Complex cylindrical bionic bone scaffold and preparation method thereof
By adopting a preparation method of combining composite cylindrical structure and multiple technologies in the bionic bone stent, the shortcomings of the existing bionic bone stent in distinguishing bone structure and improving repair effects are solved, and efficient bone defect repair is achieved.
Patent Information
- Application Number
- CN202510185172.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-27
AI Technical Summary
Due to the FDM printing accuracy problem, common bionic bone stents are difficult to effectively distinguish cortical bones and trabecular bones in the stent, which limits the degree of bionicity, and has simple structure and limited functions, which cannot effectively promote the repair of bone defects.
The preparation method of composite cylindrical bionic bone stent was adopted. By mixing ZIF-8 with polylactone powder, ZP stent was printed using an FDM three-dimensional printer, and PH stent was printed by DLP photocuring three-dimensional printing technology. Combined with melt deposition printing technology, a composite cylindrical bionic bone stent with macromicrostructure was constructed.
It has achieved effective distinction between cortical bone and trabecular bone in bone defect repair, improved the mechanical properties and biocompatibility of the stent, significantly promoted the angiogenesis and osteogenesis process, and achieved the purpose of repairing large sections of bone defects.
Smart Images
Figure CN120037447A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of bone defect repair, and in particular to a composite tubular bionic bone scaffold and a preparation method thereof. Background Art
[0002] The reconstruction of large bone defects caused by severe trauma, tumors, and congenital deformities is an international clinical challenge. Currently, the methods for treating large bone defects include autologous bone transplantation, allogeneic bone transplantation, and distraction osteogenesis. Autologous bone transplantation is considered the gold standard for bone tissue repair and regeneration. Autologous bone has good osteoinductive and bone integration properties. After being implanted into the defect site, it can quickly integrate with the body's own vascular network, will not cause immune rejection reactions, and thus form a normal physiological structure to ensure the mechanical strength of the bone defect site. However, the source of autologous bone transplantation is limited, which will cause secondary injuries to patients and it is difficult to meet the repair of large segmental bone defects. Although allogeneic bone transplantation has a wide source and will not cause secondary injuries to itself, there are problems such as poor bone integration, immune rejection, and transmission of blood diseases, which lead to transplantation failure. The distraction osteogenesis technique is prone to problems such as neurovascular injury, inflammation, and infection, resulting in poor treatment effects.
[0003] Natural bone is macroscopically composed of outer cortical bone and inner cancellous bone. Under a microscope, cortical bone is composed of bone lamellae with high density and regular arrangement, providing the main mechanical properties of bone. Cancellous bone is a reticular structure composed of plate-like or rod-like structures, with a thickness of about 200 μm. In addition, various cells and a rich extracellular matrix (ECM) are dispersed in bone tissue. This ordered hierarchical structure improves the mechanical strength and adaptability of the scaffold, promotes the interaction between various cells, and ultimately promotes bone repair. However, current research mainly focuses on bone healing. Most well-designed scaffolds either focus on using high-mechanical-strength materials for load-bearing bone repair or preferentially use degradable hydrogel composites to promote bone repair, ignoring the important role of the hierarchical structure of bone in repairing segmental bone defects. Therefore, it is very necessary to develop a bionic scaffold with a complex gradient structure similar to natural bone.
[0004] Recently, the progress of materials science and manufacturing methods has promoted the development of various bionic strategies for constructing high-performance bone tissue engineering materials, such as modular assembly, electrospinning, and 3D printing. Among these methods, 3D printing has received great attention because of its ability to precisely control the internal structure of the scaffold and quickly generate complex geometries. Many researchers have tried to use 3D printing technology to produce scaffolds that can simulate the bone tissue structure.
[0005] Regarding the above related technologies, the inventors believe that there are the following technical defects to be improved:
[0006] Common bionic bone scaffolds are limited by the FDM printing accuracy problem, making it difficult to effectively distinguish cortical bone and trabecular bone within the scaffold, which restricts the degree of bionics. At the same time, most of these scaffolds have simple structures and limited functions, and cannot effectively promote the repair of bone defects. Summary of the Invention
[0007] This application provides a composite tubular bionic bone scaffold and its preparation method to improve the following technical problems:
[0008] Common bionic bone scaffolds are limited by the FDM printing accuracy problem, making it difficult to effectively distinguish cortical bone and trabecular bone within the scaffold, which restricts the degree of bionics. At the same time, most of these scaffolds have simple structures and limited functions, and cannot effectively promote the repair of bone defects.
