Biomimetic snail shell bioactive scaffold and preparation method and application thereof

Abstract

The present application relates to a conch-like bioactive scaffold and a preparation method and application thereof. The conch-like bioactive scaffold has a spiral structure feature; the whole scaffold comprises a middle support, a peripheral wall containing a through-hole structure, and a spiral-shaped node and a spiral-shaped cavity integrally formed between the middle support and the peripheral wall.

Description

TECHNICAL FIELD

[0001] The present application relates to a conch-imitated bioactive scaffold and a preparation method and application thereof, and belongs to the field of biomaterials. BACKGROUND

[0002] Bone is one of the important organs in the human body, which plays a key role in protection, support and body movement. However, due to bone tumors, osteoarthritis (OA), a large number of osteoporosis cases caused by the increasingly serious social aging, and fractures caused by accidents, there is a huge clinical demand for current bone defect repair. However, due to the limited self-repairing ability of bone tissue, the repair of large bone defects is still a difficult problem in current clinical treatment. The main methods for repairing large bone defects at present are autologous bone transplantation, allogeneic bone transplantation and artificial bone transplantation materials. Autologous bone transplantation is the gold standard in clinical treatment, but its application is limited due to the limited source and the additional pain and trauma; allogeneic bone transplantation will inevitably face immune rejection, disease transmission and ethical issues, also limiting its further application. Therefore, artificial bone transplantation materials have become a more universal and effective bone repair method. Among them, 3D-printed porous bone tissue engineering scaffolds are considered to be an ideal strategy for bone defect repair due to their rapid and personalized manufacturing characteristics. However, the current 3D-printed scaffolds have a certain porous structure, but are mostly solid pillars stacked in cross, making it difficult for cells to migrate to the inside of the scaffold, with poor bioactivity, simple structure, less function, unable to regulate cell migration behavior, and low cell loading rate, which limits the internal osteogenesis effect, and some defect sites lack osteogenic environment, making it difficult to form bone. The commonly used extrusion type 3D printing technology can only construct a scaffold with a pillar stacking structure, and it is difficult to construct a more complex and delicate structure, and the forming precision is poor. SUMMARY

[0003] In view of the problems existing in the prior art, the present application aims to provide a conch-imitated bioactive scaffold and a preparation method and application thereof.

[0004] In one aspect, the present application provides a conch-imitated bioactive scaffold, which has a spiral structure characteristic; the whole scaffold comprises a middle pillar, a peripheral wall containing a through-hole structure, and a spiral-shaped node and a spiral-shaped cavity integrally formed between the middle pillar and the peripheral wall.

[0005] In the present application, the bioactive scaffold simulates the natural structural characteristics and growth pattern of a snail, and has a unique spiral structure. The overall structure of the scaffold composed of the intermediate strut, the peripheral wall with a through-hole structure, and the spiral-shaped node and spiral-shaped cavity can play a good supporting role, meeting the requirements of the mechanical properties of the implanted scaffold; in addition, compared with the existing traditional cross scaffold, the continuous spiral structure can guide and promote the directional migration of cells, and has enhanced cell delivery, proliferation and osteogenic differentiation induction capacity, improving the bioactivity; in vivo, it induces good directional bone regeneration from the bottom to the top, and can be used for the repair and regeneration of bone defects.

[0006] Preferably, the spiral structure, including the spiral-shaped node and the spiral-shaped cavity, extends along the axial direction of the scaffold; the pitch of the spiral-shaped node is 0.5-3 mm, and the thickness of the spiral-shaped node is 0.2-1.5 mm; the height of the spiral-shaped cavity structure is 0.3-2.8 mm.

[0007] Preferably, the intermediate strut is embedded in the spiral structure and extends along the axial direction of the scaffold; the diameter of the intermediate strut is 0.5-5 mm.

[0008] Preferably, the inner diameter of the peripheral wall with a through-hole structure is 2-30 mm, and the thickness is 0.5-15 mm.

[0009] Preferably, the through hole on the peripheral wall with a through-hole structure penetrates or partially penetrates the peripheral wall along the axial direction of the scaffold; the number of through holes with a through-hole structure is 1-20, and the diameter of each through hole is 0.2-2 mm.

[0010] In the present application, the porosity and / or mechanical properties (including compressive strength and bending strength) of the scaffold can be regulated by changing the pitch of the spiral-shaped node, the number and diameter of the through holes on the peripheral wall.

[0011] Preferably, at least one through hole on the peripheral wall with a through-hole structure is in communication with the spiral-shaped cavity structure, and the communication position can be at the height corresponding to each spiral cavity of the scaffold.

[0012] Preferably, the bottom of the snail-imitating bioactive scaffold is an open structure or a closed structure; the height of the closed structure is 0.2-2 mm.

[0013] Preferably, the material of the snail-imitating bioactive scaffold is a bioactive material; the bioactive material is a bioceramic, a bioglass, a metal or a metal oxide.

[0014] On the other hand, the present application provides a preparation method of a snail-imitating bioactive scaffold, comprising:

[0015] (1) using three-dimensional design software to design the printing model of the imitation snail bioactive scaffold; preferably, the three-dimensional design software is SolidWorks, 3ds Max, Pro / Engineer, Cinema 4D or CAD, the exported file format is STL, and then the STL file is imported into the printer for slicing and setting the printing parameters;

[0016] (2) mixing the bioactive material powder, liquid photocuring resin and additives to obtain a slurry for photocuring printing;

[0017] (3) adding the slurry for photocuring printing into a material pool, and using a photocuring printer to perform layer-by-layer curing forming according to the printing model and the set parameters to form a green body;

[0018] (4) cleaning and secondary curing the green body obtained by printing with a liquid; preferably, the liquid is ethanol or / and water, and the light source for secondary curing is ultraviolet light or blue light;

[0019] (5) performing debinding and sintering on the green body obtained after secondary curing to obtain the imitation snail bioactive scaffold.

