Patent Grant
11400027
The invention relates to a kit and a probe for detecting porous dental hydroxyapatite and a method for detecting a condition involving porous dental hydroxyapatite.
The resilience of teeth depends on a complex interplay between mineral (termed hydroxyapatite) and organic components (proteins, cells and tissues). Under normal conditions the hydroxyapatite in enamel and dentine is organised into an extraordinarily dense structure that confers the hardness and toughness required for maintenance of the tooth's integrity. Loss of mineral-density in enamel and dentine results in abnormally porous hydroxyapatite, which compromises the tooth's physical resilience and can lead to structural failure. Porous hydroxyapatite is caused by several prevalent conditions, including dental caries and developmental dental defects (DDD).
Dental caries (tooth decay) is a disease caused by bacteria that secrete acid. The acid produced by cariogenic bacteria can dissolve hydroxyapatite in a process termed demineralisation. The initial process of demineralisation (termed incipient caries) leads to discrete regions of porous hydroxyapatite termed white spot lesions. Over time, a white-spot lesion may progress to a cavity (i.e. loss of tooth material) or it may stall (termed inactive caries) and re-form a dense hydroxyapatite shell in a process called remineralisation. Before a cavity forms, the process is reversible (i.e. remineralisation), but once enamel is lost it cannot be regenerated.
Caries is diagnosed by a combination of visual inspection, physical challenge (e.g. scratching with dental probe), and X-ray radiography (to detect caries between teeth or beneath the gum line). Worryingly, these diagnostic approaches miss approximately half of early caries, and up to 13% of teeth diagnosed as carious with these methods are in fact caries-free. Recent attempts at improving diagnosis include use of equipment that measures electrical impedance, quantitative light-induced fluorescence (QLF) and infrared laser fluorescence (DIAGNOdent®), but none have found widespread use because of the cost and size of apparatus, and problems with inter-individual variation. Another approach has been the use of dyes to detect dental caries in dentine. However, these dyes are not selective for porous hydroxyapatite: they bind to proteins (presumed to be associated with infecting bacteria in dentine) or they occupy interstitial space, which reduces specificity and sensitivity. Moreover, these dyes cause the oral cavity to become discoloured, bind to healthy teeth, or require visualisation with an irradiator.
There are two main treatments for caries, the selection of which is dictated by the extent of disease. White spot lesions may be treated with remineralisation approaches (e.g. fluoride therapy or amorphous calcium phosphate stabilised with bioactive molecules). Cavities require conventional restorative dentistry (i.e. fillings).
DDD are another common cause of porous hydroxyapatite. They are disturbingly prevalent and costly, potentially afflicting over 50% of the population with multiple burdens including dental pain, disfigurement and increased caries risk. The two most prevalent DDD are dental fluorosis (characterised by diffuse opacities) and Molar/Incisor Hypomineralisation (MIH; characterised by demarcated opacities); both are caused by environmental agents (i.e. acquired defects). Another serious but rare DDD that can result in porous hydroxyapatite is the genetic disease amelogenesis imperfecta.
MIH typically affects 10-20% of children and is a major risk factor for caries, a risk factor for orthodontics, and is costly to society. MIH is thought to result from a multifactorial systemic disturbance of the enamel-forming cells. However, other than being dissociated from fluoride and linked to illness during infancy, the cause of MIH remains a mystery.
There are currently no products available that are designed to diagnose and repair MIH or other DDD. Differential diagnosis of caries and various DDD can be difficult and is largely dependent upon the experience and skill of individual dental health professionals. Current procedures and/or products developed for remineralisation of caries do not work well on MIH. Restorative treatment is frequently compromised because MIH enamel is soft, porous and poorly delineated from normal tooth tissue.
Accordingly, a need exists for new tools to diagnose, delineate and repair porous dental hydroxyapatite caused by caries and DDD. Here we address this need by detailing new technologies based on our recent discoveries of pathogenic mechanisms in conditions involving porous hydroxyapatite.
A first aspect provides a kit, when used for detecting porous dental hydroxyapatite, comprising: a protein capable of binding porous dental hydroxyapatite; or a detector that detects said protein bound to porous dental hydroxyapatite.
A second aspect provides a probe, when used for detecting porous dental hydroxyapatite, comprising: a protein capable of binding to porous dental hydroxyapatite; and a reporter.
A third aspect provides a method for producing the probe of the second aspect comprising linking (i) a protein capable of binding to porous dental hydroxyapatite and (ii) a reporter.
A fourth aspect provides a method for detecting a condition involving porous dental hydroxyapatite comprising detecting in or on a tooth or a sample of the tooth of a subject a protein bound to porous dental hydroxyapatite.
A fifth aspect provides a method for detecting a hypomineralisation DDD comprising detecting a protein whose concentration bound to test hydroxyapatite of a tooth or of a sample of the tooth is increased relative to its concentration bound to control hydroxyapatite of a control tooth or of a control sample of a tooth, and detecting amelogenin whose concentration bound to the test hydroxyapatite is near that bound to the control hydroxyapatite.
A sixth aspect provides a method for detecting intact and/or broken MIH enamel comprising detecting albumin and hemoglobin bound to MIH hydroxyapatite, wherein detection of albumin but not hemoglobin is indicative of intact MIH enamel, and wherein detection of hemoglobin is indicative of broken MIH enamel.
A seventh aspect provides a kit for removing a protein bound to porous dental hydroxyapatite comprising: (a)(i) one or more washing solutions or (ii) dry components to prepare one or more washing solutions upon admixture with water, wherein the one or more washing solutions are adapted to remove a protein bound to porous dental hydroxyapatite; and (b) a remineralisation agent or remedial mineralisation agent.
An eighth aspect provides a method for removing a protein bound to porous dental hydroxyapatite comprising washing a tooth or a sample of the tooth with one or more washing solutions.
A ninth aspect provides a kit for removing a protein bound to porous dental hydroxyapatite comprising: one or more washing solutions; or one or more dry components to prepare one or more washing solutions upon admixture with water, wherein the one or more washing solutions are adapted to remove a protein in or on a tooth or a sample of the tooth detected as having porous dental hydroxyapatite by the method of the fourth aspect.
The kit, probe or methods of the first to sixth aspects allow detection in situ or diagnosis ex situ.
The kit, probe or methods of the first to sixth aspects are useful in detecting dental caries and/or MIH/DDD and delineating carious and/or MIH/DDD boundaries in preparation for restoration of a tooth. The clinician may then specifically remove the carious or MIH tissue thus revealed, ensuring clean border preparation and improving the likelihood of restoration success.
The kit of the first aspect or the probe of the second aspect provides key tools and the method of the fourth aspect allows for routine screening for porous dental hydroxyapatite. Moreover, the kit, probe or methods of the first to sixth aspects may be used for early detection of exposed dental hydroxyapatite. In this manner, the kit, probe or methods of the first to sixth aspects may be used for routine screening of dental changes that, without detection, may ultimately lead to dental caries (a precursor to caries), enabling accurate and timely targeting of restoration and/or remineralisation to prevent caries progression and/or promote remineralisation. In some embodiments, the kit, probe or methods of the first to sixth aspects are particularly suited to routine screening of children after eruption of the first permanent molar. It follows that the kit, probe or methods of the first to sixth aspects are also suited to routine screening of teeth for early detection of porous hydroxyapatite. Routine regular screening provides an excellent opportunity to detect at the earliest practical moment dental changes that may lead to dental caries.
Furthermore, the kit, probes and methods of the first to sixth aspects also allow monitoring of any treatment, such as known remineralisation therapies including fluoride or amorphous calcium phosphate that may be stabilised with bioactive molecules, which may be undertaken.
The method of the eighth aspect and the kits of the seventh and ninth aspects enable gentle and/or specific removal of excess proteins that are strongly retained on porous hydroxyapatite, for example in MIH lesions, to be used prior to or during remineralisation treatments.
The protein of any one of the first to fifth or seventh to ninth aspects may be selected from the group: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2 chain C region; Uncharacterized protein c6orf58; and Serpin B3. Furthermore, the protein of any one of the first to fourth or seventh to ninth aspects may be Amelogenin.
The kits of the first, seventh and ninth aspects, or the probe of the second aspect, may be in alternative forms. One form designates either suitability for or restriction to a specific use and is indicated by the word “for”. Another form is restricted to a specific use only and is indicated by the words “when used for”.
The methods of the third to sixth or eighth aspects may be presented in alternative forms, for example in European form (“agent for use”) or second medical use (Swiss) form (“use of an agent in the manufacture of a medicament”).
The patent or application file contains at least one drawing executed as a photograph and in color. Copies of this patent or patent application with photographs and color drawings will be provided by the Office upon request and payment of the necessary fee.
Disclosed herein are kits, probes and methods for detecting a protein capable of binding to porous hydroxyapatite. The hydroxyapatite may be comprised in enamel or dentine. Moreover, whereas existing products stain dentine (but do not detect porous dental hydroxyapatite), for the first time disclosed herein is a product that detects defects in enamel, specifically by detecting porous dental hydroxyapatite.
