Effect of Physiotherapy on the Promotion of Bone Mineralization in Preterm Infants
Phase: NA
Conditions: Metabolic Bone Disease
Sponsor: Galaad Torró Ferrero

	    
	  
	  
	  
	  
UNIVERSITY	  OF	  MURCIA	  
	  
INTERNATIONAL	  SCHOOL	  OF	  DOCTORAL	  STUDIES	  OF	  THE	  
UNIVERISTY	  OF	  MURCIA	  (EIDUM)	  
	  
	  
STUDY	  PROTOCOL	  
	  
EFFECT	  OF	  PHYSIOTHERAPY	  ON	  THE	  PROMOTION	  
OF	  BONE	  MINERALIZATION	  IN	  PRETERM	  
INFANTS	  
	  
RESEARCH	  TEAM	  
GALAAD	  TORRÓ	  FERRERO,	  FRANCISCO	  JAVIER	  FERNÁNDEZ	  REGO,	  JUAN	  JOSÉ	  AGÜERA	  ARENAS,	  
JESICA	  PIÑERO	  PEÑALVER,	  MARIA	  DEL	  MAR	  SÁNCHEZ	  JOYA,	  ANTONIA	  GÓMEZ	  CONESA,	  
	  
Contact:	  galaad.torro@um.es	  
University	  of	  Murcia	  
	  
	  
ClinicalTrials.gov	  ID:	  NCT04356807	  
	  
	  
	  
	  
	  
	  
	  
	  
	  MURCIA,	  JULY	  2015	  
	  

	    
INDEX	  
	  
1.-­‐	  INTRODUCTION	  .........................................................................................................................................	  1	  
1.1.-­‐	  BACKGROUND	  ................................................................................................................................................	  1	  
1.2.-­‐	  JUSTIFICATION	  ................................................................................................................................................	  2	  
1.3.-­‐	  OBJECTIVE	  .....................................................................................................................................................	  3	  
2.-­‐	  METHODS	  .................................................................................................................................................	  3	  
2.1.-­‐	  DESIGN	  .........................................................................................................................................................	  3	  
2.2.-­‐	  ELIGIBILITY	  ....................................................................................................................................................	  3	  
2.3.-­‐	  SAMPLE	  SIZE	  ..................................................................................................................................................	  4	  
2.4.-­‐	  ORIGIN	  AND	  INCLUSION	  PROCEDURE	  ...................................................................................................................	  4	  
2.5.-­‐	  GROUPS	  FORMATION	  ......................................................................................................................................	  4	  
2.6.-­‐	  VARIABLES	  ....................................................................................................................................................	  5	  
2.7.-­‐	  TREATMENT	  PROGRAM	  ....................................................................................................................................	  6	  
2.8.-­‐	  STATISTICAL	  ANALYSIS	  ......................................................................................................................................	  7	  
2.9.-­‐	  ETHICAL	  ASPECTS	  ............................................................................................................................................	  7	  
3.-­‐	  EXPECTED	  RESULTS	  ...................................................................................................................................	  7	  
4.-­‐	  REFERENCES	  ..............................................................................................................................................	  8	  
	  

