Method, system and device for interrogating or investigating the oral microbiome and oral mycobiome

Abstract

A method, system, and device for interrogating the oral microbiome and oral mycobiome of a subject. The method includes implanting one or more dental devices each having a target growth surface in an oral cavity of the subject, incubating the one or more dental devices in the oral cavity of the subject, removing at least a portion of the target growth surface of at least one of the dental devices from the oral cavity of the subject at a first time point, and analyzing the microbiome and mycobiome present on the removed target growth surface.

Description

[0001] TITLE OF THE INVENTION

[0002] Method, System and Device for Interrogating or Investigating the Oral Microbiome and Oral Mycobiome

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 711,555 filed on October 24, 2024, incorporated herein by reference in its entirety.

[0004] BACKGROUND OF THE INVENTION

[0005] The oral microbiome is a complex ecosystem crucial for overall health, influencing digestion, the immune system, inflammation, metabolic regulation, detoxification, and disease prevention. A healthy oral microbiome prevents decay and disease by inhibiting harmful pathogens. This dynamic ecosystem can rapidly change due to factors like diet, oral hygiene, environmental pH, bacterial interactions, and genetic mutations. The oral microbiome exists as a biofilm, playing a vital role in maintaining oral health and protecting the oral cavity from infections. Disruption of this balance can lead to oral diseases like dental caries, endodontic infections, gingivitis, and periodontitis, as well as other non-oral diseases. Understanding how restorative materials affect the oral microbiome is important. While materials like lithium disilicate, composite resin, and zirconia have been studied for bacterial binding, comprehensive microbiome studies are lacking, especially for 3D printed crowns.

[0006] The oral microbiome consists of bacteria, fungi and viruses. Oral bacteria have been well studied and have important roles to play in oral diseases such as dental caries, periodontal disease, oral cancer etc. Additionally, oral bacteria also have very important roles in systemic diseases such as diabetes, cardiovascular disease, gut cancer, liver cancer, rheumatoid arthritis and Alzheimer’s disease. Oral fungi have also been shown to play important roles in oral mucosal disease and systemic diseases including invasive candidiasis, aspergillosis etc. to name a few, which have high morbidity and mortality. Some oral fungi like Mallessezia have been implicated in cancer. Moreover, the association between bacteria and fungi within biofilms has been shown to be important for oral diseases such as early childhood caries (ECC). In ECC, the oral fungus Candida albicans interacts with the bacterium Streptococcus mutans, resulting in an increased incidence of caries with rampant destruction of dental tissue.

[0007] The oral microbiota is highly diverse and crucial for maintaining oral and systemic health (Wade WG. Pharmacol Res. 2013;69: 137-143.). The oral cavity provides unique environments for bacterial and fungal colonization, with different surfaces like teeth, tongue, and gingiva hosting distinct microbial communities (Deo PN, DeshmukhR. J Oral Maxillofac Pathol. 2019;23: 122-128). These communities are essential for digestion, immune function, inflammation regulation, and disease prevention (Deo PN, Deshmukh R. J Oral Maxillofac Pathol. 2019;23: 122-128, Willis JR, Gabaldon T. Microorganisms. 2020;8. doi: 10.3390 / microorganisms8020308, Zhang Y et al. Biomed Pharmacother. 2018;99: 883-893.). Disruption of the microbiome balance can lead to dysbiosis, impacting Alpha and Beta diversity and contributing to conditions such as dental caries, gingivitis, and periodontitis (Willis JR, Gabaldon T. Microorganisms. 2020;8. doi: 10.3390 / microorganisms8020308). Dental caries may involve more than just 5. mutans, and it has been shown that co-infections with S. mutans and C. albicans can exacerbate carious lesions (Falsetta ML, et al. Infect Immun. 2014;82: 1968-1981, Xiang Z, et al. MBio. 2023; 14: e0276922.). The oral microbiome is also associated with systemic diseases, including Alzheimer’s, cardiovascular disease, cystic fibrosis, cancer, diabetes, and rheumatoid arthritis (Wade WG. Pharmacol Res. 2013;69: 137-143., Willis JR, Gabaldon T. Microorganisms. 2020;8. doi: 10.3390 / microorganisms8020308, Sampaio-Maia B, et al. Adv Appl Microbiol. 2016;97: 171-210.).

[0008] The oral mycobiome, composed of fungal organisms, is less understood due to challenges in culturing and genetic complexity, with fungi representing less than 0.1% of the oral microbiome (Bandara HMHN, et al. Oral Dis. 2019;25: 363-371., Diaz PI, Dongari-Bagtzoglou A. J Dent Res. 2021; 100: 133-140). Advances in Next Gen sequencing and ITS (Internal Transcribed Spacer) sequencing have improved identification of fungi (Tsang C-C, et al. J Fungi (Basel). 2021;7. doi:10.3390 / jof7080636, Gweon HS, et al. Methods Ecol Evol. 2015;6: 973-980., Yang R-H, et al. PLoS One. 2018; 13 : e0206428.). The ITS2 region is particularly effective for revealing fungal community specifics (Yang R-H, et al. PLoS One. 2018; 13 : e0206428.). Analysis indicates 193 genus-level taxa in the oral mycobiome, though many are transient or present in low levels. Dominant genera include Candida and Malassezia, with Candida linked to oral candidiasis and potential cancer risks associated with Malassezia (Bandara HMHN, et al. Oral Dis. 2019;25 : 363-371., Diaz PI, Dongari-Bagtzoglou A. J Dent Res. 2021;100: 133-140., Gamal A, et al. Cancers . 2022;14. doi:10.3390 / cancersl4122875). Interactions between fungal and bacterial communities, such as between S. oralis and C. albicans, are known to enhance biofilm formation and increase virulence factor expression, which can have clinical implications (Diaz PI, Dongari-Bagtzoglou A. JDentRes. 2021;100: 133-140., Chevalier M, et al. Med Mycol.

[0009] 2018;56: 653-667.).

[0010] Bacterial adhesion to oral surfaces involves complex interactions influenced by surface properties like charge, wettability, and roughness (Kreve S, Reis ACD. Jpn Dent Sci Rev. 2021;57: 85-96., Kreve S, Dos Reis AC. J Esthet Restor Dent. 2022;34: 461-472., Zheng S, et al. Front Bioeng Biotechnol. 2021;9: 643722.). Surface roughness correlates with bacterial adherence, and polishing techniques can reduce bacterial adhesion (Aykent F, et al. J Prosthet Dent. 2010; 103: 221-227., Hahnel S, et al. Dent Mater. 2009;25: 969-975., SongF, et al. JDentRes. 2015;94: 1027-1034., Vo DT, et al. J Prosthet Dent. 2015; 114: 696-701.). Properly finished materials can achieve smoothness similar to natural teeth, potentially lowering S. mutans adhesion and reducing caries risk (KozmosM, et al. Molecules. 2021;26. doi: 10.3390 / molecules26041152).

[0011] Dental resin composites, used for tooth restoration, often suffer from shrinkage and marginal leakage, leading to recurrent decay (Kozmos M, et al. Molecules. 2021;26. doi: 10.3390 / molecules26041152). Compared to composites, dental ceramics like lithium disilicate and zirconia induce lower biofilm density, though finishing techniques impact surface roughness (Vo DT, et al. J Prosthet Dent. 2015; 114: 696-701., Contreras-Guerrero P, et al. Am J Dent. 2020;33: 59-63., Souza JCM, et al. Braz Dent J. 2016;27: 141-147.) Zirconia shows lower bacterial adherence than lithium disilicate (Bremer F, et al. Quintessence Int. 2011;42: 565-574.).

[0012] 3D printers, used for various dental applications, offer rapid production with minimal waste, though printed restorations may exhibit lower mechanical strength compared to milled ones (Tian Y, et al. Scanning. 2021;2021: 9950131., Valenti C, et al. J Prosthet Dent. 2022. doi: 10.3390 / dj 10100181). Common 3D printing technologies include SLA, DLP, and LCD, each with specific light curing methods (Tsolakis IA, Dent J. 2022;10. doi: 10.3390 / dj 10100181). Orientation during printing affects accuracy and surface properties, with 0-degree orientation providing higher accuracy (Revilla-Leon M, et al. J Prosthet Dent. 2023. doi: 10.1016 / j.prosdent.2023.03.020, Shim JS, et al. J Prosthet Dent. 2020; 124: 468-475., Yacob N, et al. J Prosthet Dent. 2023; 130: 131. el-13 Le7.). Comparative studies have shown varied bacterial adhesion based on printing orientation and material (Ozer NE, et al. Odontology. 2024; 112: 460-471.).

[0013] There remains a need in the art for sampling the oral microbiome and oral mycobiome.

[0014] SUMMARY OF THE INVENTION

[0015] In some aspects, the invention relates to a method for interrogating the oral microbiome and oral mycobiome of a subject.

[0016] In some embodiments, the method includes the steps of implanting one or more dental devices each having a target growth surface in an oral cavity of the subject, incubating the one or more dental devices in the oral cavity of the subject, removing at least a first portion of the target growth surface of at least one of the dental devices from the oral cavity of the subject at a first time point, and analyzing the microbiome and mycobiome present on the removed target growth surface.

[0017] In some embodiments, the method includes the steps of removing at least a second portion of the target growth surface from the oral cavity of the subject at a second timepoint, and analyzing the microbiome and mycobiome contained on the at least second part of the one or more dental devices.

[0018] In some embodiments, the dental device includes a permanent portion.

[0019] In some embodiments, implanting the one or more dental devices includes adhering the one or more dental devices to a tooth. In some embodiments, the at least first portion of the target growth surface of the one or more dental devices is incubated in the oral cavity for a period of time less than 11 week, less than 2 weeks, less than 3 weeks, or less than 4 weeks.

[0020] In some embodiments, analyzing the microbiome and mycobiome present on the removed target growth surface includes sequencing the microbiome and mycobiome present on the removed target growth surface.

[0021] In some embodiments, analyzing the microbiome and mycobiome present on the removed target growth surface includes determining the species diversity of the microbiome and mycobiome present on the removed target growth surface.

[0022] In some embodiments, the method includes administering at a treatment to a subject based on the analysis.

[0023] In some aspects, the invention relates to a dental device.

[0024] In some embodiments, the dental device includes a body having an exterior surface and configured to releasably engage a portion of an oral cavity of a subject, and at least one target growth surface on the exterior surface of the body

[0025] In some embodiments, the target growth surface is configured to promote or enhance microbial growth thereon.

[0026] In some embodiments, the target growth surface is recessed.

[0027] In some embodiments, at least a portion of the dental device containing at least a portion of the target growth surface is detachable.

[0028] In some embodiments, the target growth surface is positively charged.

[0029] In some embodiments, the target growth surface includes a resin or resin composite.

