A bionic corn cob core stent with piezoelectric antibacterial, osteogenic and healing-promoting functions

Abstract

This invention discloses a biomimetic corncob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions, comprising: extraction of corncob cellulose nanofibers (CNF); preparation of 3D printing ink; 3D printing of a porous scaffold; loading of antimicrobial peptides and BMP-2 onto a GelMA+PAAM composite hydrogel; and composite preparation of the scaffold and hydrogel. Under external stimulation, the scaffold generates a microcurrent / surface charge, directly disrupting bacterial cell membranes; it avoids drug release and resistance issues, employing a physical mechanism of action with a sustainable response. The microcurrent / electric field generated by the piezoelectric effect simulates the bioelectric environment of natural bone, directly stimulating osteoblast proliferation and differentiation, and promoting mineralization; it mimics the natural anisotropic porous structure of the corncob, facilitating nutrient transport and cell migration; the piezoelectric material itself provides both antibacterial and osteogenic functions, achieving synergistic and unified mechanisms; it is suitable for harsh environments, biodegradable, and has flexible activation methods.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to a biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions. Background Technology

[0002] Bone defect repair is a significant challenge in clinical orthopedics, especially in complex situations such as war trauma, severe infection, or tumor resection, which often involve substantial bone loss and deterioration of the local microenvironment, presenting numerous limitations to traditional repair methods. Currently, commonly used bone repair materials include autologous bone, allogeneic bone, and synthetic materials. While autologous bone transplantation offers good biocompatibility and osteoinductive properties, it suffers from limited donor sites, bone harvesting trauma, and potential complications. Allogeneic bone transplantation, on the other hand, faces risks of immune rejection and disease transmission. Synthetic materials such as hydroxyapatite and tricalcium phosphate, although widely available, still require improvement in bioactivity, osteoconductivity, and integration with host bone, and often lack the function of actively promoting bone regeneration.

[0003] In the process of bone defect repair, infection is one of the key factors leading to treatment failure. Although traditional local or systemic application of antibiotics can control infection to some extent, long-term use can easily lead to bacterial resistance, and systemic administration often results in local drug concentrations that are difficult to achieve effective bactericidal levels, while also potentially producing toxic side effects. Therefore, developing bone repair materials with intrinsic antibacterial properties to reduce or avoid the use of antibiotics is currently a hot research topic.

[0004] Furthermore, natural bone tissue possesses a unique bioelectrical microenvironment, where osteocyte proliferation, differentiation, and the synthesis and mineralization of the extracellular matrix are all regulated by bioelectrical signals. However, most existing bone repair materials lack the ability to mimic this bioelectrical microenvironment, making it difficult to actively stimulate the bone regeneration process. Although some studies have attempted to assist bone repair through external electrical stimulation, this method is complex to operate and requires continuous equipment support, limiting its application in resource-constrained or wartime environments.

[0005] Porous structures are a key characteristic of ideal bone repair scaffolds, providing space for cell growth and facilitating nutrient exchange and waste removal. Natural biomaterials such as coral and wood, with their unique porous structures, are often used as inspiration for biomimetic scaffold design. However, effectively combining their natural structural advantages with functional properties (such as antibacterial and osteogenic induction) and achieving precise control over the scaffold structure through controllable fabrication methods (such as 3D printing) remains a pressing issue. Corn cobs, as an abundant source of agricultural waste, offer a good structural template for biomimetic bone scaffold fabrication due to their naturally anisotropic porous structure. However, they lack antibacterial and osteogenic activity, requiring functional modification to impart the necessary biological properties.

[0006] Hydrogels, as materials with high water content and good biocompatibility, are often used to load bioactive factors (such as growth factors and antimicrobial peptides) to regulate cell behavior. However, single hydrogels often suffer from insufficient mechanical properties and difficulty in controlling the release of bioactive factors. By combining hydrogels with different properties (such as gelatin methacryloyl (GelMA) and polyacrylamide (PAAM)) with scaffold materials, it is hoped that a synergistic composite system can be constructed to achieve the continuous and stable release of bioactive factors, while improving the overall mechanical properties and biological functions of the material.

[0007] In summary, the current field of bone defect repair urgently needs a multifunctional bone repair scaffold that can simultaneously address infection control, promote bone regeneration, achieve structural biomimicry, and adapt to special environments (such as wartime). Developing a method for preparing a biomimetic porous structure based on natural biomass (such as corn cob), possessing piezoelectric effects for physical antibacterial action and bioelectric stimulation of osteogenic formation, combining 3D printing technology for precise fabrication, and loading bioactive factors onto a composite functional hydrogel, will provide new ideas and technical approaches to solving these problems. Summary of the Invention

