A bone-like callus bone adhesive as well as a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a bone callus-like bone adhesive as well as a preparation method and application thereof. The bone callus-like bone adhesive is obtained by a second polymerization reaction between hydrogen atoms in type I collagen fibers and isocyanate groups in bone callus-like polyurethane bone adhesive prepolymer; the bone callus-like polyurethane bone adhesive prepolymer is obtained by a polyaddition reaction between hydroxyl groups in nano-hydroxyapatite and isocyanate groups in hexamethylene diisocyanate trimer; and the type I collagen fibers are a new type of biological soft segment, which significantly improves the biocompatibility of the material. Meanwhile, in the polymerization process of the material, it can be found through thermal infrared characterization that the material does not generate heat higher than the body temperature, and thus does not cause thermal damage to the tissue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a bone callus-like bone adhesive as well as a preparation method and application thereof. BACKGROUND

[0002] The traditional treatment method for fracture patients is internal fixation, which mainly fixes the fracture ends by applying pressure through intramedullary nails, metal plates and the like. Although the traditional fixation method can achieve good results, it still has the following defects: ① limited use environment: cannot be used for severe comminuted or complex fractures; ② damage to remaining bone tissue: the use of screws and intramedullary nails can cause bone fracture stress problems and affect the growth of remaining tissue; ③ disadvantages of metal implants: metal is prone to wear and breakage, requires secondary surgery for removal and cannot be biodegraded. Therefore, a bone adhesive that can be used for various types of fractures, does not need to be removed twice and promotes bone tissue growth is developed.

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

[0004] To solve the above technical problems, the present application provides a bone callus-like bone adhesive as well as a preparation method and application thereof.

[0005] A bone callus-like bone adhesive is obtained by a second polymerization reaction between hydrogen atoms in type I collagen fibers and isocyanate groups in a bone callus-like polyurethane bone adhesive prepolymer.

[0006] The bone callus-like polyurethane bone adhesive prepolymer is obtained by a polyaddition reaction between hydroxyl groups in nanometer hydroxyapatite and isocyanate groups in hexamethylene diisocyanate isocyanurate trimer.

[0007] A preparation method of the bone callus-like bone adhesive material comprises the following steps:

[0008] Hexamethylene diisocyanate isocyanurate trimer and nanometer hydroxyapatite are mixed uniformly at a mass ratio of 1-3:1, and a polyurethane bone adhesive prepolymer is obtained by a polymerization reaction under an anaerobic environment at 60-80℃.

[0009] The bone callus-like polyurethane bone adhesive prepolymer and type I collagen fibers are mixed uniformly at a mass ratio of 1-4:100.

[0010] Preferably, the reaction is carried out under an anaerobic environment at 60-80℃ for 2-5h.

[0011] Preferably, acetyl chloride and dibutyltin dilaurate are used as catalysts during the first polymerization reaction.

[0012] Preferably, the volume ratio of the acetyl chloride and dibutyltin dilaurate is 0.75-1.25:1.

[0013] Preferably, the mass ratio of the total volume of the acetyl chloride and dibutyltin dilaurate to the hexamethylene diisocyanate isocyanurate trimer is 3 muL-6 muL:0.5 g.

[0014] The application of the bone callus-like bone adhesive in fracture fixation.

[0015] The application of the bone callus-like bone adhesive in the preparation of bone repair materials.

[0016] Preferably, the repair is at least one of osteogenesis and bone remodeling of the fracture site.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The bone adhesive of the application mainly reduces cytotoxicity through the new polyurethane biological soft segment of type I collagen fibers. Experimental results show that the material does not appear to be higher than the body temperature during polymerization. In the application, the new biological soft segment of type I collagen fibers significantly improves the biocompatibility of the material. At the same time, during the polymerization of the material, through thermal infrared characterization, it can be found that the material does not generate heat higher than the body temperature of human beings, and will not cause thermal damage to the tissue.

[0019] Enhanced biological safety: in vitro cell experiments (osteoblasts), in vivo subcutaneous implantation, and hemolysis experiments prove that the bone adhesive material simulates the components of natural bone callus, has good cell compatibility and blood compatibility.

[0020] Fracture piece fixation: animal models show that, compared with the blank control group, the bone adhesive can effectively fix the fracture pieces and prevent displacement.

[0021] Promoting rapid osteogenesis in the fracture area: in vitro cell experiments (osteoblasts) and animal model results show that the bone adhesive can promote the maturation and calcium salt deposition of osteoblasts. Compared with the blank control group, the bone density and bone volume of the fracture area using the bone adhesive are significantly increased. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 CLA synthesis is shown, wherein A is the Fourier infrared spectrum result, and B is the XPS result.

