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8/10/2019 JBJS 1986 ALK PHOS in Callus in Rats
1/6
F ig . I
V O L . 68-B . N o . 4 . A U G U S T 19 86
629
D IS T R IB U T IO N O F A L K A L IN E P H O S P H A T A S E A C T IV IT Y IN
E X P E R IM E N T A L L Y P R O D U C E D C A L L U S IN R A T S
G . V O LP IN , J . A . R E E S , S . Y . A L l, G . B E N T LE Y
F rom The Ins titu te o fO r th opaed ics , R o ya l N a tiona l O rthop aed ic H osp ita l, L ond on
E xp e rim e n ta lly p ro d u ce d fra c tu res in lo n g b o n e s s tu d ie d b y lig h t a n d e le c tro n m ic ro s c o p ic
h is to c h e m is try w e re fo u n d to h e a l b y a process o f e n c h o n d ra l c a lc if ic atio n . T h e re w as in te n s e p ro life ra tio n in
th e c e lls o f th e c a m b iu m la y er o f th e p e rio s te u m , w ith d iffe re n tia tio n to c h o n d ro b la s ts a n d o s te o b la s ts ,
s u g g e s tin g th a t th is la y e r w a s th e p rim a ry t issue
responsib le
fo r d e v e lo p m e n t o f th e c a llu s . C y to p la s m ic
p ro ces ses o f
th e h y p e rtro p h ic c h o n d ro c y te s a p p e a re d to b u d a n d p ro d u c e m a trix v e s ic le s . A lk a lin e
p h o s p h a ta s e a c tiv ity w a s d e te c te d a lo n g th e p la sm a m e m b ra n e o f th e h yp e rtro p h ic ch o n d roc y te s a n d a ro un d
th e m a trix v es ic le s , b e fo re a n y s ig n s o f m in e ra l d e p o s it io n . C a lc ific a tio n to o k p la c e b y d e p o s itio n o f
h y d ro x y a p atite c ry s ta ls in a n d a ro u n d th e s e m a trix v es ic le s w h ic h fre q u e n tly s h o w e d a lk a lin e p h o s p h a ta s e
act iv i ty .
It is s u g g e s te d th a t th e re is a c lo s e fu n c tio n a l a s s o c ia tio n b e tw ee n a lk alin e p h o s p h a ta s e a c tiv ity a n d
ca lc ific a tio n in th e p ro c e ss o f fra c tu re h e a lin g , w h ic h is a n o th e r ty p e o f e n ch o n d ra l ca lc ific a tio n m ed ia te d b y
m atr ix v e s ic le s .
P re v io u s h is to c h e m ic a l s tu d ie s u s in g l igh t
and elec tro n
mic ro s c o p y and va rio us b iochem ica l inv estiga tions have
in d ic a te d th a t
th e p ro cess
o f e nch o nd ra l ca lc if ica tio n
of
the ep ip hy sea l g row th p la te and of som e o th er n o rm a l
t is s u e is m e d ia te d b y e x tra c e llu la r m a trix v e s ic le s
(B o n u c c i 19 67 ; A n de rso n
a n d c o -a u th o rs
]969 , 197 0 ,
1 976 ; A li 197 6 ; L ew inson , T o iste r and S ilb e rm ann 1 982 ;
A li e t a l. 1 9 8 4 ); th is is a lso tru e o f p ath o lo g ica l tiss u e
calc if ication (Y am ad a 197 6 ; S e la e t a l. 19 81 ; S te in , B ab
a n d S e la 19 81 ; B ab , Se la an d S te in 198 2). T hese m a trix
v e s ic le s a p p e a r to b e the in itia l s ite fo r m in era l depos i-
t io n . It h a s a ls o b e en fo u n d tha t a lka line pho sph ata se is
e s s e n tia l fo r th is p rocess and d iffe ren t theo rie s abou t its
ro le have been sugg es ted (R ob ison 1 923 ; B achra 197 0 ;
S a lom on 1 974 ; A li 197 6 , 19 80 ; L ew in son et al. 198 2 ;
W u th ie r 1 982 ).
Th e p u rp os e o f th e presen t s tudy w as to
fo llow
th e
p a rtic ip a tio n o f m a tr ix ve s ic les a n d to co rre la te it w ith
the alk alin e pho spha ta se ac tiv ity d urin g the su ccessive
b io lo g ic a l s ta g e s o f frac tu re hea ling o f lo ng bon es . F o r
th is p u rp o se w e u s ed h is to c he m ica l m e th o ds in v o lv in g
bo th ligh t and e lec tro n m ic roscopy . O u r re su lts in d ic a te
G . V o lp in , M D , S en io r O rth o p a ed ic S u rg e o n
D epa rtm e n t o f O rth op ae d ic S urg e ry , R am ba m M e d ic a l C e n tre , H a ifa ,
Is rael.
J.
