Modeling method and application of spinal cord high pressure syndrome disease model

Abstract

The invention belongs to the technical field of animal model construction, and particularly relates to a spinal cord hypertension syndrome disease model modeling method and application. A guinea pig or a macaque is used as an experimental animal, and a composite biostimulation material and an accurate pressure regulation and control modeling system are combined to realize modeling. The composite biostimulation material is composed of a core pressure slow-release layer, a middle biocompatible coating and a surface targeting binding layer, has excellent biocompatibility, degradability and targeting property, and can reduce non-specific inflammatory response; the precise pressure regulation and control modeling system comprises a pressure sensing module, a micro-injection module, a real-time monitoring module and a main control module, and can dynamically regulate and control the pressure in the spinal cord in a closed-loop mode. The modeling method is easy and convenient to operate and good in repeatability, the model building success rate exceeds 95%, the pathological features are highly similar to clinical features and can last for 4 weeks, a reliable experimental model is provided for mechanism research, drug screening and therapeutic schedule evaluation of the spinal cord high pressure syndrome, and the modeling method is suitable for large-scale popularization.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a modeling method and application of a spinal cord hypertension syndrome disease model. Background Technology

[0002] Spinal cord hypertension syndrome is a group of neurological disorders caused by abnormally high pressure within the spinal cord. Its pathological process is complex, involving multiple injury mechanisms such as neuronal apoptosis, massive release of inflammatory factors (e.g., TNF-α, IL-6), and disruption of the blood-spinal cord barrier. It can cause irreversible damage to the spinal cord nerves, seriously threatening the nervous system health of humans and animals. Currently, there is a lack of targeted and effective treatments in clinical practice. In medical research, establishing stable and reliable animal disease models is a core prerequisite and key support for elucidating the pathogenesis of this disease, screening potential therapeutic targets, developing effective therapeutic drugs, and optimizing clinical intervention programs.

[0003] However, existing models suffer from drawbacks such as poor biocompatibility, low precision in pressure regulation, insufficient correlation between pathological features and clinical findings, and short survival periods, making it difficult to meet the needs of long-term drug intervention, treatment efficacy evaluation, and large-scale scientific research applications. Therefore, developing an animal model of spinal cord hypertension syndrome that accurately simulates clinical symptoms is crucial for advancing basic and clinical translational research on this disease. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides a modeling method for spinal cord hypertension syndrome. This method uses guinea pigs or macaques as experimental animals and combines composite biostimulation materials with a precise pressure regulation modeling system to achieve modeling. The resulting animal model can meet the needs of long-term drug intervention and treatment effect evaluation, providing a reliable experimental model for mechanism research, drug screening, and treatment plan evaluation of spinal cord hypertension syndrome.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a modeling method for a spinal cord hypertension syndrome disease model, the method comprising the following steps: S1. Preparation of composite biostimulatory materials: S11. Preparation of core pressure-relieving layer: Polycaprolactone (PCL) and polylactic acid (PLA) are dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a polymer solution with a mass fraction of 10% to 20%. Then, biodegradable carbonate microspheres are added, dispersed and mixed, and poured into a mold. After removing the solvent by programmed cooling freeze drying, the solution is then vacuum dried to constant weight to obtain a porous core layer. S12, intermediate biocompatible coating: Chitosan and gelatin are dissolved in acetic acid solution, glycerol is added, and the mixture is stirred evenly. Then, genipin solution is added and the pH is adjusted to 5.5-6.5 to obtain the coating solution. Then, the porous core layer of S11 is immersed in the coating solution by dip coating method. After immersion, the intermediate coating is formed by curing. S13, Surface Targeting Binding Layer Modification: Carboxymethylated hyaluronic acid was dissolved in PBS buffer to prepare a 1%-3% solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added. After activation, integrin αvβ3 antibody was added, and after a light-protected reaction, a targeting modification solution was obtained. Then, the core layer coated with the intermediate coating in S12 was immersed in the above modification solution, incubated, and dried to obtain the composite biostimulatory material. S2. Debugging of the Precision Pressure Regulation Modeling System: The precision pressure regulation modeling system includes a pressure sensing module, a micro-injection module, a real-time monitoring module, and a main control module, used for precise control, dynamic monitoring, and data feedback adjustment of intraspinal pressure; the pressure sensing module adopts a miniature implantable fiber optic pressure sensor; the micro-injection module consists of a high-precision infusion pump, a medical silicone injection catheter, and a micro-injection needle; the real-time monitoring module includes a temperature sensor, a pH sensor, and a data acquisition unit; and the main control module consists of an embedded processor, a touch screen, and a data storage unit. Start the system and perform a self-test to check whether the communication of each module is normal, whether the sensor accuracy meets the standard, and whether the injection pump runs smoothly. Set the preset pressure range, sampling frequency, and injection speed through the touch screen. Perform sterilization treatment on the pressure sensor probe, temperature sensor, pH sensor, and micro-injection catheter (ethylene oxide sterilization, sterilization temperature 55℃, time 6h). S3. Spinal cord exposure and device implantation: Using guinea pigs or macaques as experimental animals, after anesthesia, hair removal and disinfection pretreatment, the spinal cord dura mater was surgically exposed, and the composite biostimulation material prepared in S1 was implanted into the subdural space at a depth of 2-3 mm and an implantation dose of 5-10 mg; subsequently, the pressure sensor probe, temperature sensor and pH sensor were implanted sequentially into the parietal lobe parenchyma of the guinea pig. S4. Pressure Regulation and Model Construction: The real-time monitoring module is activated to begin data collection. The initial pressure within the spinal cord is observed through the main control module. If the initial pressure is lower than the preset range, the micro-injection module is controlled to inject sterile buffer at a set rate until the pressure reaches the preset range. Subsequently, the system enters a closed-loop regulation state. The main control module analyzes the pressure data in real time and automatically adjusts the operation of the injection pump to maintain the pressure within the preset range of 20–60 mmHg for 70–80 hours. During the regulation period, the device position and animal vital signs (body temperature, respiration, heart rate) are checked every 12 hours to ensure normal system operation. After 70–80 hours, the regulation system is shut down, all implanted devices are removed, and absorbable sutures are used to suture the dura mater, muscle, subcutaneous tissue, and skin layer by layer. Postoperative anti-infection treatment is administered (sodium penicillin 50,000 U / kg, intramuscular injection, once daily for 3 consecutive days). S5. Postoperative feeding and model maintenance: After surgery, the animals were kept in isolation until the incision healed and stable symptoms of spinal cord hypertension syndrome appeared, and a disease model of spinal cord hypertension syndrome was constructed.

[0006] Preferably, in S11, the mass ratio of PCL to PLA is 1-7:1-7; the volume ratio of dichloromethane to N,N-dimethylformamide is 3:1; the carbonate microspheres are calcium bicarbonate-magnesium carbonate composite microspheres with a mass ratio of 1-3:1, a particle size of 50-200 μm, and a loading of 20%-40% of the total mass of the core layer; the programmed cooling freeze-drying process is as follows: first, cooling to -30 to -10℃ at a rate of 4-6℃ / min and holding for 1-3 hours; then cooling to -60℃ to -40℃ at a rate of 1-3℃ / min and holding for 20-30 hours; the resulting porous core layer has a porosity of 40%-60% and a pore size of 10-50 μm.

[0007] Preferably, in S12, the mass ratio of chitosan to gelatin is 1-2:1-2; the concentration of acetic acid solution is 0.1-0.3 mol / L; the mass fraction of glycerol is 0.5%-2%; the mass fraction of genipin solution is 1%-3%; the soaking time is 4-10 min; the curing is carried out at 37°C and 60% humidity for 10-15 h; the thickness of the coated intermediate biocompatible coating is 5-20 μm, and the degree of crosslinking is 30%-50%.

[0008] Preferably, in S13, the molecular weight of carboxymethylated hyaluronic acid is 50-200 kDa; the pH of PBS buffer is 7.0-7.4; the molar ratio of EDC to NHS is 1:1, and the total concentration is 4-6 mmol / L; the activation time is 20-40 min; the loading of integrin αvβ3 antibody is 50-200 μg / mg; the reaction is carried out at 4°C in the dark for 10-15 h; the incubation temperature is 35-40°C for 4-8 h, with gentle shaking every 1 h.

[0009] Preferably, in S3, the method for exposing the guinea pig spinal cord is as follows: a longitudinal incision is made in the T8-T10 segment of the guinea pig's spine, and the skin, subcutaneous tissue, and paravertebral muscles are separated layer by layer to expose the T8-T10 segment of the spine. The T8-10 lamina is removed to clearly expose the spinal cord area, which is approximately 3×5 mm in size. The dura mater is then exposed, and the dura mater is carefully cut open (the incision length is approximately 0.5 cm) to avoid damaging the spinal cord parenchyma. The method for exposing the macaque spinal cord is as follows: a longitudinal incision is made along the sagittal direction on the midline of the macaque's head. A No. 20 scalpel is used to cut the skin and subcutaneous tissue, and the subcutaneous tissue is carefully separated to expose the skull. The periosteum is then dissected using a periosteal elevator to fully expose the skull surface. A 1 mm ball drill is then used to thin the skull at the predetermined puncture point to expose the dura mater, but perforation is avoided.

