Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration

Kotaro Sano; Michihiko Usui; Yuki Moritani; Kohji Nakazawa; Tomoya Hanatani; Hisataka Kondo; Mitsushiro Nakatomi; Satoru Onizuka; Takanori Iwata; Tsuyoshi Sato; Akifumi Togari; Wataru Ariyoshi; Tatsuji Nishihara; Keisuke Nakashima

doi:10.1016/j.reth.2019.12.008

Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration

Regen Ther. 2020 Jan 14:14:59-71. doi: 10.1016/j.reth.2019.12.008. eCollection 2020 Jun.

Affiliations

¹ Division of Periodontology, Department of Oral Function, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan.
² Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan.
³ Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
⁴ Division of Anatomy, Department of Health Improvement, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan.
⁵ Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
⁶ Department of Oral and Maxillofacial Surgery, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama 350-0495, Japan.
⁷ Division of Infection and Molecular Biology, Department of Health Improvement, Kyushu Dental University, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan.

Abstract

Introduction: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) have been known that they play important roles in homeostasis and regeneration of periodontal tissues. Additionally, spheroids are superior to monolayer-cultured cells. We investigated the characteristics and potential of periodontal tissue regeneration in co-cultured spheroids of hPDLMSCs and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo.

Methods: Co-cultured spheroids were prepared with cell ratios of hPDLMSCs: HUVECs = 1:1, 1:2, and 2:1, using microwell chips. Real-time polymerase chain reaction (PCR) analysis, Enzyme-Linked Immuno Sorbent Assay (ELISA), and nodule formation assay were performed to examine the properties of co-cultured spheroids. Periodontal tissue defects were prepared in the maxillary first molars of rats and subjected to transplantation assay.

Results: The expression levels of stemness markers, vascular endothelial growth factor (VEGF), osteogenesis-related genes were up-regulated in co-cultured spheroids, compared with monolayer and spheroid-cultured hPDLMSCs. The nodule formation was also increased in co-cultured spheroids, compared with monolayer and spheroid cultures of hPDLMSCs. Treatment with co-cultured spheroids enhanced new cementum formation after 4 or 8 weeks of transplantation, although there was no significant difference in the new bone formation between co-cultured spheroids and hPDLMSC spheroids.

Conclusions: We found that co-cultured spheroids enhance the periodontal tissue regeneration. Co-cultured spheroids of hPDLMSCs and HUVECs may be a useful therapy that can induce periodontal tissue regeneration.

Keywords: Periodontal ligament mesenchymal stem cells; Periodontal tissue regeneration; Spheroid.