2022 / 04

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DOI: 10.47183/mes.2022.037

ORIGINAL RESEARCH

Fabrication of cartilage tissue substitutes from cells with induced pluripotency

Eremeev AV1,3, Pikina AS1,2, Ruchko ES1,3, Sidorov VS1, Ragozin AO1
About authors

1 Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow, Russia

2 Lomonosov Moscow State University, Moscow, Russia

3 Koltzov Institute of Developmental Biology, Moscow, Russia

Correspondence should be addressed: Artem V. Eremeev
Malaya Pirogovskaya, 1a, 119435, Moscow, Russia; ur.xednay@veemere-tra

About paper

Funding: the study was supported with an allocation #22-15-00250 by the Russian Science Foundation.

Acknowledgments: the authors thank Corresponding Member of the Russian Academy of Sciences M.A. Lagarkova for providing research facilities for the study

Author contribution: Eremeev AV — design of the experiment, general guidance, article authoring; Pikina AS — literature review, collection of the material, participation in the experimental part of the work, analysis of the resulting data; Ruchko ES — participation in the experimental part of the work; Sidorov VS, Ragozin AO — provision of material for the experiment.

Compliance with ethical standards: the study was performed in conformity with the principles of the Declaration of Helsinki (2000) and its subsequent revisions.

Received: 2022-10-12 Accepted: 2022-10-28 Published online: 2022-11-23
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Fig. 1. General sequence of chondrogenesis processes. A. Migration of the mesenchyme. B. Prechondrogenic mesenchymal condensation. C. Formation of cell contacts, synthesis of ECM. D. Intercellular space and microenvironment of differentiating cells. E. Beginning of chondrogenic differentiation. F. Cartilage formation. G. Phase contrast microscopy pictures, magnification x10: 1-5 — spheroids after 1, 2, 3, 4 and 5 weeks of cultivation, 3D cultivation condition. ×100
Fig. 2. Immunocytochemical analysis of monolayer cultures. A. Use of TGFβ1, BMP2 and 10% FBS. B. Using Chir99021 and RA. C. Articular chondrocyte culture, positive control. E. FD4S iPSC culture, negative control. 1 — aggrecan (green) and Sox9 (red), 2 — type I collagen (red), 3 — type II collagen (red), 4 — CD105 (red). Scale bar — 200 microns
Fig. 3. Indicators of gene expression of chondrogenic markers in monolayer cultures. Error bar is the standard deviation. Significance of differences between groups: ns, p > 0.05; * — p < 0.05; * * — p < 0.01; * * * — p < 0.001
Fig. 4. Immunocytochemical analysis of aggrecan (green) and Sox9 (red) synthesis in 3D spheroid cultures of different protocols. A–D. Differentiation protocols: "long" (A), "short" (B), "combined" (C), with conditioned medium (D). E. Spheroids of the positive control group. F. Spheroids of the negative control group. Duration of differentiation: 1 — 14 days, 2 — 21 days, 3 — 28 days, 4 — 35 days. Scale bar — 200 microns
Fig. 5. A. ACAN expression indicators in 3D spheroid cultures of various protocols. B. SOX9 expression indicators. Abscissa, duration of differentiation; ordinate, value of normalized expression. Error bar is the standard deviation. Significance of differences between groups: ns, p > 0.05; * — p < 0.05; * * — p < 0.01; * * * — p < 0.001
Fig. 6. А. COL1A2 expression indicators in 3D spheroid cultures of various protocols. B. COL2A1 expression indicators. Abscissa, duration of differentiation; ordinate, value of normalized expression. Error bar is the standard deviation. Significance of differences between groups: ns, p > 0.05; * — p < 0.05; * * — p < 0.01; * * * — p < 0.001
Fig. 7. Histological analysis of 3D spheroid cultures of different differentiation protocols. Differentiation protocols: "long" (A), "short" (B), "combined" (C), with conditioned medium (D). Control groups: positive control (fragments of articular cartilage) (E), negative control (F). Type of histological staining: 1 — hematoxylin-eosin, 2 — picrosirius red, 3 — safranin O
Table 1. Chondrogenic differentiation protocols
Table 2. Primers used in the work