Preparation method of injectable composite support material for denasal endoscopic skull base reconstruction

Abstract

The invention relates to the technical field of biomedical materials, and discloses a preparation method of an injectable composite support material for denasal endoscopic skull base reconstruction, and the method comprises the following steps: preparing a component A aqueous solution and a component B non-aqueous phase paste; the component A comprises a hyaluronic acid derivative grafted with dopamine and alginate. The component B is paste in which calcium salt solid-phase particles, enzyme, polymer microspheres and nanoscale fumed silica are dispersed in a non-aqueous phase dispersion medium, and the nanoscale fumed silica is used for inhibiting solid-phase particle sedimentation. According to the method, the prepared component A and the prepared component B are respectively packaged in a double-component application device. According to the invention, active components are isolated in a non-aqueous phase, and controllable operation time, firm adhesion to tissues and mechanical support provided by polymer microspheres are realized through a staged curing mechanism of ionic crosslinking and enzymatic crosslinking during use; the problem that an existing material is difficult to consider storage stability, operation convenience and composite functionality at the same time is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for preparing an injectable composite support material for transnasal endoscopic skull base reconstruction. Background Technology

[0002] Endoscopic skull base surgery, especially when removing pituitary tumors or other lesions in the sellar region, often results in defects in the sellar floor bone and dura mater. Without effective reconstruction, these defects can lead to complications such as cerebrospinal fluid rhinorrhea or retraction of the diaphragm sellae.

[0003] Currently, the main clinical approach to repairing these types of defects is multi-layered packing reconstruction. This technique typically combines multiple materials, such as autologous fat or fascia for filling, and covers the wound with an artificial dura mater or a pedicled mucosal flap to close it. However, precisely placing and fixing multiple layers of material within the narrow surgical field provided by a transnasal endoscope requires a high level of skill and is relatively complex.

[0004] Furthermore, this layered physical stacking makes it difficult to ensure a seamless interface between materials, potentially creating leakage gaps that affect the reliability of the watertight seal. Simultaneously, the mechanical support properties of materials such as autologous soft tissue or flexible membranes are limited, insufficient to effectively resist brain tissue pressure and prevent collapse of the diaphragm sellae in the long term. Using rigid materials such as autologous bone for support presents challenges in shaping and achieving stable fixation at the defect site. Existing auxiliary sealing materials such as bio-protein adhesives, while providing some adhesion, typically lack structural support capabilities, and their adhesion strength and durability in humid and dynamic environments also face challenges.

[0005] Therefore, developing a skull base repair material that is both easy to apply and provides reliable watertight sealing and effective mechanical support is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The technical problem solved by this invention is that existing biomaterials for skull base reconstruction are inadequate in terms of storage stability, matching of gelation time and operability time, adhesion strength to moist bone tissue, and mechanical support properties after curing, making it difficult to simultaneously meet the various requirements of clinical application.

[0007] To address the above problems, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing an injectable composite support material for transnasal endoscopic skull base reconstruction, the method comprising the following steps:

[0009] (a) Prepare an aqueous solution of component A, which contains a hyaluronic acid derivative grafted with dopamine and alginate;

[0010] (b) A non-aqueous paste of component B is prepared, comprising: calcium salt solid particles as ionic crosslinking agents, an enzyme as a covalent crosslinking catalyst, polymer microspheres as reinforcing phase, and nanoscale fumed silica as a physical stabilizer dispersed in a non-aqueous dispersion medium; wherein the nanoscale fumed silica is used to construct a three-dimensional network to suppress the sedimentation of calcium salt solid particles and polymer microspheres;

[0011] (c) The aqueous solution of component A and the non-aqueous paste of component B are respectively packaged in a two-component application device.

[0012] By adopting the above technical solution, the present invention solves the aforementioned technical problem by constructing a system containing two independent components, A and B, and by specifically designing each component and its function in the system.

[0013] First, this invention stably disperses solid-phase reactants (calcium salts, enzymes, microspheres) in a non-aqueous medium to form a component B paste. Nanoscale fumed silica forms a physical three-dimensional hydrogen-bonded network in the non-aqueous medium through its surface silanol groups. This network imparts high static viscosity and yield stress to the component B paste, thereby preventing sedimentation and stratification of solid particles with a density greater than the dispersion medium during long-term storage. This ensures the physical stability of the product during its shelf life and guarantees the accuracy of the mixing ratio of components A and B during use.

[0014] Secondly, this invention designs a staged, dual-crosslinking curing mechanism, achieving a distinction between the gelation time and the operable time window. The curing process is as follows:

[0015] Stage 1: Formation of the ionic cross-linking network. When the aqueous solution of component A is mixed with the non-aqueous paste of component B, water molecules permeate into component B, causing the solid calcium salt to slowly dissolve and release calcium ions. These calcium ions coordinate with the alginate molecular chains in component A, forming a physical cross-linking network. The rate of this process is jointly controlled by the rates of water permeation and calcium salt dissolution, causing the material to transform from a liquid to a gel with a preliminary morphology within approximately 60–120 seconds, losing its fluidity.

[0016] The second stage: formation of a covalent cross-linked network. Within the hydrogel environment formed in the first stage, the enzymes originally dispersed in component B are activated and begin catalyzing the oxidation of dopamine groups on the hyaluronic acid derivative in component A. Specifically, the catechol groups of dopamine are oxidized to highly reactive benzoquinone groups. These quinone groups can further react, forming covalent bonds between polymer chains and also covalently linking to nucleophilic groups on the surface proteins of the bone tissue to be repaired. This enzymatic reaction is relatively slow, providing a 3–5 minute window of operability for shaping and non-adhesive instruments after initial gelation, ultimately endowing the material with tissue adhesion capabilities.

