Skull tissue engineering scaffold prepared by adopting double cold extraction freeze-drying process and preparation method of skull tissue engineering scaffold

Abstract

The invention discloses a skull tissue engineering scaffold prepared by adopting a dual cold extraction freeze-drying process and a preparation method thereof, the dual cold extraction freeze-drying process is creatively proposed for the first time to realize a mild treatment process of printing, drying, crosslinking and re-drying, and while the morphology precision of the scaffold is kept, the skull tissue engineering scaffold prepared by adopting the dual cold extraction freeze-drying process is prepared. The mechanical property and the structural stability are remarkably enhanced, a brand new thought and technical support are provided for clinical conversion of 3D printing bone tissue engineering materials, and the method has a wide application prospect in the field of clinical bone repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cranial tissue engineering scaffold prepared using a dual cold extraction freeze-drying process and its preparation method. Background Technology

[0002] Skull defects are a common sequela of traumatic brain injury, and in severe cases can lead to brain tissue exposure, changes in intracranial pressure, brain dysfunction, and cosmetic deformities. Currently, clinical practice demands a dual approach to skull repair materials: on the one hand, materials must possess good mechanical strength to support intracranial and extracranial pressure; on the other hand, they must exhibit excellent biocompatibility and osteogenic induction to promote bone regeneration and fusion in the defect area.

[0003] Existing cranioplasty materials mainly include rigid artificial materials such as titanium alloys and polyetheretherketone (PEEK). While these materials possess high strength, they suffer from limitations such as significant foreign body rejection, non-degradability and poor bone integration. Hydrogel materials, due to their excellent biomimetic properties and three-dimensional molding capabilities, have become a hot research area in cranioplasty tissue engineering in recent years. In particular, hydrogel scaffolds based on 3D printing technology can achieve personalized customization and precise filling of complex defect areas, showing broad application prospects.

[0004] However, in the process of industrialization, these 3D printed scaffolds generally face the following key challenges: (1) the mechanical properties of hydrogels are low, making it difficult to match the high load requirements of the skull; (2) skull defects are often accompanied by inflammatory responses of traumatic brain injury (TBI), and the dense structure of hydrogels may hinder blood vessel ingrowth; (3) hydrogels usually require post-processing steps such as secondary cross-linking, freeze-drying, or sintering after printing, which may introduce defects (such as cracking and shrinkage deformation) during scale-up production. The lack of unified industry standards makes it difficult to control the stable performance of products.

[0005] Based on the above challenges, this invention proposes for the first time to introduce a "dual cold extraction freeze-drying process" to achieve a mild processing procedure of "printing-drying-crosslinking-re-drying". While preserving the morphological accuracy of the scaffold, it significantly enhances its mechanical properties and structural stability, providing a new approach and technical support for the clinical translation of 3D printed bone tissue engineering materials, and has broad application prospects in the field of clinical bone repair. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems of existing hydrogel scaffolds, the present invention aims to provide a cranial tissue engineering scaffold prepared by a dual cold extraction and freeze-drying process and its preparation method.

[0007] Most bone repair products on the market are currently hydroxyapatite or collagen-based, while GelMA is a modified gelatin with lower immunogenicity compared to collagen. Furthermore, GelMA provides a suitable growth environment for cells, which is beneficial for bone repair. Hydrogel scaffolds based on 3D printing technology can achieve personalized customization and precise filling of complex defect areas, showing broad application prospects. However, current hydrogel scaffolds face problems such as low mechanical strength, easy degradation, and high difficulty in transformation. This invention uses a dual cold extraction and freeze-drying process to prepare a skull tissue engineering scaffold. The production process is simple and easy to control, and the produced scaffold has high bioactivity and high compressive strength. The freeze-dried scaffold has uniform internal pores, suitable for the growth of cells around the skull, accelerating bone repair, and has broad application prospects.

[0008] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0009] A first aspect of the present invention provides a 3D-printed skull tissue engineering scaffold.

[0010] Furthermore, the skull tissue engineering scaffold is prepared from the following raw materials: GelMA, lithium magnesium silicate, photoinitiator, and light blocker.

[0011] Furthermore, the cranial tissue engineering scaffold is prepared by 3D printing and dual cold extraction freeze-drying processes from a composite hydrogel of GelMA and lithium magnesium silicate.

[0012] Furthermore, the concentration of GelMA in the composite hydrogel is 0.001-0.02 g / mL;

[0013] Optionally, the concentration of lithium magnesium silicate in the composite hydrogel is 0.0005-0.02 g / mL.

[0014] Furthermore, the concentration of GelMA in the composite hydrogel is 0.15 g / mL;

[0015] Optionally, the concentration of lithium magnesium silicate in the composite hydrogel is 0.02 g / mL.

[0016] Furthermore, the molecular formula of the lithium magnesium silicate is Na. + 0.7 [(Si8Mg 5.5 Li 0.3 O 20 (OH)4)] - 0.7 ;

[0017] Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 10-50 nm and a thickness of 0.05-5 nm;

[0018] Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 25 nm and a thickness of 1 nm;

[0019] Optionally, the disk is composed of two layers of tetrahedral silica coated with an octahedral crystal layer in the middle;

[0020] Optionally, the photoinitiator includes LAP, Irgacure 2959, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, or a ruthenium photoinitiator;

[0021] Optionally, the light-blocking agent includes UVA, carbon black, TiO2, ZnO, iron oxide, zinc oxide, CaCO3, phthalocyanine blue, phthalocyanine green, or benzophenone compounds.

