Mouth on a chip

Abstract

A tissue model, comprising: a portion of gingival epithelium; a vascular network, the portion of gingival epithelium being superposed over the vascular network, and the vascular network being in fluid communication with the portion of vascular gingival epithelium; and at least one first model tooth organ, the at least one first model tooth organ contacting the portion of gingival epithelium, the at least one first model tooth organ optionally comprising hydroxyapatite, and optionally, a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ. Also provided are other tissue models as well as methods.

Description

MOUTH ON A CHIPRELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of United States patent application no. 63 / 549,972, “Mouth On A Chip,” filed February 5, 2024. All foregoing applications are incorporated herein by reference in their entireties for any and all purposes.TECHNICAL FIELD

[0002] The present disclosure relates to the field of tissue engineering and to the field of oral tissue models.BACKGROUND

[0003] Although existing tissue models provide users with the ability to recapitulate certain tissues and / or organs, existing models have not to date provided a model of the mouth that integrates the various tissues found in and near the mouth. Accordingly, there is a long-felt need in the art for such a model.SUMMARY

[0004] In meeting the described long-felt needs, the present disclosure provides a tissue model, comprising: a portion of gingival epithelium; a vascular network, the portion of gingival epithelium being superposed over the vascular network, and the vascular network being in fluid communication with the portion of vascular gingival epithelium; and at least one first model tooth organ, the at least one first model tooth organ contacting the portion of gingival epithelium, the at least one first model tooth organ optionally comprising hydroxyapatite, and, optionally, a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ.

[0005] Also provided is a modular tissue model, comprising: a first portion of gingival epithelium; a first model tooth organ that contacts the first portion of gingival epithelium and optionally comprises hydroxyapatite; and a second model tooth organcontacting the first portion of gingival epithelium, the second model tooth organ differing from the first model tooth organ in at least one of composition and size. The second tooth organ model can comprise hydroxyapatite.

[0006] Further provided is a modular tissue model, comprising: a first tissue module comprising a (i) first portion of gingival epithelium and (ii) a first model tooth organ comprising hydroxyapatite and contacting the first portion of gingival epithelium; a second tissue module comprising (i) a second portion of gingival epithelium and (ii) a second model tooth organ comprising hydroxyapatite and contacting the second portion of gingival epithelium; and a first vascular network, the vascular network in fluid communication with at least the first module.

[0007] Also provided is a method, comprising: contacting a first agent to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with a vascular network; and determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent.

[0008] Additionally provided is a method, comprising: contacting at least one microbe to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with the vascular network; and determining any one or more of a presence of the at least one microbe, an absence of the at least one microbe, or an effect of the at least one microbe.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals having different letter suffixes may represent different instances of similar components The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0010] FIGs. 1A-1Q provide example engineering of a Mouth on a chip (MOC) device. FIGs. 1 A-1B: Morphological and anatomical features of gingival tissue around thetooth. FIG. 1C: High magnification image demonstrates the vicinity of stratified gingival epithelial with tooth enamel and underling vascular tissue. FIG. ID: Demonstration of the natural feature of gingival tissue in MOC device as we have a vascularized tissue underneath the differentiated and stratified gingival epithelial cell. FIG. IE: Cross-section of MOC device. In MOC device the teeth are substitute with hydroxyapatite (main mineral component of tooth enamel). Micro beads locate at the edge of gingival tissue similar to configuration of teeth in our mouth. FIG. IF: To stablish the MOC device, after fabrication of the chip with casting and curing the PDMS in mold, lower chamber (1) is injected with a mixture of endothelial and gingival fibroblast in fibrin gel (2). After gelation of Fibrin, medium channels in lower chamber are seeded with endothelial cell s(3 ) . After 14 days of culture perfusable vascular network is formed (4). Next, gingival epithelial cells are seeded on porous membrane in upper chamber (5). After cells form a confluent layer, gingival epithelial cells are airlifted (5) and cultured for 12 days in Airliquid interface. In the last step microbeads are placed in MOC device (6). FIG. 1G: Topview and cross-sectional views of MOC device demonstrate different compartments of HOC device and different steps of preparation and culture of the device in single or array form. FIG. 1H: Images of vascular development in MOC over time from day one in form of single cell to day 14 in form of complete vessels. FIG. II: After 14 days of cultured a perfusable vascular network is formed in MOC device. FIGs. 1J-1M: Vascular network is characterized and the changes in total number of junction, vessel length, vessel area and average vessel diameter is quantified. FIGs. IN- IQ: Vascular network connectivity and perfusability is tested by perfusing the fluorescent dextran (70 kD) solution thorough the vessels and is imaged at different time point. High magnification images illustrates the vessels filled with fluorescent dye.