[0030] In some embodiments, the target growth surface is hydrophilic.

[0031] In some embodiments, the body of the dental device includes a permanent portion configured to inhibit microbial growth. In some embodiments, the permanent portion is negatively charged or neutral. In some embodiments, the permanent portion includes a ceramic.

[0032] In some embodiments, the permanent portion is hydrophobic.

[0033] In some aspects, the invention relates to a dental implant.

[0034] In some embodiments, the dental implant includes a frame configured to releasably engage a portion of an oral cavity of a subject, a plurality of sampling devices releasably secured to the frame, wherein each sampling device includes a target growth surface on an exterior surface of the sampling device, and wherein the target growth surface of each sampling device is configured to promote or enhance microbial growth thereon.

[0035] In some embodiments, the dental implant includes at least one hinge.

[0036] In some embodiments, the target growth surface of the dental implant is recessed. In some embodiments, the target growth surface of the dental implant is positively charge.

[0037] In some embodiments, the target growth surface of the dental implant includes a resin or resin composite.

[0038] In some embodiments, the target growth surface of the dental implant is hydrophilic.

[0039] In some embodiments, the dental implant includes a connector portion, wherein the connector portion releasably secures at least one of the plurality of sampling devices to the frame, and wherein the connector portion is frangible.

[0040] In some embodiments, the connector portion of the dental implant is perforated. In some aspects, the invention relates to system for interrogating the oral microbiome and oral my cobiome of a subject.

[0041] In some embodiments, the system includes a dental device and a dental device applicator tool comprising a base and a set of dental device engagement arms, wherein the dental device engagement arms are configured to grip at least a portion of the dental device and leave exposed a portion of the dental device.

[0042] In some embodiments, the system includes a dental device remover tool configured to grip at least a portion of the dental device.

[0043] In some embodiments, the system includes a dental implant and a dental device remover tool configured to grip at least a portion of the at least one connector portion of the dental implant. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0045] Figure 1 depicts a schematic of an exemplary method for interrogating the oral my cobiome and / or oral microbiome of a subject.

[0046] Figure 2 depicts an exemplary embodiment of an implanted dental device.

[0047] Figure 3A through Figure 3B depict an exemplary embodiment of a dental device applicator tool. Figure 3A depicts an exemplary embodiment of a dental device applicator tool holding an exemplary embodiment of a dental device. Figure 3B depicts an exemplary embodiment of a dental device applicator tool applying an exemplary embodiment of a dental device to a tooth.

[0048] Figure 4A through Figure 4B depict an exemplary embodiment of an implanted dental device.

[0049] Figure 5A through Figure 5C depict an exemplary embodiment of a dental device. Figure 5A depicts an exemplary embodiment of an implanted dental device adhered to a tooth. Figure 5B depicts a view of the smooth side of an exemplary embodiment of a dental device. Figure 5C depicts an exemplary embodiment of an implanted dental device.

[0050] Figure 6 depicts a flow chart of the Illumina MiSeq used for sequencing the ITS region for mycobiome analysis.

[0051] Figure 7 depicts a flow chart of the bioinformatics analysis of amplicons following sequencing with the illumina Miseq.

[0052] Figure 8 depicts the total number of reads of the 16S rRNA sequencing for microbiome analysis per dental crown sample separated by group.

[0053] Figure 9A through Figure 9E depict the bacteria identified in the microbiome analysis illustrating the most abundant bacteria identified in the various groups of dental crown samples. The data indicates that there is more similarity in the types of bacteria identified among the various groups. Figure 9A depicts the identified bacteria by phylum. Figure 9B depicts the identified bacteria by class. Figure 9C depicts the identified bacteria by order. Figure 9D depicts the identified bacteria by family. Figure 9E depicts the identified bacteria by genus.

[0054] Figure 10 depicts the bacterial alpha diversity in dental crown samples and their respective groups.

[0055] Figure 11 depicts the beta-diversity of bacterial composition for all dental crown samples.

[0056] Figure 12 depicts total number of reads of the ITS2 region following mycobiome sequencing of dental crown samples.

[0057] Figure 13 A through 13E depict the fungi identified in the mycobiome analysis illustrating the most abundant fungi identified in the various groups of dental crown samples. The data indicates that there is less similarity in the types of fungi identified among the various groups of dental crown samples. Figure 13A depicts the fungi identified by phylum. Figure 13B depicts the fungi identified by class. Figure 13C depicts the fungi identified by class. Figure 13D depicts the fungi identified by family. Figure 13E depicts the fungi identified by genus.

[0058] Figure 14 depicts the alpha diversity of fungi in the respective dental crown sample groups.

[0059] Figure 15 depicts the beta-diversity of fungal composition for all dental crown samples.

[0060] Figure 16 depicts dental crowns fabricated through 3D printing a resin.

[0061] Figure 17A through Figure 17D depict an exemplary embodiment of a dental device and associated frame. Figure 17A depicts an exemplary embodiment of an implanted dental device and associated frame adhered to a tooth. Figure 17B depicts a view of the surface configured to adhere to a tooth of an exemplary embodiment of a frame associated with a dental device. Figure 17C depicts a view of the surface opposite the surface configured to adhere to a tooth of an exemplary embodiment of an implanted dental device and associated frame. Figure 17D depicts a view of an internal cross section of an exemplary embodiment of a dental device and associated frame. DETAILED DESCRIPTION

[0062] The following discussion omits or only briefly describes conventional features of methods, systems, and devices for interrogating the oral microbiome and oral mycobiome that are apparent to those skilled in the art. Those of ordinary skill may thus recognize that other elements may be desirable and / or necessary to implement the devices, systems, and methods described herein. It is noted that various examples are described in detail with reference to the drawings. Reference to these various examples does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are intended to be non-limiting and merely set forth some of the many possible implementations for the appended claims. Further, particular features described herein can be used in combination with other described features in each of the various possible combinations and permutations. As such, it is understood that the detailed description is exemplary and explanatory only and is not restrictive of the broad inventive concepts upon which the examples disclosed herein are based.

[0063] Unless otherwise specifically defined herein, all terms are to be given their broadest reasonable interpretation. This includes meanings implied from the specification as well as meanings understood by those skilled in the art and / or as defined in dictionaries, treatises, etc.

[0064] It is noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless otherwise specified. The terms “includes” and / or “including,” when used in this specification, specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0065] Relative terms such as “horizontal,” “vertical,” “up,” “down,” “top,” and “bottom” as well as derivatives thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then-described or as shown in the drawing figure under discussion. These relative terms are for convenience of description and normally are not intended to require a particular orientation in actuality. Terms including “inwardly” versus “outwardly,” “longitudinal” versus “lateral” and the like are to be interpreted relative to one another or relative to an axis of elongation, or an axis or center of rotation, as appropriate. Terms concerning attachments, coupling and the like, such as “connected” and “interconnected,” refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. The phrases “operatively” or “operably connected” indicates such an attachment, coupling or connection that allows the pertinent structures to operate as intended by virtue of that relationship.

[0066] Reference throughout the specification to “exemplary”, “one example”, “an example” or “some examples” means that a particular feature, structure, or characteristic described in connection with at least one example of the subject matter disclosed. Thus, the appearance of the phrases “in one example”, “in an example” or “in some examples” in various places throughout the specification is not necessarily referring to the same example. Further, the particular features, structures or characteristics of “one example”, “an example” or “some examples” may be combined in any suitable manner with each other to form additional examples of such combinations. It is intended that examples of the disclosed subject matter cover modifications and variations thereof. Terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and / or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0067] Moreover, throughout this disclosure, various aspects can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments there between. This applies regardless of the breadth of the range. As used herein, the term “about” in reference to a measurable value, such as an amount, a temporal duration, and the like, is meant to encompass variations of plus or minus 20%, plus or minus 10%, plus or minus 5%, plus or minus 1%, and plus or minus 0.1% of the specified value, as such variations are appropriate.

[0068] The terms “proximal,” “distal,” “anterior,” “posterior,” “medial,” “lateral,” “superior,” and “inferior” are defined by their standard usage indicating a directional term of reference. For example, “proximal” refers to a position that is situated nearer to the center of a body or point of attachment or interest, while “distal” refers to a position that is situated away from the center of the body or point of attachment or interest. In another example, “anterior” refers to the front of a body or structure, while “posterior” refers to the rear of a body or structure, in relation to a relative viewpoint. In another example, “medial” refers to the direction towards the midline of a body or structure, and “lateral” refers to the direction away from the midline of a body or structure. In some examples, “lateral” or “laterally” may refer to any sideways direction. In another example, “superior” refers to the top of a body or structure, while “inferior” refers to the bottom of a body or structure. It should be understood, however, that the directional term of reference may be interpreted within the context of a specific body or structure, such that a directional term referring to a location in the context of the reference body or structure may remain consistent as the orientation of the body or structure changes.

[0069] Described herein are novel systems, methods, and devices for interrogating the oral microbiome and oral mycobiome. Although exemplary devices, methods, and systems for interrogating the oral microbiome and oral mycobiome are disclosed, it should be appreciated that aspects of the devices, systems, and methods disclosed herein may be operated in conjunction with other systems, devices, or methods including any dental procedures or treatment, any oral surgeries, any microbiome or mycobiome sampling techniques, and any microbiome or mycobiome analysis techniques.

[0070] In some aspects, the invention relates to a method of interrogating the oral microbiome and / or oral mycobiome of a patient. Referring now to Figure 1 shown is an exemplary method 100 for interrogating the oral microbiome and / or oral mycobiome. In some embodiments, method 100 comprises the steps of 110 implanting a dental device having an outer surface in an oral cavity of a subject; 120 incubating the dental device in the oral cavity of the subject for a certain period of time; 125 removing at least portion of the outer surface of the dental device from the oral cavity; and 130 analyzing the microbiome and / or mycobiome from the outer surface of the device. In some embodiments, the method also includes the step of 140A determining a relationship between a characteristic of the subject and analysis of the microbiome and / or mycobiome. In some embodiments, the method also includes the step of 140B diagnosing a subject based on the analysis of the microbiome and / or mycobiome.

[0071] In some aspects, the invention relates to a dental device. The dental device may be implantable in an oral cavity of a subject. The dental device may have one or more target growth surfaces forming at least part of an outer surface of the dental device. The one or more target growth surfaces may be configured to promote, increase and / or accelerate the adherence, formation, or growth of a biofilm during incubation in an oral cavity of a subject. The one or more target growth surfaces, optionally including an associated biofilm, may be removable from the rest of the dental device at any incubation timepoint. In some examples, the target growth surface may be positioned on a removable portion of the device. The associated biofilm formed on a target growth surface may be separated or extracted from the target growth surface.

