IADR Abstract Archives
A mechanical strain model for the assessment of periodontal ligament cell endoplasmic reticulum stress in three-dimensional culture
Objectives: A balanced cellular homeostatic response is essential for safe and controlled orthodontic tooth movement. The endoplasmic reticulum (ER) plays a major role in maintaining homeostasis, with ER stress activating the unfolded protein response (UPR), potentially resulting in apoptotic cell death. This study examines human periodontal ligament cell (hPDL) viability, apoptosis, and mRNA expression levels of strain-responsive and UPR/ER stress genes when cultured under mechanical strain.
Methods: Three primary hPDL cell lines were cultured as a monolayer or encapsulated in hydrogel, on flexible-bottomed culture plates for 7 days before being mechanically strained at a level of 18% elongation for 24 hours. Proliferation was measured over the 7 days and cell viability, caspase activity and mRNA quantity levels of a number of mechanically-responsive and UPR/ER stress genes were measured following strain.
Results: The quantity of viable hPDLs in monolayer culture increased to a greater extent compared to cells cultured in hydrogel, which initially decreased in viability from day 1 to 3 (-34%; P = 2x10-8) before recovering from day 5 to 7 (+12%; P = 1x10-4). Cell viability significantly reduced following strain in monolayer cultures (P = 0.043), while in hydrogel constructs no significant differences in the number of cells, caspase activity, or mRNA quantity levels were found when compared to the unstrained controls. For monolayer samples, the gene LOX (involved in cross-linking of collagen and elastin) demonstrated a response to mechanical strain, and was upregulated compared to unstrained controls.
Conclusions: hPDLs cultured as monolayers proliferated rapidly and are affected by mechanical strain; cells cultured in a hydrogel proliferate slowly and are not significantly affected by static 24-hour 18% strain. The failure to detect a gene response for the encapsulated cells suggests that hydrogel dampens the propagation of mechanical strain. Future experiments should include longer strain times and varying strain magnitudes for encapsulated hPDLs.
Methods: Three primary hPDL cell lines were cultured as a monolayer or encapsulated in hydrogel, on flexible-bottomed culture plates for 7 days before being mechanically strained at a level of 18% elongation for 24 hours. Proliferation was measured over the 7 days and cell viability, caspase activity and mRNA quantity levels of a number of mechanically-responsive and UPR/ER stress genes were measured following strain.
Results: The quantity of viable hPDLs in monolayer culture increased to a greater extent compared to cells cultured in hydrogel, which initially decreased in viability from day 1 to 3 (-34%; P = 2x10-8) before recovering from day 5 to 7 (+12%; P = 1x10-4). Cell viability significantly reduced following strain in monolayer cultures (P = 0.043), while in hydrogel constructs no significant differences in the number of cells, caspase activity, or mRNA quantity levels were found when compared to the unstrained controls. For monolayer samples, the gene LOX (involved in cross-linking of collagen and elastin) demonstrated a response to mechanical strain, and was upregulated compared to unstrained controls.
Conclusions: hPDLs cultured as monolayers proliferated rapidly and are affected by mechanical strain; cells cultured in a hydrogel proliferate slowly and are not significantly affected by static 24-hour 18% strain. The failure to detect a gene response for the encapsulated cells suggests that hydrogel dampens the propagation of mechanical strain. Future experiments should include longer strain times and varying strain magnitudes for encapsulated hPDLs.