Compositions and methods for dental tissue engineering

Abstract

A method for regenerating a tooth comprising periodontal ligament, cementum, pulp, dentin, and enamel tissues. The method comprises forming a composition of dentin matrix proteins, forming a mixture, and implanting the mixture into a socket site of jawbone for a predetermined time duration. The mixture comprises a plurality of passaged dental pulp stem cells (DPSCs) with a concentration between 103 and 108 (cells / mL), the composition of dentin matrix proteins with a concentration between 2% and 10% (w / v), and a calcium hydroxide paste comprising calcium hydroxide with a concentration between 20% (w / w) and 40% (w / w).

Description

COMPOSITIONS AND METHODS FOR DENTAL TISSUE ENGINEERINGCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Patent Application Ser. No. 63 / 534,124, filed on August 23, 2023, entitled “STEM CELL DIFFERENTIATION COMPONENT IN TISSUE ENGINEERING FOR TOOTH REGENERATION” which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure is generally related to an exemplary method for dental tissue engineering, and more particularly to an exemplary method for preparing exemplary compositions for dental tissue engineering.BACKGROUND

[0003] Dental caries and tooth loss constitute common clinical pathologies within the realm of dentistry. As global life expectancy continues to rise, there is a growing need for the regeneration of dental tissues and entire teeth. Conventional or gold-standard dental procedures encompass restorations, root canal therapy, tooth replacement using diverse prostheses, and implantation. Each of these treatments presents its unique benefits and drawbacks. Consequently, conventional procedures may not entirely replicate optimal functionality and aesthetic appeal of a natural tooth.

[0004] Endodontic procedures, specifically root canal therapy, are primary treatments for dental infections that may penetrate a pulp. This process may involve extraction of natural tooth pulp, which may be subsequently replaced with synthetic materials to fill root canal. Despite being a standard practice in modem dentistry, endodontic or root canal treatment is not without its shortcomings. These limitations may adversely impact a patient's quality of life. Thereby,there is need for creation of novel therapeutic approaches to facilitate dental tissue repair and regeneration and to address dental diseases.SUMMARY

[0005] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0006] One or more exemplary embodiments describe an exemplary method for regenerating an exemplary tooth comprising exemplary periodontal ligament, cementum, pulp, dentin, and enamel tissues. In an exemplary embodiment, an exemplary method may comprise forming an exemplary composition of dentin matrix proteins. In an exemplary embodiment, forming an exemplary composition of dentin matrix proteins may include: obtaining an exemplary dentin tissue by removing an exemplary enamel tissue, an exemplary cement tissue, and an exemplary pulp tissue from at least one exemplary tooth; and demineralizing an exemplary dentin tissue of at least one exemplary tooth by placing an exemplary dentin tissue in an exemplary acid solution with an exemplary pH level between 2 and 3.5 for a time duration between 10 days and 20 days.

[0007] In an exemplary embodiment, an exemplary method may further comprise forming an exemplary mixture, and implanting an exemplary mixture into an exemplary socket site of jawbone for an exemplary time duration between 20 days and 60 days. In an exemplary embodiment, an exemplary mixture may comprise a plurality of exemplary passaged dental pulp stem cells (DPSCs) with a concentration between 103and 108(cells / mL), an exemplarycomposition of dentin matrix proteins with a concentration between 2% and 10% (w / v), and an exemplary calcium hydroxide paste. In an exemplary embodiment, an exemplary calcium hydroxide paste may comprise calcium hydroxide with a concentration between 20% (w / w) and 40% (w / w). In an exemplary embodiment, an exemplary calcium hydroxide paste may further comprise iodoform with a concentration between 30% (w / w) and 45% (w / w), and an exemplary silicone oil with a concentration between 10% (w / w) and 30% (w / w).

[0008] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential / key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which an exemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Exemplary embodiments will now be described by way of example in association with the accompanying drawings in which:

[0010] FIG. 1 illustrates a flowchart of an exemplary method for regenerating an exemplary tooth comprising exemplary periodontal ligament, cementum, pulp, dentin, and enamel tissues, consistent with one or more exemplary embodiments of the present disclosure;

[0011] FIG. 2 illustrates an exemplary chart of cell viability analysis of human dental pulp stem cells (hDPSCs) after incubation with dentin matrix proteins and demineralized freeze-dried bone allograft (DFDBA), consistent with one or more exemplary embodiments of the present disclosure;

