Application of Local Melatonin for Controlling Peri-implant Diseases: a Randomized Clinical Trial
Phase: NA
Conditions: Periimplantitis
Sponsor: Universidad Complutense de Madrid

EFFECT  OF  MELATONIN  IN  THE  TREATMENT  OF
Department  of  Oral  and  Maxillofacial  Medicine  and  Surgery
PERI-IMPLANT  DISEASES
Sponsor:  Dr.  Carlos  Manuel  Cobo  Vázquez  
Principal  Investigator:  Dr.  Juan  López-Quiles  Martínez  
Code:  MEL19
FACULTY  OF  DENTISTRY
COMPLUTENSE  UNIVERSITY  OF  MADRID
EudraCT  number  2019-000828-18
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July 17th 2019

7.  METHODS ................................................. .................................................. .......................................13
3.  JUSTIFICATION  OF  THE  STUDY.......................................................................................................................7
A.  Population .......................................................................................................................................10
ANNEX  I ................................................ .................................................. ............................................22
B.  Sample .................................................................................................................................................11
ANNEX  II ................................................ .................................................. ............................................24
1.  MAIN  DATA.......................................................................................................................................4
6.  MATERIAL............................................... .................................................. .......................................10
9.  SCHEDULE ................................................... .................................................. ..................................17
6.1.  POPULATION  AND  SAMPLE ................................................................................................................10
10.  BIBLIOGRAPHY ................................................................................................................................18
4.  HYPOTHESIS .......................................................................................................................................8
7.1.  INTERVENTION  AND  STUDY  GROUPS .......................................................................................................13  
7.2.  SAMPLE  COLLECTION ..........................................................................................................................14  
7.3.  ANALYSIS ..........................................................................................................................................15
5.  OBJECTIVES .........................................................................................................................................9
8.  LIMITATIONS.................................................................................................................................16
6.2.  HUMAN  RESOURCES ................................................................................................................................11
2.  BACKGROUND.......................................................................................................................................5
6.3.  PHARMACOLOGICAL  MATERIAL..........................................................................................................11  
6.4.  SURGICAL  MATERIAL...........................................................................................................................11  
6.5.  MATERIAL  FOR  SAMPLE  ANALYSIS........................................................................................................12
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•  Principal  Investigator:
Carlos  Manuel  Cobo  Vazquez
Juan  Lopez-Quiles  Martinez
•  Center  where  the  research  is  carried  out:
PERI-IMPLANT  DISEASES
•  Promoter:
•  Title:  EFFECT  OF  MELATONIN  IN  THE  TREATMENT  OF
1.  MAIN  DATA
Department  of  Oral  and  Maxillofacial  Medicine  and  Surgery.  Faculty  of  Dentistry.
Complutense  University  of  Madrid
Gregorio  Marañón  General  University  Hospital,  Madrid
•  Contact  information:  91  394  1964
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608  61  28  38
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“Peri-implant  mucositis”  was  the  term  proposed  for  the  reversible  inflammation  of  the  soft  
tissues  surrounding  functioning  implants  (Albrektsson  and  Isidor,  The  First  European  
Workshop  on  Periodontology,  1994)3 .
,  mostly  caused  by  IL-  7,8
“Peri-implantitis”  was  introduced  in  the  late  1980s  and  was  later  defined  as  an  inflammatory  
process  affecting  the  soft  and  hard  tissues  surrounding  a  functioning  osseointegrated  
implant,  resulting  in  loss  of  supporting  bone.
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2.  BACKGROUND
:
The  etiology  of  this  disease  is  not  clear,  although  the  increased  presence  of  gram-negative  
microorganisms  and  spirochetes  has  been  determined  when  associated  with  pathogenic  
bacteria.  In  other  cases,  bone  loss  can  be  caused  by  occlusal  trauma  or  peri-implant  
overload.  Genetic  and  systemic  factors  have  also  been  described,  as  well  as  toxic  habits  
that  could  influence  the  development  and  progression  of  this  disease.  Peri-implantitis  is  the  
main  challenge  facing  dentists  today.  Bone  loss  occurs  progressively  and  can  cause  early  
implant  failure  if  appropriate  treatment  is  not  established.
Two  types  of  peri-implant  diseases  can  be  considered:
It  is  known  that  the  large  increase  in  interleukins  (IL-1,  IL-6,  IL-8  and  IL-17)  from  cell  
types  in  the  injured  area  triggers  the  production  of  the  first  symptoms  derived  from  
inflammation6
.
.
Peri-implant  mucositis  and  peri-implantitis  have  become  an  increasing  problem  in  recent  
years,  due  to  the  increased  and  more  frequent  use  of  endosseous  dental  implants  as  part  of  
prosthetic  rehabilitation.  According  to  the  Sixth  European  Consensus  on  Periodontology  
(2008),  its  incidence  is  between  28-56%.
.  IL-6  plays  an  important  role  in  leukocyte  recruitment  during  the  acute  inflammation  
phase8,9 .  IL-6  also  stimulates  bone  resorption  and  appears  to  be  involved  in  the  early  
stages  of  osteoclastogenesis10 .
Peri-implant  diseases  are  defined  as  the  host  response  to  the  formation  of  a  biofilm  
on  the  implant,  which  initially  includes  various  inflammatory  reactions  in  the  soft  tissue  
surrounding  the  implant,  and  which  can  subsequently  progress  and  cause  loss  of  bone  
support;  that  is,  it  includes  inflammatory  pathological  changes  in  the  tissues  surrounding  an  
implant  subjected  to  load.
