Abstract
Objective : To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures.Material and Methods : hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity were performed. Also, cell morphology and immunolabeling for alkaline phosphatase (ALP) and osteopontin (OPN) were assessed by epifluorescence and confocal microscopy. The expression of osteogenic markers, including alkaline phosphatase, osteopontin, osteocalcin (OC), collagen I (COL I) and runt-related transcription factor 2 (RUNX2), were analyzed using real-time polymerase chain reaction (RT-PCR). Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay.Results : Experimental cultures produced an increase in cell proliferation. Immunolabeling for OPN and ALP in hPDLF were increased and ALP activity was inhibited by three-dimensional conditions. OPN and RUNX2 gene expression was significantly higher on 3D culture when compared with control surface. Moreover, ALP and COL I gene expression were significantly higher in three-dimensional collagen than in 2D cultures at 7 days. However, at 14 days, 3D cultures exhibited ALP and COL I gene expression significantly lower than the control, and the COL I gene expression was also significantly lower in 3D than in 2D cultures. Significant calcium mineralization was detected and quantified by alizarin red assay, and calcified nodule formation was not affected by tridimensionality.Conclusion : This study suggests that the 3D cultures are able to support hPDLF proliferation and favor the differentiation and mineralized matrix formation, which may be a potential periodontal regenerative therapy.
Highlights
Periodontitis is an inflammatory disease caused by a microbial challenge in a susceptible host
This study focused on evaluating the effect of three-dimensionality on human periodontal ligament fibroblast (hPDLF) behavior when cultured on 2D and 3D collagen gel, whether the collagen could induce hPDLF to an osteoblastic phenotype
Alkaline phosphatase activity A significant lower degree of alkaline phosphatase (ALP) activity was observed on 3D cultures when compared with 2D (p
Summary
Periodontitis is an inflammatory disease caused by a microbial challenge in a susceptible host. Periodontal ligament (PDL), bone and cementum are destroyed, which eventually might lead to tooth loss. Current treatments of periodontitis consist of removal of plaque and calculus, which prevents further disease progression, but these treatments do not regenerate the lost tissues. Histological studies have shown epithelial downgrowth, indicating it is a mere reparative type of healing. Many recent studies focus on treatment strategies to achieve actual regeneration of the PDL rather than repair.
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