WO2021128767A1 - Periodontal biological module for 3d biological printing, construction method, and application - Google Patents

Abstract

A periodontal biological module for 3D biological printing, a construction method, and an application, relating to the technical field of oral regenerative medicine. The module is provided with a lattice structure, a columnar structure, and a bio-ink; the lattice structure is formed by interweaving N hollow grids; the columnar structure is provided with M columnar bodies and a bottom plate; the columnar bodies are fixed to the bottom plate in parallel and integrally formed with the bottom plate; the positions of the columnar bodies on the bottom plate correspond to the positions of grids in the lattice structure; the lattice structure and the columnar structure are embedded integrally; the holes are formed between inner walls of the grids and outer walls of the columnar bodies, the diameter of the grids being greater than that of the columnar bodies. According to the construction method for the periodontal biological module, an active bio-ink is formed by photo curing, the constructed periodontal biological module enables cells to have special and specific arrangement orders spatially and structurally while providing good adhesion and growth microenvironments for the cells, can keep microstructures stable for a long term, and can well repair periodontal defects in laboratory animals.

Description

A periodontal biological module for 3D bioprinting and its construction method and application Technical field

The invention belongs to the technical field of oral regenerative medicine, and in particular relates to a periodontal biological module for 3D bioprinting, a construction method and application.

Background technique

Teeth are important human organs, which are responsible for the functions of chewing, pronunciation, and maintaining the shape of the maxillofacial region. Periodontal tissue, as the supporting structure of the teeth, has the function of maintaining the teeth upright, turning the chewing pressure into traction, fixing the teeth and root tips, and preventing the roots of the teeth. Move to the crown and other important roles. Therefore, inflammation and loss of periodontal tissue is one of the important reasons for tooth loss. Inflammation and defects of periodontal tissue caused by various reasons have become a common phenomenon. The repair of periodontal tissue provides a way or need for the treatment of periodontal disease and loose teeth.

Clinically, traditional repair and treatment methods have been used to repair periodontal tissue defects, such as periodontal bone transplantation, guided tissue regeneration, guided bone regeneration, and local slow release of growth factors. However, there is a technical problem that the repair of the periodontal defect tissue after the inflammation is controlled depends on the number and function of the remaining healthy mesenchymal stem cells in the patient's body. It is only suitable for the mild periodontal inflammation or the remaining healthy periodontal tissue. Many patients.

Constructing a bionic structure of periodontal tissue with complete structure and function is the necessary trend and goal of repairing periodontal defects.

With the development of tissue engineering technology, it is increasingly used to repair and treat periodontal tissue inflammation and defects. It is currently the most widely used and most promising technology, which is expected to achieve complete regeneration of periodontal tissue. Periodontal tissue has complex components and three-dimensional structures. It is not only composed of a "sandwich" structure composed of cementum, periodontal ligament, and alveolar bone, and the periodontal ligament fibers are microscopically composed of multiple groups. The periodontal ligament fiber bundles in different and specific directions are composed. Therefore, there are also technical problems in tissue engineering technology that it is difficult to simulate the above-mentioned complex microstructure.

Some tissue engineering research uses biomaterials combined with dental stem cells or cell membranes to successfully regenerate the "sandwich structure" of periodontal tissue, including new alveolar bone, cementum and periodontal fibrous tissue. However, in this new fiber, the fiber has no specific arrangement direction, and the regeneration effect is unstable.

Someone also used the technology of 3D printing PCL scaffold and compound periodontal ligament stem cells to simulate the arrangement direction of periodontal fibers. Although it was found that there were fibrous tissues arranged in a certain direction, the fibrous structure was discontinuous and discontinuous, and regenerated The fiber direction is single, and there is an obvious interface between the three-layer structure in the regenerated "sandwich structure", which cannot guarantee the integrity of the final structure, and the production method is complicated. However, the clinical trials conducted by this method showed that the periodontal defect was not well repaired.

Chinese invention CN201910465240.1 discloses a gradient material for guiding the regeneration of periodontal hard and soft tissues and a preparation method thereof, including a 3D printing scaffold layer and an electrospun fiber membrane layer. The content of hydroxyapatite in the 3D printing scaffold layer is higher than that of the electrostatic Spinning fiber membrane layer, the 3D printing stent layer has a larger pore size than the electrospinning fiber membrane layer, the 3D printing stent layer has a pore size of 100-1000 μm, and the electrospinning fiber membrane layer has a fiber diameter of 300 ~5000nm, the structure of the electrospun fiber membrane layer is randomly distributed or oriented arrangement or grid-like structure, and the thickness of the electrospun fiber membrane layer is 0.08-1mm. The method has simple and stable preparation process, integrates periodontal guided tissue regeneration membrane and alveolar bone repair scaffold material, and has the potential to realize clinical customized treatment. The gradient material has a good in vivo guided hard and soft tissue repair effect, and can be better applied to the synchronous regeneration and repair of alveolar bone and periodontal tissue. However, in this patent, it is necessary to construct three-layer structure separately, and there is an obvious interface between each layer structure, which cannot generate a complete periodontal "sandwich structure"; at the same time, it fails to generate a oriented periodontal fiber structure; in addition, there is a manufacturing method Cumbersome technical issues.

Chinese invention CN201710463999.7 discloses a 3D printing scaffold material and its preparation method and application. The material includes the following components: stromal cell-derived factor-1 (SDF-1) and bone morphogenetic protein 2 (BMP2). SDF-1 plays an important role in the reconstruction of periodontal tissues, and plays a key role in the recruitment and proliferation of BMSCs, PDLSCs and other stem cells existing in periodontal supporting tissues. SDF-1 plays a key role in the whole process of BMSCs mobilization, migration and recruitment. Not only that, SDF-1 can increase the expression of BMP2, and the two components of SDF-1 and BMP2 work together to promote periodontal tissue regeneration. The three-layer structure of periodontal tissue is not completely constructed in this patent.

Therefore, it is still necessary to regenerate the periodontal ligament fibers that have a complete "sandwich structure" and the periodontal ligament fibers arranged in different directions at the same time, that is, to construct a periodontal functional tissue module that mimics the periodontal fiber structure.

Summary of the invention

In order to solve the above technical problems, the present invention provides a periodontal biological module for 3D bioprinting and a construction method and application. The module imitates the periodontal fiber structure and realizes hard tissue (ie alveolar bone, cementum) and soft tissue ( That is, the periodontal ligament fibers) regenerate at the same time, and the periodontal ligament fibers have specific and different directions, which are very similar to natural periodontal ligament. The three-layer structure in the new periodontal tissue is continuous as a whole without obvious interfaces. The preparation method is simple and operability. Strong.

