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Effect of NaOH solutions on planktonic bacteria, biofilms, and lipopolysaccharide Mo, Anthony John 2016

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	  Effect	  of	  NaOH	  Solutions	  on	  Planktonic	  Bacteria,	  Biofilms,	  and	  Lipopolysaccharide	  	  by  Anthony John Mo  BSc., The University of Alberta 2002  DMD., The University of Sydney 2006   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   JUNE 2016  © Anthony John Mo, 2016  	  	  	  	  	   ii	  Abstract	  	  Background:	   Eradicating	   bacteria,	   biofilms	   and	   harmful	   by-­‐products	   such	   as	   LPS	  from	   the	   root	   canal	   system	   is	   important	   in	   providing	   successful	   endodontic	  treatment.	  	  	  To	  date	  no	  irrigant	  or	  medicament	  used	  in	  endodontics	  has	  been	  able	  to	  completely	  eradicate	  bacteria	  in	  the	  root	  canal	  system	  or	  detoxify	  all	  LPS.	  	  Aim:	  It	  is	  proposed	   that	   a	   novel	   solution	   containing	   NaOH	   (Sodium	   hydroxide),	   Sodium	  dodecyl	   sulfate	   (SDS)	   and	   an	   alcohol	   may	   have	   unique	   disinfective	   properties	  against	   bacterial	   factors	   highly	   relevant	   in	   endodontic	   treatment.	   	  Materials	   and	  Methods:	   Combinations	   of	   the	   proposed	   solution	   were	   tested	   and	   compared	   to	  NaOCl	  (sodium	  hypochlorite)	  against	  1-­‐	  Planktonic	  E.	  faecalis	   in	  direct	  contact	  and	  quantified	  by	  CFU	   counts	  2	   -­‐	   Polymicrobial	   biofilms	   in	   an	  open	  model	   exposed	   to	  solutions	  and	  visualized	  with	  CLSM	  (Confocal	  Laser	  Scanning	  Microscopy)	  3	  –	  LPS	  using	   a	   biofunctional	   assay,	   stimulating	   IL-­‐1ß	   production	   in	   RAW	   264.7	  macrophages	  with	  treated	  LPS	  aliquots	  and	  analyzed	  by	  ELISA.	  	  Results:	  Planktonic	  killing	   tests	   with	   E.	   faecalis	   showed	   that	   NaOCl	   was	   more	   effective	   than	   NaOH	  solutions.	   	  With	  potency	   from	  highest	   to	   lowest	   as	   follows:	   6%	  NaOCl,	   2%	  NaOCl,	  NaOH/SDS/Propanol,	  NaOH/Propanol.	  	  Biofilm	  tests	  showed	  that	  NaOCl	  killed	  more	  biofilm	   bacteria,	   however	   NaOH/SDS	   combinations	   removed	   more	   biofilm	   mass.	  	  Results	  showed	  that	  LPS	  samples	  treated	  with	  either	  6%	  or	  2%	  NaOCl	  produced	  no	  IL-­‐1ß.	  	  Samples	  treated	  with	  NaOH/SDS	  combinations	  produced	  inconsistent	  results	  regarding	   IL-­‐1ß	   	   release	   due	   to	   inefficient	   dialysis	   removal	   of	   toxic	   irrigants.	  	  	  	  	  	  	   iii	  Conclusions:	  Results	  suggest	  that	  NaOCl	  remains	  the	  irrigant	  of	  choice	  as	  it	  is	  most	  effective	   in	   killing	   bacteria	   either	   planktonically	   or	   within	   biofilm	   systems.	   	   The	  results	   of	   this	   study	   suggest	   that	   NaOCl	   is	   indeed	   effective	   in	   LPS	   detoxification	  which	   is	   contrary	   to	   suggestions	   by	   several	   previous	   studies.	   	   NaOH/SDS	  combinations	   while	   less	   effective	   in	   killing,	   appear	   to	   remove	  more	   biofilm	  mass	  when	   compared	   to	  NaOCl.	   	   This	  may	  be	   attributed	   to	   the	   surfactant	   properties	   of	  SDS.	  	  	  	   	  	  	  	  	  	   iv	  Preface	  All	   thesis	  work	  was	  completed	  by	  Dr.	  Anthony	   J.	  Mo.	   	  The	  relative	  research	  contribution	  by	  Dr.	  Mo	  was	  90%.	  	  The	  research	  abstract	  and	  poster	  were	  presented	  at	  UBC	  Faculty	  of	  Dentistry	  Research	  Day	  2016	  and	  at	  the	  Pacific	  Dental	  Conference	  2016,	  Vancouver,	  Canada.	  Research	   was	   in	   part	   funded	   by	   a	   grant	   from	   The	   Canadian	   Academy	   of	  Endodontists	   and	   also	   presented	   at	   The	   Canadian	  Academy	  of	   Endodontists	   2015	  Annual	  Session	  in	  Banff,	  Canada.	  	  	  	   	  	  	  	  	  	   v	  	  Table	  of	  Contents	  	  Abstract	  ..........................................................................................................................................................	  ii	  Preface	  ...........................................................................................................................................................	  iv	  Table	  of	  Contents	  .......................................................................................................................................	  v	  List	  of	  Tables	  ..............................................................................................................................................	  vii	  List	  of	  Figures	  ..........................................................................................................................................	  viii	  List	  of	  Abbreviations	  ................................................................................................................................	  x	  Acknowledgements	  .................................................................................................................................	  xii	  Dedication	  .................................................................................................................................................	  xiii	  1.	   Introduction:	  .......................................................................................................................................	  1	  2.	   Review	  of	  the	  literature	  ..................................................................................................................	  4	  2.1	  Bacterial	  factors	  and	  relevance	  to	  AP	  ...................................................................................	  4	  2.1.1	  E.	  faecalis	  .......................................................................................................................................	  4	  2.1.2	  Biofilms	  ..........................................................................................................................................	  6	  2.1.3	  Lipopolysaccharides	  .................................................................................................................	  9	  2.1.3.1	  LPS	  structure	  .......................................................................................................................	  9	  2.1.3.2	  Immune	  activation	  by	  LPS	  ..........................................................................................	  10	  2.1.3.3	  Role	  of	  endotoxin	  in	  periapical	  pathology	  ...........................................................	  12	  2.1.3.4	  Endotoxin	  detection	  ......................................................................................................	  13	  2.2	  Basis	  for	  irrigants	  to	  be	  tested	  and	  use	  in	  endodontic	  therapy	  ..............................	  15	  2.2.1	  Sodium	  hypochlorite	  (NaOCl)	  ......................................................................................	  16	  2.2.2	  	  Calcium	  hydroxide	  (Ca(OH)2)	  ......................................................................................	  19	  3.	   Aims	  and	  hypothesis	  .....................................................................................................................	  22	  3.1	  Aims	  ..................................................................................................................................................	  22	  3.2	  Hypothesis	  .....................................................................................................................................	  23	  4.	   Materials	  and	  methods	  ................................................................................................................	  24	  4.1	  Planktonic	  killing	  tests	  .............................................................................................................	  24	  4.2	  LPS	  detoxification	  tests	  ............................................................................................................	  26	  4.2.1	  Dose	  response	  .....................................................................................................................	  27	  	  	  	  	  	   vi	  4.2.2	  Detoxification	  tests	  ............................................................................................................	  27	  4.3	   Biofilm	  tests	  ..............................................................................................................................	  28	  5.	   Results:	  ................................................................................................................................................	  31	  5.1	  Planktonic	  killing	  tests	  .............................................................................................................	  31	  5.2	  LPS	  detoxification	  .......................................................................................................................	  31	  5.3	  Biofilm	  tests	  ..................................................................................................................................	  32	  5.3.1	   One	  week	  old	  polymicrobial	  biofilms	  ....................................................................	  32	  5.3.2	   Three	  week	  old	  polymicrobial	  biofilms	  ................................................................	  33	  6.	   Discussion	  ..........................................................................................................................................	  34	  6.1	  Planktonic	  killing	  tests	  .............................................................................................................	  34	  6.2	  LPS	  detoxification	  tests	  ............................................................................................................	  36	  6.3	  Biofilm	  tests	  ..................................................................................................................................	  37	  6.3.1	   Effectiveness	  of	  killing	  biofilm	  microbes	  .............................................................	  37	  6.3.2	   Biovolume	  assessment	  ................................................................................................	  38	  7.	   Limitations	  of	  the	  study	  ...............................................................................................................	  41	  8.	   Conclusions	  .......................................................................................................................................	  42	  9.	  Tables	  and	  figures	  ..............................................................................................................................	  43	  References	  ..................................................................................................................................................	  53	  	  	  	  	    	  	  	  	  	   vii	  List	  of	  Tables	   Table	  1	  Planktonic	  killing	  test	  1	  (%	  Killed)	  .................................................................................	  43	  Table	  2	  Planktonic	  killing	  test	  2	  (%	  Killed)	  .................................................................................	  43	  Table	  3	  LPS	  detoxification	  test	  (IL-­‐1ß	  Detection)	  ....................................................................	  47	  Table	  4	  Biofilm	  tests:	  1	  &	  3	  week	  biofilms	  (%	  killed	  and	  biovolume)	  .............................	  50	  	   	  	  	  	  	  	   viii	  List	  of	  Figures	  	  	  Figure	  1	  Planktonic	  killing	  tests:	  CFU	  concentration	  2.5x105/mL.	  ..................................	  44	  Figure	  2	  Planktonic	  killing	  tests:	  CFU	  concentration	  1.75x106/mL.	  ...............................	  44	  Figure	  3	  E.	  coli	  LPS	  from	  Sigma-­‐Aldrich	  Canada	  (0111:B4	  Lot#043M4104V)	  ............	  45	  Figure	  4	  Schematic	  presentation	  of	  the	  Quantikine	  ELISA	  method	  	  .................................	  45	  Figure	  5	  LPS	  detoxification	  tests	  ......................................................................................................	  46	  Figure	  6	  Dialysis	  procedure	  using	  Slide-­‐Alyzer	  cassettes	  (0.5ml,	  2kDA	  MWCO)	  .......	  46	  Figure	  7	  IL-­‐1ß	  standard	  curve	  ..........................................................................................................	  47	  Figure	  8	  Cell	  morphology	  of	  untreated	  mouse	  macrophages	  .............................................	  48	  Figure	  9	  Macrophage	  cell	  morphology	  after	  exposure	  to	  2%	  and	  6%	  NaOCl	  ..............	  48	  Figure	  10	  Macrophage	  cell	  morphology	  after	  exposure	  to	  LPS	  ..........................................	  