Expression of osteoblastic phenotype in periodontal ligament fibroblasts cultured in three-dimensional collagen gel

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.

Electrospun ultrathin PBAT/nHAp fibers influenced the in vitro and in vivo osteogenesis and improved the mechanical properties of neoformed bone

“…Similar results were obtained in previous studies in three-dimensional scaffolds, [47,48] where ALP activity was higher at 7 days with subsequent reduction due to the beginning of extracellular matrix mineralization. The slight ALP increase at 21 days corroborates with Runx2 expression in the same period, and may be related to the renewal of the cell cycle [49]. The expression of Runt-related transcription factor 2 (Runx2) is associated with osteoblastic differentiation, and was decreased at 7 days of cell culture in the scaffold groups, regarding other periods and the control group (p < 0.001).…”

“…We suggest that the low Runx2 expression observed in the scaffold groups at 7 days may be justified due to the mature phenotype in this period, as confirmed by high cell activity and by elevated ALP expression in the scaffold groups in the same period. Furthermore, the renewal of the cell cycle may explain the increased late expression of Runx2 at 21 days; [49] nHAp-incorporated scaffold groups had higher Runx2 expression due to the influence of nHAp in the modification of the surface wettability and then to promote osteoblastic differentiation. The expression of Collagen type 1 (Col I), an abundant organic component of the extracellular matrix, was similar at 7 and 14 days of cell culture with higher control group expression (p > 0.001).…”

Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons

“…Human ESC lines H1, H7, and H9 (passages [25][26][27][28][29][30][31][32][33][34][35]; WiCell Research Institute, Madison, Wisconsin, USA, https://www.wicell.org) were cultured as previously described [27]. The step-wise processing is shown diagrammatically in Figure 1.…”

Spheroid formation and stemness preservation of human periodontal ligament cells on chitosan films

“…Recently, Alves et al. reported that PDL cells cultured in 3D collagen gel presented an increased proliferation rate over time (Alves et al., ). However, compared with the monolayer cell culture and the above‐mentioned 3D collagen gel culture system, PDL cells exhibited decreased proliferation after spheroid formation, which is in agreement with some earlier studies performed on spheroids of human ASCs (Cheng et al., ) and hepatocellular carcinoma cell line (Meli, Jordan, Clark, Linhardt, & Dordick, ).…”