DISTRIBUTION

OF

ALKALINE

EXPERIMENTALLY

PRODUCED

G. VOLPIN,

From

The

Institute

PHOSPHATASE

J. A.

REES,

ofOrthopaedics,

Royal

ACTIVITY

CALLUS

S. Y. ALl,

G.

National

Experimentally
produced
fractures
in long bones
histochemistry
were found to heal by a process of enchondral

IN

IN

RATS

BENTLEY

Orthopaedic

studied
by
calcification.

Hospital,

light
There

London

and electron
microscopic
was intense proliferation
in

the cells of the cambium
layer of the periosteum,
with differentiation
to chondroblasts
and osteoblasts,
suggesting
that this layer was the primary
tissue responsible
for development
of the callus. Cytoplasmic
processes
of the hypertrophic
chondrocytes
appeared
to bud and produce
matrix
vesicles.
Alkaline
phosphatase
activity
was detected
along the plasma membrane
of the hypertrophic
chondrocytes
and around
the matrix
vesicles, before any signs of mineral
deposition.
Calcification
took place by deposition
of
hydroxyapatite

crystals

in and around

these matrix

vesicles

which

frequently

showed

alkaline

phosphatase

activity.
It is suggested
that there is a close functional
calcification
in the process of fracture
healing,
which
matrix

Previous

association
is another

between alkaline
type of enchondral

phosphatase
calcification

activity
mediated

and
by

vesicles.

histochemical

studies

using

and

light

electron

microscopy
and various
biochemical
investigations
have
indicated
that the process
of enchondral
calcification
of
the epiphyseal
growth
plate
and of some
other
normal
tissue
is mediated
by
(Bonucci
1967; Anderson
1976;
Ali

extracellular
and

Ali 1976; Lewinson,
Toister
et al. 1984); this is also true

calcification

(Yamada

and Sela 1981;
vesicles appear
tion.

1976;

Bab, Sela
to be the

It has also been found

1974;
1982).

Ali

purpose

The
participation
the alkaline

and Silbermann
of pathological

Sela

et al.

that

alkaline

and different
(Robison

1976,

1980;

of the present

of matrix
phosphatase

FRCS,

study

for reprints

should

Professor

68-B.

No.

4. AUGUST

is

1982;

to follow

the

of long bones.
For
methods
involving
Our results indicate

Medical

1986

of Bone

Centre,

Pathology

ofOrthopaedic

Surgery
Orthopaedic
England.

be sent to Professor

( 1986 British
Editorial
Society
0301 620X864107
S2.00

VOL.

et al.

was

Surgeon
Rambam

tor ofClinical
Studies
and Research
The Institute
of Orthopaedics,
Royal
National
Brockley
Hill. Stanmore,
Middlesex
HA7 4LP,
Requests

Fig.

vesicles
and to correlate
it with
activity
during
the successive

MD, Senior Orthopaedic
of Orthopaedic
Surgery,

ChM,

Bab

matrix
deposi-

phosphatase

J. A. Rees. BSe, PhD,
Electron
Microscopist
S. Y. Ali. BSc, DIC.
PhD, Director,
Experimental

G. Bentley.

Stein,

theories
about
its
1923; Bachra
1970;

Lewinson

biological
stages of fracture
healing
this purpose
we used histochemical
both light and electron
microscopy.

G. Volpin,
Department
Israel.

1981;

1982;
tissue

and Stein
1982). These
initial
site for mineral

essential for this process
role have
been suggested
Salomon
Wuthier

matrix
vesicles
]969,
1970,

co-authors

Haifa,

Unit

and DirecHospital,

S. Y. Ali.

and Joint

___________

Surgery

____________

I

Radiograph
of the fractured
radius (r) at three weeks showing
the position of the fracture
in the mid-diaphysis
and anatomical
alignment
of
the fragments.
The ulna (u) was left intact
to splint the fractured
radius.

specific
vesicles,

temporal
alkaline

of initial
long

crystals

and
focal
associations
phosphatase
activity
and
of apatite

during

of matrix
the formation

fracture

healing

of

bones.

