The present study provides a wide characterization of the in vitro osteogenic properties of two commercial surfaces, TiOblast and OsseoSpeed. TiOblast was the first moderately roughened implant surface with 10 years followup reported in the literature and the precursor of the OsseoSpeed surface [18
]. The OsseoSpeed surface is a further development introduced in 2004 that incorporates small amounts of fluoride ions in the oxide layer, a slight increase on the micrometer scale in surface roughness, and the appearance of a nanoscale topography. In the present work, we showed the topographical differences between the two surfaces, mainly an increase in the Sa
value in the OsseoSpeed surface, but also changes in surface skewness, kurtosis, and the core fluid retention index. The obtained results confirm that OsseoSpeed surface show an increased micrometer-scale surface roughness, together with the formation of nanostructures, as reported in earlier studies [11
]. The presence of micro- and nanoscale topography in OsseoSpeed compared to TiOblast surface and the addition of fluoride, did not change the biocompatibility of the implants and the initial attachment and proliferation of the MC3T3-E1 cells. However, it was observed that OsseoSpeed surfaces induced a more branched cell morphology. It has been reported that this cell shape may increase the contractility of the cytoskeleton and lead to preferential osteoblastic differentiation [21
], which has been found in the present study for the OsseoSpeed surface compared to TiOblast. The increase found in the LDH activity on the TCP surface after 14 days is most probably due to the higher proliferation and/or cellular activity at this later time point on TCP.
MC3T3-E1 osteoblast-like cells undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture [22
]. Osteoblast maturation in vitro is characterised by changes in gene expression at each developmental stage [23
]. Modulation of these expressed genes is subjected to a transcriptional control regulated by growth factors and cytokines [23
]. BMP-2 is a highly potent growth/differentiation factor that induces differentiation of progenitor cells into the osteoblast lineage, and exhibits this osteogenic action by activating Smad signaling and by regulating transcription of osteogenic genes. Thus, the higher release of BMP-2 found in the OsseoSpeed group could initiate osteogenic differentiation through the regulation of transcriptional factors.
Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic gene expression that is necessary for the osteoblast lineage commitment and, as well, regulates osteoblast differentiation [27
]. Here, we did not determine Runx2 mRNA levels since, as we have previously reported that Runx2 mRNA expression is constant during osteoblast differentiation [28
], probably due to the fact that MC3T3-E1 cells are already committed to the osteoblast lineage. Nevertheless, we have analysed the expression of different transcription factors that have been described to interact with Runx2: Dlx2, a downstream target of BMP-2 that is thought to directly activate Runx2 and Osterix genes [29
], and Hes1 that can stimulate the transactivating function of Runx2 [30
], although it negatively regulates bone phenotypic maturation and its expression decreases during osteoblast differentiation [31
]. We have previously reported that Hes1 and Dlx2 are early responsive genes to roughness and fluoride treatment of titanium implants [32
]. In our previous report, both genes were downregulated by fluoride treatment of rough titanium implants after one day in primary human osteoblasts. Here, no differential regulation was found for these two genes among the two surfaces analysed, neither when compared to TCP. The difference between the results may have been caused by differences in the surfaces used for comparison and differences in the cell model.
The Smad family of proteins has been identified as the downstream propagators of BMP signals [33
]. BMP-activated Smads induce Runx2 gene expression and Smads interact physically with the Runx2 protein to induce osteoblast differentiation [34
]. In particular, Smad1 and Smad5 are necessary for BMP-mediated Runx2 acetylation [35
]. We found no important changes on Smad1 and Smad5 at the different time points and groups analysed; only Smad1 showed higher significant levels in OsseoSpeed compared to TCP. Thus, although Smad expression patterns are informative, future studies should investigate their phosphorylation stage to find out whether their activity is regulated in the different surfaces.