	  
1	  
1.-­‐	  INTRODUCTION	  
1.1.-­‐	  Background	  
According	  to	  the	  World	  Health	  Organization	  (WHO),	  a	  pretemr	  infant	  is	  considered	  to	  be	  any	  
baby	  born	  alive	  before	  37	  weeks	  of	  gestational	  age	  (ga),	  regardless	  of	  birth	  weight1.	  
Prematurity	  is	  the	  neonatal	  condition	  that	  occurs	  most	  frequently,	  carrying	  with	  it	  a	  high	  
risk	  of	  disability2.	  For	  this	  reason,	  the	  WHO	  considers	  prevention	  and	  care	  of	  its	  
complications	  as	  top	  priorities	  for	  premature	  babies3;	  because	  those	  who	  survive	  face	  a	  
lifetime	  of	  significant	  disability	  in	  many	  cases,	  special	  care	  is	  required	  and	  are	  at	  increased	  
risk	  for	  serious	  health	  problems4.	  
In	   this	   sense,	   one	   of	   the	   characteristic	   pathologies	   in	   this	   population	   is	   osteopenia	   of	  
prematurity,	  it	  is	  estimated	  that	  between	  30%	  and	  42%	  of	  premature	  children	  worldwide	  
suffer	   osteopenia5.	   This	   entity	   is	   multifactorial,	   progressive	   and	   of	   variable	   severity,	   of	   a	  
pathogenesis	  not	  yet	  well	  described,	  characterized	  mainly	  by	  a	  bone	  mineral	  deficiency.	  It	  
appears	   in	   30%	   of	   children	   under	   1500	   g	   and	   occurs	   in	   more	   than	   50%	   of	   newborns	  
weighing	  less	  than	  1000	  g5	  
One	   of	   the	   causes	   for	   which	   it	   occurs	   can	   be	   explained	   by	   the	   early	   termination	   of	  
pregnancy.	   It	   is	   estimated	   that	   during	   the	   third	   trimester	   the	   mother	   contributes	   to	   the	  
fetus	  about	  80%	  of	  the	  total	  calcium5.	  In	  this	  way,	  premature	  birth	  deprives	  the	  baby	  from	  
the	   supply	   of	   nutrients,	   proteins	   and	   minerals,	   essential	   for	   the	   formation	   of	   the	   bone	  
matrix5.	  This	  is	  why	  infants	  born	  prematurely,	  even	  in	  the	  last	  trimester	  (30-­‐36	  +	  6	  ga),	  have	  
smaller	  bones	  and	  a	  lower	  level	  of	  bone	  mineralization	  compared	  to	  infants	  born	  at	  term6.	  
In	   this	   sense,	   it	   has	   been	   observed	   in	   different	   studies	   that	   in	   preterm	   infants,	  
mineralization	  is	  much	  lower	  than	  expected	  intrauterine	  bone	  mineralization7.	  It	  has	  been	  
observed	   that	   these	   rates	   of	   poor	   mineralization	   are	   maintained	   in	   children	   and	   young	  
adults	  born	  prematurely8,9,	  a	  situation	  that	  leads	  to	  a	  reduction	  in	  maximum	  bone	  mass,	  
weaker	  bones,	  shorter	  height,	  and	  a	  higher	  rate	  of	  fractures	  in	  comparison	  with	  those	  born	  
at	  term10.	  	  
On	  the	  other	  hand,	  some	  of	  the	  reasons	  why	  an	  osteopenic	  condition	  may	  be	  aggravated	  in	  
the	  premature,	  can	  be	  found	  in	  the	  nutritional	  conditions	  (prolonged	  parenteral	  nutrition);	  
postnatal	  morbidity,	  such	  as	  bronchopulmonary	  dysplasia	  and	  necrotizing	  enterocolitis;	  by	  
diuretic	  drugs;	  by	  steroid	  drugs	  and	  by	  caffeine	  (commonly	  used	  to	  treat	  apnea)11,12.	  Finally,	  
other	  aspects	  that	  influence	  this	  are	  the	  baby's	  own	  inactivity	  caused	  by	  her	  lack	  of	  tone	  and	  
weakness;	  in	  addition	  to	  the	  usual	  care	  in	  relation	  to	  promoting	  development	  established	  in	  
Neonatal	  Intensive	  Care	  Units	  (NICUs),	  which	  include	  minimal	  management	  of	  the	  newborn,	  
with	  nest	  containments	  that	  limit	  their	  movements	  and	  deprive	  them	  of	  both	  sensory	  and	  
physical	  stimuli13.	  
All	   these	   aspects	   cause	   immobility	   of	   the	   baby	   with	   an	   increase	   in	   bone	   resorption,	  
decreased	  bone	  formation,	  and	  therefore	  a	  deficiency	  in	  mineral	  density	  that	  can	  lead	  to	  a	  
picture	  of	  osteopenia14.	  	  	  

