WO2011128931A1

WO2011128931A1 - Pulpitis diagnostic marker and pulpitis diagnostic system

Info

Publication number: WO2011128931A1
Application number: PCT/JP2010/002653
Authority: WO
Inventors: 石田敬雄; 中島美砂子
Original assignee: 財団法人ヒューマンサイエンス振興財団
Priority date: 2010-04-12
Filing date: 2010-04-12
Publication date: 2011-10-20

Abstract

Provided is a pulpitis diagnostic system with which pulpitis can be diagnosed correctly and objectively. The pulpitis diagnostic system (300) includes an imaging fiber device (200) and a pulpitis diagnostic marker (100). The pulpitis diagnostic marker (100) includes an E-selectin antibody conjugated to the outer surface of a liposome which encapsulates indocyanine green. The imaging fiber (210) has a silicone tube (420) attached to the fiber tip. The silicone tube (420) is pressed against the lateral section of the tooth that has pulpitis. The pulpitis diagnostic marker (100) binds specifically with the inflammatory site caused by pulpitis. The fluorescent light emitted by indocyanine green is observed with a monitor (370). Thus, pulpitis can be diagnosed correctly and objectively.

Description

Pulpitis diagnosis marker and pulpitis diagnosis system

The present invention relates to a pulpitis diagnostic marker used for diagnosing the condition of a dental pulp to be examined, and a pulpitis diagnostic system using the pulpitis diagnostic marker.

Pulpitis is classified into acute pulpitis and chronic pulpitis, for example, and is often caused by touch. In the initial symptoms of acute pulpitis, degeneration of odontoblasts, hyperemia, serous exudation, etc. occur at the site of inflammation. For deciduous teeth and young people who have a fast progress of touching, the dentin is destroyed almost before and after acute purulent pulpitis, and open total purulent pulpitis occurs, resulting in pulp necrosis. If you have pulpitis, if you leave it untreated, it may progress to periodontitis.

If a bacterial infection spreads to the root dental pulp and falls into irreversible total gingivitis, a pulpectomy is performed. Extraction is the action of removing the pulp present inside the tooth. Since all pulps that are infected or possibly infected are removed by extraction, the spread of inflammation to the periodontal tissue can be prevented, the affected tooth is made harmless to the periodontal tissue, and the chewing function is restored again It becomes possible.

However, after removal of the dental pulp, the substance metabolism of the teeth (dentin / dental pulp) by the blood vessels is cut off, so that the dentin becomes brittle and easily broken. In addition, since there is no nerve to convey pain, no subjective symptoms can be obtained when the caries progresses again, and there is also a problem that the caries deteriorates unexpectedly because there is no infection resistance. Therefore, in recent years, it is considered that it is preferable to preserve the pulp in order to maintain and maintain the health of the teeth, and it is preferable to distinguish between a site infected with pulpitis and a healthy site as much as possible, There is a need for a method for diagnosing pulpitis that leaves healthy sites.

As a conventional method for diagnosing pulpitis, there are thermodiagnosis described in Non-Patent Documents 1 to 3. Thermodiagnosis is a method in which a cold stimulus or a thermal stimulus is applied to a tooth, and the inflammatory state of the pulp is judged based on the duration of pain after the removal of the temperature stimulus or the response to the cold stimulus after the temperature stimulus. For cold stimulation, dry ice, dichlorodifluoromethane, ethyl chloride, ice, tetrafluoroethane, or the like is used. A heating gutta percha is used for thermal stimulation. However, thermodiagnosis is based solely on the patient's senses and cannot be diagnosed objectively. In addition, the reaction varies depending on the condition of the tooth (the state of stenosis of the dental pulp cavity, the presence or absence of a restoration or prosthesis, and the type of the material), the stimulation time at the examination, and the temperature, and lesions occur in the pulp However, there is a problem that it may not show pain.

Non-patent document 4 describes electrodiagnosis as a method for diagnosing pulpitis. Electrodiagnosis is a method of applying an electrical stimulus (dent tester, analytic pulp tester) to the tooth surface, and examining the life or death of the pulp or the condition of the pulp based on the occurrence of pain. There is an advantage of high reliability. However, in electrodiagnosis, the electrical threshold value is increased or there is no reaction in the unfinished root tooth, the tooth in the middle of eruption, and the tooth immediately after the trauma. In the case of a multiple root tooth, even if one root is inactivated, it reacts if another root is alive. Even if inactivated, there may be pain sensations if the root canal is filled with electrolyte liquid. Furthermore, when there is a metal restoration, it cannot be used if an electric current flows through the gingiva. Furthermore, it is contraindicated for those who are wearing pacemakers. Moreover, it cannot be said that the pain occurrence threshold directly reflects the condition of the dental pulp.

