Preparation method of dynamic connection bone extracellular matrix simulation hydrogel and application thereof in bone defect repair

Abstract

The application provides a preparation method of a dynamically connected bone extracellular matrix simulation hydrogel and application thereof in bone defect repair, and belongs to the technical field of medical hydrogels. A thiol-disulfide bond dynamic crosslinking network is formed by preparing L-cysteine ethyl ester functionalized hyaluronic acid and allicin, a sacrificial pre-vascularization template is constructed by combining collagen protein threads loaded with vascular endothelial growth factor, vascularization guide microchannels are formed by using the enzymatic properties of collagen protein, inflammatory reactions are inhibited by regulating macrophage polarization through hyaluronic acid, bone marrow mesenchymal stem cell adhesion and osteogenic differentiation are promoted by means of functionalized surfaces, the synergistic effect of vascularization, anti-inflammation and bone integration is realized, and finally, the dynamically connected bone extracellular matrix simulation hydrogel with a porous interconnected structure and biological activity is formed, so that the technical problems of insufficient vascularization, inflammatory interference and poor bone integration of traditional bone defect repair materials are solved.

Description

Technical Field

[0001] This invention belongs to the field of medical hydrogel technology, specifically, it relates to a method for preparing a dynamically connected bone cell extracellular matrix-simulated hydrogel and its application in bone defect repair. Background Technology

[0002] Bone defect repair is a significant challenge in clinical orthopedics and neurosurgery. The core difficulties lie in: the limited regenerative capacity of bone tissue (especially for large defects), the susceptibility to postoperative infection and chronic inflammation, and poor integration of repair materials with the host bone. Traditional repair methods such as autologous bone grafting, allogeneic bone grafting, and synthetic material implantation have significant limitations.

[0003] 1. Autologous bone transplantation: The donor site is highly invasive and the amount of bone available is limited, which can easily lead to complications such as donor site pain and infection;

[0004] 2. Allogeneic bone transplantation: carries the risk of immune rejection, may transmit pathogens, and bone induction activity decreases with storage time;

[0005] 3. Synthetic materials (such as metals, ceramics, and traditional polymers): have poor biocompatibility, are difficult to simulate the biochemical and mechanical microenvironment of bone ECM, are prone to foreign body reactions, and cannot promote the synergistic process of vascularization and osteoogenesis.

[0006] In current bone tissue engineering applications, traditional materials lack effective vascularization induction mechanisms, making it difficult to quickly establish a vascular network after implantation. This leads to insufficient nutrient supply and accumulation of metabolic products in the central region of the material. Simultaneously, the material surface easily activates macrophages, generating a persistent inflammatory response that interferes with the normal bone repair process. Furthermore, the lack of an effective biological integration interface between the material and the host bone tissue results in poor bone integration and low repair efficiency. In other words, existing technologies suffer from technical problems such as insufficient vascularization, inflammatory interference, and poor bone integration in traditional bone defect repair materials. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing a dynamically connected extracellular matrix-simulated hydrogel and its application in bone defect repair, which can solve the technical problems of insufficient vascularization, inflammatory interference and poor osseointegration in traditional bone defect repair materials in the prior art.

[0008] The present invention is achieved as follows: The first aspect of the present invention provides a method for preparing a dynamically connected extracellular matrix-simulated hydrogel, comprising the steps of preparing L-cysteine ​​ethyl ester-functionalized hyaluronic acid, preparing collagen I threads loaded with vascular endothelial growth factor, preparing a dynamic cross-linked network structure, detecting the concentration of thiol groups and the molar ratio of allicin, and injecting the mixture into a polytetrafluoroethylene mold and monitoring the change in cross-linking degree in real time during gelation at 37°C. The L-cysteine ​​ethyl ester-functionalized hyaluronic acid forms a thiol-disulfide bond dynamic cross-linked network with allicin, and the collagen I threads loaded with vascular endothelial growth factor serve as a sacrificial pre-vascularization template. Hyaluronic acid is used to regulate macrophage polarization towards the M2 type to inhibit the inflammatory response, and the functionalized surface promotes the adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby achieving a synergistic effect of vascularization, anti-inflammation, and bone integration.

[0009] Specifically, the step of preparing L-cysteine ​​ethyl ester functionalized hyaluronic acid involves dissolving hyaluronic acid in deionized water to prepare a 0.4% solution, adjusting the pH to 5.5 with 0.1M hydrochloric acid, adding N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH to 5.5, stirring for 15 minutes, adding L-cysteine ​​ethyl ester hydrochloride to the solution, stirring the reaction at room temperature for 4 hours, transferring the reaction solution to a dialysis bag, and dialyzing with physiological saline to obtain L-cysteine ​​ethyl ester functionalized hyaluronic acid.

[0010] Specifically, the L-cysteine ​​ethyl ester-functionalized hyaluronic acid is grafted onto the hyaluronic acid backbone via amide bonds, introducing thiol functional groups to provide reaction sites for subsequent dynamic cross-linking. Hyaluronic acid, as a natural polysaccharide component of the extracellular matrix of bone cells, has biocompatibility and biodegradability.

[0011] Specifically, the step of preparing collagen I threads loaded with vascular endothelial growth factor involves injecting a 1 wt% collagen I solution into a 50°C water bath using a 16G needle at an injection rate, gelling for 30 minutes to form a thread-like structure, immersing the collagen I threads in a vascular endothelial growth factor solution, incubating at 4°C for 24 hours to load vascular endothelial growth factor onto the thread surface, and then pre-freezing at -80°C for 2 hours followed by vacuum freeze-drying for 24 hours.

[0012] Specifically, the step of preparing the dynamic cross-linked network structure involves dispersing L-cysteine ​​ethyl ester-functionalized hyaluronic acid solution, allicin, and collagen I thread loaded with vascular endothelial growth factor in deionized water under a nitrogen atmosphere, and stirring vigorously until the mixture is homogeneous.

[0013] The thiol-disulfide dynamic crosslinking refers to the redox reaction between the thiol group and the allyl disulfide group in allicin to form a reversible disulfide crosslinking network. Dynamic crosslinking endows the hydrogel with viscoelastic adaptive ability, which can adjust the network structure according to changes in external stress.

[0014] The sacrificial prevascularization template refers to the collagen I thread being enzymatically hydrolyzed in vivo by matrix metalloproteinases to form a microchannel structure loaded with vascular endothelial growth factor. The microchannel provides a guiding path for the migration of vascular endothelial cells and the formation of lumen, promoting the formation of new blood vessels.

[0015] The second aspect of this invention provides the application of the hydrogel prepared by the above-described method in the repair of bone defects, including skull defects, long bone defects, and maxillofacial bone defects; the hydrogel repairs bone defects through the following mechanisms: ① VEGF microchannels formed by sacrificial template enzymatic hydrolysis guide the migration of vascular endothelial cells and lumen formation; ② HACys network simulates bone ECM to promote bone marrow mesenchymal stem cell adhesion and osteogenic differentiation; ③ regulates macrophage polarization towards the M2 type and inhibits excessive inflammatory response.

[0016] The hydrogel can be prepared as any one of an injection, implant, or topical gel formulation.

[0017] The hydrogel can be used in conjunction with autologous bone, allogeneic bone, or synthetic bone scaffolds in bone defect repair.

[0018] Specifically, the step of detecting the molar ratio of thiol concentration to allicin involves adjusting the allicin concentration when the molar ratio of thiol to allicin reaches 2:1, so that the cross-linking density of thiol-disulfide bonds is controlled within the optimal range.

