Visual bionic dental pulp chip based on microfluidic device as well as preparation method and application of visual bionic dental pulp chip

Abstract

The invention discloses a visual bionic dental pulp chip based on a microfluidic device, and a preparation method and application thereof, and belongs to the technical field of biomedicine. The visual bionic dental pulp chip comprises a micro-fluidic chip and a 3D cell reaction unit arranged on the micro-fluidic chip, a perfusion channel, a tube cavity and a liquid storage tank are arranged on the micro-fluidic chip; the 3D cell reaction unit comprises a light-cured gel coating which is coated at the bottom of the liquid storage tank and carries dentin matrix particles, and a bionic dental pulp tissue arranged on the surface of the gel coating. The invention discloses a preparation method and application of the visual bionic dental pulp chip. Based on the principle of organ chips, the micro-fluidic device is introduced into the construction of the bionic dental pulp chip, and the ecological niche of natural dental pulp tissue is simulated in vitro to the greatest extent.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a visualized bionic dental pulp chip based on a microfluidic device, and a preparation method and application thereof. Background Art

[0002] Pulp disease and periapical disease are common oral diseases that are mainly caused by bacterial infection and can cause irreversible necrosis of the dental pulp. However, the oral microbial environment is complex, and there are many types of bacteria in saliva. The pathogenic mechanisms of various bacteria on pulp tissue are still unclear. Like other organs, teeth will initiate complex biological processes to repair and respond after being damaged, including various changes and mutual regulation mechanisms at the cellular and molecular levels. In addition, the pulp tissue is located in a pulp cavity with hard walls, and blood circulation can only pass through the tiny apical foramen, which is in a relatively closed space in terms of anatomical structure. This makes it difficult to intuitively observe the biological or pathological reactions of the pulp and the morphological and metabolic conditions of pulp cells. Therefore, establishing an appropriate pulp disease research model is crucial to explore the pathogenesis of pulp diseases and the response process of pulp cells to chemical drugs or biological agents from the root.

[0003] At the current clinical and scientific research level, the study of the pathogenicity of pathogenic microorganisms to the dental pulp mainly relies on establishing a dental pulp inflammation model in experimental animals. This method mainly opens the pulp cavity by mechanical means to expose the dental pulp tissue to the oral microenvironment. However, its construction time is relatively long and there are differences between different species. For example, the tip of the root of the rat's front teeth has the potential to grow throughout life, so that the rat's teeth can continue to extend to replace the worn part. The oral flora and microenvironment of experimental animals lack sufficient similarity with the human oral cavity. In addition, statistics show that 80% of the drugs that pass preclinical animal trials are actually not effective in treating the target diseases expected by humans, as described in the literature "An analysis of the attrition of drug candidates from four major pharmaceutical companies (Waring MJ, Arrowsmith J, Leach AR, Leeson PD, Weir A.. Nature Reviews Drug Discovery 2015, 14 (7): 475.)". Therefore, the in vivo research model relying on experimental animals is difficult to fully reflect the biological characteristics of human dental pulp. It is a necessary trend to transfer the research platform of dental pulp diseases from in vivo to in vitro in the future.

[0004] According to the document “Establishment of an in vitro culture model of the dental pulp-dentin complex (Chen Ying et al. Journal of Shanghai Jiao Tong University (Medical Edition) 2006, 26(11): 1256-1259.)”, slices of human ex vivo teeth were cultured in vitro to establish an in vitro culture model of the dental pulp-dentin complex. However, after 21 days of in vitro culture, some cells inside the slices began to degenerate, making it difficult to maintain the long-term survival of the cells in vitro. Moreover, this study relied on the acquisition of ex vivo teeth, which had obvious limitations in terms of source and clinical ethics. With the development of interdisciplinary fields in medicine and engineering, new tissue engineering technologies such as 3D bioprinting and organoid culture have been tried to be applied to the construction of in vitro disease research models equipped with pluripotent stem cells, and have gradually become a necessary supplement to animal models in disease research. Based on the above research status, there is an urgent need to develop an in vitro dental pulp disease research platform equipped with stem cells in clinical practice to solve the problem of the lack of effective dental pulp disease models. Summary of the invention

[0005] One of the purposes of the present invention is to provide a visualized bionic dental pulp chip based on a microfluidic device, which is based on the organ chip technology, constructs a bionic dental pulp tissue in vitro and contains a perfusable vascular chamber and a gel shell that can simulate the microenvironment of the pulp cavity, and can be used for 3D perfusion culture. Using this visualized bionic dental pulp chip, the pathogenic process of pathogenic microorganisms on dental pulp tissue and the pharmacological process of drugs acting on inflammatory dental pulp can be intuitively observed. This visualized bionic dental pulp chip can also be used for pre-vascularization of regenerative dental pulp tissue before in vivo transplantation. In addition, this chip can also be used to study the mutual regulation of the co-culture system of dental pulp stem cells and other cells or components.

[0006] The second object of the present invention is to provide a method for preparing the visualized bionic dental pulp chip.

[0007] The third objective of the present invention is to provide an application of a visualized bionic dental pulp chip.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses a visualized bionic dental pulp chip based on a microfluidic device, comprising a microfluidic chip and a 3D cell reaction unit arranged on the microfluidic chip;

[0010] The microfluidic chip comprises a chip body, on which a perfusion channel and a liquid storage unit connected to the perfusion channel are arranged; the liquid storage unit comprises a lumen connected to the perfusion channel, and a liquid storage tank connected to the end of the lumen;

[0011] The 3D cell reaction unit comprises a light-curing gel coating applied to the bottom of a liquid reservoir for carrying dentin matrix particles, and a bionic dental pulp tissue arranged on the surface of the light-curing gel coating.

[0012] In some embodiments of the present invention, there are multiple liquid storage units, and the liquid storage units are distributed in pairs, and the two liquid storage units in the same pair of liquid storage units are distributed on both sides of the perfusion channel in a mirror-symmetrical manner;

[0013] Preferably, the two liquid storage tanks in the same pair of liquid storage units are of the same size and shape;

[0014] Preferably, the two lumens in the same pair of liquid storage units have the same size and shape;

[0015] Preferably, the bottom of the perfusion channel is a semicircular structure;

[0016] Preferably, the bottom of the liquid storage tank is a semicircular structure.