[0009] In the first aspect, this application provides a preparation method for a composite tubular bionic bone scaffold, adopting the following technical solution:
[0010] A preparation method for a composite tubular bionic bone scaffold includes the following steps:
[0011] Step 1, preparation of regenerated material: Dissolve 2-methylimidazole powder in methanol solution to form 2-methylimidazole solution, and then dissolve zinc nitrate hexahydrate crystals in methanol solution to form zinc nitrate solution. Pour the 2-methylimidazole solution into a volumetric flask, and stir it with a magnetic stirrer at room temperature until it is fully dissolved. Then, drop the zinc nitrate solution into the volumetric flask containing the 2-methylimidazole solution, and stir it evenly at room temperature for 3 - 5 hours. After centrifugation, pour in an equal amount of methanol solution, and wash it by repeated oscillation 2 - 4 times. After the washing is completed, perform drying treatment for 8 - 12 hours to obtain the dried regenerated material ZIF-8. Take out the regenerated material ZIF-8 and grind it into a fine powder, and store it sealed at room temperature;
[0012] Step 2, preparation of ZP scaffold: Use 3D mapping software to draw the stl format drawing of the ZP scaffold. Mix the regenerated material ZIF-8 and polycaprolactone powder in a ratio of 1:9, heat it to 100 - 120 °C using a heating platform, stir and melt it, and then press it into a ZIF-8 / PCL tablet. Place the ZIF-8 / PCL tablet in the feeding trough of an FDM 3D printer, and use the motor boost micro-injection technology to print it into a ZP scaffold. Heat it at a temperature of 100 - 120 °C for 20 - 30 minutes. After printing, rinse the ZP scaffold with deionized water, and perform mineralization treatment on the ZP scaffold;
[0013] Step 3, Preparation of composite hydrogel: Take polyethylene glycol and nitrobenzene and dissolve them in anhydrous dichloromethane. Stir the obtained mixture under nitrogen for 20 - 28 hours. Then remove the dichloromethane in the mixture, dissolve it in 5 deionized water, transfer the solution to a dialysis bag, dialyze it in deionized water for 4 - 6 days, and freeze-dry to obtain PEGNB gel; completely dissolve the HA hyaluronic acid solution in phosphate buffer solution at a temperature of 55 - 65 °C, then add methacrylic anhydride, and react for 1.5 - 2.5 hours under light-shielded conditions. After the reaction, dialyze at room temperature for 6 - 8 days, and freeze-dry to obtain HAMA gel; dissolve the PEGNB gel at a concentration of 10% w / v, the HAMA gel at a concentration of 2% w / v, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate at a concentration of 0.3% w / v, and the lemon yellow light absorber at a concentration of 0.1% w / v in phosphate buffer solution to obtain a composite hydrogel solution.
[0014] Step 4, Preparation of PH scaffold: Use 3D mapping software to draw the stl format drawing of the PH scaffold. Place the composite hydrogel solution in the feed trough of a DLP stereolithography 3D printer to print the PH scaffold. Put the obtained PH scaffold into phosphate buffer solution for cleaning and store it at a temperature of 3 - 5 °C for later use;
[0015] Step 5, Scaffold assembly: Perform ultraviolet disinfection treatment on the ZP scaffold and the PH scaffold, then rinse and treat them with phosphate buffer solution, and then combine the ZP scaffold and the PH scaffold to form a macro-micro structure composite tubular bionic bone scaffold.
[0016] In an implementable technical solution of the present application, in Step 1, the proportion of the active ingredient in the methanol solution used is greater than 99%. Among them, the 2-methylimidazole solution contains 1 g of 2-methylimidazole powder and 30 mL of methanol solution, and the zinc nitrate solution contains 300 mg of zinc nitrate hexahydrate and 10 mL of methanol solution.
[0017] In an implementable technical solution of the present application, in Step 2, the printing parameters set in the control software of the FDM 3D printer are as follows:
[0018] The nozzle temperature is 120 °C, the nozzle aperture is 0.3 mm, the spacing is 0.75 mm, the speed is 10 mm / s, the extrusion speed is 0.01 mm / s. After the scaffold printing is completed, drill holes on the side. The diameter of the drilled holes is 1 mm, and the spacing between adjacent drilled holes is 1.5 mm.
