showed some progress in repair but was still incomplete, while the tissue morphology of the two BP-treated groups further matured, and the cell arrangement tended to be normal, showing better tissue differentiation and integration capabilities. (Marshon staining results) Figure 32 Further analysis revealed the collagen deposition situation: at week 4, the control group had few collagen fibers, the PCPL-MgO group had some collagen formation, while the BP group had significantly richer collagen deposition; at week 8, the BP group not only had a significant increase in collagen amount, but also had neat and orderly fiber arrangement, reflecting good collagen remodeling and tissue maturity.

[0156] Micro-CT reconstructed images showed that at 4 weeks post-surgery, the defect area in the PCPL-MgO-BP2 group had formed a continuous and dense bone bridge structure, while the control group and the PCPL-MgO group still showed obvious defect cavities. Figure 33 (A) By week 8, the defect area in the PCPL-MgO-BP2 group had basically completed bony healing, and the new bone structure was well integrated with the surrounding primary bone tissue. Figure 35 A in the text). Quantitative analysis of bone regeneration showed ( Figure 33 B in Figure 35 In group B), the bone mineral density of the PCPL-MgO-BP2 group reached 271.6±25.7 mgHA / cm³ and 373.8±31.2 mgHA / cm³ at weeks 4 and 8 postoperatively, respectively, and the bone volume fraction (BV / TV) reached 65.2±5.9% at week 8, which was significantly higher than that of other groups (P<0.0001).

[0157] Results of electroencephalogram (ECoG) monitoring in neurological function monitoring ( Figure 34 , Figure 36 The results showed a high correlation between the efficiency of bone repair and the overall brain structure reconstruction. The PCPL-MgO-BP2 group exhibited the highest bone repair efficiency, providing a superior structural foundation for neural signal transmission and demonstrating excellent electrophysiological signal acquisition performance due to the complete reconstruction of the skull structure. At week 4 post-surgery, the ECoG signal amplitude recorded in the PCPL-MgO-BP2 group was significantly higher than that in other groups, with spectral analysis showing coverage of the delta to beta frequency band (0.5-30 Hz), indicating active neural activity in the cerebral cortex (Figure 34). By week 8, the signal quality had further improved, with stable waveforms and clear rhythms. Figure 36In contrast, the signal amplitudes of both the control group and the PCPL-MgO group were below 20 μV, and their spectral distribution was limited to the low-frequency range. Notably, correlation analysis showed a significant positive correlation between bone regeneration indices (BMD and BV / TV) and ECoG signal intensity (R²>0.85, P<0.05), indicating a close relationship between the complete reconstruction of skull structure and the recovery of neurological function.

[0158] The above results indicate that the introduction of BP significantly enhances the tissue repair capacity of PCPL-MgO-based materials. From a materials science perspective, the PVA-Col-PL composite matrix provides degradable physical support, MgO regulates the cellular microenvironment by releasing magnesium ions, while BP, with its excellent bioactive surface and the ability of its degradation products to participate in energy metabolism or signal regulation, synergistically promotes cell adhesion, proliferation, and collagen synthesis.

[0159] Dynamic monitoring of the skull defect repair process using Micro-CT clearly revealed the time-dependent relationship between different material groups in promoting bone regeneration. At week 4 post-surgery, the control group still maintained a significant cavity structure in the defect area; the PCPL-MgO group showed initial signs of repair; the PCPL-MgO-BP1 group exhibited a more active osteogenesis process; while the PCPL-MgO-BP2 group demonstrated the best early repair effect. By week 8, the PCPL-MgO-BP2 group showed the most complete bone regeneration, with the newly formed bone comparable to the surrounding host bone in terms of thickness, density, and trabecular structure, achieving functional repair.

[0160] The superior repair performance of the PCPL-MgO-BP2 group is mainly attributed to the following factors: the optimized porous structure (porosity 66.4%, pore size 120-180 μm) provides an ideal three-dimensional space for osteoblast migration and angiogenesis; according to in vitro degradation experimental data, its Mg 2+ The release rate remained consistently between 80-200 μg / mL over 56 days, effectively promoting the expression of osteogenic differentiation-related genes. Phosphate and calcium ions generated from BP degradation jointly promoted the deposition of hydroxyapatite. Notably, the degradation rate of the material showed a good match with the rate of new bone formation, providing stable mechanical support in the early stages of repair and promptly creating space for new bone tissue in the later stages.

[0161] The PCPL-MgO-BP2 hydrogel scaffold, with its optimized composition and structural characteristics, exhibits significant promoting effects and ideal time-dependent properties in the repair of skull defects. Simultaneously, it enables high-quality neural signal monitoring, demonstrating its potential as an integrated bone repair-neural monitoring platform. This composite material system shows promising prospects for tissue engineering applications, and its specific molecular mechanisms, such as the regulation of key cytokines and osteogenic signaling pathways, warrant further in-depth investigation.

[0162] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A flexible conductive hydrogel scaffold that combines cranial bone repair and nerve signal monitoring functions, characterized in that, It is prepared by the following method: (1) Preparation of composite precursor solution: Polyvinyl alcohol was dissolved in deionized water, and then collagen, magnesium oxide nanocomposite coated with polylysine and black phosphorus nano dispersion were added and mixed to obtain composite precursor solution. (2) Preparation of flexible conductive hydrogel scaffold: The composite precursor solution is injected into the mold and placed on a cold source for freeze-thaw cycles 1-5 times. Then, the flexible conductive hydrogel scaffold is obtained by vacuum freeze-drying.

2. The flexible conductive hydrogel scaffold according to claim 1, characterized in that, The components in a 100mL composite precursor solution are: 5-15g polyvinyl alcohol, 0.1-1g collagen, 0.2-6g magnesium oxide nanocomposite encapsulated with polylysine, and 10-40mg black phosphorus.

3. The flexible conductive hydrogel scaffold according to claim 1, characterized in that, The black phosphorus nano-dispersion is obtained by dispersing black phosphorus nanosheets in N-methylpyrrolidone, and the mass concentration of black phosphorus in the black phosphorus nano-dispersion is 0.1-1%.

4. The flexible conductive hydrogel scaffold according to claim 1, characterized in that, The magnesium oxide nanocomposite encapsulated with polylysine was prepared by in-situ coprecipitation. Specifically, MgCl2 solution and polylysine were mixed under alkaline conditions, and NaOH solution was slowly added dropwise until no more precipitate was formed. After continuing the reaction for 2-3 hours, the product was obtained by centrifugation and washing.

5. The flexible conductive hydrogel scaffold according to claim 4, characterized in that, The amount of MgCl2 solution and polylysine added is measured and added according to the mass ratio of magnesium oxide to polylysine = 1:

1.

6. The flexible conductive hydrogel scaffold according to claim 1, characterized in that, The freeze-thaw cycle is set as follows: freezing is unidirectional freezing for 4-24 hours, and thawing is thawing at room temperature for 4-12 hours.

7. The flexible conductive hydrogel scaffold according to claim 6, characterized in that, The unidirectional freezing cold energy is transferred longitudinally from the bottom to the top of the mold, and the temperature of the bottom cold source is -10~-80℃.

8. The flexible conductive hydrogel scaffold according to claim 1, characterized in that, The vacuum freeze-drying temperature is -50~-60℃, and the time is 36-60 hours.

Images