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A correlation between the distribution of biological apatite and amino acid sequence of type I collagen Maitland, Murray E. 1989

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A C O R R E L A T I O N BETWEEN THE D I S T R I B U T I O N OF B I O L O G I C A L AND AMINO A C I D SEQUENCE OF T Y P E I C O L L A G E N  APATITE  By MURRAY E.  MAITLAND  B.S.R.(P.T. and O.T.), U n i v e r s i t y of B r i t i s h Columbia,  A THESIS  1984  SUBMITTED IN P A R T I A L F U L F I L L M E N T REQUIREMENTS FOR THE DEGREE OF MASTER'S OF S C I E N C E  OF  THE  in THE  We  accept  this  F A C U L T Y OF GRADUATE S T U D I E S (Department of Anatomy)  thesis  THE  as c o n f o r m i n g  to the require  U N I V E R S I T Y OF B R I T I S H COLUMBIA M a r c h 1989 ®Murray E. M a i t l a n d  standard  In  presenting this  degree  at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  head of  that  copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  _  Murray E. Maitland  Department  of  A  n  a  t  o  m  y  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  A p r i l 25, 1989  i i  ABSTRACT  The  present  distribution fibrils of  of  of  was  dark  bright  field  both  electron  data  computer  microscopic  in  both  the  computer  images  was  density. on  density of  The  and  C-  of  on  the for  gap  known  but  each  mineral  proportion  of  of  hydrophobic  amino  acids.  hydrophobic  e f f e c t s and  The the  are  discussed.  In  addition,  the  deposition  of  apatite  in  with  chick These  apatite  early  stages  corresponds  low  in  of  the  of  collagen  hydrophobic the  overlap  areas  of  corresponded regions  density  with  mineral  accommodation zone  high to a  of  interactions  overlap  to  deposition  than  had  which  of  theoretical  high  zones  process  the  that  mineral  of  possible  the  from  sequence  Conversely,  were  was  modelling.  at  hydrophobic  acids  density.  mineral  of  areas  these  amino  and  and  which  acid  stages  is less  hydrophobic  low  amino  from  collagen  orientation within  comparisons  early  zone  pattern  molecular  Based  zones  I  acids  illustrated  overlap  asymmetric  i s excluded  average of  gap  to  determined  apatite  techniques  i n an IM-  by  amino  the  localization  microscopy  sequence  fibril.  high  The  I collagen  collagen  sites  by  type  tendons.  of  polarized  zone  field  leg  within  classes  mineralization  that  turkey  examine  specific  occurs  it  apatite  to  with  electron  the  undertaken  determined  selected-area correlated  was  biological  calcifying  apatite  type  study  between  deposition of is  collagen  to  discussed.  i i i  TABLE  OF  CONTENTS  Page  Numb n  Abstract Abbreviations List  of Figures  Acknoiuledgements Historical  Review  Introduction Materials  and  v y i 1 28  Methods  32  Results  37  Discussion  gg  Conclusions  58  References  7Q  iv  LIST  Figure  OF  FIGURES  Title  Page  Number  1.  Hodge  and P e t r u s k a  2. E l e c t r o n turkey  micrographs  5. S t a g e s  39  of g l y c e r o l  of c a l c i f i c a t i o n  Analysis  Electron and  9.  of collagen  alcoholic stained  hydrophobic  10.  the  i n collagen.  50  orientation  tendon.  diffraction  A comparison  near  46  7.  mineralized  42  treated  front.  L o c a l i z a t i o n of apatite  8.  fibrils.  45  6.  in  of collagen  tendon.  mineralization  16  non-mineralized  staining pattern  Lou m a g n i f i c a t i o n turkey  of  packing model.  l e g tendon.  3. P e r i o d i c  4.  (1963)  54  i n unstained mineralized  of the d i s t r i b u t i o n amino  acids  D i s t r i b u t i o n of p r o l i n e  mineralized tendon.  54  of  and m i n e r a l  apatite.  and h y d r o x y p r o l i n e .  59  59  ABBREVIATIONS Amino  Acids  A  Alanine  K  Lysine  R  Arginine  C  Cysteine  L  Leucine  S  Serine  D  Aspartic  Acid  1*1  Methionine  T  Threonine  E  Glutamic  Acid  N  Asparagine  U  Hydroxylysine  Phenylalanine  0  Hydroxyproline  V  Valine  F G  Glycine  P  Proline  Ui  Tryptophan  H  Histidine  Q  Glutamine  Y  Tyrosin  I  Isoleucine  Other  Abbreviations  a  " a " bands  of collagen  staining  pattern  b  "b" bands  of collagen  staining  pattern  " c " bands  of collagen  staining  pattern  c  -  C-  Carboxy  d  " d " band  D  Axial  e  "e" bands  h  Hour  min  Minute Amino  terminus  of peptide  of collagen  period  of type  of collagen  terminus  nanometer  PTA  Phosphotungstic  SADF  Selected-area  UA  Uranyl  jqm  Micrometer  Acetate  staining I  pattern  collagen staining  of peptide  nm  chain  pattern  chain  acid  dark  field  electron  microscopy  vi ACKNOWLEDGEMENTS A t t h i s t i m e I w o u l d l i k e t o t h a n k t w o i n d i v i d u a l s who w e r e i n s t r u m e n t a l i n my c o m p l e t i o n o f t h i s t h e s i s . D r . A r s e n a u l t has c r e a t e d a wonderful environment w i t h i n h i s l a b through h i s p a t i e n t guidance and s u p e r v i s i o n . A l s o , h i s c r e a t i v e i d e a s and p r o d u c t i v i t y a r e c h a r a c t e r i s t i c s which i n s p i r e a n d m o t i v a t e . L i s a , my w i f e , w a s l a r g e l y r e s p o n s i b l e f o r t h e s u c c e s s o f t h i s e n d e a v o u r b e c a u s e s h e c o n s i d e r e d my g o a l s i m p o r t a n t e n o u g h t o e n d u r e my many y e a r s a s a s t u d e n t . In a d d i t i o n , I would l i k e t o c o l l e c t i v e l y thank the' g r a d u a t e s t u d e n t s , p r o f e s s o r s a n d r e s e a r c h e r s who s u f f e r e d my many q u e s t i o n s a n d a s s i s t e d me i n l e a r n i n g t h r o u g h o u t t h i s program. F i n a n c i a l l y t h i s w o r k was s u p p o r t e d by a g r a n t t o D r . A.L. A r s e n a u l t f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d a s c h o l a r s h i p t o M. E . M a i t l a n d f r o m t h e P h y s i o t h e r a p y Foundation o f Canada.  1  HISTORICAL  REVIEW  Introduction As loose most  a major  component  and dense  connective  prevalent  importance in  works  of this  about  their  collagen  These  collagen  structure,  works  research  have  on c o l l a g e n that  directly  related  entire  world  turkey  tendon  development,  this  review  of m i n e r a l i z a t i o n  some  for this  i n order  to avoid  formed  previous  type  areas  to  The v o l u m e o f tissues which are  explore  i s a model  the since the  o f bone  differences In t h i s  specifically  unneccessary  collagen  i n order  However,  be d i s c u s s e d .  refer  I  several  i n various  and  current  protein  area.  research.  structure  our  microscopy  than  but i t s The  apatite  within  i n this  study  will  will  understood.  morphology,  of the s i m i l a r i t i e s  t h e two t i s s u e s  protein,  have  rather  i s the  has r e s u l t e d  be s e l e c t i v e o f p a p e r s  to the thesis  used  processes  c o l l e c t e d from  of ideas  of  I t s recognized  structure,  and e l e c t r o n  types  I collagen  i s t o examine  and m i n e r a l i z a t i o n  use of " c o l l a g e n "  collagen  been  including  biochemistry,  requires  the  poorly  review  and other  this  mineralization  the development  between  about  i n t e r - r e l a t i o n s h i p s which  fibrils.  works  remains  molecular  of b i o l o g i c a l  assess  of b i o l o g i c a l  historical  concepts  of  t i s s u e s , type  of information  i n mineralization  purpose  tendon  i n vertebrates.  i n a variety  an a b u n d a n c e  role  and  protein  of bone,  thesis  to type  I  r e p e t i t i o n . The  term  2 "biological  a p a t i t e " i s used  differences  between  model  for apatite  biological  recently 1 984;  hierarchy  molecule;  collagen  collagen  many  fibers  have  wishes  been  best  and t h e a c t u a l further  published  and Hulmes,  1984; Bonnucci,  responsible different  consist  from  tissues, collagen  from  chains  the molecular form  molecules  microfibrils  with  form  o f many  other  molecules  These  to the  combine  t o form  a collagen  f o r microns diameters  o f t h e e y e t o 500nm  a l . , 1978).  extracellular  In bone, matrix  network,  a  fibril;  Differences  and  result  of the  extracellular  prior  properties collagen  to  support  from  i n mature  collagen  of the  fibrils  or centimeters  vary  of osteoid  and  architectures are, i n part,  t i s s u e s . For example,  tissue. Fibril  has a  the collagen  fibrils.  f o r the biomechanical  extend  tissue  post-translational modifications  i n t h e body.  a n d may  defined  and d i f f e r e n c e s  r e s u l t i n g i n a v a r i e t y of architectures  tissues  humour  order  peptide  tissue-specific  matrix,  et  reviews  a l l connective  several  interactions  the  good  I f the reader  has s i m i l a r i t i e s  Three  microfibril;  long  structure.  of structural  microscopic.  the  tendon,  1 9 8 4 ; Chapman  tissue. Within  from  c r y s t a l s i n turkey  the  K'uhn, 1 9 8 7 ) . Collagen  to  t o emphasize the  considered  several  (Miller,  thesis  hydroxyapatite,  crystal  information  i n this  1Onm  depending  on  tendon  (Parry  within the  are interwoven,  to mineralization.  very  i n the vitreous  rat tail  fibrils  are  in a  Following  poorly  3  remodelling within  the  of  bone  are  shear  lamella.  fibrils  close  to  within  90  bone,  parallel  "twisted  the  fibrils  which  fibrils  one  present, fibrils  the  within  sinusoidal fibrils  may  In  course,  forces. rapid  The  minimum Turkey  of of  leg  the  lamella  within  nested to  to  arcs.  of  muscle.  organized The  forces  and  which  this  into  a  is model  the  patterns of  They  a  follow occur  collagen  and  as  tensile  tendons  by  these  deformation  permit  contractile loads  (Silver, a  1 988).  parallel, high  of  by  are  (Giraud-Guille,  generated  mineralize  In  arrangement  sustain  transmit  angle  continuously  general  contain  of  fibrils  d i r e c t i o n of  rotates  two  an  same s p e c i m e n ,  fibrils.  composition  Tendons  loss  tendons,  and  can  direction  of  direction  which  are  organization  complex.  tendons  into  by  h e l i c o i d a l bundles  bone,  structure  the  the  next  of  are  these  to  the  another  change  fibrils  energy  and  lamella  local  transmission  elements  another  i s very  abruptly  densely-packed  one  three-dimensional  bone  contrast  fibrils  Although  local  organized  the  architecture  lamella  angle.  are  fibrils  In  lamella  occur  of  Collagen  d i f f e r e n t forms.  fibrils  one  collagen  r e f l e c t i o n of  alternative  each  series  from  An  plywood"  superimposed  constant  from  of  each  to  may  within  is a  two  model,  degrees.  architecture  distractive forces.  Within  changes  the  a  matrix  and  plywood"  predominantly the  laminated  distributed in  "orthogonal distinct  the  mineralized  compressive, in  bone,  part  with  a  1987). of  normal  the  4  growth  after  9 weeks  of  age,  have  microscopic  i n v e s t i g a t i o n s of  because  linear,  the  analysis and  along  Engstrbm,  individual 1965;  Collagen, also  Evidence  for  biological from  of  i n bone,  the  close  of  defects  the  mechanisms The  primary  give  composition integrate  of new  gap  zones  these  of  In  leg  of  and  of  function  most  a  stems  are  the due  part,  about  skeletal to  1987).  function  work to  i s the  the  from  amino an  of  the  concepts  Recent  acid  information within  stages  1989).  As  an  need  to  of  (Arsenault,  early  localization  apparent  apatite crystals  (Arsenault,  in  relationship.  current  fibrils  and  heterogeneous  (Hollister,  i s known  this  collagen  instances,  i n the  this  i n the  Myers  tendon.  comes,  imperfecta  respect  collagen  zones  turkey  imperfecta,  interaction.  gradient  a l . , 1960;  of  bone  to  findings with  distribution  developmental within  with  collagen  collagen/mineral  et  p h y s i o l o g i c a l process  collagen  rise  permits  is  and  little  interest  apatite crystals  fibrils  role,  I collagen  of  t i s s u e s very which  (Nylen  of  r e l a t i o n s h i p between  importance  mineralized  and  dentin  type  electron  i t s biomechanical  osteogenesis of  in  1988).  the  inherited diseases.  molecular  periodic  in  osteogenesis  m a n i f e s t a t i o n s " of  Despite  fibrils  a p a t i t e i n normal  studies  group  arrangement  Arsenault,  intimately involved  used  collagen mineralization  i n a d d i t i o n to  mineralization  of  parallel  been  suggests the  1988),  a  overlap and  a  of m i n e r a l i z a t i o n extension  of  5  these the  works  the present  study  periodic distribution  of apatite.  