[0072] The dental device may be a dental crown, a dental filling, a tooth implant, a denture, a retainer, an attachment to a tooth, gum or other tissue within the oral cavity, or any kind of implantable dental device. In some embodiments, the dental device, for example a dental crown, a dental filling or a tooth implant, is configured to be implanted temporarily. In some embodiments, the dental device is configured for everyday wear by a subject for any period of time. For example, the dental device may be configured for everyday wear for a period of time of 4-6 weeks or longer.

[0073] The dental device may be constructed of any material. In some examples, the dental device is a dental crown constructed of any known materials used in dental crowns, for example, any metals, any porcelain, any ceramic, or any resin. In some embodiments, the dental device is a dental crown constructed of Lithium Disilicate (LD), composite resin, a printed resin, a printed composite resin, ceramic crown resin, a printed ceramic crown resin, SprintRay ceramic crown resin, 3M Filtek composite, zirconia, or any combination of materials.

[0074] The dental device may be fabricated by any method. In some embodiments, the dental device is designed using computer-aided design. In some embodiments, the dental device is constructed of lithium disilicate, zirconia, or any porcelain or ceramic and is milled. Dental devices may be milled using any mill and from any shape starting material, for example a block shape. For example, lithium disilicate samples may be milled from lithium disilicate blocks. Milled dental devices may be polished by any method including using a lathe or dental ceramic polish burs. The polishing or any other finishing technique may be optimized for a desired surface roughness.

[0075] In some embodiments, the dental device is a dental crown and is constructed of a resin, or a composite resin and is molded or cured. For example, a stint or mold may be filled with a packable composite resin or packable resin, for example 3M Filtek packable composite. The filled mold is then placed on the tooth on which it will later be implanted. The packed resin is then cured in any manner, for example light cured. In some embodiments, the tooth is prepared with Vaseline, any petroleum jelly, or an oil-based substance to prevent adherence of the crown to the tooth while the packed resin cures. In some embodiments, the cured crown is removed from the oral cavity and from the mold after curing to repair any molding defects before being implanted on the tooth. In some embodiments, the molded crown is polished, for example with Dentsply Sirona PoGo system before being implanted. The polishing or any other finishing technique may be optimized for a desired surface roughness.

[0076] In some embodiments, the dental device is constructed of a resin, for example any ceramic crown resin or SprintRay ceramic crown resin, and fabricated by printing, optionally 3D printing, as in Figure 16. The dental device may be printed using any 3D printing technology including SLA, DLP and LCD. The dental device may be printed using any light curing method. The dental device may be printed using any printer, for example a SprintRay 55 printer. The printed dental device may be a dental crown or tooth implant and printed such that occlusal surfaces are parallel to a build platform. The printed dental device may be printed at any orientation. The printing orientation may be optimized for printing accuracy. For example a O-degree printing orientation may provide high accuracy. The printing orientation may also be optimized for desired surface properties of the dental device. For example, some printing orientations may increase or decrease dental device surface roughness. The printing orientation and composite material may also be optimized for desired bacterial adherence, for example some printing orientations may promote or inhibit bacterial adherence to the device implant. Printing orientations and composite materials that increase roughness may also increase bacterial adherence.

[0077] After printing, the dental device may be washed to remove all uncured resin, for example from the intaglio surface of a printed dental crown or printed tooth implant. The dental device may be washed with an isopropyl alcohol solution with any percentage isopropyl alcohol, for example 95%. The dental device may be washed in a bath with mechanical jets, for example the SprintRay ProWash2. The dental device may also be handwashed. The printed dental devices may be finished using any finishing technique. The printed dental devices may be polished by any method, for example by using a lathe and polish wheel. The printed dental devices may be glazed using any coating. The coating may be light curable, for GC Optiglaze. The coating may be cured for any amount of time using any wavelength or intensity light. For example, the coating may be cured for about 40 seconds using high intensity light of a wavelength ranging from 385 nm to 515 nm. The polishing, glazing, or any other finishing technique may be optimized for a desired surface roughness. The desired surface roughness may be chosen for desired bacterial adherence properties.

[0078] In some embodiments, the dental device may be derived from a donor subject or animal for example a tooth, part of a tooth, a bone, or a part of a bone. In some embodiments, a tooth or bone may be shaped using a mill or any other method before implantation. In some embodiments, a tooth or bone may be smoothed, polished, or sanded to achieve a desired surface roughness before implantation. The desired surface roughness may be chosen for desired bacterial adherence properties. In some embodiments, the dental device is machined, 3D printed, extruded, or injection molded.

[0079] In some embodiments, the dental device includes one or more target growth surfaces forming at least part of any exterior surface of the dental device after it is implanted. For example, a target growth surface may be on a surface of a dental implant that faces the oral cavity. In some embodiments, the whole exterior surface of the dental device is a target growth surface. In some examples, the target growth surface promotes biofilm formation, bacterial adherence, and / or fungal adherence. In some examples, the target growth surface accelerates and / or increases biofilm formation, bacterial adherence, and / or fungal adherence. In some examples, a dental device includes target growth surfaces as well as surfaces that inhibit biofilm formation, biofilm density, bacterial adherence, and / or fungal adherence. Surfaces that inhibit biofilm formation, biofilm density, bacterial adherence and / or fungal adherence may be desirable for portions of a dental device that may remain in an oral cavity indefinitely.

[0080] In some embodiments, the dental device includes one or more removable portions. In some examples, the dental device may include one or more portions configured to remain in the oral cavity of a subject and may function to treat a subject. Examples of subject treatments may include filling a tooth, implanting a crown on a tooth, or implanting a tooth implant. In some examples, the dental device may include removable portions in addition to portions designed to remain in the oral cavity of a subject indefinitely. The removable portions of a dental device may include target growth surfaces and may be removable after any incubation time such that the microbiome and / or mycobiome of the removable portion may be sampled and analyzed. There may be a plurality of separate removable portions included in a dental device. Removable portions of a dental device may removed at different timepoints after implantation in the oral cavity such that the oral microbiome and / or oral mycobiome may be sampled over time or at any desired timepoints. Portions of the dental device may be constructed of the same material or different materials. Portions of the dental device may be fabricated by the same method or different methods. Portions of the dental device may be acquired from the same source, for example a human or animal donor, or a different sources. A dental device or a surface of a dental device including a target growth surface may have any material properties, for example, charge, wettability, roughness, porosity, and mechanical strength. In some examples, a target growth surface may be a biomimetic surface or a mimetic surface such that the biomimetic surface promotes biofilm formation, bacterial adherence or fungal adherence. A biomimetic surface mimics any material properties of chemical composition of a tooth or any other surface naturally found in the oral cavity. A biomimetic or mimetic surface may be 3D printed. Mimetic materials may be 3D printed to decrease cost. 3D printing may allow for the generation of complex mimetic materials. Portions of a dental device or dental device surface may have different material properties. Material properties may be chosen, engineered, or designed for desired bacterial and / or fungal adherence. For example, positively charged surfaces, increased wettability, and increased roughness may increase bacterial adherence and promote biofilm formation or density. For example, hydrophilic surfaces may increase bacterial adherence and promote biofilm formation or density. Neutral or negatively charged surfaces, decreased wettability, and decreased roughness may decrease bacterial adherence and decrease biofilm formation or density. For example, hydrophobic surfaces may decrease bacterial adherence and decrease biofilm formation or density. The fabrication method or the material that the dental device or portions thereof is constructed of may be chosen to achieve a desired level of bacterial adherence and biofilm formation or density. For example, dental devices or portions constructed of resin or resin composites may promote bacterial adherence and biofilm formation or density, while dental devices or portions constructed of a ceramic may decrease bacterial adherence and biofilm formation or density. In some embodiments, the permanent component of a dental device is designed to inhibit bacterial adherence and biofilm formation or density for the promotion of subject health. In some embodiments, the temporary component of a dental device is designed to promote bacterial adherence and biofilm formation or density to promote the sampling of the oral microbiome and / or oral mycobiome after removal of the temporary component. In some embodiments, micropillars or any other surface micropattern may be included on the device.

[0081] In some embodiments, the dental device or portions thereof including a target growth surface, removable portions, or permanent portions may incorporate any substance. Substances may be incorporated within the dental device or portions of the dental device or may be coated onto any surface or surface portion of the dental device including a target growth surface. A substance may be included to promote or inhibit bacterial adherence or biofilm formation or density. Increasing bacterial adherence or biofilm formation or density may be useful for microbiome and / or mycobiome sampling, for example on a target growth surface. Decreasing bacterial adherence or biofilm formation or density may be useful for the long-term usefulness of a permanent portion of a dental device. A substance may be included also for the purpose of determining the substance’s effect on the microbiome and / or mycobiome that forms on the dental device. The same subject may have two dental devices implanted, for example one control dental device and one dental device with an included substance. In some embodiments the substance is a bioactive molecule, for example an antifungal, an antibiotic, or any other bioactive molecule. In some embodiments the substance is a bacteria, fungi, a bacterial colony, a fungal colony, a biofilm, a microbiome sample, and / or a mycobiome sample. In some embodiments, a microbiome and / or mycobiome sample of a second subject is included in the dental device. In some embodiment, the first and second subject have different health and disease states. In some embodiments the microbiome and / or mycobiome of a first subject is included in the dental device before being implanted in a second subject.

[0082] Now referring to Figure 2. In some embodiments, the dental device 200 may be adhered to a tooth 10 of a subject. As contemplated herein, the dental device 200 may be any oral microbiome and / or mycobiome sampling device. As contemplated herein, tooth 10 may be a natural tooth of a subject, or alternatively it may be an implant. For example, the dental device 200 may have a flat side such that it can be flush against a tooth 10 when it is adhered. In other words, an implanted dental device 200 may be adhered to a tooth 10 and may form a continuous surface in contact with the tooth 10. In some embodiments, continuous contact with the tooth 10 prevents any unintended damage including decay to the tooth or to the dental device. The dental device 200 may be sufficiently flexible such that it can be in continuous surface contact with a tooth 10 after it is applied. For example, the dental device 200 may be sufficiently flexible such that its flat surface can match the curvature of a tooth 10 on which it as applied. In some embodiments, the dental device has a high surface area to volume ratio such that a large biofilm area may be captured on the dental device or in some examples specifically on a target growth surface. In some embodiments, as in Figure 2, a plurality of dental devices 200 may be adhered to the same tooth 10. In some embodiments, a plurality of dental devices 200 may be adhered to different teeth in any locations of an oral cavity. Each dental device 200 may be adhered to a tooth 10 using any method or any substance. The adherence method or the adherence substance may allow for temporary adherence.