[0022] Optionally, the photoinitiator is a 0.01-0.10 g / mL LAP solution;

[0023] Optionally, the photoinitiator is a 0.03 g / mL LAP solution;

[0024] Optionally, the light-blocking agent is a 0.01-0.10 g / mL UVA solution;

[0025] Optionally, the light-blocking agent is a 0.03 g / mL UVA solution.

[0026] In this invention, GelMA refers to methacrylamide gelatin, a derivative of gelatin, which can be cured and molded by ultraviolet / visible light. It combines the characteristics of both natural and synthetic materials, and can form 3D structures adapted to cell growth. It exhibits excellent biocompatibility, provides cell adhesion sites, has controllable degradation, and possesses adjustable mechanical properties, temperature-sensitive gel properties, and multiple viscoelastic properties. It has been widely used in 3D cell culture, tissue engineering, and bioprinting, and is a strong candidate to replace traditional adhesive raw materials. In this invention, GelMA is combined with lithium magnesium silicate to prepare a cranial tissue engineering scaffold, providing a suitable environment for cell growth and promoting bone repair.

[0027] In a specific embodiment of the present invention, the photoinitiator is LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine), which is a key triggering substance for the photocuring reaction. During photocuring 3D printing, when irradiated with a specific wavelength (such as ultraviolet or visible light), LAP triggers a cross-linking reaction of the double bonds in GelMA (methacryloyl gelatin) molecules, causing the liquid GelMA composite solution to rapidly solidify and form a scaffold structure with a specific shape. Its function is to ensure the accuracy of "layer-by-layer curing" during 3D printing, ensuring that the morphology of the scaffold is consistent with the design model, and it is a core component for realizing photocuring 3D printing technology.

[0028] In a specific embodiment of the present invention, the light-blocking agent is UVA. UVA, as a light-blocking agent, is mainly used to regulate the range and rate of the photocuring reaction. In the formulation of bioprinting inks, UVA is used in conjunction with a photoinitiator (LAP) to avoid unintended cross-linking caused by excessive light penetration or diffusion (such as premature curing of unprinted areas in adjacent layers). This controls the curing reaction to occur only within the illuminated area, ensuring the precision and dimensional stability of the scaffold structure during 3D printing and reducing defects such as scaffold deformation and cracking caused by over-curing.

[0029] A second aspect of the present invention provides a method for preparing a skull tissue engineering scaffold using a dual cold extraction freeze-drying process.

[0030] Furthermore, the method includes the following steps:

[0031] (1) Preparation of bioprinting ink: Photoinitiator and light blocker are added to a mixed solution obtained by mixing GelMA and lithium magnesium silicate to obtain bioprinting ink;

[0032] (2) 3D printing: The initial blank of the skull framework was obtained by using photopolymerization 3D printing technology;

[0033] (3) First freeze drying: The cleaned skull framework is pre-frozen and then freeze-dried under vacuum;

[0034] (4) Immersion in GelMA solution: Immerse the freeze-dried skull scaffold in GelMA solution;

[0035] (5) -80℃ cold extraction: The soaked skull support is placed in -80℃ for cold extraction;

[0036] (6) Re-curing: Curing the cold-extracted skull framework;

[0037] (7) Second freeze-drying: The solidified skull scaffold is freeze-dried again under vacuum to obtain a skull tissue engineering scaffold.

[0038] Furthermore, the concentration of GelMA in the mixed solution described in step (1) is 0.001-0.02 g / mL;

[0039] Optionally, the concentration of lithium magnesium silicate in the mixed solution in step (1) is 0.0005-0.02 g / mL;

[0040] Optionally, the concentration of GelMA in the mixed solution in step (1) is 0.15 g / mL;

[0041] Optionally, the concentration of lithium magnesium silicate in the mixed solution in step (1) is 0.02 g / mL;

[0042] Optionally, the molecular formula of the lithium magnesium silicate is Na.+ 0.7 [(Si8Mg 5.5 Li 0.3 O 20 (OH)4)] - 0.7 ;

[0043] Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 10-50 nm and a thickness of 0.05-5 nm;

[0044] Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 25 nm and a thickness of 1 nm;

[0045] Optionally, the disk is composed of two layers of tetrahedral silica coated with an octahedral crystal layer in the middle;

[0046] Optionally, the photoinitiator in step (1) includes LAP, Irgacure 2959, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, or ruthenium photoinitiator;

[0047] Optionally, the light blocking agent in step (1) includes UVA, carbon black, TiO2, ZnO, iron oxide, zinc oxide, CaCO3, phthalocyanine blue, phthalocyanine green or benzophenone compounds.

[0048] Optionally, the photoinitiator in step (1) is a 0.01-0.10 g / mL LAP solution;

[0049] Optionally, the photoinitiator in step (1) is a 0.03 g / mL LAP solution;

[0050] Optionally, the light-blocking agent mentioned in step (1) is a 0.01-0.10 g / mL UVA solution;

[0051] Optionally, the light-blocking agent in step (1) is a 0.03 g / mL UVA solution;

[0052] Optionally, the volume of the mixed solution used in step (1) is 5-35 mL;

[0053] Optionally, the volume of the mixed solution used in step (1) is 20 mL;

[0054] Optionally, the amount of photoinitiator used in step (1) is 0.5-5 mL;

[0055] Optionally, the amount of photoinitiator used in step (1) is 1.2 mL;

[0056] Optionally, the amount of light blocker used in step (1) is 100-600 μL;

[0057] Optionally, the amount of light-blocking agent used in step (1) is 340 μL.