[0017] Finally, by introducing polymer microspheres as a reinforcing phase into component B, these microspheres are uniformly dispersed in the final double-network hydrogel matrix, serving as the main mechanical load-bearing unit. This improves the compressive strength and modulus of the composite material after curing, enabling it to resist cerebrospinal fluid pressure and provide effective mechanical support for the defect area.

[0018] Preferably, the method for preparing the dopamine-grafted hyaluronic acid derivative includes: activating the carboxyl group of hyaluronic acid with a chemical coupling agent, then reacting it with dopamine, and obtaining the derivative after purification.

[0019] By adopting the above technical solution, a feasible preparation route is provided for preparing functional polymer components with enzymatic reactivity.

[0020] Preferably, in the aqueous solution of component A, the concentration of the dopamine-grafted hyaluronic acid derivative is 0.5–1.5% w / v, and the concentration of the alginate is 3.0–5.0% w / v.

[0021] By employing the above technical solution, the concentrations of the two functional polymers in component A are limited. This concentration range ensures that component A has a suitable viscosity for encapsulation and extrusion, while providing sufficient reaction sites and network framework for the subsequent dual crosslinking reaction.

[0022] Preferably, in the non-aqueous phase paste of component B, the content of nano-sized fumed silica is 0.5-2.0% by total mass of component B, the content of polymer microspheres is 40-60%, the content of calcium salt solid particles is 10-20%, and the content of enzyme is 0.01-0.1%.

[0023] By adopting the above technical solution, this ratio range ensures that the B component paste has physical stability, the material has mechanical strength within the target range after curing, and the double cross-linking reaction can proceed at a preset rate and extent.

[0024] Preferably, the non-aqueous dispersion medium is anhydrous glycerol or polyethylene glycol; the calcium salt is calcium sulfate hemihydrate; and the enzyme is laccase or tyrosinase. The polymer microspheres are poly(L-lactic acid-co-ε-caprolactone) copolymer microspheres.

[0025] By adopting the above technical solution, the specific material selection of each component has been clarified. These components all have good biocompatibility, and the combination has been proven to achieve the technical effects of the present invention.

[0026] Preferably, the encapsulation volume ratio of the aqueous solution of component A to the non-aqueous paste of component B is 1:(1 to 1.5).

[0027] By adopting the above technical solution, the mixing ratio of the two components during clinical application is defined, which ensures that the reactants meet in the correct stoichiometric ratio, thereby obtaining the expected curing behavior and final performance.

[0028] Preferably, the preparation process of the non-aqueous paste of component B in step (b) includes: firstly, adding the nano-sized fumed silica to the non-aqueous dispersion medium and performing high-speed shear stirring to form a thixotropic matrix; subsequently, after forming the thixotropic matrix, maintaining medium-low speed stirring, adding the polymer microspheres and the calcium salt solid particles in sequence, and finally adding the enzyme; after all components have been added, homogenization is performed by planetary centrifugal mixing or three-roll milling.

[0029] By adopting the above technical solution, a preparation process for component B is provided. This process first constructs a stable dispersion system framework, and then adds other solid components sequentially. This avoids particle agglomeration, ensures the uniformity of dispersion of each component, and finally eliminates air bubbles through efficient homogenization to obtain a uniform paste product.

[0030] In a second aspect, the present invention also provides a method for using an injectable composite support material prepared according to the method described in the first aspect, the method comprising the following steps:

[0031] (1) Preoperative preparation: The two-component application device containing the A component and the B component is equilibrated at a predetermined temperature of 20-25°C.

[0032] (2) Mixing: Connect the static mixing tube to the front end of the two-component application device, and apply pressure to force components A and B to be extruded and mixed through the static mixing tube;

[0033] (3) Application: Under endoscopic guidance, the mixed material from step (2) is applied to the skull base defect area through the static mixing tube to cover the defect;

[0034] (4) Shaping: Within the operable time window after the material has completed its initial gelation but before its adhesion is significantly enhanced, the applied material is shaped using surgical instruments to conform it to the tissue contour of the defect area, and then the material is cured in situ.

[0035] In summary, the present invention has at least one of the following beneficial technical effects:

[0036] 1. This invention pre-disperses solid-phase active components such as ionic crosslinking agents and enzymes in a non-aqueous medium and utilizes nanoscale fumed silica to construct a physically stable network, thus preparing an independent component B. This design avoids reactions that occur during material storage due to contact between the components and water, while also inhibiting the sedimentation and stratification of solid particles, giving the product good storage stability and ensuring consistent performance throughout its shelf life.

[0037] 2. This invention utilizes two reactions with different rates—ionic crosslinking and enzymatic covalent crosslinking—to construct a staged curing process. After the materials are mixed, ionic crosslinking rapidly forms a non-flowing preliminary gel; the subsequent enzymatic reaction provides a workable time window, allowing the user to shape the material. This design, which separates rapid shaping from a workable window, solves the technical problems in clinical applications where excessively rapid gelation leads to operational difficulties or excessively slow gelation leads to material loss.

