Remodeling of Bone in Tissue Culture
PAUL GOLDHABER
Harvard School of Dental Afedicine, Boston, M1assachusetts
The in vitro cultivation of bone cells, bone
tissue, and whole limb buds has attracted the
attention of many investigators who prefer a
living system wherein morphologic and bio-
chemical changes may be observed, meas-
ured, and correlated in response to experi-
mental modifications of the environment.
Much significant data has been obtained on
bone resorption or formation by using
culturing technics in which one or the other
process was stimulated by the experimental
conditions employed. Such studies have
been reviewed by Fell,' Goldhaber,2 and
Bassett.' To date, however, none of the sys-
tems has been successfully used to demon-
strate the simultaneous occurrence of bone
resorption, osteoid formation, and calcifica-
tion to any appreciable extent. The purpose
of the present report is to describe some
observations of such a tissue culture system
which, in the presence of suitable environ-
mental factors, is capable of undergoing
rapid remodeling, bringing into play many
of the cellular and extracellular phenomena
associated with bone repair.
Materials and Methods
Although most of our published studies
to date concerning the behavior of bone in
tissue culture have emphasized the mor-
phologic and biochemical alterations associ-
ated with bone resorption as influenced by
various factors, it was recognized initially
that our tissue culture approach provided an
opportunity to study the mechanisms of
bone formation and calcification, since
young mouse calvaria exposed to high con-
centrations of oxygen maintained the addi-
tional capacity to form small amounts of
new osteoid within the tissue after the
initial phase of rapid bone resorption had
This work was supported in part by Research Contract
DA-49-193-MD-2018 from the U.S. Army Medical Research &
Development Command, Washington, D.C.; U.S. Public Re-
search Grant DE 01298 from the National Institute of Dental
Research, National Institutes of Health, Bethesda, Md.; and
U.S. Public Health Service Career Development Award 2-KE-
DE-752 from the National Institute of Dental Research, Na-
tional Institutes of Health, Bethesda, Md.
490
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ceased.4 Attempts to make this osteoid
calcify in culture were unsuccessful.' During
the past several years, to improve and re-
fine our tissue culture system for the study
of bone resorption, two successive modifica-
tions in technic were instituted.
The first change consisted of placing cul-
ture tubes in a roller mechanism during in-
cubation and using lower oxygen tensions
(30 to 50 per cent) in the gas phase instead
of utilizing stationary culture tubes in which
the gas phase contained 95 per cent 02 with
5 per cent CO2 or 100 per cent 02. The
medium was the same as that used previous-
ly, consisting of Gey's balanced salt solution,
heated horse serum, and chicken embryo
extract (6:2:2). Under these conditions,
good resorption occurred despite the lower
oxygen tensions in the gas phase.
In the second modification (still using
the roller tubes), embryo extract was elimi-
nated from the medium, which then con-
sisted of Gey's balanced salt solution and
horse serum (2:8). Under these conditions,
even in the presence of 50 per cent 02 in the
gas phase, bone resorption was significantly
inhibited. The addition of 0.5 unit of
parathyroid extract* per milliliter of horse
serum medium, however, resulted in massive
bone resorption that almost completely de-
stroyed the bone by 2 weeks in culture.5'-
Enhanced bone resorption could also be
demonstrated by the addition of high con-
centrations of crystalline vitamin A or vita-
min D in place of the parathyroid extract.
Bone formation, however, was never seen in
the horse serum medium (which lacked
embryo extract) regardless of the presence
or absence of parathyroid extract, crystal-
line vitamin A, or vitamin D. The combined
use of the original embryo extract-contain-
ing medium in roller-tube cultures under an
atmosphere of 50 per cent 02 has provided
our best tissue culture system to date for the
study of bone remodeling.
* Eli Lilly & Co., Indianapolis, Ind.
Vol. 45, 1966 REMODELING OF BONE IN TISSUE CULTURE 491