A periodontal biological module for 3D bioprinting in the present invention that solves the above technical problems is characterized in that: the module is provided with a grid structure and a columnar structure, and the grid structure is formed by interweaving N small grids. The grid is hollow; the columnar structure is provided with M columnar bodies and a bottom plate, and the columnar bodies are fixed on the bottom plate in parallel to form an integral body; one end of the mesh structure is fixed with the bottom plate, so that the mesh structure and the columnar structure are integrated into one body, and the columnar body is The position on the bottom plate corresponds to the position of the small grid in the network structure, and the columnar body is located in the small grid; there are pores between the inner wall of the small grid and the outer wall of the columnar body, and the diameter of the small grid>the diameter of the columnar body.

The columnar body and the small grid are a whole, the height of the two is the same, and the length value range simulates the length of the natural periodontal ligament fiber, that is, the width of the periodontal ligament.

The small cells are square, rectangular or circular.

The columnar body is a cylinder or a rectangular parallelepiped.

The module is composed of active bio-ink, which includes gelatin methacrylated hydrogel, mesenchymal stem cells and photocrosslinking agent LAP.

The diameter of the columnar body=80-180μm, the small grid side width (grid-like support width)=20-50μm, the module thickness is 300-600μm, 300-500μm in the optimized scheme, and the pore is 30-40μm. The module thickness is the overall thickness.

In the present invention, the columnar body is located in the middle of the mesh body, and the pores provided between the columnar body and the mesh body are provided for cell nutrient exchange and waste metabolism. Gelatin methacrylated hydrogel and mesenchymal stem cells constitute active bio-ink, and bio-ink is used for printing, so the overall structure is gelatin methacrylated hydrogel and mesenchymal stem cells. When the module is placed in the culture medium, the nutrients in the culture medium can fill the gaps, so as to ensure that the cells in the structure can evenly receive nutrients.

The invention uses a columnar structure with a diameter of 80-180 μm to simulate periodontal ligament fibers, and the mesenchymal stem cells in the structure can stretch, grow, and proliferate along the columnar structure. Subsequently, in order to realize the implantation in the body, in the design of the structure, a grid structure of 20-40μm was added as a support structure on the basis of the columnar structure. At the same time, a 30-40μm pore was formed between the grid and the columnar structure to facilitate the structure. The internal cells exchange nutrients.

In bio-printing, the composite structure of mesh and column is printed at the same time, and the thickness of the entire printed module is 300-600 μm to simulate the width of human natural periodontal ligament. The top view of the overall structure is a columnar and grid composite structure. In the longitudinal direction, the cells inside the structure can proliferate along the longitudinal structure. The side view is the fiber-like structure formed by the longitudinal proliferation of the cells to simulate periodontal ligament fibers.

The periodontal biological module of the present invention is constructed by light curing 3D biological printing technology based on a digital light processing system; it is composed of a columnar structure and a grid structure and retains nutrient exchange pores. It can maintain the microstructure inside the module for a long time and has good structural stability; maintain the high activity of mesenchymal stem cells for a long time, guide the stem cells to proliferate in a certain direction in space, and make the stem cells have a special and specific arrangement in space and structure Sequence; simulates the microstructure of periodontal fibers to make mesenchymal stem cells proliferate and arrange fibrously; it can repair animal periodontal tissue defects in vivo, including hard tissue defects and periodontal fibrous tissue defects with specific directions.

The top view of this structure is a columnar and net-like composite structure, but the key point is that it is a three-dimensional structure. The cells in the columnar structure in the longitudinal direction can stretch, grow, and proliferate along the columnar structure, thereby forming a fiber-like structure in the longitudinal direction, which can be guided The growth of periodontal ligament fibers in a specific direction.

Mesenchymal stem cells have multiple differentiation characteristics, which can differentiate into blood vessels, nerves, fibers, bones, etc., and can simultaneously regenerate the sandwich structure.

By applying the module of the present invention, a sandwich structure can be generated, and the fiber structure in the generated sandwich structure can be arranged in a specific direction. Mesenchymal stem cells have multiple differentiation characteristics, which can differentiate into blood vessels, nerves, fibers, bones, etc., so they can form a sandwich structure. This is due to the multidirectional differentiation characteristics of stem cells; the modular structure guides the cells to proliferate in a certain direction in the longitudinal direction, forming a press The fibers arranged in a certain direction generate periodontal ligament fibers arranged in a certain direction.

In the process of applying the module, cells grow, proliferate, and material degrade, and the proliferated cells gradually replace the original material structure to form an integral module with a specific structure composed of cells, thereby regenerating the periodontal period. The hydrogel itself is a porous structure, which is conducive to cell adhesion and growth, has good biocompatibility, and has a dense layer and a loose porous layer.

A periodontal biological module for 3D bioprinting and a construction method of the present invention is characterized in that it includes the following steps:

(1) Preparation of mesenchymal stem cells: The mesenchymal stem cells can be derived from rats, beagles, miniature pigs, humans, etc. The obtained mesenchymal stem cells are preferably odontogenic stem cells or bone marrow mesenchymal stem cells, as required The obtained mesenchymal stem cells have good cell activity and various differentiation characteristics such as osteogenic, neurogenic, fibroblast, and osteogenesis;

(2) Preparation of hydrogel solution: under dark conditions, use cell culture medium to prepare 5-15% by mass of gelatin methacrylated hydrogel solution, and then add 0.5-1% by mass of photocrosslinking agent LAP Put it in a 37°C water bath and let it stand for 10-15 minutes until the gelatin methacrylated hydrogel powder is completely dissolved into a hydrogel solution; protect from light when preparing the hydrogel solution.

The gelatin methacrylated hydrogel solution with a mass percentage of 5-15% means that the hydrogel solution contains 5-15% gelatin methacrylic acid. Gelatin methacrylic acid, namely methacrylic acid anhydride gelatin, is a photosensitive biological hydrogel material. The material has excellent biocompatibility, and can be activated by ultraviolet light or visible light to form a solidification reaction to form a three-dimensional structure suitable for cell growth and differentiation with a certain strength. Methacrylic acid is a chemical group and the basis for the photocrosslinking reaction of hydrogels.

The cell culture medium is a complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin.

The LAP photocrosslinking agent can cause the photocrosslinking reaction of the hydrogel, that is, the photocuring 3D bioprinting process. The photocrosslinking agent LAP plays a role in causing the photocrosslinking reaction of the hydrogel, and the photocrosslinking is photocuring.

(3) Preparation of active bio-ink: Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters and add the methacrylated hydrogel obtained in step (2), Pipette to resuspend the cells, and evenly mix the mesenchymal stem cells in the hydrogel solution to obtain active bio-ink; centrifugation conditions: rotation speed 1000rpm (rotation per minute), time 5 minutes;

The active bio-ink can maintain the low viscosity of the original gelatin methacrylated hydrogel in step (2), can be cured and cross-linked by light, and other related characteristics;

(4) Modular structure design: Computer-aided design of the required periodontal biological module structure; computer-aided design of the three-dimensional structure model of the functional module, the designed structure is a columnar structure with a diameter of about 80-180μm and a mesh with a width of about 20-50μm The structure is a composite structure with 30-40μm pores for cell nutrient exchange, and the thickness is about 300-500μm. After design, import the designed structure into the bioprinter system for printing.