Osterix is another transcription factor downstream of Runx2 which is required for the ongoing differentiation within the osteogenic pathway [36
], being involved in the differentiation step from preosteoblast to fully functional osteoblast [37
]. Here we found higher Osx mRNA levels in cells cultured onto OsseoSpeed implants compared to TiOblast, in agreement with earlier observations [8
Other osteogenic markers were analysed during osteoblast differentiation. Type I collagen is expressed in high levels in the early proliferation stage, which is gradually decreased as the cell matures. This downregulation was only observed in the TCP group, while TiOblast and OsseoSpeed showed higher coll-1 mRNA levels. In agreement with these results, Masaki et al. [10
] also found higher mRNA levels of coll-I in human palatal mesenchymal stem cells cultured on TiOblast and OsseoSpeed than on TCP after 3 days, although these differences were not significant. Alkaline phosphatase increases during extracellular matrix maturation then decreases when mineralization is well progressed and bone sialoprotein is transiently expressed very early and then upregulated again in differentiated osteoblasts at the onset of mineralization [23
]. These two markers, that reflect a more advanced stage of osteoblast differentiation, showed higher mRNA levels on TCP up to the first week, while OsseoSpeed surfaces increased their levels greatly after 14 days, indicating that extracellular matrix of cells seeded on TCP was mature and competent for mineralization after 1 week and on OsseoSpeed surfaces after 2 weeks. This also indicates that differentiation of MC3T3-E1 cells on TiOblast was delayed compared to OsseoSpeed surface. These results are in line with those obtained in the present study for the higher ALP activity and number of crystals deposited in the cell monolayer of OsseoSpeed surface. Using the same in vitro model, another study [8
] did not observe significant differences in BSP between the two titanium surfaces, although in this case the roughness was similar, and the titanium particles for grit-blastingwas smaller. Finally, CD44 was analysed as this marker has been indicated to be expressed in higher levels in osteocytes [38
]. However, the analysis did not reveal important differences between the two titanium surfaces investigated, only for the TCP group.
Besides these bone-specific markers, the effect of the different titanium implant surfaces on the expression of different growth factors and cytokines involved in bone formation was analysed. IL-6 is a cytokine produced by cells of the osteoblast and osteoclast lineages that not only has a role in inflammation but also increases bone resorption and possibly bone remodeling [39
]. Both the mRNA levels and the secretion of IL-6 decreased over the time in cell culture, but in a lesser extent for TiOblast surface. IGF-I induces osteoblast proliferation, bone collagen, and matrix synthesis [40
] and stimulates the activity of alkaline phosphatase [42
]. Similar to other osteogenic markers commented before, this growth factor was significantly upregulated in OsseoSpeed surface compared to both TCP and TiOblast. Similar results have been found in a previous study in vivo [16
], suggesting that IGF-I might play an important role stimulating bone formation when administered in combination with fluoride [43
Osteoblasts exert a crucial function in osteoclast activation and differentiation, through the production of specific biological mediators such as the activator of nuclear factor k B ligand (RANKL) and its antagonist osteoprotegerin (OPG). In the present work, the total amount of RANKL produced by MC3T3-E1 osteoblasts was below the detection limit of the Luminex assay (3
pg/mL), while OPG was detected in the supernatant during the whole culture period, was similar to the reported results by Guida and coworkers using ELISA [44
]. However, OPG production found in this study was similar between TiOblast and OsseoSpeed group in MC3T3-E1 osteoblasts, with higher levels on titanium when compared to TCP. As a trend, TiOblast surfaces showed higher levels than OsseoSpeed, opposite to the results obtained in human bone marrow mesenchymal stem cells [44
]. Due to the lack of differences in OPG/RANKL mRNA levels and the secretion of OPG, we conclude that in MC3T3-E1 cells, the levels of OPG/RANKL are not regulated by the different surfaces used in the study. Other authors have reported increased OPG levels in response to rough surfaces [45
] and different chemical composition [46
In conclusion, the results from the present study demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation. The enhanced in vitro osteogenic properties are in line with the improved osseointegrating properties and clinical performance of fluoride-modified titanium implants [47