	  
2	  
Bone	   is	   a	   living,	   dynamic	   tissue	   that	   is	   continually	   creating	   and	   destroying	   itself.	   This	  
dynamism	   is	   mediated	   by	   the	   action	   of	   osteoblasts,	   responsible	   for	   formation	   and	  
osteoclasts,	   responsible	   for	   resorption	   (destruction).	   The	   amount	   of	   bone	   mass	   that	   an	  
individual	  has	  at	  a	  given	  time	  will	  depend	  on	  the	  proportion	  between	  these	  activities,	  that	  
is,	  on	  the	  rate	  of	  bone	  turnover15.	  Under	  normal	  conditions,	  bone	  metabolism	  is	  in	  balance	  
to	  maintain	  the	  mass	  and	  microstructure	  of	  the	  skeleton.	  In	  this	  sense,	  quantitative	  changes	  
in	  bone	  metabolism	  can	  be	  assessed	  by	  measuring	  biochemical	  markers	  of	  bone	  remodeling	  
in	  serum	  or	  urine,	  resulting	  from	  activity	  in	  bone	  throughout	  the	  entire	  skeleton.	  In	  contrast,	  
bone	  mass	  measurements	  and	  radiographs	  provide	  a	  static	  picture	  of	  a	  specific	  site	  of	  the	  
skeleton.	  
1.2.-­‐	  Justification	  	  
An	  estimated	  15	  million	  babies	  are	  born	  worldwide	  each	  year	  before	  37	  ga,	  that	  is,	  more	  
than	  10%	  of	  babies	  born	  are	  premature16.	  In	  almost	  all	  countries	  with	  reliable	  data,	  the	  rate	  
of	   premature	   births	   is	   increasing.	   Of	   these	   countries,	   all	   but	   three	   have	   registered	   an	  
increase	   in	   prematurity	   rates	   in	   the	   last	   20	   years.	   An	   estimated	   8.6%	   of	   live	   births	   in	  
developed	  countries	  are	  premature17.	  
In	  Spain,	  we	  have	  gone	  from	  having	  in	  1996	  a	  5.9%	  of	  preterm	  births	  to	  6.5%	  in	  2013,	  with	  a	  
total	  of	  27,015	  preterm	  births,	  according	  to	  data	  from	  the	  National	  Institute	  of	  Statistics18.	  
Of	  which,	  most	  are	  premature	  32-­‐36	  sg	  
Despite	  the	  fact	  that	  the	  data	  on	  prematurity	  have	  been	  reduced	  since	  2008,	  they	  are	  still	  a	  
high	   number	   if	   we	   think	   about	   the	   cost	   of	   premature	   births.	   In	   the	   United	   States,	   the	  
average	  medical	  cost	  of	  the	  first	  year	  of	  life	  for	  premature	  babies,	  including	  both	  hospital	  
and	   outpatient	   care,	   was	   about	   10	   times	   higher	   for	   premature	   babies	   than	   for	   full-­‐term	  
newborns.	  In	  addition,	  hospital	  stay	  was	  also	  higher	  on	  average	  in	  the	  premature	  than	  in	  the	  
full-­‐term	  newborn	  (about	  six	  times	  higher	  on	  average).	  In	  total,	  the	  cost	  of	  hospital	  stay	  for	  
premature	  babies	  reaches	  5.8	  billion	  dollars,	  which	  represents	  47%	  of	  the	  cost	  of	  all	  infant	  
hospitalizations	  and	  27%	  of	  all	  pediatric	  hospitalizations19.	  On	  the	  other	  hand,	  the	  rates	  of	  
large	  premature	  babies	  have	  not	  been	  reduced,	  but	  have	  been	  maintained	  over	  time.	  
Taking	  these	  data	  into	  account,	  it	  is	  necessary	  to	  develop	  therapeutic	  strategies	  that	  address	  
the	  morbidity	  associated	  with	  prematurity.	  
As	   previously	   mentioned,	   osteopenia	   is	   one	   of	   the	   characteristic	   pathologies	   of	   the	  
premature	  population.	  In	  this	  sense,	  mechanical	  stress	  has	  been	  shown	  to	  be	  one	  of	  the	  
most	   stimulating	   factors	   in	   bone	   formation	   and	   growth,	   thus	   increasing	   bone	   mass	   in	  
children,	   adolescents	   and	   adults20,21;	   while	   the	   inactivity	   facilitated	   by	   the	   usual	  
management	   practices	   for	   the	   newborn	   favors	   bone	   resorption	   and	   a	   decrease	   in	   bone	  
mineral	  density22.	  
Currently,	  Physiotherapy	  has	  proven	  to	  be	  one	  of	  the	  therapeutic	  resources	  with	  which	  the	  
best	  results	  have	  been	  obtained	  in	  the	  approach	  to	  osteopenia	  in	  the	  premature	  infant.	  The	  
physiotherapy	   techniques	   used	   in	   the	   treatment	   of	   this	   clinical	   entity	   have	   been	   mainly	  