MATERIALS

AND

METHODS

This study was performed
in 40 young
albino
weighing
50 to 60 g at the onset of the experiment.

rats

each
Under

ether anaesthesia,
experimental
fractures
were made with
scissors in the middle
of the diaphysis
of both radii. The
ulnae were left intact
to serve as splints
for the fractured
radii
bone

(Fig. I). The
was observed

rats each
days and
mical

position
and degree
of healing
of each
by weekly
radiographs.
Groups
of I 0

were killed
the fractured

studies

by light

successively
radii were

after
8, 14, 21 and 28
removed
for histoche-

and

microscopy.

electron

Light microscopy.
The fractured
radii
least 24 hours
in neutral
10% formol

were fixed
saline. The

for at
speci629

x 40).630 G. cartilage cells of differentiation were present in callus. Sections were examined unstained. (1982) was used. Figure 3 Section through the callus at two weeks showing different degrees of differentiation of cartilage cells (cr) with production and secretion of proteoglycans and further development of new bone trabeculae (bt). immersed buffer in for tetroxide in through graded specimens were purposes. Successive stages of found in the cartilaginous eration of chondroblasts. nitrate at 40 mM-tris/HCI medium 5 mM-MgCl2.5% glutaraldehyde in buffer and sliced as as possible (about 200 lsm) using new razor blades a dissecting microscope. BENTLEY Fig. Tissue pieces were across the fracture site the formation of a cartilaginous callus in a V-shape was observed. layer formed (Fig. neutral red and alcian demonstrate the cartilage microscopy. ALl. changes were found in the cambium Rapid proliferation of the cells differgroups groups layer in this lifted the periosteum away from the bone a collar-shaped callus around the fracture and site 2). ent degrees of throughout the varied between The initial of the periosteum. or after staining with saturated aqueous uranyl acetate for I 5 minutes and Reynolds lead citrate for 5 minutes. 2 3 Figure 2 Section through the callus during early stages of repair (7 days) showing proliferation of the osteoprogenitor cells (op) of the cambium layer of the periosteum (ep) with formation of cartilaginous callus (cr) and of new bone trabeculae (bi) adherent to the compact bone. Y. Identification of the bone trabeculae was achieved using the Sayers alcian blue and haematoxylin method (Sayers I 973). x 40).6 mM-lead prepared incubation fl-glycerophosphate. A. The outer part of the callus showed differentiaof these cells into osteoblasts with the formation of bone trabeculae. ethanols and finally callus across and the pieces were M-cacodylate I% minutes at 370C To specimens morphological was osmium After dehydration 1:2 epoxypropane.085 post-fixed in buffer for one hour. S. In the central peripheral area and uncoated 600-mesh copper grids or on Parlodion-coated 200-mesh grids. could be observed. 3). cationic dyes such blue were used to proteoglycans. buffer at pH 9) for (Fig. The different stages of development of the callus were initially assessed using the haematoxylin and eosin staining method. observed by layer the along OF BONE appearance the cell AND JOINT microof membrane SURGERY an . Thin to 8Onm) were cut on a Reichert OM-U2 tome using diamond knives and collected I tm knives sections (60 ultramicroon either test for false the same as for those observation. Slices excised in a longitudinal plane general morphology 2. tion new methods successive of staining and the stages of fracture radio- healing At each stage. VOLPIN. After two hours the tissue were washed in cacodylate buffer overnight and immersed in freshly (9 mM-sodium 3. mens were and sodium then decalcified formate fluid in Kristensen’s for 48 to 72 hours. G. enchondral calcification were callus. J. Electron 30 non-enzymatic deposition of reaction product. Different cationic dyes demonstrated the production of the extracellular matrix with secretion of proteoglycans from the chondrocytes immersed chondrocytes and 5). New thinly under slices then in I . embedded in Spurr resin. REES. Note the blood clot (ci) in the fracture site (fr) (haematoxylin and neutral red.5% glutaraldehyde these For two hours and of in 0. Alkaline scopic studies. For the visualisation of alkaline phosphatase activity at the ultrastructural level the metal salt method of Lewinson Ct al. Various as azure A. For orientation sections were cut for light microscopy bone were fracture site. on a Philips EM 300 at 60 kV or 80 kV. The area occupied by these specimens. Fig. After washing in buffer for 10 minutes the preparation of these prepared for general RESULTS Using graphic the different evaluations. endosteal cells. electron-dense phosphatase was precipitate THE JOURNAL in the electron activity. consisting of prolifdevelopment of hypertrophic and calcification bone trabeculae on the ofcartilage were also necrotic ends of matrix formed the (Figs 4 by the bone frag- ments. control tissue slices were incubated in medium which had 2 mM-levamisole added as an inhibitor of alkaline phosphatase activity. using glass and stained with Humphrey’s solution. formic The acid speci- mens were then embedded in wax and cut on a sledge microtome set at 7 im thickness. Note the cambium layer ofthe periosteum (cp) (azure A.

