Cryo-printed scaffolds with synergistic signaling molecules, their fabrication methods and applications

Abstract

The application belongs to the field of bone tissue engineering, and particularly relates to a frozen 3D printing scaffold with a synergistic signal molecule, a preparation method and application thereof, and an ink composition with the synergistic signal molecule, which comprises a first porous carrier loaded with a CO molecule prodrug and a second porous carrier loaded with a NO molecule prodrug, the surface of the first porous carrier is coated with a first encapsulating layer, the first encapsulating layer is used for plugging the CO molecule prodrug in the pores of the first porous carrier, and the first encapsulating layer can self-degrade and release the CO molecule under high oxygen activity conditions; the surface of the second porous carrier is coated with a second encapsulating layer, the second encapsulating layer is used for plugging the NO molecule prodrug in the pores of the second porous carrier, and the second encapsulating layer can self-degrade and release the NO molecule under acidic conditions. The method of the application can obtain a scaffold which is relatively complete in structure, and which is timely vascularized and stable in bone formation.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering, and particularly relates to a cryogenic 3D printed scaffold with synergistic signaling molecules, its preparation method and application. Background Technology

[0002] Bone tissue engineering is crucial for the structural and functional repair of bone defects. The ultimate goal of bone tissue engineering is to reconstruct the functional structure of the bone defect site and achieve biomimetic results in the scaffold structure and mechanical properties of normal bone tissue. Soft and hard tissue injuries often occur simultaneously in severe bone defects, cartilage damage, tooth loss, or tendon injuries, and current methods of repairing only soft or hard tissue are far from sufficient.

[0003] Choosing the right scaffold material is one of the key factors for successful bone repair. Currently, the main clinical treatments for bone defects include autologous bone grafting and allogeneic bone grafting. Autologous bone is considered an ideal material due to its biocompatibility, strength, elasticity, and good shapeability; however, the quantity of autologous bone is limited, and the harvesting site may cause pain or discomfort, leading to complications. Allogeneic bone grafting is an alternative to autologous bone grafting, but it carries the risk of transmitting donor diseases and causing infections, and may also lead to immune rejection in the recipient organism.

[0004] The main problems with current bone tissue engineering scaffolds include poor mechanical properties, excessively rapid or slow degradation rates, poor biofunctionality, and susceptibility to inflammatory responses. Therefore, research into using tissue engineering to construct composite materials that mimic the structure of human bone tissue is receiving increasing attention. Ideal bone repair materials can not only mimic the tissue microenvironment but also regulate the activity of multiple cells to achieve desired biological functions. Designing bone repair scaffolds that mimic the natural layered structure of bone for both soft and hard tissue repair helps improve the biocompatibility of bone scaffolds and their ability to induce osteogenic differentiation of stem cells in vitro.

[0005] Bone immunomodulation is a novel strategy developed in recent years to promote bone immune homeostasis by regulating the interaction between immune cells and osteocytes. It involves the interaction between bone regeneration and immune system function. After implantation of a scaffold with good immunomodulatory properties, the immune system is activated, the body releases bioactive molecules, regulates local inflammatory responses, and promotes the differentiation of bone marrow stem cells into osteoblasts, thereby promoting bone growth and repair. Bone repair is a precisely regulated process involving multiple steps such as inflammatory response, angiogenesis, bone resorption, and new bone formation. Macrophages are among the most important effector cells in the body's immune response, playing a dynamic regulatory role in balancing osteoclast-osteogenesis.

[0006] The key to balancing osteoclast and osteogenic activity in macrophages during bone repair lies in their phenotypic transformation and the different cytokines they release. Macrophages can transform into different phenotypes according to different physiological needs, such as classically activated M1 macrophages and selectively activated M2 macrophages. M1 macrophages mainly participate in inflammatory responses and osteoclastogenesis by producing pro-inflammatory cytokines, activating and recruiting other immune cells, and destroying old bone to clear dead bone and debris from the injury site. M2 macrophages, on the other hand, reduce inflammation by producing anti-inflammatory cytokines, thereby promoting tissue repair and osteogenic activity and promoting new bone formation. In the early stages of bone repair, an appropriate inflammatory response is necessary to clear dead cells and debris from the injury site. However, as bone repair progresses, macrophages need to transform from M1 to M2 to promote bone tissue formation and repair. Therefore, regulating the M1 / M2 balance of macrophages is crucial for regulating osteoclast and osteogenic activities.

[0007] Regulating the M1 / M2 phenotypic transformation of macrophages is crucial for bone repair. In the early stages of scaffold implantation, the goal is to upregulate the expression of inflammatory genes and promote the transformation of macrophages to the M1 phenotype, thereby clearing inflammation at the site of bone injury. However, maintaining the M1 phenotype may subsequently kill newly generated bone cells. Therefore, how to regulate macrophage phenotype has become an urgent problem to be solved.