[0008] In view of this, the present invention provides a biomimetic corn cob scaffold with piezoelectric antibacterial, osteogenic and healing-promoting functions.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions, comprising: (1) Extraction of cellulose nanofibers (CNF) from corn cob pith Select corn cobs, crush and grind them into granules, rinse and dry them; Dry corn cob particles were added to a sodium hydroxide solution and stirred in a water bath. After the reaction was complete, the mixture was filtered, the residue was collected, washed, and dried to obtain delignified corn cob cellulose. The cellulose was added to deionized water in a certain proportion to form a uniform suspension; homogenization was performed, centrifugation was carried out, the supernatant was collected as CNF dispersion, and vacuum freeze-drying was performed to obtain dry CNF powder, which was then sealed and stored. (2) Preparation of 3D printing ink Hydroxyapatite (HAP) was dried in a muffle furnace, and 2-hydroxypropyltrimethylammonium chloride chitosan (HACC) was dried under vacuum conditions. Prepare the HACC solution and determine the CNF:HAP:HACC mass ratio; (3) 3D printing to prepare porous scaffolds Set the parameters for the 3D printer; coat the printing platform with polyethylene glycol (PEG) solution; monitor the molding quality in real time during the printing process; after printing, place the support in a vacuum drying oven to dry and obtain a preliminary molded support. Post-stent processing; (4) GelMA+PAAM composite hydrogel loaded with antimicrobial peptides and BMP-2 Add appropriate amounts of acrylamide (AAM) and N,N'-methylenebisacrylamide (MBAA) sequentially to a centrifuge tube, and then add them to a 0.25% phenyl-2,4,6-trimethylbenzoyl lithium phosphine sulfate (LAP) solution. Weigh out GelMA and add it to the above mixture. Heat in a 50°C water bath for 25 minutes, shaking several times during the process to ensure thorough mixing. The above steps require exploration of appropriate mixing ratios to determine the optimal compatibility, biocompatibility, biocompatibility, and drug release rate. Different concentrations of antimicrobial peptide LL-37 stock solution and BMP-2 stock solution were added sequentially to the base prepolymer solution to determine an appropriate ratio; viscosity was adjusted and degassing was performed. (5) Preparation of composite scaffold and hydrogel The dried and sterilized 3D printed porous scaffold is placed into a 24-well plate. The viscosity-adjusted composite prepolymer solution is slowly dripped onto the surface of the scaffold until it is completely submerged. The entire 24-well plate is then transferred to a vacuum dryer. The air in the pores of the scaffold is extracted under a low vacuum. The negative pressure is used to drive the prepolymer solution to fully penetrate into all the micropores of the scaffold. The vacuum is then slowly released, allowing atmospheric pressure to further promote the filling of the prepolymer solution. Remove the soaked scaffold with tweezers and absorb any excess prepolymer solution from the surface. Place the scaffold under 405nm blue light for 15-30 seconds to allow the GelMA / PAAM prepolymer solution to completely crosslink and solidify inside and on the surface of the scaffold, forming a stable composite network.

[0010] Preferably, step (1) specifically includes the following steps: ① Raw material pretreatment: Select corn cobs, crush and grind them into granules, rinse with water to remove surface dust and soluble impurities, and place the rinsed granules in a forced-air drying oven to dry; ② Delignin treatment: Add sodium hydroxide to the solution of dried corn cob particles and stir in a water bath; after the reaction is complete, filter with a Buchner funnel, collect the filter residue, wash with deionized water to remove residual alkali and lignin; dry the washed filter residue in a forced-air drying oven to obtain delignin-treated corn cob cellulose. ③ Preparation of cellulose nanofibers: The above cellulose was added to deionized water in proportion and a uniform suspension was formed by high-speed disperser; then homogenized by high-pressure homogenizer to decompose cellulose into nanofibers under high pressure shear; the homogenized suspension was centrifuged at 8000 r / min for 20 min to remove undecomposed coarse fibers, and the supernatant was collected as CNF dispersion. Finally, the CNF powder was obtained by vacuum freeze drying and sealed for storage. Preferably, in step (2), the method for preparing the HACC solution is as follows: add dried HACC to deionized water, stir and dissolve in a water bath, and cool to room temperature for later use; prepare the HAP dispersion according to the proportion, add sodium dodecylbenzenesulfonate as a dispersant, and use ultrasound to make it uniformly dispersed.

[0011] Preferably, in step (2), the method for determining the CNF:HAP:HACC mass ratio is as follows: CNF powder is first added to HACC solutions of different concentrations and magnetically stirred to ensure full dispersion; then HAP dispersions of different mass ratios are added and stirring is continued, with a glass rod used for stirring every 20 minutes to improve uniformity; the mixture is degassed under vacuum to obtain uniform and stable 3D printing ink, and a suitable ratio for printing, biomechanical strength and porosity is summarized; the ink viscosity is detected using a rotational viscometer and adjusted to be within the range of 10000–20000 mPa·s.

[0012] Preferably, in step (3), the method for setting the 3D printer parameters is as follows: using a fused deposition modeling (FDM) 3D printer, filling the ink barrel with ink, setting the barrel temperature and nozzle room temperature; based on the target support size and porosity, constructing a three-dimensional model using SolidWorks, adopting a layered staggered structure, exploring layer thickness and printing speed in groups, controlling porosity by adjusting the line spacing, converting the model into G-code and importing it into the printer, and rehearsing the printing path.

[0013] Preferably, in step (3), the post-treatment method of the stent is as follows: the dried stent is cross-linked with glutaraldehyde, rinsed with deionized water and then vacuum dried again. In order to improve the mechanical properties, it is heat-treated in N2 atmosphere to promote the cross-linking and curing of components. After the material preparation is completed, it is sterilized with Co60 for later use. Preferably, in step (4), the viscosity adjustment and degassing method is as follows: measure the viscosity; if adjustment is needed, add sterile deionized water and record the amount added; by adding sterile deionized water, the viscosity of the prepolymer solution is adjusted from 10000-20000 mPa. Adjust s to 5000-8000mPa The viscosity was measured using a rotational viscometer at 25°C and 50 r / min. The reduced viscosity can decrease the flow resistance of the hydrogel in the pores of the scaffold, ensuring full filling. The adjusted composite prepolymer solution needs to be left to stand in a sterile laminar flow hood to observe whether stratification or bubbles occur. If bubbles are present, ultrasonic degassing is required again to ensure that the prepolymer solution is uniform and free of bubbles, thus avoiding pore defects in the subsequent composite material. Preferably, in step (4), the method of storage and use is as follows: the prepared composite prepolymer solution is immediately placed in 37°C and stored in the dark, and is used up within 1 hour; during this period, it is gently blown and mixed once every 15 minutes with a sterile pipette to prevent the sedimentation of biological factors.