	  
3	  
based	  on	  the	  use	  of	  passive	  mobilizations	  with	  joint	  pressure,	  described,	  obtaining	  favorable	  
results.23.	  
The	  results	  of	  some	  studies	  show	  that,	  for	  the	  treatment	  of	  osteopenia,	  active	  mobilizations	  
will	  be	  more	  effective	  than	  passive	  ones24,	  and	  to	  achieve	  the	  latter,	  we	  will	  need	  methods	  
that	  do	  not	  require	  the	  subject's	  will	  to	  achieve	  active	  movement,	  due	  to	  the	  young	  age	  of	  
the	   same.	   In	   this	   sense,	   reflex	   locomotion	   therapy	   (RLT)25	   may	   be	   a	   suitable	   method	   to	  
achieve	   this	   goal,	   since	   it	   generates	   involuntary	   activity,	   developing	   an	   active-­‐resisted	  
movement	  in	  the	  population	  of	  preterm	  infants.	  
With	   the	   application	   of	   this	   technique,	   we	   achieve	   a	   stimulation	   of	   the	   central	   nervous	  
system	  (CNS),	  and	  an	  activation	  of	  the	  locomotion	  patterns	  of	  the	  first	  year	  of	  life.	  These	  
patterns	   are	   triggered	   reflexively,	   placing	   the	   baby	   in	   certain	   positions,	   and	   generating	  
proprioceptive	   stimuli	   through	   the	   pressure	   exerted	   by	   our	   fingers	   on	   specific	   areas25–27,	  
thus	  producing	  a	  series	  of	  synergistic	  muscle	  contractions	  in	  an	  exactly	  defined	  coordination	  
for	  all	  the	  skeletal	  muscles,	  also	  producing	  a	  specific	  active	  movement25–27.	  This	  is	  because	  
through	  RLT,	  CNS	  activation	  takes	  place	  from	  the	  spinal	  level	  to	  the	  subcortical	  and	  cortical	  
areas27.	  
Knowing	  the	  effect	  of	  RLT,	  we	  think	  that	  it	  can	  be	  effective	  in	  the	  treatment	  of	  osteopenia,	  
since	   by	   applying	   it,	   we	   would	   be	   causing	   an	   active-­‐resisted	   muscular	   contraction	   of	   the	  
entire	  body,	  which	  is	  what	  is	  intended	  in	  any	  treatment	  for	  osteoporosis.	  .	  Although	  these	  
are	  reflexive	  and	  non-­‐voluntary	  active	  mobilizations,	  they	  are	  perhaps	  more	  effective	  than	  
passive	  mobilizations.	  
1.3.-­‐	  Objective	  
To	  determine	  whether	  RLT	  is	  effective	  for	  the	  prevention	  of	  osteopenia	  in	  preterm	  infants	  
and	  compare	  its	  effectiveness	  over	  other	  physiotherapeutic	  methods	  
2.-­‐	  METHODS	  
2.1.-­‐	  Design	  
Multicenter	  randomized	  clinical	  trial,	  consisting	  of	  three	  intervention	  groups,	  two	  treatment	  
and	  one	  control;	  and	  three	  measures:	  pre,	  inter	  and	  post.	  
This	   project	   will	   be	   carried	   out	   at	   the	   Virgen	   de	   la	   Arrixaca	   University	   Hospital,	   the	  
Torrecárdenas	   Hospital	   in	   Almería	   and	   the	   Hospital	   General	   Universitario	   in	   Elche.	   The	  
Virgen	  de	  la	  Arrixaca	  University	  Hospital	  is	  the	  coordinating	  center	  of	  the	  study.	  
2.2.-­‐	  Eligibility	  
Inclusion	  Criteria:	  
• Preterm	  infants	  
• 26	  to	  34	  weeks	  of	  gestational	  age	  
• Admitted	  in	  neonates	  

	  
4	  
• Hemodynamically	  stable	  
• Complete	  enteral	  nutrition	  
• Parents	  or	  guardians	  signed	  an	  informed	  consent	  authorizing	  the	  participation	  of	  the	  
baby	  in	  this	  study.	  
Exclusion	  Criteria:	  
• Neurological	  disorders	  
• Mechanical	  ventilation	  
• Bronchopulmonary	  dysplasia	  
• Congenital	  malformations	  
• Metabolic	  diseases	  
• Genetic	  diseases	  
• Intraventricular	  hemorrhage	  III-­‐IV,	  
• Diuretic	  medication	  or	  corticosteroids	  
• Bone	  fractures	  at	  the	  time	  of	  inclusion.	  
2.3.-­‐	  Sample	  size	  
Sample	  size	  has	  been	  calculated	  considering	  the	  f2	  with	  Cohen’s	  criteria,	  using	  a	  f2	  value	  
between	   moderated	   and	   low	   0.15,	   between	   0,10	   (low)	   and	   0,25	   (moderated).	   We	   will	  
assume	  a	  5%	  significance	  level,	  a	  80%	  statistical	  power	  with	  three	  levels	  for	  each	  ones	  of	  the	  
factors	  iner	  and	  intra	  group.	  This	  way	  it	  is	  projected	  a	  sample	  size	  of	  93	  participants,	  31	  per	  
group.	   The	   software	   program	   was	   nQuery	   Advisor	   version	   7.028,29.	   We	   use	   this	   method	  
because	  there	  are	  no	  previous	  studies	  with	  tree	  groups30.	  
Assuming	  possible	  deviations	  from	  these	  premises,	  we	  opted	  for	  a	  sample	  of	  33	  patients	  per	  
group.	  As	  the	  present	  study	  requires	  three	  treatment	  groups,	  the	  final	  sample	  size	  will	  be	  99	  
participants	  who,	  distributed	  among	  the	  three	  hospitals,	  give	  us	  a	  sample	  of	  33	  babies	  per	  
hospital.	  For	  its	  part,	  each	  hospital	  will	  have	  the	  same	  number	  of	  participants	  per	  treatment	  
group,	  so	  11	  patients	  will	  be	  distributed	  by	  each	  group	  in	  each	  hospital.	  
2.4.-­‐	  Origin	  and	  inclusion	  procedure	  
All	  subjets	  will	  be	  infants	  admitted	  to	  the	  Virgen	  de	  la	  Arrixaca	  University	  Hospital	  (HUVA),	  
the	   Hospital	   Torrecárdenas	   of	   Almería	   Hospital	   and	   the	   Hospital	   General	   Universitario	   of	  
Elche,	  dividing	  the	  population	  into	  groups	  of	  33	  patients	  for	  each	  hospital.	  
2.5.-­‐	  Groups	  formation	  
The	   groups	   will	   be	   formed	   by	   simple	   randomization.	   The	   randomization	   procedure	   will	  
consist	   of	   sealed	   envelope	   labels	   containing	   a	   number	   for	   each	   group.	   A	   non-­‐research	  
person	  drew	  a	  random	  number	  from	  the	  envelope	  each	  time	  a	  new	  patient	  was	  proposed	  
for	   treatment	   and	   made	   the	   assignment.	   For	   ethical	   reasons,	   twins	   and	   triplets	   were	  
assigned	  to	  the	  same	  group	  with	  the	  same	  number.	  