Melatonin  can  be  defined  as  a  hormone  with  paracrine,  autocrine  and  antioxidant  
actions  that  exerts  several  actions  dependent  and  independent  of  its  receptors11,12 .  Its  
first  effects  were  described  in  1917  by  McCord  and  Allen,  but  it  was  not  until  1958  when  it  
was  isolated  and  identified  by  Lerner,  Case  and  Takahashi12 .  This  molecule  is  mainly  
synthesized  in  the  pineal  gland  until  30  years  of  age.  Melatonin  production  is  genetically  
determined  and  varies  between  individuals,  and  can  reach  200  pg/ml  during  the  dark  phase,
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Other  studies  have  shown  that  exogenous  administration  of  melatonin  above  the  body's  basal  levels  
stimulates  the  immune  system21,22
Some  studies  in  oral  surgery  support  the  usefulness  of  melatonin  as  an  anti-inflammatory  and  
protector  against  infections.  Although  its  action  would  be  amplified  when  the  free  radicals  of  NO  
(nitric  oxide)  resulting  from  tissue  damage  are  already  present  in  the  injured  area18 .  In  addition,  the  
application  of  melatonin  in  the  post-extraction  alveolus  favors  tissue  healing  due  to  the  properties  of  
this  molecule19,20 .
increasing  the  proliferation  of  T21  lymphocytes
This  stimulation  of  the  immune  and  inflammatory  system  dependent  on  the  concentration  of  
melatonin35,36  would  be  responsible  for  the  inflammatory  response23  and  the  defense  of  the  body  
against  bacterial  and  viral  infections35,38 .
Melatonin  has  already  been  used  successfully  after  radical  oral  surgery  interventions  due  to  
its  antioxidant  action  and  the  stimulating  and  protective  effects  of  intracellular  enzymes  involved  in  
the  healing  process12,17 .
It  has  also  been  shown  to  regulate  functions  unrelated  to  the  endocrine  system  and  plays  an  
important  role  in  pain  modulation.  Several  mechanisms  have  been  proposed  for  its  function,  related  
to  membrane  receptors,  nuclear  receptors,  and  by  scavenging  free  radicals  in  a  receptor-independent  
manner15 .
One  of  the  main  physical-chemical  characteristics  of  melatonin  is  its  high  diffusion  capacity,  since  it  
is  a  very  lipophilic  molecule.  This  property  allows  melatonin  to  cross  all  morphophysiological  barriers  
and  penetrate  all  cells  and  cellular  spaces13,14 .  It  also  has  a  very  high  redox  potential  of  0.74  V,  
which  determines  an  antioxidant  activity  far  superior  to  that  of  other  known  antioxidants11,15,16 .  
There  is  general  agreement  that  melatonin  is  a  non-toxic  molecule.  It  produces  intense  anti-apoptotic  
signals  even  in  situations  of  ischemia  and  has  cytoprotective  properties  that  have  been  described  for  
the  treatment  of  neurodegenerative  disorders.  It  also  stimulates  the  immune  system  and  has  
oncosuppressive  properties  that  still  today  represent  an  extensive  field  of  research13 .
Its  basal  daytime  level  is  about  10  pg/ml.  It  is  also  synthesized  in  other  regions  of  the  body  such  as  
the  retina,  the  gastrointestinal  tract,  the  skin,  the  bone  marrow  and  lymphocytes13 .
The  result  of  these  studies  shows  that,  in  general,  melatonin  could  act  by  stimulating  the  immune  
system  in  basal  conditions  or  states  of  immunosuppression,  for  a  more  effective  early  immune  
response.  However,  in  the  presence  of  an  exacerbated  immune  or  inflammatory  response,  melatonin  
could  exert  a  negative  regulation  by  acting  as  an  anti-inflammatory.  Therefore,  melatonin  could  be  
considered  a  pleiotropic  molecule  with  varied  and  complex  effects  on  the  immune  system.
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NK  cells20,21  and  granulocytes,  and  the  
antibody  response26,27 .  The  activation  of  T  lymphocytes  increases  the  release  of  inflammatory  
cytokines  such  as  interferon-ÿ,  TNF-ÿ,  IL-1,  IL-1ÿ,  IL-2,  IL-6  and  IL-  1228,29,30,31,32,33,34.  In  
addition,  it  has  been  proposed  that  it  induces  the  production  of  NO35 .
,
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pathological  changes  in  the  soft  tissue  surrounding  the  loaded  implant,  which  may  
subsequently  progress  and  lead  to  loss  of  bone  support.
Peri-implant  diseases  are  defined  as  those  inflammatory  reactions
The  diagnosis  of  this  pathology  should  be  confirmed  by  evaluating  the  clinical  signs  of  mucosal  inflammation,  the  
presence  of  bleeding  and/or  suppuration  on  careful  probing,  increased  probing  depth  compared  to  previous  
records,  and  bone  loss  of  the  crestal  bone  greater  than  the  initial  bone  remodeling  processes.
Treatment  protocols  for  peri-implantitis  are  not  clearly  established,  even  less  so  
when  the  etiology  of  this  disease  is  difficult  to  determine  and  in  many  cases  could  be  
multifactorial.  As  a  standard,  it  has  been  proposed  to  eliminate  all  occlusal  traumatic  
factors  that  could  cause  progression  of  bone  loss  due  to  overload.  A  basic  periodontal  
treatment  should  then  be  carried  out  by  disinfecting  the  surface  of  the  implants  and  
removing  granulation  tissue  if  present.  In  this  way,  the  prostheses  are  temporarily  removed  
for  a  variable  period  of  between  1  and  2  weeks  to  avoid  loading  the  implant.