[0058] In a specific embodiment of the present invention, GelMA powder and nano-clay (i.e., lithium magnesium silicate) powder are dissolved in deionized water or a buffer solution and thoroughly stirred to prepare a uniform and stable composite bio-ink. An appropriate amount of photoinitiator (such as LAP, Irgacure 2959) may be added.

[0059] Furthermore, the printing conditions described in step (2) are: light intensity 5-30 mw / cm². 2 Exposure time 3-10s, number of substrate exposure layers 2-8, substrate exposure time 2-8s, film thickness 70-160μm.

[0060] In a specific embodiment of the present invention, the bioprinting ink prepared in step (1) is loaded into the barrel of a photopolymerizable bio-3D printer, and layer-by-layer printing is performed according to a preset skull defect model; during the printing process, each layer of ink is printed at a specific wavelength (e.g., ~365nm or ~405nm) and intensity (e.g., 5-30mW / cm). 2 The tissue cross-links and solidifies under light irradiation, layer by layer, eventually forming a preliminary blank of a skull tissue engineering scaffold with a specific macroscopic shape and internal microstructure.

[0061] Optionally, the printing conditions in step (2) are: light intensity 10 mw / cm². 2 Exposure time 7s, number of substrate layers 5, substrate exposure time 5s, film thickness 125μm;

[0062] Optionally, the cleaning conditions in step (3) are cleaning at 35-60℃ for 0-2 hours;

[0063] In a specific embodiment of the present invention, the initial skull scaffold blank is immersed in warm deionized water or a buffer solution for cleaning to remove unreacted monomers, photoinitiator residues, and some soluble impurities; the cleaning temperature is 35-60°C, and the cleaning time is 0-2 hours (Note: 0 hours indicates that this step can be omitted depending on the purity of the material). After cleaning, the scaffold is freeze-dried to obtain a first-stage freeze-dried scaffold.

[0064] Optionally, the pre-freezing conditions in step (3) are overnight pre-freezing at -(10-80)℃;

[0065] Optionally, the pre-freezing conditions in step (3) are overnight pre-freezing at -20°C or -80°C;

[0066] Optionally, the concentration of the GelMA solution in step (4) is 0.001-0.02 g / mL;

[0067] Optionally, the concentration of the GelMA solution in step (4) is 0.05 g / mL, 0.075 g / mL, or 0.1 g / mL;

[0068] Optionally, the soaking conditions in step (4) are constant temperature at 30-50℃, protected from light, and soaked for 5-12 hours;

[0069] Optionally, the soaking conditions in step (4) are: constant temperature at 40°C, protected from light, and soaked for 8 hours.

[0070] Optionally, the cold extraction conditions described in step (5) are -80°C freezing overnight;

[0071] In a specific embodiment of the present invention, the initially lyophilized stent is completely immersed in a GelMA solution with a concentration of 0.001-0.02 g / mL for 0-12 hours (Note: 0 hours means the stent can be briefly immersed and then immediately proceed to the next step). After removing the immersion solution, the immersed stent is placed in an environment of -80°C (such as an ultra-low temperature freezer) for cold extraction for 2-24 hours. This step aims to utilize low temperature to induce the GelMA solution to form a specific phase in the internal pores and surface of the stent.

[0072] Optionally, the curing conditions in step (6) are curing under 405nm or 365nm ultraviolet light for 5-30 minutes.

[0073] In a specific embodiment of the present invention, the cold-extracted scaffold is subjected to a specific wavelength (e.g., ~365nm or ~405nm) and intensity (e.g., 5-30mW / cm). 2 The material undergoes secondary cross-linking and curing under light irradiation for 5-30 minutes. This step aims to fix the structure formed after cold extraction and enhance network stability.

[0074] Furthermore, step (7) involves secondary freeze-drying and obtaining the final product. The cured scaffold is subjected to secondary freeze-drying to completely remove moisture. The final product is a GelMA / nanoclay composite tissue engineering scaffold with stable structure, uniform internal pore height, excellent mechanical strength, and good biocompatibility, suitable for cranial repair.

[0075] A third aspect of the present invention provides a bioprinting ink for 3D printing skull tissue engineering scaffolds.

[0076] Furthermore, the bioprinting ink is the bioprinting ink described in the second aspect of the present invention.

[0077] The bioprinting ink described in this invention is a key material for 3D printing skull tissue engineering scaffolds. It is composed of a specific ratio of GelMA, lithium magnesium silicate, photoinitiator (LAP), and UVA inhibitor. This bioprinting ink plays a crucial role in the fabrication of skull tissue engineering scaffolds, and its compositional characteristics and interactions are essential to the scaffold's performance and the final skull repair effect.

[0078] Among them, GelMA is a modified gelatin with good biocompatibility, which can provide a suitable environment for cell growth and can be cured and molded by ultraviolet / visible light to form a 3D structure adapted to cell growth; the magnesium lithium silicate has a special microstructure, and its main degradation products, silicate and magnesium ions, can promote mineralization, bone formation and angiogenesis.

[0079] The bioprinting ink described in this invention enables personalized customization and precise filling of complex defect areas. Based on 3D printing technology, it can print a scaffold prototype that matches the specific shape and size of the skull defect. The synergistic effect of GelMA and lithium magnesium silicate gives the printed scaffold high bioactivity, which is beneficial to the growth of cells around the skull and accelerates bone repair. Simultaneously, the scaffold printed with this ink undergoes a dual cold extraction and freeze-drying process, giving it high compressive strength and a uniform internal pore structure, better meeting the requirements of skull repair for material mechanical properties and microstructure.