[0012] FIG. 3 illustrates exemplary microscopic images of hDPSCs analyzed by immunocytochemical (ICC) staining with dentin sialopho sphoprotein (DSPP) antibodies, consistent with one or more exemplary embodiments of the present disclosure;

[0013] FIG. 4 illustrates an exemplary bar chart representing percentage of DSPP marker expression in pulp tissue mesenchymal stem cells of an exemplary group (i) of hDPSCs on days 3, 7, and 10 in comparison to an exemplary negative control group (i.e., group (iii) of hDPSCs), consistent with one or more exemplary embodiments of the present disclosure;

[0014] FIG. 5 illustrates exemplary ICC images representing bone sialoprotein (BSP) and 4',6-diamidino-2-phenylindole (DAPI) expression in hDPSCs of group (ii), consistent with one or more exemplary embodiments of the present disclosure;

[0015] FIG. 6 illustrates an exemplary bar chart representing percentage of BSP marker expression in pulp tissue mesenchymal stem cells of exemplary group (ii) of hDPSCs on days 3, 7, and 10 in comparison to an exemplary negative control group (i.e., group (iii) of hDPSCs), consistent with one or more exemplary embodiments of the present disclosure;

[0016] FIG. 7 illustrates an exemplary image of excised rabbit maxilla on 30th day of implantation, showcasing an intact right maxillary incisor and a region on left where a tooth is extracted, consistent with one or more exemplary embodiment of the present disclosure;

[0017] FIG. 8 illustrates an exemplary three-dimensional cone beam computed tomography (CBCT) image of excised rabbit maxillary bone, consistent with one or more exemplary embodiment of the present disclosure;

[0018] FIG. 9 illustrates exemplary histological images showcasing regenerated tooth bud (subject of study) and an exemplary normal tooth (control group), consistent with one or more exemplary embodiments of the present disclosure;

[0019] FIG. 10A illustrates exemplary immunohistochemistry images of tooth bud(subject of study) and an exemplary normal tooth (control group) using anti-DSPP antibodies, consistent with one or more exemplary embodiments of the present disclosure; and

[0020] FIG. 10B illustrates exemplary immunohistochemistry images of tooth bud (subject of study) and an exemplary normal tooth (control group) using anti-amelogenin antibodies, consistent with one or more exemplary embodiments of the present disclosure.DETAILED DESCRIPTION

[0021] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0022] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0023] Tissue engineering may offer potential for full biological restoration of dental and pulpal tissues in the field of regenerative dentistry. Dental pulp stem cells (DPSCs) are a heterogeneous population of cells derived from human permanent third molar pulp. DPSCs may possess similar properties to those of mesenchymal stem cells (MSCs), with the major distinction being that DPSCs may have a capacity to differentiate into odontoblast-like cells, which may be capable of forming dentin. DPSCs may present a distinct advantage over mesenchymal bone marrow stem cells due to their greater association with dental tissues. Consequently, DPSCs may be advantageous for employment in the realm of dental engineering.

[0024] Disclosed herein is an exemplary method for dental tissue engineering. In particular, one or more exemplary embodiments may describe an exemplary cost-effective method for regenerating an exemplary tooth comprising periodontal ligament, cementum, pulp, dentin, and enamel tissues. In an exemplary embodiment, an exemplary construct comprising exemplary DPSCs and exemplary dentin matrix proteins may be used for regeneration of an exemplary tooth in an exemplary socket site of jawbone. It may be appreciated that exemplary embodiments pertaining to exemplary compositions, exemplary medical treatment methods, and exemplary kits related to an exemplary method described in one or more exemplary embodiments may also fall into the context of the present disclosure.

[0025] FIG. 1 illustrates a flowchart of exemplary method 100 for regenerating an exemplary tooth comprising exemplary periodontal ligament, cementum, pulp, dentin, and enamel tissues, consistent with one or more exemplary embodiments of the present disclosure. In an exemplary embodiment, exemplary method 100 may include: forming an exemplary composition of dentin matrix proteins (step 102); forming an exemplary mixture comprising a plurality of exemplary passaged DPSCs, an exemplary composition of dentin matrix proteins,and an exemplary calcium hydroxide paste (step 104); and implanting an exemplary mixture into an exemplary socket site of jawbone for a predetermined time duration (step 106).