[0011] FIGs. 2A-2E Illustrates enablement of fibroblast growth factor binding activity. Acts upstream of or within positive regulation of cell population proliferation and positive regulation of fibroblast growth factor receptor signaling pathway. Located in cell surface and extracellular region. Is expressed in several structures, including alimentary system; brain; genitourinary system; meninges; and skin. Orthologous to human FGFBP1 (fibroblast growth factor binding protein 1). FIG. 2A: Top view of gingival epithelial cell stained with KRT14. FIG. 2B: Immunofluorescent staining of gingival epithelia cells at different time point of day 1, day 7, and day 14 of culture. Tissues are stained withdifferentiation markers antibodies of KRT14, Claudin, Occludin, ZO-1 at different time point and fluorescent intensity is quantified as a measure of differentiation. FIG. 2C: Cross sectional view of differentiated and stratified gingival tissue at day 14 stained with H&E, DAPI, CRT14, and ZO-1. TEER and Permeability of the gingival tissue at different time of day 1, 7, 14 is measured and plotted. FIG. 2D: Single-cell RNA sequencing of vascularized human gingival tissue from MOC device. UMAP projection of 13 clusters representing distinct cell populations in vascularized gingival tissue produced in MOC device. FIG. 2E: Top indicator gene utilized for clustering the cells.

[0012] FIGs 3A-3K provide example illustration of micro mechanisms of CA and SM growth and infection of gingival tissue and example disclosure related to Sma-seq of infected tissue with CA+SM.

[0013] FIGs. 4A-4I provide example disclosure related to infection of MOC with CA and SM separately.

[0014] FIGs. 5A-5H provide example disclosure related to CO VID infection to MOC tissues.

[0015] FIG.s 6A-6H provide example disclosure related to an MOC with saliva flow.

[0016] FIG. 7 provides example disclosure related to MOC devices.

[0017] FIG. 8 provides exemplary disclosure related to MOC devices.

[0018] FIG. 9 provides exemplary disclosure related to MOC devices, showing a perfusable vascular network connected with gingival epithelial culture.

[0019] FIG. 10 provides exemplary disclosure related to MOC devices, showing a tooth surrogate (model tooth organ) contacting epithelial tissue connected to tissue- engineered blood vessels.

[0020] FIG. 11 provides exemplary disclosure related to model tooth organs.

[0021] FIG. 12 provides exemplary disclosure related to model tooth organs with various coatings

[0022] FIG. 13 provides exemplary disclosure related to performing multiplexed analyses on the sane MOC, as different tooth organs can be used to assess different agents and / or conditions.

[0023] FIG. 14 provides exemplary disclosure related to using HA beads as model tooth organs.

[0024] FIG. 15 provides exemplary disclosure related to using imaging to study biofilm dynamics in a MOC.

[0025] FIG. 16 provides exemplary disclosure related to cross-kingdom (bacteria, fungi) development in a MOC.

[0026] FIG. 17 provides exemplary disclosure related to cross-kingdom (bacteria, fungi) development in a MOC.

[0027] FIG. 18 provides exemplary disclosure related to cross-kingdom (bacteria, fungi) development in a MOC.

[0028] FIG. 19 provides exemplary disclosure related to tracking fungal growth and spreading in a MOC.

[0029] FIG. 20 provides exemplary disclosure related to tracking detachment in a MOC

[0030] FIG. 21 provides exemplary disclosure related to tracking detachment in a MOC

[0031] FIG. 22 provides exemplary disclosure related to tracking offspring release and spreading in a MOC.

