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RGD-Modified Gellan Gum L. Stevensa,b,  C.  Ferrisa,b,  E.  Mumec,  D.  Kirchamajera,b,  K.  Gilmorea,  I.  Greguricc,  S.  Smithc,  G.  Wallacea  and  M.  i.  h.  Panhuisa,b,     aIntelligent  Polymer  Research  InsDtute,  ARC  Centre  of  Excellence  for  Electromaterials  Science,  AIIM  Facility,  University  of  Wollongong,  Wollongong,  NSW  2522,  Australia.   bSoL  Materials  Group,  School  of  Chemistry,  University  of  Wollongong,  Wollongong,  NSW  2522,  Australia.   cAustralian  Nuclear  Science  and  Technology  OrganisaDon,  Lucas  Heights,  NSW  2234,  Australia.  

lrs849@uowmail.edu.au

Cell Response

Introduction Tissue   engineering   is   a   field   of   research   that   has   developed   over   several   decades   to   now   be   on   the   cusp   of   providing   treatments   for   a   wide   range   of   Dssue   damage,   from   burns   to   organ   failure.   Materials   used   in   Dssue   engineering  constructs  are  required  to  conform  to  very  high  standards  of  both   performance  and  safety,  requirements  that  are  frequently  applicaDon  specific.   As  such  there  is  an  ongoing  need  for  idenDfying  new  materials  that  combine   strength,  processability  and  favourable  cell  interacDons.   Gellan   gum   is   an   anionic   polysaccharide   that   forms   ionic   hydrogels   under   physiological  condiDons.  These  materials  are  known  to  be  cytocompaDble,  but   a  lack  of  cell  recogniDon  sites  limits  its  potenDal  as  a  scaffold  for  aXachment   dependent  cells,  which  are  a  criDcal  component  of  most  Dssues.  In  this  work   we   demonstrate   a   chemical   modificaDon   procedure   for   gellan   gum,   covalently   linking   the   gellan   gum   chain   to   a   pepDde   sequence   RGD,   which   has   been   shown  mediate  cell  binding  in  similar  systems1.  The  impact  of  modificaDon  is   examined  using  the  aXachment  dependent  cell  lines  C2C12  and  PC12.    

Gellan gum   hydrogels   were   formed   by   ionic   cross-­‐linking   with   Ca2+   added   to   DMEM   culture   media.   These   hydrogels   were   seeded   with   rat   adrenal   (PC12)   and  mouse  skeletal  muscle  (C2C12)  cell  lines,  which  are  widely  used  models  of   nerve  and  muscle  cell  behaviour,  respecDvely.  On  unmodified  gellan  hydrogels   (Fig  2:  A,B,E,F)  both  cell  lines  were  observed  to  form  large  cell  clusters,  with   liXle   observable   interacDon   with   the   hydrogel   substrate.   RGD-­‐modifed   surfaces  (Fig  2:  C,D,G,H)  by  contrast,  enabled  substanDally  more  cell  spreading   and   encouraged   typical   proliferaDon   cell   morphologies,   especially   in   the   C2C12  cell  line.  Under  differenDaDon  condiDons  (data  not  shown)  C2C12  cells   rapidly  responded  by  contracDng  into  large  fibers,  with  some  mulDnucleated   myofibers   present   by   5   days   of   differenDaDon.   PC12   cells   however,   did   not   differenDate   during   exposure   nerve   growth   factor,   which   typically   prompts   terminal   differenDaDon.   OpDmisaDon   of   the   density   of   cell   aXachment   sites   may  be  needed  to  facilitate  PC12  differenDaDon.  

A

Coupling Reaction Gellan gum   was   modified   with   the   short   pepDde   sequence   GGGGRGDSY   by   carboiimide  chemistry  similar  to  the  procedure  outlined  by  Rowley  et  al.2-­‐3  for   alginate   modificaDon.   The   reacDve   EDC   intermediate   is   stabilised   by   the   addiDon   of   a   labile   amine,   SulfoNHS   (Scheme   1),   limiDng   hydrolysis.   The   pepDde   chain   then   binds   through   the   terminal   amine   by   subsDtuDon.   It   was   found  during  iniDal  reacDon  aXempts  that  the  divalent  caDons  in  as  received   gellan   inhibited   EDC   binding   by   occupying   carboxylic   residues   on   the   gellan   backbone.   Gellan   was   thereaLer   purified   by   heaDng   gellan   soluDons   in   the   presence   of   Dowex   50W-­‐X8   caDon   exchange   resin,   followed   by   regeneraDon   with   NaOH.   Ion   content   was   assessed   by   flame   atomic   absorpDon   spectroscopy   and   found   to   have   greatly   reduced   concentraDons   of   Ca2+   and   Mg2+   (Table   1).   When   purified   gellan   gum   was   used,   coupling   efficiencies   of   approximately  40%  were  obtainable.  The  pepDde  coupled  gellan  was  refined   and  isolated  by  dialysis  against  H2O,  and  lyophilisaDon.  

Scheme 1   (Above):   Simplified   reacDon   schemaDc   for   the   coupling   of   the   terminal   amine   of   the   GGGGRGDSY  pepDde  sequence  with  the  carboxyl   residues  on  the  gellan  gum  polysaccharide.  