leading ofchondrocytes However. the appeared to be around (Figs 6 and 8). was in the matrix 0. 7 around hyper- . indicating increased metabolic activity (Fig. 6). granules. matrix vesicles. These in lacunae inside the cartilage to calcil during most some electron-dense differintense Large early hypertrophic cells. 7). In a few of the mitochondria. In many cells. Golgi apparatus and many mitochondria. Precipitate of lead phosphate indicating enzyme activity (up) is seen along the plasma membrane of the cell. stained for alkaline phosphatase activity. Note the hyperplasia of the endoplasmic reticulum (er).tm to 0. glycogen (gI) and absence of enzymatic activity along the plasma membrane of the cell (pm). Membrane-bound in diameter. No. many mitochondria (ni). AUGUST 1986 Fig. observed scattered extracellular observed. many mitochondria (ni) and intracellular areas of glycogen (go. precipitation chondrocytes found is also evident. accumulation of Figure 6 EM section through early hypertrophic chondrocyte within its lacuna.DISTRIBUTION OF ALKALINE PHOSPHATASE ACTIVITY IN EXPERIMENTALLY PRODUCED CALLUS 631 IN RATS 4’1 - cellular matrix (cc) and cytoplasmic processes ent stages of differentiation. had in diameter well- VOL. gen appeared as masses ofelectron-dense somes developed cytoplasmic organelles such as rough endoplasmic reticulum (RER). amounts ofthe and greater adjacent in electron RER (Figs gen 6 fibres were of the density early than the and glycolarger ribo- 6 and 7). amorphous of glycogen were material found mature hypertrophic chondrocytes. 1 l. Note the hyperplasia of the endoplasmic reticulum (er). Figure 7 EM section through an early hypertrophic chondrocyte stained for alkaline phosphatase activity and inhibited by levamisole (control staining for Fig. 68 B.2 iim between the collaand Fig. which were matrix. 4. The extracellular matrix consists predominantly of collagen fibres.

alkaline phosphatase activity could not be observed in the intercellular matrix because of the calcific deposits. Stripping of the periosteum from the surface of the fracture fragment was found to decrease the osteogenic potential of the bone (McKibbin 1978) and might therefore fracture. 9.‘ -‘. Our study also tiation healing ofcartilaginous process. either osteoblastic that reports gave rise to two or chondroblastic. Figure 9 EM section ofthe extracellular matrix ofthe cartilaginous callus stained for general morphology showing many matrix vesicles (mi) scattered between collagen fibres (/). - . 9 Fig.-- . 13). . The typical cells in that layer of cells.. The result in delayed showed different tissue amount external callus of a long bone to be greater in lower animals produced fractures where JOURNAL degrees at each stage of cartilage of the of differenofthe present fracture in the has been shown previously and also in experimentally unrestricted fracture fragments was allowed also been reported that fractures reduction and internal fixation THE or non-union OF movement of the (Anderson 1965). ofthe study present may heal through where the initial suggest that a process hydroxyapatite of fractures of enchondral deposition takes place within and around extracellular matrix vesicles showing alkaline phosphatase activity.. . 13). “. In the alkaline sections treated by general morphological differentiation vesicles could reaction phosphatase levamisole) observations ofenzymatic groups (those prepared for stages of cell control and those similar and mineralisation be observed. BENTLEY I cf . a . It has treated by anatomical under compression may BONE AND JOINT SURGERY . ALl. 10 Figure 8 EM section through a hypertrophic chondrocyte stained for alkaline phosphatase activity.. A. crystal growth continued and the matrix became calcified (Fig. . VOLPIN. .. 8 Fig. showing an intense reaction along the plasma membrane of the cell (up) and around extracellular matrix vesicles (my). . Our findings indicating that these cambium many source of osteogenic (Kernek and Wray 1973). .. and found of the in the pen- matrix took place needle-like crystals of of these matrix vesicles and around them. Kernek and Wray 1973.e . Y. . As mineral accumulation progressed. McKibbin 1978). around of the intracellular of the cells to mature The enzymatic part of or the reaction whole matrix and hypertrophic matrix vesicles in the intercellular lacunar regions (Figs 8 and 10).’#{231} k my .-. but there products The findings long bones calcification. 632 J.EM section ofthe extracellular matrix ofcartilaginous callus stained for alkaline phosphatase activity. It appeared that these matrix vesicles were formed by budding from the cytoplasmic processes of chondrocytes.G. trophic chondrocytes before any sign of alkaline vesicles phosphatase activity in and around these matrix could be detected only in the stage of further organisation ferentiation cytes. showing intense alkaline phosphatase reaction products around a part ofthe membrane or the whole membrane ofmatrix vesicles (arrows). progenitor cells” (Young different types suggesting responsible for the support previous cells are the pri- cells during fracture healing they have been termed “osteo1962. Fig. S. activity inside was no (Figs the matrix evidence of 7..-! . . The early stages consisted of an intense proliferative response in the cambium layer of the periosteum. the initial signs of tissue of the callus. #{149}J 2im . Note the budding of a matrix vesicle from a longitudinal cytoplasmic process ofthe cell (arrow). REES. At this stage of mineralisation. Note the initial deposition ofneedle-like crystals ofhydroxyapatite (arrows) inside a few matrix vesicles. 12.‘ S . G. Figure 10 . 4. However. # . Calcification of the by progressive hydroxyapatite matrix. but only in those vesicles that showed alkaline phosphatase activity (Figs I I and 12). but could be seen along the cell membrane of the degenerating chondrocytes and in the perilacunar areas. is the main the periosteum development extracellulan deposition of within the centre further difchondro- products were the membrane of DISCUSSION mineral deposition (Figs 8 and 9).

