This study demonstrated in vivo and in vitro beneficial biological effects of the implant surface texturing at the micro- and nano-scales and functionalized with strontium element. This metallic element in ionic form has been known to play a role in regulating bone metabolism as a dual-action agent, stimulating both pre-osteoblast and osteoblast differentiation and inhibiting osteoclast differentiation and activation [13,16,22,23]. This regulation of bone remodeling, by promoting bone formation while simultaneously inhibiting bone resorption, is widely recognized as effective, even in the context of peri-implant regeneration [13-15,24]. Recent systematic reviews have shown that local administration of strontium from functionalized implant surfaces allows for higher localized dosing, avoiding systemic side effects, and improving implant osseointegration [25-27].
In general, the present study demonstrated that implants with treated surface exhibit osteogenic properties, due to the micro- and nano-scale structures and the biological effects of Sr, observed by the biomechanical, histomorphometric, and gene expression results, which are consistent with numerous other evaluations [2,10,12,17,18,20,28-33]. In this context, to assess the bond strength between bone and threaded dental implants, the removal torque is one of the most appropriate approaches for obtaining information about the bone-implant interface, as it reflects the anchoring capacity of the implant34. In our study model, removal torque values demonstrated superior effects on bone formation when compared to machined implants, implying enhanced early-stage osseointegration of the treated implants. It is noteworthy that a significant (p<0.05) increase in removal torque values within these groups was observed for the two observation periods, while the same was not found for the control group. Furthermore, at 45 days, all the treated groups showed a significant increase in relation to the control group, demonstrating the beneficial effects of micro-nanoscale topographies in longer-term osseointegration. A systematic review and meta-analysis work [27] examined the impact of strontium incorporation into titanium implants on bone apposition when implanted in animal models. Among the included studies, nine reported biomechanical test results such as pull-out tests or removal torque tests. In all these studies, a significant increase in implant fixation was observed, with substantially higher removal torque values, maximum push-out force, and/or shear strength when compared to control groups.
It is worth noting that the values of BIC and BAFO in cortical and cancellous bone were investigated separately in this study. This separation was undertaken due to their differences in vascularity, density, and mechanical properties [29]. In this present study, histomorphometric analysis revealed superior results for BIC% in the cortical threads of the functionalized surfaces at the 15 and 45-day time points, with statistical significance differences observed for MN and MNSr groups (Figure 6 and Annex 1 – Tables 2 and 3). This finding is of great significance, as BIC% is widely recognized as the gold standard for assessing bone formation around metallic implants. It is well-established that contact osteogenesis plays a pivotal role in the success of implant therapy [1,6]. These results may be interpreted as an acceleration in the bone healing process, which can be particularly beneficial in complex clinical scenarios [3]. Experimental implants containing Sr+2 produced by hydrothermal treatment also showed significantly higher BIC percentages in rabbit tibias and femurs compared to control implants [11,30]. Despite the BAFO results in our study (Figure 6 and Annex 1 – Table 4 and 5) demonstrating an increase between the two observation periods in the cortical bone area, yielded comparable results among the groups after both time intervals, both in the cortical and cancellous bone areas. These findings support the hypothesis of a localized effect measurable near the surface due to the direct release of strontium at the implant-tissue interface, as no significant differences were observed at a considerable distance from the surface in the period investigated. Similarly, other studies have found analogous findings in the assessment of late-stage osseointegration of Sr-functionalized surfaces in healthy animal models, at 6 weeks [11] and 12 weeks [10]. In these reports, no statistical differences were observed in terms of bone area, while an increase in BIC was evidenced for the functionalized surfaces compared to controls. An increased proportion of bone area (%) in the histomorphological analysis at 4 weeks days was also reported in another study when comparing rough titanium surfaces with hydrothermal incorporation of strontium on the surface of SLA vs. Sr-SLA surfaces [12]. Our findings demonstrated an increased BIC% value soon after two weeks of implantation, demonstrating the importance of surface treatment micro and nano textured to accelerating the osseointegration of implants. Reaching levels of BIC in 15 days comparable to 45 days of implantation, when the osseointegration is practically ended.
Shi et al. (2017) [27] in their review also examined studies that utilized μ-CT in quantitative analyses. Among the included studies, four revealed that strontium-enhanced implants exhibited a more substantial impact on all μ-CT parameters, such as bone volume to total volume (BV/TV), three-dimensional bone distribution, including trabecular number (Tb.N), thickness (Tb.Th), and/or spacing (Tb.Sp), and/or connectivity density (Conn.D), when compared to control implants. Meanwhile, three other studies did not identify statistically significant differences in some of these parameters [27]. In our study, comparable results between the groups were also observed between the two time periods for most of the analyzed parameters, although a statistically significant decrease in Tb.N in the cortical and cancellous bone area of the MNSr group and in the cortical area of the MNSr+ group between the two evaluation periods was observed. This can be justified by an increase in bone formation and trabecular fusion, rendering the bone denser and with a reduced number of trabeculae detected by μ-CT.