[0020] Preferably, the mixing method is ball milling, the rotation speed of the ball milling is 200-1000 r / min, and the time is 1-24 h.

[0021] Preferably, the mass fraction of the bioactive material powder in the slurry for photocuring printing is 30%-70%.

[0022] Preferably, the photocuring printer is in a DLP forming or SLA forming mode. According to the present application, the structure of the prepared imitation snail bioactive scaffold can be accurately controlled and kept intact by using the photocuring 3D printing technology.

[0023] In another aspect, the present application also provides an application of the imitation snail bioactive scaffold in preparing in-vitro cell culture materials or preparing bone defect repair and bone regeneration materials.

[0024] Advantages:

[0025] In the present application, the bioactive scaffold simulates the natural structure and growth mode of a snail, can guide and promote the directional migration of cells, and has enhanced abilities of cell delivery, proliferation and induction of osteogenic differentiation, improves the bioactivity, induces good directional bone regeneration from the bottom to the top in the body, and can be used for repairing and regenerating large bone defects. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1The structure of the different pitch parameters (P) of the conchiform bioactive scaffold (P = 1.6, 2.0, 2.4) and the traditional cross strut scaffold (Cross). (a1-d1) are digital photos (scale 2 mm), (a2-d2) are cross-sectional images reconstructed by Micro-CT (scale 2 mm), (a3-d3) are SEM images (scale 400 μm) and (e-g) are SEM images (scale 400 μm for e, f and scale 5 μm for g). (a1-d1) are the overall structures of the four scaffolds, (a2-d2) are the vertical cross sections showing the spiral structure of the conchiform bioactive scaffold with different pitches, the middle strut and the peripheral wall structure containing through holes, (a3-d3) are 1 / 4 of the cross strut or spiral structure of the four scaffolds, (e) is the through hole structure on the peripheral wall connected to the middle spiral cavity of the scaffold, (f) is the peripheral wall and the through hole structure thereon, and (g) is the microstructure of the surface of the sintered conchiform bioactive scaffold. As can be seen from the digital photos, Micro-CT reconstructed images and SEM images, the conchiform bioactive scaffold has a unique spiral structure, including spiral nodes and cavities; the middle strut of the conchiform bioactive scaffold, the peripheral wall containing through hole structure and the structure of the spiral nodes and cavities are of good structure and are microscopically dense after sintering.

[0027] Figure 2 (a-c) are Micro-CT reconstructed cross-sectional images of the conchiform bioactive scaffold with 8, 12 and 16 through holes, respectively, and (d-f) are Micro-CT reconstructed cross-sectional images of the conchiform bioactive scaffold with 1.0, 1.2 and 1.5 mm diameter through holes, respectively, (d1-f1) are horizontal cross sections and (d2-f2) are vertical cross sections (scale 2 mm).

[0028] Figure 3 The porosities of different scaffolds. (A) are the porosities of the conchiform bioactive scaffolds with different pitches and the traditional cross strut scaffold, (B) are the porosities of the conchiform bioactive scaffolds with different numbers of through holes and (C) are the porosities of the conchiform bioactive scaffolds with different diameters of through holes. The results show that the porosity of the conchiform bioactive scaffold can be regulated by changing the pitch, the number and diameter of the through holes on the peripheral wall and other parameters.

[0029] Figure 4The mechanical strengths of different scaffolds are shown in Figure 1. (A) Compressive strength of a traditional cross-pillar scaffold and a snail-inspired bioactive scaffold with different pitches. (B) Compressive strength of a snail-inspired bioactive scaffold with different numbers of through holes and (C) different through hole diameters. (D) Bending strength of a traditional cross-pillar scaffold and a snail-inspired bioactive scaffold with different pitches. (E) Bending strength of a snail-inspired bioactive scaffold with different numbers of through holes and (F) different through hole diameters. The results indicate that the compressive and bending strengths of the snail-inspired bioactive scaffold can be controlled by changing parameters such as the pitch, the number and diameter of through holes on the outer wall, etc., and compared with the traditional cross-pillar scaffold, the snail-inspired bioactive scaffold exhibits enhanced compressive and bending strength.

[0030] Figure 5 Images of bone marrow mesenchymal stem cells (MSCs) on four scaffolds (Cross, P=1.6, P=2.0, and P=2.4) after 24 hours of adhesion (A, B) show cell morphology and distribution on the scaffolds, and (C) cell loading efficiency of each scaffold after 4, 12, and 24 hours of adhesion (scale bar 500 μm, bars from left to right in the figure represent Cross, P=1.6, P=2.0, and P=2.4). The results indicate that stem cells adhered and spread well on all scaffolds, and the snail-inspired bioactive scaffold can load and deliver more cells compared to the traditional cross-pillar scaffold, exhibiting higher cell loading and delivery efficiency.

[0031] Figure 6 The figures show the proliferation results of bone marrow mesenchymal stem cells cultured on four scaffolds (Cross, P=1.6, P=2.0, and P=2.4) for 1, 3, and 7 days, respectively. The bars in the figure, from left to right, represent Cross, P=1.6, P=2.0, and P=2.4. The results indicate that the snail-inspired bioactive scaffold with P=2.4 is more conducive to cell proliferation than the traditional solid cross-pillar scaffold (Cross).

[0032] Figure 7 The results show the cell migration of bone marrow mesenchymal stem cells after 10 days of culture on four different scaffolds (Cross, P=1.6, P=2.0, and P=2.4) (scale bar 500 μm). The results indicate that the snail-inspired bioactive scaffold significantly promotes stem cell migration in both vertical and horizontal directions compared to the traditional cross-pillar scaffold.