[0019] In an implementable technical solution of the present application, in Step 2, the specific operation of rinsing the ZP scaffold is as follows:
[0020] Prepare 20 mL of 0.1 mol / L CaCl 2 solution and 20 mL of 0.1 mol / L K2 HPO 4 solution. First, immerse the ZP scaffold in the above-mentioned CaCl 2 solution for 4 - 6 minutes, then immerse the ZP scaffold in deionized water for 25 - 35 seconds, and then immerse the ZP scaffold in the above-mentioned K 2 HPO 4 solution for 4 - 6 minutes, and finally immerse the ZP scaffold in deionized water for 25 - 35 seconds.
[0021] In an implementable technical solution of the present application, in step two, the specific operation of mineralizing the ZP scaffold is as follows:
[0022] Immerse the ZP scaffold in SBF simulated body fluid to deposit biomimetic hydroxyapatite, and change the SBF simulated body fluid every day to keep the ionic strength consistent. After soaking for 2 - 4 days, gently rinse the ZP scaffold with deionized water to remove the SBF simulated body fluid on the surface, and then vacuum freeze-dry for 20 - 30 hours.
[0023] In an implementable technical solution of the present application, in step three, the volume of the deionized water used is the same as that of the anhydrous dichloromethane used. The mixture contains 0.1 mmol of polyethylene glycol, 0.4 mmol of nitrobenzene, and 50 mL of anhydrous dichloromethane. The concentration of the HA hyaluronic acid solution is 10% w / v, the concentration of the phosphate buffer solution is 0.01 mol / L, and the amount of methacrylic anhydride used is 1 mL.
[0024] In an implementable technical solution of the present application, in step four, the printing parameters set in the control software of the DLP stereolithography 3D printer are as follows:
[0025] Light intensity: 14 mW / cm 3 , light source wavelength is 405 nm, exposure time is 27 s, number of base layers is 5, base layer exposure time is 30 s, peeling distance is 6 mm, lifting height is 0 mm, peeling speed is 25 mm / min, lifting speed is 100 mm / min, peeling recovery speed is 180 mm / min, and slice layer height is 50 um.
[0026] Second, the present application provides a composite tubular biomimetic bone scaffold, adopting the following technical solution:
[0027] A composite tubular biomimetic bone scaffold is made by the above preparation method.
[0028] In an implementable technical solution of the present application, it includes a ZP scaffold with a cylindrical structure and a PH scaffold with a square tubular structure. The outer peripheral wall of the ZP scaffold is inscribed in the four inner side walls of the PH scaffold.
[0029] In an implementable technical solution of the present application, a plurality of first holes arranged along the central axis direction and a plurality of second holes arranged along the radial direction are provided on the ZP bracket, and square / circular / elongated third holes are provided on the four peripheral walls of the ZP bracket.
[0030] In summary, the present application includes at least one of the following beneficial technical effects:
[0031] ZIF-8 is a nanoparticle with a regular dodecahedron structure. Under the electron microscope, its diameter is about 100 nm. Through elemental analysis, it can be seen that it contains Zn and N elements inside. ZIF-8 is formed by zinc ions (Zn 2+ ) and 2-methylimidazole, and it is one of the notable metal-organic framework MOF materials. Zn 2+ is one of the most abundant transition metals in biology, and the imidazole group is an important part of amino acids. Compared with other metal-organic framework MOF materials, this unique composition makes ZIF-8 have excellent biocompatibility, making it particularly suitable for tissue repair;
[0032] In simulated body fluid SBF, a large number of active metal cations and phosphates perform anion exchange with ZIF-8, promoting the uniform deposition of a thick enough hydroxyapatite layer on polymer materials lacking functional groups or charged regions. This process can significantly improve osteogenesis and biocompatibility in vitro and in vivo. At the same time, the bone scaffold with polycaprolactone as the main body improves the mechanical properties of the scaffold;
[0033] Tetra-armed polyethylene glycol with o-nitrobenzyl alcohol (NB) ends (PEGNB; 3.5NB molecules / PEG) and methacrylate-grafted, highly rigid hyaluronic acid (HAMA, grafting degree 117%) are introduced into the composite hydrogel. Through photo-triggered instantaneous radical and persistent radical coupling reactions (PTPC reactions), HAMA is polymerized into hard particles and covalently crosslinked with the PEGNB polymer to form a tough hydrogel. This composite hydrogel also exhibits good photocrosslinking properties, making it suitable for manufacturing complex porous trabecular structures;