[0026] In further detail with respect to step 102, step 102 may include forming an exemplary composition of dentin matrix proteins. In an exemplary embodiment, forming an exemplary composition of dentin matrix proteins may comprise an exemplary process including: i) obtaining an exemplary dentin tissue by removing exemplary enamel, cement, and pulp tissues from one or more exemplary healthy teeth; and ii) demineralizing an exemplary dentin tissue of one or more exemplary healthy teeth by placing an exemplary dentin tissue in an exemplary acid solution with a pH level between about 2 and 3.5 for a time duration between about 10 and 20 days. In an exemplary embodiment, obtaining an exemplary dentin tissue by removing exemplary enamel, cement, and pulp tissues from one or more exemplary healthy teeth may include removing exemplary enamel and cement tissues from one or more exemplary healthy teeth using an exemplary diamond fissure followed by water spraying exemplary remained dental tissues in an exemplary laboratory container. In an exemplary embodiment, an exemplary laboratory container may include, but is not limited to, beakers, tins, flasks, bottles, buckets, basins, bowls, vials, tubes, barrels, cannisters, etc. In an exemplary embodiment, obtaining an exemplary dentin tissue by removing exemplary enamel, cement, and pulp tissues from one or more exemplary healthy teeth may further include removing an exemplary pulp tissue from exemplary remained dental tissues in an exemplary laboratory container (e.g., a bowl or a beaker). In an exemplary embodiment, obtaining an exemplary dentin tissue may further include forming one or more exemplary dentin blocks by cutting an exemplary obtained dentin tissue into exemplary blocks (with a size of about 1x1 mm) in an exemplary laboratory container (e.g., a bowl or a beaker). In an exemplary embodiment, “healthy tooth” may refer to an exemplary tooth that may not be affected by periodontaldisease, caries, or any other oral diseases, and may not contain restorations or fillings (such as amalgam or composite).

[0027] In an exemplary embodiment, demineralizing an exemplary dentin tissue of one or more exemplary healthy teeth by placing an exemplary dentin tissue in an exemplary acid solution with a pH level between about 2 and 3.5 for a time duration between about 10 and 20 days may include placing one or more exemplary dentin blocks (e.g., 1x1 mm in size) in an exemplary laboratory container (e.g., bowel, beaker, etc.) containing an exemplary acid solution with a pH level between about 2 and 3.5 for a time duration between about 12 and 16 days. In an exemplary embodiment, placing one or more exemplary dentin blocks (e.g., 1x1 mm in size) in an exemplary acid solution with a pH level between about 2 and 3.5 for a time duration between about 10 and 20 days may include placing one or more exemplary dentin blocks (e.g., 1x1 mm in size) in an exemplary laboratory container (e.g., bowel, beaker, etc.) containing an exemplary acetic acid solution with a concentration between about 3.5% (v / v) and 7.5% (v / v) for a time duration between about 10 days and 20 days. In an exemplary embodiment, placing one or more exemplary dentin blocks (e.g., 1x1 mm in size) in an exemplary laboratory container (e.g., bowel, beaker, etc.) containing an exemplary acetic acid solution with a concentration between about 3.5% (v / v) and 7.5% (v / v) for a time duration between about 10 days and 20 days may include placing one or more exemplary dentin blocks (e.g., 1x1 mm in size) in an exemplary laboratory container (e.g., bowel, beaker, etc.) containing an exemplary acetic acid solution with a concentration of about 5% (v / v) for a time duration of about 14 days. In an exemplary embodiment, forming about 100 mL of an exemplary acetic acid solution with a concentration of about 5% (v / v) may include: transferring about 5 mL of acetic acid into an exemplary laboratory container, such as volumetric flask or graduated cylinder, using an exemplary pipette or burette; adding water (e.g., distilled water and / or double-distilled water) to an exemplary laboratory container (e.g., volumetric flask orgraduated cylinder) containing acetic acid until reaching a final volume of about 100 mL; gently swirling an exemplary laboratory container containing an exemplary mixture of acetic acid and water (e.g., distilled water and / or double-distilled water). “Double distilled water” or “DDW” may refer to water obtained after two distillation processes. Double distilled water may have very low organic matter, inorganic salts, volatile impurities, soluble gases, and microorganisms.