[0017] In some embodiments of the present invention, the thickness of the gel coating is 5 to 10 μm; preferably 10 μm;

[0018] The bionic dental pulp tissue occupies more than 40% of the surface area of ​​the gel coating.

[0019] In the present invention, the size of the microfluidic chip, the size of the perfusion channel, the number and size of the liquid reservoirs, and the size of the lumen can all be designed according to specific experimental requirements.

[0020] In some embodiments of the present invention, the hollow perfusion channel has a length of 40 to 60 mm, preferably 50 mm, and a diameter of 3 to 7 mm, preferably 5 mm.

[0021] In some embodiments of the present invention, the diameter of the liquid reservoir is 4 to 8 mm, preferably 6 mm; the height is 8 to 12 mm, preferably 10 mm.

[0022] In some embodiments of the present invention, the length of the multiple lumens connecting each group of liquid reservoirs and the central recessed perfusion channel is 6 to 10 mm, preferably 8 mm; the pore diameter is 2 to 4 mm, preferably 3 mm.

[0023] In some embodiments of the present invention, the gel coating is formed by photocuring a photocurable gel solution containing dentin matrix particles, and the content of the dentin matrix particles in the gel solution is 200-400 mg / ml, preferably 200 mg / ml;

[0024] The photocurable gel comprises methacryloyl hyaluronic acid gel, the concentration of which in the gel solution is 5-10% w / v, preferably 5% (w / v).

[0025] The gel coating of the present invention has low adhesion and can provide a 3D culture environment for bionic dental pulp tissue inside the chip.

[0026] In some other embodiments of the present invention, the thickness of the gel coating is 0.5-1 mm, preferably 1 mm.

[0027] In some embodiments of the present invention, the method for preparing the light-curable gel solution containing dentin matrix particles comprises the following steps:

[0028] Step a. preparing dentin matrix particles and sterilizing them;

[0029] Step b. adding the dentin matrix particles prepared in step a and methacryloyl hyaluronic acid to α-MEM culture medium and mixing well to obtain;

[0030] Preferably, the sterilization is radiation sterilization;

[0031] Preferably, the gel solution is prepared by adding 0.5-1 g of methacryloyl hyaluronic acid and 2-4 g of dentin matrix particles per 10 ml of α-MEM culture medium.

[0032] The preparation method of the dentin matrix particles is an existing technology. The crown, root tip and periodontal tissue, cementum, pulp and pre-dentin covering the surface of clinically discarded teeth are removed; after demineralization, freeze-dried, ground into powder, and irradiated for sterilization, the dentin matrix particles are obtained.

[0033] In some embodiments of the present invention, grinding into a powder that can reach a 50-mesh cell sieve;

[0034] In some embodiments of the present invention, cobalt 60 irradiation sterilization is used.

[0035] In some embodiments of the present invention, gradient demineralization is performed using aqueous solutions of ethylenediaminetetraacetic acid (EDTA) of different concentrations; preferably, demineralization is performed in the following manner: 17% EDTA demineralization for 30 min→10% EDTA demineralization for 20 min→5% EDTA demineralization for 15 min→rinsing with deionized water for 10 min.

[0036] In some embodiments of the present invention, the bionic dental pulp tissue includes dental pulp stem cell aggregates cultured in 3D without a scaffold, and dental pulp stem cells loaded on a tissue engineering scaffold;

[0037] Preferably, the scaffold-free 3D cultured dental pulp stem cell aggregates include at least one of cell membrane sheets and stem cell pellets;

[0038] Preferably, the tissue engineering scaffold loaded with dental pulp stem cells comprises dental pulp stem cells based on gelatin, decellularized matrix or hydrogel;

[0039] The method for preparing a visualized bionic dental pulp chip based on a microfluidic device disclosed in the present invention comprises the following steps:

[0040] S1. First, design the structure of the microfluidic chip and make the mold;

[0041] S2. Prepare a microfluidic chip according to the mold;

[0042] S3. perfusing the light-curable gel solution containing dentin matrix particles through the perfusion channel, and light-curing the gel solution adhered to the bottom of the reservoir to form a microenvironment coating;

[0043] S4. The bionic dental pulp tissue is slowly and evenly perfused through the perfusion channel, and the perfusion is allowed to stand until the bionic dental pulp tissue is deposited on the microenvironment coating at the bottom of the reservoir, thereby obtaining the visualized bionic dental pulp chip.

[0044] In some embodiments of the present invention, in S1, the structure of the microfluidic chip is designed using CAD, and the metal positive mold is manufactured using precision metal processing.

[0045] In S2, a mixture of polydimethylsiloxane prepolymer and curing agent is poured into a mold, cured, demoulded, and cut to obtain a microfluidic chip. It is a prior art to pour a mixture of polydimethylsiloxane prepolymer and curing agent into a mold to prepare a corresponding object.

[0046] In some embodiments of the present invention, the weight ratio of the polydimethylsiloxane prepolymer to the curing agent is (8-12):1; more preferably 10:1.

[0047] The present invention discloses the use of the visualized bionic dental pulp chip based on a microfluidic device in the in vitro bionic construction of dental pulp tissue, or / and dental pulp vascularization reconstruction, or / and prevascularization of dental pulp tissue for transplantation, or / and construction of an in vitro three-dimensional dental pulp model;

[0048] Preferably, the in vitro dental pulp three-dimensional model includes a vascularized regeneration in vitro dental pulp three-dimensional model and an in vitro pulpitis model.

[0049] The application of the visualized bionic dental pulp chip based on the microfluidic device disclosed in the present invention is in the testing and screening of oral materials or drugs.

[0050] In some embodiments of the present invention, porous engineered blood vessels or vascular organoids are inserted through the hollow perfusion channels of the chip body, or the channel surface is modified with materials to directly inoculate endothelial cells to perform vascularization or pre-vascularization of bionic dental pulp tissue; and then complete culture medium is perfused through the hollow perfusion channels of the chip body to fill the entire chip.

[0051] The porous engineered blood vessels are constructed by 3D bioprinting;

[0052] The material modification of the channel surface is to modify the surface of the methacrylylated hyaluronic acid gel by RGD, and the cell density of the inoculated vascular endothelial cells is 1.5×10 5 ~2×10 6 cells / ml.