[0034] The DLP stereolithography three-dimensional printing technology provides a promising method for constructing microchannels and complex scaffold trabecular structures due to its high printing accuracy. Therefore, the PH scaffold has a cortical layer with a Haversian canal system and a trabecular layer with a microstructure, promoting osteogenesis and angiogenesis through gradient density. Moreover, the PH scaffold in the present application is mainly composed of hydrogel and has a good postoperative degradable effect. Therefore, the present application combines the fused deposition printing technology and the DLP printing technology to construct a macro-microstructure composite tubular bionic bone scaffold for implanting it into an animal body to participate in in-situ regeneration repair to achieve the purpose of repairing large segment bone defects;
[0035] The ZP scaffold was prepared by using motor micro-injection (MAM) printing based on fused deposition printing technology. The degradation of ZIF-8 and the competitive binding of metal cations / inorganic anions in SBF simulated body fluid promoted the mineralization of polycaprolactone, thereby fabricating a biomimetic bone cortex scaffold with osteoinductive ability (i.e., the ZP scaffold). In addition, inspired by the structure of cancellous bone, with the help of DLP stereolithography three-dimensional printing technology, a biomimetic cancellous bone scaffold containing vascular channels (i.e., the PH scaffold) was prepared using a PEGNB / HAMA composite hydrogel. And through inoculating endothelial cells and perfusion in the PH scaffold, pre-vascularization of the biomimetic cancellous bone scaffold was achieved. Finally, the ZP scaffold and the PH scaffold were combined to form a vascularized composite tubular biomimetic bone scaffold containing double internal and external biomimetic structures. In vivo experiments showed that the above scaffolds had satisfactory biocompatibility and significantly promoted angiogenesis and osteogenesis processes. Description of the Drawings
[0036] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0037] Figure 1 It is a schematic flowchart of the preparation method of the composite tubular biomimetic bone scaffold in the embodiments of the present application.
[0038] Figure 2 It is a schematic structural diagram of the composite tubular biomimetic bone scaffold in the embodiments of the present application.
[0039] Figure 3 It is a transmission electron microscope image and energy spectrum analysis diagram of the regenerative material ZIF-8 powder in the embodiments of the present application.
[0040] Figure 4 It is an electron microscope image and printing accuracy test diagram of the composite hydrogel in the embodiments of the present application.
[0041] Figure 5 It is a solid diagram of the composite tubular biomimetic bone scaffold in the embodiments of the present application.
[0042] Figure 6 It is an animal experiment diagram of the composite tubular biomimetic bone scaffold in the embodiments of the present application.
[0043] Figure 7 It is an animal experiment result diagram of the composite tubular biomimetic bone scaffold in the embodiments of the present application.
[0044] Description of the Reference Numerals:
[0045] 100, ZP bracket; 200, PH bracket. Detailed implementation mode
[0046] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer and more understandable, the following further details this application in combination with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.
[0047] It should be noted that when an element is referred to as "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.
[0048] It should be understood that the orientation or positional relationship indicated by the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation to this application.
[0049] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, "a plurality" means two or more, unless otherwise specifically defined.
[0050] The following further details this application in combination with the attached Figure 1-7 drawings.
[0051] The embodiment of this application discloses a composite cylindrical bionic bone bracket and its preparation method. Refer to Figure 1 - Figure 7 , the preparation method of the composite cylindrical bionic bone bracket includes the following steps:
[0052] Step 1, Preparation of regenerated material: Take 2-methylimidazole powder and dissolve it in methanol solution to form 2-methylimidazole solution. Then take zinc nitrate hexahydrate crystals and dissolve them in methanol solution to form zinc nitrate solution. Pour the 2-methylimidazole solution into a volumetric flask, and stir it with a magnetic stirrer at room temperature until it is fully dissolved. Then add the zinc nitrate solution dropwise to the volumetric flask containing the 2-methylimidazole solution, and stir evenly at room temperature for 3 - 5 hours. After centrifugation, pour in an equal amount of methanol solution, and wash it by repeated oscillation 2 - 4 times. After the washing is completed, dry it for 8 - 12 hours to obtain the dry regenerated material ZIF-8. Take out the regenerated material ZIF-8 and grind it into a fine powder, and store it sealed at room temperature;
[0053] The proportion of the active ingredient in the methanol solution used is greater than 99%. Among them, the 2-methylimidazole solution contains 1 g of 2-methylimidazole powder and 30 mL of methanol solution, and the zinc nitrate solution contains 300 mg of zinc nitrate hexahydrate and 10 mL of methanol solution.
