Cellular Proliferation, Dermal Repair, and Microbiological Effectiveness of Ultrasound Assisted Wound Debridement (UAW) Versus Standard Wound Treatment in Complicated Diabetic Foot Ulcers (DFU): A Randomized Controlled Trial
Phase: NA
Conditions: Diabetic Foot Ulcer
Sponsor: Universidad Complutense de Madrid

STUDY	  PROTOCOL	  	  AND	  STATISTICAL	  ANALYSIS	  PLAN	  
	  
	  
Document	  date:	  20.01.2020	  
	  
Title	  
study:	  
Cellular	   Proliferation,	   Dermal	   Repair,	   and	   Microbiological	  
Effectiveness	  of	  Ultrasound-­‐AssistedWound	  Debridement	  (UAW)	  Versus	  Standard	  
Wound	   Treatment	   in	   Complicated	   Diabetic	   Foot	   Ulcers	   (DFU):	   An	   Open-­‐Label	  
Randomized	  Controlled	  Trial.	  
	  
An	   open-­‐label	   randomized	   and	   controlled	   parallel	   clinical	   trial	   was	   performed	  
involving	  51	  outpatients	  with	  complicated	  DFU	  that	  were	  admitted	  to	  specialized	  
diabetic	  foot	  unit	  between	  November	  2017	  to	  December	  2019.	  This	  study	  protocol	  
received	  full	  approval	  from	  the	  local	  Ethics	  Committee	  of	  the	  Hospital	  Clínico	  San	  
Carlos,	  Madrid,	  Spain	  (C.P.-­‐C.I.	  16/484-­‐P).	  All	  patients	  provided	  written	  informed	  
consent	   before	   inclusion.	   The	   present	   study	   was	   registered	   retrospectively	   in	  
ClinicalTrial.gov	  (Registration	  no.:	  NCT04633642).	  
	  
2.	  Methods	  
2.1.	  Trial	  Design	  
An	   open-­‐label	   randomized	   and	   controlled	   parallel	   clinical	   trial	   was	   performed	  
involving	  51	  outpatients	  with	  complicated	  DFU	  that	  were	  admitted	  to	  specialized	  
diabetic	  foot	  unit	  between	  November	  2017	  to	  December	  2019.	  	  
	  
2.2.	  Participant	  
We	  enrolled	  patients	  in	  which	  the	  following	  inclusion	  criteria	  were	  implemented:	  
•	  Male	  and	  female	  patients	  over	  18	  years	  old	  
Type	  1	  or	  type	  2	  diabetes	  with	  levels	  of	  HbA1c	  ≤85.8	  mmol/mol	  (10%)	  within	  30	  
days	  of	  the	  beginning	  of	  the	  study,	  based	  on	  a	  previous	  international,	  multicenter,	  
randomized	  controlled	  trial	  [11]	  
•	  Wound	  stages	  IB,	  IIB,	  ID,	  and	  IID	  according	  to	  the	  University	  of	  Texas	  Diabetic	  
Wound	  Classification	  [12]	  
•	  Wound	  duration	  of	  1–24	  months	  
•	  Wound	  size	  among	  1–30	  cm2	  after	  debridement	  

•	  Diabetic	  foot	  ulcers	  showing	  mild	  or	  moderate	  infection,	  according	  to	  the	  criteria	  
of	  the	  European	  Wound	  Management	  Association	  (EWMA)	  [13]	  and	  the	  Infectious	  
Disease	  Society	  of	  America	  Guidelines	  [14]	  
•	  Ankle-­‐brachial	  index	  (ABI)	  >	  0.9	  or	  ABI	  ≤	  0.9	  and	  ankle	  systolic	  blood	  pressure	  
(ASBP)	  "	  70	  mmHg,	  or	  toe	  systolic	  blood	  pressure	  (TSBP)	  50	  mmHg.	  In	  patients	  
with	   medial	   arterial	   calcification	   (ABI	   >	   1.4)	   we	   considered	   Peripheral	   Arterial	  
Disease	  (PAD)	  a	  toe–brachial	  index	  (TBI)	  <	  0.7	  [15,16]	  
	  
We	  considered	  exclusion	  criteria:	  
•	   Chronic	   kidney	   disease	   (glomerular	   filtration	   rate	   <	   60mL/min	   per	   1.73	   m2	  
during	  at	  least	  three	  months)	  or	  dialysis	  [17]	  
•	  Non-­‐treated	  osteomyelitis	  
•	  Necrotizing	  soft	  tissue	  infections	  
•	   Critical	   limb	   ischemia	   patients	   with	   ABI	   <	   	   0.5	   and	   ASBP	   <	   70mmHg	   or	   <	  
50mmHg	  [15,16]	  
	  
Life	  expectancy	  <	  6	  months	  due	  to	  malignant	  DFU	  
•	  Pregnancy	  and	  lactation	  
•	  Patients	  diagnosed	  with	  human	  immunodeficiency	  virus	  (HIV)	  or	  hepatitis	  
•	   Patients	   showing	   local	   or	   systemic	   conditions	   that	   could	   impair	   tissue	  
regeneration.	  
	  
