New method

Abstract

The present invention relates to a method for obtaining a denture including a crown and a root. The method comprises a step of inducing at least one undifferentiated or keratinized cell, and a three-dimensional printing step, preferably a 3D bioprinting step of the denture. Furthermore, the present invention relates to a denture obtained by the method, and a bioink suitable for three-dimensional (3D) printing, preferably suitable for 3D bioprinting.

Description

Field of the Invention

[0001] The present invention relates to a method for obtaining a denture including a dental crown and a tooth root. Background

[0002] Human teeth perform various functions such as chewing food and being able to pronounce correctly. Furthermore, teeth play a major role in the human aesthetic appearance. In fact, white, healthy, and well-arranged teeth are considered the ideal beauty and are regarded as an aesthetic symbol of youth and success.

[0003] Various preventive measures such as frequently using a toothbrush and dental floss or ingesting fluoridated water and iodinated water are widely recommended and practiced by many humans. However, ultimately, the majority of humans will eventually fall into a situation where they have to deal with dental fillings, restorative implants, and / or dentures.

[0004] The main objective of dental medicine is to prevent tooth loss or delay it as much as possible. Another objective is, of course, to provide comfortable, widely applicable, and long-lasting dentures.

[0005] Generally, the number of available restorative and prosthetic options is limited. When the tooth root and the periodontal tissues including it are healthy and sufficient to support these partial prostheses, fillings, crowns, and dental crowns are used. Conventionally, when the original tooth becomes unusable, bridges and prefabricated bone prosthetic implants have been recommended. In this case, various negative aspects have to be endured. In order to create the support structure of the bridge, the adjacent teeth on both sides are shaved, and the healthy enamel is partially destroyed. In addition, bone prosthetic implants are highly invasive, and the interface between the gingiva and the implant often causes chronic local infections. Furthermore, all of the above repair and prosthetic options have a limited average service life. Removable dentures are certainly a decisive prosthetic option.

[0006] When a tooth is partially damaged due to cavities, trauma, or other reasons, it is usually necessary to repair the missing portion. This is the preferred solution as long as the tooth has sufficient structural strength to support prostheses such as inlays or crowns. However, if the loss of tooth structure is severe, this solution may not be applicable. In such cases, a bridge can be fitted, with full awareness of the aforementioned disadvantages. Another option is to replace the tooth with an implant.

[0007] There are many methods and options for replacing missing teeth. One of these options is a prefabricated dental bone prosthesis implant. Bone prosthesis means that the surface of the implant is in direct contact with the bone, and there is no connecting tissue between them (natural teeth are usually not in direct contact with the bone, but are connected to the bone by periodontal ligaments). The use of such dental implants involves various implant designs and materials, use in different locations in the oral cavity, and the use of various surgical methods.

[0008] Currently, implants are typically made of cylindrical or conical titanium (and more recently, zirconia), with screw threads, other fastening mechanisms, and a structured surface that has been specially treated to enhance volume and bone grafting. The abutment (sometimes integrated with the screw) is fixed onto the implant and supports a crown that mimics a natural tooth.

[0009] Traditional implant clinical protocols are typically very lengthy and complex (generally requiring around 10 visits), and therefore expensive. While immediate loading implants are now used, they are only applicable to very rare cases where all appropriate clinical conditions are met, and even then, the results are not always optimal.

[0010] Furthermore, one-piece implants made of zirconia also exist. This material is not biologically derived, and one-piece implants are limited to the screw and abutment, with the crown treated as a separate component in both cases.

[0011] Regarding bone grafting, bone regeneration techniques are now widely available and include methods of increasing bone volume using the patient's own bone or by using highly biocompatible foreign bone.