[0054] Step 2, Preparation of ZP scaffold: Use 3D drawing software to draw the stl format drawing of the ZP scaffold. Mix the regenerated material ZIF-8 and polycaprolactone powder in a ratio of 1:9, heat it to 100 - 120 °C using a heating platform, stir and melt it, and then press it into a ZIF-8 / PCL tablet. Place the ZIF-8 / PCL tablet in the feeding trough of an FDM 3D printer, and use the motor-boosted micro-injection technology to print it into a ZP scaffold. Heat it at a temperature of 100 - 120 °C for 20 - 30 minutes. After printing, rinse the ZP scaffold with deionized water. The specific operation of rinsing the ZP scaffold is as follows: Prepare 20 mL of 0.1 mol / L CaCl 2 solution and 20 mL of 0.1 mol / L K 2 HPO 4 solution. First, immerse the ZP scaffold in the above-mentioned CaCl 2 solution for 4 - 6 minutes, then immerse the ZP scaffold in deionized water for 25 - 35 seconds, then immerse the ZP scaffold in the above-mentioned K 2 HPO 4 solution for 4 - 6 minutes, and finally immerse the ZP scaffold in deionized water for 25 - 35 seconds; And carry out mineralization treatment on the ZP scaffold. The specific operation of mineralizing the ZP scaffold is as follows: Immerse the ZP scaffold in SBF simulated body fluid to deposit biomimetic hydroxyapatite, and change the SBF simulated body fluid every day to keep the ionic strength consistent. After soaking for 2 - 4 days, gently rinse the ZP scaffold with deionized water to remove the SBF simulated body fluid on the surface, and then vacuum freeze-dry it for 20 - 30 hours;
[0055] The printing parameters set in the control software of the FDM 3D printer are as follows: the nozzle temperature is 120 °C, the nozzle aperture is 0.3 mm, the spacing is 0.75 mm, the speed is 10 mm / s, the extrusion speed is 0.01 mm / s. After the support is printed, drill holes on the side, the diameter of the drilled holes is 1 mm, and the spacing between adjacent drilled holes is 1.5 mm.
[0056] Step 3, preparation of composite hydrogel: Dissolve polyethylene glycol and nitrobenzene in anhydrous dichloromethane, stir the obtained mixture with nitrogen for 20 - 28 hours, then remove the dichloromethane in the mixture, dissolve it in 5 deionized water again, transfer the solution to a dialysis bag, dialyze it in deionized water for 4 - 6 days, and freeze-dry to obtain PEGNB gel; completely dissolve the HA hyaluronic acid solution in phosphate buffer solution at a temperature of 55 - 65 °C, then add methacrylic anhydride, react for 1.5 - 2.5 hours under light-shielded conditions. After the reaction, dialyze at room temperature for 6 - 8 days, and freeze-dry to obtain HAMA gel; dissolve PEGNB gel at a concentration of 10% w / v, HAMA gel at a concentration of 2% w / v, lithium phenyl(2,4,6-trimethylbenzoyl)phosphate at a concentration of 0.3% w / v, and lemon yellow light absorber at a concentration of 0.1% w / v in phosphate buffer solution to obtain a composite hydrogel solution;
[0057] The volume of the used deionized water is the same as that of the used anhydrous dichloromethane. The mixture contains 0.1 mmol of polyethylene glycol, 0.4 mmol of nitrobenzene, and 50 mL of anhydrous dichloromethane. The concentration of the HA hyaluronic acid solution is 10% w / v, the concentration of the phosphate buffer solution is 0.01 mol / L, and the dosage of methacrylic anhydride is 1 mL.
[0058] Step 4, preparation of PH scaffold: Use 3D mapping software to draw the stl format drawing of the PH scaffold, place the composite hydrogel solution in the feed trough of a DLP stereolithography 3D printer, print to obtain the PH scaffold, put the obtained PH scaffold into phosphate buffer solution for cleaning, and store it at a temperature of 3 - 5 °C for later use. The printing parameters set in the control software of the DLP stereolithography 3D printer are as follows:
[0059] Light intensity 14 mW / cm 3 , the light source wavelength is 405 nm, the exposure time is 27 s, the number of base layers is 5, the base exposure time is 30 s, the peeling distance is 6 mm, the lifting height is 0 mm, the peeling speed is 25 mm / min, the lifting speed is 100 mm / min, the peeling recovery speed is 180 mm / min, and the slice layer height is 50 um.