2.3.	  DFU	  Assessment	  
A	  senior	  clinician	  in	  the	  management	  of	  diabetic	  foot	  (F.J.Á.-­‐A.)	  always	  carried	  the	  
baseline	   assessment	   of	   patients’	   DFU.	   Sensorimotor	   neuropathy	   of	   DFUs	   was	  
diagnosed	  using	  a	  biothesiometer	  (both	  from	  Novalab	  Iberica,	  Madrid,	  Spain)	  and	  
Semmes-­‐Weinstein	  5.07/10g	  monofilament.	  Patients	  who	  did	  not	  feel	  one	  of	  the	  
two	  tests	  were	  diagnosed	  with	  neuropathy	  [18,19].	  
Brachial	  and	  ankle	  systolic	  pressure	  were	  evaluated	  using	  a	  manual	  8MHz	  Doppler	  
(Doppler	   II,	   Huntleigh	   Healthcare	   Ltd.,	   Cardi↵,	   UK).	   Toe	   systolic	   pressure	   was	  
taken	  via	  digital	  plethysmography	  (Systoe,	  Atys	  Medical,	  Madrid,	  Spain).	  Wound	  
tissue	   oxygen	   levels	   were	   measured	   using	   transcutaneous	   oxygen	   readings	  
(Radiometer	  Medical,	  Brønshøj,	  Denmark).	  

2.4.	  Intervention	  
2.4.1.	  DFU	  Debridement	  andWound	  Management	  Patients	  were	  randomly	  assigned	  
to	  receive	  either	  surgical	  debridement	  or	  UAWdebridement	  every	  week	  during	  a	  
six-­‐week	  treatment	  period.	  All	  debridement	  procedures	  were	  performed	  by	  the	  
same	  surgeon	  (J.L.L.-­‐M.),	  who	  is	  specialist	  in	  the	  field	  of	  diabetic	  foot	  surgery	  with	  
more	  than	  21	  years	  of	  experience.	  
Surgical	  debridement	  involved	  removal	  of	  all	  necrotic	  and	  devitalized	  tissue	  that	  
was	  incompatible	  with	  healing,	  as	  well	  as	  surrounding	  callus.	  
UAWdebridement	   was	   performed	   using	   anUAWSONOCA	   185	   device	   (Söring	  
GmbH,	  Quickborn,	  Germany)	  by	  a	  senior	  clinician	  (J.L.L.-­‐M.)	  with	  more	  than	  three	  
years	   of	   experience	   applying	   this	   type	   of	   debridement.	   The	   UAW	   device	   is	  
equipped	   with	   three	   UAW	   instruments	   with	   di↵erent	   sonotrode	   shapes	   and	  
generates	   an	   ultrasound	   low	   frequency	   of	   25	   kHz.	   The	   choice	   of	   sonotrode	  
depends	   on	   ulcer	   depth	   (ranges	   from	   superficial	   to	   deep).	   The	   UAWdevice	  
piezoelectrically	   transforms	   the	   electrical	   energy	   delivered	   from	   the	   UAWdevice	  
into	  mechanical	  oscillations	  in	  the	  sonotrode	  tip.	  In	  the	  majority	  
of	   diabetic	   foot	   ulcers	   in	   the	   UAW	   group,	   a	   two-­‐minute	   treatment	   with	   40%	  
intensity	   was	   made	   by	   holding	   the	   sonotrode	   in	   contact	   mode,	   holding	   it	  
perpendicular	  to	  the	  wound	  bed	  and	  moving	  it	  across	  in	  an	  up-­‐and-­‐down	  pattern.	  
For	   diabetic	   foot	   ulcers	   measuring	   >	   15	   cm2,	   the	   debridement	   treatment	   was	  
increased	  to	  three	  minutes.	  In	  addition	  to	  UAWdebridement,	  a	  scalpel	  was	  used	  for	  
careful	  tissue	  removal,	  but	  only	  in	  cases	  where	  periwound	  tissue	  exhibited	  calluses	  
or	  maceration.	  
Between	  debridement	  sessions,	  sterile	  saline	  was	  used	  to	  clean	  all	  wounds	  prior	  to	  
evaluation	  and	  all	  patients	  received	  standard	  of	  care	  for	  their	  diabetic	  foot	  ulcers,	  
which	  consisted	  of	  moist	  wound	  dressings	  for	  wound	  management	  and	  proper	  o↵-­‐
loading	  (a	  removable	  walker	  cast	  based	  on	  the	  functioning	  and	  ambulatory	  status	  
of	   the	   patient)	   as	   per	   the	   InternationalWorking	   Group	   of	   the	   Diabetic	   Foot	  
guidelines	   [1,20].	   When	   necessary,	   patients	   with	   moderate	   infections	   took	  
empirical	   antibiotics	   during	   the	   treatment	   period,	   based	   on	   IDSA	   guideline	  
recommendations	  [14],	  until	  the	  results	  from	  deep	  tissue	  culture	  were	  available	  
[21].	  After	  we	  received	  tissue	  culture	  results,	  we	  adjusted	  the	  antibiotic	  therapy	  to	  
target	  the	  bacteria	  that	  were	  isolated	  during	  tissue	  culture.	  