The μ-CT results generally exhibited similar trends to the histomorphometric analysis, although statistical significance was not reached. It is worth noting, however, that information loss during sectioning and histological preparation procedures can lead to uncertain outcomes, as previously described [23,35,36]. Additionally, μ-CT analysis has disadvantages such as metallic artifacts caused by a combination of beam hardening, scatter, and non-linear partial volume effect. Therefore, while it is a desirable method for measuring bone parameters, special care is required when assessing osseointegration due to various limitations [35,36]. Differences between μ-CT and histology may be particularly observed due to the challenge of metal-induced artifacts with μ-CT systems or partly because of the substantial intra-group variation (up to 35%) among histological sections [37], which was not observed in the present study. Furthermore, the concurrent evaluation of removal torque is opportune to assess a potential correlation between increased bone apposition in the implant vicinity and potential improvement in mechanical stability. Regarding the promotion of the osseous response by functionalized surfaces, for our treated and doped-strontium surfaces, the results of BIC% and removal torque, when considered together, may suggest an acceleration of osseointegration.
Another distinctive feature of our study was the in-depth assessment of osteogenesis-related bone markers using the PCR Array technique. Eighty-four markers associated with this pathway were evaluated for the 15-day period to elucidate the transcriptional events in the early phases of osseointegration (Figure 7 and Annex 2). Initially, the upregulation of certain genes in the Sr-doped groups draws attention. In the MNSr+ group, eight genes were upregulated, while in the MNSr group, five genes exhibited upregulation.
Among the upregulated genes, matrix metalloproteinase 8 (Mmp-8) stands out as it was overexpressed in all three treatment groups. Also known as collagenase-2, it is an enzyme that plays a role in extracellular matrix remodeling and the body's inflammatory response. MMP-8 substrates include collagen I, II, III, and IV, which are among the important proteins in the soft tissue surrounding implants [38]. In addition, it is known that MMPs can modulate bone resorption through the activation and differentiation of osteoclasts, in addition to the direct degradation of bone collagen matrix. Therefore, even though MMP-8 is not typically directly associated with bone formation, it may play an important role in the regulation of the bone extracellular matrix, as it is involved in the degradation and remodeling of collagen during physiological processes such as wound healing and bone remodeling [38-41]. Interestingly, collagen markers were also upregulated in the treated groups, such as Collagen type X alpha 1 (Col10a1) and Collagen type XIV alpha 1 (Col14a1); Collagen type III alpha 1 (Col3a1) and Collagen type VI alpha 1 (Col6a1), suggesting increased bone remodeling activity in the treated groups, especially in the MNSr+ group (Figure 8).
Itgam (Integrin alpha M), a gene related to cell adhesion molecules and cell-matrix adhesion [42], was upregulated in the MNSr+ group (3.62-fold), although other genes related to cell adhesion showed comparable results between the groups. Fibronectin 1 (Fn1) also had its expression increased in this group (2.32-fold). This gene plays a significant role in various biological processes, including cell migration, cell adhesion, and cytoskeletal organization. Previous reports have indicated that osteoblasts produce FN-1 during the stages of proliferation and differentiation, concomitant with the synthesis of collagen type I, suggesting that osteoblasts generate FN-1 during active bone formation processes [43,44].
Many down-expressed genes were observed in the treatment groups compared to the polished control, and these genes are associated with various functions, including cell adhesion and growth differential, skeletal development, bone mineralization, ossification, and the production of extracellular matrix proteins. Our assumption is 15-day time point can be relatively late when attempting to assess differences in gene expression during the initial phase of cell differentiation and bone formation around the implant. Therefore, the negative regulation of osteogenic markers in the treated groups can indicate the biological events related to osteogenesis were anticipated since many osseointegration events have already occurred in the first week post-implantation at the surgical site. Thus, the 15-day period may be untimely to capture the peak of positive regulation of early adhesion and osteogenesis markers, which may have been slower on the machined surface. For example, the secretion of growth factors and platelet absorption at the site, the adhesion of undifferentiated osteoblasts to the implant surface aided by fibronectin, migration of pluripotent mesenchymal cells along the implant surface, local ischemia and subsequent necrosis due to neutrophil dominance, and later by macrophages, activation of Runx2 and Op, necrotic bone resorption, the onset of some bone-implant interface and new bone formation and mineralization, as well as matrix remodeling, are all events occurring fast in the first week after implantation [45].