[0008] Due to the complex and diverse shapes and structures of bone defects, current research increasingly utilizes 3D printing technology to construct bone graft substitutes. 3D printing, commonly known as additive manufacturing, refers to the process of designing a 3D model using computer-aided design (CAD) software and then stacking and printing it layer by layer. Each layer allows for precise control over the position and thickness of the material, facilitating the fabrication of high-quality scaffolds. 3D printing enables personalized customization, allowing scaffolds to be tailored to the specific bone defect site based on the patient's needs. This technology also helps in the precise control of the scaffold's microstructure, including pore size, total pore volume, and shape. Although currently lacking clinical evidence and regulatory approval, 3D printing technology can meet the personalized needs of patients, printing three-dimensional scaffolds with controllable structures and pore sizes to promote cell migration and bone growth.

[0009] Currently, scaffolds obtained through traditional extrusion 3D printing may form pores in the material due to the characteristics of the printing technology itself, and the processing precision is difficult to control, affecting the porosity, pore size and connectivity of the scaffold. During the printing process, it is difficult to process and shape slurries with good fluidity, and the printed scaffolds are prone to structural collapse and side hole collapse, affecting the integrity and functionality of the bone scaffold structure.

[0010] Compared to traditional extrusion 3D printing, cryogenic 3D printing can control the flowability of materials at low temperatures, overcoming problems such as easy collapse and deformation during the process of general extrusion 3D printing scaffolds, and achieving more precise control over shape and structure. However, existing cryogenic 3D printing still struggles to obtain bone repair scaffold materials with good performance. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides a cryo-3D printed scaffold with synergistic signaling molecules, its preparation method, and its application, which can yield a scaffold with a relatively complete structure, timely vascularization, and stable bone formation.

[0012] This invention provides the following technical solution:

[0013] In a first aspect, the present invention provides an ink composition having synergistic signaling molecules, the ink composition comprising a first porous carrier loaded with a CO molecule prodrug and a second porous carrier loaded with an NO molecule prodrug, the surface of the first porous carrier being coated with a first encapsulation layer, the first encapsulation layer being used to seal the CO molecule prodrug in the pores of the first porous carrier, and the first encapsulation layer being capable of self-degradation and releasing CO molecules under high oxygen activity conditions; the surface of the second porous carrier being coated with a second encapsulation layer, the second encapsulation layer being used to seal the NO molecule prodrug in the pores of the second porous carrier, and the second encapsulation layer being capable of self-degradation and releasing NO molecules under acidic conditions.

[0014] According to an embodiment of the present invention, the ink composition further includes hydroxyapatite, bioactive glass, polylactic acid, polyvinyl alcohol, and chitosan.

[0015] According to an embodiment of the present invention, the ink composition further includes a solvent, the solvent including one or more of deionized water, ethanol, propanol, dichloromethane, dimethyl sulfoxide, dimethylformamide, and glycerol.

[0016] According to an embodiment of the present invention, the CO molecule prodrug is selected from materials that can release CO molecules in body fluids, preferably selected from one or more of the following: carbon-based manganese and other metal carbonyl compounds, carbonyl imidazoles, nitryl compounds (such as silver nitrate, iron nitrate, etc.), heme derivatives (such as thioheme, etc.), and CO metal complexes, for example, carbon-based manganese.

[0017] According to an embodiment of the present invention, the NO molecule prodrug is selected from materials that can release NO molecules in body fluids, preferably selected from one or more of arginine, citrulline, dimethylarginine, nitro drugs (such as sodium nitroprusside, nitrobenzene, etc.), organic nitrate esters (such as glycerol trinitrate, isosorbide mononitrate, etc.), nitrosothiolate compounds, metal-nitrosoyl complexes, and diol diazepinenium salts, for example, arginine.

[0018] According to an embodiment of the present invention, the first porous carrier is selected from biocompatible mesoporous materials, preferably one or more of mesoporous hydroxyapatite, mesoporous bioactive glass, mesoporous silica, mesoporous carbon, metal-organic frameworks (MOFs, such as ZIF series), and metal oxide mesoporous materials (such as mesoporous titanium dioxide, mesoporous iron oxide, etc.), for example, mesoporous hydroxyapatite.

[0019] According to an embodiment of the present invention, the second porous carrier is selected from biocompatible mesoporous materials, preferably one or more selected from mesoporous bioactive glass, mesoporous hydroxyapatite, mesoporous silica, mesoporous carbon, metal-organic frameworks (MOFs, such as ZIF series), and metal oxide mesoporous materials (such as mesoporous titanium dioxide, mesoporous iron oxide, etc.), for example, mesoporous bioactive glass.

[0020] According to an embodiment of the present invention, the first encapsulation layer is selected from one or more of chitosan, polylactic acid, polyvinyl alcohol, tannic acid, sodium alginate, and gelatin, for example, chitosan.

[0021] According to an embodiment of the present invention, the second encapsulation layer is selected from one or more of tannic acid, chitosan, polylactic acid, polyvinyl alcohol, sodium alginate, and gelatin, for example, tannic acid.

[0022] According to an embodiment of the present invention, the mass ratio of the first porous carrier loaded with CO molecule prodrug to the second porous carrier loaded with NO molecule prodrug is 1 (1-5):1 (1-5), for example, 5:1.