(5) 3D bioprinting: Using a 3D bioprinter based on a digital light processing system, according to the required periodontal bio-module structure designed in step (4), the active bio-ink obtained in step (3) is subjected to photo-curing molding, That is 3D bioprinting. The bio-ink is placed on the printer and then cured by light.

The mesenchymal stem cells are derived from rats, beagle dogs, miniature pigs and humans, and the mesenchymal stem cells are pluripotent mesenchymal cells, specifically P2 to 5 generation stem cells; the mesenchymal stem cells obtained in the optimization scheme are Dental-derived stem cells or bone marrow mesenchymal stem cells; in the further optimization plan, the dental-derived stem cells are specifically dental follicle stem cells, periodontal ligament stem cells, exfoliated deciduous tooth stem cells, and dental pulp stem cells.

The concentration of intermediate mesenchymal stem cells in the step (3)=1.3×10 ⁶ -2×10 ⁶ cells/ml.

When the concentration of mesenchymal stem cells is lower than 1.3×10 ⁶ cells/ml, the uniform distribution of mesenchymal stem cells in the printed structure cannot be guaranteed; when the concentration is higher than 2×10 ⁶ cells/ml, the good proliferation of mesenchymal stem cells cannot be guaranteed Speed and growth status.

The concentration of mesenchymal stem cells in the optimized scheme = 1.8×10 ⁶ ～2×10 ⁶ cells/ml.

The DLP light-curing 3D bioprinter has a resolution of 1824×1140, a wavelength of 365-405 nm, and a light source with a maximum power of 50W.

The periodontal biological module constructed by the present invention provides a good adhesion and growth microenvironment for the cells, and at the same time enables the cells to have a special and specific arrangement order in space and structure, and can maintain the stability of the long-term microstructure, and has a periodontal structure. The repair function can well repair periodontal defects in experimental animals.

Wherein, the periodontal functional tissue module imitating the periodontal fiber structure prepared by applying any of the above methods all belong to the protection scope of the present invention. All the features disclosed in this specification, or all disclosed methods or steps in the process, except for mutually exclusive features and/or steps, can be combined in any manner.

Any feature disclosed in this specification (including any appended claims, abstract), unless specifically stated, can be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise stated, each feature is just one example of a series of equivalent or similar features.

In the present invention, the bioprinting step is to print stem cells and hydrogel at the same time. The printing step is continuous. The printed structure is a complete and continuous whole in space. There is no interface in the overall structure. Stem cells can continuously grow and proliferate in the structure. .

3D printing refers to printing materials or biological materials, and 3D bioprinting refers to printing cells and biological materials at the same time, and the focus is on whether the cells are printed.

Description of the drawings

The present invention will be described in further detail below in conjunction with the drawings and specific embodiments:

Fig. 1 is a schematic diagram of the top view of the periodontal biological module of the present invention

Figure 2 is a schematic diagram of the three-dimensional structure of the periodontal biological module of the present invention

Figure 3 is a schematic diagram of the 3D bioprinting periodontal function module and a fluorescent entity diagram after printing in the present invention

Figure 4 is a physical light microscope diagram of the 3D bioprinting periodontal function module of the present invention

Fig. 5 is a comparison diagram of cell survival in the process of intermediate filling culture of the scaffold in the present invention

(Figure A, Figure B, Figure C are the living cell distribution of the cells in the periodontal function module after bioprinting at 3, 7, and 14 days)

Figure 6 is a diagram of the spatial distribution and spatial arrangement of dental follicle cells in the scaffold of the present invention on the 3rd to 14th days

Figure 7 is a diagram showing the positional relationship between the distribution of dental follicle cells in the fluorescent scaffold on the 14th and 21st days and the scaffold in the present invention

Fig. 8 is a diagram showing the regeneration of the 3D bioprinted periodontal function module of the present invention after implantation of the periodontal defect in SD rats and the detection of new bone density

Figure 9 is a histological examination diagram of the regenerated tissue of the 3D bioprinted periodontal function module of the present invention after implantation of the SD rat periodontal defect

Figure 10 is a diagram of the periodontal ligament fibers with specific and different directions in the regenerated tissue of the 3D bioprinted periodontal function module implanted in the SD rat periodontal defect in the present invention

Figure 11 is a diagram showing the expression of periodontal-related proteins in the regenerated tissue of the 3D bioprinted periodontal function module of the present invention after implantation of the SD rat periodontal defect

Figure 12 is the restoration diagram of the periodontal defect model of the 3D bioprinting periodontal function module of the present invention

Fig. 13 is a clinical detection diagram of periodontal related indexes after restoration of the 3D bioprinting periodontal function module of the Beagle dog periodontal defect model of the present invention

Figures 14 and 15 are comparison diagrams of growth conditions when the cell density ratio is different in the present invention

Detailed ways

The following further describes the present utility model in combination with specific embodiments:

Example 1

(1) Obtain P3 generation rat dental follicle stem cells: select the rats 8 days after birth, take the upper and lower jaws, separate the tooth germ, separate the dental follicle tissue from the isolated tooth germ, and use the conventional primary tissue to adhere to the wall. Obtain rat primary dental follicle stem cells and culture them to the P3 generation;

(2) Preparation of hydrogel solution: Use complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare 15% gelatin methacrylated hydrogel solution, and add 1% photocrosslinker LAP ，Place it in a 37℃ water bath and keep it still, away from light;

(3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters, add the methacrylated hydrogel obtained in step (2), and blow to make the cells heavy Suspend, mix the mesenchymal stem cells evenly in the hydrogel solution to obtain the active bio-ink, adjust the cell concentration to 1.3×10 ⁶ cells/ml; centrifuge conditions: rotation speed 1000rpm (rotation per minute), time 5 minutes;

(4) Computer-aided structure design: The designed structure is composed of a composite structure of a columnar structure with a diameter of about 100 μm and a mesh structure with a side width of about 40 μm, and there are pores with a width of about 40 μm for cell nutrient exchange and waste metabolism. ；

(5) Light-curing molding: DLP light-curing 3D bioprinter with a wavelength of 360nm is used, and the structure is designed according to step (4), and the light-curing molding is performed, and the curing time is 20s;

The principle of DLP light-curing bioprinter is used. The printing process is mainly as follows: design the structure through computer software, and transfer the designed structure to the digital optical chip in the bioprinter. The digital optical chip emits a specific light source by controlling the projection optical system to affect the hydrogel and cells on the base. The hybrid bio-ink is light-cured according to a specific structure.

(6) There is no difference between the structure of the bionic periodontal function module obtained by observing step (5) under the microscope and the structure designed by the computer after light curing;

(7) The bionic periodontal function module obtained in the in vitro culture step (5) was observed under the microscope after 14 days, and the microstructure was stable;

(8) Construct a SD rat periodontal defect model, and implant the bionic periodontal function module after in vitro culture in step (7): Select the buccal distal area of the first molar of SD rat to construct a length of 3mm, width of 2mm, and thickness of 700μm. The periodontal defect model was implanted in a bionic periodontal functional module after 14 days of in vitro culture; imaging examination found that the rat periodontal defect was well repaired after 2 months.