Also, there is laser Doppler blood flow measurement as a method for diagnosing pulpitis. Trial of dental pulp diagnosis using laser Doppler blood flow measurement in endodontic treatment began in Gazelius B. et al., 1986 and Wilder-Smith PE. Et al., 1988 、, and the group of Matthews et al. (Matthews B. et al., 1993; Soo-ampon S. et al., 2003). The measurement principle is that when a laser beam irradiated on a tissue hits an erythrocyte moving in a blood vessel, the laser beam is reflected with a frequency deviation due to the Doppler effect. By analyzing the frequency deviation, the motion speed of red blood cells, that is, the blood flow speed circulating in the tissue is vector-calculated and analyzed. Unlike the above-mentioned thermodiagnosis and electrodiagnosis of the nerve reaction of the dental pulp, the laser Doppler blood flow measurement measures the presence or absence of dental pulp blood flow, so it can be applied even in cases where it is difficult to measure the nerve reaction. is there. However, there are disadvantages such as an expensive apparatus, being susceptible to measurement errors, poor reproducibility of measurement values, and taking time for measurement techniques. In addition, when blood flow is derived from the surface of the tooth, it is doubtful whether the reflected light is derived from the pulp and the blood flow of the gingiva is also mixed. It often ends up. Non-Patent Document 5 describes a device that analyzes transmitted light, not reflected light, and transmits laser light from the labial surface of the tooth and receives the transmitted light on a straight lingual tooth surface. A device that can prevent misdiagnosis of inactivated teeth is described, and it is effective for the diagnosis of the anterior teeth of young people with a wide pulp cavity, but it is insufficient for the diagnosis of the teeth and molars of the elderly There is a point.

Also, as described in Non-Patent Document 6, there is a nuclear magnetic resonance imaging (MRI) method as a method for diagnosing pulpitis. Nuclear magnetic resonance imaging uses a nuclear magnetic resonance phenomenon of hydrogen nuclei to create and diagnose an image, and is determined by an energy relaxation time T1 and a phase relaxation time T2. When the T1-weighted image is used, the vital dental pulp is emphasized brightly and the inactivated dental pulp is darkened. The advantages of the nuclear magnetic resonance imaging (MRI) method are that it is less exposed to radiation, is non-invasive, takes cross-sectional images in any direction, and has good soft tissue contrast. However, there are problems that the apparatus is expensive and cannot be applied to a patient wearing magnetic metal (attachment or metal restoration).

Furthermore, in Patent Document 1, a patient is asked whether or not the dental pain that has confirmed reversible pulp inflammation has been stopped by applying a solution of potassium oxalate dihydrate to the cavity surface of the interface of the dental restoration. A method for diagnosing pulpitis to be pointed out is described. However, the diagnostic method described in Patent Document 1 is based on the patient's senses and has a problem that cannot be objectively diagnosed.

Japanese translation of PCT publication 2003-528800 (page 13)

The present invention has been made in view of the above problems, and provides a pulpitis diagnostic marker that can accurately indicate the inflammation site of pulpitis with high sensitivity and accurately uses the pulpitis diagnostic marker. It is another object of the present invention to provide a pulpitis diagnosis system that can objectively diagnose pulpitis.

In order to achieve the above object, a pulpitis diagnosis marker according to the first aspect of the present invention provides pulpitis on the outer surface of a liposome encapsulating a fluorescent drug containing at least one of indocyanine green and its derivatives. A binding substance that specifically binds to the inflammatory site is bound.

In the above case, the binding substance is CD62E antibody, CD54 antibody, CD50 antibody, CD106 antibody, MHC class II antibody, PAF antibody, IL-8 antibody, MIP-1B antibody, E-selectin ligand, and E- It preferably contains at least one derivative of a ligand of selectin.

Further, the binding substance is preferably bound to the outer surface of the liposome via a linker protein.

In order to achieve the above object, a pulpitis diagnosis system according to the second aspect of the present invention is provided on the outer surface of a liposome encapsulating a fluorescent agent containing at least one of indocyanine green and its derivative, A pulpitis diagnosis marker characterized by binding a binding substance that specifically binds to an inflammation site of pulpitis, a light source unit that emits excitation light for exciting the fluorescent agent, and a light source unit The excitation light is guided to the condensing lens through a filter, and the input optical system that makes the light condensed by the condensing lens enter the end of the image fiber, the light detection unit that detects the optical signal, and the imaging fiber The fluorescence light of the pulpitis diagnostic marker attached to the affected part of pulpitis returning to the end is guided to a condenser lens through a filter, and the light collected by the condenser lens is An output optical system for guiding the serial light detection section, and having an imaging fiber device having an image display unit for displaying the diagnosis target pulp image based on the fluorescence light detected by the light detecting unit.

Further, it is preferable that a silicon tube that is pressed against the side of the tooth having the pulpitis inflammation site is attached to the fiber tip of the imaging fiber.

The wavelength of the excitation light is preferably 770 nm or more and 805 nm or less, the wavelength of the fluorescence light is 810 nm or more and 850 nm or less, and the difference in wavelength between the excitation light and the fluorescence light is preferably 15 nm or more and 50 nm or less. .

Moreover, it is preferable that the tip of the silicon tube has a concave curved shape on the inside.

The pulpitis diagnosis marker of the present invention can indicate the inflammation site of pulpitis with high sensitivity and accuracy. Also, the pulpitis diagnosis system of the present invention can accurately and objectively diagnose pulpitis.

