RU2841741C1 - Nutrient medium for temporary storage and transportation of teeth - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, particularly to a culture medium for temporary storage and transportation of teeth and periodontal ligament cells. Said medium includes minimum medium α-MEM containing amino acids, vitamins, inorganic salts, minor components and D-glucose, and additional components in the following concentrations: trehalose – 750 mg/l; eicosapentaenoic acid – 0.5 mg/l; succinic acid – 5.0 mg/l; epigallocatechin gallate – 0.25 mg/l; astaxanthin – 0.15 mg/l; vancomycin – 50 units/ml; amphotericin B – 50 units/ml.

EFFECT: present invention provides an increase in the viability of the cells of the periodontal ligament of the tooth and a longer period of storage and transportation at ambient temperature.

1 cl, 2 tbl, 4 ex

Description

The invention relates to the pharmaceutical industry, namely to the composition of a nutrient medium for temporary storage and transportation of teeth - periodontal ligament cells.

Tooth dislocation is a complex traumatic impact characterized by a complete loss of contact between the tooth and the body, which is accompanied by serious damage to the surrounding tissues, vascular and nerve structures of the alveolar process of the jaw and requires a quick and correct response. In such injuries, the best treatment method is tooth replantation to restore the natural dentition and chewing function of the jaw, while preventing necrosis of the periodontal ligament and reducing the risk of inflammatory processes in the jaw. Immediate replantation of dislocated/extracted teeth has a positive effect on the viability of the cells of the periodontal ligament and increases the positive prognosis of the result of such treatment. The possibility of practically immediate replantation is not always available, and, in practice, a patient who has lost a tooth as a result of an injury gets to a specialist, at best, several hours after the injury. After just 1 hour of storing a dislocated/extracted tooth in a dry form, a handkerchief or simply in water, the cells of the dental ligament of the tooth - periodontium - fibroblasts will completely die. The most important task is the correct storage of the dislocated/extracted tooth, which is necessary to prevent necrosis of the periodontal ligament, and therefore increases the positive prognosis of such treatment.

To ensure maximum viability of periodontal ligament cells (fibroblasts), the avulsed/extracted tooth should be protected from desiccation by storing in media with optimal osmolarity and pH values. The ideal preservative medium should: (1) have antimicrobial properties; (2) be able to maintain the viability of fibroblasts for an acceptable period of time; (3) promote the proliferative capacity of periodontal ligament cells; (4) not interact with biological fluids; (5) not cause antigen-antibody reactions; (6) reduce the risk of root resorption after replantation [1].

The literature describes various types of media for storing avulsed/extracted teeth, such as physiological solution, saliva, pasteurized milk, Hanks balanced salt solution (HBSS), ViaSpan medium, Eagle's culture medium, Gatorade oral rehydration fluid, propolis, egg white, coconut water, green tea extract, ascorbic acid. It has been experimentally established that the proposed compositions can be arranged in a row according to their preservation efficiency:

Eagle's culture medium=ViaSpan=HBSS > pasteurized milk ≥ propolis ≥ green tea extract ≥ egg white ≥ coconut water [1].

A number of technical solutions are known aimed at obtaining compositions for preserving living mammalian cells, including fibroblasts.

Thus, a nutrient medium for accumulating a cell sample for subsequent cytological and/or immunocytochemical analysis is known, protected by Russian patent No. 2246110, G01N 33/48, A01N 33/53, A01N 1/02, published 10.02.2005, containing the salts NaCl, KCl, anhydrous CaCl ₂ , MgSO⋅6H ₂ O, MgCl ₂ ⋅6H ₂ O, Na ₂ HPO⋅2H ₂ O, KH ₂ PO ₄ , NaHCO ₃ , glucose and Hanks' solution, 10% albumin solution, polyglucin at a ratio of 1:1:1. The invention provides for increased cell survival. The known invention is not without drawbacks, the main one of which is low antimicrobial activity.

Also known is a method for culturing dental pulp cells and a method for transferring an extracted tooth for storage (RU Patent No. 2499609, A61L 2/00, C12N 5/00, A61K 33/52, published on 27.11.2013). The medium or preservative solution used may be a medium or preservative solution that are generally used for culturing and preserving cells. The use of an α-MEM medium containing 20% fetal bovine serum (FBS), 100 μM L (+)-ascorbic acid, penicillin (50 U/ml)/streptomycin (50 μg/ml) is most preferred. In this case, the dental pulp cells can be preserved in an almost unchanged state for 24 to 48 hours. A disadvantage of the described environment is the presence of a large number of foreign proteins, in particular protein components of serum, which can cause an immune response.