structure  and t h e p r o p e r t i e s o f b i o l o g i c a l  The  given  Collagen The  chains and  helical Crick,  interact  apatite  of t h i s  c o n s i s t s of three  t o form  1955; Rich  chains  and a r e c o i l e d  1955).  Piez  a triple  and C r i c k ,  polypeptide  coil  proportions  molecular  thesis.  e t a l . (1963)  of the three unit  peptide  helix  have  each  determined chains  individual  ( R i c h and the r e l a t i v e  and d e s c r i b e d t h e  molecular  peptides  and one  2(l) peptide.  collag'en  molecule  was f i r s t  described  by B o e d t k e r  (1956) as a r i g i d  r o d 300nm  i n length  and h a v i n g  1.25nm. L a t e r , when  resolved, one  i t was  a t each  terminal both These  found  as c o n s i s t i n g  et al.,1955).  their  other  t h e amino there  t h e N- a n d Cconformations  prediction  based  and shape  sequence  1(1)  of the and  a  Doty  diameter  was  being  a r e two n o n - h e l i c a l p o r t i o n s ,  end o f t h e m o l e c u l e .  portions called  o f two i d e n t i c a l  The s i z e  acid  peptide  (Ramachandran  1 9 5 5 ; Cowan  each  about  helical  collagen  of  crystals  Molecule  which  three  of collagen  t o the development  collagen molecule  Kartha,  These  rise  analyze  I t i s therefore  to review  have  concepts  to further  necessary  which  some  attempts  These  are the  a n d C-  t e l o p e p t i d e s . The c o n f o r m a t i o n s  t e l o p e p t i d e s a r e n o t known a t are modelled  on e n e r g y  using  and s t e r i c  secondary  of  present. structure  calculations.  Several  6  stereochemical this  method  models  (Jones  may  and  of  may  not  the  be  semiflexible molecules on  their  side  would  the  within  the  interactions. dependent pitch  will  Although is  on  the  The  pitch  be  between  or  the  to  et  of 29 al.  a  single  rat  tail  tendon  45  residues  and  (1983)  value  of  the  the  the  side  chains  be of  the  edge.  collagen  molecule  intramolecular agreed  been  1976).  pitch  acid  intramolecular  been  has  (Miller,  calculated  acid  reaction  yet  edges  chains  would  and  collagen  portion  amino  side  the  the  not  rigid  a  alteration  inter-  has  as  of  amino  any  pitch  a  determine  edges  of  as  reaction  acid  would  the  and  the  via  position  the  of  the  determining  other  determine  pitch  of  from  cylindrical  amino  composition  determination  interactions,  Fraser  helical  of  while  would  regions  helical  described  cylinder  composition  change  critical  the  helix  The  triple  Two  the  groups  of  these  molecule  be  each  on  interactions  triple  The  also  with  depending  exterior  collagen  (K'uhn, 1 9 8 2 ) .  Functional  intermolecular  can  interact  surfaces  the  for  1987).  correct.  molecule  cylinder  chains.  towards  of  entirely  collagen  possible  Miller,  Conceptualization rod  be  to  upon.  reported The  be  study 30  to of  residues  8.68nm. Individual  flexibility fluctuations without  collagen  (Veis, are  molecules  1985a).  This  accommodated  appreciable  changes  in  can  by  show  occur  changes  free  substantial because in  eneregy  the  lateral segmental  triple  (Okuyama  helix  et a l . ,  7  1981).  Flexibility  motions  and  regions  located  they  are  2)  hinge  the  stability  about  the  N-C  restricted.  because  Thus,  of  flexible the  simplistic.  backbone  and  suggest  that  involve  a  domains.  myriad  of  et  collagen  of  molecule  as  molecular flexibility  of  the  molecules  these  long  axis  and  38Kb  is  by  is a  two  of  et  the in  peptide  1987)  molecules  in  fluid-like cause  transverse  axis  more  and  fibrils  in mineralization I  issues  of  the  local  explore  relevant  fully  of  in  I collagen length  of  are  which  the  properties would  protein translation  Genes type  be  NMR  a l . ,  would  also  to  T y p e _I C o l l a g e n  about  there  adjacent  changes  The  are  rotation  c y l i n d e r may  changes  post-translational modifications.  for  a  Sarkar  and  genes  in  C-C=0 b o n d  flanked  observed  like  The  which  element  molecule  motions  conformational  order  discuss  because  prevents the  length  now  They  to  In  250nm  i n t e r a c t i o n s between  i n both  helix.  collagen  hinge  residues  major  structure  a l . , 1983;  Accommodation  distortions triple  the  The  chain  (Sarkar  is a  flexural  915-939  hydroxyproline  r o t a t i o n about  about  slow  suggested  81-102 and  ring  the  collagen  side  1)  regions.  somewhat  experiments  the  forms (1984)  Proline  the  within  region  Viewing  residues  helix.  and  two  Veis  p r o l i n e and  bond  semiflexible relatively  of  in  regions.  triple  helix  occur  between  devoid  stabilize  may  large 10%  and  is a  complex. coding  a sequence many the  o f a b o u t 50 e x o n s  exons have ancestral  (K'uhn, 1 9 8 4 ) .  an i d e n t i c a l  gene  arose  o f 54bp  unit  sixfold  r e p e t i t i o n of a oligonucleotide  pattern  sizes  recombinational  may  events  shows  molecule  b a s e d on " b i o c h e m i c a l of collagens  to  acid  amino  acids  peptide, amino  acids  several  Seyer  bovine acids  acid  chick  I  of the  collagen  species  which  (1985) used  type  a n d human  I  collagen  collagen.  They  (R a n d K; s e e letter  amino  were  codes),  retained.  acids,  and 66% o f t h e h y d r o p h o b i c Overall  amino  microscopic  and Kang  single  i n the a l ( l ) peptide  unsubstituted.  to  91/5 o f  (D a n d E ) a n d 7 9 % o f h y d r o p h o b i c  96% of t h e b a s i c  acids,  this  Nordwig and  of the type  t o compare  amino  f o r amino  a c i d i c amino  of  i n protein  species.  and e l e c t r o n  level.  data  100% of the b a s i c  "Abbreviations" the  structures  a combined sequence from  found  a  f o r the  f o r the invariance  i s o l a t e d from  i n evolutionary amino  between  evidence  and q u a t e r n a r y  available  coding  from  degree of conservation'in  tertiary  differ  arisen  et a l . , 1985).  a high  (1969) presented  studies  have  due t o an i n t o l e r a n c e  sequence and s t r u c t u r e  Hayduk  may  r e f l e c t e d by a c h a n g e  (De C r o m b r u g g h e  Collagen acid  i n turn  that  single  g l y c i n e - p r o l i n e - p r o l i n e . The f i x a t i o n  o f exon  function  which  that  suggests  by a m p l i f i c a t i o n o f a  genetic  tripeptide  o f 54bp  length  The f i n d i n g  homology  between  In thea2 ( l )  87% of the a c i d i c amino  acids  were  the sequence of  9  the a l ( l ) and  calf  chains was  of  found  Consistency prerequisite inorganic for  this  from  a  comes n o t  Amino  Following be and at  subdivided sequence the  which  reticulum  of  the  sites  regions  the  the N-  may  between  be  changes  matrix.  or  alterations. the  a  2  (Hollister,  chain  but  These be  also  such  genetic  and  and  Evidence  formation  i n the  a  organic  i n t e r s p e c i e s homology  Small  rat  a l . , 1982).  code  p a t h o l o g i c a l bone  as  code  changes as  small  as  1987).  the  The  These  first  p r o t e i n to  adjacent  carboxy  because  propeptides. C-  are  from  from  differing  is a the  the  across  peptides  described  region  move  It i s cleaved  with  and  procollagen  six functional regions  It i s considered  along  by  from  of  terminus.  molecule, of  extracellular  transcription,  1981 ) . The  assembly  of  f o l l o w i n g movement  propeptide.  genetic  interaction  the  et  from  Sequence  (rER).  immediately (Kriel,  collagen  the a 1  into  enables  sequence  (Highberger  characteristics.  amino  combined  normal  mutation  Acid  a  biomechanical  either  point  and 93.4%  the  only  studies  i n major  single  be  imperfecta.  involve  The  of  components  osteogenesis  may  to  f o r the  genetic  results  chick  rER  functional region i n the  propeptide,  crossbridges Following  propeptide  starting  signal  rough  size  peptide  endoplasmic  propeptide  the  important  in  can  at  are  the  specific  the  membrane i s the  amino  molecular opposite  end  formed  at  specific  cleavage  of  these  proteinases  in  the  10 extracellular  matrix,  crossbridges.  Adjacent  telopeptide, length.  in  length The  a  (Miller, amino  The  acid  length  The  stages  following  amino  triplet  acid  position helical  D period,  responsible  structure  repeat  acid  except  suggested  stereochemically position  was  more  residues  i s characterized  with  repeat where  234 a m i n o  acids i n  s t a i n i n g and a l s o  I t i s considered  organization.  i n every  i s responsible  sequence  analysed  The  third  f o r the and  triple Kartha,  i s often  or tryptophan.  Jones  t h e X and Y p o s i t i o n s  involved  Other  region  t o be t h e  G i s g l y c i n e , a n d X o r Y c a n be a n y  i n the t r i p l e  interactions.  i n early  t i s s u e , due t o t h e  glycine  of t h i s  cysteine that  by  are f u n c t i o n a l l y  for fibrillar  chain  t h e X p o s i t i o n was  were  in  acid  i s 25  of the molecule- (Ramachandran  The t r i p l e t G-X-Y  16 r e s i d u e s  i s v i s u a l i z e d i n the electron  of a p a t i t e .  of the peptide  abbreviated  while  which  heavy metal  distribution unit  (1985)  patterns  repeating  structural  amino  telopeptide  of m i n e r a l i z a t i o n , i n unstained  periodic  t h e amino  i s 1014 amino  sequence of collagen  (Hulmes e t a l . , 1973),  microscope  1955).  carboxy  segment,  disulphide  1984).  v/ariety of repeating  significant.  portion  t o form  propeptide,  non-helical  helical  i n length.  remains  t o t h e amino  i s a short  The t r i p l e  residues  no c y s t e i n e  helix  such  differ  that  i n intermolecular  involved  more  potential repeating by Hofrnann  et a l .  the Y  interactions  i n intramolecular units  o f amino  e t a l . (1 9 8 0 ) .  They  acid found  11  several D,  2D,  patterns 3D,  unlikely  D/3,  to  functional been and  occur  and  place  may  for  of  to  polypeptides reticulum,  modify  molecular  procollagen  the  the  large  number  and  to  to  and  the  units  are  have  some  has  long  sequence  biomechanical  yet  peptide  there the  has  amino  exist  site  of  i n d i v i d u a l peptides  with  not acid  three  in  while the  1982).  but  of  are  organization  also  of  endoplasmic  Th i s  conformation  hydroxylation.  is a  (Chorpra  of  The  conformational  i s necessary  only  unhydroxylated  conformations  Hydroxyproline not  individual  the  1978).  proline  which  the  rough  g-turn  chains  stabilization  molecule  events  modifications  fibrillar  segments  hydroxylation  the  Ananthanarayanan,  several  These  for  Ananthanarayanan,  proline  of  the  protein.  of  of  collagen  acid  collagen  assembly,  of  i n the  presumed  importance  assembly,  result  role  are  r e l a t i o n s h i p between  associated  specific  intertwining  statistically  amino  of  repeat  Modifications  molecules  (Brahmachari  the  and  The  biomineralization.  molecular are  a  the  chains.  4D/11  t r a n s l a t i o n of  influence  Prior  be  chance  paramount  Post-Translational  may  and  functions  which  essential and  peptide  mineralization.  Following take  D/13  by  of  analysis  sequence  two  s i g n i f i c a n c e . The  considered  an  the  D/2,  physiologic  been  in  i t s assembly  p r i o r to  the  and  plays the  change  an  important  triple-helical into  microfibrils  1 2  (Nemethy always  Scheraga,  i s the  portion that  and  1986).  Y position  (Bornstein  hydrogen  and  bonds  (Gly-X-Y)  Traub,  between  and  et  Hyd r o x y p r o l i n e  the  1975).  structural  (Prockop  bonds the  the  The  between  degree tissues  of and  and  glycosylation  is  immediately  endoplasmic  to  the  polypeptide at  t o be  glycosylating  prior  of  D-galactose  0-glycosidic appears  between (Bansal  essential  a t body  for  temperature  et a l . (1973)  of  the  and  has hydrogen  further  stabilize  ages.  The  role  triple  linkage a high enzymes  degree due  to  and of  of  1980).  the  of  Hydroxy l y s i n e  formation at  to  residues  v i a an  Cunningham,  1966).  few  specific  continue  the  covalently  for  the  1973).  modifications  specificity  is  crosslink  et a l . ,  are  helix  are  lysine  disaccharides  hydroxylysine (Butler  of  helix  (Kivirikko  D-glucose  triple  importance  (Kivirikko, to  to  residues  hydroxylation  mono- a n d and  prior  lysine  post-translational  attachment  molecule.  