[0019] The real-time monitoring of crosslinking degree changes includes: if the crosslinking degree change rate exceeds a first threshold and the duration is greater than a second threshold, then a rapid response adjustment is immediately executed; if the crosslinking degree change rate exceeds the first threshold but the duration is less than the second threshold, then a slow response adjustment is executed; when three consecutive test results show crosslinking abnormalities, it is determined to be a continuous abnormal state and an emergency gel reprocessing procedure is initiated; when only a single test result is abnormal, the test frequency is increased for secondary confirmation.

[0020] The process also includes steps to experimentally determine the first and second thresholds, preparing L-cysteine ​​ethyl ester-functionalized hyaluronic acid solutions with different thiol concentrations, mixing them with allicin solutions of a fixed concentration, monitoring the change in crosslinking degree in real time during gelation at 37°C, recording the critical condition corresponding to when the crosslinking degree change rate exceeds 15% / second, repeating the experiment 15 times and taking the average value as the basic data for the first threshold, and observing the stability of the hydrogel network by changing the duration under the condition of a fixed crosslinking degree change rate.

[0021] The first threshold and the second threshold are calculated by a threshold optimization function, which is used to determine the optimal response threshold based on experimental data. The inputs include the crosslinking degree change rate, temperature, pH value and ionic strength, and the output is the optimized first threshold and the second threshold.

[0022] The threshold optimization function is expressed as follows: , ,in The first threshold, The second threshold, Based on the rate of change of crosslinking degree, Based on the duration, The experimental temperature, Standard temperature For the experimental pH value, Standard pH value, For the experimental ionic strength, Standard ionic strength.

[0023] The rapid response regulation refers to immediately increasing the concentration of allicin solution and simultaneously decreasing the gelation temperature after detecting an abnormal change in the degree of crosslinking, so as to quickly stabilize the crosslinked network structure. The slow response regulation refers to gradually adjusting the concentration of allicin while maintaining a constant temperature, so as to restore the crosslinking balance in a gentle manner.

[0024] The emergency gel reprocessing process refers to stopping the current gelation process when a continuous abnormal state is detected, re-preparing the L-cysteine ​​ethyl ester functionalized hyaluronic acid solution and allicin solution, and restarting the preparation process. The crosslinking abnormality judgment is based on a comprehensive evaluation of the first threshold and the second threshold.

[0025] The final product is a dynamically connected osteoblast extracellular matrix-mimicking hydrogel with a porous interconnected structure. The hydrogel guides the migration of vascular endothelial cells and the formation of lumens through VEGF microchannels formed by sacrificial template enzymatic hydrolysis. The HACys network simulates bone ECM to promote the adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, regulates the polarization of macrophages towards the M2 type, and inhibits excessive inflammatory response.

[0026] This invention addresses the technical problems of insufficient vascularization, inflammatory interference, and poor osteointegration by constructing a sacrificial pre-vascularization template loaded with vascular endothelial growth factor (VEGF), establishing a dynamic disulfide bond cross-linking network, and optimizing the material's surface properties. Utilizing the characteristic that collagen threads are specifically hydrolyzed by matrix metalloproteinases in vivo, this invention forms a directional microchannel structure loaded with VEGF, providing a three-dimensional guiding path for vascular endothelial cell migration and lumen formation, significantly enhancing the material's vascularization capacity. Simultaneously, the hyaluronic acid matrix effectively inhibits excessive inflammatory responses by regulating macrophage polarization towards the M2 anti-inflammatory phenotype. L-cysteine ​​ethyl ester functionalization modification gives the material surface a biochemical environment similar to the bone matrix, promoting the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells. Through a multi-component synergistic mechanism, this invention promotes rapid vascularization while regulating the inflammatory microenvironment and optimizing osteocyte behavior, achieving an integrated solution for vascularization, anti-inflammation, and osteointegration. In summary, this invention solves the technical problems of insufficient vascularization, inflammatory interference, and poor osteointegration mentioned in the background art for traditional bone defect repair materials. Attached Figure Description

[0027] Figure 1 This is a flowchart of the method of the present invention.

[0028] Figure 2 Here is the 1H NMR spectrum of HACys.

[0029] Figure 3 Characterization of HACys-VEGF@Coll-A, including sub-figures: (A) SEM and EDS-mapping images of HACys-VEGF@Coll-A; (C) Digital photograph of the collagenized HACys-VEGF@Coll-A hydrogel composite (blue indicates collagen 1 loaded with VEGF); (D) FTIR spectrum of HACys-VEGF@Coll-A; (E) X-ray diffraction pattern of HACys-VEGF@Coll-A; (F) Energy dispersive spectroscopy (EDS) analysis of HACys-VEGF@Coll-A.

[0030] Figure 4 The graph shows the sacrificial performance of the thread hydrogel.

[0031] Figure 5 The in vitro biocompatibility assessment of HACys-VEGF@Coll-A includes the following sub-figures: (A) Cell viability of bone marrow mesenchymal stem cells (BMSCs) after 24 hours of incubation with different samples. (B) Cell viability of human umbilical vein endothelial cells (HUVECs) after 24 hours of incubation with different samples. (C) Representative fluorescence images of BMSCs and HUVECs stained with Calcein-AM / PI after different sample treatments.

[0032] Figure 6 The in vitro angiogenesis assessment of HACys-VEGF@Coll-A includes subplots: (A) Mastocyte angiogenesis assay; (B) Angiogenesis characteristics assessed by wound healing assay; (C, D) Quantitative analysis of cell node and network numbers; (D) Quantitative percentage of migration regions in HUVECs; (n=3, *p<0.05, compared with control group).

[0033] Figure 7 The in vitro osteogenic capacity assessment of HACys-VEGF@Coll-A includes subplots: (A) Alkaline phosphatase (ALP) and basic chromatin reaction (ARS) staining of HACys-VEGF@Coll-A. (B) Relative quantitative assessment of ALP and ARS. (n=3, *p<0.05).

[0034] Figure 8 The in vitro anti-inflammatory capacity assessment diagram of HACys-VEGF@Coll-A includes subplots: (A) The presence of M1 macrophages (characterized by CD86 expression) and M2 macrophages (characterized by CD206 expression) was assessed by flow cytometry; (B) Immunofluorescence micrographs of CD86 (M1 phenotype) and CD206 (M2 phenotype). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0036] like Figure 1 The diagram shown is a flowchart of a method for preparing a dynamically connected extracellular matrix simulated hydrogel provided by the present invention. This method includes the following steps:

[0037] S01. Preparation of L-cysteine ​​ethyl ester functionalized hyaluronic acid: Hyaluronic acid was dissolved in deionized water to prepare a 0.4% solution. The pH was adjusted to 5.5 with 0.1M hydrochloric acid. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to a final concentration of 50mM. After adjusting the pH to 5.5, the mixture was stirred for 15 minutes.

[0038] S02. Add 0.5g of L-cysteine ​​ethyl ester hydrochloride to the above solution, stir at room temperature for 4 hours, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8-14kDa, dialyze with 5L of physiological saline for more than 6 hours, and change the physiological saline every 2 hours to obtain L-cysteine ​​ethyl ester functionalized hyaluronic acid.

[0039] S03. Prepare collagen I threads loaded with vascular endothelial growth factor. Inject 1 wt% collagen I solution into a 50°C water bath through a 16G needle at an injection rate of 0.5-1 mL / min and gel for 30 minutes to form a thread-like structure.

[0040] S04. Immerse the collagen I threads in a 50-200 ng / mL vascular endothelial growth factor solution and incubate at 4°C for 24 hours to load vascular endothelial growth factor onto the thread surface. Then pre-freeze at -80°C for 2 hours and freeze-dry under vacuum for 24 hours.