[0032] FIG. 23 provides exemplary disclosure related to tracking offspring release and spreading in a MOC

[0033] FIG. 24 provides exemplary disclosure related to MOC systems.

[0034] FIGs. 25A-25H provide metabolomic analysis of the epithelial tissue of VMOC infected with Ca FIG. 25 A: Schematic demonstrating the process of sample collection from vascular compartment of VMOC infected with Ca and the instrument that was used for global untargeted metabolite profiling of vascular compartment in infected VMOC versus healthy VMOC. FIG. 25B: Principal component analysis plot illustrates the distribution of quantifiable metabolites (n = 596) from five biological replicates for each tested condition. Each data point denotes an individual biological replicate. FIG. 25C: Heatmap depicting the metabolites that significantly changed as the result of Ca infection (T-test / ANOVA). Metabolite types are color-coded in their labels -peptide metabolism (blue), lipids (brown), carbohydrate metabolism (green), and nucleotide metabolism (purple). The color gradient of the scale bar indicates the relative abundance of metabolites, with red and blue representing higher and lower concentrations, respectively FIG 25D: Comparison of normalized concentrations of select metaboliteswhich their levels significantly changed under Ca infection. FIG. 25E: Scatter plot highlighting significantly changed metabolic pathways identified by pathway impact analysis of metabolites that their levels changed under Ca infection. FIG 25F: List of the top 25 metabolic pathways that significantly altered due to Ca infection compared to healthy VMOC. The p-value is displayed in a negative loglO scale. FIG. 25G: ROC curves for biomarker prediction models aiming to distinguish between Ca infected metabolites versus healthy condition. Different colors represent models with varying numbers of constituent metabolite features FIG. 25H: The list of top metabolites generated by the 25-feature prediction model is shown in g and is ranked based on their predictive accuracy. FIG. 251: Box plots demonstrate change in level of selected metabolites (highlighted in red) from the list of top 25-feature prediction models that can be used as markers of Ca infection in VMOC. N = 5 for each group, *P < 0.05, **P < 0.01, ***P < 0.001.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0035] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0037] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of' and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words thatrequire the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of and "consisting essentially of the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps

[0039] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0040] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0041] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0042] As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In atleast some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9- 1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4.

[0043] Further, the term “comprising” should be understood as having its open- ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

[0044] Any embodiment or aspect provided herein is illustrative only and does not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more embodiments or aspects can be combined with any part or parts of any one or more other embodiments or aspects.

[0045] Aspects

[0046] The following embodiments are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.

[0047] Aspect 1. A tissue model, comprising: a portion of gingival epithelium; a vascular network, the portion of gingival epithelium being superposed over the vascular network, and the vascular network being in fluid communication with the portion of vascular gingival epithelium; and at least one first model tooth organ, the at least one first model tooth organ contacting the portion of gingival epithelium, the at least one first model tooth organ optionally comprising hydroxyapatite, and, optionally, a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ. An example such tissue model is shown in FIG 1, which figure also provides an exemplary method for forming such a tissue model.

[0048] Without being bound to any particular theory or embodiment, the disclosed tissue model is unique in that the model includes both gingival tissue and a tooth organ, as well as vascular tissue. It is desirable to include these kinds of tissue in a model. For example, as an infection sometimes starts in the tooth organ and spreads into the other tissue and vascular tissue, and an accurate model of such tissues provides the best way to study such spreading.

[0049] It should be understood that one can use the techniques and approaches described herein to generate a network of lymphatic vessels, as opposed to blood vessels. It should also be understood that the vascularized tissue also contains other types of cells, such as fibroblasts, whose activity can be perturbed or modulated by external stimuli for certain applications; as an example, fibroblasts can become activated in the event of injury to repair wound or dysregulation of this activation process can lead to scarring and fibrosis. Similarly, the disclosed models can be engineered in such a way that blood-borne cells are included in the perfusate moving through the blood vessels and / or lymphatic vessels.

[0050] Aspect 2. The tissue model of Aspect 1, wherein the vascular network is disposed in a first channel, and the first channel is in fluid communication with at least one supply channel. The supply channel can carry a nutritive media or other media. Such media can be perfused into the vascular network.