suggested  water-bridged  i s i n the  reticulum  Intracellular the  collagen  number  formation  with  been  t o be  is  helical  occur  h y d r o x y p r o l i n e which  to collagen  rough  triple  adjacent helix  i s known  specific  hydroxylysine  formed  an  peptide translation  a variable  hydroxylated. variable  on  found  helix.  Following formation  with  of  of  the  I t has  Ramachandran  existence  associated  triple  1976).  1979).  group  integrity  et a l . ,  suggested  a C=0  of  triple-helices  hydroxyproline al.,  H y d r o x y p r o l i n e when  attached  There  the sites  to  which  1 3  the  carbohydrates  found  no  studies  glycosylation  of  attach  (Miller,  directed  at  the  collagen  i n bone  1976).  To  function  date  of  I  have  the  f o r m a t i o n or  mineral  deposition.  Self  assembly Following  extracellular and  secretion  environment,  asymmetrical  molecules  are  the  collagen  the  molecular  of  where  molecules  organized fibrils  into  and  believed  polymerize  Dirk,  1958).  Furthermore,  form  forms  are  dissolution  side  Thus,  chains  directing  on  stabilization side and  chains positive  1974).  been  adjacent  collagen of  molecules  fibril  due  charges  Alternating  to  assembly  have  the  i n the  charges,  G-X-Y  and  a major  Ionic  and  native subsequent  et  role al.,  and  among in  1973)  clustering  position  triplet  positive  the  collagen,  interactions  (Hulmes  relative  in  (Schmitt et a l . ,  that  structure.  of  of  are  (Gross  s p a c i n g and  conditions  suggested  collagen  occurs  by  specific  i t has  fibrils  in vitro  fibrils  1953).  local  the  long  under  the  studies  l o n g s p a c i n g , segmented  of  function  in vitro  interconvertible  these  a  on  fibrous  reaggregation  1985a),  be  within  to  the  elongated  Collagen molecules  spontaneously  based  into  architecture  initially  1988).  as  (l/eis, The  solubility  (Giraud-Guille,  matrix  behave  fibrils.  d e r i v e d may  geometry  extracellular  they  in solution  environment to  procollagen molecules  (Doyle  of  and  of  negative  et a l ,  n e g a t i v e , may  occur  1 A  linearly  to  provide  three-dimensions molecules have  hydrophobic  less  this  The  of  an  groups  aggregation  environment  which  association  of  the  in  i n the  of  c r o s s l i n k s . Hulmes by  orderly  1975).  than  arrangement  of  ions  periodic  spacing  (Piez  and  groups  would  lead  polar  them  collagen (1983)  prior  to  that  concentric  the  1980).  would  suggested  of  of  (Tanford,  fibril  and  addition,  forces  acids  In  two  In  amino  accretion  Torchia,  crystal.  strong  hydrophobic  stability  and  hydrophobic  surrounds  to  occurs  an  of  (Piez  r e s u l t i n more  ionic  occur  d i s r u p t i o n of  growth  would  c o n t r i b u t i n g to  properties  1978).  stability  Trus,  aqueous The  close  therefore the  lead  formation  collagen  layers  to  of  fibril collagen  molecules. Procollagen  processing  interactions  with  suggested  as  factors  diameter.  Aminopropeptides  and  may  1983). been  regulate  Procollagen  (Nowack  et  formation  may  on  alternative  the  tissue  and  hypothesis  to of  by  Procollagen  the  the  control  been fibril  et a l . ,  I collagen  have  immunofluorescence  polymerization  remove  or  fibrillogenesis  type  a l . , 1983).  a l . , 1985). fibrils  of  immunoelectron  the  have  (Fleischmajer  fibrils  et  a l . , 1983)  specific  participate in  collagen  involve et  the  diameter  (Fleischmajer  (Fleischmajer act  limiting  et  (Wood, 1960)  aminopropeptides  a l . , 1976)  techniques  then  proteoglycans  fibril  demonstrated  (Fleischmajer  microscopic Therefore, of  aminoprocollagen  N-proteinase  amino of  fibril  would  propeptide.  fibril  size  is  An  1 5  derived  from  in vitro  interactions  which  related  to  present  (Parry  The  the  period  due  to  1952).  Early  determined al.,  values et  al.,  al.,  changes  in D  dependent  The  in fibril  type  pattern  feature  which of  of  the  x-ray  true  collagen but  local  D  charged  period  the  degree  i t may  are also  collagen  aggregates  (Hodge  and  led to  (i960) defined  for  collagen  thesis.  has  Hodge  and  fibril  the  longitudinally  space  (see Figure  1).  (Bear,  (Hall  yield  to  et  greater (Fraser in  artifactual specific  or  of  repeating  the  in theories  In a d d i t i o n ,  " a , b,  banding  units  This  of  of the  electron  quarter-staggered packing  c,  which  P e t r u s k a (1963)  which  nm  remains  tissue  resulted  Schmitt, 1960).  Schmitt  acids  stretch  two-dimensional packing arrangment. approach  D  environment.  vitro  microscopic  i s the  collagen 64  unit  be  microscopic analysis  the  of  prone  electron  model  of  methods  value for this fibrils  collagen  amino  t o be  diffraction on  of  r e p e a t s i n each  The  molecular  diameter  of p r o t e o g l y c a n  microscopic studies  length  the  and  localization  length  on  changes  1982).  staining  However,  because  proteoglycan/collagen  ultrastructural  electron  1983).  of  found  (67.8nm) d e p e n d i n g  debate  in  et  the  the  1942).  have  concentration  striking  cross-striated  studies  d and  e"  will  be  first  Hodge  nomenclature used  suggested  a d j a c e n t m o l e c u l e s were  and  in  this  a model  s e p a r a t e d by  in a  1 6  D=67nm  D=234  OVER| P ZONE  GAP, ZONt  RESIDUES  A  •'.•'.•'.•'.•'.•'A  it  •  ^••••••••Vr]  ESsd  •>::.:>>::v^^  t  K-WX-K  AD  .6D 40.2nm  26.8nm  44D=300nrn  Figure type  rods  staggered  adjacent  different packed  densities  overlap  available.  zone  Second,  be a n a l y z e d  sequence for  First,  repeating model.  These  This  t h e model within  units  are depicted as  r e s i d u e s , o r D,  theperiodic  sequence  to  has two i m p o r t a n t  predicts  a n da gap zone  according  acid  model o f  two zones o f  therepeating D unit,  by computer  acid  packing  molecules  model  with  thesis  using  o f c o l l a g e n were  densely space  o f amino  acids  the primary  thesingle  t o t h e Hodge  a  potential  localization  modelling  ofcollagen. Inthis  t h e amino  (1963)  by 234 amino  molecule.  consequences.  can  andP e t r u s k a  I collagen molecules.  linear the  1. T h e Hodge  letter  organized  and Petruska  codes into  (1963)  17  The  Hodge  consequence different contain would the  and P e t r u s k a  that  contain  A closely  fitting  (1963)  four  between  has been  model  the electron microscopic  fibril be  amino  used  i n this  microscopic model  acid  (Meek  Despite  and r i g i d .  the i l l u s i o n  In r e a l i t y  There  the l e v e l  about axis the  of the f i b r i l .  the molecular a n d i n some  fibril appears  of  t h e gap zone  However, this  illustration  the c o i l  about  o f t h e way  to the scale  the width  gap z o n e s  this  molecules  levels  the  of  in coiling  the  coil  microfibrillar  are coiled  within  In a d d i t i o n , i n Figure  Figure  holes 1 was  at the  ( o r 0.5mm). So small  based  1,  level  constructed.  of the a c t u a l molecule,  o f t h e gap zone  i s very  will  that the  of the chain,  are l a r g e , porous  of the molecule  individual  coil  the m i c r o f i b r i l s  because  t o be m o r e  or four  et a l . , 1979).  as i f there  x the length the  cases,  (Ruggeri  it  axis,  The  This  the  space  are three  and  to the current  s t r u c t u r e of  s t r u c t u r e of the collagen  to  Hodge  i n electron  three-dimensional i s complex.  The  of  e t a l . , 1979) which  i n v e s t i g a t i o n s the f i b r i l l a r  are linear  because  analysis of collagen  i t s utility  i s oversimplified. I t gives  molecules  zone  s t r u c t u r e and h i s t o c h e m i s t r y .  composition  study.  would  gap  molecules  important  of collagen  of  zone  molecules.  very  important  zones  overlap  understanding permits  has t h e  molecules.The  f o r every  separation model  packed  parallel  one s p a c e  longitudinal  Petruska  model  i t p r e d i c t s two a l t e r n a t i n g  densities.  tightly  (1963)  would  be  5/1000  t h e volume on t h i s  in  of model.  1 8  For  example,  shaped sides of  space 1.5nm  i f we with  this  about  3,000nm^,  see below)  gaps,  two i d e a s  have  aligned  i n three  (Weiner  and Traub,  arrangment order.  Second,  built  the  primary  zones.  into  they  Current  been The  H'ohling  views  of crystal  fibrils  exhibit  support  the concept  may  be  space  predict  (1963)  packing  n o t be i n t h e that  staggered  larger  spaces  and n o t t h e gap  dimensional  packing  theories but neither  packing  crystalline  order.  qualities  of periodicity  have  There  may  of collagen  i n  several  In c r o s s - s e c t i o n i n some  regions  which  three-dimensions.  a r e n o t c o n s i s t e n t . I n some  the cross-section pattern areas.  arrangement  i s n o t known d e s p i t e  portions of the f i b r i l  irregular  individual  demonstrated.  at defining a regular  However,  would  formation  these  of  300nm^ t o  o r g a n i z a t i o n and these a r e  of the three  do n o t e x c l u d e  to explain the  sufficient  suggested  the m i c r o f i b r i l l a r  within the f i b r i l  attempts  theory  would  (1 9 8 0)  three-dimensional  molecules  regions  molecules  location  arrangement  to provide  volume  non-collagenous  t h e gap z o n e s  i n t h e Hodge and P e t r u s k a because  are  First,  This  the total  (minimum  and t h e volume  dimensions  and t h e o t h e r  In order  of mineral  emerged.  1986).  40nm  no o t h e r  space.  the volume  gap as a box  1963) then  be 90nm^ a s s u m i n g  between  distortions  dimension  and P e t r u s k a ,  are occupying  difference  the individual  the longest  (Hodge  one gap w o u l d  molecules  consider  i s distorted  a l s o be s h a r p  over  discontinuities  to  1 9  the  lattice-like  close  packed,  optimal  quasi-hexagonal Miller, 1976)  1979)  may  a  consistent regions  fibrils  i s considered  crystal  since  growth  to  evidence  distance  Katz to  collagens. in  vary They  needs  reasons.  different  in  crosslinks  to  and  Li  short not  be  between found  tissues  In  the  appropriate  (Mechanic  et  the  collagen  apatite  environment have  crystal  There  organization  is  is  some  tissue  intermolecular  and  non-mineralized spaces  non-mineralizing  inhibited in  tissues  to  of  of  volume.  intermolecular  addition,  various  growth  space  are  perfectly  the  found  in  a  Piez,  there  structure  appropriate  than be  to  and  and  but  for  mineralized  that  (Hulmes  (Trus  distances  conform  Therefore,  the  arrangement  sufficient  (1972)  as  arrangement  important  m i n e r a l i z a t i o n may  steric  a l . , 1985).  such  three-dimensional  and  mineralizing  that  do  biologically the  et  three-dimensional  formation  that  specific.  The  there  the  over  which  pattern.  crystal  packing  microfibril  regular  for  (Hulmes  arrangements  molecular  or  be  significant  structure  are  greater  tissues  certain tissues  the  geometry  of  packing  and  result in different  may  and for be  a l . , 1987).  Crosslinkinq Covalent important connective specific  c r o s s l i n k s between  determinants tissue  of  matrices  the and  collagen  mechanical are  an  molecules properties  example  p o s t - t r a n s l a t i o n a l heterogeneity  of  of  the  type  I  are of tissue  20  collagen.  Crosslinking  aldehydes, peptides,  i n t h e amino with  amino  i n the t r i p l e  et  1986).  demonstrated  Two  sclera  and r a t t a i l  most  tendons  labile  (Eyre,  (Tanzer  reducible  Glimcher, crosslinks  1973).  o f bone  1974).  collagen  These  crosslinks  pyridinoline chemistry  undergo  (Fujii  i n type  and T a n z e r ,  I collagen  residues  from  o t h e r c h a i n s form  Although  i ti s believed  involved.  Banes  can r e a r r a n g e t o ( E y r e and reduced  chains,  that  ages  The  crosslinking  initially  involves  but subsequently,  t r i and t e t r a - c h a i n  the crosslinks  as t o the nature  e t a l . (1983)  bone  to nonreducible  and n o n - m i n e r a l i z e d c o l l a g e n debate  (aldimine)  As n o r m a l  1974).  fibrils  two c o l l a g e n  some  participate in  The two p r i n c i p a l  a transition  from  remains  l i g a m e n t and  are dihydroxylysinonorleucine  residues  mineralized  cornea,  collagen, the  crosslinks  (DHLNL) and h y d r o x y l y s i n o n o r l e u c i n e . DHLNL  on  on h y d r o x y l y s i n e  also  I n bone  1971).  been  skin,  i n bone, may  (Yamauchi  - one b a s e d  are Schiff-base  ketoamine  1972; Mechanic,  have  i n adult  Histidine  et a l . ,  stable  hydroxylysine  the o t h e r based  crosslinks  specific  nonhelical  linking  collagens  predominates  1987).  