.. 1982). AUGUST 1986 . It was characterised by successive stages of proliferation and hypertrophy together with synthesis. However. of or synovial experimentally observations 633 IN RATS of which.: _. 614 B. Fig. b 02 im Q. Wuthier 1982). have demonstrated that the formation ofmatnix vesicles in the epiphyseal plate occurred by budding (blebbing) from the membrane of the cytoplasmic processes of proliferating chondrocytes (Ali I 980. 1982) and are typi- cal ofenchondral calcification. Wuthier 1982). 4. 1976. enchondral calcification healing in long bones (Bonucci 1970.DISTRIBUTION heal without (Anderson OF ALKALINE external 1965 callus Glimcher have suggested that the metaplasia of the cambium low oxygen tension creased blood supply The progressive external electron due PHOSPHATASE as an et al. 12 13 Figure 11 EM section stained for alkaline phosphatase activity showing initial sign of needle-like hydroxyapatite crystals (arrows) inside the matrix vesicle. No.. It has also been suggested that release of calcium from .. I I Fig. 1980: Boskey 1981. 1980. evidence of intracellular . . mitochondria of tion matrix vesicles play an important of these chondrocytes these cells is the and trigger for that role the forma- mitochondria in matrix of calcifica- laginous callus during the process of fracture healing of long bones.. the and alveolar bone 1967. . These findings resemble those described previously in many other normal calcifying tissues. showing pattern of calcium deposition within and around extracellular matrix vesicles. Figure 12 EM section through the callus stained for general morphology. skeletal have also includneoplasms Alkaline the plasma stages may phosphatase membrane during the process of fracture therefore support these views.. cartilage callus was clearly demonstrated by our light and microscopy. took place within and around extracellular matrix vesicles. . development blood vessels McKibbin of the and in of chondrocytes. These matrix vesicles contained the earliest recognised deposits of hydroxyapatite and were probably the initial site for calcification of the carti- PRODUCED Our CALLUS 1981) and also after grafts (Bab et al. Fig.. Wuthier free grafts produced some amorphous electron-dense material compared with findings of other authors. ACTIVITY intermediate 1980). . stage Other (Sela et al. when could be calhypentrophic and Hunt studies 1978.’ . mandibular condyle. 1976. possibly together with the removal or exclusion of calcification inhibitors (Ali 1976. Similar findings been described in some pathological ing calcified tendons (Yamada 1976). penichondrial authors main factor for this type of layer of the periosteum was to torn (Ham 1930. Calcification of the cartilage. .. during and development ofdifferentiation nous callus. secretion and various components of the intercellular IN EXPERIMENTALLY transplantation 1982) also showed (Ali et al. activity was observed around ofchondnocytes. Ali the epiphyseal predentine growth plate. Boskey 1981. Other authors have suggested that matrix vesicles may promote calcification by the concentration of calcium and phosphate. Anderson 1969. Note the absence of enzyme reaction products in the extracellular matrix and along the membrane of the hypertrophic chondrocytes. which was found to be associated with degeneration of these hypertrophic chondrocytes. I. we could not activity of successive ofthe find this cartilagi- histochemical enzyme in the 4. - VOL.1 . . Anderson et al.: w.. Boskey I 98 1 Wuthier 1982). --‘ h . . defects accumulation Brighton Previous 1977.wI . It was also found that these structures were formed by budding from the cytoplasmic processes of early hypertrophic cartilage cells.I ‘. mainly collagen and proteoglycans.. such as tion (Brighton and Hunt 1978. cium inside the mitochondria of the early chondrocytes Boskey 1981. (Stein et al. . Note the intense enzymatic activity around the matrix vesicles. 1981) into of articular cartilage. Figure 13 EM section through the cartilaginous callus stained for alkaline phosphatase activity inhibited by levamisole (control staining for Figs 10 to 12) showing advanced stages of calcification (m). de- 1978). Our observations of neonatal long bones. conditions. organisation of organic matrix... Lewinson et al.