[0010] More preferably, in the procedure of exposing the guinea pig spinal cord, the removal of the T8-10 lamina is performed using a micro-drill at a speed of 5000-8000 r / min. During the drilling process, sterile saline is continuously dripped to cool the spinal cord tissue and avoid high temperature damage.

[0011] Preferably, in S3, the implantation of the composite biostimulation material and device is controlled by a micromanipulator with a positioning accuracy of ±0.01mm to ensure precise implantation location.

[0012] Preferably, in S4, the preset pressure range includes three gradients: low pressure gradient 20–30 mmHg, medium pressure gradient 30–45 mmHg, and high pressure gradient 45–60 mmHg. The corresponding pressure gradient can be selected for modeling according to experimental requirements. The buffer injection volume ranges corresponding to different pressure gradients are: low pressure gradient injection volume 20–50 μL, medium pressure gradient injection volume 50–100 μL, and high pressure gradient injection volume 100–150 μL.

[0013] Preferably, in S2, the probe diameter of the miniature implantable fiber optic pressure sensor is ≤0.5mm, the detection accuracy is ±0.1mmHg, the detection range is 0~100mmHg, and the response time is ≤10ms. The probe surface is coated with polyethylene glycol (PEG). In the micro-injection module, the injection pump has an injection accuracy of ±0.1μL, and the injection speed can be continuously adjusted within the range of 0.1~10μL / min, supporting both constant-speed injection and pulse injection modes. The medical silicone injection catheter has an inner diameter of 0.2~0.5mm and an outer diameter of 0.8~1.2mm, and its surface is treated with heparin coating to prevent blood clotting and clogging of the catheter. The micro-injection needle is a blunt-tipped design. With a diameter ≤0.8mm to avoid damaging the spinal cord parenchyma, one end of the catheter is connected to an infusion pump, and the other end extends into the subdural space of the spinal cord through an injection needle for injecting sterile buffer solution (PBS buffer, pH 7.4) to regulate intraspinal pressure. In the real-time monitoring module, the temperature sensor has a detection range of 35–40℃ with an accuracy of ±0.1℃, and the pH sensor has a detection range of 6.5–8.0 with an accuracy of ±0.01℃. The data acquisition unit integrates a multi-channel data acquisition card with an adjustable sampling frequency within the range of 10–100Hz, capable of simultaneously acquiring pressure, temperature, and pH parameters. The data is transmitted to the main control module after analog-to-digital conversion. The main control module uses an ARM embedded processor. The Cortex-M4 core enables real-time analysis of the collected pressure data. When the detected pressure deviates from the preset range, it automatically generates adjustment commands to control the micro-injection module to start or stop injection, thus achieving closed-loop pressure regulation. The touch screen is used for parameter setting (preset pressure range, sampling frequency, injection speed, etc.) and real-time data display. The data storage unit supports SD card expansion and can store at least 72 hours of continuous monitoring data for subsequent traceability analysis.

[0014] Preferably, in S5, postoperative feeding conditions are: temperature 22–25℃, humidity 50%–60%, 12h light / 12h dark cycle, free access to food and water, and supplementation of feed with vitamin C (e.g., 0.1% by mass) to enhance immunity; postoperative wound care involves covering the wound with sterile gauze and changing it once daily until the wound heals (usually 7–10 days).

[0015] Preferably, the guinea pigs are 6-month-old guinea pigs weighing 700-1200 g. Before model construction, the guinea pigs are acclimatized for 7 days in an SPF-grade animal room with the temperature controlled at 20-25℃, the relative humidity maintained at 40-70%, and the light cycle alternating between 12 hours of light and 12 hours of darkness, with free access to food and water.

[0016] Preferably, the anesthesia, hair removal and disinfection methods for guinea pigs are as follows: after a brief induction anesthesia of guinea pigs with 4% isoflurane, 1.5% isoflurane is used for continuous anesthesia. The hair removal area is 2 cm on each side of the spine on the back. Disinfection is carried out by iodine-alcohol gradient disinfection (wiping with iodine 3 times and removing iodine with 75% alcohol 2 times).

[0017] The second aspect of the present invention also provides applications of the spinal cord hypertension syndrome disease model constructed using the modeling method described in the first aspect, including: as an animal model for studying the pathological mechanism of spinal cord hypertension syndrome; or as an animal model for screening drugs for spinal cord hypertension syndrome; or as an animal model for evaluating treatment regimens for spinal cord hypertension syndrome.

[0018] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses a modeling method for spinal cord hypertension syndrome. The method uses guinea pigs or macaques as experimental animals and combines a composite biostimulant material with a precise pressure regulation modeling system to achieve modeling. The composite biostimulant material has a three-layer structure, consisting of a core pressure-releasing layer, a middle biocompatible coating, and a surface targeted binding layer. It possesses excellent biocompatibility, biodegradability, and targeting, reducing non-specific inflammatory responses. The precise pressure regulation modeling system includes a pressure sensing module, a micro-injection module, a real-time monitoring module, and a main control module, enabling closed-loop dynamic regulation of intraspinal pressure and simultaneous monitoring of multiple parameters. This modeling method is simple to operate, has good repeatability, a model construction success rate exceeding 95%, high postoperative animal survival rate, and pathological characteristics highly similar to clinical findings that can be maintained for up to 4 weeks. It meets the needs of long-term drug intervention and treatment efficacy evaluation, providing a reliable experimental model for mechanism research, drug screening, and treatment plan evaluation of spinal cord hypertension syndrome, and is suitable for large-scale promotion.

[0019] Specifically, the present invention has the following advantages: (1) The composite biostimulant material used in this invention has excellent biocompatibility, biodegradability and targeting. After in vitro cytotoxicity test, the cell survival rate is ≥90%. There is no obvious foreign body reaction after implantation. It can reduce non-specific inflammatory response and secondary damage. The model construction success rate can reach more than 95%. The pathological characteristics last for up to 4 weeks, which meets the needs of long-term drug intervention and treatment effect evaluation. (2) The precision pressure regulation system used in this invention realizes high-precision, closed-loop dynamic regulation and multi-parameter synchronous monitoring of intraspinal pressure. The pressure fluctuation coefficient is ≤4.5%. It can construct disease models with low, medium and high pressure gradients according to experimental needs, providing a flexible and reliable experimental platform for studying the influence of pressure factors on the pathogenesis of spinal cord hypertension syndrome. (3) The modeling method of the present invention is scientific and standardized, easy to operate, the required experimental equipment is easy to obtain, the parameters of each step are clear, the repeatability is good, and it is suitable for large-scale promotion and application. It can effectively reduce the operational difficulty and experience dependence of experimental personnel. (4) The pathological features of the animal model constructed by the method of the present invention are highly similar to those of clinical spinal cord hypertension syndrome, including the neuronal apoptosis rate maintained at 28%-45%, the expression level of inflammatory factors (TNF-α, IL-6) moderate and stable, and the degree of blood-spinal cord barrier damage controllable. It can accurately simulate the pathological process of clinical diseases and provide a reliable experimental model for drug screening and treatment plan evaluation, which has important scientific research value and application prospects. (5) All components of the precision pressure control system used in this invention have been biocompatible modified and are implanted in a minimally invasive manner, causing little trauma to guinea pigs. The survival rate ≥85% after 4 weeks of surgery meets the requirements of animal ethics. Detailed Implementation

[0020] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0022] In the following embodiments, the precision pressure regulation modeling system used includes a pressure sensing module, a micro-injection module, a real-time monitoring module, and a main control module. The modules work together to achieve precise control, dynamic monitoring, and data feedback regulation of intraspinal pressure.

[0023] The structure and parameters of each component module of the precise pressure control modeling system are as follows: (1) Pressure sensing module: A miniature implantable fiber optic pressure sensor is used. The sensor probe diameter is ≤0.5mm, the detection accuracy is ±0.1mmHg, the detection range is 0-100mmHg, and the response time is ≤10ms. The surface of the sensor probe is modified with polyethylene glycol (PEG) coating, which has good biocompatibility and can avoid adhesion and inflammatory reaction with spinal cord tissue. The probe is implanted into the subdural space of the spinal cord in a minimally invasive manner and is arranged adjacent to the composite biostimulation material to collect local pressure data in the spinal cord in real time. (2) Micro-injection module: It consists of a high-precision injection pump, a medical silicone catheter and a micro-injection needle. The injection accuracy of the injection pump is ±0.1μL, and the injection speed can be continuously adjusted within the range of 0.1-10μL / min. It supports two modes: constant speed injection and pulse injection. The medical silicone catheter has an inner diameter of 0.2-0.5mm and an outer diameter of 0.8-1.2mm. The surface is treated with heparin coating to prevent blood coagulation and blockage of the catheter. The micro-injection needle is blunt-tipped with a diameter ≤0.8mm to avoid damage to the spinal cord parenchyma. One end of the catheter is connected to the injection pump, and the other end extends to the subdural space of the spinal cord through the injection needle for injecting sterile buffer (PBS buffer, pH 7.4) to regulate the pressure inside the spinal cord. (3) Real-time monitoring module: including temperature sensor, pH sensor and data acquisition unit. The temperature sensor has a detection range of 35-40℃ and an accuracy of ±0.1℃. The pH sensor has a detection range of 6.5-8.0 and an accuracy of ±0.01. The data acquisition unit integrates a multi-channel data acquisition card. The sampling frequency can be adjusted in the range of 10-100Hz. It can simultaneously collect three parameters: pressure, temperature and pH. The data is transmitted to the main control module after analog-to-digital conversion. (4) Main control module: It consists of an embedded processor, a touch screen and a data storage unit. The embedded processor adopts the ARM Cortex-M4 core, which can analyze the collected pressure data in real time. When the detected pressure deviates from the preset range, it automatically generates adjustment instructions to control the micro-injection module to start or stop injection, so as to realize closed-loop pressure regulation. The touch screen is used for parameter setting (preset pressure range, sampling frequency, injection speed, etc.) and real-time data display. The data storage unit supports SD card expansion and can store at least 72 hours of continuous monitoring data, which is convenient for subsequent traceability analysis.

