Cell survival and gene expression under compressive stress in a three-dimensional in vitro human periodontal ligament-like tissue model

Abstract

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-L-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm(2) compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic "double-peak" expression at 15 and 35 g/cm(2) on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm(2) on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.

Keywords: Cell living condition; Compressive stress; Gene expression; Human periodontal ligament-like tissue; Orthodontic force.

Fig. 1

Microscope image (a) of the poly-l-lactide (PLLA) matrix. Scanning electron microscopic image of PLLA matrix without seeded cells (b) and with cells grown for 14 days (c)

Fig. 2

Method used to apply compression stress to mimic orthodontic force on the PDL. Pre-cultured HPdLLTs were compressed continuously in vitro using metal weights loaded on a piece of thin cover glass placed over the HPdLLTs in each well of a 24-well plate. The metal weight mimicked the orthodontic force and the in vitro HPdLLTs mimicked the PDL tissue

Fig. 3

Surface SEM observation of in vitro HPdLLTs (×100) after application of various compressive stress conditions. In all figures, spindle-shaped cells (arrow A) and extracellular matrix (arrow B) can be observed on the surface of each in vitro HPdLLT, with no relationship to compressive load duration or compressive stress intensity

Fig. 4

Cross-sectional SEM observation of HPdLLTs (×1,000) after in vitro application of various compressive stress conditions. In all figures, similar filamentous extracellular matrix and spindle-shaped cells can be observed inside each HPdLLT with no relationship to compressive load duration or compressive stress intensity. This figure is a local magnification of Fig. 5 (×250), which shows the extracellular matrix and spindle-shaped cells observed from the surface to the rear side of each in vitro HPdLLT

Fig. 5

Cross-sectional SEM images of HPdLLTs (×250) following the application of various compressive stress conditions. These images reveal the extracellular matrix and spindle-shaped cells observed from the surface to the rear side of each in vitro HPdLLT

Fig. 6

Caspase-3 and caspase-7 detection following the application of various compressive stress conditions. In all figures, similar density of green fluorescence was observed among the in vitro HPdLLTs without any relationship to the compressive load duration or compressive stress intensity

Fig. 7

Gene expression of HPdLLTs following application of various compressive stress conditions. The RANKL gene showed a characteristic double peak on day 7 and day 14 at 15 and 35 g/cm² compressive stress. BMP-2 gene showed the double peak only on day 14 at 15 and 35 g/cm². PLAP-1 and ALP showed expression peaks on day 14 at 5 g/cm². OPG and FGF-2 showed similar gene expression, with significantly higher levels on day 14 compared to earlier days. COL1 expression on days 7 and 14 was significantly higher than that on days 1 and 3 at all compressive stress levels