A . R ee s . B S e , P h D , E lec tro n M ic ro s co p is t
S. Y . A li . B S c , D IC . P h D , D ire c to r, E xp erim e n ta l P a th o lo g y U n it
G . B en tle y . C hM , F R C S , P ro fess o r o fO rtho pa e d ic S u rg e ry an d D ire c -
to r o fC lin ic a l S tu d ie s a nd R es ea rch
T h e In s t itu te o f O rthop ae d ic s , R oy a l N a t ion a l O rth opa ed ic H os p ita l,
B roc k le y H ill. S tan m o re , M idd le sex H A 7 4 LP , E ng lan d .
R e qu es ts fo r rep rin ts sho u ld be s en t to P ro fe ss o r S . Y . A li.
19 86 B rit ish E d ito ria l S oc ie ty o f B o ne a nd Jo in t S u rg e ry
0301 62 0X864107 S2.00 _ __ ___ __ __ _ _ __ __ __ __ __ _
R a d io g raph o f the frac tu red rad ius r ) a t th re e w ee ks sh ow ing the po s i-
t ion o f the frac tu re in th e m id -d iaph ys is a nd an a to m ica l a lignm e n t o f
th e frag m e n ts . T he u ln a u ) w a s le ft in tac t to sp lin t the fra c tu re d ra d iu s .
specif ic tem pora l an d fo ca l a sso cia tion s o f m a trix
vesicles , a lka lin e p h o sp h a ta se a c tiv ity a n d the fo rm atio n
of
in it ia l c rysta ls o f ap atite du ring fractu re h ea ling of
lon g b on es .
M A T E R IA L S A N D M E T H O D S
T h is s tu d y w a s pe rfo rm ed in 40 you ng a lb ino ra ts each
weigh ing 5 0 to 6 0 g at th e
on se t o f
th e ex pe rim en t. U n de r
e the r anaesthesia , exp erim en ta l frac tu res w ere m ade w ith
sc iss o rs in th e m id d le o f th e d ia p h ys is o f b o th rad ii. T h e
u lnae w ere lef t in tact to se rv e as s p lin ts fo r th e fra ctu re d
radii (F ig . I). T he po sition and d eg ree o f hea ling of e a c h
bon e w as observed by w eek ly rad io g rap hs. G roup s o f I 0
ra ts each w ere k illed successive ly a fter 8 , 1 4 , 21 an d 28
days and the frac tu red rad ii w e re rem o ved fo r h istoche -
m i ca l
s tud ies
b y ligh t an d e lec tron m ic ro scopy .
L ig h t m ic ro s c o p y . T h e fra c tu re d ra d ii w e re fix e d fo r a t
le a s t 24 ho urs in neu tra l 10%
fo rm o l sa lin e . T h e s p e c i-
8/10/2019 JBJS 1986 ALK PHOS in Callus in Rats
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F ig .
2
F ig . 3
F igu re 2 S e ction th rou gh th e c a llu s du rin g e a rly s tag es o f rep a ir (7 d ays ) sh ow ing p ro life ra t ion o f the o s te op rog en ito r ce l ls o p) o f the ca m bium
la ye r o f the p e r ios teu m e p) w ith fo rm a tio n o f ca rtilag ino us c a llus c r) an d o f ne w b on e tra be cu lae b i) adh eren t to the c om pa ct bo ne . N o te the
b loo d c lo t c i) in the frac tu re s ite f r) (h ae m a to xy l in a nd ne u tra l re d , x 4 0 ). F igu re 3 S e ction th roug h th e ca llus a t tw o w e eks sh ow in g d iffe ren t
d eg ree s o f d iffe ren tia t ion o f c a rt ilag e c e lls (c r) w ith p ro du ctio n a nd s ec re t ion o f p ro te og lyca ns a nd fu rth e r d ev e lo pm en t o f ne w bo ne trabe cu lae
bt) . N o te th e ca m b iu m lay e r o fthe pe rios teu m c p) (az u re A , x 40 ).
63 0
G . V O L P IN J . A . R E E S S . Y . A L l G . B E N T L E Y
T H E J O U R N A L O F B O N E A N D J O IN T S U R G E R Y
m e n s w e re th e n d e ca lc ifie d in K ris te n se n s fo rm ic a c id
and sod ium fo rm ate flu id fo r 48 to 7 2 ho urs . T he sp eci-
m e n s w e re th e n
em bedd ed in w ax an d cu t on a sledg e
m ic ro to m e se t a t 7 m th ick n e ss .
T he d iffe ren t stag es o f d e v e lo p m e n t o f th e c a llu s
w e re in itia lly a s s e s s e d u s in g th e h a e m a to x y lin a n d e o s in
s ta in in g m eth o d . Id e n tif ic a tio n of the bon e trab ecu lae
w a s a ch ie v e d u s in g th e S a y e rs a lc ia n b lu e a n d h a e m a -
to x y lin m eth o d (S ay e rs I 973 ). V a rio us ca tio n ic dy es such
a s a zu re A . n e u tra l re d a n d a lc ia n b lu e w e re u se d to
d e m on stra te th e c a rtila g e p ro te o g lyc a n s .