[0038] 3. This invention integrates dopamine groups with tissue adhesion function with polymer microspheres that provide mechanical support into the same system. The hydrogel matrix formed after curing can form an interfacial adhesion with the surrounding bone tissue through covalent bonding, achieving watertight sealing of the defect area. At the same time, the polymer microspheres dispersed in the matrix act as a reinforcing phase, improving the overall compressive modulus of the composite material, enabling it to provide semi-rigid support against physiological pressure. Detailed Implementation

[0039] To further clarify the objectives, technical solutions, and advantages of this invention, the invention will be described in detail below with reference to specific preparation examples, embodiments, comparative examples, and test examples. It should be understood that these specific embodiments are only for explaining the invention and are not intended to limit the scope of protection of the invention.

[0040] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0041] Sodium alginate, CAS No.: 9005-38-3, pharmaceutical grade; the weight-average molecular weight of the product used ranges from 150 to 250 kDa, and the M / G ratio ranges from 0.8 to 1.2.

[0042] Sodium hyaluronate, CAS No.: 9067-32-7, pharmaceutical grade; the weight-average molecular weight range of the product used is 50-150 kDa.

[0043] Hyaluronic acid-dopamine conjugate (HA-DOPA) is a graft copolymer formed by linking the amino groups of dopamine molecules to some carboxyl groups on the hyaluronic acid molecular chain via amide bonds. For details on its preparation method, please refer to the preparation examples.

[0044] Poly(L-lactic acid-co-ε-caprolactone) copolymer microspheres, medical-grade biodegradable; a random copolymer of L-lactic acid and ε-caprolactone; the molar ratio of L-lactic acid to ε-caprolactone in the product ranges from 70:30 to 80:20; the microspheres are spherical with a particle size distribution range of 50–150 μm.

[0045] Calcium sulfate hemihydrate, CAS No.: 10034-76-1, pharmaceutical grade powder, particle size range 10-50 μm.

[0046] The enzyme catalyst is a freeze-dried powder and can be selected from: laccase (CAS No.: 80498-15-3), derived from Polyporus velutipes, with an enzyme activity of not less than 10 U / mg; or tyrosinase (CAS No.: 9002-10-2), derived from Agaricus bisporus, with an enzyme activity of not less than 1000 U / mg.

[0047] Nanoscale fumed silica, CAS No.: 112945-52-5, hydrophilic, pharmaceutical grade; its specific surface area ranges from 150 to 250 m². 2 / g.

[0048] The dispersion medium is pharmaceutical grade with a water content of no more than 0.5%, and can be selected from: anhydrous glycerol (CAS No.: 56-81-5); or polyethylene glycol 400 (CAS No.: 25322-68-3).

[0049] Preparation Example 1:

[0050] This preparation example provides a method for preparing a hyaluronic acid-dopamine conjugate, including the following steps:

[0051] (1) Weigh 1.0g of sodium hyaluronate with a weight average molecular weight of 70kDa and dissolve it completely in 100mL of 0.1M MES buffer (pH 5.5) to obtain a hyaluronic acid solution.

[0052] (2) Add EDC and NHS to the above solution so that the molar ratio of hyaluronic acid repeating unit, EDC and NHS is 1:3:1.5. Stir magnetically for 45 minutes at room temperature to activate the carboxyl group.

[0053] (3) Dissolve dopamine hydrochloride in a small amount of MES buffer and add it to the reaction system of step (2) so that the molar ratio of hyaluronic acid repeating unit to dopamine is 1:3. Then slowly adjust the pH of the reaction system to 8.0 with 1M NaOH solution, and then stir the reaction continuously for 18 hours at room temperature and in the dark.

[0054] (4) After the reaction is complete, adjust the pH of the system to 3.5 with 1M HCl solution. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 7.0kDa), dialyze it with hydrochloric acid aqueous solution at pH 3.5 for 1 day, and then dialyze it with deionized water for 3 days, changing the water 4 times a day during the period.

[0055] (5) The solution purified by dialysis was freeze-dried to obtain a white flocculent solid, namely HA-DOPA-1. The dopamine grafting rate (the molar percentage of the grafted HA repeating units to the total repeating units) of the product was determined to be 10-15% by detecting the characteristic absorption peak of dopamine at 280 nm and comparing it with the standard curve using ultraviolet-visible spectrophotometry.

[0056] Preparation Example 2:

[0057] This preparation example provides a method for preparing a hyaluronic acid-dopamine conjugate, including the following steps:

[0058] (1) Weigh 1.0g of sodium hyaluronate with a weight average molecular weight of 120kDa and dissolve it completely in 100mL of 0.1M MES buffer (pH 5.5) to obtain a hyaluronic acid solution.

[0059] (2) Add EDC and NHS to the above solution to make the molar ratio of hyaluronic acid repeating unit, EDC and NHS 1:5:2.5, and stir magnetically for 45 minutes at room temperature to activate the carboxyl group.

[0060] (3) Dissolve dopamine hydrochloride in a small amount of MES buffer and add it to the reaction system of step (2) so that the molar ratio of hyaluronic acid repeating unit to dopamine is 1:5. Then slowly adjust the pH of the reaction system to 8.0 with 1M NaOH solution, and then stir the reaction continuously for 18 hours at room temperature and in the dark.

[0061] (4) After the reaction is complete, adjust the pH of the system to 3.5 with 1M HCl solution. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 7.0kDa), dialyze it with hydrochloric acid aqueous solution at pH 3.5 for 1 day, and then dialyze it with deionized water for 3 days, changing the water 4 times a day during the period.

[0062] (5) The solution purified by dialysis was freeze-dried to obtain a white flocculent solid, namely HA-DOPA-2. The dopamine grafting rate of the product was determined to be 18-23% by detecting the characteristic absorption peak of dopamine at 280 nm and comparing it with the standard curve using ultraviolet-visible spectrophotometry.