and  of  regions of molecules  of cross  tendon;  et a l . ,  (Mechanic  terminal  predominates  which  reducible  compounds form  which  pathway  crosslinks  helical  f o r the f i b r i l l a r  aldehydes  the reaction  of lysine  pathways  lysine  aldehyde  from  and c a r b o x y  groups  located al.,  results  links.  i n the  are different  there  of the structures  d i d not find  pyridinoline i n  21  the  mineralized  collagen  o f bone  non-mineralized  portion.  They  physically  inhibits  approximation found for  that  suggested  of the collagen particle  resulted  m o l e c u l e s . Wu size  i n protease  found  used  They  mineralized  and n o n - m i n e r a l i z e d t i s s u e . t o b e o f some  process.  At t h i s  further  clarification.  Biological  having  time  apatite  (Jackson  et a l . ,  rod-like  structure  field field  difficulties a wide  measurements a mean  Robinson et  (Myers  dark  characteristic  given  al.,  thin,  1978; Weiner  bright  selected-area  Direct  of  (1 9 8 8 ) bone  pyridinoline  i n pyridinoline I  between  expect  in mineralization  there remains  crystals  size  a need f o r  1978; L a n d i s  been  and Traub,  have  been  electron electron  range  1965).  measured  microscopy microscopy with  field  by  x-ray  and (SADF).  Due t o  this  been r e p o r t e d . microscopy  have  (Robinson, 1952;  1952; S t e v e - B o c c i a r e l l i , and G l i m c h e r ,  shape  The  studying  electron  3D t o 70nm  either  1986) o r as a  o f v a l u e s have  by b r i g h t  described  elongated, plate-like  associated  between  and Watson,  have  and Engstrom,  of the c r y s t a l s  diffraction,  technical  by p r e v e n t i n g  i n powdering  significance  though,  an i r r e g u l a r ,  dimensions  mineralization  Apatite  Individual as  no d i f f e r e n c e  i n the  and Eyre  digestion  crosslinks.  crosslinking  that  formation of p y r i d i n o l i n e  the small  analysis  b u t i t was f o u n d  1970; Jackson  1978; Weiner  and  Price,  22  1986).  However,  electron  visual  micrographs  background  electron  differences Hunziker,  interpretation  results density  i n detection  1988).  interpretation  of c r y s t a l s  crystals.  Second,  position  leading  to larger  small  section  crystal  contains  probability the  X-ray from  that  t o 36nm  Engstrbm,  1985;  Grynpas small  which  crystals.  the cumulative crystal  (Myers  has been  reported  even  size.  (Arsenault  the thinnest the  density  o f many  and Grynpas, forcrystal  i n the l i t e r a t u r e as  et a l . ,  et a l . ,  and Grynpas,  to i t s large  1988).  length  Jackson  e t a l . , 1983; Boskey  i s related  Due t o  e t a l . , 1989) and  electron  results  more  in crystal  increases  e t a l . , 1986; Arsenault size  biased  of larger,  and Engstrb'm, 1 965;  1972; Bonar  crystal  a  and  of c r y s t a l  This  First,  results i n  be o v e r l a p  (Hunziker  d i f f r a c t i o n has produced  1978;  The  many  problems.  expect  cross-sectional size,  i s a single  1 Dnm  may  estimates  of overprojection  perception  crystals  one w o u l d  there  field  (Arsenault  due t o s e l e c t i o n  obvious  the  i n two b a s i c  of the specimen  Therefore,  of size  of bright  1988).  surface  area  between  2 85-265m 1963;  /g ( R o b i n s o n  Miller,  Grynpas, extremely and  may  1 9 5 2 ; Hodge a n d  and P r i c e ,  The b i o l o g i c a l  important  Studies size  1984; Weiner  1988).  crystal  and Watson,  control  i n determining  Petruska,  1986; A r s e n a u l t of c r y s t a l  biomechanical  and  size i s properties  chemistry. which  mislead  report  the reader  single into  mean  values  assuming  that  for crystal there  i s a  23  narrow  range  standard  i n the  sample.  deviations  reported  heterogeneous  population.  of  this  range  all  of  large the  maturity. on  the  stage  depend are to  on  of  the  emphasize the  (Arsenault crystals  apatite  for  literature,  preparation associated Arsenault  leads  to  apatite electron  gap  s i z e and  1988).  at  that  would  the  For  not  of  times  crystals  seem  and  like to  Petruska  collagen  example,  l e a s t ten  also  I would  Hodge  packing  depend  I t may  adjacent  do  in  tissue  remodelling.  in  two-dimensional  are  of  shape  dimensions  zone  sources  common p r o b l e m  different levels.  crystal  the  a  possible  standardization  such  large  the  the  width  width  of  of  the  the  zones. microscopic  major i t has  ultrastructure  aqueous  the  at  A  the  for  several  sizes.  crystal  Grynpas,  electron pose  evidence are  environment  s i z e of  gap  There  i s the  growth  measured  predicted In  in  and  are  contrary,  m i n e r a l i z a t i o n and  that  (1963) model  the  crystal  that  local  restricted  reflect  of  techniques I assume  On  are  and with  and  stains  the  c h a r a c t e r i s t i c s of  t e c h n i c a l problems.  Often,  been  changes  a  suggested  r e s u l t of  specimen the  totally  ambiguities c r y s t a l s are  that  the  methods  visualization.  solubility  Hunziker  microscopy  studies  (1988)  showed  demineralize  i n what labile  i s being to  (Arsenault,  of  problem  is  conventional sections  stained.  As  tissue  apatite.  r a d i a t i o n damage 1988).  mineral  for  such  that thin  the  in  used  One  properties  in  a  and  In  this  addition,  during  consequence  of  24  these  technical  difficulties,  methods  quick-freezing/freeze-substitution 1988;  Arsenault  (Landis  et  developed  to  differences  there  may  between  model  the  be  calcium  two.  biological structure  and  been  the  analyses  ideal  carbonate  (Hagen,  These  stages  of  geological  or  reveal  result  synthetic  Second,  relationships in  other  may  mineralization. must  that  First,  1973).  arrangements  crystal structure  some  fluoride,  stoichiometric which  are  formula.  and  phosphorus  considered  there  including  various  Hunziker,  techniques  have  i n bone  alternative  apatite of  Chemical  from  arrangements.  at  a l ; 1980)  exist  chloride, also  important  anhydrous  for biological apatite  between  crystalline  ( A r s e n a u l t and  h y d r o x y a p a t i t e , Ca^^(P0^)gOH, 4s  impurities  magnesium,  et  as  a r t i f a c t u a l changes.  differs considerably  numerous  and  Landis  minimize  idealized  bone  a l . , 1988)  a l . , 1977;  Although the  et  such  be Therefore,  deviate  from  hydroxyapatite  the  of  ideal  composition. The crystal the been  unit  symmetry  length the  and  described  c.  of  structure.  apatite, and  cell  the  The of  as  unit  apatite  I t has  chemical a  cell  positions  axes, of  the  a l l the  elements,  i s defined  their  are  of  the  by  three  axes  labelled according  The  unit  cell  the including  c r y s t a l and  (Hagen,  i n c l i n a t i o n to  atoms.  throughout  characteristics,  s t r u c t u r a l molecule  c r y s t a l planes the  i s repeated  one of  1973).  In  labelled to  another, the  has  a,b  the and  crystal in  25  calcified  turkey  a=Q.942nm  a n d c=0.688nm  basic  cells  consistent of  tendon  of c r y s t a l  i s hexagonal (Myers  diffracted electrons  selected-area  dark  Apatite/Collagen Biological  apatite  the non-collagenous  their  (Glimcher,  preferred  within  the  in a  visualization  crystal  planes i n  microscopy.  the collagen  and Traub,  of the c r y s t a l s within  suggested  location  that  t h e gap zone  f o r mineral  1986).  (1963)  and  Yet the  remains the  be t h e  within  fibrils  because  of the p o t e n t i a l space  a v a i l a b l e . Since  that  certain  studies  this  et a l . ,  1977).  localized  overlap  localization focussed result  zone.  view  (1983)  i n t h e gap zone  The p r i m a r y  recent  arrangement  information  deposition  believed  are within  both  that  i s  throughout  f o r the s t i l l  remains  t o e x i s t as a  of collagen  suggests  time,  the mineral  but spreads  o f c r y s t a l s i n t h e gap zone  of the staggered  (White  states  explanation  on t h e p o t e n t i a l s p a c e  However, mineral  supported  A l t e r n a t i v e l y , Glimcher  initially the  have  the  i n proposing  would  deposition  leg  outside  the f i b r i l s  Hodge and P e t r u s k a  turkey  fibrils  e x t r a c e l l u l a r matrix  1959; Weiner  of debate.  model  These  electrons  crystals i n mineralizing  within  subject  1965).  Relationships  and bone a r e f o u n d  localization  specific  electron  tendon  fibrils  diffract  makes p o s s i b l e  from  field  the dimensions  and Engstrom,  organization  manner and t h i s  with  early  molecules. sites  t h e gap and o v e r l a p  of zones  26  (Arsenault, of  mineral  overlap  1 988).  is initially  zone  progresses. the  but  this  a  previously The  to  of  specifically  the  viewed  by  observed  alternate  to  This  arrangement  of  the  axial  i s most of  the  axial  Conclusion  to  arrangement  proportion  zone  as  the  than  in  the  mineralization overlap  collagen  explanation  of  space  or  crystal in  than  zone  then  fibril has  been  the due  dark  collagen  molecules  planes  run  are  electron  crystals is  length  the  to  (Engstrom  field  apatite  fibril to  tendon  crystal  o r i e n t a t i o n of along  i s considered  turkey  selected-area  likely  acid  (Arsenault,  three-dimensional such  that  the  structural  rotation  properties  the  because spaces.  of  the  fibrils of  collagen  localization  First,  collagen  negative  Review  sequence  and  reasons.  intrafibrillar positive  the  When a p a t i t e  Historical  amino  several  potential  in  the  fibril.  biomineralization for  gap  equal  orientation reflects  collagen  The  within  apatite  1951).  the  apatite  the  more  molecules  microscopy,  of  in  is localized  complex  collagen  Zetterstrom,  1988).  reported  described.  c-axis  parallel  greater  apatite  much m o r e  (1 9 8 9 )  becomes  If apatite  i n t e g r a t i o n of  requires  and  Arsenault  e f f e c t s on  of  play  mineral  a  may  result in  the  amino  nucleation  in  packing  regions  flexibility  specific  role  apatite  three-dimensional  regional  Second,  may  of  or  acids or  may  have  growth  of  27  apatite such  crystals.  Thirdly,  as h y d r o x y l a t i o n ,  factors  which  specific  locations  mineralization literature.  where  collagen  fibril  amino  apatite? the  and t h e r o l e  acid  D period? sequence  Are there  mineralization  acid  of type  some  precise  t h e mechanisms of e x i s t s i n the  unanswered. F o r  i n relation  to the  study  between  and t h e l o c a t i o n o f which  i n the overlap  the following  of  sequence.  of collagen  of these  be  Fourth,  process  i s the r e l a t i o n s h i p  of collagen  of apatite  at  remain  located  What  in-the  I collagen  questions  properties  to understand  collagen  regarding  i s the mineral  localization  attempt  basic  of a p a t i t e .  involved  by t h e a m i n o  uncertainty  Numerous  example,  proteins  i n t e r a c t with  determined  Considerable  the  the l o c a l i z a t i o n  non-collagenous may  modifications  c r o s s l i n k i n g a n d g l y c o s y l a t i o n may  determine  mineralization  post-translational  aspects  zone?  can  explain  I n an  of  was c a r r i e d o u t .  28  INTRODUCTION  Type numerous  I collagen connective  mineralization collagen formed al.,  molecule  portion  terminal, that  organized rise  o f bone,  1963; Ueis,  showed  tissues  i n a regular  adjacent  ends  and each  neighbor  by 67nm,  there  for  (1963)  are l o n g i t u d i n a l l y fashion  which  separation  The r e p e a t i n g  was e n v i s i o n e d overlap  region, where  molecules. would  there  between  would  lateral  residues  of the  two r e g i o n s ,  i n length,  a  and a "gap"  be p o t e n t i a l  space  between l o n g i t u d i n a l l y  Hodge and P e t r u s k a  (1963)  suggested  that  be t h e p r e f e r e n t i a l l o c a t i o n o f a p a t i t e  mineralization  growth.  D period  as having .4D  gives  are linearly  acid  the r e s u l t of the separation  crystal  Hodge and P e t r u s k a  t o 234 amino  .60 i n l e n g t h ,  during  N- a n d  corresponding  region,  crystals  triple  to i t s  et a l . , 1973).  gap r e g i o n  a  (Piez et  and s m a l l  The m o l e c u l e s i s a 35nm  chains  i s staggered  packed  arranged  have  The  i n length  molecule  densely  the  molecules  i n the  l e g tendons.  residues)  67nm, D - s t a g g e r e d  period.  that  acid  to  involved  peptide  molecules  portions.  