It is explanatory drawing explaining the outline of a pulpitis diagnostic marker. It is explanatory drawing explaining the state which a pulpitis diagnostic marker couple | bonds specifically with a pulpitis inflammation site | part. It is explanatory drawing explaining the outline of an imaging fiber apparatus. It is explanatory drawing explaining the front-end | tip of an imaging fiber, and is the state before attaching an elastic tube. It is explanatory drawing explaining the front-end | tip of an imaging fiber, and is a state which covered the objective lens with the elastic tube. It is sectional drawing of the fiber main body of an imaging fiber. 1 is an overall view illustrating an outline of a pulpitis diagnosis system. It is a flowchart explaining the example of 1 use aspect of a pulpitis diagnosis system. It is explanatory drawing which shows another embodiment of the shape of the front-end | tip part of a silicon tube. It is explanatory drawing which shows another embodiment of the shape of the front-end | tip part of a silicon tube, and is explanatory drawing which shows the state pressed against the side surface of the tooth which has an inflammation part. It is a schematic diagram of partial serous pulpitis. It is a schematic diagram of total suppurative pulpitis. It is a schematic diagram of partial suppurative pulpitis. It is a schematic diagram of total serous pulpitis.

[Pulmonary pulpitis diagnostic marker]
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. FIG. 1 is a schematic diagram of a pulpitis diagnosis marker 100 according to the present embodiment, and FIG. 2 is an explanatory diagram for explaining how the pulpitis diagnosis marker 100 binds to a pulpitis inflammation site.

As shown in FIG. 1, in the pulpitis diagnostic marker 100, the binding substance 120 is bound to the outer surface of the liposome 110 encapsulating indocyanine green (ICG) 140 via the linker protein 130.

Indocyanine green 140, which is a kind of indocyanine compound, is a structural formula represented by Formula 1, and is a substance that emits dark green-blue fluorescence by exhibiting specific absorption by infrared irradiation.

As the derivative of indocyanine green 140, a derivative that modifies the mother nucleus of indocyanine green 140 or a compound into which a reactive group is introduced can be used. Reactive indocyanine green derivatives into which reactive groups have been introduced may be sparingly soluble in water, but indocyanine green derivatives are not used after being dissolved in an organic solvent or the like, but are used in liposome 110. It can be used without difficulty. The indocyanine green derivative is not particularly limited, but for example, the one represented by Formula 2 can be used.

Here, R1 and R2 each independently represent a hydrogen atom, an alkyl group, an aryl group, a sulfonic acid group, or an alkoxy group, and R3 represents an alkyl group, a sulfonic acid alkyl group, or an aminoalkyl group. As the alkyl group, linear or branched lower alkyl having 1 to 6 carbon atoms can be used.

The liposome 110 is a spherical particle (closed vesicle) in which a solvent is encapsulated inside a membrane composed of one or more lipid bilayers. The lipid composition, charged state, density, weight, particle size and the like of the liposome 110 can be appropriately designed according to the properties of the binding substance and the like that bind to the outer surface of the liposome 110.

The components of the lipid bilayer membrane of the liposome 110 are not particularly limited, and are, for example, phospholipids and glycolipids. Phospholipids include, for example, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, lecithin, lysolecithin, phosphatidylcholine, phosphatidylserine, sphingomyelin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristolphosphatidylcholine, dioleylphosphatidylcholine, Distearoyl phosphatidylserine, distearoyl phosphatidylglycerol, and mixtures thereof. The glycolipid is, for example, glycerolipid such as galactosylceramide, galactosylceramide sulfate, digalactosyl diglyceride, galactosyl diglyceride sulfate, lactosylceramide, sphingoglycolipid such as ganglioside G7, ganglioside G6, ganglioside G4, and the like. It is a mixture.

Other substances can be added to the liposome 110 as necessary as a membrane-constituting substance. For example, sitosterol, cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, stigmasterol, campesterol, cholestanol, lanosterol as a membrane stabilizer. , 1-O-sterol glucoside, 1-O-sterol maltoside, and mixtures thereof.

The liposome 110 can be positively charged, negatively charged, or electrically neutral as a whole. When charging to a negative charge, for example, dicetyl phosphate which is a negatively charged substance can be added, and when charging to a positive charge, an aliphatic amine such as stearylamine can be added.

The weight of the liposome 110 is not particularly limited, and can be increased by filling the liposome with a high-density solution such as cesium chloride. The density of the liposome 110 can be increased by incorporating a polysaccharide such as dextran sulfate into the liposome.

In order for the pulpitis diagnostic marker 100 to appropriately reach the inflammation site of pulpitis, it is preferable that the pulpitis diagnosis marker 100 is not easily captured from the phagocytic cells of the cell endothelial tissue. A polyalkylene oxide chain (polyoxyalkylene chain) or PEG chain, which is an inert hydrophilic polymer, can be attached to the liposome surface in order to make it difficult to be captured from phagocytic cells of cell endothelial tissue. PEG is preferably polyethylene glycol having 10 to 3500 oxyethylene units. The amount of PEG used can be 0.1 to 30% by mass with respect to the lipid constituting the liposome.

The average inner diameter of the liposome 110 in the capsule can be appropriately adjusted in consideration of stability and the like, and can be, for example, 30 to 600 nm, preferably 50 to 500 nm, more preferably 60 nm to 400 nm. Here, the average inner diameter in the capsule of the liposome 110 is measured by the digital Coulter principle.

The binding substance 120 that specifically binds to the inflammation site of pulpitis is not particularly limited. For example, CD62E antibody, CD54 antibody, CD50 antibody, CD106 antibody, MHC class II antibody, PAF antibody, IL-8 Antibodies, MIP-1B antibodies, ligands for E-selectin, derivatives of ligands for E-selectin, and mixtures thereof.

