Preparation method and application of a bioactive scaffold with a spatial polyhedral structure

Abstract

The present application relates to a kind of preparation method and application of bioactive scaffold with spatial polyhedral structure.The bioactive scaffold includes: the plane base at the bottom and the spatial polyhedral structure of upper portion by a plurality of pillars connection accumulation;The bioactive scaffold with spatial polyhedral structure can be applied to promote osteogenic differentiation, angiogenesis and neurogenesis in vitro, and promote neurovascularization bone regeneration in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a method for preparing a bioactive scaffold with a spatial polyhedral structure and its application in bone repair. Background Technology

[0002] As one of the most important organs and tissues in the human body, bone has a highly complex composition and multi-level structure. Structurally, bone tissue can be divided into external cortical bone and internal cancellous bone. The external cortical bone provides mechanical support, while the internal cancellous bone is a porous network structure composed of needle-like or sheet-like trabeculae. It not only provides adhesion sites for various cells, forming cellular "niches," but also helps absorb energy from external impacts. However, this porous network structure is irregular and difficult to simulate through programmed design. Therefore, designing bone tissue engineering scaffolds that can simulate the structure of cancellous bone remains a significant challenge.

[0003] Bone growth, development, and repair are also closely regulated by other systems such as the immune system, vascular system, and nervous system. For example, the vascular network in the vascular system can provide bone-related cells with sufficient oxygen and nutrients while removing metabolic waste, thereby accelerating bone regeneration. Sensory nerves and sympathetic neural networks in the nervous system can secrete various neurotransmitters (CGRP, SP, etc.) and neurotrophic factors (NGF, BDNF, and GDNF, etc.) to participate in bone repair, regeneration, and homeostasis. Therefore, reconstructing the neurovascular network at bone defects remains crucial for high-quality bone regeneration.

[0004] Existing technical documents:

[0005] [1]A.Marrella,TYLee,DHLee,S.Karuthedom,D.Syla,A.Chawla,A.Khademhosseini,HLJang.Engineering vascularized and innervated bonebiomaterials for improved skeletal tissue regeneration[J].Materials Today,2018,21,362.

[0006] [2] Chen Qin, Hongjian Zhang, Lei Chen, Meng Zhang, Jingge Ma, Hui Zhuang, Zhiguang Huan, Yin Xiao, and Chengtie Wu. Cell-Laden Scaffolds for Vascular-Innervated Bone Regeneration[J]. Advanced Healthcare Materials. 2023, 2201923.

[0007] [3]Li WT,Miao WQ,Liu YH,et al.Bioprinted Constructs that Mimic theOssification Center Microenvironment for Targeted Innervation in BoneRegeneration[J].Advanced Functional Materials,2022,32(9):2109871.

[0008] [4]Egan PF, Gonella VC, Engensperger M, et al. Computationally designed lattices with tuned properties for tissue engineering using 3D printing[J]. PloS One, 2017, 12(8):e0182902.

[0009] [5]Egan PF, Ferguson SJ, Shea K. Design of Hierarchical Three-Dimensional Printed Scaffolds Considering Mechanical and Biological Factors for Bone Tissue Engineering[J]. Journal of Mechanical Design, 2017, 139(6):061401. Summary of the Invention

[0010] To address the above problems, this invention provides a bioactive scaffold with a spatial polyhedral structure, its preparation method, and its application.

[0011] In this invention, a bioactive scaffold with a spatial polyhedral structure is created by using photopolymerization 3D printing technology to arrange and connect pillars in different ways on a spatial structure. This spatial polyhedral structure provides a good solution and reference value for designing bone tissue engineering scaffolds that simulate the structure of cancellous bone. Simultaneously, the spatial effects of the spatial polyhedral structure offer new insights into cell adhesion, osteogenic differentiation, and the induction of host angiogenesis and neural network integration.

[0012] In a first aspect, the present invention provides a bioactive scaffold with a spatial polyhedral structure, the bioactive scaffold comprising: a planar abutment at the bottom and a spatial polyhedral structure at the top formed by multiple pillars connected and stacked together.

[0013] A spatial polyhedron is a polyhedron that can seamlessly fill space, with the sum of its dihedral angles across all shared edges being 360°. Different spatial polyhedra exhibit significant differences in their three-dimensional structure and mechanical properties. The complex structures of three-dimensional spatial polyhedra share certain similarities with the porous network structure of cancellous bone, providing new insights for the design of bone tissue engineering scaffolds. Furthermore, the spatial effects of spatial polyhedra can regulate cell differentiation, offering a potential solution for achieving high-quality bone regeneration.

[0014] Preferably, the diameter of the planar abutment is 2–20 mm and the height is 0.1–1 mm; the three-dimensional dimensions of each unit cell of the spatial polyhedral structure are 0.5–2 mm × 0.5–2 mm × 0.5–2 mm; the diameter of each support is 100–500 μm; and the overall diameter of the bioactive scaffold is 2–20 mm and the height is 0.5–20 mm.

[0015] Preferably, the bioactive scaffold is made of bioactive bioceramics, polymers, metals, or metal oxides; the bioceramic is β-phase tricalcium phosphate or hydroxyapatite, preferably β-phase tricalcium phosphate.

