markers, nor hearing loss as measured by auditory brainstem response. (ABR) or distoruon product oto-‐acousuc emissions (DPOAE), between wild type and ...

Parallel  Mechanisms  Suppress  Cochlear  Bone  Remodeling  to  Protect  Hearing   Emmanuel  Jáuregui,  BA1;  Omar  Akil,  PhD2;  Claire  Acevedo,  PhD1,3;  Faith  Hall-­‐Glenn,  PhD1;  Be?y  S.  Tsai,  MD2;  Hrishikesh  A.  Bale,  PhD3;  Ellen  Liebenberg,  MS1     Mary  Beth  Humphrey,  MD,  PhD5;  Robert  O.  Ritchie,  PhD3;  Lawrence  R.  LusOg,  MD2;  Tamara  Alliston,  PhD1      


1Department  of  Orthopaedic  Surgery,  University  of  California,  San  Francisco  ,    2Department  of  Otolaryngology—Head  &  Neck  Surgery,  University  of  California,  San  Francisco,  University  of  California,  San  Francisco,  CA    

3Materials  Science  Division,  Lawrence  Berkeley  NaOonal  Laboratory,  Berkeley,  CA,  4Department  of  Otorhinolaryngology,  University  of  Oklahoma  Health  Sciences  Center,  5Department  of  Medicine,  University  of  Oklahoma  Health  Sciences  Center    




Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provide unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13-/-).   Therefore, we seek to investigate the cellular and molecular mechanisms that maintain cochlear bone in a mouse model deficient in MMP13, with defective long bone PLR, to evaluate whether the cochlea is uniquely dependent on MMP13mediated osteocytic PLR to maintain cochlear bone to maintain normal hearing. RANKL














Figure 2. Normal inner ear structures and ossicular chain in wild type and MMP13-/- mice. (A-F) Plastic embedded sections of cochleae showing cochlea in sagittal plane of WT (A) and MMP13-/- mice (D). Organ of Corti (B) and spiral neuroganglion (C) in WT and MMP13-/- (E and F) reveal no bony compression of neuroganglia. Toluidine blue stain of WT (G) and MMP13-/- (H) show no bony deformation. Micro-CT reconstruction show normal ossicular chain (I) and cochlea (J) in WT and MMP13-/- (K and L).


Figure 3. Hearing is maintained despite lack of MMP13. Auditory brainstem responses (ABR) on wild-type and MMP13-/- and distortion product otoacoustic emissions (DPOAE) on wild-type and MMP13-/mice at 6 months of age reveal increased variability, however no significant difference between the two groups.

Cochlear  bone  is  singular  in  its  regulaOon  of  remodeling  by  expressing   high  levels  of  osteoprotegerin.  Since  cochlear  bone  expresses  a  key  PLR   enzyme,  MMP13,  we  examined  whether  cochlear  bone  relies  on,  or  is   protected   from,   osteocyte-­‐mediated   PLR   to   maintain   hearing   and   bone   quality   using   a   mouse   model   lacking   MMP13   (MMP13-­‐/-­‐).   We   invesOgated   the   canalicular   network,   collagen   organizaOon,   lacunar   v o l u m e   v i a   m i c r o -­‐ c o m p u t e d   t o m o g r a p h y ,   a n d   d y n a m i c   histomorphometry.   Despite   finding   defects   in   these   hallmarks   of   PLR   in   MMP13-­‐/-­‐   long   bones,   cochlear   bone   revealed   no   differences   in   these   markers,   nor   hearing   loss   as   measured   by   auditory   brainstem   response   (ABR)   or   distorOon   product   oto-­‐acousOc   emissions   (DPOAE),   between   wild   type   and   MMP13-­‐/-­‐   mice.   Dynamic   histomorphometry   revealed   abundant  PLR  by  Obial  osteocytes,  but  near  absence  in  cochlear  bone.   Cochlear   suppression   of   PLR   corresponds   to   repression   of   several   key   PLR   genes   in   the   cochlea   relaOve   to   long   bones.   These   data   suggest   cochlear   bone   uniquely   maintains   bone   quality   and   hearing   independent   of   MMP13-­‐mediated   osteocyOc   PLR.   Furthermore,   the   cochlea   employs   parallel   mechanisms   to   inhibit   remodeling   by   osteoclasts   and   osteoblasts,   and   by   osteocytes,   to   protect   hearing.   Understanding   the   cellular   and   molecular   mechanisms   that   confer   site-­‐ specific  control  of  bone  remodeling  have  the  potenOal  to  elucidate  new   pathways  that  are  deregulated  in  skeletal  disease.      