[0028] In further detail with respect to step 104, step 104 may include forming an exemplary mixture comprising a plurality of exemplary passaged DPSCs, an exemplary composition of dentin matrix proteins, and an exemplary calcium hydroxide paste. “Passaging” may refer to an exemplary technique that may provide for harvesting cells from a culture and transferring them to one or more vessels containing an exemplary fresh growth medium to keep cells alive and growing for extended periods of time. In an exemplary embodiment, forming an exemplary mixture comprising a plurality of exemplary passaged DPSCs, an exemplary composition of dentin matrix proteins, and an exemplary calcium hydroxide paste may include adding a plurality of exemplary fourth-passage DPSCs with a final concentration between about 103and 108(cells / mL) with respect to final volume of an exemplary mixture (e.g., using a sampler, graduated cylinder / tube, etc.), an exemplary composition of dentin matrix proteins (e.g., in powder form) with a final concentration between 2% and 10% (w / v) with respect to final volume of an exemplary mixture (e.g., using a spatula), and an exemplary calcium hydroxide paste (e.g., 3-7 mL) to an exemplary laboratory container, such as a beaker or bowel, while mixing (e.g., using a spatula, stirring rod, etc.) at a temperature level of about 22-25 °C (i.e., room temperature). In an exemplary embodiment, exemplary fourth-passage DPSCs may refer to exemplary DPSCs that may have undergone four consecutive rounds of sub-culturing or passaging.

[0029] In further detail with respect to step 104, in an exemplary embodiment, an exemplary calcium hydroxide paste may refer to an exemplary alkaline substance with a pH of about 12.5-12.8 which may give it antibacterial properties, making it effective in treating various dental issues. An exemplary calcium hydroxide paste may be used in endodontics for pulp capping, root canal treatments and as an intracanal medicament due to its capability to stimulate hard tissue formation and its excellent antimicrobial activity. An exemplary calcium hydroxide paste may promote healing and regeneration within a tooth structure by facilitating formation of mineralized tissues. In an exemplary embodiment, an exemplary calcium hydroxide paste may comprise calcium hydroxide (Ca(OH)2) with a concentration of about 20- 40% (w / w). In an exemplary embodiment, an exemplary calcium hydroxide paste may further comprise iodoform with a concentration of about 30-45% (w / w), and silicone oil with a concentration of about 10-30% (w / w). In an exemplary embodiment, an exemplary calcium hydroxide paste may comprise calcium hydroxide (Ca(OH)2) with a concentration of about 30.3% (w / w), iodoform with a concentration of about 40.4% (w / w), and silicone oil with a concentration of about 22.4% (w / w). In an exemplary embodiment, forming 100 mL of an exemplary calcium hydroxide paste (containing 30.3% (w / w) calcium hydroxide, 40.4% (w / w) iodoform, and 22.4% (w / w) silicone oil) may include: i) adding about 30.3 g calcium hydroxide powder and about 40.4 g iodoform powder (e.g., using a spatula) to an exemplary laboratory container (e.g., a beaker); ii) adding an exemplary volume (e.g., 20 mL) of water (e.g., distilled water or DDW) to an exemplary mixture of calcium hydroxide and iodoform powders followed by stirring using a magnetic stirrer until a uniform mixture is achieved; iii) adding dropwise about 23.1 mL of silicone oil (with a density of about 0.97 g / mL) to an exemplary obtained uniform mixture while stirring using a magnetic stirrer; iv) adjusting final volume of an exemplary mixture formed in step (iii) with water (e.g., distilled water or DDW) up to 100 mLwhile stirring (using a magnetic stirrer) until an exemplary smooth paste without any visible lumps or clumps is formed.

[0030] In further detail with respect to step 106, step 106 may include implanting an exemplary mixture into an exemplary socket site of jawbone for a predetermined time duration. In an exemplary embodiment, implanting an exemplary mixture into an exemplary socket site of jawbone for a predetermined time duration may include implanting a predetermined volume of an exemplary mixture (as set forth in step 104) into an exemplary socket site of jawbone for a time duration between 20 days and 60 days using an exemplary delivery system (e.g., a needle tube delivery system). “Socket site” refers to a location or cavity in jawbone where a tooth was previously located. In particular, socket site may refer to a hole left in jawbone after tooth extraction or loss. In an exemplary embodiment, an exemplary mixture may be implanted in an exemplary socket site of a subject’s jawbone. In an exemplary embodiment, “subject” may refer to an exemplary mammal, such as human, rabbit, rodent (e.g., rat, mouse, hamster, guinea pig, gerbil), ferret, livestock (e.g., goats, horses, pigs, sheep, cows), camel, lama, and monkey. In an exemplary embodiment, an exemplary predetermined volume of an exemplary mixture may be determined with respect to size and shape of an exemplary socket site of an exemplary subject’s jawbone. For example, in an exemplary embodiment, implanting an exemplary mixture into an exemplary socket site of jawbone for a predetermined time duration may include implanting about 5 mL of an exemplary mixture into an exemplary socket site of a rabbit’s jawbone for a time duration between 25 days and 35 days.EXAMPLES