CD62E antibody is an antibody that binds to E-selectin. E-selectin belongs to the selectin family of cell adhesion molecules, is specifically expressed in vascular endothelial cells (endothelium), is synthesized by inflammatory stimuli such as pulpitis, and is expressed on the cell surface. The CD54 antibody is an antibody that binds to ICAM-1 (intercellular adhesion molecule-1), and ICAM-1 is an integrin ligand that appears on the surface of vascular endothelial cells upon inflammation stimulation such as pulpitis. CD106 antibody is an antibody that binds to VCAM-1 (vascular cell adhesion molecule-1), and VCAM-1 is an integrin ligand that appears behind ICAM-1 on the surface of vascular endothelial cells due to inflammation stimulation such as pulpitis. is there. The CD50 antibody is an antibody that binds to ICAM-3 (intercellular adhesion molecule-3). MHC class II antibodies are antibodies against MHC class II molecules. There are two polymers of MHC class II molecules, α chain and β chain, each consisting of two extracellular region, transmembrane region and intracellular region. There are three types of MHC class II molecules in humans: HLA-DR, HLA-DQ, and HLA-DP.

PAF antibody is an antibody that specifically binds to PAF (Platelet-activating factor), which is one of inflammatory mediators. IL-8 antibody is an antibody that specifically binds to interleukin-8. Interleukin 8 is a basic polypeptide having a molecular weight of 8 KDa and is a chemotactic factor that acts on neutrophils and T lymphocytes during selection. The MIP-1B antibody is an antibody that specifically binds to MIP-1B, which is a ligand of the chemokine receptor CCR5.

The ligand of E-selectin is a glycoprotein ligand such as PSGL-1, a glycolipid ligand, and a sugar chain ligand which is a terminal structure thereof. Examples of the sugar chain ligand include sialyl Lewis X (SLeX), Lewis X, sulfate sugar and the like. SLeX is a sugar chain present at the sugar chain terminal of the above glycoprotein.

The ligand derivatives of E-selectin include glycoproteins, glycolipids and sugar chain derivatives which are selectin ligands.

The binding of the binding substance 120 that specifically binds to the inflammation site of pulpitis is not particularly limited. For example, as shown in FIG. 1, the binding substance 120 binds via the linker protein 130. It is preferable to do.

As the linker protein, for example, serum albumin is preferably used, and more specifically, human serum albumin, bovine serum albumin and the like are used.

Next, an example of a method for producing the above-described pulpitis diagnostic marker 100 will be described.

(Preparation of indocyanine green encapsulated liposomes)
Liquid carbon dioxide is added in the presence of membrane components such as phospholipids, sterols, and lecithins, and mixed with cationic substances, sterols, glycols, etc. at a pressure of 50 to 500 kg / cm ² and a temperature of 31 to 200 ° C., for example. And dissolve. Subsequently, an aqueous solution containing indocyanine green is continuously added to form an aqueous / carbon dioxide emulsion. In this emulsion system, the lipid component is in the form of micelles and separates and collects. Further, the aqueous solution is continuously added until the carbon dioxide phase and the aqueous phase are separated. As the aqueous phase increases, the phase transition of the system occurs. When the system is depressurized and carbon dioxide is discharged, an aqueous dispersion in which the liposome 110 encapsulating indocyanine green 140 is dispersed is generated. Then, it is also possible to perform a microbial treatment as needed.

The content of the fluorescent drug in the liposome 110 is 1 to 15, preferably 3 to 10, more preferably 5 to 8 with respect to the liposome membrane lipid weight. desirable. This is because if the weight ratio of the fluorescent drug in the liposome 110 is less than 1, the delivery efficiency of the fluorescent drug to the pulpitis inflammation site may deteriorate. On the other hand, if the weight ratio of the fluorescent agent in the liposome 110 exceeds 10, the liposome may be structurally unstable.

(Hydrophilization on the liposomal lipid membrane surface)
Subsequently, a crosslinking reagent such as bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) is added and stirred to form a chemical bond between the lipid on the liposome membrane and BS ³ . Next, for example, after adding tris (hydroxymethyl) aminomethane and stirring, the liposome in which indocyanine green is encapsulated by completing the chemical bonding reaction between BS ³ bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane is completed. Hydrophilicity is achieved by the coordination of the hydroxyl group of tris (hydroxymethyl) aminomethane on the membrane lipid dipalmitoylphosphatidylethanolamine.

(Linker protein binding)
Next, for example, sodium metaperiodate is added and stirred to oxidize the liposomes, human serum albumin is added to the oxidized liposomes and stirred, and human serum albumin is bound by a coupling reaction.

(Binding of a binding substance that specifically binds to the inflammation site of pulpitis)
Subsequently, the E-selectin antibody is added to an NH ₄ HCO ₃ aqueous solution and stirred, and then a glycosylamine compound of the E-selectin antibody is obtained. Next, 1 mg of the crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate (DTSSP; Pierce Co., USA) is added to the liposome solution to which human serum albumin is bound, and the mixture is stirred to bind the DTSSP to the HSA on the liposome. Liposomes are obtained. Next, the glycosylamine compound of the above E-selectin antibody is added to this liposome solution and stirred for a certain period of time, and the E-selectin antibody is bound to DTSSP on the liposome membrane surface-bound human serum albumin. In this way, a pulpitis diagnostic marker in which an E-selectin antibody is bound to the surface of a liposome encapsulating indocyanine green is obtained.

