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Abstract: The aim of this study was to evaluate the effect of the topical Antonio Cutando1, Gerardo
application of melatonin on osteointegration of dental implants in Beagle Gómez-Moreno1, Carlos Arana1,
dogs 14 days after their insertion. In preparation for subsequent insertion of Fernando Muñoz2, Mónica Lopez-
dental implants, upper and lower premolars and molars were extracted from Peña2, Jean Stephenson3 and
12 Beagle dogs. Each mandible received cylindrical screw implants of Russel J. Reiter4
1
3.25 mm in diameter and 10 mm in length. The implants were randomly Department of Special Care in Dentistry,
assigned to the mesial and distal sites on each side of the mandible. Prior to School of Dentistry, University of Granada,
Granada, Spain; 2Department of Clinical
implanting, 1.2 mg lyophylized powdered melatonin was applied to one bone Veterinary Sciences, University of Santiago de
hole at each side of the mandible. None was applied at the control sites. Eight Compostela, Lugo, Spain; 3Department of
histological sections per implant were obtained for histomorphometric English, Facultad de Filosofı́a y Letras,
University of Granada, Granada, Spain;
studies. After a 2-wk treatment period, melatonin significantly increased the 4
Department of Cellular and Structural Biology,
perimeter of bone that was in direct contact with the treated implants Health Science Center, University of Texas,
(P < 0.0001), bone density (P < 0.0001), new bone formation (P < 0.0001) San Antonio, TX, USA
Introduction upon any specific target organ. It reaches all tissues and due
to its amphiphilicity, it enters all subcellular compartments
Melatonin influences numerous aspects of circadian and [15, 16]. Moreover, several organelles including the nucleus
circannual rhythms, including sleep, actions that are and the mitochondria may accumulate melatonin [5, 17].
mediated by the binding of the indoleamine to membrane Numerous studies have documented that melatonin is an
receptors [1–4]. Subsequent studies have established actions important mediator in bone formation and stimulation [18].
of melatonin with aspects of intracellular functions, which At micromolar concentrations, melatonin stimulates the
depend on mechanisms that are independent of the action proliferation and synthesis of type I collagen fibres in
of the molecule on membrane receptors. For these actions, human osteoblasts in vitro [19]. Moreover, in preosteoblast
nuclear receptors for melatonin in peripheral organs [5, 6], cultures from rats, melatonin, in a dose-dependent manner,
and in cells of the central nervous system have been promotes development of bone sialoprotein and of other
identified [7]. Melatonin is also capable of binding to protein bone markers, including alkaline phosphatase,
cytosolic proteins including kinase C [8], calmodulin [9] and osteopontine, and osteocalcine, and reduces their period
calreticulin [10], and probably quinone reductase-2 [11]. of differentiation into osteoblasts from 21 days (which is
Additionally, some functions of melatonin seem to involve normal) to 12 days. This reaction seems to be mediated by
nuclear receptors [12]. the membrane receptors for the indole [20]. With regard to
In view of these findings, melatonin is not a hormone in bone metabolism, melatonin acts directly on the osteoclast,
the classic sense, but functions as a cell protector and an a multinucleated cell, which resorbs the extracellular matrix
antioxidant [13]. It is known that the enzymes required for by various mechanisms, including the production of free
the biosynthesis of melatonin are found in tissues other radicals. Melatonin, with its antioxidant properties and its
than the pineal gland, and it is also known that several of ability to detoxify free radicals [21], may interfere in this
these tissues, amongst them the retina, thymus, spleen, function of the osteoclast and thereby inhibit bone resorp-
B-lymphocytes, ovaries, testicles, and the intestine, all tion [22]. The inhibition of bone resorption may be
produce melatonin. Extrapineal melatonin produced by enhanced by a reaction of indolamine in osteoclastogenesis.
specific organs is used locally as a paracoid or autocoid and It has been observed that melatonin, at pharmacological
does not enter the circulation [14]. Melatonin does not act doses, increases bone mass by suppressing resorption
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Melatonin and osteointegration
175
Cutando et al.