[0053] The complete culture medium is α-MEM supplemented with 10% fetal bovine serum.

[0054] In some embodiments of the present invention, after the microfluidic chip is prepared in step S2, a glass cover plate package is loaded on the chip body or a porous membrane is loaded and a bacterial infection / drug screening chip is installed on the porous membrane, wherein the loading of the glass cover plate package can be used for:

[0055] a. Pre-vascularization treatment of dental pulp stem cells for vascularization regeneration of dental pulp tissue;

[0056] b. 2D or 3D co-culture of dental pulp stem cells and vascular endothelial cells for the study of promoting vascularization and regeneration of dental pulp tissue using the co-culture system.

[0057] Loading a porous membrane on the chip body and adding a bacterial infection / drug screening chip on the porous membrane can be used for:

[0058] a. Installing a bacterial infection chip on top of the porous membrane can be used for in vitro construction of pulpitis models and pathogenicity research;

[0059] b. A drug screening chip is installed above the porous membrane, which can be used for in vitro screening of pulpitis treatment drugs and optimal drug concentrations.

[0060] Among them, the bacterial infection / drug screening chip has a multi-channel microfluidic system, a "total-separation" channel, and independent dual inlets and multiple outlets, which can automatically sort the bacterial suspension / drug solution according to the concentration gradient.

[0061] Preferably, the porous membrane is a polyamide porous membrane.

[0062] The "% (w / v)" mentioned in the present invention represents the mass volume concentration. For example, if the concentration of methacrylated hyaluronic acid hydrogel is 5% (w / v), it means that 100 ml of aqueous solution contains 5 g of methacrylated hyaluronic acid hydrogel.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] The present invention is scientifically designed and ingeniously conceived. Based on the principle of organ chips, microfluidic devices are introduced into the construction of bionic dental pulp chips. A gel coating that can simulate the microenvironment of the pulp cavity, a bionic dental pulp tissue that can simulate natural dental pulp tissue, and a vascularization device that provides blood supply to it are constructed separately, and the three are integrated into a dimethylsiloxane chip with multiple sets of symmetrically distributed liquid reservoirs and hollow blood perfusion channels. Finally, a visualized bionic dental pulp chip is formed, which contains multiple sets of symmetrically distributed "3D cell reaction units" to simulate the ecological niche of natural dental pulp tissue in vitro to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is a top view of the bionic dental pulp chip of the present invention; wherein the names corresponding to the reference numerals are: 1-chip body, 2-perfusion channel, 3-liquid reservoir, 4-lumen;

[0066] Figure 2 The graph is a result of the TDM and TDM particles used to construct the microenvironment coating of the present invention and the protein release curve thereof; wherein Figure 2 A is the appearance of the dentin matrix. Figure 2 B is a picture of dentin matrix particles. Figure 2 C is the dentin matrix granule protein release curve result diagram;

[0067] Figure 3 The figure is a flow chart of the preparation of dental pulp stem cell pellets (DPSCS) of the present invention;

[0068] Figure 4 The diagram is a result diagram of the DPSCS under a microscope (left) and a result diagram of the sphere diameter statistics (right);

[0069] Figure 5 The results of cell live-death staining (left) and survival rate statistics (right) of DPSCS of the present invention on days 3 and 7;

[0070] Figure 6 This is a diagram showing the results of immunofluorescence staining of the DPSCS cytoskeleton of the present invention;

[0071] Figure 7 This is a diagram showing the results of immunofluorescence staining of DPSCS protein expression of the present invention;

[0072] Figure 8 The digital general image of the engineered blood vessel in the embodiment of the present invention (left) and the perfusion experiment result image (right);

[0073] Fig. 9 Schematic diagram of a bacterial infection / drug screening chip in an embodiment of the present invention;

[0074] Fig.10This is a flow chart of the preparation of a bacterial infection / drug screening chip according to an embodiment of the present invention;

[0075] Fig.11 A digital schematic diagram of a bacterial infection / drug screening chip according to an embodiment of the present invention;

[0076] Fig.12 This is a diagram showing the results of a perfusion experiment of a bacterial infection / drug screening chip in an embodiment of the present invention;

[0077] Fig.13 It is a CAD design drawing of a metal positive mold for constructing a bionic dental pulp chip of the present invention;

[0078] Fig.14 A general digital image of a metal positive mold for constructing a bionic dental pulp chip according to the present invention;

[0079] Fig.15 A general digital image of a metal groove for constructing a bionic dental pulp chip of the present invention;

[0080] Fig.16 A physical picture of the PDMS chip for integrating the substructure of the bionic dental pulp chip of the present invention;

[0081] Fig.17 This is a schematic diagram of the process of HAMA modification by RGD in an embodiment of the present invention;

[0082] Fig.18 This is a digital general image of RGD-HAMA and HAMA after photocuring in an embodiment of the present invention;

[0083] Fig.19 The scanning electron microscopy results of RGD-HAMA and HAMA after photocuring in the embodiment of the present invention are shown;

[0084] Fig. 20 The light microscopic results of cell adhesion of RGD-HAMA and HAMA after light curing in the embodiment of the present invention;

[0085] Fig.21 The results of cell live-death staining of RGD-HAMA and HAMA surface cells after light curing (upper) and the results of cytoskeleton immunofluorescence staining (lower) in the embodiment of the present invention are shown;

[0086] Fig. 22 Schematic diagram (left) and top view (right) of the assembly of the bacterial infection / drug screening chip and the bionic dental pulp chip in an embodiment of the present invention. DETAILED DESCRIPTION

[0087] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0088] If the specific experimental steps or conditions are not specified in the embodiments, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without indicating the manufacturer are all conventional reagent products that can be purchased on the market. The PDMS prepolymer solution and curing agent in the embodiments of the present invention are matching commercial products, such as Dow Corning's 184PDMS prepolymer product, which includes a PDMS prepolymer solution and a curing agent.