[0041] S05. To prepare a dynamic cross-linked network structure, under a nitrogen atmosphere, 2.5 wt% of L-cysteine ​​ethyl ester-functionalized hyaluronic acid solution, 0.2 wt% of allicin and collagen I thread loaded with vascular endothelial growth factor were dispersed in deionized water and stirred vigorously at a rate of 300-500 rpm until the mixture was homogeneous.

[0042] S06. Detect the concentration of thiol and the molar ratio of allicin. When the molar ratio of thiol to allicin reaches 2:1, adjust the concentration of allicin to control the cross-linking density of thiol-disulfide bonds within the optimal range.

[0043] S07. Inject the mixture into a polytetrafluoroethylene mold and monitor the change in crosslinking degree in real time during the gelation process at 37°C. If the rate of change in crosslinking degree exceeds the first threshold and the duration is greater than the second threshold, execute a rapid response adjustment immediately. If the rate of change in crosslinking degree exceeds the first threshold but the duration is less than the second threshold, execute a slow response adjustment. When three consecutive test results show abnormal crosslinking, it is determined to be a continuous abnormal state and an emergency gel reprocessing procedure is initiated. When only a single test result is abnormal, increase the testing frequency for secondary confirmation. Finally, a dynamically connected extracellular matrix simulated hydrogel with a porous interconnected structure is formed.

[0044] L-cysteine ​​ethyl ester-functionalized hyaluronic acid involves grafting L-cysteine ​​ethyl ester onto the hyaluronic acid backbone via amide bonds, introducing thiol functional groups to provide reaction sites for subsequent dynamic cross-linking. Hyaluronic acid, as a natural polysaccharide component of the bone cell extracellular matrix, exhibits excellent biocompatibility and biodegradability.

[0045] The dynamic cross-linking of thiol-disulfide bonds refers to the redox reaction between thiol groups and allyl disulfide groups in allicin, forming a reversible disulfide cross-linking network. This dynamic cross-linking endows the hydrogel with viscoelastic adaptive capabilities, enabling it to adjust its network structure according to changes in external stress.

[0046] Sacrificial prevascularization templates refer to the enzymatic degradation of collagen I threads in vivo by matrix metalloproteinases, forming microchannel structures loaded with vascular endothelial growth factor. These microchannels provide a guiding pathway for the migration of vascular endothelial cells and lumen formation, promoting angiogenesis.

[0047] Step S07 further includes the experimental determination of a first threshold and a second threshold. The determination of the first threshold includes: preparing L-cysteine ​​ethyl ester-functionalized hyaluronic acid solutions with different thiol concentrations (1.0-4.0 mM), mixing them with a fixed concentration of allicin solution, monitoring the change in crosslinking degree in real time during gelation at 37°C, recording the critical condition corresponding to a crosslinking degree change rate exceeding 15% / second, repeating the experiment 15 times, and taking the average value as the basic data for the first threshold. The determination of the second threshold includes: under a fixed crosslinking degree change rate condition, changing the duration from 5 seconds to 60 seconds, observing the stability of the hydrogel network, and setting 30 seconds as the basic data for the second threshold when the duration exceeds 30 seconds and irreversible changes occur in the network structure.

[0048] The first and second thresholds are calculated using a threshold optimization function. This function determines the optimal response threshold based on experimental data. Inputs include the crosslinking degree change rate, temperature, pH value, and ionic strength; the output is the optimized first and second thresholds. The threshold optimization function is described below:

[0049] ;

[0050] ;

[0051] in The first threshold is expressed as % / second. The second threshold is in seconds. Based on a crosslinking degree change rate of 15% / second, The base duration is 30 seconds. The experimental temperature, The standard temperature is 37℃. For the experimental pH value, The standard pH value is 7.4. For the experimental ionic strength, The standard ionic strength is 0.15 M.

[0052] The principle of the first threshold formula is based on the thermodynamic and kinetic properties of the thiol-disulfide crosslinking reaction. Increased temperature promotes molecular thermal motion and reaction rate; therefore, a linear proportional relationship is adopted. The rate of change in the basic degree of crosslinking was corrected. pH affects the dissociation state and reactivity of thiol groups; increased pH promotes thiol deprotonation and enhances crosslinking reactivity. A linear proportional relationship was adopted. Corrections are made. Ionic strength affects the conformation and electrostatic interactions of macromolecular chains; high ionic strength shields electrostatic repulsion and promotes molecular approach. Therefore, an inverse relationship is adopted. A correction is made to reduce the threshold as the ionic strength increases.

[0053] The principle behind the second threshold formula is based on the time dependence and environmental sensitivity of cross-linked network formation. The effect of temperature on the time threshold is expressed as a power of 0.5. This reflects the diffusion-controlled characteristics of the cross-linking reaction; increasing temperature accelerates molecular diffusion, but its effect on the time scale decays non-linearly. The pH value is corrected for the time threshold using a power of 0.3. This reflects the mild effect of pH changes on the time it takes for the reaction to reach equilibrium. Ionic strength is expressed as an inverse relationship to the power of 0.2. This indicates a weak negative correlation between ion strength and network stability establishment time.

[0054] The first threshold formula establishes a quantitative relationship between environmental conditions and crosslinking rate sensitivity, enabling the system to adaptively adjust the monitoring standard for crosslinking degree change rate according to actual process conditions. Through a multi-factor coupling correction mechanism, the formula ensures accurate identification of abnormal crosslinking behavior under different temperature, pH, and ionic strength conditions, avoiding misjudgments and over-adjustments caused by environmental fluctuations. The formula enhances the environmental adaptability of crosslinking degree monitoring, making the triggering conditions for rapid and slow response adjustments more precise, thereby ensuring the stability and consistency of the hydrogel preparation process.

[0055] The second threshold formula serves to determine the criteria for judging the duration of crosslinking anomalies, avoiding interference from instantaneous fluctuations in the preparation process through dynamic adjustment over time. The formula considers the environmental sensitivity during the maturation of the crosslinked network, enabling the duration threshold to automatically adjust with changes in process conditions, ensuring the accuracy and timeliness of anomaly detection. The formula improves the timeliness and reliability of anomaly detection, responding promptly to genuine anomalies while effectively filtering out transient system fluctuations, avoiding unnecessary process interruptions and resource waste, ultimately achieving intelligent monitoring and precise adjustment of the crosslinking process.

[0056] Rapid response regulation refers to immediately increasing the allicin solution concentration to 0.3 wt% and simultaneously decreasing the gelation temperature to 35°C upon detecting an abnormal change in crosslinking degree, in order to quickly stabilize the crosslinked network structure. Slow response regulation refers to gradually adjusting the allicin concentration to 0.25 wt% while maintaining a constant temperature, thereby restoring crosslinking equilibrium through a gentler approach.

[0057] The emergency gel reprocessing procedure refers to stopping the current gelation process when a persistent abnormal state is detected, re-preparing the L-cysteine ​​ethyl ester-functionalized hyaluronic acid solution and allicin solution, and restarting the preparation process according to step S05. The crosslinking abnormality determination is based on a comprehensive evaluation of the first and second thresholds to ensure the stability and consistency of the gelation process.

[0058] The specific implementation methods of the above steps are described in detail below.