In the studies to be reported here, These islands seemed to grow and fuse,
calvaria from 5-day-old mice of two differ- forming various trabecular patterns that at
ent strains (Webster and Charles River times appeared to be more opaque to trans-
CD 1) were utilized. Each calvarium was re- mitted light than the remaining original
moved aseptically, the occipital bone was bone. In the 9-day living culture shown
eliminated, and the remaining portion (Fig. 1), new trabeculae have formed medial
(frontal and parietal bones) was placed on a to the original bone, extending partially into
rectangular glass coverslip and covered with the resorbed area. In view of our previous
a thin mixture of chicken plasma and findings using stationary tubes of new oste-
chicken embryo extract. After clotting, each oid in embryo extract-containing media,2' 4 it
coverslip with its calvarium was inserted was assumed that these dark trabeculae de-
into the flat well of a Leighton tube, covered veloping in the living cultures represented
with 2 ml. of a supernatant medium com- new osteoid that appeared opaque because
posed of Gey's balanced salt solution, heated of its thickness and density. As expected,
horse serum, and 11- or 13-day chicken horizontal histologic sections through a de-
embryo extract in a ratio of 6:2:2. Each calcified 14-day culture revealed osteoclastic
milliliter of medium contained 100 units of resorption of old bone (Fig. 2) and the
penicillin and 100 mg. of streptomycin. The presence of new osteoid (Fig. 3). In some
air in the tubes was replaced by brief gassing regions, however, the new osteoid appeared
with a mixture of 50 per cent 02 and 50 per to be calcifying (Fig. 4). The clear space
cent N2. After stoppering, the tubes were around the osteoclasts (Fig. 2, 4) were here-
placed in a horizontal position in a roller tofore assumed to represent shrinkage arti-
drum (1 revolution/5 minutes) that was facts. By correlating histologic sections with
kept in an incubator at 370 C. The cultures the appearance of osteoclasts containing
were refed and gassed every 2 to 3 days for giant vacuoles in the living cultures, we now
periods up to approximately 3 weeks. Micro- realize that this space is real and correlates
scopic observations of the living cultures with the giant vacuoles that are found in
were made daily and, at the conclusion of osteoclasts that are or have been function-
each experiment, the cultures were fixed in ing at maximum capacity.7
10 per cent neutral formalin, embedded in
paraffin, and sectioned horizontally or in New Osteoid Formation
cross section for routine staining with and Calcification
hematoxylin and eosin, or von Kossa's stain.
Other specimens were decalcified prior to Although the histologic differences be-
paraffin embedding and routine staining. tween the original bone and new osteoid
appeared quite obvious (particularly on
Bone Resorption and Formation cross section), it was deemed worthwhile to
demonstrate in some other manner that new
Bone resorption was first noted in the bone collagen was being formed
sagittal suture area of the frontal bone at tem. Accordingly, tritiated prolinein (0.1the sys-
gc./
about to 3 days. The resorption continued ml. of medium) was added to the supernatant
2
rapidly in most cultures so that by 5 to 6 fluid of each feeding. After 23
days the entire suture area was destroyed. ture, autoradiographs of the days in cul-
decalcified
In vigorously resorbing cultures, resorption bone were
progressed laterally from the midline, clear- radiograph ofprepared. An unstained auto-
such a culture, sectioned hori-
ing out a strip of bone one-fifth to one- zontally, is shown (Fig. 5). The exposed
fourth the width of the calvarium. In such grains clearly correspond
cultures, resorption of bone around the cellular bone collagen that tohasthe new extra-
formed
frontoparietal sutures urther decreased the sagittal suture area of the frontal in
f the
bone.
bone mass, frequently leading to contraction
and collapse of the remaining calvarium. By Along this line, other biochemical studies
6 to 7 days, the process of resorption had utilizing this tissue culture system with air
slowed and it was noted that the "clear in the gas phase have shown that the addi-
areas"l were becoming more dense and less tion of C'4-proline to the medium resulted
translucent. By 8 to 9 days, a number of in the formation of C14-hydroxyproline,
cultures had developed small, discrete, dark thereby indicating that new collagen had
islands within the resorbed suture areas. been synthesized.8

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 ---as 7z.w.>.ix;.

FIG. 1.-Frontal bone of calvarium, 9 days in culture. Pale strip in center is the midline (M). Solid dark
areas bordering on upper and lower edges of photomicrograph are the original bone (OB) that has not been
resorbed. The new trabeculae (NT) are more opaque and extend into the previously resorbed area, which is
becoming more dense.
FIG. 2.-A decalcified hematoxylin and eosin-stained horizontal section through a 14-day culture, showing
resorption of the darkly stained original bone (OB) by at least three osteoclasts (OCL). Note the clear space
around several osteoclasts. This corresponds to the giant vacuoles associated with these cells. In addition,
note the "free" osteoclast in the connective tissue.
FIG. 3.-Same culture as in Figure 2, showing new uncalcified osteoid (OI) on either side of midline (M).
FIG. 4.-Same culture as in Figure 2, showing new osteoid undergoing calcification (COI). The paler
osteoid below has not yet calcified, whereas the darker area above is presumably more heavily mineralized.
Note the numerous osteoclasts immediately adjacent to all three types of osteoid and the vacuolar space
associated with several of these cells.
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 I
100op/I li 5 O, .
i- .. .

