Bone-like adhesive as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a callus-imitating bone adhesive as well as a preparation method and application thereof. And carrying out second polymerization reaction on hydrogen atoms in the type I collagenous fibers and isocyanate groups in the callus-imitating polyurethane bone adhesive prepolymer to obtain the callus-imitating bone adhesive, wherein the callus-imitating polyurethane bone adhesive prepolymer is obtained by carrying out a polyaddition reaction on hydroxyl groups in nano-hydroxyapatite and isocyanate groups in a hexamethylene diisocyanate tripolymer, and the biocompatibility of the material is obviously improved through a novel biological soft segment, namely the I-type collagenous fiber. Meanwhile, in the polymerization process of the material, thermal infrared characterization shows that the material does not generate heat higher than the temperature of a human body and does not cause thermal damage to tissues.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a callus-imitation bone adhesive and a preparation method and application thereof. Background Art

[0002] The traditional treatment for fractures is internal fixation, which primarily involves applying pressure to stabilize the fracture ends using intramedullary nails or metal plates. While these traditional fixation methods can achieve good results, they still have the following drawbacks: 1. Limited use environments: they cannot be used for severe comminuted or complex fractures; 2. Damage to remaining bone tissue: the use of screws and intramedullary nails can cause stress in the fracture, hindering the growth of remaining tissue; and 3. The drawbacks of metal implants: metal easily wears and breaks, requiring secondary surgical removal and being non-biodegradable. Therefore, a bone adhesive that can be used for a variety of fracture types, eliminates the need for secondary removal, and promotes bone growth is being developed.

[0003] However, traditional bone adhesives, such as PMMA adhesives, release a large amount of heat during application, causing tissue burns. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a callus-imitation bone adhesive and a preparation method and application thereof.

[0005] A callus-mimicking bone adhesive is obtained by a second polymerization reaction between hydrogen atoms in type I collagen fibers and isocyanate groups in a callus-mimicking polyurethane bone adhesive prepolymer; The callus-mimicking polyurethane bone adhesive prepolymer is obtained by a polyaddition reaction between the hydroxyl groups in nano-hydroxyapatite and the isocyanate groups in hexamethylene diisocyanate trimer.

[0006] A method for preparing the simulated callus bone adhesive material comprises the following steps: Hexamethylene diisocyanate isocyanurate trimer and nano-hydroxyapatite are uniformly mixed in a mass ratio of 1 to 3:1, and then polymerized in an oxygen-free environment at 60° C. to 80° C. to obtain a callus-mimicking polyurethane bone adhesive prepolymer. The callus-mimicking polyurethane bone adhesive prepolymer and type I collagen fibers were mixed at a mass ratio of 1 to 4:100.

[0007] Preferably, the reaction is carried out in an oxygen-free environment at 60° C. to 80° C. for 2 h to 5 h.

[0008] Preferably, acetyl chloride and dibutyltin dilaurate are used as catalysts during the first polymerization reaction.

[0009] Preferably, the volume ratio of acetyl chloride to dibutyltin dilaurate is 0.75-1.25:1.

[0010] Preferably, the mass ratio of the total volume of the acetyl chloride and dibutyltin dilaurate to the hexamethylene diisocyanate isocyanurate trimer is 3 μL-6 μL:0.5 g.

[0011] Application of the callus-like bone adhesive in fracture fixation.

[0012] Application of the callus-mimicking bone adhesive in the preparation of bone repair materials.

[0013] Preferably, the repair is at least one of osteogenesis and bone remodeling at the fracture site.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The bone adhesive of this invention primarily reduces cytotoxicity through the use of a novel bio-soft segment of polyurethane, type I collagen fibers. Experiments have shown that the material does not reach temperatures above body temperature during polymerization. The use of this novel bio-soft segment significantly improves the biocompatibility of the material. Furthermore, thermal infrared characterization during the polymerization process reveals that the material does not generate heat above human body temperature, preventing thermal damage to tissues.

[0015] Enhanced biosafety: In vitro cell experiments (osteoblasts), in vivo subcutaneous implantation, and hemolysis experiments have demonstrated that the bone adhesive material simulates the components of natural callus and has good cell and blood compatibility.

[0016] Fracture fragment fixation: Animal models show that bone adhesive can effectively fix fracture fragments and prevent displacement compared with the blank control group.

[0017] Promote rapid osteogenesis in the fracture area: The results of in vitro cell experiments (osteoblasts) and animal models showed that bone adhesives can promote the maturation of osteoblasts and the deposition of calcium salts. Compared with the blank control group, the bone density and bone volume in the fracture area of ​​the group using bone adhesives were significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Showing the successful synthesis of CLA, where A is the Fourier transform infrared spectroscopy result and B is the XPS result.

