JP4458373B2 - Dental aqueous solution for acid-resistant film formation and method for producing the same - Google Patents

Description

  The present invention relates to a method of reducing dentin sensitivity, acid permeability and discomfort by occluding dentin tubules using an acid resistant film-forming liner material.

  It has often been reported that after surgery, the dentin becomes more sensitive or painful, and sudden heat stimulation is suddenly applied to specific teeth or groups of teeth. The output of the system (light, heat, etc.) after replacing the restoration part due to recurrent caries symptoms below the restoration part placed before the existing amalgam alloy or dental resin composite restoration part, or whitening the teeth ) The above symptoms may occur after the supporting tooth bleaching process. The patient is simply alerted to the dentist to note that a fast air jet, cold drinks, and the painfulness of other factors such as occlusal force and acid food may suddenly increase. Not too much. When there is cold water, cold air, a gradual change in osmotic pressure, or stimulation of sweet or acidic solution around the surface of the restoration cavity, dentin pain response immediately increases. The dentist may refer to this picture phenomenon simply as the patient's dentin pain (postoperative sensitivity / DPH) or simply as dental discomfort. The patient is often told the dentist to simply wait 2-3 days or 2-3 weeks, and the discomfort distress is gradually reduced and eventually said to disappear.

Amalgam alloy or resin composite restorations are applied to live dentin treated with conventional dentin liners such as calcium hydroxide Ca (OH) ₂ materials such as Dycal (Dycal (r)) or Life (Life (r)). Among many recent patients, dentine acute, sharp and stinging dentin pain is often a very common complaint. The postoperative hypersensitivity of dentin is due to normal physiological degradation of the applied layer, or removal of the applied layer around the sinus surface by changing the oral fluid from acidic pH 2.7 to neutral pH 6.0. Usually occurs.

  If a dentist uses any type of instrument, for example a rotating instrument with a drill or drilling tool, it will be scraped or polished with some kind of manual instrument and scraped onto the surface of the tooth, called the application layer. Will leave a layer. When the coating layer is damaged by physiological action or by the dentist, the dentinal tubule group opens and is exposed to the flow of fluid flowing in both directions from the dentine pulp. It is because of this increased bidirectional fluid flow that the patient's dentin becomes post-operatively sensitive to cold or fast airflow.

In many patients, the existing amalgam alloys and resin composite restorations and the underlying Dical (r) and Life (r) Ca (OH) ₂ bases are washed out and removed, and the dentin provides a biological seal. If you lose, you will experience postoperative hypersensitivity of the dentin, or you will feel uncomfortable pain by touching it prematurely or by touching extreme heat or cold.

  The physiological mechanism for dentin pain after placement of an amalgam alloy or resin composite restoration is due to the increased flow of pulp fluid through the microchannels as soon as the coating layer is broken or lost. Has been described (Pashley, et al. 1984 Arch. Oral. Biol. 29: 65-68). The flow increase may be 94% greater than the normal physiological flow of fluid through the normal dentin matrix.

  The Pashley et al. Document discloses a hydrodynamic theory of the flow and content of dentin tubule content under various conditions. Painful stimuli are transmitted to the neural structure as the force moves hydraulically through the tubules in the dentin. A conventional method for removing pain during the dental restoration process is to create a cavity in the tooth and place the restoration material in the cavity liner or cavity varnish. The restoration material prevents the attack by contaminants from the oral fluid that attempts to ooze into the cavity even if a minor amount of leakage occurs, i.e., when the restoration material leaks around the edge of the tooth. It is claimed to reduce the permeability of the dentin to other materials placed in the cavity during. Prior art cavity varnishes often contain organic rubber dissolved in an organic solvent. The organic solvent evaporates, leaving a film of organic rubber on the dentin.

  These hollow liner materials are mainly composed of water and soluble organic materials, and the outer liner is often placed on a liquid layer covering the surface of the dentin without bonding. This can weaken the adherence of the cavity varnish to the tooth surface, but can also cause leakage.

  A hollow natural liner is a microcrystalline strip that is found on the surface of the cut dentin and is called the coating layer. The coating layer blocks the opening of the dentin tube and prevents bacteria from entering the tubule. However, the coating layer is often destroyed by acidification in the oral cavity and the presence of microleakage around the filler that contacts the cavity varnish.