[0012] In addition to the advantages of currently available dentures, the literature also reports data on the environmental impact of various materials used in the field of dentistry. These materials include yttria-stabilized tetragonal zirconia polycrystalline material (Y-TZP), pure titanium (cp-Ti), and alpha-beta titanium alloy (Ti6Al4V). Considering the ecological footprint of these materials, the amount of CO2 emitted during the manufacturing process of cp-Ti and Ti6Al4V alloys is estimated to reach 39-46 kg CO2 / kg, while the production of zirconia is estimated to reach 4.83 kg CO2 / kg. Furthermore, the total energy required for processing these materials should also be considered, ranging from 89.5 MJ / kg for zirconia to 685.5 MJ / kg for Ti6Al4V alloys.

[0013] Therefore, there is a strong need to obtain bio-dentures using technologies that can significantly reduce CO2 emissions and energy consumption, minimize material waste, optimize resource use, and thus contribute to environmental sustainability and a circular economy in the fields of medicine and dental prosthetics. [Overview of the project]

[0014] A first aspect of the present invention relates to a method for obtaining a denture including a crown and a root. The method preferably includes the following steps: a) A step of inducing undifferentiated cells to form an odontoblast-like phenotype, b) A step of inducing undifferentiated cells or keratinocytes to form an ameloblast-like phenotype, c) Steps for preparing bioink, d) Preferably using 3D bioprinting to print three-dimensionally the cells obtained in step a), the cells obtained in step b), and the bioink from step c), to obtain a denture, and e) A step of maintaining the dentures obtained in step d) in a culture medium.

[0015] Preferably, the undifferentiated cells are mesenchymal cells, and more preferably, the mesenchymal cells are isolated from human dental pulp.

[0016] Preferably, the keratinocytes are human-derived cells, originating from one donor, either an adult or a newborn.

[0017] In one embodiment, in step a), the undifferentiated cells are maintained in a culture medium containing a specific medium for mesenchymal cells based on their origin.

[0018] In one embodiment, in steps a) and b), the cells are maintained in the culture medium for at least 12 days, preferably at least 14 days.

[0019] In further embodiments, in steps a) and b), the undifferentiated cells or keratinocytes are maintained in the culture medium for at least 20 days, preferably at least 25 days, in order to completely differentiate them into odontoblasts and / or ameloblasts.

[0020] Preferably, the bio-ink is chitosan, acrylic group-modified chitosan, methacrylic group-modified chitosan, sugar-modified chitosan, alginate, acrylic group-modified alginate, methacrylic group-modified alginate, sugar-modified alginate, cellulose and its derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, polylactide, polyglycol, polycaprolactone, polyanhydride, polyamide, polyurethane, polyesteramide, polyether, polydioxanone, polyacetal, polyketal, polycarbonate, polyorthocarbonate, polyhol The bioink comprises biodegradable, bioabsorbable or bioerosive materials selected from the group consisting of sphazene, polyhydroxybutyrate, polyhydroxyvalerate, polyalkylene oxalate, polyalkylene succinate, polymeric acid, polyamino acid, polymethyl vinyl ether, chitin, various collagens, gelatin, methacrylate-modified gelatin, proteoglycans, chondroitin sulfate, silk protein, keratan sulfate, dermatan sulfate, glycosaminoglycans and copolymers, polypropylene glycol alginate, polyglycol and polylactic acid, ternary copolymers, and any combination thereof. Preferably, the bioink comprises at least one filler, preferably hydroxide apatite, nanohydroxyapatite, and / or bioglass.

[0021] Preferably, step d) of the three-dimensional printing includes a sub-step d1) of preparing at least one bio-ink for three-dimensional printing, wherein the bio-ink contains 1 million to 15 million ameloblasts and 1 million to 15 million odontoblasts per 1 mL of ink. More preferably, step d) of the three-dimensional printing is a 3D bioprinting step. Furthermore, the denture is preferably subjected to a decellularization step, preferably a decellularization step by hot air treatment.

[0022] A second aspect of the present invention relates to dentures obtained by the above method.