The protein capable of binding to porous hydroxyapatite may be a human protein. For example, the protein may be selected from the group: Serum albumin (P02768); Complement C3 beta chain (P01024); Alpha-1-antitrypsin (P01009); Protein S100-A9 (P06702); Lactotransferrin (P02788); Leukocyte elastase inhibitor (P30740); Antithrombin-III (P01008); Hemoglobin subunit alpha (P69905); Hemoglobin subunit beta (P68871); Hemoglobin subunit delta (P02042); Prolactin-inducible protein (P12273); Alpha amylase 1 (P04745); Ig kappa chain V-III region SIE (P01620); Ig alpha-2 chain C region (P01877); Uncharacterized protein c6orf58 (Q6P5S2); Serpin B3 (P29508), where the term in parentheses indicates the unique SwissProt accession identifier (as listed in Table 1, SEQ ID NOs: 1 to 16 and
In some embodiments of the first to fourth and seventh to ninth aspects, the protein may be albumin, hemoglobin or a subunit thereof, or amelogenin.
In one embodiment of the fourth aspect, the method comprises detecting the protein which is other than amelogenin and detecting amelogenin, wherein presence of the protein and absence of amelogenin is indicative of MIH, and presence of amelogenin is indicative of hypomaturation defects including types of** amelogenesis imperfecta or dental fluorosis. The protein which is “other than amelogenin” is any one selected from: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2 chain C region; Uncharacterized protein c6orf58; and Serpin B3.
As used herein, “porous” or “porosity” refers to dental hydroxyapatite that is either hypomineralised or demineralised. Increased “porosity” is due to reduction in extent of mineral density, leading to increased space between mineral crystals.
“Hypomineralisation” as used herein, refers to incomplete development of dental enamel, resulting in decreased mineral density (increased enamel porosity) and mechanical strength. “Hypomineralisation” is caused by a genetic (e.g. amelogenesis imperfecta) or acquired (e.g. MIH, fluorosis) disruption of dental development. “Hypomineralisation” is distinct from “demineralisation”, which occurs in caries for example. In caries, developmentally normal (or abnormal) enamel is subsequently demineralised. “Demineralisation” is distinct from “hypomineralisation”, which refers to enamel that never achieved normal mineral content due to disrupted development.
As used herein, “remineralisation” refers to the return of minerals to the molecular structure of the tooth. The predominant mineral of teeth is hydroxyapatite. In some remineralisation processes, the hydroxyl group is substituted for a fluoro group to produce fluoroapatite, which is more acid-resistant than hydroxyapatite.
As used herein, “remedial mineralisation” refers to the use of remineralisation therapies on DDD (i.e. porous hydroxyapatite caused by incomplete mineralisation). Use of the term “remineralisation” is inappropriate in the DDD context because the porous hydroxyapatite was not caused by demineralisation.
As used herein, “caries” or “tooth decay” refers to reduction or loss of tooth enamel and dentine due to acid, particularly acid produced by infecting bacteria. “Caries” is defined by the process of demineralisation, and may be corrected using remineralisation methods if caught early.
As used herein, a “condition involving porous dental hydroxyapatite” includes dental caries, Molar/Incisor Hypomineralisation (MIH), amelogenesis imperfecta, dental fluorosis and other DDD manifesting as hypomineralised enamel (i.e. diffuse or demarcated opacities).
As used herein, “Molar/Incisor Hypomineralisation” or “MIH” refers to a DDD that results in incompletely hardened (hypomineralised) enamel, usually on the occlusal or incisal third of first permanent molars and incisors, respectively.
MIH and fluorosis are both characterised by subsurface porosity, whereas active caries can have a porous surface (inactive caries can form a sealed surface due to remineralisation).
As used herein, “exposed” enamel refers to sub-surface tissue that has been revealed due to loss of its protective surface layer. “Exposed” enamel may be normal or porous; there are many instances of surface breakdown on teeth that are not affected by MIH, or any other condition for that matter (e.g. otherwise normal teeth can fracture upon biting a hard object).
As used herein, “binds”, “binding” or “bound” refers to a chemical interaction between a protein and hydroxyapatite that arrests the protein in relation to the hydroxyapatite. The interaction may be ionic, covalent, non-covalent, polar or non-polar.
As used herein, the term “detector” refers to any chemical, biochemical or biological substance that interacts specifically with a protein disclosed herein and generates an effect in response to the interaction. For example, the response may be visualisation of a coloured reporter, and thus visualisation of the protein. A “detector” may comprise a “reporter” or an antibody.
The term “detect” or “detecting” refers to identifying the response from the detector.
In one embodiment of the kit of the first aspect, the detector comprises a coloured reporter. In the probe of the second aspect, the detector is a reporter. In one embodiment of the probe, the reporter comprises a coloured reporter. When the detector comprises a coloured reporter, detecting the coloured reporter would involve visualising the coloured reporter and therefore the protein of interest. Alternatively, a reporter may be radio-opaque.
As used herein, the term “probe” refers to an agent such as a protein disclosed herein that can infiltrate porous enamel and that can specifically and tightly bind to hydroxyapatite and upon binding enable such binding to be detected. In other words, the probe comprises a specific “hydroxyapatite-targeting” molecule. A “probe” comprises a protein as disclosed herein and a reporter. According to this disclosure, a “probe” may not be an antibody.
Similarly, the term “specific” or “specifically” refers to binding where one substance binds to a particular second substance without substantially binding to any other substance. Such binding is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. As used herein, “specific” or “specifically” binding may refer to (i) the protein binding specifically to hydroxyapatite, (ii) the detector specifically binding to the protein, or (iii) the reporter specifically binding to the detector or protein.
In particular, specific binding refers to a substance having a Kd at least 2-fold less than that of a non-specific target, for example, a substance having a Kd at least 4-fold, 6-fold, 8-fold, 10-fold, or more than 10-fold less than that of a non-specific target. Alternatively, specific binding can be expressed as a molecule having a Kd for the target of at most about 10−4 M, for example, about 10−5 M, about 10−6 M, about 10−7 M, about 10−8 M, about 10−9 M, about 10−10 M, about 10−11 M, about 10−12 M, or less.
In one embodiment of the kit of the first aspect, the detector comprises a reporter.
When used in situ, the detector or probe is non-toxic to the subject.
As used herein, a “reporter” refers to any chemical, biochemical or biological substance that generates a detectable effect. The “reporter” may specifically bind to or be linked to the detector or protein. The reporter may comprise biotin or streptavidin for use in a high affinity, non-covalent biotin-streptavidin bond. The reporter may exploit another high affinity, non-covalent bond.
In one embodiment of the kit of the first aspect or the probe of the second aspect, the reporter may be a coloured reporter. In other embodiments of the first or second aspect, the reporter may be a pigment, or a luminescent (including fluorescent or phosphorescent), radioactive, chemiluminescent substance, enzyme, or x-ray contrast molecule. A reporter comprising an X-ray contrast molecule (e.g. 5-amino-2,4,6-triiodoisophthalic acid; 3I) may be of use for sensitively detecting early-stage interproximal caries (a major challenge for current methods) using existing clinical radiographic equipment.
As used herein, the term “coloured reporter” refers to any coloured substance that absorbs some wavelengths of visible light preferentially.
Thus, when the reporter is a coloured reporter, the detectable effect is visualisation of a colour.
The coloured reporter may be any coloured substance that is amenable to linking, coupling or conjugating to the protein, whilst maintaining its characteristic as a coloured reporter. In one embodiment of the kit of the first aspect or probe of the second aspect, the coloured reporter is amido black. In one example, the probe comprises the protein (i.e. a hydroxyapatite-binding-protein) linked or coupled to a coloured reporter. Any protein from Table 1 may be linked or coupled to a coloured reporter and function to target the coloured reporter to porous hydroxyapatite. In one example, the protein is hemoglobin. Thus, in one example, the probe comprises hemoglobin linked to amido black.
A probe comprising a protein as disclosed herein, e.g. haemoglobin, is adsorbed cumulatively to porous dental hydroxyapatite. The probe will competitively bind to hydroxyapatite in the presence of other proteins because it is able to displace any species possessing lower affinity for hydroxyapatite. Such a probe may comprise amido black or 3I.