  In addition, as disclosed in US Pat. No. 4,057,621, the prior art uses oxalate to desensitize the sensitive dentin or the cement surface on the teeth. U.S. Pat. No. 4,057,621 discloses a two-step method for reducing the permeability of dental cavities prepared for placing restorations using different oxalate salts. In this method, oxalate is sequentially applied to the coating layer. First, after applying a 1-30% weight to volume neutral oxalate solution, eg, dipotassium oxide, within 0.5 minutes, the 0.5% weight to volume percent acid oxalate solution, eg, monopotassium Apply hydrogen oxalate, etc. Neutral oxalate forms large crystals of calcium oxalate on the dentin surface, while acidic oxalate forms a uniform crystal by forming small crystals around previously precipitated large crystals. A crystal layer is formed.

  US Pat. No. 2,746,905 discloses the use of dihydroacetic acid and soluble salts to maintain a mouth pH of about 5.2 to prevent inorganic tooth enamel materials from dissolving. In this method, the acid resistance of teeth is improved by using oxalate in the composition as an enamel protective material.

  In contrast to the above documents and patents, the present invention utilizes a specific oxalate, 99% potassium oxalate dihydrate, and when applied to the tooth surface, the dihydrate Objects penetrate the tubules and fibrils of the dentin layer. Potassium oxalate dihydrate, which can also be called potassium oxalate dihydrate, eliminates fluid movement in the tubules, and thus painful irritation in the form of dentin and fluid movement Can be prevented from transmitting to the pulp. Therefore, the patient. There is no pain or discomfort over a long period of time.

  The present invention uses 99% potassium oxalate dihydrate, hereinafter referred to as potassium oxalate dihydrate, to react with calcium ions in the dentin fluid, and to form sodium oxalate including dentinal tubules. A soluble white precipitate is formed. This action reduced the permeability of the dentin, thereby reducing the permeability of the dentine, reducing the acid permeability of the dentin, and reducing the sensitivity of the dentin. Potassium oxalate dihydrate contains about 1.5 to about 10% potassium oxalate dihydrate, with a pH ranging from about 2.4 to about 4.0.

An object of the present invention is to reduce dentin permeability using a potassium oxalate dihydrate solution.
Another object of the present invention is to reduce the sensitivity of dentin using a potassium oxalate dihydrate solution.

Another object of the present invention is to reduce the acid permeability of dentin using a potassium oxalate dihydrate solution.
Yet another object of the present invention is to provide a simple diagnostic test method for determining whether dental pain or discomfort is reversible or irreversible.
Still another object of the present invention is to provide a method for solubilizing soluble potassium oxalate dihydrate in water and making the dihydrate available in a dosage form used as a susceptibility reducing agent. It is.

  The mechanistic action and effect of potassium oxalate in reducing dental sensitivity is described in Pashley, DH et al. Z (1983): “Dentis Permeability-Effects of Dessensitizing Dentrificeds In Vitro. Dentin permeability and effects on desensitization of tubules), J. Periodontol, 55: 522-525; Pashley, DH & Galloway (1985): The Effects of Oxalate Treatment on the Smear Layer of Ground Surfaces of Human Dentine Effect of oxalate treatment on the coating layer of the dentin grinding surface). Arch. Oral. Biol., 30: 731-737; Pashley, DH (1989): Dentin; A dynamic Substrate Dentin)-A Review.Scanning Micros 3: 161-176; Pashley, EL et al. (1989) Dentin Permeability and Bond Strengths after Various Surface Treatments. Dent. Mater. 5: 373-378.

  Pashley et al. Disclose a protective layer in the form of potassium oxalate and occludes open tubules against insoluble calcium oxalate on the exposed dentin surface. This occlusion reduces hydroconductivity and tubule permeability, reduces acid permeability, and ultimately reduces dentin sensitivity. U.S. Pat. No. 4,057,621 discloses potassium oxalate compounds useful in the present invention, including methods for reducing the sensitivity of sensitive dentin and cementum. In this method, an effective amount of a compound selected from the group consisting of mono- or disubstituted alkali metals and ammonium oxalate in an aqueous solution is applied to the dentin and cementum with an aqueous solution to reduce the sensitivity in the same region. Yes. The compounds disclosed in this patent include the following and also show their solubility in water. These are described in Chemical / Physical Handbook 54th Edition and 75th Edition (1973-74, 1995-96).

