Composition of nutrient medium for temporary storage of teeth with pandanusamaryllifolius extract

Abstract

FIELD: pharmaceutical industry.SUBSTANCE: compositions of nutrient media for the temporary storage of teeth lost as a result of injury or removed for surgical indications, containing cells of the periodontal ligament of teeth, in particular fibroblasts. The proposed composition of the nutrient medium includes Hanks' balanced salt solution (HBSS), which additionally contains L-glutamine, L-arginine hydrochloride, para-aminobenzoic acid and dry extract of Pandanus amaryllifolius in the following concentrations, mg / l: L-glutamine – 250.0, L-arginine hydrochloride – 150.0, para-aminobenzoic acid – 0.52, dry Pandanus amaryllifolius extract – 2000.EFFECT: use of the proposed nutrient medium composition, which has antibacterial properties due to the use of a plant extract, ensures high viability of dental ligament fibroblasts during storage and transportation of teeth.1 cl, 2 dwg, 1 ex

Description

[0001] The invention relates to medicine and the pharmaceutical industry, specifically to the composition of nutrient media for the temporary storage of teeth, in the event of their extraction as a result of trauma, or removed for surgical indications, containing cells of the periodontal ligament of teeth, in particular fibroblasts.

[0002] The prevalence of dental trauma in children is 9.75%, which is the second most common dental disease after caries and its complications (69.02%) [1]. Thus, on average, every 10th child experiences clinically significant dental trauma.

[0003] Replantation is an effective treatment for dental trauma, such as complete luxation, extraction, crown fracture, and root fracture. However, the prognosis for the survival of replanted teeth depends on factors such as the time interval between the loss of tooth attachment to the bone tissue and replantation, the type of tooth storage (dry storage or nutrient medium), the viability of the periodontal tissues and pulp, and the type, kind, and timing of tooth fixation (splinting). The most important factor influencing the postoperative survival prognosis for teeth dislocated as a result of trauma or removed for surgical indications after their replantation is the use of properly selected nutrient media [2].

[0004] A number of technical solutions are known aimed at obtaining nutrient media for storing periodontal cells in vitro. Thus, a nutrient medium is known for temporary storage and transportation of teeth (periodontal ligament cells), including a minimal α-MEM medium containing amino acids, vitamins, inorganic salts, minor components and D-glucose, trehalose, eicosapentaenoic acid, succinic acid, epigallocatechin gallate, astaxanthin, vancomycin and amphotericin B (RU 2841741 C1, A01N 1 / 10; C12N 5 / 00; published 17.06.2025). The above-described nutrient medium allows to increase the shelf life of a sample of cellular material to 72 hours.

[0005] The nutrient medium of the described composition is not without its drawbacks, the main ones being the presence of fat-soluble compounds in its composition, which creates difficulties in its homogenization, and the presence of antibiotics in the composition.

[0006] A liquid for the preservation and preservation of periodontal ligament cells is known, containing 200 units / ml of penicillin, 100 units / ml of streptomycin, 200 mg / ml of dexamethasone and 272 mg / ml of L-glutamine in Hank's solution (CN 109526940 A, A01N 1 / 02, published 03 / 29 / 2019). Experiments have shown that periodontal ligament cells in this solution retain high viability and the ability to reproduce for 24 hours. The main disadvantages of the drug can be considered the insufficient survival of periodontal cells during preservation for more than 24 hours, as well as the use of antibiotics.

[0007] The closest analogue can be considered a nutrient medium for temporary storage of teeth dislocated as a result of injury or removed for surgical indications (RU 2831471 C1, A01N 1 / 02, published 09.12.2024), which consists of Hanks' balanced salt solution with the following additives: L-glutamine - 250.0 mg / l, L-arginine hydrochloride - 150.0 mg / l, green tea extract - 0.8 vol. %, para-aminobenzoic acid - 0.52 mg / l, docosahexaenoic acid - 0.75 mg / l, vancomycin - 50 units / ml and amphotericin B - 50 units / ml.

[0008] The main drawback of this medium is the use of glycopeptide (vancomycin) and polyene macrolide (amphotericin B) antibiotics. Amphotericin B is indicated exclusively for severe disseminated mycoses in cases where alternative antifungal agents are resistant or unavailable. However, this therapeutic approach is associated with a high risk of life-threatening adverse events, primarily irreversible nephrotoxicity.