48	  Figure	  11	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Dialyzed	  solution	  (NaOCl,	  NaOH,	  Propanol)	  ............................................................................................................................	  49	  Figure	  12	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Double	  Solution	  ................	  49	  Figure	  13	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Triple	  Solution	  ..................	  49	  Figure	  14	  Representative	  3D	  biofilm	  reconstructions	  	  ..........................................................	  50	  Figure	  15	  Biovolume	  and	  proportions	  of	  live	  and	  dead	  bacteria	  in	  1	  week	  old	  biofilms	  after	  indicated	  treatment.	  (Logarithmic	  Scale	  in	  3D	  units)	  ......................	  51	  Figure	  16	  Proportions	  (%)	  of	  live	  and	  dead	  bacteria	  in	  1	  week	  old	  biofilms	  after	  the	  various	  treatments	  .......................................................................................................................	  51	  	  	  	  	  	   ix	  Figure	  17	  Biovolume	  and	  proportions	  of	  live	  and	  dead	  bacteria	  in	  3	  week	  old	  biofilms	  after	  indicated	  treatment.	  (Logarithmic	  Scale	  in	  3D	  units)	  ......................	  52	  Figure	  18	  Proportions	  (%)	  of	  live	  and	  dead	  bacteria	  in	  3	  week	  old	  biofilms	  after	  the	  various	  treatments	  .......................................................................................................................	  52	  	  	  	    	  	  	  	  	   x	  List	  of	  Abbreviations	  3-­‐HMA	  ……………………………………………………………………………	  3	  –	  hydroxymyristic	  acid	  	  AAE	  ……………………………………………………….…..	  American	  Association	  of	  Endodontists	  	  AP	  …………………………………………………………………………………..........	  apical	  periodontitis	  	  BHI	  ……………………………………………………………………………..	  brain	  heart	  infusion	  broth	  	  Ca(OH)2	  …………………………………………………………………………………	  calcium	  hydroxide	  	  CD-­‐14	  ……………………………………………………………………….	  cluster	  of	  differentiation	  14	  	  CFU	  ………………………………………………………………………….…………	  colony	  forming	  units	  	  CHX	  ………………………………………………………………………………………………	  chlorhexidine	  	  CLSM	  ……………………………………………………………	  confocal	  laser	  scanning	  microscopy	  	  DMEM	  …………………………………………………...….	  Dulbecco's modified Eagle's medium	  	  DNA	  …………………………………………………………………………..……...	  deoxyribonucleic	  acid	  	  EDTA	  ………………………………………………………………….	  ethylenediaminetetraacetic	  acid	  	  ELISA	  …………………………………………………......…	  enzyme-­‐linked	  immunosorbent	  assay	  	  EPS	  ……………………………………………………………....…	  extracellular	  polymeric	  substance	  	  EU	  ……………………………………………………………………………………………...	  endotoxin	  units	  	  FDA	  …………………………………………………………………	  food	  &	  drug	  administration	  (USA)	  	  HA	  ………………………………………………………………………………………………	  hydroxyapatite	  	  HOCl	  ……………………………………………………………………………………...	  hypochlorous	  acid	  	  H20	  …………………………………………………………………………………………………………...	  water	  	  Il-­‐1ß	  ………………………………………………………………………………………	  interleukin-­‐1-­‐beta	  	  LAL	  ……………………………………………………………………..	  limulus	  amebocyte	  lysate	  assay	  	  	  	  	  	   xi	  	  LBP	  …………………………………………………………………………………….	  lipid	  binding	  protein	  	  LPS	  ………………………………………………………………………………………..	  lipopolysaccharide	  	  LTA	  ……………………………………………………………………………………………	  lipoteichoic	  acid	  	  MWCO	  ………………………………………………………………………….	  molecular	  weight	  cut-­‐off	  	  NaOCl	  …………………………………………………………………………………	  sodium	  hypochlorite	  	  NaOH	  ……………………………………………………………………………………...	  sodium	  hydroxide	  	  PBS	  ……………………………………………………………………………..	  phosphate	  buffered	  saline	  	  PCR	  …………………………………………………………………………....	  polymerase	  chain	  reaction	  	  SEM	  ………………………………………………………………………	  scanning	  electron	  microscopy	  	  SDS	  ………………………………………………………………………………….	  sodium	  dodecyl	  sulfate	  	  TLR	  -­‐2/4	  …………………………………………………………………………….	  toll-­‐like	  receptor	  2/4	  	  TNF-­‐α………………………………………………………………………	  tumor	  necrosis	  factor	  –	  alpha	  	  TSA	  …………………………………………………………………………………………….	  tryptic	  soy	  agar	  	   	  	  	  	  	  	   xii	   Acknowledgements	  It	  has	  been	  one	  of	  my	   life’s	  great	  experiences	  and	  richest	  blessings	  to	  work	  and	  associate	  with	  some	  of	  the	  most	  inspired	  and	  talented	  individuals	  in	  our	  field.	  	  I	  am	   deeply	   grateful	   to	   each	   of	   them	   for	   providing	   support,	   insight	   and	  encouragement	  to	  broaden	  my	  horizons	  during	  the	  course	  of	  my	  research	  journey.	  Firstly,	   to	  my	   supervisor	   Professor	  Markus	   Haapasalo,	   whose	  wisdom	   and	  encouragement	  have	  been	  my	  beacon	  throughout	  my	  time	  at	  UBC.	  	  His	  example	  and	  willingness	  to	  share	  his	   time	  and	  expertise	  have	  taught	  me	  so	  much	   in	  the	   field	  of	  endodontics,	  and	  further	  in	  valuable	  life	  lessons.	  	  	  My	  heartfelt	  gratitude	  to	  Dr.	  Ya	  Shen,	  who’s	  kind	  smile	  and	  genuine	  support	  do	  so	  much	   for	  each	  of	   the	  residents	  who	  progress	   from	  novices	   to	  experts	  under	  her	  tutelage	  in	  research	  and	  throughout	  the	  program.	  	  To	   Dr.	   Jeff	   Coil,	   our	   program	   director,	   your	   clinical	   prowess	   and	   critical	  reasoning	  have	  done	  so	  much	  to	  make	  me	  a	  better	  clinician.	  	  	  My	  sincere	  thanks	  to	  Dr.	  Lari	  Hakkinen,	  who’s	  door	  was	  always	  open	  to	  me	  when	  exploring	  new	  ideas,	  or	  solving	  problems	  in	  my	  research.	  	  A	   big	   thank-­‐you	   to	   Dr.	   Zhe-­‐Jun	   Wang	   and	   Dr.	   Hazuki	   Maezono,	   my	   lab	  buddies,	  for	  introducing	  me	  to	  the	  various	  lab	  techniques	  used	  within	  this	  project.	  	  I	  could	  not	  have	  done	  it	  without	  you!	  And	   lastly,	   to	  UBC	  and	  the	  Faculty	  of	  Dentistry	   for	   truly	  creating	   ‘a	  place	  of	  mind’	  in	  a	  beautiful	  setting,	  the	  ideal	  place	  to	  ‘broaden	  one’s	  horizons’.	  	  	  	  	  	   xiii	  Dedication	  	   To	  my	  parents,	  thank	  you	  for	  laying	  the	  foundation	  for	  everything	  that	  I	  am!	  	  As	   parents,	   your	   commitment,	   selflessness	   and	   love	   have	   been	   limitless	   and	   I	   am	  forever	  indebted	  to	  you.	  	  	  	  	   To	   my	   wife	   and	   son,	   for	   you	   I	   tirelessly	   find	   the	   need	   to	   improve	   and	   do	  everything	  I	  do.	  	  Thank-­‐you	  for	  allowing	  me	  to	  pursue	  my	  dream.	  	  	  	   To	   all	   my	   teachers	   (in	   every	   field),	   friends	   and	   family	   along	   the	   way.	   	   My	  experience	  is	  enriched	  by	  each	  of	  you.	  	   	  	  	  	  	  	   1	   1. Introduction:	  	   Apical	   periodontitis	   is	   caused	   by	   inflammatory	   bone	   resorption	   stimulated	   by	  interactions	  between	  the	  host	  defense	  and	  bacterial	  factors	  emanating	  from	  the	  root	  canal	  system	  (Nair	  2004).	  Thus	  the	  ability	  to	  eradicate	  or	  inactivate	  virulent	  factors	  that	   bacterial	   species	   might	   possess	   is	   paramount	   to	   success	   in	   endodontic	  treatments.	   	   The	   role	   of	   bacteria	   in	   AP	   has	   been	   unequivocally	   established	  (Kakehashi	  et	  al.	  1965,	  Sundqvist	  1976),	  however	  the	  precise	  mechanisms	  by	  which	  bone	   resorptive	   processes	   are	   initiated	   by	   bacteria	   in	   root	   canals	   are	   not	   so	  well	  elucidated	   (Siqueira	   2008).	   	   	   Bacterial	   species	  may	   possess	   abilities	   that	   promote	  survival	  in	  the	  unique	  root	  canal	  environment.	  	  Further	  complicating	  the	  issue	  is	  the	  recognition	   that	  biofilms	  are	  responsible	   for	   the	  chronic	  root	  canal	   infection	  (Nair	  1987,	   Siqueira	   and	   Rocas	   2009,	   Ricucci	   and	   Siqueira	   2010).	   This	   communal	  colonization	   imparts	   many	   advantages	   for	   intra-­‐canal	   survival,	   such	   as	   barrier	  protection	   (i.e.	   EPS),	   nutrient	   storage	   and	   accumulation	   of	   by-­‐products	   that	   are	  harmful	  to	  the	  host	  defense,	  such	  as	  LPS	  (Costerton	  et	  al.	  1999,	  2007).	  	  	   To	  date	  no	   irrigant	  or	  medicament	  used	   in	   endodontics	  has	  been	   shown	   to	  be	  able	   to	   completely	   eradicate	   bacteria/biofilm	  or	   detoxify	   LPS	   (AAE	  Colleagues	   for	  Excellence	   2011).	   	   The	   most	   widely	   used	   irrigant,	   NaOCl	   possesses	   excellent	  antibacterial	   and	   tissue	   dissolving	   properties,	   and	   is	   used	   worldwide	   in	  	  	  	  	  	   2	  concentrations	   ranging	   from	   0.5%-­‐6%	   (Haapasalo	   et	   al.	   2005).	   	   A	   possible	  disadvantage	   of	  NaOCl	   is	   the	   suggestion	   from	  various	   studies	   that	   it	   is	   ineffective	  against	  LPS	  (Buttler	  &	  Crawford	  1992,	  Buck	  et	  al.	  2001,	  Dias	  de	  Oliveira	  et	  al.	  2006,	  Martinho	  et	  al.	  2007,	  Gomes	  et	  al.	  2009,	  Maekawa	  et	  al.	  2011).	  	  This	  premise	  coupled	  with	  the	  observation	  that	  LPS	  is	  positively	  correlated	  with	  bone	  resorption,	  abscess	  formation	  and	  pain,	  (Sousa	  et	  al.	  2014)	  raises	  a	  relevant	  clinical	  question.	  	  Another	  interesting	   irrigant	   more	   recently	   emerging	   is	   Q-­‐Mix	   (Tulsa-­‐Dentsply)	   which	   has	  also	  demonstrated	  superior	  antibacterial/biofilm	  properties	  and	  further	  combining	  components	  that	  may	  aid	  in	  smear	  layer	  removal	  among	  other	  benefits.	  (Stojicic	  et	  al.	   2012)	   	   The	  most	  widely	   used	   intra-­‐canal	  medicament	   in	   endodontics,	   Ca(OH)2	  (calcium	  hydroxide)	  has	  been	  shown	  to	  maintain	  sufficiently	  high	  pH	  to	  kill	  bacteria	  (Bystrom	  et	   al.	   1985),	   detoxify	  LPS	   (Safavi	   et	   al.	   1993)	   and	  promote	   apexification	  and	   regeneration	  of	  pulpal/periapical	   tissues	   (Paula-­‐Silva	   et	   al.	   2010).	   	  Therefore,	  modern	  endodontic	  procedures	  often	  involve	  multiple	  appointments	  and	  irrigation	  steps	  in	  addition	  to	  instrumentation	  to	  achieve	  the	  biological	  objective.	  	  	  	   A	   recent	   publication	   from	   the	   field	   of	   virology	   suggested	   a	   novel	   solution	  containing	  a	  combination	  of	  NaOH,	  SDS	  and	  an	  alcohol	  may	  have	  fast,	  broad	  range	  disinfective	   properties	   on	  bacteria,	   fungi,	   and	   viral	   prion	  proteins.	   	   At	   the	   time	  of	  this	  writing,	   to	  date	   there	  has	  been	  no	   report	   of	   research	  performed	  on	   solutions	  containing	   NaOH	   in	   the	   endodontic	   literature.	   	   However,	   opinions	   have	   been	  expressed	   that	   NaOH	   is	   unsafe	   and	   therefore	   unsuitable	   for	   use	   in	   endodontics	  	  	  	  	  	   3	  (Siqueira	  &	  Lopes	  1999).	  	  The	  aim	  of	  this	  project	  will	  be	  to	  compare	  a	  combination	  of	   NaOH,	   SDS	   and	   n-­‐propanol	   with	   the	   current	   gold	   standard,	   NaOCl	   at	   varying	  concentrations	   in-­‐vitro	   for	   efficacy	   against	   planktonic	   bacteria	   (E.	   faecalis),	  polymicrobial	  biofilms	  and	  LPS	  (E.	  coli).	  	  	   	  	  	  	  	  	   4	  2. Review	  of	  the	  literature	  	  2.1	  Bacterial	  factors	  and	  relevance	  to	  AP	  	   The	   pathogenesis	   of	   AP	   is	   invariably	   linked	   to	   bacteria,	   as	   previously	  mentioned,	   and	   this	  has	  been	  attributed	   to	  multiple	   factors	  possessed	  by	  bacteria	  displaying	  virulence.	  	  Among	  these,	  the	  most	  studied	  species	  in	  endodontics	  may	  be	  E.	   faecalis	   which	   may	   be	   considered	   a	   'model	   species'	   for	   reasons	   that	   will	   be	  reviewed.	  	  Further	  the	  organization	  of	  intracanal	  flora	  into	  sessile	  biofilms	  is	  an	  area	  of	  developing	  research	  giving	  insight	   into	  the	  complexity	  of	  bacterial	  colonizations	  and	   reasons	   for	   infective	   persistence.	   	   	   LPS,	   a	   widely	   studied	   molecule	   in	  endodontics,	   has	   been	   demonstrated	   to	   have	   perhaps	   the	   biggest	   impact	   in	  periapical	  lesion	  development	  in	  studies	  of	  pathogenesis.	  	  The	  significance	  of	  LPS	  is	  a	   prime	   example	   of	   a	   bacterial	   by-­‐product	   capable	   of	   causing	   tissue	   destruction.	  	  Thus,	   the	   test	  methodology	   in	   this	   project	  will	   be	  developed	  using	   this	   context	   to	  provide	  insight	  relative	  to	  the	  materials	  tested	  and	  relevance	  in	  clinical	  therapy.	  	  