Next, an example of use of the above-described pulpitis diagnosis marker 100 will be described.

First, an aqueous solution in which the pulpitis diagnosis marker 100 is dispersed is injected into a subject by intravenous injection or the like. The amount to be injected into the subject is not particularly limited. For example, the amount of indocyanine green per kilogram of the subject's body weight can be 0.2 to 1.0 mg. On the other hand, as shown in FIG. 2, in the subject, E-selectin is synthesized in the body by stimulation based on pulpitis, and is expressed on the blood vessel surface of the inflammation site 190 of pulpitis. Since the E-selectin antibody of the pulpitis diagnosis marker 100 has an action of specifically binding to E-selectin, the pulpitis diagnosis marker 100 is transferred from the blood vessel 180 near the pulpitis inflammation site 190 to the pulpitis inflammation site 190. Bind specifically. Thereafter, the indocyanine green 140 of the pulpitis diagnosis marker 100 emits fluorescence by irradiating the vicinity of the pulpitis inflammation site 190 with excitation light. Thereby, the pulpitis inflammation site 190 can be grasped with high sensitivity and accuracy.

[Pulmonitis diagnosis system]
Next, an embodiment of a pulpitis diagnosis system according to the present embodiment will be specifically described with reference to the accompanying drawings. The pulpitis diagnosis system 300 includes the above-described pulpitis diagnosis marker 100 and the imaging fiber device 200.

FIG. 3 is a schematic diagram of the imaging fiber device 200. The imaging fiber device 200 includes a light source unit 260 that emits excitation light, an input optical system, an output optical system, a light detection unit 340 that detects an optical signal, and an image display unit 370. The input optical system guides the excitation light from the light source unit 260 to the condensing lens 220 through the filter 240 and causes the light condensed by the condensing lens 220 to enter the end portion 211 of the image fiber 210. The wavelength of the excitation light is, for example, 770 to 805 nm, preferably 780 to 795 nm. The output optical system guides the fluorescent light returning to the end 211 of the imaging fiber 210 to the condenser lens 330 through the filter 310 and guides the light collected by the condenser lens 330 to the light detection unit 340. The wavelength of the fluorescent light is, for example, 810 to 850 nm, preferably 820 to 840 nm. The image display unit (monitor) 370 displays a dental pulp image to be diagnosed based on the fluorescent light detected by the light detection unit 340. In addition, the imaging fiber device 200 includes a light source control unit 270, a control unit 280, an operation unit 360, a recording unit 380, and an outer box 390.

As shown in FIG. 4A, the imaging fiber 210 has a fiber main body 213, an objective lens 215, and a cylindrical member 214, and a cylindrical elastic tube 420 is attached to the tip. The objective lens 215 is a biconvex lens whose both end surfaces are polished and mirror-finished. The objective lens 215 is bonded and fixed to one end side of the cylindrical member 214. The objective lens 215 takes in light from the outside and forms an image on the light receiving end face of the fiber bundle. As shown in FIG. 5, the fiber main body 213 is formed of a fiber bundle in which a plurality of optical fibers 216 are bundled, for example, 10,000 to 30,000 and stored in a light leakage preventing coating tube. The diameter of this fiber bundle is, for example, 1.0 to 2.5 mm. The fiber main body 213 has a certain elasticity and can be bent so as not to be less than a predetermined curvature. The optical fiber 216 is not particularly limited, and is, for example, a quartz optical fiber, a plastic optical fiber, or the like. The diameter of the optical fiber 216 is, for example, 2 to 4 μm. The cylindrical member 214 has an inner diameter in which the objective lens 215 and the fiber bundle can be accommodated, the objective lens 215 is attached to one end side, and the fiber bundle is attached to the other end side, and the objective lens 215 and the fiber bundle are integrated. . As shown in FIG. 4B, the tubular member 214 is covered with an elastic tube 420. As will be described later, the elastic tube 420 is used to fix the objective lens 215 by being pressed against the side of the tooth 191 where the pulpitis has occurred. The elastic tube 420 is preferably made of a material having elasticity and biocompatibility, such as a silicon tube. The length and outer diameter of the elastic tube 420 are not particularly limited, but are appropriately designed depending on the size of the teeth to be pressed, and the length 420L is, for example, 5 to 6 mm.

3, the operation of the imaging fiber device 200 will be described. The control unit 280 sends a light source activation control signal to the light source control unit 270 based on an instruction from the operation unit 360. The light source control unit 270 activates the light source unit 260 such as a xenon lamp. The light from the light source unit 260 is collected by the condenser lens 250, excited by the excitation filter 240, condensed by the condenser lens 220, and guided to the end 211 of the imaging fiber 210.

The excitation light sent to the end 211 of the image fiber 210 is irradiated with evanescent light from the fiber tip 212. When the fluorescence from indocyanine green 140 is received by the fiber tip 212 of the image fiber 210, the fluorescence is emitted from the end 211 of the image fiber 210 and enters the condenser lens 220 and the dichroic mirror 230. The dichroic mirror 230 reflects the wavelength band including fluorescence and is guided to the filter 310 through the convex lens 290. The filter 310 removes noise portions other than the fluorescent region, and the fluorescence from which the noise has been removed enters the photodetector 340 via the reflecting mirror 320 and the condenser lens 330. The difference in wavelength between the excitation light and the fluorescence light is, for example, 15 nm to 50 nm.