[0083] In some embodiments, a dental device may include a recessed collection area 210 that is recessed such that the collection area has a volume. A recessed collection area 210 may present a surface on which biofdm formation can occur, for example a target growth surface 201. The dental device may have a lip 220 that extends from the dental device into the oral cavity. The lip 220 may surround the recessed collection area 210. The collection area may be sufficiently recessed such that a target growth surface 201 and any associated biofilm is protected from any accidental abrasion from tooth brushing, flossing, or from tongue movements. For example, a lip 220 may sufficiently extend such that the lip 220 can protect the recessed collection area 210. In some examples, a dental device 200 includes an outer lip 220 forming a recessed collection area 210 within which is a target growth surface 201.

[0084] In some embodiments, a recessed collection area 210 may be filled or coated with any material. The filling material may be a biomimetic material. In some embodiments, a recessed collection area 210 is filled or coated with a material before or after application of the dental device 200 to an oral cavity. In some embodiments, a recessed collection area 210 is of any dimensions. For example, the recess may be any depth, any length, or any width. In some embodiments, the depth of a recessed collection area 210 is shorter than 3 mm. In some embodiments, the depth of a recessed collection area 210 is about 3 mm, 2.5mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, or 0.1 mm. In some embodiments, the depth of a recessed collection area 210 is in the range of 0.1 mm to 3 mm, 0.1 mm to 1 mm, 1 mm to 2 mm, 2 mm to 3 mm, 1 mm to 3 mm, or 1.5 mm to 2.5 mm. In some embodiments, the depth of a recessed collection area 210 is any depth within the range of about 3 mm to about 0.1 mm. In some embodiments, a recessed collection area 210 is a square, a rectangle, a circle, or an ellipse. In some embodiments, a recessed collection area 210 is square with dimensions less than about 1 cm by 1 cm. In some embodiments, a recessed collection area 210 is rectangular with a largest dimension less than about 1 cm. In some embodiments, a recessed collection area 210 is a circle with a diameter less than about 1 cm. In some embodiments, a recessed collection area 210 is an ellipse with a major axis less than about 1 cm. In some embodiments, a recessed collection area 210 is designed with any dimensions such that it may fit easily on a tooth 10 surface. For example, the recessed collection area 210 may have a surface area that is less than the surface area of a tooth 10. In some embodiments, an entire dental device 200 is less than the surface area of a tooth 10 surface such that it may fit easily on a tooth 10 surface.

[0085] In some embodiments, the dental device 200 may be a portion of or may be adhered to a tooth implant 10. For example, as shown in Figure 2, the dental device 200 may be incorporated into a tooth implant 10 before implantation. In some embodiments, the dental device 200 may be releasably engaged to the tooth implant 10. For example, the tooth implant 10 may include an inset or recess in which the dental device 200 may be placed. The tooth implant 10, which may be a standard tooth implant, may be milled such that a plurality of recesses are formed. The tooth implant 10 may be 3D printed and may be designed with a plurality of recesses. The dental device 200 may be attached to a tooth implant 10. The dental device 200 may snap or fit into place and may be maintained in place by friction or by the use of an adhesive. The dental device 200 may be attached to a tooth implant 10 recess such that a lip 220 does not extend past the height of the tooth implant 10. In some embodiments, the dental device 200 can include a component to aid removability. For example, the dental device 200 may include a pull tab, eyelet, or any other structure to aid its removal from the tooth implant.

[0086] In some embodiments, a plurality of dental devices 200 or any sampling devices may be applied to an oral cavity or to a tooth 10 in an oral cavity. A first dental device 200 may be removed at a first timepoint. A second dental device 200 may be removed at a second timepoint. A third dental device 200 may be removed at a third timepoint. A fourth dental device 200 may be removed at a fourth timepoint. Any sequential dental devices 200 may be removed at any sequential timepoints.

[0087] In some aspects, the invention relates to an applicator tool. In some embodiments of the method 100, an applicator tool is used to position and apply a dental device to a tooth 10. Now referring to Figure 3A through Figure 3B, in some embodiments, an applicator tool 300 includes base 310 and a set of dental device engagement arms 320 extending from the base 310. In some examples a dental device 200, a plurality of dental devices 200, or any portion of a dental device 200 are releasably secured by the dental device engagement arms 320. In some embodiments, an applicator tool base 310 itself is shaped so that it can hold a dental device 200, a plurality of dental devices 200, or any portion of a dental device 200. An applicator tool base 310 or dental device engagement arms 320 may be shaped such that it can hold any dental device 200 embodiment.

[0088] In some embodiments, the applicator tool base 310 or dental device engagement arms 320 holds a dental device 200 such that a portion of the dental device 200 is exposed. In some embodiments, the exposed portion of a dental device 200 may be a smooth or flat side that is designed for continuous surface contact to a tooth 10. The applicator tool 300 can be manipulated such that an exposed portion of a dental device 200 contacts a tooth 10 for application as in Figure 3B. An adhesive may be applied at any point such that an exposed portion of a dental device 200 contains an adhesive. The applicator tool 300 may be used to apply pressure to a dental device 200 after an exposed portion of a dental device 200 contacts a tooth 10. For example, an operator of the applicator tool 300 can apply pressure or force using the applicator tool 300 such that the dental device 200 contacts, presses against, and / or adheres to a tooth 10 or tooth implant 10 as in Figure 3B. The adhesive, applied pressure, or combination of adhesive and applied pressure may aid the attachment of a dental device 200 to a tooth 10. For example, the applicator tool 300 may be pulled away from the oral cavity after being used to contact a dental device 200 to a tooth 10 leaving behind the dental device 200 attached to a tooth 10 or adhered to a tooth 10 surface. In some embodiments, the applicator tool 300 may hold a plurality of dental devices 200 or portions of a dental device 200 in a desired pattern such that the dental devices 200 are applied to a tooth 10 with a desired spacing or pattern.

[0089] Dental device engagement arms may releasably secure a dental device 200, a plurality of dental devices 200, or any portion of a dental device 200 by any method. The securing method may be a friction fit or gripping mechanism. The securing method may be a releasable clamp. Dental device engagement arms may release a dental device 200, a plurality of dental devices 200, or any portion of a dental device 200 by any method. Any number of additional tools may be used to apply an applicator tool 300 to a location in the oral cavity. For example, a handle or another device may be attached to the applicator tool 300 such that it can be handled or manipulated by an operator. Likewise, any number of additional tools may be used to add additional portions to an applied dental device 200. Additionally, any number of additional tools may be used to remove a dental device 200 or portions thereof after they are applied to a tooth 10 for analysis.

[0090] Now referring to Figure 4A through Figure 4B. In some embodiments any number of dental devices 200 or any sampling devices may be connected by any number of frangible connectors 230. Dental devices 200 connected by frangible connectors 230 may similarly include a lip 220, a recessed collection area 210 within which there is a target growth surface 201. Frangible connectors 230 may function to allow for the removal of individual dental devices 200, and optionally each device’s associated biofilm, from the oral cavity at different times. Frangible connectors 230 may also allow for a plurality of dental devices 200 to be removed from the oral cavity together and remain attached. The plurality of dental devices 200 may be detached from each other after removal from the oral cavity such that different methods may be used to analyze the microbiome and / or mycobiome of each dental device 200. Frangible connectors 230 may be designed such that they are easily broken off or detached from the tooth or from other portions of the dental device 200. For example, frangible connectors 230 may be thin or designed with a geometry that can be easily broken off, snapped, or disassembled. For example, frangible connectors 230 may include perforations or through holes. In some embodiments, frangible connectors 230 are of a different material than the other portions of the dental device 200 such that the frangible connectors 230 are more easily broken off, snapped, degraded, or disassembled.

[0091] Reference is now made to Figures 5A through 5C. In some embodiments, a plurality of dental devices 200 or any sampling devices may be attached to a frame 250. Frame 250 may make up a border around a plurality of dental devices 200. In some embodiments, frame 250 is adhered to a tooth. Frame 250 may be adhered to a tooth using any methods for seating a dental device in a subject, for example any bonding agent and / or dental cement may be used. In some embodiments, only frame 250 is adhered to a tooth while dental device 200 is adhered or removably attached to frame 250. Therefore, in some embodiments, bonding agents, dental cements, or other compositions useful for adherence do not contact the one or more dental device 200 directly. This may be advantageous to reduce contamination of collected dental devices 200 by any bonding agent, dental cement, or any other composition for adherence used. In some embodiments, frame 250 may include flexible sections 260. Flexible sections may aid the continuous contact of a frame flat surface 270 to a tooth. For example, flexible sections may allow a frame flat surface 270 to match the curvature of a tooth. Flexible sections 260 may be attached to and positioned in between multiple dental devices 200. For example, frame 250 may support a grid of dental devices 200 and flexible sections 260 may be positioned to form a grid in between the dental devices 200. Flexible sections 250 may hinge, flex, or be of a flexible material. Flexible sections 250 may be positioned along principal axes of the frame 250.

[0092] In some embodiments, the plurality of dental devices 200 may be attached to frame 250 through a frangible connector 230. In some embodiments, the dental devices 200 are not themselves directly adhered to a tooth, but attached to frame 250 which may be adhered to a tooth. In some embodiments, the frangible connecters 230 are thin such that they may be broken by a rotational force. The frangible connectors 230 may be suspended above the tooth by their attachment to the frame 250 and a dental device 200. In some embodiments, the frangible connectors 230 are shaped such that they may fit a tool that may apply a rotational force. The tool may be a remover tool. In some embodiments, frame 250 is removed at some time point after one or more dental devices 200 have been removed.

[0093] Reference is now made to Figures 17A through 17D. In some embodiments, frame 250 may comprise a first surface 280 configured to be positioned against a tooth and a frame recess 290 opposite the first surface 280. First surface 280 may be smooth or substantially smooth. For example, a smooth surface may aid in positioning first surface 280 such that it may be positioned flush or substantially flush against a tooth. First surface 280 may be adhered to the tooth. Frame recess 290 may be configured such that one or more dental devices 200 or any sampling devices may be positioned within frame recess 290. In some embodiments, one dental device 200 is positioned within frame recess 290. Dental device 200 may be removably attached to frame recess 290. In some embodiments, dental device 200 is attached to frame recess 290 via a frangible component such that dental device 200 may be removed from frame 250 by an operator optionally via a remover tool. Dental device 200 may comprise a target growth surface 201.