The coloured reporter may be selected, based on desired features that would be known to a person skilled in the art, from the group: Acetyl yellow (Fast yellow); Acid black 1 (Amido black 10B); Acid blue 22 (Water blue I); Acid blue 93 (Methyl blue); Acid fuchsin (Acid fuchsin); Acid green (Light green SF yellowish); Acid green 1 (Naphthol green B); Acid green 5 (Light green SF yellowish); Acid magenta (Acid fuchsin); Acid orange 10 (Orange G); Acid red 4 (Azo-eosin); Acid red 26 (Xylidine ponceau); Acid red 29 (Chromotrope 2R); Acid red 44 (Ponceau 6R); Acid red 51 (Erythrosin B); Acid red 52 (Lissamine rhodamine B); Acid red 66 (Biebrich scarlet); Acid red 73 (Woodstain scarlet); Acid red 87 (Eosin Y ws); Acid red 91 (Eosin B); Acid red 92 (Phloxine B); Acid red 94 (Rose bengal); Acid red 101 (Azocarmine G); Acid red 103 (Azocarmine B); Acid roseine (Acid fuchsin); Acid rubin (Acid fuchsin); Acid violet 19 (Acid fuchsin); Acid yellow 1 (Naphthol yellow S); Acid yellow 7 (Lissamine flavine FF); Acid yellow 9 (Fast yellow); Acid yellow 23 (Tartrazine); Acid yellow 24 (Martius yellow); Acid yellow 36 (Metanil yellow); Acid yellow 73 (Fluorescein); Acid yellow 85 (Coomassie fast yellow G); Acid yellow S (Naphthol yellow S); Acid yellow T (Tartrazine); Acridine orange (Acridine orange); Acridine red (Acridine red); Acriflavine (Acriflavine); Alcian blue (Alcian blue 8GX); Alcian yellow (Alcian yellow); Alcohol soluble eosin (Ethyl eosin); Alizarin (Alizarin); Alizarin blue (Alizarin blue); Alizarin blue 2RC (Anthracene blue SWR); Alizarin carmine (Alizarin red S); Alizarin cyanin BBS (Alizarin cyanin BBS); Alizarol cyanin R (Chromoxane cyanin R); Alizarin red S (Alizarin red S); Alizarin purpurin (Purpurin); Alkali blue 4B, 5B (Alkali blue 5B); Aluminon (Chrome violet CG); Amido black 10B (Amido black 10B); Amidonaphthol red (Azophloxine); Amidoschwarz (Amido black 10B); Aniline blue WS (Aniline blue WS); Aniline purple (Mauveine); Anthracene blue SWR (Anthracene blue SWR); Anthracene blue SWX (Alizarin cyanin BBS); Auramine O (Auramine O); Azo-eosin (Azo-eosin); Azocarmine B (Azocarmine B); Azocarmine G (Azocarmine B); Azoeosin G (Azo-eosin); Azoic diazo 5 (Fast red B); Azoic diazo 48 (Fast blue B); Azophloxine (Azophloxine); Azovan blue (Evans blue); Azure A (Azure A); Azure B (Azure B); Azure C (Azure C); Basic blue 8 (Victoria blue 4R); Basic blue 9 (Methylene blue); Basic blue 12 (Nile blue A); Basic blue 15 (Night blue); Basic blue 17 (Toluidine blue O); Basic blue 20 (Methyl green); Basic blue 26 (Victoria blue B); Basic brown 1 (Bismarck brown Y); Basic fuchsin (Basic fuchsin); Basic green 4 (Malachite green); Basic green 5 (Methylene green); Basic orange 14 (Acridine orange); Basic red 2 (Safranin O); Basic red 5 (Neutral red); Basic red 9 (Pararosanilin); Basic violet 2 (New fuchsin); Basic violet 3 (Crystal violet); Basic violet 4 (Ethyl violet); Basic violet 10 (Rhodamine B); Basic violet 14 (Rosanilin); Basic yellow 1 (Thioflavine T); Basic yellow 2 (Auramine O); Biebrich scarlet (Biebrich scarlet); Biebrich scarlet R (Sudan IV); Bismarck brown Y (Bismarck brown Y); Blauschwarz (Naphalene blue black CS); Brazilein (Brazilein); Brazilin (Brazilin); Brilliant crocein (Woodstain scarlet); Brilliant crystal scarlet 6R (Ponceau 6R); Brilliant green (Brilliant green); Calcium red (Nuclear fast red); Carmine (Carmine); Carminic acid (Carmine); Carmoisine 6R (Chromotrope 2R); Celestine blue B (Celestine blue B); China blue (Aniline blue); Chlorantine fast red 5B (Sirius red 4B); Chicago blue 4B (Pontamine sky blue 5B); Chrome fast yellow 8GL (Chrome fast yellow 8GL); Chrome luxine yellow 8G (Chrome fast yellow 8GL); Chrome violet CG (Chrome violet CG); Chromotrope 2R (Chromotrope 2R); Chromoxane cyanin R (Chromoxane cyanin R); Cochineal (Carmine); Coelestine blue (Celestine blue B); Congo corinth (Congo corinth); Congo red (Congo red); Coomassie fast yellow G (Coomassie fast yellow G); Cotton blue (Methyl blue); Cotton red (Congo red); Croceine scarlet (Biebrich scarlet); Crocein scarlet 3B (Woodstain scarlet); Crocein scarlet MOO (Woodstain scarlet); Crocin (Saffron); Crystal ponceau 6R (Ponceau 6R); Crystal scarlet (Ponceau 6R); Crystal violet (Crystal violet); Dahlia (Hoffman's violet); Diamond green B (Malachite green); Direct blue 14 (Trypan blue); Direct blue 58 (Evans blue); Direct red (Congo red); Direct red 10 (Congo corinth); Direct red 28 (Congo red); Direct red 80 (Sirius red F3B); Direct red 81 (Sirius red 4B); Direct yellow 7 (Thioflavine S); Direct yellow 11 (Sun yellow); Durazol blue 4R (Durazol blue 4R); Durazol blue 8G (Durazol blue 8G); Eosin B (Eosin B); Eosin Bluish (Eosin B); Eosin (Eosin Y ws); Eosin Y (Eosin Y ws); Eosin yellowish (Eosin Y ws); Eosinol (Eosinol); Erie garnet B (Congo corinth); Eriochrome cyanin R (Chromoxane cyanin R); Erythrosin B (Erythrosin B); Ethyl eosin (Ethyl eosin); Ethyl green (Ethyl green); Ethyl violet (Ethyl violet); Evans blue (Evans blue); Fast blue B (Fast blue B); Fast green FCF (Fast green FCF); Fast red B (Fast red B); Fast yellow (Fast yellow); Fast yellow extra (Fast yellow); Fast yellow G (Fast yellow); Fat black HB (Sudan black B); Fluorescein (Fluorescein); Food green 3 (Fast green FCF); Gallein (Gallein); Gallamine blue (Gallamine blue); Gallocyanin (Gallocyanin); Gentian violet (Methyl violet 2B); Guinee green (Guinee green B); Haematein (Hematein); Haematine (Hematein); Haematoxylin (Hematoxylin); Helio fast rubin BBL (Nuclear fast red); Helvetia blue (Methyl blue); Hematein (Hematein); Hematine (Hematein); Hematoxylin (Hematoxylin); Hoffman's violet (Hoffman's violet); Hydrazine yellow (Tartrazine); Indigo carmine (Indigo carmine); Imperial red (Eosin B); Ingrain blue 1 (Alcian blue 8GX); Ingrain yellow 1 (Alcian yellow); INT (lodonitrotetrazolium); Iodine green (Iodine green); Kermes (Kermes); Kermesic acid (Kermes); Kernechtrot (Nuclear fast red); Kiton rhodamine B (Lissamine rhodamine B); Lac (Laccaic acid); Laccaic acid (Laccaic acid); Lauth's violet (Thionin); Light green (Light green SF yellowish); Lissamine fast yellow (Lissamine fast yellow); Lissamine flavine FF (Lissamine flavine FF); Lissamine green SF (Light green SF yellowish); Lissamine rhodamine B (Lissamine rhodamine B); Luxine pure yellow 6G (Chrome fast yellow 8GL); Luxol fast blue (Luxol fast blue MBS); Magenta 0 (Pararosanilin); Magenta I (Rosanilin); Magenta II (Magenta II); Magenta III (New fuchsin); Malachite green (Malachite green); Manchester brown (Bismarck brown Y); Martius yellow (Martius yellow); Mauve (Mauveine); Mauveine (Mauveine); Merbromin (Mercurochrome 220); Mercurochrome (Mercurochrome 220); Metanil yellow (Metanil yellow); Methyl blue (Methyl blue); Methyl green (Methyl green); Methyl violet (Methyl violet 2B); Methyl violet 2B (Methyl violet 2B); Methyl violet 10B (Crystal violet); Methylene azure A (Azure A); Methylene azure B (Azure B); Methylene azure C (Azure C); Methylene blue (Methylene blue); Methylene green (Methylene green); Milling yellow 3G (Milling yellow 3G); Mordant blue 3 (Chromoxane cyanin R); Mordant blue 10 (Gallocyanin); Mordant blue 14 (Celestine blue B); Mordant blue 23 (Alizarin cyanin BBS); Mordant blue 32 (Anthracene blue SWR); Mordant blue 45 (Gallamine blue); Mordant red 3 (Alizarin red S); Mordant red 11 (Alizarin); Mordant violet 25 (Gallein); Mordant violet 39 (Chrome violet CG); Mordant yellow 33 (Chrome fast yellow 8GL); Naphthalene blue black (Naphalene blue black CS); Naphthol blue black (Amido black 10B); Naphthol green B (Naphthol green B); Naphthol yellow S (Naphthol yellow S); Natural black 1 (Hematein); Natural red (Purpurin); Natural red 3 (Kermes); Natural red 4 (Carmine); Natural red 8 (Purpurin); Natural red 16 (Purpurin); Natural red 24 (Brazilin); Natural red 25 (Laccaic acid); Natural red 28 (Orcein); Natural yellow 6 (Saffron); NBT (Nitro blue tetrazolium); Neutral red (Neutral red); New fuchsin (New fuchsin); Niagara blue 3B (Trypan blue); Night blue (Night blue); Nile blue (Nile blue A); Nile blue A (Nile blue A); Nile blue sulphate (Nile blue A); Nile red (Nile red); Nitro BT (Nitro blue tetrazolium); Nitro blue tetrazolium (Nitro blue tetrazolium); Nuclear fast red (Nuclear fast red); Oil red O (Oil red O); Orange G (Orange G); Orcein (Orcein); Pararosanilin (Pararosanilin); Perkin's violet (Mauveine); Phloxine