[0014] Preferably, in step (5), post-processing and storage: the prepared composite scaffold is gently rinsed twice with sterile PBS to remove a small amount of unreacted monomers. It is then placed in fresh PBS and stored at 4°C for subsequent biological performance testing.

[0015] Application of a piezoelectric antibacterial, osteogenic, and healing-promoting multifunctional biomimetic corn cob scaffold preparation method in special wartime medical environments.

[0016] The present invention achieves the following technical effects compared to the prior art: This invention uses cellulose nanofibers (CNF) extracted from corn cob pith, combined with hydroxyapatite (HAP) and 2-hydroxypropyltrimethylammonium chloride chitosan (HACC) to prepare 3D printing ink, thereby printing a biomimetic porous scaffold. This scaffold not only utilizes the natural anisotropic porous structure of corn cob to provide an ideal space for cell growth, but also endows the scaffold with good biocompatibility, osteoconductivity and certain mechanical properties through the synergistic effect of CNF, HAP and HACC. Subsequently, antimicrobial peptide LL-37 and bone morphogenetic protein-2 (BMP-2) were loaded onto a GelMA and PAAM composite hydrogel, and the viscosity of the prepolymer solution was precisely adjusted to 5000-8000 mPa. The s (measured at 25℃ and 50r / min) ensures that the hydrogel can fully penetrate and fill the pores of the scaffold, and forms a stable composite network after cross-linking and curing by blue light. This composite scaffold integrates the physical antibacterial and bioelectric stimulation osteogenic effects brought about by the piezoelectric effect (derived from CNF and other components). The antimicrobial peptides loaded on the hydrogel can effectively inhibit infection, while BMP-2 can actively promote bone regeneration. Meanwhile, the scaffold preparation process is controllable, the composite prepolymer solution is stored at 37°C in the dark and used within 1 hour, and the sterility and stability of the scaffold are ensured by PBS rinsing and storage at 4°C. Overall, the multifunctional biomimetic scaffold prepared by this method achieves a synergistic effect of structural biomimicry, antibacterial properties, piezoelectric stimulation of osteogenic formation, and sustained release of bioactive factors. It can effectively address the challenges of infection control and bone regeneration in the repair of complex bone defects such as wartime trauma, and is especially suitable for special medical environments with limited resources during wartime. It provides an efficient, multifunctional, and practical solution for bone defect repair. Attached Figure Description

[0017] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention discloses a biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions, comprising: (1) Extraction of cellulose nanofibers (CNF) from corn cob pith ① Raw material pretreatment: Select corn cobs, crush and grind them into granules, and rinse with water to remove surface dust and soluble impurities. Place the rinsed granules in a forced-air drying oven to dry.

[0020] ② Delignin treatment: Add sodium hydroxide to the solution of dried corn cob particles and stir in a water bath. After the reaction is complete, filter through a Buchner funnel, collect the filter residue, and wash with deionized water to remove residual alkali and lignin. Dry the washed filter residue in a forced-air drying oven to obtain deligninated corn cob cellulose.

[0021] ③ Preparation of cellulose nanofibers: The cellulose was added to deionized water in the above proportion and a homogeneous suspension was formed using a high-speed disperser. Subsequently, a high-pressure homogenizer was used for homogenization, causing the cellulose to decompose into nanofibers under high-pressure shear. The homogenized suspension was centrifuged at 8000 r / min for 20 min to remove undecomposed coarse fibers, and the supernatant, i.e., the CNF dispersion, was collected. Finally, the CNF powder was obtained by vacuum freeze-drying and stored in a sealed container.

[0022] (2) Preparation of 3D printing ink ① Raw material preparation: Hydroxyapatite (HAP) is dried in a muffle furnace, and 2-hydroxypropyltrimethylammonium chloride chitosan (HACC) is dried under vacuum to remove moisture.

[0023] ② Preparation of solutions for each component: Prepare the HACC solution: Add dried HACC to deionized water, stir to dissolve in a water bath, and cool to room temperature for later use. Prepare the HAP dispersion according to the specified ratio, adding sodium dodecylbenzenesulfonate as a dispersant, and use ultrasound to ensure uniform dispersion.

[0024] ③ Ink Compounding and Control: Exploring the CNF:HAP:HACC mass ratio to summarize suitable printing ink formulations. First, CNF powder was added to HACC solutions of different concentrations and magnetically stirred to ensure thorough dispersion. Then, HAP dispersions of different mass ratios were added, and stirring continued, with a glass rod used every 20 minutes to improve homogeneity. The mixture was then degassed under vacuum to obtain a uniform and stable 3D printing ink, and a suitable ratio for printing, biomechanical strength, and porosity was determined. The ink viscosity was measured using a rotational viscometer and controlled within the range of 10000–20000 mPa·s.

[0025] (3) 3D printing to prepare porous scaffolds ① 3D Printer Parameter Settings: A Fused Deposition Modeling (FDM) 3D printer is used. Ink is loaded into the barrel, and the barrel temperature and nozzle room temperature are set. Based on the target support size and porosity, a 3D model is built using SolidWorks, employing a layered, staggered structure. Layer thickness and printing speed are explored in groups, and porosity is controlled by adjusting the line spacing. The model is converted to G-code and imported into the printer to preview the printing path.