Sajdera tides in calcifying cartilage matrix. New York etc: Academic Press. J Cell Biol suggested vesicles has a very wide a variety of phosphate have supported in the transfer (Bachra 1970. C. Compression plate of internal fixation on 1965:47-A: 19 1-208. Arch and specialization J Cell Biol 1962: JOURNAL OF BONE studies Histol on Jap calcifiand the calci1976:39: during enchondral 14:357 70. plate Joint and ATPase of isolated Res 1976:22: 1 -7. The possible significance of hexosephosphoric fication. epiphyseal methods. J Bone Joint Surg[Am] l98O:62-A:96473. ed. Toister Z. matrix vesicles of calcifying cartilage (Ali 1976. eds. 17:286-93. Fine structure Res 1967:20:33-SO. Felix for hydrolysis of phosincrease in the concentra- such as sodium glycerophosphate. Ralphs and Mr D. Phosphatases of studied by electron microscopic cytochemical chem Cytochem 1971 . In: Hall DA. An intense reaction was also observed around the membrane of the extracellular matrix vesicles before any signs of mineral deposition. Bab IA. C/in early proliferation Orthop 1973 phases of bone repair. Y. and their role in the calcification 1976:35: 135-42. of early Boskey AL. Ellis RD. of the physiology 1981 : 157:225-57. REES. Ali Analysis epiphyseal enzyme around and 1981. Clin Orthop Brighton CT. in the formation : 197-209. Ham AW. Vesicles associated physeal cartilage. as was described al. Ultrastructural and of the tendon-bone Young RW. of calciSurg cartilage Glimcher MJ. A review of the primary mechanism of enchondral cation with special emphasis on the role ofcells. followed by deposition of calcium. SY. Vol. VOLPIN. Salomon 1982). Cancer 1981 :48: 1602-10. nucleus or cytoplasm of proliferating J. Wray JR. Wuthier (1982) suggested that the enzyme acts primarily as an inorganic phosphate-binding is that protein the matrix vesicles. Calcification took place only within and around matrix vesicles showing enzymatic activity. DS. Matsuzawa vesicles Anderson 1971. Sela J. Wisby A. Matsuzawa T. of Anderson HC. made Boskey similar Au s. Bachra BN. Goodfellow J. AND JOINT SURGERY . alternative pyrophosphate. 1980. The fication as demonstrated [Am] l978:60-A:630-9. Fleisch H. In: Bourne GH. The reaction of alkaline phosphatase appeared These observations suggest that alkaline phosplays an essential role in calcification of carticallus during fracture healing of long bones. The biology Surg[Br] 1978. 1982). crystals in of the of cartilaginous callus development and emphasises the functional association between alkaline phosphatase activity and enchondral calcification mediated by extracellular matrix vesicles during the process of experimental fracture healing. 91 Lewinson D. Au s. J Microsc 1977. (1982) in mandibular condyles. and distributional during the matura30: 26 1-9. eds. Sela J. Current concepts calcification. A general staining technique bone. cartilage calcification. A histological study of the JBoneJoint Surg 1930. 1980) found that alkaline phosphatase of matrix vesicles cartilage. Cell proliferation osteogenesis in young rats. or in association with hydroxyapatite deposition. Kernek CB. phosphate in the organic matrix (Robison 1923. Extracellular human osteogenic neoplasms: an ultrastructural study. Biochem J 1923. We are deeply indebted to Dr J. C’ell Tissue Res 1981 :214:449-54. fication 347-78. Stein H. 619-30. Stein H. Matsuzawa T. 15:201-12. Acta Anat l982. J Bone R. role process of enchondral but several theories explain that phate this the of alkaline enzyme during the calcification is not yet established have been proposed in an attempt association. Mechanism of calcification. Felix and Fleisch 1976). Adams for typing the manuscript. Other authors have 1974. A. Boskey 1981). Scient(ficfoundations oforthopaedics and traumatology. New York etc: Academic Press. esters. Fed Proc Anderson HC. 1970: 165-208. Joint in ossi- activity Tissue for the demonstration of Res Sayers DCJ. London: Heinemann Medical. 