[0023] Figure 2 CLA morphology and element spectrum analysis are shown, wherein 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 element spectrum analysis.

[0024] Figure 3TEM images of CLA.

[0025] Figure 4 Surface pore diameter of CLA.

[0026] Figure 5 Pore area of CLA, where A is CLA-1, B is CLA-2, and C is CLA-4.

[0027] Figure 6 Partial basic properties of CLA, where A is water contact angle, A(1) is representative sample images of each group of CLA, A(2) is histogram analysis of water contact angle data of each group of CLA, B is compression modulus, B(1) is compression modulus results, B(2) is compression modulus histogram, C is tensile modulus, C(1) is tensile modulus results, and C(2) is tensile modulus histogram.

[0028] Figure 7 Biological safety results, where A is in vitro cell experiment, and B is hemolysis experiment.

[0029] Figure 8 In vivo subcutaneous implantation experiment, where A is white blood cell count, B is lymphocyte percentage, C is red blood cell count, D is hemoglobin concentration, E is platelet count, and F is neutrophil percentage.

[0030] Figure 9 Biological safety results, where A is blood routine test results, and B is histological section results.

[0031] Figure 10 Adhesion effect of CLA on cancellous bone and compact bone in dry, wet, and blood environments, where a is a schematic diagram of CLA use, b is shear adhesion strength of CLA in dry environment, c is shear adhesion strength of CLA in wet environment, d is shear adhesion strength of CLA in blood environment, e is tensile adhesion strength of CLA in dry environment, f is tensile adhesion strength of CLA in wet environment, and g is tensile adhesion strength of CLA in blood environment. 2

[0032] Figure 11 Construction of rat skull fracture model and schematic diagram of CLA use.

[0033] Figure 12 In vitro and in vivo osteogenesis effect of CLA, where A is alizarin red staining of osteoblasts induced in vitro for 21 days (top) and alkaline phosphatase staining of osteoblasts induced for 14 days (bottom), B is CT detection results of CLA used for rat fracture treatment, and C is histological section results of CLA used for rat fracture treatment.​

[0034] Figure 13 Thermal infrared characterization of CLA. DETAILED DESCRIPTION

[0035] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0036] 1. Material system design: ① Construction of nano-hydroxyapatite (nHAP)-hexamethylene diisocyanate trimer (Tri-HDI): Using the polymerization reaction of hydroxyl groups in nHAP and isocyanate groups in Tri-HDI, a bone-like callus polyurethane bone adhesive pre-polymer (Pre-CLA) is constructed. By adjusting the content of nHAP, the mechanical strength of Pre-CLA is controlled, and the optimal construction ratio is selected; ② Construction of bone-like callus polyurethane bone adhesive (CLA): Through the further polymerization reaction of active hydrogen atoms such as hydroxyl and amino groups in type I collagen fiber (Col I) and the remaining isocyanate groups in Pre-CLA, a bone-like callus polyurethane bone adhesive CLA is formed. By regulating the content of Col I, a regulation system of CLA is constructed, realizing the regulation system of the pore size, hydrophilic performance, mechanical performance, etc. of CLA.

[0037] 2. Preparation method: ① Synthesis of bone-like callus polyurethane bone adhesive pre-polymer (Pre-CLA): First, hexamethylene diisocyanate isocyanurate trimer (HDI) and nano-hydroxyapatite (nHAP) are mixed uniformly according to the ratio (1-3:1), and then Pre-CLA is obtained by reaction in an anaerobic environment at 75°C; ② Construction of bone-like callus polyurethane bone adhesive (CLA): Pre-CLA and Col I are uniformly mixed at 20°C~25°C (1-4:100), the influence law and mechanism of material composition, concentration ratio and synthesis conditions on the properties such as pore, bonding performance and mechanical strength of the prepared polyurethane bone adhesive are explored and clarified, and the bone-like callus polyurethane bone adhesive with excellent comprehensive performance is selected, providing experimental basis for subsequent related research.

[0038] (1) Titration of isocyanate group (-NCO): The NCO content in Tri-HDI was determined by toluene-di-n-butylamine titration method. 0.1 g of Tri-HDI was taken in a beaker, its weight was measured and recorded as M (g) with data accurate to three decimal places. 2 mL of toluene was added to the beaker and continued to stir until completely melted, 3 mL of di-n-butylamine-toluene solution with a concentration of 0.1 M was added, and after the reaction was complete, 0.1% bromocresol green indicator was added to make the liquid in the beaker dark blue. Start stirring titration with 0.1 M hydrochloric acid solution, when the liquid in the beaker gradually changes from dark blue to green, and finally completely changes to golden yellow and the color does not change within 1 min, the titration is complete, and the volume of hydrochloric acid solution used for titration is recorded as V2 (unit: mL). Another set without Tri-HDI was taken as a blank group, and the volume of hydrochloric acid solution consumed in the blank group was recorded as V1 (unit: mL). The NCO group content in the Tri-HDI sample was calculated according to the formula: NCO (%) = 0.1 (V1-V2) x 0.042 / M x 100%, and the average value was obtained by repeating the test multiple times until the error was small.