[0026] ② 3D printing process of the scaffold: The printing platform is coated with polyethylene glycol (PEG) solution to enhance adhesion. The molding quality is monitored in real time during the printing process. After printing, the scaffold is placed in a vacuum drying oven to dry, obtaining a preliminary scaffold.

[0027] ③ Post-treatment of the stent: The dried stent is cross-linked with glutaraldehyde, rinsed with deionized water, and then vacuum dried again. To improve mechanical properties, it is heat-treated in an N2 atmosphere to promote cross-linking and curing of the components. After the material preparation is completed, it is sterilized with Co60 for later use.

[0028] (4) GelMA+PAAM composite hydrogel loaded with antimicrobial peptides and BMP-2 ① Preparation of a basic prepolymer solution free of biological agents: Add appropriate amounts of acrylamide (AAM) and N,N'-methylenebisacrylamide (MBAA) sequentially to a centrifuge tube, then add to a 0.25% phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP) solution. Weigh GelMA and add it to the above mixture. Heat in a 50°C water bath for 25 minutes, shaking several times during the process to ensure thorough mixing. The above steps require exploration to determine the optimal mixing ratio to achieve the best biocompatibility, biocompatibility, and drug release rate.

[0029] ② Add bioactive factors: Add different concentrations of antimicrobial peptide LL-37 stock solution and BMP-2 stock solution to the basic prepolymer solution sequentially to explore the appropriate ratio; gently pipette to ensure thorough mixing and avoid generating air bubbles. Accurately bring the volume to 5.0 mL with pre-cooled PBS and gently mix again.

[0030] ③ Viscosity Adjustment and Degassing: Measure the viscosity. If adjustment is needed, add sterile deionized water dropwise and record the amount added. By adding sterile deionized water, the viscosity of the prepolymer solution can be adjusted from 10,000-20,000 mPa. Adjust s to 5000-8000mPa The viscosity (s) is measured using a rotational viscometer at 25°C and 50 rpm. Lower viscosity reduces the flow resistance of the hydrogel within the scaffold pores, ensuring adequate filling. The adjusted composite prepolymer solution needs to be allowed to stand in a sterile laminar flow hood to observe for stratification or bubbles. If bubbles are present, ultrasonic degassing must be performed again to ensure the prepolymer solution is uniform and bubble-free, preventing pore defects in the subsequent composite material.

[0031] ④ Storage and Use: The prepared composite prepolymer solution should be immediately stored at 37°C in the dark and used within 1 hour. During this period, gently mix the solution every 15 minutes using a sterile pipette to prevent sedimentation of biological agents.

[0032] (5) Preparation of composite scaffold and hydrogel ① Scaffold pretreatment: The dried and sterilized 3D printed porous scaffold is placed into a 24-well plate.

[0033] ② Vacuum-assisted impregnation: Slowly add the viscosity-adjusted composite prepolymer solution to the scaffold surface using a pipette until it is completely submerged. Transfer the entire 24-well plate to a vacuum dryer, and extract the air from the scaffold pores under a low vacuum (e.g., 5-10 kPa). The negative pressure drives the prepolymer solution to fully penetrate all the micropores of the scaffold. Slowly release the vacuum, allowing atmospheric pressure to further promote the filling of the prepolymer solution.

[0034] ③ Photocrosslinking and curing: Carefully remove the impregnated scaffold with tweezers and absorb any excess prepolymer solution from the surface. Place the scaffold under 405nm blue light for 15-30 seconds to allow the GelMA / PAAM prepolymer solution to completely crosslink and cure inside and on the surface of the scaffold, forming a stable composite network.

[0035] ④ Post-processing and storage: Gently rinse the prepared composite scaffold twice with sterile PBS to remove any unreacted monomers. Place it in fresh PBS and store at 4°C for subsequent biological performance testing.

[0036] Mechanical strength and porosity testing: ① The flexural strength (FS) and elastic modulus (EM) of each group of composite supports were measured using a universal testing machine: 10mm × 10mm × 8mm cuboid specimens were prepared, freeze-dried, and tested using a universal testing machine with a span of 20mm and a loading speed of 1mm·min⁻¹. The maximum load (F) was recorded in N. FS and EM were calculated using the formulas: FS = 3FL / 2bh², EM = FL³ / 4bdh³, where L (mm) is the support spacing, b (mm) is the specimen width, h (mm) is the specimen thickness, and d is the FS corresponding to the load F (N).

[0037] ② SEM observation of the cross-sectional morphology of each group of composite resins: The fracture surface morphology of the specimens after the three-point bending test was observed by SEM.

[0038] ③ Vickers microhardness test: Prepare a cylindrical specimen with a diameter of 6 mm and a height of 4 mm. After storing it in distilled water at 37℃ in the dark for 24 h, measure the surface hardness using a microhardness tester (loaded force 980 mN, loading time 10 s). The unit is HV.

[0039] ④ Porosity detection: The porosity of the prepared scaffold was detected by mercury intrusion porosimetry.

[0040] Research on wartime medical environment adaptability: The porous scaffold and its composite hydrogel system based on corn cob cellulose nanofibers (CNF) developed in this invention not only focus on its biological and mechanical properties, but also prospectively evaluate its application potential and adaptability in special medical environments during wartime.