[0023] A third aspect of the present invention relates to a bioink suitable for three-dimensional (3D) printing, preferably 3D bioprinting, comprising at least one alginate, at least one gelatin, and optionally at least one filler. Here, the at least one alginate is selected from acrylate-modified alginate, methacrylate-modified alginate, and glycosyl-modified alginate. Also, the at least one filler is selected from hydroxyapatite, nano-hydroxyapatite, and / or bioglass. Preferably, the at least one alginate is methacrylate-modified alginate, and the at least one filler is hydroxyapatite.

Brief Description of Drawings

[0024] Figure 1 shows the results of analyzing the viability of MG63 cells embedded in the bioink according to the present invention using an alamar assay. The data are presented as mean ± standard deviation (n = 7).

[0025] Figure 2 shows the results of a live / dead test on MG63 cells embedded in the bioink according to the present invention. The images were acquired with a fluorescence microscope at 4× magnification. Viable cells were stained green and non-viable cells were stained red.

[0026] Figure 3 shows the results of analyzing the viability of MG63 cells embedded in the bioink containing hydroxyapatite according to the present invention using an alamar assay. The data are presented as mean ± standard deviation (n = 7).

[0027] Figure 4 shows the results of a biocompatibility test (LDH test) on MG63 cells of the bioink containing hydroxyapatite according to the present invention.

[0028] Figure 5 shows the results of a biocompatibility test (LDH test) performed on 3T3 cells to confirm the biocompatibility of the bioink without filler. Detailed Description of the Invention

[0029] The first aspect of the present invention relates to a method for obtaining a prosthetic tooth including a crown and a root.

[0030] In one embodiment, the method includes the following steps: a) inducing undifferentiated cells into an odontoblast-like phenotype; b) inducing undifferentiated cells or keratinocytes into an ameloblast-like phenotype; c) preparing a bioink; d) three-dimensionally printing the cells obtained in step a), the cells obtained in step b), and the bioink from step c), preferably using 3D bioprinting, to obtain a prosthetic tooth; and e) maintaining the prosthetic tooth obtained in step d) in a culture medium.

[0031] Steps a) and b) are, in other words, steps of inducing undifferentiated cells or keratinocytes into the phenotypes of odontoblasts and ameloblasts.

[0032] In one embodiment, steps a) and b) are steps of completely differentiating undifferentiated cells or keratinocytes into odontoblasts and ameloblasts. Preferably, the cultures of ameloblasts and odontoblasts are obtained by differentiating undifferentiated cells, and more preferably by differentiating mesenchymal cells or keratinocytes.

[0033] In one embodiment, the mesenchymal cells are derived from a donor tissue, and more preferably isolated from dental pulp.

[0034] The term "isolated" means that the mesenchymal cells of dental pulp are cells isolated from a natural source or its progeny, for example, derived from cell proliferation. The mesenchymal cells of dental pulp used are preferably derived from dental pulp tissue or donor tissue of a tooth. Preferably, the mesenchymal cells of dental pulp are primary cells that have not been transformed or immortalized. In a further embodiment, the mesenchymal cells of dental pulp are primary cells that have been transformed and / or immortalized.

[0035] In particular, the mesenchymal cells used in the present invention are derived from and extracted from dental pulp. Preferably, the adult mesenchymal cells of dental pulp are derived from the non-embryonic tissue of the dental pulp of a donor tooth, and preferably are autologous. The mesenchymal cells of dental pulp are derived from the dental pulp tissue or tissue differentiated into dental pulp tissue of any tooth. The mesenchymal cells of dental pulp used in the method of the present invention are preferably human dental pulp cells. These cells are extracted from the molars of patients. In one embodiment, after extraction, the teeth are left in a buffer containing at least one antibiotic, preferably a phosphate buffer (PBS) containing penicillin / streptomycin.

[0036] In one embodiment, dental pulp collected from a human is mechanically dissociated to obtain small pieces of dental pulp, which are then preferably washed with a balanced saline solution supplemented with an enzyme solution for digesting tooth bud tissue, and then undifferentiated stem cells are extracted.