[0016] Preferably, the shape of the spatial polyhedron structure is a truncated octahedron, a truncated half-cube, a truncated cube, or a truncated tetrahedron.

[0017] Secondly, the present invention also provides a method for preparing the above-mentioned bioactive scaffold with a spatial polyhedral structure, comprising:

[0018] (1) Design a printed model with a spatial polyhedral structure using 3D modeling software;

[0019] (2) Prepare photocurable printing ink by mixing bioactive material powder and photosensitive resin and ball milling.

[0020] (3) Printing the green blank holder;

[0021] (4) High-temperature sintering.

[0022] Preferably, in step (2), the mass fraction of bioactive material powder in the photocurable printing ink is 20-80%, more preferably 30-50%.

[0023] Preferably, in step (2), the preparation method of the photosensitive resin includes: mixing 20-60g of 1,6-hexanediol diacrylate, 20-60g of polyethylene glycol diacrylate, 3-6g of Triton X-100 and 1-2g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide evenly to obtain the photosensitive resin.

[0024] Preferably, in step (3), the photocurable printing ink obtained in step (2) is introduced into the photocurable 3D printer, and the printing model in step (1) is printed and cured layer by layer to obtain a green bracket.

[0025] Preferably, in step (4), the high-temperature sintering temperature is 1000-1200℃ and the time is 2-8 hours.

[0026] Thirdly, the present invention provides an application of the above-mentioned bioactive scaffold with a spatial polyhedral structure in promoting osteogenic differentiation, angiogenesis and neurogenicity in vitro, and in promoting neurovascularized bone regeneration in vivo.

[0027] Preferably, the osteogenic differentiated cells are bone marrow mesenchymal stem cells, the angiogenic cells are human umbilical vein endothelial cells, and the neuralized cells are Schwann cells.

[0028] In this invention, the three-dimensional polyhedral structure of the bioactive scaffold can not only promote the ingrowth of surrounding bone tissue, but also induce the ingrowth of blood vessels and neural networks in the host, thereby accelerating the bone repair process.

[0029] Beneficial effects:

[0030] The bioactive scaffold with a spatial polyhedral structure designed in this invention, based on its spatial effect, can not only promote the ingrowth of surrounding bone tissue, but also induce the ingrowth of blood vessels and neural networks in the host, thereby accelerating the bone repair process. Furthermore, this bioactive scaffold can effectively promote the expression of related genes during osteogenic, vascularization, and neuralization processes. Attached Figure Description

[0031] Figure 1Schematic diagrams of 3D-printed traditional cross-structured scaffolds (Ctr76 and Ctr66) and different spatial polyhedral bioactive scaffolds (Tt, To, Tc, and Ct) prepared according to this invention; wherein Ctr76 is a traditional cross-structured bioactive scaffold with a porosity of 76%, Tt is a truncated tetrahedral spatial polyhedral bioactive scaffold with a porosity of 76%, To is a truncated octahedral spatial polyhedral bioactive scaffold with a porosity of 76%, and Tc is a truncated cubic spatial polyhedral bioactive scaffold with a porosity of 76%. Ctr66 is a traditional cross-structure bioactive scaffold with a porosity of 76%, and Ct is a truncated cubic spatial polyhedral bioactive scaffold with a porosity of 66%. Among them, (A) is an overall model diagram of the traditional cross-structure and spatial polyhedral scaffolds; (B) is a schematic diagram of a single-layer structure of the traditional cross-structure and spatial polyhedral scaffolds; (C) is a SEM scanning electron microscope image of the traditional cross-structure and spatial polyhedral scaffolds; and (D) is a Micro-CT reconstruction image of the traditional cross-structure and spatial polyhedral scaffolds.

[0032] Figure 2 The compressive modulus of 3D-printed conventional cross-structure scaffolds (Ctr76 and Ctr66) and spatial polyhedral bioactive scaffolds (Tt, To, Tc and Ct) with different structures prepared by the present invention.

[0033] Figure 3 Porosity of 3D-printed conventional cross-structure scaffolds (Ctr76 and Ctr66) and spatial polyhedral bioactive scaffolds (Tt, To, Tc and Ct) with different structures prepared by the present invention.

[0034] Figure 4 The results show the cell adhesion, proliferation, and osteogenic gene expression of bone marrow mesenchymal stem cells in 3D-printed conventional cross-structure scaffolds (Ctr76 and Ctr66) and spatial polyhedral bioactive scaffolds (Tt, To, Tc, and Ct) with different structures prepared in this invention. Among them, (A) is a fluorescence image of bone marrow mesenchymal stem cells after culturing on 6 different scaffolds for 1 day, which is used to characterize the spread of cells on the scaffolds; (B) is the proliferation result of bone marrow mesenchymal stem cells after culturing on 6 different scaffold structures for 1, 3, and 7 days; (C) is the gene expression result of osteogenic differentiation-related genes OCN, OPN, RUNX2, ALP, and BMP2 of bone marrow mesenchymal stem cells after culturing on 6 different scaffold structures for 7 days.

