JBJS 1986 ALK PHOS in Callus in Rats

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-


2/6

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


3/6

- 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 ) .


4/6

I

m y .

.

S

.

a

.

.

.

c f

.

4.- . #{231} ..,.-

k

..

- , .-

2 m

#{149 }J

..

. . -- .

.

. - .

F ig . 8

Fig. 10

63 2


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


5/6

--

..,

: . .

_..,,.

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 .


6/6

63 4


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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