2.1.1	  E.	  faecalis	  	  E.	   faecalis	   is	   the	   most	   commonly	   isolated	   or	   detected	   species	   from	   oral	  infections,	   including	   infected	   root	   canals	   (Engstrom	   1964).	   	   It	   is	   predominantly	  associated	   with	   failed	   endodontic	   treatment	   and	   is	   seldom	   isolated	   in	   primary	  	  	  	  	  	   5	  endodontic	   infections	   (Sundqvist	   et	   al.	   1989,	   1998).	   	   While	   its	   association	   is	  persistent	   infections	   strong,	   a	   direct	   link	   to	   how	   it	   may	   be	   involved	   in	   post-­‐treatment	  AP	  has	  not	  yet	  been	  established	  (Siqueira	  &	  Rocas	  2009).	  A	  study	  using	  PCR	   analysis	   of	   isolates	   from	   different	   cases	   showed	   that	   E.	   faecalis	   was	   more	  associated	  with	  asymptomatic	  cases	   than	  symptomatic	  cases,	  and	  again	  confirmed	  its	   prevalence	   in	   failed	   cases	   vs.	   primary	   cases	   (Rocas	   et	   al.	   2004).	   Clinically,	   the	  elimination	   of	   E.	   faecalis	   is	   challenging	   and	   the	   subject	   of	   much	   research	   in	  endodontics.	  	  	  A	  systematic	  review	  and	  meta-­‐analysis	  associating	  E.	  faecalis	  and	  the	  use	   of	   NaOCl	   or	   CHX	   found	   that	   both	   irrigants	   showed	   low	   ability	   to	   eliminate	  E.	  faecalis	  when	  evaluated	  by	  either	  culture	  or	  PCR	  techniques	  (Estrela	  et	  al.	  2008)	  	  E.	   faecalis	   possesses	   a	   number	   of	   virulence	   factors	   including:	   aggregation	  substance,	  gelatinase,	  cytolysin	  toxin,	  extracellular	  superoxide	  production,	  capsular	  polysaccharides	  and	  antibiotic	  resistance	  determinant	  (Portenier	  et	  al.	  2003).	  These	  factors	  combined	  contribute	  to	  the	  species’	  superior	  ability	  to	  survive	  the	  harsh	  root	  canal	   environment	   both	   during	   and	   after	   treatment.	   	   A	   series	   of	   pathobiological	  studies	   indicated	   E.	   faecalis	   to	   be	   the	   only	   species	   able	   to	   sustain	   a	   biofilm	   in	  monoculture,	  whereas	   all	   other	   species	   required	   a	   polymicrobial	   environment	   for	  sustenance	   (Fabricius	   et	   al.	   1982).	   	   Various	   studies	   have	   also	   demonstrated	   the	  unique	  response	  by	  E.	  faecalis	   in	  response	  to	  different	  environmental	  stresses	  (e.g.	  starvation,	  acidic	  environment,	  heat)	  allowing	  it	  to	  grow	  and	  persist	  in	  a	  very	  harsh	  conditions	  	  (Portenier	  et	  al.	  2005,	  Shen	  et	  al.	  2010).	  Other	  characteristics	  that	  help	  E.	  	  	  	  	  	   6	  faecalis	   to	   survive	  may	   include:	   ability	   to	   tolerate	  high	  pH	  by	  virtue	  of	  membrane	  proton	   pumps	   (Evans	   et	   al.	   2002),	   genetic	   transference	   of	   resistant	   phenotypes	  (Sedgley	   &	   Clewell	   2004),	   and	   recruitment	   of	   polymorphonuclear	   cells	   (Ma	   et	   al.	  2011).	  Thus,	  E.	   faecalis	   is	   useful	   as	   a	   test	   species	   due	   to	   its	   high	   association	  with	  endodontic	   pathology	   and	   its	   superior	   ability	   to	   survive	   challenging	   ecological	  environment.	   Such	   characteristics	   of	  E.	   faecalis	  help	   validate	   the	   testing	   for	  useful	  antibacterial	  solutions	  for	  clinical	  therapy.	  	  2.1.2	  Biofilms	  	  	  	   Biofilms	  may	  be	  defined	  as	  communities	  of	  microorganisms	  embedded	  in	  an	  extra-­‐cellular	  matrix.	   	  This	  environment	  provides	  a	  physical	  barrier	  for	  protection,	  nutrient	   storage,	   enhanced	   cell-­‐cell	   communication	   and	   may	   accelerate	   genetic	  element	  exchange.	  	  The	  composition	  of	  biofilms	  has	  been	  characterized	  as	  being	  by	  volume	  85%	  extracellular	  polymeric	  matrix	  material	  (EPS),	  and	  15%	  cells	  (Fleming	  et	  al.	  2007).	  	  The	  EPS	  matrix	  consists	  of	  polysaccharides,	  proteins,	  nucleic	  acids	  and	  salts	   that	   surround	   the	   bacterial	   colonies	   and	   anchor	   them	   to	   a	   solid	   substrate	  (Whitchurch	  et	  al.	  2002).	  	  	  	   Unfortunately,	  while	  advantageous	  to	  bacteria,	  biofilm	  formation	  provides	  a	  unique	  challenge	  for	  eradication	  of	  infections	  from	  the	  root	  canal	  environment.	  	  The	  observation	   that	   biofilm	   bacteria	   are	   up	   to	   1000-­‐fold	   more	   resistant	   to	  	  	  	  	  	   7	  phagocytosis,	   antibodies	   and	   antibiotics	   than	   planktonic	   bacteria	   (Costerton	   et	   al.	  1999)	   demonstrates	   the	   challenge	   posed	   by	   biofilm	   mediated	   infections.	   	   The	  following	   criteria	   have	  been	  defined	   for	   characterization	  of	   biofilm	   infections:	   1	   –	  bacteria	  are	  adherents	  of	  host	  surface	  structure;	  2	  –	  infected	  tissues	  show	  bacterial	  microcolonies	   embedded	   in	   EPS;	   3	   –	   infection	   is	   confined	   locally,	   though	  dissemination	   of	   biofilms	   may	   occur	   secondarily;	   4	   –	   eradication	   of	   infection	   is	  difficult,	   even	   with	   antimicrobial	   agents	   that	   are	   otherwise	   effective	   in	   killing	   of	  bacteria	  in	  their	  planktonic	  state	  (Parsek	  &	  Singh	  2003).	  	  	  	   Within	  the	  biofilm	  itself	  a	  dynamic	  environment	  with	  varying	  conditions	  has	  also	   been	   found,	   explaining	   the	   observation	   of	   bacteria	   in	   different	   phases	   and	  phenotypic	  expression	  with	  more	  virulent	  forms	  (Costerton	  et	  al.	  1987).	  The	  maturity	  of	  biofilms	  by	  oral	  bacteria	  may	  be	  more	  significant	   than	  the	  type	  of	  bacteria	  present,	  in	  biofilm	  resistance.	  	  Studies	  have	  demonstrated	  that	  at	  3	  weeks	  a	  significant	   change	   in	   biofilms	   lead	   to	   greatly	   increased	   resistance	   to	   antibacterial	  agents,	   irrespective	  of	  the	  source	  in	  six	  different	  polymicrobial	  biofilms	  (Stojicic	  et	  al.	  2013).	  This	   is	  an	   important	  observation	  given	  the	  chronic	  nature	  of	  endodontic	  infections.	  	  	  	  Biofilm	   constituents	   alone	   may	   also	   contribute	   to	   pathogenicity,	   irrespective	   of	  bacteria.	   	  The	  EPS	  matrix	  in	  biofilms	  consists	  of	  structural	  components	  of	  bacterial	  cell	  walls,	   such	   as	   LPS	   and	  LTA	   (lipoteichoic	   acid),	   structural	   proteins,	  membrane	  vesicles,	   lipoproteins,	   polysaccharides,	   and	   bacterial	   DNA.	   	   Any	   of	   these	   bacterial	  	  	  	  	  	   8	  byproducts	   have	   been	   demonstrated	   to	   activate	   immune	   responses	   and	   may	  potentially	  be	  involved	  in	  the	  inflammatory	  cascade	  leading	  to	  AP	  (Siqueira	  &	  Rocas	  2007).	  Therefore,	  even	  if	  all	  bacteria	  in	  the	  biofilm	  were	  killed,	  the	  biofilm	  can	  lead	  to	  disease	  if	  left	  in	  situ.	  	  In	   periapical	   disease,	   there	   is	   significant	   and	   increasing	   evidence	   that	  infection	   is	   at	   least	   in	   part	   biofilm-­‐induced	   (Siqueira	  &	   Rocas	   2009).	   	   It	  was	   first	  demonstrated	  using	  light	  and	  SEM	  techniques	  that	  colonies	  of	  bacteria	  ‘resembling	  dental	   plaque’	   	   	   were	   adherent	   to	   root	   canal	   walls	   in	   infected	   roots	   (Nair	   1987).	  	  Ricucci	  and	  Siqueira	  (2010)	   found	  biofilms	   in	  the	  apical	  segment	  of	  80%	  of	  canals	  with	  primary	  infection	  and	  74%	  of	  canals	  with	  persistent	  disease.	  	  A	  correlation	  was	  also	  found	  between	  the	  presence	  of	  biofilms	  and	  larger	  bony	  lesions	  and	  even	  cysts.	  	  	  	   Various	  methods	  have	  been	  used	  to	  test	  biofilm	  susceptibility	  to	  endodontic	  materials	  in-­‐vitro.	  	  Studies	  using	  CLSM	  to	  visualize	  treated	  biofilms	  grown	  on	  either	  collagen-­‐coated	  hydroxyapatite	  discs	  (Shen	  et	  al.	  2009)	  or	  within	  dentin	  canals	  in	  a	  simulated	   closed	   root	   canal	   system	   (Wang	   et	   al.	   2012)	   have	   assessed	   various	  irrigants	  for	  their	  ability	  to	  kill	  biofilm	  microbes.	  	  The	  results	  have	  shown	  relatively	  large	  differences	  between	  the	  substances,	  depending	  on	  their	  concentration,	  time	  of	  exposure	   and	   chemistry.	   A	   few	   recent	   studies	   suggest	   that	   the	   most	   effective	  solutions	  for	  biofilm	  eradication	  include	  high	  concentration	  NaOCl	  and	  QMix	  (Wang	  et	  al.	  2012,	  Stojicic	  et	  al.	  2012).	  	  	  	  	  	  	  	   9	  2.1.3	  Lipopolysaccharides	  	  Lipopolysaccharides	  are	  a	  major	  component	  of	   the	  outer	  cell	  wall	  of	  Gram-­‐negative	   bacterial	   species.	   They	   are	   a	   class	   of	   molecules	   with	   significant	  microbiological	  and	  immunological	  importance	  as	  they	  are	  potent	  activators	  of	  the	  immune	   system,	  potentially	   causing	   inflammation	  and	   tissue	  damage.	  The	   specific	  role	  of	  LPS	  was	  first discovered	  at	  the	  end	  of	  the	  19th	  century	  by	  Richard	  Friedrich	  Johannes	   Pfeiffer	   (Fildes	   1956),	   a	   significant	   researcher	   credited	   for	   many	  important	   medical	   discoveries	   (H.	   influenzae,	   cocci	   characterization,	   vaccinations	  against	  typhus,	  pest	  and	  cholera).	   	  Originally	  LPS	  was	  thought	  to	  be	  released	  upon	  bacterial	   cell	   death	   and	   cell	  wall	   lysis,	   it	   is	   now	   known	   also	   to	   be	   part	   of	   normal	  membrane	   vesicle	   trafficking	   and	   are	   even	   secreted	   in	   vesicles.	   (Reitschel	   et	   al.	  1994)	   Because	   of	   this	   unique	   property	   and	   the	   ability	   of	   LPS	   to	   instigate	   potent	  immune	  response,	  it	  has	  been	  a	  popular	  target	  for	  research	  and	  is	  often	  considered	  independently	  from	  its	  particular	  donor	  bacterial	  species.	  	  2.1.3.1	  LPS	  structure	  Lipopolysaccharides	   are	   ubiquitous	   within	   the	   outer	   cell	   wall	   of	   Gram-­‐negative	   bacteria,	   and	   as	   such	   they	   stabilize	   the	   cell	   wall	   structure.	   While	   their	  structure	   varies	   within	   different	   Gram-­‐negative	   species,	   they	   generally	   share	   a	  common	   structure	   comprising	   of	   a	   lipid	   A	   moiety,	   a	   core	   oligosaccharide,	   and	   O	  antigen.	  	  	  	  	  	   10	  The	   O	   antigen,	   as	   its	   name	   suggests	   is	   a	   possible	   target	   for	   immune	  antibodies	  and	  aids	  in	  classification	  of	  the	  particular	  LPS	  as	  rough	  or	  smooth.	  This	  is	  of	  significance	  as	  the	  rough	  and	  smooth	  parts	  of	  the	  molecule	  impart	  different	  level	  of	   hydrophobicity.	   	   Rough	   LPS	   is	   more	   hydrophobic	   and	   as	   such	   Gram-­‐negatives	  with	   rough	   LPS	   are	   more	   susceptible	   to	   hydrophobic	   or	   lipophilic	   antibiotics	  (Reitschel	  et	  al.	  1994).	  Lipid	   A	   is	   a	   unique	   phospholipid	   structure	   mainly	   responsible	   for	   LPS’s	  immunological	  activity.	   	  Lipid	  A	  alone	  released	   into	  circulation	  has	  been	  shown	  to	  cause	  fever,	  diarrhea	  and	  septicemic	  shock	  (Raetz	  &	  Whitfield	  2002).	  This	  portion	  of	  the	  LPS	  molecule	  is	  the	  most	  commonly	  shared	  LPS	  structure	  across	  different	  Gram-­‐negative	  species,	  and	  thus	  Lipid	  A	  alone	  is	  often	  referred	  to	  as	  endotoxin.	  As	  will	  be	  discussed	  in	  more	  detail	  subsequently,	  the	  potent	  activation	  of	  immune	  cascades	  by	  lipid	  A	   is	  considered	  a	  major	  virulence	   factor	  of	  Gram-­‐negative	  bacteria	   leading	  to	  acute	  abscesses	  or	  even	  more	  severe	  systemic	  consequences.	  2.1.3.2	  Immune	  activation	  by	  LPS	  Extensive	   work	   in	   the	   field	   of	   endotoxin	   research	   has	   revealed	   multiple	  pathways	   whereby	   immune	   activation	   is	   effected	   by	   LPS,	   which	   is	   also	   a	   known	  pyrogen	   (fever	   inducer).	   A	   dose	   of	   1µg/kg	   is	   sufficient	   to	   induce	   septic	   shock	   in	  humans	  (Raetz	  &	  Whitfield	  2002).	  Activation	  of	  Toll-­‐like	   receptors	   expressed	  by	  macrophages,	   dendritic	   cells,	  monocytes	  and	  B	   lymphocytes	   is	   the	  most	   significant	   immune	  activation	  pathway.	  	  	  	  	  	   11	  The	  cell	  surface	  receptor	  TLR4	  recognizes	  lipid	  A	  via	  CD14/LBP	  protein	  complex	  to	  signal	   intracellular	   pathways	   leading	   to	   proinflammatory	   cytokine	   production.	  (Raetz	   &	   Whitfield	   2002)	   Il-­‐1ß	   and	   TNF-­‐α	   are	   important	   cytokines	   secreted	   by	  monocyte	  delineated	  cells	  (macrophages,	  dendritic	  cells)	  and	  may	  cause	  a	  plethora	  of	   immune	   activity	   such	   as	   enzyme	   secretion,	   tissue	   destruction,	   fever	   induction,	  immune	   cell	   migration,	   T	   &	   B-­‐cell	   activation.	   	   Endothelial	   cells	   activated	   by	  TLR4/lipid	   A/CD14	   again	   may	   cause	   secretion	   of	   tissue	   factor	   and	   thereby	  activation	  of	  the	  extrinsic	  clotting	  cascade.	  (Wang	  et	  al.	  2010)	  TLR4	  is	  expressed	  by	  many	  different	  cells	  and	   thus	   the	  actions	  and	   implications	  of	  endotoxin	  are	  varied	  and	  severe.	  Ongoing	  research	  continues	  to	  reveal	  the	  importance	  of	  TLR4	  activation	  in	  systemic	  and	  local	  inflammatory	  processes,	  thus	  an	  understanding	  of	  the	  role	  of	  endotoxin	  in	  infections	  by	  gram-­‐negative	  bacteria	  is	  emphasized.	  Other	   pathways	   such	   as	   antigenic	   recognition	   of	   LPS	   components	   (e.g.	   O	  antigen)	  are	  also	  pathways	  whereby	  the	  immune	  response	  is	  rallied	  and	  multiplied	  in	   the	   presence	   of	   LPS.	   In	   this	   case	   the	   variability	   in	   structure	   may	   account	   for	  differences	   in	   immune	   stimulation	   by	   various	  microbial	   species.	   For	   example,	   the	  variability	  in	  lipid	  A	  fatty	  acid	  chains	  has	  been	  shown	  to	  produce	  entirely	  different	  effects	  and	  even	  different	  receptor	  activation	  (ie.TLR2)	  (Netea	  et	  al.	  2002).	  The	  large	  variability	   in	   the	   O-­‐antigen	   component	   of	   LPS	   is	   also	   an	   example	   of	   how	   specific	  immunity	  is	  adept	  in	  recognizing	  variability	  in	  structure	  and	  thereby	  effecting	  host	  defense.	  	  	  	  	  	   12	  2.1.3.3	  Role	  of	  endotoxin	  in	  periapical	  pathology	  In	  endodontic	   lesions,	   such	  as	  apical	  periodontitis,	  endotoxin	  has	  also	  been	  demonstrated	   to	   be	   of	   important	   consideration.	   Schein	   &	   Shilder	   (1975)	  demonstrated	   the	   presence	   of	   endotoxin	   in	   root	   canals,	   suggesting	   a	   possible	  aetiology	   for	   apical	   periodontitis.	   	   Schonfeld	   et	   al.	   (1982)	   showed	   a	   positive	  correlation	  between	  the	  presence	  of	  LPS	  and	  tissue	  inflammation.	  	  Pitts	  et	  al.	  (1985)	  showed	  that	  endotoxin	  placed	  in	  root	  canals	  in	  dogs	  accelerated	  the	  progression	  of	  apical	  lesions.	  The	  aggressiveness	  of	  these	  processes	  may	  be	  dose	  dependent	  and/or	  directly	  related	  to	  the	  potency	  of	  the	  endotoxin	  reaction.	  Most	  recently,	  in	  a	   	  study	  by	   Sousa	   et	   al.	   (2014)	   a	   strong	   association	   between	   high	   levels	   of	   endotoxin	  detection	  and	  acute	  apical	  abscesses	  was	  found.	  Further,	  it	  was	  shown	  that	  a	  linear	  relationship	   in	   quantities	   of	   endotoxin	   isolated	   from	   abscess	   exudate	   and	   root	  canals	  existed	  (i.e.	  1	  EU	  in	  root	  canal	  =	  1	  EU	  in	  abscess	  exudate).	  	  Studies	  have	  also	  correlated	  an	  increased	  amount	  of	  endotoxin	  in	  clinical	  cases	  with	  a	  predominance	  of	  gram-­‐negative	  species	  such	  as	  P.	  nigrescens,	  P.	  endodontalis	  and	  T.	  socranskii.	   	  A	  positive	   correlation	   was	   also	   noted	   between	   higher	   levels	   of	   IL-­‐1ß,	   TNF-­‐a	   and	  lesions	  of	  larger	  size.	  	  (Martinho	  et	  al.	  2010).	  	  Thus,	  it	  seems	  that	  the	  severity	  of	  the	  inflammatory	  reaction	  in	  apical	  periodontitis	  may	  be	  directly	  related	  to	  quantities	  of	  endotoxin	  present	  or	  produced	  in	  the	  root	  canal	  space	  by	  gram-­‐negative	  bacteria.	  Removal	  and	  detoxification	  of	  LPS	  seems	  to	  be	  a	  difficult	  task	  and	  of	  interest	  in	  endodontic	  treatment.	  Endotoxin	  structure	  appears	  not	  to	  be	  susceptible	  to	  some	  of	   the	   most	   commonly	   used	   irrigants/disinfectants	   in	   endodontics.	   Sodium	  	  	  	  	  	   13	  hypochlorite	   and	   chlorhexidine	   have	   both	   been	   shown	   to	   have	   little	   effect	   on	  endotoxin.	  (Buttler	  et	  al.	  1982,	  Tanomaru	  et	  al.	  2003,	  Gomes	  et	  al.	  2009)	  Thus	  far,	  only	   Ca(OH)2	   and	   perhaps	   Polymyxin	   B	   have	   been	   the	   only	   documented	  medicaments	  with	   detoxification	   effect	   on	   LPS	   (de	  Oliveira	   et	   al.	   2007).	  However,	  combination	  of	  chemo-­‐mechanical	  debridement	  has	  been	  demonstrated	  as	  playing	  the	  major	   role	   in	   removal	   of	   the	   toxin	   from	   infected	   root	   canals.	   Instrumentation	  and	   irrigation	  with	   sterile	   saline	   alone	   can	   be	   attributed	   to	   over	   95%	   removal	   of	  detectable	  endotoxin,	  whereas	  changing	  from	  saline	  to	  hypochlorite	  only	  improves	  this	   figure	   marginally	   (Gomes	   et	   al.	   2009).	   Thus	   it	   seems	   that	   physical	   removal	  rather	   than	   chemical	   action,	   at	   least	   in	   the	   case	   of	   endotoxin,	   has	   been	   the	  more	  important	   factor	   in	   successful	   treatment.	   This	   gives	   rise	   to	   the	   question	  whether	  addition	   of	   a	   chemically	   detoxifying	   step,	   such	   as	   inter-­‐appointment	   Ca(OH)2	  dressing,	  may	   improve	  success	  rates	  and	   treatment	  outcomes	  (Xavier	  et	  al.	  2013).	  Overall,	   it	   is	  clear	   that	  endotoxin	  plays	  a	  role	   in	  endodontic	  pathogenesis	  and	  that	  attention	  must	  be	  given	   to	   effectively	  debride	  and	  detoxify	   root	   canals	   to	  procure	  healing.	  2.1.3.4	  Endotoxin	  detection	  Finally,	   as	   endotoxin	   as	   a	   molecule	   has	   the	   ability	   of	   provoking	   immune	  reaction	  without	  the	  need	  for	  the	  presence	  of	  active/viable	  bacteria,	  its	  detection	  is	  a	   worthy	   topic	   for	   discussion.	   The	   FDA	   has	   set	   maximum	   acceptable	   endotoxin	  levels	  for	  drugs	  and	  potable	  water.	  Levels	  from	  0.2	  EU/ml	  or	  kg	  (water/intrathecal	  drugs)	   to	   a	  maximum	   of	   5	   EU/kg	   is	   permissible.	   The	  most	  widely	   used	   detection	  	  	  	  	  	   14	  method	   is	   the	   Limulus	  Amebocyte	   Lysate	   (LAL)	   assay.	   Based	   on	   the	   reaction	   that	  blood	  from	  the	  horseshoe	  crab	  coagulates	  rapidly	  upon	  contact	  with	  endotoxin,	  the	  purified	   coagulum	   component	   is	   widely	   available	   in	   test	   kits	   for	   endotoxin	  detection.	  Reactions	  are	  calibrated	  to	  Endotoxin	  Units	  (EU)	  rather	  than	  w/w	  values	  to	  reflect	  relative	  toxic	  potency,	  and	  the	  LAL	  assay	  may	  be	  quantified	  by	  a	  variety	  of	  methods	  including	  gel	  clot,	  colorimetric	  and	  turbidimetric	  with	  varying	  but	  overall	  high	  sensitivities.	  However,	  in	  the	  context	  of	  deactivated	  LPS/endotoxin	  the	  LAL	  assay	  may	  be	  of	   limited	  usefulness.	  Studies	  have	  shown	  that	  deacylated/dephosphorylated	  Lipid	  A	  may	  still	   react	  with	   the	   lysate	  and	  provide	  a	  potential	   false	  positive	  when	  using	  LAL,	  since	  clotting	  in	  the	  horseshoe	  crab	  may	  not	  equate	  to	  immune	  provocation	  in	  humans.	   (Takayama	   et	   al.	   1984)	  Thus,	   there	   is	   room	   for	   other	   detection	  methods	  that	   may	   correlate	   more	   directly	   with	   the	   relevant	   action	   of	   endotoxin.	   Bio-­‐functional	   tests	   using	   ELISA	   may	   be	   a	   more	   direct	   indication	   of	   immunological	  activity.	  Using	   endotoxin	   to	   stimulate	  macrophage	  production	   of	   IL-­‐1ß,	   in	   a	   dose-­‐response	  manner,	   contaminated	   samples	   can	   be	   analyzed	   using	   ELISA	  with	   IL-­‐1ß	  specific	  antigen	  and	  thus	  provide	  a	  very	  direct	  quantification	  of	  immune	  response	  to	  suspected	  LPS	  quantity.	  This	  method	  also	  allows	  for	  testing	  of	  efficacy	  for	  detoxified	  LPS	   since	   an	   immunologically	   inert	   molecule	   should	   fail	   to	   provoke	   macrophage	  activation.	  In	  endodontic	  therapy	  the	  aim	  would	  be	  to	  provide	  a	  disinfectant	  that	  can	  successfully	  remove	  the	  pathogenic	  potential	  of	  LPS.	  	  	  	  	  	  	   15	  	  2.2	  Basis	  for	  irrigants	  to	  be	  tested	  and	  use	  in	  endodontic	  therapy	  	   Modern	   endodontic	   therapy	   relies	   on	   a	   combination	   of	   mechanical	   and	  chemical	  debridement.	   	  Three	  dimensional	   analysis	   and	   computational	   techniques	  have	  brought	  about	  a	  greater	  appreciation	  for	  anatomical	  complexities	  of	  root	  canal	  systems	  and	   the	  difficulties	   involved	   in	  effective	  debridement	  (Rhodes	  et	  al.	  1999,	  Peters	   et	   al.	   2003).	   	   	   Multistep	   root	   canal	   therapies	   involving	   mechanical	  instrumentation	   and	   various	   irrigant	   delivery	   have	   been	   shown	   to	   greatly	   reduce	  bacterial	   load,	  LPS	  content	  and	  debris	  effectively	  up	  to	  99%	  (Martinho	  et	  al.	  2010,	  Xavier	   et	   al.	   2013,	   Sousa	   et	   al.	   2014).	   	   However	   these	   studies	   report	   cultivable	  bacteria	  or	  LPS	  (LAL-­‐detected)	  by	  paper	  point	  sampling	  and	  may	  not	  fully	  represent	  the	   sterility	   of	   root	   canals	   following	   treatment.	   	   Nevertheless,	   the	   importance	   of	  effective	  delivery	  of	  irrigants	  with	  multiple	  properties	  is	  stressed.	   	  Haapasalo	  et	  al.	  (2010)	   outlined	   properties	   of	   an	   ideal	   irrigating	   solution	   as	   follows:	   1	   –	  washing	  action	  to	  aid	  in	  debris	  removal;	  2	  –	  lubricant	  ability	  to	  reduce	  instrument	  friction;	  3	  –	  	  ability	  to	  dissolve	  organic	  tissue	  (pulp,	  collagen)	  and	  inorganic	  matter	  (dentin);	  4	  –	  be	  non-­‐irritating	  or	  non-­‐toxic	   to	  periradicular	   tissue;	  5	  –	  kill	  bacteria	  and	  yeasts	  (also	  in	  biofilm);	  6	  –	  do	  not	  weaken	  tooth	  structure.	  	  However,	  as	  no	  single	  irrigant	  currently	  may	  meet	  these	  requirements,	  thus	  generally	  a	  combination	  of	  irrigants	  is	  used	  to	  achieve	  the	  desired	  functions	  necessary	  for	  successful	  treatment.	  	  	  	  	  	  	  	   16	  	  2.2.1	  Sodium	  hypochlorite	  (NaOCl)	  	   Sodium	  hypochlorite	  has	  been	  used	  as	  a	  universal	  disinfectant	   for	  almost	  a	  100	   years.	   	   In	   endodontics,	   reports	   as	   early	   as	   1920	  have	   suggested	   its	   use	   as	   an	  irrigant	  solution	  for	  microbial	  disinfection	  (Crane	  1920,	  Walker	  1936).	  	  Currently,	  it	  is	   the	   most	   widely	   used	   solution	   for	   root	   canal	   therapies	   with	   concentrations	  ranging	   from	   0.5%	   -­‐	   6%	   (Vianna	   et	   al.	   2004,	   Stojicic	   et	   al.	   2012).	   	   Its	   unique	  properties	   as	   an	   irrigant	   are	   due	   to	   a	   combination	   of	   dissolution	   of	  organic/inorganic	  debris	  and	  antibacterial	  activity.	  	  NaOCl	  readily	  dissolves	  in	  water	  via	  the	  following	  reaction:	  NaOCl	  +	  H20	  <=>	  NaOH	  +	  HOCl	  <=>	  Na+	  +	  OH-­‐	  +	  H+	  +	  OCl-­‐	  The	   hypochlorous	   acid	   ion	   formed	   is	   proposed	   as	   having	   much	   to	   do	   with	   its	  antibacterial	  effect	  (Haapasalo	  et	  al.	  2005).	   	   It	  has	  been	   found	  to	  disrupt	  oxidative	  phosphorylation	  of	  cell	  membranes	  and	  affect	  DNA	  synthesis	  (Mcdonnell	  &	  Russell	  1999).	   	   Other	   reactions	   have	   been	   suggested	   for	   its	   varied	   properties	   including	   a	  saponification	  reaction	  via	  NaOH	  producing	  a	  soap	  and	  glycerol,	  which	  may	  aid	   in	  lubrication	   and	   surface	   activation	   during	   chemical	   debridement	   (Estrela	   et	   al.	  2002).	   	   Further,	   chloramination	   and	   neutralization	   of	   amino	   acids	   has	   been	  suggested	  as	  partially	  responsible	  for	   its	  overall	  tissue	  dissolving	  and	  antibacterial	  effects	  (Estrela	  et	  al	  2002).	  	  	  	  	  	  	  	  	   17	  	  	  2.2.1.1	  Efficacy	  of	  NaOCl	  	   In-­‐vitro	   studies	   of	   the	   efficacy	   of	  NaOCl	   in	   planktonic	   and	  biofilm	  bacterial	  systems	  have	  been	  conducted	  demonstrating	  its	  superior	  killing	  capacity.	  	  