2.4.2.	  Analysis	  of	  Tissue	  Samples	  
Soft	  tissue	  punch	  biopsies	  (3	  mm)	  were	  taken	  after	  wound	  debridement	  sessions	  
at	  week	  zero	  (day	  0)	  and	  week	  six	  (day	  42).	  After	  tissue	  collection,	  samples	  were	  
immediately	   transported	   to	   the	   laboratory	   for	   cellular	   proliferation	   and	  
microbiological	  analyses.	  
	  
2.5.	  Outcome	  Measures	  
2.5.1.	   Main	   Outcome	   Measure:	   Cellular	   Proliferation	   Analysis	   of	   Wound	   Tissue	  
Samples.	  	  The	  same	  senior	  clinician	  interpreted	  all	  samples	  to	  evaluate	  the	  cellular	  
proliferation.	   The	   microvascular	   structure	   of	   CD31,	   an	   endothelial	   marker,	   was	  
subjected	  to	  immunohistochemical	  analysis	  and	  quantification	  to	  understand	  the	  
e↵ects	  of	  debridement	  on	  neo-­‐angiogenesis,	  [22].	  Sections	  of	  tissue	  sample	  were	  
immunohistochemically	  stained	  with	  the	  CD31	  marker.	  Light	  microscopy	  was	  used	  
to	  count	  the	  number	  of	  microvessels/endothelial	  cells	  in	  a	  standardized	  grid,	  with	  
the	   results	   expressed	   as	   microvessel	   density	   (Leica	   DMD	   800	   morphometric	  
system).	   Microvessel	   density	   was	   scored	   in	   proportion	   to	   the	   following	   scale:	   0	  
(absent),	   1	   (low,	   at	   least	   one	   microvessel),	   2	   (moderate)	   and	   3	   (more	   than	   two	  
microvessels).	  	  
To	   differentiate	   collagen	   content	   from	   other	   components,	   such	   as	   muscle	   fibrin	  
and	  erythrocytes,	  in	  tissue	  samples	  we	  used	  Massons’s	  trichome	  staining.	  Collagen	  
content	   was	   scored	   according	   to	   the	   following	   scale:	   0	   (absent),	   1	   (mild),	   2	  
(moderate)	  and	  3	  (severe)	  [23].	  
Actin	   staining	   was	   used	   to	   evaluate	   the	   presence	   of	   myofibroblasts	   involved	   in	  
wound	  healing.	  These	  cells	  increase	  in	  number	  during	  wound	  healing.	  The	  number	  
of	  stained	  cells	  was	  semi-­‐quantitatively	  analyzed	  using	  a	  0–3	  scaling	  score	  (0	  =	  no	  
myofibroblasts,	   1	   =	   myofibroblasts	   in	   low	   quantities,	   2	   =	   myofibroblasts	   in	  
moderate	  quantities,	  3	  =	  myofibroblasts	  in	  high	  quantities).	  
	  
2.5.2.	   Secondary	   Outcome	   Measure:	   Microbiological	   Analysis	   of	   Wound	   Tissue	  
Samples	   Specimens	   of	   wound	   tissue	   were	   homogenized	   in	   0.5	   mL	   volumes	   of	  
sterile	   phosphate	   buffered	   saline	   (PBS,	   Sigma	   Aldrich,	   St	   Louis,	   MO).	   After	  
mechanical	   homogenization,	   the	   specimens	   were	   seeded	   in	   Columbia	   agar	   (BD,	  
Sparks,	  MD),	  MacConkey	  agar	  (BD),	  Sabouraud	  dextrose	  agar	  (BD)	  and	  Columbia	  

agar	   supplemented	   with	   nalidixic	   acid	   and	   colistin	   (BD)	   using	   a	   spiral	   plater	  
Workstation	   (Don	   Whitley	   Scientific,	   Shipley,	   UK).	   Quantitative	   and	   qualitative	  
microbiological	  analyses	  were	  performed	  after	  incubation	  of	  plates	  at	  37	  #C	  for	  24	  
h.	  Isolated	  microorganisms	  were	  identified	  by	  standard	  methods	  and	  susceptibility	  
testing	  was	  performed	  in	  accordance	  with	  Clinical	  and	  Laboratory	  Standards	  by	  
the	   disk	   di↵usion	   method	   [24].	   The	   results	   were	   expressed	   as	   CFU	   per	   gram	   of	  
tissue	  (CFU/g)	  and	  the	  limit	  of	  detection	  was	  10	  colony-­‐forming	  units	  (CFU).	  
	  