[0023] Secondly, the present invention also provides a method for cryogenic 3D grading printing a scaffold using the above-mentioned ink composition, comprising the following steps:

[0024] (1) Mix polylactic acid and hydroxyapatite powder to print a substrate scaffold;

[0025] (2) The first porous carrier loaded with CO molecule prodrug, hydroxyapatite, bioactive glass powder, polyvinyl alcohol and chitosan solution were mixed and cryogenically 3D printed to obtain a drug-loaded scaffold.

[0026] (3) Fix the base scaffold and the drug-loaded scaffold to obtain the first composite scaffold.

[0027] According to an embodiment of the present invention, step (3) includes the following steps: immersing the substrate scaffold in an organic solvent to completely cover the substrate scaffold with the organic solvent, dissolving the polylactic acid on the surface of the substrate scaffold, contacting and bonding the substrate scaffold with the drug-loaded scaffold, and drying to obtain the first composite scaffold.

[0028] As an example, step (3) includes the following steps: the drug-loaded stent and the base stent are composited using the organic solvent dichloromethane. The base stent is immersed in dichloromethane to uniformly cover its surface with a layer of dichloromethane. It is then removed and placed on the drug-loaded stent to ensure that the two are in contact and bonded together. The stent is then placed in a ventilated environment to dry. As the solvent dichloromethane evaporates, PLA will re-solidify to form a solid bonding layer, thereby firmly bonding the base stent and the drug-loaded stent together to obtain the first composite stent.

[0029] According to an embodiment of the present invention, after step (2) and before step (3), the following step is also included: immersing the drug-loaded stent in sodium alginate solution and drying it.

[0030] According to an embodiment of the present invention, the concentration of sodium alginate is 5-60 g / L, preferably 10-60 g / L, for example 10 g / L, 20 g / L, 30 g / L, or 40 g / L.

[0031] According to an embodiment of the present invention, the drying is freeze drying, the freeze drying temperature is -30℃ to -10℃, and the time is 15 to 35 hours, for example, the freeze drying temperature is -20℃ and the time is 24 hours.

[0032] According to an embodiment of the present invention, step (3) is followed by step (4): dissolving the second porous support loaded with NO molecular prodrug in a solvent and electrospinning it to obtain a fiber membrane; dispersing the fiber membrane in a dispersion liquid and homogenizing it at high speed to obtain a nanofiber dispersion solution; immersing the first composite scaffold in the nanofiber dispersion solution and then drying it to obtain the second composite scaffold.

[0033] According to an embodiment of the present invention, the drying is freeze drying, for example, freeze drying at -20°C.

[0034] According to an embodiment of the present invention, step (4) includes the following steps: forming a suspension of mesoporous bioactive glass + arginine / tannic acid powder in anhydrous ethanol, preparing a bioactive glass + arginine / tannic acid fiber membrane by electrospinning, dispersing the fiber membrane in tert-butanol solvent and homogenizing it at high speed to obtain a nanofiber dispersion solution, immersing the first composite scaffold in the nanofiber dispersion solution, and freeze-drying it at -20°C to obtain the second composite scaffold.

[0035] According to an embodiment of the present invention, the concentration of arginine in the suspension of step (4) is 10 to 50 μg / mL, for example, 25 μg / mL.

[0036] According to an embodiment of the present invention, the conditions for electrospinning in step (4) are as follows: using an electrospinning machine at a voltage of 17kV, a flow rate of 1mL / h, and a working distance of 20cm to obtain a fiber membrane.

[0037] According to an embodiment of the present invention, in step (1), the mass ratio of polylactic acid to hydroxyapatite is 1:(1)3, for example, 4:6.

[0038] According to an embodiment of the present invention, step (2) includes the following steps:

[0039] a) The CO molecule prodrug is loaded into the first carrier, and the CO molecule prodrug-first carrier is encapsulated with the first encapsulating material to form a first porous carrier loaded with the CO molecule prodrug.

[0040] b) The NO molecule prodrug is loaded into the second carrier, and the NO molecule prodrug-second carrier is encapsulated with the second encapsulating material to form a second porous carrier loaded with the NO molecule prodrug.

[0041] c) Polyvinyl alcohol solution, chitosan solution, a first porous carrier loaded with CO molecule prodrug, a second porous carrier loaded with NO molecule prodrug, hydroxyapatite powder, and bioactive glass powder are mixed to prepare printing ink. The ink is then printed layer by layer using cryogenic 3D printing technology to obtain a drug-loaded scaffold.

[0042] According to an embodiment of the present invention, in step a), the molar ratio of the CO molecule prodrug to the first carrier is 1:(1-2), for example, the molar ratio of mesoporous hydroxyapatite and carbonyl manganese is 1:1.

[0043] According to an embodiment of the present invention, filling carbonyl manganese powder into mesoporous hydroxyapatite includes the following steps: adding carbonyl manganese powder and mesoporous hydroxyapatite in a 1:1 ratio to PBS-Tween 80 (1% wt) and mixing.

[0044] According to an embodiment of the present invention, in step a), the molar ratio of the first encapsulating material to the CO molecule prodrug-first carrier is 1:(1 to 3), for example, 3:5.

[0045] As an example, the process of coating mesoporous hydroxyapatite loaded with carbonyl manganese with chitosan includes the following steps: mixing chitosan and mesoporous hydroxyapatite-carbonyl manganese in a Tris-HCl solution at a ratio of 3:5.

