Effects of Estradiol on the Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells 4

Discussion

 

Dental tissue possesses several functional, developmental similarities to bone. For example, the maintenance of hard tissue in the tooth and bone depends upon the stimulation of morphologically and functionally related cells indentified as odontoblasts and osteoblasts. Studies have demonstrated that osteoblast activity that enables hard tissue to form is promoted by gonadal steroid hormones such as androgens and estrogens. Steroid hormone activity within bone is facilitated by locally produced cytokines, including hepatocyte growth factor (HGF) (Fuller et al, 1995; Grano et al., 1996; Spelsberg et al., 1999). By comparison, odontoblasts mediate hard-tissue (dentin) formation in teeth: however, the steroid hormone- and cytokine-dependence of this phenomenon has yet to be established in odontoblast (Dale et al., 2002).

Although hormonal regulation of human odontoblast function has not been directly shown in vivo, steroid hormone do affect dental health, as evidenced by compromised dentition resulting from the menopausal-associated loss in ovarian estradiol 17-beta production and altered patterns of dentin apposition in teeth from ovariectomized rats (Hietaia and Larmas, 1992; Krall et al., 1997). Also, the apparent androgen-dependence of tooth morphogenesis during human development was reported (Molsted et al., 1997). There are possibilities that steroid hormones and cytokines support tooth integrity by modulating odontoblast activity.

 Estrogen, a multifunctional sex steroid, effectively regulates the stemness characteristics of adult and embryonic stem cells (Balston et al., 2010). Its powerful capacity to regulate stem cells and bone marrow mesenchymal stem cells (MSC) proliferation and differentiation has been recently described in many reports (Cha et al., 2008; Asselin-Labat et al., 2010). Also, estrogen substantially participates in the regulation of bone metabolism by inhibiting bone resorption and increasing bone formation. Estrogen exerts an osteogenic function in bone formation via release or upregulation of a number of cytokine (interlukin-1 and 6), prostaglandin, and osteogenic growth factor (bone morphogenic proteins, transforming growth factor-beta1, and insulin-like growth factor) (Yamamoto et al., 2002). These cytokine, hormones, and growth factors further promote proliferation and differentiation of osteo-progenitor cells and MSCs through autocrine or paracrine mechanisms (Cha et al., 2008). However, effects of estradiol on proliferation or differentiation in HDPCs were uncertain until now.  

Dental pulp stem cells as clonogenic cells are capable of both self-renewal and multi-lineage differentiation. The most notable characteristic of HDPCs is to regenerate dentin-pulp-like complexes. These cells can be induced into odontogenic differentiation under appropriate culture conditions. The typical odontoblastic differentiation inductive medium is a combination of glycerophosphate and ascorbic acid.

In the present study, the effect of estradiol on proliferation in HDPCs was examined in non differentiative induction medium. The result showed that estradiol increased cell viability and number of BrdU positive cells, and enhanced PCNA mRNA expression, indicating that estradiol stimulates proliferation of HDPCs because specific markers, containing PCNA and BrdU have been used to analyse cell proliferation during odontogenesis in animals and humans (Matulova et al., 2002). These results are similar to data shown in stimulated PDL cell proliferation by estrogen (Mamalis et al., 2010). However, there are some experiments have proven the decreased proliferation of osteoblast-like immortalized calvatial cell lines, trabecular bone cells, rat calvaria osteoblast-like cells and ROS 17/2.8 cell in response to estrogen (Gray et al., 1989) and other evidence showed no influence of estrogen on the proliferation of osteoblasts (Liel et al., 1992). To date, the differences observed between these cell lines in response to estrogen remain unclear. It is possible that these divergent results could be ascribed to variability in the number of estrogen receptors, stage of cell differentiation or production of growth factors by various cell lines.