B (Phloxine B); Picric acid (Picric acid); Ponceau 2R (Xylidine ponceau); Ponceau 6R (Ponceau 6R); Ponceau B (Biebrich scarlet); Ponceau de Xylidine (Xylidine ponceau); Ponceau S (Ponceau S); Pontamine sky blue 5B (Pontamine sky blue 5B); Primula (Hoffman's violet); Primuline (Primuline); Purpurin (Purpurin); Pyronin B (Pyronin B); Pyronin G (Pyronin Y); Pyronin Y (Pyronin Y); Rhodamine B (Rhodamine B); Rosanilin (Rosanilin); Rose bengal (Rose bengal); Saffron (Saffron); Safranin O (Safranin O); Scarlet R (Sudan IV); Scarlet red (Sudan IV); Scharlach R (Sudan IV); Shellac (Laccaic acid); Sirius red F3B (Sirius red F3B); Sirius red 4B (Sirius red 4B); Sirius supra blue F3R (Durazol blue 4R); Solochrome cyanin R (Chromoxane cyanin R); Soluble blue (Aniline blue); Solvent black 3 (Sudan black B); Solvent blue 38 (Luxol fast blue MBS); Solvent red 23 (Sudan III); Solvent red 24 (Sudan IV); Solvent red 27 (Oil red O); Solvent red 45 (Ethyl eosin); Solvent yellow 94 (Fluorescein); Spirit soluble eosin (Ethyl eosin); Sudan III (Sudan III); Sudan IV (Sudan IV); Sudan black B (Sudan black B); Sudan red BK (Sudan III); Sulfur yellow S (Naphthol yellow S); Sulpho rhodamine B (Lissamine rhodamine B); Sun yellow (Sun yellow); Swiss blue (Methylene blue); Tartrazine (Tartrazine); Thioflavine S (Thioflavine S); Thioflavine T (Thioflavine T); Thionin (Thionin); Toluidine blue (Toluidine blue O); Toluyline red (Neutral red); Tropaeolin G (Metanil yellow); Trypaflavine (Acriflavine); Trypan blue (Trypan blue); Uranin (Fluorescein); Victoria blue 4R (Victoria blue 4R); Victoria blue B (Victoria blue B); Victoria blue R (Victoria blue R); Victoria green B (Malachite green); Water blue I (Water blue I); Water soluble eosin (Eosin Y ws); Woodstain scarlet (Woodstain scarlet); Xylene red B (Lissamine rhodamine B); Xylidine ponceau (Xylidine ponceau); and Yellowish eosin (Eosin Y ws). The desired features to be considered by the skilled addressee include compatibility with a protein and/or a linker to be used according to this disclosure, non-toxicity, and maintenance of protein binding to porous dental hydroxyapatite, for example.
In alternative embodiments of the first to fifth or seventh to ninth aspects, the protein is not listed in the examples of Table 1, but is known to the skilled addressee to bind to hydroxyapatite, for example osteocalcin or decorin. Use of the leucine-rich repeat domains 4-5 from decorin may provide a specific targeting mechanism for porous hydroxyapatite in dentine.
Alternatively, in embodiments of the first to fifth or seventh to ninth aspects, the protein may be a peptide or protein fragment, provided that the peptide or protein fragment retains its ability to bind to porous dental hydroxyapatite.
Alternatively, the skilled addressee will be aware of small molecules (or polymers thereof), for example tetracycline or amino bisphosphonate, that can bind to hydroxyapatite, which may be of more use in terms of ability to penetrate micro-porous regions, and in terms of stability (e.g. product shelf-life). Amino-bisphosphonate may produce a compound with qualities suited to detecting and delineating caries (small, high-affinity probe for penetrating porous enamel surface and strongly binding to demineralised enamel).
In some embodiments of the kit of the first aspect or probe of the second aspect, the detector or probe further comprises a linker linking the reporter and the detector or protein. The linker may be a heterobifunctional cross-linker. For example, the heterobifunctional linker may be succinimidyl 4-[N-maleimidomethyl]cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC). Other examples of linking agents that may be used in accordance with this disclosure include succinimidyl-6-[β-maleimidopropionamido] hexanoate (SMPH), N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), and N,N-dicyclohexylcarbodiimide (DCC).
Other types of molecules may be used as a linker. For example, high affinity, non-covalent bonds such as biotin-streptavidin are also contemplated herein.
The skilled addressee will be aware of many cross-linking agents that are available with various reactive chemistries and spacer-arm lengths, further increasing the flexibility of this approach.
In one embodiment of the kit of the first aspect or probe of the second aspect, the reporter and the protein may be provided already linked. Alternatively, the reporter and protein may be provided separately for subsequent linkage. The kit or probe may comprise a linker. The protein, reporter and linker of the kit or probe may be presented in any possible combination. For example, when the reporter and protein are linked via a linker, the probe may be “ready-to-use”, i.e. the three components may be linked. Alternatively, the protein and the linker may be linked and provided separately to the reporter. Alternatively, the linker and the reporter may be linked and provided separately to the protein. Alternatively, the protein, the reporter, and the linker may be provided as separate components. In one embodiment of the first aspect, the kit will comprise the reporter and the linker, but not the protein.
In some embodiments of the kit of the first aspect, the detector comprises an antibody that specifically binds the protein. In another embodiment, the detector may comprise biotin or streptavidin for use in a high affinity, non-covalent biotin-streptavidin bond. The detector may exploit another high affinity, non-covalent bond. For example, the detector may comprise an antibody alternative, such as a peptide-based protein ligand. A peptide-based protein ligand known in the art is a synbody.
The term “antibody” is used in the broadest sense and specifically covers, for example, polyclonal antibodies, monoclonal antibodies (including antagonist and neutralizing antibodies), antibody compositions with polyepitopic specificity, single chain antibodies, and fragments of antibodies, provided that they exhibit the desired biological or immunological activity. The antibody may be a conjugated antibody or any other type of antibody known to the person skilled in the art.
The antibody may be detected by any method known to the person skilled in the art. The primary antibody may comprise a reporter. Alternatively, a secondary antibody targeting the primary antibody may comprise a reporter.
The antibody may be any antibody known by the skilled addressee to specifically bind to a protein selected from the group: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2 chain C region; Uncharacterized protein c6orf58; and Serpin B3. In one embodiment, the antibody may specifically bind to an amelogenin.
In one embodiment of the kit of the first aspect, an anti-serum albumin monoclonal antibody may be selected from the group: AL-01; 1.6.731; 1A9; 6611; OCH1E5; 1C8; 1G2; 2B2; 2B3; 2B6; 14E7; 15C7; Alb1; and a mouse monoclonal IgG1 antibody with product code sc-70340 (Santa Cruz Biotechnology Inc). In one embodiment, an anti Complement C3 beta chain monoclonal antibody may be clone 755. In another embodiment, an anti-human C3 monoclonal antibody that cross-reacts with Complement C3 beta chain may be used and may be clone 11H9. In one embodiment, an anti-Alpha-1-antitrypsin monoclonal antibody may be selected from the group: 51312; 703; 704; 8A0; B9; and G11. In one embodiment, an anti-Protein S100-A9 monoclonal antibody may be selected from the group: 0.N.390A; 47-8D3; NO.134; NO.19; and S32.2. In one embodiment, an anti-Lactotransferrin monoclonal antibody may be selected from the group: 106; 2B8; B97; CLB-13.17; and 1A1. In one embodiment, an anti-Antithrombin-III monoclonal antibody may be 4B3 or BDI205. In one embodiment, an anti-Hemoglobin subunit alpha antibody may be a goat polyclonal IgG antibody with product code sc-70340 (Santa Cruz Biotechnology Inc). In one embodiment, an anti-Hemoglobin subunit beta antibody may be a mouse monoclonal IgG1 antibody with product code sc-21757 (Santa Cruz Biotechnology Inc). In one embodiment, an anti-Ig alpha-2 chain C region monoclonal antibody may be clone 14AS (also referred to as anti-human IgA2). In one embodiment, an anti-Amelogenin X antibody may be a rabbit polyclonal IgG antibody with product code sc-32892 (Santa Cruz Biotechnology Inc). The skilled addressee will appreciate that other suitable antibodies are available.