[0039] (2) Titration of amino group (-NH2): The amino content in Col I was determined by sodium nitrite titration method. 0.0025 g of Col I was weighed, 1 mL of 6 M hydrochloric acid solution was added, and the collagen fiber was completely dissolved by heating at 60°C for 3 min. Then 200 mL of pure water, 1 mL of analytical pure glacial acetic acid and 0.5 g of analytical pure potassium bromide were added, the beaker was placed in ice water and stirred, the temperature was controlled at 0-5°C, then 0.1 M sodium nitrite standard solution was started to be added, the sodium nitrite was diazotized with amino compounds under the conditions of hydrochloric acid and low temperature, a certain amount was titrated at a time, and starch potassium iodide test paper was used to indicate the end point of titration. Slightly excessive NaNO2 will oxidize KI in an acidic environment to precipitate I2, making the test paper blue. The volume of sodium nitrite standard solution consumed was recorded as V, unit: mL. Therefore, the amino group in 1 g of collagen fiber is:

[0040] NH2 (mol / g) = 0.1 x V / (1000 x 0.0025)

[0041] The average value was obtained by repeating the test multiple times until the error was small.

[0042] According to the total amount of amine group in Col I and the content of isocyanate group in Pre-CLA determined in (1) and (2), the mass required for the complete equivalent of the two was calculated, which was used as the basis to adjust the content of Col I.

[0043] Example 1

[0044] The preparation method of the bone callus-like bone adhesive material comprises the following steps:

[0045] Synthesis of Pre-CLA

[0046] 0.5g of Tri-HDI and 0.33g of nHAP were uniformly mixed, 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, a viscous prepolymer was obtained under the condition of nitrogen at 75℃ for 3min, which was recorded as Pre-CLA.

[0047] Synthesis of CLA

[0048] 0.005g of Col I was weighed and uniformly mixed into the prepared Pre-CLA at room temperature to obtain a bone adhesive with a collagen mass ratio of 1% to Tri-HDI, which was named CLA-1.

[0049] Example 2

[0050] The preparation method of the bone callus-like bone adhesive material comprises the following steps:

[0051] Synthesis of Pre-CLA

[0052] 0.5g of Tri-HDI and 0.33g of nHAP were uniformly mixed, 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, a viscous prepolymer was obtained under the condition of nitrogen at 75℃ for 3min, which was recorded as Pre-CLA.

[0053] Synthesis of CLA

[0054] 0.01g of Col I was weighed and uniformly mixed into the prepared Pre-CLA at room temperature to obtain a bone adhesive with a collagen mass ratio of 2% to Tri-HDI, which was named CLA-2.

[0055] Example 3

[0056] The preparation method of the bone callus-like bone adhesive material comprises the following steps:

[0057] Synthesis of Pre-CLA

[0058] 0.5g of Tri-HDI and 0.33g of nHAP were uniformly mixed, 2.5μL of acetyl chloride and 2.5μL of dibutyltin dilaurate were added as catalysts, and after uniform stirring, a viscous prepolymer was obtained under the condition of nitrogen at 75℃ for 3min, which was recorded as Pre-CLA.

[0059] Synthesis of CLA

[0060] Take 0.02 g of Col I, add it to the prepared Pre-CLA at room temperature, mix uniformly, get the bone adhesive with collagen mass accounting for 4% of Tri-HDI, named CLA-4.

[0061] Characterize and test the performance of CLA-1, CLA-2 and CLA-4, and the results are as follows:

[0062] Figure 1 The successful synthesis of CLA is proved.

[0063] Figure 2 The uncured and cured morphology of CLA is shown, and the element content and distribution on the surface of CLA are scanned by SEM-EDS technology, and it can be seen that the element distribution on the surface of CLA is uniform.

[0064] As shown in Figure 3 , CLA contains more pores, and the diameter increases with the increase of Col I, and the pore area increases, as shown in Figure 4 and Figure 5 , the pores are necessary conditions for cells to enter the material from the bone surface, and large pores can promote the formation of new bone and promote the rapid healing of fractures; CLA-4 group has better ductility than CLA-1, as shown in Figure 6 , which helps the anti-force effect of CLA in vivo and protects the stable healing of the fracture repair site.