Among them, A and B in the figure are schematic diagrams of the structure simulation of the designed periodontal function module. Then use the gelMA hydrogel containing fluorescence to mix the mesenchymal stem cells for bioprinting. The periodontal function module after printing according to the designed specific structure in Figure C, you can clearly see the columnar fiber structure and the grid-like support. Structure and nutrient exchange gap between the two. At the same time, in order to explore whether the designed structure is effective, a bio-ink block that directly solidifies the mixed bio-ink of cells and GelMA hydrogel is used as a control, as shown in Figure D.

The module can maintain the high activity of mesenchymal stem cells for a long time, and guide the stem cells to proliferate in a certain direction in space, so that the stem cells have a special and specific arrangement order in space and structure. It can simulate the microstructure of periodontal fibers and make mesenchymal stem cells proliferate and arrange fibrously. It can repair animal periodontal tissue defects in vivo, including hard tissue defects and periodontal fibrous tissue defects with specific directions.

Example 2

(1) Obtain P3 generation of human periodontal ligament stem cells: take the young permanent premolars that need to be removed due to orthodontics with the consent of the patient, and culture human periodontal ligament stem cells by conventional tissue adhesion method, and culture to the third generation;

(2) Preparation of hydrogel solution: Use complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare methacrylated hydrogel hydrogel solution, add light crosslinking agent LAP, and set Let stand in a 37°C water bath, away from light. Prepare a methacrylated hydrogel hydrogel solution including 15% methacrylated hydrogel and 0.5% LAP photo-crosslinking agent;

(3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters, add the methacrylated hydrogel obtained in step (2), and blow to make the cells heavy Suspend, mix the mesenchymal stem cells evenly in the hydrogel solution to obtain the active bio-ink, adjust the cell concentration to 1.5×10 ⁶ cells/ml; Centrifuge conditions: rotation speed 1000 rpm (rotation speed per minute), time 5 minutes;

In step (3), the active bio-ink can maintain the low viscosity of the original methyl methacrylate hydrogel in step (2), and can be cross-linked by light curing.

(4) Computer-aided structural design: The designed structure is composed of a composite structure of a columnar structure with a diameter of about 100μm and a network structure with a side width of about 40μm, and a 30μm wide pore for cell nutrient exchange and waste metabolism is retained. ；

(5) Light-curing molding: A DLP light-curing 3D bioprinter with a wavelength of 360nm is used, and the structure is designed according to step (4), and the light-curing molding is performed, and the curing time is 20s.

The structure of the bionic periodontal function module obtained by observation under the microscope is not different from the structure designed by the computer after light curing; the bionic periodontal function module obtained by in vitro culture is observed under the microscope after 14 days, and the microstructure is stable;

(6) Construct SD rat periodontal defect model: make animal periodontal defect. The bionic periodontal function module cultured in vitro was implanted, and the buccal distal area of the first molar of SD rats was selected to construct a periodontal defect model with a length of 3mm, a width of 2mm and a thickness of 700μm, and implant the bionic periodontal function module after 14 days of culture in vitro ; Two months later, the imaging examination found that the rat periodontal defect was well repaired.

In the present invention, the active biological ink is placed under a light-curing 3D biological printer based on a digital light processing system (DLP) and is formed by light curing. The constructed periodontal biological module provides cells with a good adhesion and growth microenvironment, and at the same time enables the cells to have a special and specific arrangement order in space and structure, and can maintain long-term microstructure stability, and can be used in experimental animals. The periodontal defect is repaired well in the body.

Example 3

(1) Obtain P3 generation of Beagle dog dental follicle stem cells: take the tooth germ that has not erupted from the 3-month-old beagle dog, separate the dental sac tissue from the isolated tooth germ, and use the conventional primary tissue to attach to the wall to obtain the primary rat tooth Capsule stem cells, and cultivated to the P3 generation;

(2) Preparation of hydrogel solution: Use complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare methacrylated hydrogel hydrogel solution, add light crosslinking agent LAP, and set Let stand in a 37°C water bath, away from light. Prepare a methacrylated hydrogel hydrogel solution including 10% methacrylated hydrogel and 0.5% LAP photocrosslinking agent;

(3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters, add the methacrylated hydrogel obtained in step (2), and blow to make the cells heavy Suspend, mix the mesenchymal stem cells evenly in the hydrogel solution to obtain the active bio-ink, adjust the cell concentration to 2×10 ⁶ cells/ml; Centrifuge conditions: rotation speed 1000 rpm (rotation speed per minute), time 5 minutes;

(4) Computer-aided structure design: The designed structure is composed of a composite structure of a columnar structure with a diameter of about 100μm and a network structure with a side width of about 30μm, and there are pores with a width of about 30μm for cell nutrient exchange and waste metabolism. ；

(5) Light-curing molding: A DLP light-curing 3D bioprinter with a wavelength of 360nm is used, and the structure is designed according to step (4), and the light-curing molding is performed, and the curing time is 15s.

The structure of the bionic periodontal function module obtained by observation under the microscope is not different from the structure designed by the computer after light curing; the bionic periodontal function module obtained in the in vitro culture step (5) is observed under the microscope after 14 days, and the microstructure is stable;

(6) Constructing the Beagle dog periodontal defect model and implanting the bionic periodontal function module after in vitro culture: select the mesial roots of the second and third premolars of the Beagle dog to construct a periodontal defect with a size of about 6mm×5mm×3mm The model was implanted in the bionic periodontal biological module after 14 days of culture in vitro; imaging examination found that the periodontal defect of the Beagle was well repaired after 3 months.

Example 4

(1) Obtain P4 generation of deciduous deciduous tooth stem cells: take the remaining deciduous teeth with pulp tissue that need to be removed with the consent of the patient due to the retention of deciduous teeth, and culture the human deciduous deciduous tooth cells by conventional tissue adhesion method, and culture to the third generation;

(2) Preparation of hydrogel solution: Use complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare methacrylated hydrogel hydrogel solution, add light crosslinking agent LAP, and set Let stand in a 37°C water bath, away from light. Prepare a methacrylated hydrogel hydrogel solution including 10% methacrylated hydrogel and 0.5% LAP photocrosslinking agent;

(3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters, add the methacrylated hydrogel obtained in step (2), and blow to make the cells heavy Suspend, mix the mesenchymal stem cells evenly in the hydrogel solution to obtain the active bio-ink, adjust the cell concentration to 1.8×10 ⁶ cells/ml; Centrifuge conditions: rotation speed 1000 rpm (rotation speed per minute), time 5 minutes;

(4) Computer-aided structural design: The designed structure is composed of a composite structure of a columnar structure with a diameter of about 180 μm and a mesh structure with a side width of about 40 μm, with a width of about 30 μm for cell nutrient exchange and waste metabolism.