[0041] ① Low Cost and Resource Availability Assessment: The core raw material, corn cob, is agricultural waste, widely available, and extremely low-cost, even zero-cost. This study will systematically analyze the material costs (including chemicals and energy consumption) from raw materials to the final scaffold, and compare them with traditional polymeric scaffold materials (such as polycaprolactone PCL and polylactic acid PLA) to quantify its cost advantages. To cope with wartime supply chain disruptions, the feasibility of alternative solutions will be assessed. For example, in the delignification process, the treatment effects of different concentrations of alkaline solutions (such as sodium hydroxide and potassium hydroxide) will be investigated, as well as the impact of using low-purity industrial-grade chemicals on the performance of the final product, ensuring that qualified materials can still be produced when high-purity reagents are unavailable. The consumption of deionized water in all process steps will be recorded, and simple rainwater collection, filtration, and distillation schemes will be designed to assess the feasibility of using simple purified water for key steps (such as rinsing and solution preparation) in the absence of pure water preparation equipment.

[0042] ② Portability and Rapid Deployment Research: Field hospitals or frontline aid stations require lightweight equipment and simple processes. The weight of composite materials of equal volume will be compared with that of bone cement, aiming to achieve a lighter weight than traditional materials like bone cement for easier portability. The final prepared dry porous scaffold (before hydrogel impregnation) will undergo accelerated aging experiments (e.g., high temperature and high humidity environments) to test its mechanical property retention and sterilization stability after long-term storage, in order to determine its suitability for the storage and transportation of wartime supplies.

[0043] Example 1: In vitro experiment (1) Experimental materials and model construction: ① Cell culture: Mouse osteogenic progenitor cells (MC3T3-E1) were selected as the research object. After revival, they were cultured in osteogenic induction medium and placed in a 37℃, 5% CO2 incubator. The medium was changed every 2 days, and cells in the logarithmic growth phase were used for experiments.

[0044] ② Preparation of composite material samples: Prepare materials according to the method described above and group them as follows: a. scaffold group; b. GelMA / PAAM@scaffold group; c. GelMA / PAAM / LL-37@scaffold group; d. GelMA / PAAM / LL-37 / BMP-2@scaffold group. Prepare 3 parallel samples for each group, with a size of 10mm×10mm×6mm. After sterilization, they are ready for use.

[0045] ③ Establishment of cell seeding and culture system: Place each group of samples in a 24-well plate, add 1 ml of osteogenic induction medium to each well, and pre-incubate for 24 h; then discard the medium and seed 5 × 10⁶ cells per well. 4 Use 10 cells and gently shake the culture plate to distribute the cells evenly on the sample surface. Set up 3 replicates for each group at each time point.

[0046] (2) Detection of biocompatibility and osteogenic related indicators ①Cell proliferation was detected using the CCK-8 assay: Detection was performed on days 1, 3, and 5 of culture. 100 μL of CCK-8 working solution was added to each well, and the cells were incubated at 37°C for 2 hours. Then, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance (OD value) was measured at 450 nm using a microplate reader. The relative cell proliferation rate (%) was calculated using control group 2 as a baseline.

[0047] ②Inflammatory response: The reaction was detected on days 1, 3, and 5 of culture. The expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) in each group were detected using the ELISA kit.

[0048] ③ Cell morphology: Rhodamine-labeled phalloidin was used to stain cytoskeleton microfilaments (green area) and DAPI was used to stain cell nuclei (blue area). The adhesion of cells to the composite material was observed using a fluorescence microscope after 24h and 48h.

[0049] ④ Alkaline phosphatase (ALP) activity assay: Samples were collected on days 7 and 14 of culture. After washing twice with PBS, 200 μL of cell lysis buffer was added, and the cells were lysed at 4°C with shaking for 30 minutes. The lysate was collected. 50 μL of the lysate was taken, and p-nitrophenyl phosphate solution was added according to the ALP assay kit instructions. The reaction was terminated by adding stop solution, and the OD value was measured at 405 nm. Simultaneously, the total protein concentration in the lysate was determined using a BCA protein quantification kit, and ALP activity was standardized to "U / mg protein".

[0050] ⑤ Alizarin Red S Method for Detection and Quantitative Analysis of Calcified Nodules: Detection was performed on days 14 and 21 of culture. The culture medium was discarded, and the nodules were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes. 0.1% Alizarin Red S staining solution was added, and staining was performed at room temperature for 30 minutes. The nodules were repeatedly washed with PBS until no excess staining solution remained. The formation of calcium nodules was observed and photographed under an optical microscope, and the area ratio of calcium nodules was analyzed using ImageJ software. After staining, the nodules were soaked in 10% acetic acid solution for 24 hours. After dissolution by shaking, the supernatant was collected by centrifugation. The OD value was measured at 570 nm according to the instructions of the calcium content detection kit (o-crestyrene complex ketone method), and the calcium content was calculated based on the standard curve.

[0051] ⑥ Osteocalcin (OCN) secretion detection: On days 14 and 21 of culture, the supernatant of the culture medium from each well was collected, and cell debris was removed after centrifugation. The concentration of OCN in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA) according to the instructions of the OCN detection kit.

[0052] ⑦ Immunofluorescence detection of type I collagen (Col-I) expression: On day 5 of culture, samples were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked at room temperature for 1 hour. Rabbit anti-mouse Col-I primary antibody (dilution ratio 1:200) was added, and the samples were incubated overnight at 4°C. After washing three times with PBS, goat anti-rabbit secondary antibody labeled with Alexa Fluor 594 (dilution ratio 1:500) was added, and the samples were incubated at room temperature in the dark for 1 hour. After nuclear staining with DAPI, the expression and distribution of Col-I were observed under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed using ImageJ software.