The closest analogue of the proposed nutrient medium is a medium for tissue storage, irrigation and infusion, as well as methods of use (patent WO 2023205339 A1, A61M 3/02, C12N 5/0735, C12N 5/074, published on 26.10.2023). According to the invention, the medium for infusion and/or irrigation is a modified Eagle Dulbecco's medium (DMEM) or an essential α-MEM medium containing additional components, in particular fetal bovine serum, antibiotics selected from cefazolin, vancomycin, tobramycin, gentamicin, amphotericin; salts of organic acids, for example, lactates, acetates, gluconates; organic acids such as 3-hydroxybutyric, citric, formic, malic, malonic, succinic and uric acids; fatty acids ( _C4 - _C24 ), such as palmitic, stearic, oleic, arachidonic, eicosapentaenoic and docosahexaenoic acids; zinc, copper, vanadium, strontium, selenium and nickel salts; minor nutrients (para-aminobenzoic acid, inositol, carnitine, taurine and L-amino-N-butyric acid); nitrogenous bases and nucleosides such as adenine, adenosine, guanine, guanosine, cytosine, thymine, thymidine and uracil. The proposed composition has an osmolarity of 170 to 406 mOsm/l and a pH of 6.0 to 8.8.

Using mesenchymal stem cells as an example, it was shown that the death of cells stored in the medium according to the described invention for 24 hours was insignificant, compared to their storage in a physiological solution and a 5% semi-saline dextrose solution. In addition, when stored in this medium for 1 hour, mesenchymal stem cells successfully proliferated.

The main disadvantages of the medium according to the invention WO 2023205339 A1 are its multi-component composition and proven effectiveness in terms of cell survival for no more than 24 hours. Longer periods of tissue storage in the proposed medium have not been investigated in the described invention.

The technical problem solved by the proposed invention is the creation of a nutrient medium that promotes high survival of cells of the periodontal ligament of the tooth using various technologies for preserving dislocated or extracted teeth.

The technical result of using the invention consists in increasing the viability of the cells of the periodontal ligament of the tooth and achieving long periods of their storage and transportation at ambient temperature and immediate availability of this solution.

The specified technical result is achieved by the fact that the nutrient medium for temporary storage and transportation of teeth (periodontal ligament cells), containing the basic minimum medium α-MEM, additionally includes components at the following concentrations:

trehalose - 750 mg/l

eicosapentaenoic acid - 0.5 mg/l

succinic acid - 5.0 mg/l

epigallocatechin gallate - 0.25 mg/l

astaxanthin - 0.15 mg/l

vancomycin - 50 units/ml

amphotericin B - 50 units/ml.

The pH value of the nutrient medium of the specified composition is at the level of 1.2-1. A.

The claimed composition of the nutrient medium is optimal in terms of obtaining the best results in terms of the survival of periodontal fibroblasts.

To prepare a nutrient medium for temporary storage and transportation of periodontal ligament cells of the specified composition, all components are mixed in the specified concentrations. The nutrient medium must be stored in a refrigerator at a temperature of (4±2)°C. Storage and transportation of the tooth - periodontal cells placed in the nutrient medium of the specified composition can be carried out at ambient temperature for 72 hours.

The nutrient medium, according to the claimed invention, can be used for preserving permanent or baby teeth. Teeth dislocated as a result of traumatic impacts, as well as teeth removed in medical institutions during dental procedures, can be temporarily stored in the said nutrient medium.

The nutrient medium of the stated composition is used as follows. For temporary storage of periodontal cells, sterile culture tubes with a screw-on hermetic cap are used. The required volume of the nutrient medium for temporary storage is placed in the culture tube, the dislocated/extracted tooth is immersed there, after which the cap is screwed on and the tube is transported at ambient temperature for 72 hours.

The invention is illustrated by the following examples.