Therefore, investigations conducted at shorter time intervals could have a greater capacity to highlight distinctions between the surfaces subjected to treatment. In this context, a supplementary study was conducted in an in vitro environment using discs that underwent the same surface treatments for a period of seven days, both in terms of gene expression and protein expression of osteogenic markers. BMP-2 is one of the key cytokines involved in regulating the differentiation and maturation of precursor cells into osteoblasts [46], and its expression was increased in all treated surface groups (p < 0.05) (Figure 9a). Similarly, an elevation in the protein expression of this marker was also observed through ELISA assays on micro-nanotextured surfaces, although a statistically significant increase was only observed for the MNSr+ group (Figure 9b). Interestingly, bmp-2 was upregulated at 15 days in the in vivo study for the MN group, corroborating our hypothesis that downregulated genes observed in the PCR surpassed the pronounced expression due to the advancement of effects induced by the surface treatments on the implant surfaces. Also positive regulations of bmp-2 on Sr-loaded surfaces were also observed in other studies [47,48].
Similar to Bmp-2, Spp1, which is involved in the synthesis and regulation of osteopontin, had its expression increased in the Sr groups, with a prominent increase compared to both, grinded surface topography with aligned mark and to standard micro-nano surface. In line with this, protein quantification also demonstrated an increase in the Sr-treated groups (Figure 9b). This protein plays an important role in the regulation of the extracellular matrix, bone mineralization, the immune response, and various biological processes comprising the osseintegration [49]. Geng et al. (2022) [48] also obtained similar results and observed that Sr-loaded surface promoted an increase in osteopontin expression in vitro at four and seven days. Ibsp, which encodes bone sialoprotein-binding integrin, a multifunctional extracellular matrix protein present in bone, cementum, and dentin [50], followed the same pattern and was overexpressed in the MN and MNSr+ groups, while MNSr group was similar to control.
Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein (BGLAP), is the most abundant non-collagenous protein found in bone and is commonly used as a biomarker of bone turnover [49,51]. This gene also had its expression increased in the Sr-doped groups, especially in the MNSr+ group, which showed superiority in expression compared to both the control (p=0.0317) and the standard micro-nano texutured substrate (p<0.0034), highlighting the positive aspects of Sr+2 ion release in modulating non-collagenous proteins, suggesting gains in the biological and mechanical functions of bone tissue. On the other hand, it was downregulated at 15 days in the animal study in the MNSr group, reinforcing our observations. The incorporation of Sr through hydrothermal treatment also increased osteocalcin expression assessed with in vitro models, researching osteoporotic mesenchymal stem cells from bone marrow after the 4th and 7th day of culture [48], and human osteoblast-like cells (MG63) after 14 days30. Similarly, collagen type I, the primary protein of the organic matrix (80-90%) [51], despite no statistical significance presented tendency for expression in the micro-nano surface group at 7 days. In the in vivo results at 15 days, it was already upregulated compared to the control (-2.42 FC; Figure 8d). In the MNSr group, the expression was reduced compared to the other groups, and at 15 days in the in vivo study, it was similar among the groups. The Alpl gene, which encodes the alkaline phosphatase enzyme, important in the process of bone tissue mineralization, also exhibited increased expression. Another report, using the same cell type, also observed an increase in Alpl expression on Sr-functionalized surface at 7 days of culture [52]. A study conducted by Parker et al. (2010) [30], using MG63 cells on a Sr-containing oxide layer, the authors observed that alkaline phosphatase (ALP) activity was significantly increased within one week. However, in Shimuzi et al.'s study (2020) [52], despite an average ALP activity higher compared to non-Sr-treated group, there was no statistically significant difference. In summary, these results are consistent with similar literature that have demonstrated the stimulating effect of Sr on osteoblastic cell behavior [16,53]. The potential for improved bone formation by incorporating Sr into biomaterials has also been well-documented in in vitro and in vivo studies [23,54-57]. However, some limitations should be noted in this study. For example, in vitro assays using only one cell type, as utilized in our study, have the potential to evaluate individual biological processes in a controlled manner but cannot fully replicate the complex in vivo environments, especially when investigating the effects of multipotent drugs like Sr+2 [52]. So the animal models are complex studies with multiple variants and provide important highlights found in vitro, despite the limitations imposed by the site of implantation (long bone instead of maxillary) and euthanasia periods, intrinsec for surgery protocols.