E le c tro n m ic ro s c o p y . S lic e s o f c a llu s a n d b o n e w e re
e x c is e d in a lon g itud in a l p lane ac ross the frac tu re s ite .
F o r g en era l m orp ho lo gy th ese p ieces
wer e
im m ersed in
2 .5
g lu tara ldehy de in
0 .085
M -cacod y la te bu ffe r fo r
tw o
h o u rs a n d p o s t-f ix e d in I o s m iu m te tro x id e in
b u ffe r fo r o n e h o u r. A fte r d e h y d ratio n th ro u g h g ra d e d
e th a n o ls a n d fin a lly 1 :2 e p o xy p ro p a n e , s p e c im e n s w e re
e m b e d d e d in S purr re sin . F o r o rien ta tion purpo ses, I m
s ec tio n s w e re cu t fo r lig h t m ic ro s co p y u s in g g las s kn iv es
and sta ined w ith H um p hrey s so lu tion . T h in se c tio n s (6 0
to 8 O n m ) w e re cu t o n a R e ic h e rt O M -U 2 u ltra m ic ro -
to m e u s in g d ia m o n d k n iv e s and co llec ted o n e ith e r
u n c o a te d 6 0 0 -m es h c o p p e r g rid s o r o n P a rlo d io n -c o a te d
2 0 0 -me s h grids. S ectio ns w ere ex am ined uns tain ed , o r
a fte r s ta in in g w ith sa tu ra te d a q u e o u s u ra n y l a ce ta te for
I 5 m inu tes and R eyn o lds lead c itra te fo r 5 m in u te s, on a
Phil ips E M 30 0 a t 60 kV or 8 0 k V .
Fo r t he v is ua li sa tio n of alk alin e ph osp ha tase ac tiv -
ity a t th e u ltra s tru c tu ra l
leve l the m e ta l sa lt m e tho d
of
L ew i n so n Ct a l. (19 82) w as used . T issue p ieces w ere
imme rs e d
in I
.5
g lu ta ra ldehy de in buffe r an d s liced as
th in ly a s p o s s ib le (abo u t 200 lsm ) usin g new razor b lades
un de r a d issec ting m icroscop e . A fte r tw o hou rs th e tissu e
s lic e s w e re w as h e d in caco dy la te bu ffer ove rn igh t and
then im m ersed in fre sh ly p repa red in cuba tion m ed iu m
(9 m M -sod ium fl-g lyce rop hospha te , 5 m M - M g C l 2 ,
3 .6 m M -lead n itra te a t 40 m M -tris /H C I bu ffe r at p H 9 ) fo r
3 0 m inu tes a t 3 70C T o test fo r false n on -enzym atic
d e p o s it io n o f re a c tio n p ro d u c t, c o n tro l t is s u e s lic e s w e re
in cu ba ted in m ed ium w hich had 2 m M -lev am iso le add ed
as an in h ib ito r o f alk alin e ph osp ha tase ac tiv ity . A fte r
w ash i n g in b u ffe r fo r 1 0 m in u te s th e p rep a ra tio n o f th e se
specim en s w as th e sam e as fo r th ose p rep ared fo r gene ral
m orpho log ica l observa tion .
R E S U L T S
U s in g the d iffe ren t m e tho ds o f sta in ing an d th e rad io -
g rap h ic ev alu atio ns, success ive s tag es
o f fra ctu re hea l ing
co u ld b e observed . A t each stag e, car tilage ce lls o f d iffe r-
e n t d eg re e s o f d iffe re n tia tio n w e re p re s e n t
in g rou ps
th rou gh ou t the ca llu s . T he a rea occu p ied by th ese g roup s
v a rie d b e tw ee n sp ec i m en s .
T he in itia l changes w ere fo und in the cam b ium laye r
o f the pe rio steum . R a p id p ro life ra tio n o f the ce lls in th is
laye r lifted the pe rio steum aw ay from th e bo ne and
fo rm ed a co lla r-shaped ca llu s a ro und the fractu re site
(F ig .
2) .
T h e o u te r p a rt o f th e ca llu s s h o w e d d iffe re n tia -
t io n o f these c e lls in to o s te o b la s ts w ith th e fo rm a tio n o f
ne w b o ne tra b e cu la e . In the ce n tra l p e rip h e ra l a rea and
across th e fra c tu re s ite th e fo rm a tio n o f a ca rtila g in o u s
ca llu s in a V -shape w as
o b se rv ed . D iffe ren t c a tion ic
dyes
d e m o n stra te d th e p rod u c tio n o f th e e x tra c e llu la r m a trix
w ith sec retio n o f p ro teog lycan s from the ch ond ro cy tes
(F ig . 3 ) .