[0024] In the following examples, the preparation of carboxymethylated hyaluronic acid was as follows: 10g of hyaluronic acid (molecular weight 100kDa, purity ≥98%) was dissolved in 500mL of deionized water and stirred until completely dissolved (300r / min, room temperature, 2h) to obtain an aqueous hyaluronic acid solution; 20g of chloroacetic acid was dissolved in 100mL of isopropanol and stirred evenly before being slowly added dropwise to the above aqueous hyaluronic acid solution at a dropping rate of 1mL / min. Simultaneously, 40% sodium hydroxide solution was added to adjust the pH of the system to 8.5-9.0, and the reaction temperature was maintained at 50℃. The reaction was stirred for 6 hours. After the reaction, the pH of the system was adjusted to 6.5-7.0 with 1 mol / L hydrochloric acid, and 3 times the volume of anhydrous ethanol was added for precipitation. After standing for 24 hours, the mixture was filtered and the precipitate was collected. The precipitate was washed three times with 70% ethanol solution (100 mL each time) to remove unreacted chloroacetic acid and impurities. Then it was placed in a vacuum drying oven (60℃, vacuum degree ≤10Pa) and dried to constant weight to obtain carboxymethylated hyaluronic acid. The product was found to have a degree of substitution ≥80% and a molecular weight of 50-200 kDa, which meets the requirements for surface targeted binding layer modification.

[0025] Example 1: This embodiment uses guinea pigs as an example to provide a disease modeling method for spinal cord hypertension syndrome, which includes the following steps: 1. Preparation of composite biostimulatory materials The composite biostimulant material used in this embodiment consists of a core pressure-releasing layer, an intermediate biocompatible coating, and a surface-targeting binding layer. The core pressure-releasing layer is a porous composite material of polycaprolactone-polylactic acid copolymer (PCL-PLA) loaded with biodegradable carbonate microspheres; the intermediate biocompatible coating is a chitosan-gelatin composite gel; and the surface-targeting binding layer is a hyaluronic acid derivative modified with integrin αvβ3 antibody.

[0026] The composite biostimulating material is prepared as follows: (1) Preparation of core pressure sustained-release layer: 10g of PCL and PLA were weighed at a mass ratio of 1:1 and dissolved together in 200mL of a mixed solvent of dichloromethane and N,N-dimethylformamide (volume ratio 3:1). The mixture was stirred until completely dissolved to prepare a polymer solution with a mass fraction of 15%. 9g of calcium bicarbonate-magnesium carbonate composite microspheres (mass ratio 2:1, particle size 100μm) were then added to the solution. The loading of the microspheres was 30% of the total mass of the core layer. After ultrasonic dispersion for 30min (power 200W, frequency 40kHz), the resulting mixed solution was poured into a polytetrafluoroethylene mold (size 1cm×0.5cm×0.3cm). The solution was subjected to programmed cooling freeze drying treatment (-20℃ for 2h, -50℃ for 24h), and then vacuum dried to constant weight to obtain a porous core layer with a porosity of 50% and a pore size of 30μm. (2) Coating of intermediate biocompatibility coating: Weigh 2g of chitosan and 2g of gelatin at a mass ratio of 1:1, dissolve them together in 100mL of 0.1mol / L acetic acid solution, add 1mL of 1% glycerol, stir evenly (300r / min, 30min), add 1mL of 1% genipin solution, and adjust the pH of the system to 6.0 to obtain the coating solution; then coat the intermediate biocompatibility coating by dip coating method: immerse the core layer in the coating solution for 5min, take it out and place it under the conditions of 37℃ and 60% humidity for 12h to form an intermediate coating with a thickness of 10μm and a crosslinking degree of 40%. Finally, rinse the surface with sterile physiological saline to remove residual solution and air dry for later use. (3) Surface targeting binding layer modification: 2g of carboxymethylated hyaluronic acid was dissolved in 100mL of PBS buffer (pH 7.4) to prepare a 2% mass fraction solution. EDC and NHS (molar ratio 1:1, total concentration 5mmol / L) were added to the solution. After activation for 30min, 200μg of integrin αvβ3 antibody (purchased from Beijing Baipusaisi Biotechnology Co., Ltd.) was added to obtain a hyaluronic acid derivative with an antibody loading of 100μg / mg. After reacting at 4℃ and in the dark for 12h, the targeting modification solution was obtained. The core layer treated with the intermediate coating was then immersed in the above targeting modification solution and incubated at 37℃ for 6h. During the incubation, the core layer was shaken once every 1h. After the incubation was completed, the core layer was washed 3 times with PBS buffer (10min each time) and then freeze-dried under vacuum to obtain the composite biostimulation material. The material was stored in a sterile environment for later use.

[0027] 2. Modeling methods: (1) Pretreatment of experimental guinea pigs: Twenty healthy adult guinea pigs weighing 700-1200 g (purchased from Zhuhai Baishitong Experimental Animal Co., Ltd., 6 months old) were selected, 10 males and 10 females. After acclimatization for 1 week, they were anesthetized by intraperitoneal injection of 10% chloral hydrate (30 mg / kg). Then, hair was removed from a 2 cm area on both sides of the spine on the back, and disinfected with iodine-alcohol gradient: First, sterile cotton wool was picked up with sterile forceps that had been sterilized by high pressure steam (121℃, 30 minutes), soaked in a sufficient amount of 0.5% iodine, and hair was removed from the T8-T10 segment of the spine on the back as the center. Wipe the area (2cm on each side of the spine) in a spiral motion; replace with a new sterile absorbent cotton and repeat the wiping in the same direction once; replace the sterile absorbent cotton again and focus on wiping the pre-planned incision line and the surrounding 0.5cm area, quickly wiping the remaining areas; after the iodine solution has dried naturally for 30 seconds, use a new sterile absorbent cotton soaked in 75% medical alcohol to wipe the entire disinfected area in a spiral direction "from the center of the incision outwards" to remove iodine residue and perform secondary sterilization; replace the sterile absorbent cotton and repeat the wiping operation to ensure that there are no yellow iodine residues on the skin surface.

[0028] (2) Debugging of the precise pressure control modeling system: Start the precise pressure control modeling system. After the self-test is completed, set the following parameters: preset pressure range 30-45 mmHg (medium pressure gradient), sampling frequency 50 Hz, injection speed 2 μL / min, and buffer solution is sterile PBS buffer (pH 7.4); and sterilize the pressure sensor probe, temperature sensor, pH sensor and micro-injection catheter with ethylene oxide (55℃, 6h). (3) Spinal cord exposure and device implantation: A longitudinal incision (3 cm in length) was made in the T8-T10 segment of the guinea pig's spine. The skin, subcutaneous tissue, and paravertebral muscles were separated layer by layer to expose the spine. The T9 segment lamina (0.8 cm in diameter) was slowly removed using a micro-drill (6000 r / min). Sterile saline was continuously dripped to cool the spine during the drilling process. The dura mater (0.5 cm in length) was carefully cut open. Under the control of a micromanipulator (Sutter Instrument MP-285), 5 mg of composite biostimulant material was precisely implanted into the subdural space of the spinal cord to a depth of 2.5 mm. Subsequently, a 1.5 cm incision was made along the midline of the guinea pig's skull. The connective tissue and the periosteum covering the skull were separated in sequence to expose the skull. Then, a 5 mm × 5 mm incision was made at a pre-set location (2 mm anterior to the left frontal coronal suture and 3 mm lateral to the midline) using a micro-drill. A bone window with an area of ​​mm was created. During the procedure, care was taken to protect the integrity of the dura mater. The pressure sensor probe, temperature sensor, and pH sensor were implanted sequentially and placed into the parietal parenchyma of the left parietal lobe through the bone window, to a depth of about 2 mm below the cortex.

[0029] (4) Pressure regulation and model construction: The real-time monitoring module was activated to put the system into a closed-loop regulation state, maintaining the intraspinal pressure at 30-45 mmHg for 72 hours. During the regulation period, the device position and guinea pig vital signs were checked every 12 hours. After 72 hours, the regulation system was turned off, all devices were removed, and the dura mater, muscle, subcutaneous tissue and skin were sutured layer by layer with absorbable sutures. 50,000 U / kg of penicillin sodium was injected intramuscularly to fight infection once a day for 3 consecutive days. (5) Postoperative feeding: After surgery, the guinea pigs were fed under the conditions of 23±1℃ temperature, 55%±5% humidity, and 12h light / 12h dark cycle. They were allowed free access to food and water. 0.1% vitamin C was added to the feed. During this period, the incision was covered with sterile gauze and changed once a day until the incision healed.