[0053] (3) Research on antibacterial mechanism ① Detection of antimicrobial peptide LL-37 release kinetics: The experimental composite material was placed in a centrifuge tube, and 5 ml of PBS was added and incubated in a shaker at 37℃. 1 mL of release solution was collected on days 1, 5, 9, 11, 14, and 21, followed by the addition of 1 mL of PBS. The collected sample was immediately added to a microcentrifuge tube, and 10 μL of 10% acetic acid was added to lower the pH, inactivate the protease activity, and terminate any possible release reaction. The collected sample was centrifuged at 13,000 rpm for 10 minutes (4℃), and the supernatant was collected. The supernatant was transferred to a low-adsorption HPLC vial for high-performance liquid chromatography-fluorescence detection (HPLC-FLD).

[0054] ② Antibacterial performance test: Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶ with PBS. 6 CFU / mL. Mix 1 mL of bacterial suspension with 0.1 mL of antimicrobial peptide release solution and incubate at 37°C for 1 hour. A blank control was also provided: bacterial suspension + PBS. After centrifugation, collect the bacteria, wash twice with PBS, and add the SYTO9 / PI double staining kit (dilution ratio 1:1000). Incubate at room temperature in the dark for 15 minutes. Observe the bacterial staining under a fluorescence microscope and count the ratio of live to dead bacteria.

[0055] ③ Long-term antibacterial experiment: The three samples from the experimental group and the control group were respectively subjected to 1×10 6 The bacterial suspensions were co-cultured with CFU / mL bacterial culture, and the number of viable bacteria was determined by colony counting after 24 hours to calculate the inhibition rate.

[0056] (4) Study on osteogenic mechanism ① BMP-2 release kinetics assay: The experimental composite material was placed in 5 mL of sterile PBS and incubated in a shaker at 37 °C. 1 mL of the release solution was collected at days 1, 3, 7, 14, and 21, and 1 mL of fresh PBS was added. Following the instructions of the BMP-2 ELISA kit, the concentration of BMP-2 in the release solution at each time point was measured, and release curves were plotted.

[0057] ② Western Blot detection of BMP-2 / Smad signaling pathway proteins: Cells were collected on day 3 of culture, washed twice with PBS, and lysed on ice for 30 minutes with RIPA lysis buffer. The supernatant was collected by centrifugation, and protein concentration was determined by BCA method. 30 μg of protein sample was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk for 1 hour. Rabbit anti-mouse BMPR-II, p-Smad1 / 5 / 8, Smad1 / 5 / 8, and Runx2 primary antibodies were added, and incubated overnight at 4°C. After washing with TBST, HRP-labeled secondary antibody was added, and the cells were incubated at room temperature for 1 hour. Development was performed using ECL chemiluminescence, and the gray values ​​of the protein bands were analyzed using ImageJ software.

[0058] ③qRT-PCR detection of osteogenic-related gene expression: Cells were collected on days 7 and 14 of culture, and total RNA was extracted using the Trizol method. RNA purity and concentration were then determined. 1 μg of RNA was reverse transcribed into cDNA. Using GAPDH as an internal reference gene, qPCR was performed using SYBR Green PCRMaster Mix under the following conditions: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds; 60℃ annealing for 30 seconds, for 40 cycles. The relative expression levels of Runx2, ALP, Col-I, and OCN genes were calculated using the 2^(-ΔΔCt) method.

[0059] ④ Sirius red staining to detect extracellular matrix (ECM) synthesis: After 21 days of culture, the cell slides were washed three times with PBS, then fixed with 4% paraformaldehyde fixative for 15 minutes at room temperature, followed by three washes with PBS. 0.1% Triton X-100 was added for permeation at room temperature for 5 minutes, followed by three washes with PBS. DAPI staining solution was added, and the slides were stained for 5 minutes at room temperature in the dark, followed by three washes with PBS. 0.1% Sirius red picric acid solution was added to the cell slides, and the slides were stained for 30 minutes at room temperature in the dark. The distribution and arrangement of collagen fibers were observed under a microscope, and the area ratio of collagen fibers was quantitatively analyzed using ImageJ software to assess ECM synthesis.

[0060] ⑤ Detection of mineralization-related enzyme activity: Cell lysates were collected at 7 and 14 days of culture, and the activity of mineralization-related enzymes was determined using an ALP and osteopontin (OPN) detection kit.

[0061] Example 2: In vivo experiment (1) Preparatory work for the experiment ① Experimental animals and ethical approval: Animal selection: SD rats were selected; the ethics complied with the requirements of the hospital's ethics committee.

[0062] ② Preparation of experimental materials and reagents: Materials were prepared as described above and grouped into the following groups: a. scaffold group; b. GelMA / PAAM@scaffold group; c. GelMA / PAAM / LL-37@scaffold group; d. GelMA / PAAM / LL-37 / BMP-2@scaffold group; e. blank control group.

[0063] (2) Construction of infectious bone defect model ① Animal grouping and preoperative preparation: Grouping: 50 SD rats were randomly divided into 5 groups of 10 each: scaffold group; GelMA / PAAM@scaffold group; GelMA / PAAM / LL-37@scaffold group; GelMA / PAAM / LL-37 / BMP-2@scaffold group; and blank control group.