[0035] Figure 5The results show the cell proliferation and vascularization-related gene expression of human umbilical vein endothelial cells in 3D-printed conventional cross-structure scaffolds (Ctr76 and Ctr66) and spatial polyhedral bioactive scaffolds (Tt, To, Tc and Ct) with different structures prepared in this invention; wherein (A) shows the proliferation results of human umbilical vein endothelial cells cultured on 6 different scaffold structures for 1, 3 and 7 days; (B) shows the gene expression results of the vascularization-related gene VEGF of human umbilical vein endothelial cells after 3 days of culture on 6 different scaffold structures.

[0036] Figure 6 The results show the cell proliferation and neurotrophic gene expression of Schwann cells in 3D-printed conventional cross-structure scaffolds (Ctr76 and Ctr66) and spatial polyhedral bioactive scaffolds (Tt, To, Tc and Ct) with different structures prepared in this invention; where (A) shows the proliferation results of Schwann cells cultured on 6 different scaffolds for 1, 3 and 7 days; (B) shows the gene expression results of neurotrophic genes NGF, BDNF, GDNF and S100 of Schwann cells after 3 days of culture on 6 different scaffolds.

[0037] Figure 7 The in vivo bone repair results are shown for a 3D-printed traditional cross-structure scaffold (Ctr76) and a spatial polyhedral bioactive scaffold (Tt and To) with different structures prepared in this invention; where (A) are Micro-CT reconstructed images of bone defects at 4 and 10 weeks after implantation for different groups; (B) are the new bone volume / total score at 4 weeks after implantation for different groups; and (C) are the new bone volume / total score at 10 weeks after implantation for different groups.

[0038] Figure 8 The images show the early in vivo neurovascular innervation results of a 3D-printed conventional cross-structure scaffold (Ctr76) and different spatial polyhedral bioactive scaffolds (Tt and To) prepared according to this invention; (A) shows immunofluorescence staining images of nerve fibers and blood vessels at the bone defect site in different groups at 4 weeks after implantation; (B) shows the statistical results of nerve fibers at the bone defect site in different groups at 4 weeks after implantation; and (C) shows the statistical results of neovascularization at the bone defect site in different groups at 4 weeks after implantation.

[0039] Figure 9 Results of late-stage bone regeneration for 3D-printed conventional cross-structure scaffold (Ctr76) and spatial polyhedral bioactive scaffolds (Tt and To) with different structures prepared in this invention; wherein (A) is VG-stained image of newly formed bone in different groups at 10 weeks after implantation; (B) is the statistical result of newly formed bone in different groups at 10 weeks after implantation. Detailed Implementation

[0040] To further illustrate the invention's content, features, and practical effects, the invention will be described in detail below with reference to embodiments. It should be noted that the modification methods of the invention are not limited to these specific implementation methods. Equivalent substitutions and modifications made by those skilled in the art based on their reading of the invention's content, without departing from the spirit and essence of the invention, are also within the scope of protection claimed by this invention.

[0041] The bioactive scaffold with a spatial polyhedral structure provided by this invention can simulate the porous structure of cancellous bone, and its spatial structure and combination can be flexibly controlled. Furthermore, different spatial structures affect paracrine effects and intercellular communication, thereby influencing the bone defect repair process.

[0042] The bioactive scaffold with a spatial polyhedral structure provided by the present invention includes: a planar abutment at the bottom and a spatial polyhedral structure at the top formed by multiple pillars connected and stacked together.

[0043] The diameter of the planar base is 2–20 mm, and the height is 0.1–1 mm. If the diameter is less than 2 mm, the complexity of the upper spatial polyhedral structure will be reduced. If the diameter is greater than 20 mm, the support will be difficult to remove from the printing substrate after printing, making it prone to breakage and hindering the overall integrity of the support. The three-dimensional dimensions of each unit cell of the spatial polyhedral structure are 0.5–2 mm × 0.5–2 mm × 0.5–2 mm. If the dimension is less than 0.5 mm, the spatial complexity will be reduced, and the printing accuracy will decrease, which is not conducive to support preparation and subsequent cleaning. If the dimension is greater than 2 mm, it will be difficult to demonstrate the advantages of the spatial structure. The diameter of each support column is 100–500 μm. If the support column diameter is less than 100 μm, it will be difficult to print due to the limitations of printer accuracy and will be prone to breakage, which is not conducive to maintaining the integrity of the support structure. If the support column diameter is greater than 500 μm, the overall porosity of the support will be low, the structure will be simple, and it will be difficult to demonstrate the advantages of the spatial polyhedral structure. The overall diameter of the bioactive scaffold is 2–20 mm, and the height is 0.5–20 mm. If the diameter is less than 2 mm, the spatial structure of the scaffold will be too simple, failing to demonstrate its spatial effect advantages. If the diameter is greater than 20 mm, it will increase the printing difficulty and the defect rate will rise. If the height of the scaffold is less than 0.5 mm, it will be difficult to achieve complex spatial structures. If the diameter is greater than 20 mm, it will increase the printing difficulty, making it difficult to achieve high-precision stacking between layers, and subsequent cleaning will remain a challenge.