[0030] Example 2: This embodiment uses guinea pigs as an example to provide a disease modeling method for spinal cord hypertension syndrome, which includes the following steps: 1. The preparation of composite biostimulant materials is as follows: (1) Preparation of core pressure sustained-release layer: 6g of PCL and 14g of PLA were weighed at a mass ratio of 3:7 and dissolved together in 200mL of a mixed solvent of dichloromethane and N,N-dimethylformamide (volume ratio 3:1). The mixture was stirred until completely dissolved to prepare a polymer solution with a mass fraction of 10%. 4g of calcium bicarbonate-magnesium carbonate composite microspheres (mass ratio 1:1, particle size 50μm) were then added to the solution, with a loading amount of 20% of the total mass of the core layer. After ultrasonic dispersion for 30min, the resulting mixed solution was poured into a polytetrafluoroethylene mold (size 1cm×0.5cm×0.3cm), and subjected to programmed cooling freeze drying treatment (-20℃ for 2h, -50℃ for 24h), followed by vacuum drying (vacuum degree ≤10Pa) to constant weight to obtain a porous core layer with a porosity of 40% and a pore size of 10μm. (2) Coating of intermediate biocompatibility layer: Weigh 1.33g of chitosan and 2.67g of gelatin at a mass ratio of 1:2, dissolve them together in 100mL of 0.1mol / L acetic acid solution, add 0.5% glycerol (0.5mL), stir evenly (300r / min, 30min), add 1mL of 1% genipin solution, and adjust the pH of the system to 5.5 to obtain the coating solution; then coat the intermediate biocompatibility layer by dip coating method: immerse the core layer in the coating solution for 5min, take it out and place it under the conditions of 37℃ and 60% humidity for 12h to form an intermediate coating with a thickness of 5μm and a crosslinking degree of 30%. Finally, rinse the surface with sterile physiological saline to remove residual solution and air dry for later use. (3) Surface targeting binding layer modification: 1g of carboxymethylated hyaluronic acid was dissolved in 100mL of PBS buffer (pH7.4) to prepare a 1% mass fraction solution. EDC and NHS (molar ratio 1:1, total concentration 5mmol / L) were added to the solution. After activation for 30min, 50μg of integrin αvβ3 antibody was added to obtain a hyaluronic acid derivative with an antibody loading of 50μg / mg. After reacting at 4℃ and in the dark for 12h, the targeting modification solution was obtained. The core layer treated with the intermediate coating was then immersed in the above targeting modification solution and incubated at 37℃ for 6h. During the incubation, the core layer was shaken once every 1h. After the incubation was completed, the core layer was washed 3 times with PBS buffer (10min each time) and then freeze-dried under vacuum to obtain the composite biostimulation material. The material was stored in a sterile environment for later use.

[0031] 2. Modeling methods: (1) Pretreatment of experimental guinea pigs: 20 healthy adult guinea pigs weighing 250±20g were selected, 10 males and 10 females. After acclimatization for 1 week, they were anesthetized, hair removed and disinfected according to the method in Example 1. (2) Debugging of the precise pressure control modeling system: Start the precise pressure control modeling system. After the self-test is completed, set the following parameters: preset pressure range 20-30 mmHg (low pressure gradient), sampling frequency 50 Hz, injection speed 1 μL / min, and buffer solution is sterile PBS buffer (pH 7.4); the subsequent device sterilization process is the same as in Example 1. (3) Spinal cord exposure and device implantation: A longitudinal incision (3cm in length) was made in the T8-T10 segment of the guinea pig's spine. The skin, subcutaneous tissue and paravertebral muscles were separated layer by layer to expose the spine. The T9 segment lamina (0.8cm in diameter) was slowly removed using a micro-drill (5000r / min). Sterile saline was continuously dripped to cool the lamina during the drilling process. The dura mater (0.5cm in length) was carefully cut open. Under the control of a micromanipulator, 8mg of composite biostimulant material was precisely implanted into the subdural space of the spinal cord to a depth of 2mm. The subsequent device implantation position and fixation method were the same as in Example 1. (4) Pressure regulation and model construction: Start the real-time monitoring module to put the system into a closed-loop regulation state, maintain the intraspinal pressure at 20-30 mmHg, and continue to regulate for 72 hours. During the regulation period, check the device position and guinea pig vital signs every 12 hours. After 72 hours, postoperative anti-infection and nursing care are the same as in Example 1. (5) Postoperative feeding: After the operation, the guinea pigs were fed under the conditions of 22±1℃ temperature, 50%±5% humidity, and 12h light / 12h dark cycle. The other conditions were the same as in Example 1.

[0032] Example 3: This embodiment uses guinea pigs as an example to provide a disease modeling method for spinal cord hypertension syndrome, which includes the following steps: 1. The preparation of composite biostimulant materials is as follows: (1) Preparation of core pressure-relieving layer: 14g of PCL and 6g of PLA were weighed at a mass ratio of 7:3 and dissolved together in 200mL of a mixed solvent of dichloromethane and N,N-dimethylformamide (volume ratio 3:1). The mixture was stirred until completely dissolved to prepare a polymer solution with a mass fraction of 20%. 8g of calcium bicarbonate-magnesium carbonate composite microspheres (mass ratio 3:1, particle size 200μm) were then added to the solution, with a loading of 40% of the total mass of the core layer. After ultrasonic dispersion for 30min (power 200W, frequency 40kHz), the resulting mixed solution was poured into a polytetrafluoroethylene mold (size 1cm×0.5cm×0.3cm), and subjected to programmed cooling freeze-drying treatment (-20℃ for 2h, -50℃ for 24h). The solution was then vacuum dried to constant weight to obtain a porous core layer with a porosity of 60% and a pore size of 50μm. (2) Coating of intermediate biocompatibility layer: Weigh 2.67g of chitosan and 1.33g of gelatin at a mass ratio of 2:1, dissolve them together in 100mL of 0.1mol / L acetic acid solution, add 2% glycerol (2mL), stir evenly (300r / min, 30min), add 1mL of 1% genipin solution, and adjust the pH of the system to 6.5 to obtain the coating solution; then coat the intermediate biocompatibility layer by dip coating method: immerse the core layer in the coating solution for 5min, take it out and place it under the conditions of 37℃ and 60% humidity for 12h to form an intermediate coating with a thickness of 20μm and a crosslinking degree of 50%. Finally, rinse the surface with sterile physiological saline to remove residual solution and air dry for later use. (3) Surface targeting binding layer modification: 3g of carboxymethylated hyaluronic acid was dissolved in 100mL of PBS buffer (pH7.4) to prepare a 3% mass fraction solution. EDC and NHS (molar ratio 1:1, total concentration 5mmol / L) were added to the solution. After activation for 30min, 600μg of integrin αvβ3 antibody was added to obtain a hyaluronic acid derivative with an antibody loading of 200μg / mg. After reacting at 4℃ and in the dark for 12h, the targeting modification solution was obtained. The core layer treated with the intermediate coating was then immersed in the above targeting modification solution and incubated at 37℃ for 6h. During the incubation, the core layer was shaken once every 1h. After the incubation was completed, the core layer was washed 3 times with PBS buffer (10min each time) and then freeze-dried under vacuum to obtain the composite biostimulation material. The material was stored in a sterile environment for later use.

[0033] 2. Modeling methods: (1) Pretreatment of experimental guinea pigs: 20 healthy adult guinea pigs weighing 350±20g were selected, 10 males and 10 females. After acclimatization for 1 week, they were anesthetized, hair removed and disinfected according to the method in Example 1. (2) Debugging of the precise pressure control modeling system: Start the precise pressure control modeling system. After the self-test is completed, set the following parameters: preset pressure range 45-60 mmHg (high pressure gradient), sampling frequency 50 Hz, injection speed 3 μL / min, and buffer solution is sterile PBS buffer (pH 7.4); the subsequent device sterilization process is the same as in Example 1. (3) Spinal cord exposure and device implantation: A longitudinal incision (3cm in length) was made in the T8-T10 segment of the guinea pig's spine. The skin, subcutaneous tissue and paravertebral muscles were separated layer by layer to expose the spine. The T9 segment lamina (0.8cm in diameter) was slowly removed using a micro-drill (8000r / min). Sterile saline was continuously dripped to cool the spine during the drilling process. The dura mater (0.5cm in length) was carefully cut open. Under the control of a micromanipulator, 10mg of composite biostimulant material was precisely implanted into the subdural space of the spinal cord to a depth of 3mm. The device implantation position and fixation method were the same as in Example 1. (4) Pressure regulation and model construction: Start the real-time monitoring module to put the system into a closed-loop regulation state, maintain the intraspinal pressure at 45-60 mmHg, and continue to regulate for 72 hours. During the regulation period, check the device position and guinea pig vital signs every 12 hours. After 72 hours, postoperative anti-infection and nursing care are the same as in Example 1. (5) Postoperative feeding: After the operation, the guinea pigs were fed under the conditions of 25±1℃ temperature, 60%±5% humidity, and 12h light / 12h dark cycle. The other conditions were the same as in Example 1.