Cochlear  Bone   ñOPG      -­‐-­‐|  Osteoclast  

       ???          -­‐-­‐|  Osteocytes  

Mineralized Bone Matrix

Figure  4.  MMP13  is  necessary  for  collagen  organizaHon  in  long  bones,  not  in  cochlear  bone.  Polarized  light   birefringence   of   cochleae   and   femora   reveal   collagen   fibril   organizaOon.   Both   WT   and   MMP13-­‐/-­‐   cochleae   reveal   well-­‐organized   collagen   fibrils   (A-­‐C).   Plot   of   the   distribuOon   of   orientaOon   of   collagen   fibrils   in   cochlear  bone  (D)  reveal  no  differences  between  MMP13-­‐/-­‐  (36.6±10.4o)  and  WT  mice  (33.5±7.2o),  p>0.05.  In   femora   (E-­‐G),   collagen   organizaOon   is   not   well   organized   throughout   MMP13-­‐/-­‐   mice   (G)   compared   to   WT   (F).   For   MMP13-­‐/-­‐   femora,   the   distribuOon   of   collagen   orientaOon   shows   a   wider,   less   well-­‐defined   peak   (H),   suggesOng  poorer  organizaOon  by  width  at  half-­‐maximum  analysis,  (71.8±30.6  vs.  34.3±4.8o,  MMP13-­‐/-­‐  and   WT,  respecOvely;  p=0.0002).    




Tibia Femur Cochlea




+*    +*  



Figure 1: MMP13 is expressed at multiple stages of development in wild-type mouse cochlea. Immunohistochemical stain for MMP13 protein in wild-type mice cochlea from embryonic day (E)15 to postnatal day (P)60. Black arrows show areas of most intense staining of MMP13 in cochlear bone. Plus IgG negative control stain of cochlea at P60.





Figure  6.  CriHcal  PLR  factors  are   expressed  to  a  lesser  degree  in   the   cochlea   compared   to   long   bones.   qPCR   expression   levels   from   p60   WT   male   mice.   The   relaOve   expression   levels   of   MMP13   and   other   important   PLR   genes   (CatK,   ATPd2,   MMP14)   in   the   cochlea   are   lower   than   in   other   bones,   suggesOng   suppressed   PLR   acOvity   in   the   cochlea   relaOve   to  long  bones.  

Figure   5.   Cochlear   canalicular   network   is   maintained   without   MMP13,   however   is   required   in   femur.   Lacuno-­‐canalicular  networks  in  cochleae  (A-­‐B)  and  femora  (D-­‐E)  stained  with  silver  nitrate  (in  black)  in  male   p60   mice.   QuanOficaOon   of   the   lacuno-­‐canalicular   area,   using   ImageJ,   reveals   no   significant   difference   between   the   two   groups   (0.260±0.04µm2   vs.   0.235±0.08µm2,   MMP13-­‐/-­‐   and   WT,   respecOvely,   p>0.05)   (C).   Silver  nitrate  staining  of  axial  femur  secOon  reveals  a  robust,  radiaOng  canalicular  network  from  lacunae  in   WT  femora  (D).  MMP13-­‐/-­‐  mice  reveal  a  less  extensive,  blunted  canalicular  network  (E).  MMP13-­‐/-­‐  mice  have  a   significantly  smaller  lacuno-­‐canalicular  area  (0.143±0.04  µm2)  normalized  to  bone  area,  compared  to  WT  mice   (0.256±0.10  µm2;  p=0.0002)  (F).     0.002  

Lacunae/BV  (nb/mm3)  


ñSOST  -­‐-­‐|  Osteoblast  




0.001   0.0005  


Osteocyte Perilacunar Remodeling Markers

MMP13 Collagen org. Mineralization Canalicular Network org.



+/+ ✓ ✓

-/✗ ✗

+/+ ✓ ✓

-/✓ ✓

Methods Detailed   methods   are   described   with   these   references:   Tang4,   Chang5,   and   Busse6.  Briefly,  the  procedures  include:     Histological   analysis:   Cochleae   were   collected,   decalcified,   embedded   in   paraffin,   secOoned,   and   stained   with   picro-­‐sirius   red   for   polarized-­‐ birefringence  analysis  of  collagen  orientaOon  and  analysed  with  OrientaOonJ   an   ImageJ   plug-­‐in.   Silver   nitrate   stain   was   used   to   visualize   the   lacuno-­‐ canalicular   network   and     quanOfied   with   ImageJ.   MMP13   protein   was   examined  via  immunohistochemistry.     High-­‐resoluHon  micro  computed  tomography  was  used  to  scan  cochleae  and   to  determined  lacunar  volume.   Auditory   brainstem   responses   &   distorHon   product   oto-­‐acusHc   emissions   were  measured  to  assess  hearing  in  6-­‐month-­‐old  mice.  


0   0  

100   200   300   Lacunar  Volume  (mm3)  


Figure   7.   Lacunar   volume   in   cochlear   bone   is   unaffected   by   MMP13   deficiency.   RepresentaOve   3D  volume  rendered  image  of  micro-­‐CT  scan  of  the   cochlea.   No   significant   differences   in   the   average   lacunar   volumes   in   the   cochlea   despite   the   lack   of   MMP13  was  found.  

Funding   sources:   Hearing   Research,   Inc.   compeOOve   award.   NIH   R01   DE019284  (to  Alliston).   References 1.   Zehnder  A,  et  al.  The  Laryngoscope,  2005.   2.   van  Bezooijen  R,  et  al.  Journal  of  Experimental  Medicine,  2004.   3.   Wysolmerski  J.  Bone,  2013.   4.   Tang  S,  et  al.  Journal  of  Bone  and  Mineral  Research,  2012.   5.   Chang  J,  et  al.  EMBO  Rep,  2010.   6.   Busse  B,  et  al.  Science  Transla?onal  Medicine,  2013.  

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