[0060] Step 5, scaffold assembly: Perform ultraviolet disinfection treatment on the ZP scaffold and the PH scaffold, then rinse and treat them with phosphate buffer solution. Seed endothelial cells at a density of 1×10 5mL was implanted into the PH stent and perfused for culture for 6 - 8 days. Then, the ZP stent was combined with the PH stent implanted with endothelial cells to form a macro - microstructured composite tubular bionic bone stent.
[0061] This application also provides a composite tubular bionic bone stent, which is made by the above - mentioned preparation method.
[0062] The composite tubular bionic bone stent includes a ZP stent with a cylindrical structure and a PH stent with a square - tubular structure. The outer peripheral wall of the ZP stent is inscribed in the four inner side walls of the PH stent. Moreover, a plurality of first holes arranged along the central axis direction and a plurality of second holes arranged along the radial direction are provided on the ZP stent. Square / circular / strip - shaped third holes are provided on the four peripheral walls of the ZP stent. See Figure 2 and Figure 5 .
[0063] The regenerated material ZIF - 8 powder sample was observed by transmission electron microscopy and its elements were analyzed. It can be seen that ZIF - 8 is nanoparticles with a regular dodecahedron structure. Its diameter is about 100 nm under the electron microscope. Through element analysis, it can be seen that it contains Zn and N elements. See Figure 3 .
[0064] As Figure 4 shown, the left figure is the electron micrograph of the composite hydrogel; the right figure is the test chart of the printing accuracy of the composite hydrogel. It can be seen that the composite hydrogel can successfully print a fine structure with a diameter of 200 um.
[0065] As Figure 5 shown, Figure A is the mineralized bionic bone cortical stent - ZP stent; Figure B is the bionic bone cancellous stent - PH stent. Combining the two stents can form an inner - outer double - bionic composite tubular bionic bone stent.
[0066] As Figure 6 shown, the animal experiment is as follows:
[0067] New Zealand white rabbits weighing 2.5 - 3 kg were selected, weighed, and anesthetized by intramuscular injection of Shu Tai - 50 at a dose of 0.15 ml / kg. After anesthesia, the forelimbs were sheared and prepared for skin. The rabbit was fixed supine with its four limbs, and the skin was disinfected twice with iodophor after covering with a drape. A surgical field of 1.5 - 2 cm was opened with a blade, and the lateral muscle space of the radius was bluntly dissected to expose the radius. An electric drill was used to excise about 1.5 cm of bone defect, that is, a critical bone defect model, and the above - mentioned composite tubular bionic bone stent was implanted. The wound was rinsed with normal saline, and the surgical incision was sutured layer by layer and then disinfected with iodophor. Postoperatively, gentamicin injection was intramuscularly injected at a dose of 0.5 ml / rabbit for 3 days to prevent infection. The results of the 0.625 - thin - layer CT scan of the forelimb reconstruction of the New Zealand white rabbit radius defect model at 1 month.
[0068] As Figure 7 shown, it can be seen that the bone repair effect of the experimental group is significantly better than that of the blank control group.
[0069] The beneficial technical effects of the composite tubular bionic bone scaffold and its preparation method according to the embodiments of the present application are roughly as follows:
[0070] ZIF-8 is a nanoparticle with a regular dodecahedron structure. Under the electron microscope, its diameter is about 100 nm. Through elemental analysis, it can be seen that it contains Zn and N elements inside. ZIF-8 is formed by zinc ions (Zn 2+ ) and 2-methylimidazole, and it is one of the notable metal-organic framework MOF materials. Zn 2+ is one of the most abundant transition metals in biology, and the imidazole group is an important part of amino acids. Compared with other metal-organic framework MOF materials, this unique composition makes ZIF-8 have excellent biocompatibility, making it particularly suitable for tissue repair;
[0071] In simulated body fluid SBF, a large number of active metal cations and phosphates perform anion exchange with ZIF-8, promoting the uniform deposition of a thick enough hydroxyapatite layer on polymer materials lacking functional groups or charged regions. This process can significantly improve osteogenicity and biocompatibility in vitro and in vivo. At the same time, the bone scaffold with polycaprolactone as the main body improves the mechanical properties of the scaffold;
[0072] Tetra-arm polyethylene glycol (PEGNB; 3.5NB molecules / PEG) with o-nitrobenzyl alcohol (NB) ends and methacrylate-grafted, highly rigid hyaluronic acid (HAMA, grafting degree 117%) are introduced into the composite hydrogel. Through photo-triggered instantaneous free radical and persistent free radical coupling reaction (PTPC reaction), HAMA is polymerized into hard particles and covalently crosslinked with the PEGNB polymer to form a tough hydrogel. This composite hydrogel also exhibits good photocrosslinking characteristics, making it suitable for manufacturing complex porous trabecular structures;
