METHOD OF PRODUCING BIOLOGICAL PRODUCTS FROM PRIMARY TEETH Russian patent published in 2022 - IPC C12N5/775 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, which involves obtaining biomaterials for regenerative medicine, particularly to a method for biotechnological treatment of primary teeth. For implementation of the specified method there used are naturally fallen primary teeth or primary teeth extracted for orthodontic indications. First, the obtained material is transported and washed. Then, after primary three-fold washing of tooth with sterile Hanks solution with antibiotics, it is additionally treated by means of ultrasonic bath with frequency of ultrasonic waves 40 kHz for 2 minutes, using sterile Hanks solution with Gentamycin 8 mcg/ml. Further, after aspiration of the tooth canal content into a syringe, it is transferred into a centrifuge tube and incubated for 2 minutes in a thermal shaker at temperature of 37°C. Complete nutrient medium, in which the cell pellet is resuspended, contains 10% FBS, which is tested for maintenance of MSC growth. After placing the cells in a culture flask, it is placed for 1 hour in a thermal shaker at a temperature of +37 °C, speed 50 rpm. Further, the state of the cells is examined using an inverted microscope. When culturing in a CO₂ incubator, the medium is changed every 5 days; at 4^th passage, the number and viability of cells in Goryaev’s chamber are determined using 0.4 % trypan blue solution. Thereafter, MSCs are cryopreserved. After aspiration of the canal contents to obtain MSC, the tooth is treated with 3 % sodium hypochlorite solution, 3 % hydrogen peroxide solution, enamel and cement are removed, parapulpal dentin is exposed using a water-cooled diamond bur. Then material is subjected to low-frequency ultrasonic treatment with frequency of 24–40 kHz. Before demineralisation, the state of the material surfaces is monitored using Raman spectroscopy, determining the ratio of the intensity of the phosphate ion line 960 cm^-1 to the intensity of the Amide I line 1660 cm^-1, which has value from 11 to 17, and the ratio of the intensity of the carbonate ion line of 1070 cm^-1 to the intensity of the Amide I line of 1660 cm^-1, which has value from 2.6 to 3.3. Material is demineralised in 2.4H–4.8H hydrochloric acid solution. After demineralisation, its efficiency is evaluated by examining the surface of the material using Raman spectroscopy, determining the ratio of the intensity of the phosphate ion line 960 cm^-1 to the intensity of the Amide I line 1660 cm^-1, which takes value from 0.25 to 0.65, and the ratio of the intensity of the line of carbonate ion 1070 cm^-1 to the intensity of the line of Amide I 1660 cm^-1, which takes value from 0.4 to 0.55. Demineralised dentin is washed in pyrogen-free water, frozen at a temperature of -60 °C, lyophilized, packed, packed and sterilized by radiation method.

EFFECT: present invention enables to simultaneously obtain a culture of mesenchymal stromal cells (MSC) and demineralised dentin from primary teeth.

1 cl, 2 ex