[0034] Example 4: This embodiment uses a rhesus monkey as an example to provide a disease modeling method for spinal cord hypertension syndrome, which includes the following steps: 1. The preparation of the composite biostimulant material is the same as in Example 1.

[0035] 2. Modeling methods: (1) Pretreatment of experimental macaques: Male macaques aged 6-8 years (8-12 kg, purchased from Guangzhou Landao Biotechnology Co., Ltd.) were selected as experimental animals and sedated with ketamine 5 mg / kg combined with dexmedetomidine 0.0075 mg / kg via intramuscular injection. Anesthesia was then induced by slow intravenous injection of propofol 2-5 mg / kg. A 5 mm endotracheal tube was used for assisted ventilation, and the correct position of the endotracheal tube was confirmed by a stethoscope. Atropine (0.04 mg / kg) was used to control oral and respiratory secretions before intubation. Anesthesia was then maintained using an anesthesia machine with 2-4% isoflurane. Additional 0.9% sodium chloride solution was administered for fluid supplementation at a rate of 5-10 mL / kg / h. A heating pad was used to maintain anesthesia and prevent hypothermia. Anesthesia depth, heart rate, blood oxygen, and body temperature were monitored continuously.

[0036] (2) Debugging of the precision pressure control modeling system: The operation is the same as in Example 1; (3) Spinal cord exposure and device implantation: Fasting for 12 hours and abstaining from water for 4 hours were required before the experiment. A longitudinal incision of about 6–8 cm was made along the midline of the spine, centered on the spinous processes of the eighth to tenth thoracic vertebrae. After incising the skin and subcutaneous tissue, the longus dorsalis muscles on both sides were bluntly dissected using small curved hemostatic forceps or micro forceps, and dissected along the lateral edge of the spinous process to the lateral edge of the lamina. Electrocoagulation was used to maintain a clear surgical field. The spinous processes and the upper half of the lamina of the target segment were removed sequentially using a micro-bone drill and bone forceps to expose the spinal canal. The yellow ligaments in the spinal canal were gently peeled off to expose the dura mater, and the surrounding tissues were protected with sterile gauze. After confirming that the dura mater was intact, the composite biostimulation material was precisely implanted into the subdural space of the spinal cord using a micromanipulator (positioning accuracy ±0.01 mm) to a depth of 3 mm and an implantation dose of 10 mg. Hair was then removed from the top of the skull and the back of the T6–L1 segment of the experimental monkeys using depilatory cream. Disinfect the area three times alternately with povidone-iodine and 75% alcohol, and cover the surgical area with a sterile drape, strictly adhering to aseptic techniques. Make a longitudinal incision of approximately 6 cm along the sagittal direction on the midline of the head, using a 20-gauge scalpel to cut the skin and subcutaneous tissue. Carefully dissect the subcutaneous tissue to expose the skull. Use a periosteal elevator to dissect the periosteum, fully exposing the skull surface. Use a 1 mm ball drill to thin the skull at the predetermined puncture point, exposing the dura mater but avoiding perforation. Insert the pressure sensor probe, temperature sensor, and pH sensor vertically into the lateral ventricle in sequence. After cerebrospinal fluid outflow, fix the puncture needle. Connect the needle to the pressure sensor and data acquisition system via a catheter. Monitor intracranial pressure changes by recording sensor data in real time.

[0037] (4) The pressure regulation and model construction operations are the same as in Example 1; (5) Postoperative feeding: After the operation, the macaques were fed under the conditions of temperature 23±1℃, humidity 55%±5%, and 12h light / 12h dark cycle. They were allowed to eat and drink freely. 0.1% vitamin C was added to the feed. During this period, the incision was covered with sterile gauze and changed once a day until the incision healed.

[0038] Comparative Example 1 (lacking surface-targeting binding layer): The difference from Example 1 is that the composite biostimulant material does not have a surface targeting binding layer, but consists only of a core pressure-releasing layer and an intermediate biocompatible coating. That is, the surface modification step (3) is missing. The rest of the preparation process, system parameters and modeling methods are the same as those in Example 1.

[0039] Comparative Example 2 (lacking intermediate biocompatible coating): The difference from Example 1 is that the composite biostimulant material does not have an intermediate biocompatible coating. It consists only of a core pressure-releasing layer and a surface-targeting binding layer. That is, the coating step (2) is missing. The core layer is directly modified with surface targeting. The rest of the preparation process, system parameters and modeling methods are the same as those in Example 1.

[0040] Comparative Example 3 (core layer is a single PCL material, without carbonate microspheres): The difference from Example 1 is that the core pressure-releasing layer uses only PCL as the substrate (20g of PCL is weighed), without adding PLA, and without loading biodegradable carbonate microspheres. The porosity is adjusted to 50% by freeze-drying process. The rest of the preparation process, system parameters and modeling methods are the same as those in Example 1.

[0041] Comparative Example 4 (core layer is a single PLA material, without carbonate microspheres): The difference from Example 1 is that the core pressure-releasing layer uses only PLA as the substrate (weighing 20g PLA), without adding PCL, and without loading biodegradable carbonate microspheres. The porosity is adjusted to 50% by freeze-drying process. The rest of the preparation process, system parameters and modeling methods are the same as in Example 1.

[0042] Comparative Example 5 (carbonate microspheres are made of calcium bicarbonate alone): The difference from Example 1 is that the biodegradable carbonate microspheres used in the core pressure release layer are single calcium bicarbonate microspheres (particle size 100 μm), without the addition of magnesium carbonate, with an addition amount of 9 g and a loading of 30%. The rest of the preparation process, system parameters and modeling methods are the same as in Example 1.

[0043] Comparative Example 6 (the carbonate microspheres are made of magnesium carbonate alone): The difference from Example 1 is that the biodegradable carbonate microspheres used in the core pressure release layer are single magnesium carbonate microspheres (particle size 100 μm), without the addition of calcium bicarbonate, with an addition amount of 9 g and a loading of 30%. The rest of the preparation process, system parameters and modeling methods are the same as those in Example 1.

[0044] Comparative Example 7 (pressure was manually adjusted without using a precise pressure control system): The difference from Example 1 is that: no precise pressure control system was used in the modeling process. The pressure was adjusted by manually injecting buffer solution with a micro-syringe. The pressure was detected every 1 hour by a pressure sensor (implanted separately), and the injection was manually supplemented according to the detection results. No real-time monitoring and closed-loop adjustment were performed. The rest of the preparation process and modeling method were the same as in Example 1.

[0045] Comparative Example 8 (antibody without surface targeting binding layer modification): The difference from Example 1 is that the surface targeting binding layer is carboxymethylated hyaluronic acid of unmodified integrin αvβ3 antibody, that is, integrin αvβ3 antibody is not added in step (3). The rest of the preparation process, system parameters and modeling methods are the same as those in Example 1.

[0046] Comparative Example 9 (intermediate coating not cross-linked): The difference from Example 1 is that the intermediate biocompatible coating was not cross-linked with genipin, that is, no genipin solution was added in step (2), and the coating solution consisted only of chitosan, gelatin, glycerol and acetic acid solution. The rest of the preparation process, system parameters and modeling methods were the same as those in Example 1.

[0047] Experimental Example: Model Performance Testing I. Testing Methods The disease models constructed in Examples 1-3 and Comparative Examples 1-9 were subjected to the following performance tests. The test time points were 1 week, 2 weeks and 4 weeks after modeling. At each time point, 5 guinea pigs were selected from each group for testing, and the average value was taken. The testing process strictly followed animal ethics guidelines.

[0048] 1. Spinal cord pressure stability testing: (1) Test preparation: At the corresponding time points after modeling (1 week, 2 weeks, 4 weeks), guinea pigs were anesthetized by intraperitoneal injection of 10% chloral hydrate (30mg / kg), fixed on a constant temperature operating table (maintaining body temperature of 37±0.5℃), and the back incision was cut layer by layer to expose the pressure sensor wire implanted in the subdural space of the spinal cord, ensuring that the sensor communicates normally with the main control module.

[0049] (2) Data acquisition: Spinal cord pressure data were continuously collected for 1 hour using a precision pressure control system (Examples 1-3 and comparative examples except Comparative Example 7) or an intracranial pressure detector (Shanghai Yuyan Scientific Instruments Co., Ltd., YAN6000; applicable to Comparative Example 7, sampling frequency 50Hz, accuracy ±0.01mmHg), with 1 data point recorded every 1 second, for a total of 3600 data points.

[0050] (3) Result calculation: SPSS software was used to collect data, calculate the average pressure and standard deviation, and calculate the fluctuation coefficient according to the formula "fluctuation coefficient = (standard deviation / average value) × 100%". Five guinea pigs were used in each group, and the average value was taken as the final result. The smaller the fluctuation coefficient, the better the pressure stability.

[0051] 2. Detection of nerve cell apoptosis rate: (1) Tissue processing: After euthanizing the guinea pig, quickly remove the spinal cord injury tissue (about 0.1g, precisely locate the T9 segment injury area), rinse twice in pre-cooled PBS buffer (pH 7.4) to remove blood and impurities; cut the tissue into 1mm pieces. 3 Add 1 mL of digestion solution containing 0.25% trypsin to the sample, and digest at 37°C with shaking for 30 min (150 r / min). Gently pipette once every 10 min during digestion. After digestion is stopped, filter through a 200-mesh cell sieve and collect the single-cell suspension.