On  the  other  hand,  melatonin  seems  to  contribute  to  the  neoformation  of  bone  since  it  
stimulates  the  differentiation  of  preosteoblasts,  which  are  transported  from  the  bone  
marrow  to  the  alveolar  bed  through  the  vascular  system.  In  addition,  it  stimulates  the  gene  
expression  of  certain  proteins  of  the  bone  matrix  and  stimulates  the  activity  of  
osteoblasts40,41 .
3.  JUSTIFICATION  OF  THE  STUDY
There  are  studies  applied  to  osseointegrated  implants  that  have  observed  an  earlier  
differentiation  of  osteoblasts  caused  by  melatonin,  which  accelerates  the  synthesis  and  
mineralization  of  the  osteoid  matrix  after  2  weeks27  and  4  weeks40,  as  well  as  a  significant  
increase  in  the  width  and  length  of  cortical  bone .  42,43  This  rapid  increase  in  bone  
formation  indicates  that  melatonin  acts  during  bone  remodeling  at  two  levels  simultaneously.  
42,43,44  In  histological  analysis,  the  local  application  of  3  mg  of  melatonin  has  been  shown  
to  increase  trabecular  formation  by  directly  stimulating  osteoblasts  from  the  endosteum .  
45,46
immune  and  inflammatory  system.  These  immunomodulatory  and  anti-inflammatory  actions  
of  melatonin  have  been  successfully  studied  in  several  experimental  models,  and  there  is  
general  agreement  that  melatonin  can  be  administered  with  insignificant  side  effects39 .
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Peri-implant  bone  loss  due  to  infection  is  the  most  common  and  complex  problem  in  
implantology.  This  bone  loss,  caused  by  the  presence  of  pathogenic  microorganisms,  is  
associated  with  localized  tissue  inflammation.
irreversible.  The  problems  resulting  from  this  pathology  make  the  prognosis  of  implants  
difficult,  leading  in  many  cases  to  their  loss.  Furthermore,  there  is  currently  no  treatment  
for  this  disease.  Its  diagnosis  is  made  by  peri-implant  probing  and  radiographic  evaluation  
of  the  bone  level.
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There  is  sufficient  scientific  evidence  about  the  usefulness  and  safety  of  melatonin  as  an  
antibacterial,  anti-inflammatory  and  analgesic  in  situations  of  inflammatory  pain  and  as  an  
osteodifferentiator  and  early  osteogenic.
previous  studies,  investigated  using  different  study  models  and  through  various  evaluation  
methods,  the  present  investigation  of  its  antimicrobial,  anti-inflammatory  and  osteogenic  
properties  in  patients  is  recommended.
This  research  will  provide  the  quantification  of  antimicrobial  and  anti-inflammatory  properties  
in  a  known  model  of  inflammation  caused  by  infection  in  patients  with  implants.  It  also  aims  
to  offer  a  therapeutic  alternative  in  terms  of  bone  formation  in  a  situation  that  is  currently  
difficult  to  predict,  such  as  peri-implant  bone  defects.
4.  HYPOTHESIS
The  etiology  of  the  disease  may  determine  that  the  process  of  bone  loss  continues  
to  occur,  although  at  a  slower  rate.  The  establishment  of  the  disease  as  well  as  its  
progression  may  be  due  to  systemic  diseases  and  patient  conditions,  systemic  medications,  
tissue  healing  processes,  tissue  renewal  and  tissue  response  to  clinical  interventions,  
trauma  to  orofacial  structures,  local  diseases  affecting  teeth,  periodontium,  bone  and  
mucosa,  biomechanical  factors,  tissue  morphology  and  phenotype  and  iatrogenic  factors.
Considering  the  beneficial  results  of  melatonin  obtained  in  the  studies
In  some  cases,  bone  substitutes  and  biomaterials  have  been  proposed  to  try  to  
recover  bone  loss  in  order  to  regenerate  the  bone  defect  that  has  occurred.  However,  
predictable  results  have  not  been  found  in  all  situations  and  long-term  results  are  still  
controversial.
implant  and  promote  local  hygiene  and  adequate  healing  of  peri-implant  tissue.
There  is  an  increased  incidence  of  bone  loss  in  osseointegrated  implants.
The  presence  of  Gram-negative  pathogenic  bacteria  is  the  main  etiological  factor  in  peri-
implant  bone  loss.
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The  dissemination  of  the  results  of  this  research  would  allow  us  to  offer  the  first
effective,  simple  and  economical  therapeutic  alternative  with  a  high  success  rate  that  achieves  peri-implant  bone  
regeneration,  a  decrease  in  inflammation  and  pathogenic  bacterial  species.  The  single  application  of  local  
melatonin  in  the  same  session  as  the  peri-implant  treatment  would  allow  the  reduction  of  pharmaceutical  and  
biomaterial  costs  as  well  as  the  patient's  recovery  time.  In  this  way,  the  antibacterial,  anti-inflammatory  and  early  
osteogenic  properties  will  allow  patients  with  destructive  bone  pathology  caused  by  bacteria  to  benefit.
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could  improve  local  bone  tissue  conditions,  especially  bone  density,  which  is  the  most  important  factor  for  
proper  osseointegration.
control.
The  main  objective  of  this  research  protocol  is  to  evaluate  the  demonstrated  effects  of  melatonin  on  the  peri-
implant  sulcus  of  patients  requiring  treatment  of  peri-implant  pathology  with  bone  loss.