[0089] Example 1

[0090] As attached Figure 1 As shown, this embodiment discloses a visualized bionic dental pulp chip based on a microfluidic device, including a microfluidic chip and a 3D cell reaction unit arranged on the microfluidic chip;

[0091] The microfluidic chip comprises a chip body 1, on which a perfusion channel 2 and a liquid storage unit connected to the perfusion channel 2 are arranged; the liquid storage unit comprises a lumen 4 connected to the perfusion channel 2, and a liquid storage tank 3 connected to the end of the lumen 4;

[0092] The 3D cell reaction unit includes a light-cured gel coating applied to the bottom of the reservoir 3 for carrying dentin matrix particles, and a bionic dental pulp tissue disposed on the surface of the light-cured gel coating. There are multiple reservoir units, and the reservoir units are distributed in pairs, and the two reservoir units in the same pair of reservoir units are mirror-symmetrically distributed on both sides of the perfusion channel 2;

[0093] The two liquid storage tanks 3 in the same pair of liquid storage units are of the same size and shape; the two cavities 4 in the same pair of liquid storage units are of the same size and shape; the bottom of the perfusion channel 2 is a semicircular structure; the bottom of the liquid storage tank 3 is a semicircular structure.

[0094] The thickness of the gel coating is 5-10 μm; the bionic dental pulp tissue occupies more than 40% of the surface area of ​​the gel coating.

[0095] The gel coating is formed by photocuring a photocurable gel solution containing dentin matrix particles, the content of the dentin matrix particles in the gel solution is 200-400 mg / ml, and the photocurable gel includes methacrylated hyaluronic acid gel, and its concentration in the gel solution is 5-10% w / v.

[0096] Example 2

[0097] This embodiment discloses a method for preparing a light-curable gel solution containing dentin matrix particles. The details are as follows:

[0098] 1. Preparation of Dentin Matrix (TDM) Particles

[0099] Freshly extracted teeth (human orthodontic teeth collected clinically) were repeatedly rinsed with PBS buffer and immersed in a PBS solution with double antibodies (100U / mL penicillin and 100μg / mL streptomycin). The crown, root apex, and periodontal tissue and cementum covering the surface of the obtained tooth tissue were removed using a dental high-speed turbine. The pulp tissue in the excised tooth was completely extracted using a pulp extraction needle, and the residual pulp tissue and pre-dentin were ground off using a high-speed turbine. The appearance was further trimmed to meet the requirements for transplantation. The prepared dentin scaffold material was transferred into a centrifuge tube containing 50 ml of deionized water, and the centrifuge tube was placed in an ultrasonic cleaner for shaking and cleaning for 20 minutes; the cleaned dentin scaffold material was placed in a 100 ml wide-mouth bottle containing different concentrations of ethylenediaminetetraacetic acid (EDTA), and the wide-mouth bottle was placed on a magnetic stirrer for stirring and demineralization in the following order: 17% EDTA demineralization for 30 minutes → 10% EDTA demineralization for 20 minutes → 5% EDTA demineralization for 15 minutes → deionized water rinsing for 10 minutes, and finally TDM was obtained, such as Figure 2 As shown in A.

[0100] Preparation of TDM particles: First, the prepared TDM was freeze-dried using a freeze dryer, and then ground into TDM powder using a high-throughput tissue grinder until all the powder could pass through a 50-mesh cell sieve. The TDM powder was irradiated and sterilized using cobalt 60. The obtained TDM particles were as follows: Figure 2 As shown in B.

[0101] 2. Preparation of Gel Solution

[0102] Preparation of methacryloyl hyaluronic acid (HAMA) loaded with TDM particles: Add 0.5 g methacryloyl hyaluronic acid (HAMA) and 2 g TDM powder per 10 ml α-MEM culture medium, shake and mix at a constant temperature of 37° C. to obtain the gel solution.

[0103] In this example, the obtained gel solution was photocured, and then the release of protein inside the gel after photocuring was measured by BCA method. The specific measurement method is as follows:

[0104] 1) Place the light-cured gel in 4 ml PBS and pipette 20 μl into an EP tube every 2 hours as the sample to be tested;

[0105] 2) Setting standard wells in a 96-well plate;

[0106] 3) Set up sample wells, take 10 μl of the sample to be tested and dilute it to 100 μl, and add 20 μl of the diluted sample to be tested to each well;

[0107] 4) Prepare the color developing solution according to the ratio of solution A: solution B = 50:1 in the instruction manual and shake to mix;

[0108] 5) Add 200 μl of color development solution to each well;

[0109] 6) After shaking and mixing the 96-well plate in an ELISA reader, incubate at 37°C for 30 min;

[0110] 7) Measure the absorbance (OD) value of each well under 562 nm excitation light;

[0111] 8) Draw a standard curve based on the OD value of each well.

[0112] BCA test results Figure 2 As shown in C: The results show that TDM particles can be gradually released in HAMA after light curing.

[0113] Example 3

[0114] This example discloses a method for preparing scaffold-free 3D cultured dental pulp stem cell spheres (DPSCS), and investigates their microscopic characteristics and cytological properties.

[0115] 1. Preparation of dental pulp stem cell pellets

[0116] The preparation process of dental pulp stem cell pellets (DPSCS) is as follows Figure 3 As shown, the specific steps are as follows:

[0117] 1) Isolation and culture of DPSCs: Natural rat dental pulp tissue was obtained and digested with 3 mg / mL type I collagenase and 4 mg / mL neutral collagenase at a ratio of 1:1 at 37°C for 30 min to prepare a single cell suspension, and 2-4 generation DPSCs were selected.

[0118] 2) Preparation of DPSCs:

[0119] a. Plate package processing: SpheroX TM Add anti-adhesion coating solution to the 96Ukit well plate, 100uL per well, avoid bubbles when adding liquid, then put the well plate into the CO2 incubator and let it stand overnight, take out the culture plate, completely aspirate and discard the coating solution before inoculating cells;

[0120] b. DPSCS culture: Add 200uL / well of cell suspension to the coated well plate, 1×10 4cells / well, let it stand for 48 hours, and after the sphere is formed, use a 200uL pipette tip to gently change the medium to ensure the integrity of the cell sphere. When changing the medium, keep about 50uL of culture medium in the well to avoid sucking away the sphere.

[0121] 2. Microscopic characteristics of dental pulp stem cell spheroids

[0122] The sphering of DPSCs was observed under an optical microscope from 0 to 48 hours. The results showed that DPSCs could be spherical in 48 hours under the above preparation method. Figure 4 shown.

