CC BY
13B-O-7
Scaffolds structural heterogeneity influence on the efficiency of stem cells osteogenic differentiation
Shchechkin I.D. 12, Rodimova S.A. 12, Elagin V.V. 1, Karabut M.M. 1, Minaev N.V. 3, Shpichka A.I. 3, Timashev P.S. 3, Zagaynova E.V. 12, Kuznetsova D.S. 12
1- Research Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research
Medical University, Minin and Pozharsky Sq., 10/1, Nizhny Novgorod, Russia;
2- Nizhny Novgorod National Research State University N.I. Lobachevsky, Gagarin Ave., 23, Nizhny
Novgorod, Russia;
3- Institute of Regenerative Medicine, Sechenov University, Trubetskaya st., Moscow, Russia.
Iliahasa1992@gmail.com
One of the promising approaches for the restoration of damaged tissues is the implantation of mesenchymal stem cells (MSCs) in injuries. However, this approach does not provide complete defect recovery. In this regard, the use of 3D scaffolds is more effective, contributing to a better restoration of the integrity of the damaged tissue. The effectiveness of the recovery process depends on many factors; the effect of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage has not been fully studied.
The aim of this work was to study the effect of the structure of scaffolds on the efficiency of osteogenic differentiation.
By the method of two-photon polymerization, scaffolds with a homogeneous (equal pore size) and heterogeneous (larger pore size of the central layer of scaffolds) structure were obtained. For each type, cultivation was carried out in a standard medium and medium for osteogenic induction for 28 days. The metabolic status of MSCs was analyzed by fluorescence lifetime imaging microscopy (FLIM) based on the data on the lifetimes of free and bound forms of NAD(P)H, as well as open and closed forms of FAD and their contributions. The efficiency of osteogenic differentiation was confirmed by staining with alizarin red C and fluorometric analysis for the content of secreted alkaline phosphatase (ALP). Staining with alizarin red showed that MSCs cultured on homogeneous scaffolds in a standard medium do not proceed to spontaneous differentiation, while single differentiated cells were observed on heterogeneous scaffolds. When cultivated in an osteogenic medium, scaffolds of both types have the same red coloration, which indicates an effective induction of osteogenic differentiation. Analysis for ALP showed a significant increase on day 28 for MSCs on heterogeneous scaffolds, which indicates a greater efficiency of osteogenic differentiation. The FLIM method showed an increase in the contributions of the bound form of NAD(P)H and the closed form of FAD in the osteogenic medium, the efficiency of differentiation for heterogeneous scaffolds was higher. An increase in the contributions of the bound form of NAD(P)H and the closed form of FAD was also observed for heterogeneous scafflods and in a standard medium, which indicates the induction of spontaneous differentiation. It is worth noting that the results of the analysis of standard methods and methods of fluorescence bioimaging agreed with each other.
Acknowledgments. This work was supported by the Russian Science Foundation, grant no. 19-7510008.