[0051] Aspect 3. The tissue model of Aspect 2, further comprising comprises a separator, the separator disposed between the vascular network and the portion of gingival epithelium. A separator can be, for example, a porous membrane, such as a polystyrene porous membrane. Pores can be in the range of, for example, about 0.4 to about 12 pm.

[0052] In one non-limiting embodiment, one can use a semi-permeable membrane made out of polystyrene that contains 400nm, lum, 3um, or 8 um pores. Pore size can be varied depending on the user’s objectives. As an example, for the purposes of generating tissues, for instance, a pore size of 400 nm can be suitable. If the objective were to mimic the process of white blood cells being recruited from blood vessels to the site of infection or inflammation, one might use pore sizes of 3-8 pm to allow those cells to pass through.

[0053] Aspect 4. The tissue model of any one of Aspects 1-3, wherein the tissue model further comprises a frame configured to maintain the at least one first model toothorgan in position relative to the portion of gingival epithelium. An example such frame is shown in FIG. 1; a frame can include grooves or other recesses to maintain a model tooth organ in position.

[0054] It should be understood, however, that one can use other types of oral tissues, including, for example, tongue epithelium with chemoreceptors, and skin epithelium as would be found on the surface of lips.

[0055] Aspect 5. The tissue model of any one of Aspect 1-4, wherein the at least one first model tooth organ comprises a hydroxyapatite particle.

[0056] Aspect 6. The tissue model of any one of Aspects 1-4, wherein the at least one first model tooth organ is any one or more of (i) at least partially coated with hydroxyapatite, (ii) configured to resemble any one or more of a dental implant, a crown, and a dental restoration, (iii) at least partially coated with an antimicrobial coating, (iv) at least partially coated with an antifouling material, (v) at least partially coated with any one or more of a fungus, a bacteria, and a virus. As an example, a model tooth organ can comprise a polymeric or other particle that is at least partially coated with hydroxyapatite.

[0057] Aspect 7. The tissue model of any one of Aspects 1-6, wherein the tissue model further comprises any one or more of a fungus, a bacteria, and a virus, the fungus optionally including any one or more of a Candida genus fungus, Aspergillus, Malassezia, Cladosporium, Aureobasidium, Saccharomyces, Fusarium, Cryptococcus, Penicillium, Schizophyllum, Rhodotorula, and Gibberella, the bacteria optionally including any one or more of Firmicutes (genera Streptococcus and Granulicatella), Fusobacteria (genus Fusobacterium), Proteobacteria (genera Neisseria and Haemophilus), Actinobacteria (genera Corynebacterium, Rothia, and Actinomyces), Bacteroidetes (genera Prevotella, Capnocytophaga, and Porphyromonas), and the virus optionally comprising any one or more of SARS, influenza, HIV, and HPV.

[0058] Aspect 8. The tissue model of Aspect 7, wherein the at least one of the fungus, virus, or bacteria is disposed on the gingival epithelium The at least one of the fungus, virus, bacteria can also be disposed on or in the vascular network. For example, a fungus and a bacteria can be disposed on or in the vascular network; two different fungi can be disposed on the vascular network.

[0059] Aspect 9. The tissue model Aspect 7, wherein the at least one of the fungus, virus, or bacteria is disposed on the at least one first model tooth organ. As anexample, a fungus and a virus can be disposed on the model tooth organ. As another example, two different fungi can be disposed on the model tooth organ.

[0060] Aspect 10. The tissue model of any one of Aspects 1-9, wherein the tissue model further comprises a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ, the fluid optionally being at least one of saliva, a saliva substitute, or gingival crevicular fluid (GCF). Other fluids include, for example, PBS, cell culture medium, and the like A fluid can also be a mouthwash, an antimicrobial, and the like.

[0061] A fluid can include media or saliva containing chemicals used in consumer products, such as toothpaste, whitening strips, and the like. A fluid can include orally administered drugs; dietary supplements - such as vitamins; and food additives - such as preservatives and food coloring. A fluid can include tobacco or electronic cigarette smoke extracts, alcohol, as well as gaseous compounds used in clinical settings or even in the settings of occupational or environmental exposures, tragic accidents, as well as media or saliva containing their biochemical byproducts.