[0123] The diameter of DPSCS was counted at 48 hours, and the statistical results showed that the size distribution of DPSCS was 300±20μm, with uniform size and good morphology.

[0124] 3. Cytological characteristics investigation

[0125] After culturing DPSCs in vitro for 3 and 7 days, Live / Dead TM The viability / cytotoxicity kit was used to detect the viability of DPSC cells in the module. All samples were observed under a laser scanning confocal microscope, and three samples were randomly selected from each group for quantitative analysis. The results showed that the cell viability of DPSCs could be maintained above 99.5% after 3 and 7 days of in vitro culture. Figure 5 shown.

[0126] To further detect the morphology of DPSCs, immunofluorescence (IF) staining was performed on the 3rd and 7th days of culture to detect the cell morphology in DPSCs. The results showed that the DPSCs in DPSCs were in an extended state and the cells were in good growth state. Figure 6 shown.

[0127] In order to explore the differences between DPSCS and 2D cultured DPSC, equal cell numbers of DPSCS and adherent DPSC were cultured, and after 7 days of culture, the expression of pluripotency and proliferation-related proteins was analyzed by IF, including Sox 2 (1:200 dilution, Abcam, UK), Nanog (1:200 dilution, Abcam, UK), Oct 4 (1:200 dilution, Abcam, UK) and Ki 67 (1:200 dilution, Abcam, UK). The results showed that DPSCS positively expressed pluripotency-related and proliferation marker genes Sox 2, Nanog, Oct 4 and Ki67, while 2D cultured DPSC did not show obvious expression, such as Figure 7 shown.

[0128] Example 4

[0129] This embodiment discloses a method for constructing a porous engineered blood vessel.

[0130] First, DLP-based 3D printing technology was used to prepare non-porous engineered blood vessels, and the lumen was perfused to observe whether it leaked. Figure 8 As shown, its perfusion capacity was investigated. The results showed that the engineered blood vessels constructed in this experimental example had structural integrity and could meet the perfusion requirements of nutrients, such as Figure 8 Subsequently, the inlet lumen of the non-porous engineered blood vessels corresponding to the reservoirs was perforated to meet the nutrient exchange between each group of reservoirs and the central perfusion channel.

[0131] The steps of preparing non-porous engineered blood vessels using DLP-based 3D printing technology are as follows:

[0132] 1. Use L-edit software to design a hollow vascular structure with a length of 5 cm, a wall of 1 mm, and a lumen diameter of 3 mm;

[0133] 2. Using a DLP 3D bioprinter with a wavelength of 360-405nm, the photocurable hydrogel is printed and formed according to the designed structure to obtain non-porous engineered blood vessels.

[0134] Among them, methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) are preferred as photocurable hydrogels.

[0135] Example 5

[0136] This embodiment discloses the construction and identification of a bacterial infection / drug screening chip used in conjunction with the visualized bionic dental pulp chip of the present invention.

[0137] First, if Fig. 9 As shown, a special microfluidic channel structure with 2 inlets, 6 outlets, fusion first and then sorting was designed using CAD software. The inner diameter of the channel was 3 mm, and a drug screening chip with a serpentine structure was combined.

[0138] Subsequently, the PDMS bacterial infection / drug screening chip was prepared by secondary molding. The preparation process is as follows: Fig.10 As shown in the figure, the digital general photo of the constructed bacterial infection / drug screening chip is as follows Fig.11 shown.

[0139] The preparation process is as follows:

[0140] 1). Preparation of silicon wafer microchannel template: According to the designed channel structure, a micro-concave channel template is printed on the silicon wafer using 3D printing technology; the prepared silicon wafer microchannel template is subjected to hydrophobic treatment, and the steps mainly include: Plasma treatment of the silicon wafer with a plasma machine to clean the template surface; immediately after treatment, the template is placed in a vacuum drying oven, and 10uL FAS is taken in the drying oven with a pipette gun, and kept in a vacuum environment of 80℃ for 5h, and a fluorosilane hydrophobic layer is modified on the template surface to prevent PDMS from sticking to the template and affecting subsequent demolding.

[0141] 2). Preparation of PDMS positive mold: prepare a simple groove mold with metal to prevent the PDMS liquid from flowing out; place the silicon wafer microchannel template at the bottom; mix the PDMS prepolymer and curing agent in a ratio of 10:1, pour it into the groove mold, put it in a vacuum drying oven, vacuum to remove bubbles, and cure it at 37°C for 24 hours; take out the cured PDMS positive mold for use.

[0142] 3). Preparation of PDMS chip: PDMS prepolymer and curing agent were mixed in a ratio of 10:1, poured into the groove of the PDMS positive mold, cured at 37°C for 24 hours, and after molding, the PDMS bacterial infection / drug screening chip was peeled off from the PDMS positive mold to obtain.

[0143] In order to explore the channel integrity of the bacterial infection / drug screening chip and its ability to sort bacteria / drug concentration gradients, a perfusion experiment was conducted in this test case. Red pigment and yellow pigment solutions were perfused at the same rate from inlets-2 and 3 respectively to simulate drug concentration gradient sorting. The results showed that the bacterial infection / drug screening chip has channel integrity and can be effectively fused and sorted at the center of the distance between inlets-2 and-3. Fig.12 shown.

[0144] Example 6-Example 8 discloses the preparation of a visualized bionic dental pulp chip based on a microfluidic device.

[0145] Example 6

[0146] This embodiment discloses the preparation of a visualized bionic dental pulp chip based on a microfluidic device of the present invention, and the specific steps are as follows:

[0147] S1. Use CAD software to design the microfluidic chip structure. The design diagram is as shown in the attached Fig.13 As shown, the metal positive mold is made by precision metal processing, and the mold is as shown in the attached Fig.14 and attached Fig.15 shown.

[0148] S2, polydimethylsiloxane prepolymer (PDMS prepolymer) and curing agent are mixed in a mass ratio of 10:1 to prepare a prepolymer mixture, and the prepolymer mixture is poured into a metal positive mold, and then cured, demolded, and cut to obtain a PDMS microfluidic chip, such as Fig.16 As shown: the central depression of the chip body forms a hollow perfusion channel 2, and the raised parts on both sides are provided with 6 groups of symmetrically distributed liquid storage units, the liquid storage units include a lumen 4 connected to the perfusion channel 2, and a liquid storage tank 3 connected to the end of the lumen 4; the bottom of the liquid storage tank 3 and the perfusion channel 2 are both "U-shaped";

[0149] S3, pour the light-curable gel solution containing dentin matrix particles into the inlet-1 through the hollow perfusion channel of the microfluidic chip body. When the gel solution is observed to flow out from the outlet-1, the perfusion is stopped, the remaining gel solution is sucked out, and the gel solution adhering to the bottom of the reservoir is light-cured to form a microenvironment coating with a coating thickness of 1 mm.