[0033] Figures 8A-8C Osteogenic differentiation results of bone marrow mesenchymal stem cells after 7 days of culture on Cross, P=1.6, P=2.0, and P=2.4 scaffolds (scale bar 75 μm). Figure 8A PCR quantification results showed osteogenic protein ( Figure 8B) BSP and ( Figure 8C ) CLSM images of immunofluorescent staining of OCN. The results show that the conchiform bioactive scaffold significantly induced osteogenic differentiation of stem cells compared to the traditional cross strut scaffold (Cross).

[0034] Figure 9 are digital photos of different groups of animals in the rabbit femur experiment, 2D and 3D reconstruction images of Micro-CT scanning. (Different colors represent different components, white: primary bone tissue; red: implanted scaffold; green: bone tissue) The results show that the conchiform bioactive scaffold induced more new bone tissue to form in the scaffold (including the protruding top of the scaffold) compared to the traditional cross strut scaffold (Cross), and had a better bone regeneration effect.

[0035] Figure 10 are the osteogenesis quantitative data statistics of the whole (A, the column chart in the figure from left to right is blank, Cross and P = 2.4 respectively) and the protruding part of the top of the scaffold (B, the column chart in the figure from left to right is Cross and P = 2.4 respectively) of different components in the rabbit femur experiment.

[0036] Figure 11 are Van Gieson histological section staining analysis of the embedded part and the protruding part of the top of the scaffold of different components at 8 weeks and 12 weeks in the rabbit femur experiment. They are histological section staining images of the blank group, Cross and P = 2.4 embedded part at 8 weeks (A-C) and 12 weeks (D-F), respectively; Cross and P = 2.4 top protruding part at 8 weeks (G-H) and 12 weeks (I-J) ; (a-j) are local enlarged images of (A-J), and the blue arrow points to the new bone tissue. (Different colors represent different components, black: implanted scaffold; red: bone tissue) The results show that due to its unique spiral structure, the conchiform bioactive scaffold (P = 2.4) induced more new bone tissue to form in the scaffold (including the embedded part of the scaffold and the protruding part of the top of the scaffold) compared to the traditional cross strut scaffold (Cross), and more new bone tissue formed on the top with the increase of time, and had a better effect of inducing bone regeneration. DETAILED DESCRIPTION

[0037] The application is further illustrated by the following embodiments, which should be understood as merely illustrative of the application, but not limiting the application.

[0038] In the present disclosure, the imitation snail bioactive scaffold (hereinafter referred to as the biomimetic scaffold) is prepared by simulating the macroscopic structural characteristics and growth mode of snails. The macroscopic structural characteristics of the snails involved in the present application are as follows: the spiral structure can accommodate the living soft tissue of the snails and allow them to freely stretch and contract in the spiral cavity; the peripheral wall and the intermediate support play the roles of protection and mechanical support; and the growth mode of the snails is that the shell continuously extends in the spiral direction over time.

[0039] Figure 1 and Figure 2 The structure of the scaffold according to an embodiment of the present application is shown. As shown in Figure 1 and Figure 2 The biomimetic scaffold includes a spiral structure, an intermediate support and a peripheral wall structure containing through holes. The above-mentioned structures of the scaffold are integrated, but the size and proportion of each part of the structure are adjustable.

[0040] In the spiral structure, spiral segments and cavities are included. The pitch of the spiral segments is fixed or variable. For example, the pitch of the spiral segments can range from 0.5 to 3 mm, the vertical thickness of the segments can range from 0.2 to 1.5 mm, and the height of the spiral cavity can range from 0.3 to 2.8 mm.

[0041] For the intermediate support, it is embedded in the spiral structure and extends along the axial direction of the scaffold. The diameter of the intermediate support can be adjusted as needed, for example, it can be 0.5-5 mm.

[0042] In the peripheral wall structure containing through holes, the structure of the peripheral wall and its size and overall shape can be adjusted as needed. In one example, the overall shape of the peripheral wall is a hollow column. The size of the peripheral wall is adjustable, for example, the inner diameter of the peripheral wall can be 2-30 mm, the wall thickness can be 0.5-15 mm, and the height can be 3-100 mm.

[0043] For the through holes on the peripheral wall, they penetrate or partially penetrate the peripheral wall along the axial direction of the scaffold. The size and position of the through holes are also adjustable, for example, the number of through holes can be 1-20, the diameter can be 0.2-2 mm, and the through holes are distributed on the peripheral wall in axial symmetry.

[0044] The bottom of the biomimetic scaffold can be open or closed. The height of the closed structure can be 0.2-2 mm. After defatting and sintering, the closed structure at the bottom of the biomimetic scaffold will not produce cracks or openings, so as to ensure that the added cell suspension does not flow out from the bottom and ensure the accuracy of the number of cell inoculation.

[0045] One of the plurality of through holes on the peripheral wall serves as a cell addition hole, and communicates with the spiral cavity in the middle of the scaffold. The communication position can be adjusted as needed, and can be adjusted at a suitable height corresponding to each spiral cavity of the scaffold to control and ensure the operability and accuracy of the position of the cell suspension added in the spiral structure of the scaffold.

[0046] The porosity and / or mechanical properties of the biomimetic scaffold can be regulated by changing the pitch of the spiral segment, the number and diameter of the through holes on the peripheral wall, and other structural parameters of the biomimetic scaffold, such as the size of the peripheral wall, the diameter of the middle strut, and the thickness of the spiral segment. In some embodiments, the porosity of the biomimetic scaffold can be 33.4%-43.8%, the compressive strength can be 11.2-38.7 MPa, and the bending strength can be 2.7-9.9 MPa.