(5) Light-curing molding: A DLP light-curing 3D bioprinter with a wavelength of 405nm is used, and the structure is designed according to step (4), and the light-curing molding is performed, and the curing time is 10s.

The structure of the bionic periodontal function module obtained in step (5) under the microscope is not different from the structure designed by the computer after light curing; the bionic periodontal function module obtained in step (5) of in vitro culture is observed under the microscope after 14 days, and the microstructure is stable;

(6) Construction of SD rat periodontal defect model and implanted in vitro cultured bionic periodontal function module: select the buccal distal area of SD rat’s first molar to construct a periodontal defect model with a length of 3mm and a width of 2mm and a thickness of 700μm. The bionic periodontal function module was implanted in vitro cultured for 14 days; after 2 months, the imaging examination found that the rat periodontal defect was well repaired.

Example 5

(1) Obtain P4 generation human dental follicle stem cells: take the incompletely erupted human third molars that need to be extracted for clinical reasons with the consent of the patient, then separate the dental follicles, and culture the human primary dental follicle cells using conventional tissue adherence methods. To the third generation;

(2) Preparation of hydrogel solution: Use complete α-MEM medium containing 10% FBS and 1% penicillin/streptomycin to prepare methacrylated hydrogel hydrogel solution, add light crosslinking agent LAP, and set Let stand in a 37°C water bath, away from light. Prepare a methacrylated hydrogel hydrogel solution including 10% methacrylated hydrogel and 0.5% LAP photocrosslinking agent;

(3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters, add the methacrylated hydrogel obtained in step (2), and blow to make the cells heavy Suspend, mix the mesenchymal stem cells evenly in the hydrogel solution to obtain the active bio-ink, adjust the cell concentration to 1.8×10 ⁶ cells/ml; centrifugation conditions: rotation speed 1000rpm (rotation speed per minute), time 5 minutes;

(4) Computer-aided structure design: The designed structure is composed of a composite structure of a columnar structure with a diameter of about 120μm and a mesh structure with a side width of about 40μm, and a 30μm wide pore for cell nutrient exchange and waste metabolism is retained. ；

(5) Light-curing molding: DLP light-curing 3D bioprinter with a wavelength of 360nm is adopted, and the structure is designed according to step (4), and the light-curing molding is performed, and the curing time is 15s;

The structure of the bionic periodontal function module obtained in step (5) under the microscope is not different from the structure designed by the computer after light curing; the bionic periodontal function module obtained in step (5) of in vitro culture is observed under the microscope after 14 days, and the microstructure is stable;

(6) Construct a SD rat periodontal defect model and implant the bionic periodontal functional module after in vitro culture in step (7): Select the buccal distal area of the SD rat’s first molar to construct a 3mm wide, 2mm thick 700μm The periodontal defect model was implanted in a bionic periodontal function module after 14 days of in vitro culture; imaging examination found that the rat periodontal defect was well repaired after 2 months.

Example 6

The module in the present invention is provided with a grid structure and a columnar structure. The grid structure is formed by interweaving N cells, which are hollow; the columnar structure is provided with M columnar bodies and a bottom plate, and the columnar bodies are fixed in parallel on the bottom plate and formed by the bottom plate. One end of the mesh structure is fixed with the bottom plate, so that the mesh structure and the columnar structure are integrated into one body, and the position of the columnar body on the bottom plate corresponds to the position of the small grid in the network structure, and the columnar body is located in the small grid; the inner wall of the small grid and the columnar There are pores between the external walls, and the diameter of the small cell is greater than the diameter of the columnar body.

The columnar body and the small grid are a whole, the height of the two is the same, and the length value range simulates the length of the natural periodontal ligament fiber, that is, the width of the periodontal ligament. The small grid is rectangular, and the columnar body is a cylinder. The module is composed of active bio-ink, which includes gelatin methacrylated hydrogel, mesenchymal stem cells and photocrosslinker LAP.

The diameter of the columnar body=100μm, the width of the small grid side=30μm, the thickness of the module is 450μm, which is 300～500μm in the optimized scheme; the pore is 35μm. The module thickness is the overall thickness.

Example 7

The module is equipped with a grid structure and a columnar structure. The grid structure is formed by interweaving N small grids, which are hollow; the columnar structure is equipped with M columnar bodies and a bottom plate. The columnar bodies are fixed side by side on the bottom plate and form an integral body with the bottom plate; One end of the shaped structure is fixed to the bottom plate, so that the mesh structure and the columnar structure are integrated into one body, and the position of the columnar body on the bottom plate corresponds to the position of the small grid in the network structure. The columnar body is located in the small grid; the inner wall of the small grid and the outer wall of the columnar There are pores between them, and the diameter of the small cell>the diameter of the columnar body.

The columnar body and the small grid are a whole, the height of the two is the same, and the length value range simulates the length of the natural periodontal ligament fiber, that is, the width of the periodontal ligament.

The small grid is square or round, and the columnar body is a rectangular parallelepiped. The module is composed of active bio-ink, which includes gelatin methacrylated hydrogel, mesenchymal stem cells and photocrosslinker LAP.

The diameter of the columnar body = 80 or 180 μm, the side width of the small grid = 20 or 50 μm, the thickness of the module is 300 or 600 μm; the pore is 30 or 40 μm. The module thickness is the overall thickness.

Example 8

The module is equipped with a grid structure and a columnar structure. The grid structure is formed by interweaving N small grids, which are hollow; the columnar structure is equipped with M columnar bodies and a bottom plate. The columnar bodies are fixed side by side on the bottom plate and form an integral body with the bottom plate; One end of the shaped structure is fixed to the bottom plate, so that the mesh structure and the columnar structure are integrated into one body, and the position of the columnar body on the bottom plate corresponds to the position of the small grid in the network structure. The columnar body is located in the small grid; the inner wall of the small grid and the outer wall of the columnar There are pores between them, and the diameter of the small cell>the diameter of the columnar body.

The columnar body and the small grid are a whole, the height of the two is the same, and the length value range simulates the length of the natural periodontal ligament fiber, that is, the width of the periodontal ligament. The small grid is rectangular, and the columnar body is a cylinder. The module is composed of active bio-ink, which includes gelatin methacrylated hydrogel, mesenchymal stem cells and photocrosslinker LAP.

The diameter of the columnar body=120μm, the width of the small cell side=45μm, the thickness of the module is 300 or 500μm; the pore is 38μm.

Test 1 Detection of the survival rate of live and dead cells

The periodontal biology module in Example 5 was cultured and tested.

Fluorescence staining method was used to detect the activity of mesenchymal stem cells within 3, 7, and 14 days of culturing the biological module.

The reagents used are as follows: calcein CaAM (company: Invitrogen, article number: C3100MP), propidium iodide nucleic acid dye PI (company: Invitrogen, article number: P1304MP).

CaAM is used to stain live cells, can label cell esterases, and display green fluorescence. How to use: Dissolve 50ug CaAM with 10ul DMSO, then add 10ml PBS and mix. The final concentration of the calcium CaAM working solution in the obtained solution is 5 mmol/l.