[0150] The preparation method of the gel solution is prepared according to the method of Example 2.

[0151] S4, uniformly and slowly perfuse complete medium containing scaffold-free 3D cultured dental pulp stem cell spheres through the hollow perfusion channel of the chip body, with a cell sphere concentration of 200 spheres / ml; perfuse once into inlet-1, and stop perfusing when the cell sphere solution is observed to flow out from outlet-1; let it stand until the dental pulp stem cell spheres are deposited on the microenvironment coating at the bottom of the reservoir; and obtain a visualized bionic dental pulp chip. The preparation method of the scaffold-free 3D cultured dental pulp stem cell spheres (DPSCS) in this embodiment is prepared according to the method of Example 3.

[0152] The visualized bionic dental pulp chip prepared in this embodiment is used to perform prevascularization of bionic dental pulp tissue. The specific operation is as follows: after the microfluidic chip is prepared in step S2, a glass sheet is loaded on the chip body; then the above steps S3 and S4 are performed, and then the following steps (1) and (2) are performed.

[0153] (1) Vascular organoids (DPSCS prepared according to the method of Example 3) are placed through the hollow perfusion channel of the chip body to perform pre-vascularization of bionic dental pulp tissue; the specific perfusion method is the same as step S4 of Example 6.

[0154] (2) Complete culture medium is perfused through the hollow perfusion channel of the chip body to fill the entire chip.

[0155] The present embodiment constructs a bionic dental pulp chip that contains microenvironmental encapsulation, scaffold-free 3D cultured dental pulp stem cell spheroids and vascular organoids, and integrates the three into one. While providing a pulp cavity microenvironment for in vitro bionic dental pulp tissue, it simulates the blood supply of natural dental pulp tissue and simulates natural dental pulp tissue to the greatest extent under 3D perfusion culture, thereby realizing the vascularization of dental pulp tissue.

[0156] This embodiment can be used for: in vitro bionic construction of dental pulp tissue, reconstruction of dental pulp vascularization, prevascularization of dental pulp tissue for transplantation, etc.

[0157] Example 7

[0158] This embodiment discloses the preparation of a visualized bionic dental pulp chip based on a microfluidic device of the present invention, and the specific steps are as follows:

[0159] S1. Use CAD to design the microfluidic chip structure, and use precision metal processing to make a metal positive mold. The mold is as shown in the attached Fig.14 and attached Fig.15 shown.

[0160] S2, polydimethylsiloxane prepolymer (PDMS prepolymer) and curing agent are mixed in a mass ratio of 8:1 to prepare a prepolymer mixture, the prepolymer mixture is poured into a metal positive mold, and the prepolymer mixture is cured, demolded, and cut to obtain a PDMS microfluidic chip, such as Fig.16 As shown: the central depression of the chip body forms a hollow perfusion channel, and the raised parts on both sides are provided with 6 groups of symmetrically distributed liquid reservoirs, and the bottom of the liquid reservoir is "U-shaped", and each group of liquid reservoirs is composed of two perfusion liquid reservoirs respectively opened in the raised parts, and the two perfusion liquid reservoirs are symmetrically arranged relative to the depressed part, and are connected to the central depressed perfusion channel through the lumen;

[0161] S3, pouring a light-curable gel solution containing dentin matrix particles through the hollow of the microfluidic chip body, and light-curing the gel solution adhering to the bottom of the reservoir to form a microenvironment coating; the coating thickness is 1 mm;

[0162] The preparation method of the gel solution is the same as that of Example 2, except that the amounts of the dentin matrix particles and the methacryloyl hyaluronic acid are different. In this example, 0.5 g of methacryloyl hyaluronic acid and 4 g of dentin matrix particles are added to every 10 ml of α-MEM medium.

[0163] S4, uniformly and slowly perfuse the scaffold-free 3D cultured dental pulp stem cell pellets through the hollow perfusion channel of the chip body, and let it stand until the stem cell pellets are deposited on the microenvironment coating at the bottom of the reservoir; thus, a visualized bionic dental pulp chip is obtained. The preparation method of the scaffold-free 3D cultured dental pulp stem cell pellets (DPSCS) in this embodiment is prepared according to the method of Example 3.

[0164] The visualized bionic dental pulp chip prepared in this embodiment is used to perform prevascularization of bionic dental pulp tissue, and the specific operations are as follows:

[0165] After the microfluidic chip is prepared in step S2, a polyamide porous membrane is loaded on the chip body, and a bacterial infection / drug screening chip (the bacterial infection / drug screening chip is prepared according to the method of Example 5) is installed on the porous membrane; then the above steps S3 and S4 are performed, and then the following steps are performed:

[0166] (1) A solution containing porous engineered blood vessels is uniformly and slowly perfused through the hollow perfusion channel of the chip body to perform prevascularization of biomimetic dental pulp tissue; the porous engineered blood vessels of this embodiment are prepared according to the method of Example 4, and a porous structure is added in the design. The specific perfusion operation is: perfuse once into inlet-1, and stop perfusing when the solution is observed to flow out from outlet-1; let it stand until the porous engineered blood vessels are deposited on the microenvironment coating at the bottom of the reservoir. (2) Complete culture medium is perfused through the hollow perfusion channel of the chip body to fill the entire chip.

[0167] This embodiment constructs a bionic dental pulp chip that contains a microenvironmental shell, scaffold-free 3D cultured dental pulp stem cell pellets and porous engineered blood vessels, and integrates the three into one. While providing a pulp cavity microenvironment for in vitro bionic dental pulp tissue, it simulates the blood supply of natural dental pulp tissue and simulates natural dental pulp tissue to the greatest extent under 3D perfusion culture.

[0168] This embodiment can be used for: in vitro bionic construction of pulp inflammation model, screening of pulpitis drugs and effective drug concentrations, etc.