[0052] (2) Centrifugation and washing: Place the single-cell suspension in a centrifuge tube, centrifuge at 1500 r / min for 5 min (4℃), discard the supernatant, add 1 mL of pre-cooled PBS buffer to resuspend the cells, repeat the centrifugation and washing twice, and adjust the cell concentration to 1×10⁻⁶. 6 per mL.

[0053] (3) Staining reaction: Add 100 μL of cell suspension to a flow cytometer, add 5 μL of Annexin V-FITC reagent and 5 μL of PI staining solution in sequence, mix gently, incubate at room temperature in the dark for 15 min, and then add 400 μL of PBS buffer to terminate the reaction.

[0054] (4) Detection and analysis: Flow cytometry was used for detection. The excitation wavelength was 488 nm, and the emission wavelengths were 530 nm (FITC) and 617 nm (PI). The data were analyzed using FlowJo software to count the number of apoptotic cells (Annexin V). + / PI - Early apoptotic cells + Annexin V + / PI + The proportion of late-stage apoptotic cells to the total number of cells is the neuronal apoptosis rate. The average value was taken from 5 guinea pigs in each group.

[0055] 3. Detection of inflammatory factor expression levels: The levels of TNF-α and IL-6 were detected using ELISA. The specific steps are as follows: (1) Sample preparation: Take 0.1g of tissue from the spinal cord injury site, add 900μL of pre-cooled physiological saline, grind it into a homogenate in an ice bath, centrifuge at 4℃ and 12000r / min for 10min, take the supernatant as the test sample, and store it in a -80℃ refrigerator for later use.

[0056] (2) Kit preparation: Take out the TNF-α ELISA kit (Auscare Biotechnology (Shanghai) Co., Ltd.) and the IL-6 ELISA kit (Renjie Biotechnology), let them warm to room temperature for 30 min, and prepare the standards (concentration gradient: TNF-α 0, 15.625, 31.25, 62.5, 125, 250 pg / mL; IL-6 0, 7.8125, 15.625, 31.25, 62.5, 125 pg / mL), washing buffer, enzyme-labeled working solution and stop solution according to the instructions.

[0057] (3) Sample addition reaction: Add 100 μL of standard and sample to each well of the ELISA plate in sequence, add 100 μL of PBS buffer to the blank well, and incubate at 37℃ for 60 min; discard the liquid in the well, add 300 μL of washing buffer to each well, let stand for 30 s and then discard, repeat the washing 5 times; add 100 μL of enzyme label working solution to each well, incubate at 37℃ for 30 min, and wash 5 times again in the above manner; add 100 μL of colorimetric solution to each well, incubate at 37℃ in the dark for 15 min, and finally add 50 μL of stop solution, mix gently, and complete the detection within 10 min.

[0058] (4) Calculation of results: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader. A standard curve was plotted with the concentration of the standard as the x-axis and the OD value as the y-axis. The expression levels of TNF-α and IL-6 (pg / mL) were calculated by substituting the OD values ​​of the samples. Five guinea pigs were used in each group, and the average value was taken.

[0059] 4. Blood-spinal barrier integrity test: (1) Evans blue (EB) injection: At the corresponding time point after modeling, inject 2% EB solution (5mL / kg) into the tail vein of guinea pigs. The injection speed is controlled at 0.2mL / min to avoid leakage of the drug solution.

[0060] (2) Circulation and perfusion: After the injection, the guinea pig was allowed to survive for 2 hours. Then, it was anesthetized with 10% chloral hydrate, the chest cavity was opened to expose the heart, the perfusion needle was inserted through the left ventricle, and the right atrium was cut open to bleed. First, it was rapidly perfused with physiological saline (flow rate 10 mL / min) until the outflow was bloodless (about 50 mL), and then it was perfused and fixed with 4% paraformaldehyde solution for 30 min.

[0061] (3) Sample processing: Quickly extract tissue (0.1g) from the spinal cord injury site, remove residual blood from the surface, weigh accurately and place it in a centrifuge tube, add 1mL of formamide solution, and incubate at 60℃ for 24h. During this period, gently shake once every 4h to ensure that EB is fully dissolved.

[0062] (4) Detection and calculation: After incubation, centrifuge at 4℃ and 12000r / min for 10min, take the supernatant, and measure the OD value at 590nm wavelength using an ELISA reader; at the same time, prepare EB standard curve (concentration gradient 0, 0.1, 0.2, 0.4, 0.8, 1.6μg / mL), substitute the OD value of the sample to calculate the EB content (μg), and then convert it to the EB content per gram of tissue (μg / g). The higher the EB content, the more severe the blood-spinal cord barrier is damaged. Five guinea pigs were used in each group, and the average value was taken.

[0063] 5. Model survival rate and stability assessment: (1) Survival rate statistics: After modeling, observe the mental state, diet and water intake, activity ability and wound healing of guinea pigs every day, record the number of guinea pigs surviving in each group within 4 weeks, and calculate the survival rate according to the formula "survival rate = (number of surviving guinea pigs / total number of guinea pigs) × 100%".

[0064] (2) Assessment of the duration of pathological features: The apoptosis rate of nerve cells, the expression level of inflammatory factors, and the integrity of the blood-spinal cord barrier were tested at 1, 2 and 4 weeks after modeling. The results of healthy guinea pigs were used as normal reference values ​​(apoptosis rate ≤5%, TNF-α ≤20pg / mL, IL-6 ≤15pg / mL, EB content ≤2μg / g). When all three indicators returned to the normal reference range at a certain time point, the pathological features were considered to have disappeared. The time from the completion of modeling to the disappearance of pathological features was recorded as the duration of pathological features. The longer the duration, the better the stability of the model. Five guinea pigs were used in each group, and the average value was taken.

[0065] 6. Material biocompatibility testing: (1) Tissue fixation and embedding: Two weeks after modeling, guinea pigs were euthanized, and tissue from the spinal cord injury site (including tissue surrounding the material) was removed and fixed in 4% paraformaldehyde solution for 24 hours. Then, after gradient dehydration (70%, 80%, 90%, 95%, 100% ethanol sequentially, 1 hour each), clearing (xylene twice, 30 minutes each time), paraffin immersion (60℃ paraffin immersion for 3 hours), and embedding, 5 μm thick paraffin sections were prepared.

[0066] (2) HE staining: Dewax the paraffin sections to water (xylene twice, 10 min each time; 100%, 95%, 90%, 80%, 70% ethanol sequentially, 5 min each step; rinse with distilled water for 5 min); stain with hematoxylin for 5 min, rinse with tap water for 10 min to return to blue; differentiate with 1% hydrochloric acid alcohol for 30 s, rinse with tap water for 5 min; stain with eosin for 2 min, rinse with tap water for 3 min; after gradient dehydration and clearing again, mount with neutral resin.

[0067] (3) Observation and statistics: The slides were observed using a laser confocal microscope. Five fields of view were randomly selected. The total area and inflammatory infiltration area of ​​each field of view were measured using ImageJ software (based on the area where inflammatory cells are gathered). The percentage was calculated according to the formula "Inflammation infiltration area percentage = (Inflammation infiltration area / Total field of view area) × 100%". The smaller the percentage, the better the biocompatibility. Five guinea pigs were used in each group, and the average value was taken.

[0068] II. Test Results 1. Core pathological marker: Abnormally elevated intraspinal pressure (the core of the disease definition) The core characteristic of spinal cord hypertension syndrome is that the intraspinal pressure exceeds the normal range (the intraspinal pressure in healthy guinea pigs is usually ≤15 mmHg), and test data clearly show this. Examples 1-3 show that through precise pressure regulation, the intraspinal pressure was stably maintained at 20-60 mmHg (low, medium, and high pressure gradients), and the pressure fluctuation coefficient within 4 weeks was only 3.2%-4.5% (Table 1), maintaining a continuous pathological high-pressure state; Although the pressure stability of each control group differed greatly, the intraspinal pressure after modeling was significantly higher than normal (although the pressure fluctuation of the manually adjusted control group in control group 7 was large, the average pressure was still ≥40 mmHg, Table 1), which met the core prerequisite of "hypertension" for the disease.

[0069] 2. Characteristic pathological damage: Significantly increased apoptosis rate of nerve cells. Clinical spinal cord hypertension syndrome can lead to ischemic hypoxia and apoptosis of nerve cells due to high pressure, as confirmed by test data: The apoptosis rate of nerve cells in Examples 1-3 was 25.3%-42.8% (Table 2), and the apoptosis process showed a slow downward trend (the decrease in 4 weeks was only 13%-15% compared to 1 week), which is consistent with the characteristics of chronic damage in the disease. Although the apoptosis rates of each control group were excessively high (e.g., 72.4%-81.5% in control group 7) or insufficient (with no obvious abnormalities) due to pressure regulation or material defects, they all deviated from the normal range and were essentially different manifestations of high-pressure-related pathological damage (Table 2).