In one embodiment of the kit, probe or method of the first, second or fourth aspects, a first protein is selected from the group: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2 chain C region; Uncharacterized protein c6orf58; and Serpin B3 may be detected, and a second protein may be detected, wherein the second protein is amelogenin. It follows that a kit of the first aspect may also comprise a second detector that detects amelogenin. The second detector may be an anti-amelogenin antibody.
Alternatively, detecting may comprise immunodetection, chromatography, electrophoresis, mass spectrometry, or microscopy. Immunodetection may comprise enzyme-linked immunosorbent assay (ELISA), Western Blot, dot blot, slot blot, or flow cytometry, for example. Microscopy may comprise confocal laser, fluorescence or electron microscopy, for example.
The detector or probe may be applied in different ways, for example in a liquid, gel, capsule, tablet, aqueous solution, aqueous or oily suspension, lozenge, troche, powder, granule, emulsion, syrup or elixir.
In one embodiment of the kit of the first aspect or probe of the second aspect, the detector or probe comprises a solvent in which the detector or probe is dissolved, suspended or emulsified. The solvent may be one that is used generally in medicine or industry or similar. Examples include water, ethanol, n-propanol, 2-butyl alcohol, isobutyl alcohol, n-amyl alcohol, isoamyl alcohol, ethylene glycol, 2-methoxyethanol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, trimethylene glycol, 1,2-butanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,5-pentanediol, ethylene glycol monomethyl ether, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether, ethylene glycol diethylether, ethyleneglycolmonoethyletheracetate, ethylene glycol isopropyl ether, ethylene glycol monobutyl ether, ethylene glycol dibutyl ether, ethylene glycol monoacetate, ethylene glycol diacetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monoethyl ether acetate, diethylene glycol monobutyl ether, diethylene glycolmonobutyl ether acetate, diethylene glycol dimethyl ether, diethylene glycol methylethyl ether, diethylene glycol diethyl ether, diethylene glycol acetate, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether,ee tripropylene glycol monomethyl ether, glycerin, tetrahydrofuran, dimethylformamide, dioxane, acetone, and dimethoxyethane.
In some embodiments, the solvent comprises water, ethanol, glycerin, isobutyl alcohol, ethylene glycol, diethylene glycol, triethylene glycol, acetone, or propylene glycol, which are compatible with humans.
One solvent may be used singly or two or more solvents may be used in admixture.
The detector may be compounded with a thickener to increase its viscosity to about 50 to about 2 000 mPa·s, for example 100, 200, 300, 400, 500, 750, 1000, 1250, 1500, or 1750 mPa s (at 25° C.), thereby forming a gel. In gel form, applying the detector with a toothbrush enables simultaneous cleaning of the tooth and application of the detector.
Examples of thickeners that may be used include: synthetic additives such as sodium alginate, propylene glycol alginate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch, sodium starch phosphate, sodium polyacrylate, methyl cellulose, hydroxypropyl cellulose, and polyvinylpyrrolidone; natural thickeners such as cyamoposis gum, Carob bean gum, Tara gum, Tamarind seed gum, gum arabic, tragacanth gum, Karaya gum, alginic acid, carrageenan, xanthan gum, gellan gum, curdlan, chitin, chitosan, and chitosamine; and inorganic thickeners such as calcium carbonate, calcium silicate, silica powder, amorphous hydrous silicate, and hydrophobic silica.
In order to obtain viscosity in the range of about 50 to about 2 000 mPa·s, the compounding amount of the thickener varies depending on the kind of the thickener. For example, when sodium carboxymethyl cellulose having a large thickening effect, the compounding amount may be about 0.5 to 4% by weight, and when methyl cellulose, the compounding amount may be about 10 to 30% by weight.
Furthermore, the detector or probe may comprise additives such as sweeteners, flavours, and preservatives. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. A tablet may contain the detector in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
In another embodiment, the kit of the first aspect further comprises one or more washing solutions.
The kit of the seventh or ninth aspect comprises one or more washing solutions.
A washing solution of the kit of the first, seventh or ninth aspect may comprise a solution to remove any protein not specifically bound to porous hydroxyapatite, i.e. non-desorbing. For example, a washing solution that does not desorb a protein bound to hydroxyapatite may be water, saline, Tris buffer, or mild detergent etc. As the oral cavity contains abundant proteins including many of the proteins that bind hydroxyapatite, a washing solution allows protein not specifically bound to hydroxyapatite to be removed from the tooth or sample thereof prior to application of the detector.
In other embodiments of the kit of the first, seventh or ninth aspect, the washing solution comprises magnesium ions (Mg2+), dihydrogenphosphate ions (H2PO4−), hydrogenphosphate ions (HPO42−), or phosphate ions (PO43−) (collectively “PO4”), or may comprise a plurality of washing solutions that may each comprise magnesium ions (Mg2+), dihydrogenphosphate ions (H2PO4−), hydrogenphosphate ions (HPO42−), or phosphate ions (PO43−), administrable sequentially. Any soluble magnesium salt may be used and any soluble dihydrogenphosphate, hydrogenphosphate ions (HPO42−), or phosphate salt may be used, provided that it is non-toxic if applied in situ. In one embodiment, the washing solution comprises magnesium chloride or sodium dihydrogenphosphate. The skilled addressee will appreciate that other washing solutions capable of desorbing protein from hydroxyapatite are available.
A washing solution may comprise hypochlorous acid (HOCl), hypochlorite (NaOCl) or calcium hypochlorite (Ca(OCl)2) (collectively “bleach”).
A washing solution may be provided ready to use. Alternatively, the washing solution may be provided as a concentrate to prepare the washing solution upon dilution with water. Alternatively, the washing solution may be provided as one or more dry components to prepare the washing solution upon admixture with water.
The washing solution may comprise less than 1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM or more than 10 mM magnesium ions. The washing solution may comprise less than 0.1 M, about 0.1 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 2 M, about 10 M or more than 10 M magnesium ions. The washing solution may comprise less than 0.04 M, about 0.04 M, about 0.08 M, about 0.09 M, about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.5 M, about 2 M, about 4 M, or more than 4 M dihydrogenphosphate, hydrogenphosphate or phosphate ions.
The washing solution may comprise about 10% bleach (about 0.4% −OCl), neat or undiluted bleach (about 4% −OCl), or may comprise about 20% (about 0.8% −OCl), about 30% (about 1.2% −OCl), about 40% (about 1.6% −OCl), about 50% (about 2.0% −OCl), about 60% (about 2.4% −OCl), about 70% (about 2.8% −OCl), about 80% (about 3.2% −OCl), about 90% (about 3.6% −OCl) or about 95% bleach (about 3.8% −OCl).
While not wishing to be bound to any particular theory, it is thought that providing a plurality of washing solutions with a step-wise concentration gradient of magnesium and/or phosphate removes more proteins than a single concentration magnesium solution. It is thought that bleach (−OCl) non-specifically strips bound proteins from hydroxyapatite.
Thus, in one embodiment of the kit of the first, seventh or ninth aspect, the one or more washing solutions, or plurality of washing solutions, may comprise a solution of about 5 mM magnesium chloride, a solution of about 1 M magnesium chloride, and/or a solution of about 0.4 M sodium dihydrogenphosphate.
Where one or more (a plurality) of washing solutions is applied, the washing solutions may be applied in any order. Alternatively, where one or more (a plurality) of washing solutions is applied, the washing solutions may be applied sequentially in the order of low magnesium concentration (e.g. 5 mM), high magnesium concentration (e.g. 1 M), dihydrogenphosphate (e.g. 0.4 M; or hydrogenphosphate or phosphate). Alternatively, a washing solution may comprise in combination magnesium and phosphate, for example, about 1 M magnesium concentration and about 0.4 M dihydrogenphosphate, hydrogenphosphate or phosphate.
Washing may occur before detecting, or after detecting, or before and after detecting.
As used herein, “removes” or “removing” refers to a reduction in the concentration of protein bound to hydroxyapatite.
As used herein, a “sample” is a portion or part of the tooth to be used for detection or diagnosis of porous hydroxyapatite. A “control sample” is a portion or part of the tooth known to be healthy and free of porous hydroxyapatite and is used for reference purposes when detecting or diagnosing porosity in test hydroxyapatite. A “sample” may be obtained by wiping, swabbing, scraping, chipping, drilling or similar. A sampler may be adapted for obtaining a sample by swabbing, wiping or any other method of collection known to the skilled addressee.