[0062] Such a fluidic train can include, for example, a pump, tubing, and the like. A fluidic train can be configured to deliver and / or withdraw fluid.

[0063] Aspect 11 The tissue model of any one of Aspects 1-10, wherein the tissue model further comprises a second model tooth organ, the second model tooth organ differing from the first model tooth organ in at least one of composition and size.

[0064] Aspect 12. The tissue model of any one of Aspects 1-11, wherein the first tooth organ is configured to resemble at least one of a dental implant, crown and a dental restoration. A tooth organ can include, for example, resin, ceramic, and / or metal. In this way, one can recapitulate a dental implant, crown or dental restoration, which dental implant, crown or dental restoration can be incorporated into the disclosed tissue model.

[0065] Aspect 13 The tissue model of any one of Aspects 1-12, wherein the tissue model is in fluid communication with a tissue model that recapitulates tissue other than oral tissue. Such a tissue model can be, for example, a model of respiratory tissue, a model of placental tissue, salivary gland, lung, and the like. In this way, one can introduce an agent to a tissue model according to the present disclosure and evaluate the effect of that agent on another simulated organ or tissue in communication with the tissue model.

[0066] Aspect 14. A modular tissue model, comprising: a first portion of gingival epithelium; a first model tooth organ that contacts the first portion of gingival epithelium and optionally comprises hydroxyapatite; and a second model tooth organ contacting the first portion of gingival epithelium, the second model tooth organ differing from the first model tooth organ in at least one of composition and size. The second tooth organ model can comprise hydroxyapatite.

[0067] As an example, the first model tooth organ can include a first coating, and the second model tooth organ can include a second coating. A coating can be, for example, antimicrobial, a chemical - such as a varnish or a sealant; a coating can also include one or more of a fungus, a bacteria, and a virus. The coatings on different tooth organ models can differ from one another; as but one example, the first coating can harbor bacteria, and the second can harbor bacteria and fungi. Also, different materials, for example the first can be tooth and second could be an implant or crown.

[0068] Aspect 15. A modular tissue model, comprising: a first tissue module comprising a (i) first portion of gingival epithelium and (ii) a first model tooth organ comprising hydroxyapatite and contacting the first portion of gingival epithelium; a second tissue module comprising (i) a second portion of gingival epithelium and (ii) a second model tooth organ comprising hydroxyapatite and contacting the second portion of gingival epithelium; and a first vascular network, the vascular network in fluid communication with at least the first module.

[0069] Thus, one can construct two or more gingival / tooth organ compartments that share a common vascular bed, in which case communication between the different oral compartments would be mediated by the common vasculature.

[0070] It should be understood that one can use a vascular network; one can also use a fluidic conduit lined with vascular endothelial cells or even acellular tubing.

[0071] In this way, one can construct a multiplexed tissue model that includes multiple MOCs MOCs of the multiplexed model can be in fluid communication with one another, but this is not a requirement, as one can construct a multiplexed tissue model that includes two or more independent MOCs. One can thus construct a model that includes a first MOC having certain characteristics - such as the presence of a certain antimicrobial - and a second MOC having certain other characteristics - such as model tooth organs that are formulated to represent a decayed state - and evaluate the effect of the same ordifferent agents on the different MOCs. Thus, one can construct a system that includes two or more MOCs, each of which MOCs includes tooth organ models, gingival tissue, and underlying vessels. A tooth organ model can comprise a dental implant, a crown, and the like; a tooth organ model can include abiotic materials and can even be abiotic in shape or other configuration. These separate MOCs can, for example, be connected simply by a tube that does not contain any cells or by a fabricated fluidic conduit or network that can be lined with cells. One can thus, for example use several MOCs in tandem or even in parallel to evaluate multiple variable and multiple conditions simultaneously.

[0072] Aspect 16. The tissue model of Aspect 15, wherein the first vascular network is further in fluid communication with the second module.