[0046] According to an embodiment of the present invention, in step b), the molar ratio of the NO molecule prodrug to the second carrier is 1:(1-2), for example, the molar ratio of mesoporous bioactive glass to arginine is 1:1.

[0047] According to an embodiment of the present invention, filling arginine powder into mesoporous bioactive glass includes the following steps: adding arginine powder and mesoporous bioactive glass to PBS-Tween 80 (1% wt) at a ratio of 1:1.

[0048] According to an embodiment of the present invention, step b) of encapsulating the NO molecule prodrug-second carrier with a second encapsulating material includes the following steps: mixing tannic acid and mesoporous bioactive glass-arginine in a Tris-HCl solution at a ratio of 3:5.

[0049] According to an embodiment of the present invention, in step c), the mass ratio of polyvinyl alcohol, chitosan and deionized water is 10:(0.1-1)0.5:90(80-100).

[0050] According to an embodiment of the present invention, in step c), the mass ratio of the first porous support loaded with CO molecule prodrug, the first porous support loaded with NO molecule prodrug, hydroxyapatite, bioactive glass, polyvinyl alcohol, and chitosan is 0.00125:0.00025:3:3:3.75:0.25.

[0051] According to an embodiment of the present invention, the mixing temperature in step c) is 60 to 120 degrees Celsius, and the mixing time is 1 to 5 hours, for example, the mixing temperature is 90 degrees Celsius and the time is 2 hours.

[0052] Preferably, a first porous support loaded with carbonyl manganese and a second porous support loaded with arginine are added in a quantitative manner to maintain the concentrations of carbonyl manganese and arginine at 125 μg / mL and 25 μg / mL, respectively.

[0053] According to an embodiment of the present invention, in step (2), the fill density of the cryogenic 3D printing is 40%-60%.

[0054] According to an embodiment of the present invention, the hydroxyapatite powder is prepared by the following method: calcium nitrate tetrahydrate, diammonium hydrogen phosphate, ammonia and deionized water are mixed in a molar ratio of 10:6:8:(800-1200), and the mixture is reacted at 70°C for 2 hours by co-precipitation to obtain hydroxyapatite powder.

[0055] According to an embodiment of the present invention, the mesoporous bioactive glass is prepared by the following method: hexadecyltrimethylammonium bromide, triethanolamine, tetraethyl orthosilicate, cyclohexane and triethyl phosphate are mixed in a mass ratio of 12:0.36:12:48:1 to synthesize SPN particles (95% mol SiO2: 5% mol P2O5), the SPN particles are mixed with calcium nitrate tetrahydrate in a molar ratio of 1:2, and the mixture is stirred for 12 h using a template method to obtain the mesoporous bioactive glass.

[0056] As an example, step (2) includes the following steps: filling carbonyl manganese powder into mesoporous hydroxyapatite, coating the carbonyl manganese-loaded mesoporous hydroxyapatite with chitosan to obtain mesoporous hydroxyapatite + carbonyl manganese / chitosan powder; filling arginine powder into mesoporous bioactive glass, coating the arginine-loaded mesoporous bioactive glass with tannic acid to obtain mesoporous bioactive glass + arginine / tannic acid powder; mixing polyvinyl alcohol solution, chitosan solution, mesoporous hydroxyapatite + carbonyl manganese / chitosan powder, mesoporous bioactive glass + arginine / tannic acid powder, hydroxyapatite powder, and bioactive glass powder to prepare printing ink; and printing the ink layer by layer using cryogenic 3D printing technology to obtain a drug-loaded scaffold.

[0057] Thirdly, the present invention provides a stent prepared by the above method, the stent comprising a base stent, a drug-loaded stent attached thereon, and the surfaces of the base stent and the drug-loaded stent being coated with short nanofibers.

[0058] According to an embodiment of the present invention, the substrate scaffold, drug-loaded scaffold, and nanofibers have the definitions described above.

[0059] Beneficial effects

[0060] 1) Nitric oxide (NO) and carbon monoxide (CO) are two important endogenous gaseous signaling molecules that play crucial roles in regulating macrophage phenotypic transformation. NO and CO synergistically regulate the M1 / M2 phenotypic balance of macrophages. Reports indicate that NO plays a vital role in vasodilation, angiogenesis, immune response, and wound healing. NO can accelerate wound healing through various mechanisms, such as increasing myofibroblasts, wound contraction, and collagen deposition. Simultaneously, NO can promote macrophage polarization towards the M1 phenotype to clear dead cells from the injury site. CO can inhibit inflammatory responses, promote macrophage polarization towards the M2 phenotype, and promote bone tissue repair. Therefore, macrophage function and metabolism can be regulated by altering the interaction between NO and CO, thereby affecting macrophage polarization. The synergistic effect of NO and CO can promote macrophage transformation to specific polarization states, regulate the M1 / M2 balance of macrophages, and enhance their immune surveillance and immunomodulatory functions.

