A SERM increasing the expression of the osteoblastogenesis and mineralization-related proteins and improving quality of bone tissue in an experimental model of osteoporosis

Abstract Raloxifene is an antiresorptive drug, selective estrogen receptor modulator (SERM) used in the treatment of osteoporosis. Objective To evaluate proteins related to bone repair at the peri-implant bone in a rat model of osteoporosis treated with raloxifene. Material and Methods 72 rats were divided into three groups: SHAM (healthy animals), OVX (ovariectomized animals), and RLX (ovariectomized animals treated with raloxifene). Raloxifene was administered by gavage (1 mg/kg/day). Tibial implantation was performed 30 days after ovariectomy, and animals were euthanized at 14, 42, and 60 days postoperatively. Samples were collected and analyzed by immunohistochemical reactions, molecular analysis, and microtomographic parameters. Results RLX showed intense staining of all investigated proteins at both time points except for RUNX2. These results were similar to SHAM and opposite to OVX, showing mild staining. The PCR gene expression of OC and ALP values for RLX (P<0.05) followed by SHAM and OVX groups. For BSP data, the highest expression was observed in the RLX groups and the lowest expression was observed in the OVX groups (P<0.05). For RUNX2 data, RLX and SHAM groups showed greater values compared to OVX (P<0.05). At 60 days postoperatively, microtomography parameters, related to closed porosity, showed higher values for (Po.N), (Po.V), and (Po) in RLX and SHAM groups, whereas OVX groups showed lower results (P<0.05); (BV) values (P=0.009); regarding total porosity (Po.tot), RLX group had statistically significant lower values than OVX and SHAM groups (P=0.009). Regarding the open porosity (Po.V and Po), the SHAM group presented the highest values, followed by OVX and RLX groups (P<0.05). The Structural Model Index (SMI), RLX group showed a value closer to zero than SHAM group (P<0.05). Conclusions Raloxifene had a positive effect on the expression of osteoblastogenesis/mineralization-related proteins and on micro-CT parameters related to peri-implant bone healing.


Introduction
Post-menopausal osteoporosis is the main skeletal disorder with high incidence found in society, being characterized by reduced bone mass due to estrogen deficiency and decreased intestinal absorption of calcium, resulting in increased bone fragility and fracture susceptibility 19 . Studies evidenced that one of the signaling pathways of these proteins, as the canonical pathway, has very importance in the knowledge of osteoporosis and drug action used in their treatment 2 . The animals were divided into three groups: SHAMrats submitted to sham surgery and fed a balanced diet; OVX -rats submitted to bilateral ovariectomy and fed a low calcium diet (osteoporotic) without medical treatment; RLX -rats submitted to bilateral ovariectomy and fed a low calcium diet (osteoporotic) with raloxifene treatment.
The animals were kept in cages and fed a balanced diet (NUVILAB, Curitiba, PR, Brazil) containing 1.4% Ca ++ and 0.8% P, and given water ad libitum. After completion of sham surgeries and ovariectomies, animals in the SHAM group continued to receive rations of the balanced diet, whereas animals in the other two groups were switched to a diet containing 0.1% Ca ++ and 0.5% P (RHOSTER Ind. Com., Vargem Grande Paulista, SP, Brazil).

Estrous cycle classification
The rats were placed in individual cages for daily This fact confirmed the presence of osteopenia in the latter group, which is characteristic of the osteoporotic rat model.

Bilateral ovariectomy
RLX and OVX rats were anesthetized, and incisions in both flanks were subsequently performed to expose the ovaries, which were then surgically removed. Rats from the SHAM group were subjected to the same procedure. However, the ovaries were exposed and not removed.   For BSP data, the highest expression was observed in RLX groups and the lowest expression, in OVX groups Values of closed pores surface were higher for SHAM group followed by OVX and RLX groups (P<0.05) ( Figure 8F).
Regarding the porosity open (Po.V and Po), we observed higher values for SHAM group followed by OVX and RLX groups (P<0.05) (Figures 8G and H).
As for SMI data, RLX group showed values closer to zero, while SHAM group showed values closer to three, which means more parallel trabeculae and higher bone density (P<0.05) (Figure 9).

Discussion
We suggested that raloxifene can stimulate the growing of pre-osteoblastics cells and promote the mineralization of bone extracellular matrix by increasing the immunolabeling of osteoblastogenesis-  However, Wnt/β-catenin levels showed that raloxifene treatment stimulated the osteoblastic differentiation process, and consequently bone formation, around the tibial implants, compared to the ovariectomized animals that received no treatment, which showed low osteoblastic activity.
Despite the benefits of the drug regarding osteoblastic activity, it is important that the neo-formed   Thus, we opted to analyze the bone microstructure parameters in this period (60 days), such as bone volume and porosity, verifying if that bone would be able to receive the implants to rehabilitation.
A very important analysis to provide the safety use of the drugs would be to investigate the adverse effects. In this study, we did not perform any analysis about the systemic effect of raloxifene, such as metabolizin organs evaluation (heart, liver, brain), to show some adverse effects of this drug. Although there was no observed clinical change in the animals during the therapy with raloxifene, due to the reestablishment of metabolism induced by drug therapy, it was observed

Conclusions
Regarding these findings, and despite all limitations of this study, it is possible to conclude that raloxifene treatment in ovariectomized rats provided a positive effect on tibial peri-implant repair by stimulating  We also demonstrated that, by investigating bone matrix mineralization and microarchitecture characteristics, this newly formed bone is of high quality. We therefore believe that raloxifene must be considered in the treatment of osteoporosis, not only for its benefits in preventing fractures, but also as an adjunct in the dental implant rehabilitation process.