[0044] The bioactive scaffold is made of bioactive materials including bioceramics, polymers, metals, or metal oxides. The bioceramics include β-phase tricalcium phosphate (Ca3(PO4)2) or hydroxyapatite (Ca5(PO4)3(OH)), preferably β-phase tricalcium phosphate (Ca3(PO4)2) ceramic. β-phase tricalcium phosphate (Ca3(PO4)2) exhibits good biocompatibility, biodegradability, and osteoinductive properties, effectively promoting cell adhesion and spread. The sustained-release bioactive calcium and phosphate ions from the material can promote osteogenic differentiation of bone marrow mesenchymal stem cells and osteoblasts, and promote high expression of osteogenic-related genes such as BMP2, OCN, and OPN.

[0045] The spatial polyhedron is a polyhedron capable of seamlessly filling space, with the sum of its dihedral angles across all shared edges being 360°. The shape of the spatial polyhedron can be a truncated octahedron, a truncated half-cube, a truncated cube, or a truncated tetrahedron. Based on its spatial effect, the bioactive scaffold of the spatial polyhedron structure can provide suitable pressure for attached cells, which is beneficial for osteogenic differentiation of bone marrow mesenchymal stem cells and promotes new bone formation. Different space-filling polyhedra have significantly different structural and mechanical properties. For example, a cubic structure has high elasticity and low shear modulus, while an octahedral structure has high shear modulus and surface area-to-volume ratio, but low permeability. In terms of bone tissue engineering scaffolds, the truncated octahedron has a higher surface area-to-volume ratio than other truncated polyhedra at the same porosity, which is more conducive to nutrient transport, thus exhibiting superior properties in promoting osteogenic, vascularized, and neuralized processes.

[0046] The bioactive scaffold is prepared using one of the following technologies: photopolymerization 3D printing, extrusion 3D printing, and selective laser sintering 3D printing; preferably, photopolymerization 3D printing.

[0047] The spatial polyhedral structure in this invention is an open, porous network structure that mimics cancellous bone. Utilizing its spatial effects, it regulates the proliferation and differentiation behavior of various cells, promoting bone regeneration while inducing the ingrowth of the host's neurovascular network. The porosity and / or mechanical strength of the bioactive scaffold with the spatial polyhedral structure can be controlled by altering parameters such as the spatial structure and the diameter of the support pillars. Compared to traditional bioactive scaffolds, the spatial polyhedral scaffold generates suitable wall pressure in fluids, thus facilitating the adhesion of various cells in the tissue fluid. Furthermore, the spatial polyhedral structure exhibits good adsorption of various proteins and growth factors in the tissue fluid, significantly enhancing the scaffold's bioactivity and functionality. Therefore, the bioactive scaffold with the spatial polyhedral structure demonstrates superior effects in promoting osteogenic, vascularized, and neuralized processes. In some embodiments, the compressive strength of the bioactive scaffold with the spatial polyhedral structure can be 1–8 MPa, and the porosity can be 40–80%.

[0048] The following exemplarily illustrates a method for preparing a bioactive scaffold with a spatial polyhedral structure provided by the present invention.

[0049] A 3D modeling software was used to design a printable model with a spatial polyhedral structure. The file format was STL, and then the model was imported into the printer to set the slicing parameters.

[0050] In an optional implementation, the 3D model design software may be SolidWorks, 3ds Max, or CAD.

[0051] Photocurable printing ink is prepared by rapidly ball milling and mixing bioactive material powder and photosensitive resin.

[0052] In an optional embodiment, the bioactive material powder has a mass fraction of 20-80%, preferably 30-50%.

[0053] The preparation method of the photosensitive resin includes: mixing 20-60g of 1,6-hexanediol diacrylate, 20-60g of polyethylene glycol diacrylate, 3-6g of Triton X-100 and 1-2g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide evenly to obtain the photosensitive resin.

[0054] The ball mill can rotate at a speed of 300 to 800 rpm and last for 1 to 10 hours.

[0055] The photocurable printing ink is introduced into the material pool of the printer, and then printed and cured layer by layer according to the printing model and the set parameters to obtain a green bracket.

[0056] In an optional implementation, the thickness of the slice is set to 25–100 μm.

[0057] After thorough cleaning, the obtained green scaffold is sintered at high temperature to obtain the bioactive scaffold with a spatial polyhedral structure.

[0058] In an optional embodiment, the high-temperature sintering temperature is 1000-1200°C and the time is 2-8 hours.

[0059] This study investigated the regulatory effects of cell proliferation and differentiation on a bioactive scaffold with a spatial polyhedral structure by seeding cell suspensions onto it, using a 3D-printed scaffold with a traditional cross-shaped structure as a control. For example, bone marrow mesenchymal stem cells were loaded onto the scaffold to explore the regulatory effect of the spatial polyhedral structure on osteogenic differentiation. Alternatively, endothelial cells could be replaced with bone marrow mesenchymal stem cells while other conditions remained unchanged to explore the regulatory effect of the spatial polyhedral structure on angiogenesis, or nerve cells could be replaced to explore the regulatory effect of the spatial polyhedral structure on neurogenic differentiation. Furthermore, a rat femoral critical bone defect repair experiment was conducted to investigate the effect of this spatial polyhedral scaffold on bone regeneration and its role in inducing host neurovascular network integration. The results show that, compared with traditional cross-shaped scaffolds, the bioactive scaffold with a spatial polyhedral structure of the present invention can better promote the proliferation of bone marrow mesenchymal stem cells, the high expression of osteogenic genes such as BMP2, ALP, OCN, and OPN, the proliferation of human umbilical vein endothelial cells and the expression of vascularization-related gene VEGF, and the proliferation of neurogenic Schwann cells and the high expression of neurotrophic factors NGF, BDNF, GDNF, and S100. In vivo studies show that the bioactive scaffold with a spatial polyhedral structure can promote early neurovascular network integration and subsequent bone regeneration, thereby achieving a high level of neurovascularized bone regeneration. In summary, the spatial polyhedral bioactive scaffold with a porous mesh structure mimicking cancellous bone can achieve neurovascularized bone regeneration and has a better bone repair effect.