[0061] Arginine (Arg), as a prodrug of NO, can be converted into L-citrulline in vivo under the stimulation of the damaged bone microenvironment, producing an equimolar amount of NO. This polarizes macrophages towards the M1 phenotype, thereby clearing dead cells from the damaged site. It can significantly reduce the levels of pro-inflammatory cytokines, increase the levels of anti-inflammatory factors, and promote angiogenesis. NO accelerates the metabolism of intracellular glutathione (GSH), reducing the clearance of reactive free radicals that can induce apoptosis. NO can also react with superoxide ions (O3). 2·– ) generates peroxynitrite anion (ONOO) – Arginine, a major reactive nitrogen species (RNS), is more effective than most reactive oxygen species (ROS) in inducing apoptosis through the peroxidation and nitration of biomolecules. Furthermore, the guanidinium group in arginine can be oxidized by endogenous H2O2 abundant in the damaged bone microenvironment to produce NO, inducing bacterial apoptosis to resist bone infection. Arginine, through its conversion to NO, can reduce oxidative stress during bone healing; its antioxidant effect helps protect cells from oxidative damage, promotes bone healing, and NO can also increase the expression of vascular endothelial growth factor (VEGF), promoting angiogenesis.

[0062] Manganese carbonyl (MnCO), as an excellent CO molecular prodrug, can rapidly release CO gas and Mn under the stimulation of a highly reactive oxygen-rich inflammatory microenvironment. 2+ CO polarizes macrophages towards the M2 phenotype. CO inactivates Toll-like receptor 4 (TLR4) by inhibiting NADPH oxidase activity, downregulating the expression of inflammation-related genes, and thus significantly improving the inflammatory response. The endogenous H2O2 abundant in the damaged bone microenvironment undergoes a Fenton-like reaction with MnCO, continuously releasing CO and MnCO. 2+ It upregulates the M2 macrophage phenotype and significantly reduces the inflammatory response. Furthermore, Mn... 2+ MBGs particles can synergistically promote the secretion of VEGF by M2 macrophages and activate the HIF-1α pathway, inhibiting osteoclast differentiation and further promoting angiogenesis.

[0063] During an inflammatory response, lactic acid buildup occurs due to the production of ROS, changes in cell metabolism, and insufficient oxygen supply, causing the pH of the in vivo environment to become acidic. When the inflammation subsides, the in vivo environment returns to alkaline or neutral. This invention addresses this by coating a NO release carrier with a layer of tannic acid (TA). The nanocarrier encapsulation layer formed by tannic acid can spontaneously degrade under acidic conditions, enabling drug release in an acidic microenvironment. Furthermore, the introduction of the cross-linking agent tetraethylenepentamine (TEPA) allows the self-assembled polymer to dissociate into a loose state without degradation under acidic conditions. When the solution returns to alkaline or neutral, the dissociated polymer can be reassembled into a closed state by adjusting the protonation and deprotonation of TEPA. This allows the drug to stop releasing when the in vivo environment returns to normal, and macrophages to stop transforming into the M1 phenotype.

[0064] 2) This invention employs a preliminary rapid low-temperature freezing followed by freeze-drying during the printing process. This allows the solvent inside the ink to quickly change from a liquid to a gaseous state, removing ice crystals and forming a porous structure in the material. This facilitates better cell adhesion and angiogenesis. The fibrous membrane obtained through electrospinning in this application possesses a nanofiber network that exhibits significant capabilities in controlling cell behavior, such as cell adhesion and proliferation. This is crucial for scaffold-tissue integration. Compared to dense scaffolds, scaffolds mimicking the extracellular matrix (ECM) can better promote oxygen and nutrient transport and tissue growth. Electrospinning is a widely used method for manufacturing nanofiber structures similar to natural ECM. Combining electrospinning with 3D printing technology, short BGs fibers are manufactured. A hierarchical scaffold is constructed by utilizing the different degradation rates of electrospun nanofibers and 3D-printed microfilaments. Ions are sequentially released from the biomimetic scaffold for timely vascularization and stable bone formation. Attached Figure Description

[0065] Figure 1 A photograph of the composite scaffold prepared in Example 1;

[0066] Figure 2 The images show the actual printed products of different proportions of inorganic hydroxyapatite and bioactive glass in Experiment Example 1.

[0067] Figure 3 The Pr values ​​of inks containing different proportions of inorganic hydroxyapatite and bioactive glass in Experiment Example 1;

[0068] Figure 4 The relationship between ink viscosity and shear rate in different proportions of inorganic hydroxyapatite and bioactive glass in Experiment Example 1;

[0069] Figure 5 A comparison of the loss modulus (G”) and storage modulus (G’) of scaffolds prepared by inks containing different proportions of inorganic hydroxyapatite and bioactive glass in Experiment Example 1.

[0070] Figure 6 The cyclic strain test diagrams show the scaffolds prepared by inks containing different proportions of inorganic hydroxyapatite and bioactive glass in Experimental Example 1.