[0080] The fourth aspect of the present invention provides the application of a skull tissue engineering scaffold prepared by the method described in the second aspect of the present invention in the preparation of skull repair materials.

[0081] In this invention, cranial repair refers to the clinical treatment scenario of cranial defects, that is, by using the cranial tissue engineering scaffold prepared by this invention to fill the cranial defect area caused by trauma, disease, etc., to achieve the following objectives: (1) Support intracranial and extracranial pressure, provide mechanical strength matching that of normal skull, and maintain the integrity and stability of skull; (2) Utilize the good biocompatibility and osteogenic induction of the scaffold (such as the mineralization, osteogenic and angiogenesis promoted by the degradation products of lithium magnesium silicate, etc.) to promote the regeneration and fusion of bone tissue in the defect area, and realize the repair of cranial structure and function; (3) Combine the personalized customization capability of 3D printing technology to accurately adapt to complex cranial defect morphology and solve the problem that traditional repair materials are difficult to fill irregular defect areas.

[0082] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0083] (1) This invention is the first to creatively propose the introduction of a "dual cold extraction freeze-drying process" to achieve a mild processing procedure of "printing-drying-crosslinking-re-drying," which significantly enhances the mechanical properties and structural stability of the scaffold while preserving its morphological accuracy. The tissue engineering scaffold prepared using the method provided by this invention has uniform internal pores, high mechanical strength, and good biocompatibility, making it suitable for cranial bone repair. This invention provides a novel approach and technical support for the clinical translation of 3D-printed bone tissue engineering materials, and has broad application prospects in the field of clinical bone repair.

[0084] (2) Conventional 3D printed hydrogel scaffolds suffer from low crosslinking density and fragile networks, resulting in industrialization bottlenecks such as insufficient mechanical strength (compressive strength <1MPa), rapid in vivo degradation (<2 weeks), and poor process stability (e.g., swelling and deformation). This invention innovatively proposes a "dual cold extraction and freeze-drying enhancement process," which overcomes the above difficulties through three core technologies:

[0085] (a) Cold extraction-induced micro / nano reinforcement: In an ultra-low temperature environment of -80℃, directional phase separation occurs inside the scaffold immersed in GelMA solution, forming a microcrystalline reinforced framework;

[0086] (b) Secondary photocrosslinking and curing: After cold extraction, in-situ photocuring is used to construct a high-density covalent network, which increases the compressive strength to >5MPa (reaching the level of cancellous bone);

[0087] (c) Dual freeze-drying to control pores: The first freeze-drying constructs the basic pores, and the second freeze-drying repairs structural defects, forming an ideal cell migration channel with uniform pore size (80±20μm) and connectivity >95%.

[0088] (3) The GelMA / nanoclay composite scaffold produced by the process provided by this invention has three clinical advantages: the physical cross-linking of nanoclay and the synergistic toughening effect of cold-extracted microcrystals ensure that the load-bearing capacity meets the stress requirements of the skull; the degradation cycle can be controlled within 6-8 months, which is synchronized with the new bone growth rate; the uniform porous structure promotes blood vessel ingrowth and osteoblast infiltration, and animal experiments show that the bone regeneration rate is increased by more than 40%. The entire process does not require organic solvents or high-temperature sintering, but only conventional ultra-low temperature freezers and ultraviolet light sources. The production process is simple and has high parameter tolerance (such as cleaning temperature 35-60℃, cold extraction time 2-24h), which significantly reduces the transformation threshold and provides a technical foundation for the large-scale clinical application of personalized skull repair products. Attached Figure Description

[0089] Figure 1 The structural formula of GelMA;

[0090] Figure 2 The structural formula of naoclay;

[0091] Figure 3 Printed model;

[0092] Figure 4 : Appearance of the finished product from the cold extraction rack with 5% immersion solution;

[0093] Figure 5 Microstructure of the cold-extracted support in 5% immersion solution;

[0094] Figure 6 : 5% soaking solution cold extraction scaffold cell proliferation experiment;

[0095] Figure 7 : 7.5% soaking solution cold extraction of the finished support;

[0096] Figure 8 Microstructure of the cold-extraction scaffold in 7.5% immersion solution;

[0097] Figure 9 7.5% soaking solution cold extraction scaffold cell proliferation experiment;

[0098] Figure 10 : 10% soaking solution cold extraction of the finished support frame;

[0099] Figure 11 Microstructure of the cold extraction support in 10% immersion solution;

[0100] Figure 12 : Cold extraction of scaffold cells with 10% soaking solution for cell proliferation experiment;

[0101] Figure 13 Appearance of non-cold brewing support products;

[0102] Figure 14 Microstructure of non-cold extraction scaffold;

[0103] Figure 15 Non-cold-extracted scaffold cell proliferation experiment. Detailed Implementation

[0104] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0105] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0106] Example 1: A skull tissue engineering scaffold prepared using a dual cold extraction freeze-drying process and its preparation method. 1. A skull tissue engineering scaffold prepared using a dual cold extraction freeze-drying process.

[0107] Composition: 0.15 g / mL GelMA and 0.02 g / mL nanoclay (lithium magnesium silicate (Laponite XLG-XR), nanoclay) composite hydrogel.

[0108] Methacrylamide gelatin (GelMA) is one of its derivatives, cured under UV / Vis light, combining the properties of natural and synthetic materials to form 3D structures adapted for cell growth. This hydrogel not only exhibits excellent biocompatibility, providing cell adhesion sites and controllable degradation, but also possesses tunable mechanical properties, temperature-sensitive gel characteristics, and multiple viscoelastic properties. It has been widely used in 3D cell culture, tissue engineering, and bioprinting, becoming a strong candidate to replace traditional gel raw materials. The structural formula of GelMA is shown below. Figure 1 As shown.