One	  study	  comparing	   QMix,	   a	   novel	   irrigant,	   with	   NaOCl,	   CHX	   2%	   and	   MTAD	   found	   that	   in	  planktonic	   solution	   QMix	   and	   NaOCl	   	   1%	   were	   equally	   sufficient	   in	   killing	   all	   E.	  faecalis	  within	  5	  seconds,	  whereas	  CHX	  and	  MTAD	  were	  unable	  to	  produce	  the	  same	  effect	   (Stojicic	   et	   al.	   2012).	   	   The	   same	   study	   using	   a	   biofilm	   model	   grown	   on	  hydroxyapatite	  or	  dentin	  discs	  showed	  superior	  killing	  on	  biofilm	  bacteria	  of	  both	  QMix	   and	  NaOCl	   2%	  of	   up	   to	   12	   times	  more	   than	  NaOCl	   1%,	   CHX	  2%	  and	  MTAD	  (Stojicic	   et	   al.	   2012).	   	   Another	   study	   using	   a	   biofilm	   model	   with	   E.	   faecalis	  centrifuged	  into	  dentin	  tubules,	  more	  closely	  simulating	  a	  closed	  root	  canal	  system,	  showed	  superior	  penetration	  and	  killing	  efficacy	  of	  6%	  NaOCl	  and	  QMix	  on	  biofilms	  in	  the	  dentin	  canals	  (Wang	  et	  al	  2012).	  	  A	  further	  difference	  between	  young	  and	  old	  biofilms	  was	  noted	  within	  this	  study,	  illustrating	  the	  increased	  resistance	  as	  biofilms	  mature.	   	  These	  studies	  combined	  affirm	  the	  superior	  antibacterial	  ability	  of	  NaOCl.	  	  However,	   to	   be	   noted	   is	   the	   difference	   in	   effect	   measured	   with	   different	  concentrations	  of	  the	  solution.	  	  As	  to	  be	  expected	  with	  any	  disinfectant	  solution,	  the	  amount	   of	   reactive	   substance	   present	   will	   have	   effect	   on	   its	   potency	   and	   overall	  action.	   	   In	   the	   case	   of	  NaOCl,	   in-­‐vivo	   studies	  have	   failed	   to	  demonstrate	   improved	  antibacterial	   effect	   as	   the	   concentration	   increases	   (Bystrom	   &	   Sundqvist	   1983).	  	  This	   may	   be	   due	   to	   the	   in-­‐vivo	   complexity	   	   allowing	   less	   access	   and	   contact	   to	  	  	  	  	  	   18	  bacteria	  and	  also	  the	  significant	  buffering	  effect	  that	  dentin	  has	  on	  various	  solutions	  i.e.	  CHX	  and	  in	  particular	  NaOCl	  (Haapasalo	  et	  al.	  2005).	  	  	  	   The	   effect	   of	   NaOCl	   on	   LPS	   has	   also	   been	   debated	   within	   the	   literature.	  	  Various	   studies	   evaluate	   detoxification	   of	   LPS	   by	   quantifying	   levels	   of	   free	   fatty	  acids	   (3-­‐HMA)	   following	   treatment	  with	   chemicals	   (Safavi	   et	   al.	   1993,	   Buck	   et	   al.	  2001).	   	   Findings	   suggest	   that	   high	   alkaline	   materials	   are	   responsible	   for	   de-­‐esterification	   of	   fatty	   acids	   within	   the	   lipid	   A	   moiety,	   and	   thus	   detoxification	   is	  assumed	   (Safavi	   et	   al.	   1993).	   	   Buck	   et	   al.	   (2001)	   correlated	   findings	   that	   Ca(OH)2	  caused	   significant	   release	   of	   3-­‐HMA,	   but	   NaOCl,	   EDTA	   and	   CHX	   did	   not.	   	   Further	  studies	  simulating	  root	  canal	  debridement	  in-­‐vitro	  and	  endotoxin	  detection	  via	  LAL	  assay	  appear	  to	  corroborate	  the	  previous	  conclusion.	  	  Dias	  de	  Oliveira	  et	  al.	  (2008)	  concluded	  that	  2.5%	  and	  5%	  NaOCl	  and	  2%	  CHX	  did	  not	  detoxify	  LPS,	  while	  Ca(OH)2	  and	   polymyxin	   B	   did.	   	   These	   results	   used	   LAL	   detection	   of	   LPS	   as	   well	   as	   B-­‐lymphocyte	   antibody	   production.	   	   A	   similar	   study	   by	  Maekawa	   et	   al.	   (2011)	   also	  found	  only	  significant	  reduction	  in	  detectable	  endotoxin	  levels	  following	  intracanal	  Ca(OH)2	  application.	   	  A	  series	  of	  clinical	  studies	  evaluating	  endotoxin	  and	  bacteria	  levels	  by	  paper	  point	  sampling,	  LAL	  detection	  and	  culture,	  unequivocally	  show	  that	  chemo-­‐mechanical	  debridement	  of	  infected	  root	  canals	  does	  remove	  significant	  LPS	  and	   bacteria	   (Martinho	   &	   Gomes	   2008,	   Gomes	   et	   al.	   2009,	   Martinho	   et	   al.	   2010,	  Xavier	   et	   al.	   2013,	   Sousa	   et	   al.	   2014).	   However,	   in	   all	   cases	   endotoxin	   remains	  detectable	  and	  levels	  did	  not	  significantly	  change	  when	  different	  concentrations	  of	  	  	  	  	  	   19	  NaOCl	  were	  used,	  or	  when	  simply	  using	  saline	  as	  the	  irrigant.	   	  Only	  the	  addition	  of	  Ca(OH)2	   as	   an	   interappointment	   medication	   appeared	   to	   significantly	   affect	  endotoxin	   levels.	   	   The	   various	   authors	   agree	   that	   reduction	   in	   endotoxin	   levels	  within	   infected	   root	   canals	   is	   attributed	   to	   the	   overall	   chemo-­‐mechanical	  debridement	   process,	   including	   interappointment	   Ca(OH)2,	   but	   is	   not	   significantly	  affected	  by	  the	  use	  of	  NaOCl	  at	  various	  concentration.	  	  	  	  2.2.2	  	  Calcium	  hydroxide	  (Ca(OH)2)	  	   	  The	   use	   of	   Ca(OH)2	   as	   a	   medicament	   is	   well	   documented.	   	   Introduced	   in	  1920	   as	   an	   endodontic	  medicament	   by	  Hermann,	   it	   is	   now	   the	  most	  widely	   used	  intracanal	  medicament.	   	   Ca(OH)2	  maintains	   a	   very	   high	   pH	   of	   approximately	   12.5	  which	  most	   endodontic	   pathogens	   are	   unable	   to	   survive	   (Heithersay	   1975).	   	   This	  antimicrobial	   activity	   is	   attributed	   to	   the	   release	   of	   hydroxyl	   ions	   in	   aqueous	  solution,	  which	  are	  highly	  oxidant	  free	  radicals.	  	  The	  free	  hydroxyl	  ion	  may	  directly	  damage	   the	   bacterial	   cytoplasmic	   membrane,	   denature	   proteins	   and	   damage	  bacterial	  DNA	  (Siqueira	  &	  Lopes	  1999).	  The	   effect	   of	   Ca(OH)2	  within	   tubular	   dentin	   is	   related	   to	   diffusability	   of	   hydroxyl	  ions	   and	  may	   persist	   for	   extended	   periods.	   	   Tronstad	   et	   al.	   (1981)	   noted	   that	   pH	  levels	  within	  monkey	  dentin	   remained	  elevated	   for	  up	   to	  4	  weeks.	   	  Nerwich	  et	  al.	  (1993)	  found	  that	  pH	  levels	  were	  related	  to	  the	  level	  of	  application	  and	  time,	  noting	  that	   pH	   levels	   peaked	   at	   2	  weeks	   and	  were	   higher	   cervically	   than	   apically.	   	   	   The	  effect	  on	  bacteria	  may	  also	  be	   time	  dependent	  as	   it	  has	  been	  observed	   that	   it	   can	  take	  up	  to	  10	  days	   to	  disinfect	   tubules	   infected	  by	   facultative	  bacteria	  (Ørstavik	  &	  	  	  	  	  	   20	  Haapasalo	  1990).	  	  Further	  the	  buffering	  capacity	  of	  dentin	  by	  proton	  donors	  such	  as	  H2PO4,	  H2CO3	  and	  HCO3	  may	  dampen	  the	  pH	  effect	  of	  Ca(OH)2	  (Wang	  &	  Hume	  1988).	  	  Several	  species	  have	  been	  found	  to	  persist	   following	  treatment	  periods	  of	  Ca(OH)2	  with	   different	   preparations:	   E.	   faecalis	   following	   Calasept	   (Nordiska	   Dental,	  Sweden)	   was	   found	   in	   superficial	   tubules	   (Haapasalo	   &	   Ørstavik	   1987),	   and	   E.	  faecium	   following	   extended	   application	   of	   Ca(OH)2	   in	   saline	   (Safavi	   et	   al	   1990).	  	  	  There	  is	  also	  disagreement	  regarding	  the	  optimal	  time	  period	  with	  varying	  reports	  and	  times	  showing	  either	  complete	  or	  incomplete	  disinfection	  (Bystrom	  et	  al.	  1985,	  Reit	  &	  Dahlen	  1988,	  Sjogren	  et	  al	  1991,	  Ørstavik	  et	  al.	  1991).	  	  Further	  as	  previously	  reviewed,	   the	   effect	   of	   Ca(OH)2	   on	   LPS	   detoxification	   is	   highly	   regarded.	   	   Studies	  agree	  that	  while	  the	  effect	  of	   irrigants	  on	  LPS	  is	  debatable,	   the	  effect	  of	  Ca(OH)2	   is	  unequivocal	  (Safavi	  &	  Nichols	  1993,	  Buck	  et	  al.	  2001,	  Martinho	  et	  al.	  2008,	  Martinho	  et	   al.	   2010).	   	   This	   detoxification	   is	   attributed	   to	   alkaline	   hydrolysis	   of	   the	   LPS	  structure.	  	  	  	   To	  date	   there	  have	  been	   few	  reports	   in	  endodontic	   literature	  regarding	   the	  use	  of	  NaOH.	  	  At	  the	  time	  of	  this	  writing,	  only	  one	  study	  using	  NaOH	  in	  combination	  with	   CHX	   and	   ethanol	   was	   found	   showing	   a	   positive	   detoxification	   effect	   on	  endotoxin	   in-­‐vitro	  (Buck	  et	  al.	  2001).	   	   	  A	  publication	  from	  the	  field	  of	  virology	  has	  suggested	   that	   a	   combination	  of	   low	   concentration	  NaOH	  combined	  with	   SDS	   and	  propanol	  was	  shown	  to	  act	  synergistically	  achieving	  fast,	  broad-­‐range	  disinfection	  of	  bacteria,	  fungi,	  viruses	  and	  prions	  (Beekes	  et	  al.	  2010).	  	  Various	  combinations	  with	  	  	  	  	  	   21	  alcohols	  in	  this	  study	  were	  used,	  which	  improved	  the	  disinfective	  properties	  up	  to	  6	  log	   reduction	   against	   enterococci	   and	   mycobacteria.	   	   Against	   fungal	   spores	  NaOH/SDS,	  ethanol	  and	  propanol	  individually	  did	  not	  improve	  antimicrobial	  action,	  however	  when	   used	   in	   combination	   an	   over	   5	   log	   reduction	   in	   spore	   counts	  was	  observed.	  	  	  	   Thus,	   the	   combination	  of	  different	   irrigants	   to	  achieve	  multiple	  properties	  and	  synergism	  may	  be	  a	  useful	  strategy	  in	  endodontic	  therapy.	  	  A	  prime	  example	  of	  this	  is	   the	   strong	   antibacterial,	   antibiofilm	   and	   smear	   layer	   removal	   effects	  demonstrated	  by	  QMix	  (Stojicic	  et	  al.	  2012,	  Wang	  et	  al.	  2012).	  	   	  	  	  	  	  	   22	  3. Aims	  and	  hypothesis	  	  3.1	  Aims	  	   Specific	  aims	  of	  this	  project	  were	  to:	  1	   -­‐	   Evaluate	   and	   compare	   solutions	   containing	   combinations	   of	   NaOH,	   SDS	   and	  Propanol	   to	   the	   current	   gold	   standard	   in	   endodontic	   irrigation,	   NaOCl,	   for	   their	  effect	   on	   relevant	   bacterial	   factors	   in	   root	   canal	   infections;	   namely:	   Planktonic	  E.	  faecalis,	  bacterial	  LPS	  and	  polymicrobial	  biofilms.	  	  2	  –	  To	  develop	  and	  refine	  a	  useful	  protocol	  for	  testing	  detoxification	  of	  bacterial	  LPS	  using	  a	  biofunctional	  assay.	  	  The	  stepwise	  test	  protocol	  is	  outlined	  as	  follows:	  	  a)	  LPS	  is	  treated	  with	  potential	  disinfecting/detoxifying	  agent	  b)	  Toxic	  substances	  are	  removed	  by	  microdialysis	  c)	   Remaining	   or	   modified	   LPS	   is	   tested	   for	   immunogenicity	   by	   incubating	   with	  macrophages	  to	  stimulate	  cytokine	  production	  (IL-­‐1ß)	  d)	  Cytokine	  production	  is	  quantified	  via	  ELISA	  for	  specific	  cytokine	  (IL-­‐1ß)	  	   The	   universal	   test	   and	   detection	  method	   for	   LPS/endotoxin	   is	   the	   Limulus	  Amebocyte	  Lysate	  assay	  (LAL),	  which	  uses	  isolate	  from	  blood	  of	  the	  Horseshoe	  crab	  and	   its	   coagulant	   reaction	  with	  endotoxin.	   	  The	   reaction	   is	   then	  quantified	  via	   gel	  	  	  	  	  	   23	  clot,	   turbidimetric	   or	   colorimetric	   methods	   and	   standardized	   in	   endotoxin	   units	  (EU).	  	  	  	   The	  rationale	  for	  a	  different	  test	  methodology	  is	  to	  correlate	  the	  presence	  of	  LPS/endotoxin	  with	   clinically	   relevant	   immune	  markers,	   i.e.	   cytokines	   as	   a	   direct	  indication	  of	  immunogenic	  potential	  in	  inflammatory	  bone	  resorption.	  	  It	  is	  possible	  that	  chemically	  modified	  LPS	  or	  byproducts	  may	  stimulate	  immune	  reactions	  which	  may	  or	  may	  not	  be	  detected	  by	  LAL	  assay.	  	  3.2	  Hypothesis	  	   The	  null	  hypothesis	  for	  this	  study	  is	  that	  NaOH/SDS/Propanol	  solutions	  will	  be	  equal	   to	   NaOCl	   in	   killing	   planktonic	   E.	   faecalis	   and	   microbes	   in	   polymicrobial	  biofilms,	  and	  in	  detoxifying	  LPS.	  	   	  	  	  	  	  	   24	  4. Materials	  and	  methods	  	   Test	  solutions	  were	  prepared	  as	  follows:	  	  -­‐	  6%	  NaOCl	  (Clorox,	  Oakland,	  CA,	  USA)	  stock	  solution	  verified	  by	  sodium	  thiosulfate	  titration	  -­‐	  2%	  NaOCl	  by	  3:1	  dilution	  in	  sterile	  water	  -­‐	   Triple	   solution:	   0.3%	   NaOH,	   0.2%	   SDS,	   20%	   n-­‐propanol	   in	   sterile	   water	   (from	  stock	   solutions	   of	   30%	   NaOH,	   20%	   SDS	   and	   99%	   n-­‐propanol	   all	   obtained	   from	  Sigma-­‐Aldrich	  Chemical,	  St	  Louis,	  Missouri,	  USA)	  -­‐	  Double	   solution:	   0.3%	  NaOH,	   0.2%	  SDS	   in	   sterile	  water	   (from	   stock	   solutions	   of	  30%	  NaOH,	  20%	  SDS	  from	  Sigma-­‐Aldrich	  Chemical)	  Note:	   NaOH	  mixed	  with	   SDS	   alone	   formed	   a	   precipitate	   that	   required	   addition	   of	  propanol	  and/or	  water	  to	  dissolve.	  	  