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To  this  end,  the  following  secondary  objectives  are  established:
Regenerative  treatment  of  bone  defects  is  not  predictable  using  conventional  techniques  and  biomaterials.
5.  OBJECTIVES
-  Assess  the  appearance  of  complications  associated  with  melatonin  in  the
Based  on  the  previous  hypothesis,  this  phase  IV  randomized  pilot  clinical  study  is  proposed  to  be  carried  out.
postoperative.
Based  on  the  above,  we  formulate  the  following  main  hypothesis:
-  To  compare  whether  there  are  statistically  significant  differences  in  probing  depth  after  peri-implant  
treatment  depending  on  whether  local  melatonin  or  the  control  treatment  is  applied.
Melatonin  applied  locally  to  peri-implant  defects  produces  favorable  differences  in  the  treatment  of  peri-implant  
diseases  in  terms  of  the  reduction  of  pathogenic  bacterial  species,  decrease  in  mediators  and  signs  of  
inflammation,  and  acceleration  of  local  early  osteogenesis  with  respect  to  conventional  peri-implant  treatment.
-  To  contrast  whether  there  are  statistically  significant  differences  in  the  levels  of  peri-implant  crestal  bone  
after  peri-implant  regeneration  depending  on  whether  the  treatment  is  performed  with  local  melatonin  
or  without  it.
-  To  establish  whether  there  are  statistically  significant  differences  in  the  presence  of  bacterial  species  after  
applying  local  melatonin  compared  to  the  control.
Several  studies  have  shown  that  melatonin  has  short-term  anti-inflammatory  and  antimicrobial  properties.  In  
addition,  due  to  its  early  osteogenic  potential
-  To  determine  whether  there  are  statistically  significant  differences  in  the  levels  of  interleukin-1ÿ,  2,  and  6  
depending  on  the  application  of  local  melatonin  versus  the
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A.  Population
-  Ages  between  40  and  85  years.
-  Not  suffer  from  coagulation  or  healing  disorders.
Exclusion  criteria
-  Health  status  (ASA  Category  I  and  II).
-  Tobacco  use.
-  Pregnant  or  breastfeeding  women
6.  MATERIAL
Inclusion  criteria
-  Meet  the  inclusion  criteria  and  agree  to  sign  the  consent
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-  Patients  of  both  sexes  with  implants  with  peri-implant  disease  according  to  the  criteria  of  the  
Classification  of  Periodontal  and  Peri-implant  Diseases  and  Conditions  of  the  2017  World  Workshop.
informed.
The  subjects  who  will  participate  in  the  study  will  be  patients  selected  from  the
-  Implants  of  the  same  diameter,  distributed  bilaterally.
-  Chronic  disorders,  and  history  of  allergies  or  adverse  effects
Department  of  Oral  and  Maxillofacial  Medicine  and  Surgery  of  the  Faculty  of  Dentistry  of  the  Complutense  
University  of  Madrid.  The  cases  will  be  those  patients  with  an  indication  for  peri-implant  treatment,  in  good  
health  (Category  I  and  II  of  the  American  Society  of  Anesthesiologists  [ASA]),  of  both  sexes,  aged  between  
40  and  85  years.
-  Patients  with  one  or  more  fully  erupted  teeth.
-  No  history  or  metabolic  or  systemic  diseases  that  affect  the  bone  or  the  healing  process  of  the  
surgical  area,  such  as  diabetes  or  osteogenesis  imperfecta.
-  Use  of  contraceptive  or  hormonal  medications.
associated  with  the  medications  that  were  used.
6.1.  POPULATION  AND  SAMPLE
-  Not  suffer  from  autoimmune  diseases.
-  Use  of  anti-inflammatory  drugs,  corticosteroids,  immunosuppressants,  analgesics,  antidiabetics  or  
bisphosphonates  regularly  or  recently.
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B.  Sample
For  the  supervision  and  advice  of  the  Research  project  in  the  most  specific  phases,  the  following  
qualified  personnel  will  be  required:
Pure  melatonin  without  excipients:  1.9  mg  of  melatonin  powder,  Farmacia  La  Botica  de  Argensola  
(Madrid,  Spain).
Straight  handpiece  with  saline  irrigation  and  round  steel  burs.
A  biochemist  from  the  Clinical  Analysis  Service  of  the  Complutense  University  of  Madrid  who  is  
able  to  process  and  analyze  the  crevicular  fluid  samples  obtained  according  to  the  laboratory  
techniques  indicated  for  the  established  biochemical  parameters.
Surgical  aspirators.
Periodontal  probes.
-  Completely  edentulous  patients  (absence  of  erupted  teeth).
6.2.  HUMAN  RESOURCES
Carpule-type  metal  anesthesia  syringes  and  disposable  needles.
All  procedures,  from  diagnosis,  treatment  of  patients,  obtaining  samples  and  complementary  
tests,  as  well  as  sample  processing  and  statistical  analysis  will  be  carried  out  by  the  doctoral  
student.
Syndesmototomes  and  Bard-Parker  type  scalpels  with  No.  15  blades.
The  aim  is  to  include  and  study  30  individuals  with  an  indication  for  peri-implant  treatment  
in  implants,  in  a  healthy  state  (Category  I  and  II  of  the  ASA)  who  attend  the  Department  of  Oral  
and  Maxillofacial  Medicine  and  Surgery  of  the  Faculty  of  Dentistry  of  the  Complutense  University  
of  Madrid.