[0047] In the present application, the biomimetic scaffold is made of a bioactive material, such as a bioceramic, a bioglass, a metal, and a metal oxide. The bioceramic can be, for example, tricalcium phosphate (TCP), hydroxyapatite (Ca 10 (PO4)6(OH)2), hardystite (Ca2MgSi2O7), xonotlite (Ca2SiO4), calcium dihydrogen phosphate (Ca(H2PO4)2), or dicalcium hydrogen phosphate (CaHPO4·2H2O). The bioglass can be, for example, 45S5. The metal can be, for example, iron / stainless steel, titanium, tantalum, alloys containing titanium and / or magnesium, etc. The metal oxide can be, for example, alumina (Al2O3) or zirconia (ZrO2).

[0048] In one embodiment of the present application, the biomimetic scaffold is prepared using a light-curing 3D printing technology. Hereinafter, the preparation method of the above-mentioned biomimetic scaffold will be described as an example.

[0049] A 3D printing file that can be recognized by a printer is obtained for light-curing 3D printing. In one embodiment, a three-dimensional design software can be used to design a printing model, and the design model data is exported in STL format. Then, the STL file is imported into the processing software of the light-curing printer to set the relevant parameters and perform slicing, and the relevant file is exported in a 3D printing file format that can be recognized by the printer, such as TDP, etc. The three-dimensional design software can be, for example, SolidWorks, 3ds Max, Pro / Engineer, Cinema 4D, or CAD, etc.

[0050] A slurry for light-cured printing is prepared. The printing slurry is prepared by mixing the bioactive material, the light-cured resin and other additives (including diluents, dispersants, initiators and / or inhibitors, etc.) in a proportion by ball milling. In one example, the bioactive material, the light-cured resin and other additives account for 30%-70%, 70%-30% and 0-30% of the total slurry by mass. The light-cured resin can be a photosensitive resin known in the art, such as epoxy resin, acrylate, epoxy acrylate, etc. The other additives can be diluents (such as acrylate, vinyl, vinyl ether and / or epoxy, etc.), dispersants, initiators and / or inhibitors, etc., such as hexanediol diacrylate (HDDA), boric acid, KH570, oleic acid, TPO, Triton X-100, etc. The rotation speed of the ball mill can be 200-1000 r / min, and the ball milling time can be 1-24 h.

[0051] A light-cured 3D printing is performed to obtain a printed green body. In one example, a 3D printing file is imported into a light-cured 3D printer, printing parameters are set, and then the prepared slurry is added to the material pool of the light-cured printer, and the light-cured printer is used to perform layer-by-layer curing and forming according to the printing model and the set parameters to form a green body. In one example, the forming method of the light-cured 3D printer is DLP forming or SLA forming, etc.

[0052] The printed scaffold green body obtained after printing is subjected to cleaning and / or secondary curing and other post-processing. In one example, the obtained scaffold green body is cleaned with a liquid to remove the uncured printing slurry attached to the scaffold green body, and then the cleaned printing green body is subjected to secondary curing and other post-processing with a curing light source to ensure the accuracy of the scaffold printing structure and the quality of subsequent sintering. In one example, the liquid used for cleaning the printed scaffold green body can be ethanol, water and / or a solution formed by mixing the two liquids in a proportion, and the secondary curing light source can be an ultraviolet lamp or a blue light lamp.

[0053] The post-processed printing green body is subjected to debinding and sintering to obtain a biomimetic scaffold, and the debinding and sintering conditions can be selected according to the components of the printing slurry. In one example, the debinding conditions of the scaffold can be calcination at 350-750°C (e.g., 650°C) for 1-6 hours, and the sintering conditions of the scaffold can be calcination at 1000-1350°C (e.g., 1150°C) for 1-6 hours.

[0054] Suitable stem cell (as stem cell with osteogenic potential: bone marrow mesenchymal stem cell) suspension can be added into the spiral structure of the imitation snail bioactive scaffold with open or sealed bottom through the cell addition hole on the outer wall of the bracket, and the cell loading delivery capacity, cell proliferation, migration and osteogenic differentiation of the biomimetic scaffold are studied by taking the traditional 3D printed cross solid pillar type scaffold as a control, and the in-vivo osteogenic capacity of the scaffold is studied through the half-embedded rabbit femoral defect experiment. The results show that, compared with the traditional 3D printed cross pillar type scaffold, by changing the pitch, the biomimetic scaffold has higher cell loading delivery efficiency, can promote cell proliferation, enhance the expression of osteogenic related genes and proteins such as BSP, OCN, Runx2, BMP2, etc., and has better in-vitro induced osteogenic differentiation capacity. More importantly, by changing the pitch, the biomimetic scaffold can significantly promote the vertical and horizontal migration of stem cells on the 3D scaffold, and can also guide the migration of endogenous bone marrow mesenchymal stem cells to the inside and top of the scaffold in-vivo, and affect the osteogenic differentiation of the endogenous bone marrow mesenchymal stem cells, thereby inducing good directional bone regeneration from the bottom to the top of the scaffold, forming more new bone tissue, and the repair effect of the bone defect is better. In summary, compared with the traditional 3D printed cross pillar type scaffold, the imitation snail bioactive scaffold prepared in the application is more conducive to the migration of stem cells, and can also improve cell adhesion to improve the cell delivery efficiency of the scaffold, promote cell proliferation and induce osteogenic differentiation, improve the biological activity of the scaffold, have better in-vivo osteogenic effect, and be more suitable for the repair of large bone defects.