Thus	  solutions	  should	  be	  agitated	  briefly	  before	  use.	  All	  data	  were	  analyzed	  statistically	  using	  ANOVA	  (analysis	  of	  variance).	  	  	  4.1	  Planktonic	  killing	  tests	  	  E.	  faecalis	  strain	  VP-­‐181	  was	  subcultured	  overnight	  on	  tryptic	  soy	  agar	  (TSA)	  plates	   (Difco	  &	   Becton,	   Dickinson,	   Sparks,	  MD,	   USA).	   	   A	   bacterial	   suspension	  was	  created	  using	  sterile	  water	  and	  the	  subcultured	  E.	  faecalis	  to	  an	  OD	  value	  of	  0.20+/-­‐	  0.05	  @	  405nm.	  	  	  	  	  	  	  	  	   25	  	  Test	  1	  	  Fifty	  µl	  of	  bacterial	  suspension	  (OD	  0.249)	  was	  mixed	  with	  450	  µl	  each	  of	  2%	  NaOCl,	  Triple	  solution,	  Double	  solution,	  and	  sterile	  water	  for	  5	  seconds,	  30	  seconds	  and	   1	   minute.	   	   Following	   appropriate	   time	   of	   exposure	   the	   samples	   were	  immediately	   serially	   diluted	   5	   by	   10	   fold	   in	   brain	   heart	   infusion	   (BHI;	   Difco	   &	  Becton,	   Dickinson)	   broth	   to	   negate	   the	   carry	   over	   killing	   effect	   of	   the	   solutions.	  	  Following	  dilutions	  10:l	  of	  each	  of	  the	  diluted	  series	  were	  plated	  in	  triplicate	  on	  TSA	  plates	   and	   incubated	   for	   24	   hours.	   	   Colony	   forming	   unit	   (CFU)	   counts	   were	  evaluated	   from	   the	   plated	   series	   in	   which	   colonies	   were	   numerous	   but	   separate	  enough	  to	  be	  counted	  accurately	  and	  compared	  with	  controls	  of	   the	  same	  dilution	  series.	  Test	  2	  	  Two-­‐hundred	  and	  fifty	  µl	  of	  bacterial	  suspension	  (OD	  0.22)	  was	  mixed	  with	  250ul	   each	   of	   6%	   NaOCl,	   2%	   NaOCl,	   Triple	   solution,	   Double	   solution,	   and	   sterile	  water	  for	  5	  seconds,	  30	  seconds	  and	  1	  minute.	  	  Following	  appropriate	  time	  exposure	  samples	  were	   immediately	  serially	  diluted	  5	  by	  10	   fold	   in	  BHI	   to	  negate	   the	  carry	  over	   killing	   effect	   of	   the	   solutions.	   	   Following	  dilutions	   10:l	   of	   each	   of	   the	   diluted	  series	   were	   plated	   in	   triplicate	   on	   TSA	   plates	   and	   incubated	   for	   24	   hours.	   	   CFU	  counts	  were	  evaluated	  from	  the	  plated	  series	  in	  which	  colonies	  were	  numerous	  but	  separate	  enough	  to	  be	  counted	  accurately	  and	  compared	  with	  controls	  of	  the	  same	  dilution	  series.	  	  	  	  	  	  	   26	  	   This	   subsequent	   test	   was	   performed	   in	   hopes	   of	   saturating	   the	   bacterial	  concentration	  and	   thereby	   showing	  a	   relative	  difference	   in	  potency	  of	   the	  various	  solutions.	  	  In	  addition,	  6%	  NaOCl	  was	  added	  to	  the	  test	  groups.	  	  4.2	  LPS	  detoxification	  tests	  	  E.	  coli	  LPS	  (Batch	  0111:B4,	  Lot	  No:043M4104V,	  Sigma-­‐Aldrich	  Chemical)	  was	  used	  for	  all	  tests	   	  (Fig.	  3).	   	  The	  basis	  for	  the	  method	  of	  LPS	  testing	  is	  based	  on	  the	  known	   stimulation	   of	   IL-­‐1ß	   production	   by	   monocyte	   delineated	   cells	   i.e.	  macrophages.	   	   The	   secretion	   of	   IL-­‐1ß	   is	   an	   important	   cytokine	   involved	   in	   the	  inflammatory	  reaction	  leading	  to	  bone	  resorption	  by	  osteoclasts.	  	  	  LPS	  solutions	  were	  treated	  with	  the	  various	  irrigants	  for	  the	  possible	  detoxification,	  then	  subjected	  to	  microdialysis	  for	  removal	  of	  toxic	  irrigants.	  	  The	  molecular	  weight	  of	   LPS	   is	   generally	   in	   the	   range	   of	   20	   kDa	   and	   thus	   would	   be	   retained	   by	   low	  molecular	  weight	  cut	  off	  dialysis	  membranes	  i.e.	  2kDa,	  whereas	  substances	  such	  as	  NaOCl,	  NaOH,	  and	  propanol	  should	  all	  pass	  through	  and	  be	  removed	  from	  solution.	  	  The	  remaining	  LPS	  then	  is	  used	  to	  stimulate	  macrophages	  for	  the	  production	  of	  IL-­‐1ß,	  which	  can	  then	  be	  quantified	  by	  ELISA.	  (Fig.	  4)	  	  The	  amount	  of	  IL-­‐1ß	  produced	  theoretically	   should	   be	   proportional	   to	   the	   remaining	   amount	   of	   LPS	   or	  immunologically	  active	  components	  of	  LPS,	  i.e.	  Lipid	  A.	  	  	  	  	  	  	  	  	   27	  	  4.2.1	  Dose	  response	  	   Mouse	   macrophages	   RAW	   264.7	   were	   cultured	   in	   Dulbecco’s	   modified	  Eagle's	   minimal	   essential	   medium	   (DMEM,	   ThermoFisher	   Scientific,	   Waltham,	  Massachusetts,	   U.S.A.)	   in	   75cm	   tissue	   culture	   flasks	   	   until	   confluency,	   whereupon	  they	  were	   seeded	  at	   approximately	  200,000	   cells	   per	  well	   in	   a	  96	  well	   plate	  with	  200:l	  culture	  media.	  Fifty	  µl	  of	  LPS	  solution	  in	  PBS	  at	  concentrations	  of	  each	  of	  250	  µg/ml,	   50	   µg/ml,	   10	   µg/ml,	   2	   µg/ml,	   0.5	   µg/ml	   and	   0.16	   µg/ml	   were	   added	   in	  triplicate	  to	  stimulate	  IL-­‐1ß	  production.	  	  OD	  values	  at	  420nm	  were	  obtained	  using	  a	  spectrophotometer.	  	  IL-­‐1ß	  production	  was	  then	  quantified	  using	  Quantikine	  ELISA	  for	   mouse	   IL-­‐1ß	   (R&D	   Systems,	   Minneapolis,	   Minnesota,	   U.S.A.)	   and	   averages	  compared	   to	   the	   IL-­‐1ß	   standard	   results	  provided	  within	   the	  kit.	   	  Results	  of	   these	  tests	  suggested	  that	  the	  maximum	  concentration	  of	  LPS	  tested	  (250µg/ml)	  provided	  stimulation	   of	   IL-­‐1ß	   in	   the	   range	   of	   200-­‐400	   pg/ml,	   which	   corresponded	   to	   the	  middle	  range	  of	  the	  detection	  spectrum	  of	  results.	  	  	  	  4.2.2	  Detoxification	  tests	  	   A	  solution	  of	  2mg/ml	  of	  LPS	  in	  PBS	  was	  prepared.	  	  0.5ml	  of	  LPS	  solution	  was	  then	  mixed	  thoroughly	  with	  0.5ml	  of	  6%	  NaOCl,	  2%	  NaOCl,	  Triple	  solution,	  Double	  solution	  and	  PBS	  (control)	  for	  5	  minutes	  under	  agitation.	  	  0.5ml	  of	  treated	  solutions	  were	   then	  added	   to	  Slide-­‐A-­‐Lyzer	  cassettes	   (0.5	  ml,	  2kDA	  MWCO,	  Thermo	  Fischer	  Scientific)	  for	  microdialysis	  in	  1	  L	  of	  PBS	  for	  3	  successive	  cycles	  2	  hours	  each.	  (See	  	  	  	  	  	   28	  Fig	  6).	   	   	  The	  dialyzed	  solution	  was	  then	  removed	  and	  50	  µl	  in	  5	  replicates	  for	  each	  group	  were	   added	   to	   96	  well	   plate	  wells	   containing	   200	   µl	   of	   culture	  media	   and	  previously	   seeded	   macrophages	   (ca.	   200,000	   cells/well	   +	   200µg/ml	   LPS	   if	  unmodified).	  	  These	  were	  incubated	  at	  37oC	  for	  24	  hours	  to	  allow	  stimulation	  of	  IL-­‐1ß.	  	  Following	  this	  period	  aliquots	  of	  each	  well	  were	  tested	  using	  Quantikine	  ELISA	  for	   mouse	   IL-­‐1ß	   (R&D	   systems)	   as	   per	   manufacturer’s	   instructions.	   (Fig.	   5).	   OD	  values	  at	  420	  nm	  were	  obtained	  using	  a	  spectrophotometer	  and	  averages	  correlated	  to	  standard	  curve	  values	  produced	  by	  known	  concentrations	  of	  IL-­‐1ß	  (provided	  in	  ELISA	  kit	  @	  800pg/ml	  and	  diluted	  serially).	  	  	  The	  following	  controls	  were	  tested	  in	  a	  similar	   fashion:	   LPS	   dialyzed,	   LPS	   non-­‐dialyzed,	   irrigant	   solutions	   dialyzed.	   	   Cell	  morphology	   was	   visualized	   following	   incubation	   under	   light	   microscope	   and	  photographed.	  (Figs.	  8-­‐13).	  	   It	  should	  be	  noted	  that	  similar	  tests	  were	  previously	  performed	  using	  different	  dialysis	  times/protocol	  and	  LPS	  concentrations.	  	  The	  results	  of	  these	  varied	  greatly	  and	  were	  grossly	  inconsistent,	  such	  as	  producing	  negative	  results	  in	  positive	  control	  groups.	  	  Challenges	  arising	  in	  developing	  a	  successful	  test	  protocol	  will	  be	  described	  in	  the	  discussion	  subsequently.	  	   4.3 Biofilm	  tests	  	  Sterile	  hydroxyapatite	  (HA)	  discs (Lot# 300314 Size: 0.50" Dia. X 0.04-0.06" thick)	  (Clarkson Chromatography Products Inc., South Williamsport, PA, USA)	  were	  	  	  	  	  	   29	  soaked	  in	  2	  ml	  of	  dermal	  type	  I	  collagen	  (10µg/ml	  in	  0.012	  N	  HCl	  in	  water;	  Sigma-­‐Aldrich	  Chemical)	  overnight	  at	  4oC.	  	  Plaque	  samples	  from	  2	  volunteers	  were	  pooled	  and	  suspended	  in	  BHI	  broth,	  where-­‐upon	  HA	  discs	  were	  soaked	  in	  the	  broth	  (2	  ml)	  in	  wells	  of	  a	  24-­‐well	  plate	  and	  incubated	  at	  37oC	  anaerobically	  for	  either	  1	  or	  3	  weeks.	  	  BHI	  solution	  was	  refreshed	  weekly	  in	  the	  3	  week	  old	  discs.	  	  Biofilm	  discs	  were	  rinsed	  in	  PBS	  twice	  by	  immersion	  to	  remove	  culture	  broth	  then	  treated	  with	  100	  µl	  of	  	  solutions:	  2%	  NaOCl,	  Double	  Solution,	  Triple	  Solution	  or	  sterile	  water	  for	  3	  minutes.	  	  Discs	  were	  then	  rinsed	  again	  by	  immersion	  in	  2ml	  PBS	  in	  24-­‐well	  plates.	  	  LIVE/DEAD	  Bac-­‐Light	  Bacterial	  Viability	  stain	  (kit	  L-­‐7012;	  Molecular	  Probes,	  Eugene,	  OR,	  USA)	  containing	  two	  component	  dyes	  in	  equal	  proportions	  of	  SYTO	  9	  and	  propidium	  iodide	  was	  used	  to	  stain	  the	  biofilm	  for	  15	  minutes	  and	  following	  a	  rinse	  were	  immediately	  visualized	  using	  a	  confocal	  laser	  scanning	  microscope	  (CLSM)	  (Nikon	  Eclipse	  C1;	  Nikon,	  Mississauga,	  ON,	  Canada).	  	  The	  mounted	  specimens	  were	  observed	  using	  10x	  lenses	  with	  numerical	  aperture	  0.30	  and	  confocal	  pinhole	  set	  at	  30um	  diameter	  without	  a	  coverslip.	  	  CLSM	  images	  were	  acquired	  by	  EZ-­‐C1	  v.	  3.40	  build	  691(Nikon)	  at	  a	  pixel	  resolution	  of	  2.5	  um	  and	  field	  resolution	  of	  512x512	  pixels.	  	  Step	  sizes	  ranged	  from	  0.4-­‐0.6µm.	  	  Images	  were	  then	  analyzed	  and	  quantified	  using	  Imaris	  x64	  V.7.3.0	  software	  (Bitplane	  Inc,	  St	  Paul,	  MN,	  USA).	  	  Live	  bacteria	  appeared	  green	  and	  dead	  bacteria	  red	  and	  thus	  proportion	  red	  fluorescence	  was	  used	  to	  calculate	  the	  percentage	  of	  dead	  cells	  in	  biofilm,	  and	  total	  biovolume	  was	  calculated	  by	  combining	  total	  red	  and	  green	  fluorescence.	  	  Each	  test	  group	  contained	  2	  HA	  biofilm	  discs,	  from	  which	  6-­‐10	  stacks	  of	  images	  were	  obtained	  	  	  	  	  	   30	  and	  analyzed.	  	   	  	  	  	  	  	   31	  	  5. Results:	  5.1	  Planktonic	  killing	  tests	  	   At	  CFU	  concentration	  2.5x105/ml	  (approx.)	  E.	  faecalis	  VP-­‐181	  was	  killed	  up	  to	   between	   98%-­‐100%.	   	   No	   significant	   difference	   was	   found	   between	   groups	   at	  different	   times	   of	   exposure;	   5s,	   30s	   or	   1min	   or	   different	   chemicals;	   2%NaOCl,	  Double	  and	  Triple	  solutions.	  	  (Table	  1,	  Fig.	  1)	  	   At	  CFU	  concentration	  1.75x106/ml	  (approx.)	  E.	  faecalis	  VP-­‐181	  was	  killed	  up	  to	  100%	  only	  by	  6%	  NaOCl	  at	  exposure	  time	  of	  1min.	  	  NaOCl	  efficacy	  was	  dependent	  on	  time	  as	  well	  as	  on	  concentration.	   	  Both	  6%	  and	  2%	  NaOCl	  were	  more	  effective	  than	   the	   two	   NaOH	   solutions,	   and	   no	   significant	   difference	   was	   found	   between	  killing	  by	  Double	  or	  Triple	  solutions.	  	  (Table	  2	  and	  Fig.	  2)	  	  5.2	  LPS	  detoxification	  	  	   ELISA	  results	  (@450nm	  w/correction	  @420nm)	  indicated	  the	  amount	  of	  IL-­‐1ß	  present	  after	  stimulation	  by	  the	  various	  solutions.	  	  	   6%	   NaOCl,	   2%	   NaOCl	   and	   Double	   solutions	   resulted	   in	   negligible	   IL-­‐1ß	  values	   (<12.5pg/ml)	   suggesting	   no	   remaining	   LPS	   effect	   after	   exposure	   to	   these	  solutions.	   	  Negative	   controls	   i.e.	   non-­‐treated	   cells	   (no	  LPS)	   and	  dialyzed	   solutions	  	  	  	  	  	   32	  only	   also	   produced	   similar	   results.	   	   The	   Triple	   solution	   group	   produced	   an	  unexpected	   maximum	   level	   of	   >800pg/ml	   of	   IL-­‐1ß.	   	   Positive	   controls;	   LPS	   and	  Dialyzed	   LPS	   produced	   IL-­‐1ß	   around	   100pg/ml	   with	   no	   significant	   difference	  between	  Dialyzed/Non-­‐Dialyzed	  groups.	  	  (Table	  3	  and	  Fig.	  7)	  	  The	  cellular	  morphology	  of	  the	  microbes	  under	  light	  microscopy	  indicated	  that	  cells	  treated	  with	  Triple	  solution	  appeared	  different	  than	  all	  other	  cells.	   	  