the collagen  such  as  These  (1014 amino  nonhelical  fibril  and t u r k e y  one a 2 ( l )  1985a).  arranged  collagen  and i s d i r e c t l y  i s a s e m i f l e x i b l e r o d , 300nm  to i t s axial  (Hulmes  structural integrity  dentin  by t w o a 1 ( I ) a n d  helical C-  provides  due t o t h e s p a c e  available  29 The results  regular  i n the periodic  specific 1952). amino  D-stagger  Meek acid  e t a l . (1979) were sequence,  of type  individual  amino  coefficient  I collagen acids  density  of their  amino  Hulmes,  approach mineral  1 9 8 4 ; Meek  into  collagen  relative  present  study,  density  analyzed  and compared  have  used t h e  with  specific 1982b; An  Chapman  the impregnation  amino  acid  t o heavy  of  of apatite  composition. metal  and d i f f r a c t e d e l e c t r o n s t o models  classes  analogous  by t h e l o c a l i a t i o n  i n addition  electron  correlation  and t h e t h e o r e t i c a l  and Chapman, 1 9 8 5 ) .  t o t h e known  of  the e l e c t r o n - o p t i c a l  e t a l . , 1982a,  fibrils  and t h e s t a i n i n g  to determine the  stains  to understand  (Bear,  t h e known  A high  studies  of collagen  (Tzaphilidou  c a n be u s e d  crystals this  data  with  the l o c a l i z a t i o n  collagen  of electron-dense  acids  to integrate  between  Subsequent  also  acids  implications  the f i b r i l .  found  stained  model.  sequence  localization  and  o f amino  t o model  within  ( r = . 9 2 B ) was  of positively  acid  able  molecules  structural information  density  of  localization  s t r u c t u r a l and h i s t o c h e m i c a l  patterns  amino  of the collagen  In  s t a i n i n g , the  of apatite are  of collagen  amino  acid  sequences. The  role  mineralization suggestion reported exclusion  and i n t e r a c t i o n s o f type process  remains  an e n i g m a .  by Hodge a n d P e t r u s k a the occurrence from  (1963)  of apatite  the overlap  zone  I collagen  Corroborating  several  authors  i n t h e gap zone  (White  i n the the have  and i t s  e t a l . , 1977;  30  Berthet-Colominas initial  localization  zone  followed  zone  (Glimcher,  earliest the  1983).  zone  and t h a t  overlap 1989)  zones  . There  collagen  remain  an i n t e r m e d i a r y  tissue  proper  collagen  analyses  individual The  present  acid  the  the  proceeds  (Arsenault,  the role of  between  apatite  collagen  and  an i n t e r a c t i v e p r o c e s s  tendon  thereby,  force?  Many  instead  high  or  studies  o f bone  degree of  permitting  of the m i n e r a l i z a t i o n  collagen  was u n d e r t a k e n  localization of type  of apatite  t o be r e l a t e d  inverse  i n t h e gap and  of b i o l o g i c a l  of the tendon's  study  sequence  organization found  within  ease of  process  along  fibrils.  intrafibrillar amino  turkey  that the  (Arsenault,  concerning  i s the dominant  alignment,  the  overlap  indicates  gap zone  of mineral  association,  because  fibril  sequential  to'the  questions  the mineralized  the  crystals are localized  the association  whether  used  throughout  i n t h e mechanism  i s a passive  describe  t o be e x c l u s i v e l y i n t h e gap  as m i n e r a l i z a t i o n  many  whether  authors  information  the proportion  mineral  have  Recent  apatite  changes  Other  spread  i n addition  structure  deposition-  of mineral  by p r o g r e s s i v e  detectable  overlap  1988)  et a l . , 1979).  fibril  localization  of apatite  in relation  I collagen.  within  The  the collagen  to the polarized as determined  r e l a t i o n s h i p between  to investigate the  asymmetric D period  t o C-  was  d i r e c t i o n of  by t h e s t a i n i n g p a t t e r n .  the l o c a l i z a t i o n  of hydrophobic  to the  amino  acids  of apatite  was f o u n d  A  and  t o be  31  evident  i n both  conclusion effect, serves  of  which to  this  determined  by  controlled  influences.  gap  and  thesis  o v e r l a p zones.  therefore,  i s important to  influence  distribution  be  the  of  the  the by  the  crystals  gap other  region  i s that  collagen  distribution does  not  primary  the  hydrophobic  structure,  of a p a t i t e . appear  i n collagen  c o l l a g e n o u s or  The  to  fibrils  also The  be  solely  but  non-collagenous  could  32  MATERIALS  Tissue  Preparation  Small tendons  of  solution 0.05M then  10  of  f o r 15  sodium  of  old  1%  pH  changes each  w/v  osmium  7.4, of  followed placed 100%  water by  in a  3  2%  w/v  f o r 2h.  0.05M  followed  by  1h  was  (50%,  70%, of  s e r i e s of  r e s i n ) f o r one  sucrose  resin  i t was  (gold  i n t e r f e r e n c e c o l o r ) were  mesh  or  with  Reynolds UA  200  polymerized  mesh  i n 75%  lead  glycerol under  f o r 2h.  these  pH  portions  turkeys  60  C  were  These  of  for leg  excised  Non-mineralized  c o n d i t i o n s and  f o r 16  so  methanol  in  f o r 15  (3:1,  Thin  min  each  then 1:1,  1:3,  of  100%  sections  formvar-coated  1963)  75  stained  f o r 6 min  and  3%  min.  tendons and  of  from  placed  tendon  areas  0.05M  placing  s e c t i o n s were  6  a  overnight  h.  on  (Reynolds, 4.2  by  2%  and in  and  f r e s h change  placed  grids.  citrate  methanol  Mineralized domestic  copper  at  a  out  resin  Following  tissues with  t i s s u e s were  cacodylate  of  a  and  t i s s u e s were  acetone:Spurr's each.  in  sucrose  3x100%)  The  leg  post-fixation  carried  90%,  acetone.  hour  the  80%,  placed  The  sodium  t e t r o x i d e , 2%  Dehydration  changes  i n f i l t r a t i o n ' of  old  were  tissues in increasing concentrations  distilled  w/v  non-mineralized  turkeys  g l u t a r a l d e h y de,  min  cacodylate.  excised,  domestic  cacodylate, 3  METHODS  Visualization  freshly  v/v  rinsed with  solution  of  week  2.3%  sodium  sucrose  the  pieces  and  AND  10 in  becomes interest  to  14  week  100% transparent near  the  33  mineralization mineralized  front,  regions  glycerinated  unmineralized  c a n be  tendon  were  tendon  identified.  placed  ( 3 : 1 , 1:1,  100%  3 changes of acetone  tissues  were  then  Sections tendons  with  depending  on  were  collagen  banding  selected-area  were  dark  water.  75 m e s h  electron  collagen  on  these  the  uranyl  molecular  their  banding  o r 600 field  min  analysis on  stained  with  were  1%  o r i e n t a t i o n of collagen and  to maintain  to  In order fibrils the  bright  were  and  f o r 10 m i n .  1988).  and  aqueous  f o r 15 m i n  found  on  visualize  produce the conventional they  on  grids.  sections to  and  color  microscopy  In order  of  distilled  placed  copper  of mineralized  (Arsenault,  patterns  t o be e m p l o y e d .  quickly  4.2  the  localization  electron  3.2  The  thicknesses  color  hexagonal  were  each.  along  interference  were  ( U A ) a t pH  I collagen  l e g tendons  gray  ( P T A ) a t pH  acetate  of type  3 changes of  microscopic  of mineral  sections  sections  acid  technique  sections.  s t a i n i n g methods  pattern turkey  dark  unstained  banding,  by  of  r e s i n as above.  interference  were  microscopy  phosphotungstic aqueous  field  These  Selected-area  performed  gold  of  series  f o r 15  in different  for electron  for analysis  formvar-coated  field  knife  stained  Sections  followed  i n Spurr's  the v i s u a l i z a t i o n  t o be  distilled  1:3)  heavily  pieces  cut i n l o n g i t u d i n a l planes  a diamond  Sections  water.  embedded  Small  i n a graded  glycerol:methanol m e t h a n o l and  and  1%  Although  banding demineralize to by  native  visualize analysis  of  34  distribution sections Bright using The  the  field  stained  300 e l e c t r o n  o f SADF  beam  microscope  positioned  from  This  selected  In this  collect  planes  electrons  of apatite  technique  the objective  specific  study  a t 80 k V .  (Jackson uses  aperture.  tilting  correspond  of  are  With the beam  c a n be c o l l e c t e d  diffraction  pattern  aperture  d i f f r a c t e d from  et  mode t h e t i l t e d  electrons  the objective  which  performed  diffracted electrons  the c h a r a c t e r i s t i c electron  apatite.  f o r 5 min.  operating  elsewhere  i n the d i f f r a c t i o n  so t h a t  mineralized  a n d SADF w e r e  microscope  1988).  through  and  i n 100% methanol  i s described  so t h a t  to pass  electron  1 % UA  microscopy  1978; A r s e n a u l t , main  non-mineralized  with  electron  a Philips  allowed  to  were  technique  al.,  is  of apatite,  was  of  positioned  the c - a x i a l  lattice  to the (002) d  lattice  spacing.  Computer The  Modelling system  of modelling  fibrils  used  by  et a l . (1979).  Meek  (Highberger chick  here  each  collagen  i s an a d a p t a t i o n The amino  acid  were  translated  as a s i n g l e  acid  sequences  were  turkey  i s unavailable  either  turkey  or chick  into  string used  data  into  because  at this  sequence  i n collagen  of the system  e t a l . , 1982) a n d a 2 ( l )  collagen  entered  amino  time.  sequences  (Boedtker single a text amino  developed for a l ( l )  e t a l . , 1985)  letter  codes and  editor. acid  Chick  data f o r  The r e s u l t s when  are expected  to d i f f e r  using  only  35  minimally homology single amino  because there between  positions to  by  fifty  regions  acids  1984).  stacked  to simulate  four  were  i n width.  deviate  then  identified.  amino  pattern  of collagen  abbreviations  spaces. and  then  size  D,E,R,K  of electron  determined  reduced  of collagen by f o u r  from  classes  p. i v ) were acids  were  i m a g e s . The  stained  with  a l t e r n a t i v e methods  amino  acids  (K,R a n d H ) ; 3) w i t h collagen;  et a l . (1982b).  their  o f amino the  i n their  replaced  by  by c o m p u t e r  to the equivalent molecular  of the d i s t r i b u t i o n  charged  Tzaphilidou  and  of comparison:  (D a n d E ) ; 2 ) w i t h  stained  because  a l c o h o l i c UA w a s  acids  PTA/UA  left  was r e d u c e d  amino  aqueous  i n length  to represent  charged  of  repeating  linearity  photographically  a t h e o r e t i c a l model  assumed  and H ( s e e t a b l e of  amino  microscopic  were  f o r the positive staining  The r e s u l t a n t d i s t r i b u t i o n further  relative  D units  c a n be e x p e c t e d  In order  acids,  a l l other  The  The  i n the l o c a l i z a t i o n  Functional  responsible  f o r amino  while  orientation  with  acids  error  the greatest  a r e n o t known.  charged  positions  Some  i n the telopeptides  conformations  chains  and Hulmes,  molecules  these  2(l)  a  These  D u n i t s , 234  i n width.  (Chapman  then  acids  repeating  and 5 m o l e c u l e s a  amino  et a l . , 1985).  into  o f t h e two 1 ( I ) and t h e  were  degree of sequence  (Boedtker  organized  i n length  be a 1 - a 2 - a 1  units  species  s t r i n g s were acids  i s a large  1)  of negatively  a model  of  a scaled  and 4) w i t h  positively micrograph  data  The p e r i o d i c d i s t r i b u t i o n  from of the  36  hydrophobic and  amino  I , has been  structure acids  acids  considered  (Hofmann  were  having  long  t o be  e t a l . , 1978)  selected  and  then  side-chains,  important  reduced  to the scale  collagen  fibrils  for direct  The  contrast  of t h i s  image  hydrophobic groups  as w h i t e .  considered helix  groups  t o be  stability  as b l a c k  and  Hydroxyproline important were  also  represents  areas and  high  amino of  comparisons. areas  which  flexibility  in this  \J, L  low i n  i n hydrophobic  praline,  i n molecular selected  visual  M,  fibril  These hydrophobic  mineralized reverse  in  F,  fashion.  are and  37 RESULTS  Electron  microscopic  glutaraldehyde tetroxide  this  an e f f i c i e n t  a r e seen  cell The  system  Tenocytes  extracellular  because  t o have  the section  Intracellular  Rough  f o r studying  These  extend  features of these  i n a parallel fibrils  microns.  Their  70nm  alternating  bodies  axial light  separated  of c e l l s  a r e about  the c e l l  fibrils.  i n width. vary  and i t s p r o c e s s e s . secretory  and m i c r o t u b u l e s a r e  matrix,  appear  collagen f i b r i l s the c e l l s .  t o vary  from  are  Individual f o r many  approximately  low m a g n i f i c a t i o n t h e  of these  and dark  through  i n the tenocytes  c a n be f o l l o w e d l o n g i t u d i n a l l y  repeat  diameters  and  60nm  by  elongated  to collagen  f a s h i o n between  diameters  makes  cells.  