[0055] In the application, the imitation snail bioactive scaffold with spiral structure prepared by using light-cured 3D printing technology has a complex structure which can be accurately controlled and maintained intact, and the biomimetic scaffold has better in-vitro cell loading delivery, migration, proliferation and osteogenic differentiation effect, and effectively promotes the bone regeneration effect of new bone from the bottom to the top of the scaffold in-vivo, and can be used for the repair and regeneration of large bone defects.

[0056] The following further examples are used to illustrate the application in detail. It should also be understood that the following examples are only used to further illustrate the application, and cannot be understood as limiting the protection scope of the application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application all belong to the protection scope of the application. The specific process parameters in the following examples are only one example in the appropriate range, i.e. those skilled in the art can select within the appropriate range through the description herein, and are not limited to the specific values in the following examples.

[0057] Example 1

[0058] The model of the bionic stent was designed using SolidWorks software. A cylindrical bionic shell-shaped bioactive stent with a diameter of 13 mm and a height of 15 mm was designed. The outer wall had 12 through holes with a diameter of 1.2 mm, and the helical pitch of the spiral-shaped segments was 1.6, 2.0, and 2.4 mm, respectively. The designed bionic stent model data was output in STL format and imported into the processing software of a DLP type light-curing 3D printer (purchased from Beijing Shivi Technology Co., Ltd., model AUTOCERA-M) to set parameters and slice. The 3D printing file format TDP recognized by the printer was exported, and then the TDP file was imported into the DLP type light-curing 3D printer.

[0059] β-TCP powder (purchased from Kunshan Huaguo Technology New Material Co., Ltd.) 40 g, 17.5 g of photosensitive resin (epoxy acrylate resin, purchased from Jinhua Wanhuo Fittings Co., Ltd.), 10 g of 1,6-hexanediol diacrylate (HDDA, purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.), 3 g of Triton X-100 (purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.), 2 g of KH570 (purchased from Sinopharm Chemical Reagent Co., Ltd.), and 0.25 g of (2,4,6-trimethylbenzoyl) diphenyl phosphine oxide (DPO, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were mixed at a speed of 500 r / min for 8 hours to obtain a light-curing printing slurry.

[0060] The printing slurry was added to the material pool of the light-curing printer, and the DLP type light-curing printer was used to perform layer-by-layer curing and forming according to the printing model and the set parameters to obtain a printing green body. The printing green body was washed twice with anhydrous ethanol, and then placed under a blue light lamp for 1 hour of secondary curing to complete the post-processing of the printing green body.

[0061] The post-processed printing green body was first calcined at 650°C for 2 hours for debinding, and then calcined at 1150°C for 3 hours to completely sinter into a bionic shell-shaped bioactive stent.

[0062] Example 2

[0063] The model of the bionic stent was designed by using SolidWorks software. The cylindrical bionic conch bioactive stent with a diameter size of 13 mm and a height of 15 and 30 mm was designed. The pitch of the spiral-shaped node was 2.4 mm, and the diameter of the through hole of the peripheral wall was 1.2 mm, and the number of through holes was 8, 12, and 16. When the number of through holes of the peripheral wall was 12, the diameter was 1.0, 1.2, and 1.5 mm. One of the through holes of the peripheral wall of the above stent was replaced by a hole with a diameter of 1.5 mm to communicate with the spiral cavity in the middle of the stent as a cell addition hole of the stent. The designed bionic stent model data was output as an STL format file, which was imported into the processing software of the DLP type light curing 3D printer to set parameters and slicing, and exported as a 3D printing file format TDP that could be recognized by the printer. Then the TDP file was imported into the DLP type light curing 3D printer.

[0064] β-TCP powder 80 g, 35 g photosensitive resin, 20 g HDDA, 6 g Triton X-100, 4 g KH570 and 0.5 g DPO were ball milled at a speed of 500 r / min for 8 hours to obtain a light curing printing slurry.

[0065] The printing slurry was added to the material pool of the light curing printer, and the DLP type light curing printer was used to perform layer-by-layer curing forming according to the printing model and the set parameters to obtain a printing green body. The printing green body was washed twice with anhydrous ethanol, and then placed under a blue light lamp for secondary curing for 1 hour to complete the post-processing of the printing green body.

[0066] The post-processed printing green body was first calcined at 650°C for 2 hours for degreasing, and then calcined at 1150°C for 3 hours to completely sinter into a bionic conch bioactive stent.

[0067] Example 3

[0068] The model of the bionic stent was designed by using SolidWorks software. The cylindrical bionic conch bioactive stent with a diameter size of 13 mm and a height of 5 mm and a diameter size of 13 mm and a height of 15 mm was designed. The number of through holes of the peripheral wall was 12, and the diameter was 1.2 mm, and the pitch of the spiral-shaped node was 1.6, 2.0, and 2.4 mm. One of the through holes of the peripheral wall of the above stent was replaced by a hole with a diameter of 1.5 mm to communicate with the spiral cavity in the middle of the stent as a cell addition hole of the stent. The designed bionic stent model data was output as an STL format file, which was imported into the processing software of the DLP type light curing 3D printer to set parameters and slicing, and exported as a 3D printing file format TDP that could be recognized by the printer. Then the TDP file was imported into the DLP type light curing 3D printer.

[0069] β-TCP powder 80 g, mixed with 35 g photosensitive resin, 20 g HDDA, 6 g Triton X-100, 4 g KH570 and 0.5 g DPO at a rotation speed of 500 r / min for 8 hours to obtain a light-cured printing slurry.

[0070] The printing slurry was added to the material pool of a light-cured printer, and layer-by-layer curing was performed according to the printing model and the set parameters by using a DLP type light-cured printer to obtain a printing green body. The printing green body was washed twice with anhydrous ethanol, and then was placed under a blue light lamp light source for 1 hour of secondary curing to complete the post-processing of the printing green body.

