Abstract
The aim of the study was to evaluate in vitro the effect of native and deproteinized compact and spongy allogenic bone matrices on the characteristics of adipose mesenchymal stromal cells (ASC) in combined tissue engineering.Material and Methods. 24 samples of native and deproteinized compact and spongy bone were examined, which were exposed to mechanical treatment, modeling, followed by sterilization of the samples by ionizing radiation and bacteriological control of sterilization. Some of the samples underwent deproteinization. The characterized cultures of human ASC were used as test cultures to assess the interaction with the bone samples. The Cytation-5 fluorescent imager and Hoechst 3334 fluorochromes (BD Pharmingen™) and calcein (Calcein AM, BD Pharmingen™) were used to characterize the degree of adhesion, migration, and viability of ASC on bone matrix samples. Matrix cytotoxicity was evaluated by MTT assay on days 1 and 7 of extraction.Results. The bone matrix samples are characterized by the absence of cytotoxicity (rank 1). ASC demonstrated good adhesion and migration on any surface of the bone matrix and preservation of cell viability during 7 days of observation. Nuclei sizes of the cells adhered to the deproteinized bone matrix of the spongy structure increased by 25–30% compared to other samples. The cells on deproteinized bone matrix had greater size (the size of the cells from nuclei 8.8 to 11.5 μm, the average size of cells nuclei from an 86.3 μm to 129,0 μm, the average perimeter of the cells nuclei from 30.7 μm to 40.7 μm) than in the native bone matrix samples.Conclusion. The results of the study of various allogeneic bone matrices demonstrate that deep purification of the bone matrix determines the absence of cytotoxicity and the most favorable conditions for the adhesion, migration, proliferation and viability of ASC. Also makes it possible to use tissue engineering based on bone matrices of different structures. Deproteinized spongy bone matrices are best suited for this purpose.
Highlights
Задачи исследования: 1) изучение с использованием цифрового имиджера Cytation 5 адгезии мезенхимальных стромальных клеток (МСК) к костному матриксу; 2) изучение миграции МСК в межбалочные пространства; 3) изучение жизнеспособности МСК на костном матриксе
The aim of the study was to evaluate in vitro the effect
which were exposed to mechanical treatment
Summary
Для получения клеточной культуры ДФЧ использовали метод тканевых эксплантатов [14]. Биоптаты кожи забирали в стерильных условиях операционной во флаконы со стерильной ростовой средой 199 с добавлением 1% раствора антибиотиков (пенициллин/стрептомицин). Образцы кожи тщательно промывали средой с антибиотиками (среда 199, 1% пенициллина/ стрептомицина для культур клеток), очищали от подкожно-жировой клетчатки и подвергали ферментативной обработке. После ферментативной обработки кусочки кожи механически измельчали до размеров 0,1 см и в шахматном порядке раскладывали в культуральные флаконы Costar 25 см. В качестве ростовой среды использовали среду ДМЕМ с добавлением 1% антибиотиков, 2% глутамина и 10% телячьей эмбриональной сыворотки. Культивирование проходило в условиях СО2инкубатора при 5% СО2, температуре 37oС, абсолютной влажности. Ежедневный контроль формирования монослоя и стерильности культуры клеток осуществляли с помощью инвертированного микроскопа Leica DMI 3000 B (Германия). Для исследований in vitro использовали культуры ДФЧ на 4–6-м пассаже.
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