Anesthesia  carpules:  articaine  4%  with  adrenaline  1:100,000.
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Patients  will  be  randomized  by  computer  into  a  melatonin  treatment  group  or  a  control  group.
6.4.  SURGICAL  MATERIAL
A  Data  Analyst  specializing  in  Biostatistics  to  apply  appropriate  statistical  formulas  to  the  data  
obtained,  ensuring  the  objectives  of  the  study.
Ultrasound.
-  Patients  who  refused  to  participate  in  the  study,  who  did  not  wish  to  sign  the  informed  
consent  and  those  who  did  not  allow  follow-up  as  well  as  the  collection  of  samples.
6.3.  PHARMACOLOGICAL  MATERIAL
Stainless  steel  periodontal  curettes.
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Sterile  transport  media
Perio-paper  strips
Human  IL-1ÿ,  2,  6  ELISA  BLK  4-213  and  BLK  4-231TEN  Kit  79010055  (LETI  Laboratories,  
Barcelona,  Spain).
Microbiological  culture  Petri  dishes.
6.5.  MATERIAL  FOR  SAMPLE  ANALYSIS
Sterile  paper  points
Pre-mounted  atraumatic  suture  needle  and  4/0  monofilament  silk  thread  on  3/8  semicircular  
needle.
Langenbeck  type  retractors,  straight  and  curved  cutting  scissors,  Adson  forceps,  curved  
mosquito  forceps  and  Mayo  needle  holders.
Graduated  pipettes  of  5  and  10  ml,  and  tips.
Ivymen  System  2100-C  ELISA  plate  reader.
SPSS  computer  program  (Static  Package  for  the  Social  Sciences,  version  21.0,  SPSS  Inc.,  
Chicago,  Ill.,  USA).
Nuaire  Freezer  -85ºC  Ultralow  freezer.
Periotest  reader  for  Perio-paper  samples.
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sutured  with  4/0  monofilament  silk  on  a  pre-mounted  3/8  semicircular  needle.
A  prior  radiological  study  will  be  performed  on  the  same  day  of  the  intervention.  The  inclusion  of  
the  patient  in  the  melatonin  treatment  group  or  in  the  control  group  will  be  randomized  by  
computer,  assigning  a  different  code  to  the  cases  of  application  of  melatonin  and  to  the  control  
cases  that  will  be  unknown  to  the  patient  and  during  the  data  analysis.  The  peri-implant  treatment  
and  mechanical  debridement  will  be  performed  in  all  cases  under  conventional  local  anesthesia.  
The  procedure  will  follow  the  usual  sequence  in  all  cases.  Surgical  access  will  be  made  through  
an  intrasulcular  and  linear  incision,  and  the  detachment  of  a  mucoperiosteal  flap.  The  peri-implant  
treatment  will  be  performed  with  an  ultrasound  tip  under  irrigation,  complemented  by  the  use  of  
curettes  if  necessary.  Subsequently,  in  the  cases  of  the  corresponding  group,  melatonin  (1.9  mg)  
will  be  applied  to  the  peri-implant  bed.
All  patients  will  be  prescribed  the  same  pharmacological  therapy  consisting  of:  Paracetamol  1000  
mg  1  tablet/8  hours  (in  case  of  pain).
After  7  days,  the  healing  process  will  be  reviewed  and  the  sutures  will  be  removed.
7.1.  INTERVENTION  AND  STUDY  GROUPS
A  complete  peri-implant  diagnosis  will  be  performed,  with  peri-implant  probing  and  recording  of  
plaque  and  bleeding  indices.  A  sample  of  crevicular  fluid  will  be  obtained  from  two  implants  for  
each  patient  and  will  be  coded  so  that  it  cannot  be  known.
The  clinical  history  used  in  the  Department  of  Stomatology  III  of  the  Faculty  of  Dentistry  
will  be  taken  first.  The  informed  consent  of  the  patients  for  the  surgical  intervention  will  be  
obtained,  as  well  as  for  the  application  of  melatonin  and  the  obtaining  of  samples  of  crevicular  
fluid  and  digital  images.
7.  METHODS
New  samples  of  crevicular  fluid  will  be  taken  from  the  implants  in  a  coded  manner.  After  21  days,  a  new  
complete  peri-implant  record  will  be  made  with  a  new  crevicular  fluid  sample.  After  60  days,  a  new  radiological  
study  and  a  complete  peri-implant  record  and  crevicular  fluid  sample  will  be  performed.
All  clinical  information  recorded,  as  well  as  the  information  required  to  carry  out  the  study,  will  be  
treated  in  such  a  way  that  the  patient  cannot  be  identified.
13
All  patients  will  be  provided  with  a  24-hour  telephone  number  to  monitor  their  postoperative  status  
and  progress.  Assessment  of  the  patient's  perception  of  inflammation  and  subjective  improvement  
will  be  carried  out  through  communication  with  the  patient.  In  addition,  the  occurrence  of  adverse  
events  will  be  recorded  for  a  period  of  two  months  after  treatment.
To  do  this,  a  history  number  and  a  case  number  will  be  assigned  to  each  patient,  without  using  
any  information  that  would  allow  them  to  be  identified.
Machine Translated by Google

Crevicular  fluid  will  be  obtained  using  sterile  paper  points.  These  will  be  transferred  to  a  
vial  of  reduced  transport  liquid,  which  allows  the  anaerobic  state  of  the  samples  to  be  
preserved.  The  samples  will  be  transported  immediately  after  obtaining  them  to  the  
Research  Laboratory  of  the  Faculty  of  Dentistry  of  the  Complutense  University  of  Madrid.