[0019] Figure 2 is the morphology and elemental energy spectrum analysis of CLA, where A is the uncured morphology, B is the thickness of the cured morphology, C is the diameter of the cured morphology, and C is the elemental energy spectrum analysis.

[0020] Figure 3 This is an electron microscope image of CLA.

[0021] Figure 4 is the surface pore diameter of CLA.

[0022] Figure 5 is the pore area of ​​CLA, where A is CLA-1, B is CLA-2, and C is CLA-4.

[0023] Figure 6 Some basic properties of CLA, where A is the water contact angle, A(1) is the representative sample of each group of CLA, A(2) is the histogram analysis of the water contact angle data of each group of CLA, B is the compression modulus, B(1) is the compression modulus result, B(2) is the compression modulus histogram, C is the tensile modulus, C(1) is the tensile modulus result, and C(2) is the tensile modulus histogram.

[0024] Figure 7 These are the biosafety results, where A is the in vitro cell test and B is the hemolysis test.

[0025] Figure 8 This is an in vivo subcutaneous implantation experiment, where A is the white blood cell count, B is the lymphocyte percentage, C is the red blood cell count, D is the hemoglobin concentration, E is the platelet count, and F is the neutrophil percentage.

[0026] Figure 9 Biosafety results, where A is the result of routine blood test and B is the result of tissue section.

[0027] Figure 10 The bonding effect of CLA on cancellous bone and compact bone in dry, wet and blood environments, where a is the schematic diagram of CLA use, and the bonding force diagram (bonding area is 4mm 2 When the density of CLA is less than 5 kg, the compact bone can withstand a weight of 5 kg). b is the shear bonding strength of CLA in a dry environment, c is the shear bonding strength of CLA in a wet environment, d is the shear bonding strength of CLA in a blood environment, e is the tensile bonding strength of CLA in a dry environment, f is the tensile bonding strength of CLA in a wet environment, and g is the tensile bonding strength of CLA in a blood environment.

[0028] Figure 11 Schematic diagram of the construction of the rat skull fracture model and the use of CLA.

[0029] Figure 12 Figure 3 is the in vitro and in vivo osteogenic effect of CLA, where A is the alizarin red staining of osteoblasts induced for 21 days in vitro (upper) and alkaline phosphatase staining of osteoblasts induced for 14 days in vitro (lower), B is the CT examination result after CLA was used to treat rat fractures, and C is the tissue section result after CLA was used to treat rat fractures.

[0030] Figure 13 Thermal infrared characterization of CLA. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0032] 1. Material System Design: ① Construction of nanohydroxyapatite (nHAP)-hexamethylene diisocyanate trimer (Tri-HDI): A callus-mimicking polyurethane bone adhesive prepolymer (Pre-CLA) was constructed through the polymerization reaction of the hydroxyl groups in nHAP with the isocyanate groups in Tri-HDI. The mechanical strength of Pre-CLA was controlled by adjusting the nHAP content, and the optimal construction ratio was selected. ② Construction of callus-mimicking polyurethane bone adhesive (CLA): The callus-mimicking polyurethane bone adhesive CLA was formed through the further polymerization reaction of active hydrogen atoms such as hydroxyl and amino groups in type I collagen fibers (Col I) with the remaining isocyanate groups in Pre-CLA. By adjusting the Col I content, a control system for CLA was established, achieving regulation of CLA's pore size, hydrophilicity, and mechanical properties.

[0033] 2. Preparation method: ① Synthesis of callus-mimicking polyurethane bone adhesive prepolymer (Pre-CLA): First, hexamethylene diisocyanate isocyanurate trimer (HDI) and nanohydroxyapatite (nHAP) were uniformly mixed in a ratio of (1-3:1) by polymerization reaction, and then reacted in an oxygen-free environment at 75°C to obtain Pre-CLA; ② Construction of callus-mimicking polyurethane bone adhesive (CLA): Pre-CLA and Col I were uniformly mixed (1-4:100) at 20°C-25°C, and the influence of material composition, concentration ratio and synthesis conditions on the porosity, bonding properties, mechanical strength and other properties of the prepared polyurethane bone adhesive was explored and clarified, and the mechanism of action was clarified, so as to screen out callus-mimicking polyurethane bone adhesive with excellent comprehensive performance, providing an experimental basis for subsequent related research.

[0034] (1) Titration of isocyanate group (-NCO): The NCO content in Tri-HDI is determined by toluene-di-n-butylamine titration. Take 0.1g Tri-HDI in a beaker, weigh it and record it as M (g), with the data accurate to three decimal places. Add 2mL of toluene to the beaker and continue stirring until it is completely melted. Add 3mL of 0.1M di-n-butylamine-toluene solution. After the reaction is complete, add 0.1% bromocresol green indicator to make the liquid in the beaker dark blue. Start stirring and titrating with 0.1M hydrochloric acid solution. When the liquid in the beaker gradually changes from dark blue to cyan and finally completely changes color to golden yellow and the color does not change within 1 minute, the titration is completed. Record the volume of hydrochloric acid solution used for the titration as V2 (unit: mL). In addition, a group without Tri-HDI was carried out as a blank group. The volume of hydrochloric acid solution consumed in the blank group was recorded as V1 (unit: mL). The calculation formula for the NCO group content in the Tri-HDI sample is: NCO (%) = 0.1 (V1-V2) × 0.042 / M × 100%. The experiment was repeated many times and the average value was calculated when the error was small.