[0064] ② Surgical procedure: Male SD rats weighing approximately 200g-220g were used to construct an infectious skull defect model. The specific model preparation method is as follows: Anesthesia was performed by intraperitoneal injection of 1.5% sodium pentobarbital (30mg / kg). After anesthesia, the skin of the rat skull was prepared at the surgical site using a shaving razor. After preparation, the rat was fixed in a prone position on the operating table, and the surgical area was thoroughly disinfected with povidone-iodine disinfectant. After disinfection, a 1cm long incision was made along the long axis of the midline of the rat skull, and the subcutaneous soft tissue was separated layer by layer to fully expose the skull. Then, a full-thickness skull defect was carefully and slowly constructed on both sides of the midline of the skull using a 6mm diameter hollow trephine. After completion, a pre-prepared collagen sheet containing 100 μl of bacterial suspension (Staphylococcus aureus, 107 CFU / ml) was placed at the defect site. The subcutaneous tissue and skin were sutured layer by layer using silk sutures. The skin was then disinfected after suturing, completing the first surgery. Seven days after the first surgery, the rats were anesthetized, disinfected, and prepared using the same method. The skin was incised along the original incision, and the layers were dissected until the skull was exposed. The collagen sheet from the defect was removed, and implants were placed according to the assigned group. The wound was then sutured, completing the second surgery.

[0065] (3) Validation of in vivo anti-infective properties ① General observation and sample collection: On postoperative days 1, 3, 7, 14, 21, and 28, the mental state (activity level, responsiveness), weight changes (weighed weekly), wound healing (redness, swelling, oozing, crusting), and limb movement (lameness, limb swelling) of rats in each group were observed. The infection rate of each group was also recorded. Sample collection: At 4 and 8 weeks postoperatively, 5 rats from each group were randomly selected and euthanized under an overdose of sodium pentobarbital. The entire skull was separated under aseptic conditions, and the surrounding soft tissue was ground for bacterial culture and counting. The specimens were fixed with 4% paraformaldehyde for Micro-CT detection, and the specimens were then subjected to histological analysis.

[0066] ② Methods for testing anti-infection performance: Bone tissue bacterial count: Skull samples were collected at 4 and 8 weeks post-surgery. Under aseptic conditions, tissue from the bone defect area was excised, placed in a sterile grinding tube, and 1 mL of sterile saline was added. The tissue was then homogenized. The homogenate was serially diluted, and different dilutions were spread onto agar plates. After 24 hours of incubation, the number of colonies was counted, and the differences in bacterial load among the groups were compared. Histological inflammation analysis (HE staining): Fixed skull samples were decalcified with EDTA decalcification solution for 4 weeks. After decalcification, the samples were dehydrated, paraffin-embedded, sectioned, and stained with HE. The infiltration of inflammatory cells (neutrophils, lymphocytes) in the bone defect area was observed under an optical microscope. The number of inflammatory cells was counted using ImageJ software to assess the degree of inflammatory response.

[0067] (4) Verification of osteogenic properties in vivo ① Micro-CT Imaging Examination: At 4 and 8 weeks post-surgery, the skulls of sacrificed rats were harvested, soft tissues were removed, and micro-CT scans were performed. Image reconstruction was performed, and osteogenic parameters of the bone defect area were analyzed: Bone volume fraction (BV / TV): the percentage of bone tissue volume to the total volume of the region of interest (ROI); Trabecular bone number (Tb.N, mm). - ¹): Number of trabeculae per unit length; Trabecular thickness (Tb.Th, mm): Average thickness of trabeculae; Bone defect healing rate (%).

[0068] ② Histological staining analysis: HE staining: observe the morphology of newly formed bone tissue, trabecular structure, and material degradation in the bone defect area to assess the degree of bone tissue repair; Masson staining: observe the distribution of collagen fibers under a microscope and calculate the proportion of collagen fiber area using ImageJ software; Safranin O-Fix Green staining (distinguishing between cartilage and bone tissue): after staining, mount the sections and observe the ratio of cartilage tissue (red) to bone tissue (green).

[0069] ③ Osteogenesis-related gene and protein expression detection: Immunohistochemistry was used to detect osteogenic protein expression, and positive expression areas (brownish-yellow) were observed under an optical microscope. ImageJ software was used to calculate the percentage of positive expression area.

[0070] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions, characterized in that, include: (1) Extraction of cellulose nanofibers (CNF) from corn cob pith Select corn cobs, crush and grind them into granules, rinse and dry them; Dry corn cob particles were added to a sodium hydroxide solution and stirred in a water bath. After the reaction was complete, the mixture was filtered, the residue was collected, washed, and dried to obtain delignified corn cob cellulose. The cellulose was added to deionized water in a certain proportion to form a uniform suspension; homogenization was performed, centrifugation was carried out, the supernatant was collected as CNF dispersion, and vacuum freeze-drying was performed to obtain dry CNF powder, which was then sealed and stored. (2) Preparation of 3D printing ink Hydroxyapatite (HAP) was dried in a muffle furnace, and 2-hydroxypropyltrimethylammonium chloride chitosan (HACC) was dried under vacuum conditions. Prepare the HACC solution and determine the CNF:HAP:HACC mass ratio; (3) 3D printing to prepare porous scaffolds Set the parameters for the 3D printer; coat the printing platform with polyethylene glycol (PEG) solution; monitor the molding quality in real time during the printing process; after printing, place the support in a vacuum drying oven to dry and obtain a preliminary molded support. Post-stent processing; (4) GelMA+PAAM composite hydrogel loaded with antimicrobial peptides and BMP-2 Add appropriate amounts of acrylamide (AAM) and N,N'-methylenebisacrylamide (MBAA) sequentially to a centrifuge tube, and then add them to a 0.25% phenyl-2,4,6-trimethylbenzoyl lithium phosphine sulfate (LAP) solution. Weigh out GelMA and add it to the above mixture. Heat in a 50°C water bath for 25 minutes, shaking several times during the process to ensure thorough mixing. The above steps require exploration of appropriate mixing ratios to determine the optimal compatibility, biocompatibility, biocompatibility, and drug release rate. Different concentrations of antimicrobial peptide LL-37 stock solution and BMP-2 stock solution were added sequentially to the base prepolymer solution to determine an appropriate ratio; viscosity was adjusted and degassing was performed. (5) Preparation of composite scaffold and hydrogel The dried and sterilized 3D printed porous scaffold is placed into a 24-well plate. The viscosity-adjusted composite prepolymer solution is slowly dripped onto the surface of the scaffold until it is completely submerged. The entire 24-well plate is then transferred to a vacuum dryer. The air in the pores of the scaffold is extracted under a low vacuum. The negative pressure is used to drive the prepolymer solution to fully penetrate into all the micropores of the scaffold. The vacuum is then slowly released, allowing atmospheric pressure to further promote the filling of the prepolymer solution. Remove the soaked scaffold with tweezers and absorb any excess prepolymer solution from the surface. Place the scaffold under 405nm blue light for 15-30 seconds to allow the GelMA / PAAM prepolymer solution to completely crosslink and solidify inside and on the surface of the scaffold, forming a stable composite network.