[0071] Figure 7 The effect of different concentrations of sodium alginate crosslinking on the mechanical properties of the scaffold in Experiment Example 2. Detailed Implementation

[0072] The ink composition, its preparation method, and its application of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0073] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0074] Example 1

[0075] (1) Dissolve 0.5062 mol Ca(NO3)2·4H2O and 0.3104 mol (NH4)2HPO4 in 500 mL of deionized water, respectively, and then stir magnetically at room temperature until completely dissolved to prepare solutions A and B. Then, introduce solution B into solution A to obtain solution C and heat it slightly to 20-50℃. Add ammonia water to solution C and adjust the pH of solution C to 9.5-10 using a pH meter. Then heat solution C in a 70℃ water bath and stir vigorously for 2 h. After the reaction is complete, filter the obtained white precipitate and wash it 3 times with deionized water and 3 times with anhydrous ethanol. Take the filtered product and dry it at 60℃ for 24 h and sinter it at 80℃ for 2 h to remove impurities. Finally, hydroxyapatite powder is obtained.

[0076] (2) Mix polylactic acid with hydroxyapatite powder from step (1) at a mass ratio of 4:6 to obtain printing ink. Use modeling software to construct cylindrical models of Φ15×5mm and Φ15×30mm and export slice files using slicing software. The layer thickness is 0.6mm and the filling density is 50%. Then, use an extrusion 3D printer to print the "ink" layer by layer under the control of gcode code to obtain PLA-HA bracket.

[0077] (3) 2.14 g of diamine hydrogen phosphate was added to 300 mL of deionized water containing 2 g of CTAB (hexadecyltrimethylammonium bromide), and the pH was adjusted to 10. Then, 100 mL of aqueous solution containing 6.3747 g of calcium nitrate tetrahydrate was slowly added, and the pH was adjusted to 10 again. The reaction was carried out at 90 °C for 24 h. After the reaction was completed, the mixture was centrifuged at 5000 rpm and washed three times with ethanol and water. After drying, the mixture was sintered at 500 °C for 2 h to remove CTAB, and finally mesoporous hydroxyapatite (MHA) was obtained.

[0078] (4) 0.5g MHA and 0.5g MnCO powder were sonicated in 100ml PBS-Tween 80 (1wt%) for 10min to remove air bubbles in the mesopores. The mixture was stirred at 37℃ in the dark for 12h, centrifuged at 5000rpm, and the precipitate was freeze-dried to obtain MHA / MnCO powder.

[0079] (5) Dissolve 2g of CS (chitosan) powder in 100ml of deionized water and stir until dissolved. Add 1ml (1%) of glacial acetic acid dropwise, seal and stir for 1h to obtain CS solution (1%). Disperse 1g of MHA / MnCO in 100ml of deionized water and stir for 20min. Slowly add the CS solution to MHA / MnCO (while stirring), seal and stir for 12h, then centrifuge, wash and freeze dry to obtain MHA-MnCO / CS powder.

[0080] (6) Dissolve 12g CTAB and 0.36ml TEA (triethanolamine) in 108ml deionized water to obtain water-CTAB-TEA. Stir at 60℃ for 1h. Mix 12ml TEOS and 48ml cyclohexane evenly and slowly add to water-CTAB-TEA. Stir at 60℃ for 3h, then add 0.921ml TEP (triethyl phosphate) and react at 60℃ for 9h. After the reaction is complete, centrifuge at 5000rpm, wash three times with ethanol and water, and freeze-dry to obtain SPN particles (95% mol SiO2: 5% mol P2O5).

[0081] (7) Dissolve 0.704 g of calcium nitrate tetrahydrate in 40 ml of ethanol, add 1 g of SPN particles, stir for 12 h until the ethanol evaporates, sinter at 600 °C for 5 h to remove the template, sieve, and finally obtain mesoporous bioactive glass (MBG).

[0082] (8) 0.5 g MBG and 0.5 g Arg powder were sonicated in 100 ml PBS-Tween 80 (1 wt%) for 10 min to remove air bubbles in the mesopores. The mixture was stirred at 37 °C for 12 h, centrifuged at 5000 rpm, and the precipitate was freeze-dried to obtain MBG-Arg powder.

[0083] (9) 2g of MBG-Arg powder was ultrasonically dispersed in Tris-HCl buffer solution (100ml, pH 8.5), TA (50mg) and TEPA (tetraethylenepentamine) (20ml) were added, and the mixture was stirred in the dark for 8h. After centrifugation, washing and freeze-drying, MBG-Arg / TA powder was obtained.

[0084] (10) Weigh 3g HA, 0.0025g MHA-MnCO / CS powder, 3g BG, and 0.0005g MBG-Arg / TA powder, dissolve them in 26mL of an aqueous solution with a polyvinyl alcohol content of 3.75g and a chitosan content of 0.25g, and stir evenly to obtain a frozen printing ink.

[0085] (11) Cylindrical models of Φ15×5mm and Φ15×30mm were constructed using modeling software, and slice files were exported using slicing software. The layer thickness was 0.6mm and the infill density was 50%. Then, the "ink" in step (10) was printed layer by layer under the control of gcode code using an extrusion 3D printer to obtain the scaffold. After printing, the scaffold was immersed in sodium alginate solutions of different concentrations (10, 20, 30, 40 g / L) for 2 hours to obtain polyvinyl alcohol-hydroxyapatite / chitosan-bioactive glass / tannic acid scaffold. The scaffold was freeze-dried in a freeze dryer for 48 hours and named PVA-MHA / CS-MBG / TA (hereinafter referred to as drug-loaded scaffold).