	  
5	  
2.6.-­‐	  Variables	  	  
Those	   variables	   that	   we	   intend	   to	   measure	   in	   our	   study	   are	   bone	   mineralization,	   bone	  
formation,	   bone	   resorption	   and	   anthropometry.	   The	   main	   variable	   being	   bone	  
mineralization	  obtained	  with	  the	  tibial	  sound	  velocity.	  
In	   order	   to	   measure	   bone	   mineralization,	   the	   tibial	   speed	   of	   sound	   (Tibial-­‐SOS)	   will	   be	  
recorded	  using	  a	  quantitative	  ultrasound	  device	  (QUS)	  (Sunlight	  Omnisense	  7000)31–33.	  It	  will	  
be	   measured	   on	   the	   third	   lower	   part	   of	   the	   left	   tibia,	   keeping	   the	   knee	   bended	   to	   a	   90	  
degree	  angle.	  The	  measurement	  point	  is	  made	  perpendicular	  to	  the	  direction	  of	  the	  bone.	  
Three	   to	   five	   consecutive	   measurements	   are	   going	   to	   be	   made,	   and	   the	   average	   will	   be	  
calculated	  to	  determine	  the	  Tibial-­‐SOS	  (m/s).	  This	  variable	  will	  only	  be	  taken	  at	  the	  Virgen	  
de	  la	  Arrixaca	  University	  Hospital.	  
To	   measure	   bone	   formation	   and	   resorption,	   we	   will	   use	   serum	   and	   urine	   biomarkers,	  
respectively,	  to	  reveal	  analytical	  data	  on	  the	  speed	  of	  bone	  formation	  and	  its	  metabolism34.	  
Specifically,	   in	   bone	   formation,	   we	   will	   use	   bone-­‐specific	   phosphatase	   markers,	   and	  
osteocalcin	   markers,	   since	   at	   present,	   they	   are	   the	   most	   sensitive	   formation	   markers15.	  
Resorption	  biomarkers	  are	  of	  special	  interest	  to	  us,	  because	  it	  is	  observed	  that	  osteopenia	  is	  
more	  caused	  by	  bone	  resorption7,	  therefore	  studying	  this	  variable	  is	  of	  great	  interest.	  We	  
will	   specifically	   use	   the	   N-­‐telopeptides	   of	   collagen	   bonds	   (NTx)	   and	   Beta-­‐cross	   Laps	   (BC)	  
since	  they	  are	  the	  most	  sensitive	  and	  specific	  markers	  to	  measure	  this	  quality15,35.	  
Tests	  of	  the	  urine	  biomarkers	  and	  the	  Tibial	  SOS	  will	  be	  carried	  out	  one	  day	  before	  starting	  
treatment	  sessions,	  two	  weeks	  laterand	  at	  the	  end	  of	  treatment.	  The	  Tibial	  SOS,	  since	  it	  is	  
probable	  that	  in	  the	  other	  hospitals	  they	  do	  not	  have	  this	  measurement	  system,	  it	  will	  only	  
be	  carried	  out	  in	  the	  HUCVA.	  
Serum	  biomarker	  tests	  will	  be	  performed	  one	  day	  before	  starting	  sessions	  and	  at	  the	  end	  of	  
treatment.	  The	  serum	  biomarkers,	  during	  the	  hospitalization	  period,	  will	  always	  be	  taken	  
coinciding	  with	  blood	  collection	  guidelines	  already	  ordered	  by	  their	  doctor,	  in	  any	  case	  will	  
blood	  samples	  be	  taken	  exclusively	  for	  the	  purposes	  of	  this	  study.	  
Then	  for	  anthropometry,	  measurements	  on	  weight,	  length	  and	  head	  circumference	  will	  be	  
collected	  with	  a	  scale	  and	  a	  tape	  measure,	  these	  being	  the	  same	  for	  all	  babies	  and	  for	  all	  
measurements.	  Anthropometric	  measures	  will	  be	  taken	  from	  one	  day	  before	  starting	  the	  
treatment	  to	  one	  day	  after	  finishing	  it,	  collecting	  them	  in	  alternating	  days,	  according	  to	  the	  
nursing	  protocol,	  and	  will	  be	  carried	  out	  by	  such	  staff.	  For	  our	  analysis,	  we	  will	  use	  those	  
that	  coincide	  with	  the	  day	  the	  Tibial-­‐SOS	  and	  biomarkers	  are	  measured,	  or,	  failing	  that,	  the	  
last	  measurement	  made	  before	  that	  day.	  
All	  the	  personnel	  in	  charge	  of	  carrying	  out	  the	  measurement	  tests	  are	  external	  to	  the	  study	  
and	   will	   be	   masked	   to	   which	   intervention	   group	   the	   patients	   belonged	   to.	   Likewise,	  
participants,	  family,	  and	  data	  analysts	  will	  be	  also	  masked.	  The	  physiotherapists	  in	  charge	  of	  
performing	  the	  treatments	  will	  be	  masked	  against	  the	  objectives	  of	  the	  study.	  