[0031] Hereinafter, one or more exemplary embodiments will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure.Example 1: Preparation of Demineralized Dentin Matrix Proteins

[0032] In this example, exemplary dentin matrix proteins were extracted from one or more exemplary healthy teeth. In an exemplary embodiment, exemplary teeth were first disinfected using sodium hypochlorite solution. Enamel and cement tissues were then removed with an exemplary diamond fissure bur (008) and water spray, while an exemplary pulp tissue was removed with an excavator. An exemplary dentin tissue was then cut into 1x1 mm blocks and placed in 5% (v / v) acetic acid for 14 days until it was fully demineralized and only an exemplary dentin protein matrix remained. A freeze dryer device was used to dry and powderize exemplary dentin protein matrix while preserving its proteins. An exemplary freeze- drying process involved freezing an exemplary dentin blocks followed by a sublimation process which converted water from solid to gas, maintaining exemplary chemical and physical characteristics of an exemplary dentin protein matrix.Example 2: Differentiation of Human Dental Pulp Stem Cells to Odontoblast Cells

[0033] In this example, human dental pulp stem cells (hDPSCs) were differentiated to odontoblast cells using dentin matrix proteins. In an exemplary embodiment, fourth subculture of hDPSCs (e.g., an exemplary commercial cell line of hDPSCs) may be utilized to induce stem cell differentiation. During an exemplary initial subculture, exemplary hDPSCs were seeded in a 25-mL flask containing alpha minimum essential medium (alpha-MEM) and embryonic stem cell-fetal calf serum (FCS), then incubated at a temperature level of about 37 °C, a humidity level of about 95%, and a CO2 concentration of about 5%. Cell culture media was replaced every two to three days over roughly a two-week period until an appropriate density was achieved.

[0034] Upon achieving full flask floor coverage, cell subculturing was carried out with Trypsin / Ethylenediaminetetraacetic acid (EDTA). Following four consecutive subcultures and subsequent purification processes, cells were detached from flask floor and quantified usingtrypan blue staining and nicobar slide assessment. Subsequently, each group was provided with a 25-mL flask containing 2 x 105cells for further experimentation. Subsequently, exemplary fourth-passage cells were introduced to an exemplary medium comprising of 50% ES-FCS, 10% dimethyl sulfoxide (DMSO), and 40% mesenchymal stem cell culture medium. Exemplary passaged hDPSCs were then relocated to a nitrogen tank for preservation.

[0035] Differentiation of hDPSCs into osteoblasts and odontoblasts was initiated by culturing hDPSCs (at a density of about 100 cells / cm2) in exemplary six-well plates. Following the attainment of 70-80% confluency on the flask floor, an exemplary culture medium was substituted with dulbecco's modified eagle medium (DMEM), which comprised 15% ES-FCS, 50 pg / mL ascorbic acid-2 phosphate, dexamethasone at a concentration of aboutlO-8M, 10 mM of glycerol phosphate, and dentin matrix proteins (0.7 mL from a 100 pg / mL solution of dentin matrix proteins). Exemplary hDPSCs were maintained in this culture for a period of ten days, during which an exemplary differentiation medium was refreshed every three days. Differentiation of exemplary hDPSCs into osteoblasts and odontoblasts was assessed via light microscopy following immunocytochemical (ICC) staining. In this example, three exemplary culture groups were studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and ICC assays.

[0036] In an exemplary embodiment, exemplary three culture groups include: i) flasks with colonies of hDPSCs amounting to 200,000 cells that were subjected to treatment with dentin matrix proteins (0.7 mL from a 100 pg / mL solution of dentin matrix proteins); and ii) flasks containing hDPSCs (200,000 cells) treated with about 0.7 mL of demineralized freeze- dried bone allograft (DFDBA) with a concentration of about 100 pg / mL. DFDBA is an allograft comprising demineralized bone matrix (DBM) which is achieved through an exemplary process of demineralization applied to a previously freeze-dried bone allograft(FDBA). Exemplary three culture groups may further include group (iii), i.e., negative controlgroup, that may consist of exemplary flasks housing only hDPSCs colonies (200,000 cells), without any additional treatment. Following intervals of 3, 7 and 10 days respectively, exemplary purified cells were isolated. Subsequently, cell vitality percentages were determined using an exemplary MTT assay method. Additionally, cellular differentiation and exemplary expression markers for osteoblast and odontoblast cells were assessed via an exemplary ICC method.