t o 300nm. A t a r e l a t i v e l y  pronounced  columns  of cross-sectional  structures,  the extracellular  collagen  (fibroblast-like  reticulum, mitochondria,  prominent  clearly  are generally  parallel  and between  filamentous  aligned  cells  processes  vacuoles,  osmium  the mineralization  into  organelles visualized  endoplasmic  with  ( F i g u r e s 2a and b) w h i c h  i s through  which  to cell  Within  post-fixed  and l e a d c i t r a t e ,  a variety  smallest of these  cell  UA  are organized  matrix.  processes  from  tendon,  of non-mineralized,  the organization of tenocytes  and c o l l a g e n f i b r i l s  process.  and  turkey  and s t a i n e d w i t h  illustrates cells)  fixed  examination  fibrils  regions  which  consists  of  a r e due t o t h e  38  Figure  2a a n d b. E l e c t r o n  tendon.  Parallel  processes (F).  columns o f t e n o c y t e s (T) and  endoplasmic  reticulum  and  microtubules (arrow)  are  commonly  observed  extend  analysis tetroxide  of non-mineralized  ( C P ) a r e s e p a r a t e d by p a r a l l e l  Rough  fibrils  micrographs  along  longitudinally  fixed,  b=2DD; a X 2 5 . 0 0 0 ;  cells.  X50,000.  citrate  fibrils  m i t o c h o n d r i a (ffl), organelles  The p a r a l l e l  f o r many  fibrils.  UA a n d l e a d b  collagen  are intracellular  i n these  individual  (rER),  their  microns  which  collagen  permitting  Glutaraldehyde/osmium stained.  Bars  a=400nm,  40  specific  staining  molecules  within  magnification composed been  1960).  an  Each  intensity, The  fibrils which  i n Figure  a^»  and  not  t o be  are  very  a^)  single  (e^  62)  and  molecular the  band  a^  c^  with  well  defined  lines  other  distance separating  borders;  tend  (b^ and have  a  b^) well  apparent  i s an the "e"  separated  a r e due  the l i g h t  s t u d i e s have  which  bands  f r o m ea'ch  a narrow l u c e n t space.  stained regions  d are within  PTA/UA  " c " bands; f o r  "d" band  narrowly by  used;  i s readily  the s i n g l e  are separated  c and  they  and  are f a i n t ;  these  morphological  bands  " a " and  only  bands.  " a " bands ( a ^ ,  the adjacent c^)  of  spaced bands  separated,  have  staining  aqueous The  The  also  between  level  follows:  and  3 the densely  b, w h i l e  to each band.  be  Schmitt,  to other  stained with  "b"  well  and  methods  d e l i n e a t e d ; t h e two  are d i s t i n c t  and  been  a r e as  ( c ^ , C2>  intense  Figure  have  to  which  structure,  the s t a i n i n g  are narrow, c l o s e l y  the others  other,  i s shown  i t srelative  therefore the f i n e  bands  distinct border  by  collagen  higher  repeat  relationships  definition  clearly  " c " bands  and  spatial  3a  clear  a^,  while  At a  of narrow bands  identified  d e p e n d i n g upon  of these  the  and  and  details  defined  c a n be  width  gives  each a x i a l  ordered  " a , b , c , d , e " n o m e n c l a t u r e (Hodge  band  varies  structure.  asymmetric series  intensity,  bands  the f i b r i l  ( F i g u r e 3a)  o f an  given  p r o p e r t i e s of the highly  shown  t o bands  r e g i o n . At that  t h e gap  e,  In a  the zone,  -  41  Figure  3 a . The p e r i o d i c  fibrils. to  The D r e p e a t i n g  the recognizable  intensity c,  staining pattern  and t h e i r  d , a n d e" p e r m i t  unit,  series  defined  the determination  displayed  image  display  characteristic resulting the  bands  N- t o C-  displayed collagen both PTA  of p o s i t i v e l y  image  a n d UA  alignments  the s p a t i a l of these  a r e l a b e l l e d from  d i r e c t i o n i s from  image  images  depicts  from  Figure  of Figure  fibril.  and n e g a t i v e l y  i s s h o w n i n t h e same m o l e c u l a r  This  charged  distribution  charged  amino  amino Figure  and acids; the  "e t o a " i n d i c a t i n g  left  to right.  3b i s s c a l e d  3a ( i n s e t ) .  Bar=10Qnm;  " a , b,  3b. A computer  o r i e n t a t i o n as  X13D,QQ0.  that  A computer  and a l i g n e d  The b a n d i n g  l i ei n r e g i s t r a t i o n . Glycerol stained;  their  o f t h e N- t o C-  o r i e n t a t i o n of the collagen  3a.  by  according  r e l a t i o n s h i p s . The b a n d s  molecular  acids  collagen  ( D ) c a n be l a b e l l e d  of bands  spatial  of  to the  patterns  treated,  aqueous  of  42  3b  43  longitudinally a., _ . , e„ of  C2  ^» The  . , d and  b2  ^ and  banding  distribution therefore, known  align  and  acid  c, while  a^  (Hodge  collagen  permit  unambiguous  The  t o C-  with  asymmetric  pattern found  of bands that  scaled 3a  a"  and  fibrils  or of opposite  derived and  orientation. orientation of  insert).  i n turkey  o r i e n t a t i o n and  orientation within  to the a n a l y s i s  and  image  presentation 3b  of  and  of the bands  molecular  the  acids  pattern  (Figure  of collagen  acids;  from  amino  to the stained  (Figure  adjacent  of molecular  i s critical  c, b,  then  the  In our  banding  was  intensity  determination  of amino  charged  fibril  correspond  t h e " e , d,  o f t h e same  determination  3b)  d i r e c t i o n of collagen  correlates  i t was  and  represents  I collagen.  model  collagen  registration  computer modelling  fibril  This  consists  constructed  t h e " a t o e"  fibrils.  The  be  (Figure  to give  groups  c a n be  of  1963).  I collagen  sequence of type model  consists  zone  Petruska,  of charged  inset).  may  and  density  to the stained  study  the overlap  of type  aligned  by  molecules  pattern  generated  vertically  native  collagen  t h e o r e t i c a l models  amino  computer  arranged  of a p a t i t e  the  this In  this  tendon thus,  the  collagen  localization.  44  Figure  4. Low m a g n i f i c a t i o n o f g l y c e r o l  distal  to the mineralization front.  appear  white  translucent. (Min).  the  dense,  least  regions  stages  i n unstained  mineral  density  of c a l c i f i c a t i o n  (arrow)  proportion of mineralized matrix  more  c  are evident  regions  increases  within the collagen  of non-mineralized  heavily mineralized region  X5Q,0Q0.  localized,  electron-lucent, non-mineralized  (arrowheads)  subsequent  m i n e r a l i z a t i o n was stages  calcification.  electron within  matrix.  5b.  and p e r i o d i c  fibrils  matrix  but w i l l  Bars=200nm;  near  s e c t i o n s . 5a I n  small  The  Small  proximal  of m i n e r a l i z a t i o n present  otherwise  5c.  to  a n a l y s i s of early  the  banding  are  d e p o s i t i o n . Bar=0.5mm; X 2 5 .  5. D i f f e r i n g  with  distal  just  regions  regions  of visible  f o r electron microscopic  mineralization front  regions  precedes  a t the boundary  apatite crystal  Figure  The c a l c i f i e d  the non-mineralized  Mineralization  Tissue  selected of  while  t r e a t e d tendon  i s present.  (*) r e m a i n  be f i l l e d  a X45,000, b  in a  i n by X40,QQQ,  47  Treatment with  of mineralized  g l y c e r o l makes  regions  collagen  easily recognizable  insertion  of the tendons  mineralized. decreases  The d e g r e e  away  from  domestic  translucent (Figure  into  which  was  for electron  microscope the of  electron  dense  the  matrix  which  do n o t h a v e  which  pattern  which  mineral  At sections  heavily  remains.  progressive  occurs  D period  within  exibits  with  nature  there  In t h i s  of mineral,  magnification  at this  seemingly  unstained,  at the mineralization  of  are  study  front  regions  I am  pattern  i s most l i k e l y  (Figure  of  the  e x t r a c e l l u l a r matrix.  feature  gradient  5b). In addition  a periodic  deposition  at  a periodicity  (Figure  pattern.  mineralized  The main  spread  higher  of the  regions  5 a ) . As t h e d e g r e e  has a p e r i o d i c  the non-collagenous a r e more  analysis  localized  are evident  banding  a banding  mineral  non-periodic  within  region  i t an a p p a r e n t  small,  (Figure  to the collagen  considering  of the  to  longitudinal section  has w i t h i n  Initially  increases  the mineral  which  i t disappears  microscopic  unstained,  calcification  electron-lucent  to  front  spread.  equivalent  progressively  i s representative  an i n d i v i d u a l ,  calcification  are heavily  f r o n t . When v i s u a l i z e d i n t h e e l e c t r o n  mineralization mineral  mineralized  to the  the tendons  the i n s e r t i o n u n t i l this  mineralization  and t h e  of m i n e r a l i z a t i o n  observation;  l e g tendons  4 ) . Near  bone,  gross  prepared  turkey  only  because occurring  In  regions  5c) the banding stage  i s the  i n a l l dimensions. longitudinal  of turkey  l e g tendons  48  (Figure  6a)  electron  reveal  density  a distinct  of a p a t i t e  non-mineralized  regions  demonstrate  structural  any  mineralization, zone; the  however,  overlap  zone  has  random  most  zone.  on  More  The  side,  the  lucent  area.  occurs  i n the mid-portion.  in  contrast  marked.  detail.  Within  similar between  by  occurs  zone  present that  neither  appears  to  be  zone  gap  gap  is  the  the  i n the  electron-lucent  In r e g i o n s  early of  are  observed  and  overlap  the  early  which  asymmetrically  features  of  observes  i s a single overlap  to  not  i s also  density  an  do  In regions  one  due  contrast,  section  material  gap  pattern  In  density  of e l e c t r o n  either zone  same  specifically,  within  deposition  gap  the  electron  homogeneous.  delineated,  crystals.  electron-dense  a pattern  nor  of  repeating  line  in each  and  positioned  mineralization  increased but zones  the  mineral difference  i s less  49  Figure  6.  The  bright  field  localization image  mineralized  turkey  pattern  to  due  compared  to  a  less  (OZ).  The  zones  demarcated  gap  zones  space 6b  and  c.  unstained of  has by  a  bordered arrows).  Two  collagen  the  mineral  content  i n gap  an  i n the  lucent  space  (white  both  sides  to  is identified and  by  Non-mineralized  as  white  overlap  the  lengths)  bright field  (GZ)  overlap  zones  within  Also,  narrow  lucent  of  of  which  X120,000.  the  matrix  (*).  mineralized  specific  at  gap This  fibril.  localization  appear  in  the  micrographs.  h i g h l i g h t s w i t h i n both (OZ)  as  arrowheads).  portion  electron  zones  m i n e r a l i z a t i o n . Bars=100nm;  a  images  showing  (c-axial  Apatite  (GZ)  on  given  of  banding  zones  distribution  A  section  inherent  interspaced  asymmetric  fibrils  contrast  of  longitudinal  showing  r e p r e s e n t a t i v e SADF  apatite crystals  zones  unstained,  tendon  mineral  reverse  gap  an  a p a t i t e i n c o l l a g e n . 6a.  i s consistent within a  are  (white  leg  high  mineral  distribution  of  of  the  earliest  the stages  50  51  SADF apatite 6b);  these  pattern  c-axially  of mineral  electron  both  and d i r e c t l y  gap and o v e r l a p  field  images.  deposition b y SADF  order  within  t h e gap and  the pattern  to relate  of apatite  the stained  Certain  technical difficulties  because  i t i s known t h a t  demineralize adjacent  sections.  to mineral as regions  structure  distribution alcoholic  computer  deposition  with  this  was  patterns model  areas must  UA  fibrils  found  be  regions i n their  t o enhance t h e  were  PTA/UA  and a l i g n e d  to a  region  of a collagen  a different s t a i n s . So  of  charged  representative  fibril  fibril  with  o r i e n t a t i o n of  a t h e o r e t i c a l model  scaled  native  stained  s t a i n i n g produced  the molecular  of the stained  totally  and t o r e t a i n t h e sections  of  overcome  that  t o t h e aqueous  stain,  pattern  o n t h e same  a r e not as c l e a r  crystals,  determine  i t i s  banding  more d i s t a n t . T h e r e f o r e ,  as compared  non-mineralized banding  ,field  s t a i n i n g procedures  i t was  T h e a l c o h o l i c UA  pattern  acids  Also,  of apatite  UA.  mineralized  aqueous  of collagen  unambiguously  amino  with  collagen  fibril.  collagen  the  orientation of  deposition  areas  to  that  overlap  a s by b r i g h t  the molecular  non-mineralized  banding  reverse  I t i s apparent  i s t h e same  to correlate  with  necessary  fine  in  (Figure  microscopy.  collagen  banding  visualizes  zones  oriented' c r y s t a l s appear  to the bright  demonstrated  In  identifies  crystals within  contrast  zones  specifically  (Figure  and t h e  are i n r e g i s t r a t i o n , thereby  7a).. The  scaled  enabling  the  52  determination  o f IM- t o C-  directed  left  with  a l c o h o l i c UA  PTA/UA bands The  from  stained within  d band  However,  pattern  also  toward  banding  pattern  of apatite  asymmetric  Figures  distribution  C-  portion  adjacent  fibrils  (Figure  i n these  determined  by t h e i r  spaces  (Figure 7b).  