[0169] Example 8

[0170] This embodiment discloses the preparation of a visualized bionic dental pulp chip based on a microfluidic device of the present invention, and the specific steps are as follows:

[0171] S1. Use CAD to design the microfluidic chip structure and use precision metal processing to make the metal positive mold, such as Fig.14 as shown, and metal grooves, such as Fig.15 As shown;

[0172] S2, polydimethylsiloxane prepolymer (PDMS prepolymer) and curing agent are mixed in a mass ratio of 12:1 to prepare a prepolymer mixture, the prepolymer mixture is poured into a metal positive mold, and the prepolymer mixture is cured, demolded, and cut to obtain a PDMS microfluidic chip, such as Fig.16As shown: the central depression of the chip body forms a hollow perfusion channel, and the raised parts on both sides are provided with 6 groups of symmetrically distributed liquid reservoirs, and the bottom of the liquid reservoir is "U-shaped", and each group of liquid reservoirs is composed of two perfusion liquid reservoirs respectively opened in the raised parts, and the two perfusion liquid reservoirs are symmetrically arranged relative to the depressed part, and are connected to the central depressed perfusion channel through the lumen;

[0173] S3, perfusing the light-curable gel solution containing dentin matrix particles through the hollow perfusion channel of the microfluidic chip body, and light-curing the gel solution adhering to the bottom of the reservoir to form a microenvironment coating with a coating thickness of 1 mm;

[0174] The preparation method of the gel solution is the same as that of Example 2, except that the amounts of the dentin matrix particles and the methacryloyl hyaluronic acid are different. In this example, 1 g of methacryloyl hyaluronic acid and 2 g of dentin matrix particles are added to every 10 ml of α-MEM medium.

[0175] S4. The methacrylated gelatin solution carrying dental pulp stem cells was perfused slowly and evenly through the hollow perfusion channel of the chip body, and the cell concentration was controlled to be 2×10 6 cells / ml, and allowed to stand until the bionic dental pulp tissue was deposited on the microenvironment coating at the bottom of the reservoir; thus, a visualized bionic dental pulp chip was obtained.

[0176] Wherein, the preparation method of methacrylated gelatin solution is as follows:

[0177] The gel solution was prepared by adding 1 g of methacryloyl gelatin (GelMA) powder to every 10 ml of α-MEM culture medium, shaking and mixing at a constant temperature of 37° C.

[0178] The visualized bionic dental pulp chip prepared in this embodiment is used to inoculate vascular endothelial cells to perform prevascularization of bionic dental pulp tissue. The specific operation is as follows:

[0179] After the microfluidic chip is prepared in step S2, a glass cover is loaded on the chip body for packaging; then the above steps S3 and S4 are performed, and then the following steps are performed:

[0180] (1) The HAMA on the surface of the hollow perfusion channel of the chip body is modified with RGD by acryloylation. The process of RGD modification is as shown in the attached Fig.17 As shown, digital macrophotographs of RGD-HAMA and HAMA are shown Fig.18 The electron microscope scanning image is shown in the attached Fig.19 Then, cells at a concentration of 2×10 6 cells / ml of umbilical vein endothelial cell (HUVEC) suspension, and seeding HUVEC on the surface of the channel to perform prevascularization of biomimetic dental pulp tissue;

[0181] The steps of modifying the HAMA surface with RGD by acryloylation are as follows:

[0182] ① Use 405nm light source to photocuring HAMA hydrogel for 5s;

[0183] ② Add sterile RGD peptide modification solution to the surface of HAMA hydrogel and irradiate with 405nm light source for 30s to complete the surface modification;

[0184] ③ Aspirate and discard the residual RGD modification solution.

[0185] (2) Complete culture medium is perfused through the hollow perfusion channel of the chip body to fill the entire chip.

[0186] This embodiment constructs a bionic dental pulp chip that includes a microenvironment shell, a bionic dental pulp module, and porous engineered blood vessels, and integrates the three into one. While providing a pulp cavity microenvironment for in vitro bionic dental pulp tissue, it simulates the blood supply of natural dental pulp tissue and simulates natural dental pulp tissue to the greatest extent under 3D perfusion culture.

[0187] This embodiment can be used for: in vitro bionic construction of dental pulp tissue, reconstruction of dental pulp vascularization, prevascularization of dental pulp tissue for transplantation, etc.

[0188] Test Example 1

[0189] This test example discloses the cell compatibility test of the RGD-modified HAMA of Example 8. After inoculation of endothelial cells, the cell adhesion ability, survival rate and proliferation rate were investigated respectively:

[0190] a. Cell adhesion detection: After cell inoculation, HUVECs were subjected to cytoskeleton IF staining after 3 and 7 days of culture, and the cell morphology of HUVECs on the surface of RGD-HAMA was observed and photographed under a confocal microscope;

[0191] b. Cell survival rate detection: After cell inoculation, the cells were cultured for 3 and 7 days, live and dead cell staining was performed to observe the cell survival status, and the cells were observed and photographed under a confocal microscope. The fluorescent bright spots in 100 images of each group were taken to calculate the cell survival rate, and the cell survival rate curve over time was drawn;

[0192] c. Cell proliferation detection: After cell inoculation, the cells were cultured for 3 and 7 days, and the cell proliferation rate was detected by CCK-8 and BrdU method. The cell proliferation status of HUVEC was observed and photographed under a confocal microscope.

[0193] The results showed that RGD-modified HAMA exhibited good cell adhesion, and HUVEC on its surface showed an obvious tendency to form tubes on the first day. Fig. 20As shown; the results of cell live-death staining and cytoskeleton staining showed that cells can maintain good survival rate and extension state after adhering to the RGD-HAMA surface, as shown Fig.21 shown.

[0194] Test Example 2

[0195] This test example demonstrates the application of the bacterial infection / drug screening chip, taking the construction of a rat pulpitis model and drug screening as an example. The steps are as follows:

[0196] ① Punch, package and bond the bacterial infection / drug screening chip in the following order from top to bottom: glass plate, bacterial infection / drug screening chip, polyamide porous membrane, visualized bionic dental pulp chip, glass plate, and metal frame. Fig. 22 The visualized bionic dental pulp chip is prepared according to the method of Example 7, and the bacterial infection / drug screening chip is prepared according to the method of Example 5.