[0060] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0061] Example 1

[0062] (1) A truncated tetrahedral spatial polyhedral bioactive scaffold with a porosity of 76% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. The three-dimensional dimensions of each unit cell of the upper integrated spatial polyhedral structure are 1.7 mm × 1.7 mm × 1.7 mm. The diameter of the pillars within the unit cell is 320 μm, and the length of each pillar within the same unit cell is the same.

[0063] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China) and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled at 500rpm for 1h to obtain a photosensitive ceramic slurry.

[0064] (3) Import the obtained printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain a green bracket; set the thickness of the slice to 25μm.

[0065] (4) After the obtained green scaffold is thoroughly cleaned, it is placed in a high-temperature furnace and sintered at 1150°C for 3 hours at a heating rate of 2°C / min to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure.

[0066] Example 2

[0067] (1) A truncated octahedral spatial polyhedral bioactive scaffold with a porosity of 76% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. The three-dimensional dimensions of each unit cell of the upper integrated spatial polyhedral structure are 1.7 mm × 1.7 mm × 1.7 mm. The diameter of the pillars within the unit cell is 320 μm, and the length of each pillar within the same unit cell is the same.

[0068] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China) and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled at 500rpm for 1h to obtain a photosensitive ceramic slurry.

[0069] (3) Import the obtained printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain a green bracket; set the thickness of the slice to 25μm.

[0070] (4) After the obtained green scaffold is thoroughly cleaned, it is placed in a high-temperature furnace and sintered at 1150°C for 3 hours at a heating rate of 2°C / min to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure.

[0071] Example 3

[0072] (1) A truncated cubic spatial polyhedral bioactive scaffold with a porosity of 76% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. The three-dimensional dimensions of each unit cell of the upper integrated spatial polyhedral structure are 1.7 mm × 1.7 mm × 1.7 mm. The diameter of the pillars within the unit cell is 320 μm, and the length of each pillar within the same unit cell is the same.

[0073] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China) and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled at 500rpm for 1h to obtain a photosensitive ceramic slurry.

[0074] (3) Import the obtained printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain a green bracket; set the thickness of the slice to 25μm.

[0075] (4) After the green scaffold is thoroughly cleaned, it is placed in a high-temperature furnace and heated to 1150°C for 3 hours at a heating rate of 2°C / min to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure.

[0076] Example 4

[0077] (1) A semi-cubic spatial polyhedral bioactive scaffold with a porosity of 66% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. The three-dimensional dimensions of each unit cell of the upper integrated spatial polyhedral structure are 1.7 mm × 1.7 mm × 1.7 mm. The diameter of the pillars within the unit cell is 320 μm, and the length of each pillar within the same unit cell is the same.

[0078] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China) and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled at 500rpm for 1h to obtain a photosensitive ceramic slurry.

[0079] (3) Import the obtained printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain a green bracket; set the thickness of the slice to 25μm.

[0080] (4) After the green scaffold is thoroughly cleaned, it is placed in a high-temperature furnace and heated to 1150°C at a heating rate of 2°C / min for 3 hours to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure.

[0081] Comparative Example 1

[0082] (1) A bioactive scaffold with a traditional cross structure and a porosity of 76% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. Each support of the upper integrated cross structure is 150 μm wide and 400 μm high, and the spacing between two adjacent supports in the same layer is 1 mm.

[0083] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China), and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled to obtain a photosensitive ceramic slurry. The ball milling speed was 500 rpm, and the ball milling time was 1 h.

[0084] (3) Import the printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain the green bracket; set the thickness of the slice to 25μm.

[0085] (4) After thorough cleaning, the obtained green scaffold is placed in a high-temperature furnace for sintering to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure. The cured scaffold is then calcined at 1150°C for 3 hours at a heating rate of 2°C / min to obtain the scaffold.

[0086] Comparative Example 2

[0087] (1) A bioactive scaffold with a traditional cross structure and a porosity of 66% was designed using 3ds Max software. The overall scaffold has a diameter of 6 mm and a height of 3 mm. The base has a diameter of 6 mm and a height of 0.5 mm. Each support of the upper integrated cross structure is 250 μm wide and 400 μm high, and the spacing between two adjacent supports in the same layer is 1 mm.