[0059] The specific implementation of step S01 is as follows: First, hyaluronic acid powder is slowly added to deionized water at room temperature. A magnetic stirrer is used to continuously stir the solution at 200 rpm for 30 minutes until completely dissolved, forming a homogeneous 0.4% hyaluronic acid solution. Then, the pH value of the solution is continuously monitored using a pH meter. 0.1M hydrochloric acid solution is added dropwise using a micro-pump, with each addition not exceeding 0.1 mL. The pH value is measured after stirring for 10 seconds. This process is repeated until the pH value stabilizes within the range of 5.5 ± 0.1. Next, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are accurately weighed using a balance and dissolved separately in a small amount of deionized water to prepare stock solutions. These stock solutions are then added to the hyaluronic acid solution according to the stoichiometric ratio to achieve a final concentration of 50 mmol / L. This step utilizes the amide coupling reaction principle, activating the carboxyl group in the hyaluronic acid molecule through a carbodiimide-mediated activation mechanism to form an active ester intermediate, creating conditions for the subsequent coupling reaction of amino compounds. Precise control of the solution pH is crucial for maintaining the activity of carbodiimide and avoiding side reactions. Too high a pH will lead to the hydrolysis and inactivation of carbodiimide, while too low a pH will affect the degree of deprotonation of the carboxyl group.

[0060] The specific implementation of step S02 involves adding 0.5 g of pre-weighed L-cysteine ​​ethyl ester hydrochloride to the activated hyaluronic acid solution, rapidly mixing it using a vortex mixer for 30 seconds, and then transferring it to a magnetic stirrer for continuous stirring at 150 rpm for 4 hours at room temperature. During the reaction, pH values ​​are sampled every hour to monitor changes, and if necessary, the pH is fine-tuned to 5.5 ± 0.2 using dilute hydrochloric acid or sodium hydroxide solution. After the reaction is complete, the solution is transferred to a dialysis bag with a molecular weight cutoff of 8000 to 14000 Daltons, using the molecular weight cutoff principle to remove unreacted small molecule compounds and reaction byproducts. Dialysis is performed using 5 liters of physiological saline as the dialysis medium at 4°C, with the dialysate changed every 2 hours, for at least 6 hours until the conductivity of the dialysate is similar to that of pure physiological saline. This step covalently binds L-cysteine ​​ethyl ester molecules to the hyaluronic acid backbone through an amide bond formation reaction, and the introduced thiol functional groups provide active sites for subsequent dynamic cross-linking reactions. The dialysis purification process effectively removes free coupling reagents and unreacted L-cysteine ​​ethyl ester, ensuring product purity and the controllability of subsequent reactions.

[0061] The specific implementation of step S03 involves first equilibrating the collagen I solution overnight at 4°C to ensure complete hydration and uniform dispersion of collagen molecules. Using a precision injection pump system, a 1% (w / w) collagen I solution is injected through a 16-gauge stainless steel needle into a preheated 50°C constant-temperature water bath at a constant flow rate of 0.5 to 1 ml per minute. During injection, the needle is inserted vertically to a depth of 2 to 3 cm below the water surface, creating a continuous linear flow path. Upon contact with the hot water, the collagen solution undergoes a thermo-gelation reaction, and the triple helix structure of the collagen molecules reassembles to form a fibrous network structure. Maintaining a constant temperature of 50°C for 30 minutes allows the gelation process to complete, forming a thread-like collagen structure with a certain mechanical strength. This process is based on the temperature-sensitive gelation mechanism of collagen, triggering a phase transition from a sol to a gel state through rapid temperature changes, forming a thread template with the microstructural characteristics of natural collagen fibers. A constant injection rate ensures uniformity of thread diameter, while appropriate gelation temperature and time guarantee the stability and integrity of the thread structure.

[0062] The specific implementation of step S04 involves carefully removing the newly prepared collagen I threads and placing them in a sterile culture dish. A vascular endothelial growth factor (VEGF) solution with a concentration of 50 to 200 nanograms per milliliter is then prepared, using phosphate-buffered saline (PBGS) as a dilution medium to maintain protein activity. The collagen threads are completely immersed in the VEGF solution and incubated at 4°C for 24 hours, with the culture dish gently shaken every 6 hours to promote uniform distribution and adsorption of the growth factor. The incubation process is based on the principle of non-covalent interaction between proteins and the collagen matrix; VEGF binds to collagen fibers through electrostatic interactions, hydrogen bonds, and hydrophobic interactions. Low-temperature incubation conditions help maintain the biological activity of the growth factor and slow down its degradation rate. After incubation, the growth factor-loaded threads are transferred to a liquid nitrogen container and pre-frozen at -80°C for 2 hours, allowing the water in the tissue to rapidly crystallize into small ice crystals, preventing large ice crystals from damaging the collagen structure. After pre-freezing, the product is transferred to a vacuum freeze dryer for 24 hours of sublimation drying. The water vapor converted from ice crystals is removed by the principle of depressurized sublimation, and finally a porous structure is obtained while maintaining the loading state of growth factors.

[0063] The specific implementation of step S05 is to operate in a nitrogen-filled glove box environment to avoid interference from the oxidation reaction. First, the purified L-cysteine ​​ethyl ester-functionalized hyaluronic acid is accurately weighed and dissolved in deionized water to prepare a 2.5% (w / w) stock solution. Simultaneously, a 0.2% (w / w) allicin stock solution is prepared. The allicin solution should be prepared fresh each time to maintain its oxidative activity. The pre-prepared collagen I threads loaded with vascular endothelial growth factor are uniformly dispersed in deionized water at a predetermined density, controlled at 10 to 20 threads per cubic centimeter to form a suitable porous network. The three components are mixed using a high-speed disperser at a speed of 300 to 500 rpm for 5 to 10 minutes until the system reaches a macroscopically homogeneous state. The viscosity of the system is monitored in real time during mixing. When the viscosity begins to increase significantly, stirring is immediately stopped to avoid excessive shearing that could damage the formed cross-linked structure. The core principle of this step is to achieve uniform distribution of each component at the microscale through mechanical dispersion, creating sufficient contact opportunities for subsequent chemical cross-linking reactions, while nitrogen gas protects the environment to prevent accidental oxidation of thiol groups.

[0064] The specific implementation of step S06 involves quantitatively detecting the concentration of free thiol groups in the mixed system using the Elman reagent colorimetric method. Specifically, 0.1 mL of sample is mixed with 1 mL of Elman reagent solution, reacted at room temperature for 15 minutes, and then the absorbance is measured at 412 nm using a UV-Vis spectrophotometer. The molar concentration of thiol groups is calculated based on a pre-established standard curve, and the actual concentration of allicin is detected by high-performance liquid chromatography (HPLC). The molar ratio of thiol groups to allicin is calculated. When this ratio deviates from the theoretical value of 2:1, the allicin concentration needs to be adjusted. Concentration adjustment is performed using a stepwise addition method, with each addition of allicin stock solution not exceeding 1% of the total volume. After addition, the mixture is immediately stirred, and the molar ratio is re-measured until the target range is reached. This step is based on the stoichiometric principle of the thiol-disulfide bond crosslinking reaction. An ideal 2:1 molar ratio ensures that each allicin molecule can react with two thiol groups to form stable disulfide bond crosslinking points. Precise control of the molar ratio is crucial for obtaining the optimal crosslinking density. A ratio that is too high will lead to over-crosslinking, making the gel too rigid, while a ratio that is too low will result in insufficient crosslinking, affecting gel strength.