Successive s ta g e s o f e n ch o n d ra l ca lc ific a tio n w er e
foun d in the ca rtilag ino us ca llu s , con sis ting of p ro lif-
e ratio n of ch ond ro b la sts , d ev elo pm en t o f hyp ertroph ic
ch ond ro cy tes an d ca lcif icatio n ofca r tilag e
m a trix (F ig s 4
an d 5) . N e w b o n e trabecu lae w ere also fo rm ed b y th e
en dos tea l cells ,
on
th e n e cro tic e n d s o f th e b o n e fra g-
m en t s .
A lka line ph osp ha tase ac tiv ity , in th e e le c tro n m icro -
s c o p ic stu die s , w as o bse rv ed b y th e ap pearance o f an
e lectron -d en se p recip itate lay er a lon g the ce ll m em b rane
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- ce llu la r m atr ix (cc) is a lso e v iden t, lea d ing to ca lc i l
1
F ig . 6 F ig . 7
D IS T R IB U T IO N O F A L K A L IN E P H O S P H A T A SE A C T IV IT Y IN E X P E R IM E N T A L L Y P R O D U C E D C A L L U S IN R A T S
63 1
V O L . 68 B .
N o . 4 .
A U G U S T 1 986
and cy top la sm ic p ro cesse s o fch ond ro cy tes during d iffe r-
en t stages of d ifferen tia tion . H o w eve r , the m o st in tense
p re cip ita tio n a p p e a re d to b e arou nd early h ype rtrop h ic
c h o n d ro c y te s (F ig s 6 and 8). T hese ce lls , w h ich w ere
fo u n d in la c u n a e in s id e the ca rtilag e m a trix , had w e ll-
deve lop ed cy top la sm ic o rgane lle s su ch as ro ugh en do-
p la sm ic re ticu lu m (R E R ), G o lg i ap pa ra tus and m an y
mito c h o n d r ia ,
ind ica ting inc rea sed m etabo lic ac tiv ity
(F ig . 7 ).
In a few o f th e m ito ch o n d ria , a c cu m u la tio n o f
som e ele ctron -d en se am orp h ou s
material w as ob se rve d.
L arge am ou n ts o f g ly cogen w ere fo un d in the ea rly an d
m atu re hy pe rtroph ic chon drocy te s . In m a ny ce lls , g lyco -
gen ap peared as m asses o fe lec tron -d en se g ranu les , large r
in d ia m ete r an d grea te r in e lectron d en sity than the ribo -
so m es o fth e a d ja ce n t R E R (F ig s 6 an d 7 ).
M e m b r a n e - b o u n d
m atrix v es icle s , 0 . 1 l .tm to 0 .2 iim
in d iam e te r, w e re obse rved sca tte red be tw een the co lla-
gen fib re s
of
th e ex tracellu lar m a tr ix and a rou nd hyp e r-
F i g ur e 6 E M se ctio n th rou gh ea rly
h yp e rtrop h ic cho nd roc y te w ith in its
la cu na , s ta in ed fo r a lk a line ph os pha ta se
act iv ity . P re c ip ita te o f le ad p ho sph a te
indicat ing e nzy m e ac tiv ity u p) is s ee n
a lo ng the p la sm a m em bra ne o f the ce ll.
N o te the hyp erp la s ia o f the end op las m ic
ret iculum er ) . m any m itoc hon dr ia (n i)
and in tra ce llu la r a re as o f g lyc og en g o.
T h e e xtra ce llu la r m a tr ix co ns is ts p re dom i-
nan tly o f c o lla ge n fib res . F igu re 7 E M
sec tio n th rou gh a n ea rly h yp e rtrop h ic
ch on drocy te s ta in ed fo r a lka line ph os ph a-
tas e a ct iv ity an d inh ib ited by leva m iso le
(c on tro l s ta in ing fo r F ig . 6 ). N o te the
hyp erp la s ia o f the en do p la sm ic re t icu lum
er ) , m a ny m ito ch on dria n i). g ly c ogen g I)
an d ab sen ce o f e nzy m a tic ac tiv ity a long
the p la sm a m em b ran e o f the ce ll p m ) .
8/10/2019 JBJS 1986 ALK PHOS in Callus in Rats
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I
m y .
.
S
.
a
.
.
.
c f
.
4.- . #{231} ..,.-
k
..
- , .-
2 m
#{149 }J
..
. . -- .
.
. - .