[0073] The DLP photocuring three-dimensional printing technology provides a promising method for constructing microchannels and complex scaffold trabecular structures with its high printing accuracy. Therefore, the PH scaffold has a cortical layer with a Haversian canal system and a trabecular layer with microstructures, promoting bone formation and angiogenesis through gradient density. Moreover, the PH scaffold in this application is mainly composed of hydrogel and has a good postoperative degradable effect. Therefore, this application combines the fused deposition printing technology and the DLP printing technology to construct a composite tubular bionic bone scaffold with macro-micro structures, so as to implant it into an animal body to participate in in-situ regeneration repair to achieve the purpose of repairing large segment bone defects;
[0074] The ZP scaffold was prepared by motor-assisted microinjection (MAM) printing based on fused deposition modeling (FDM) technology. The degradation of ZIF-8 and the competitive binding of metal cations / inorganic anions in simulated body fluid (SBF) promoted the mineralization of polycaprolactone, thereby fabricating a biomimetic cortical bone scaffold with osteoinductive ability (i.e., the ZP scaffold). In addition, inspired by the structure of cancellous bone, a biomimetic cancellous bone scaffold containing vascular channels (i.e., the PH scaffold) was prepared using a PEGNB / HAMA composite hydrogel by digital light processing (DLP) three-dimensional printing technology. The pre-vascularization of the biomimetic cancellous bone scaffold was achieved by inoculating endothelial cells and perfusion in the PH scaffold. Finally, the ZP scaffold and the PH scaffold were combined to form a vascularized composite tubular biomimetic bone scaffold with dual internal and external biomimicry. In vivo experiments showed that the above scaffolds had satisfactory biocompatibility and significantly promoted angiogenesis and osteogenesis.
[0075] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the application shall be included in the protection scope of the present application.
Claims
1. A method for preparing a composite cylindrical bionic bone scaffold, characterized in that: The following steps are involved: Step 1, preparation of regenerated material: dissolve 2-methylimidazole powder in methanol solution to form 2-methylimidazole solution, dissolve zinc nitrate hexahydrate crystals in methanol solution to form zinc nitrate solution, pour the 2-methylimidazole solution into a volumetric flask, stir with a magnetic stirrer at room temperature until fully dissolved, then drop the zinc nitrate solution into the volumetric flask loaded with the 2-methylimidazole solution, stir at room temperature for 3-5 hours, pour into an equal amount of methanol solution after centrifugation, repeatedly shake and wash for 2-4 times, dry for 8-12 hours after washing to obtain dry regenerated material ZIF-8, take out the regenerated material ZIF-8, grind it into fine powder, and seal and store it at room temperature; Step 2, ZP scaffold preparation: Use 3D drawing software to draw the stl format drawing of the ZP scaffold, mix the recycled material ZIF-8 and polycaprolactone powder in a ratio of 1:9, heat to 100-120°C using a heating platform, stir to melt and then press into a ZIF-8 / PCL tablet, place the ZIF-8 / PCL tablet in the feed slot of the FDM 3D printer, use the motor-assisted microinjection technology to print into a ZP scaffold, heat at 100-120°C for 20-30 minutes, rinse the ZP scaffold with deionized water after printing, and mineralize the ZP scaffold; Step 3, preparation of composite hydrogel: polyethylene glycol and nitrobenzene are dissolved in anhydrous dichloromethane, and the mixture is stirred with nitrogen for 20-28 hours, and then the dichloromethane in the mixture is removed, and then dissolved in 5 deionized water, and then the solution is transferred to a dialysis bag, dialyzed in deionized water for 4-6 days, and freeze-dried to obtain PEGNB gel; the HA hyaluronic acid solution is completely dissolved in a phosphate buffer solution at a temperature of 55-65°C, and then methacrylic anhydride is added, and the reaction is carried out under light-proof conditions for 1.5-2.5 hours. After the reaction is completed, it is permeated at room temperature for 6-8 days, and freeze-dried to obtain HAMA gel; PEGNB gel is dissolved in a phosphate buffer solution at a concentration of 10% w / v, HAMA gel is dissolved in a concentration of 2% w / v, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is dissolved in a concentration of 0.3% w / v, and lemon yellow light absorber is dissolved in a concentration of 0.1% w / v to obtain a composite hydrogel solution. Step 4, preparation of PH scaffold: use 3D drawing software to draw the stl format drawing of PH scaffold, put the composite hydrogel solution into the feed slot of DLP light-curing 3D printer, print to obtain PH scaffold, put the obtained PH scaffold into phosphate buffer solution for cleaning, and store at 3-5°C for future use; Step 5, scaffold assembly: the ZP scaffold and the PH scaffold are subjected to ultraviolet disinfection, and then rinsed with a phosphate buffer solution, and then the ZP scaffold and the PH scaffold are combined to form a macro-microstructure composite cylindrical bionic bone scaffold.