[0035] (2) Titration of amino group (-NH2): The amino group content in Col I was determined by sodium nitrite titration. Weigh 0.0025g of Col I, add 1mL of 6M hydrochloric acid solution, heat at 60℃ and stir continuously for 3min until the collagen fibers are completely dissolved. Then add 200mL of pure water, 1mL of analytical grade glacial acetic acid and 0.5g of analytical grade potassium bromide, place the beaker in ice water and stir, control the temperature at 0-5℃, and start adding 0.1M sodium nitrite standard solution. Sodium nitrite reacts with amino compounds under hydrochloric acid and low temperature conditions to undergo diazotization. Starch potassium iodide test paper is used to indicate the titration end point when a certain amount is titrated. A slightly excess of NaNO2 will oxidize KI in an acidic environment, precipitating I2 and turning the test paper blue. The volume of sodium nitrite standard solution consumed is recorded as V, in mL. Therefore, the amino group content per 1g of collagen fiber is:

[0036] NH2(mol / g)=0.1×V / (1000×0.0025) Repeat the test several times until the error is small and calculate the average value.

[0037] Based on the total amount of amino groups in Col I and the content of isocyanate groups in Pre-CLA determined in parts (1) and (2), the mass required for the two to be fully equivalent was calculated and used as the basis for adjusting the Col I content.

[0038] Example 1 The preparation method of the imitation callus bone adhesive material comprises the following steps: Synthesis of Pre-CLA 0.5 g of Tri-HDI and 0.33 g of nHAP were evenly mixed, 2.5 μL of acetyl chloride and 2.5 μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, the mixture was reacted at 75°C for 3 min under nitrogen to obtain a viscous prepolymer, which was recorded as Pre-CLA.

[0039] Synthesis of CLA 0.005 g of Col I was weighed and added to the prepared Pre-CLA at room temperature and mixed evenly to obtain a bone adhesive in which the mass of collagen accounted for 1% of Tri-HDI. The HDI was named CLA-1.

[0040] Example 2 The preparation method of the imitation callus bone adhesive material comprises the following steps: Synthesis of Pre-CLA 0.5 g of Tri-HDI and 0.33 g of nHAP were evenly mixed, 2.5 μL of acetyl chloride and 2.5 μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, the mixture was reacted at 75°C for 3 min under nitrogen to obtain a viscous prepolymer, which was recorded as Pre-CLA.

[0041] Synthesis of CLA 0.01 g of Col I was weighed and added to the prepared Pre-CLA at room temperature and mixed evenly to obtain a bone adhesive in which the mass of collagen accounted for 2% of Tri-HDI, which was named CLA-2.

[0042] Example 3 The preparation method of the imitation callus bone adhesive material comprises the following steps: Synthesis of Pre-CLA 0.5 g of Tri-HDI and 0.33 g of nHAP were evenly mixed, 2.5 μL of acetyl chloride and 2.5 μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, the mixture was reacted at 75°C for 3 min under nitrogen to obtain a viscous prepolymer, which was recorded as Pre-CLA.

[0043] Synthesis of CLA 0.02 g of Col I was weighed and added to the prepared Pre-CLA at room temperature and mixed evenly to obtain a bone adhesive in which the mass of collagen accounted for 4% of Tri-HDI, which was named CLA-4.

[0044] CLA-1, CLA-2, and CLA-4 were characterized and tested for performance, with the following results: Figure 1 The successful synthesis of CLA was demonstrated.

[0045] Figure 2The uncured and cured morphologies of CLA are shown. The element content and distribution on the surface of CLA were scanned using SEM-EDS technology, and it can be seen that the elements on the surface of CLA are evenly distributed.

[0046] like Figure 3 The CLA shown contains many pores, whose diameters become larger and the pore areas become larger as the amount of Col I increases, e.g. Figure 4 and Figure 5 Pores are a necessary condition for cells to enter the material from the bone surface. Large pores can promote the formation of new bone and promote rapid healing of fractures. The CLA-4 group has better ductility than the CLA-1 group. Figure 6 As shown, it helps CLA resist the stress in the body and protects the stable healing of the fracture repair site.

[0047] Figure 7-9 It shows that CLA has excellent cell compatibility and blood compatibility. Subcutaneous implantation sections show that surrounding tissues can enter the interior of CLA through the pores.