[0065] The specific implementation of step S07 involves rapidly injecting the prepared mixture into a polytetrafluoroethylene mold preheated to 37°C. The inner surface of the mold is pre-coated with silicone oil to prevent adhesion. During gelation, an online rheometer continuously monitors the change in crosslinking degree, with a measurement frequency set to once every 10 seconds, while simultaneously recording the real-time values ​​of storage modulus and loss modulus. The system calculates a first threshold and a second threshold under the current environmental conditions based on a preset threshold optimization algorithm. The first threshold is calculated using the current experimental temperature, pH value, and ionic strength parameters. The baseline crosslinking degree change rate of 15% per second is corrected by multiplying the temperature ratio, pH ratio, and inverse ionic strength ratio to obtain the environmentally corrected threshold. The second threshold is calculated using a baseline duration of 30 seconds, corrected by powers of 0.5 (temperature ratio), 0.3 (pH ratio), and 0.2 (inverse ionic strength ratio). When the crosslinking degree change rate exceeds the first threshold and the duration exceeds the second threshold, the system automatically performs a rapid response adjustment, increasing the allicin concentration from 0.2% to 0.3% and simultaneously decreasing the gelation temperature from 37°C to 35°C. If the rate of change in crosslinking degree exceeds the first threshold but the duration is less than the second threshold, a slow response adjustment is executed, gradually adjusting the allicin concentration to 0.25% while keeping the temperature constant. An abnormal state is determined using a continuous detection algorithm. When three consecutive detections show abnormal crosslinking, it is considered a persistent abnormal state, and an emergency re-processing procedure is initiated. If only a single abnormal detection is detected, the detection frequency is increased to once every 5 seconds for secondary confirmation. The entire monitoring and adjustment system is based on the real-time feedback control principle, achieving adaptive optimization of the gelation process through a closed-loop control mechanism of sensor-controller-actuator, ensuring the structural consistency and performance stability of the final product.

[0066] It should be noted that the first key technical concept of this invention is the construction of a dynamic cross-linking mechanism of thiol-disulfide bonds. Traditional hydrogels mostly employ irreversible covalent cross-linking or physical cross-linking, lacking the ability to respond to changes in the external environment. This invention introduces thiol functional groups by grafting L-cysteine ​​ethyl ester onto the hyaluronic acid backbone, and then reacts with the allyl disulfide groups in allicin through a redox reaction to form a reversible disulfide bond cross-linking network. This dynamic cross-linking mechanism endows the hydrogel with viscoelastic adaptive capabilities, enabling it to automatically adjust its network structure according to changes in external stress. Under mechanical loads, molecular chain segment rearrangement occurs to disperse stress, and the original conformation is restored after the load is removed. Compared to traditional static cross-linking networks, dynamic cross-linking significantly improves the toughness and fatigue resistance of the material, better mimicking the dynamic characteristics of the natural extracellular matrix.

[0067] The second key technological approach is the establishment of a real-time monitoring and adaptive adjustment system. Traditional gelation processes lack process control and rely on empirical parameters, leading to poor product consistency. This invention constructs a real-time monitoring system based on rheological parameters, which assesses the crosslinking process in real time by detecting changes in storage modulus and loss modulus online. More importantly, an environmentally sensitive threshold optimization algorithm has been developed, dynamically adjusting the monitoring threshold based on environmental factors such as temperature, pH, and ionic strength, enabling the anomaly detection criteria to adapt to different process conditions. When an anomaly is detected, the system automatically executes a graded response adjustment, selecting a rapid or slow adjustment strategy based on the severity of the anomaly, avoiding the lag and subjectivity of manual intervention. This intelligent monitoring mechanism significantly improves the stability and reproducibility of the production process.

[0068] The third key technological approach is the design of a sacrificial prevascularization template. Traditional tissue engineering scaffolds struggle to rapidly establish a vascular network in the early stages of implantation, leading to malnutrition and necrosis of cells in the central region. This invention uses collagen I threads as sacrificial templates, with vascular endothelial growth factor (VEGF) loaded on the thread surface. After implantation, these threads undergo enzymatic degradation by matrix metalloproteinases, forming growth factor-loaded microchannel structures at the original thread locations. These prefabricated microchannels provide migration pathways and spatial guidance for vascular endothelial cell formation, accelerating angiogenesis and maturation. Compared to traditional random pore structures, this ordered channel network more effectively mimics the distribution characteristics of the natural vascular system, providing a superior blood supply foundation for tissue regeneration.

[0069] The synergistic effect of these three key technological approaches has yielded significant systemic advantages. The dynamic cross-linking mechanism provides environmental adaptability to the material, the real-time monitoring system ensures the controllability of the preparation process, and the pre-vascularized template solves key challenges in application. Together, they constitute a complete technological solution. The plasticity of the dynamic cross-linking network facilitates the migration of vascular endothelial cells and luminal remodeling, the monitoring and regulation system ensures precise control of the cross-linking density, thereby optimizing the material's mechanical properties and biocompatibility, and the pre-vascularized design accelerates the vascularization process after implantation. This synergistic effect of multi-technology integration results in hydrogels that not only possess excellent biomimetic properties but also have the process stability for large-scale production, providing a comprehensive and high-performance biomaterial platform for bone tissue engineering applications.

[0070] It should be noted that this invention also solves the following technical problems: The lack of quality control and batch consistency assurance in traditional hydrogel preparation processes. Existing hydrogel preparation processes mostly employ simple mixed crosslinking methods, lacking means for real-time monitoring and dynamic adjustment of the crosslinking reaction. Environmental factors such as temperature fluctuations, pH changes, and differences in ionic strength can easily affect the formation of the crosslinking network, leading to significant performance differences between batches and failing to meet the stringent quality stability requirements of medical materials. This invention establishes a real-time monitoring system based on changes in the degree of crosslinking of thiol-disulfide bonds. Through a dynamic judgment mechanism of first and second thresholds, it can promptly identify abnormal changes in the crosslinking process and employ rapid response adjustment, slow response adjustment, or emergency reprocessing procedures according to the degree and duration of the abnormality, ensuring that the crosslinking network structure and performance parameters of each batch of products remain within a preset range. Simultaneously, the threshold optimization function adaptively adjusts the monitoring standards according to actual process conditions, making the quality control system environmentally adaptable and effectively solving the batch consistency problem. The invention also addresses the technical problem of traditional bone repair materials having limited functionality and lacking integration of multiple bioactive components. Conventional materials often only achieve a single function, such as scaffold materials that only provide support or drug carriers that only release growth factors. They struggle to simultaneously meet multiple biological needs, including vascularization induction, inflammation regulation, and bone integration, thus limiting repair efficacy. This invention achieves functional integration through a multi-component design. Hyaluronic acid matrix provides a biocompatibility basis and regulates the inflammatory microenvironment; collagen threads construct a vascularization template and load vascular endothelial growth factor; L-cysteine ​​ethyl ester functionalization optimizes cell adhesion and differentiation behavior; and dynamic disulfide cross-linking endows the material with adaptive mechanical properties. Each component, while performing its specific function, forms a synergistic effect, achieving the organic integration of multiple biological activities and significantly improving the overall effect of bone defect repair.

[0071] Specifically, the principle of this invention is as follows: The fundamental reason why the technical solution of this invention can solve the problems of insufficient vascularization, inflammatory interference, and poor bone integration lies in the establishment of a multi-level synergistic mechanism of biological functions. Regarding the vascularization mechanism, collagen threads serve as sacrificial templates. Their enzymatic hydrolysis in vivo follows the specific recognition and cleavage rules of matrix metalloproteinases. The microchannels generated by enzymatic hydrolysis have a directional spatial structure, providing a three-dimensional migration path for vascular endothelial cells. Vascular endothelial growth factor is loaded onto the thread surface through physical adsorption and is released spatiotemporally and controllably during the enzymatic hydrolysis process, maintaining synchronization with the microchannel formation process in time and space, forming a spatiotemporal coupling effect induced by vascularization. Regarding the anti-inflammatory regulation mechanism, hyaluronic acid, as a natural component of the extracellular matrix, activates intracellular anti-inflammatory signaling pathways by specifically binding to CD44 receptors on the surface of macrophages. This promotes the polarization of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type. M2 macrophages secrete anti-inflammatory factors such as IL-10 and TGF-β, establishing a microenvironment conducive to tissue repair. In terms of bone integration mechanisms, the surface of the biomaterial formed by L-cysteine ​​ethyl ester-functionalized hyaluronic acid linked by amide bonds mimics the biochemical characteristics of glycosaminoglycans in the natural bone matrix, providing specific adhesion sites for bone marrow mesenchymal stem cells. Cells activate the expression of osteogenic-related genes through integrin-mediated signal transduction. At the same time, the viscoelastic adaptive properties of the dynamic disulfide bond cross-linking network enable the material to respond to cell contractile forces, providing a suitable mechanical microenvironment for cell differentiation. This dual effect of biochemical and mechanical signals promotes the directed differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0072] To better understand and implement this invention, several specific application scenarios of this invention are provided below.