2. The biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, Step (1) specifically includes the following steps: ① Raw material pretreatment: Select corn cobs, crush and grind them into granules, rinse with water to remove surface dust and soluble impurities, and place the rinsed granules in a forced-air drying oven to dry; ② Delignin treatment: Add sodium hydroxide to the solution of dried corn cob particles and stir in a water bath; after the reaction is complete, filter with a Buchner funnel, collect the filter residue, wash with deionized water to remove residual alkali and lignin; dry the washed filter residue in a forced-air drying oven to obtain delignin-treated corn cob cellulose. ③ Preparation of cellulose nanofibers: The above-mentioned cellulose was added to deionized water in proportion and a uniform suspension was formed by high-speed dispersion. Then, a high-pressure homogenizer was used for homogenization to decompose the cellulose into nanofibers under high pressure shear. The homogenized suspension was centrifuged at 8000 r / min for 20 min to remove undecomposed coarse fibers. The supernatant was collected as CNF dispersion. Finally, the CNF powder was obtained by vacuum freeze drying and sealed for storage.

3. The biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (2), the method for preparing the HACC solution is as follows: add dried HACC to deionized water, stir and dissolve in a water bath, and cool to room temperature for later use; prepare the HAP dispersion according to the ratio, add sodium dodecylbenzenesulfonate as a dispersant, and use ultrasound to make it uniformly dispersed.

4. The biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (2), the method for determining the CNF:HAP:HACC mass ratio is as follows: CNF powder is first added to HACC solutions of different concentrations and magnetically stirred to ensure full dispersion; then HAP dispersions of different mass ratios are added and stirring is continued, with a glass rod used for stirring every 20 minutes to improve uniformity; the mixture is degassed under vacuum to obtain uniform and stable 3D printing ink, and a suitable ratio for printing, biomechanical strength and porosity is summarized; the ink viscosity is detected using a rotational viscometer and adjusted to be within the range of 10000–20000 mPa·s.

5. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (3), the method for setting the parameters of the 3D printer is as follows: using a fused deposition modeling (FDM) 3D printer, filling the ink into the barrel, setting the barrel temperature and nozzle room temperature; based on the target support size and porosity, constructing a three-dimensional model using SolidWorks, adopting a layered staggered structure, exploring layer thickness and printing speed in groups, controlling porosity by adjusting the line spacing, converting the model into G-code and importing it into the printer, and rehearsing the printing path.

6. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (3), the post-treatment method of the stent is as follows: the dried stent is cross-linked with glutaraldehyde, rinsed with deionized water and then vacuum dried again. In order to improve the mechanical properties, it is heat-treated in N2 atmosphere to promote the cross-linking and curing of components. After the material preparation is completed, it is sterilized with Co60 for later use.

7. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (4), the viscosity adjustment and degassing method is as follows: Measure the viscosity; if adjustment is needed, add sterile deionized water and record the amount added. By adding sterile deionized water, the viscosity of the prepolymer solution can be adjusted from 10,000-20,000 mPa. Adjust s to 5000-8000mPa The viscosity was measured using a rotational viscometer at 25°C and 50 r / min. The reduced viscosity can decrease the flow resistance of the hydrogel in the pores of the scaffold, ensuring full filling. The adjusted composite prepolymer solution needs to be left to stand in a sterile clean bench to observe whether stratification or bubbles occur. If bubbles are present, ultrasonic degassing is required again to ensure that the prepolymer solution is uniform and free of bubbles, thus avoiding pore defects in the subsequent composite material.

8. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (4), the method of storage and use is as follows: the prepared composite prepolymer solution is immediately placed in 37°C and stored in the dark, and is used up within 1 hour; during this period, it is gently blown and mixed once every 15 minutes with a sterile pipette to prevent the sedimentation of biological factors.

9. A biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions according to claim 1, characterized in that, In step (5), post-processing and storage: The prepared composite scaffold is gently rinsed twice with sterile PBS to remove any unreacted monomers. It is then placed in fresh PBS and stored at 4°C for subsequent biological performance testing.

10. The application of the biomimetic corn cob scaffold with piezoelectric, antibacterial, osteogenic, and healing-promoting functions as described in claims 1-9 in special medical environments during wartime.

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