[0094] First surface 280 may be adhered to a tooth by any method, for example any method for seating a dental device described herein. For example, a bonding agent such as a dental cement or any other composition useful for adherence may be used to adhere frame 250 to a tooth, optionally via adherence of first surface 280 to a tooth. In some embodiments, compositions useful for tooth adherence only contact frame 250, therefore reducing the risk of any contamination of dental device 200 samples by a composition for adherence. In other words, dental device 200 may be removed from frame 250 for any downstream processing and / or analysis while frame 250 remains adhered to the tooth. Frame 250 may be removed from the tooth shortly thereafter dental device 200 is removed or at any later timepoint. Removing dental device 200 and frame 250 from the tooth in separate steps may aid in reducing contamination of dental device 200 with any bonding agent, dental cement, or other composition used to adhere frame 250 to the tooth. Positioning dental device 200 within frame recess 290 may also aid in reducing the risk of fracture of dental device 200 and / or frame 250 upon removal and / or upon normal wear of dental device 200 and / or frame 250. Additionally, positioning dental device 200 within frame recess 290 may aid in reducing the risk of losing or leaving behind any portion of dental device 200 upon accidental fracturing of dental device 200. Positioning dental device 200 within frame recess 290 may aid in reducing the risk of contact of dental device 200 with any other mouth or tooth locations upon accidental fracturing of dental device 200. For example, upon accidental dental device 200 fracture, fractured portions of dental device 200 may remain positioned within frame recess 290. This may be due to shielding provided by frame recess 290.

[0095] In some embodiments, frame 250 further comprises one or more frame juts 255. A frame jut 255 may comprise any geometry that substantially extends outward along the tooth surface from frame 250. One or more frame just 255 may aid in the removal of the frame by an operator optionally via a remover tool. For example, frame jut 255 may be configured to be gripped by an operator optionally via a remover tool. Frame just 255 may be configured to aid in the application of rotational force that may break the adherence of frame 250 to a tooth. In some embodiments, frame 250 may be pried off of a tooth via undercuts on first surface 280.

[0096] Dental device 200 may be adhered to a surface of frame recess 290. In some embodiments, dental device 200 is removably attached to a surface of frame recess 290. In some embodiments, the attachment points or attachment surfaces between dental device 200 and frame recess 290 is frangible such that dental device 200 may be detached. In some embodiments, dental device 200 comprises any number of notches 205. Notches 205 may be configured such that dental device 200 may be more easily removed from frame 250. For example, notches 205 may be configured to be easily gripped by an operator or a remover tool. Notches 205 may be configured such that an operator, optionally via a remover tool, may more easily apply a rotational force to dental device 200 to promote the detachment of dental device 200 from frame 250.

[0097] Now referring to Figure 17D, depicted is an exemplary internal cross-sectional view of dental device 200 and frame 250. Attachment surfaces between dental device 200 and frame 250 may comprises any number of spacings 295. A spacing 295 may be configured such that the connection between dental device 200 and frame 250 is frangible. Any portion of the attachment surface between dental device 200 and frame 250 may comprise spacings 295. The locations of spacings 295 or the overall amount of of the surface area of frame 250 and dental device 200 covered by spacings 295 may be configured such that frame 250 and dental device 200 are not detached under everyday use but may be detached by an operator optionally via a remover tool.

[0098] Any dental device 200, any portion of a dental device 200, any connectors, any frames, or any other structures that may hold, adhere, or be implanted along with a dental device 200 may be fabricated together or may be fabricated separately and adhered to each other before implantation. They may be adhered using a glue or any other adhesive. The adhesive may be light sensitive, thermal sensitive, or sensitive to a force such that dental devices 200, frames, connectors, or portions thereof may be removed as desired without disturbing any nearby portions that are desired to remain implanted.

[0099] Dental devices 200, frames, connectors, or portions thereof may be adhered to each other or a tooth using a dental cement, or a temporary dental cement. For example, separate dental devices 200 may be fabricated separately and adhered using a method that allows for each dental device 200 to be removed at separate timepoints. Any adhesive used may be designed to lose adherence over time such that any devices, portions, or components may be removed at desired timepoints. Dental devices 200, frames, connectors, or portions thereof may be detachable by brushing, picking, flossing, or any other method. A dental pick, a toothbrush, a waterpick, or any other device may be used to detach any dental devices 200, frames, connectors, or portions thereof.

[0100] In some embodiments, the flat surface of a dental device 200, or frame may be substituted with any other surface geometry that promotes the adherence to a tooth. For example, a surface of the dental device or frame may be based on the shape of a subject’s tooth. For example, the surface may be shaped to match a recess in a subject’s tooth similar to a dental filling.

[0101] Referring again to Figure 1 and embodiments of the method 100, a dental device is implanted in any subject. In some embodiments, the subject is a healthy subject or the subject may have a disease. In some embodiments, the subject is chosen to have or engineered for any characteristic such that the characteristic’s effect on or the characteristic’s correlation with the oral microbiome and / or oral mycobiome can be determined. In some examples, the subject may be a person having any desired characteristic. Subject characteristics may include any physical characteristic, any occupation, any diet, use of any oral hygiene practices, use of any recreational drugs, use of any medications, any allergies, any genetic background, any socioeconomic characteristics, any genetic mutations, or be of any age. The subject characteristic may include tobacco use status, for example the subject may use or smoke tobacco. The subject characteristic may be the presence or history of any oral diseases including dental caries, endodontic infections, gingivitis, periodontitis, peri-implantitis, any mucosal disease including lichen planus and leukoplakia, oral cancer, and / or denture stomatitis. The subject characteristic may be the presence or history of any non-oral diseases or systemic diseases. The subject characteristic may be the presence or history of any disease or condition including any metabolic disease, diabetes, any autoimmune disease, Alzheimer’s disease, any cardiovascular disease, cystic fibrosis, rheumatoid arthritis, any oral mucosal disease, invasive candidiasis, aspergillosis, and / or any cancer including pancreatic cancer, esophageal cancer, gut cancer, liver cancer, and / or colorectal cancer. In some embodiments, the subject is given a treatment or other intervention such that the treatment’s effect on or correlation with the oral microbiome and / or oral mycobiome can be determined. The treatment may be any treatment including a bioactive molecule, a chemotherapy, a diet, an exercise regimen, a surgery, an oral surgery, or any dental procedure. The subject may be recovering from a surgery, an oral surgery, or any dental procedure.

[0102] In some embodiments, the subject may be any animal subject. For example, the subject may be a mouse, a rat, a dog, a cat, any primate, or any dentate animal. In some embodiments, the subject is any animal that has an oral cavity suitable for the implantation of a dental device. In some examples, an animal subject is an animal such that the oral microbiome and / or oral mycobiome may be interrogated or investigated as part of any experimental study. In some examples, the subject may be a model organism or a genetically engineered model organism. A model organism may be chosen or engineered such that the model organism has any desired characteristic. In some examples, the subject is a pet. In some embodiments, a subject that is a pet or any other animal or any model organism may have any characteristic contemplated for human subjects.

[0103] In some embodiments of the method 100, the dental device or plurality of dental devices are applied to the oral cavity of the subject in any manner. A dental device may be applied using any methods for implanting a tooth, filling a tooth, implanting a dental crown, applying a retainer, or applying a denture. In some embodiments, for example in the case of a dental device being a retainer or a denture, the device is removably applied. In some embodiments, a dental device may be seated using a dental cement, for example a temporary dental cement, Kerr Temp-Bond, carboxylate luting cement or 3M Durelon carboxylate luting cement. In some embodiments, a dental device may become loose, detached, fall out, or become lost, for example in the case of a temporary dental filling, temporary dental crown, or any embodiment of a temporary dental device. In some embodiments, a detached dental device is reseated or reapplied. In some embodiments, the subject is provided instructions to reseat a detached dental device using a temporary dental cement. In some embodiments, a dental device is applied while the patient is undergoing any dental procedure. For example, a dental device may be applied while the patient is undergoing endodontic treatment, a tooth implant, dental carie filling, or any oral surgery. In some examples, a dental device is a temporary tooth implant, a temporary dental crown, or a temporary dental filling. In some examples, a dental device is a temporary crown applied to tooth that has undergone endodontic treatment. In some embodiments, a dental device is a temporary dental filling applied to a tooth recess. In some examples, an applied dental device is removed from the oral cavity and replaced by a permanent tooth implant, a permanent dental crown, or a permanent dental filling some time after being applied. In some embodiments, a dental device is applied in any relative location to an oral cancer, a dental carie, denture stomatitis, endodontic infection, gingivitis, or periodontitis such that the oral microbiome and / or mycobiome within any distance of a dental infection or cancer can be determined. For example, any number of dental devices may be applied to a subject at different locations in the oral cavity relative to an infection or cancer such that the infection or cancer’s effect on or correlation with the oral microbiome and / or mycobiome can be determined. In some embodiments, the dental device is an embodiment of a dental device 200 or multiple dental devices 200. In some embodiments of the method 100, the dental device or plurality of dental devices are incubated in the oral cavity. In some embodiments, the dental device, plurality of dental devices, or portions of dental devices are incubated for any amount of time. In some embodiments, the dental device, plurality of dental devices, or portions of dental devices are incubated in the oral cavity for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 week, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the dental device, plurality of dental devices, or portions of dental devices are incubated in the oral cavity for a time period in the range of 1 day to 12 months, 1 week to 8 weeks, 4 weeks to 6 weeks, 1 week to 12 months, 1 month to 12 months, or 2 months to 6 months. In some embodiments, properties of a target growth surface allow for rapid biofilm formation and may necessitate only short incubation times. In some embodiments, multiple dental devices are incubated in the same oral cavity for different lengths of time. In some embodiments, one or more portions of a dental device, connectors, or frames are incubated in an oral cavity for the same length of time or different lengths of time. In some embodiments, one of multiple dental devices or dental device components is removed before a subject begins, stops, or changes a treatment, while others are allowed to remain incubating and are removed at a later timepoint. In this embodiment, the microbiome and / or mycobiome of the dental devices or dental device components may be compared to determine the effect of the treatment on the microbiome and / or mycobiome.

[0104] In some embodiments of the method 100, the dental device may be swabbed to retrieve a sample of the oral microbiome and / or oral mycobiome. In some embodiments of the method 100, a dental device or at least a part of a dental device or dental device component may be removed in any manner to retrieve a sample of the oral microbiome and / or oral mycobiome that is associated with the dental device. In some embodiments, only a target growth surface of a dental device is retrieved. In some embodiments, a portion of a dental device that includes a target growth surface is retrieved. In some embodiments, only a collection area of a dental device is removed. In some embodiments, the retrieved portion of a dental device contains a biofilm. The biofilm may contain bacteria and / or fungi and may be representative of the oral microbiome and / or my cobiome of the subject in which the dental device is implanted.

[0105] The dental device may be removed by a surgery, for example in the case of the dental device being a tooth implant. In some embodiments, for example in the case of the dental device being a dental crown, a dental filling, any dental implant with a temporary component, the dental device, a component of the dental device, a portion of a component of a dental device may be removed or detached by brushing, picking, flossing, or any other method. A dental pick, a toothbrush, floss, or a waterpick may be used to remove the dental device. In some embodiments, any tool may be used to remove a portion of the dental device. In some examples, the tool is a custom tool. In some embodiments, the dental device is a retainer or denture and may be removably attached and detached.