Dipotassium oxalate (K ₂ C ₂ O ₄ -H ₂ O) 33.0 Hot water solubility Potassium hydrogen oxalate (KHC ₂ O ₄ ) 16.7 Hot water solubility Sodium oxalate (Na ₂ C ₂ O ₄ ) 6.33 Hot water solubility Shu Sodium hydrogen oxyhydrogen (Na ₂ C ₂ O ₄ -H ₂ O) 21.0 Hot water solubility Lithium oxalate (Li ₂ C ₂ O ₄ ) 8.0 Cold water solubility Lithium hydrogen oxalate (KHC ₂ O ₄ -H ₂ O) No reading Shu Ammonium acid ((NH) ₂ C ₂ O ₄ -H ₂ O) 11.8 Hydrothermal solubility Hydrogen oxalate (NH ₄ K ₂ C ₂ O ₄ -H ₂ O) Not read

The active ingredient of the present invention is 99% potassium oxalate dihydrate, the molecular weight is 254.19, and the chemical formula is C ₄ H ₃ KO ₈ -2H ₂ O. Here, 99% potassium oxalate dihydrate is potassium oxalate dihydrate, which is a white crystalline powder that is slightly soluble in water and has a solubility of 29 Gm / liter. Potassium oxalate hydrate is preferably used in an aqueous solution. Dissolving potassium oxalate dihydrate in water may be difficult with conventional practice, but the product of the present invention disperses large-sized crystals of potassium oxalate in water by exposure to ultrasound. And solubilized in water. This treatment brings the potassium dihydrate to a particle size that is necessary and sufficient for the purposes of the present invention. Any treatment that solubilizes potassium oxalate dihydrate in water is satisfactory, but it is preferred to use various high frequency acoustic waves.

  To obtain the product of the present invention, use double-distilled water and deionized water having a water purity of 1,000,000 to 5,000,000 ohms according to the standardized test method of the US National Bureau of Standards. High resistance is comparable to high purity. Other forms of pure water can be used, but deionized water distilled twice is preferred. A sufficient amount of 99% crystals of potassium oxalate dihydrate is added to the water so that the final solution concentration ranges from 1.5% to 4.0 weight to volume. Preferably, the amount is about 2.9% by weight in the final product. The water and crystals are then exposed to various high frequency effects to make the crystals very small particles and form a solution. Typically, the above steps are performed using an ultrasonic cell disrupter, but any means may be used to solubilize potassium oxalate dihydrate. Preferably, a Branson ultrasonic device or an ultrasonic cell crusher specified as an equivalent can be used. An ultrasonic device converts electrical energy from a power source into mechanical vibration. In this device, water and potassium oxalate dihydrate crystals are placed in a mixing vessel and attached to a pump device. Start the pump and circulate the water as a continuous stream at about 1/2 liter as a continuous stream. Circulate water and crystals through the tube chamber for about 30 minutes. In this method, various super-high frequencies are directly applied to crystals in water with a small chamber. The mechanical vibration can be up to 2000 Hz. In use, preferable vibration is vibration that dissociates with ultrasonic waves at a frequency of about 16,000 Hz to about 20,000 Hz at the tip of the ultrasonic horn. The reason is that this breaks the crystals and breaks them down into very small particles that are released into the solution. The mixture of water and crystals passes through the ultrasonic horn several times, and the crystals are continuously reduced to smaller particles each time the mixture passes. When viewed through a 100X microscope, the 1 liter final product preferably has a particle size of about 1 to about 4 microns. About 60% of the particles fall into this size. The remaining particles can range from about 5 to about 10 microns. After solubilization, particles were not seen with the naked eye regardless of the microscope even after 24 hours. Larger particle sizes are acceptable, but a particle size in the range of about 1 micron to about 10 microns is preferred, with a particle size of about 1 to 4 microns being most preferred.

  The acidic solution has a pH of about 2.0 to 4.0, with a preferred range of about 2.7 to about 3.0. Most preferably, the pH is 3.0. The pH of the acidic solution is controlled by the amount of potassium oxalate dihydrate used in the formulation. The higher the amount of potassium oxalate dihydrate, the lower the pH.