[0073] Aspect 17. The tissue model of any one of Aspects 15-16, wherein the modular tissue model further comprises a second vascular network, the second vascular network being in fluid communication with the second module.

[0074] Aspect 18. A method, comprising: contacting a first agent to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with a vascular network; and determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent.

[0075] The methods can be useful in, for example, evaluating therapeutic activity or cytotoxicity of different agents Such agents can be, for example, materials or solutions. The methods can also be useful in pharmaceutical screening of new drugs or medicine.

[0076] Aspect 19. The method of Aspect 18, wherein the first agent comprises at least one of an antimicrobial agent, an anti-inflammatory agent, an immunomodulatory agent, a microbe, and a biologic. Biologies can include, for example, enzymes, prebiotics, probiotics, and the like. In some embodiments, one or more microbes - apart from the agent - is present on one or more of the first portion of gingival epithelium, the first model tooth organ, and the vascular network.

[0077] Aspect 20. The method of any one of Aspects 18-19, further wherein contacting the first agent comprises at least one of contacting the first agent to the firstmodel tooth organ, introducing the first agent to a fluid contacting the first model tooth organ, and contacting the first agent to the first portion of gingival epithelium.

[0078] Aspect 21. The method of any one of Aspects 18-20, wherein the method further comprises contacting a second agent to at least one of (i) the first portion of gingival epithelium, (ii) the first model tooth organ, and (iii) the vascular network.

[0079] Aspect 22. The method of any one of Aspects 18-21, wherein the method further comprises contacting a second agent to at least one of (i) a second portion of gingival epithelium and (ii) a second model tooth organ, the second model tooth organ in contact with the second portion of gingival epithelium.

[0080] Aspect 23. The method of Aspect 22, wherein the method further comprises determining any one or more of a presence of the second agent, an absence of the second agent, or an effect of the second agent.

[0081] Aspect 24. The method of any one of Aspects 18-23, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect of a microbe on any one or more of the first portion of the gingival epithelium, the gingival tissue, and the vascular network.

[0082] Microbes can be, for example, bacteria, fungi, and viruses. One can use multiple microbes; such multiple microbes can be microbes of the same time and / or microbes of different types. As an example, one can utilize two bacteria. One can also intermix a bacteria and a fungus. All combinations and permutations of microbes can be utilized.

[0083] Aspect 25. The method of any one of Aspects 18-24, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an immune response from at least one of the first portion of gingival tissue and the vascularized network in response to a microbe.

[0084] Aspect 26. The method of any one of Aspects 18-25, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect on a microbe from an immune response of at least one of the first portion of gingival tissue and the vascularized network.

[0085] Aspect 27. The method of any one of Aspects 18-26, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining a presence of a microbe in at least one of the gingival epithelium and the vascular network.

[0086] Aspect 28. The method of any one of Aspects 18-27, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect of the first agent on at least one oral microbe. As described elsewhere herein, the at least one oral microbe can comprise any one or more of a bacteria, a fungus, and a vims.

[0087] Aspect 29. The method of any one of Aspects 18-28, wherein at least one of the first portion of gingival epithelium and vascular network is in fluid communication with a tissue model that recapitulates tissue other than oral tissue.

[0088] In some embodiments, the methods can include delivering a fluid - such as saliva or a buffer - to any one or more of the gingival epithelium, the model tooth organ, and the vascular network. One can, for example, change the rate of saliva or media flow to simulate fluid pressure and shear stress generated during chewing or other oral tissue movements.

[0089] Aspect 30 A method, comprising: contacting at least one microbe to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with the vascular network; and determining any one or more of a presence of the at least one microbe, an absence of the at least one microbe, or an effect of the at least one microbe. In this way, one can evaluate the effect of a microbe on any one or more of (i) the first portion of gingival epithelium (ii) the first model tooth organ, and (iii) the vascular network. One can also evaluate the effect of an agent - such as a mouth rinse - and / or the effect of a material coating or other variable - such as the material from which a tooth organ model is made - on the microbe.

[0090] Aspect 31. The method of Aspect 30, wherein the microbe comprises any one or more of a bacteria, a fungus, and a virus.