In one embodiment of the method of the fourth to sixth aspect, the tooth is first cleaned by brushing or other means. The tooth may be dried.
In one embodiment of the kit of the first aspect, probe of the second aspect, or method of the fourth to sixth aspect, the detector is applied using a brush, toothbrush, a cotton swab, a cotton ball or by dropping from a nozzle-equipped container.
As used herein, “applied”, “applying” or “application” has its ordinary meaning of bringing into contact the detector or probe or washing solution and the tooth or sample thereof, or bringing into contact the reporter and the detector or protein.
After application of the detector or probe, the detector or probe is incubated on the tooth for a period of time sufficient for binding of the detector to the protein or for binding of the probe to the hydroxyapatite. The incubation period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 s. Alternatively, the incubation time may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 min. Alternatively, the incubation time may be more than 5 min, such as 10, 15 or 20 min. After incubation, excess detector or probe may be disgorged from the mouth with or without washing using water or a washing solution.
The method or use may be performed in or on the tooth in situ in a subject. Alternatively, the method or use may be performed in or on the tooth or a sample of the tooth after removal from a subject.
The subject includes a mammal. The mammal may be a human. The human may be any age. The human may be under about 12 years of age. The human may be about 2 to about 12 years of age, about 4 to about 10 years of age, or about 6 to about 10 years of age. Alternatively, the subject may be 12 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 years of age.
The subject may develop porous hydroxyapatite after normal hydroxyapatite and normal enamel has developed, or the subject may have porous hydroxyapatite throughout development.
Alternatively, the subject may be a domestic, zoo, or companion animal. While it is particularly contemplated that the methods and uses herein are suitable for humans, they are also applicable to primates, companion animals such as dogs and cats, domestic animals such as horses, cattle, sheep and goats, zoo animals such as felids, canids, bovids, and ungulates, or laboratory animals such as lagomorphs and rodents. A subject may be afflicted with a dental disorder, or may not be afflicted with a dental disorder (i.e., free of detectable disease).
The diagnostic power of the kits or methods disclosed herein is based on conditions of porous hydroxyapatite (DDD and caries) having distinguishable protein profiles (e.g. MIH: abundant proteins from Table 1, little or no amelogenin; mature fluorosis: trace amounts of albumin and amelogenin; hypomaturation amelogenesis imperfecta: abundant amelogenin). Different defects may require different wash procedures before remedial mineralisation, or different restoration methods and materials (or influence the choice thereof). Protein concentration in test enamel of a tooth or sample thereof may be assessed by various means, and the condition involving porous hydroxyapatite can be diagnosed based on the identity of proteins with elevated abundance relative to control.
A further application of the present disclosure is to categorise the MIH lesion sub-type (e.g. as intact or broken), which may impact the type of treatment required (e.g. different protein compositions may need different wash procedures before remedial mineralisation).
As used herein, “intact” has its ordinary meaning of undisrupted, uninjured or unaltered and is used in relation to the surface of tooth enamel. “Intact” here refers to a lesion covered with a shell of harder enamel at the tooth surface, and is referred to as a subsurface lesion, indicating a stratified structure.
In contrast, “broken” here refers to an MIH lesion whose hard enamel shell either has become disrupted due to mechanical forces, or was not present initially (perhaps lost during tooth eruption, or not produced during development).
As used herein, “permeable surface” refers to intact or broken enamel that allows access of oral fluid or any other solution (and associated components including proteins) into subsurface regions. Conversely, an “impermeable surface” refers to intact or broken enamel that blocks such access.
MIH lesions comprising intact enamel, despite comprising porous hydroxyapatite, may or may not present porous dental hydroxyapatite amenable to detection (i.e. may have a permeable or impermeable surface). Therefore, in some embodiments of the first, seventh or ninth aspects, a kit may comprise a permeabilising agent, or a method of the fourth to sixth aspects (e.g. mechanical permeabilisation) may comprise permeabilising the tooth or a sample of the tooth. Such an agent or method will be used in pre-treating a lesion that has an impermeable surface. Alternatively, such an agent may be used to access a lesion previously subject to remineralisation or remedial mineralisation.
As used herein, “permeabilise” or “permeabilising” refers to opening pores in impermeable enamel of sufficient dimension to enable the detector and/or the washing solution access to the porous hydroxyapatite, and/or to enable protein removal.
The permeabilisation agent is a formulation capable of permeabilising the surface layer of enamel (e.g. it may comprise an acid or some other agent known by the skilled addressee to permeabilise enamel). The permeabilisation agent may be in the form of a solution or a gel, for example.
In some embodiments of the first, seventh or ninth aspect, a kit may comprise a remineralisation agent, or the method may comprise remineralising the tooth or sample thereof. A remineralisation agent may comprise fluoride, soluble calcium phosphate or amorphous calcium phosphate, which may be stabilised with bioactive molecules.
Materials and Methods
Specimens
Human and Sprague-Dawley rat specimens were obtained with appropriate ethical approvals, and stored at −80° C. MIH was diagnosed according to standard criteria (Weerheijm, 2003). After extraction, MIH teeth were water-rinsed to remove visible blood, then blotted dry and stored frozen immediately. Whole saliva, stimulated by chewing on wax, was clarified by centrifugation (20,000 g, 5 min) before storage. Serum and erythrocytes were prepared conventionally from blood of 6-day-old rats. Secretory enamel matrix was isolated from developing rat teeth as before (Hubbard, 1996) except using 5-day-old first molars.
Profiling of Enamel Proteins
Overt MIH lesions were collected from freshly thawed specimens by scraping with a scalpel, taking care to avoid carious enamel and dentine. Normal enamel was sampled using a slowly rotating dental bur (No. 6). Immediately afterwards, enamel samples (2-5 μl packed vol) were suspended in 10% trifluoroacetic acid (10 volumes, 10 min at room temperature with vortexing and bath sonication), then centrifuged (20,000 g, 4° C., 5 min) to sediment acid-insoluble protein. Pellets were solubilized in gel-loading buffer containing 2% SDS and 100 mmol/L dithiothreitol (Hubbard, 1996), with additional protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, 5 μg/mL pepstatin, 5 μg/mL leupeptin) where indicated. SDS extracts were quantified by dot blotting with Amido Black and subjected to mini SDS-PAGE with Coomassie Blue staining or immunoblotting (Hubbard, 1995). Amelogenin antiserum was raised conventionally in rabbits, using recombinant mouse amelogenin (SEQ ID NO: 19) as immunogen.
Proteomics Analysis
Gel bands were subjected to trypsinolysis and tandem mass spectrometry as before (Mangum et al., 2006) except using an ion-trap instrument with chip-based nanospray (Chip-LC/MSD XCT, from Agilent Technologies, Santa Clara, Calif., USA). Proteins were identified using the MASCOT search engine and SwissProt human database with strict acceptance criteria (minimally two sequence tags (Mangum et al., 2006)).
Mineral-Binding Assays
Mock oral fluid was prepared by empirically spiking saliva with serum and erythrocyte lysate so that major proteins from all three components were similarly abundant (
Results
MIH Enamel is Enriched with Non-Amelogenin Proteins
Profiling of enamel proteins has provided useful insights to the pathogenesis of fluorosis and amelogenesis imperfecta, particularly by linking amelogenin levels with clinical properties. Accordingly, unfixed MIH enamel specimens were investigated using an SDS-PAGE approach. Unlike normal enamel, MIH enamel gave visible precipitates when dissolved in acid, suggesting a relatively high protein content. As shown in
Body Fluid Proteins Predominate in MIH Enamel
To identify the major protein constituents of MIH enamel, SDS-PAGE bands were subjected to proteomic analysis. As shown in
Intact and Broken MIH Lesions have Distinct Protein Profiles
Given the clinical diversity of MIH lesions (colour, consistency, size, surface integrity), it was investigated whether the different presentations have distinct protein compositions. Appraisal of the protein profiles (
The stability of the protein profiles was also queried, noting evidence of protein degradation (
Protein Composition of MIH Enamel Varies with Surface Integrity
It is known that MIH lesions exhibit sub-surface porosity and that albumin and haemoglobin bind avidly to hydroxyapatite. Accordingly, it was posited that oral-fluid proteins permeate MIH enamel and selectively bind to hydroxyapatite crystals, subject to absence of an intact surface layer. When broken lesions were compared with saliva, serum and erythrocytes, collective similarities in the protein-banding patterns were found (
When mock oral fluid comprising albumin and hemoglobin was applied to hydroxyapatite, albumin and hemoglobin bound to the hydroxyapatite (
Discussion
Given growing concerns about MIH worldwide, a pressing need exists to elucidate the protein composition of hypomineralized enamel. It is disclosed herein that MIH enamel has substantially higher protein content than normal, but a near-normal level of residual amelogenins. This characteristic distinguishes MIH from hypomaturation defects that contain high residual amelogenins (amelogenesis imperfecta, fluorosis) and in turn typifies MIH as a hypocalcification defect. Secondly, MIH enamel was found to have accumulated various proteins from oral fluid and blood, with differential incorporation depending on integrity of the enamel surface. Pathogenically, these results point to a pre-eruptive disturbance of mineralisation involving albumin and, in cases with post-eruptive breakdown, subsequent protein adsorption to the exposed hydroxyapatite matrix. These insights to the pathogenesis and properties of MIH enamel hold significance for the prevention, diagnosis and treatment of MIH.
