Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA

Maorui Zhang; Yuta Fukushima; Kosuke Nozaki; Hideyuki Nakanishi; Jia Deng; Noriyuki Wakabayashi; Keiji Itaka

doi:10.1186/s41232-023-00285-3

Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA

Inflamm Regen. 2023 Jun 20;43(1):32. doi: 10.1186/s41232-023-00285-3.

Authors

Maorui Zhang^{1

2

3}, Yuta Fukushima¹, Kosuke Nozaki², Hideyuki Nakanishi¹, Jia Deng^{1

4}, Noriyuki Wakabayashi², Keiji Itaka^{5

6}

Affiliations

¹ Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), Tokyo, 1010062, Japan.
² Department of Advanced Prosthodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, 1138549, Japan.
³ Department of Oral Implantology, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou, 646000, People's Republic of China.
⁴ Department of Masticatory Function and Health Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, 113-8549, Japan.
⁵ Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), Tokyo, 1010062, Japan. itaka.bif@tmd.ac.jp.
⁶ Clinical Biotechnology Team, Center for Infectious Disease Education and Research (CiDER), Osaka University, Osaka, 565-0871, Japan. itaka.bif@tmd.ac.jp.

Abstract

Background: Bone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible.

Methods: The mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (μCT) imaging, and histologic analyses.

Results: Osteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation.

Conclusions: These results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Keywords: Angiogenesis; Bone regeneration; Mandible bone defect; Polyplex nanomicelle; Runt-related transcription factor 2; Vascular endothelial growth factor; mRNA medicine.

Abstract

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