[0088] (2) Preparation of ink for photopolymer printing: 40g of 1,6-hexanediol diacrylate (HDDA, Maclean, China), 40g of polyethylene glycol diacrylate (PEGDA, molecular weight 200, Maclean, China), 4.8g of Triton X-100 (Maclean, China), and 1.6g of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO, Maclean, China) were mixed on a magnetic stirrer until homogeneous to obtain a photopolymer resin. Then, 80g of the photopolymer resin was mixed with 120g of β-tricalcium phosphate (β-TCP) and ball-milled to obtain a photosensitive ceramic slurry. The ball milling speed was 500 rpm, and the ball milling time was 1 h.

[0089] (3) Import the printing ink into the material pool of the printer, print and solidify layer by layer according to the printing model and the set parameters to obtain the green bracket; set the thickness of the slice to 25μm.

[0090] (4) After thorough cleaning, the obtained green scaffold is placed in a high-temperature furnace for sintering to remove organic matter and impurities, thereby obtaining the bioactive scaffold with a spatial polyhedral structure. The cured scaffold is then calcined at 1150°C for 3 hours at a heating rate of 2°C / min to obtain the scaffold.

[0091] The bioactive scaffolds prepared in Comparative Example 1, Comparative Example 2, and Examples 1-4 were subjected to physicochemical and biological evaluations, including structure, SEM scanning analysis, Micro-CT reconstruction, mechanical strength testing, porosity, in vitro biological effects (including cell proliferation and differentiation), and in vivo animal experiments on rabbit femoral defect repair, as detailed below:

[0092] (I) Structural, SEM scan analysis, and Micro-CT reconstruction

[0093] Figure 1 Schematic diagrams of 3D-printed traditional cross-structure scaffolds (Ctr76 and Ctr66) and different spatial polyhedral bioactive scaffolds (Tt, To, Tc, and Ct) prepared according to this invention; wherein Ctr76 is a traditional cross-structure scaffold with a porosity of 76%, Ctr66 is a traditional cross-structure scaffold with a porosity of 76%, Tt is a truncated tetrahedral spatial polyhedral bioactive scaffold with a porosity of 76%, To is a truncated octahedral spatial polyhedral bioactive scaffold with a porosity of 76%, and Tc is a pore size distribution. A truncated cubic spatial polyhedral bioactive scaffold with a porosity of 76% and a truncated half-cubic spatial polyhedral bioactive scaffold with a porosity of 66% are shown in Figures (A). Figure (B) shows the overall model of the traditional cross-structure and spatial polyhedral scaffolds; Figure (C) shows the single-layer structure of the traditional cross-structure and spatial polyhedral scaffolds; Figure (D) shows the SEM images of the traditional cross-structure and spatial polyhedral scaffolds; and Figure (D) shows the Micro-CT reconstruction images of the traditional cross-structure and spatial polyhedral scaffolds. As shown in the figures, the scaffold structure after printing and sintering is consistent with the design model, demonstrating the accuracy of photopolymerization 3D printing technology and achieving high-fidelity fabrication of various scaffolds.

[0094] (II) Mechanical Strength Test

[0095] The compressive strength of the bioactive scaffold was characterized using a universal testing machine (INSTRON-1195, Instrand, USA). The loading rate for the compressive strength test was 0.5 mm / min until the scaffold completely broke, and the maximum stress F was recorded. The compressive strength of the scaffold was then calculated using a formula. The porosity of the scaffold was characterized using Archimedes' method of drainage.

[0096] Figure 2The compressive modulus of the 3D-printed traditional cross-structure scaffolds (Ctr76 and Ctr66) and the spatial polyhedral bioactive scaffolds (Tt, To, Tc, and Ct) with different structures prepared in this invention are shown in the figure. As can be seen from the figure, the mechanical strength of the scaffolds with different structures varies. Specifically, the compressive strength of the polyhedral scaffolds Tt, To, and Tc with a porosity of 76% is significantly higher than that of the control group Ctr76, and the compressive strength of the polyhedral scaffold Ct with a porosity of 66% is significantly higher than that of the control group Ctr66. This indicates that under the same porosity conditions, the polyhedral scaffolds have better mechanical strength than the traditional cross-structure scaffolds.

[0097] Figure 3 The porosity of 3D-printed traditional cross-structure scaffolds (Ctr76 and Ctr66) and different spatial polyhedral bioactive scaffolds (Tt, To, Tc, and Ct) prepared according to this invention is shown in the figure. As can be seen, the porosity of the control group Ctr76 is not significantly different from that of the spatial polyhedral scaffolds Tt, To, and Tc, consistent with the designed porosity of 76%. The porosity of Ctr66 and Ct also shows no significant difference, consistent with the designed porosity of 66%.

[0098] (III) In vitro biological evaluation (including cell proliferation and differentiation)

[0099] Bone marrow mesenchymal stem cells, human umbilical vein endothelial cells, and Schwann cells were selected as model cells to investigate cell proliferation and differentiation behavior, as detailed below:

[0100] Appropriate amounts of bone marrow mesenchymal stem cells were seeded onto 3D-printed conventional cross-structure scaffolds and bioactive scaffold materials with spatial polyhedral structures, respectively. The cells were cultured for one day in a DMEM complete medium in an incubator (37℃, 5% CO2), followed by fixation with 4% paraformaldehyde. The nuclei and cytoskeleton of the cells on the scaffolds were stained with DAPI and fluorescein isothiocyanate, respectively. Cell morphology was then observed using a laser scanning confocal microscope. Figure 4 As shown in (A), the cells spread well on the surface of each group of scaffolds, forming a dense cell network, indicating that each group of bioactive scaffolds has good biocompatibility.