[0037] The viability of cells in each exemplary group was assessed after a 24-hour treatment period using an exemplary MTT assay. An exemplary MTT procedure involved initially discarding an exemplary culture medium from plate surfaces and subsequently washing them twice with phosphate-buffered saline (PBS) buffer. Following this, each well was supplemented with 100 pL of serum-free culture medium and 10 pL of MTT solution, which was then incubated for two hours at conditions of 37 °C, 5% CO2, and 90% relative humidity over a span of four hours. Post-incubation, each well received an addition of 100 pL isopropanol acid at a concentration of 0.4%, followed by an incubation period lasting 10 minutes. The optical density (OD) for each well was then measured using an exemplary enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 492 nm. Furthermore, to calculate the percentage of viable cells in each group, a standard curve was plotted and analyzed statistically (p-value < 0.05). FIG. 2 illustrates chart 200 of cell viability analysis of hDPSCs after incubation with dentin matrix proteins and DFDB A, consistent with one or more exemplary embodiments of the present disclosure. In further detail with respect to chart 200, vitality of cells in groups (i), (ii), and (iii) exhibited a mean ± standard deviation (SD) of 0.13 ± 0.03, 0.12 ± 0.01, and 0.10 ± 0.02 respectively. Statistical analysis revealed no significant difference among these groups (p-value = 0.465). Consequently, MTT assay demonstrated that all three groups had a similar vitality rate.

[0038] Quality assessment of exemplary developed dentin and bone tissues, constructed from differentiation of odontoblasts and osteoblasts derived from dental pulp stem cells, was conducted utilizing ICC staining (using anti-dentin sialophosphoprotein (DSPP) and anti-bone sialoprotein (BSP) antibodies). FIG. 3 illustrates microscopic images 300 of hDPSCs analyzed by ICC staining with DSPP antibodies, consistent with one or more exemplary embodiments of the present disclosure. As illustrated in microscopic images 300, DSPP marker expression was analyzed in culture group (i) (exemplary hDPSCs treated with dentin matrix proteins) at three distinct time intervals — day 3, day 7, and day 10. A comparative analysis was also conducted with a control group, which solely comprised of hDPSCs. Microscopic images 300 demonstrate elevated expression of DSPP on seventh and tenth days. This observation may be potentially attributed to extended exposure of hDPSCs to dentin matrix proteins.

[0039] FIG. 4 illustrates bar chart 400 representing percentage of DSPP marker expression in pulp tissue mesenchymal stem cells of an exemplary group (i) of hDPSCs on days 3, 7, and 10 in comparison to an exemplary negative control group (i.e., group (iii) of hDPSCs), consistent with one or more exemplary embodiments of the present disclosure. In further detail with respect to bar chart 400, average percentage of DSPP marker expression in group (i), as well as an exemplary negative control across days 3, 7, and 10, demonstrated statistical significance (p-value < 0.001). Consequently, when conducting pairwise comparisons among group (i) and group (iii) (i.e., negative control), the average expression percentage of DSPP marker exhibited a significant deviation in every instance (p-value < 0.001 in all cases, i.e., 3, 7, and 10 days). FIG. 5 illustrates ICC images 500 representing BSP and 4',6-diamidino-2-phenylindole (DAPI) expression in hDPSCs of group (ii), consistent with one or more exemplary embodiments of the present disclosure. As illustrated in ICC images 500, expression level of BSP marker in group (ii) of hDPSCs increased on days 3, 7, and 10. A similar increase was observed within an exemplary negative control group (i.e., group (iii)) ondays 7 and 10. The heightened expression of BSP marker may be attributed to an extended exposure of hDPSCs to DFDBA. FIG. 6 illustrates bar chart 600 representing percentage of BSP marker expression in pulp tissue mesenchymal stem cells of exemplary group (ii) of hDPSCs on days 3, 7, and 10 in comparison to an exemplary negative control group (i.e., group (iii) of hDPSCs), consistent with one or more exemplary embodiments of the present disclosure. In further detail with respect to bar chart 600, the average percentage of BSP marker expression in group (ii) and negative control on days 3, 7, and 10 showed a statistically significant difference (p-value < 0.0001). Consequently, when conducting pairwise comparisons among group (ii) and group (iii) (i.e., negative control), the average percentage of BSP marker expression displayed a substantial disparity in all instances (p-value < 0.001 in each case, i.e., 3, 7, and 10 days).Example 3: Tooth Regeneration in Rabbits

