Short term sodium alendronate administration improves the peri-implant bone quality in osteoporotic animals

Abstract Sodium alendronate is a bisphosphonate drug that exerts antiresorptive action and is used to treat osteoporosis. Objective The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin). Material and Methods A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level. Results Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days. Conclusion There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats.


Introduction
Rehabilitation with dental implants has become widespread in society and most of the patients demanding this treatment are above 60 years old, a life stage in which osteoporosis is very common, especially in women 7 . Studies regarding dental implants in osteoporotic patients claim that the osteoporotic bone is similar to the proposed model of type IV bone, that is, residual bone formed by a thin layer of cortical bone surrounding the low density cancellous bone. quality, ranging from type I to IV 13 . Because of this, some authors contraindicate the use of implants in patients with osteoporosis, while others believe it is not a determining factor to contraindicate therapy with dental implants since the professional performs an larger diameter, implant surface treatment and a longer waiting period for prosthetic load application 13,14 .
Regarding to the impact of osteoporosis on dental implant survival, Chen, et al. 8 (2013) and Busenlechner, et al. 5 (2014) reported that since there is no statistically significant differences between osteoporotic and non-osteoporotic patients that had been rehabilitated with dental implants, there is a strong correlation between this bone metabolism condition and implant survival, mainly in the mandible. These studies did not include a feasible number of patients to establish a comparison between osteoporotic and non-osteoporotic patients. Besides that, these clinical studies did not show indicate drugs used for osteoporosis.
Currently, several therapies are available for the treatment of osteoporosis, however, they present some The role of estrogen in bone integrity maintenance has been recognized for a while, although estrogen including vascular events and breast carcinoma 5 . Some drugs act by decreasing bone resorption and therefore delaying bone loss rate (antiresorptive therapy), such as bisphosphonates, calcitonin and the human monoclonal antibody, denosumab, or by promoting bone formation (anabolic therapy), such as teriparatide 1, 3,14,18,19,28 .
Sodium alendronate is part of the second generation of bisphosphonates, exhibiting less collateral effects in antiresorptive drug due to its low cost in comparison with other drugs 17 . The class of these drugs has a characteristics similar to pyrophosphate, that is, they are inhibitors of calcium hydroxyapatite crystal growth and exert their antiresorptive activity by inhibiting osteoclast development and migratory activity, and also by promoting their apoptosis 4,31 .
Several studies investigated the effects of the oral bisphosphonates in osteoporosis conditions related to 1,4,6,14 , bone mineral density 3,6,9,20,28  and fed with a balanced diet containing 1.4% Ca, 0.8% P and water ad libitum); OST (rats submitted to bilateral ovariectomies and fed with a low calcium diet containing 0.1% Ca, 0.5% P and water ad libitum, without medical treatment); and ALE (rats submitted to bilateral ovariectomies, fed with a low calcium diet and treated with sodium alendronate).
For the quantitative analysis (micro CT and PCR), after the Power Test calculation, the sample number for each group was a minimum of 6 (power test=0.8).
Thus, as PCR was made in quadruplicate, we elected 4 animals (left tibia) for this evaluation. For the micro CT evaluation, 6 animals were selected (4 right tibia belonging to the PCR groups plus 2 animals) with a total number of 18 animals.

Estrous cycle evaluation
The evaluation of the estrous cycle was performed according to the method described by Long and Evans 16 (1922), in which rats were separated into individual cages and their estrous cycles were assessed daily, for eight days.
Bilateral ovariectomy and low calcium diet (osteoporosis induction) All rats were initially anesthetized with xylazine hydrochloride (Dopaser -Laboratório Calier do Brasil, Dodge Animal Health Ltd, Campinas, SP, Brazil) to ovaries from the rats of the OST and ALE groups.
From the CTL group, only the ovary exposure was performed.
The rats from the OST and ALE groups were fed with a low calcium diet (containing 0.1% Ca, 0.5% P and water ad libitum) and the CTL group was fed with a balanced diet containing 1.4% Ca, 0.8% P and water ad libitum.
The low calcium and phosphate diet was used in order to simulate a real osteoporosis situation.
According to previous studies 21,26,27 , when rats were subjected to bilateral ovariectomies and fed with low calcium diet, there was a decrease of bone mineral density up to two higher greater. Thus, only those undergoing ovariectomy surgery could have an osteopenia condition.

Sodium alendronate
Eight days after ovariectomy, the drug therapy sodium alendronate dissolved in an aqueous solution to the ALE group, as designed by Paz, et al. 20 (2001), and gavage with saline solution to the OST and CTL groups. This treatment was conducted until the end of the experiment.

Implants
After fasting for eight hours, the animals were In order to collect material, the animals were anesthetized following the anesthesia protocol for implant placement. Then, the implants of left tibias were removed by counter-torque and the bone material that was previously in contact with the implant was collected for RT-PCR analysis (real time polymerase chain reaction). At this moment, the rats were euthanized as outlined by Ramalho-Ferreira, et al. 22 (2015) and the right tibias were removed and reduced with margins of about 1 cm to perform the Micro-CT analysis.

Microtomography evaluation (Micro-CT)
For the three-dimensional analysis of animals from the CTL, OST and ALE groups after euthanasia, at the 42-day period, the right tibias that had been removed      is about maintaining these characteristics over time. The molecular and immunohistochemical data and, consequently, a cellular characteristic that is unfavorable to the process of bone tissue formation in treatments with alendronate. These results suggest that in the long term, there is a detriment in the quality of bone formed during the osseointegration process, which seems to be correlated with the reduced production of the proteins that favor bone tissue formation.
In addition to these probable limitations, the literature also describes the possibility of this treatment to cause maxillary osteonecrosis 23 . Even though alendronate is an oral bisphosphonate that is less powerful when compared to other intravenous osteonecrosis induction, both in tooth extraction surgery and in loss of dental implants that have been, sometimes, installed for decades 17,31 .
This study was statically conducted on peri-implant is related to dynamic changes in the bone, mainly due to physiological changes observed during mastication loads on implant-supported prostheses. Given the statements above, new studies must be designed, with a primary transcutaneous load application on the implants after bone tissue maturation (42 days), with micromotion devices, as described by Wazen 30 (2013).
As previously emphasized, another issue to be investigated concerns the question: what is the behavior of osteoporotic bone treated with ALE in the long term? Thus, future research should examine the same conditions of this study, but at 6 month and more studies are necessary in order to evaluate the quality of the peri-implant bone under long-term sodium alendronate administration in the presence of the osteoporotic condition.