The  first  sample  of  crevicular  fluid  will  be  obtained  by  direct  extraction  from  the  peri-implant  groove  in  
the  diagnostic  phase.  The  second  sample  of  crevicular  fluid  will  be  obtained  7  days  after  the  intervention.  
Subsequently,
The  samples  will  be  placed  at  room  temperature  at  the  time  the  determinations  are  carried  out  following  the  
instructions  of  the  kit  used.
The  determinations  will  be  read  using  the  ELISA  reader  (Ivymen  System  2100-C).
7.2.  SAMPLE  COLLECTION
stored  in  sterile  13x75mm  x  2  ml  BD  Vacutainer®  tubes.
.  Radiographic  evaluation
The  samples  will  be  transported  immediately  after  collection  to  the  Research  Laboratory  of  the  Faculty  of  Dentistry  
of  the  Complutense  University  of  Madrid.  The  samples  will  not  be  treated  with  any  additives,  so  that  60-90  minutes  
after  collection  they  will  be  processed  using  the  Perio-Tron  device  for  the  analysis  of  interleukins.
To  obtain  the  radiographic  images,  periapical  radiographs  will  be  taken  using  a  parallelizing  device  so  that  they  are  
always  taken  in  14
Crevicular  fluid  samples  at  21  days  and  60  days  after  treatment.
The  samples  are  then  dispersed,  diluted  and  inoculated  into  different  selective  and  enriched  culture  media.  The  
plates  are  incubated  in  the  appropriate  atmosphere  for  the  required  time,  at  which  time  the  plates  are  examined  
for  morphologically  suspicious  colonies.
Crevicular  fluid  will  be  obtained  using  Prio-paper  strips.  The  sample  will  be
They  are  subsequently  isolated  and  identified  using  microscopic  and  biochemical  techniques.
Microbiological  analysis
Enzyme-linked  immunosorbent  assay  (ELISA)
The  first  sample  of  crevicular  fluid  will  be  obtained  by  direct  extraction  from  the  peri-implant  sulcus  during  the  
diagnostic  phase.  The  second  sample  of  crevicular  fluid  will  be  obtained  7  days  after  the  intervention.  Subsequently,  
crevicular  fluid  samples  will  be  obtained  at  21  days  and  60  days  after  treatment.
Machine Translated by Google

The  processing  and  obtaining  of  results  from  crevicular  fluid  samples  will  be
7.3.  ANALYSIS
carried  out  by  the  doctoral  student  at  the  Research  Laboratory  of  the  Faculty  of  Dentistry  of  the  Complutense  
University  of  Madrid.
Dependent  variables  are  those  whose  behavior  is  explained  or  predicted  by  one  or  more  independent  variables.  
Dependent  variables  may  be  considered  to  be  the  concentration  of  interleukin-1ÿ,  2  and  6,  microbiological  
culture  determined  in  crevicular  fluid  samples,  probing  depth,  as  well  as  the  amount  of  bone  determined  in  
diagnostic  imaging  tests.
The  evaluation  of  the  peri-implant  crestal  bone  level  will  be  carried  out  by  evolutionary  comparison  of  
the  bone  level  with  respect  to  the  implant  threads.
The  results  obtained  will  be  compared,  contrasting  the  levels  of  change  in  patients  in  whom  melatonin  
is  administered  versus  control  patients.
prior  to  treatment,  superimposing  the  x-rays  taken  subsequently  in  subsequent  visits.
orthogonal  position  at  the  diagnostic  visit,  at  7  days,  at  21  days  and  at  60  days.  Panoramic  radiographs  will  be  
taken  at  the  diagnostic  appointment  and  at  60  days.
Data  will  be  analyzed  using  the  computer  program  SPSS  (Statistical  Package  for  the  Social  Sciences,  version  
21.0,  SPSS  Inc.,  Chicago,  Ill.,  USA).
Variables
The  independent  variables  studied  will  be  the  characteristics  in  which  the  objects  of  study  differ,  which  can  be  
specified  as  the  case  group,  treated  with  melatonin,  compared  to  the  control  group,  treated  conventionally.
15
The  statistical  processing  of  the  results  obtained  will  be  carried  out  by  the  doctoral  student  under  the  supervision  
of  a  Data  Analyst  specialized  in  Biostatistics.
Machine Translated by Google

There  is  controversy  regarding  the  effects  of  melatonin  described.  It  is  described  as  a  pleiotropic  molecule  whose  
effects  depend  on  the  previous  state  of  the  receptors.  In  this  regard,  there  are  few  previous  studies  detailing  the  
properties  of  this  hormone  in  situations  such  as  those  raised  in  the  present  study  locally  in  patients.  On  the  other  
hand,  systemic  changes  in  the  individual,  as  well  as  their  habits,  can  locally  influence  the  study  variables.
Despite  the  scientific  evidence  available,  there  are  no  previous  studies  on  the  use  of  melatonin  in  a  study  model  
such  as  the  one  proposed.  Nor  are  there  any  studies  using  the  doses  chosen  in  the  present  research  protocol.
Although  the  present  study  model  is  considered  ideal  for  assessing  the  properties  of  melatonin,  there  may  be  
intersubject  and  even  intrasubject  differences  due  to  peri-implant  bone  defects.
This  research  protocol  presents  a  series  of  limitations  that  are  detailed  below.
enough  to  assume  the  results  as  sufficient  scientific  evidence.