[0091] Aspect 32. The method of any one of Aspects 30-31, wherein an agent is present on any one or more of (i) the first portion of gingival epithelium (ii) the first model tooth organ, and (iii) the vascular network.

[0092] Aspect 33. The method of any one of Aspects 30-32, wherein the method further comprises contacting a treatment to the at least one microbe.

[0093] Aspect 34. The method of Aspect 33, wherein the treatment resides on any one or more of (i) the first portion of gingival epithelium (ii) the first model tooth organ, and (iii) the vascular network.

[0094] The disclosed technology thus allows one to study a number of different situations. One can, for example, use the disclosed models and technology to evaluate the effect on one or more microbes of any one or more (1) an agent, such as a mouth rinse; (2) a coating - such as an antimicrobial coating; and (3) a material - such as an anti-fouling surface. One can add and subtract variables - such as subjecting a microbe first to a mouth rise and then to a mouth rinse and an antibacterial coating to determine the effect of adding, subtracting, and changing variables.

[0095] The disclosed technology also allows one to study the effect of one or more microbes on a variety of subjects. For example, one can study the effect of one or more microbes on any one or more of a tooth organ model - such as a hydroxyapatite particle; gingival tissue; and vasculature. I

Claims

What is Claimed:

1. A tissue model, comprising: a portion of gingival epithelium; a vascular network, the portion of gingival epithelium being superposed over the vascular network, and the vascular network being in fluid communication with the portion of vascular gingival epithelium; and at least one first model tooth organ, the at least one first model tooth organ contacting the portion of gingival epithelium, the at least one first model tooth organ optionally comprising hydroxyapatite, and optionally, a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ2. The tissue model of claim 1, wherein the vascular network is disposed in a first channel, and wherein the first channel is in fluid communication with at least one supply channel3. The tissue model of claim 2, further comprising a separator, the separator disposed between the vascular network and the portion of gingival epithelium.

4. The tissue model of any one of claims 1-3, further comprising a frame configured to maintain the at least one first model tooth organ in position relative to the portion of gingival epithelium.

5. The tissue model of any one of claims 1-3, wherein the at least one first model tooth organ comprises a hydroxyapatite particle.

6. The tissue model of any one of claims 1-3, wherein the at least one first model tooth organ i any one or more of (i) at least partially coated with hydroxyapatite, (ii) configured to resemble any one or more of a dental implant, a crown, and a dental restoration, (iii) at least partially coated with an antimicrobial coating, (iv) at least partially coated with an antifouling material, (v) at least partially coated with any one or more of a fungus, a bacteria, and a virus.

7. The tissue model of any one of claims 1-3, further comprising any one or more of a fungus, a bacteria, and a virus, the fungus optionally including any one or more of a Candida genus fungus, Aspergillus, Malassezia, Cladosponum, Aureobasidium, Saccharomyces, Fusarium, Cryptococcus, Penicillium, Schizophyllum, Rhodotorula, and Gibberella, the bacteria optionally including any one or more of Firmicutes (genera Streptococcus and Granulicatella), Fusobacteria(genus Fusobacterium), Proteobacteria (genera Neisseria and Haemophilus), Actinobacteria (genera Corynebacterium, Rothia, and Actinomyces), Bacteroidetes (genera Prevotella, Capnocytophaga, and Porphyromonas), and the virus optionally comprising any one or more of SARS, influenza, HIV, and HPV.

8. The tissue model of claim 7, wherein the at least one of the fungus, virus, bacteria is disposed on the gingival epithelium.

9. The tissue model of claim 7, wherein the at least one of the fungus or bacteria is disposed on the at least one first model tooth organ.

10. The tissue model of any one of claims 1-3, further comprising a fluidic train configured to deliver a fluid to at least one of the portion of gingival epithelium and the at least one first model tooth organ, the fluid optionally being at least one of saliva, a saliva substitute, or gingival crevicular fluid (GCF).

11. The tissue model of any one of claims 1-3, further comprising a second model tooth organ, the second model tooth organ differing from the first model tooth organ in at least one of composition and size.