  In treatment, the use of potassium oxalate dihydrate is a one-step method that immediately stops cold and air sensitivity. Useful as a diagnostic aid to assist dentists in distinguishing between reversible fluid flow in dentin and non-pulp swelling and irreversible fluid flow resulting in pulp swelling . Using forceps, place a clean Dappen dish with about 3-6 drops of potassium oxalate dihydrate, fill a small sterile hemp pallet with potassium oxalate dihydrate, and then affect for at least 30 seconds. Gently rub or gently rub against the received teeth. The solution can also be slowly rubbed against the cementum periphery of the crown, above or on the exposed root surface, and against the exposed root of teeth sensitive to color and air irritation. The product need not be applied to the tooth surface with a brush and should not. Rinse is not necessary. After application, the solution can also be evaporated from the area by gently applying an air dispersion stream to the surface. This leaves a frosty white surface that is an acid-resistant mineral layer. The mineral layer stops fluid movement or stops cold and air-stimulated movement to stop dentin sensitivity. Thus, the solution can be removed, so that it is not necessary to blow air strongly.

  Before and after the mouth is prophylactically sanitized to clean and remove tartar, the product of the present invention can be applied on the tooth structure prepared in live dentin or the like. The product can be used as a one-step arrangement under all crowns and fittings in the form of a lamellar plate. The product can be used on dentin in all cases where cavities have been created for amalgam alloys and resin composite restorations. The acid resistant film forming liner material can also comprise an adhesive material that can be applied directly to the surface for adhering to the restorative material. Depending on whether the bleaching process is performed at the dentist office or if the patient uses a bleaching kit at home, the liner material can be applied to the tooth surface following the bleaching process. Furthermore, potassium oxalate dihydrate can also be used as a diagnostic tool for distinguishing between acute dentin pain or chronic pulp pain. Acute dentin pain is generally referred to as reversible tooth pain. For the dentist and patient this means that the defect is located in the dentin matrix and not in the nerve in the pulp. The problem is reversible, requiring no invasive endodontic treatment. Instead, chronic tooth pain is an irreversible stimulus that indicates that the nerves in the dental pulp of the teeth are peeled off and the nerves must be removed by some biomechanical endodontic instrument. The potassium oxalate dihydrate of the present invention provides dentists with a simple one-step diagnostic tool that makes it possible to distinguish between reversible and irreversible dental pain. Patient complains of cold and air, but X-ray does not have diagnostic features to determine if there is a root or other obvious clinical problem that is destroyed by X-ray permeability around the apex In some cases, the dentist simply rubs the potassium oxalate dihydrate of the present invention to the edge or cavity edge of the dental restoration interface. If the patient says that the tooth pain has stopped immediately, the dentist can complete the diagnosis that the problem is a flow of dentine fluid or a minor leak. This confirms reversible pulp inflammation and can be treated by repairing the restoration, without removing the pulp.

  In order to explain the operation mechanism of the present invention, for example, the operation mode of the present invention used in the repair process will be described below. However, this mode of operation is similar for all applications. The acidic solution of potassium oxalate dihydrate of the present invention first breaks the coating layer and opens an opening in the substrate of dentin as well as enamel and cement. The buffering action acts on the pH of potassium oxalate dihydrate, and as the reaction proceeds, the pH of the solution moves to neutral. At the same time, calcium particles settle across the cavity, in addition to any physiological small cracks that are usually present in the surface of the enamel and / or cementum of the adult root. When the particle sediment is dried, it becomes an acid-resistant lining layer that chemically bonds to the surface and hollow dentin tubule Tubule. Once granular crystals are formed, the patient immediately senses the barrier effect. Even with the naked eye, a slightly whitish coating can be seen on the cavities and tooth surfaces.

(Example 1: Preparation of solution)
29 g of 99% of potassium oxalate dihydrate, a white crystalline substance, was added to 1 liter of deionized water distilled twice in a mixing vessel. The container was capped and attached to a Branson ultrasonic device made by Ultrasonic in Danbury. A supply and return hose was connected between the container and the sonicator. The ultrasonic pump was started and water was circulated at a rate of 1/2 liter / min. The sonicator was started at .9 or 18,000 Hz with a fixed duty cycle, hold time and power control.
The water was subjected to ultrasonic dissociation or vibration for 30 minutes. The water was left as it was for 30 minutes, and the sample was taken out and observed with a 100 × microscope. The crystal size was about 10 microns.