First,  since  this  is  a  pilot  study,  the  patient  sample  may  not  be
8.  LIMITATIONS
16
As  regards  methodology,  the  laboratory  processes  and  the  measurement  and  comparison  of  radiographic  images  
will  be  carried  out  by  qualified  personnel.  However,  the  possibility  of  individual  variability  must  be  taken  into  account.
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FEB  
2019
SEA-
SEPT-
SEPT  
2019
MAY  
2019
OCT-
MAR  
2021
SEPT-
MAY  
2019
NOV  
2018
JUN-
DEC  
2019-
NOV  
2019
APR-
FEB  
2020
SEA-
APR-
DEC  
2020
JUN  
2021
DEC  
2018-
JUL  
2019
JAN-
Ethics  
Committee  
Approval  
Request
Writing  the  
Manuscript-
Thesis
Validation  of  
methodology  and  
drugs
Preparation  of  the  
presentation
9.  SCHEDULE
Preparation  and  
correction  of  the  
Protocol
Data  organization
Drug  
Agency  
Approval  
Request
Interpretation  of  
results
Patient  recruitment:  
Intervention,  re-
evaluation  and  
second  re-evaluation
Sample  processing
Measurement  of  radiographic  tests
Purchase  of  kits  
and  
pharmacological  products.
Dissemination  of  
results
Testing  of  materials
17
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21
Machine Translated by Google

Two  digital  panoramic  images  will  also  be  required,  obtained  in  two  visits  and  a  periapical  
series  in  the  four  scheduled  visits.  The  first  panoramic  image  will  be  performed  prior  to  the  
treatment  of  peri-implant  bone  loss,  on  the  same  day  of  the  intervention.  The  second  will  
be  performed  60  days  later  during  the  review  of  the  healing  process.
Aim:
Expected  benefits  for  the  patient  or  society:
The  benefit  is  obtaining  a  natural  substance  that  can  be  used  in  the  same  surgical  session  
to  reduce  inflammation  and  the  risk  of  infection  from  the  very  beginning,  as  well  as  
promoting  the  formation  of  adequate  and  sufficient  bone  in  less  time.
ANNEX  I
Phase  IV  randomized  clinical  trial  study  with  analysis  of  biological  variables.
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Measurement  of  certain  molecules  indicating  inflammation  and  infection  in  crevicular  fluid  using  biochemical  
enzyme  immunoassay  and  culture  techniques  and  measurement  of  bone  quantity  using  a  computerized  system  
for  counting  surfaces  obtained  in  an  orthopantomographic  image  and  periapical  series,  comparing  the  
measurements  with  those  obtained  from  the  control.
The  Research  Project  "EFFECT  OF  MELATONIN  IN  THE  TREATMENT  OF  PERI-
IMPLANT  DISEASES"  aims  to  provide  an  effective  alternative  in  the  treatment  of  peri-
implant  bone  loss  by  measuring  biochemical  and  microbiological  parameters  obtained  
from  crevicular  fluid  samples.  It  also  aims  to  demonstrate  its  usefulness  in  bone  
regeneration,  obtaining  bone  of  adequate  volume  and  density  in  a  shorter  period  of  time  
through  digital  comparison  of  images  obtained  by  orthopantomography  and  periapical  
series.
The  only  inconvenience  is  that  of  attending  the  check-up,  since  obtaining  samples  of  
crevicular  fluid  is  a  procedure  that  is  performed  without  discomfort  for  the  patient.
Methodology  used:
Possible  adverse  events:
Discomforts  and  risks  arising  from  the  study:
INFORMATION  SHEET  FOR  POTENTIAL  PARTICIPANTS  (RD  561/93)
Two  samples  of  crevicular  fluid  will  need  to  be  obtained  from  each  peri-implant  groove,  so  two  samples  will  be  
processed  from  each  side  immediately  before  surgery  and  two  more  from  each  side  at  7,  21  and  60  days  after  
surgery.
Machine Translated by Google

There  is  no  financial  compensation.
Method  of  financial  compensation  and  treatment  in  the  event  of  damage  or  injury  due  to  participation  in  
the  trial,  as  set  out  in  the  Medicines  Law:
Researcher  responsible  for  the  Research  Project  and  for  informing  the  subject  and  answering  his/her  
questions  and  doubts,  and  how  to  contact  him/her  in  case  of  emergency:
The  principal  investigator  of  the  Research  Project  is  Dr.  Carlos  Cobo  Vázquez,  from  the  Faculty  of  Dentistry  of  
the  Complutense  University  of  Madrid.  In  the  Department  of  Stomatology  III  (Medicine  and  Oral  and  Facial  
Surgery),  the  coordinating  investigator  is  Dr.  Juan  López-Quiles  Martínez,  whose  contact  telephone  number  is  91  
394  1964.
Data  protection:
Your  personal  data  will  be  accessed,  with  your  prior  consent,  by  healthcare  professionals  who  are  involved  in  the  
treatment  of  peri-implant  bone  loss  and  in  obtaining  crevicular  fluid  samples  in  the  Department  of  Stomatology  III  
(Buccofacial  Medicine  and  Surgery)  of  the  Faculty  of  Dentistry  at  the  Complutense  University  of  Madrid,  
coordinated  by  Dr.  Carlos  Cobo.  The  data  collected  in  the  Medical  History,  as  well  as  the  results  of  the  study  of  
the  saliva  samples  and  the  panoramic  X-ray  images,  will  be  separated  from  your  personal  identity,  assigning  the  
same  code  to  the  Medical  History  and  the  results  of  the  study,  so  that  it  will  not  be  possible  for  research  staff  
outside  the  Department  of  Stomatology  III  (Buccofacial  Medicine  and  Surgery)  of  the  Faculty  of  Dentistry  to  
associate  them  without  express  authorization.