[0070] 3. Key inflammatory responses: Specifically elevated levels of inflammatory factor expression. Spinal cord hypertension can trigger a local inflammatory response, clinically characterized by elevated levels of inflammatory factors such as TNF-α and IL-6. Data shows that: The TNF-α expression levels in Examples 1-3 ranged from 60.2 to 92.8 pg / mL, and the IL-6 levels ranged from 42.1 to 68.5 pg / mL (Table 3), which were significantly higher than those in healthy guinea pigs (TNF-α ≤ 20 pg / mL, IL-6 ≤ 15 pg / mL). Moreover, the levels decreased slowly with the progression of the disease, and there were no nonspecific inflammatory outbreaks. The levels of inflammatory factors in each control group were significantly higher than the normal range (up to 220.8 pg / mL in control group 7). Even in the control groups with poor biocompatibility (such as control groups 2, 3-4; Table 6), the inflammatory response was essentially a secondary injury induced by high pressure, consistent with the pathological mechanism of the disease (Table 3).

[0071] 4. Typical complication: disruption of the blood-spinal cord barrier Clinical spinal cord hypertension syndrome is often accompanied by impaired blood-spinal cord barrier integrity, as confirmed by EB leakage data: The EB content in Examples 1-3 was 5.6-9.8 μg / g, which was significantly higher than that in healthy guinea pigs (≤2 μg / g), and gradually decreased over time (Table 4), reflecting the repair trend after barrier damage, which is consistent with the disease progression pattern; The EB content of each comparative example was ≥12.5 μg / g (the highest being 33.2 μg / g in comparative example 7). The degree of barrier damage was related to pressure stability and material biocompatibility, but all met the disease characteristics of "barrier damage caused by high pressure" (Table 4).

[0072] 5. Model stability: The duration of pathological features closely matches the disease cycle. Clinical spinal cord hypertension syndrome is a chronic disease, and the model needs to have long-term stable pathological characteristics. Data shows that: The pathological features (high pressure, apoptosis, inflammation, and barrier damage) of Examples 1-3 lasted for 4 weeks (Table 5), which can simulate the chronic process of the disease and meet the needs of long-term research. Although the duration of pathological features was shortened (1.5-3.2 weeks) in each pair due to technical defects, the core pathological indicators (high pressure, apoptosis, inflammation, and barrier damage) were present synchronously during the duration, which is consistent with the pathological manifestations of the disease (Table 5).

[0073] The above analysis shows that: The core pathological chain of spinal cord hypertension syndrome is "spinal cord hypertension → neuronal apoptosis + inflammatory response → blood-spinal cord barrier disruption". The detection data of Examples 1-3 and each comparative example completely cover this chain. (1) Pressure data proves that the premise of "high pressure" is valid; (2) Data on apoptosis rate, inflammatory factors, and EB content prove the existence of characteristic pathological damage induced by high pressure; (3) The changing trends of each indicator over time (slow decline, repair) are consistent with the chronic process of the disease; (4) By adjusting a single variable (such as pressure regulation, material structure, or targeted modification), the proportion only changes the degree and stability of pathological damage, without changing the core pathological logic of "high pressure → damage", thus proving the consistency between the model and the nature of the disease.

[0074] In comparison, Examples 1-3 differ from Comparative Examples 1-7 in the following ways: (1) Intraspinal pressure stability: Table 1 shows that the pressure fluctuation coefficients of Examples 1-3 remained consistently within the range of 3.2%-4.5%, and the increase in fluctuation over time (1 to 4 weeks) was less than 1.3 percentage points, demonstrating excellent pressure stability. This advantage stems from two core technologies: first, the closed-loop regulation function of the precise pressure control system, which collects data in real time through a miniature fiber optic pressure sensor, and the embedded processor responds quickly and automatically regulates the micro-injection module to achieve dynamic pressure balance; second, the hierarchical design of the composite biostimulant material, where the porous structure of the core layer PCL-PLA copolymer and the slow degradation characteristics of the calcium bicarbonate-magnesium carbonate composite microspheres form a pressure-releasing buffer system, preventing sudden increases and decreases in pressure.

[0075] In contrast, the pressure stability of each comparative example was significantly worse than that of the example: Comparative example 7, lacking a precise pressure control system and relying on manual intermittent adjustment, exhibited a fluctuation coefficient as high as 18.6%-25.8%, with the fluctuation amplitude exceeding that of example 1 by 22 percentage points after 4 weeks, directly confirming the necessity of a closed-loop control system; Comparative example 2, lacking an intermediate biocompatible coating, experienced adhesion between the material and spinal cord tissue, triggering a local stress response, resulting in a fluctuation coefficient of 8.9%-11.5%; Comparative examples 3-4, using a single polymer core layer without carbonate microspheres, lacked a pressure-releasing mechanism, leading to a fluctuation coefficient that climbed to 12.5%-17.8%; Comparative examples 5-6, using a single carbonate microsphere, suffered from an unbalanced degradation rate (calcium bicarbonate too fast, magnesium carbonate too slow), maintaining a fluctuation coefficient of 7.8%-10.5%. Furthermore, the fluctuation coefficient of comparative example 9 (without cross-linked intermediate coating) also exceeded 6.9%, further illustrating the crucial impact of the appropriate selection of modeling materials on pressure stability.

[0076] (2) Neuronal apoptosis and inflammatory response: The data in Tables 2 and 3 show that the pathological state of the models in Examples 1-3 is more consistent with clinical reality: the apoptosis rate of nerve cells was controlled at 25.3%-42.8%, and decreased slowly over time (the decrease was 13.1%-14.7% after 4 weeks compared to 1 week); the expression levels of TNF-α and IL-6 were maintained at 60.2-92.8 pg / mL and 42.1-68.5 pg / mL, respectively, also showing a gradient decreasing trend, without a violent inflammatory outbreak, demonstrating a controllable pathological damage process. This is due to the optimized design of the composite biostimulatory material: the intermediate chitosan-gelatin composite coating, after being cross-linked with genipin, significantly improved biocompatibility and reduced non-specific inflammation; the surface targeting binding layer, after being modified with integrin αvβ3 antibody, achieved specific binding of the material to nerve cells at the site of spinal cord injury, accurately inducing pathological damage and avoiding accidental damage to normal tissues.

[0077] All comparative examples exhibited excessively high apoptosis rates and uncontrolled inflammatory responses due to the use of different process methods to prepare the modeling materials or the absence of a precise pressure control system. Comparative Example 7 (manual pressure adjustment) showed an apoptosis rate of 72.4%-81.5%, with inflammatory factor levels 1.2-1.9 times higher than Example 1, and traumatic inflammation caused by pressure fluctuations was the main inducing factor. Comparative Example 2 (lacking the intermediate coating) and Comparative Examples 3-4 (single core layer) showed apoptosis rates exceeding 58.7%, with inflammatory factor levels more than 40% higher, due to insufficient biocompatibility of the materials leading to an aggravated foreign body reaction. Comparative Example 1 (lacking the targeting layer) and Comparative Example 8 (unmodified antibody) showed apoptosis rates of 45.6%-54.7%, with inflammatory factor levels 20%-30% higher, due to insufficient targeting leading to an expanded damage area.

[0078] (3) Extended performance analysis: blood-spinal barrier integrity, model survival rate and biocompatibility Combining the results of blood-spinal barrier integrity testing (EB leakage method), model survival rate, and biocompatibility testing, the comprehensive performance advantages of Examples 1-3 are further highlighted: the EB content is 5.6-9.8 μg / g, and the decrease at 4 weeks compared to 1 week is 35.7%-41.3%, indicating that the degree of blood-spinal barrier damage is controllable and shows a repair trend; the 4-week survival rate is ≥86.0%, and the pathological characteristics last for up to 4 weeks, far exceeding the level of existing models; the proportion of inflammatory infiltration area is only 3.8%-4.5%, and the biocompatibility is excellent.

[0079] In the comparative examples, Comparative Example 7 (manually adjusted pressure) had the highest EB content of 33.2 μg / g, a survival rate of less than 58.0%, and an inflammatory infiltration area of ​​20.8%. Pressure fluctuations and traumatic inflammation together led to severe barrier damage and deterioration of guinea pig survival. Comparative Examples 3-4 (single core layer) and Comparative Example 9 (coating not cross-linked) had EB contents exceeding 18.6 μg / g, a survival rate of less than 62.0%, and an inflammatory infiltration area exceeding 15.6%. Persistent inflammation caused by defects in the modeling material structure was the core reason for the unsatisfactory performance indicators. Although the indicators of Comparative Examples 1, 5-6, and 8 were better than the above extreme groups, they were still significantly worse than the examples, further verifying that the spinal cord hypertension model constructed using the synergistic effect of the present invention is more in line with clinical practice.

[0080] Furthermore, according to the data from Example 4 in Tables 1 to 6, the modeling method for the spinal cord hypertension syndrome disease model of the present invention is also applicable to rhesus monkeys.