Example 1

The nutrient medium for the preservation and transportation of periodontal ligament cells is prepared by mixing the components in distilled water in the following concentrations:

A distinctive feature of the nutrient medium of the stated composition from the well-known basic minimum medium α-MEM is the inclusion in its composition of an additional carbon source (trehalose), a polyunsaturated essential fatty acid (eicosapentaenoic acid), an organic acid involved in the processes of energy metabolism of living cells (succinic acid), components of antioxidant action (epigallocatechin gallate, astaxanthin), as well as components of antibacterial (vancomycin) and antifungal (amphotericin B) action.

Example 2

To confirm the ability of the nutrient medium of the stated composition to ensure high viability of periodontal ligament cells, the following experiment was conducted. The required volume of the nutrient medium prepared according to Example 1 was placed in a culture tube, then the extracted tooth was immersed in this tube, after which the lid was screwed on, and the tube was kept at ambient temperature for 72 hours. The extracted tooth was incubated in a commercial DentoSafe solution (manufacturer Hager Werken, Duisburg, Germany) in a similar manner.

At specified storage periods (24, 36, 48, 60 hours) and at the end of preservation (72 hours), the viability of periodontal ligament cells was determined for both samples (test and control) according to the protocol described by Rajendrann et al. [2]. Each sample was washed with PBS buffer solution and then added to a 15 ml test tube containing collagenase type 4 and trypsin solution for 10 minutes. The sample was incubated at 37°C in CO ₂ (5%) atmosphere for 7 minutes. During the final 2-3 minutes of incubation, the sample was resuspended to stimulate cell detachment. After incubation, 1 ml of the suspension with detached cells in collagenase type 4 and trypsin solution was placed in a 2 ml test tube. 10 μl of fetal bovine serum (FBS) were added to this volume. The solutions were then centrifuged at 90 g for 4 minutes. Upon completion of centrifugation, the supernatant was removed and the sediment was dissolved in 400 μl of PBS buffer. The cell mass was stained using a 0.4% trypan blue solution in a 1:1 ratio (100 μl of the test solution and 100 μl of trypan blue). After this, the samples were examined under a microscope using a Leica DM 3000 inverted light microscope (Leica Camera AG, Germany) with 40-fold magnification, and cell viability was counted using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific, USA).

The results of the assessment of the viability of periodontal cells after incubation in a nutrient medium of the stated composition, in comparison with the control solution, for 24, 36, 48, 60 and 72 hours are shown in Table 1.

According to the data in Table 1, the nutrient medium of the declared composition ensures high survival (not less than 90%) of periodontal ligament cells for up to 48-50 hours, after which a decrease in cell survival is noted. The experimental results are comparable with those obtained for the DentoSafe control solution. It should be noted that storing a tooth (periodontal cells) for more than 72 hours for replantation purposes is inappropriate, since during this period of time, changes occur in the socket of the dislocated tooth, which lead to a sharp deterioration in the prognosis for the survival of replanted teeth.

Thus, the nutrient medium for temporary storage and transportation of a tooth (periodontal ligament cells) claimed in this invention has advantages over the closest analogue. Firstly, high viability of periodontal cells (not less than 90%) when stored in the medium of the proposed composition for 48-50-72 hours. Secondly, the possibility of temporary storage and transportation of a tooth (periodontal cells) in the described nutrient medium at ambient temperature. Thirdly, a simpler component composition. Fourthly, almost instant availability of a container with this solution.

Literature:

1. Clinical and practical implications of storage media used for tooth avulsion / V.I. Khinda, G. Kaur, G. S Brar, et al. // International Journal of Clinical Pediatric Dentistry. - 2017. -Vol.10(2). - P. 158-165.

2. Rajendran, P. Evaluation, using extracted human teeth, of Ricetral as a storage medium for avulsions - an in vitro study / P. Rajendran, N.O. Varghese, J.M. Varughese, E. Murugaian // Dental Traumatology. - 2011. - Vol.27. - P. 217-220.

Claims (8)

A nutrient medium for temporary storage and transportation of teeth and periodontal ligament cells, including a minimum α-MEM medium containing amino acids, vitamins, inorganic salts, minor components and D-glucose, characterized in that it includes additional components at the following concentrations: - trehalose - 750 mg/l; - eicosapentaenoic acid - 0.5 mg/l; - succinic acid - 5.0 mg/l; - epigallocatechin gallate - 0.25 mg/l; - astaxanthin - 0.15 mg/l; - vancomycin - 50 units/ml; - amphotericin B - 50 units/ml.