PI is used to stain dead cells and can label cell nuclei and display red fluorescence.

Usage: Dilute PI to 1mg/ml with double-distilled water and use it as a storage solution; then dilute the storage solution with PBS at a ratio of 1:3000 to a final concentration of 500nM and use it as a working solution.

The dyeing method is as follows:

The prepared bionic periodontal biological modules were cultured for 3, 7, and 14 days respectively, and then taken out, placed in the center of a confocal petri dish, and dripped 1ml of the above CaAM working solution, incubated at 37℃ for 1h; then added 1ml of propidium iodide nucleic acid dye , Staining for 15min. After that, use a laser confocal microscope to observe the staining results of the periodontal biological module from the top view.

The survival results of hDFC cells in the bionic module after 3, 7, and 14 days of culture are shown in A, B, and C of Figure 5, respectively. It can be seen from Figure 5 that in the periodontal biological module at 3, 7, and 14 days, the internal cells proliferate uniformly, and the cell survival rate and proliferation rate are stable (Figure 5 and Figure 6).

Experiment 2 Live cell distribution observation experiment

The periodontal biology module in Example 5 was cultured and tested. The experimental group and the control group are set up. The experimental group is the periodontal biological module designed in Example 5, that is, the internal cells are arranged in an orderly manner; the control group is directly light-cured from the mixture of the same cells and hydrogel in Example 5 Then, there is no modular design, and the internal cells are arranged in disorder.

Fluorescence staining method was used to detect the distribution of mesenchymal stem cells in the biological modules of the experimental group and the control group for 3 and 14 days respectively.

The reagents used and the method of use are as in the first experiment.

The dyeing method is as follows:

The prepared experimental group and the control module were cultured for 3-14 days and then taken out, placed in the center of a confocal petri dish, and 1ml of the above CaAM working solution was added dropwise, and incubated at 37°C for 1h. After that, a laser confocal microscope is used to scan and perform three-dimensional reconstruction, and then detect the staining results of different layers of the periodontal biological module through different viewing angles.

The distribution of mesenchymal stem cells in the experimental group and the control group after culturing for 3-14 days is shown in Figure 6. It can be seen from Figure 6 that in the periodontal biology module at 3-14 days, the internal cells proliferate uniformly, the cell survival rate and proliferation rate are stable, and at 14 days, the cells in the experimental group show fibrous distribution; while the control group is at 3-14 days , Its internal cells proliferate unevenly, and the cells are distributed in disorder at 14 days (Figure 6). The cells of the experimental group and the control group can maintain a cell survival rate of 90-95% for 3-14 days, but the cell proliferation rate of the experimental group is faster than that of the control group (Figure 6).

Experiment 3 The positional relationship between the distribution of mesenchymal stem cells on the 14th and 21st days of the fluorescent scaffold and the scaffold

After culturing the biological module in Example 4 for 14 and 21 days, samples were taken. At room temperature, wash the biological module with PBS, fix it with 4% paraformaldehyde solution for 15 minutes, then permeabilize it with 0.5% TritionX-100 solution for 5 minutes, wash 3 times with PBS, 10 minutes each time, take 200μl of the prepared FITC-labeled ghost pen Cyclic peptide (FITC labeled Phalloidin) working solution, incubate at room temperature for 30min in the dark, wash with PBS 3 times, 5min each time, use 200μl DAPI solution (concentration: 100mM) to counter-stain the cell nucleus, about 30s, wash with PBS, add dropwise anti-fluorescence quenching Extinguishant. After that, a confocal laser microscope was used to observe the staining of the biological module. The results showed that the biological module under long-term culture can still maintain the original microscopic shape, and the cells in the scaffold can maintain a good growth state. As shown in Figure 7, after 14-21 days, it can be seen that as the scaffold material degrades, the internal stem cells can replace the degraded material to maintain the original microscopic shape of the structure. And after 60 days of culture in vitro, it can be seen that after the structure of the hydrogel scaffold is completely degraded, the stem cells in it can maintain the original fibrous microstructure without deformation. It is proved that the periodontal biological module can maintain the bionic periodontal fibrous structure for a long time, and can ensure that the fibrous tissues arranged in a specific direction are generated according to the original microscopic structure after transplantation in the animal body.

Test 4 The regeneration of periodontal functional modules in animals and the detection of new alveolar bone bone density

The periodontal biological module in Example 5 was tested in vivo.

Set up experimental group, control group, blank group and natural group. The experimental group was implanted with 3D bioprinted periodontal biological modules, that is, the internal cells are arranged in an orderly manner; the control group was implanted with a bio-ink block that was randomly mixed and light-cured with stem cells and hydrogel after surgery, that is, no module design , The internal cells are arranged in disorder; the blank group is the non-implantation group after the operation; the natural group is the non-surgery group.

The distal middle of the first molar of SD rats was selected to construct a 3mmx2mmx1mm periodontal defect model, and the experimental group, control group and blank group were implanted to observe the differentiation of biological modules in vivo. After 8 weeks of implantation, samples were taken. Perform Micro CT scan. Afterwards, SCANCO Evaluation software was used to select the operation area for bone mineral density data analysis.

As shown in Figure 8, the test results showed that after 8 weeks of implantation, a large amount of alveolar bone was formed in the experimental group, and the alveolar ridge was recovered well, which was close to the height of the alveolar ridge of natural alveolar bone; while the control group only saw a small amount of alveolar ridge. Osteogenesis; in the blank group, progressive alveolar bone resorption occurred, and the alveolar bone resorbed to the apex. The bone density test on the right shows that the average bone density of the experimental group is much higher than that of the control and blank groups. The bioprinted periodontal functional module has achieved good results in periodontal restoration in animals.

Test 5 Histological examination of regenerative tissue

Set up experimental group, control group, blank group and natural group. The experimental group is a periodontal biological module constructed by implanting GFP-labeled 3D bioprinting, that is, the internal cells are arranged in an orderly manner; the control group is a bio-ink block after the operation is implanted with stem cells and hydrogel disorderly mixed and light-cured, namely No module design, disorderly arrangement of internal cells.

The distal middle of the first molar of SD rats was selected to construct a 3mmx2mmx1mm periodontal defect model, and the experimental group, control group and blank group were implanted to observe the differentiation of biological modules in vivo. After 8 weeks of implantation, the material was taken, fixed in 4% paraformaldehyde, and then placed in EDTA for demineralization for 2 months, washed under running water and overnight, and then dehydrated with gradient alcohol (75% ethanol→85% ethanol→95% Ethanol (I) → 95% ethanol (II) → 95% ethanol (Ⅲ) → 100% ethanol (I) → 100% ethanol (II) → 100% ethanol (Ⅲ) → xylene (I) → xylene (II) ) 1 hour each), embed in paraffin overnight, prepare 5μm tissue sections for hematoxylin-eosin staining (H&E staining) and MASSON staining and observe the sections with fluorescence detection to observe the GFP-labeled implanted teeth GFP expression of Zhou Biological Module.