	  
6	  
The	  data	  will	  be	  collected	  in	  digital	  format	  on	  a	  computer,	  where	  it	  will	  be	  stored	  in	  external	  
memory	   and	   where	   the	   name	   and	   data	   of	   the	   patients	   will	   be	   protected	   in	   code,	   to	  
guarantee	  their	  confidentiality.	  
2.7.-­‐	  Treatment	  program	  
The	   program	   will	   be	   applied	   the	   same	   in	   all	   centers,	   as	   described	   below,	   by	   different	  
physical	   therapists	   (one	   in	   each	   hospital)	   with	   experience	   in	   the	   different	   treatment	  
modalities.	   For	   a	   better	   standardization	   of	   the	   study	   in	   all	   hospitals,	   regarding	   the	  
nutritional	  aspect,	  the	  recommendations	  proposed	  in	  the	  protocols	  of	  the	  Spanish	  Society	  
of	  Neonatology	  (SENeo)	  will	  be	  followed,	  with	  the	  variations	  that	  may	  occur	  in	  terms	  of	  
the	  specific	  characteristics	  of	  each	  patient.	  
The	  participants	  in	  this	  study	  will	  be	  divided	  into	  three	  groups	  that,	  along	  with	  the	  usual	  
nursing	  care,	  will	  recieve	  different	  Physiotherapy	  treatments.	  
Control	  group	  (CG),	  will	  be	  given	  limb	  and	  core	  massage,	  with	  gentle	  deep	  pressures	  and	  
caresses;	  lasting	  15	  minutes	  a	  day	  in	  a	  single	  Physiotherapy	  session,	  5	  days	  per	  week,	  for	  4	  
weeks;	   considering	   it	   a	   placebo	   since	   this	   intervention	   has	   no	   influence	   on	   bone	  
mineralization36–41.	  
Experimental	   group	   (EGpmc),	   with	   passive	   movements	   with	   gentle	   compression	   (PMC),	  
described	  by	  Moyer-­‐Mileur,	  et	  al.23	  and	  with	  the	  adaptations	  of	  Vignochi,	  et	  al.42	  in	  a	  15	  
minutes	  Physiotherapy	  session,	  5	  days	  per	  week	  for	  4	  weeks.	  These	  mobilizations	  consist	  of	  
flexion	  and	  extension	  movements	  in	  all	  the	  joints	  of	  both	  the	  upper	  and	  lower	  extremities	  
and	  ending	  with	  chest	  movements	  following	  the	  baby's	  respiratory	  pace.	  
Experimental	  group	  (EGrlt),	  with	  RLT	  according	  to	  the	  procedures	  used	  by	  other	  authors25–
27,	  for	  16	  minutes	  divided	  into	  two	  Physiotherapy	  sessions	  of	  8	  minutes	  each,	  5	  days	  per	  
week	  for	  4	  weeks.	  The	  exercises	  corresponding	  to	  the	  motor	  complexes	  of	  the	  1st	  phase	  of	  
reflex	  rolling	  and	  the	  reflex	  creeping	  will	  be	  performed,	  dedicating	  one	  minute	  to	  each	  side	  
and	  performing	  two	  repetitions	  in	  each	  session.	  
For	   the	   1st	   phase	   of	   reflex	   rolling,	   the	   child	   is	   placed	   in	   dorsal	   decubitus,	   with	   the	   head	  
turned	  to	  one	  side	  at	  an	  angle	  of	  30º,	  the	  spine	  as	  aligned	  as	  possible,	  and	  the	  limbs	  relaxed.	  
The	  physiotherapist	  makes	  gentle	  pressure	  with	  his	  thumb,	  at	  the	  point	  of	  intersection	  of	  
the	   mammillary	   line	   with	   the	   diaphragm,	   between	   the	   6th-­‐7th	   intercostal	   space,	   in	   the	  
hemithorax	   on	   the	   side	   towards	   which	   the	   head	   rotates,	   with	   a	   dorsal-­‐medial-­‐cranial	  
direction,	  while	  resisting	  with	  the	  other	  hand	  the	  turning	  of	  the	  head	  towards	  the	  other	  
side25.	  
For	   reflex	   creeping,	   the	   child	   is	   placed	   proned,	   passively	   bringing	   the	   head	   to	   axial	   neck	  
extension	  and	  30	  degrees	  of	  rotation.	  The	  upper	  limb,	  on	  the	  side	  which	  the	  head	  is	  turned,	  
is	  placed	  in	  a	  position	  of	  shoulder	  flexion	  between	  120	  degrees	  and	  135	  degrees,	  with	  30	  
degrees	   of	   abduction,	   leaving	   the	   epitrochlea	   supported;	   the	   wrist	   is	   aligned	   with	   the	  
shoulder,	  the	  forearm	  rests	  on	  the	  palmar	  face,	  and	  the	  longitudinal	  axis	  of	  the	  humerus	  