EEO nJs~j Z

FIG. 5.-Autoradiograph of horizontal section of decalcified calvarium which has been in tissue culture for
23 days with H3-proline (0.1 Ac./ml.) added to the medium at each feeding. The slide has not been stained.
Note that the heavy concentration of grains on either side of the midline corresponds to the pattern of new
bone collagen (NBC).
FIG. 6.-Horizontal section through an undecalcified calvarium (in tissue culture for 22 days) stained
with the Von Kossa reaction and counterstained with safranin 0. Note the pale, uncalcified new osteoid
(01) and the dark-staining Von Kossa "positive" areas, which represent remnants of original bone and new
osteoid in various stages of mineralization (COI).
FIG. 7.-Hematoxylin and eosin-stained cross section through a calvarium 21 days in culture, showing
a broad strip of new osteoid (01) which is undergoing calcification at one end (COI).
FIG. 8.-Hematoxylin and eosin-stained cross section through a calvarium 14 days in culture, showing a
band of new osteoid (OI) undergoing central calcification (COI).
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494 GOLDHA BER
As mentioned previously, some of the
hematoxylin-and-eosin-stained sections indi-
cated that, although most of the new osteoid
did not appear mineralized, some regions of
new osteoid took on the basophilic staining
characteristics associated with calcification.
This observation was confirmed by subject-
ing undecalcified tissue sections to the von
Kossa reaction: the new, calcifying osteoid
stained dark brown (Fig. 6). As seen in the
hematoxylin-and-eosin-stained cross sec-
tions (Fig. 7, 8), calcification within the
calvarium occurred selectively within the
collagen fibers of the new osteoid. It was not
clear why only a minor portion of the avail-
able new osteoid was calcifying, however.
These histologic findings suggested the
possibility that the dark trabeculae develop-
ing in the cultures were calcifying or calci-
fied osteoid rather than uncalcified osteoid
as originally thought, thereby probably
accounting for their opaqueness when
viewed with transmitted light. Further evi-
dence suggesting that this idea was correct
was obtained by subjecting calvaria (which
had developed new trabeculae in tissue cul-
ture) to brief fixation in 10 per cent forma-
lin, followed by distilled water rinses and
immersion in a decalcifying solution of
sodium citrate and formic acid. Under these
conditions, the new dark trabeculae disap-
peared almost instantaneously, leaving be-
hind a faint outline of the former trabeculae
that failed to disappear completely on pro-
longed immersion in the decalcifying solu-
tion. In the sagittal suture areas of the
frontal bone and parietal bones from a single
living culture (Fig. 9, 10), the previously
resorbed regions are partially filled with
new, dark trabeculae. The effects of the de-
calcifying solution on these respective
regions are shown (Fig. 11, 12) within sev-
eral minutes after solution was added to the
tube. These results indicate that the new
dark trabeculae seen in the living cultures
were definitely mineralized. In addition, the
rapid dissolution suggests that their crystals
were of small size and highly reactive. The
appearance was not appreciably altered
after immersion in the decalcifying solution
for 18 hours (Fig. 13). With regard to the
"residual" density remaining after the
decalcifying solution has acted, this could be
accounted for by the presence of the osteoid
matrix. This increased density might also
represent in part a significant alteration in
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J. dent. Res. Supplement to No. 3
the osteoid matrix that makes it calcifiable.
If so, it remains to be shown whether this
increased density represents a "maturation"
of the collagen within the osteoid matrix,9
or the influx of some mucopolysaccharide'O
or a lipid.1' Each of these factors has been
implicated in the calcification mechanism.
Although calcification within the calvarium
is limited to the collagen fibers of the osteoid
matrix, thereby supporting the idea of a
physicochemical mechanism for the initia-
tion of mineralization based on the stereo-
chemistry of collagen,'2 the fact that most
of the osteoid in these cultures appeared un-
mineralized suggests that such osteoid had
not yet "matured," or that it lacked the
presence of some other calcifying agent.
Apparently, there was an adequate supply
of minerals available in the medium. As seen
in Figure 14 (a gross photograph of the
tissue culture tube and calvarium shown in
Figure 1), the whitish material surrounding
the calvarium was an accumulation of cal-
cium salts that had precipitated into the
plasma clot. This heavy precipitate had
failed to take place in the clot overlying the
calvarium, and it did not begin immediately
adjacent to the calvarium but left a precipi-
tate-free zone which, at higher magnifica-
tion, may be seen to be completely filled
with outgrowing fibroblast-like cells (Fig.
15). From previous x-ray diffraction studies,
it has been shown that the inorganic reflec-
tions of this material are consistent with an
apatite-like lattice. On the basis of pH indi-
cator studies (using 0.3 to 1.0 per cent
phenol red), it was postulated that mineral
salts precipitate more readily in the plasma
clot rather than within the bone because the
rapidly metabolizing calvarium has a more
acid pH than the surrounding supernatant
medium and clot.2 Whether pyrophosphate
inhibition of calcification and pyrophos-
phatase"3 play a role in this system remains
to be seen.
Remodeling of New Trabeculae
Continued observation of cultures form-
ing new trabeculae in the resorbed sites
revealed that the amount and pattern of
formation was completely unpredictable.
Some cultures did manage to fill in most of
the defect so that the gap between the re-
maining portions of original bone was essen-
tially bridged. Close observation, however,
Vol. 45, 1966
revealed that the new trabeculae were
rapidly remodeling, growing, and fusing in
some regions and resorbing in others. This
remodeling process may be demonstrated by
periodically photographing the same area.
The new dark trabeculae that formed in the
REMODELING OF BONE IN TISSUE CULTURE 495
resorbed sagittal suture area of the frontal
bone after 10 days in culture are shown
(Fig. 16), as in the remodeling that occurred
by 11, 12, and 14 days, respectively (Fig.
17-19). By 16 days, much of the new bone
formed previously had resorbed (Fig. 20).