[0037] Preferably, the enzyme solution, which also contains an antibiotic, comprises at least one enzyme selected from the group of collagenase or dispase. More preferably, the enzyme solution comprises collagenase and dispase. According to a preferred embodiment, the collagenase is of type I and is present in a concentration range of about 5 mg / 25 mL to about 100 mg / 25 mL. Preferably, the dispase is of type I and is present in a concentration range of about 3 mg / 25 mL to about 100 mg / 25 mL.

[0038] In one embodiment, pulp dissociation is performed by a mechanical process, for example, by mechanical stirring for 30 to 120 minutes at a temperature of 30°C to 40°C (preferably about 37°C). Preferably, at the end of the culture period, the enzyme solution is inhibited in a culture medium, for example, DMEM-HG medium containing fetal bovine serum (FBS), and the cells are collected after centrifugation. Preferably, the cell precipitate obtained by centrifugation is suspended in DMEM-HG medium supplemented with 100 μM ascorbic acid or a specific medium for mesenchymal stem cells.

[0039] In one embodiment, the keratinocytes are obtained from donor tissue, preferably from donor gingival tissue.

[0040] In one embodiment, the ameloblast culture and the odontoblast culture are maintained separately, i.e., they are not co-cultured.

[0041] In one embodiment, in steps a) and b), the cells are maintained in the culture medium for at least 12 days, more preferably at least 14 days. During this period, the undifferentiated cells are induced into an odontoblast-like phenotype in step a) and into an ameloblast-like phenotype in step b).

[0042] In one embodiment, cells obtained in steps a) and b), i.e., cells induced to have an odontoblast-like phenotype or an ameloblast-like phenotype, are mixed with a bioink from step c), printed with a 3D printer in step d), and then maintained in a culture medium to achieve complete differentiation into odontoblasts and ameloblasts.

[0043] In further embodiments, in steps a) and b), the cells are maintained in the culture medium for at least 20 days, more preferably at least 25 days, to achieve complete differentiation of undifferentiated or keratinocytes into odontoblasts or ameloblasts. In other words, steps a) and b) are then extended to achieve complete differentiation of undifferentiated or keratinocytes for mixing with the bioink and printing three-dimensionally.

[0044] In one embodiment, odontoblasts are cultured by methods well known to those skilled in the art. Preferably, pulp mesenchymal cells are isolated and cultured in a culture medium as described above.

[0045] Preferably, the mesenchymal cells are maintained in an adherent state (two-dimensional) using a standard medium. In one embodiment, MEM (Minimum Essential Medium) or standard DMEM (Dulbeccoo's Modified Eagle Medium), or fetal bovine serum (FBS) or calf serum (FCS) is used. The serum is preferably present at a concentration of 5-15% (by volume), more preferably 10% FBS. In another embodiment, the serum addition concentration can be gradually increased from about 5% to about 15% each time the culture medium is changed.

[0046] In one embodiment, the culture medium comprises at least one antibiotic, and essential amino acids and / or non-essential amino acids and / or a calcium source.

[0047] Preferably, to obtain a culture system for odontoblasts, mesenchymal cells are maintained in a culture medium containing growth factors and / or differentiation factors selected from ascorbic acid, β-glycerophosphate, and dexamethasone at a concentration of 0.1 to 500 μM.

[0048] In one embodiment, the culture medium for ameloblasts is obtained by methods of the art well known to those skilled in the art. Preferably, keratinocytes derived from adult or neonatal humans are cultured in a culture medium. For example, the keratinocytes are obtained by separating the epidermis from the dermis using at least one enzyme or a mixture of enzymes. The keratinocytes are preferably separated from the collected tissue by mechanical stirring and / or magnetic stirring, or by density gradient centrifugation, or by gravity-assisted cell sorting (GACS) based on passive filtration of a keratinocyte suspension through a special nylon filter, or by specific marker antibodies, or by enzymatic digestion using a combination of collagenase and dispase at a concentration of 0.1 to 10 mg / mL, more preferably 2 mg / mL. The enzymatic digestion is carried out at a temperature of 30 to 40°C (more preferably 37°C) for 2 hours. Subsequently, the tissue is further digested with trypsin-EDTA at a concentration of 0.01-1%, more preferably trypsin at a concentration of 0.05%, at a temperature of 30-40°C, more preferably 37°C, for 5 minutes.

