shRNA knockdown of integrin-linked kinase on hPDLCs migration, proliferation, and apoptosis under cyclic tensile stress.

OBJECTIVE: To investigate the roles of integrin-linked kinase (ILK) in mediating the cell migration, proliferation, and apoptosis of human periodontal ligament cells (hPDLCs) in response to cyclic tensile stress. METHODS: Primary hPDLCs were obtained through the enzyme digestion and tissue culture method. Short hairpin ILK-expressing hPDLCs were constructed using a recombinant lentiviral vector that specifically targeted ILK gene expression. The silencing of the ILK gene was identified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The hPDLCs were seeded on a flexible substrate and loaded with cyclic tensile stress at 0.5 Hz for 0, 2, 4, and 8 hr, consecutively, with the Flexcell Tension System. The response of cell migration was tested by the scratch assay. Cell proliferation was characterized by optical density (OD) value of cell counting kit-8 (CCK-8) test and Ki67 mRNA expression of qRT-PCR. Cell apoptosis was determined by flow cytometry and Caspase-3 mRNA expression of qRT-PCR. RESULTS: Knocking down ILK substantially reduces migration and proliferation as well as regulates the sensitivity of hPDLCs to apoptosis under cyclic tensile stress. CONCLUSIONS: ILK can promote the proliferation and migration as well as inhibit apoptosis of hPDLCs under cyclic tensile stress.

Effect of ILK on small-molecule metabolism of human periodontal ligament fibroblasts with mechanical stretching.

BACKGROUND: Mechanical stimuli can cause periodontal tissue reconstruction. Studies have found that changes in metabolites can be the terminal effect of integrin-mediated mechanical signaling. As a key kinase in integrin regulation, integrin-linked kinase (ILK) mediates mechanical signal transduction, which may contribute to metabolite changes. Defining the components of small-molecule metabolites can optimize mechanical stimuli and periodontal tissue reconstruction. Our purpose is to detect the effect of ILK-mediated mechanical signaling on intracellular small-molecule metabolites (amino acids and organic acids) in human periodontal ligament fibroblasts (HPDLFs). METHODS: Primary HPDLFs were isolated by enzyme digestion method. Tensile stresses were applied on HPDLFs in vitro using a Flexcell system. ILK gene in HPDLFs was knocked down by RNA interference (RNAi). Twenty common amino acids and seven organic acids in HPDLFs were analyzed by gas chromatography/mass spectrometry technique. RESULTS: Five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) were found to be changed remarkably after mechanical stretching. In addition, baseline levels of four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) were significantly different in ILK knockdown compared with wild-type HPDLFs. CONCLUSION: This study suggests that five amino acids (ie, alanine, glutamine, glutamate, glycine, and threonine) and three organic acids (ie, pyruvate, lactate, and citric acid) may act as cellular mediators for mechanical signals in HPDLFs. Among them, four amino acids (ie, glutamate, glutamine, threonine, and glycine) and two organic acids (ie, lactate and citric acid) may be closely linked to ILK.

Combining enamel matrix proteins with mechanical stimuli potentiates human periodontal ligament fibroblasts proliferation and periodontium remodeling.

BACKGROUND: Collagen I (Col-I) and matrix metalloproteinase-1 (MMP-1) have been implicated in the regeneration and remodeling of the periodontium. Studies have shown that enamel matrix proteins (EMPs) and mechanical stimuli can promote the synthesis and degradation, respectively, of Col-I and MMP-1. However, the effects of the combination of EMPs and mechanical stimuli on human periodontal ligament are not known. OBJECTIVE: Our aim was to test the combined effects of EMPs and mechanical stimuli on the proliferation of human periodontal ligament fibroblasts (HPDLFs) and Col-I and MMP-1 mRNA expression. METHODS: Primary HPDLFs were isolated using an enzyme digestion method. To select the optimum EMP concentration and the optimum magnitude and loading time of mechanical stimuli, HPDLFs were stimulated with gradient concentration of EMPs (0 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL and 200 µg/mL) and mechanical stimuli (0 kPa, 25 kPa, 50 kPa, 100 kPa, and 200 kPa for 0 h, 3 h, 6 h, 12 h, and 24 h), respectively. The cell proliferative response was tested by the MTT assay. The impact of EMPs combined with mechanical stimuli on Col-I and MMP-1 mRNA expression were measured by reverse transcription polymerase chain reaction. RESULTS: 100 µg/mL of EMPs and a 50 kPa mechanical stimulus were chosen as the optimum parameters due to the higher proliferation rates than other doses. The combination of 100 µg/mL of EMPs and a 50 kPa mechanical stimulus significantly stimulated HPDLFs proliferation and increased Col-I and MMP-1 expression levels compared with incubation with two factors alone. CONCLUSIONS: We concluded that the combination of EMPs and mechanical stimulus have synergistic effects on cell growth, cell number, collagen turnover, and periodontium remodeling.