Cells	  appeared	  hazy	  and	  non-­‐spherical.	  	  (See	  Photo	  8-­‐13)	  A	  further	  test	  was	  performed	  with	  2%	  NaOCl,	  NaOH,	  SDS	  and	  propanol	  separately	  to	  ascertain	   likely	   cause	   for	   results	   exhibited	  by	   the	  Triple	   solution	  group.	   	  This	   test	  confirmed	  that	  propanol	  alone	  produced	  IL-­‐1ß	  stimulation.	  	  All	  other	  groups	  failed	  to	  elicit	  an	  IL-­‐1ß	  response.	  	  	  	  5.3	  Biofilm	  tests	  	  5.3.1 One	  week	  old	  polymicrobial	  biofilms	  	  	  The	   results	   indicated	   that	   at	   the	   3	   min	   exposure	   by	   2%	   NaOCl	   killed	  approximately	   90%	  of	  microbial	   cells,	  whereas	  Double	   and	  Triple	   solutions	   killed	  only	  42%	  and	  28%,	   respectively.	   	  Negative	   controls	   (untreated	  biofilm)	   showed	  a	  12%	  dead	  cell	  population	  on	  the	  average.	   	  Differences	  were	  statistically	  significant	  for	  all	  groups,	  except	  for	  between	  Double	  and	  Triple	  solution	  groups.	  (Table	  4	  and	  Figure	  16)	  The	  average	  biofilm	  biovolumes	  after	  exposure	  were	  noticeably	  different	  amongst	  the	  groups.	  	  When	  compared	  to	  controls,	  there	  was	  approximately	  10	  fold	  reduction	  	  	  	  	  	   33	  in	  remaining	  biofilm	  mass	  after	   treatment	  with	  2%	  NaOCl.	   	  For	  Double	  and	  Triple	  solutions	  a	  biovolume	  reduction	  of	  100-­‐200	  fold	  was	  noted.	   	  However,	  due	  to	  high	  standard	   deviations	   in	   biovolume,	   statistically	   significant	   differences	   were	   found	  only	  between	  test	  and	  control	  groups	  and	  not	  between	  test	  groups	  specifically.	  	   5.3.2 Three	  week	  old	  polymicrobial	  biofilms	  	  The	  results	  showed	  that	  at	  a	  3	  min	  exposure	  2%	  with	  NaOCl	  killed	  75%	  of	   the	  biofilm	  cells,	  Double	  solution	  killed	  32%	  and	  Triple	  solution	  killed	  44%.	   	  Negative	  controls	  without	  treatment	  showed	  that	  only	  2%	  of	  the	  cells	  were	  dead.	  	  Statistically	  significant	   differences	   were	   noted	   between	   all	   groups	   and	   controls,	   except	   for	  between	  Double	  and	  Triple	  solution	  groups.	  	  (Table	  4	  and	  Figure	  18).	  Biovolume	  averages	  again	  reflected	  a	  similar	  trend	  as	  noticed	  in	  experiments	  with	  one	  week	  old	  biofilms.	  	  Biovolume	  averages	  showed	  between	  10-­‐20	  fold	  reductions	  from	  controls	  when	  treated	  with	  2%	  NaOCl,	  and	  again	  100-­‐200	  fold	  reductions	  for	  solution	  mixtures	  containing	  NaOH.	  	  Biovolume	  averages	  in	  three	  week	  old	  biofilms	  were	  greater	   than	  1	  week	  averages	   for	  controls	  and	  all	   treatment	  groups.	   	   (Figure	  17).	  	   	  	  	  	  	  	   34	  6. Discussion	  This	   study	   represents	   the	   first	   of	   its	   kind	   in	   evaluating	   the	   effect	   of	   solutions	  containing	  NaOH/SDS	   and	  propanol	   combinations	   against	   biofilms	   and	   endotoxin.	  	  The	  use	  of	  NaOH	  may	  have	  controversial	  safety	  implications	  for	  use	  in	  endodontics.	  However,	  at	  the	  NaOH	  concentrations	  presented	  herein,	  pH	  levels	  were	  comparable	  to	   NaOCl.	   	   Further,	   the	   aim	   of	   endodontic	   irrigation	   is	   to	   effectively	   debride	   and	  disinfect	  intra-­‐canal	  contents	  while	  minimizing	  extrusion	  and	  damage	  to	  periapical	  tissues.	   	   NaOCl	   also	   may	   be	   considered	   cytotoxic	   to	   surrounding	   tissues.	  	  Contraindications	   for	   the	   use	   of	   NaOH	   in	   endodontics	   may	   require	   further	  substantiation.	  	  	  	  	  6.1	  Planktonic	  killing	  tests	  	   Planktonic	   killing	   of	   E.	   faecalis	   has	   been	   described	   in	   numerous	   previous	  studies	  (Stojicic	  et	  al.	  2012,	  Wang	  et	  al.	  2012).	  	  Stojicic	  et	  al.	  (2013)	  reported	  100%	  killing	  by	  2%	  NaOCl	  and	  QMix	  at	  all	  exposure	  times	  (5s,	  30s,	  1min).	   	  Test	  1	  results	  were	   in	   line	   with	   this	   finding	   at	   99%	   (±	   S.D.)	   for	   all	   groups	   and	   times.	   	   Minor	  variations	  may	  be	  accounted	  for	  by	  differences	  in	  concentration	  as	  measured	  by	  OD	  values	  and	  CFU	  counts	  from	  control	  groups.	  	  Test	  2	  was	  performed	  at	  concentration	  7	  times	  higher	  than	  in	  Test	  1,	  thereby	  increasing	  the	  killing	  ability	  of	  the	  solutions.	  	  The	   differences	   between	   the	   antimicrobial	   regimens	   tested	   represented	   a	   relative	  potency	   between	   solutions	   that	   showed	   a	   proportional	   increase	   with	   increased	  concentration	  and	  exposure	   times.	   	  The	  present	   results	   suggested	   that	   in	  order	  of	  	  	  	  	  	   35	  effectiveness	   (from	   strong	   to	   weak);	   6%	   NaOCl	   >	   2%	   NaOCl	   >	   Double/Triple	  solutions.	   	   There	   was	   no	   statistically	   significant	   difference	   between	   Double	   and	  Triple	   solutions	   in	   their	   effectiveness	   to	   kill	   planktonic	  E.	   faecalis	  cells.	   	   	   A	   larger	  difference	  in	  killing	  was	  observed	  between	  5s	  and	  30s,	  more	  than	  between	  30s	  and	  1	  min	  exposure	  times.	  	  This	  may	  suggest	  that	  the	  optimal	  exposure	  time	  for	  irrigants	  is	  30s	  in	  planktonic	  killing,	  and	  that	  refreshing	  the	  solution	  at	  this	  time	  may	  increase	  killing	  efficacy.	  	  	  The	  clinical	  relevance	  of	  planktonic	  killing	  tests	  is	  still	  uncertain	  for	  several	  reasons:	  1.	   Types	   of	   bacteria	   and	   concentrations	   present	   in	   the	   root	   canal	   are	   difficult	   to	  ascertain,	  and	  most	  in-­‐vivo	  studies	  to	  date	  rely	  on	  either	  PCR	  or	  culturing	  methods	  that	   produce	   varied	   results	   (Estrela	   et	   al.	   2008).	   	   2.	   The	   efficacy	   of	   the	   solutions	  relies	  on	  direct	  exposure	  to	  bacteria	  which	  in	  a	  clinical	  situation	  is	  complicated	  by	  variable	  anatomy,	  dentin	  tubule	  penetration,	  root	  canal	  geometry	  and	  formation	  of	  protective	  biofilms	  (Torabinejad	  et	  al.	  2002),	  3.	  Planktonic	  killing	  tests	  are	  generally	  well	   standardized	   but	   lack	   practical	   application,	   and	   therefore	   Capacity	   tests	   and	  Practical	   tests	   offer	   more	   precise	   information	   on	   real	   applications	   (Reybrouck	  1998).	   	   Test	   2	   was	   an	   attempt	   at	   evaluating	   specific	   capacity	   of	   the	   different	  solutions.	   	   An	   example	   of	   a	   practical	   test	   in	   endodontic	   irrigation	   is	   the	   model	  proposed	  by	  Ma	  et	  al.	  (2011)	  using	  a	  ‘closed’	  dentin	  model	  and	  centrifuging	  bacteria	  into	   tubules	   for	   testing	  with	   irrigants	  and	  chemicals.	   	  Results	   from	   this	   study	  also	  suggest	  that	  6%	  NaOCl	  was	  superior	  in	  bacterial	  killing.	  	  	  	  	  	  	  	  	   36	  	  6.2	  LPS	  detoxification	  tests	  	   The	   test	   methodology	   proposed	   for	   the	   assessment	   of	   LPS	   detoxification	  within	  this	  study	  is	  novel,	  and	  to	  date	  no	  similar	  or	  corresponding	  methodology	  has	  been	  published	  in	  the	  literature.	  	  A	  number	  of	  analogous	  studies	  have	  demonstrated	  the	   use	   of	   cytokine	   detection	   by	   ELISA	   as	   an	   indicator	   of	   immune	   potential	   of	  treated	  bacterial	  factors.	   	  Baik	  et	  al.	  (2008)	  assessed	  Lipoteichoic	  Acid	  inactivation	  by	   Ca(OH)2	   using	   RAW	   264.7	   macrophages	   and	   TNF-­‐α	   production	   via	   ELISA.	  	  Shrestha	   et	   al.	   (2015)	   evaluated	   endotoxin	   inactivation	   by	   Ca(OH)2	   and	   Chitosan	  particles	   also	  using	  ELISA	   for	  TNF-­‐α	  and	   IL-­‐6.	   	  The	   latter	   study	   found	   that	  due	   to	  cytotoxicity	  of	  Ca(OH)2	  on	  macrophage	  cells,	  a	   false	  positive	  result	   for	   inactivation	  was	  produced.	  	  The	  results	  of	  the	  present	  study	  also	  should	  be	  read	  with	  caution,	  as	  the	   test	   protocol	   requires	   complete	   dialysis	   of	   harmful	   chemicals	   prior	   to	  macrophage	   stimulation	   to	   accurately	   assess	   remaining	   LPS	   activity.	   	  Macrophage	  morphology	  (Fig.	  12-­‐13)	  from	  aliquots	  containing	  combinations	  with	  SDS	  suggested	  that	   cells	   were	   altered	   physically	   by	   the	   dialyzed	   solutions	   and	   cell	   membranes	  appeared	   hazy	   and	   poorly	   defined.	   	   SDS	   in	   solution	   will	   form	   micelles	   that	   may	  exceed	  10	  kDa,	  and	  therefore	  would	  not	  be	  removed	  by	  dialysis.	  	  Incomplete	  dialysis	  of	  SDS	  may	  explain	  the	  observed	  altered	  morphology,	  and	  if	  cells	  were	  non-­‐viable	  a	  similar	   false	   positive	   result	   as	   observed	   by	   Shrestha	   et	   al.	   (2010)	   would	   be	  observed.	   	   Baek	   et	   al.	   (2008)	   also	   noted	   that	   testing	   with	   Ca(OH)2	   resulted	   in	  residual	  cytotoxic	  effect	  on	  cells	  and	  was	  a	  potential	  confounding	  variable.	  	  	  	  	  	  	  	   37	  Another	   interesting	   finding	   was	   observed	   in	   the	   experiments	   with	   the	   Triple	  solution.	   	   IL-­‐1ß	   production	   here	   was	   even	   higher	   than	   the	   LPS	   only	   (positive	  control)	  groups.	  	  When	  individual	  chemicals	  were	  tested	  subsequently	  non-­‐dialyzed	  propanol	  was	  able	  to	  stimulate	  IL-­‐1ß	  though	  not	  as	  pronounced.	  	  It	  is	  hypothesized	  that	  the	  presence	  of	  propanol	  in	  combination	  with	  SDS	  may	  provide	  premature	  lysis	  of	  cells	  and	  release	  of	  IL-­‐1ß	  in	  these	  groups.	  	  	  NaOCl	   groups	   clearly	   provided	   negligible	   IL-­‐1ß	   stimulation	   which	  may	   be	  interpreted	   as	   either	   a	   strong	   LPS	   detoxification	   result,	   or	   again	   as	   previously	  mentioned	   an	   incomplete	   dialysis	   and	   cytotoxic	   effect	   on	   viable	   cells	   resulting	   in	  false	   positive.	   	   In	   the	  NaOCl	  wells,	   cell	  morphology	  was	   not	   significantly	   different	  from	  untreated	  cells	  (Figs.	  8-­‐9).	  	  Further,	  the	  pH	  levels	  remained	  neutral,	  similar	  to	  DMEM,	   for	   all	   aliquots	   suggesting	   that	   dialysis	   at	   least	   removed	  NaOH	   and	  NaOCl	  from	  solutions,	  otherwise	  an	  increase	  in	  pH	  would	  have	  been	  observed	  (Fig.	  5,	  right	  caption).	   	   Considering	   these	   findings	   it	   can	   be	   concluded	   that	   NaOCl,	   contrary	   to	  suggestions	  from	  previous	  studies,	  in	  fact	  is	  able	  to	  detoxify	  LPS.	  	  	  6.3	  Biofilm	  tests	  	   6.3.1 Effectiveness	  of	  killing	  biofilm	  microbes	  	   The	  results	  from	  testing	  on	  polymicrobial	  biofilms	  grown	  on	  collagen	  coated	  HA	  discs	  suggests,	  again	  similar	  to	  planktonic	  tests,	  that	  NaOCl	  is	  superior	  to	  the	  NaOH	  containing	   combinations	   in	   bacterial	   killing.	   	   Only	   2%	   NaOCl	   was	   used	   based	   on	  	  	  	  	  	   38	  findings	   from	  previous	  biofilm	  killing	  experiments	   showing	   that	  with	  6%	  NaOCl	   a	  detachment	   of	   biofilm	   samples	   impeded	   proper	   assessment.	   	   2%	   NaOCl	   killed	  approximately	  90%	  and	  75%	  of	  biofilm	  bacteria	  in	  1	  week	  and	  3	  week	  old	  biofilms,	  respectively.	   	  There	  was	  no	  statistically	  significant	  difference	  between	  Double	  and	  Triple	  solutions,	  and	  the	  killing	  efficacy	  ranged	  from	  30-­‐45%.	  	  Various	  studies	  have	  demonstrated	  the	  superior	  antibiofilm	  properties	  of	  NaOCl	  using	  live/dead	  staining	  and	   confocal	   microscopy	   (del	   Carpio-­‐Perochena	   et	   al.	   2011,	   Stojicic	   et	   al.	   2012).	  	  Stojicic	  et	  al.	  (2012)	  reported	  approximately	  25%	  killing	  of	  biofilm	  microbes	  with	  3	  min	   exposure	   to	   1%	   NaOCl	   using	   biofilms	   grown	   for	   3	   weeks	   on	   HA	   discs.	   	   The	  results	   are	   comparable	  with	   the	   present	   study	  when	   corrected	   for	   error	   in	   the	   3	  week	   group	   for	   this	   study.	   	   Other	   studies	   are	   difficult	   to	   compare	   as	   the	   biofilms	  have	  been	  grown	  in	  various	  conditions	  on	  different	  materials.	  