[0063] Example 1:

[0064] This embodiment provides a method for preparing an injectable composite support material for transnasal endoscopic skull base reconstruction, including the following steps:

[0065] (1) Preparation of component A: Weigh HA-DOPA-1 and sodium alginate obtained in Preparation Example 1, dissolve them in deionized water and stir continuously until a homogeneous solution is formed, so that the final concentration of HA-DOPA-1 in the solution is 0.5% (w / v) and the final concentration of sodium alginate is 3.0% (w / v).

[0066] (2) Preparation of component B: Add anhydrous glycerol to a stirring container, turn on high-speed shear stirring (500 rpm), slowly add 0.5% of nano-sized fumed silica of the total mass of component B, and continue stirring for 15 minutes to form a thixotropic matrix; maintain medium-low speed stirring (200 rpm), and add 40% of poly(L-lactic acid-co-ε-caprolactone) (molar ratio 70:30) copolymer microspheres of the total mass of component B and 10% of calcium sulfate hemihydrate powder of the total mass of component B in sequence; reduce the stirring speed to 50 rpm, add 0.01% of laccase lyophilized powder of the total mass of component B, and gently stir to disperse it evenly; the remainder is anhydrous glycerol; finally, degas and homogenize the mixture at 2000 rpm for 5 minutes using a planetary centrifuge to obtain the paste of component B.

[0067] (3) Packaging: The A component solution obtained in step (1) and the B component paste obtained in step (2) are filled into the two independent cavities of the double-barrel syringe in a 1:1 volume ratio and then sealed.

[0068] Example 2:

[0069] This embodiment provides a method for preparing an injectable composite support material for transnasal endoscopic skull base reconstruction, including the following steps:

[0070] (1) Preparation of component A: Weigh HA-DOPA-2 and sodium alginate obtained in preparation example 2, dissolve them in deionized water and stir continuously until a homogeneous solution is formed, so that the final concentration of HA-DOPA-2 in the solution is 1.5% (w / v) and the final concentration of sodium alginate is 5.0% (w / v).

[0071] (2) Preparation of component B: Polyethylene glycol 400 was added to a mixing container, and high-speed shear stirring (1500 rpm) was started. Nanoscale fumed silica, accounting for 2.0% of the total mass of component B, was slowly added and stirred for 30 minutes to form a thixotropic matrix. The stirring speed was maintained at medium to low speed (800 rpm), and poly(L-lactic acid-co-ε-caprolactone) (molar ratio 80:20) copolymer microspheres, accounting for 60% of the total mass of component B, and calcium sulfate hemihydrate powder, accounting for 20% of the total mass of component B, were added in sequence. The stirring speed was reduced to 200 rpm, and tyrosinase lyophilized powder, accounting for 0.1% of the total mass of component B, was added and gently stirred to disperse it evenly. The remainder was polyethylene glycol 400. Finally, the mixture was ground twice by a three-roll mill to obtain the paste of component B.

[0072] (3) Packaging: The A component solution obtained in step (1) and the B component paste obtained in step (2) are filled into the two independent cavities of the double-barrel syringe at a volume ratio of 1:1.5 and then sealed.

[0073] Example 3:

[0074] This embodiment provides a method for preparing an injectable composite support material for transnasal endoscopic skull base reconstruction, including the following steps:

[0075] (1) Preparation of component A: Weigh HA-DOPA-1 and sodium alginate obtained in Preparation Example 1, dissolve them in deionized water and stir continuously until a homogeneous solution is formed, so that the final concentration of HA-DOPA-1 in the solution is 1.0% (w / v) and the final concentration of sodium alginate is 4.0% (w / v).

[0076] (2) Preparation of component B: Add anhydrous glycerol to a stirring container, turn on high-speed shear stirring (1000 rpm), slowly add 1.0% of nano-sized fumed silica of the total mass of component B, and continue stirring for 20 minutes to form a thixotropic matrix; maintain medium-low speed stirring (500 rpm), add 50% of poly(L-lactic acid-co-ε-caprolactone) (molar ratio 70:30) copolymer microspheres of the total mass of component B and 15% of calcium sulfate hemihydrate powder of the total mass of component B in sequence; reduce the stirring speed to 100 rpm, add 0.05% of laccase lyophilized powder of the total mass of component B, and gently stir to disperse it evenly; the remainder is anhydrous glycerol; finally, degas and homogenize the mixture at 2200 rpm for 8 minutes using a planetary centrifuge to obtain the paste of component B.

[0077] (3) Packaging: The A component solution obtained in step (1) and the B component paste obtained in step (2) are filled into the two independent cavities of the double-barrel syringe at a volume ratio of 1:1.2 and then sealed.

[0078] Comparative Example 1:

[0079] Compared with Example 3, the difference is that no laccase lyophilized powder was added during the preparation of component B, while the rest were the same.

[0080] Comparative Example 2:

[0081] Compared with Example 3, the difference is that no nano-sized fumed silica is added during the preparation of component B, while the rest are the same.

[0082] Comparative Example 3:

[0083] Compared with Example 3, the difference is that in the preparation of component A, sodium hyaluronate (weight average molecular weight 70kDa) of the same mass as HA-DOPA-1 without dopamine grafting was used to replace HA-DOPA-1, and all other aspects were the same.

[0084] Comparative Example 4:

[0085] Compared with Example 3, the difference is that poly(L-lactic acid-co-ε-caprolactone) copolymer microspheres are not added during the preparation of component B, and their mass is replaced by an equal amount of anhydrous glycerol, while the rest are the same.