[0109] Among them, lithium magnesium silicate (nanoclay) is represented by the molecular formula Na. + 0.7 [(Si8Mg 5.5 Li 0.3 O 20 (OH)4)] - 0.7The microstructure of lithium magnesium silicate is a disk with a diameter of 25 nm and a thickness of about 1 nm. The disk consists of two layers of tetrahedral silicate encapsulating an octahedral crystalline layer in the middle, resembling a sandwich. The octahedral crystals in the middle layer contain magnesium ions, which can be randomly replaced by lithium ions. This substitution effect gives the upper and lower surfaces of the disk a negative charge. In the dry powder state, lithium magnesium silicate particles are stacked together. After dissolving in water, the negative charge on the disk surface is balanced by the positive charge carried by sodium ions distributed inside and around the disk. Due to the relatively weak charge on the disk surface, a large amount of water molecules can be absorbed between the two surfaces, and the upper and lower surfaces of the disk can even be completely separated, resulting in the water-absorbing and swelling properties of lithium magnesium silicate. The edges of the disk can carry a positive charge or a weak negative charge. When the solution pH < 9, the exposed hydroxyl groups at the edges can be protonated, resulting in a positive charge at the edges, with the positive charge being approximately 10% of the negative charge. The surface structure of the particles allows for uniform dispersion and easy binding with substances. The main degradation products of lithium magnesium silicate, silicate and magnesium ions, have been shown to promote mineralization, bone formation, and angiogenesis. The release of ions occurs simultaneously with the degradation of lithium magnesium silicate, and is influenced by the system's pH and particle concentration. The structural formula of nanoclay is as follows: Figure 2 As shown.

[0110] 2. Preparation of skull tissue engineering scaffolds

[0111] 2.1 Instruments, Equipment and Consumables

[0112] 2.1.1 Instruments, Equipment and Consumables

[0113] Electronic balance (MTW 120g / 0.0001g); Centrifuge tube benchtop air bath shaker (LC-DIS-20H); SONOBIO ultrasonic homogenizer (split type 24kHz, 1200W); UV curing 3D printer 8601pro (Suzhou Yongqinquan Intelligent Equipment Co., Ltd.); Vacuum freeze dryer (Shinzhi); UV curing chamber (UVGO-120W).

[0114] 2.1.2 Reagents

[0115] GelMA (Sigma, XXV900863); nanolay (Laponite XLG-XR, BYK, Germany); photoinitiator (LAP, Sigma); water-soluble light blocker (UVA, Suzhou Yongqinquan Intelligent Equipment Co., Ltd., product number: EFL-UVAW-001).

[0116] 2.2 Solution Preparation

[0117] 2.2.1 Preparation of 0.02 g / mL nanoclay (lithium magnesium silicate) solution

[0118] Weigh 0.4997 g of nanocaly, place it in a centrifuge tube, add 25 mL of purified water, and sonicate for 3 min at 120 Hz to prepare a 0.02 g / mL lithium magnesium silicate solution.

[0119] 2.2.2, 0.02 g / mL nanoclay + 0.15 g / mL GelMA mixed solution

[0120] Weigh 3.0107g of GelMA and add 20mL of 0.02g / mL nanoclay solution. Shake the solution in a benchtop air bath shaker at 40-50℃ and 100-200rpm / min for 2 hours. If the solution is found to be homogeneous, transparent and free of lumps, then a 0.02g / mL nanoclay + 0.15g / mL GelMA mixed solution is obtained.

[0121] 2.2.3 Preparation of 0.03 g / mL LAP solution

[0122] Weigh 3.001g of LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine), add purified water, and dissolve at 40℃ for 1-2 hours. When the white powder is completely dissolved and the solution is clear and transparent, it is considered completely dissolved. Filter using a 0.22μm sterile filter membrane, dispense into 2mL EP tubes, protect from light, and freeze at -20℃ for later use.

[0123] 2.2.4 Preparation of 0.03 g / mL UVA solution

[0124] Weigh 0.0251g of water-soluble light-blocking agent (UVA, Suzhou Yongqinquan Intelligent Equipment Co., Ltd., product number: EFL-UVAW-001) powder, add 8.33mL of purified water, mix well, and observe that the solution is clear and transparent, thus obtaining a 0.03g / mL UVA solution. Filter through a 0.22μm sterile filter membrane, dispense into 2mL EP tubes, protect from light, and freeze at -20℃ for later use.

[0125] 2.2.5 Preparation of 0.1 g / mL GelMA solution

[0126] Weigh 2.042g of GelMA, add 20mL of purified water, and shake the solution in a benchtop air bath shaker at 40-50℃ and 100-200rpm / min for 2 hours. If the solution is found to be uniform, transparent, and free of lumps, then a 0.1g / mL GelMA solution is obtained.

[0127] 2.2.6 Preparation of Bioprinting Ink

[0128] Measure 20 mL of a mixture of 0.02 g / mL nanoclay and 0.15 g / mL GelMA, add 1.2 mL of 0.03 g / mL LAP solution (60 μL / mL) and 340 μL of 0.03 g / mL UVA solution (17 μL / mL), and mix thoroughly.

