An isolator for establishing an animal model of bone nonunion and its preparation method.

Abstract

This invention discloses an isolator for establishing an animal model of bone nonunion and its preparation method, relating to the field of medical device technology, including the following steps: Step 1: Obtaining a hydrogen-containing silicone oil prepolymer with Si-H bonds in the side chain; Step 2: Obtaining a silicone macromonomer with epoxy groups in the side chain; Step 3: Obtaining an anti-inflammatory functionalized silicone macromonomer; Step 4: Three-dimensionally reconstructing a bone model to create an isolator model at the defect site; Step 5: Adding the anti-inflammatory functionalized silicone macromonomer obtained in Step 3 and an active diluent to a mixing tank, adding a crosslinking agent under mechanical stirring, adding polyether-modified polydimethylsiloxane, stirring for 30 minutes, degassing under 500Pa vacuum for 2 hours, and then allowing it to stand and mature in a dark room; Step 6: Printing the isolator using a multi-cartridge DLP photopolymerization 3D printer. This invention fundamentally reduces interference with the observation of the pathological process of bone nonunion and achieves high-fidelity modeling and construction.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to an isolator for establishing an animal model of bone nonunion and its preparation method. Background Technology

[0002] Nonunion is a serious complication of fracture healing failure, characterized by the lack of continuous callus formation at the fracture ends and closure of the medullary canal. To further investigate its pathological mechanisms and screen for effective treatments, it is crucial to construct animal models that highly mimic the human pathological state.

[0003] Currently, the common method for establishing animal models of nonunion is to combine "critical-size bone defects" with "physical isolation." The core idea of ​​this method is to create a sufficiently large bone defect and implant an isolation material to prevent osteoblasts and osteogenic factors from surrounding soft tissues from entering the defect area, thereby blocking natural healing. However, existing isolation materials and designs have significant drawbacks:

[0004] Poor morphological matching: Commercial tubing or sheeting cannot perfectly fit the anatomical shape of the radius, and gaps are easily present between it and the bone surface, leading to the invasion of fibrous connective tissue or callus, which reduces the success rate of the model.

[0005] Insecure fixation: Mismatched shapes can easily lead to displacement of the isolation device during or after surgery, resulting in loss of isolation effect;

[0006] Insufficient biocompatibility of materials leads to persistent inflammatory interference: Traditionally widely used rigid materials such as polymethyl methacrylate (PMMA) will trigger a typical foreign body reaction after implantation. This process is accompanied by continuous macrophage activation, foreign body giant cell formation, and the release of pro-inflammatory factors (such as TNF-α and IL-1β), resulting in chronic inflammation and the formation of thick fibrous encapsulation. This inflammatory microenvironment caused by the material itself will directly inhibit osteoblast activity, confusing the true cause of bone nonunion—whether it is caused by mechanical isolation or by chronic inflammation caused by the material. This reduces the signal-to-noise ratio of experimental observations and casts doubt on the reliability of the conclusions.

[0007] Mechanical property mismatch causes additional physical stimulation: The Young's modulus (hardness) of existing materials (such as PMMA and titanium alloys) is much higher than that of surrounding soft tissues (such as muscles and fascia), resulting in a serious mechanical mismatch. This "hard material-soft tissue" interface will produce local stress concentration, continuously compress the surrounding tissues, affect local microcirculation, and may abnormally activate immune cells through mechanical signaling pathways. This continuous physical stimulation itself is an important factor in inducing fibrosis and chronic inflammation, interfering with the normal healing environment.

[0008] Low standardization and repeatability: Manual cutting and shaping make it difficult to ensure consistency of each model, affecting the reliability and comparability of experimental results.

[0009] In summary, the existence of an isolator that can highly match the morphology of bone defects, achieve firm fixation, and whose material itself has excellent biocompatibility to avoid chronic inflammation, while also possessing mechanical properties that match soft tissue to eliminate physical stimulation, has hindered the construction of high-quality, standardized, and reproducible animal models of bone nonunion. Summary of the Invention

[0010] The purpose of this invention is to address the problems and shortcomings described in the background art by providing an isolator for establishing an animal model of bone nonunion and its preparation method.