Perform H&E staining according to the following steps:

(1) Dewaxing; 10 minutes each in xylene (I) and xylene (II);

(2) Hydration: absolute ethanol (I) 10 min → absolute ethanol (II) 10 min → 95% ethanol (I) 5 min → 95% ethanol (II) 5 min → 85% ethanol 5 min → 75% ethanol 5 min;

(3) Wash with PBS buffer for 5 minutes, 3 times;

(4) Staining: hematoxylin staining for 5 min → washing with water for 1 min → 1% hydrochloric acid alcohol for 10 s → washing with running water for 5 min → eosin staining for 1 min → washing with water for 30 s;

(5) Dehydration: 85% ethanol for 1 min → 90% ethanol for 2 min → 95% ethanol (I) for 2 min → 95% ethanol

(II) 2min→Anhydrous ethanol (I) 2min→Anhydrous ethanol (II) 2min→Xylene(I)5min→Xylene(II)5min;

(6) Mounting: Neutral resin sealing, observation under a microscope.

Masson Tricolor Staining Kit (China) was used to stain tissue slices. The kit consists of: Weigert iron hematoxylin A solution, Weigert iron hematoxylin B solution, Ponceau acid fuchsin staining solution, phosphomolybdic acid solution, aniline blue staining liquid:

(1) Dewax the film to hydration according to the above steps;

(2) Weigert iron hematoxylin A solution and Weigert iron hematoxylin B solution are mixed in equal proportions to prepare working solution, dye the tablets for 5-10 minutes, and rinse with running water;

(3) The 1% hydrochloric acid alcohol is differentiated for 10s and rinsed with running water;

(4) Dye with Ponceau red acid fuchsin dye solution for 5-10 minutes, rinse with running water;

(5) Treatment with phosphomolybdic acid solution for about 5 minutes, followed by counter-staining with aniline blue dye solution for 5 minutes;

(6) 1% glacial acetic acid treatment for 2 minutes, gradient alcohol dehydration for 2 minutes each;

(7) Xylene is transparent, and the film is sealed with neutral resin, and observed under a microscope.

From the histological staining in Figure 9, the results of HE and MASSON showed that the new fibers in the experimental group were arranged in an orderly manner, and at a certain angle to the root, which was similar to the periodontal ligament fibers in the natural group; while the new fibers in the control group were arranged disorderly.

Perform fluorescence detection on the slices to observe the GFP expression of the implanted periodontal biological modules marked with GFP:

(1) Dewax the film to hydration according to the above steps;

(2) Mount the coverslip and place it under a microscope for observation.

From Figure 10, the fluorescence results show that the new fibers in the experimental group intersect the surface of the root in a specific and different direction from the neck to the root, which is very similar to the natural periodontal ligament.

It shows that the periodontal bio-modules of the experimental group-3D bioprinting can regenerate periodontal ligament fibers in an orderly and specific direction, and the regenerated periodontal ligament fibers are at a certain angle with the tooth root, which is similar to natural periodontal ligament. It proves that the periodontal biological module constructed by the present invention can not only repair periodontal defects, generate periodontal sandwich structure, but also regenerate periodontal ligament fibers arranged in a specific direction, that is, complete regeneration of periodontal tissues similar to natural periodontal tissues. Newborn periodontal tissue.

Test 6 Protein expression related detection of regenerated tissue

Set up experimental group, control group and natural group. The experimental group was implanted with a periodontal bio-module constructed by 3D bioprinting, that is, the internal cells are arranged in an orderly manner; the control group was a bio-ink block that was randomly mixed and light-cured with implanted stem cells and hydrogel after surgery, that is, there was no internal cell. Sequence arrangement.

The distal middle of the first molar of SD rats was selected to construct a 3mmx2mmx1mm periodontal defect model, and the experimental group, control group and blank group were implanted to observe the differentiation of biological modules in vivo. After 8 weeks of implantation, the material was taken, fixed in 4% paraformaldehyde, and then placed in EDTA for demineralization for 2 months, washed under running water and overnight, and then dehydrated with gradient alcohol (75% ethanol→85% ethanol→95% Ethanol (I) → 95% ethanol (II) → 95% ethanol (Ⅲ) → 100% ethanol (I) → 100% ethanol (II) → 100% ethanol (Ⅲ) → xylene (I) → xylene (II) ) 1 hour each), embedded in paraffin overnight, prepared into 5 μm tissue sections and stained with immunofluorescence protein to identify the expression of periodontal-related proteins in the new tissues.

Perform immunofluorescence protein staining as follows:

(1) Wash with PBS buffer for 5 minutes, 3 times;

(2) 3% hydrogen peroxide treatment: wipe off the fluid around the tissue, add dropwise 3% hydrogen peroxide solution, and incubate for 20 minutes in the dark; take out the section, rinse with PBS buffer for 5 minutes, 3 times;

(3) Antigen retrieval: Wipe off the fluid around the tissue, drop the frozen section fast antigen retrieval solution (dilute the storage solution with double distilled water 1:5 before use), incubate at room temperature for 8 minutes; rinse with PBS buffer for 5 minutes, 3 times;

(4) Sealing: Take out the section, draw a circle in immunohistochemistry, add goat serum dropwise, and place it in a wet box at 37°C for 30 minutes;

(5) Primary antibody incubation: Pour out the blocking solution, dry it slightly, add the prepared primary antibodies COL-1, Fibronectin, CAP, DSP (1% BSA 1:200 dilution), and incubate overnight at 4°C in a humidified box ；

(6) Secondary antibody incubation: discard the primary antibody, wash with PBS three times, add the secondary antibody dropwise, incubate at 37°C in the dark for 1 hour, wash with PBS 3 times, 5 min/time;

(7) DAPI staining, washing with PBS three times;

(8) Mounting and taking pictures: mounting the slides with anti-fluorescence quencher, taking pictures under a confocal microscope.

From the expression of periodontal-related protein in the regenerated tissue in Figure 11, it can be seen that in the periodontal tissue regenerated by the experimental group, a large number of periodontal-related proteins can be expressed, which proves that the regenerated fibrous tissue is the unique fiber of periodontal tissue. . And the protein expression is greater than the control group, and generates more blood vessels than the control group. In addition, the experimental group can see the obvious expression of neurogenesis-related proteins, while the control group only has the expression of tiny blood vessels and nerve fibers.

This experiment proves that the 3D bioprinted periodontal biological module of the present invention can regenerate periodontal tissue, promote the expression of periodontal related proteins, and can generate abundant blood vessels and nerves.

Experiment 7: Repair of periodontal defects of large animal Beagles with periodontal biological module

A large animal in vivo experiment was performed on the periodontal biological module in Example 3.

Set up experimental group, control group, blank group and natural group. The experimental group was implanted with a periodontal bio-module constructed by 3D bioprinting, that is, the internal cells are arranged in an orderly manner; the control group was a bio-ink block that was randomly mixed and light-cured with implanted stem cells and hydrogel after surgery, that is, there was no internal cell. Arranged in order; the blank group is the non-implantation group after the operation; the natural group is the non-surgery group.