[0073] Example 1: Synthesis of L-cysteine ​​ethyl ester-functionalized hyaluronic acid (HACys)

[0074] Reagent preparation: Hyaluronic acid (HA, molecular weight 150kDa, Aladdin), EDC-HCl (purity 98%, Aladdin), NHS (purity 98%, Aladdin), L-cysteine ​​ethyl ester hydrochloride (purity 99%, Aladdin), 0.1M hydrochloric acid, physiological saline;

[0075] Operating steps:

[0076] 1. Dissolve 0.4g of HA in 100mL of deionized water, stir in a 37℃ water bath for 2 hours until completely dissolved, and adjust the pH to 5.5 with 0.1M hydrochloric acid;

[0077] 2. Add 0.95g EDC-HCl and 0.57g NHS, stir for 5 minutes, then adjust the pH to 5.5 with 0.1M hydrochloric acid and stir at room temperature for 15 minutes;

[0078] 3. Add 0.5g L-cysteine ​​ethyl ester hydrochloride (Cys), and stir magnetically at room temperature for 4 hours to obtain a pale yellow viscous liquid;

[0079] 4. Transfer the liquid to a dialysis tube with a molecular weight cutoff of 10 kDa, dialyze with 5 L of physiological saline for 6 hours (replace the physiological saline every 2 hours), take 10 mL of the solution after dialysis, freeze dry, weigh and calculate the yield (approximately 75%).

[0080] 5. Threshold determination: HACys solutions with different thiol concentrations (1.0-4.0 mM) were prepared and mixed with 0.2 wt% allicin solution. The crosslinking degree was monitored in real time at 37℃. The storage modulus G' change was recorded every 10 seconds using a rheometer (Anton Paar MCR 302). The critical condition was defined as the rate of change of G' exceeding 15% / second. The experiment was repeated 15 times, and the average value was taken as the basic data for the first threshold. Under a fixed rate of change in crosslinking degree, the duration was varied from 5 seconds to 60 seconds. When the duration exceeded 30 seconds and irreversible changes occurred in the network structure, a second threshold data was set. ).

[0081] 6. Threshold optimization function calculation: Based on the standard conditions of experimental temperature 37℃, pH value 7.4, and ionic strength 0.15M, the threshold optimization function was used to calculate the following:

[0082] ;

[0083] ;

[0084] Characterization and verification: Characteristic peaks of HACys were detected using 1H NMR (Bruker AVANCE III 400MHz). Characteristic peaks appeared at 1.3 ppm (ethyl ester methyl) and 3.0 ppm (thiol-adjacent methylene), confirming successful grafting of L-cysteine ​​ethyl ester. Figure 2 ).

[0085] Example 2: Preparation of VEGF-loaded collagen I thread

[0086] Reagent preparation: Collagen I (rat tail tendon, 98% purity, Corning), VEGF (human recombinant, 95% purity, PeproTech), 0.1M acetic acid, 0.1M NaOH, PBS buffer;

[0087] Operating steps:

[0088] 1. Dissolve 1g of collagen I in 100mL of 0.1M acetic acid, stir overnight at 4℃ until completely dissolved, and adjust the pH to 7.4 with 0.1M NaOH to obtain a 1wt% collagen solution;

[0089] 2. Load the collagen solution into a 10mL syringe, attach a 16G needle, fix it vertically on the support, and inject the solution into a 50℃ constant temperature water bath (in a petri dish) at a rate of 0.8mL / min to form a continuous thread. Let it stand for 30 minutes to gel.

[0090] 3. Carefully remove the thread with tweezers and immerse it in 50 mL of PBS solution containing 100 ng / mL VEGF. Incubate at 4°C for 24 hours.

[0091] 4. Transfer the thread to a freeze-drying bottle, pre-freeze at -80℃ for 2 hours, then freeze-dry in a vacuum freeze dryer (Labconco FreeZone 6) for 24 hours to obtain a diameter... White thread, sealed and stored at -20℃;

[0092] 5. Quality Control Testing: Quality testing was performed on each batch of VEGF-collagen threads prepared. VEGF loading was determined using an ELISA kit (R&D Systems), requiring a VEGF loading of [value missing] per gram of threads. The tensile strength of the threads was tested using a universal testing machine (Instron 5944). Requirements: SEM was used to observe the surface morphology of the threads, requiring a smooth surface without obvious defects. Only threads that passed the quality inspection could be used for subsequent experiments.

[0093] 6. Storage Stability Verification: Lyophilized threads were stored at -20℃, 4℃, and room temperature for 1 week and 1 month, respectively, and the VEGF activity retention rate was periodically monitored. Cell proliferation assays were used to verify VEGF biological activity, requiring a certain activity retention rate after 1 month of storage. .

[0094] Example 3: Preparation and characterization of HACys-VEGF@Coll-A composite hydrogel

[0095] Reagent preparation: HACys prepared in Example 1, VEGF-collagen thread prepared in Example 2, allicin (98% purity, Aladdin), deionized water;

[0096] Hydrogel preparation:

[0097] 1. In a nitrogen glove box, dissolve 2.5g of HA-Cys in 90mL of deionized water, add 0.2g of allicin, and stir magnetically for 30 minutes until clear;

[0098] 2. Add 0.5g of freeze-dried VEGF-collagen threads and stir with a tissue homogenizer (1000rpm) for 10 minutes to disperse the threads evenly;

[0099] 3. Inject the mixture into a 24-well plate (1 mL per well), place the plate in a 37°C incubator, and use a rheometer probe inserted into the hydrogel surface to record the storage modulus G' every 10 seconds. Calculate the rate of change in crosslinking degree: The monitoring program is set to automatically determine:

[0100] When the change rate of crosslinking degree And duration When needed, initiate rapid response adjustment;

[0101] When the change rate of crosslinking degree But duration At that time, slow response adjustment is initiated;

[0102] If three consecutive abnormal detections are detected, the emergency reprocessing procedure will be initiated.

[0103] 4. Response adjustment execution:

[0104] Rapid response: Immediately inject the pre-prepared 0.3wt% allicin solution into the incubator. At the same time, adjust the temperature to 35℃;

[0105] Slow response: Add 0.25wt% allicin solution in 3 portions, each time... 5-minute intervals;

[0106] Emergency reprocessing: Stop the current preparation, clean the petri dish, and prepare again according to steps 1-2;

[0107] 5. Determination of gelation completion: When the G' value stabilizes within the target range ( And the rate of change over 30 consecutive minutes At this point, gelation is considered complete. The hydrogel is observed to be milky white, elastic, and does not detach when inverted.