[0049] Preferably, the keratinocytes are maintained in a standard medium in an adherent state on a layer of already differentiated mesenchymal cells, preferably a layer of pulp differentiated into odontoblasts. In one embodiment, MEM medium (Minimum Essential Medium) or DMEM medium (Dulbecc's Modified Eagle Medium) containing fetal bovine or calf serum (FBS or FCS) is used. The serum is preferably present at a concentration of 5-15% (by volume), more preferably at a concentration of 10% FBS. In another embodiment, the concentration of added serum can be gradually increased from about 5% to about 15% each time the culture medium is changed.

[0050] In one embodiment, the culture medium comprises at least one antibiotic, and essential amino acids and / or non-essential amino acids and / or a calcium source. Preferably, the calcium concentration is 0.1 mM to 5 mM, or the calcium concentration of a specific medium for keratinocytes is 0.1 mM to 5 mM.

[0051] Preferably, to obtain a culture system for ameloblasts, keratinocytes are maintained in a culture medium containing growth factors and / or differentiation factors, and / or preferably recombinant proteins selected from FGF-8, SHH, and BMP4 at a concentration of 10-100 μg / mL.

[0052] In one embodiment, the bio-ink comprises a material of natural or synthetic origin.

[0053] Preferably, the bio-ink is chitosan, acrylic group-modified chitosan, methacrylic group-modified chitosan, sugar-modified chitosan, alginate, acrylic group-modified alginate, methacrylic group-modified alginate, sugar-modified alginate, cellulose and its derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, polylactide, polyglycol, polycaprolactone, polyanhydride, polyamide, polyurethane, polyesteramide, polyether, polydioxanone, polyacetal, polyketal, polycarbonate, polyorthocarbonate, poly This material includes biodegradable, bioabsorbable, or bioerosive materials selected from riphosphazene, polyhydroxybutyrate, polyhydroxyvalerate, polyalkylene oxalate, polyalkylene succinate, polymeric acid, polyamino acid, polymethyl vinyl ether, chitin, various collagens, gelatin, methacrylate-modified gelatin, proteoglycans, chondroitin sulfate, silk protein, keratan sulfate, dermatan sulfate, glycosaminoglycans and copolymers, polypropylene glycol alginate, polyglycol and polylactic acid, terpolymers, and any combination thereof.

[0054] In preferred embodiments, the bioink is a chitosan-based, gelatin-based, or alginate-based bioink. Preferably, the bioink comprises at least one filler, preferably apatite hydroxide, nanoapatite hydroxide, and / or bioglass.

[0055] In one embodiment, step d) of the three-dimensional printing includes a sub-step d1) of preparing at least one bioink for three-dimensional printing. Preferably, the bioink further comprises ameloblasts and odontoblasts obtained in steps a) and b).

[0056] In a preferred embodiment of the present invention, the bioink contains 1 million to 15 million cells / mL, preferably about 10 million cells / mL of ameloblasts and 1 million to 15 million cells / mL, preferably about 10 million cells / mL of odontoblasts.

[0057] In one embodiment, step d) includes three-dimensional printing of the denture, preferably bioprinting, and more preferably using a 3D printer suitable for bioprinting.

[0058] Preferably, the printing of the denture is performed after an image of the human oral cavity or a part thereof, preferably the dental arch or a part thereof, in which the denture will subsequently be implanted, has been obtained.

[0059] In one embodiment, computed tomography (CT) or conical beam computed tomography (CBCT) is used to acquire at least one image of a desired area, preferably of the oral cavity.

[0060] In a further embodiment, an image of the oral cavity is obtained by a human tooth model combined with at least one acquired image.

[0061] Preferably, the acquired images are imported into implant treatment planning software.