[0071] The post-processed printing green body was first calcined at 650°C for 2 hours for debinding, and then calcined at 1150°C for 3 hours for complete sintering into a conchiform bioactive scaffold.

[0072] The obtained conchiform scaffold was subjected to physicochemical and biological evaluation, including structure, porosity, mechanical properties, cell activity (including adhesion, proliferation and osteogenic differentiation) and cell migration, and rabbit femoral defect animal experiments for evaluation, as follows.

[0073] Figure 1 Structural characterization of conchiform β-TCP bioceramic scaffolds with different pitches. (b), (c), (d) are conchiform scaffolds with a pitch (P) of 1.6, 2.0 and 2.4 mm, respectively, designed in Example 1, (e) is a through hole on the outer wall connected to the middle spiral cavity, i.e. a cell addition hole, (f) is the outer wall structure containing the through hole, (g) is the sintering condition of the scaffold, it can be seen that the conchiform bioceramic scaffold has a spiral structure, the overall structure of the conchiform scaffold (including the middle spiral-shaped node and cavity, the middle strut and the outer wall containing the through hole structure) is good, and the microstructure is dense after sintering.

[0074] Figure 2 Cross-sectional images of the conchiform scaffold reconstructed by Micro-CT scanning. (a-c) are scaffolds designed in Example 2, with the diameter of the through hole of the outer wall being 1.2 mm, and the number of through holes being 8, 12 and 16, respectively. Similarly, (d-f) are three conchiform scaffolds designed in Example 2, with the number of through holes of the outer wall being 12, and the diameters of the through holes being 1.0, 1.2 and 1.5 mm, respectively, (d1-f1) are images of horizontal sections, and (d2-f2) are images of vertical sections, which can further show that the conchiform bioceramic scaffold has a spiral structure.

[0075] Figure 3The porosity test results of different scaffolds. The porosity of the scaffold was tested by the following method: (all the scaffolds used for characterization and testing were sintered) Micro-CT was used to scan at a resolution of 8.9 μm, and then porosity analysis was performed, and the number of samples in each group was 3. Figure 3 (A) is the porosity test of traditional 3D printed cross-brace type scaffold and different pitch of imitation snail bioceramic scaffold, it can be seen that the porosity of the scaffold is 36.98%, 33.36%, 36.57%, 38.23% respectively, the porosity of the imitation snail bioceramic scaffold increases with the increase of the pitch. Figure 3 (B) is the porosity test of imitation snail bioceramic scaffold with different number of through holes, it can be seen that the porosity of the scaffold is 36.91%, 38.23%, 41.98% respectively, the porosity of the imitation snail bioceramic scaffold increases with the increase of the number of through holes. Figure 3 (C) is the porosity test of imitation snail bioceramic scaffold with different through hole diameters, it can be seen that the porosity of the scaffold is 35.52%, 38.23%, 43.77% respectively, the porosity of the imitation snail bioceramic scaffold increases with the increase of the through hole diameter. The above results collectively show that by changing the structural parameters, the porosity of the biomimetic bioceramic scaffold can be well regulated in the range of 33.4-43.8%.

[0076] Figure 4The mechanical compression and bending strength test results of different scaffolds. The scaffold designed in Example 2. The compression strength of the scaffold was tested by the following method: the scaffold was made into a cylindrical sample with a diameter of 13 mm and a height of 15 mm (the size of the model is the designed size, and the size of the 3D printed printing blank is about 9.8 mm in diameter and about 11 mm in height after sintering, and all the scaffolds used for characterization and testing are after sintering), the diameter D of the scaffold was measured, and the scaffold was uniformly loaded at a loading rate of 0.5 mm / min to the scaffold completely broken by a material mechanics universal testing machine (INSTRON-5566, USA), and the maximum stress F was recorded, then the calculation formula of the compression strength was бc=4F / πD2, and the number of samples in each group was 6. Similarly, the bending strength of the scaffold was tested by the following method: the scaffold was made into a cylindrical sample with a diameter of 13 mm and a height of 30 mm (the size of the model is the designed size, and the size of the 3D printed printing blank is about 9.8 mm in diameter and about 22 mm in height after sintering, and all the scaffolds used for characterization and testing are after sintering), the bending strength of the scaffold was tested by the three-point bending test method, for the three-point bending test, the span L was fixed at 18 mm, the diameter D of the scaffold was measured first, and the scaffold was uniformly loaded at a loading rate of 0.5 mm / min to the scaffold fracture by a material mechanics universal testing machine (INSTRON-5566, USA), and the maximum stress F was recorded, then the calculation formula of the bending strength was бS3P=8FL / πD3, and the number of samples in each group was 6. Figure 4 (A) is the compression strength of the traditional 3D printed cross strut type scaffold and the imitation seashell bioceramic scaffold with different pitches, which is 15, 31.1, 24.8, 25.6 MPa, respectively. It can be seen that the compression strength of the imitation seashell bioceramic scaffold is significantly higher than that of the traditional scaffold. Figure 4 (B) is the compression strength of the imitation seashell bioceramic scaffold with different numbers of through holes, which is 11.20, 25.63, 20.62 MPa, respectively. It can be seen that the compression strength of the imitation seashell bioceramic scaffold with 12 through holes is the highest. Figure 4 (C) is the compression strength of the imitation seashell bioceramic scaffold with different through hole diameters, which is 38.7, 30.68, 22.65 MPa, respectively. It can be seen that the compression strength of the imitation seashell bioceramic scaffold decreases with the increase of the through hole diameter. Figure 4 (D) is the bending strength test of the traditional 3D printed cross strut type scaffold and the imitation seashell bioceramic scaffold with different pitches. It can be seen that the bending strength of the scaffold is 4.19, 9.95, 8.41, 4.01 GPa, respectively, indicating that the bending strength of the imitation seashell bioceramic scaffold decreases with the increase of the pitch, but is significantly higher than that of the traditional scaffold with similar porosity. Figure 4(E) is the bending strength of the imitation snail bioceramic scaffold with different numbers of through holes, which are 9.45, 4.01, and 2.71 GPa, respectively. It can be seen that the bending strength of the imitation snail bioceramic scaffold decreases with the increase of the number of through holes. Figure 4 (F) is the bending strength of the imitation snail bioceramic scaffold with different diameters of through holes, which are 7.97, 4.01, and 6.91 MPa, respectively. It can be seen that the bending strength of the imitation snail bioceramic scaffold with a through hole diameter of 1.2 mm is lower. The results show that by changing the structural parameters, the mechanical compressive strength and bending strength of the biomimetic bioceramic scaffold can be well regulated in the range of 11.2-38.7 MPa and 2.71-9.45 GPa, respectively, and compared with the traditional 3D printed cross pillar type scaffold, the mechanical compressive strength and bending strength of the biomimetic bioceramic scaffold are improved.