The photodetector 340 is in a state where fluorescence can be detected by receiving the start signal and the timing signal from the photodetector controller 350. The fluorescence detected by the photodetector 340 is converted into an electrical signal and amplified by the photodetector controller 350. The control unit 280 performs light source control and measurement operation by an arithmetic element such as a CPU based on a control program, A / D converts the received output of the photodetector control unit 350, and controls the indocyanine green 140 based on a predetermined formula. Calculate the fluorescence intensity. Then, the abundance of indocyanine green 140 is calculated based on the fluorescence intensity. The monitor 370 displays a menu screen for measurement operation, and displays the measured fluorescence intensity and the amount of indocyanine green 140 present. The recording unit 380 records changes in fluorescence intensity of indocyanine green 140 over time.

Next, how the pulpitis diagnosis system 300 according to this embodiment is used will be described with reference to FIGS. FIG. 6 is a schematic diagram of a usage mode of the pulpitis diagnosis system 300. FIG. 7 is a flowchart showing how the pulpitis diagnosis system 300 is used. FIG. 8A is an explanatory view showing another embodiment of the shape of the tip of the silicon tube. FIG. 8B is an explanatory view showing another embodiment of the shape of the distal end portion of the silicon tube, and is an explanatory view showing a state of pressing against the side surface of a tooth having an inflammatory site. FIG. 9A is a schematic diagram of partial serous pulpitis. FIG. 9B is a schematic diagram of total suppurative pulpitis. FIG. 9C is a schematic diagram of partial purulent pulpitis. FIG. 9D is a schematic diagram of total serous pulpitis.

First, the elastic tube 420 attached to the fiber tip 212 is pressed against the side of the tooth 191 having a pulpitis inflammation site (S001). Since the elastic tube 420 has elasticity, the fiber tip 212 is fixed by being pressed against the teeth 191. In addition, as for the shape of the elastic tube 420, as shown to FIG. 8A, it is preferable that the front-end | tip part 421 of the elastic tube 420 is a concave curved surface shape inside. With this configuration, as shown in FIG. 8B, the distal end portion 421 of the elastic tube 420 can be brought into contact with the side surface of the tooth 191 having the pulpitis inflammation site with a small gap.

Next, the imaging fiber device 200 is turned on and irradiated with excitation light (S002). Then, the returning fluorescent light is detected by the photodetector 340 (S003), and this light output is confirmed by the monitor 370 to determine whether or not the excitation light output is stable (S004). If the excitation light output is not stable, the excitation light irradiation is appropriately adjusted.

After confirming that the output is stable, the pulpitis diagnosis marker 100 is intravenously injected to the subject by injection 410 (S005). In order to appropriately reach the pulpitis diagnostic marker 100 to the inflamed site, it is desirable that the injection 410 is performed on the gum where the tooth 191 having the inflamed site of pulpitis is located. The injected pulpitis diagnostic marker 100 binds to the pulpitis inflammation site, and the fluorescent light intensity is displayed on the monitor 370 (S006). Here, it is determined whether or not the amount of insertion of the pulpitis diagnosis marker 100 is sufficient (S007). If it is determined to be insufficient, the pulpitis diagnosis marker 100 is appropriately supplemented and injected.

Wait for a predetermined time after injection (S008). Then, it is determined whether or not the fluorescence intensity is observed after a predetermined time (S009). If the fluorescence intensity is not observed, it does not correspond to pulpitis (S010). If the subject does not have an inflammatory site, indocyanine green is bound to lipoproteins in the blood and transported to the liver, where it is ingested by hepatocytes while passing through the sinusoids and excreted in bile without being conjugated. Because. On the other hand, when the fluorescence intensity is observed, a further diagnosis can be made by further classifying the pulpitis from the degree of the fluorescence intensity, and it is determined whether or not the fluorescence is observed in the entire pulp (S011). When fluorescence is not observed in the entire pulp but partially observed, it is diagnosed as partial serous pulpitis schematically shown in FIG. 9A (S012). Se represents a serous inflammation part. Next, although fluorescence intensity is observed throughout the dental pulp, it is determined whether or not there is a difference in fluorescence intensity (S013). If so, all suppurative pulpitis is diagnosed as schematically shown in FIG. 9B (S014). In addition, Su shows a purulent inflammation part. Next, if there is a partial difference in the fluorescence intensity, it is determined whether or not there is a strong fluorescence intensity only in the medullary corner (S015), and the fluorescence intensity is not limited to only the medullary corner. In this case, as shown schematically in FIG. 9C, partial purulent pulpitis is diagnosed (S016). On the other hand, when only the medullary corner has a strong fluorescence intensity, as shown schematically in FIG. 9D, all serous pulpitis is diagnosed (S017).