[0086] Comparative Example 5:

[0087] Compared with Example 3, the difference is that: component A is the same; component B is not in paste form, but is prepared as an aqueous solution containing calcium chloride with an equimolar amount of calcium ions as calcium sulfate hemihydrate in Example 3, and dissolved in deionized water.

[0088] Test Example 1:

[0089] The experimental steps are as follows:

[0090] 1. The B component pastes prepared in Example 3 and Comparative Example 2 were respectively placed into 50mL stoppered centrifuge tubes, with a filling volume of approximately 40g. The centrifuge tubes were sealed and stored vertically in a constant temperature incubator at 25°C for 3 months.

[0091] 2. After the storage period, keep the centrifuge tube vertical and use a spatula to take samples from the top, middle, and bottom of the tube. Take approximately 1g from each location and place it in a pre-weighed weighing bottle. Weigh and record the initial sample mass. Repeat the sampling operation for each location 3 times.

[0092] 3. Add 20 mL of anhydrous ethanol to each weighing bottle and sonicate for 5 minutes. Centrifuge the resulting suspension at 4000 rpm for 10 minutes and discard the supernatant. Repeat the washing-centrifugation process twice with ethanol. Finally, place the weighing bottles containing the solid precipitate in a 60℃ vacuum oven and dry to constant weight.

[0093] 4. Weigh the weighing bottle and the total mass of the solid phase after drying, and calculate the solid phase mass. Calculate the solid phase content of each sample using the formula: Solid phase content (w / w%) = (Mass of solid phase after drying / Initial sample mass) × 100%. Finally, calculate the average and standard deviation of the solid phase content at the top, middle, and bottom positions for each test object.

[0094] The experimental results are shown in Table 1.

[0095] Table 1. Solid content stability test data of component B paste after standing at 25℃ for 3 months:

[0096]

[0097] According to the data in Table 1, after 3 months of storage, the average solid content of component B of the paste in Example 3 was 66.02% at the top, middle, and bottom positions, with a standard deviation of 0.081, indicating that its component distribution was uniform. In contrast, component B of Comparative Example 2 exhibited solid-liquid stratification, with a solid content of 15.21% in the upper layer and 87.35% in the lower layer, with a standard deviation of 37.76, indicating an uneven component distribution.

[0098] Data shows that nano-sized fumed silica is an essential component for maintaining the physical stability of the B-component paste. Its mechanism of action lies in the formation of a three-dimensional hydrogen-bonded network of silanol groups on the surface of the nano-silica particles within anhydrous glycerol media. This network structure provides sufficient yield stress to resist the sedimentation of high-density solid particles such as polymer microspheres and calcium sulfate due to gravity. In Comparative Example 2, the lack of this stabilizing network prevented the dispersion medium from suspending the solid particles for an extended period, leading to sedimentation and stratification.

[0099] The physical stability of component B is fundamental to the product's shelf life and reliability in clinical applications. If component separation occurs, the stoichiometric ratio of key components such as the crosslinking agent in the mixture of components A and B extruded through a dual-barrel syringe will deviate significantly from the preset values. This will lead to uncontrollable key indicators such as gelation time, mechanical properties, and tissue adhesion, affecting the final performance. Therefore, introducing nanoscale fumed silica to construct a stable solid-liquid dispersion system is the technical foundation for ensuring the reliable implementation of this solution.

[0100] Test Example 2:

[0101] The experimental steps are as follows:

[0102] 1. Place the encapsulated double-barreled syringes from Examples 1, 2, and 3 at 25°C for at least 1 hour to equilibrate.

[0103] 2. Take out a syringe and install a uniform static mixing tube (12 mixing units, 2.5mm inner diameter) at the tip. Fix the syringe vertically onto the fixture of the universal testing machine.

[0104] 3. Adjust the compression head of the testing machine so that it just contacts the top of the syringe piston rod, and apply a preload of 0.1N to eliminate the gap.

[0105] 4. Start the testing machine and press the piston rod downward at a constant rate of 10 mm / min, setting the total stroke to 50 mm. The testing machine software synchronously records the thrust-displacement data throughout the entire process.

[0106] 5. Extract thrust data within the displacement range of 10mm to 40mm (corresponding to 20% to 80% of the total stroke), and calculate their arithmetic mean, which is the average extrusion thrust of the sample.

[0107] 6. Each set of examples was tested three times, all data were recorded and the final average was calculated.

[0108] The experimental results are shown in Table 2.

[0109] Table 2. Average extrusion thrust of the two-component materials in each embodiment:

[0110]

[0111] According to the data in Table 2, the average extrusion thrusts of Examples 1, 2, and 3 were 30.2 N, 71.2 N, and 44.9 N, respectively. The thrust values ​​of all samples were within the acceptable range for conventional manual injection, confirming the injectability of this two-component material for clinical application using standard medical devices.

[0112] The differences in extrusion thrust mainly stem from the different inherent rheological properties of components A and B in the formulations of each example. The viscosity of component B is the main contributor to extrusion resistance. In Example 2, the total solid content of component B (polymer microspheres and calcium sulfate) reaches 80% (w / w, relative to the total mass of solid phase and dispersion medium), higher than 50% in Example 1 and 65% in Example 3. The increase in solid volume fraction leads to enhanced interparticle interactions, thereby increasing the apparent viscosity and flow resistance of the paste. Meanwhile, the concentration of polymers (sodium alginate and HA-DOPA) in component A also affects the overall extrusion resistance. In Example 2, the total polymer concentration of component A is the highest (6.5% w / v), and its solution viscosity is greater than that of Example 3 (5.0% w / v) and Example 1 (3.5% w / v), which also contributes to the increase in total extrusion force.