[0077] The interaction of the imitation snail β-TCP bioceramic scaffold with rabbit bone marrow mesenchymal stem cells, including the influence of the biomimetic scaffold on cell adhesion, cell loading delivery efficiency, proliferation, migration and osteogenic differentiation related gene expression, is the scaffold designed in Example 3.

[0078] The bone marrow mesenchymal stem cells were inoculated on the 3D printed traditional cross pillar type scaffold and the imitation snail bioceramic scaffold with different pitches through the cell addition hole, and after adhesion for 24 hours, the morphology and distribution of the stem cells on the different scaffolds were observed by CLSM after staining. For the characterization of the cell loading efficiency of the scaffold, the CCK-8 method was used for testing, specifically: the cell loading rate of the stem cells adhered to the blank hole plate for 4 hours was taken as 100%, the standard curve of absorbance and cell number was drawn by the absorbance of CCK-8 and the inoculation number gradient of the cells in the blank hole plate, the number of cells loaded on the scaffold was tested by measuring the absorbance of the scaffold at 4, 12 and 24 hours, and then divided by the initial number of cells, and then converted to the cell loading rate of the scaffold, and the number of samples in each group was 6. As shown in Figure 5 the results show that the stem cells adhere and spread well on each scaffold, the stem cells on the imitation snail bioceramic scaffold spiral segment are distributed in 3D spiral, and the imitation snail bioceramic active scaffold can load and deliver more cells than the traditional cross pillar type scaffold (Cross), and has higher cell loading and delivery efficiency. Among them, the cell loading efficiency of each scaffold after adhesion for 24 hours is P=2.4 (51.69%), P=2.0 (35.36%), Cross (28.65%) and P=1.6 (25.67%) from high to low.

[0079] Bone marrow mesenchymal stem cells were inoculated on 3D-printed traditional cross-strut scaffolds and different-pitch conchiform bioceramic scaffolds through cell addition holes, and the proliferation of different scaffolds was tested by CCK-8 method after 1, 3 and 7 days of culture, with 6 samples in each group. Figure 6 The proliferation results of stem cells on the four scaffolds are shown in the table. The results show that compared with the traditional solid cross-strut scaffold Cross, the conchiform bioceramic scaffold with P = 2.4 is more conducive to cell proliferation.

[0080] Bone marrow mesenchymal stem cells were inoculated on 3D-printed traditional cross-strut scaffolds and different-pitch conchiform bioceramic scaffolds through cell addition holes, and the proliferation of different scaffolds was tested by CCK-8 method after 1, 3 and 7 days of culture, with 6 samples in each group. Figure 7 The cell migration results of bone marrow mesenchymal stem cells on the four scaffolds (Cross, P = 1.6, P = 2.0 and P = 2.4) after 10 days of culture are shown in the table. Among them, the migration of cells on the scaffolds in the vertical direction after 10 days is P = 2.4 (15.4 printing layer thickness), P = 2.0 (8.8 printing layer thickness), P = 1.6 (4.2 printing layer thickness) and Cross (3.2 printing layer thickness) from high to low; the migration in the horizontal direction is P = 2.4 (111.0°), P = 2.0 (79.2°), P = 1.6 (47.3°) and Cross (18°) from high to low, indicating that the conchiform bioceramic scaffold significantly promotes the migration of stem cells in the vertical and horizontal directions compared with the traditional cross-strut scaffold (Cross).

[0081] Bone marrow mesenchymal stem cells were seeded on 3D-printed traditional cross strut scaffolds and different pitch conchiform bioactive ceramic scaffolds through the cell addition hole, respectively. After 7 days of culture, the expression of osteogenic related genes of stem cells on different scaffolds was tested by RT-qPCR, and the number of samples in each group was 4. The expression of osteogenic related genes of stem cells on different scaffolds was further tested by immunofluorescence staining of BSP and OCN and imaging by CLSM. (A) PCR quantitative results, immunofluorescence staining of (B) BSP and (C) OCN CLSM images. The results show that the conchiform bioactive scaffold improves the expression level of osteogenic related genes compared with the traditional cross strut scaffold (Cross), and significantly induces the osteogenic differentiation of stem cells.

[0082] The above results show that compared with the traditional cross solid strut scaffold, the conchiform bioactive scaffold can better guide and promote cell migration, and has enhanced cell delivery, proliferation and induced osteogenic differentiation ability in vitro, improving the bioactivity of the scaffold.

[0083] Osteogenic ability of conchiform β-TCP bioactive ceramic scaffolds in animals.