[0040] In this example, an exemplary construct comprising dental pulp stem cells (DPSCs), dentin matrix proteins, and an exemplary scaffold (e.g., calcium hydroxide paste) was used to regenerate tooth (including periodontal ligament, cementum, pulp, dentin, and enamel) in rabbits based on a method similar to exemplary method 100. In an exemplary embodiment, an exemplary albino rabbit, aged between one and two years and weighing approximately 2.5 kg, was subjected to anesthesia via an intra peritoneal (IP) injection consisting of 60 mg / kg ketamine and 0.12 xylazine. Subsequently, the left maxillary central tooth of an exemplary anesthetized rabbit was extracted. The resulting socket was then filled with an exemplary composition comprising of DPSCs at fourth passage (about 5 x 106DPSCs), dentin matrix proteins (about 0.2 g), and an exemplary calcium hydroxide paste (about 5 mL) that may comprise about 40.4% (w / w) iodoform, 30.3% (w / w) calcium hydroxide, and 22.4%(w / w) silicone oil.

[0041] On thirtieth day, an exemplary subject rabbit was humanely euthanized using carbon dioxide gas, after which rabbit’s maxilla was meticulously detached with a chisel and mallet. FIG. 7 illustrates image 700 of excised rabbit maxilla on 30th day of implantation, showcasing an intact right maxillary incisor and a region on left where a tooth is extracted, consistent with one or more exemplary embodiment of the present disclosure, subsequently, an exemplary specimen (i.e., an exemplary excised tooth) was inspected by radiology for obtaining a cone beam computed tomography (CBCT) image and to conduct a histopathological analysis. FIG. 8 illustrates three-dimensional CBCT image 800 of excised rabbit maxillary bone, consistent with one or more exemplary embodiment of the present disclosure. In further detail with respect to CBCT image 800, an ovoid, opaque structure was found centrally in upper left socket of rabbit’s maxilla. This structure exhibited a density comparable to enamel of an undamaged right incisor, measuring approximately 1 cm in length and 4 mm in diameter. This observation suggested the presence of what appeared to be a newly developed tooth (bud), exhibiting noticeably higher density than adjacent bone tissue.

[0042] FIG. 9 illustrates histological images 900 showcasing regenerated tooth bud (subject of study) and an exemplary normal tooth (control group), consistent with one or more exemplary embodiments of the present disclosure. In further detail with respect to histological images 900 (obtained from hematoxylin and eosin (H&E) staining), healthy right tooth served as a control (labeled as 902), while an exemplary treated socket was examined as a case study (labeled as 904). No signs of inflammation were observed in an exemplary control tooth. Furthermore, all structures including arterioles, nerves, pulp connective tissue, enamel space, and tubular dentin were found to be in their normal state. Cementum and periodontal ligament were distinctly visible between an exemplary tooth and adjacent alveolar bone, with an exemplary tooth maintaining its regular morphological shape. An exemplary case study of left socket displayed a post-Bell stage tooth bud. “Bell stage” may refer to a stage in which dentallamina undergoes a process of disintegration, thereby preparing itself for subsequent formation of dental hard tissues. Furthermore, as illustrated in histological images 900, pulp tissue was akin to that of a normal tooth with arterioles and nerves being distinctly observable. Enamel tissue manifested as a space in its natural state while dentin texture presented as regular tubular dentin. Additionally, an unbroken odontoblastic layer was clearly discernible.

[0043] With further reference to FIG. 9, histological images 900 revealed the presence of cementum and periodontal ligament interposed between an exemplary tooth bud and an exemplary encasing alveolar bone, with residual follicular fragments discernible in a superior region. Notably, an inflammation was identified in upper segment of an exemplary bud's crown. This inflammatory response was characterized as chronic and moderate, exhibiting a sparse population of neutrophils but a higher concentration of lymphocytes. Overall, morphological characteristics of an exemplary tooth bud appeared typical and exhibited a developmental stage beyond that of Bell stage.

