FGF-9 accelerates epithelial invagination for ectodermal organogenesis in real time bioengineered organ manipulation

Abstract

Background: Epithelial invagination is important for initiation of ectodermal organogenesis. Although many factors regulate ectodermal organogenesis, there is not any report about their functions in real-time study. Electric cell-substrate impedance sensing (ECIS), a non-invasive, real-time surveillance system, had been used to detect changes in organ cell layer thickness through quantitative monitoring of the impedance of a cell-to-microelectrode interface over time. It was shown to be a good method for identifying significant real-time changes of cells. The purpose of this study is to establish a combined bioengineered organ-ECIS model for investigating the real time effects of fibroblast growth factor-9 (FGF-9) on epithelial invagination in bioengineered ectodermal organs. We dissected epithelial and mesenchymal cells from stage E14.5 murine molar tooth germs and identified the real-time effects of FGF-9 on epithelial-mesenchymal interactions using this combined bioengineered organ-ECIS model.

Results: Measurement of bioengineered ectodermal organ thickness showed that Fibroblast growth factor-9 (FGF-9) accelerates epithelial invagination in reaggregated mesenchymal cell layer within 3 days. Gene expression analysis revealed that FGF-9 stimulates and sustains early Ameloblastin and Amelogenin expression during odontogenesis.

Conclusions: This is the first real-time study to show that, FGF-9 plays an important role in epithelial invagination and initiates ectodermal organogenesis. Based on these findings, we suggest FGF-9 can be applied for further study in ectodermal organ regeneration, and we also proposed that the 'FGF-BMP balancing system' is important for manipulating the morphogenesis of ectodermal organs. The combined bioengineered organ-ECIS model is a promising method for ectodermal organ engineering and regeneration research.

Figure 1

Tooth germ dissection and dissociation. First molar tooth germ dissection from the mandibular arch of an E14.5 mouse embryo and its dissociation into single cells using the non-serum protocol.

Figure 2

The combined bioengineered organ-ECIS model for analysis of epithelial invagination.

Figure 3

MTT assay. MTT assay for the in vitro culture of dissociated mesenchymal and epithelial cells to determine the optimal Fibroblast growth factor-9 (FGF-9) concentration. (A) MTT data for mesenchymal cells (n = 4, p < 0.5): FGF-9 concentrations of 25 and 40 ng/ml were both suitable for cell culture in the first 7 days. # : 40 ng/ml FGF-9 outperformed 25 ng/ml after 14 d of culture, (B) MTT for epithelial cells (n = 4, p < 0.5): 40 ng/ml is optimal for epithelial cell culture within the first 183 h (> 7 days). Thus, we chose 40 ng/ml FGF-9 for further experimentations.

Figure 4

ECIS Assay. We chose the ECIS Z8 system, with four wells chosen as the control group, and the other four wells as the FGF-9 group. The ECIS parameters include a frequency of 15,000 Hz and impedance data were recorded every 90 s. The black line represents the control group and the pink line represents the FGF-9 group. The vertical (dash) line represents the time of medium change. (A) Before adding epithelial cells and FGF-9 (40 ng/ml), it was necessary to allow mesenchymal cells to reaggregate as a stable layer in each well of the ECIS kit. We then added 2 × 10⁵ mesenchymal cells to each well. Impedance data showed no difference between the two groups in the first 25 h, indicating no significant proliferation or death of mesenchymal cells. (B) The invasion test began after the addition of epithelial cells and FGF-9 (40 ng/ml). We added 2 × 10⁵ epithelial cells to each well of the 8W10E kit. We added ordinary fresh medium to the control group (black line) and fresh medium with FGF-9 (40 ng/ml) to the FGF-9 group (pink line). The vertical (Dash) line:1. Start invasion. 2–4. Change half medium in each well. 5. Stop observation at 163 h. Red arrow: significant time point (approximately 72–74 h) of impedance change in the FGF-9 group, when epithelial invagination starts to result in a thinner layer of bioengineered organ.

Figure 5

Real-time PCR measurement of Ameloblastin and Amelogenin gene expression. (A) Real-time PCR Ameloblastin expression at different time points in the bioengineered organ culture. Ameloblastin expression peaked at 60 h. Ameloblastin expression was significantly higher at 72, 84 and 150 h in the FGF-9 group compared with the control group. (B) Real-time PCR Amelogenin expression at different time points of the bioengineered organ culture. Amelogenin expression was significantly higher in the FGF-9 group compared with the control group at 1 h and peaked at 72 h, coinciding with a significant change in ECIS assay (Figure 4B) and due to epithelial invagination. These two graphs show that the peak Ameloblastin expression at 60 h is followed by a peak Amelogenin expression at 72 h, in agreement with previous research [21]. Significantly higher Amelogenin expression also coincides with early lower impedance data (Figure 4B) and earlier differentiation of tooth germ evident in early organ culture (Additional file 2). These results indicate that FGF-9 can initiate early phenotypic and morphological characteristics of ameloblasts within the epithelium, which coincides with epithelial invagination evident from the ECIS assay (Figure 4B). After invagination, the FGF-9 group still exhibits higher Ameloblastin and Amelogenin expressions than the control group. Because of the clear differences, no mark is necessary to specify the results of the FGF-9 group.

Figure 6

Proposed mechanism of FGF-BMP balancing system. In this study, FGF-9 upregulates Ameloblastin and Amelogenin, and speeds up epithelial invagination. No direct relationship between Sonic hedgehog (Shh), Jagged (Jag2) and Ameloblastin has been elucidated until now. Other genes (such as Paired box 9) were found to support antagonistic interactions between FGFs and BMPs [32] but are not shown in this graph. We propose an ‘FGF-BMP balancing system’ which manipulates the morphogenesis of ectodermal organs. “→” is the upregulate pathway proved by previous researches and our study. “⊥” is the downregulated pathway proved by previous research. “?” is the suggested pathway need to be investigated further.