	  
7	  
points	   towards	   the	   vertex	   of	   the	   lumbosacral	   hinge.	   The	   opposite	   arm	   is	   placed	   relaxed	  
parallel	  towards	  the	  longitudinal	  axis	  of	  the	  body.	  
The	  leg	  on	  the	  side	  where	  the	  child's	  head	  is	  turned,	  is	  supported	  extended	  and	  relaxed.	  The	  
other	  leg	  is	  placed	  with	  the	  hip	  in	  external	  rotation	  and	  abduction,	  leaving	  the	  support	  on	  
the	  internal	  condyle	  of	  the	  femur,	  the	  knee	  slightly	  flexed,	  and	  the	  heel	  aligned	  with	  the	  
ischium.	  The	  stimulation	  is	  carried	  out,	  with	  the	  index	  finger	  of	  one	  hand,	  on	  the	  lateral	  
tuberosity	  of	  the	  calcaneus,	  in	  the	  ventral-­‐cranial-­‐medial	  direction	  of	  the	  leg	  opposite	  to	  the	  
turn	  of	  the	  head,	  and	  with	  the	  index	  finger	  of	  the	  other	  hand,	  on	  the	  epitrochlea	  of	  the	  arm	  
towards	  which	  the	  head	  is	  turned,	  in	  a	  dorsal-­‐medial-­‐cranial	  direction43.	  
2.8.-­‐	  Statistical	  analysis	  
The	  qualitative	  baseline	  sex	  characteristics	  of	  the	  infants	  will	  be	  compared	  using	  a	  crosstab	  
and	  a	  Chi-­‐square	  test	  for	  its	  analysis.	  Quantitative	  variables	  (gestational	  age,	  birth	  weight,	  
gestational	  age	  at	  baseline,	  weight,	  height,	  head	  circumference,	  Tibial-­‐SOS	  at	  baseline	  and	  
serum	  and	  urine	  biomarkers	  at	  baseline),	  are	  going	  to	  be	  analized	  performing	  a	  one-­‐way	  
analysis	  of	  variance.	  Repeated	  measures	  analysis	  of	  variance	  will	  be	  performed	  to	  compare	  
the	  effect	  of	  the	  intervention	  on	  anthropometric,	  Tibial-­‐SOS	  and	  biomarkers	  measures	  using	  
the	  time	  of	  measurement	  as	  the	  intra-­‐subject	  factor	  and	  the	  treatment	  group	  as	  the	  inter-­‐
subject	  factor.	  All	  analyzes	  will	  be	  performed	  using	  SPSS	  (Statistical	  Package	  for	  the	  Social	  
Sciences)	  for	  Windows	  (v.22.0)44.	  Statistical	  analysis	  with	  intention	  to	  treat	  is	  going	  to	  be	  
performed	  for	  all	  variables.	  Statistical	  significance	  is	  stipulated	  with	  p	  <	  0.05.	  For	  the	  effect	  
size,	   the	   partial	   eta	   square	   (ηp
2)	   will	   be	   calculated,	   considering	   a	   value	   >	   0.14	   as	   high;	  
moderate	   with	   values	   between	   0.14	   and	   0.06;	   and	   small	   values	   between	   0.06	   and	  
0.0129,45,46.	  Data	  will	  be	  presented	  as	  mean	  ±	  standard	  deviation.	  
2.9.-­‐	  Ethical	  aspects	  
All	  parents	  or	  guardians	  of	  infants	  participating	  in	  the	  study	  must	  sign	  the	  informed	  consent	  	  
The	  project	  will	  follow	  the	  Declarations	  of	  the	  1975	  Helsinki	  World	  Medical	  Association	  and	  
must	  be	  approved	  by	  the	  ethical	  committee	  of	  each	  hospital,	  that	  is,	  of	  the	  Virgen	  de	  la	  
Arrixaca	  University	  Hospital,	  the	  Torrecárdenas	  Hospital	  in	  Almería	  and	  the	  Hospital	  General	  
Universitario	  of	  Elche.	  All	  parents/guardians	  will	  be	  informed	  about	  the	  nature	  of	  the	  study,	  
voluntary	  participation	  in	  it,	  the	  proposed	  objectives,	  as	  well	  as	  any	  possible	  adverse	  effects	  
that	  may	  take	  place	  in	  its	  conduct.	  Before	  beginning	  the	  study,	  each	  parent	  or	  guardian	  will	  
be	  asked	  to	  consent	  to	  participate	  in	  the	  study.	  The	  baby	  will	  leave	  the	  study	  at	  any	  time,	  if	  
the	  parent	  or	  guardian	  so	  wishes.	  
Those	  patients	  whose	  parents	  or	  guardians	  do	  not	  sign	  the	  informed	  consent	  will	  not	  be	  
able	  to	  participate	  in	  the	  study.	  
In	  the	  study,	  the	  ethical	  principles	  of	  beneficence,	  non-­‐maleficence,	  justice	  and	  autonomy	  
and	  those	  principles	  dictated	  by	  the	  Clinical	  Research	  Ethics	  Committee	  will	  be	  followed	  at	  
all	  times.	  
3.-­‐	  EXPECTED	  RESULTS	  