12. The tissue model of any one of claims 1-3, wherein the at least one first model tooth organ i configured to resemble at least one of a dental implant and a dental restoration.

13. The tissue model of any one of claims 1-3, wherein the tissue model is in fluid communication with a tissue model that recapitulates tissue other than oral tissue.

14. A modular tissue model, comprising: a first portion of gingival epithelium; a first model tooth organ comprising contacting the first portion of gingival epithelium and the first model tooth organ optionally comprising hydroxyapatite; and a second model tooth organ contacting the first portion of gingival epithelium, the second model tooth organ differing from the first model tooth organ in at least one of composition and size.

15. A modular tissue model, comprising: a first tissue module comprising a (i) first portion of gingival epithelium and (ii) a first model tooth organ contacting the first portion of gingival epithelium and optionally comprising hydroxyapatite; a second tissue module comprising (i) a second portion of gingival epithelium and (ii) a second model tooth organ and contacting the second portion of gingival epithelium; and a first vascular network, the first vascular network in fluid communication with at least the first tissue module.

16. The modular tissue model of claim 15, wherein the first vascular network is further in fluid communication with the second tissue module.

17. The modular tissue model of claim 15, further comprising a second vascular network, the second vascular network being in fluid communication with the second tissue module.

18. A method, comprising: contacting a first agent to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with the vascular network; and determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent19. The method of claim 18, wherein the first agent comprises at least one of an antimicrobial agent, an anti-inflammatory agent, an immunomodulatory agent, a microbe, and a biologic.

20. The method of any one of claims 18-19, wherein contacting the first agent comprises at least one of contacting the first agent to the first model tooth organ, introducing the first agent to a fluid contacting the first model tooth organ, and contacting the first agent to the first portion of gingival epithelium.

21. The method of any one of claims 18-19, further comprising contacting a second agent to at least one of (i) the first portion of gingival epithelium, (ii) the first model tooth organ, and (iii) the vascular network.

22. The method of any one of claims 18-1 , further comprising contacting a second agent to at least one of (i) a second portion of gingival epithelium and (ii) a second model tooth organ, the second model tooth organ in contact with the second portion of gingival epithelium.

23. The method of claim 22, further comprising determining any one or more of a presence of the second agent, an absence of the second agent, or an effect of the second agent.

24. The method of any one of claims 18-19, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect of a microbe on any one or more of the first portion of gingival epithelium, the first model tooth organ, the gingival epithelium, and the vascular network.

25. The method of any one of claims 18-1 , wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an immune response from at least one of the first portion of gingival tissue and the vascularized network in response to a microbe.

26. The method of any one of claims 18-19, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect on a microbe from an immune response of at least one of the first portion of gingival tissue and the vascularized network.

27. The method of any one of claims 18-19, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining a presence of a microbe in at least one of the gingival epithelium and the vascular network.

28. The method of any one of claims 18-19, wherein determining any one or more of a presence of the first agent, an absence of the first agent, or an effect of the first agent comprises determining an effect of the first agent on an oral microbe.

29. The method of any one of claims 18-19, wherein at least one of the first portion of gingival epithelium and vascular network is in fluid communication with a tissue model that recapitulates tissue other than oral tissue.

30. A method, comprising: contacting at least one microbe to at least one of (i) a first portion of gingival epithelium (ii) a first model tooth organ, and (iii) a vascular network, the a first model tooth organ in contact with the first portion of gingival epithelium, and the first portion of gingival epithelium being in fluid communication with the vascular network; and determining any one or more of a presence of the at least one microbe, an absence of the at least one microbe, or an effect of the at least one microbe.

31. The method of claim 30, wherein the microbe comprises any one or more of a bacteria, a fungus, and a virus.

32. The method of any one of claims 30-31, wherein an agent is present on any one or more of (i) the first portion of gingival epithelium (ii) the first model tooth organ, and (iii) the vascular network.

33. The method of any one of claims 30-31, further comprising contacting a treatment to the at least one microbe.

34. The method of claim 33, wherein the treatment resides on any one or more of (i) the first portion of gingival epithelium (ii) the first model tooth organ, and (iii) the vascular network.

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