[0106] In some embodiments, the dental device and contained microbiome and / or mycobiome may be preserved after it is removed by any method, for example by freezing or by fixing. For example, the dental device may be removed and placed in container with a sterile solution for freezing. In some embodiments, the dental device is submerged in a solution after it is removed such that a biofilm may be separated from the dental device. In some embodiments, the dental device surface is designed such that it promotes biofilm adherence in the oral cavity but releases the biofilm after removal upon exposure to a solution. In some embodiments, the dental device and contained microbiome may be preserved or fixed by submersion in formalin or any fixative agent. Fixing of the contained microbiome on a dental device may be useful for imaging analyses including light microscopy, confocal microscopy, or electron microscopy.

[0107] In some aspects, the invention relates to a dental device remover tool. In some embodiments of the method 100, the dental device or a part of the dental device is removed using a remover tool. In some embodiments, the remover tool is shaped such that it can compress, apply pressure, or shield any dental devices 200 or portions thereof while leaving other dental devices 200 or other portions of a dental device 200 amenable to manipulation. The dental devices 200 or portions thereof that are shielded, compressed, and / or on which pressure is applied may remain undisturbed while the dental device 200 or portion thereof desired to be removed is removed in any manner. In some embodiments, a remover tool may wrap around an entire tooth to apply even pressure or compression to the dental devices 200 or portions thereof applied to the tooth. In some embodiments, the remover tool is designed such that the tool may apply a rotation to any portions of a dental device 200 including frangible connectors 230. For example, a rotational force applied using a remover tool may promote the snapping, breaking, or disassembly of frangible connectors 230 from a frame 250, from dental devices 200, from flexible sections 260 or any other portions of an applied dental device 200. In some embodiments, frangible connectors 230, frame 250, flexible sections 260 or any dental device 200 portions may have capture features such that they can be gripped by a remover tool. A capture feature may be an inlet, a groove, or a crevasse. In some embodiments, a capture feature may extend outward such that it can be gripped by a remover tool.

[0108] In some embodiments of the method 100, the microbiome and / or my cobiome sample retrieved from the dental device is analyzed. In some examples, the relative abundance of individual species of the microbiome and / or mycobiome samples are determined by any method. In some embodiments, the microbiome and / or mycobiome is sequenced by any method. In some embodiments, the microscopy techniques are used to view and / or image the adhered bacteria and fungi, the microbiome and / or mycobiome, and / or a biofdm on the dental device surface. Microscopy techniques may also include any histological techniques including immunofluorescence microscopy. In some embodiments, mass spectrometry may be used to determine the molecular content of the biofdm, microbiome, and / or mycobiome on the surface of the dental device. For example, the glycoprotein, polysaccharide, or other molecular content of a biofdm may be determined by mass spectrometry. In some embodiments, any molecular biology technique may be used to analyze the dental device surface microbiome and / or mycobiome, for example flow cytometry, real-time PCR, or western blotting. For example, protein content, any post-translational modifications, or the level of expression of any number of genes may be determined using any technique. In some embodiments of the method 100, any microbiome and / or mycobiome sequencing method is used to determine the fungal and / or bacterial species content of the sample contained on the dental device. In some embodiments, Illumina MiSeq protocol is used as in Figure 6. In some embodiments, microbiome sequencing and / or mycobiome sequencing results are analyzed using any method. In some embodiments, any metric of diversity, richness, or evenness within a microbiome and / or mycobiome, for example alpha diversity, is calculated based on the sequencing results as in Figure 7. In some embodiments, any metric of similarity among microbiome and / or mycobiome samples, for example beta diversity, is calculated based on the sequencing results as in Figure 7.

[0109] In some embodiments of the method 100, a relationship between a characteristic of a subject and the sampled microbiome and / or mycobiome is determined. For example, any characteristic of any number of subjects may be compared to any characteristic of the sample contained on the dental device of the corresponding subject. The determined relationship between a characteristic of a subject and the sampled microbiome and / or mycobiome of the dental device of the subject may be used to inform the prediction of a subject characteristic based on a sampled microbiome and / or mycobiome of a dental device. In some examples, the characteristic is a disease state or the subject’s risk of acquiring any disease. In this example, the determined relationship may be further used to diagnose a subject based on the sampled microbiome and / or mycobiome of the device. In some embodiments, a saliva sample, gastric lavage sample, sputum sample, blood sample, or any other sample is gathered from a subject at any time. In some embodiments, any characteristic of the sample contained on the dental device is compared to a saliva sample, gastric lavage sample, sputum sample, blood sample, or other sample of the subject. For example, the fungal and / or bacterial species diversity or species content of the sample contained on the dental device can be correlated with the fungal and / or bacterial species diversity in a saliva sample. In some examples, comparison of the species content between the sample contained on the dental device and a saliva sample may aid in determining any characteristic of pathogen release into the saliva from the microbiome and / or mycobiome contained on a tooth or other surface of the oral cavity. For example, a rate of pathogen release, the likeliest or least likely pathogen species to be released, or any other property of pathogen release may be determined. In some examples, the method may be used to better understand how any characteristics of any patient sample can be used to predict any characteristic of the oral microbiome or oral my cobiome of a subject. In some examples, any characteristic of the sample contained on the dental device is compared to any other sample derived from a subject to determine the correlation of characteristics between the oral microbiome and / or mycobiome sample and other patient samples.

[0110] In some embodiments of the method 100, a subject is diagnosed based on the sampled microbiome and / or mycobiome of the dental device. The subject may be diagnosed based on any characteristic of the sampled microbiome and / or mycobiome of the dental device. For example, the result of any analysis of the sampled microbiome and / or mycobiome may be used to diagnose a subject. The diagnosis may include any disease or condition. The diagnosis may include predicting future disease risk of a subject. In some embodiments, the diagnosis includes predicting a subject’s recovery from a disease, injury, or any medical procedure. In some embodiments, the diagnosis includes monitoring a subject’s recovery from a procedure or response to a treatment. In some embodiments, treatment of a subject is based on the diagnosis.

[0111] A diagnosis may relate to any oral disease including dental caries, endodontic infections, gingivitis, periodontitis, peri-implantitis, any mucosal disease including lichen planus and leukoplakia, oral cancer, and / or denture stomatitis. A diagnosis may relate to any disease including any metabolic disease, diabetes, any autoimmune disease, Alzheimer’s disease, any cardiovascular disease, cystic fibrosis, rheumatoid arthritis, any oral mucosal disease, invasive candidiasis, aspergillosis, and / or any cancer including pancreatic cancer, esophageal cancer, gut cancer, liver cancer, and / or colorectal cancer.

[0112] In some embodiments of the method 100, the microbiome and / or mycobiome of the dental device may be recovered and cultured. The recovered microbiome and / or mycobiome may be cultured for any length of time on any culture substrate. In some examples, any molecule or any number of molecules are administered to the culture of the recovered microbiome and / or mycobiome such that the effect of the molecule on any characteristic the sampled microbiome and / or mycobiome can be determined. For example, the effect of the molecule or molecules on the species contained in the sample or the diversity of species in the sample is determined. In some examples, the molecule or molecules are an antibiotic, an antifungal, any therapeutic, any sugar, or any molecules found in a potential diet or dietary supplement of a subject. In some embodiments, the subject is treated based on the response of a sampled microbiome and / or sampled mycobiome culture to an administered molecule or any number of administered molecules. For example, a subject may be prescribed an antibiotic or an antifungal or prescribed a diet based on the response of the culture to a molecule. In some embodiments, the culture may be grown and passaged onto any number of individual substrates or the culture may be apportioned into any number of small units such that any number of molecules can be screened for their effect on the culture.

[0113] The disclosures of each and every patent, patent application, and publication cited herein are hereby each incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0114] EXPERIMENTAL EXAMPLES

[0115] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0116] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore specifically point out exemplary embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

[0117] Example 1 : Comparison of the microbiome and mycobiome of crowns made from lithicum disilicate, composite resin, and 3D printed resin.

[0118] Microorganisms adhering to Ceramic Crown Resin compared to lithium disilicate and composite resin in the same oral environment were analyzed. The bacterial and fungal organisms on all samples were evaluated by comparing the 16S rRNA genes and the ITS2 regions on the samples. The study was the first to perform a microbiome analysis on dental crowns in patients. The bacterial (microbiome) and fungal (mycobiome) communities on temporary dental crowns in groups (N=10): lithium disilicate, composite resin, and 3D printed SprintRay Ceramic Resin were identified.

[0119] The findings provide new insights into how different crown materials affect the oral microbiome. Understanding these interactions is crucial for developing materials that support oral health and prevent disease. The results highlight the impact of 3D printed materials on the oral microbiome. The results allow the development of restorative options that do not disrupt the delicate balance of the vital oral microbiome and oral mycobiome ecosystem. A foundation for research on the microbiome associated with different dental materials is established. The findings emphasize the importance of considering microbial communities in dental restoration development.

[0120] The methods are now described.

[0121] Sample Collections

[0122] The study protocol was approved by the Medical University of South Carolina Institutional Review Board (IRB), ID: Pro00127365. Patients were recruited for the study using inclusion and exclusion criteria(Table 1, below). Patients with chronic diseases, periodontitis and / or rampant caries, and patients undergoing antibiotic treatment within 30 days were excluded from the study. Patients between the age of 18-75 years, male or female, with no systemic disease undergoing full arch rehabilitation with full coverage restorations were included. Informed consent was obtained. The teeth were prepared under local anesthesia and temporized using three different crowns materials: Group 1 (N=10): lithium disilicate (LD), Group 2 (N=10): Composite (Comp), and Group 3 (N=10): Printed Resin (Pr). The printed resin samples were placed into two subgroups based on the technique used to finish the crowns; Group 3.1 (N=5) printed polished (PP) and Group 3.2 (N=5) printed glazed (PG). The temporaries were then seated using 3M Durelon carboxylate luting cement. The patients had the temporary crowns for 4-6 weeks in their oral cavity. After this period the samples were removed, placed in sterile phosphate buffered saline (PBS) in a 15 ml sterile plastic tube, and stored at -80°C. The samples were shipped to Microbiome Insights Inc. (Vancouver, Canada) for microbiome and mycobiome analysis.