[0108] Characterization analysis:

[0109] SEM: The lyophilized hydrogel was sputter-coated with gold and observed using a Hitachi S-4800 SEM. A porous interconnected structure and pore size distribution were visible. This meets the requirements for cell infiltration;

[0110] EDS: Elemental mapping shows that C, O, N, and S are evenly distributed, and the S elemental signal confirms the existence of dynamic cross-linking bonds;

[0111] FTIR: Nicolet iS50 FTIR detection, (OH stretching) (Amide I band, collagen characteristic) (SH extension / retraction) The presence of characteristic peaks (COC glycosidic bond) confirms the successful recombination of the components;

[0112] XRD: Bruker D8 Advance XRD test. The presence of broadened amorphous peaks and the absence of sharp crystalline peaks confirms that the hydrogel has an amorphous structure and possesses good mechanical adaptability. Figure 3 )

[0113] Example 4: Sacrificial performance experiment of thread hydrogel

[0114] Experimental procedure:

[0115] 1. After gelling in the mold, the hydrogel complex was treated with 0.1% trypsin for 30 min;

[0116] 2. HUVECs were cultured with a prevascularized hydrogel complex for 24 h, stained with Calcein-AM and DAPI, and observed under a confocal microscope;

[0117] 3. Monitoring of the sacrificial process: During trypsin treatment, samples were taken every 10 minutes to observe thread degradation. Changes in thread diameter were recorded using a microscope; when the thread diameter decreased to 20-30% of its original size, an ideal microchannel structure was formed. pH changes were monitored and maintained within the range of 7.2-7.6.

[0118] 4. Microchannel quality assessment: After trypsin treatment, fluorescent microspheres (diameter...) were used... Infuse microchannels to verify channel connectivity. Required channel connectivity rate. Channel diameter uniformity coefficient of variation .

[0119] Results: Representative fluorescence microscopy images of human umbilical vein endothelial cells (HUVECs) on hydrogels with and without collagen threads after 24 hours of co-culture. Only on hyaluronic acid-collagen complexes (HACys) were HUVECs sparsely distributed and without obvious alignment. Conversely, in constructs containing sacrificial collagen thread templates, continuous, densely distributed, elongated, and viable bands of HUVECs were observed forming along the gaps left by the threads. This alignment and linear coverage were consistent with contact guidance of vascular endothelial growth factor (VEGF) along topographic cues and / or from the HACys matrix into the newly formed channels at local concentration gradients. Figure 4 )

[0120] Example 5: Biocompatibility and Functional Testing of Hydrogels

[0121] 5.1 Biocompatibility test (CCK-8 assay)

[0122] Cell preparation: BMSCs (rat bone marrow-derived, P3 generation) and HUVECs (human umbilical vein-derived, P4 generation) were cultured in DMEM medium containing 10% FBS.

[0123] Experimental groups: control group (culture medium only); HACys group (2.5wt% HACys solution); VEGF@Coll group; HACys-VEGF@Coll-A group (hydrogel extract, prepared by soaking 1g hydrogel in 10mL culture medium for 24h).

[0124] Testing steps:

[0125] 1. The cells were prepared using... Inoculate 10 cells / well into a 96-well plate, add 10 cells / well to each well. The culture medium was replaced with the extract for each group after 24 hours of incubation.

[0126] 2. Incubate for 1 day and 3 days respectively, adding [the following ingredient] to each well. CCK-8 reagent, incubated at 37℃ for 2 hours, absorbance measured at 450 nm using a microplate reader (BioTek Synergy H1);

[0127] 3. Extract Quality Control: During the preparation of the extract, pH, osmotic pressure, and endotoxin levels are monitored in real time. The pH is controlled within the range of 7.2-7.6, and the osmotic pressure is controlled within [specific parameters]. , endotoxin levels Each batch of extract is subjected to sterility testing after preparation to ensure it is free from bacterial and fungal contamination.

[0128] 4. Real-time cell viability monitoring: In addition to CCK-8 assay, cell viability was assessed every 24 hours using a Live / Dead staining kit, and the ratio of live to dead cells was observed using a fluorescence microscope. When the proportion of dead cells... At that time, it was determined to be positive for cytotoxicity.

[0129] Results: After 3 days of culture, the viability of BMSCs in the HACys-VEGF@Coll-A group was [missing information]. HUVECs activity is It has no significant toxicity. Figure 5 )

[0130] 5.2 Angiogenesis Capacity Experiment

[0131] Experimental procedure:

[0132] 1. Apply matrix gel (Corning Incorporated, USA) to the wells of a 48-well plate to culture cells. HUVECs were inoculated into 48-well plates and incubated at 37°C with 5% [temperature missing]. HUVECs were cultured in a serum-free medium in an incubator. After 6 hours, the morphology of HUVECs was observed using an optical microscope. The number of connecting points and vessels in the reticular structure within the field of view was quantitatively analyzed using ImageJ software.

[0133] 2. Scratch assays were performed using HUVECs cell lines. In a typical experiment, the HUVECs cell line was used to perform scratch assays. HUVECs were inoculated into 6-well plates and incubated at 37°C with 5% [missing information]. Complete culture medium was used in the incubator. When the cell density reached 100%, the medium was replaced with serum-free medium, and a line was drawn in each well. After 24 hours, the morphology of HUVECs was observed using an optical microscope.

[0134] 3. Dynamic monitoring of vascular network formation: The vascular network formation process was captured every 2 hours using a live-cell imaging system (IncuCyte S3) and observed continuously for 24 hours. The time points of network formation were recorded as follows: initial connection (2-4 hours), network expansion (4-8 hours), network maturation (8-12 hours), and network stabilization (12-24 hours).

[0135] 4. Real-time analysis of vascular network parameters: The ImageJ Angiogenesis Analyzer plugin is used to automatically calculate parameters such as the number of connection points, the number of grids, the total length of the main vessel, and the number of branch points. A threshold is set for the number of connection points. Number of grids The result was determined to be positive.

[0136] Results: On matrix gel, the medium cultured with the VEGF construct induced rapid formation of a dense and interconnected capillary-like network (Fig. 6A). In contrast, the control medium and the extract containing only HACys produced sparse, discontinuous cord-like structures with fewer connection points and meshes. Quantitative analysis confirmed that the number of connection points and meshes under VEGF conditions was significantly increased compared to the control group and the HACys-only group (Fig. 6C, D). These indicators are typical surrogate indicators of network complexity, directly reflecting the morphogenetic potential of the provided signal. The comparable performance of the VEGF-containing group suggests that embedding VEGF in HACys maintains its bioactivity and does not render it inactive; conversely, the hydrogel microenvironment may stabilize VEGF and support its sustained release, thereby maintaining the effectiveness of induced morphogenesis.

[0137] Scratch assays yielded similar results. In the scratch healing assay, VEGF-containing medium accelerated wound closure within 24 hours; the control group and HACys extract only supported limited edge advancement (Fig. 6B). The quantitative percentage of closure was significantly higher only in the VEGF group (Fig. 6E), consistent with the requirement that sustained local VEGF signaling is essential for driving directional migration and budding of endothelial cells. Given this sacrificial template design, this migratory ability is crucial for the rapid colonization of newly formed channels by endothelial cells and the formation of anastomoses on the scaffold. Figure 6 )

[0138] 5.3 Osteogenic differentiation capacity test (ALP and ARS staining)

[0139] Experimental procedure:

[0140] 1. Osteogenic differentiation was assessed according to established procedures. MC3T3-E1 cells were cultured in osteogenic induction medium containing hydrogel. The formation of mineralized nodules and alkaline phosphatase activity were assessed by Alizarin Red S (ARS) staining and alkaline phosphatase (ALP) staining, respectively, and the results were analyzed by relative quantification using ImageJ.

[0141] 2. BMSCs Inoculate one cell per well into a 6-well plate, and after 24 hours of culture, replace with osteogenic induction medium (containing 10 mM... -Sodium glycerophosphate, Ascorbic acid, 10 nM dexamethasone), and added to each group of hydrogels (Transwell chambers, (aperture);

[0142] 3. After 7 days of culture, staining was performed using an ALP staining kit (Sigma-Aldrich); after 21 days, staining was performed using an ARS staining kit (Sigma-Aldrich).