[0129] 2.3 Printing of Skull Tissue Engineering Scaffolds

[0130] Use a UV-curing printer for printing, under the following conditions:

[0131] Light intensity: 10mw / cm 2 Exposure time: 7s, number of substrate layers: 5, substrate exposure time: 5s, film thickness: 125μm. Print according to the above parameters, the printed model is as follows. Figure 3 As shown.

[0132] Place the printed skull tissue engineering scaffold in PBS buffer solution at 40°C and wash for 1-2 hours (note that the specific time can be adjusted according to the sample size). Discard the PBS buffer solution and allow the surface of the skull tissue engineering scaffold to dry.

[0133] 2.4 Freeze-drying

[0134] The above-treated skull tissue engineering scaffold was pre-frozen overnight at -20℃ or -80℃ (it should be noted that any temperature within the range of -20 to -80℃ is acceptable), and then freeze-dried in a vacuum freeze dryer (vacuum degree 110kPa, freezing temperature -55℃, time 4-72h) (it should be noted that the specific freezing time can be adjusted according to the sample size).

[0135] 2.5, Immersion in 0.001-0.2 g / mL GelMA solution

[0136] The freeze-dried skull tissue-engineered scaffold was placed in a 24-well plate, and 2 mL of 0.1 g / mL GelMA solution was added to each well to completely immerse the scaffold in the solution. The plate was incubated at a constant temperature of 40°C for 8 hours, avoiding light during the soaking process. After soaking, any residual solution on the surface of the skull tissue-engineered scaffold was removed.

[0137] 2.6, -80℃ cold smelting

[0138] Place the bone flap processed in step 2.5 above in a -80°C freezer and freeze overnight.

[0139] 2.7 Re-curing

[0140] The cold-extracted skull tissue engineering scaffold was cured under 405nm ultraviolet light for 5-30 minutes, and then placed in a vacuum freeze dryer for freeze drying (vacuum degree 110kPa, freezing temperature -55℃, time 4-72h) (it should be noted that the specific freezing time can be adjusted according to the sample size) to obtain the final skull tissue engineering scaffold.

[0141] Example 2: A skull tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process and its performance testing.

[0142] This embodiment provides a cranial tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process, and its preparation method. The soaking solution is a 5% GelMA solution (wherein, 5% (w / v)), i.e., a GelMA solution of 0.05 g / mL. Other steps are consistent with those described in Example 1. It meets the requirements for a hydrogel product used in cranial repair. The cranial tissue engineering scaffold prepared by this method has advantages such as good mechanical properties, simple production process, certain degradation properties, and good biocompatibility.

[0143] 1. Experimental Methods

[0144] The prepared skull tissue engineering scaffold was subjected to performance testing using conventional testing methods known in the art. The performance testing included appearance (visual inspection), bonding effect (SEM), mechanical property testing, and biocompatibility testing.

[0145] 2. Experimental Results

[0146] 2.1 Appearance

[0147] The appearance of the finished product of the 5% immersion solution cold extraction support is shown in the figure. Figure 4 As shown in the figure, the inorganic scaffold is a light yellow porous scaffold.

[0148] 2.2 SEM

[0149] Microstructure of the cold extraction scaffold in 5% immersion solution as follows Figure 5 As shown in the figure, the results indicate that the pore size is uniform and the pore connectivity is good.

[0150] 2.3 Mechanical Properties

[0151] Mechanical property testing revealed that the compressive strength of the 5% immersion solution cold extraction support was 4.62 MPa, indicating strong resistance to damage.

[0152] 2.4 Biocompatibility

[0153] Results of the 5% immersion solution cold extraction scaffold cell proliferation experiment are as follows: Figure 6As shown, the results indicate that the cells proliferated normally, demonstrating that the 5% immersion solution cold-extracted scaffold has good biocompatibility.

[0154] Example 3: A skull tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process and its performance testing.

[0155] This embodiment provides a skull tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process, and its preparation method. The soaking solution is a 7.5% GelMA solution (wherein, 7.5% (w / v)), i.e., a GelMA solution of 0.75 g / mL. Other steps are consistent with those described in Example 1. It meets the requirements for a hydrogel product used in skull repair. The skull tissue engineering scaffold prepared by this method has advantages such as good mechanical properties, simple production process, certain degradation properties, and good biocompatibility.

[0156] 1. Experimental Methods

[0157] The prepared skull tissue engineering scaffold was subjected to performance testing using conventional testing methods known in the art. The performance testing included appearance (visual inspection), bonding effect (SEM), mechanical property testing, and biocompatibility testing.

[0158] 2. Experimental Results

[0159] 2.1 Appearance

[0160] The appearance of the finished product of the 7.5% immersion solution cold extraction support is shown in the figure below. Figure 7 As shown in the figure, the inorganic scaffold is a light yellow porous scaffold.

[0161] 2.2 SEM

[0162] Microstructure of the cold extraction scaffold in 7.5% immersion solution as follows Figure 8 As shown, the results indicate that the pores are uniform in size and have good pore connectivity, forming a "coral" or "foam" structural feature.

[0163] 2.3 Mechanical Properties

[0164] Mechanical property testing revealed that the compressive strength of the 7.5% immersion solution cold extraction support was 6.50 MPa, indicating strong resistance to damage.

[0165] 2.4 Biocompatibility

[0166] Results of the 7.5% soaking solution cold extraction scaffold cell proliferation experiment are as follows: Figure 9 As shown, the results indicate that the cells proliferated normally, demonstrating that the 7.5% immersion solution cold-extracted scaffold has good biocompatibility.

[0167] Example 4: A skull tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process and its performance testing.