[0011] A method for preparing an isolator for establishing an animal model of bone nonunion includes the following steps:

[0012] Step 1: Octamethylcyclotetrasiloxane, 1,3,5,7-tetramethylcyclotetrasiloxane, and concentrated sulfuric acid are added to a three-necked flask for reaction, so that octamethylcyclotetrasiloxane and 1,3,5,7-tetramethylcyclotetrasiloxane undergo ring-opening copolymerization to obtain a crude prepolymer. After standing and separating into layers, the upper organic phase is washed with deionized water and then distilled under reduced pressure at 80°C / 10mmHg to remove unreacted monomers and low-boiling substances, to obtain a hydrogen-containing silicone oil prepolymer with a certain proportion of Si-H bonds in the side chain.

[0013] Step 2: Dissolve the hydrogen-containing silicone oil prepolymer from Step 1 in anhydrous toluene, and react it with excess allyl glycidyl ether in the presence of a platinum catalyst to undergo a hydrosilylation reaction. After the reaction is complete, purify the product by vacuum distillation to obtain a silica macromonomer with epoxy groups on the side chain.

[0014] Step 3: Dissolve dexamethasone and the epoxy-containing silica macromonomer obtained in Step 2 in dichloromethane to obtain mixture A. Cool mixture A in an ice-salt bath to -5°C to 0°C and purge with nitrogen. Add boron trifluoride diethyl ether to dichloromethane and mix. When mixture A gradually changes from colorless to pale yellow, continue the reaction at 0°C for 4 hours. Remove the ice-salt bath and allow the reaction to naturally warm to room temperature. After reacting at room temperature for a period of time, separate, extract, purify and dry to obtain the anti-inflammatory functionalized silica macromonomer.

[0015] Step 4: Reconstruct the skeletal model in 3D and create an isolator model for the defect area;

[0016] Step 5: Add 60wt% of the anti-inflammatory functionalized silicone macromonomer and 25wt% of the active diluent obtained in Step 3 to the mixing tank, add 10wt% of the crosslinking agent under mechanical stirring, and continue stirring for 1 hour. Add 4wt% of the photoinitiator in 3 portions, with an interval of 10 minutes between each addition. Add 1wt% of the polyether-modified polydimethylsiloxane, stir for 30 minutes, and degas under a vacuum of 500Pa for 2 hours. After degassing, let it stand and mature in a dark room at a temperature of 25±2°C for 2 hours.

[0017] Step Six: Print the isolator using a multi-cartridge DLP photopolymerization 3D printer.

[0018] Preferably, the reaction conditions in step one are as follows: heat the mixture to 60°C, stir and premix for 15 minutes, then heat the reaction system to 80-85°C and continue stirring for 4-6 hours.

[0019] Preferably, the reaction conditions in step two are as follows: the reaction system is slowly heated to 80°C at a rate of 1°C / min under nitrogen protection and the reaction is continued for 6-8 hours.

[0020] Preferably, in step six: the printing parameters are: layer thickness 50μm, bottom layer exposure time 35s, normal layer exposure time 10s, lifting height 8mm, lifting speed 2mm / s, return speed 4mm / s, and waiting time 3s.

[0021] Preferably, in step six, the multi-cartridge DLP photopolymerization 3D printer is divided into cartridge A and cartridge B. Cartridge A is filled with anti-inflammatory functionalized silicone macromolecular monomer slurry, and cartridge B is filled with commercial 60A hardness silicone resin material. Cartridge A is used when printing the inner layer of the isolator, and the printer automatically switches to cartridge B when printing the outer layer of the isolator. The printing is performed layer by layer. After printing is completed, the isolator is cleaned with isopropyl alcohol and then a second UV curing is performed.