[0123] Table 1. A summary of the inclusion and exclusion criteria

[0124] Inclusion criteria Exclusion criteria

[0125] • Ages of 18-75 years • Patients with chronic diseases, • Male or Female periodontitis, and / or rampant caries • No systemic disease • Patients undergoing antibiotic • Undergoing full arch rehabilitation treatment within 30 days were with full coverage restorations excluded from the study

[0126]

[0127] Dental crown fabrication

[0128] The crowns were designed in Exocad and saved as standard tessellation language (STL) files, which were then used to make the samples. The LD samples were milled using a Roland DWX-42W mill and were milled from GC LiSi blocks. The LD samples were polished using a lathe and dental ceramic polish burs before being seated intraorally. The Comp samples were made by creating a clear stint from a 3D model of the digitally designed arch. Vaseline was used to cover the prepped tooth and the stint was then filled with 3M Filtek packable composite and seated on the preparation and light cured to form the temporary restorations. The temporary was then removed and 3M Filtek flowable composite was used to repair deficiencies, then the temporary was polished with Dentsply Sirona PoGo system before being seated intraorally. The Pr samples were printed on a SprintRay 55 printer using SprintRay Ceramic Crown resin. The samples were printed at a 0° angle with the occlusal surfaces parallel to the build platform. The samples were then washed in the SprintRay ProWash 2 using 95% isopropyl alcohol, and a hand wash was performed using 95% isopropyl alcohol to ensure all resin was removed from the intaglio surface. Then the samples were cured using SprintRay Procure 2 with the preprogrammed Ceramic Crown resin curing setting. The samples were then separated into subgroups depending on finishing technique. PP were polished using a lathe and polish wheel before being seated. The PG were glazed using GC Optiglaze and then light cured for 40 secs using a Vaio light prior to being seated intraorally. Table 2 breaks down and illustrates the sample groups (Table 2, below).

[0129] Table 2. A summary of the different groups and subgroups being used in the study Milled from GC Li Si Blocks and Group 1 Lithium Disilicate (LD) polished with a lathe and ceramic polish burs

[0130] Made using a combination of packable and flowable composite, and a clear Group 2 Composite (Comp)

[0131] stint to mold the composite over the prepped tooth Printed from SprintRay Ceramic crown resin using a SprintRay 55 Printed and Group 3 Printed Resin (Pr)

[0132] washing and curing according to the manufacturer Group 3.1 Printed Polished (PP) Finished with lathe and polish wheel Group 3.2 Printed Glazed (PG) Finished with GC Optiglaze

[0133]

[0134] Microbiome and Mycobiome analysis

[0135] The dental crowns were placed into a 15 ml sterile tube with glass beads and lysis buffer (MoBio PowerMag Soil DNA Isolation kit, VWR, Atlanta, GA), and bead beating was performed to extract genomic DNA from the biofilm attached to the dental crowns. DNA was extracted using MoBio’s instructions on a King Fisher robot. Bacterial 16S rRNA genes were PCR-amplified with dual-barcoded primers targeting the V4 region (515F 5’ - GTGCCAGCMGCCGCGGTAA-3’, and 806R 5’-GGACTACHVGGGTWTCTAAT-3’), as per the protocol of Kozich (Kozich JJ, et al. Appl Environ Microbiol. 2013;79: 5112-5120.). Fungal ITS2 region was sequenced on an Illumina MiSeq (v. 3 chemistry) using the dual barcoding protocol (Kozich JJ, et al. Appl Environ Microbiol. 2013;79: 5112-5120.). Primers (ITSF 5’-CCTCCGCTTATTGATATGC-3’, ITSR 5’-CCGTGARTCATCGAATCTTTG-3’) and PCR conditions used for 16S sequencing are identical to those of Kozich; those used for ITS2 sequencing were described by Gweon (Gweon HS, et al. Methods Ecol Evol. 2015;6: 973-980., Kozich JJ, et al. Appl Environ Microbiol. 2013;79: 5112-5120.). Amplicons were sequenced with an Illumina MiSeq (Figure 6) using the 300-bp paired-end kit (v.3). Sequences were denoised, taxonomically classified using Silva (v. 138) as the reference database, and clustered into 97%-similarity operational taxonomic units (OTUs) with the mothur software package (v. 1.48.0) (Schloss PD, Appl Environ Microbiol. 2009;75: 7537-7541.). The potential for contamination was addressed by cosequencing DNA amplified from specimens and from template-free controls (negative control) and extraction kit reagents processed the same way as the specimens. A positive control from ‘S00Z1-’ samples consisting of cloned SUP05 DNA was also included. Operational taxonomic units were considered putative contaminants (and were removed) if their mean abundance in controls reached or exceeded 25% of their mean abundance in specimens.

[0136] Data Analysis

[0137] The flowchart for bioinformatic analysis is depicted in Figure 7. Alpha diversity was estimated with the Shannon index on raw OTU abundance tables after filtering out contaminants. The significance of diversity differences was tested with ANOVAor linear mixed model depending on the study design. To estimate beta diversity across samples, OTUs occurring with a count of less than 3 in at least 5% of the samples were excluded and then Bray-Curtis indices were computed. Beta diversity, emphasizing differences across samples, was visualized using Principal Coordinate Analysis (PCoA) ordination. Vanation in community structure was assessed with permutational multivariate analyses of variance (PERMANOVA) with treatment group as the main fixed factor and using 999 permutations for significance testing. A dot-dash circle in the ordination plot represents the center of each cluster. All analyses were conducted in the R environment.

[0138] The results are now described.

[0139] Bacterial 16S rRNA Genes Sequence Curation and metric

[0140] 16Sv4 amplicons were generated from dental crown samples on a MiSeq. MiSeq-generated Fastq files were quality -filtered and clustered into 97% similarity operational taxonomic unit (OTUs) using the mothur software package. The resulting dataset had 2036 OTUs (not including those occurring total count < 3). An average of 37089 quality-filtered reads were generated per sample. Figure 8 illustrates the total number of quality reads filtered per sample. These reads reflect the total number of high-quality sequences that align with 16Sv4, clustered into OTUs and were assigned taxonomic classification. Any ambiguous or low-quality data were discarded from the subsequent analyses. High quality reads were classified using Silva v. 138.1 as the reference database. The OTUs were then aggregated into each taxonomic rank and plotted the relative abundance of the most abundant ones.

[0141] The read counts when examining the 16S rRNA gene were ranging from 9555 reads to 57398 reads, with an average of 37089 reads. Read counts for each sample are broken down in Table 3 (Table 3, below). Slight variations were noticed between the groups with the printed crowns seeming to have higher reads, followed by composite, and then finally lithium disilicate having the fewest reads.

[0142] Table 3. A table of each sample crown, the material type and the quantity of 16SrRNA gene reads that were found in the sample crown

[0143] Composite Lithium Disilicate Printed 27776 9555 30389 37425 20114 57398 45367 12570 32160

[0144]

[0145] 53679 26504 46322 30547 31168 56821 43815 45654 42846 33059 32800 42596 34674 17391 56705 30783 32227 46975 44009 36813 54533

[0146]

[0147] Bacterial 16S rRNA Genes Taxonomic Composition

[0148] High quality reads were classified using Silva v. 138.1 as the reference database. The OTUs were aggregated into each taxonomic rank and the relative abundance of the most abundant ones was plotted. Figure 9 shows the bacterial taxonomy breakdown of each sample into their relative phylum, class, order, family, and genus. The most dominant bacterial phyla identified in the samples were Firmicutes, Actinobacteriota, and Fusobacteriota. The most dominant bacterial classes identified were Bacilli and Actinobacteria. The most abundant orders appearing were Lactobacillales, Actinomycetales, and Fusobacteriales. The most abundant families identified were Streptococcaceae and Actinomycetaceae. The most abundant genera identified were Streptococcus and Actinomyces. These groups did vary between certain crowns exhibiting higher groups of other phyla, class, order, family, and genus and this could be due to each patient's individual microbiome inhabiting their oral cavity. It is expected that there is great variation in microbiomes between patients just as there is great variation in an individual's microbiome. This could be due to multiple host factors such as diet, pH of the mouth, oral hygiene, frequency of eating, genetics and many other environmental factors.

[0149] Bacterial 16S rRNA Genes Alpha Diversity

[0150] Alpha diversity (Shannon index) was computed and illustrated for each sample. This is a measure of richness (how many OTUs) and evenness (how evenly distributed these OTUS are) in a sample. Figure 10 is a Box and Whisker plot of the Shannon of each sample broken down into their 3 groups. Composite had a Shannon Mean of 2.896 with an SD of 0.585. Lithium Disilicate (LD) had a Shannon mean of 2.778 with a SD of 0.748. Printed had a Shannon mean of 2.683 and an SD of 0.488. An ANOVAtest was run and a p value of 0.744 was obtained. The ANOVA determined no significant differences in the Shannon diversity index. According to our study the type of material does not impact the bacterial diversity around the environmental niche of the dental crown meaning that none of the material tested selectively influences certain bacteria to dominate the niche.

[0151] Bacterial 16S rRNA Genes Beta Diversity

[0152] To obtain a graphical representation of microbiome composition similarity among samples the OTU abundances were summarized into Bray-Curtis dissimilarities and a pcoa ordination was performed (Figure 11). Permutational analysis of variance gave a p-value of 0.325 and determined no significant differences in beta-diversity between the groups. Since there is no significant diversity between the samples in a specific material group it indicates no material tested in having selection for specific bacteria.

[0153] Fungal ITS2 Region Sequence Curation and Metrics

[0154] Sequenced ITS2 amplicons were generated from dental crown samples on a MiSeq. MiSeq-generated Fastq files were quality-filtered and clustered into 97% similarity operational taxonomic unit (OTUs) using the mothur software package. The resulting dataset had 221 OTUs (not including those occurring total count < 3). An average of 50785 quality-filtered reads were generated per sample. The following box-and-whisker plot (Figure 12) illustrates the total number of quality filtered per sample. These reads reflect the total number of high-quality sequences that align with ITS2, clustered into OTUs and were assigned taxonomic classification. Any ambiguous or low-quality data were discarded from the subsequent analyses. The variation in the reads of the ITS2 region were much greater than the variation noted in the reads of the 16SrRNA gene. For the ITS2 region it ranged from 22 reads to 152198 reads (Table 4, below) Table 4. A table of each sample crown, the material type and the quantity of TTS2 sequence reads that were found in the sample crown

[0155] Composite Eithium Disilicate Printed

[0156] 95815 6235 94071

[0157] 114583 76850 2867

[0158] 125405 28952 49

[0159] 927 22 152198

[0160] 685 108 82417

[0161] 603 666 109187

[0162] 113363 56326 643

[0163] 129545 40904 4236

[0164] 12739 93825 87245

[0165]

[0166] 2167 89455 1476

[0167] Fungal ITS2 Region Taxonomic Composition

[0168] High quality reads were classified using UNITE (v. 8.1) as the reference database. OTUs were aggregated into each taxonomic rank and the relative abundance of the most abundant ones were plotted. Figure 13 shows the Taxonomy breakdown of each sample into their relative phylum, class, order, family, genus, and species. The most prevalent phylum was Ascomycota, the most prevalent class was Saccharomycetes, the most prevalent order was Saccharomycetales, the most prevalent family was Saccharomycetales incertae sedis, the most prevalent genus was Candida, and the most prevalent species was Candida duliniensis.