2.5.3.	  Third	  Outcome	  Measure:	  Evaluation	  of	  Wound	  Conditions	  
Diabetic	  foot	  ulcers	  were	  evaluated	  at	  patient	  admission	  and	  weekly	  before	  and	  
after	  each	  debridement	  treatment.	  A	  validated	  wound	  scoring	  system	  was	  used	  to	  
assess	   the	   wound	   bed	   tissue	   according	   to	   quality,	   presence	   and	   consistency	   of	  
granulation	   tissue	   [25].	   Furthermore,	   diabetic	   foot	   ulcers	   were	   evaluated	   for	  
amounts	   of	   wound	   exudate	   and	   periwound	   skin	   conditions	   such	   as	   skin	  
maceration	  by	  the	  same	  senior	  clinician	  (F.J.Á.-­‐A.)	  according	  to	  the	  triangle	  wound	  
assessment	  [26].	  
Wound	  healing	  was	  supervised	  weekly	  during	  the	  treatment	  period	  (6	  weeks)	  and	  
the	   wound	   size	   was	   assessed	   using	   Visitrak	   (Smith	   &	   Nephew,	   Hull,	   UK),	  
determining	  the	  area	  of	  the	  lesion	  with	  an	  
approximation	  of	  ±	  5	  mm2.	  
	  
2.6.	  Follow-­‐Up	  
Patients	   were	   followed	   up	   for	   6	   months	   after	   inclusion.	   During	   the	   follow-­‐up	  
period,	   we	   recorded	   ulcer	   healing.	   Ulcer	   healing	   was	   defined	   as	   complete	  
epithelialization	  without	  any	  sustained	  drainage	  up	  to	  24	  weeks	  after	  the	  end	  of	  
the	  study	  follow-­‐up.	  
	  
2.7.	  Sample	  Size	  
Granmov.12	  program	  (Municipal	  Institute	  of	  Medical	  Research,	  Barcelona,	  Spain)	  
(https://www.imim.	   cat/ofertadeserveis/software-­‐public/granmo/)	   was	   used	   to	  
calculate	  the	  sample	  size.	  Thus,we	  analyzed	  51	  patients	  (24	  in	  surgical	  group	  and	  
27	   in	   UAW	   group)	   with	   a	   statistical	   power	   of	   0.80	   and	   an	   alpha	   of	   0.05,	   with	   a	  

power	  of	  the	  clinical	  di↵erence	  of	  37%	  to	  detect	  a	  statistically	  significant	  between	  
groups.	  
	  
2.8.	  Randomization	  
A	   computer-­‐generated	   random	   number	   table	   was	   used	   to	   carry	   out	   the	  
randomization	   of	   the	   patients	   into	   the	   two	   groups	   by	   an	   investigator	   who	   was	  
unaware	  of	  the	  identity	  of	  the	  participants.	  
	  
2.9.	  Blinding	  
None	  of	  the	  participants,	  care	  providers	  and	  outcome	  adjudicators	  were	  blinded	  to	  
the	  interventions	  after	  assignment.	  
	  
2.10.	  Statistical	  analysis	  
Data	  were	  analyzed,	  based	  upon	  an	  intention-­‐to-­‐treat	  analysis,	  using	  the	  software	  
package	   SPSS	   for	   IOs	   version	   21.0	   (SPSS,	   Inc.	   Chicago,	   IL,	   USA).	   Kolmogorov–
Smirnov	   test	   was	   used	   to	   verify	   the	   assumption	   of	   normality	   of	   all	   continuous	  
variables.	  Chi-­‐square	  test	  was	  performed	  to	  calculate	  differences	  between	  groups	  
and,	  if	  applicable,	  Fisher’s	  exact	  test	  for	  categorical	  variables.	  Student’s	  t-­‐test	  and	  
Mann–Whitney	   U	   test	   were	   performed	   for	   normally	   and	   abnormally	   distributed	  
quantitative	   variables,	   respectively.	   Graphics	   to	   evaluate	   the	   differences	   among	  
decrease	   in	   bacterial	   load	   and	   cellular	   proliferation	   between	   groups	   were	   done	  
using	  GraphPad®	  for	  Mac	  OS.	  
	  
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