Example 2 Preparation of Solution with Heated Water 1 liter of double distilled water was heated in the solution until the temperature was changed from 85 ° F. to 100 ° F. and mixed with a stir bar. A hot plate was used to heat the water. 29 g of 99% potassium oxalate dihydrate was added to the container, mixed with a stir bar, and 99% white opaque potassium oxalate dihydrate was clear and relatively transparent as viewed with the naked eye . When the solution became relatively clear, the potassium salt was suspended. As described in Example 1, the solution was further added to an ultrasonic device, and 99% potassium oxalate dihydrate was dissociated with ultrasonic waves. The solution was treated with 18,000 Hz ultrasound for 40 minutes to obtain a clear solution.

Example 3 Clinical Evaluation In order to demonstrate the desensitization properties of the potassium oxalate dihydrate of the present invention, an amalgam repair patient is used as a commercially available hollow varnish commonly used before placing the amalgam or the production of the present invention. I was treated with things. Prior to anesthesia for pretreatment evaluation and during the first week of treatment, the patient responded to all distress questionnaires. Patients were evaluated at 1, 3, and 6 weeks after surgery. The results of the study have demonstrated that patients treated with the potassium oxalate dihydrate of the present invention have reduced post-treatment sensitivity, particularly to coldness.

(Materials and methods)
A total of 65 teeth with active caries symptoms were selected with the exception of acute or chronic post-surgery hypersensitivity symptoms and repaired with a commercially available amalgam alloy titin (Tytin (r)). Only patients who selected the amalgam repair method with UAB were used in this study. For each tooth, preliminary anesthesia was evaluated for postoperative hypersensitivity of the dentin by cold stimulation and air blowing for the thermal test. For the cold ice test, a plastic needle cover was filled with water and frozen in a freezer in the refrigerator and used as a standard cold stimulus.

  For air stimulation, a standard baseline air jet from a syringe was used to blow an air stream directly onto the attacking tooth and the defect restoration. A random number chart was used to select teeth that were controlled by Copalite (r) varnish and teeth that were treated with the product of the invention, potassium oxalate dihydrate.

Preparation of amalgam After pre-processing data was collected and anesthesia was performed, each tooth was opened with a class I or class II cavity in a normal crown.
Spraying water on 35 teeth controlled by Copalite® controlled and 30 teeth treated with the product potassium oxalate dihydrate, a total of 65 teeth Then, while discharging at high speed, a class I or class H cavity was drilled with a new # 245 or # 330 carbide drilling tool at ultra high speed. After the cavities were opened, they were rinsed and gently dried with air, and the entire perforated surface was treated with Copalite® without modifying or removing the coating layer. Each cavity of the control was treated with 3 layers of copal varnish and air was slightly dispersed in each layer with a tip syringe before applying the following Copalite® coat. The metal matrix was placed over all of the class II cavities and the teeth were restored to an anatomical shape with the dispersed phase spherical amalgam alloy Tytin®.

  All other clinical preparation procedures, test criteria and recovery methods were identical to those used for 39 teeth treated with the product of the present invention. In addition, 30 new teeth were treated with the product of the invention, potassium oxalate dihydrate. After removing the cavity by opening a class I or class H cavity in the crown, the cavity is cleaned with sterilized water and gently scattered with air, and the resulting cavity surface is diluted with potassium oxalate dihydrate. Processed once. The potassium oxalate dihydrate solution was taken up in a clean Dappen dish and absorbed into sterilized cotton pellets. The entire surface area of the prepared enamel and dentin surface cavities was mechanically cleaned for about 2 minutes, sprinkled with air and treated again as before. The surface was gently dried with air, the matrix was placed, and the cavity was repaired with Tytin®.

  Before anesthesia for pre-treatment evaluation and during the first week of treatment, the patient is brought back to the dentist to “feel” or “feel” for various stimuli including coldness, heat, sweetness, biting and brushing. Fill in the format for "response". In addition, post-operative patient hypersensitivity after 1 week, 3 weeks and 6 weeks is evaluated. When the patient feels in pain, collect subjective considerations of patient data by crossing the 10 cm line at that point. At each hour, attention was paid to the McGill visual similarity scale. Thermal tests were conducted for ice and air sprays and data was recorded for all previous tests.