F ig . 8
Fig. 10
63 2
G . V O L P IN J . A . R E E S S . Y . A L l G . B E N T L E Y
T H E JO U R N A L O F B O N E A N D JO IN T S U R G E R Y
;e
F ig . 9
F i g ur e 8 E M sec tio n th ro ug h a h yp ertrop h ic c ho nd roc y te s ta in ed fo r a lka line p ho sp ha tas e ac tiv ity , sho w ing a n in te ns e re ac tio n a lon g th e p la sm a
m em bra ne o f the ce ll u p) a nd a ro und e xtrace llu la r m a trix v es ic le s my ) . N ote th e bu dd ing o f a m atrix v es ic le fro m a lo ng itu d in a l c y to p la sm ic p ro -
c ess o fthe c e ll (a r row ). F igu re 9 E M se ction o fthe e x trace llu la r m a trix o fth e ca rtilag ino us ca llus s ta ine d fo r ge ne ra l m orp ho log y sho w ing m a ny
m atr ix ve sic le s m i) s ca tte red be tw ee n c o llage n fib re s ). N o te the in itia l de po s ition o fne ed le - like cry s ta ls o fhyd ro xya pa tite (a rro w s ) in s id e a few
m atr ix ve s ic les . F ig u re 1 0
E M se ctio n o fth e e x trace llu la r m atr ix o fc ar tila g in o u s c a llu s s ta in ed fo r a lk a l in e p h o s p h ata se ac t iv ity , sh o w in g in ten se
a lka line pho sp ha tas e re ac tion p rod ucts a ro un d a p art o f the m e m b ran e o r th e w ho le m em bra ne o fm a trix ves ic les (a rro w s ).
t ro p h ic cho ndrocy te s b efo re any sig n o f m ine ral
d e p o s i t io n (F ig s 8 and 9). I t ap peared tha t the se m a trix
ve s ic le s w e re fo rm ed b y bu dd in g fro m the cy to p la sm ic
p ro c e s s e s of cho ndrocy te s . H o w eve r , the in itia l s ig n s o f
a lk a lin e p h o s p h a ta s e a c tiv ity in and aroun d th ese m a trix
vesic les cou ld be d etec ted on ly in the stage o f fu r the r
o rgan isa tion of the in trace llu la r m a trix an d fu rthe r d if-
ferentia tion of the ce lls to m a tu re hyp ertro ph ic ch ond ro -
cy tes . T h e en zy m a tic reac tion p ro duc ts w ere fo und
a ro und p a rt o f o r th e w h o le o f th e m e m b ra n e o f th e
m atrix v e s ic le s in t he i nt er ce ll ul ar m atrix , and in th e p en-
la c u n a r re g io n s (F ig s 8 a n d 1 0).
C alc if ica tion o f th e ex trace llu lan m a trix too k p lace
by
p ro gre ss ive de p o s itio n o f n e e d le -lik e crys ta ls o f
hyd ro xyapa tite w ith in th e cen tre o f th ese m atr ix vesic les
and a ro und th em , b u t o n ly in tho se vesic le s th at show ed
a lk a lin e p h o s p h a ta s e activ ity (F igs I I an d 12). A s
m in era l accu m ula tion prog re ssed , c rysta l g ro w th
con -
tinued and the m a tr ix becam e ca lc ified (F ig . 13 ) . A t th is
s ta g e o f m in e ra lis a tio n , a lk a lin e p h o s p h ata s e a c tiv ity
c o u ld n o t be obse rved in the in te rce llu lar m a trix b ecause
o f th e ca lc ific d e p o s its ,
bu t
cou ld
be seen alo ng
th e c e ll
m em b r an e of the degene ratin g cho ndrocy te s an d in th e
p e r i la c u n a r
areas.
In the alk alin e ph osph a tase con tro l g ro u p s (th o s e
s e c t io n s trea ted b y levam iso le ) and those p repa red fo r
g en era l m o rp ho lo g ical ob serva tion s sim ila r stages o f ce ll
d ifferen tia tion and m ine ralisa tio n in side th e m a trix
ve s ic le s co u ld b e o b se rv e d , bu t the re w as n o ev idence o f
re ac tio n p ro du cts ofen zy m a tic ac tiv ity (F ig s 7 , 9 , 12 , 13 ).
D I S C U S S I O N
T h e find in gs o fthe p re sen t
s tudy
sugg es t th a t fractu re s
of
long bo nes m ay
hea l
th rou gh a p ro c e ss o f enchondral
calc if ica tion , w h ere the in itial hy dro xyap atite d ep ositio n
tak es p lace w ith in and a roun d ex trace llu la r m atr ix
vesic les sho w in g a lk a line p hospha ta se ac tiv ity .
T h e ea rly stages co nsis ted o f an in ten se p ro life ra tiv e
re spo nse in the cam b ium laye r o f the p erio steum . T he
typ ic a l c e lls in th a t lay e r g a v e rise to tw o d iffe re n t ty pe s
of cells , e ithe r o steo b la stic o r cho ndrob lastic , sugg esting
tha t th e p erio steu m is the m a in tissue respon sib le
fo r
th e
deve lop m en t o f the ca llu s .
