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Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix

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Abstract

Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson’s trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.

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Authors and Affiliations

  1. School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 151-742, Republic of Korea

    Hyunuk Ro, Jungha Park, Jiyong Kim, Hyun-Gu Yim, Giyoung Jung & Nathaniel S. Hwang

  2. The NBio Institute of Seoul National University, Seoul, 151-742, Republic of Korea

    Nathaniel S. Hwang

  3. Department of Biotechnology, Yonsei University, Seoul, 120-749, Republic of Korea

    Kisuk Yang & Seung-Woo Cho

  4. College of Pharmacy, Ewha Womans University, Seoul, 120-750, Republic of Korea

    Giyoung Jung & Hyukjin Lee

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  1. Hyunuk Ro
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  2. Jungha Park
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  5. Hyun-Gu Yim
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Corresponding author

Correspondence to Nathaniel S. Hwang.

Additional information

Hyunuk Ro and Jungha Park contributed equally to this work.

This research was supported by the Basic Science Research Program (grant no. 0458–20120013) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (MSIP). This study was also partially supported by a grant (2009–0083522) from the Translational Research Center for Protein Function Control (TRCP) funded by the Ministry of Science, ICT, and Future Planning (MSIP), Republic of Korea.

Electronic supplementary material

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Supplementary Figure 1

Surface roughness of silicon wafer immersed in 10 mM TRIS-HCl buffer (Control) for 4 days. 3D rendering of surface roughness of control silicon wafer (a). Graphical analysis of surface roughness of control silicon wafer (b). (GIF 72 kb)

High resolution image (TIFF 4,022 kb)

Supplementary Figure 2

Surface roughness of silicon wafer immersed in simulated body fluid (SBF) for 4 days. 3D rendering of surface roughness of SBF silicon wafer (a). Graphical analysis of surface roughness of SBF silicon wafer (b). (GIF 80 kb)

High resolution image (TIFF 4,184 kb)

Supplementary Figure 3

Surface roughness of silicon wafer immersed in mixture of simulated body fluid, polydopamine, and gelatin (SBF/pDA/Gel) for 4 days. 3D rendering of surface roughness of SBF/pDA/Gel silicon wafer (a). Graphical analysis of surface roughness of SBF/pDA/Gel silicon wafer (b). (GIF 70 kb)

High resolution image (TIFF 3,067 kb)

Supplementary Figure 4

Real-time PCR analysis of BMP2 and BMP receptors such as BMPR1A and BMPR2 of C3H10T1/2 cells following 1 day (D1) and 6 days (D6) of exposure to chondrocyte-conditioned medium (CM). *P < 0.05, **P < 0.01 (GIF 19 kb)

High resolution image (TIFF 945 kb)

Supplementary Figure 5

Polarized light microscopy image of original bone area in the mouse skull (positive control for Fig. 6f-f’’’). Bar 200 μm. (GIF 476 kb)

High resolution image (TIFF 20,509 kb)

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Ro, H., Park, J., Yang, K. et al. Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix. Cell Tissue Res 362, 115–126 (2015). https://doi.org/10.1007/s00441-015-2195-7

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  • DOI: https://doi.org/10.1007/s00441-015-2195-7

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