[0169] Fungal ITS2 Region Alpha Diversity

[0170] Alpha diversity (Shannon index) was computed and illustrated for each sample. This is a measure of richness (how many OTUs) and evenness (how evenly distributed these OTUs are) in a sample. Figure 14 is a box and whisker plot of the Shannon of each sample broken down into their 3 groups. Composite had a Shannon Mean of 0.823 with an SD of 0.849. LD had a Shannon mean of 0.823 with a SD of 0.885. Printed had a Shannon mean of 0.398 and an SD of 0.533. An ANOVAtest was run and a p value of 0.413 was obtained. The ANOVA determined no significant differences in the Shannon diversity index.

[0171] The alpha diversity, for fungi, between the different materials was found to have no significant differences which indicates that fungi do not preferentially adhere to any of the three materials tested.

[0172] Fungal ITS2 Region Beta Diversity

[0173] To obtain a graphical representation of microbiome composition similarity among samples, MicroBio insights Inc. summarized OTU abundances into Bray-Curtis dissimilarities and performed a pcoa ordination (Figure 15). Permutational analysis of variance gave a p-value of 0.925 and determined no significant differences in betadiversity. The results of the study show that diversity between the crowns within a material group had no significant difference which indicates none of the materials tested have unique fungal species selectively adhering to them.

[0174] A discussion of the results is now described.

[0175] The intricate relationship of the oral microbiome and how it relates to healthy and diseased states is still relatively unknown (Gamal A, et al. Cancers. 2022; 14. doi:10.3390 / cancersl4122875). It is known that a biofilm forms in the oral cavity creating a complex and interlinked community of bacteria, fungi, viruses, and other microorganisms whose interactions and presence can have beneficial and harmful effects on the host (Wade WG. Pharmacol Res. 2013 ;69 : 137-143., Deo PN, Deshmukh R. J Oral Maxillofac Pathol. 2019;23: 122-128, Willis JR, Gabaldon T. Microorganisms. 2020;8. doi: 10.3390 / microorganisms8020308, Gamal A, et al. Cancers. 2022;14. doi: 10.3390 / cancersl4122875). Skewing the Alpha and Beta-diversity of the environment can lead to overexpression of a few types of microorganisms that have the potential to cause diseased states such as caries, gingivitis, and periodontitis (Willis JR, Gabaldon T. Microorganisms. 2020;8. doi: 10.3390 / microorganisms8020308).

[0176] Understanding how restorative materials influence the microbiome is crucial for dentists, as certain materials can promote harmful microorganisms causing caries, gingivitis, periodontitis, or other systemic diseases. Kozmos found that various restorative materials had roughness comparable or less than tooth structure, with tetragonal poly crystalline zirconia ceramic being the smoothest and exhibiting the least bacterial adhesion over 10 hours (Kozmos M, et al. Molecules. 2021;26. doi:

[0177] 10.3390 / molecules26041152). Vo's study on lithium disilicate samples, manufactured via traditional press or computer-automated milling, showed significant differences in surface roughness and bacterial adherence, with a strong correlation between the two (Vo DT, et al. J Prosthet Dent. 2015; 114: 696-701.). Surface roughness and topography are key in dental biofilm studies, as increased roughness correlates with increased bacterial attachment due to more contact area (Kreve S, et al. J Esthet Restor Dent. 2022;34: 461-472., Song F, et al. J Dent Res. 2015;94: 1027-1034.). Finishing techniques also significantly influence the surface roughness of restorative materials (Aykent F, et al. J Prosthet Dent. 2010;103: 221-227.).

[0178] With the advent of 3D printing and new materials, it is imperative to study both the manufacturing and materials to ensure no negative impact on a patient's oral hygiene. Shim's comparison of mechanical properties and microbial adhesion of printed polymethyl methacrylate (PMMA) found that sample orientation affected both mechanical properties and microbial adhesion (Shim JS, et al. J Prosthet Dent. 2020;124: 468-475.). Yacob's study confirmed that print orientation impacts surface roughness and bacterial adhesion, and also found that printed denture resin could have lower microbial adhesion than heat-pressed resin (Yacob N, JProsthet Dent. 2023;130: 131. el-131. e7.).

[0179] Consequently, all sample crowns in the current study were printed in the same orientation to minimize variations due to different printing orientations.

[0180] Previous studies on the impact of dental restorative materials on microbes have often been conducted extra-orally in optimized environments or environments attempting to replicate intra-oral conditions. Some studies, like those by Bremer and Engle, examined the intra-oral impact of restorative materials on the microbiome using retainers with material samples, but these were removable and worn for up to 72 hours (Bremer F, et al. Quintessence Int. 2011;42: 565-574., Engel A-S, et al. BMC Oral Health. 2020;20: 162.). The current study replicated a real clinical scenario by using the materials as temporary crowns, allowing for continuous wear and simulating true daily use and intra-oral environments for the sample crowns.

[0181] The read counts of the 16S rRNA gene of the samples were strong across all samples. However, the ITS2 region read counts were more scattered within their respective groups, though the distribution between material groups was similar, possibly due to variations in fungal communities between patients. The taxonomic breakdown revealed high diversity, reflecting the highly diverse nature of the oral microbiome (Wade WG. Pharmacol Res. 2013;69: 137-143.). Individual differences in taxonomic diversity were expected due to unique oral microbiomes. Most samples across all groups were dominated by the genus Candida, consistent with reports that Candida and Malassezia are the most dominant oral fungi (Bandara HMHN, et al. Oral Dis. 2019;25: 363-371., Diaz PI, Dongari-Bagtzoglou A. IDentRes. 2021;100: 133-140., DiazPl, et al. Virulence. 2017;8: 313-323.). The absence of Malassezia could be due to the small patient pool.

[0182] C. dubliniensis was the most prevalent fungal species found on sample crowns, which is concerning since both C. albicans and C. dubliniensis are common during active oral infections. C. dubliniensis has been correlated with the severity of caries, whereas C. albicans is found in cases of severe caries (Diaz PI, Dongari-Bagtzoglou A. J Dent Res.

[0183] 2021 ; 100: 133-140.). Despite having lower virulence than C. albicans, C. dubliniensis can form mature biofilms faster and cover larger areas when combined with C. albicans in dual-species biofilms (Pathirana RU, Front Microbiol. 2019;10: 1188.).

[0184] Alpha diversity analysis showed no significant difference in bacterial or fungal diversity between crown materials, indicating that ceramic crown samples did not selectively promote adverse bacteria. Beta diversity analysis also showed no significant differences within material groups, indicating no unique microbial adherence to the ceramic crown resin group.

Claims

CLAIMSWhat is claimed is:

1. A method for interrogating the oral microbiome and oral my cobiome of a subject comprising the steps of:implanting one or more dental devices each having a target growth surface in an oral cavity of the subject;incubating the one or more dental devices in the oral cavity of the subject; removing at least a first portion of the target growth surface of at least one of the dental devices from the oral cavity of the subject at a first time point; and analyzing the microbiome and mycobiome present on the removed target growth surface.

2. The method of claim 1 further comprising the steps of:removing at least a second portion of the target growth surface from the oral cavity of the subject at a second timepoint; andanalyzing the microbiome and mycobiome contained on the at least second portion of the one or more dental devices.

3. The method of claim 1, wherein the one or more dental devices further comprise a permanent portion.

4. The method of claim 1, wherein implanting the one or more dental devices comprises adhering the one or more dental devices to a tooth.

5. The method of claim 1, wherein the at least first portion of the target growth surface of at least one of the dental devices is incubated in the oral cavity for a period of time less than 1 week, less than 2 weeks, less than 3 weeks, or less than 4 weeks.

6. The method of claim 1, wherein the analysis comprises sequencing the microbiome and mycobiome present on the removed target growth surface.

7. The method of claim 1, wherein the analysis comprises determining the species diversity of the microbiome and mycobiome present on the removed target growth surface.

8. The method of claim 1, further comprising the step of:determining a relationship between the microbiome and mycobiome present on the removed target growth and a characteristic of the subject based on the analysis.

9. The method of claim 1, further comprising administering a treatment to the subject based on the analysis.

10. A dental device comprising:a body having an exterior surface and configured to releasably engage a portion of an oral cavity of a subject; andat least one target growth surface on the exterior surface of the body; wherein the at least one target growth surface is configured to promote or enhance microbial growth thereon.

11. The dental device of claim 10, wherein the target growth surface is recessed.

12. The dental device of claim 10, wherein at least a portion of the dental device containing at least a portion of the target growth surface is detachable.

13. The dental device of claim 10, wherein the target growth surface is positively charged.

14. The dental device of claim 10, wherein the target growth surface comprises a resin or resin composite.

15. The dental device of claim 10, wherein the target growth surface is hydrophilic.

16. The dental device of claim 10, wherein the body further comprises a permanent portion, wherein the permanent portion is configured to inhibit microbial growth thereon.

17. The dental device of claim 16, wherein the permanent portion is negatively charged or neutral.

18. The dental device of claim 16, wherein the permanent portion is comprised of a ceramic.

19. The dental device of claim 16, wherein the permanent portion is hydrophobic.

20. The method of claim 1, wherein the one or more dental devices are the dental device of claim 10.

21. A dental implant comprising:a frame configured to releasably engage a portion of an oral cavity of a subject; anda plurality of sampling devices releasably secured to the frame, wherein each sampling device includes a target growth surface on an exterior surface of the sampling device; andwherein the target growth surface of each sampling device is configured to promote or enhance microbial growth thereon.

22. The dental implant of claim 21, further comprising at least one hinge.

23. The dental implant of claim 21, wherein the target growth surface is recessed.

24. The dental implant of claim 21, wherein the target growth surface is positively charged.

25. The dental implant of claim 21, wherein the target growth surface comprises a resin or resin composite.

26. The dental implant of claim 21, wherein the target growth surface is hydrophilic.

27. The dental implant of claim 21, further comprising:a connector portion;wherein the connector portion releasably secures at least one of the plurality of sampling devices to the frame; andwherein the connector portion is frangible.

28. The dental implant of claim 27, wherein the connector portion is perforated.

29. The dental implant of claim 21, wherein the target growth surface is configured to prevent or reduce contamination by a bonding agent.

30. A system for interrogating the oral microbiome and oral mycobiome of a subject comprising:the dental device of claim 10; anda dental device applicator tool comprising a base and a set of dental device engagement arms, wherein the dental device engagement arms are configured to grip at least a portion of the dental device and leave exposed a portion of the dental device.

31. A system for interrogating the oral microbiome and oral mycobiome of a subject comprising:the dental device of claim 10; anda dental device remover tool configured to grip at least a portion of the dental device.

32. A system for interrogating the oral microbiome and oral mycobiome of a subject comprising:the dental implant of claim 27; anda dental device remover tool configured to grip at least a portion of the connector portion.

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