[0022] Preferably, in step four, the inner layer of the isolator is designed as a porous structure with a pore size of 10μm, and the outer layer of the isolator is integrally formed on the outer wall of the inner layer of the isolator, with the upper and lower ends of the outer layer of the isolator extending to the outer side of the inner layer of the isolator.

[0023] Preferably, the outer wall of the isolator has multiple isolator grooves that guide fibroblasts to arrange themselves in an orderly manner along a specific direction.

[0024] An isolator for establishing an animal model of bone nonunion is prepared by a method for preparing an isolator for establishing an animal model of bone nonunion.

[0025] The beneficial effects of this invention are:

[0026] This invention fundamentally reduces interference with the observation of the pathological process of bone nonunion, achieves high-fidelity modeling, and effectively inhibits foreign body reaction and chronic inflammation around the implant through active anti-inflammatory / anti-fibrotic functional modification of the material (covalent grafting of dexamethasone anti-inflammatory molecules), avoiding osteogenic inhibition caused by the material itself. The biomimetic mechanical design with a Shore A hardness of 30A makes the isolator match the mechanical properties of the surrounding soft tissue, reducing stress concentration, tissue compression and abnormal mechanical stimulation caused by mechanical mismatch, and eliminating another important inflammatory factor from a physical level. The synergistic effect of the above-mentioned "chemical inertness" and "mechanical biomimetic" greatly improves the signal-to-noise ratio and reliability of experimental data.

[0027] The 3D printing technology based on medical imaging in this invention ensures that the isolator is perfectly matched with the skeletal anatomy of each experimental animal, achieving precision and personalization of physical isolation and avoiding model failure due to morphological mismatch. The robust personalized fixation design (such as multi-material printed suture wings) ensures the stability of the isolator throughout the experimental cycle, preventing displacement or detachment, thereby ensuring the consistency of the construction conditions for each model.

[0028] This invention provides a highly standardized digital manufacturing process from digital model to physical entity, avoiding individual differences and uncertainties caused by manual production. The isolator is made of soft material, which is easy to implant and suture tissue, reducing the difficulty of surgery. It also has strong scalability and provides a multifunctional platform for bone healing research. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the preparation process of the hydrogen-containing silicone oil prepolymer with side chains in this invention.

[0030] Figure 2 This is a flowchart illustrating the preparation process of silica macromonomers with epoxy groups on the side chains in this invention.

[0031] Figure 3 This is a flowchart illustrating the preparation process of the anti-inflammatory functionalized silicone macromonomer in this invention.

[0032] Figure 4 This is a structural diagram of the isolator of the present invention;

[0033] Figure 5 A schematic diagram of the isolator of the present invention implanted at the radial bone defect site in a rabbit;

[0034] Figure 6 Micro-CT 3D reconstruction image of a conventional critical bone defect model 12 weeks post-operation.

[0035] Figure 7 This is a Micro-CT three-dimensional reconstruction image of the material of this invention 12 weeks after surgery;

[0036] Figure 8 X-ray image 12 weeks after traditional borderline bone defect modeling method;

[0037] Figure 9 X-ray image of the material of this invention 12 weeks after surgery.

[0038] In the picture:

[0039] 1: Octamethylcyclotetrasiloxane; 2: 1,3,5,7-Tetramethylcyclotetrasiloxane; 3: Hydrogen-containing silicone oil prepolymer; 4: Allyl glycidyl ether; 5: Silica macromonomer with epoxy groups on the side chain; 6: Dexamethasone; 7: Anti-inflammatory functionalized silica macromonomer; 81: Inner layer of isolator; 82: Outer layer of isolator. Detailed Implementation

[0040] To better illustrate the preparation process involved in this invention and its advantages over the prior art, further explanation will be provided with reference to the accompanying drawings.