In serous pulpitis, exudates mainly consist of exudate cells such as plasma cells, lymphocytes, leukocytes, etc. and serous components, and the pulpitis is weak in tissue destruction. Pulpitis with strong tissue destruction (consisting of neutrophils and concentrates that have undergone fat metamorphosis), and purulent inflammatory parts with stronger tissue destruction than with serous inflammatory parts with weaker tissue destruction, such as E-selectin Because of the large amount of occurrence, the purulent inflammatory part has a stronger fluorescence intensity than the serous inflammatory part. In FIG. 9D, purulent inflammation is present in the medullary horn in spite of all serous pulpitis. This is because secondary dentin is difficult to produce and bacteria are likely to invade, and purulent inflammation is likely to occur in the horn.

Conventionally, the classification or diagnosis of pulpitis is based on thermodiagnosis (partial serous pulpitis if only cold water pain, all suppurative pulpitis if only warm water pain, cold water pain> all serous if warm water pain) Although it was caused by pulpitis, cold water pain <partial purulent pulpitis if warm water pain), etc., the pulpitis diagnosis system according to this embodiment can make an objective and accurate judgment regardless of such a subjective judgment method. Or can be diagnosed.

The classification or diagnosis of pulpitis is not limited to such an embodiment. For example, whether pulp extraction is performed by observing whether the fluorescence intensity exists only in the crown pulp or even in the root pulp. It is possible to accurately determine whether or not. That is, when the inflammation is limited to only the crown pulp and does not reach the root pulp, only the crown pulp is removed and a spinal cord method is performed to preserve the root pulp tissue. However, if the inflammation has spread to the root pulp, the entire pulp tissue is removed and prevented from spreading to the periodontal tissue. Furthermore, with the pulpitis diagnosis system according to the present embodiment, when the inflammation extends to the root pulp, treatment can be performed after the patient is convinced by showing the fluorescence intensity image, which contributes to informed medicine. .

In addition, pulpitis generally spreads from the crown pulp to the root pulp, but the highly advanced marginal periodontitis of the lesion or the apical periodontal of the adjacent tooth. When purulent pulpitis such as inflammation develops near the root apex of a healthy tooth, inflammation spreads through the root apex of the healthy tooth or the side branch of the root canal into the root pulp tissue, and further, the crown pulp Spreads to tissues (ascending pulpitis). Thus, when the inflammation expands in the opposite direction to the general direction of pulpitis, it is called ascending pulpitis, but by using the pulpitis diagnosis system according to this embodiment, from the root pulp tissue. When strong fluorescence intensity is observed in the coronal pulp tissue, it can be determined as ascending pulpitis. Note that the pulpitis determination method in one specific example of the present invention described above is not uniquely determined by observing the fluorescence intensity, and the final determination is based on the experience of the dentist. However, it has tremendous benefits in that it helps the dentist to accurately identify cases.

As described above, according to the pulpitis diagnosis system according to the present invention, it is possible to objectively diagnose the pathology of pulpitis that has conventionally relied on the reaction of the nerve or the subjective sense of the patient, and directly in deep caries or pulpitis. Criteria for selection of pulp capping, vital pulp cutting, and pulp extraction can be obtained, and it is possible to accurately identify cases that can be left without removing the pulp, leading to the prolongation of the pulp and the life of the tooth. it can. It can also be used as a diagnostic criterion before, during, and after treatment when regenerating dental pulp.

Hereinafter, an example of manufacturing experiment of the pulpitis diagnostic marker 100 will be described.

(1) Preparation of liposomes Liposomes were prepared using a cholic acid dialysis method. That is, dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, ganglioside and dipalmitoyl phosphatidylethanolamine are each in a molar ratio of 36: 40: 4: 15: 5 to give a total lipid amount of 45.6 mg. Then, 47.1 mg of sodium cholate was added and dissolved in 2.9 ml of chloroform / methanol solution.

The solution was evaporated and the precipitate was dried in vacuum to obtain a lipid membrane. The obtained lipid membrane was suspended in 2.9 ml of TAPS buffer (pH 8.5) and sonicated to obtain 2.9 ml of a transparent micelle suspension. To this micelle suspension, PBS buffer (pH 7.1) was added to make 4.9 ml, and then ICG completely dissolved with TAPS buffer (pH 8.3) to 2250 mg / 6 ml was slowly stirred with stirring. The ICG-containing micelle suspension was subjected to ultrafiltration using a PM10 membrane (Amicon Co., USA) and a TAPS buffer (pH 8.3) to obtain uniform ICG-encapsulated liposomes. 89 ml was prepared.

(2) Hydrophilization 9.89 ml of the ICG-encapsulated liposome solution prepared in (1) was subjected to ultrafiltration using XM300 membrane (Amicon Co., USA) and CBS buffer (pH 8.6) to adjust the pH of the solution to 8 .6. Next, 10 ml of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at 25 ° C. for 2 hours. Thereafter, the mixture was further stirred overnight at 6.7 ° C. to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS ³ on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.6).

Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of CBS buffer (pH 8.6) was added to 9.9 ml of liposome solution, stirred at 25 ° C. for 2 hours, and then stirred at 6.8 ° C. overnight to prepare liposome membrane. The chemical binding reaction between BS ³ bound to the above lipid and tris (hydroxymethyl) aminomethane was completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the ICG-encapsulated liposome membrane to make it hydrated and hydrophilic.