[0009] Furthermore, it is known from the literature that Pandanus amaryllifolius extracts possess a broad spectrum of biological activity, including antioxidant, hypolipidemic, and anticancer effects in humans [3]. A number of authors also point to the presence of antibacterial properties in the absence of a pronounced toxic effect on human fibroblasts [4]. Thus, Pandanus amaryllifolius extract can be successfully used to replace antibiotics in the composition for the storage and transportation of dislocated and extracted teeth to ensure the preservation of periodontal ligament fibroblasts.

[0010] The technical problem solved by the proposed invention is the creation of a nutrient medium with antibacterial properties provided by the use of plant extracts, capable of ensuring high viability of the periodontium of a tooth containing fibroblasts.

[0011] The technical result of using the invention consists in providing antibacterial properties of the nutrient medium for maintaining the viability of dental ligament fibroblasts without the use of antibiotics through the use of a plant extract.

[0012] The specified technical result is achieved in that the nutrient medium for the temporary storage of teeth dislocated as a result of injury or removed for surgical indications, the periodontium of which contains fibroblasts, includes Hanks' balanced salt solution (HBSS) and additional components at the following concentrations, mg / l:

[0013] L-glutamine 250,0 L-arginine hydrochloride 150,0 para-aminobenzoic acid 0,52 Pandanus amaryllifolius dry extract 2000

[0014] The pH value of the nutrient medium of the specified composition is at the level of 7.2-7.4.

[0015] The claimed composition of the nutrient medium is optimal in terms of obtaining the best results in terms of the survival of tissues containing fibroblasts.

[0016] The invention is illustrated in Fig. 1, which shows the results of an assessment of the viability of cells of an extracted tooth after incubation in a nutrient medium of the claimed composition (a) and in a control commercial medium (b), as well as Fig. 2, which shows the antibacterial effect of the preparation on Porphyromonas gingivalis, as a typical representative of the pathogenic microflora of the oral cavity.

[0017] To prepare a nutrient medium of the specified composition for storing teeth with periodontal tissue containing fibroblasts, all components are mixed in the specified proportions. The storage temperature of the nutrient medium is +4 ± 2°C. Teeth with periodontal tissue containing fibroblasts can be stored and transported in the nutrient medium of the stated composition for 72 hours at a temperature of +4 ± 2°C. A ready-made Pandanus amaryllifolius extract in powder form is used to prepare the solution. If necessary, the extract can be obtained by ethanol maceration at room temperature for 5-7 days, followed by filtration of the extract and distillation of the solvent.

[0018] For ease of use, the culture medium is sterilized and poured into sealed 10-25 ml containers with screw-on lids, providing the necessary level of protection against contamination. The prepared medium is stored in the refrigerator at a temperature not exceeding +4 ±2°C until use. If necessary, open the container and insert the dislocated / extracted tooth, then screw the lid back on. The container is stored at +4 ±2°C for 72 hours.

[0019] An example of using a nutrient medium of the stated composition is presented below.

[0020] A nutrient medium for storing teeth whose roots contain fibroblasts is prepared by mixing the components in distilled water in the following concentrations, mg / l:

[0021] sodium chloride 8000,0 potassium chloride 400,0 potassium dihydrogen phosphate 60,0 sodium hydrogen phosphate 48,0 sodium bicarbonate 350,0 magnesium chloride hexahydrate 100,0 magnesium sulfate heptahydrate 100,0 calcium chloride anhydrous 140,0 D-glucose 1000,0 L-glutamine 250,0 L-arginine hydrochloride 150,0 para-aminobenzoic acid 0,52 Pandanus amaryllifolius dry extract 2000

[0022] The difference between the nutrient medium of the stated composition and the well-known Hanks' balanced salt solution (HBSS) is the inclusion of nitrogen sources (L-glutamine, L-arginine hydrochloride), an amino acid involved in the synthesis of purine and pyrimidine bases (para-aminobenzoic acid), as well as a component with an antibacterial effect - Pandanus amaryllifolius extract.

[0023] To confirm the ability of the stated nutrient medium to ensure the viability of avulsed / extracted teeth, an experiment was conducted. The required volume of nutrient medium prepared as described in Example 1 was placed in a culture tube. An extracted tooth was then immersed in the tube, the cap was screwed on, and the tube was maintained at ambient temperature for 72 hours. The extracted tooth was similarly incubated in the commercial DentoSafe solution (manufacturer: Hager Werken, Duisburg, Germany).