O u r fin d in g s s u p p ort p re v io u s
reports
ind ica ting tha t the se cam biu m ce lls a re the p ri-
m any sou rce
o f o s te o g e n ic ce lls d u rin g fra c tu re h e a lin g
(K ernek an d W ray 1 973 ); th ey h ave been te rm ed osteo -
p ro gen ito r ce lls (Y o ung 196 2 ; K ernek and W ray 197 3 ;
M cK ib b in 19 78). S tripp ing of the p erio s teu m fro m the
s u rfa c e o f th e fra c tu re fra g m e n t w a s fo u n d to d e c re a s e
th e osteogen ic po ten tial o f th e b o n e (M cK ibb in 19 78)
an d m ig h t the refo re re su lt in d e la ye d o r n o n -u n io n o f th e
fracture.
O ur study
a lso s ho w e d d iffe ren t d e g re es o f d iffe re n -
tia tion o fca rtilag in ous tissu e a t
e ac h s tag e o fthe fra c tu re
heal ing
process .
T h e am o u n t o f c a rtila g e p re s en t in th e
ex terna l ca llu s
o f a lo ng b o n e h a s b ee n s h o w n p re v io u s ly
to b e g re a te r in lo w e r a n im a ls a n d a lso in e x p e rim e n ta lly
pro du ced fractu re s w h ere un re str icted m ovem en t o f the
frac tu re fragm en ts w as a llow ed (A n de rso n 1 965 ). It h as
a lso b een rep orted tha t fractu re s treated b y ana tom ica l
reduc tion and in te rna l f ixa tion u nde r com p ressio n m ay
8/10/2019 JBJS 1986 ALK PHOS in Callus in Rats
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--
..,
: . .
_..,,.
I.
. 4,,
w
h .
.1
02 m
.,I
wI
.
b
Q
Fig. I I Fig. 12 F ig . 13
V O L . 614 B . N o . 4 . A U G U S T 1986
D IS T R IB U T IO N O F A L K A L IN E P H O S P H A T A S E A C T IV IT Y IN E X P E R IM E N T A L L Y P R O D U C E D C A L L U S IN R A T S
63 3
h ea l w ith o u t e x te rn a l c a llu s a s a n in te rm e d ia te s ta g e
(A n d e rs o n 1 9 6 5 G lim c h e r e t a l. 1 980 ). O th e r au tho rs
hav e su ggested th a t the m a in facto r fo r th is typ e of
m e ta p la s ia o f th e cam b ium lay e r o f the p erio s teu m w as
lo w o xyg e n te n s io n d u e to to rn b lo od ve ss e ls a n d d e -
c re a s e d b lo o d sup p ly (H am 19 30 ; M cK ibb in 197 8).
T he p ro gress ive d ev elo pm en t o f the cartilage in
ex terna l ca llu s w as c lear ly dem o nstrated b y ou r lig h t and
e le c tro n m ic ro s c o p y . It w a s ch a ra c te ris e d b y s u c c e s s iv e
s ta g e s o f p ro life ra tio n a n d h yp e rtro p h y o f ch o n d ro cy te s ,
to g e th e r
w ith s yn the s is , se cre tio n
and organ isa tion o f
v a r ious
c o m p o n e n ts o f th e in tercellu lar o rgan ic m a tr ix ,
m ain ly co lla g e n a nd p ro te og ly ca ns . C a lc ific a tio n o f th e
c a r tila g e. w h ic h w a s fo u n d to b e a s s o c ia te d w ith
d e g e n e ra tio n o f th e s e h y p e rtro p h ic c h o n d ro c y te s , to o k
p la c e
w ith in
and a rou nd ex trace llu la r m atr ix vesic le s . I t
w a s a lso fo u n d th a t th e se s tru c tu re s w e re fo rm ed by
b u d d in g fro m the cy top la sm ic p ro cesse s o f ea r ly
hyper-
tro p h ic ca rtila g e ce lls . T h e se m a tr ix ve s ic le s co n ta in e d
th e ea rlies t recog n ised d ep osits o f h ydrox yapa tite and
w e re p ro b a b ly th e
in itia l s ite fo r ca lc ifica tion of th e ca rti-
l a g in o u s c a llu s d u rin g th e p ro c e ss o f fra c tu re h e a lin g o f
lo n g b o n es . T h e s e fin d in g s re se m b le th o se d escribed p re -
v io u s ly in m a n y o th e r n o rm a l c a lc ify in g tis s u es , s u c h a s
neonatal lo ng b o n e s, th e ep ip hysea l g row th p la te, th e
m a n d ib u la r c o n d y le . p re d e n tin e a n d a lv e o lar b o n e
(B o n u cc i 1 9 6 7 ; A n d e rso n 1 96 9 , 1 9 7 6 ; A n d e rso n e t a l.