[0168] This embodiment provides a skull tissue engineering scaffold prepared using a dual cold extraction and freeze-drying process, and its preparation method. The soaking solution is a 10% GelMA solution (wherein, 10% (w / v)), i.e., a 0.1 g / mL GelMA solution. Other steps are consistent with those described in Example 1. It meets the requirements for a hydrogel product used in skull repair. The skull tissue engineering scaffold prepared by this method has advantages such as good mechanical properties, simple production process, certain degradation properties, and good biocompatibility.

[0169] 1. Experimental Methods

[0170] The prepared skull tissue engineering scaffold was subjected to performance testing using conventional testing methods known in the art. The performance testing included appearance (visual inspection), bonding effect (SEM), mechanical property testing, and biocompatibility testing.

[0171] 2. Experimental Results

[0172] 2.1 Appearance

[0173] The appearance of the finished product of the 10% immersion solution cold extraction support is shown in the figure below. Figure 10 As shown in the figure, the inorganic scaffold is a light yellow porous scaffold.

[0174] 2.2 SEM

[0175] Microstructure of the cold extraction support in 10% immersion solution as follows Figure 11 As shown in the figure, the results indicate that the pore size is uniform, the pore connectivity is good, and a uniformly distributed honeycomb porous structure is formed.

[0176] 2.3 Mechanical Properties

[0177] Mechanical property testing revealed that the compressive strength of the 10% immersion solution cold extraction support was 9.92 MPa, indicating strong resistance to damage.

[0178] 2.4 Biocompatibility

[0179] Results of cold extraction scaffold cell proliferation experiment with 10% soaking solution as follows: Figure 12 As shown, the results indicate that the cells proliferated normally, suggesting that the 10% immersion solution cold-extracted scaffold has good biocompatibility.

[0180] A comparative study of the performance of skull tissue-engineered scaffolds prepared by the method described in this invention and skull tissue-engineered scaffolds prepared by conventional methods commonly used in the art.

[0181] Compared to the current mainstream biomimetic mineralized collagen technology (which relies on collagen self-assembly and random deposition of hydroxyapatite, resulting in two major drawbacks: uncontrollable structure and unbalanced mechanical properties), this invention achieves a generational breakthrough through a photopolymerization 3D printing-cold extraction freeze-drying synergistic process: biomimetic mineralized collagen suffers from low pore connectivity and limited cell migration depth due to the randomness of mineralization, while this invention optimizes the above limitations by using 3D printing of pre-set gradient channels and dual freeze-drying for directional pore formation.

[0182] Biomimetic mineralized collagen faces a rigidity-toughness contradiction: "insufficient mineralization leads to brittleness, while excessive mineralization leads to stiffness," and its long degradation cycle is mismatched with bone regeneration. This invention utilizes nano-clay toughening and cold-extracted microcrystal reinforcement to achieve a compressive strength >5MPa (reaching the level of cancellous bone) and precisely matches the degradation cycle to 3-6 months through secondary photocrosslinking density regulation.

[0183] This comparative example uses an experimental control test without cold extraction as a comparison example (except for the absence of cold extraction, the other experimental steps are consistent with those described in Example 1), such as direct light curing followed by immersion.

[0184] 1. Experimental Methods

[0185] The prepared skull tissue engineering scaffold was subjected to performance testing using conventional testing methods known in the art. The performance testing included appearance (visual inspection), bonding effect (SEM), mechanical property testing, and biocompatibility testing.

[0186] 2. Experimental Results

[0187] 2.1 Appearance

[0188] The appearance of the non-cold brewing support product is shown in the image below. Figure 13 As shown in the figure, the inorganic scaffold is a light yellow porous scaffold.

[0189] 2.2 SEM

[0190] Microstructure of non-cold-extracting scaffolds Figure 14 As shown, the results indicate that the non-cold-extracted scaffold has poor pore connectivity and is classified as a "closed-pore" hydrogel for improving the permeability of imaging materials.

[0191] 2.3 Mechanical Properties

[0192] Mechanical performance testing revealed that the compressive strength of the non-cold-extracting scaffold was 0.38 MPa, indicating weak resistance to damage and making it difficult to provide mechanical support.

[0193] 2.4 Biocompatibility

[0194] Results of non-cold-extracted scaffold cell proliferation experiments are as follows: Figure 15 As shown in the figure, the results indicate that the cells proliferated normally, demonstrating good biocompatibility.

Claims

1. A 3D-printed skull tissue engineering scaffold, characterized in that, The skull tissue engineering scaffold is prepared from the following raw materials: GelMA, lithium magnesium silicate, photoinitiator and light blocker.

2. The skull tissue engineering scaffold according to claim 1, characterized in that, The skull tissue engineering scaffold is prepared by 3D printing and dual cold extraction freeze-drying process using a composite hydrogel of GelMA and lithium magnesium silicate.

3. The skull tissue engineering scaffold according to claim 2, characterized in that, The concentration of GelMA in the composite hydrogel is 0.001-0.02 g / mL; Optionally, the concentration of lithium magnesium silicate in the composite hydrogel is 0.0005-0.02 g / mL.

4. The skull tissue engineering scaffold according to claim 3, characterized in that, The concentration of GelMA in the composite hydrogel is 0.15 g / mL; Optionally, the concentration of lithium magnesium silicate in the composite hydrogel is 0.02 g / mL.