[0086] (12) By using organic solvent dichloromethane to composite, the PLA-HA substrate scaffold is immersed in dichloromethane to uniformly cover its surface with a layer of dichloromethane. It is then removed and placed on the drug-loaded scaffold, ensuring that the two are in contact and bonded together. It is then placed in a ventilated environment to dry. As the solvent dichloromethane evaporates, the PLA will re-solidify to form a solid bonding layer, thus firmly bonding the substrate scaffold and the drug-loaded scaffold together to obtain the first composite scaffold.

[0087] (13) Add MBG-Arg / TA powder to anhydrous ethanol (maintain the arginine content in the suspension at 25 μg / ml) and stir until uniform to obtain a stable bioactive glass ethanol suspension, and prepare an electrospinning solution.

[0088] (14) MBG-Arg / TA fiber membranes were obtained by electrospinning at 17kV voltage, 1mL / h flow rate and 20cm working distance. The membranes were sintered at 800℃ for 1h and dispersed in tert-butanol and homogenized in a homogenizer for 5min (104rpm) to obtain a nanofiber dispersion solution. The first composite scaffold was immersed in the nanofiber dispersion solution and freeze-dried at -20℃ to obtain the second composite scaffold. The scaffold was freeze-dried and named polylactic acid-polyvinyl alcohol-hydroxyapatite / carbonyl manganese-bioactive glass / arginine scaffold.

[0089] Comparative Example 1

[0090] To verify the effect of the introduction of carbonyl manganese and arginine on the printability of the scaffold, this comparative example is the same as Example 1 except that steps (4)-(5) and (8)-(9) are removed and hydroxyapatite powder is used to replace MHA-MnCO / CS powder and bioactive glass powder is used to replace MBG-Arg / CS in step (10). That is, carbonyl manganese and arginine are not added in this comparative example 1.

[0091] To prepare a PVA-MHA-MBG ink that does not contain carbonyl manganese and arginine, 3.0025g of prepared hydroxyapatite powder and 3.0005g of bioactive glass powder were dispersed in an aqueous solution with a solid content of 3.75g of polyvinyl alcohol and a solid content of 0.25g of chitosan. The mixture was stirred evenly with a glass rod to form a composite ink, which was named PVA-MHA-MBG.

[0092] Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slice files were exported using slice parts. The layer thickness was 0.6mm, and the infill density was 50%. Then, "ink" was printed layer by layer using an extrusion 3D printer under the control of gcode.

[0093] Experimental Example 1

[0094] In this study, our aim was to explore the impact of bio-ink formulations on scaffold printability and to optimize the proportions of inks with the best printability. The printability of bio-inks was assessed using a semi-quantitative approach based on rheological measurements and image analysis.

[0095] In this study, the printability of the scaffolds prepared in Example 1 and Comparative Example 1 was measured based on fidelity (Pr):

[0096] Pr = L 2 / 16A

[0097] Where L represents the perimeter of the square grid formed by the support, and A represents the area enclosed. Each set of supports is photographed (n=5), and the supports are observed using an optical microscope to measure the perimeter and area of ​​the grid.

[0098] Experimental results show that the printing effects of scaffolds with different ratios (changing the ratio of inorganic hydroxyapatite to bioactive glass in the scaffold printing slurry while keeping other conditions constant: 1:1-10%, 1:1-15%, 1:2-10%, 1:2-15%, 2:1-10%, 2:1-15%, where the percentages represent the concentrations of organic polyvinyl alcohol and chitosan) in Example 1 varied. The Pr values ​​further validated this result; a Pr value closer to 1 indicates better scaffold printability. (See [link to relevant documentation]). Figure 2 and Figure 3 As shown, when the ratio of inorganic hydroxyapatite to bioactive glass in the scaffold printing slurry is 1:1 and the concentration of organic phases polyvinyl alcohol and chitosan is 15%, the Pr value is closest to 1. At this time, the printed mesh is obvious and there is no breakage.

[0099] The rheological properties of ink are also a key factor in evaluating printability. This experimental example further explores the relationship between the viscosity and shear rate of bio-inks, revealing pseudoplastic behavior characterized by shear thinning. See [link to relevant documentation]. Figure 4 and Figure 5 As shown, further analysis of the ink modulus reveals that, across the entire strain range, the loss modulus (G”) of the scaffolds in Examples 1 (2:1-15% and 1:2-10%) remains higher than the storage modulus (G’), revealing its viscous-to-elasticity characteristic. Therefore, it is prone to collapse after extrusion, consistent with the Pr value structure. Conversely, at low strain, the G’ of the 2:1-10%, 1:1-10%, 1:2-15%, and 1:1-15% scaffolds in Examples 1 exceeds G”, indicating that the elasticity of the bio-ink predominates after extrusion, which is beneficial for maintaining a stable structure without collapse during post-extrusion.

[0100] The rapid self-healing capability of the ink in the examples was then evaluated; see [link to relevant documentation]. Figure 6 As shown, cyclic strain demonstrated that the four optimal ratios of bio-inks in Example 1 (1:1-10%, 1:1-10%, 1:2-15%, and 1:1-15%) exhibited alternating transitions of G' and G”, which implies a reversible transition from a gel to a fluid state. However, the 2:1-15% and 1:2-10% scaffolds in Example 1 lacked the ability to recover to a gel state under low strain, and therefore lacked printability.