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Melatonin and osteointegration
In our study, the increase in osteoblast proliferation taneously caused bone absorbed during this process, the
brought about by melatonin is seen in the production of a increase in osteointegratoin parameters would probably not
greater number of these cells in the peri-implant zone; also, have been so notable in such a short space of time (2 wk).
early cell differentiation accelerated considerably the syn- Such a rapid increase in bone formation suggests that
thesis and mineralization of the osteoid matrix. This may melatonin was acting at two different levels simultaneously
explain the greater quantity of mineralized bone matrix in the remodelling process.
around the melatonin-treated implants, as well as the It is well known that osteoclasts, multinucleate cells
significant increase in BIC and in total peri-implant bone responsible for bone resorption, contain superoxide dismu-
after 2 wk. Bone in the peri-implant area is subjected to an tase and produce reactive oxygen species in the micro-
intense remodelling process after implant insertion; thus, a environment of bone; this may contribute to the
large amount of this bone is at a more advanced stage of degradation of components of the bone matrix since
development, in the form of already mineralized bone structural molecules of the matrix, such as collagen or
matrix, while peri-implanted bone is to be found at the as hyaluronic acid, are susceptible to oxidative damage by free
yet nonmineralized osteoid stage [32, 33]. The osteointe- radicals [36]. Moreover, osteoclasts secrete another enzyme,
gration process also includes, therefore, not only the acid phosphatase that is resistant to tartrate (TRAP), which
remodelling of existing bone, but also the formation of has a binucleate center with an active iron which reacts with
new bone around the implant, especially in the inter-thread hydrogen peroxide and, via the Fenton reaction, produces
zone [34, 35]. Melatonin would appear to contribute to the the hydroxyl radical (•OH). The ferric ion formed in this
neoformation of bone around implants as it stimulates the reaction also interacts with H2O2 to produce the peroxide
differentiation of new preosteoblasts, which are transported anion radical and a ferrous ion. Hence, a sequence of
from bone marrow to the alveolar bed via the vascular reactions occurs at the osteoclast level, which give rise to
system. Another action of melatonin at the preosteoblast HO• and O2–• through the continuous oxidation and
level, which enhances new bone tissue formation is its reduction of the active iron present in the TRAP;
stimulation of gene expression of certain proteins in the this allows the generation of large quantities of reactive
bone matrix [31]. In this sense, it has been demonstrated species depending on the availability of H2O2 in the
that melatonin, after a period of 5–9 days, promoted environment [37].
expression of genes for bone sialoprotein (BSP), alkaline It appears that melatonin acts at the level of the
phosphatase (ALP) and osteocalcine (OC) [20]. osteoclast lacuna, due to its antioxidant properties and its
Our results, which show a greater percentage of inter- ability to neutralize reactive species, thereby inhibiting bone
thread bone and new bone formation around the implants resorption [22, 26]. After implant placement, despite the
treated with topical melatonin, are in line with previously care with which the surgical procedure is performed, bone
published findings. Support for this relationship is to be necrosis often occurs around the implant and there is an
found in the fact that the genes of a large portion of bone inflammatory reaction as a direct consequence of surgery
matrix [BSP, ALP, OC, SPARC: secreted protein, acidic, [32, 38]. Macrophages and leukocytes from peri-implant
cysteine-rich (osteonectin)] contain the sequence of bases blood vessels promote an increase in free radicals [38, 39],
(RGGTCA) necessary for the nuclear receptor of melatonin which stimulate bone resorption by the osteoclast [40, 41].