1 970 ; A li 1 976 , 19 80 ; L ew inson et al. 19 82) and a re typ i-
ca l o fe n ch o n d ra l ca lc ifica tio n . S im ila r f ind in gs h ave a lso
b een d esc ribed in so m e p ath o log ica l cond itions , in c lud -
in g c a lc ifie d te n d o n s (Y a m a d a 1 976 ), ske le tal n eo p la sm s
(S e la et al. 198 1) an d also
a fte r tra n sp lan ta tion o f fre e
pen icho ndria l g ra fts (B ab e t a l. 1 982 ) o r sy nov ial g raf ts
(S tein e t a l. 19 81) in to ex pe rim en tally p ro duced de fec ts
o f a r ticu la r car tilag e .
O u r o b s erva tio n s a ls o s h o w e d a cc u m u la tio n o f
som e am orpho us e lectron -dense m a te ria l w h ic h , w h e n
com pared w ith f ind in gs o f o the r au th o rs , cou ld be ca l-
c iu m in s id e th e m ito c h o n d r ia
of
th e e a rly h yp e n tro p h ic
chondrocy tes
(A l i et
al . 1977 ; B rig h ton and H un t 1 978 ;
B osk ey 1 981 ; W u t h ie r 19 82). P rev iou s stu d ie s have
dem onstra ted tha t th e fo rm a tion ofm atn ix vesic les in th e
ep ip hysea l p late occu rred by bu dd ing (b leb b ing ) fro m
the m em bra ne
of
th e cy top la sm ic p roces ses o f p ro life rat-
in g
chondrocy tes (A l i
I 98 0 ; B o s ke y I 9 8 1 ; W u t h i e r
1982) .
It ha s also been sugg ested th a t re lease o f ca lciu m from
m itoc ho nd ria o f th e se ce lls is th e tr ig ge r fo r the fo rm a -
tio n o f th e s e m a trix ve s ic le s a n d th a t m ito ch o n d ria o f
chon dro cy te s p lay an im po rtan t ro le in m a tr ix ca lcif ica-
tio n (B righ ton and H un t 19 78 ; B osk ey 19 81 ; W u th ie r
198 2). O the r au tho rs ha v e su g g e s ted th a t m a trix v es ic le s
m ay p ro m ote calc if ica tion by the concen tratio n o f cal-
c iu m and ph osph ate , p ossib ly to ge the r w ith the rem ova l
o r exc lu s io n o f ca lc ifica tio n in h ib ito rs (A li 19 76 , 1 980 :
Bos k ey 1 9 8 1 ; W u t h i e r
1 9 8 2 ). O u r o b s e rv a tio n s o f
en ch ond ra l calc if ica tion d uring the p ro cess o f frac tu re
healin g in lon g bon es m ay the re fo re sup port the se v iew s .
A lk alin e p hosp hatase ac tiv ity w as o bse rv ed arou nd
the p la sm a m em bran e ofcho nd nocy te s, du rin g su ccessiv e
s ta ge s o fd if fe re nt ia ti on
a n d d e ve lo p m e n t o fth e ca rtila g i-
n ou s ca llu s. H o w e ve r, w e c ou ld n o t fin d h is to ch e m ic a l
ev id en ce
of in tracellu lar ac tiv ity
of
th is enzym e in th e
..
,
,,,,
F igu re 1 1 E M s ec tion s ta in ed fo r a lka lin e ph osp ha tas e a ctiv ity sh ow ing in itia l s ign o f ne ed le -like h yd rox yap a t ite c rys ta ls (a rro w s) in s id e th e
m a trix ve s ic le . N o te the in te ns e e nzy m atic a c tiv ity a ro und the m atr ix v es ic le s . F igu re 1 2 E M sec tio n th roug h the ca llus s ta ine d fo r ge ne ra l
m o rph o lo gy . sh ow ing p a tte rn o f c a lc ium d ep os ition w ith in a nd aroun d extrac e llu la r m a trix ve s ic les . F igu re 1 3 - EM se c tio n th roug h th e c a rt i-
lag ino us ca llus s ta in ed fo r a lka line ph os ph a ta se ac tiv ity in h ib ite d by leva m iso le (co n tro l s ta in ing fo r F igs 10 to 1 2 ) s ho w ing ad va nce d stage s o f
calc if icat ion m ). N o te th e a bs en ce o f en zym e rea ction p ro du cts in the ex tra ce llu la r m a tr ix a nd a lon g the m em b ran e o f the hy pe rtro ph ic
c hondr oc y tes .