The present results help to explain the clinical and biophysical properties of MIH enamel. The observed 3-fold to 15-fold elevation in protein content is similar to reports for amelogenesis imperfecta and fluorosis (2.5-fold to 30-fold) and appears sufficient to account for the characteristic mechanical weakness of MIH enamel. The low residual content of amelogenins likens MIH enamel to hypocalcified types of amelogenesis imperfecta. Enamel from the latter disorders is described clinically as markedly softer than normal and friable or cheesy, which coincides with descriptions of MIH enamel. At the protein level, MIH enamel appears distinguishable from hypocalcified types of amelogenesis imperfecta and fluorosis, particularly based on its uniquely high content of albumin. However, all conditions are characterised by porous hydroxyapatite.
These results also elucidate the pathogenesis of MIH, pointing to pre- and post-eruptive steps that are mechanistically distinct. Pre-eruptively, the normal thickness and low amelogenin content of MIH enamel (<0.2% of secretion-phase level) indicates that amelogenins are secreted and then removed effectively. It follows that MIH is not a maturation defect primarily. By analogy to hypocalcified amelogenesis imperfecta, attention therefore turns to defective initiation of mineralisation. Protein profiling indicated that albumin accumulates in MIH enamel despite near-complete removal of amelogenins.
In other words, hypocalcification is a subtype of hypomineralisation, the other subtype being hypomaturation. As shown herein, MIH and some types of amelogenesis imperfecta, and probably some types of fluorosis too, are distinguished as hypocalcification defects in that they have low amounts of amelogenin. That is, the normal process of amelogenin removal (enamel maturation) has occurred, but calcification has not occurred. In hypomaturation defects, however, (immature types of amelogenesis imperfecta and fluorosis), amelogenin removal (enamel maturation) has not occurred to a major degree and it is the continued presence of amelogenin that impedes calcification.
Amelogenin levels are relatively low in hypocalcification types of DDD/hypomineralisation, but closer to normal levels in hypomaturation types of DDD/hypomineralisation (such as some types of amelogenesis imperfecta and dental fluorosis). Therefore, variations in levels both of amelogenin and the remaining proteins disclosed herein bound to porous dental hydroxyapatite could be informative (e.g. diagnostic) individually or in combination, for example as a ratio.
For the first time, these results demonstrate extravasated albumin being accumulated in malforming human enamel. Notably, intact lesions were found to contain albumin but not numerous oral-fluid proteins with demonstrated hydroxyapatite-binding potential. That albumin but not haemoglobin was prominent may be attributed either to a minor vascular leak of serum rather than whole blood, or to high proteolytic stability of albumin relative to haemoglobin and other blood proteins during enamel maturation. Indeed, albumin is resistant to kallikrein-related peptidase 4, the major protease implicated in amelogenin removal.
These results also imply that another pathogenetic step follows post-eruptive breakdown of the enamel surface. This second step involves relatively promiscuous binding of oral-fluid proteins to the exposed hydroxyapatite matrix.
The proteins identified herein have potential utility as biomarkers for characterizing MIH lesions clinically.
SMCC (succinimidyl 4-[N-maleimidomethyl] cyclohexanecarboxylic acid N-hydroxysuccinimide ester; CAS#: 64987-85-5) is a non-cleavable heterobifunctional cross-linker with amine and sulfhydryl reactivity separated by a spacer arm of 8.3 Å. Amido black (CAS#: 1064-48-8) is a common blue/black stain used here as a coloured reporter which contains a primary amine group. Hemoglobin from cow (CAS#: 9008-02-0) is a heterotetramer consisting of 2 pairs of polypeptide chains (α and β; SEQ ID NOs: 20 and 21, respectively). The β-chain has a single solvent-exposed sulfhydryl-containing cysteine residue, while the α-chain has no cysteines.
SMCC (75 mM in dimethyl sulfoxide) was added to 9-volumes of amido black (37.5 mM in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, pH 7.4)) and incubated 30 minutes at 21° C. The 5-fold molar excess of Amido black ensured maximal labelling of SMCC (creating a maleimide-activated coloured reporter,
Hemoglobin (20 mg/ml; 0.65 μmole cysteine-thiol/ml) was prepared by dissolving in PBS that contained 10 mM TCEP (tris(2-carboxyethyl)phosphine, a non-thiol reducing agent used to maintain cystine-sulfhydryl state) and 5 mM EDTA (ethylenediaminetetraacetic acid, a metal chelator used to reduce potential for oxidant/radical catalysis and subsequent thiol oxidation). After a 30 minute incubation at 21° C., reduced hemoglobin was dialysed against 1,000-volumes of PBS for 2 hours to deplete TCEP and EDTA (this step may be optional). The hemoglobin was taken to the next step immediately to minimise cysteine-thiol oxidation.
The desiccated maleimide-activated Amido black was dissolved in Hemoglobin at a Amido black:thiol molar ratio of 10:1 to ensure maximal labelling of hemoglobin. After incubating for 2 hours at 21° C., Amido black-conjugated hemoglobin was dialysed extensively against PBS (until dialysate remained uncoloured, for 1 ml this took 24 to 48 hours) to remove non-covalently bound amido black. After dialysis, the probe was ready for use.
Results
Within 5 min of applying the probe, hydroxyapatite changed in colour from white to dark blue (
Discussion
A key design requirement was the preservation of hemoglobin's hydroxyapatite-binding function after conjugation to the coloured reporter. Hemoglobin's cysteine-thiols were targeted because two of the four protein subunits carry a single cysteine (not at binding interfaces); the other two subunits lack cysteine. The resulting tetramer probe therefore contains two unmodified protein subunits, thereby maintaining at least half the native hydroxyapatite-binding sites per functional unit. A 2-step method was exemplified: the first produced a coloured reporter-SMCC conjugate (
Methods
To test whether the probe of Example 2 binds to porous enamel specifically, a complex carious lesion was coated with the probe then washed thoroughly.
A human first molar that had a large region of caries (porous enamel, white opaque region) was photographed before and after application of the probe (
Results
Normal enamel was not labelled.
Regions of overt caries were labelled strongly and specifically, but labelling was patchy in some places. The unlabelled carious regions exhibited a shiny surface that was resistant to scratching, whereas labelled regions had a dull surface that could be scratched readily. This indicates that the areas of patchy unlabelled caries may be due to remineralisation of the surface layer. Thus, the probe is capable of discriminating between active and inactive caries.
Regions of enamel broken during extraction of the tooth (forceps imprints) were also labelled indicating that the probe can detect regions of enamel that have a breached surface.
The probe provided a stable level of labelling, independent of water-rinsing time.
Methods
To test whether the probe of Example 2 can specifically detect early caries, artificial carious lesions were produced on normal surface enamel using spots of strong acid (before application of probe).
A human first molar was shown by photography before and after application of the probe to be caries-free prior to acid-treatment (
Results
The probe did not bind to any regions of the caries-free enamel.
Acid etch treatment yielded three regions of slightly opaque/dull enamel, which followed a dose-dependent severity profile (10>3>1 min). The three etched regions were all detected by the probe, in a severity-dependent manner; un-etched enamel was not labelled.
Probe binding resisted washing in water, although signal intensity diminished slightly. The probe could be quantitatively removed by application of 10% bleach for 10 seconds.
Enamel from Example 4 (
Methods (A)
To rule out a protein-staining mechanism, the probe was applied to etched enamel that had been bleach-treated (i.e. protein stripped).
Results (A)
Bleached enamel was labelled by the probe similarly to unbleached (
Methods (B)
It was proposed that, if the probe's mechanism of action is hydroxyapatite-binding, then BSA pre-treatment should block probe binding (competitive inhibition).
Enamel from Panel A was exposed to a known hydroxyapatite-binding protein (10% bovine serum albumin, BSA) by applying with a brush for one minute, followed by water rinsing for one minute. After BSA treatment, the probe was applied as before. BSA was stripped by bleach treatment, and the probe re-applied.
Results (B)
Application of BSA did not alter appearance of the enamel (
Methods
To test whether the probe of Example 2 could be used to delineate abnormal dental tissues, a portion of tooth that contained normal and abnormal enamel & dentine was treated with the probe.
