In vivo cell proliferation analysis and cell-tracing reveal the global cellular dynamics of periodontal ligament cells under mechanical-loading

Abstract

Periodontal ligament (PDL) is a uniquely differentiated tissue that anchors the tooth to the alveolar bone socket and plays key roles in oral function. PDL cells can respond rapidly to mechanical stimuli, resulting in accelerated tissue remodeling. Cell proliferation is an initial event in tissue remodeling and participates in maintaining the cell supply; therefore, analyzing cell-proliferative activity might provide a comprehensive view of cellular dynamics at the tissue level. In this study, we investigated proliferating cells in mouse molar PDL during orthodontic tooth movement (OTM)-induced tissue remodeling. Our results demonstrated that the mechanical stimuli evoked a dynamic change in the proliferative-cell profile at the entire PDL. Additionally, cell-tracing analysis revealed that the proliferated cells underwent further division and subsequently contributed to tissue remodeling. Moreover, OTM-induced proliferating cells expressed various molecular markers that most likely arise from a wide range of cell types, indicating the lineage plasticity of PDL cells in vivo. Although further studies are required, these findings partially elucidated the global views of the cell trajectory in mouse molar PDL under mechanical-loading conditions, which is vital for understanding the cellular dynamics of the PDL and beneficial for dental treatment in humans.

Acellular Extrinsic Fiber Cementum Is Invariably Present in the Superficial Layer of Apical Cementum in Mouse Molar

“…Immunohistochemistry was performed as described in previous studies. 12,15,16 The following primary antibodies were used for the analysis: anti-dentin matrix protein-1 (anti-Dmp1; 1:800; M176; Takara Bio, Kusatsu, Japan), anti-osteopontin (1:200; 25715-1-AP; Prot-eintech, Rosemont, IL), anti-β-catenin (1:2,000; 51067-2-AP; Proteintech), and anti-mouse SOST/sclerostin antibody (1:50; AF1589; R&D Systems, Minneapolis, MN). Biotinylated anti-goat IgG (H+L) (1:200; BA-5000; Vector Laboratories, Burlingame, CA) and biotinyla-ted anti-rabbit IgG (H+L) (1:200; BA-1000; Vector Laboratories) were the secondary antibodies used.…”

Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress

“…Mice were anesthetized with intraperitoneal injections of chloral hydrate (400 mg/kg body weight). Ni-Ti closed-coil springs (25 gf; TOMY International Inc., Tokyo, Japan) were inserted between the left maxillary 1 st molars and incisors to induce mesial movement of the 1 st molars ( Mizukoshi et al, 2021 ). The ends of the Ni-Ti springs were tied to the molars and incisors with stainless steel wire (φ 0.09 mm) and secured with light-cure composite resin (BEAUTIFIL Flow; Shofu, Kyoto, Japan).…”

“…Micro-CT images of the maxilla were obtained with a CosmoScan GX high-resolution X-ray tomographic system (Rigaku Corporation, Tokyo, Japan) at 90 kV and 88-μA irradiation and structurally examined using Analyze software (v.12.0; AnalyzeDirect, Stilwell, KS, United States). The distance between the closest points of the 1 st and 2 nd molars was measured after the outline of the teeth was defined with a threshold intensity of between 6,000 and 12,000 Hounsfield units ( n = 5) ( Mizukoshi et al, 2021 ).…”

“…Upper 1 st molars with surrounding tissue were dissected, decalcified with 10% (w/v) EDTA for 3 weeks at 4°C, and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan) or paraffin according to a standard protocol ( Kaku et al, 2016 ; Mizukoshi et al, 2021 ). Sagittal cryosections (10-µm thick) were prepared on membrane-coated slides (Leica Biosystems, Wetzlar, Germany), and 5-µm thick sagittal paraffin sections were prepared using a microtome (REM-710+MC-802C; Yamato Kohki, Saitama, Japan).…”

“…Mechanoregulation of the ECM in the PDL is of clinical interest because controlling PDL tissue homeostasis under mechanical loading is crucial in dental practice, such as in occlusal adjustment and managing orthodontic tooth movement (OTM). Moreover, the PDL is an adequate in vivo experimental model for studying ECM organization and remodeling under mechanical loading ( Kaku and Yamauchi, 2014 ) because of its well-organized fiber alignment, rapid tissue-turnover rate ( Sodek, 1977 ), good responsiveness, and ease of controlling the mode of mechanical loading ( Nozaki et al, 2010 ; Kaku et al, 2016 ; Mizukoshi et al, 2021 ).…”