Daily melatonin administration improves osseointegration in pinealectomized rats

Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed – Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.


Introduction
Absence of melatonin or its inefficient production affects people who work on night shift, undergo changes in time zone, and suffer from insomnia 16 . In addition, melatonin interferes with bone healing in several ways: through modulation of inflammatory process, collagen fibril formation, osteoblast differentiation, and oxidative stress 6,9,10,15,26,28 .
A lower production of melatonin, either by genetic modifications or pinealectomy, results in alterations in morphology and bone metabolism 8,30 . The literature describes that its action is related to differentiation and increase in osteoblastic activity, as well as osteoprotegerin increase, thus decreasing the action of osteoclasts 18,29 . In addition, it can promote bone cell proliferation and increase in production of type I collagen 21 , thus increasing bone mass 19 . In this context, the absence of melatonin has been shown to delay alveolar repair after exodontia 9,17 . Melatonin replacement could regulate this alteration, which would emphasize the role of this neurohormone in biological responses that drive bone metabolism, especially in conditions in which a decrease occurs in the circadian release capacity of this hormone by the pineal gland 15 .
With the popularization of dental implants, the understanding of structural characteristics and the reversion of the pathophysiological bone alterations have great importance in increasing the predictability of the success of rehabilitation treatment, considering that characteristics of the bone microarchitecture influence the capacity of the bone in support transmission and the distribution of forces 1,9,11 . In addition, although studies have demonstrated the participation of melatonin, when added to biomaterials [3][4][5] , in responses related to bone healing 6,26,31 , repair of periodontal defects and implants loaded with melatonin 2,7 , no studies were performed to evaluate osseointegration during endogenous melatonin deficiency. Thus, the process of how the absence of this hormone interferes with the quality of the perimplantar bone is unclear, which may impair the results of the rehabilitation process in these patients.
Considering this, the aim of this study consists on evaluating the cellular changes and possible complications that may occur along the interface (bone/implant) in pinealectomized rats that received or did not receive daily melatonin replacement as Animals were submitted to the first surgery (pinealectomy) and euthanized 60 days after the second surgical procedure (implant installation in both tibiae). The right tibiae were used for the realization of microtomographic and fluorochromes analyses, and the left tibiae for immunohistochemical analysis.

Pinealectomy
The rats were anesthetized with ketamine (80 mg/ kg b.w., i.m.) and xylazine (10 mg/kg b.w., i.m.). The trichotomy was performed in the scalp region, and the animals were adapted to a stereotactic apparatus.
The head skin was disinfected with alcoholic iodine solution; a longitudinal incision and separation of the subcutaneous tissue until the lambda region of the visualization were performed; and scraping of the fibrous joints (serrata) was made among the parietal bones, causing the interparietal bone to be exposed.
After removal of a bone fragment (4.5 mm diameter calvaria, with a trephine drill coupled to a low-speed motor), the venous sinus (region of intersection of the sagittal and transverse sinuses) was visualized.
With the aid of tweezers, the pineal gland, which is located just below this sinus, was removed. After the extraction of the gland, the removed bone fragment was placed in its original position, and the animal was detached from the stereotactic apparatus. After hemostasis, the skin was sutured with cotton thread.      (Figures 3 and 4).
At 60 days, the staining for ALP in the CO and PNXm groups were light (++) in the bone matrix. However, the PNX group had moderate staining (+++) due to the greater presence of connective tissue (Figures 3   and 4).

Discussion
This study evidenced that melatonin treatment is related to the discrete improvement of several parameters of bone quality related to osseointegration success, especially in the condition when the pineal gland has been removed. It is important to highlight that during long periods of melatonin administration, these differences can be better stressed, due to the osteoblast activity that may be driven by extracellular matrix proteins that had better classifications considering melatonin replacement in animals 15 .
Some studies state that melatonin has been linked to osteoblastic differentiation 20 . However, the role of melatonin replacement has not yet been widely evaluated, and no responses regarding osteogenesis Therefore, the absence of the pineal gland impairs the bone repair process during osseointegration, however the daily melatonin replacement was able to partially restore this response.