Participation  is  voluntary,  and  patients  may  withdraw  from  the  study  at  any  time  without  altering  the  
doctor-patient  relationship  or  harming  their  treatment.
The  only  adverse  effects  are  those  inherent  to  the  surgical  procedure  for  treating  peri-implant  bone  loss.  
Sometimes  it  is  not  possible  to  maintain  implants  affected  by  this  disease  and  they  may  therefore  be  removed.
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24
ANNEX  II
INFORMED  CONSENTS  FOR  POTENTIAL  PARTICIPANTS
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"PERIIMPLANTARIAS",  carried  out  by  researchers  from  the  Department  of  Stomatology  III  (Buccofacial  Medicine  
and  Surgery)  of  the  Faculty  of  Dentistry  of  the  Complutense  University  of  Madrid.  The  purpose  of  this  study  is  to  
obtain  an  effective  alternative  in  the  treatment  of  inflammation  and  peri-implant  bone  loss.
of  2018.
Signed  Mr./Mrs.
Madrid,  a
STATEMENT  OF  INFORMED  CONSENT
I  have  also  been  informed  that  my  personal  data  will  be  protected  in  accordance  with  the  provisions  of  Organic  
Law  15/1999,  of  December  13,  on  the  Protection  of  Personal  Data.  The  samples  will  be  anonymized  and  the  
necessary  clinical  data  will  be  requested  from  me  at  the  time  of  the  intervention  and  I  authorize  that  they  be  
consulted  in  my  Medical  History  by  the  Doctor  of  the  Research  Team  of  the  Health  Center.  Only  the  part  of  the  
Medical  History  whose  data  is  relevant  to  the  study  will  be  accessed,  and  in  any  case,  the  Doctors  of  the  Research  
Team  undertake  to  maintain  the  strictest  confidentiality.
(Guardian,  in  case  of  incapacity)
Taking  this  into  consideration,  I  GRANT  my  CONSENT  for  this  extraction  to  take  place  and  be  used  to  meet  the  
objectives  specified  in  the  project.
25
produced  by  peri-implant  infection  with  utility  in  early  bone  regeneration,  obtaining  bone  of  adequate  volume  and  
density  in  a  shorter  period  of  time.
Optional
I  have  been  informed  of  the  possible  harm  that  obtaining  crevicular  fluid  samples  may  have  on  my  well-being  and  
health,  which  are  attributable  to  a  routine  test.
(Patient)
of
Mr./  Mrs........................................................................................................,  aged ........  years  and  with  DNI  
number.........................................................,  declares  that  he/she  has  been  informed  
by............................................................................  about  the  benefits  that  could  be  obtained  from  obtaining  
samples  of  peri-implant  crevicular  fluid  from  each  side  to  cover  the  objectives  of  the  Research  Project  entitled  
"EFFECT  OF  MELATONIN  IN  THE  TREATMENT  OF  DISEASES
Signed  Mr/Mrs.
Machine Translated by Google

to  cover  the  objectives  of  the  Research  Project  entitled  "EFFECT  OF  MELATONIN  IN  THE  TREATMENT  OF  
PERI-IMPLANT  DISEASES  ",  carried  out  by  researchers  from  the  Department  of  Stomatology  III  (Buccofacial  
Medicine  and  Surgery)  of  the  Faculty  of  Dentistry  of  the  Complutense  University  of  Madrid.  The  purpose  of  this  
study  is  to  obtain  an  effective  alternative  in  the  treatment  of  inflammation  and  peri-implant  bone  loss  caused  by  
peri-implant  infection  with  utility  in  early  bone  regeneration,  obtaining  a  bone  of  adequate  volume  and  density  in  
a  shorter  period  of  time.
of
Optional
of  2018.
STATEMENT  OF  INFORMED  CONSENT
Taking  this  into  consideration,  I  GRANT  my  CONSENT  for  this  test  to  take  place  and  be  used  to  meet  the  
objectives  specified  in  the  project.
26
Madrid,  a
I  have  been  informed  of  the  possible  harm  that  performing  two  digital  orthopantomographies  may  have  on  my  
well-being  and  health,  which  are  attributable  to  a  routine  examination.
(Patient)
I  have  also  been  informed  that  my  personal  data  will  be  protected  in  accordance  with  the  provisions  of  Organic  
Law  15/1999,  of  December  13,  on  the  Protection  of  Personal  Data.  The  images  will  be  anonymized  and  the  
necessary  clinical  data  will  be  requested  from  me  at  the  time  of  the  intervention  and  I  authorize  that  they  be  
consulted  in  my  Medical  History  by  the  Doctor  of  the  Research  Team  of  the  Health  Center.  Only  the  part  of  the  
Medical  History  whose  data  is  relevant  to  the  study  will  be  accessed,  and  in  any  case,  the  Doctors  of  the  
Research  Team  undertake  to  maintain  the  strictest  confidentiality.
(Guardian,  in  case  of  incapacity)
Signed  Mr/Mrs.
Mr./  Mrs........................................................................................................,  aged ........  years  and  with  DNI  
number..................................................,  declares  that  he/she  has  been  informed  
by............................................................................  about  the  benefits  that  could  be  derived  from  performing  two  
digital  orthopantomographies  and  periapical  series.
Signed  Mr./Mrs.
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