[0065] Figures 7-9 It is shown that CLA has excellent cell compatibility and blood compatibility, and subcutaneous implantation sections can be seen that the surrounding tissue can enter the CLA inside through the pores.

[0066] The main mechanism of CLA to play the bonding effect is the chemical combination of isocyanate groups with amine groups on the bone surface, at the same time, CLA will also produce partial mechanical interlocking with cancellous bone to assist bonding and retention. The CLA-1 group with the least Col I content showed the highest compression modulus (60.33±10.09) MPa, the largest tensile strength (21.37±2.19) MPa and the best bonding performance (10.50±1.41) MPa, which may be due to its high cohesive strength and more compact molecular structure. CLA shows excellent bonding strength in dry, wet and blood environments, and can support a weight of 5 kg when bonded for 4 mm 2 , as shown in Figure 10 .

[0067] During the polymerization of the material, it can be found through thermal infrared characterization that the material does not generate heat higher than the body temperature, and will not cause thermal damage to the tissue, as shown in Figure 13 .

[0068] Verification

[0069] (1) Skull fracture model construction: SD rats were used to establish a skull ring fracture model. After anesthesia, the rat scalp was incised along the midline using a blade, the skull periosteum was fully stripped to expose the operation area, and a complete circular bone block was cut out using a 4mm diameter trephine to form a fracture model. During the operation, the fracture pieces were kept in a wet state to prevent tissue dehydration affecting subsequent healing.

[0070] (2) In vivo osteogenesis verification: CLA-4 was used for in vivo osteogenesis verification, 20 μL of CLA-4 was evenly applied around the circular fracture piece to connect it with the surrounding bone tissue; in the control group, the bone piece without bone adhesive was directly placed back without other treatment. Each group included 10 rats. After operation, the incision was sutured with 5 / 0 absorbable suture, and no additional immobilization measures were taken. At 4 and 8 weeks after operation, the rats were sacrificed, the skull samples were removed and fixed with 4% paraformaldehyde for subsequent Micro-CT scanning and histological analysis to evaluate the fracture repair.

[0071] The results of the osteogenic induction experiment are shown in Figures 11-12 CLA-4 group. The animal experiment results also verified the osteogenic effect of CLA. In the SD rat skull fracture model, the bone pieces in the control group showed obvious bone resorption and nonunion, while the CLA-4 group showed significant new bone formation at 8w, HE staining showed good healing of the fracture site, and part of the trabecular structure was tight, indicating that CLA not only can adhere the fracture ends, but also can promote osteogenesis and bone remodeling at the fracture site.

[0072] Natural callus is mainly composed of mineralized type I collagen fibers, and the present application simulates the composition and function of natural callus to connect the fracture ends.

[0073] It should be noted that when the present application claims involve numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0074] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they understand the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0075] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A bone cement which mimics a bone callus, characterized in that, The second polymerization reaction occurs between hydrogen atoms in the collagen fibers and isocyanate groups in the bone-like callus polyurethane bone adhesive prepolymer, and the bone-like callus bone adhesive is obtained. The bone-like callus polyurethane bone adhesive prepolymer is obtained by polyaddition reaction between hydroxyl groups in nano-hydroxyapatite and isocyanate groups in hexamethylene diisocyanate isocyanurate trimer.

2. A method of preparing the bone cement material of claim 1, characterized in that The method comprises the following steps: The hexamethylene diisocyanate isocyanurate trimer and the nano-hydroxyapatite are mixed uniformly at a mass ratio of 1-3:1, and then the first polymerization reaction occurs in an oxygen-free environment at 60-80°C to obtain the bone-like callus polyurethane bone adhesive prepolymer; The bone-like callus polyurethane bone adhesive prepolymer and the collagen fibers are mixed at a mass ratio of 1-4:100 to undergo the second polymerization reaction.

3. The production method according to claim 2, characterized by, The reaction is carried out in an oxygen-free environment at 60-80°C for 2-5 hours.

4. The production method according to claim 2, characterized by, Acetyl chloride and dibutyltin dilaurate are used as catalysts during the first polymerization reaction.

5. The preparation method according to claim 4, characterized in that, The volume ratio of the acetyl chloride to the dibutyltin dilaurate is 0.75-1.25:

1.

6. The production method according to claim 5, wherein The mass ratio of the total volume of the acetyl chloride and the dibutyltin dilaurate to the hexamethylene diisocyanate isocyanurate trimer is 3-6 μL:0.5 g.

7. Use of the bone-like callus bone adhesive of claim 1 in the preparation of a bone repair material.

8. Use according to claim 7, characterized in that, The repair is at least one of osteogenesis and bone remodeling at a fracture site.

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