Behavior of Osteoblastic Lineage Cells When in the Presence of Tamoxifen: In Vitro and In Vivo Studies on Osseointegration

Abstract

Background/Objectives: Tamoxifen, a selective estrogen receptor modulator widely used as an adjunct in the treatment of breast cancer, has known effects on bone metabolism, although its impact on osseointegration and cellular responses during early bone healing remains unclear. Understanding these effects is essential given the increasing use of dental implants in cancer survivors. The study aimed to observe the influence of tamoxifen on human osteosarcoma (SAOS-2) cells lines, as well on the osseointegration of titanium implants in ovariectomized female rats. Methods: SAOS-2 cells were incubated with Dulbecco's modified growth medium. Six titanium (Ti) disks were used at each time point. The samples were divided into groups with the presence (TAM, n = 36) or not (CTR, n = 36) of tamoxifen in a concentration of 2 μM. In vivo, 72 animals were divided in groups with bilateral ovariectomy or SHAM and tamoxifen administration or not (15 mg/kg). Cell viability, mineralization rate, and collagen synthesis were assessed, as well as bone/implant contact (BIC) and bone ingrowth (BIN). Results: Tamoxifen caused a decrease in SAOS-2 viability, although an increase in the mineralization rate was observed. In vivo, the TAM groups presented higher BIC and BIN when compared to their control, but a lower percentage of mature collagen cells. Conclusions: Based on our findings, in vitro, the therapy with TAM slightly reduced the viability of SAOS-2 cells while significantly increasing the mineralization rate. In vivo, the therapy positively influenced BIC and BIN during the osseointegration phase.

Keywords: cell culture; selective estrogen receptor modulators; tamoxifen; titanium implants.

Figure 1

Schematic representation of the in vitro experimental setup, illustrating the workflow and procedures performed during the laboratory phase of the study. At time point 0, cells were seeded. Cell viability was assessed at 24, 48, 72, and 96 h post-seeding. At 96 h, samples were processed for scanning electron microscopy (SEM) analysis. Mineralization rate and collagen synthesis were evaluated at 7 and 14 days following cell seeding.

Figure 2

Study design, surgical protocol for implant installation, and delineation of regions of interest (ROIs). (A) Timeline of the in vivo experiments. Three study groups were evaluated: SHAM (sham-operated) and OVX (bilateral ovariectomy). Experiments began at week 0, which corresponds to 18 weeks post-ovariectomy. At week 0, estrous cycle monitoring was conducted over a 2-week period. At week 2, the experimental groups received treatment for 8 weeks with either saline solution (SS) or tamoxifen (TAM). Implants were placed at week 6. Animals were euthanized at weeks 7 and 10. The osseointegration phase was defined as the period between weeks 6 and 10. (B) Schematic representation of the ground section highlighting the regions of interest: ROI 1 (yellow) and ROI 2 (white).

Figure 3

(A) Means and standard deviations (Ms ± SDs) of percentage of reduced Alamar Blue in each group and in each time point and (B) Alamar Blue (AB) supernatant depiction in the 96-well plate. (C) Means and standard deviations (Ms ± SDs) of the Alizarin Red (AR) absorbance rate of the cells in each group and (D) mineralized matrix deposition depicted at the bottom of the 6-well plate. (E) Means and standard deviations (Ms ± SDs) of the Sirius Red absorbance rate of the cells in each group and (F) collagen deposition depicted at the bottom of the 6-well plate. Statistical tests: ANOVA and Tukey. Symbols: * Statistically significant difference (p ≤ 0.05). (G) SEM photomicrographs showing the morphology of the SAOS-2-line cells in the CTR and TAM groups at 96 h. Original magnification: 500×. Scale bar: 200 μm.

Figure 4

(A) Bone/implant contact for each experimental group. Means and standard deviations (Ms ± SDs) of BIC for each group and period. (B) Photomicrographs of BIC in the SHAM-SS (B.1,B.4), OVX-SS (B.2,B.5), and OVX-TAM (B.3,B.6) groups. White arrows: Areas without bone/implant contact. Staining: Basic fuchsin. Scale bar: 200 μm. Bone percentage area for each experimental group. Means and standard deviations (Ms ± SDs) of the BIN for each group and period (C). Photomicrographs showing the histological features of the peri-implant tissues for each group. (D) Photomicrographs of the BIN in the SHAM-SS (D.1,D.5), OVX-SS (D.2,D.6,D.8), and OVX-TAM (D.3,D.4,D.7) groups. Staining: Hematoxylin and eosin. Scale bars: 100 μm and 75 μm. Statistical tests: ANOVA and Bonferroni. Symbols: * Statistically significant difference (p ≤ 0.05).

Figure 5

Percentage of mature collagen fibers. Means and standard deviations (Ms ± SDs) of PSR for each group and period (A). (B) Photomicrographs of the PSR in the SHAM-SS (B.1,B.4), OVX-SS (B.2,B.5), and OVX-TAM (B.3,B.6) groups. Staining: Picrossirius Red. Scale bar: 200 μm. Statistical tests: ANOVA and Bonferroni. Symbols: * Statistically significant difference (p ≤ 0.05).