Transient Exposure to Hypoxic and Anoxic Oxygen Concentrations Promotes Either Osteogenic or Ligamentogenic Characteristics of PDL Cells

Takako Kawasaki,Kazuhiro Egashira,Hideaki Kagami,Seigo Ohba,Simon D Tran,Yoshinori Sumita,Izumi Asahina

doi:10.1089/biores.2014.0049

Takako Kawasaki, Kazuhiro Egashira + Show 5 more

Open Access

https://doi.org/10.1089/biores.2014.0049

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Abstract

The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells (MSCs) and this tissue is easily available following teeth removal procedures. However, PDL-derived cells (PDLCs) availability for tissue engineering is limited because they are heterogeneous cells at various differentiation and lineage commitments. Therefore, efficient culture conditions to increase MSCs number are needed to use PDLCs in tissue engineering. Recent reports indicate that low-oxygen conditions amplified stem/progenitor cell numbers and inhibited cell differentiation. Our aim was to establish which low-oxygen culture conditions favored bone or tendon/ligament regeneration in cultured PDLCs. Human PDLCs were cultured and exposed to either hypoxic (O2≤5%) or anoxic (O2<0.1%) oxygen conditions in low-glucose/serum-free media for 24 hours. After 24 h, as expected, cell survival was significantly less in PDLCs exposed to anoxic conditions as compared with cells under normal or hypoxic conditions. PDLCs exposed to hypoxic conditions had the highest percentages for MSC markers (CD105, CD166, Stro-1). For both hypoxic and anoxic conditions, stem cell marker genes (oct4, sox2, p75) were upregulated after 6 h. At 24 h, these stem cell markers were maintained in PDLCs under hypoxic condition. Interestingly under anoxic conditions, expression of scleraxis gene (a key transcription factor for tendo/ligamentogenesis) was upregulated markedly. When hypoxic PDLCs were subcultured into osteogenic medium, in vitro calcification and prominent in vivo bone formation in mice calvaria were observed. When anoxic PDLCs were subcultured into tendo/ligamentogenic medium, expression of aggrecan (a mature tenogenic gene) increased remarkably. No obvious differences were detectable on chondrogenic and adipogenic inducibilities. We propose that transient exposure to low-oxygen during the culture enhanced MSC population in PDL. In addition, different low-oxygen concentrations favored osteogenic or tendo/ligamentogenic inducibilities of cultured PDLCs.

Highlights

Periodontal ligament-derived cells (PDLCs) have been proposed as a cellular reservoir of mesenchymal stem cells (MSCs) for bone and periodontal tissue engineering because these cells can supply mineralized dental tissues such as alveolar bone and cementum.[1,2] For dentists, periodontal ligament (PDL) tissues can be harvested from common teeth removal procedures without morbidity, and are a convenient source of MSCs for oral/maxillofacial tissue reconstruction.a Takako Kawasaki et al 2015; Published by Mary Ann Liebert, Inc
Immunofluorescence analysis revealed that human anti-Vimentin antibody reacted with cultured PDLCs (Fig. 1B), and anti-Stro-1 antibody reacted with some cells (Fig. 1C)
This study demonstrated that short-time low-oxygen stimulation has an effect on tissue regenerative capability of cultured PDLCs

Summary

Introduction

Periodontal ligament-derived cells (PDLCs) have been proposed as a cellular reservoir of mesenchymal stem cells (MSCs) for bone and periodontal tissue engineering because these cells can supply mineralized dental tissues such as alveolar bone and cementum.[1,2] For dentists, periodontal ligament (PDL) tissues can be harvested from common teeth removal procedures without morbidity, and are a convenient source of MSCs for oral/maxillofacial tissue reconstruction. Recent studies have indicated that low-oxygen conditions, such as hypoxic or anoxic culture conditions, amplified stem/progenitor cell numbers, and inhibited cell differentiation.[11] For example, hypoxic stimulation with 3% O2 inhibited differentiation of BMMSCs and enhanced activation of pluripotent stem cell markers.[11] These facts suggest that hypoxic/anoxic stimulations to dental MSCs in culture can arrest their differentiation and enhance their plasticity More recently, it has been reported hypoxic stimulation plays an important role in maintaining the stemness and differentiation capacity of PDLCs in vitro.[12] for tissue engineering use, it has not been well ascertained what culture condition of PDLCs with low-oxygen is effective to obtain a reliable number of functional MSCs

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