2. The method for preparing the composite cylindrical bionic bone scaffold according to claim 1, characterized in that: In step 1, the effective ingredient ratio of the methanol solution used is greater than 99%, wherein the 2-methylimidazole solution contains 1 g of 2-methylimidazole powder and 30 mL of methanol solution, and the zinc nitrate solution contains 300 mg of zinc nitrate hexahydrate and 10 mL of methanol solution.
3. The method for preparing the composite cylindrical bionic bone scaffold according to claim 1, characterized in that: In step 2, the printing parameters set in the control software of the FDM 3D printer are as follows: The nozzle temperature is 120°C, the nozzle aperture is 0.3mm, the interval is 0.75mm, the speed is 10mm / s, the extrusion speed is 0.01mm / s, and the side is drilled after the bracket is printed. The diameter of the drilled hole is 1mm, and the spacing between adjacent drilled holes is 1.5mm.
4. The method for preparing the composite cylindrical bionic bone scaffold according to claim 2, characterized in that: In step 2, the specific steps for flushing the ZP stent are as follows: Prepare 20 mL of 0.1 mol / L CaCl2 solution and 20 mL of 0.1 mol / L K2HPO4 solution, first immerse the ZP stent in the above CaCl2 solution for 4-6 minutes, then immerse the ZP stent in deionized water for 25-35 seconds, then immerse the ZP stent in the above K2HPO4 solution for 4-6 minutes, and finally immerse the ZP stent in deionized water for 25-35 seconds.
5. The method for preparing the composite cylindrical bionic bone scaffold according to claim 1, characterized in that: In step 2, the specific operations of mineralizing the ZP scaffold are as follows: The ZP scaffold was immersed in SBF simulated body fluid to deposit biomimetic hydroxyapatite, and the SBF simulated body fluid was changed every day to maintain consistent ionic strength. After immersion for 2-4 days, the ZP scaffold was gently rinsed with deionized water to remove the SBF simulated body fluid on the surface, and then vacuum freeze-dried for 20-30 hours.
6. The method for preparing the composite cylindrical bionic bone scaffold according to claim 1, characterized in that: In step three, the volumes of deionized water and anhydrous dichloromethane used are the same, wherein the mixture contains 0.1 mmol of polyethylene glycol, 0.4 mmol of nitrobenzene, and 50 mL of anhydrous dichloromethane, wherein the concentration of the HA hyaluronic acid solution is 10% w / v, the concentration of the phosphate buffer solution is 0.01 mol / L, and the amount of methacrylic anhydride used is 1 mL.
7. The method for preparing the composite cylindrical bionic bone scaffold according to claim 1, characterized in that: In step 4, the printing parameters set in the control software of the DLP light-curing 3D printer are as follows: Light intensity 14mW / cm 3 , light source wavelength is 405nm, exposure time is 27s, number of base layers is 5, base layer exposure time is 30s, peeling distance is 6mm, lifting height is 0mm, peeling speed is 25mm / min, lifting speed is 100mm / min, peeling recovery speed is 180mm / min, and slice layer height is 50um.
8. A composite cylindrical bionic bone scaffold, characterized in that: The method is prepared according to any one of claims 1 to 7.
9. The composite cylindrical bionic bone scaffold according to claim 8, characterized in that: It comprises a ZP bracket with a cylindrical structure and a PH bracket with a square cylindrical structure, wherein the outer peripheral wall of the ZP bracket is inscribed on four inner side walls of the PH bracket.
10. The composite cylindrical bionic bone scaffold according to claim 9, characterized in that: The ZP bracket is provided with a plurality of first holes arranged along the central axis direction and a plurality of second holes arranged along the radial direction, and the four peripheral walls of the ZP bracket are provided with square / circular / elongated third holes.