However,	  the	  overall	  trend	  is	  that	  NaOCl	  remains	  the	  irrigant	  of	  choice	  for	  biofilm	  eradication.	  	  	  	  	   6.3.2 Biovolume	  assessment	  	   A	  trend	  was	  observed	  when	  assessing	  overall	  biovolume	  (Live/Dead	  combined)	  in	  different	  groups.	  	  Biofilm	  biovolumes	  after	  exposure	  to	  NaOCl,	  Double	  and	  Triple	  solutions,	  when	  compared	   to	  controls,	  were	  significantly	  reduced	   in	  1	  week	  and	  3	  week	  biofilms.	   	  For	  1	  week	  old	  biofilms	  a	  1	   log	  reduction	  was	  observed	  for	  NaOCl,	  and	  a	  2-­‐2.5	  log	  reduction	  for	  Double	  and	  Triple	  solutions.	  	  (Figure	  4).	  The	  difference	  between	  NaOCl,	  Double	  and	  Triple	  groups	  was	  statistically	  significant	  only	  at	  80%	  confidence	  interval.	  	  As	  the	  biovolume	  can	  vary	  greatly	  within	  biofilms	  and	  even	  at	  	  	  	  	  	   39	  different	  locations	  on	  the	  same	  disc,	  standard	  deviations	  were	  high	  and	  contributed	  to	  the	  error.	  	  Representative	  images	  (Fig.	  12)	  illustrate	  the	  treatment	  trend	  noticed.	  NaOCl	   groups	   resulted	   in	   more	   killed	   biofilm,	   however	   there	   were	   visually	   more	  voluminous	  areas	  of	  remaining	  biofilm.	  	  In	  contrast,	  with	  Double	  and	  Triple	  solution	  groups	   there	   was	   proportionately	   less	   killing	   of	   biofilm	   microbes,	   however,	   the	  remaining	  non-­‐detached	  biofilm	  areas	  were	  sparse	  and	  low	  in	  volume.	  	  	  When	  analyzing	  three	  week	  old	  biofilms,	  a	  much	  greater	  biovolume	  was	  measured	  (2	   log	   increase)	   in	  control	  groups.	   	  A	  proportional	   reduction	   in	  biovolume	   treated	  with	   the	  solutions	  was	  observed	  similar	   to	  one	  week	  old	  biofilms;	  1	   log	  reduction	  for	  NaOCl	  and	  2	  log	  reduction	  for	  Double/Triple	  groups.	  	  (Figure	  5)	  The	   addition	   of	   surface	   modifying	   agents	   has	   gained	   interest	   in	   endodontic	  irrigation	   protocols	   recently,	   with	   solutions	   such	   as	   QMix	   and	   MTAD	   containing	  proprietary	   combinations	   including	   surface	  modifiers	   such	   as	   Cetrimide	   or	   Triton	  X100.	   	   Studies	  have	   shown	   that	  QMix	   is	   as	  effective	  as	  NaOCl	   ,	   and	  more	  effective	  than	   MTAD,	   in	   killing	   bacteria	   both	   in	   planktonic	   culture	   and	   in	   biofilm	   systems	  (Stojicic	  et	  al.	  2012,	  Wang	  et	  al.	  2013).	  	  It	  is	  reasonable	  to	  assume	  that	  this	  superior	  effect	   is	  at	   least	   in	  part	  due	  to	  the	  ability	  of	  these	  solutions	  to	  dissolve	  the	  organic	  EPS	   component	   of	   biofilms	   that	   may	   act	   as	   protection	   to	   bacteria.	   	   The	   present	  results	  also	  demonstrate	  a	  different	  effect	  of	   the	  solutions	  on	  biofilm	  systems	  and	  may	   support	   the	   hypothesis	   that	   SDS	   containing	   solutions	   dissolve	   biofilms	  more	  effectively.	   	   Greater	   power	   and	   sample	   size	   to	   overcome	   the	   large	   variability	   in	  	  	  	  	  	   40	  biovolume	   analysis	  may	   add	   to	   the	   strength	   of	   these	   arguments,	   as	   currently	   this	  inference	  can	  be	  made	  as	  a	  qualitative	  observation	  only.	  	  	  	   	  	  	  	  	  	   41	  7. Limitations	  of	  the	  study	  A	   number	   of	   limitations	   should	   be	   considered	  with	   respect	   to	   the	   results	   and	  methodologies	  described	  in	  this	  study:	  • Planktonic	  killing	   tests	  do	  not	  duplicate	   clinical	   situations,	   but	   rather	   are	   a	  standardized	  method	  to	  evaluate	  disinfectant	  solutions	  relative	  to	  each	  other.	  	  • LPS	   detection	   is	   challenging	   using	   the	   proposed	   methodology	   as	  detoxification	   requires	   the	   use	   of	   chemicals	   that	   will	   also	   affect	   cell	  stimulation	  and	  viability	  if	  not	  completely	  removed.	  	  	  • Microdialysis	   of	   combination	   solutions	   may	   have	   been	   incomplete	   due	   to	  micelle	   formation	   by	   SDS,	   thus	   macrophage	   cellular	   activity/viability	   may	  have	  been	  affected.	  	  • Statistical	   analysis	   in	   biofilm	   studies	   is	   challenging	   as	   biofilm	   volume	   can	  vary	  significantly	  even	  within	  the	  same	  sample	  at	  different	  locations	  on	  each	  disc.	  	  Further,	  calculations	  using	  3D	  software	  and	  confocal	  imaging	  relies	  on	  operator	  set	  parameters	  which	  inherently	  causes	  variations.	  • The	  live/dead	  stain	  mechanism	  is	  based	  partially	  on	  membrane	  permeability,	  where	   damaged	   cells	   uptake	   propidium	   iodide	   to	   stain	   nucleic	   acids.	   	   It	   is	  possible	   that	  damaged	  but	   viable	   cells	   or	   released	  nucleic	   acids	   from	   lysed	  cells	  uptake	  dye	  and	  are	  expressed	  as	  red	  fluorescence.	  	  (Suzuki	  et	  al.	  1997)	  	   	  	  	  	  	  	   42	  	  8. Conclusions	  Within	   the	   study	   context,	   the	   Null	   hypothesis	   is	   validated	   as	   NaOH/SDS	   and	  Propanol	  combinations	  failed	  to	  produce	  significant	  results	  when	  compared	  to	  2%	  and	   6%	   NaOCl.	   	   The	   overall	   antibacterial	   properties	   of	   the	   NaOH	   containing	  mixtures	  were	  weaker	   than	  2%	  NaOCl	  on	  planktonic	  E.	  faecalis	   and	  polymicrobial	  biofilms.	  	  However,	  the	  combination	  of	  SDS	  with	  NaOH	  appears	  to	  impart	  enhanced	  biofilm	   dissolving	   properties.	   	   Based	   on	   the	   results,	   it	   is	   unclear	   whether	   the	  addition	  of	  propanol	  to	  the	  solutions	  has	  any	  added	  benefit,	  however	  it	  does	  aid	  in	  solution	   preparation	   by	   reducing	   precipitate	   formation	   between	   NaOH	   and	   SDS.	  	  Testing	   with	   LPS	   was	   unable	   to	   provide	   significant	   conclusions	   regarding	   the	  NaOH/SDS	  combinations	  due	  to	   incomplete	  dialysis.	   	  However,	   the	  results	  suggest	  that	  NaOCl	   in	   fact	   is	   able	   to	  detoxify	  LPS,	  whereas	  previous	   studies	  have	   failed	   to	  show	  a	  positive	  effect.	  	  	  Modern	   endodontic	   irrigation	   often	   involves	   the	   use	   of	   stepwise	   protocols	  including	  various	  chemicals	  to	  impart	  the	  desired	  effects	  such	  as	  tissue	  dissolution,	  disinfection,	   biofilm	  dissolution	   and	   smear	   layer	   removal.	   	   The	   results	   from	   these	  tests	  provide	  some	  useful	  insights	  for	  irrigant	  testing	  and	  development:	  the	  need	  for	  capacity	   testing	   to	   differentiate	   potency	   of	   solutions,	   the	   development	   of	   a	   useful	  test	  protocol	  for	  LPS	  detoxification	  using	  biofunctional	  assays,	  and	  the	  significance	  of	  volumetric	  analysis	  in	  biofilm	  eradication	  studies.	   	  	  	  	  	  	   43	  	  9.	  Tables	  and	  figures	  	  Table	  1	  	  Percentage	  (mean	  ±	  S.D.)	  of	  killed	  E.	  faecalis	  after	  exposure	  at	  different	  times	  	  CFU	  concentration	  2.5x105	  	   5Sec	   30Sec	   1Min	  2%	  NaOCl	   98±2	   99±1	   99±1	  Double	  solution	   99±1	   99±1	   97±1	  Triple	  solution	   99±1	   97±1	   99±1	  Difference	  between	  groups	  was	  not	  statistically	  significant.	  	  P>0.05	  (ANOVA)	  	  	  	  	  	  	  	  Table	  2	  	  Percentage	  (mean	  ±	  SD.)	  of	  killed	  E.	  faecalis	  after	  exposure	  at	  different	  times	  	  CFU	  concentration	  1.75x106	  	   5Sec	   30Sec	   1Min	  6%NaOCl	   63±3.5	   93±1	   99±1.1	  2%NaOCl	   32±10*	   75±9	   80±5.6	  Double	  sol.	   40±9*	   56±5.7*	   59±6.5*	  Triple	  sol.	  	   37±3.2*	   55±11.5*	   48±6.8*	  P<0.05	  except	  between	  groups	  noted	  with	  *	  (ANOVA)	  	  	  	  	  	  	  	  	  	  	  	  	  	  	   44	  	  	  Figure	  1	  Planktonic	  killing	  test.	  	  CFU	  concentration	  2.5x105/mL.	  	  	  	  	  Figure	  2	  Planktonic	  killing	  tests:	  CFU	  concentration	  1.75x106/mL.	  Three June%17,%2014%%250µl%Solu1on:250µl%Bacteria%July%10,%2014%Test%Oct%1,%2014%%50µl%Solu5on:%450µl%Bacteria%	  	  	  	  	   45	  	  	  	  	  Figure	  3	  E.	  coli	  LPS	  from	  Sigma-­‐Aldrich	  Canada	  (0111:B4	  Lot#043M4104V)	  	  	  Figure	   4	   Schematic	   presentation	   of	   the	  Quantikine	   ELISA	  method	   (Courtesy:	  R&D	  Systems)	   	  Quantikine ELISA for Mouse IL-1ß (R&D Systems) 	  	  	  	  	   46	  	  Figure	   5	   LPS	   detoxification	   tests.	   Left:	   Quantikine	   ELISA	   following	   stop	  solution.	  Right:	  Stimulated	  mouse	  RAW	  264.7	  cells	  for	  aliquot	  	  	  	  	  Figure	  6	  Dialysis	  procedure	  using	  Slide-­‐Alyzer	  cassettes	  (0.5ml,	  2kDA	  MWCO)	  (Thermo	  Fischer	  Scientific)	  	  	  	  	  	  	   47	  	  Table	  3.	  OD	  values	  ±	  SD	  for	  IL-­‐1ß	  (Quantikine	  ELISA)	  	  Treatment	  Group	   OD±SD	   IL-­‐1ß	  Level	  	  (from	  Standard	  Curve)	  6%	  NaOCl	   0.003±0.001	  a	   <12pg/ml	  2%	  NaOCl	   0.003±0.001	  a	   <12pg/ml	  Triple	   0.57±0.064	   >800pg/ml	  Double	   0.004±0.002	  a	   <12pg/ml	  LPS	  Dialyzed	   0.047±0.010	  b	   75pg/ml	  LPS	  Non-­‐Dialyzed	   0.056±0.011	  b	   75pg/ml	  Solution	  Dialyzed	   0.005±0.002	  a	   12pg/ml	  Untreated	  Cells	   0.007±0.002	  a	   12pg/ml	  P<0.05,	  except	  between	  groups	  marked	  with	  matching	  superscript	  (ANOVA).	  	  	  	  	  	  Figure	  7	  IL-­‐1ß	  standard	  curve	  (y	  axis	  OD;	  x-­‐axis	  concentration	  in	  pg/ml).	  0	  0.1	  0.2	  0.3	  0.4	  0.5	  0.6	  800pg/ml	  400pg/ml	  200pg/ml	  100pg/ml	  50pg/ml	  25pg/ml	  12.5pg/ml	  0pg/ml	  OD±SD	  	  	  	  	  	   48	  	  	  Figure	  8	  Cell	  morphology	  of	  untreated	  mouse	  macrophages	  	  Figure	  9	  Macrophage	  cell	  morphology	  after	  exposure	  to	  2%	  and	  6%	  NaOCl	  	  Figure	   10	   Macrophage	   cell	   morphology	   after	   exposure	   to	   LPS	  	  	  	  	   49	  	  Figure	  11	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Dialyzed	  solution	  (NaOCl,	  NaOH,	  Propanol)	  	  Figure	  12	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Double	  Solution	  	  Figure	  13	  Macrophage	  cell	  morphology	  after	  exposure	  to	  Triple	  Solution	  	  	  	  	  	  	   50	  Table	  4	  	  Percentage	  of	  dead	  cell	  volume	  (%±SD)	  and	  biovolume	  (±SD)	  in	  1	  and	  3-­‐week-­‐old	  biofilms	  after	  exposure	  to	  the	  test	  solutions.	  	  	  	  1	  Wk	  %	  Dead	   3Wk	  %	  Dead	   1Wk	  Biovolume	  3Wk	  Biovolume	  2%	  NaOCl	   90±8.5	   75±12	   6.8E4±1.4E4	  c	   3.9E6±3.6E5	  d	  Double	   42±2.6	  a	   32±7.5	  b	   4.5E3±8.9E2	  c	   3.5E5±4.7E4	  d	  Triple	   28±14a	   44±12	  b	   2.5E3±3.3E2c	   4.0E5±7.1E4	  d	  Control	   13±4.6	   2±0.5	   6.6E5±1.1E5	   6.7E7±6.1E6	  P<0.05,	  except	  between	  groups	  marked	  with	  matching	  superscript.	  c	  &	  d	  groups	  P<0.2	  (ANOVA).	  	  	  	  	  	  Figure	   14	   Representative	   3D	   biofilm	   reconstructions	   (Imaris	   x64	   V.7.3.0	  Software)	   to	   the	   indicated	   solutions.	   Green	   indicates	   live	   bacteria,	   red	  indicates	  dead	  bacteria	  	   	  NaOH/SDS( NaOH/SDS/Propanol(Control( NaOCl(2%(1(Week(Polymicrobial(Biofilm(on(HA(Discs((Tx(=3min)(3(Week(Polymicrobial(Biofilm(on(HA(Discs((Tx(=3min)(	  	  	  	  	   51	  	  	  	  	  Figure	  15	  Biovolume	  and	  proportions	  of	  live	  and	  dead	  bacteria	  in	  1	  week	  old	  biofilms	  after	  indicated	  treatment.	  (Logarithmic	  Scale	  in	  3D	  units)	  	  	  	  	  	  	  Figure	   16	   Proportions	   (%)	   of	   live	   and	   dead	   bacteria	   in	   1	  week	   old	   biofilms	  after	  the	  various	  treatments	  	  	   	  	  1	  10	  100	  1000	  10000	  100000	  1000000	  2%	  NaOCl	   Double	   Triple	   Control	  Live	  Dead	  10	  90	  2%	  NaOCl	  Live	  Dead	   58	  42	  Double	  Live	  Dead	   72	  28	  Triple	  Live	  Dead	   87	  13	  Control	  Live	  Dead	  	  	  	  	  	   52	  	  	  	  	  Figure	  17	  Biovolume	  and	  proportions	  of	  live	  and	  dead	  bacteria	  in	  3	  week	  old	  biofilms	  after	  indicated	  treatment.	  (Logarithmic	  Scale	  in	  3D	  units)	  	  	  Figure	   18	   Proportions	   (%)	   of	   live	   and	   dead	   bacteria	   in	   3	  week	   old	   biofilms	  after	  the	  various	  treatments	  	   	  1	  10	  100	  1000	  10000	  100000	  1000000	  10000000	  100000000	  2%	  NaOCl	   Double	   Triple	   Control	  Live	  Dead	  25	  75	  2%	  NaOCl	  Live	  Dead	   68	  32	   Double	  Live	  Dead	   56	  44	   Triple	  Live	  Dead	   98	  2	  Control	  Live	  Dead	  	  	  	  	  	   53	  	   References	  AAE	  (American	  Association	  of	  Endodontists)	  Colleagues	   for	  Excellence	  2011:	  Root	  Canal	 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