[0113] The test results demonstrate that the technical solution of this invention has good controllability. By adjusting the concentration of the hydrogel precursor in component A, the content of the solid filler in component B, and the type of dispersion medium, the rheological behavior of the material can be controlled within a relatively wide range. This controllability allows the material to adapt to different clinical needs. For example, for scenarios requiring higher mechanical support performance, a formulation with a higher solid content can be selected (such as Example 2), although its extrusion resistance increases accordingly; while for scenarios requiring better flow and filling properties, a formulation with a lower solid content can be selected (such as Example 1).

[0114] Test Example 3:

[0115] The experimental steps are as follows:

[0116] 1. Place components A and B of Example 3, Comparative Example 3, and Comparative Example 5 in an environment at 25°C for at least 1 hour to equilibrate.

[0117] 2. Take the sample to be tested, and squeeze 2 mL of the mixture into a 20 mL stoppered glass bottle using a double-barreled syringe and a static mixing tube. Start the timer the moment the squeezing operation is completed.

[0118] 3. Keep the glass bottle horizontal, tilt it to 90 degrees every 15 seconds, hold for 1 second, then lay it flat. Observe the flow of the mixture on the bottle wall with the naked eye.

[0119] 4. Record the time from the start of timing until the mixture no longer exhibits macroscopic flow when tilted.

[0120] 5. Each group of samples was tested three times, all data were recorded and the final average was calculated.

[0121] The experimental results are shown in Table 3.

[0122] Table 3. Gelation time of each test subject:

[0123]

[0124] According to the data in Table 3, the average gelation times of Example 3 and Comparative Example 3 are similar, at 91.7 s and 88.3 s respectively, both around 1.5 minutes. However, the gelation time of Comparative Example 5 is extremely short, at only 6.0 s.

[0125] This result reveals the crucial role of the non-aqueous ointment formulation of component B in this invention. In Example 3 and Comparative Example 3, gelation was driven by ionic cross-linking between sodium alginate in component A and calcium ions released from calcium sulfate in component B. Since calcium sulfate is dispersed in anhydrous glycerol, when it comes into contact with the aqueous phase of component A, water must first penetrate into the ointment matrix of component B before dissolving the slightly soluble calcium sulfate and releasing calcium ions. The rate of this process is controlled by both water penetration and the dissolution rate of calcium sulfate itself, thus achieving sustained-release regulation of the ionic cross-linking process, ultimately resulting in a controllable gelation time suitable for clinical use.

[0126] Comparative Example 3 did not contain HA-DOPA, but its gelation time was basically the same as that of Example 3. This indicates that the initial curing process of the material is mainly determined by the sodium alginate-calcium ion system, and the presence of HA-DOPA does not interfere with this process.

[0127] In contrast, component B of Comparative Example 5 is an aqueous solution of calcium chloride. Calcium chloride is completely ionized in water, and the calcium ions within it are freely mobile. When components A and B are mixed, a large number of free calcium ions come into instantaneous contact with sodium alginate, triggering an explosive and uncontrollable ionic cross-linking, causing the material to lose its fluidity within seconds. Such a short gelation time cannot provide users with sufficient time for application, spreading, and shaping, thus lacking clinical applicability.

[0128] Therefore, this test case demonstrates that by stably dispersing the ionic crosslinking agent (calcium sulfate) in a solid phase in a non-aqueous medium, the technical solution of this invention constructs an ion-controlled release system. This system transforms the originally instantaneous ionic crosslinking reaction into a rate-controllable gelation process, thereby providing the material with an operable time window of approximately 90 seconds. This is the technical basis for the effective application of this invention in clinical surgical procedures.

[0129] Test Example 4:

[0130] The experimental steps are as follows:

[0131] 1. Place components A and B of Example 3 and Comparative Example 1 in an environment at 25°C for at least 1 hour to equilibrate.

[0132] 2. Extrude 2 mL of the mixture using a double-barrel syringe and a static mixing tube, place it in a petri dish to form a flat, circular sample, and start the timer when extrusion is complete.

[0133] 3. Using a 1mm diameter metal probe, start timing from the beginning of mixing and perform a probe every 30 seconds. During the probe test, make vertical contact with the material surface to a depth of about 2mm, hold for 1 second, and then lift it vertically.

[0134] 4. Observe whether a continuous filament longer than 5 mm is formed between the probe and the material body. Record the time point at which this phenomenon is first observed.

[0135] 5. Each sample group is tested 3 times. If no stringing phenomenon is observed within 1800 seconds (30 minutes), it is recorded as >1800s.

[0136] The experimental results are shown in Table 4.

[0137] Table 4. Operable time window for each test object:

[0138]

[0139] According to the data in Table 4, the average operability time of Example 3 was 303.3 s. In the sample of Comparative Example 1, no stringing was observed during the observation period of 1800 s.

[0140] This difference demonstrates the decisive role of laccase in initiating the second-stage curing reaction of the material. The curing process of this invention comprises two stages: the first stage is a rapid ionic cross-linking network formed by calcium ions released from calcium sulfate and sodium alginate, which provides initial shape stability to the material; its formation time was determined by Test Example 3 (approximately 90 s). The second stage is a covalent cross-linking reaction catalyzed by laccase. The mechanism of this reaction is that laccase catalyzes the oxidation of catechol groups on the HA-DOPA molecule to highly reactive catechol groups. These quinone groups then undergo covalent cross-linking with other groups through Michael addition or Schiff base reactions, forming a covalent cross-linking network that permeates the ionic network. The formation of this covalent network significantly increases the cohesive strength and adhesion of the material, macroscopically manifested as the appearance of filamentation.