[0101] To characterize cell proliferation on different scaffolds, appropriate amounts of cells were seeded onto the scaffolds and cultured in DMEM complete medium for 1, 3, and 7 days. Then, 10% CCK-8 reagent was added, and the cells were incubated again for 1.5 hours. The absorbance of the solution was then measured to quantitatively assess cell proliferation. Figure 4 (B) It can be seen that, under the same porosity conditions, the spatial polyhedral scaffold can better promote the proliferation of bone marrow mesenchymal stem cells than the traditional cross scaffold, demonstrating the superiority of the spatial structure.

[0102] RT-PCR was used to characterize gene expression in cells. After 7 days of cell culture, RNA was extracted using TRIzol reagent, and cDNA was synthesized using the PrimeScript 1st Strand cDNA synthesis kit. RT-PCR experiments were performed using SYBR GreenQPCR Master Mix, with GAPDH as an endogenous control. Figure 4 (C) It can be seen that the expression of osteogenic genes OPN, RunX2 and ALP in the spatial polyhedral scaffold Tt and To groups was significantly higher than that in the control group Ctr76 on day 7, indicating that the polyhedral scaffolds Tt and To can promote osteogenic differentiation of bone marrow mesenchymal stem cells compared with traditional cross structure scaffolds.

[0103] Human umbilical vein endothelial cells (HUVECs) were used to characterize the pro-angiogenic effect of a spatial polyhedral scaffold. An appropriate amount of HUVECs were seeded onto the scaffold and cultured in ECM complete medium for 1, 3, and 7 days. After adding 10% CCK-8 reagent and incubating for another 1.5 hours, the absorbance of the solution was measured to quantitatively assess cell proliferation. Figure 5 (A) It can be seen that both the traditional cross-structure scaffold and the spatial polyhedral bioactive scaffold can effectively promote the proliferation of human umbilical vein endothelial cells, with the Tt and To groups showing better promotion effects in the first three days. The expression of angiogenesis-related genes was characterized using RT-qPCR. Figure 5 (B) It can be seen that VEGF expression in the To group was significantly upregulated compared to the traditional cross-structure Ctr76, indicating that the scaffold with the truncated octahedral structure is more conducive to the angiogenic differentiation of endothelial cells.

[0104] Schwann cells were used to characterize the scaffold's neurotrophic capacity. An appropriate amount of Schwann cells were seeded onto the scaffold and cultured in DMEM low-glucose medium for 1, 3, and 7 days. After adding 10% CCK-8 reagent and incubating for another 1.5 hours, the absorbance of the solution was measured to quantitatively assess cell proliferation. Figure 6 (A) It can be seen that, under the same porosity conditions, the proliferative activity of Schwann cells loaded on the polyhedral scaffold was significantly higher than that of the traditional cross-scaffold. The expression of neuronalization-related genes was characterized using RT-qPCR. Figure 6 (B) It can be seen that the expression of neuroblast genes NGF, BDNF, GDNF and S100 in the polyhedral structure Tt and To groups is significantly higher than that in the control group Ctr76, and the expression of neuroblast genes NGF, BDNF, GDNF and S100 in the polyhedral structure Ct group is significantly higher than that in the control group Ctr66, indicating that the polyhedral structure scaffold is more conducive to the neuroblast differentiation of Schwann cells.

[0105] (iv) Evaluation of biological effects in vivo

[0106] A rabbit critical femoral defect model was used to investigate the in vivo biological characteristics of a bioactive scaffold with a spatial polyhedral structure. Male New Zealand white rabbits (weighing 2.5 kg) were randomly divided into four groups: a blank group, a conventional cross scaffold group with a porosity of 76% (Ctr76), a truncated tetrahedral scaffold group with a porosity of 76% (Tt), and a truncated octahedral scaffold group with a porosity of 76% (To). A 6 mm diameter bone defect was constructed in the cancellous bone of the rabbit femur. The sterilized and dried scaffold was implanted into the defect site. During the establishment of the bone defect model, intraosseous blood vessels and nerves were also destroyed. At 4 and 10 weeks after implantation, all animals were anesthetized and sacrificed, and the femur was removed and fixed in 4% paraformaldehyde solution for 24 hours.

[0107] Femoral samples were scanned and analyzed using Micro-CT (SKYSCAN1172, Bruker, Germany), and the volume fraction of new bone was calculated using BV / TV (bone volume / total volume). Figure 7 (A) It can be seen that 4 weeks after implantation, the defect site in the control group still had a large cavity, while the scaffold group with a spatial polyhedral structure had fewer cavities and more bone tissue ingrowth. Figure 7 (B) Statistical results showed that the volume fraction of newly formed bone in the polyhedral scaffold group was approximately 8%, significantly higher than that in the control group. Micro-CT results 10 weeks after implantation showed that the defect sites in the control group still had cavities, while the defects in the polyhedral scaffold group were almost completely filled with bone tissue. Figure 7 (C) Statistical results show that the volume fraction of new bone in the polyhedral scaffold group was about 13%, which was significantly higher than that in the control group.