  For all baseline data, each patient was evaluated for coldness and air distress or sensitivity prior to treatment in this study. A total of 35 patients were treated as controls in the Copalite® group and 30 patients were treated in the potassium oxalate dihydrate group. The number of total study groups was N = 65.

  Analysis was performed by Analysis of Variance (ANOVA) at the 0.05 level according to the cell size in the treatment vs. response contingency table. Differences between various groups within ANOVA were compared using the Student T test (p <0.050).

Result (question answer)
Responses from each patient were recorded on a separate sheet and tabulated on the master sheet according to the above criteria. Raw data was recorded from a McGill Visual Analog Scale (MVAS) evaluation sheet. Data was collected from all patients. If postoperative hypersensitivity did not appear based on the patient's response at various times after surgery on day 0, 5, 7, 21, and 42 days, if some, and if there was. In each case, responses were reported for various test stimuli: cold, heat, sweetness, bite, impact, brushing and interdental brushing. Completed the evaluation form.

  Data for each McGill VA scale was tabulated and recorded on a separate sheet containing the patient's name and clinical card record number. A 10 centimeter straight line was recorded along the axis without any specific marks.

  About 12% of patients experienced some preoperative hypersensitivity in response to pain, mostly cold stimuli. However, 65% of patients complained of some preoperative response on the MVA scale. More than half (52%) of those patients showed a value of 0-1 mm on the scale on a 10 centimeter preoperative scale. Only 2% of the total population experienced some preoperative pain greater than 5 mm on a 10 centimeter MVA scale.

  After normal removal of the caries, application of potassium oxalate dihydrate or Copalite control solution, and placement of an amalgam restoration, the patient's postoperative sensitivity decreased. Patients who received control Copalite (r) treatment had a 2.3% reduction in sensitivity to various stimuli (especially cold) in response to direct questions. However, in patients treated with the potassium oxalate dihydrate of the present invention, the reported pain was reduced overall at a level of 80.3%.

  According to data collected from the MVA scale, post-operative pain was greatly reduced for teeth treated with potassium oxalate dihydrate, compared to 68.7% of patients with Copalite (r) There was a temporary decrease in postoperative pain.

  The data showed that the overall pain was greatly reduced by the Super Seal as a means of identifying coldness compared to the commercially available Copalite (r). In addition, patients who responded to the MVA survey showed that most suffered no postoperative pain with potassium oxalate dihydrate, the product of the present invention. Overall, 25.7% of patients treated with Copalite (r) and 88.5% of patients treated with the product of the invention, potassium oxalate dihydrate, There was no pain after the procedure.

  Complete test results are shown in Table 1. The product of the present invention is shown in the table as Super Seal.

  While the above disclosure highlights certain specific embodiments of the invention, changes and modifications thereto are within the spirit and scope of the invention as described herein.

Claims (9)

1.5 wt% and 10.0 wt% of oxalic acid potassium salt dihydrate _{(C 4 H 3 KO 8 -2H} 2 O), made 90% by weight of water from 98.5 wt%, 2. An aqueous solution of potassium oxalate dihydrate (C ₄ H ₃ KO ₈ -2H ₂ O) having a pH of 0 to 4.0, wherein the crystal grain size of the potassium oxalate dihydrate is: In producing an aqueous solution of potassium oxalate dihydrate (C ₄ H ₃ KO ₈ -2H ₂ O) in the range of 1 to 10 microns , oxalic acid is used to dissolve the crystals by ultrasonic vibration of the solution. A method for producing a potassium salt dihydrate solution . The method according to claim 1, wherein the potassium oxalate dihydrate is heated in an aqueous solution before being dissociated or vibrated by ultrasonic waves. The method of claim 1 or 2, wherein the potassium oxalate dihydrate solution is heated to a temperature of 85 ° F to 100 ° F. The method according to any one of claims 1 to 3, wherein a 1 liter volume of potassium oxalate dihydrate solution is heated and mixed for 40 minutes before ultrasonic dissociation. The method according to any one of claims 1 to 4, wherein the water is purified. The method according to any one of claims 1 to 5, wherein the water is distilled twice and deionized. The method according to claim 1, wherein the ultrasonic wave has an energy level of 20,000 Hz or less. The method of claim 7, wherein the energy level is from 16,000 Hz to 19,000 Hz. The method according to claim 1, wherein the solution is vibrated for 30 minutes.