[0041] As shown in the attached figure, a method for preparing an isolator for establishing an animal model of bone nonunion includes the following steps:

[0042] Step 1: Add 100g of octamethylcyclotetrasiloxane 1, 5.26g of 1,3,5,7-tetramethylcyclotetrasiloxane 2, and 1.05g of 98% concentrated sulfuric acid as a catalyst to a dry three-necked flask. Slowly heat to 60°C and stir for 15 minutes to premix. Then slowly heat the reaction system to 80-85°C and stir continuously for 4-6 hours to allow octamethylcyclotetrasiloxane 1 and 1,3,5,7-tetramethylcyclotetrasiloxane 2 to undergo ring-opening copolymerization to obtain a crude prepolymer. Finally, allow it to stand and separate into layers. Separate the lower aqueous phase and wash the upper organic phase three times with 100mL of deionized water each time. Distill under reduced pressure for 1 hour at 80°C / 10mmHg to remove unreacted monomers and low-boiling substances to obtain hydrogen-containing silicone oil prepolymer 3 with a certain proportion of Si-H bonds (5mol%) in the side chain.

[0043] Step 2: Dissolve the hydrogen-containing silicone oil prepolymer 3 from Step 1 in anhydrous toluene, and react it with excess allyl glycidyl ether 4 under a platinum catalyst and nitrogen protection at a rate of 1°C / min for 6-8 hours to carry out a hydrosilylation reaction. After the reaction is completed, purify by vacuum distillation to obtain the silica macromonomer 5 with epoxy groups on the side chain.

[0044] Step 3: Dissolve 131.8g of dexamethasone 6 and 100g of the epoxy-containing silica macromonomer 5 obtained in Step 2 in 300ml of dichloromethane to obtain mixture A. Cool mixture A in an ice-salt bath to -5°C to 0°C, and purge with nitrogen to remove oxygen and moisture. Mix 200ml of boron trifluoride ether with 200ml of dichloromethane as a catalyst and slowly add it to mixture A using a constant pressure dropping funnel. Strictly control the dropping time to 2 hours and keep the temperature below 0°C. During the dropping process, mixture A gradually changes from colorless to pale yellow. Continue the reaction at 0°C for 4 hours, then remove the ice-salt bath and allow the reaction to naturally warm to room temperature (25°C). Continue the reaction at room temperature for 16-20 hours. Finally, after separation, extraction, purification, and drying, obtain the pale yellow viscous solid product, anti-inflammatory functionalized silica macromonomer 7.

[0045] Step 4: Obtain anatomical data: Perform a CT scan of the radius of the New Zealand white rabbit, reconstruct a 3D skeletal model, and create an isolator model for the defect site;

[0046] Step 5: Printing paste preparation: Add 60wt% of the anti-inflammatory functionalized silicone macromonomer 7 and 25wt% of the reactive diluent 1,6-hexanediol diacrylate obtained in Step 3 to a 100mL mixing tank. Slowly add 10wt% of the crosslinking agent trimethylolpropane triacrylate while mechanically stirring (200rpm). Continue stirring for 1 hour until completely homogeneous and transparent. Add 4wt% of the photoinitiator 2,4,6-trimethylbenzoyl-diphenylphosphine oxide in 3 portions, with an interval of 10 minutes between each addition. Add 1wt% of the polyether-modified polydimethylsiloxane leveling agent and stir for 30 minutes. Degas under 500Pa vacuum for 2 hours. After degassing, let it stand and mature in a dark room at 25±2°C for 2 hours.

[0047] Step 6: Print the isolator using a multi-cartridge DLP photopolymerization 3D printer. The printing parameters are: layer thickness 50μm, bottom layer exposure time 35s, normal layer exposure time 10s, lift height 8mm, lift speed 2mm / s, return speed 4mm / s, and waiting time 3s.