[0048] The main mechanism by which CLA exerts its bonding effect is the chemical bonding between the isocyanate group and the amine group on the bone surface. At the same time, CLA also produces partial mechanical interlocking with the cancellous bone to assist in bonding and retention. The CLA-1 group with the lowest Col I content showed the highest compression modulus (60.33±10.09) MPa, the greatest tensile strength (21.37±2.19) MPa, and the best bonding performance (10.50±1.41) MPa, which may be due to its higher cohesive strength and tighter molecular structure. CLA showed excellent bonding strength in dry, wet and blood environments, with a bonding strength of 4mm. 2 The area can support 5kg weight, such as Figure 10 shown.

[0049] During the polymerization process of the material, thermal infrared characterization shows that the material does not generate heat higher than the human body temperature and will not cause thermal damage to the tissue, such as Figure 13 shown.

[0050] verify (1) Construction of skull fracture model: A skull ring fracture model was established using SD rats. After the rats were anesthetized, a blade was used to cut along the midline of the rat scalp, the skull periosteum was fully peeled off to expose the surgical area, and a 4 mm diameter trephine was used to cut out a complete circular bone block to form a fracture model. During the operation, the fracture fragment was kept moist to prevent tissue dehydration from affecting subsequent healing.

[0051] (2) In vivo osteogenesis verification: CLA-4 was used for in vivo osteogenesis verification. 20 μL of CLA-4 was evenly applied to the circular fracture fragment for one week to connect it with the surrounding bone tissue. In the control group, the bone fragments not coated with bone adhesive were directly returned to their original position without other treatment. Each group contained 10 rats. After surgery, the incisions were sutured with 5 / 0 absorbable sutures without additional immobilization measures. The rats were killed at 4 and 8 weeks after surgery, and the skull samples were removed and fixed with 4% paraformaldehyde for subsequent Micro-CT scanning and histological analysis to evaluate the fracture repair.

[0052] The results of osteogenic induction experiments were as follows Figure 11-12 The results showed that MC3T3 cells treated with CLA exhibited good osteogenesis and mineralization levels, especially the ARS staining results of the CLA-4 group were the most prominent. Animal experimental results also confirmed the osteogenesis-promoting effect of CLA. In the SD rat skull fracture model, the bone fragments of the control group showed obvious bone resorption and nonunion, while the CLA-4 group showed significant new bone formation at 8 weeks. HE staining showed that the fracture site healed well and some trabecular structures were compact, indicating that CLA can not only bond the fracture ends, but also promote osteogenesis and bone remodeling at the fracture site.

[0053] Natural callus is mainly composed of mineralized type I collagen fibers. The present invention simulates the components and functions of natural callus to connect the fracture ends.

[0054] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0055] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0056] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A callus-like bone adhesive, characterized in that: The callus-mimicking polyurethane bone adhesive is obtained by a second polymerization reaction between hydrogen atoms in type I collagen fibers and isocyanate groups in a callus-mimicking polyurethane bone adhesive prepolymer; The callus-mimicking polyurethane bone adhesive prepolymer is obtained by a polyaddition reaction between the hydroxyl groups in nano-hydroxyapatite and the isocyanate groups in hexamethylene diisocyanate trimer.

2. A method for preparing the simulated callus bone adhesive material according to claim 1, characterized in that: The following steps are involved: Hexamethylene diisocyanate isocyanurate trimer and nano-hydroxyapatite are uniformly mixed in a mass ratio of 1 to 3:1, and a first polymerization reaction is carried out in an oxygen-free environment at 60° C. to 80° C. to obtain a callus-mimicking polyurethane bone adhesive prepolymer; The second polymerization reaction is carried out by subjecting the callus-mimicking polyurethane bone adhesive prepolymer and type I collagen fibers to a mass ratio of 1 to 4:

100.

3. The preparation method according to claim 2, characterized in that React in an oxygen-free environment at 60℃~80℃ for 2h~5h.

4. The preparation method according to claim 2, characterized in that Acetyl chloride and dibutyltin dilaurate are used as catalysts when the first polymerization reaction occurs.

5. The preparation method according to claim 2, characterized in that The volume ratio of the acetyl chloride to dibutyltin dilaurate is 0.75-1.25:

1.

6. The preparation method according to claim 2, characterized in that The mass ratio of the total volume of the acetyl chloride and dibutyltin dilaurate to the hexamethylene diisocyanate isocyanurate trimer is 3 μL-6 μL:0.5 g.

7. Use of the callus-mimicking bone adhesive according to claim 1 in fracture fixation.

8. Use of the callus-mimicking bone adhesive according to claim 1 in the preparation of bone repair materials.

9. The use according to claim 8, characterized in that The repair is at least one of osteogenesis and bone remodeling at the fracture site.

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