D

E

G

F

H

Figure 2:  Bright  field  and  calcien-­‐stained  fluorescence  microscopy  images  of  PC12  (A-­‐D)  and  C2C12  (E-­‐H)  cells  aLer  24  hours  of  culture  on   hydrogels  formed  from  purified  (top  row)  and  RGD-­‐modified  (lower  row)  gellan  gum.  The  presence  of  the    RGD  sequence  enhances  cell-­‐ surface   interacDons   and   limits   the   formaDon   of   cell   clusters.   Under   the   test   condiDons,   this   effect   appears   to   be   more   prominent   in   the   C2C12  culture,  which  exhibited  typical  elongated  phenotypes.  PC12  cells  did  not  differenDate,  and  retained  rounded  morphologies  in  the   presence  of  nerve  growth  factor.  Scale  bars  represent  either  100  µm  (D,F,H)  or  200  µm  (A,B,C,E,G)  

Rheology and Printing One of   the   key   benefits   of   the   gellan   gum   biopolymer,   is   it’s   ability   to   rapidly   form   solid   hydrogel   structures   through   the   addiDon   of   common  divalent  caDons  such  as  Ca2+,  enabling   reacDve  prinDng  for  scaffold  formaDon  (Fig  3).     Rheological   tesDng   of   gellan   soluDons   was   conducted  using  an  Anton  Paar  Physica  MCR301   Figure  3:  A  freestanding  structure  printed  using   (w/v)  purified  gellan  gum  and  small  volumes   Rheometer.   Shear   thinning   behaviour   was   1%   of    a  concentrated  soluDon  CaCl   observed  for  all  tested  samples,  which  is  typical   for   polymer   soluDons.   Commercial   gellan   gum   was   substanDally   more   viscous   than   purified   gellan,   likely   due   to   remnant   divalent   caDons   forming   ionic   links   between   adjacent   gellan   polymer  chains,  limiDng  fluid  flow.  Notably,  the   RGD   modified   gellan   provided   minimal   resistance   to   fluid   flow   compared   to   purified   gellan,   possibly   due   to   the   pepDde   chain   4:   The   shear   dependent   viscocity   of   1%   disrupDng  gellan  chains  helix  formaDon.  Further   Figure   (w/v)   soluDons   of   as   received   (blue),   purified   study   of   this   effect   is   needed   to   assess   its   (red)   and   RGD-­‐modified   (green)   gellan   gum.   TesDng   was   conducted   at   37˚C   using   a   50mm   impact  on  RGD-­‐gellan    bioprinDng.   small  angle  cone.   2.

Figure 1   (Right):   The   conjugaDon   efficiencies   for   pepDde   coupling   reacDons   under   varied   reactant   condiDons.   To   measure   reacDon   products,   the   pepDde   sequence   was   radiolabeled   at   the   terminal   tyrosine   (Y)   residue   with   a   gamma   emipng   125I   using   established   methods4.   RadioacDvity   was   detected   with   a   Perkin   Elmer   Gamma   counter,  and  converted  to  concentraDons  using  a  calibraDon  curve.  

Table 1   (Below):   ConcentraDons   of   four   key   anions   in   gellan   gum   soluDons   determined   by   flame   absorpDon   spectroscopy.   As   received   gellan   gum   is   primarily   in   the   potassium   form   with   a   substanDal   calcium   component.   Purified   gellan   gum   is   primarily   in   a   sodium   form,   with  no  measurable  calcium  or  magnesium.  

Element  (%w/w) Na+

C

B

K+

Ca+

Mg+

Gellan Gum

0.6 ±0.1

4.5 ±0.2

1.2 ±0.1

0.11 ±0.01

Purified   Gellan  Gum

2.5 ±0.1

1.0 ±0.1

Conclusion

< LOD

< LOD

We have  presented  the  synthesis  and  iniDal  analysis  of  a  new  biomaterial  for   Dssue   engineering,   RGD-­‐gellan   gum.   Through   radiolabelling   with   I-­‐125,   the   reacDon  efficiency  has  been  determined  under  a  variety  of  condiDons.  It  was   found   that   divalent   caDons   substanDally   inhibit   -­‐COOH   acDvaDon,   and   their   removal  by  ion-­‐exchange  allowed  for  full  reacDon  efficiencies  of  around  40%.   PC12  and  C2C12  cell  lines  were  both  found  to  survive  and  proliferate  on  the   RGD-­‐modified   material   with   less   clustering   than   unmodified   gellan   surfaces,   however  C2C12  cells  appeared  to  benefit  most  from  the  RGD  under  the  test   condiDons,   showing   typical   adherent   and   differenDated   phenotypes.   These   results   indicate   that   RGD-­‐gellan   has   promise   as   a   printable,   soL   cell   scaffold   for  aXachment-­‐dependent  cells.  

References 1 -­‐  U.  Hersel,  C.  Dahmen,  and  H.  Kessler,  Biomaterials,  2003,  24,  4385–4415.   2  -­‐  J.  A.  Rowley,  G.  Madlambayan,  and  D.  J.  Mooney,  Biomaterials,  1999,  20,  45–53.   3  -­‐  J.  A.  Rowley  and  D.  J.  Mooney,  Journal  of  biomedical  materials  research,  2001,  60,  217–223.   4  -­‐  T.  N.  Schumacher  and  T.  J.  Tsomides,  Current  protocols  in  protein  science,  2001,  Chapter  3,  Unit  3.3   5  –  M.  Tako,  T.  Teruya,  Y.  Tamaki  and  T.  Konishi,  Colloid  Polymer  Science,  2009,  287,  1445-­‐1454  

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