	  
8	  
With	   the	   completion	   of	   this	   research,	   it	   is	   intended	   to	   demonstrate	   that	   children	   who	  
receive	   Physiotherapy	   treatment	   with	   RLT	   have	   a	   better	   improvement	   in	   bone	  
mineralization	  and	  resorption	  than	  children	  who	  receive	  other	  types	  of	  treatment	  and	  who	  
have	  homogeneous	  characteristics.	  
4.-­‐	  REFERENCES	  
1.	  	  
World	  Health	  Organization.	  www.who.int.	  Published	  2015.	  
2.	  	  
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DC,	  Anthony	  Costello,	  Simon	  Cousens,	  Ruth	  Davidge,	  Joseph	  de	  Graft	  Johnson,	  Sohni	  
Dean	  CH,	  Ayesha	  Iman,	  Joanne	  Katz,	  Matthais	  Keller,	  Mary	  Kinney,	  Eve	  Lackritz,	  Zhora	  
Lassi,	  Joy	  Lawn	  EM,	  Ramkumar	  Menon,	  Mario	  Merialdi,	  Ann-­‐Beth	  Moller,	  Elizabeth	  
Molyneaux,	  Mikael	  Oestergaard	  VP,	  Jennifer	  Requejo,	  Lale	  Say,	  Joel	  Segre	  S	  von	  X.	  
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7.	  	  
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8.	  	  
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9.	  	  
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peak	  bone	  mass:	  20	  year	  follow-­‐up	  of	  a	  randomized	  trial	  of	  early	  diet	  in	  infants	  born	  
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10.	  	  
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11.	  	  
Backström	  MC,	  Kuusela	  A-­‐L,	  Mäki	  R.	  Metabolic	  Bone	  Disease	  of	  Prematurity.	  Ann	  
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12.	  	  
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phosphatase	  and	  serum	  inorganic	  phosphate	  in	  metabolic	  bone	  disease	  of	  
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13.	  	  
Cabral	  LA,	  Velloso	  M.	  Comparing	  the	  effects	  of	  minimal	  handling	  protocols	  on	  the	  
physiological	  parameters	  of	  preterm	  infants	  receiving	  exogenous	  surfactant	  therapy.	  

	  
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Blencowe	  H,	  Cousens	  S,	  Oestergaard	  MZ,	  et	  al.	  National,	  regional,	  and	  worldwide	  
estimates	  of	  preterm	  birth	  rates	  in	  the	  year	  2010	  with	  time	  trends	  since	  1990	  for	  
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45.	  	  
Cárdenas	  Castro	  JM.	  Potencia	  estadística	  y	  cálculo	  del	  tamaño	  del	  efecto	  en	  G*Power:	  
complementos	  a	  las	  pruebas	  de	  significación	  estadística	  y	  su	  aplicación	  en	  psicología.	  
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46.	  	  
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2007;39(2):175-­‐191.	  doi:10.3758/BF03193146