Despite the large amount of literature on osteoblasts, there has been little assessment of whether these bone forming cells exhibit specific differences related to the sex of the animals from which they were derived. This study examined whether osteoblasts isolated from male and female rats exhibit sexual dimorphism when grown under standard cell culture conditions, as well as when grown on substrates used as implants in clinical practice. In addition, the study addressed the question of whether or not osteoblasts are sensitive to modifications in substrate microarchitecture and if they respond to osteotropic hormones in a sex-specific manner. This study shows that, under control conditions whether on TCPS or smooth or microstructured Ti, there are no apparent differences between cells taken from male or female donors. However, osteoblasts exhibit differences in their responses to oestrogen and in the robustness of their responses to 1α,25(OH)2D3 that are sex-dependent.
The present study is limited because it was conducted in vitro and used rat cells. Thus, the data cannot be correlated directly to human cells or to in vivo responses in humans. Here, all of the rats were of the same strain while, in contrast, the human population is outbred and, therefore, inter-human variability may be greater than the differences between the sexes. The goal of this study was to compare the response of male and female osteoblasts grown on implant surfaces. In our population, we have a mixture of osteoprogenitor cells and immature osteoblasts cultured from explants of frontal and parietal bones. Cells with a similar phenotype will migrate to the implant surface in vivo, allowing us to mimic the conditions crucial to osseointegration. In order to further this system, we cultured cells only in DMEM and serum and did not include any other factors traditionally used - such as dexamethasone, β-glycerophosphate or ascorbic acid. In order to minimize the potential differences due to cell numbers from individual bone samples, cells were isolated from bones in the same location of the skull, grown in the same media, seeded at the same density and treated at confluence. Thus, in this study the baseline differences between males and females are underscored. We used sexually mature rats for these experiments, so differences in exposure to sex steroids in vivo may have affected the subpopulations of osteoblasts isolated from the donor bone.
Even with these limitations, however, the results indicate that there are physiological differences in male and female osteoblasts and sexual dimorphism in the response to bone regulating hormones. This difference is more pronounced when cells are cultured on substrates other than tissue culture polystyrene. Currently, most preclinical studies of therapeutic interventions are performed in only male or only female animal models. Further investigations on sex specific differences should be performed in order to improve our understanding of cellular responses to biomaterials.
Both female and male cells exhibited a more mature phenotype when grown on SLA than when grown on TCPS or PT. This was indicated by the higher levels of osteocalcin and OPG in their conditioned media. Although we had previously shown that male cells and female cells would exhibit a more differentiated phenotype when grown on SLA in comparison to PT [9
], we had not directly compared cells from the same species cultured under near identical conditions. The present results remove this ambiguity.
In contrast, the responses of cells cultured on the microstructured surfaces to 1α,25(OH)2
were sex-specific. Male cells were more sensitive to 1α,25(OH)2
than female cells, responding at lower concentrations. Moreover, the response of the male cells to the higher concentration of 1α,25(OH)2
was more robust. In addition, only the female osteoblasts exhibited responses to E2
-BSA. This confirms our previous results, which showed that osteoblasts from human females exhibit surface-dependent responses to 17β-oestradiol [19
], but we had not determined if male osteoblasts would also exhibit a response to E2
-BSA. Our studies assessing the effects of E2
-BSA on human articular chondrocytes and rat growth plate chondrocytes demonstrated that male cells lacked a response to 17β-oestradiol and exhibited more robust responses to 1α,25(OH)2
than female chondrocytes [45
], suggesting that these are reflective of general properties of cartilage and bone cells. Another possibility is that the male and female rat osteoblasts were at different states of maturation, since response to these systemic regulators varies with the state of the cell within the osteoblast lineage [38
The mechanisms that mediate these differences are not clear. One possibility is that there are differences in vitamin D receptor (VDR) and oestrogen receptor (ER) expression in female and male cells [30
]. Alternatively, components of the signalling pathways involved may play a role. Both 1α,25(OH)2
and 17β-oestradiol act via classical steroid hormone receptors and via membrane-dependent signalling pathways. We did not specifically assess whether there were differences in the membrane signalling by 1α,25(OH)2
, but our finding that E2
-BSA, which cannot enter the cell due to its size [41
], had no effect on male osteoblasts indicates that this mechanism of oestrogen action is not operative in cells from male donors. Moreover, E2
acted only on female osteoblasts in order to reduce cell number, suggesting that even the traditional ER response was not operational in the male cells. Male rat osteoblasts possess traditional ERs [46
], further supporting the hypothesis that the sex-specific effects noted in the present study were mediated by signalling pathways not functional in male cells.
The results suggest that the level of osteotropic hormones such as 1α,25(OH)2
and 17β-oestradiol is important to the regulation of osteoblasts during implant osseointegration. In cases where the hormone level is reduced, such as in postmenopausal females and in vitamin D deficiency [24
], the success of the implant may be reduced. In the last decade, an oestrogen effect on the male skeleton was established, although not as extensively as in females. During ageing female bone loss is due to an increased rate of bone resorption, whereas male bone loss is the result of less bone formation [47
], indicating that different mechanisms are involved. There is also a sex-related difference in osteocyte lacunar density in human vertebral cancellous bone [48
]. Sexual dimorphism may be attributed to differences in ER isoform expression [49
] or in the number of receptors [46
]. Males with deficiency of ERs or aromatase had defects in skeletal phenotypes [50
], indicating that oestrogen is an important regulator for male cells. Alternatively, the higher aromatase activity present in male cells [51
] may convert oestrogen to testosterone, thereby reducing the effective concentration of the hormone in the male cells. Whether these mechanisms accounted for the lack of a response to oestrogen noted in the present study is not known.