[0062] Preferably, the denture is designed to harmonize with the surrounding teeth and to be functionally compatible.

[0063] In one embodiment, the obtained image is converted into a three-dimensional image using software well known to those skilled in the art. Preferably, the three-dimensional image is imported into computer-aided design (CAD) software to design the denture.

[0064] In one embodiment, denture data designed in CAD is imported into 3D printing software well known to those skilled in the art. The software is preferably "slicer" software. Preferably, this slicer software controls the printing settings of the denture, such as the thickness of the print layer, the filling density, the printing speed, the extrusion pressure, the temperature of the heated bed, and the volume of the denture.

[0065] In one embodiment, after step d), in step e), the resulting denture is maintained in the culture medium until enamel and dentin are formed on the denture. Preferably, the denture is maintained in the culture medium under conditions well known to those skilled in the art, more preferably in MEM medium (minimum essential medium) or standard DMEM medium (Dulbecc's modified Eagle medium) containing fetal bovine serum (FBS) or calf serum (FCS). The serum is preferably present at a concentration of 5-15% (by volume), more preferably at a concentration of 10% FBS. According to another embodiment, the concentration of serum added can be gradually increased from about 5% to about 15% each time the culture medium is changed.

[0066] In one embodiment, the culture medium comprises at least one antibiotic, and essential amino acids and / or non-essential amino acids and / or a calcium source.

[0067] Preferably, the denture is maintained in a culture medium at a temperature of 30-40°C, preferably about 37°C, for at least 5 days, more preferably at least 10 days.

[0068] At the end of step e) culturing the denture, the denture is decellularized by techniques well known to those skilled in the art. Preferably, in order to remove all cells, the denture is decellularized by hot air treatment and / or other methods of treating the denture.

[0069] In further embodiments, the method described in detail above is also suitable for preparing bone grafts for replacing dentition bone.

[0070] At the end of step e), the denture is preferably subjected to at least one quality control step and a step of cleaning and sterilizing the denture.

[0071] Remarkably, the applicant has developed a method for preparing dentures that offer many advantages over conventional commercially available dentures. First, by using biodegradable bio-ink, the environmental impact of the dentures can be significantly reduced compared to commercially available dentures made from synthetic materials.

[0072] For example, using dentures obtained by the above method can significantly reduce the number of procedures a patient needs to undergo to implant dentures, typically from 10 to 3 or 4 procedures. Furthermore, these dentures perfectly replicate the shape and color of the patient's natural teeth from an aesthetic standpoint, eliminate the risk of denture rejection or infection, and significantly reduce the risk of infiltration or lesions in the tooth roots, nerves, or maxillary sinuses surrounding the treatment site.

[0073] Furthermore, the implantation of the aforementioned denture can be performed simultaneously with tooth extraction, and can also be carried out even in the presence of an infection, as long as it is not acute.

[0074] A second aspect of the present invention relates to a denture obtained by the method described in detail above.

[0075] A third aspect of the present invention relates to a bio-ink for 3D printing. Preferably, the bio-ink is suitable for three-dimensional (3D) bioprinting. In one embodiment, the bio-ink comprises at least one alginate, at least one gelatin, and optionally at least one filler.

[0076] Preferably, the at least one alginate is selected from acrylic group-modified alginates, methacrylic group-modified alginates, and sugar group-modified alginates, and more preferably a methacrylic group-modified alginate.

[0077] In one embodiment, the bioink contains alginate in a weight ratio of 0.05 to 5%, preferably 0.5 to 2%.

[0078] In one embodiment, the bio-ink contains gelatin in a weight ratio of 5-25%, preferably 10-15%.

[0079] Preferably, the at least one filler is selected from hydroxide apatite, nanohydroxyapatite, and / or bioglass, and more preferably hydroxide apatite.

[0080] In one embodiment, the bio-ink contains a filler in a weight ratio of 0.5 to 5%, preferably 1 to 4%.