[0081] Table 1: Spinal cord pressure fluctuation coefficient (%) Continued from the previous table: Table 2: Neuronal apoptosis rate (%) Table 3: Expression levels of inflammatory factors (pg / mL) Continued from the previous table: Table 4: Blood-spinal barrier integrity (EB content, μg / g) Continued from the previous table: Table 5: Model survival rate (%) and duration of pathological features (weeks) Table 6: Material biocompatibility (percentage of inflamed infiltration area, %) The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A modeling method for a spinal cord hypertension syndrome disease model, characterized in that, Includes the following steps: S1. Preparation of composite biostimulatory materials: S11. Preparation of core pressure-relieving layer: Polycaprolactone and polylactic acid are dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide to prepare a polymer solution with a mass fraction of 10% to 20%. Then, biodegradable carbonate microspheres are added, dispersed and mixed, and poured into a mold. After removing the solvent by programmed cooling freeze drying, the solution is then vacuum dried to constant weight to obtain a porous core layer. S12. Intermediate biocompatible coating: Chitosan and gelatin are dissolved in acetic acid solution, glycerin is added, and the mixture is stirred evenly. Then, genipin solution is added, and the pH is adjusted to 5.5-6.5 to obtain the coating solution. Then, the porous core layer of S11 is immersed in the coating solution using the dip coating method, and after immersion, it is cured to form an intermediate coating. S13, Surface Targeting Binding Layer Modification: Carboxymethylated hyaluronic acid was dissolved in PBS buffer to prepare a 1%-3% (w / w) solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. After activation, integrin αvβ3 antibody was added, and after a light-protected reaction, a targeting modification solution was obtained. Then, the core layer coated with the intermediate coating in S12 was immersed in the above modification solution, incubated, and dried to obtain the composite biostimulatory material. S2. Debugging of the Precision Pressure Regulation Modeling System: The precision pressure regulation modeling system includes a pressure sensing module, a micro-injection module, a real-time monitoring module, and a main control module, used for precise control, dynamic monitoring, and data feedback adjustment of intraspinal pressure; the pressure sensing module adopts a miniature implantable fiber optic pressure sensor; the micro-injection module consists of a high-precision infusion pump, a medical silicone injection catheter, and a micro-injection needle; the real-time monitoring module includes a temperature sensor, a pH sensor, and a data acquisition unit; and the main control module consists of an embedded processor, a touch screen, and a data storage unit. Start the system and perform a self-test to check whether the communication of each module is normal, whether the sensor accuracy meets the standard, and whether the injection pump is running smoothly. Set the preset pressure range, sampling frequency and injection speed through the touch screen. Perform sterilization on the pressure sensor probe, temperature sensor, pH sensor and micro-injection catheter. S3. Spinal cord exposure and device implantation: Using guinea pigs or macaques as experimental animals, after anesthesia, hair removal and disinfection pretreatment, the spinal cord dura mater was surgically exposed, and the composite biostimulation material prepared in S1 was implanted into the subdural space at a depth of 2-3 mm and an implantation dose of 5-10 mg; subsequently, the pressure sensor probe, temperature sensor and pH sensor were implanted sequentially into the parietal lobe parenchyma of the guinea pig. S4. Pressure Regulation and Model Construction: The real-time monitoring module is activated to begin data collection. The initial pressure within the spinal cord is observed through the main control module. If the initial pressure is lower than the preset range, the micro-injection module is controlled to inject sterile buffer at a set rate until the pressure reaches the preset range. Subsequently, the system enters a closed-loop regulation state. The main control module analyzes the pressure data in real time and automatically adjusts the operation of the injection pump to maintain the pressure within the preset range of 20–60 mmHg for 70–80 hours. During the regulation period, the device position and animal vital signs are checked every 12 hours to ensure normal system operation. After 70–80 hours, the regulation system is shut down, all implanted devices are removed, and absorbable sutures are used to suture the dura mater, muscle, subcutaneous tissue, and skin layer by layer. Postoperative anti-infection treatment is performed. S5. Postoperative feeding and model maintenance: After surgery, the animals were kept in isolation until the incision healed and stable symptoms of spinal cord hypertension syndrome appeared, and a disease model of spinal cord hypertension syndrome was constructed.

2. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S11, the mass ratio of polycaprolactone to polylactic acid is 1–7:1–7; the volume ratio of dichloromethane to N,N-dimethylformamide is 3:1; the carbonate microspheres are calcium bicarbonate-magnesium carbonate composite microspheres with a mass ratio of 1–3:1, a particle size of 50–200 μm, and a loading of 20%–40% of the total mass of the core layer; the programmed cooling freeze-drying process is as follows: first, cooling to -30 to -10℃ at a rate of 4–6℃ / min and holding for 1–3 h; then cooling to -60℃ to -40℃ at a rate of 1–3℃ / min and holding for 20–30 h; the resulting porous core layer has a porosity of 40%–60% and a pore size of 10–50 μm.

3. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S12, the mass ratio of chitosan to gelatin is 1–2:1–2; the concentration of acetic acid solution is 0.1–0.3 mol / L; the mass fraction of glycerol is 0.5%–2%; the mass fraction of genipin solution is 1%–3%; the soaking time is 4–10 min; the curing is carried out at 37°C and 60% humidity for 10–15 h; the thickness of the intermediate biocompatible coating is 5–20 μm, and the degree of crosslinking is 30%–50%.

4. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S13, the molecular weight of carboxymethylated hyaluronic acid is 50-200 kDa; the pH of PBS buffer is 7.0-7.4; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1, and the total concentration is 4-6 mmol / L; the activation time is 20-40 min; the loading of integrin αvβ3 antibody is 50-200 μg / mg; the reaction is carried out at 4℃ in the dark for 10-15 h; the incubation temperature is 35-40℃ for 4-8 h, with gentle shaking every 1 h.

5. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S3, the method for exposing the spinal cord of guinea pigs is as follows: a longitudinal incision is made in the T8-T10 segment of the spine on the back of the guinea pig, and the skin, subcutaneous tissue and paravertebral muscles are separated layer by layer to expose the T8-T10 segment of the spine. The T8-10 lamina is removed to clearly expose the spinal cord area of ​​about 3×5 mm. The dura mater of the spinal cord is exposed and carefully cut open to avoid damaging the spinal cord parenchyma. The method for exposing the spinal cord of macaques is as follows: a longitudinal incision is made in the midline of the top of the macaque's head along the sagittal direction. A No. 20 scalpel is used to cut open the skin and subcutaneous tissue. The subcutaneous tissue is carefully separated to expose the skull. The periosteum is then dissected with a periosteal elevator to fully expose the surface of the skull. A 1 mm ball drill is then used to thin the skull at the predetermined puncture point to expose the dura mater but avoid perforation.

6. The modeling method for a spinal cord hypertension syndrome disease model according to claim 5, characterized in that, In the procedure of exposing the spinal cord of guinea pigs, the removal of the T8-10 lamina was performed using a micro-drill at a speed of 5000-8000 r / min. During the drilling process, sterile saline was continuously dripped to cool the spinal cord tissue and avoid high temperature damage.

7. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S3, the implantation of composite biostimulation materials and devices is controlled by a micromanipulator with a positioning accuracy of ±0.01mm, ensuring precise implantation location.

8. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S4, the preset range of the pressure to be maintained includes three gradients: low pressure gradient 20-30 mmHg, medium pressure gradient 30-45 mmHg, and high pressure gradient 45-60 mmHg.

9. The modeling method for a spinal cord hypertension syndrome disease model according to claim 1, characterized in that, In S2, the probe diameter of the miniature implantable fiber optic pressure sensor is ≤0.5mm, the detection accuracy is ±0.1mmHg, the detection range is 0~100mmHg, and the response time is ≤10ms. The probe surface is coated with polyethylene glycol. In the micro-injection module, the injection pump has an injection accuracy of ±0.1μL, and the injection speed can be continuously adjusted within the range of 0.1~10μL / min, supporting both constant-speed injection and pulse injection modes. The medical silicone injection catheter has an inner diameter of 0.2~0.5mm and an outer diameter of 0.8~1.2mm, and its surface is treated with heparin coating to prevent blood clotting and catheter blockage. The micro-injection needle is blunt-tipped. The catheter, with a diameter ≤0.8mm to avoid damaging the spinal cord parenchyma, connects to an infusion pump at one end and extends into the subdural space via an injection needle at the other end for injecting sterile buffer solution to regulate intraspinal pressure. The real-time monitoring module includes a temperature sensor with a detection range of 35–40℃ and an accuracy of ±0.1℃, and a pH sensor with a detection range of 6.5–8.0 and an accuracy of ±0.

01. The data acquisition unit integrates a multi-channel data acquisition card with an adjustable sampling frequency of 10–100Hz, capable of simultaneously acquiring pressure, temperature, and pH parameters. The data is transmitted to the main control module after analog-to-digital conversion. The main control module uses an embedded processor with an ARM Cortex-M4 core, enabling real-time analysis of the acquired pressure data. When the detected pressure deviates from the preset range, it automatically generates adjustment commands to control the micro-injection module to start or stop injection, achieving closed-loop pressure regulation. A touchscreen display is used for parameter setting and real-time data display. The data storage unit supports SD card expansion and can store at least 72 hours of continuous monitoring data for subsequent traceability and analysis.

10. The application of a spinal cord hypertension syndrome disease model constructed using the modeling method described in any one of claims 1 to 9, characterized in that, Applications include: animal models for studying the pathological mechanisms of spinal cord hypertension syndrome; animal models for screening drugs for spinal cord hypertension syndrome; and animal models for evaluating treatment regimens for spinal cord hypertension syndrome.

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