(3) Binding of human serum albumin (HSA) onto the surface of ICG-encapsulated liposome membranes Known methods (Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242, 56-65 ) Using a coupling reaction method. That is, this reaction is performed in a two-step chemical reaction. First, a metabolite obtained by dissolving ganglioside obtained in (2) on the 9.9 ml liposome membrane surface in 1 ml of TAPS buffer (pH 8.3). After adding 43 mg of sodium periodate and stirring at room temperature for 2 hours to oxidize periodate, 9.9 ml of oxidized liposome was obtained by ultrafiltration with XM300 membrane and PBS buffer (pH 8.0). . To this liposome solution, 20 mg of human serum albumin (HSA) was added and stirred at 25 ° C. for 2 hours. Next, 100 μl of 2M NaBH ₃ CN was added to PBS (pH 8.0) and stirred overnight at 10 ° C. HSA was bound by a coupling reaction between the above ganglioside and HSA. Then, after ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.6), 9.9 ml of an HSA-conjugated ICG-encapsulated liposome solution was obtained.

(4) E-selectin antibody binding and linker protein (HSA) hydrophilic treatment on ICG-encapsulated liposome membrane surface-bound human serum albumin (HSA) E-selectin antibody 48 μg was dissolved in 0.25 g NH ₄ HCO ₃ It was added to a 0.5 ml aqueous solution and stirred at 37 ° C. for 3 days, followed by filtration with a 0.45 μm filter to complete the amination reaction of the reducing end of the sugar chain to obtain 48 μg of a glycosylamine compound of an E-selectin antibody. . Next, 1 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidyl) propionate (DTSP; Pierce Co., USA) was added to 1 ml of a portion of the prednisolone phosphate-encapsulated liposome solution obtained in (3) for 2 hours at 25 ° C. Subsequently, the mixture was stirred overnight at 7 ° C., and ultrafiltered with an XM300 membrane and a CBS buffer (pH 8.6) to obtain 1 ml of liposome in which DTSSP was bound to HSA on the liposome.

Next, 48 μg of the above-mentioned E-selectin antibody glycosylamine compound was added to the liposome solution, and the mixture was stirred at 25 ° C. for 2 hours and then stirred overnight at 7 ° C., and then XM300 membrane and PBS buffer (pH 8.0). The E-selectin antibody was bound to DTSSP on liposome membrane surface-bound human serum albumin. As a result, 2 ml of ICG-encapsulated liposomes obtained by hydrophilizing linker protein (HSA) in which E-selectin antibody, human serum albumin (HSA) and liposome were bound were obtained.

Since it can be used for accurate and objective diagnosis of pulpitis, criteria for selection of a pulpectomy method can be obtained, and the cases that can be left without removing the pulp can be accurately identified.

100: pulpitis diagnostic marker 110: liposome 120: binding substance 130: linker protein 140: indocyanine green 190: pulpitis inflammation site 200: imaging fiber device 210: imaging fiber 213: fiber body 214: cylindrical member 215: objective Lens 216: Optical fiber 220: Condensing lens 230: Dichroic mirror 240: Filter 250: Condensing lens 260: Light source unit 270: Light source control unit 280: Control unit 290: Convex lens 300: Pulpitis diagnosis system 310: Filter 320: Reflection Mirror 330: Condensing lens 340: Light detection unit 350: Photo detector control unit 360: Operation unit 370: Image display unit (monitor)
380: Recording unit 390: Outer box 420: Elastic tube

Claims

A binding substance that specifically binds to an inflammation site of pulpitis is bound to the outer surface of a liposome encapsulating a fluorescent drug containing at least one of indocyanine green and its derivatives. Pulpitis diagnostic marker.
The binding substance includes CD62E antibody, CD54 antibody, CD50 antibody, CD106 antibody, MHC class II antibody, PAF antibody, IL-8 antibody, MIP-1B antibody, ligand of E-selectin, and derivative of ligand of E-selectin The pulpitis diagnostic marker according to claim 1, comprising at least one of the following.
The pulpitis diagnosis marker according to claim 1, wherein the binding substance is bound to the outer surface of the liposome via a linker protein.
A pulpitis diagnostic marker in which a binding substance that specifically binds to an inflammation site of pulpitis is bound to an outer surface of a liposome encapsulating a fluorescent agent containing at least one of indocyanine green and its derivatives A light source unit that emits excitation light for exciting the fluorescent agent, and the excitation light from the light source unit is guided to a condenser lens through a filter, and the light condensed by the condenser lens is applied to the end of the image fiber. Fluorescent light from the pulpitis diagnostic marker coupled to the pulpitis inflammation site that returns to the end of the imaging fiber is guided to the condenser lens through a filter. An output optical system for guiding the light collected by the condenser lens to the light detection unit, and a dental pulp image to be diagnosed based on the fluorescent light detected by the light detection unit. Pulpitis diagnosis system and having a imaging fiber device having an image display unit for.
5. The pulpitis diagnosis system according to claim 4, wherein a silicon tube pressed against the side of the tooth having a pulpitis inflammation site is attached to the fiber tip of the imaging fiber.
The wavelength of the excitation light is 770 nm or more and 805 nm or less, the wavelength of the fluorescence light is 810 nm or more and 850 nm or less, and the wavelength difference between the excitation light and the fluorescence light is 15 nm or more and 50 nm or less. The pulpitis diagnosis system according to claim 4.
6. The pulpitis diagnosis system according to claim 5, wherein the tip of the silicon tube has a concavely curved shape inside.