[0108] Immunofluorescence staining was used to characterize the early angiogenesis and neurogenic effects of the scaffold. Femoral samples were immersed in 10% EDTA solution for four weeks. Then, the samples were immersed in 10% sucrose solution for 5 hours and 30% sucrose solution for 24 hours, respectively. The samples were then embedded in frozen sections and cut into 20 μm sections using a cryostat. The sections were incubated overnight at 4°C with NF200 primary antibody (CH22104, Neuromics, USA) and CD31 primary antibody (NB600-562, Novus, USA), washed three times with PBS, and then co-stained with FITC-conjugated secondary antibody and DAPI. Immunofluorescence images were acquired using confocal fluorescence microscopy. Figure 8 (A) It can be seen that the polyhedral scaffold group had more new nerve fibers and blood vessels growing in, while the control group had only a small number of nerves and blood vessels. Figure 8Statistical results in (B) and (C) show that the area fraction of newly formed nerves and blood vessels in the polyhedral scaffold group was significantly higher than that in the control group. The polyhedral scaffold To group had more newly formed nerves, while the polyhedral scaffold Tt group had more newly formed blood vessels, indicating that the polyhedral structure can promote the regeneration of nerves and blood vessels inside bone defects.

[0109] To characterize the bone regeneration effect in the later stages of scaffold implantation, Van Gieson's staining was performed on hard tissue sections. First, femoral samples were dehydrated using a gradient ethanol process, embedded in methyl methacrylate, and then sectioned (500 μm thick), ground, and polished. Finally, they were stained with VG (Van Gieson's) dye. Figure 9 (A) VG staining results showed that there was a large amount of new bone ingrowth inside the polyhedral scaffold, while the control group had only a small amount of new bone. Figure 9 (B) Statistical results showed that the fraction of newly formed bone area in the defect site of the polyhedral scaffold group was significantly higher than that of the control group, indicating that the polyhedral scaffold structure can significantly promote bone tissue regeneration and ingrowth into the scaffold.

[0110] The above findings indicate that bioactive scaffolds with spatial polyhedral structures possess highly tunable structure, mechanical strength, and porosity. Compared to traditional cross-structure scaffolds, spatial polyhedral structures exhibit superior biological properties, effectively promoting the proliferation and differentiation of various cell types. Furthermore, spatial polyhedral scaffolds can induce early ingrowth of host neurovascular networks in vivo, thereby accelerating bone regeneration and potentially achieving high-quality bone defect repair and regeneration.

Claims

1. A bioactive scaffold with a spatial polyhedral structure, characterized in that, The bioactive scaffold includes: a planar abutment at the bottom and a spatial polyhedral structure at the top formed by multiple pillars connected and stacked together; The planar abutment has a diameter of 2-20 mm and a height of 0.1-1 mm; each unit cell of the spatial polyhedral structure has a three-dimensional dimension of 0.5-2 mm × 0.5-2 mm × 0.5-2 mm; each support has a diameter of 100-500 μm; the overall size of the bioactive scaffold has a diameter of 2-20 mm and a height of 0.5-20 mm; the shape of the spatial polyhedral structure is a truncated octahedron, a truncated half-cube, a truncated cube, or a truncated tetrahedron.

2. The bioactive scaffold with a spatial polyhedral structure according to claim 1, characterized in that, The bioactive scaffold is made of bioactive bioceramics, polymers, metals, or metal oxides; the bioceramics are β-phase tricalcium phosphate or hydroxyapatite.

3. The bioactive scaffold with a spatial polyhedral structure according to claim 2, characterized in that, The bioceramic is β-phase tricalcium phosphate.

4. A method for preparing a bioactive scaffold with a spatial polyhedral structure as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Design a printed model with a spatial polyhedral structure using 3D model design software; (2) Prepare photocurable printing ink by mixing bioactive material powder and photosensitive resin and ball milling; (3) Printing the green blank holder; (4) High-temperature sintering.

5. The method for preparing a bioactive scaffold with a spatial polyhedral structure according to claim 4, characterized in that, In step (2), the mass fraction of bioactive material powder in the photocurable printing ink is 20-80%.

6. The method for preparing a bioactive scaffold with a spatial polyhedral structure according to claim 5, characterized in that, The bioactive material powder in the photocurable printing ink has a mass fraction of 30-50%.

7. The method for preparing a bioactive scaffold with a spatial polyhedral structure according to claim 4 or 5, characterized in that, In step (2), the preparation method of the photosensitive resin includes: mixing 20-60 parts by weight of 1,6-hexanediol diacrylate, 20-60 parts by weight of polyethylene glycol diacrylate, 3-6 parts by weight of Triton X-100 and 1-2 parts by weight of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide uniformly to obtain the photosensitive resin.

8. The method for preparing a bioactive scaffold with a spatial polyhedral structure according to claim 4, characterized in that, In step (3), the photocurable printing ink obtained in step (2) is introduced into the photocurable 3D printer, and the printing model in step (1) is printed and cured layer by layer to obtain a green bracket.

9. The method for preparing a bioactive scaffold with a spatial polyhedral structure according to claim 4, characterized in that, In step (4), the high-temperature sintering temperature is 1000~1200℃ and the time is 2~8 hours.

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