Tooth development is a complex, regulated series of reciprocal communication between the epithelium and mesenchyme, which eventually differentiate into ameloblasts and odontoblasts, respectively, to form hard- tissue [2]. To reproduce cellular interaction, stem cells have been used for in vitro co-culture systems [8] or bioengineering for tooth regeneration [77].
However, the invasiveness of stem cells has made the transition to bedside difficult, as well as other issues including the expenses and maintenance of cell culture. CM, on the contrary, are proven to have regenerative effects and are easily obtained from cell cultures.
CM has been established from various cell types and culture conditions, and have been utilized in the induction of cell differentiation in vitro [8, 78]
and in vivo [11, 79, 80]. It is said to contain soluble factors [81-83] that create an odontogenic microenvironment favorable for cell differentiation [13].
However, the effect of cytokines and other paracrine factors on the regulation of cell function is complex, and thus, the results are not always dose- dependent. For instance, one study found that the concentration of CM supplements did not correlate with cell proliferation [84]. Others found that varying concentrations of CM had no effect on the cell behavior [40, 85].
Interestingly, the CM in our study had concentration-dependent enhancing effects on the odontogenic capacity of SHED. This could be due to our method of concentrating the CM by lyophilization. After collecting and
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freezing the CM from HERS/ERM cell line, the frozen CM was freeze-dried, and the resultant powder was dissolved in a known volume of distilled water to concentrate the CM by a factor of 16. The rationale behind this was to increase the concentration of CM supplement in differentiation medium without constituting a larger proportion of the total volume. Thus, I was able to add 40% and 80% of CM (v/v) to the differentiation medium by adding only 25 and 50 μl of CM, respectively, per milliliter of odontogenic induction medium.
In vitro differentiation of SHED with the supplementation of freeze-dried CM was examined by mineralization-related gene expression levels, namely BSP, DMP1, MEPE, RUNX2, ON, OC, and as well as the expression of DPP proteins. Previous studies state that BSP is expressed in mature osteoblasts [86, 87], and its appearance correlates to calcified nodule formation [88]. On both day 8 and 12, BSP was significantly increased in higher concentrations of CM (Figure 3), and Alizarin red S staining exhibited increased formation of mineralized nodules in differentiated SHED with 4X and 8X freeze-dried CM supplements on day 12 (Figure 4B). Similar to BSP, DMP1 is also associated with the start of mineralization [89]. As expected, the mRNA expression of DMP1 was enhanced significantly in 4X freeze-dried CM treatment; but data was not significant in 8X freeze-dried CM treatment (Figure 3A). This may be due to inconsistent effects of 8X freeze-dried CM on SHED differentiation, as observed by the slight variations between replicates in Alizarin red S staining on day 12 and 20 (Figure 4A). Also,
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MEPE is reported to be present in mineralizing tissues as its cleaved form, acidic-serine-aspartate-rich-MEPE-associated motif [90, 91]. Unlike BSP, DMP1, and MEPE, the expression of RUNX2, ON, and OC was significantly downregulated in CM treatments on day 12, which is consistent with the claim that RUNX2 expression declines in more mature odontoblasts [92], while ON and OC are inversely correlated with calcium deposition [93, 94].
Lastly, DPP proteins that are closely related to odontogenesis and mineralization in vitro [95] exhibited thicker bands in differentiated SHED compared to the non-induction control on immunoblot, and bands at higher sizes around 131 kDa seemed to disappear in correspondence to the appearance and the thickness of lower DPP bands at 97 kDa [96]. Because the monoclonal DSPP (LFMb-21) antibody binds to the DPP domain CSRGDASYNSDESKDNG, both cleaved DPP and the remaining uncleaved DSPP form should be detected on Western blot. However, there is still no accepted explanation for the multiple bands detected by DSPP (LFMb-21) antibody. Additionally, DPP bands were expressed highly in all differentiated SHED on day 20 (Figure 5). This result was also consistent with Alizarin red staining, which displayed significant levels of calcification nodules on day 20 (Figure 4A).
To confirm that the increased odontogenic effect on SHED was due to the conditioning of medium by epithelial cells for 48 hours, the unconditioned basal medium was added to the induction medium as a control variable. The basal medium was freeze-dried and used at 4X and 8X concentrations to
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compare directly with the freeze-dried CM treatments. As a result, the lack of significant differences in relative gene levels, mineralized nodule deposits, and Western blot in the basal media controls verified that the enhancement of odontogenic capacity was due to the HERS/ERM cell line conditioning of the basal medium, and not its original components.
However, it appeared that long-term treatment of freeze-dried CM resulted in cell death, and the nuclei appeared to disintegrate, as determined by DAPI staining (Figure 6B) and the decreasing β-actin expression in Western blotting (Figure 5B). This could be due to the accumulation of freeze- dried CM toxicity in long-term cell cultures, which was also observed at higher concentrations of freeze-dried CM according to the CCK-8 assay. Cell growth and viability were not greatly affected up to 8X concentration but resulted in immediate cell death at 16X and 32X concentrations on the first day of absorbance measurement (Figure 2). However, freeze-dried basal media did not result in cell death on day 18, indicating that concentrated levels of basal media components were not the cause of cellular toxicity. Thus, it could also be explained by the nature of the cytodifferentiation stage in tooth development. At terminal differentiation of odontoblasts, the cell cycle stops, and the cells elongate, polarize and secrete a dentin matrix [5]. Smooth, oval shapes of the nuclei were maintained until day 14 in 4X freeze-dried CM treatment and day 16 in 8X freeze-dried CM treatment (data not shown).
Therefore, cell death naturally occurs as cells are terminally differentiated by CM, and without further cell proliferation due to confluence, only the secreted
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pre-dentin matrix remains as asserted by the strong expression of DPP proteins even when beta-actin was reduced.
Our results suggest that the odontogenic differentiation effects of CM derived from HERS/ERM cell line on SHED were concentration-dependent up to 8X concentration factor in vitro. In-depth studies are needed to reveal the specific composition of the CM and its involvement in the odontogenic differentiation mechanism of mesenchymal cells.
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