[0048] Preferably, in step four, the isolator is modeled using C4D software. The inner layer 81 of the isolator is designed as a porous structure with a pore size of 10μm, generated using the random domain command in C4D software. The pore size is achieved by adjusting the scaling ratio of the random domain. The outer layer 82 of the isolator is integrally formed on the outer wall of the inner layer 81, and extends 5mm to the outer side of the inner layer 81 at both ends. The wall thickness of the outer layer 82 is 1mm. Multiple isolator grooves with a width of 10μm and a depth of 5μm are distributed on the outer wall of the outer layer 82. The isolator grooves can guide fibroblasts to arrange themselves in an orderly manner along a specific direction, avoiding the formation of disordered and contractile fibrous capsules, thereby reducing mechanical stimulation and pressure on the defect area, and reducing the occurrence of inflammation.

[0049] Preferably, in step six, the multi-cartridge DLP photopolymerization 3D printer is divided into cartridge A and cartridge B. Cartridge A is filled with the anti-inflammatory functionalized silicone macromolecular monomer slurry prepared in step five, and cartridge B is filled with commercial 60A hardness silicone resin material. Cartridge A is used when printing the inner layer 81 of the isolator, and the printer automatically switches to cartridge B when printing the outer layer 82 of the isolator. The printing is performed layer by layer and formed in one piece. After printing, the isolator is cleaned with isopropyl alcohol and then a second UV curing is performed to ensure that the material is completely cross-linked.

[0050] The principle of this invention:

[0051] In step two, by utilizing the chemoselectivity of hydrosilylation, epoxy groups are introduced into the side chain of polysiloxane. This functional group acts as a "chemical handle" and can further react with the hydroxyl groups (-OH) on the drug molecule dexamethasone. In addition, the inherent properties of silica gel are preserved, and the main chain remains a flexible Si-O-Si structure, ensuring the basic elasticity and hardness (30A) of the material.

[0052] In step three, the C21 hydroxyl group of dexamethasone is highly reactive. Under the action of boron fluoride ether catalysis, the hydroxyl group (nucleophile) of dexamethasone attacks the carbon atom (electrophilic center) on the epoxy ring, causing the epoxy ring to open and forming a covalent ether bond, thereby improving the anti-inflammatory properties of the material.

[0053] The inner layer 81 of the isolator uses a designed anti-inflammatory functionalized silicone macromolecular monomer 7 with a Shore A hardness of 30A, which not only effectively inhibits foreign body reaction and chronic inflammation around the implant and avoids osteogenic inhibition caused by the material itself, but also matches the mechanical properties of the isolator with those of the surrounding soft tissue, eliminating stress concentration, tissue compression and abnormal mechanical stimulation caused by mechanical mismatch, thus eliminating another important inflammatory factor from a physical perspective. The outer layer 82 of the isolator uses commercially available silicone resin material with a hardness of 60A, which ensures effective support and fixation of the isolator, thereby reducing the occurrence of loosening.

[0054] The inner layer 81 of the isolator has a porous structure with a pore size of 10μm, which is sufficient to effectively block the migration of osteoblasts and blood vessels, while facilitating the exchange of nutrients and metabolic waste, thus reducing necrosis.