[0143] 4. Monitoring of osteogenic differentiation: ALP activity and calcium deposition were measured every 3 days. ALP activity was quantitatively measured using an ALP activity assay kit (Jiancheng Bioengineering), with the requirement that ALP activity reach at least 1.5 times that of the control group by day 7. Calcium deposition was measured using a calcium ion assay kit (Jiancheng Bioengineering), with the requirement that calcium deposition reach at least twice that of the control group by day 21.

[0144] 5. Osteogenesis-related gene expression detection: RNA was extracted from cells at days 7, 14, and 21 of culture, and the expression levels of osteogenic-related genes (ALP, OCN, OPN, Runx2) were detected using RT-qPCR. GAPDH was used as an internal control gene. The method calculates the relative expression level.

[0145] Results: Given the close link between angiogenesis and bone formation during bone repair, this study investigated whether these materials support the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts. In the control group and with HACys alone, early alkaline phosphatase (ALP) activity and late mineralization deposition (Alizarin Red S, ARS) were both scarce. In contrast, the VEGF-containing groups—whether VEGF-releasing constructs assembled with collagen threads or the composite HACys-VEGF@Coll-A—showed dense ALP-positive regions and numerous continuous mineralized nodules (Fig. 7A). Quantitative analysis showed that ALP activity and ARS absorbance increased significantly only in the presence of VEGF; HACys alone showed no significant difference compared to the control group (Fig. 7B).

[0146] 5.4 Anti-inflammatory capacity experiment (macrophage polarization detection)

[0147] Experimental procedure:

[0148] 1. RAW 264.7 cells were treated with lipopolysaccharide (Thermo Fisher Scientific, USA) to induce macrophage polarization, and then cultured with different treatment groups for 24 hours.

[0149] 2. Flow cytometry and immunofluorescence staining were performed to assess the expression of CD86 and CD206. The primary antibodies used were rabbit anti-CD206 (1:400, China Bolt Biotechnology) and mouse anti-CD86 (1:400, China Bolt Biotechnology). Secondary antibodies included goat anti-rabbit Alexa Fluor 488 (1:400, China Bolt Biotechnology) and goat anti-mouse Alexa Fluor 594 (1:400, Invitrogen, USA).

[0150] 3. After staining, the cells were analyzed by flow cytometry and the nuclei were counterstained with DAPI and observed using a confocal microscope.

[0151] 4. Dynamic monitoring of macrophage polarization: Flow cytometry was used to detect the expression levels of CD86 and CD206 at 6, 12, 24, and 48 hours of culture. Calculations were then performed. The ratio is required to be the ratio of the VEGF-containing group at 24 hours. This indicates that M2-type polarization was successfully induced.

[0152] 5. Detection of inflammatory factor secretion: ELISA kits were used to detect inflammatory factor secretion in the culture supernatant. , (M1 type marking) and , (M2 type marker) secretion levels. VEGF-containing groups are required. ratio , ratio .

[0153] Results: The innate immune response determines the tissue repair process. Therefore, macrophage polarization was assessed after exposure to different materials. Flow cytometry showed that the distribution of macrophages changed under the influence of vascular endothelial growth factor (VEGF), shifting from a high CD86 / low CD206 distribution (pro-inflammatory M1 type) to a low CD86 / high CD206 distribution (repairing M2 type). The control and HACys groups were mainly concentrated in the pro-inflammatory areas, while the VEGF-containing groups—especially the complex HACys-VEGF@Coll-A—shifted towards the repair areas (Fig. 8A). Immunofluorescence imaging also confirmed this redistribution: macrophages on the VEGF-containing constructs showed reduced CD86 (green) and enhanced CD206 (red) staining, while the complex showed the highest CD206 signal and the lowest CD86 signal (Fig. 8B).

[0154] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a dynamically connected extracellular matrix-mimicking hydrogel, characterized in that, The method includes the following steps: preparing L-cysteine ​​ethyl ester-functionalized hyaluronic acid, preparing collagen I threads loaded with vascular endothelial growth factor, preparing a dynamic cross-linked network structure, detecting the concentration of thiol and the molar ratio of allicin, and monitoring the change in cross-linking degree in real time during the gelation process of injecting the mixture into a polytetrafluoroethylene mold at 37°C. The L-cysteine ​​ethyl ester-functionalized hyaluronic acid forms a thiol-disulfide bond dynamic cross-linked network with allicin. Combined with collagen I threads loaded with vascular endothelial growth factor as a sacrificial pre-vascularization template, the method utilizes hyaluronic acid to regulate macrophage polarization towards the M2 type to inhibit the inflammatory response. Through the functionalized surface, it promotes the adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells, thereby achieving a synergistic effect of vascularization, anti-inflammation and bone integration. Specifically, the step of preparing the dynamic cross-linked network structure involves dispersing L-cysteine ​​ethyl ester-functionalized hyaluronic acid solution, allicin, and collagen I thread loaded with vascular endothelial growth factor in deionized water under a nitrogen atmosphere, and stirring vigorously until the mixture is homogeneous to form a mixture. The real-time monitoring of crosslinking degree changes includes: if the crosslinking degree change rate exceeds the first threshold and the duration is greater than the second threshold, then a rapid response adjustment is immediately executed; if the crosslinking degree change rate exceeds the first threshold but the duration is less than the second threshold, then a slow response adjustment is executed; when three consecutive test results show crosslinking abnormality, it is determined to be a continuous abnormal state and an emergency gel reprocessing procedure is initiated; when only a single test result is abnormal, the test frequency is increased for secondary confirmation. It also includes the steps of experimentally determining the first threshold and the second threshold, preparing L-cysteine ​​ethyl ester functionalized hyaluronic acid solutions with different thiol concentrations, mixing them with allicin solutions of a fixed concentration, monitoring the change in crosslinking degree in real time during the gelation process at 37℃, recording the critical condition corresponding to the crosslinking degree change rate exceeding 15% / second, repeating the experiment 15 times and taking the average value as the basic data of the first threshold, and observing the stability of the hydrogel network by changing the duration under the condition of fixed crosslinking degree change rate. The first threshold and the second threshold are calculated using a threshold optimization function, which is expressed as follows: ， ， in, The first threshold is expressed as % / second. The second threshold is in seconds. Based on a crosslinking degree change rate of 15% / second, The base duration is 30 seconds. The experimental temperature, The standard temperature is 37℃. For the experimental pH value, The standard pH value is 7.

4. For the experimental ionic strength, The standard ionic strength is 0.15 M.

2. The preparation method according to claim 1, characterized in that, The steps for preparing L-cysteine ​​ethyl ester functionalized hyaluronic acid are as follows: Hyaluronic acid is dissolved in deionized water to prepare a 0.4% solution, the pH is adjusted to 5.5 with 0.1M hydrochloric acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added, the pH is adjusted to 5.5 and stirred for 15 minutes, L-cysteine ​​ethyl ester hydrochloride is added to the solution, the reaction is stirred at room temperature for 4 hours, the reaction solution is transferred to a dialysis bag and dialyzed with physiological saline to obtain L-cysteine ​​ethyl ester functionalized hyaluronic acid.

3. The preparation method according to claim 2, characterized in that, The steps for preparing collagen I threads loaded with vascular endothelial growth factor specifically involve injecting a 1 wt% collagen I solution into a 50°C water bath using a 16G needle at an injection rate of 0.5-1 mL / min, gelling for 30 minutes to form thread-like structures, immersing the collagen I threads in a vascular endothelial growth factor solution, incubating at 4°C for 24 hours to load vascular endothelial growth factor onto the thread surface, and then pre-freezing at -80°C for 2 hours followed by vacuum freeze-drying for 24 hours.

Images