[0084] The bioactive ceramic scaffolds were made into cylindrical samples with a diameter of 8 mm and a height of 10 mm (the size of the model design, and the size of the 3D-printed printing embryo, the size of the sintered bioactive ceramic scaffold is about 6 mm in diameter and about 7.5 mm in height, all the scaffolds used for characterization and testing are sintered) to perform blank group (blank control, without implanting any scaffold), Cross (implanting traditional cross strut scaffold), P=2.4 (implanting conchiform β-TCP bioactive ceramic scaffold) three groups of semi-embedded rabbit femur experiments, wherein the implanted two kinds of scaffolds are embedded in the defect site about 5 mm, and protrude from the defect site about 2.5 mm, and the osteogenesis is observed after 8 weeks and 12 weeks to evaluate the overall osteogenesis effect of different scaffolds and the effect of different scaffolds on inducing directional osteogenesis, and the number of samples in each group is 4. Figure 9 Digital photos, 2D and 3D reconstruction images of Micro-CT scans of different groups of animals in rabbit femur experiments. Figure 10 Osteogenic quantitative data statistics of different groups of whole and top protruding parts. Figure 11Van Gieson histological section staining analysis of embedded parts and overhanging parts of the top of the scaffold of different components at 8 weeks and 12 weeks. The results show that the embedding and overhanging height of the scaffold are well controlled, and the conchiform bioactive scaffold compared with the traditional cross strut type scaffold (Cross) guides and promotes more new bone tissue to form inside the scaffold, and the overhanging part of the top of the conchiform bioactive scaffold forms more new bone tissue with the increase of time, induces good directional bone tissue regeneration from the bottom to the top in vivo, and has better bone defect repair and regeneration effect.

[0085] The above study shows that the conchiform bioactive scaffold has a highly controllable structure, porosity and mechanical strength, and has obvious improvement in bioactivity compared with the traditional cross solid strut type scaffold, enhances the cell loading delivery efficiency, proliferation capacity and expression level of osteogenic differentiation related genes, and can guide and promote the movement and migration of stem cells on the 3D scaffold, has more excellent in vitro and in vivo osteogenic activity, promotes the regeneration of bone tissue, and is expected to be used for the repair and regeneration of large bone defects.

Claims

1. A conchoidal bioactive scaffold, characterized in that, The shell-shaped bioactive scaffold has a continuous spiral structure including spiral segments and spiral cavities; wherein the scaffold as a whole comprises a middle pillar, a peripheral wall having a through-hole structure, and spiral segments and spiral cavities integrally formed between the middle pillar and the peripheral wall and extending along the axial direction of the scaffold; the middle pillar is embedded in the spiral structure and extends along the axial direction of the scaffold; the through holes in the peripheral wall having the through-hole structure penetrate or partially penetrate the peripheral wall along the axial direction of the scaffold; at least one through hole in the peripheral wall having the through-hole structure serves as a cell addition hole and communicates with the spiral cavity structure; the migration of cells in the horizontal and vertical directions of the scaffold is adjusted by changing the pitch of the shell-shaped bioactive scaffold; and the shell-shaped bioactive scaffold is made of a bioactive material.

2. The conchoidal bioactive scaffold of claim 1, wherein, The pitch of the spiral segment is 0.5-3 mm, and the thickness of the spiral segment is 0.2-1.5 mm; the height of the spiral cavity is 0.3-2.8 mm.

3. The conchoidal bioactive scaffold of claim 1, wherein, The diameter of the middle pillar is 0.5-5 mm.

4. The conchoidal bioactive scaffold of claim 1, wherein, The inner diameter of the peripheral wall having the through-hole structure is 2-30 mm, and the thickness is 0.5-15 mm.

5. The conchoidal bioactive scaffold of claim 1, wherein, The number of through holes in the peripheral wall having the through-hole structure is 1-20, and the diameter of each through hole is 0.2-2 mm.

6. The conchoidal bioactive scaffold of claim 1, wherein, The bottom of the shell-shaped bioactive scaffold is an open structure or a closed structure; the height of the closed structure is 0.2-2 mm.

7. The conchoidal bioactive scaffold of claim 1, wherein, The bioactive material is a bioceramic, a bioglass, a metal, or a metal oxide.

8. A method of preparing the conchoidal bioactive scaffold according to any one of claims 1 to 7, characterized in that, The method comprises the following steps: (1) designing a printing model of the shell-shaped bioactive scaffold by using three-dimensional design software; (2) mixing bioactive material powder, liquid photopolymerization resin, and additives to obtain a slurry for photopolymerization printing; (3) adding the slurry for photopolymerization printing into a material pool, and layer by layer solidification forming by using a photopolymerization printer according to the printing model and the set parameters to form a green body; (4) cleaning and secondary solidification of the green body obtained by printing by using a liquid; (5) defatting and sintering the green body obtained after secondary solidification to obtain the shell-shaped bioactive scaffold.

9. The production method according to claim 8, characterized by, The three-dimensional design software is SolidWorks, 3dsMax, Pro / Engineer, Cinema 4D, or CAD, the export file format is STL, and then the STL file is imported into the printer for slicing and setting the printing parameters.

10. The preparation method according to claim 8, characterized in that, The mixing method is ball milling, the rotation speed of the ball milling is 200-1000 r / min, and the time is 1-24 h.

11. The preparation method according to claim 8, characterized in that, The mass fraction of the bioactive material powder in the slurry for photopolymerization printing is 30%-70%.

12. The preparation method according to claim 8, characterized in that, The liquid is ethanol or / and water, and the light source for secondary solidification is ultraviolet light or blue light.

13. Use of the shell-shaped bioactive scaffold according to any one of claims 1-7 in the preparation of in vitro cell culture materials or bone defect repair and regeneration materials.

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