[0081] In one embodiment, the bio-ink contains a solvent, preferably water, in a weight ratio of 60-80%, preferably 5-75%.

[0082] In one embodiment, the bioink includes preservatives and / or pH stabilizers that are well known to those skilled in the art.

[0083] In a preferred embodiment of the present invention, the bioink contains 1 million to 15 million cells / mL of ameloblasts and 1 million to 15 million cells / mL of odontoblasts. Preferably, the bioink contains cells induced to have an odontoblast-like and / or ameloblast-like phenotype, as described in detail above.

[0084] Preferably, the bioink is prepared by a method that involves preparing a solution containing at least one alginate dissolved in a solvent, and preparing a second solution containing gelatin dissolved in at least one solvent. The two solutions are then mixed, preferably at a temperature of 35-40°C.

[0085] Optionally, a third solution containing at least one of the aforementioned fillers dissolved in a solvent is prepared and mixed with the alginate solution and the gelatin solution.

[0086] A fourth aspect of the present invention relates to a method for treating or preventing dental diseases or conditions related to damage or loss of at least one tooth.

[0087] In one embodiment, the method includes the step of implanting at least one denture, as described in detail above, into a person requiring such a denture. [Examples]

[0088] Preparation of bioink Solution A) Dissolve 50 mg of alginate methacrylate in 3.778 mL of water. 1M Hepes 0.05 mL 0.9M mannitol 0.825 mL Solution B) (Optional) Dissolve 200 mg of hydroxide apatite in 0.825 mL of water. 1M Hepes 0.01 mL 0.9M mannitol 0.165 mL The solution was sonicated before being added to other solutions. Solution C) Dissolve 2.5 g of gelatin in 7.95 mL of water. 1M Hepes 0.1 mL 0.9M mannitol 1.65 mL

[0089] The aforementioned solutions were mixed and stirred at approximately 40°C for approximately 12 hours. Finally, a crosslinking agent was added.

[0090] The resulting bio-ink was maintained at approximately 37°C and then mixed with cells. Once the ink was complete, it was transferred to a bioprinting syringe and irradiated with ultraviolet or visible light.

[0091] Tests on printability and cell viability The viability of MG63 cells was evaluated by Alamer testing for both bioink without solution B (i.e., without hydroxyapatite) and bioink containing hydroxyapatite at various time intervals (1 day, 3 days, 7 days, 14 days, 21 days, and 28 days). The results confirmed the biocompatibility of the bioink (Figures 1 and 3).

[0092] To verify the cell viability results obtained from the Alamer test, a "live / dead assay" was performed. This assay is based on the use of a cell membrane-permeable dye that allows staining of live cells (green) and a nuclear dye that does not permeate the cell membrane of live cells, thus allowing staining of dead cells (red). The DNA of the cells mixes with the dye and passes through the damage present in the cell membrane of dead cells (Figure 2). The results obtained allowed for verification of the results obtained from the Alamer viability test.

[0093] Furthermore, to confirm the biocompatibility of bioinks containing fillers, preferably such as hydroxyapatite, and bioinks without fillers, biocompatibility tests (LDH assays) were performed.

[0094] The biocompatibility of the bioinks under test was evaluated according to the guidelines of ISO 10993. The biocompatibility of the inks was evaluated by both release (500 μL of bioink incubated in 3 mL of DMEM HG medium at 37°C for 72 hours, followed by administration to a cell population) and contact (100 μL of bioink introduced into a cell population in 500 μL of DMEM HG medium for 72 hours).

[0095] As a positive control for toxicity testing, cells were treated with a solution of DMEM-HG medium supplemented with 0.1% Triton-X100. Conversely, DMEM-HG medium alone was used as a negative control for toxicity testing. This test was performed at 24 and 72 hours after treatment. The obtained cytotoxicity data were standardized based on toxicity values ​​obtained from cell lysis. The biocompatibility of the bioink of the present invention was also compared with the biocompatibility of commercially available bioinks for osteogenic differentiation. The biocompatibility of bioinks containing a filler (preferably hydroxyapatite) was evaluated using MG63 cells, a pilot cell line for osteogenic / odontogenic differentiation. The biocompatibility of bioinks without a filler was evaluated using 3T3 cells, a pilot cell line.