[0024] At certain storage periods (24, 36, 48, 60 hours) and at the end of preservation (72 hours), the viability of the cells of the extracted tooth was determined for both samples (test and control) according to the protocol described in the work of Rajendrami et al. [5]. Each sample was washed with PBS buffer solution and then added to a 15 ml test tube containing a solution of collagenase type 4 and trypsin for 10 minutes. The sample was incubated at 37°C in a CO2 atmosphere (5%) 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 the separated cells in a solution of collagenase type 4 and trypsin was placed in a 2 ml test tube. Ten microliters of fetal bovine serum (FBS) were added to this volume. The solutions were then centrifuged at 90 g for 4 minutes. After centrifugation, the supernatant was removed and the pellet was dissolved in 400 microliters 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). The samples were then examined under a Leica DM 3000 inverted light microscope (Leica Camera AG, Germany) at 40x magnification, and cell viability was counted using a Countess IIFL Automated Cell Counter (Thermo Fisher Scientific, USA).

[0025] The results of the assessment of the viability of the cells of the removed tooth after incubation in the nutrient medium of the stated composition, in comparison with the control solution, for 24, 36, 48, 60 and 72 hours are shown in Fig. 1.

[0026] As shown in Fig. 1, the nutrient medium of the claimed composition ensures high survival (at least 89%) of periodontal cells from the extracted tooth for up to 48 hours, after which a decrease in cell survival is observed. The experimental results are comparable to those obtained with the DentoSafe control solution. It is important to note that storing extracted / extracted teeth in the nutrient medium for more than 72 hours is not advisable, as during this period, changes occur in the alveolar process of the tooth socket, leading to a sharp deterioration in the prognosis for the survival of reimplanted teeth.

[0027] The antibacterial properties of the solution were tested by assessing the formation of a biofilm with a suspension of P. aingivalis. For this purpose, the developed nutrient medium containing 10 8CFU / ml of P. gingivalis with different concentrations of Pandanus amaryllifolius extract (8, 4, 2, and 1 mg / ml). After 48 hours of incubation, the wells were washed with phosphate-buffered saline (pH 7.4), stained with 1% aqueous crystal violet for 15 min, and then washed again. Ethanol (95%) was added to decolorize the dye. Absorbance was measured at 560 nm using a microplate reader (Multiskan GO, Thermo Scientific, USA). The results are shown in Fig. 2.

[0028] According to the results, Pandanus amaryllifolius extract significantly inhibited biofilm formation by P. gingivalis strain ATCC 33277 in all experimental variants compared to the control (Fig. 2). The most effective concentration of Pandanus amaryllifolius was considered to be 2 g / L.

[0029] The given example can serve as an illustration of the advantages of the declared composition of the nutrient medium for the temporary storage of dislocated or extracted teeth, the root surface of which contains fibroblasts, namely:

[0030] - high viability of cells of removed teeth (not less than 89%) when stored in the medium of the declared composition for 48 hours; Not less than 80% within 72 hours);

[0031] - the possibility of storing and transporting teeth in the described nutrient medium at ambient temperature.

[0032] Literature

[0033] 1. The structure of traumatic injuries of primary teeth in children of Yekaterinburg I.M. Makeeva, M.V. Sarapultseva / / Perm Medical Journal - 2011 volume XXVIII No. 6 pp. 19-24.

[0034] 2. Clinical and practical implications of storage media used for tooth avulsion / VI Khinda, G. Kash, G. S Brar, et al. / / International Journal of Clinical Pediatric Dentistry. - 2017. - Vol. 10(2). - P. 158-165.

[0035] 3. Chemical composition, bioactivity, and applications of Pandanus amaryllifolius leaves: A comprehensive review / Pengyin Li, Tao Fei, Jiamu Kang, Jingtao Cui, Junxia Jiang, Enxi Fu, Weimin Zhang, Tian Li / / Trends in Food Science & Technology, Volume 172, 2026, 105682, https: / / doi.org / 10.1016 / j.tifs.2026.105682

[0036] 4. Pandanus amaryllifolius ethanolic extract promotes oral gingival fibroblast healing and inhibits Porphyromonas gingivalis: An in-vitro study / Monwilun Wilairat, Nattapon Rotpenpian / / Journal of Oral Biology and Craniofacial Research, Volume 15, Issue 5, 2025, Pages 919-924, https: / / doi.org / 10.1016 / j.jobcr.2025.06.015

[0037] 5. 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

The composition of a nutrient medium for the temporary storage of teeth dislocated as a result of injury or removed for surgical indications, including Hanks' balanced salt solution (HBSS), characterized in that it contains additional components in the following concentrations, mg / l: L-glutamine 250,0 L-arginine hydrochloride 150,0 para-aminobenzoic acid 0,52 Pandanus amaryllifolius dry extract 2000

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