FIG. 9.-Frontal bone of 13-day old living culture, showing remaining original bone (OB) and darker new
trabeculae (NT) growing into resorbed sagittal suture area. Note the two dark islands and the arrow-like
protrusion (arrows).
FIG. 10.-Parietal bone area of same living calvarium as shown in Figure 9. Note the remaining origi-
nal bone (OB) and the darker, new trabeculae (NT) which have grown into the resorbed suture area.
FIG. 11. -Appearance of same area as shown in Figure 9 after formalin fixation, distilled water rinses, and
immersion in a sodium citrate-formic acid decalcifying solution for several minutes. Note that the opaque
trabeculae have faded considerably but that some dense material still remains (arrows).
FIG. 12.-Appearance of same area as shown in Figure 10 after decalcifying solution. Dark round bodies
are air bubbles (B) in the decalcifying solution.
FIG. 13.-Same area as shown in Figures 9 and 11, after 18 hours in decalcifying solution. Very slight
change from Figure 11. Note that the trabecular matrix is still visible (arrows).

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496 GOLDHABER J.'dent. Res. Supplement to No. 3
On the following day, however, a number of that subcutaneous implantation of the
new bone-forming centers appeared to be osteoid of devitalized autogenous bone from
arising (Fig. 21). These grew significantly rachitic rats was not resorbed by giant cells
during the next 2 days (Fig. 22, 23), the im- until the osteoid had become calcified. Since
pression being obtained that trabecular decalcified implants from normal rats began
growth and resorption developed somewhat to be resorbed by multinucleated giant cells
symmetrically on both sides of the midline. sooner than the rachitic bone, it was con-
cluded that bone matrix that is or has been
Resorption of Osteoid calcified exerts a tropic effect that influences
During the past several decades, a num- such cells, whereas rachitic osteoid that has
ber of investigators have reported that the not been calcified does not.
osteoid that accumulates during rickets does modeling As stated previously, our rapidly re-
not resort.'4' 15 Furthermore, the injection
cultures contained regions of origi-
of rachitic rats with parathyroid extract nal calcified bone and calcified and calcify-
leads to the appearance of osteoclasts on ing new osteoid, as well as uncalcified new
calcified bone, whereas the osteoid remains portunity osteoid, thereby providing an excellent op-
undisturbed."5 Once the osteoid calcifies these typesfor of
determining whether any of
tissue was resistant to resorp-
during healing, however, it becomes sus- tion, particularly by
ceptible to osteoclastic action. More recent- scopic observations of osteoclasts. the living
Micro-
cultures
ly, it was shown by Irving and Handelman"7 clearly revealed that the original calcified
bone underwent osteoclastic resorption.
This was confirmed on histologic examina-
tion (Fig. 24). The resorption of the calcified
new bone trabeculae also was observed in
the living cultures (Fig. 16-23), although
typical osteoclastic activity was not obvi-
ous, except in the histologic material. Un-
calcified osteoid could not be identified
readily in the living cultures, but it was
easily recognizable histologically on cross
section. Surprisingly, it was found that this
tissue was not resistant to osteoclastic re-
sorption. In contrast to Figure 24, where an
osteoclast is shown selectively attacking
the original calcified bone in preference to
the uncalcified osteoid, Figure 25 shows an
osteoclast attacking uncalcified osteoid in
preference to the original calcified bone. An-
other example of osteoclastic resorption of
uncalcified osteoid may be seen where only
osteoid is available (Fig. 26). In some
regions, the osteoid appeared to have been
"hollowed-out" by loose fibroblast-like cells,
some of which seemed to be fused to form
"young" osteoclasts. Although examples of
osteoclastic resorption of calcified bone were
numerous, osteoclastic resorption of uncal-
FI-. 14. Gross photograph of culture shown cified osteoid was not rare. These findings
microscopically in Figure 1. Calvarium (C) is sur-
rounded by calcium salt precipitate (P) in plasma should dispel the current notion that un-
clot. Note thin, precipitate-free zone (PF) immedi- calcified osteoid is unattractive to osteo-
ately adjacent to the calvarium. clasts and resistant to resorption.
FIG. 15. Photomicrograph of same calvarium as
seen in Figure 14, showing the precipitate-free zone Summary
(PF) filled with proliferating, fibroblast-like cells,
lying between the calvarium (C) and the precipitate In the presence of a suitable environment,
(P). which includes chicken embryo extract in