the  zones  mineralized  by a  i s consistent molecules  terminus.  was  t h e gap zone t h e  i s created  space  lucent with  also sides  orientations have  the  a n d when  I t was o b s e r v e d  molecular  on o p p o s i t e  PTA/UA.  7 b ) . The  f o r unstained  staining pattern  positioned  as w i t h  gap and o v e r l a p  of mineral  opposite  relative  of the f i b r i l s  7b a n d 5 a ) . W i t h i n  to the  having  period.  by t h e c h a r a c t e r i s t i c  o r i e n t a t i o n of the collagen i s nearest  pair of  of the D  their  stained  i s replaced  The p o s i t i o n i n g o f t h i s  apparent  lucent  maintained  t h e same a s w a s o b s e r v e d  (compare  to the  i t s characteristicintensity.  distribution  of apatite  observed  of a d i s t i n c t region  not as i n t e n s e l y  pattern  bands  s i m i l a r i n appearance  the mineralized  stained  precisely  and  and c o n s i s t e d  retained  Advancing  to  The  t h e a , e, a n d c b a n d s  distribution  o r i e n t a t i o n which i s  t h e more e l e c t r o n - d e n s e  b u t were  space.  to right.  s t a i n i n g were  positions  section  molecular  that as  consistent  o f t h e gap  zone  53  Figure  7. A n a l y s i s  turkey  tendon.  7a. A non-mineralized  fibril  stained  with  insert) acids  displaying  has been  molecular  the s p a t i a l  scaled  stained  opposite  banding  mineralized apatite border  opposite  with  shows  of the unstained t h e gap zone  t h e same p e r i o d i c sections.  and w i t h i n  (8a) and u n s t a i n e d  characteristic conditions sections  pattern  are stained  that  with  UA  reflections  are readily  reflection,  and t h e combined  reflection.  by  their  areas.  The  distribution  of  spaces  t h e gap zone  lucent  spaces  consistent  mineralized  i n 100%  stained  tendon  i s observed  apatite  with  X120,000.  o f a l c o h o l i c UA  of apatite  demonstrating  adjacent  lucent  asymmetrically  diffraction  7b.  Electron  o r i e n t a t i o n . Bars=100nm;  mineralized  The  i n the non-mineralized  the  8. E l e c t r o n  t o C-  i s not  as i n d i c a t e d  are positioned  Figure  t h e N-  amino  o f t h e same  a l c o h o l i c UA.  (arrowheads) op.posite  of charged  b and d bands.  portions  orientations  patterns  portion  image (7a  to determine  by t h e p r o m i n e n t  collagen  have  A computer  collagen  s t a i n i n g but the o r i e n t a t i o n i s  and m i n e r a l i z e d  fibrils  of a  staining pattern  Non-mineralized fibril  portion  distribution  and a l i g n e d  t o t h e PTA/UA  determined  orientation i n mineralized  a l c o h o l i c UA.  o r i e n t a t i o n . This  equivalent readily  of collagen  under  i s retained methanol.  i n d e n t i f i e d : the inner  (8b).  Two  The  both  when prominent  (002)  ( 2 1 1 , 112, 300, 202, 301)  54  55  Electron mineralized  diffraction  areas  of  a l c o h o l i c UA  the  c h a r a c t e r i s t i c maxima  and  thereby After  confirming  influence the  best  was  the  reverse  collagen  fibrils  spatial  these  side  as:  of  amino  acids  the  contrast  that  densities as  white  of  hydrophobic the  positioned  gap in  terminus  of  collagen  fibril  hydrophobic intermediate  zone;  the  the  9a).  gap  are  in  zone  molecular containing  amino  acids  regions  direction  a^-a^  third  and  lies  between  groups. of  in  model  include  l i e within  from  with  the  which  c^-c^,  and  high  contrast  and  spatial  the  fibril  are  image.  border  Two  each  asymmetrically  a^  most  having  normal  e^  nearer  o r i e n t a t i o n . Regions low  These  their  collagen  acids  reverse  amino  negative),  the  regions  amino  of  by  various  observed  three  the  regions, the  these,  I t was  b).  analyzed  because  displayed  hydrophobic  areas  Of  crystals  the  hydrophobic  mineralized  acids  there  further  selected  unstained  (Figure  of  showed  and  ( p o s i t i v e and/or  structure.  amino  nature  8a  selecting for  and  were  C-  was  models  charged  collagen  model  to the  aromatic,  hydrophobic  displayed of  such  on  N-  distribution  computer  f i t to  computer  apatite  molecular  to  of  biological  dense  sections  the  non-charged,  classes  stained  determining  groups  polar  these  (Figure  apatite-collagen  acid  for  from  i t s presence  mineralized  comparison  analysis  of  of  densities the  overlap  gap zone  the the  of zone; from  c  1  to  56  a^,  and  within  the  gap  computer-generated with 9b  micrographs  and  that  inset).  apatite  spatial  the  gap  overlap and  D  gap  (1.93) the  zone  percentage  of  are: the  1*1 = 1 1 % ,  organic (see  composition  by  high  composition  of  the  collagen  hydrophobic, repeating  there are  of  129  and  100  as  o v e r l a p zone  amino  4%.  energy  of  the  the  differences  these  two  zones  140)  overall  relate  the  amino  varies zone  the  V=27%, percentages  figures  water  of  to  for an  acid hydrophobicity  differences  i n amino about  acid 0.1%.  hydrophobicity throughout  differences  in composition.  Using from  ratios  gap  the  in  twice  acids  h y d r o p h o b i c i t y by  i n average to  1= 14%.  transfer of  The  a r e : 1*1 = 9%,  o v e r l a p zone  acids  about  acids. amino  acids  amino  W i t h i n the  acids  F = 16%,  p.  amino  the  i n width  amino  has  of  shows  than  5 molecules  hydrophobic  most  a measure  1980,  average,  hydrophobic  at  D period  hydrophobic  hydrophobic  by  on  unit,  L = 34%,  Thus,  differences  occupied  acid  free  vary  areas  shown  amino  V = 25%,  Tanford,  i t i s clearly  hydrophobic  zones  solvent  (Figure  the  1=17%. W i t h i n t h e  additional  collagen  residues.  hydrophobic  L=30%, F=17%,  correlate  acid  constituent  two  images  and  amino  density  the  These  repeats align  i n those  T h e r e f o r e , the  individual  c^*  hydrophobic  I n one  zone.  to  of  o v e r l a p zone  the  between  dense  i s less  zone.  the  set of  o v e r l a p zones  in length,  within the  and  e^  unstained mineralized  this  densities  gap  that  of  In  of  from  hydrophobic  i s less  Analysis  zone  in density  and  not  to  57  The the  proportions  D u n i t were  each  zone;  zone.  This  101  o f gap and o v e r l a p  c a l c u l a t e d by c o u n t i n g f o r the overlap  results  i n the predicted  occupied  by t h e o v e r l a p  be  assuming  .568D  are  equivalent.  the  density  for  length  that  These  o f amino of each  triple  residues  acids  density  of these  the  gap zone  gap  zone  have  regions  .432D  acids for  of the D  unit  and t h e gap zone  occupied then  1 0 ) . The  by a m i n o  be u s e d and t o  density  of s t a b i l i t y  zone  there  residues  (1.13:1).  may  proportion  to  acids  to predict  standardize  compared  regions,  localized these  molecular  and p r o l i n e t o 335 w i t h i n t h e  i s a greater  i n the overlap  Two  of  of the  a r e 359 h y d r o x y p r o l i n e  Therefore,  very  to  and 133 f o r t h e gap  per molecule  within the overlap zone.  unit.  figures could  (Figure  There  overlap  These  the lengths  are a reflection  helix.  t o be  t h e amino  and p r o l i n e a r e p e r i o d i c a l l y  the D period  residues  zone  relative  zone.  Hydroxyproline along  zone  zones  proportional  zone  as compared  one a t e a c h  few p r o l i n e o r h y d r o x y p r o l i n e represent  more  flexible  to  end o f t h e residues.  portions  of the D  58  Figure amino  9.  A comparison  acids  and m i n e r a l  hydrophobic reverse left  amino  acid  high  as  white.  as b l a c k  shown  with  image  of hydrophobic  an a l i g n e d  amino  mineralized  and s c a l e d amino  reverse  acids  hydrophobic mineral  mineral amino  density,  hydrophobicity. Figure  structure  have  corresponds  acids; while correspond  Bar=1QQnm;  These  amino  of collagen  lucent  The  acids  may  residues  accomodate  computer  asymmetrical  with  low i n less  high i n  confer This  Regions  in  image  with  to the  indicates that  residues  of collagen  are considered  to changes  hydroxyproline  stability  to these  i m p l i c a t i o n s on t h e a d a p t a t i o n  hydroxyproline  areas,  i s  registration  The  o f p r o l i n e and  molecules.  growth.  fibril  image  X12Q,0Q0.  i s a periodic localization  apatite crystal  contrast  to regions  to regions  10. A computer image  distribution.  there  (density)  depicted  contrast  (inset).  i s from  fibril  acids  collagen  d e n s i t i e s o f t h e two i m a g e s c o r r e s p o n d .  localized  and  of the c o l l a g e n  of  and  orientation  and i n t h e r e v e r s e  9 b . An u n s t a i n e d  hydrophobic  i n normal  molecular  d e n s i t i e s of hydrophobic  t h e t o p image  to  t o C-  of  9 a . A computer image  groups displayed  Periodic regions  in  and  apatite.  contrast; the  to right.  contain  of the d i s t r i b u t i o n  fewer  may  molecules  p r o l i n e and  t o be m o r e  conformation.  which  flexible  59  60  DISCUSSION  The  ordered  collagen  has  distribution  been  the  interactive  the  biomechanical  distribution collagen how  limited.  of  has  of  long  date,  focussed  may  potential  within  the  gap  from  the  overlap  mineralization comes  from  (Hodge  apatite zones  during  process.  electron  diffraction  s t u d i e s (White  specific  and  imaging  has  overlap  zones  mineralization recent  findings  localization sequence  of  direct  shown at  to  1966), et  to  earliest  a  the  present  the  molecular  collagen;  and  of  This nature  of  knowledge  of  collagen i s  result  very  of  P e t r u s k a , 1963;  Glimcher,  b e l i e v e d to early  stages  support  neutron  be  excluded  of  the  this  viewpoint  1959),  electron  diffraction  a l . , 1977).  and  A recent apatite  by  present  i n both  gap  As  an  orientation  consequently  on  SADF and  of  e x t e n s i o n of  compares  x-ray  study  of  detectable stages  study  be D-stagger  (Glimcher,  be  to the  to  ( A r s e n a u l t , 1988). the  our  I  producing  the  presumed  visualization  crystals the  with  been  microscopic  (Engstrom,  the  as  Evidence  diffraction  time  space  and  has  because  tissues.  reflect  this  i n type  mineral/collagen relationships  the  zone  study  collagen in  to  interact  t h e o r i e s of  crystals  skeletal  at  on  Therefore,  and  viewed  However,  collagen molecules  1983).  mineral  been  apatite  s u b j e c t of  p r o p e r t i e s of  crystals  To  available  important  role  structure.  apatite  have  an  of  these  apatite and  offers  amino a  acid  hypothesis  61  of  collagen  both  bright  early  influence  on  field  selected-area  stages  of  characterized apatite the  of  by  had  gap  zone  of  either  side  fibril.  of  and  midportion  properties  of  Regions  gap  the of  confinement  to  involved  the  result has  a  of  in  of  these  of  apatite  D  in  period  which  position  acids  as  which  apatite gap  to  depicted have  the  regions  evidence  of for  crystals in zones.  mechanism  of  Thus,  exclusion  from  implications  on  the  gap  zone the  crystals within  the  periodic  both  zones.  least  mineral density  computer  less  of  modelling.  mineralization  hydrophobicity.  a more collagen  complicated than  hydrophobicity  these  on  the  greatest  collagen  most  d i r e c t i o n of  periodic by  of  that  have  the  along  zone.  observations  within  provide  the  terminal  of  fibril.  regions  within  fibril  the  apatite  number  from  p o s i t i o n to  of  the  regions  consisted  discrete area  that  distribution  overlap  collagen  regions  distribution  an  the  reflects  Conversely,  findings  of  by  are  collagen  mineral  were  and  the  likely  amino  These  of  imaging  specific  most  hydrophobic  in  zone nearer  zones  correlate  correlate  density  distribution  overlap  density  and  concluded  heterogeneous  to  observed  field  periodic  o r i e n t a t i o n of density  positioned  I t was  and  greater  the  dark  corresponds  the  I t was  mineralization  asymmetric  least mineral  invariably  gap  an  molecular  which  the  Areas  C-  formation.  intrafibrillar  c r y s t a l s which to  Areas  and  bone  simple may  mineralization domains.  This  the i n t e r r e l a t i o n s h i p  be as  a  work  62  between  the  mechanism  architecture molecular  of  are  high  and  correlate  or  in  prevent  density  be  determined  hydrophobic  groups. likely  Local to  apatite.  