Author(s)

Naoaki Saito^1,2,3, Hiroko Kato⁴, Yosuke Akiba⁵, Yuko Hara², Taku Kojima¹, Michiko Yoshizawa¹, Atsushi Ohazama³, Takeyasu Maeda⁴, Tadaharu Kobayashi¹, Kenji Izumi^2*

¹Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
²Division of Biomimetics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
³Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
⁴Research Center for Advanced Oral Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
⁵Division of Bio-Prosthodontics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

Objective: The enhancement of multiple functions, including osteogenesis, angiogenesis, and cell recruitment, is required for efficient bone regeneration therapy. Recently, special attention has been focused on the microenvironment of stem cells to facilitate bone regeneration. Herein, we examined the effects of various combinations of hypoxic conditions and osteogenic induction on rat mesenchymal cells, to develop a specific protocol for enhancing the multiple cellular functions beneficial to bone regeneration. Methods: Rat mesenchymal cells, isolated from bone marrow, adipose tissue, and periodontal ligament, were examined. The cells were cultured under varied conditions of O₂ tension (hypoxia) and duration and timing of hypoxic exposure, with or without osteogenic induction. Consequently, four different protocols were examined by measuring the gene expression levels of Runx2, Vegfa, and Cxcl12, indicating a capability for osteogenesis, angiogenesis, and cell recruitment, respectively. Finally, the mineralization ability of the rat mesenchymal cells was assessed by quantitating their calcified nodule formation. Results: The simultaneous application of hypoxic exposure and osteogenic induction promoted Vegfa expression in all types of cells, but suppressed Runx2. In contrast, hypoxic preconditioning, followed by osteogenic induction, did not increase the expression of these genes; in fact, Vegfa expression decreased significantly. Among the various protocols, 0.5% O₂ exposure for 12 h after osteogenic induction exhibited the largest fold changes of gene expression level, especially of Vegfa. Hypoxic post-conditioning enhanced the formation of calcified nodules in periodontal ligament-derived cells. Conclusion: Short-term hypoxic exposure after osteogenic induction could be used to improve the efficiency of mesenchymal cells for bone regeneration.

Osteogenesis, Hypoxia, Angiogenesis, Cell Recruitment, Mesenchymal Stem Cells

Saito, N. , Kato, H. , Akiba, Y. , Hara, Y. , Kojima, T. , Yoshizawa, M. , Ohazama, A. , Maeda, T. , Kobayashi, T. and Izumi, K. (2018) Application of Hypoxic Exposure Combined with Osteogenic Induction for the Enhancement of Multiple Osteoinductive Capabilities in Rat Mesenchymal Cells. Open Journal of Stomatology, 8, 53-69. doi: 10.4236/ojst.2018.82005.