1980). J Bone Joint Surg Bab I. J Ultrasiruct and biochemistry role ofmitochondria in growth in a rachitic model. Shapiro F. 2nd ed. (‘alcif 1974. Eyre DR. Silbermann M. Bullough P. Cellular ture callus in rat tibia. to tion of phosphate ions. McKibbin B.60-B: offracture 150-62. THE cytochemical joint. Muhlrad A. studies of tissues undergoing enchondral calcifi- Previous cation have shown high around the cell membrane matrix concentrations of this of chondrocytes and (Robison 1923. Yamada M. Hunt RM. of and isolated can matrix hydrolyse Anderson HC. ALl. In: Owen R. phatase laginous S. Sela J. of by Lewinson et most intensive to be around the membrane of early and mature hypertrophic chondrocytes. phosphatase Various is responsible in a local resulting authors have specificity esters ATP. of frac- . The occurrence of hydroxyapatite extracellular matrix vesicles after surgical manipulation rabbit kneejoint. mitochondria matrix vesicles. in the matrix of epi- LD.32(5): fixation fracture and the effect of different healing. 4. Calcification of connective tissue. The biochemistrt’ and physiology ofbone. Other authors the theory that the enzyme participates of phosphate groups to the organic matrix Salomon 1974. J Histochem Cytochem 1982. cartilage J HistoJBone Robison R. 1976: 135-57. 111: 65-76. SW. 1980). esters Salomon CD.634 G. Membranous parTrans N Y AcadSci 1970. All SY. matrix and vesicles in enzymatic Stein H. Bonucci E. JSci Tech 1973:17(4): 14-16. Ali 1976. chondrocytes the cartilaginous callus. Matrix vesicles of cartilage and bone. CalcfTissue Jackson Vol. Preparation ofthin cryo-sections for electron probe analysis ofcalcifying cartilage. Quantitative changes in the activity ofalkaline phosphatase tion ofcartilage. 1980: 175 84. of new Wuthier RE. Pyrophosphatase matrix vesicles. An possibility the enzyme participates in the removal of inhibitors of mineralisation (Felix and Fleisch 1976. healing in long bones. Anderson HC. 12:827-44. C/in Orthop 1982. J. 5. Wuthier observations on epiphyseal cartilage is associated with matrix vesicles and that the enzyme content may facilitate the calcification process initiated by matrix vesicles (Ali 1976. AMP and glucose-6-phosphate. Further studies are required to ascertain the exact role of this enzyme during enchondral calcification and subsequent bone formation. I 13:53-60. A fine structural study on the extracellular alkaline phosphatase and its role in calcification. 19:801 -8. Lewinson et al. The present work in calcification describes the successive mediated stages by Anderson types [Am] with calcification 1969:41 : 59-72. 169:219-42. Sayers for expert technical help and constructive comments and also to Miss L. Gray C. Transplantation of free perichondrial grafts into rabbit articular cartilage is associated with matrix vesicle calcification. Bab IA. Ali (1976. Changes in tissue morphology and collagen composition during the repair of cortical bone in the adult chicken. International review of connective tissue research. It was also suggested by Ali that over 80% of the alkaline phosphatase activity in exact BENTLEY REFERENCES isolated The G. Phillips M.