be  molecules  in  various the  the  Various  1985b),y and  associated  with  the  amino  of  site  such  or  macrornolecules  et  either  nucleation  of  and  the  phosphate  hydrophobic  these of  may  and  calcium  of  ions  are  biological  fibril  may  be  non-collagenous  acid  as  on  (Scott  association  such  carboxyglutamic  less  influence  proteoglycans of  mineral  with  positive control  sites  (Hauschka  formation  are  diffusion  collagen of  a^-e^)  i s o l a t e d from  nucleation  as  We  of  association  the  and  regions  acids.  gradients the  c^-c^  e f f e c t may  the  fibril  Conversly,  in  including  negative  specific  osteocalcin  the  by  crystal  area.  occur  localization  have  and  collagen  (a^-a^>  environment  close  for  the  initial  ions  properties  have  the  example,  Macromolecules,  collagen.  1979)  For  by  defined  inhibit  to  the  specific  critical  which  1985)  into  concentration  Also,  instrumental  growth.  a  affected  may  hydrophobic  First,  by  of  which  expected  the  as  along  acids  hydrophobic  areas.  localization  (Veis,  of  processes.  amino  growth  be  that  two  be  localized  areas  can  of  Haigh,  to  mineralization  collagen.  regions  crystal  speculating  will  of  collagen  distribution  hydrophobic  distribution spatial  crystal  ordering  Specific  of  crystal and  with  phosphoproteins  (Glimcher  a l . , 1975)  collagen-rich mineralizing  et a l . ,  have  tissues.  been  63  Secondly, by  space  apatite crystal and  energy  hydrophobic require  a  mineral.  groups net  The  these or  two  free  during  result  being  regions  state achieved groups  and  i s mutually  neither  potential  with  processes  Although  there  hydrophobic  not  does  some D  periods  visualized  within  the  zone.  gap  In  which  hypothesis. the  of  other  Several computer  regions.  Rather  class  amino  acids  must  of  to  crystal  are  not  result  of  the  medium  also  one  6).  (Figure  9)  of  show  i s depicted  linear more as  1980);  mineral  along  a^-e^  these  line  "Why  in  is  the  the and  the  mineral  proposed are  possible.  that  homogeneous w i t h i n the perfectly  of  In a d d i t i o n ,  examine  explanations  favorable  these  defined  this  of  influences.  question  to  Both  (Tanford,  the  more  must  the  points  exibit  may  interaction  exclusion  the  of  conditions  addition,  (Figure  ask  growth.  concurrent  be  will  hydrophobicity  minimizing  exceptions  images  influenced  distribution  apatite in collagen  potential  a  the  of  a  exclude  words,  i s not than  as  aqueous  do  be  environmental  s e c t i o n s not  represent  composition  of  One  mineralized  consistent?"  acid  areas  i s present  distribution  unstained,  by  there  where  First,  affect  local  seem  regions  may  dissociation  e x c l u s i v e . In  do  mineral  The  optimal  the  regions  crystals  that  whether  hydrophobic  periodic  will  of  growth  m i n e r a l i z a t i o n process  c o n s i d e r a t i o n s , occur  energy  from  the  which  processes,  energy  and  requirements.  energy  compartmentalize  size  the  amino  hydrophobic  arrangement, clumps.  This  this  64  concept  i s compatible  hydrophobicity aggregate.  with  i n that  Therefore,  thermodynamic  the hydrophobic i t may  regions  amino  a c i d s . There  acids  ( i na three-dimensional crystal  regions.  may  formation  Second,  amino  i n the density  be s u f f i c i e n t  tend  of  hydrophobic  arrangement)  of these  the three-dimensional  packing  arrangement  and d i s c o n t i n u o u s  et  a l . , 1985).  (1963) model  to hold  collagen  molecules  approximately a  70nm  thick  forming means  true  70nm  f o r the relationship  visualized thick.  s e c t i o n may  outside  be o f f s e t  of the general  thicker  the section,  regions  w i t h i n t h e gap zone  because  the space  regions) thickness space tilted  of  crystals  of the section.  a t 0°would the space  greater. longer  between  ( i nthis  pass would  At a c e r t a i n  permit  regions  to result  molecules  distortion.  there  Third, the  relative  This i s  hydrophobic to the  electrons entering the  but i f the specimen smaller  were  as t h e angle  of incidence  the space  became would  o f e l e c t r o n s . The p r e d i c t e d based  i s no  the electron lucent  the proposed  Incident  of  i n crystals  n o t be v i s u a l i z e d .  are small  angle  the passage  the hydrophobic  case  appear  n o t be  between a l l  sufficient  likely  will  through  could  p a t t e r n . However,  of this  t h e more  (Hulmes  i n a s e c t i o n which i s  Therefore,  to p r e d i c t the degree  permit  hydrophobic  c o l l a g e n i s somewhat d i s t o r t e d  expected  amino  to  of  The H o d g e - P e t r u s k a  to  to describe  non-hydrophobic  packing  w i t h i n each  of  acids  be more a c c u r a t e  these  some  as d i f f e r i n g  concepts  on t h e c o m p u t e r  no  size  models  65  (a^-a2=.12D a^-e2=.07D  o r about or about  7.8nm;  (70nm) g i v e s  the  would  mineral  within  specimen region  incident spaces, angle  a critical  no l o n g e r  positioned  t h e gap zone  angle  would  region,  o f 7.6° T h e r e f o r e , t h e gap zone  would  would  than  sectioning  becomes c r i t i c a l l y  this  the region  would  because  o f i t s more  collagen  fibrils.  Electron represents component The  microscopic  a technical while  approach  aqueous  PTA/UA  banding  pattern.  the with  bands  imaging  visualizing  groups,  more  as does  The a n g l e o f the normal  the tendon.  t o image  the associated  with  UA i n a l c o h o l  And s o  i n bone of  tissues  the mineral organic  matrix.  i s an effects of  a different  collagen  t h e p o s i t i o n i n g and r e g i s t r a t i o n of  " a t o e" a r e t h e same  charged  much  to maintain  a t an  regions  of calcified  but produces  While  predicted  be v i s i b l e  to the demineralizing  stains  electron  structural organization  challenge  staining of collagen  alternative  within  be m o r e d i f f i c u l t complex  t h e a^-e^  t h e gap zone.  important  that the  tothe  a t an  of these  no l o n g e r  within  i n direction of collagen  pattern  equal  disappear  be d e t e c t e d  a t which  Assuming  the non-mineralized  frequently  change  angle  i n parallel,  o f 3.5? The l a r g e s t  t h e c^-c^  bordering  are blocks,  9.4nm; a n d  specimen  incident  be d e t e c t a b l e .  t h e gap zone  i n thickness,  within  o r about  4.7nm) a n d a n e s t i m a t e d  thickness space  0^-0^=.14D  d u e t o t h e i n t e r a c t i o n o f UA  the relative  i n t e n s i t i e s and  66  delineations of  the  of  observed  reasons.  First,  UA  a  to  and  the  be  bands  pattern  i s not  although  charged and  Secondly,  e f f e c t s of  the of  comparison  The collagen  each  between has  not  would  alcohol  as  a  been  water  and  alcohol  and help  scales,  as  of  the to  degree  despite  thus  overlap  zones  evidence  which  crystals  within  the  for  r e l a t i o n s h i p because  periodic  and  a l . , 1982b). in  the  so  a in  there  collagen  spaces  patterns  this  the  in  precise between within  proposed  availability  of  has  assigned  not  been  calculations in  is in  degree  contradicts gap  et  hydrophobicity  collagen  molecules.  estimating is a  the  several a  the The strength  subjective  of f i t .  localization of  of  making  v i s u a l methods  the  consider  on  correlation  substantiate  impossible  of  two  negatively  solvents  more  A mathematical  hydroxyproline  scales,  both  solvent  studied  for  done.  preliminary  However,  interpretation  the  (Tzaphilidou  not  is a  explanation  staining  depending  has  been  mineral  method  The  acids  variables  methods.  these  limitation the  UA  to  hydrophobic  of  of  fibrils  relationship.  standard  works  repeating  of  on  c l e a r . An  v i s u a l i n t e r p r e t a t i o n of  region  value  pH  collagen  correlational density  as  r e a d i l y apparent  early  amino  concentration  procedure  not  cationic stain i t actually stains  positively  staining  are  of  apatite  fibrils  theories (eg.  crystals  (Arsenault,  exclusively  UJeiner  and  within 1988)  is  localizing  Traub,  1 986)  or  67  spaces  within microfibrils  comes f r o m variable  studies  and  not  (Arsenault  and  spaces  be  may  localization explain given  the  mineral  of  crystal  These  molecules  chain  flexibility  helix  can  have  its  a  accommodate  in free  and  microscopic  stability)  2)  p r o l i n e and  4)  charged  are  et  growth  form  unit.  So  densely  or  gives  how  the  form  a  et  on:  a l . , 1981). fibrils  the  apatit zone  semiflexibl  and  1985a). side  the  triple  These which  according  studies of  G-X-Y  localization;  chain  the  appreciable  flexibility 1)  crystal  (Veis,  because  biomechanical  of  order  backbone  without  be  to  overlap  as  molecule  flexibility  The  rise  packed  higher  of  not  does  conceptualized  collagen  lateral  side  space  potential  heterogeneity  a l . , 1987)  dependent  acid  and D  hydroxyproline  amino  these  which  degree  G i r a u d - G u i l l e , 1988).  gap  therefore  (Okuyama  and  the  must  fluctuations  i n t e r t w i n e to  of  is  Consideration  collagen  high  energy  size  size  do  be  the  (Sarkar  longitudinal  electron 1980;  have  crystal  evidence  they  i n t e r t w i n e to  cylinder,  that  Although  i n the  can  Other  materials  entire  peptides  1980).  in crystal  size  deposited  which  molecules  1988).  d i s c r e t e environment  Collagen  changes  the  non-collagenous  w i t h i n the  semiflexible  shown by  observations.  become  cylinders  determined  Grynpas,  of  heterogeneity deposition  have  influential  these  to  that  (Hohling,  also to  (l/iidik, collagen  (an  triplet; 3)  interactions;  crosslinking and  68  5)  hydrophobic  collagen (Veis,  molecules  by  modelling flexible  i t can  overlap  Some o f  the  overlap  zones  chain  flexible  be  built  observed that  the  of  low  into  repeating  gap  zone  gap  d e n s i t i e s of regions  density  of  hydrophobic  regions  of  high  zones  mineral  by  the fibrils.  i n gap  acids  and  localized  by  regions  hydroxyproline  to  and  regions are  discussed  that  is  density  i s bounded  correspond  hydrophobicity  flexible  differences in  zone  and  this  w i t h i n the  p r o l i n e and  amino  units  periodic  mineral  molecules  by  are  the  regions  From  i s more  overlap  explained The  10).  there  collagen  Thus,  inflexible  that  the  collagen  be  c ^ - c ^ ) . These  and  (Figure  d i f f e r e n c e between may  interactions.  It i s possible that  flexibility.  very  side  modelling  and  into  semiflexibility  and  be  zone.  accommodated  with  can  computer  regions  collagen  acid  exibit  1985a) which  depicted  the  amino  with  adjacent  (a^-a^ a  low  to  the  above.  Conclusions  In it  would  an  observational, correlational  be  relationship Based this  on  exists  previous  t h e s i s are  experimental clarify in  improper  the  to  suggest  between  studies  the  the  biologically  designs potential  m i n e r a l i z a t i o n . In  are  that  of  cause  such and  v a r i a b l e s under  as.this,  effect examination.  relationships described p l a u s i b l e but  required  role  a  study  to  conclusion,  the  alternative  substantiate  hydrophobicity  in  and  asymmetric  and flexibility and  69 periodic and  d i s t r i b u t i o n s of  overlap  zones  correspond' to distribution  of  collagen  collagen has  a  apatite  fibrils  molecular  reverse  crystals within were  the  observed  o r i e n t a t i o n . This  c o r r e l a t i o n to  the  to mineral  position  hydrophobic  groups  as  determined  by  computer  modelling  may  intrinsic  properties  of  collagen  structure  reflect  throughout  the  found  periodic  that  flexibility  gap  may  apatite  into  role  collagen  be  given  to  throughout potential  of  the  the  collagen  mechanisms  mineralized  the  of  tissues.  addition,  i t  of  proline  Therefore,  properties rather  the  gap  of  than zone.  may  and  was  and  to  collagen  Further of  b i o m i n e r a l i z a t i o n and  as  on  the should  they  vary  the  study  apatite  enhance  of  understand  focussing  localization fibrils  of  intrafibrillar  mineralization, consideration  discrete  within  in  In  p r e f e r e n t i a l accommodation  regions.  period  zones.  density  explain  between of  by  during  D  space  properties  differences  specific  the  association  overlap  determined  hydroxyproline,  of  and  gap  of  the  and  our  understanding  the  mechanics  of  70  REFERENCES A r s e n a u l t , A.L. 1988. 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