Claims

1. A method for preparing an isolator for establishing a nonunion animal model, characterized in that: Comprising the following steps: Step one: octamethylcyclotetrasiloxane, 1,3,5,7-tetramethylcyclotetrasiloxane, concentrated sulfuric acid are added to a three-necked flask for reaction, octamethylcyclotetrasiloxane and 1,3,5,7-tetramethylcyclotetrasiloxane are subjected to ring-opening copolymerization to obtain a crude prepolymer, which is allowed to stand and separate into layers, the upper organic phase is washed with deionized water, then distilled under reduced pressure at 80°C / 10mmHg to remove unreacted monomers and low-boiling substances, thereby obtaining a hydrogen-containing silicone oil prepolymer containing a certain proportion of Si-H bonds in the side chain; Step two: the hydrogen-containing silicone oil prepolymer in step one is dissolved in anhydrous toluene, and subjected to a hydrosilylation reaction with excess allyl glycidyl ether in the presence of a platinum catalyst, after the reaction is completed, the product is purified by distillation under reduced pressure to obtain a silica gel macromonomer with an epoxy group in the side chain; Step three: dexamethasone and the silica gel macromonomer with an epoxy group in the side chain obtained in step two are dissolved in dichloromethane to obtain a mixed solution A, the mixed solution A is cooled to -5°C to 0°C by placing the container in an ice-salt bath, nitrogen is introduced for protection, a mixture of boron trifluoride etherate and dichloromethane is added to the mixed solution A, when the mixed solution A gradually changes from colorless to light yellow, the reaction is continued at 0°C for 4 hours, the ice-salt bath is removed, and the reaction is allowed to naturally warm up to room temperature, after a certain period of time at room temperature, the product is obtained by separation and extraction, and then purified and dried to obtain an anti-inflammatory functional silica gel macromonomer; Step four: three-dimensional reconstruction of a bone model, creation of a defect isolator model; Step five: 60wt% of the anti-inflammatory functional silica gel macromonomer obtained in step three and 25wt% of an active diluent are added to a batching tank, 10wt% of a crosslinking agent is added under mechanical stirring, the stirring is continued for 1 hour, 4wt% of a photoinitiator is added in three portions with an interval of 10 minutes each time, 1wt% of a polyether-modified polydimethylsiloxane is added, and the stirring is continued for 30 minutes, then the product is deaerated under a vacuum of 500Pa for 2 hours, and then allowed to stand and mature in a dark room at a temperature of 25±2°C for 2 hours; Step six: an isolator is printed by using a multi-cartridge DLP photocuring 3D printer.

2. The preparation method of the isolator for establishing an animal model of bone nonunion according to claim 1, characterized in that: The reaction conditions in step one are as follows: the system is warmed up to 60°C, pre-mixed for 15 minutes, then the reaction system is warmed up to 80-85°C, and the stirring is continued for 4-6 hours.

3. The method of claim 1, wherein the method further comprises: providing a spacer having a first end and a second end; and providing a spacer having a first end and a second end. The reaction conditions in step two are as follows: the reaction system is slowly warmed up to 80°C at a rate of 1°C / min under nitrogen protection, and the reaction is continued for 6-8 hours.

4. The method of claim 1, wherein the method further comprises: preparing the isolator for establishing the nonunion animal model. In step six, the printing parameters are as follows: layer thickness 50μm, bottom layer exposure time 35s, normal layer exposure time 10s, lifting height 8mm, lifting speed 2mm / s, return speed 4mm / s, and waiting time 3s.

5. The method of claim 1, wherein the method further comprises: 5 sterilizing the isolator and the bone defect model; and 0 placing the bone defect model into the isolator. 1 In step six, the multi-cartridge DLP photocuring 3D printer is divided into cartridge A and cartridge B, cartridge A is filled with the anti-inflammatory functional silica gel macromonomer slurry, and cartridge B is filled with a commercial 60A hardness silicone resin material, cartridge A is used when printing the inner layer of the isolator, and cartridge B is automatically switched to when printing the outer layer area of the isolator, the printing is layer by layer, after the printing is completed, the isolator is cleaned with isopropyl alcohol, and then subjected to secondary ultraviolet curing. ​ 6. The method of claim 1, wherein the method further comprises: preparing the isolator. In step four, the inner layer of the separator is designed as a porous structure with a pore size of 10 μm, and the outer layer of the separator is integrally formed on the outer wall of the inner layer and extends to the outside of the inner layer at the upper and lower ends of the outer layer.

7. The method according to claim 6, wherein the method further comprises the step of: 7.

1. preparing the isolator for establishing the animal model of bone nonunion. The outer wall of the outer layer of the separator is distributed with a plurality of separator grooves for guiding fibroblasts to arrange in a specific direction in order.

8. An isolator for establishing a nonunion animal model, comprising: The preparation method of the separator for establishing an animal model of bone nonunion according to any one of claims 1 to 5.

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