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63 4
G . V O L P IN J . A . R E E S S . Y . A L l G . B E N T L E Y
T H E J O U R N A L O F B O N E A N D J O IN T S U R G E R Y
n uc leu s o r cy to p la sm o f p ro life ra ting ch on drocy te s o f
t he c a rt il ag in o us callu s , a s w as d escr ibed by L ew inson e t
a l. (1 982 ) in m an d ibu lar cond y le s. T h e m ost in tensiv e
reactio n of alk alin e pho sph ata se app ea red to b e a ro und
the m em brane of ea rly an d m atu re hy pe rtroph ic chon -
d rocy te s . A n in ten se reac tion w as also o bse rved a ro und
the m em bran e of the ex trace llu la r m a trix vesic le s be fo re
any sig ns o f m ine ra l dep osition , o r in assoc iatio n w ith
hyd ro xyapa tite depo sition . C a lcif icatio n to ok p lace on ly
w ith in an d arou n d m a trix
vesic les sho w in g en zy m a tic
a c tiv ity . T h e se o b s e rv a tio n s s u g g e s t th a t a lk a lin e phos-
pha ta se p lays an essen tia l ro le in calc if ica tion o f car ti-
lag ino us ca llu s during frac tu re hea ling of long bo nes.
P rev iou s
s tud ie s o f
tiss u es u n d e rg o in g e n ch o n d ra l ca lc ifi-
cat ion have sho w n h igh concen tratio ns o f th is enzy m e
a ro u n d th e
cell m em b rane of cho ndrocy te s an d arou nd
m a tr ix vesic les (R ob iso n 1 923 ; M atsuzaw a and
A n d e r s o n
1 971 ; S alo m on 19 74 ; B osk ey 198 1 ; W uth ie r
1982).
O th er au th ors
have m ade s im ila r ob serva tion s o n
iso la ted m a trix vesic le s
o f ca lc ify in g ca rtila g e (A li 1 97 6 ,
198 0; F e lix and F leisch 1 976 ). I t w as a lso sugg ested
by
A li tha t o ve r 80 % of the a lka line pho spha ta se ac tiv ity in
ep ip h ys ea l ca rtilage is a ssoc ia ted w ith m atr ix vesic le s and
th at th e en zy m e co n te n t m a y fa c ilita te th e c a lc ifica tio n
p ro ce ss in itia te d b y m a tr ix ve s ic le s (A li 1 9 7 6 , 1 9 8 0 ).
T h e e xa c t ro le o f a lka line ph osph a tase during th e
p ro cess o f enchon dral ca lcif icatio n is no t y et es tab lished
bu t seve ra l th e o rie s h a v e been pro po sed in an a ttem p t to
exp lain th is a sso cia tion . V ario us au th o rs h av e sug gested
tha t the en zy m e is respon sib le fo r hy dro lysis of phos -
p h at e e st er s r es ul ti ng
in a lo ca l in c re a se in the con cen tra -
tio n o f p h o s p h a te io n s , fo llo w e d b y d e p o s itio n o f
ca lciu m . p ho spha te in the o rgan ic m a trix (R ob iso n 19 23 ;
A li 1 976 , 19 80). A li (1 976 , 19 80) fo und tha t alk a line
p h os p h atas e of isolated matrix ve s ic le s h a s a v ery w id e
spec ific ity and can hyd ro lyse a va riety o f ph osph ate
e ste rs su ch a s so d iu m glyc ero p h o sp h a te , p y ro p h o s ph a te ,
A T P , A M P a n d g lu c o s e -6 -p h o s p h a te . O th e r a u th o rs
have su ppo rted th e theo ry tha t the e nzy m e part icipates
in the tra n s fe r of p h o s p h a t e g rou ps to the o rg an ic m atr ix
(B achra 1 970 ; S alo m on 19 74 ; L ew inso n e t a l. 19 82) . A n
a lte rn ativ e p oss ib ility
is tha t the enzy m e p artic ipa tes in
th e rem ov al o f inh ib ito rs o f m ine ralisa tion (F e lix an d
Fle isch 19 76 ; B osk ey 19 81). W uth ie r (1 982 ) sugg ested
that
th e en zym e a c ts
prim arily as an ino rgan ic
p h o sp h a te-b in d in g p rote in
in
ca lc ifica tio n m ed ia ted b y
th e m a trix vesic les .
Th e p re se n t w ork d escribe s th e succes sive stages o f
car tilag in o u s ca llu s d ev elo p m en t a n d em p h asise s th e
fu nc tion a l a sso cia tio n
b e tw een a lka line ph osp ha tase
ac tiv ity an d en ch o n d ra l ca lc ifica tio n m ed ia ted b y extra-
cellu lar m atr ix ves icles d u rin g
th e
p ro ces s o f e xp e r i-
m en ta l frac tu re h ea lin g . F u r th er s tud ie s a re req u ired to
as cer ta in th e
ex ac t ro le o f th is enzym e during encho ndra l
calcification a nd sub se que nt
b on e fo rm atio n.
W e are d e ep ly in d e b te d to D r J . R a lp h s an d M r D . C . J . S ay ers fo r
e xpe rt te chn ica l he lp an d co ns tru c tive c om m ents a nd a lso to M iss L .
A d am s fo r ty p ing th e m a nu sc rip t .
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