(RZR) to bind with its promoting zone. Moreover, it may MelatoninÕs antioxidant and anti-inflammatory properties
be that this increase in the formation of bone tissue is also may attenuate this reaction and constrain the production of
mediated by the membrane receptors for the indolamine, as reactive species [42–44], and therefore bone resorption,
treatment with luzindole or pertussis toxin reduces BSP and after implant surgery. This inhibition of bone resorption
ALP expression [20, 31]. Given that peri-implant bone may be reinforced by another reaction induced by melato-
undergoes a remodelling process after implant placement, nin on the osteoclastogenesis process. According to some
had melatonin stimulated bone formation but also simul- authors, the application of indoleamine at concentrations
177
Cutando et al.
ranging from 5 to 500 lm lowers, in a dose-dependent 11. Tan DX, Manchester LC, Terron MP et al. Melatonin as a
manner, the expression of mRNA from the RANK and naturally occurring co-substrate of quinone reductase-2, the
increases levels of both OPG as well of mRNA from the putative MT(3) melatonin membrane receptor: hypothesis and
OPG in preosteoblast cell lines MC3T3-E1 [23]. This significance. J Pineal Res 2007; 43:317–320.
indicates that melatonin may bring about a reduction in 12. Tomas-Zapico C, Coto-Montes A. A proposed mechanism
bone resorption and an increase in bone mass due to its to explain the stimulatory effect of melatonin on antioxidative
repression of osteoclast activation by means of RANK [22]. enzymes. J Pineal Res 2005; 39:99–104.
13. Tan DX, Manchester LC, Terron MP et al. One molecule,
These actions of melatonin on bone tissue are of interest
many derives: a never ending interaction of melatonin with
as it may be possible to apply melatonin during endo-
reactive oxygen and reactive nitrogen species? J Pineal Res
osseous dental implant surgery as a biomimetic agent [45].
2007; 42:28–42.
As a result, the process of healing may be more precise,
14. Reiter RJ. Pineal melatonin: cell biology of its synthesis and
initial conditions of receptor tissues may be enhanced, the of its physiological interactions. Endocr Rev 1991; 12:151–180.
period of osteointegration and settling of the implant may 15. Acuña-Castroviejo D, Escames G, Macı́as M et al. Cell
be reduced, and therefore the quality of life of the patient protective role of melatonin in the brain. J Pineal Res 1995;
may be improved. 19:57–63.
16. Tan DX, Manchester LC, Reiter RJ et al. Identification of
Acknowledgements highly elevated levels of melatonin in bone marrow: its origin
and significance. Biochim Biophys Acta 1999; 1472:206–214.
This study has been possible thanks to the Research Project 17. Acuna CD, Escames G, Carazo A et al. Melatonin, mito-
entitled: ÔStudy of the synergism between Melatonin and chondrial homeostasis and mitochondrial-related diseases.
Growth Hormone (GH) on osteointegration processes in Curr Top Med Chem 2002; 2:133–151.
dental implants and on bone regeneration in the oral cavityÕ, 18. Witt-Enderby PA, Radio NM, Doctor JS et al. Therapeutic
financed by the Fondo de Investigación Sanitaria, (FIS), of treatments potentially mediated by melatonin receptors:
the Ministerio de Sanidad y Consumo, Spain (PI041610); by potential clinical uses in the prevention of osteoporosis, cancer
the Ministerio de Educación y Ciencia, Spain (Proyecto and as an adjuvant therapy. J Pineal Res 2006; 41:297–305.
PETRI 95-0885-OP); by the Instituto de Salud Carlos III 19. Nakade O, Koyama H, Ariji H et al. Melatonin stimulates
(FIS G03/137); by Grupo de Investigación CTS-263 (Junta proliferation and type I collagen synthesis in human bone cells
de Andalucı́a, Spain), and Microdent Implant System in vitro. J Pineal Res 1999; 27:106–110.
20. Roth JA, Kim BG, Lin WL et al. Melatonin promotes
(Barcelona, Spain), who provided the implants.
osteoblast differentiation and bone formation. J Biol Chem
1999; 274:22041–22047.
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