[0101] Experimental Example 2

[0102] Mechanical properties are an important factor in the biomechanical grade structure of scaffolds. In this study, our aim was to explore the effect of crosslinking with different concentrations of sodium alginate on the mechanical properties of scaffolds, and to optimize the concentration of sodium alginate used for crosslinking to obtain the scaffold with the best mechanical properties. The mechanical properties of the scaffolds were evaluated by stress-strain testing.

[0103] See the experimental results. Figure 7 As shown, the mechanical properties of scaffolds with different sodium alginate crosslinking ratios (0%, 10%, 20%, 30%) in Example 1 varied, with the scaffold obtained after crosslinking at a sodium alginate concentration of 20 g / L exhibiting the best mechanical properties.

[0104] The specific embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above exemplary embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method of freezing a 3D hierarchical printed scaffold, characterized in that, The method comprises the following steps: (1) mixing polylactic acid with hydroxyapatite powder to print a base scaffold; (2) preparing a drug-loaded scaffold; (3) fixing the base scaffold and the drug-loaded scaffold to obtain a first composite scaffold; Step (2) comprises the following steps: a) loading CO molecular prodrugs into a first carrier, coating the CO molecular prodrug-first carrier with a first encapsulating material to form a first porous carrier loaded with CO molecular prodrugs; b) loading NO molecular prodrugs into a second carrier, coating the NO molecular prodrug-second carrier with a second encapsulating material to form a second porous carrier loaded with NO molecular prodrugs; c) mixing polyvinyl alcohol solution, chitosan solution, the first porous carrier loaded with CO molecular prodrugs, the second porous carrier loaded with NO molecular prodrugs, hydroxyapatite powder, and bioactive glass powder to prepare printing ink, and printing the ink layer by layer by using a freeze 3D printing technology to obtain a drug-loaded scaffold; The CO molecular prodrug is selected from a material capable of releasing CO molecules in a body fluid, and is selected from one or more of metal carbonyl compounds, carbonyl imidazole, nitroxyl compounds, heme derivatives, and CO metal complexes; The NO molecular prodrug is selected from a material capable of releasing NO molecules in a body fluid, and is selected from one or more of arginine, citrulline, dimethylarginine, nitro drugs, organic nitrates, nitrosothiol compounds, metal-nitrosyl complexes, and diazeniumdionium salts; The first porous carrier is selected from a biocompatible mesoporous material, and is selected from one or more of mesoporous hydroxyapatite, mesoporous bioactive glass, mesoporous silicon, mesoporous carbon, metal organic framework, and metal oxide mesoporous material; The second porous carrier is selected from a biocompatible mesoporous material, and is selected from one or more of mesoporous bioactive glass, mesoporous hydroxyapatite, mesoporous silicon, mesoporous carbon, metal organic framework, and metal oxide mesoporous material; The first encapsulating material and the second encapsulating material are independently selected from one or more of chitosan, polylactic acid, polyvinyl alcohol, tannic acid, sodium alginate, and gelatin.

2. The method of claim 1, wherein, Step (3) comprises the following steps: soaking the base scaffold in an organic solvent to completely cover the base scaffold with the organic solvent, dissolving polylactic acid on the surface of the base scaffold, contacting and bonding the base scaffold with the drug-loaded scaffold, and drying to obtain the first composite scaffold.

3. The method of claim 1, wherein, After step (2) and before step (3), the method further comprises the following step: soaking the drug-loaded scaffold in a sodium alginate solution and drying.

4. The method of claim 3, wherein, The concentration of the sodium alginate solution is 5-60 g / L, the drying is freeze-drying, the temperature of the freeze-drying is -30 ℃ to -10 ℃, and the time is 15-35 h.

5. The method of claim 1, wherein, Step (3) is followed by step (4): dissolving the second porous carrier loaded with NO molecular prodrugs in a solvent and electrospinning to obtain a fiber membrane, dispersing the fiber membrane in a dispersion liquid at high speed to obtain a nanometer short fiber dispersion solution, soaking the first composite scaffold in the nanometer short fiber dispersion solution, and then drying to obtain a second composite scaffold.

6. The method of claim 1-5, wherein, In step a), the molar ratio of the CO molecular prodrugs to the first carrier is 1: (1-2).

7. The method of claim 1-5, wherein, In step a), the molar ratio of the first encapsulating material to the CO molecular prodrug-first carrier is 1:(1-3).

8. The method of claim 1-5, wherein, In step b), the molar ratio of the NO molecular prodrug to the second carrier is 1:(1-2).

9. The method of frozen 3D hierarchical printing of a scaffold according to any one of claims 1-5, characterized in that, In step (2), the filling density of the 3D printing is 40%-60%.

10. The method of claim 1-5, wherein, The metal carbonyl compound is manganese carbonyl.

11. A scaffold prepared by the method of any one of claims 1-10, wherein, The stent comprises a base stent and a drug-loaded stent attached to the base stent, and the surfaces of the base stent and the drug-loaded stent are coated with nano short fibers.

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