[0197] ②Extract rat dental pulp stem cells (rDPSCs), construct scaffold-free 3D cultured rat dental pulp stem cell microspheres (rDPSCS), perfuse rDPSCS into the bionic dental pulp chip, and construct an integrated platform for rat dental pulp disease research equipped with rDPSCS for perfusion 3D culture.

[0198] ③ In the bacterial infection / drug screening chip, the same concentration of Porphyromonas endotoxin suspension was perfused at the same rate through inlets 2 and 3 to infect the rat dental pulp stem cell pellets (rDPSCS) in the lower chip to construct a rat pulpitis model.

[0199] ④ PBS solution and target chemical drug solution were perfused at the same rate through inlets 2 and 3, and the special structure of the drug screening layer was used to perform drug concentration gradient sorting. After 24, 48, and 72 hours of drug action, the rDPSCS in each group of reservoirs were obtained, and the expression of inflammatory factors such as IL-1, IL-6, IL-8, and TNF in each group of rDPSCS was detected. The results were compared and analyzed with those before drug action to verify the effectiveness of the drug and screen the effective drug concentration.

[0200] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A visualized bionic dental pulp chip based on a microfluidic device, characterized in that: It includes a microfluidic chip and a 3D cell reaction unit arranged on the microfluidic chip; The microfluidic chip comprises a chip body (1), on which a perfusion channel (2) and a liquid storage unit connected to the perfusion channel (2) are arranged; the liquid storage unit comprises a lumen (4) connected to the perfusion channel (2), and a liquid storage tank (3) connected to the end of the lumen (4); The 3D cell reaction unit comprises a light-curing gel coating applied to the bottom of a liquid storage tank (3) for carrying dentin matrix particles, and a bionic dental pulp tissue arranged on the surface of the light-curing gel coating.

2. The visualized bionic dental pulp chip based on a microfluidic device according to claim 1, characterized in that: There are multiple liquid storage units, and the liquid storage units are distributed in pairs, and the two liquid storage units in the same pair of liquid storage units are distributed on both sides of the perfusion channel (2) in a mirror-symmetrical manner; Preferably, the two liquid storage tanks (3) in the same pair of liquid storage units have the same size and shape; Preferably, the two lumens (4) in the same pair of liquid storage units have the same size and shape; Preferably, the bottom of the perfusion channel (2) is a semicircular structure; Preferably, the bottom of the liquid storage tank (3) is a semicircular structure.

3. The visualized bionic dental pulp chip based on a microfluidic device according to claim 1, characterized in that: The thickness of the gel coating is 5 to 10 μm, preferably 10 μm; The biomimetic dental pulp tissue occupied more than 40% of the surface area of ​​the gel coating.

4. The visualized bionic dental pulp chip based on a microfluidic device according to claim 1, characterized in that: The gel coating is formed by photocuring a photocurable gel solution containing dentin matrix particles, wherein the content of the dentin matrix particles in the gel solution is 200-400 mg / ml, preferably 200 mg / ml; The photocurable gel comprises methacryloyl hyaluronic acid gel, the concentration of which in the gel solution is 5-10% w / v, preferably 5% (w / v).

5. The visualized bionic dental pulp chip based on a microfluidic device according to claim 4, characterized in that: The method for preparing the light-curable gel solution containing dentin matrix particles comprises the following steps: Step a. preparing dentin matrix particles and sterilizing them; Step b. adding the dentin matrix particles prepared in step a and methacryloyl hyaluronic acid to α-MEM culture medium and mixing well to obtain; Preferably, the sterilization is radiation sterilization; Preferably, the gel solution is prepared by adding 0.5-1 g of methacryloyl hyaluronic acid and 2-4 g of dentin matrix particles per 10 ml of α-MEM culture medium.

6. A visualized bionic dental pulp chip based on a microfluidic device according to any one of claims 1 to 5, characterized in that: The bionic dental pulp tissues include dental pulp stem cell aggregates cultured in 3D without scaffolds and dental pulp stem cells loaded in tissue engineering scaffolds; Preferably, the scaffold-free 3D cultured dental pulp stem cell aggregates include at least one of cell membrane sheets and stem cell pellets; Preferably, the tissue engineering scaffold loaded with dental pulp stem cells comprises dental pulp stem cells based on gelatin, decellularized matrix or hydrogel.

7. A method for preparing a visualized bionic dental pulp chip based on a microfluidic device according to any one of claims 1 to 6, characterized in that: The steps include: S1. First, design the structure of the microfluidic chip and make the mold; S2. Prepare a microfluidic chip according to the mold; S3. perfusing the light-curable gel solution containing dentin matrix particles through the perfusion channel, and light-curing the gel solution adhered to the bottom of the reservoir to form a microenvironment coating; S4. The bionic dental pulp tissue is slowly and evenly perfused through the perfusion channel, and the bionic dental pulp tissue is allowed to stand until it is deposited on the microenvironment coating at the bottom of the liquid reservoir, thereby obtaining the visualized bionic dental pulp chip.

8. The method for preparing a visualized bionic dental pulp chip based on a microfluidic device according to claim 9, characterized in that: In S1, the structure of the microfluidic chip is designed using CAD, and a metal positive mold is made using precision metal processing; In S2, a mixture of polydimethylsiloxane prepolymer and curing agent is poured into a mold, cured, demolded, and cut to obtain a microfluidic chip; preferably, the weight ratio of polydimethylsiloxane prepolymer to curing agent is (8-12):1; more preferably, it is 10:

1.

9. Application of a visualized bionic dental pulp chip based on a microfluidic device according to any one of claims 1 to 6 in in vitro bionic construction of dental pulp tissue, or / and reconstruction of dental pulp vascularization, or / and prevascularization of dental pulp tissue for transplantation, or / and construction of an in vitro three-dimensional dental pulp model; Preferably, the in vitro dental pulp three-dimensional model includes a vascularized regeneration in vitro dental pulp three-dimensional model and an in vitro pulpitis model.

10. The application of a visualized bionic dental pulp chip based on a microfluidic device according to any one of claims 1 to 6, characterized in that: The application of the visualized bionic dental pulp chip in the testing and screening of oral materials or drugs.

Images