[0141] In Comparative Example 1, due to the lack of laccase, the covalent cross-linking reaction could not be initiated, and the material remained only in the physical gelation stage of ionic cross-linking. Its macroscopic properties did not change significantly after the initial gelation.

[0142] Therefore, the approximately 5-minute (303.3-second) workable time in Example 3 refers to the period from initial ionogelation (approximately 90 seconds) to the significant formation of the covalent cross-linked network (approximately 300 seconds). Within this window, the material has acquired a preliminary morphology but has not yet developed strong adhesion, allowing the user to precisely shape and adjust it. This dual cross-linking, staged curing design, balancing initial curing speed with sufficient workable time, is key to achieving the feasibility of clinical applications of the material.

[0143] Test Example 5:

[0144] The experimental steps are as follows:

[0145] 1. Cut the fresh, detached pig skull into pieces approximately 2.0cm x 1.0cm in size. Use sandpaper to smooth the surface of one of the 1.0cm x 1.0cm pieces, then rinse it with deionized water and dry it with compressed air for later use.

[0146] 2. Take the sample to be tested, squeeze out about 0.2 mL of the mixture through a double-barreled syringe and a static mixing tube, and evenly coat it on the surface of a polished bone block.

[0147] 3. Align the polished surface of another bone block with the surface coated with material to form a 1.0cm × 1.0cm overlap area, and apply constant pressure above the overlap area using a 100g weight.

[0148] 4. Immediately transfer the bonded sample to a constant temperature and humidity chamber at 37°C and 100% relative humidity for 30 minutes to cure.

[0149] 5. After curing, fix both ends of the sample to the tensile fixture of the universal testing machine and perform a tensile shear test at a constant rate of 2 mm / min until the interface separates. Record the maximum load at the time of separation.

[0150] 6. Using the formula: Shear strength (kPa) = Maximum load (N) / Bond area (m²) 2 Calculate the shear strength. Each sample group was tested three times.

[0151] The experimental results are shown in Table 5.

[0152] Table 5. In vitro tissue adhesion shear strength of each test subject:

[0153]

[0154] According to the data in Table 5, the average adhesive shear strength of Example 3 was 45.1 kPa. The average shear strengths of Comparative Example 1 and Comparative Example 3 were 3.8 kPa and 4.2 kPa, respectively, which were an order of magnitude lower than those of Example 3.

[0155] This result confirms that the enzymatic covalent cross-linking system composed of HA-DOPA and laccase in this invention is key to achieving strong tissue adhesion. In Example 3, laccase catalyzes the oxidation of the catechol group on the HA-DOPA molecule to form a highly reactive benzoquinone group. This quinone group can undergo Michael addition or Schiff base reactions with nucleophilic groups such as amino and thiol groups on proteins rich in collagen on the surface of bone tissue, thereby forming a stable covalent bond between the material and the tissue interface. This molecular-level chemical bonding is the fundamental reason for the high adhesion strength.

[0156] In Comparative Example 1, the system contained HA-DOPA but lacked laccase. Therefore, the catechol groups could not be effectively oxidized to quinones, and covalent bonds could not be formed. The weak adhesion force measured mainly originated from physical adsorption after material curing or mechanical interlocking with the microstructure of the tissue surface.

[0157] In Comparative Example 3, the system contained laccase, but lacked catalytic dopamine groups on the polymer chain. Laccase lacked specific reaction substrates, and the enzymatic cross-linking reaction could not be initiated. Therefore, its adhesion strength was at the same level as Comparative Example 1, also exhibiting only non-specific physical forces.

[0158] The above data collectively demonstrate that the strong tissue adhesion properties do not originate from a single component, but rather depend on the simultaneous presence of dopamine functional groups and corresponding catalytic enzymes. This specific enzymatic reaction system can rapidly and in situ form a covalent network at the material-tissue interface under physiological conditions, providing a reliable mechanical basis for sealing defects and preventing cerebrospinal fluid leakage in applications such as skull base reconstruction.

[0159] Test Example 6:

[0160] The experimental steps are as follows:

[0161] 1. The mixture of Example 3 and Comparative Example 4 was injected into a cylindrical polytetrafluoroethylene mold with an inner diameter of 10 mm and a height of 5 mm.

[0162] 2. Place the filled mold in a constant temperature and humidity chamber at 37℃ and 100% relative humidity for 24 hours to cure.

[0163] 3. After curing, carefully remove the cylindrical sample from the mold and use calipers to measure its final diameter and height.

[0164] 4. Place the sample on the testing platform of the universal testing machine and perform a uniaxial unconfined compression test at a constant rate of 1 mm / min, while simultaneously recording the load-displacement data until the strain reaches 20%.

[0165] 5. Convert the load-displacement data into a stress-strain curve based on the initial dimensions of the sample. Read the compressive stress value corresponding to 10% compressive strain from the curve.

[0166] 6. Each group of samples was tested three times, all data were recorded and the final average was calculated.

[0167] The experimental results are shown in Table 6.

[0168] Table 6. Compressive stress of each test object after curing:

[0169]

[0170] According to the data in Table 6, the stress of the cured sample in Example 3 at 10% compressive strain was 211.9 kPa, while the corresponding stress of Comparative Example 4 was only 15.5 kPa.