5. The skull tissue engineering scaffold according to claim 1, characterized in that, The molecular formula of the lithium magnesium silicate is Na. + 0.7 [(Si8Mg 5.5 Li 0.3 O 20 (OH)4)] - 0.7 ; Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 10-50 nm and a thickness of 0.05-5 nm; Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 25 nm and a thickness of 1 nm; Optionally, the disk is composed of two layers of tetrahedral silica coated with an octahedral crystal layer in the middle; Optionally, the photoinitiator includes LAP, Irgacure 2959, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, or a ruthenium photoinitiator; Optionally, the light-blocking agent includes UVA, carbon black, TiO2, ZnO, iron oxide, zinc oxide, CaCO3, phthalocyanine blue, phthalocyanine green, or benzophenone compounds. Optionally, the photoinitiator is a 0.01-0.10 g / mL LAP solution; Optionally, the photoinitiator is a 0.03 g / mL LAP solution; Optionally, the light-blocking agent is a 0.01-0.10 g / mL UVA solution; Optionally, the light-blocking agent is a 0.03 g / mL UVA solution.

6. A method for preparing a skull tissue engineering scaffold using a dual cold extraction and freeze-drying process, characterized in that, The method includes the following steps: (1) Preparation of bioprinting ink: Photoinitiator and light blocker are added to a mixed solution obtained by mixing GelMA and lithium magnesium silicate to obtain bioprinting ink; (2) 3D printing: The initial blank of the skull framework was obtained by using photopolymerization 3D printing technology; (3) First freeze drying: The cleaned skull framework is pre-frozen and then freeze-dried under vacuum; (4) Immersion in GelMA solution: Immerse the freeze-dried skull scaffold in GelMA solution; (5) -80℃ cold extraction: The soaked skull support is placed in -80℃ for cold extraction; (6) Re-curing: Curing the cold-extracted skull framework; (7) Second freeze-drying: The solidified skull scaffold is freeze-dried again under vacuum to obtain a skull tissue engineering scaffold.

7. The method according to claim 6, characterized in that, The concentration of GelMA in the mixed solution described in step (1) is 0.001-0.02 g / mL; Optionally, the concentration of lithium magnesium silicate in the mixed solution in step (1) is 0.0005-0.02 g / mL; Optionally, the concentration of GelMA in the mixed solution in step (1) is 0.15 g / mL; Optionally, the concentration of lithium magnesium silicate in the mixed solution in step (1) is 0.02 g / mL; Optionally, the molecular formula of the lithium magnesium silicate is Na. + 0.7 [(Si8Mg 5.5 Li 0.3 O 20 (OH)4)] - 0.7 ; Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 10-50 nm and a thickness of 0.05-5 nm; Optionally, the microstructure of the lithium magnesium silicate is a disk with a diameter of 25 nm and a thickness of 1 nm; Optionally, the disk is composed of two layers of tetrahedral silica coated with an octahedral crystal layer in the middle; Optionally, the photoinitiator in step (1) includes LAP, Irgacure 2959, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, or ruthenium photoinitiator; Optionally, the light blocking agent in step (1) includes UVA, carbon black, TiO2, ZnO, iron oxide, zinc oxide, CaCO3, phthalocyanine blue, phthalocyanine green or benzophenone compounds. Optionally, the photoinitiator in step (1) is a 0.01-0.10 g / mL LAP solution; Optionally, the photoinitiator in step (1) is a 0.03 g / mL LAP solution; Optionally, the light-blocking agent mentioned in step (1) is a 0.01-0.10 g / mL UVA solution; Optionally, the light-blocking agent in step (1) is a 0.03 g / mL UVA solution; Optionally, the volume of the mixed solution used in step (1) is 5-35 mL; Optionally, the volume of the mixed solution used in step (1) is 20 mL; Optionally, the amount of photoinitiator used in step (1) is 0.5-5 mL; Optionally, the amount of photoinitiator used in step (1) is 1.2 mL; Optionally, the amount of light blocker used in step (1) is 100-600 μL; Optionally, the amount of light-blocking agent used in step (1) is 340 μL.

8. The method according to claim 6, characterized in that, The printing conditions described in step (2) are: light intensity 5-30 mw / cm². 2 Exposure time 3-10s, number of substrate layers 2-8, substrate exposure time 2-8s, film thickness 70-160μm; Optionally, the printing conditions in step (2) are: light intensity 10 mw / cm². 2 Exposure time 7s, number of substrate layers 5, substrate exposure time 5s, film thickness 125μm; Optionally, the cleaning conditions in step (3) are cleaning at 35-60℃ for 0-2 hours; Optionally, the pre-freezing conditions in step (3) are overnight pre-freezing at -(10-80)℃; Optionally, the pre-freezing conditions in step (3) are overnight pre-freezing at -20°C or -80°C; Optionally, the concentration of the GelMA solution in step (4) is 0.001-0.02 g / mL; Optionally, the concentration of the GelMA solution in step (4) is 0.05 g / mL, 0.075 g / mL, or 0.1 g / mL; Optionally, the soaking conditions in step (4) are constant temperature at 30-50℃, protected from light, and soaked for 5-12 hours; Optionally, the soaking conditions in step (4) are: constant temperature at 40°C, protected from light, and soaked for 8 hours. Optionally, the cold extraction conditions described in step (5) are -80°C freezing overnight; Optionally, the curing conditions in step (6) are curing under 405nm or 365nm ultraviolet light for 5-30 minutes.

9. A bioprinting ink for 3D printing skull tissue engineering scaffolds, characterized in that, The bioprinting ink is the bioprinting ink as described in claim 6.

10. The application of the skull tissue engineering scaffold prepared by any one of claims 6-8 in the preparation of skull repair materials.

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