[0044] FIG. 10A illustrates immunohistochemistry images 1000 of tooth bud (subject of study) and an exemplary normal tooth (control group) using anti-DSPP antibodies, consistent with one or more exemplary embodiments of the present disclosure. FIG. 10B illustrates immunohistochemistry images 1002 of tooth bud (subject of study) and an exemplary normal tooth (control group) using anti-amelogenin antibodies, consistent with one or more exemplary embodiments of the present disclosure. In further detail with respect to immunohistochemistry images 1000 and 1002, immunohistochemical analysis revealed a substantial presence of enamel tissue, as indicated by detection of amelogenin marker, and a thick dentin layer as evidenced by the presence of DSPP marker in an exemplary control group. However, despite the presence of these positive markers, there was a noted reduction in the thickness of both dentin and enamel layers in an exemplary test case (i.e., tooth bud).

[0045] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0046] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0047] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0048] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0049] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been setforth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0050] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0051] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise been set forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0052] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. A method for regenerating a tooth comprising periodontal ligament, cementum, pulp, dentin, and enamel tissues, the method comprising: forming a composition of dentin matrix proteins comprising: obtaining a dentin tissue by removing an enamel tissue, a cement tissue, and a pulp tissue from at least one tooth; and demineralizing the dentin tissue of the at least one tooth by placing the dentin tissue in an acetic acid solution with a concentration between 3.5% (v / v) and 7.5% (v / v) for a time duration between 10 days and 20 days; forming a mixture comprising a plurality of passaged dental pulp stem cells (DPSCs) with a concentration between 103and 108(cells / mL), the composition of dentin matrix proteins with a concentration between 2% (w / v) and 10% (w / v), and a calcium hydroxide paste, wherein the calcium hydroxide paste comprises calcium hydroxide with a concentration between 20% (w / w) and 40% (w / w), iodoform with a concentration between 30% (w / w) and 45% (w / w), and silicone oil with a concentration between 10% (w / w) and 30% (w / w); and implanting the mixture into a socket site of jawbone for a time duration between 20 days and 60 days.

2. A method for regenerating a tooth comprising periodontal ligament, cementum, pulp, dentin, and enamel tissues, the method comprising: forming a composition of dentin matrix proteins comprising: obtaining a dentin tissue by removing an enamel tissue, a cement tissue, and a pulp tissue from at least one tooth; anddemineralizing the dentin tissue of the at least one tooth by placing the dentin tissue in an acid solution with a pH level between 2 and 3.5 for a time duration between 10 days and 20 days; forming a mixture comprising a plurality of passaged dental pulp stem cells (DPSCs) with a concentration between 103and 108(cells / mL), the composition of dentin matrix proteins with a concentration between 2% and 10% (w / v), and a calcium hydroxide paste comprising calcium hydroxide with a concentration between 20% (w / w) and 40% (w / w); and implanting the mixture into a socket site of jawbone for a predetermined time duration.

3. The method of claim 2, wherein placing the dentin tissue in the acid solution with the pH level between 2 and 3.5 for the time duration between 10 days and 20 days comprises placing the dentin tissue in an acetic acid solution with a concentration between 3.5% (v / v) and 7.5% (v / v) for the time duration between 10 days and 20 days.

4. The method of claim 3, wherein placing the dentin tissue in the acetic acid solution with the concentration between 3.5% (v / v) and 7.5% (v / v) for the time duration between 10 days and 20 days comprises placing the dentin tissue in the acetic acid solution with a concentration between 4 (v / v) and 6% (v / v) for a time duration between 12 days and 16 days.

5. The method of claim 2, wherein the mixture comprises the plurality of passaged DPSCs with a concentration between 104and 108(cells / mL).

6. The method of claim 2, wherein the mixture comprises the composition of dentin matrix proteins with a concentration between 2% and 6% (w / v).

7. The method of claim 2, wherein the calcium hydroxide paste comprises the calcium hydroxide with the concentration between 20% (w / w) and 40% (w / w), iodoform with a concentrationbetween 30% (w / w) and 45% (w / w), and silicone oil with a concentration between 10% (w / w) and 30% (w / w).

8. The method of claim 7, wherein the calcium hydroxide paste comprises the calcium hydroxide with a concentration between 27% (w / w) and 33% (w / w), the iodoform with a concentration between 37% (w / w) and 43% (w / w), and the silicone oil with a concentration between 19% (w / w) and 25% (w / w).

9. The method of claim 2, wherein implanting the mixture into the socket site of jawbone for the predetermined time duration comprises implanting the mixture into the socket site of jawbone for a time duration between 20 days and 60 days.

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