Estrogen receptors (ERs) belong to the family of the ligand-activated transcription factors that regulate the growth, differentiation, and development of cells (Evans et al., 1988) and human tooth pulp contains steroid hormone receptors, and pulp tissue would therefore be steroid-responsive. In the present study, estradiol-treated HDPCs in non-differentiative induction medium increased both ER-α and ER-β mRNA expression, demonstrating the possibility that the effects of the estrogen on HDPCs proliferation are mediated by their receptors, ER-α and ER-β although previous study reported that estrogen regulates proliferation in HPDLCs (human periodontal ligament cells) through estrogen receptor-β, but not ER-α (Mamalis et al., 2011). Precise correlation of ER in estradiol-induced proliferation of HDPCs needs further study.

In the differentiation inductive condition, β-glycerophosphate clearly serves as a phosphate donor that is necessary to build up the mineral phase and the importance of ascorbic acid in the maintenance of normal extracellular matrix has been known for many years (Pradel et al., 2008). Interestingly, in the present study, cell viability was decreased in estradiol-treated HDPCs in differentiation inductive condition. It is speculated that proliferation rates are attenuated during differentiation process induced by estradiol in HDPCs. In addition, expression of ER-β was decreased, but not ER-α in estradiol-treated HDPCs under the differentiation inductive condition. These results suggest that ERs play crucial roles via different regulating mechanism between proliferation and differentiation in HDPCs.

There are three osteoblastic developmental phases (Owen et al., 1991) : an initial proliferative phase characterized by higher type Ι collagen expression, followed by a matrix production and maturation phase in which ALP is increased, and finally, a third phase of initiation of mineralization marked by levels of OPN (Stein and Lian, 1993 ; Siggelkow et al., 1999). In odontoblast differentiation, the secretion and mineralization of dentin matrix proteins, such as the most abundant noncollagenous DMP and DSPP, are controlled by odontoblast. As DMP-1 is a noncollagenous protein present in both teeth and bone, but with higher expression levels in the latter (D’Souza et al., 1997), mature osteoblasts are embedded into the bone matrix and finally became osteocytes, which express DMP-1.

The present study investigated the effect of estradiol on odonto/osteoblastic differentiation of HDPCs. BSP, ALP, DMP-1 and DSPP, which are referred as odonto/osteoblastic differentiation markers (Fisher et al., 2003), were up-regulated in estradiol-treated HDPCs. Also, ALPase activity considered as early markers of odontoblast differentiation (Lee et al., 2006), increased in estradiol-treated HDPCs. Additionally, estradiol increased protein levels of DMP-1 and DSPP expression a dose-dependent manner. Also, The formation of mineral nodules, which was an index for odonto/osteoblastic differentiation was revealed (Yu et al., 2007). In the present study, Alizarin red S staining for calcium deposition showed a significant increase in mineralization with estradiol treatment. These results demonstrated that estradiol stimulate an early stage of odonto/osteoblast differentiation during a short period, and also enhanced mineralization which may represent during long terms osteocenic properties. Recent studies have found that MAPK signaling is involved in dental pulp cell differentiation and tooth development process (Lee et al., 2009; Simson et al., 2009). Other study reported that estradiol-treated endometrial carcinoma cells activated ERK1/2 pathway (Lili et al., 2009). However, whether estradiol could affect the MAPK signaling pathway of HDPCs have not been assessed. This study investigated activation of MAPK signaling molecules in estradiol-treated HDPCs under the differentiation inductive condition. Here, phosphorylated ERK by estradiol treatment increased in HDPCs, indicating that estradiol activated ERK signaling pathway to induce odontoblastic differentiation.

Taken together, estradiol enhanced proliferation and promoted odonto/osteoblastic differentiation of HDPCs, as evidenced by up-regulation of odonto/osteoblastic markers, enhanced ALP activity and accelerated mineralized nudules formation. Thus, estradiol is an important factor in HDPCs, with clinical implications for pulp injuries or the regeneration of human dental tissues for tissue engineering.