A fractured tooth that displayed a region of sub-surface hypomineralisation was chosen to mimic a clinically difficult case where lesion boundaries are obscure and complex. A brief pre-exposure to the probe led to demarcation of the enamel-dentine boundary. The specimen was then photographed before (left) and after (right) the probe was applied with a brush for 30 seconds (
Results
Before application of the probe, several structures could be identified: (1) normal enamel which overlaid (2) hypomineralised enamel (pink in colour with a red border in some regions), (3) apparently normal dentine (hard) and (4) abnormal dentine (soft/leathery). After application of the probe, all four types of tissue could be readily discerned.
Normal enamel and dentine were unlabelled. Hypomineralised enamel was uniformly and specifically labelled an intense violet colour, which appeared to trace a very complex border throughout the subsurface region. Abnormal dentine (potentially due to caries and/or developmental defects) was specifically and uniformly labelled a deep green colour, which appeared to trace complex borders against normal dentine. Together, these data confirm that the probe can specifically label hypomineralised enamel and abnormal dentine.
Methods
Hypomineralised enamel from Example 6 (
Results
The upper panels of
Abnormal dentine was not addressed in this example.
Methods
Abnormal dentine from Example 6 (
Results
The upper panels of
Methods
To test whether the probe's specificity for dentine, as shown in Example 8, could be improved, a bleach wash was used to reduce staining of normal dentine.
A human molar with exposed normal and abnormal dentine was exposed to the probe of Example 2 (brush application for one minute followed by water rinse for one minute) and subsequently exposed to a bleach wash (applied with brush for 10 seconds, then water rinsed for one minute). Following probe/bleach application, labelled regions were removed with a scalpel blade then re-probed/bleached to monitor progress (
Results
Abnormal dentine was preferentially detected by the probe, however background staining of normal dentine decreased confidence in border demarcation. Application of 10% bleach (0.4% NaOCl) for 10 seconds improved resolution by reducing labelling in normal dentine, but not in abnormal. Application of neat bleach (4% NaOCl) for 10 seconds completely removed labelling from normal dentine, without affecting labelling of abnormal dentine, resulting in much clearer delineation of abnormal dentine.
After neat bleach, abnormal dentine was removed (Panel 4) then re-probed/bleached (Panel 5), showing that most, but not all, abnormal dentine was removed. Another removal/re-probe/bleach step showed that abnormal dentine was completely removed. The remaining dentine was physically indistinguishable from normal dentine.
Together, these results suggest that a protein-stripping step after application of the probe can help reduce background labelling of normal dentine, reducing potential false-positive readouts.
Methods
The probe was made radio-opaque by substituting the blue chromophore (amido black) of Example 2 for 5-amino-2,4,6-triiodoisophthalic acid (3I), a precursor compound used in medical radiography (e.g. for cerebral angiography). This compound was chosen due to the availability of a single primary amine that could be used for coupling with the same cross-linker used in the blue probe.
To couple 3I to hemoglobin, the following procedure was used:
1. 1.25 mg of SMCC (cross-linker) was dissolved in 50 μl DMSO (75 mM SMCC).
2. 3I was prepared as follows: 30 mg was dissolve in 1 ml 0.1 M NaOH (50 mM 3I), 250 μl 0.1 M HEPES pH 7.0 was added, then pH was adjusted to 7 with 1 μl additions of 5 M NaOH; such that the final solution was 40 mM 3I, 20 mM HEPES pH 7.
3. 400 μl of 3I solution was added to 50 μl 75 mM SMCC in DMSO and incubated at room temperature for 30 minutes to generate 3I-activated SMCC.
4. The 3I-SMCC was then lyophilised by vacuum centrifugation.
5. The resultant pellet was taken up in 20 μl of DMSO and 100 μl of 20 mg/ml hemoglobin was added, then the solution was incubated at room temperature for 60 min to couple 3I to cysteine thiols in hemoglobin.
6. The resultant 3I-Hb was then centrifuged (20,000×g for 5 minutes) before dialysis (10-kDa MWCO) overnight against 25 mM Tris pH 7.2, 160 mM NaCl.
7. The resulting dialysate was collected and stored at −20° C.
To assess the degree of radio-opacity conferred on the probe, it was subjected to X-ray radiography (65 kV, 8 mA, 0.5 second exposure) alongside radio-opaque standards (1 and 10 mM 3I).
Results
The X-ray probe was radio-opaque to a degree between 1 and 10 mM 3I (
Methods
To examine the relative effectiveness of each wash solution, they were individually tested using an in vitro model system (
Pure hydroxyapatite (5 mg) was loaded with proteins from rat blood (100 μl of 10 mM Tris pH 7.2 which contained 10 μl Hb extract and 2 μl neat serum) for 10 minutes at room temperature with constant shaking. Protein-loaded hydroxyapatite was sedimented by centrifugation at 2,000×g for 30 seconds, the supernatant was discarded then the pellet was washed with 300 μl 10 mM Tris pH 7.2 for 30 seconds to remove unbound interstitial components.
Protein-hydroxyapatite was then exposed to 100 μl of various wash components (water, 5 mM MgCl2, 1 M MgCl2 or 0.4 M NaH2PO4) for 2 minutes at room temperature with mixing before centrifugation. Washes were collected and Protein-hydroxyapatite was washed another two times with the same washing solution. After three wash steps, the Protein-hydroxyapatite was dissolved in 100 μl 10% trifluoroacetic acid (TFA), and precipitated proteins collected by centrifugation (2,000×g for 2 minutes), and pellets were dissolved in 100 μl of 2× SoB (0.125M Tris-HCl pH 6.8, 4% SDS, 20% Glycerol). Protein content in all fractions was assessed by densitometry of dot-blots stained with Amido Black.
Results
The relative capabilities of the washing solutions to remove protein from hydroxyapatite were:
0.4 M PO4>1M Mg2+>5 mM Mg2+ (no more effective than water).
Although PO4 appeared to provide the best protein-removal, is was noted that the hydroxyapatite remained a pink colour even after 3 washes, whereas the 1M Mg2+-treated hydroxyapatite became white after a single wash. This being the case, it appears 1M MgCl2 and 0.4M NaH2PO4 are most effective at removing protein, and they have complementary activities (likely removing different classes of proteins).
Methods
Pure hydroxyapatite was loaded with proteins, then subjected to 100 μl of various wash components (water, 1 M MgCl2 or 0.4 M NaH2PO4) as for Example 11, except with two washes (instead of three) and 5 mM MgCl2 was omitted. One tube received 1M Mg2+ followed by 0.4 M PO4. After the washes, hydroxyapatite pellets were photographed to record the colour (see inset), then protein content was assessed as for Example 11.
Results
All three washing solutions performed similarly, removing the majority of proteins after two washes, unlike water (
Sequential washing with Mg2+ then PO4 produced the best result as assessed by protein removal and colour removal (inset: arrows indicate hydroxyapatite pellets after washing). It may be concluded that sequential washing with Mg2+ and PO4 provide optimal protein removal, in this hydroxyapatite model.
Methods
Pure hydroxyapatite was loaded with protein, then subjected to 100 μl of combined wash (1 M MgCl2, 0.4 M NaH2PO4) three times as for Example 11. Protein content was assessed as for Example 11. Note that results from Example 13 (
Results
The combination wash performed similarly to PO4 alone, however the hydroxyapatite turned white after the first wash (similar to 1M Mg2+ alone), indicating the activity of each wash component was retained. It may be concluded that a combined wash may be more effective in terms of the time required to achieve protein removal.
Methods
Hypomineralised enamel from intact and broken lesions was collected separately such that 3 tubes of 5 mg powder were available for each type of lesion. Enamel was exposed to 100 μl of 5 mM Mg2+, 1 M Mg2+, then 0.4 M PO4, each for 5 minutes. Samples were then treated as for the pure hydroxyapatite of Examples 11 to 13.
Results
Treatment of hypomineralised enamel with washing solutions removed a substantial amount of protein (˜¼-⅓), whereas water was barely effective (
Methods
Hypomineralised enamel from an intact lesion was collected such that 3 tubes of 5 mg was available. Enamel was exposed to 1 ml of 1 M Mg2+ for 7 hours, then 1 ml 0.4 M PO4 for a further 16 hours. Samples were then treated as for the pure hydroxyapatite of Examples 11 to 13.
Results
Proteins were quantitatively removed from hypomineralised enamel after two extended washes with washing solutions (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010901171 | Mar 2010 | AU | national |
This application is a continuation of U.S. application Ser. No. 15/422,996, filed Feb. 2, 2017, which is a divisional of U.S. application Ser. No. 13/501,676, filed Oct. 4, 2012, which is a national stage application of International Application No. PCT/AU2011/000303, filed Mar. 18, 2011, which claims priority to Australian Application No. 2010901171, filed Mar. 19, 2010; the entire contents of each are incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20190374439 A1 | Dec 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13501676 | US | |
| Child | 15422996 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15422996 | Feb 2017 | US |
| Child | 16551965 | US |