[0171] These results demonstrate that poly(L-lactic acid-co-ε-caprolactone) copolymer microspheres are the decisive component for achieving high compressive strength in the material. The cured product of Example 3 is a composite material whose structure consists of a continuous hydrogel network phase and a dispersed semi-rigid microsphere reinforcing phase. Under compressive loads, external stress is transferred from the softer hydrogel matrix to the more rigid microsphere particles. These microspheres, acting as the primary load-bearing units, effectively bear and disperse most of the load, thereby significantly improving the overall material's resistance to deformation.

[0172] Comparative Example 4, lacking a microsphere-reinforcing phase, has a cured product that is essentially a simple hydrogel. Its mechanical properties are determined solely by the concentration and cross-linking density of the polymer chains. Due to its high water content, the hydrogel naturally has a lower compressive modulus and strength, making it prone to significant deformation under compressive loads and unable to provide effective mechanical support.

[0173] In transnasal endoscopic skull base reconstruction, the implant needs sufficient mechanical strength to support the soft tissue of the repaired area and withstand fluctuations in cerebrospinal fluid pressure. The compressibility demonstrated in Example 3 confirms that it meets this clinical requirement. However, the results of Comparative Example 4 show that without the microspheres as a reinforcing phase, the hydrogel network alone cannot provide the necessary structural support. Therefore, introducing biodegradable semi-rigid microspheres as a reinforcing phase into the hydrogel matrix is ​​the key design of this invention to realize its function as a skull base support material.

[0174] The results of test examples 1 to 6 above verify the composition and effectiveness of this technical solution from different dimensions.

[0175] The results of Test Example 1 confirm that the B component paste has long-term storage physical stability, which is a technical prerequisite for ensuring accurate stoichiometry and controllable final performance of the two-component materials during mixing.

[0176] The results of Test Example 2 demonstrate that the material of this technical solution exhibits good injectability, and its rheological properties can be adjusted through formulation. This ensures that the material is suitable for manual clinical handling and has the potential for performance optimization for different application scenarios.

[0177] The results of Test Example 3 and Test Example 5 together confirm the effectiveness of the dual crosslinking system of the present invention. By dispersing the solid-phase calcium salt in a non-aqueous phase, a controllable ionic crosslinking gelation process is achieved, providing the necessary operation time for clinical applications.

[0178] The data from Test Examples 4 and 5 further demonstrate that the enzymatic reaction system composed of HA-DOPA and laccase is the reason for obtaining high tissue adhesion strength, and the introduction of this system establishes an operational window between initial gelation and final strong adhesion of the material.

[0179] The results of Test Example 6 confirm that by introducing semi-rigid microspheres as a reinforcing phase, the material has sufficient mechanical support properties after curing.

[0180] In summary, this invention, through the systematic design of a multi-component, multi-phase system, prepares a two-component biomaterial that integrates storage stability, controllable gel properties, operability, strong tissue adhesion, and mechanical support.

[0181] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the preparation of an injectable composite support material for endoscopic transnasal skull base reconstruction, characterized in that, The method comprises the following steps: (a) preparing an aqueous A component solution comprising a hyaluronic acid derivative grafted with dopamine and an alginate; (b) preparing a non-aqueous B component paste comprising: calcium salt solid-phase particles as an ionic crosslinking agent, an enzyme as a covalent crosslinking catalyst, polymer microspheres as a reinforcing phase, and nanoscale fumed silica as a physical stabilizer dispersed in a non-aqueous dispersion medium; the nanoscale fumed silica is used to build a three-dimensional network to inhibit the sedimentation of the calcium salt solid-phase particles and the polymer microspheres; (c) separately packaging the aqueous A component solution and the non-aqueous B component paste in a two-component application device.

2. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by the steps of: The hyaluronic acid derivative grafted with dopamine is prepared by the following steps: The carboxyl group of hyaluronic acid is activated by a chemical coupling agent, and then reacted with dopamine, and after purification, the hyaluronic acid derivative grafted with dopamine is obtained.

3. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by the steps of: In the aqueous A component solution, the concentration of the hyaluronic acid derivative grafted with dopamine is 0.5-1.5% w / v, and the concentration of the alginate is 3.0-5.0% w / v.

4. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, In the non-aqueous B component paste, the content of the nanoscale fumed silica is 0.5-2.0%, the content of the polymer microspheres is 40-60%, the content of the calcium salt solid-phase particles is 10-20%, and the content of the enzyme is 0.01-0.1%, based on the total mass of the B component.

5. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, The non-aqueous dispersion medium is anhydrous glycerol or polyethylene glycol; the calcium salt is calcium sulfate hemihydrate; and the enzyme is laccase or tyrosinase.

6. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, The polymer microspheres are poly(L-lactic acid-co-ε-caprolactone) copolymer microspheres.

7. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, The packaging volume ratio of the aqueous A component solution to the non-aqueous B component paste is 1:(1-1.5).

8. The method of claim 1, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, The preparation of the non-aqueous B component paste in step (b) comprises: The nanoscale fumed silica is added to the non-aqueous dispersion medium, and high-speed shearing stirring is performed to form a thixotropic matrix.

9. The method of claim 8, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, After the thixotropic matrix is formed, low-speed stirring is maintained, and the polymer microspheres and the calcium salt solid-phase particles are sequentially added, and finally the enzyme is added.

10. The method of claim 9, wherein the injectable composite support material for endoscopic transnasal skull base reconstruction is prepared by, After all the components are added, homogenization treatment is performed by planetary centrifugal mixing or three-roll grinding.

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