vtioВестник трансплантологии и искусственных органовRussian Journal of Transplantology and Artificial Organs1995-1191Academician V.I.Shumakov National Medical Research Center of Transplantology and Artificial Organs", Ministry of Health of the Russian Federation10.15825/1995-1191-2022-4-73-84vtio-1553Research ArticleРегенеративная медицина и клеточные технологииRegenerative Medicine and Cell TechnologiesБиосовместимые и функциональные свойства тканеспецифической мелкодисперсной 3D-матрицы из децеллюляризованного хряща свиньиBiocompatible and functional properties of a microdispersed tissue-specific 3D matrix from decellularized porcine cartilageНемецЕ. А.NemetsE. A.

Немец Евгений Абрамович

123182, Москва, ул. Щукинская, д. 1

Тел. (903) 579-23-79

Evgenij Nemets

1, Shchukinskaya str., Moscow, 123182, Russian Federation

Phone: (903) 579-23-79

evgnemets@yandex.ruЛажкоА. Э.LazhkoA. E.

Москва

Moscow

ГригорьевА. М.GrigorievA. M.

Москва

Moscow

БасокЮ. Б.BasokYu. B.

Москва

Moscow

КирилловаА. Д.KirillovaA. D.

Москва

Moscow

СевастьяновВ. И.SevastianovV. I.

Москва

Moscow

ФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава РоссииРоссияShumakov National Medical Research Center of Transplantology and Artificial OrgansRussian FederationФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова»РоссияMoscow State UniversityRussian FederationФГБУ «Национальный медицинский исследовательский центр трансплантологии и искусственных органов имени академика В.И. Шумакова» Минздрава России; АНО «Институт медико-биологических исследований и технологий»РоссияShumakov National Medical Research Center of Transplantology and Artificial Organs; Institute of Biomedical Research and TechnologyRussian Federation2022290720222447384Copyright © Немец Е.А., Лажко А.Э., Григорьев А.М., Басок Ю.Б., Кириллова А.Д., Севастьянов В.И., 20222022Немец Е.А., Лажко А.Э., Григорьев А.М., Басок Ю.Б., Кириллова А.Д., Севастьянов В.И.Nemets E.A., Lazhko A.E., Grigoriev A.M., Basok Y.B., Kirillova A.D., Sevastianov V.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.transpl.ru/vtio/article/view/1553

В отличие от децеллюляризации мягких тканей для использования их в качестве тканеспецифических матриц при создании тканеинженерных конструкций децеллюляризация хрящевой ткани требует привлечения нескольких методов обработки, что может отрицательно влиять на биосовместимые и функциональные свойства нативного внеклеточного матрикса.

Цель работы

Цель работы: исследование биосовместимых и функциональных свойств тканеспецифической мелкодисперсной 3D-матрицы из хряща свиньи, децеллюляризованного последовательной обработкой химическими, физическими и ферментативными методами.

Материалы и методы

Материалы и методы. Для децеллюляризации микродисперсные частицы хряща (МЧХ), полученные методом криопомола, инкубировали в растворах детергентов (додецилсульфат натрия и Triton X-100) с последующей обработкой сверхкритическим диоксидом углерода (ск-СО2) с добавлением этанола (10% по объему) и ДНКазой I типа. Для определения генотоксичности децеллюляризованных МЧХ (ДМЧХ) использовали мутационный тест Эймса на Salmonella typhimurium. Местное и общетоксическое действие, а также резорбцию ДМЧХ исследовали in vivo на половозрелых аутбредных крысах. Образцы ДМЧХ (10 мг) имплантировали в мышечную ткань бедра. Жизнеспособность мезенхимальных стромальных клеток жировой ткани человека (МСК ЖТч) при культивировании на ДМЧХ анализировали методом прижизненной микроскопии с окрашиванием флуоресцентным красителем Calcein AM. Метаболическую активность клеток оценивали с использованием PrestoBlue™ Cell Viability Reagent.

Результаты

Результаты. Доказано, что имплантированные в мышцу крысы ДМЧХ свиньи после обработки ск-СО2 не оказывают местного и общетоксического действия, не проявляют генотоксичности и отрицательного действия на репродуктивную систему животных. После 6 месяцев эксперимента in vivo резорбируется большая часть (87%) имплантированного децеллюляризованного хряща. Показали, что полученные матриксы способны поддерживать адгезию и пролиферацию МСК ЖТч.

Заключение

Заключение. Образцы ДМЧХ свиньи соответствуют требованиям, предъявляемым к биосовместимым медицинским изделиям по показателям местного и общетоксического действия, гено- и репродуктивной токсичности, и могут быть рекомендованы в качестве матрицы при создании клеточно- и тканеинженерных конструкций хрящевой ткани.

In contrast to decellularization of soft tissues for use as tissue-specific matrices in the creation of tissue-engineered constructs, decellularization of cartilage tissue requires several processing techniques, which can negatively affect the biocompatibility and functional properties of the native extracellular matrix (ECM).

Objective

Objective: to study the biocompatible and functional properties of microdispersed tissue-specific 3D matrix from a porcine cartilage that is decellularized by sequential use of chemical, physical and enzymatic techniques.

Materials and methods

Materials and methods. For decellularization, microdispersed cartilage particles (MCPs), obtained by cryomilling, were incubated in detergent solutions (sodium dodecyl sulfate and Triton X-100), then treated with supercritical carbon dioxide (scCO2) with 10% ethanol and DNase I. The Ames test (Salmonella typhimurium reverse mutation assay) was used to determine the genotoxicity of decellularized microdispersed cartilage particles (dMCPs). Local and general toxic effects, as well as resorption of dMCPs were studied in vivo on sexually mature outbred rats. Decellularized MCP specimens (10 mg) were implanted into the thigh muscle tissue. Viability of human adipose-derived mesenchymal stem/stromal cells (hAdMSCs), when cultured on dMCPs, was analyzed by in vivo microscopy, stained with fluorescent Calcein AM dye. Cell metabolic activity was assessed using PrestoBlue™ Cell Viability Reagent.

Results

Results. It has been proven that porcine dMCPs implanted in rat muscle after treatment with scCO2 do not exhibit local and general toxic effects, and do not show genotoxicity and negative effects on the reproductive system of animals. After 6 months of in vivo experiment, most (87%) of the implanted decellularized cartilage was resorbed. It was shown that the resulting matrices are able to support adhesion and proliferation of hAdMSCs. Conclusion. Porcine dMCP specimens are suitable for biocompatible medical products in terms of local and general toxic effects, genotoxicity and reproductive toxicity, and can be used as a matrix for creating cell- and tissue-engineered cartilage constructs.

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The authors declare that there are no conflicts of interest present.