[0096] As shown in Figures 4 and 5, all bioink formulations exhibit significantly lower toxicity (LDH release rate) compared to the positive control (0.1% Triton) in toxicity tests. Furthermore, the bioinks of the present invention show toxicity signals comparable to commercially available bioinks for bone formation and differentiation, as well as release signals from cells not treated with the substance.

Claims

1. A method for obtaining a denture that includes the crown and root of the tooth, (a) A step of inducing undifferentiated cells to form an odontoblast-like phenotype, (b) A step of inducing undifferentiated cells or keratinocytes into an ameloblast-like phenotype, (c) Steps for preparing bioink, (d) Preferably using 3D bioprinting to print three-dimensionally the cells obtained in step a), the cells obtained in step b), and the bioink from step c), to obtain a denture, and (e) A step of maintaining the dentures obtained in step d) in a culture medium. Methods that include...

2. The method according to claim 1, wherein the undifferentiated cells are mesenchymal cells.

3. The method according to claim 2, wherein the mesenchymal cells are isolated from human dental pulp and the keratinocytes are isolated from gingival tissue.

4. The method according to any one of claims 1 to 3, wherein in step a) and step b), the cells are maintained in the culture medium for at least 12 days, preferably at least 14 days.

5. The method according to any one of claims 1 to 3, wherein in steps a) and b), the cells are maintained in the culture medium for at least 20 days, preferably at least 25 days, to completely differentiate the undifferentiated cells or keratinocytes into odontoblasts and / or ameloblasts.

6. The bio-ink may contain chitosan, acrylic-modified chitosan, methacrylic-modified chitosan, sugar-modified chitosan, alginates, acrylic-modified alginates, methacrylic-modified alginates, sugar-modified alginates, cellulose and its derivatives, such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, polylactide, polyglycol, polycaprolactone, polyanhydride, polyamide, polyurethane, polyesteramide, polyether, polydioxanone, polyacetal, polyketal, polycarbonate, polyorthocarbonate, polyphosphazene, polyhydroxy The method according to any one of claims 1 to 5, comprising a biodegradable, bioabsorbable or bioerosive material selected from the group consisting of butyrate esters, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, polymeric acids, polyamino acids, polymethyl vinyl ethers, chitin, various collagens, gelatin, methacrylate-modified gelatin, proteoglycans, chondroitin sulfate, silk proteins, keratan sulfate, dermatan sulfate, glycosaminoglycans and copolymers, polypropylene glycol alginate, polyglycols and polylactic acid, terpolymers, and any combination thereof.

7. The method according to any one of claims 1 to 6, wherein the bio-ink comprises at least one filler, preferably hydroxide apatite, nanohydroxyapatite, and / or bioglass.

8. The method according to any one of claims 1 to 7, wherein step d) of three-dimensional printing comprises at least one substep d1) of preparing a bio-ink for three-dimensional printing, the bio-ink comprising 1 million to 15 million cells / mL of ameloblasts and 1 million to 15 million cells / mL of odontoblasts.

9. The method according to any one of claims 1 to 8, wherein the denture preferably undergoes at least one decellularization step by hot air treatment.

10. A denture obtained by the method described in any one of claims 1 to 9.

11. A bio-ink suitable for 3D bioprinting, preferably comprising at least one alginate, at least one gelatin, and optionally at least one filler, wherein the at least one alginate is selected from acrylic group-modified alginate, methacrylic group-modified alginate, and sugar group-modified alginate, and the at least one filler is selected from hydroxyapatite, nanohydroxyapatite, bioglass, and combinations thereof.

12. The bioink according to claim 11, wherein the at least one alginate is a methacrylic group-modified alginate, and the at least one filler is hydroxyapatite.

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