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 FIG. 16.-Living calvarium, 10 days in culture, showing the remaining original bone
(OB) and the new trabeculae (NT) that have formed in the previously resorbed sagittal
suture area of the frontal bone.
FIG. 17, 18, 19.-The same area as in Figure 16 after 11, 12, and 14 days in culture,
respectively. Note the continuous remodeling of the trabeculae.
FIG. 20.-The same area after 16 days in culture. Note that most of the new trabecu-
lae in the central area have completely resorbed, although the new trabeculae (NT)
adjacent to the original bone are very dense.
FIG. 21.-The same area as in Figure 20 after 17 days in culture. Note the appear-
ance of four new bone-forming centers (A, B, C, and D).
FIG. 22.-The same area as in Figure 21 after 18 days in culture. Note that each of
the new bone-forming centers (A, B, C, and D) has darkened, grown, and fused with
other trabeculae that had been present the previous day.
FIG. 23.-The same area as in Figure 22 after 19 days in culture. The new trabeculae
have continued to grow (A, B, C, and D). Note that the trabecular pattern at this stage
bears no resemblance at all to that found after 10 days in culture (Fig. 16).

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 FIG. 24. Hematoxylin and eosin-stained cross section of calvarium after 17 days in culture. New osteoid
(01) has formed on the surface of the darker staining original bone (OB), which is undergoing resorption by
osteoclasts (OCL).
FIG, 25.-Another area from the same culture as that seen in Figure 24. Note the new osteoid (01) which
has formed on the thin, original bone (OB). In this area, however, the osteoid is undergoing osteoclastic
resorption (OCL) rather than the original bone.
FIG. 26.-Another area from the same culture as that seen in Figures 24 and 25. A wide area of osteoid
(OI) is seen, and it is being resorbed by an osteoclast (OCL). No original bone is seen in this section.
FiG. 27.-Hematoxylin and eosin-stained cross section of calvarium after 21 days in culture. The broad
band of new osteoid (OI) which hasjformed appears to be "hollowing out" due to the activity of some
C"young!! osteoclasts (OCL).
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Vol. 45, 1966
the medium and 50 per cent 02 in the gas
phase, roller-tube cultures of young mouse
calvaria have been stimulated to resorb the
original bone, lay down an osteoid matrix,
calcify portions of it, and finally resorb
these newly calcified trabeculae in a con-
tinuous process of bone remodeling that
may be readily visualized in the living cul-
tures. The fact that calcification within the
tissue was limited to certain portions of the
new osteoid collagen (despite the fact that
large amounts of calcium salts precipitated
in the surrounding plasma clot) indicates
that a calcification inhibitor factor(s) is
present in the noncalcifying portion of this
tissue. Apparently, such a factor is over-
come in certain limited sites of new osteoid
that selectively undergo calcification. Of
further interest was the finding that, con-
trary to current concepts, uncalcified osteoid
may be attacked and resorbed by osteo-
clasts.
The author thanks Miss Gunta Cirulis and Miss
Lidija Trencis for their technical assistance.
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