The mesial roots of the second and third premolars of Beagle dogs were selected to construct a periodontal defect model with a size of about 6mm×5mm×3mm, and the experimental group, control group, and blank group were implanted, while the natural group was retained (Figure 12) AD).

Three months after transplantation, the material was taken and the Micro-CT scan was performed. The results showed that the alveolar bone of the experimental group was restored to a certain extent, while the control group and the blank group had a small amount of low-density bone formation, and the alveolar ridge was not well restored (Figure 12D), Figure 12F shows that the bone density of the experimental group in the bone density test was significantly higher than that of the control group and the blank group.

After that, the clinical detection of periodontal related indicators after restoration is performed, as shown in Figure 13. The clinical examination showed that the depth of periodontal probing for beagle dogs at 3 months after the operation was less than 1.5mm, which was at the depth of clinically healthy gingival sulcus probing without loss of adhesion, which proved that the constructed bioprinted periodontal functional module can be used in Clinically repair the periodontal defect of large animals.

Test Eight

The other contents are the same, suppose the mesenchymal cell concentration is (0.5-0.8)*10 ⁵ cells/ml, (0.8-1.0)*10 ⁶ cells/ml, (1.3-1.5)*10 ⁶ cells/ml and (1.8 -2.0)*10 ⁶ cells/ml experimental group, the results are shown in Figure 14 and Figure 15.

It can be seen from the above figure that when the cell concentration is low, it is impossible to ensure that the structure contains an appropriate number of cells, and when the cell concentration is too high, it is also impossible to ensure that the stem cells in the structure have a proper growth space.

The invention uses a columnar structure with a diameter of 80-180 μm to simulate periodontal ligament fibers, and the mesenchymal stem cells in the structure can stretch, grow, and proliferate along the columnar structure. Subsequently, in order to realize the implantation in the body, in the design of the structure, a grid structure of 20-40μm was added as a support structure on the basis of the columnar structure. At the same time, a 30-40μm pore was formed between the grid and the columnar structure to facilitate the structure. The internal cells exchange nutrients. In bio-printing, the composite structure of mesh and column is printed at the same time, and the thickness of the entire printed module is 300-600 μm to simulate the width of human natural periodontal ligament. The top view of the overall structure is a columnar and grid composite structure. In the longitudinal direction, the cells inside the structure can proliferate along the longitudinal structure. The side view is the fiber-like structure formed by the longitudinal proliferation of the cells to simulate periodontal ligament fibers.

The cells and materials of the present invention are printed and formed at the same time, and it only takes 5-10 seconds to build an integral module, which is a new design for tissue engineering regeneration of periodontal tissue, and provides a new strategy for the restoration of periodontal tissue defects, in order to realize the simulation of natural teeth. Peripheral tissue and periodontal ligament fibers, to achieve complete regeneration of periodontal tissue. The regenerated soft tissue and hard tissue have a specific direction, that is, the soft tissue fibers in the middle are the fibers arranged in parallel perpendicular to the upper and lower hard tissues.

The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. The foregoing embodiments and descriptions only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also There are various changes and improvements, and these changes and improvements will fall within the scope of the claimed invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. A periodontal biological module for 3D bioprinting, characterized in that: the module is provided with a grid structure and a columnar structure, the grid structure is formed by interweaving N small grids, the small grids are hollow; the columnar structure is provided with M A columnar body and a bottom plate, the columnar body is fixed side by side on the bottom plate and integrated with the bottom plate; one end of the mesh structure is fixed to the bottom plate, so that the mesh structure and the column structure are integrated into one body, and the position of the column body on the bottom plate is in the small grid of the mesh structure Correspondingly, the columnar body is located in the small grid; there are pores between the inner wall of the small grid and the outer wall of the columnar body, and the diameter of the small grid>the diameter of the columnar body.
2. The periodontal biological module for 3D bioprinting according to claim 1, wherein: the small cells are square, rectangular or circular; the columnar body is a cylinder or a rectangular parallelepiped; the module is composed of It is composed of active bio-ink, which includes gelatin methacrylate hydrogel, mesenchymal stem cells and photocrosslinker LAP.
3. The periodontal bio-module for 3D bioprinting according to claim 1, wherein the diameter of the columnar body=80-180 μm, the width of the small grid side=20-50 μm, and the module thickness=300-500 μm.
4. The periodontal biological module for 3D bioprinting according to any one of claims 1 to 3, characterized in that: pore=30-40 μm.
5. A method for constructing a periodontal biological module for 3D bioprinting, which is characterized in that it comprises the following steps:

   (1) Prepare mesenchymal stem cells;

   (2) Preparation of hydrogel solution: use cell culture medium to prepare 5-15% gelatin methacrylated hydrogel solution, then add 0.5-1% photocrosslinker LAP, and place it in a 37℃ water bath. , Protect from light;

   (3) Preparation of active bio-ink: Centrifuge the mesenchymal stem cells obtained in step (1) to obtain mesenchymal stem cell clusters; and add the gelatin methacrylated hydrogel obtained in step (2), pipetting to make Resuspend cells to obtain active bio-ink;

   (4) Modular structure design: computer-aided design and production of the required periodontal biological module structure;

   (5) 3D bioprinting: using a 3D bioprinter based on a digital light processing system, according to the required periodontal bio-module structure, the active bio-ink obtained in step (3) is light-cured and molded to perform 3D bioprinting.
6. The method for constructing a periodontal biological module for 3D bioprinting according to claim 5, wherein the mesenchymal stem cells are derived from rats, beagle dogs, mini-pigs and humans. Stem cells are pluripotent mesenchymal cells, specifically P2 to 5 generation stem cells; the mesenchymal stem cells obtained in the optimization plan are odontogenic stem cells or bone marrow mesenchymal stem cells; the dental stem cells in the further optimization plan are specifically dental follicle stem cells , Periodontal ligament stem cells, exfoliated deciduous tooth stem cells and dental pulp stem cells.
7. The method for constructing a periodontal biological module for 3D bioprinting according to claim 5, wherein the concentration of mesenchymal stem cells in the step (3) is between 1.3×10 ⁶ -2×10 ⁶ cells /ml; The concentration of intermediate mesenchymal stem cells in the optimized scheme = 1.8×10 ⁶ ～2×10 ⁶ cells/ml.
8. A method for constructing a periodontal biological module for 3D bioprinting according to claim 5, characterized in that: the DLP light-cured 3D bioprinter has a resolution of 1824×1140, a wavelength of 365 to 405 nm, and a light source with a maximum power of 50W .
9. A method for constructing a periodontal biological module for 3D bioprinting according to claim 5, wherein the curing time in the step (5) is 5-20s.
10. The application of a periodontal biological module for 3D bioprinting according to claim 1, wherein the module imitates the periodontal fiber structure and is applied to the repair of periodontal tissue defects.

Images