/ . Embryol exp. Morph. Vol. 54, pp. 185-207, 1979
Printed in Great Britain © Company of Biologists Limited 1979
185
In vitro studies on skeletogenic potential of
membrane bone periosteal cells
By PETER THOROGOOD1
Department of Zoology, University of Southampton, U.K.
SUMMARY
In the avian embryo ectomesenchyme cells, derived from the mesencephalic level of the
cranial neural crest, migrate into the presumptive maxillary region and subsequently differentiate into the membrane bones and associated secondary cartilage of the upper jaw skeleton.
The cartilage arises secondarily within the periosteum at points of articulation between
membrane bones and provides an embryonic articulating surface. The stimulus for the
differentiation of secondary cartilage is believed to be intermittent pressure and shear
created at the developing embryonic movement. The development of one such system the quadratojugal, has been analysed using organ and explant culture techniques and
studied with particular reference to the differentiation of periosteal cells into secondary
cartilage. A number of conclusions were reached.
(1) Normally only cells at discrete loci express a chondrogenic potential in vivo: the
periosteal cells at these sites of future articulation become committed to chondrogenesis
during stage 35, more than 24 h before cartilage is identifiable in vivo.
(2) However, cells with a 'latent' chondrogenic potential are widespread in membrane
bone periosteum and occur over most, if not all, of the surface area of the bone. This potential
is expressed in the 'permissive' environment created by submersion of the tissue in explant
culture or in submerged organ culture.
(3) This chondrogenic potential exists long before the time at which commitment of
cartilage-forming cells occurs and even presumptive maxillary ectomesenchyme at stage 29
has a limited ability to form cartilage in vitro.
It is suggested that spatial position is a principal factor controlling the differentiation of
secondary cartilage. Ectomesenchyme cells with the potential to form secondary cartilage are
widespread but it is only those cells whose migration from the neural crest positions them and
their progeny at the site of a presumptive joint which subsequently express this potential.
This epigenetic interpretation is discussed in the general context of development mechanisms
underlying the spatial and temporal patterns in which neural crest-derived cells differentiate
to produce bone and cartilage during the formation of the head skeleton.
INTRODUCTION
The major part of the bony and cartilaginous components of the head
skeleton in the vertebrate groups studied to date, are formed by ectomesenchyme
cells derived from the neural crest (Horstadius, 1950; Weston, 1970; Le Lievre &
Le Douarin, 1975; Le Lievre, 1978). Whereas 'mesodermal-derived' skeleto1
Author's present address: Department of Biology, Medical and Biological Sciences
Building, Bassett Crescent East, Southampton, SO9 3TU, U.K.
186
P. THOROGOOD
Fig. 1. A diagrammatic summary of the developmental history of the cells which
comprise the quadratojugal (QJ): (A) Stages 10-11: Cells from the mesencephalic
NC migrate rostrally and ventrally. (B) Stage 16: NC-derived cells arrive at presumptive maxillary region. (C) Stage 30: Condensation of ectomesenchyme cells to form
QJ primordium followed shortly by membrane bone formation. (D) Stage 37:
Secondary cartilage formation starts on the anterior and posterior aspects of the
'hook' of the QJ. (Not drawn to scale).
genie cells may be regarded as differentiating in situ, mesenchyme cells derived
from the neural crest (N.C.) undergo precise migration patterns before differentiation and it is generally thought that it is the pathway along which NC cells
migrate that determines the nature of their subsequent differentiation (Le
Douarin & Teillet, 1974). In the case of skeletogenic cells from the N . C , some
differentiate directly into bone, i.e. membrane bone, and some into primary
cartilaginous models (e.g. Meckel's cartilage and quadrate) which may or may
not undergo a degree of bone replacement. The role of migration pathways,
and of the range of subsequent cell-cell and cell-matrix associations generated
by different migration routes, in the differentiation of skeletogenic NC cells has
been discussed and reviewed elsewhere (Morris & Thorogood, 1978).
In this paper I shall describe an analysis of one such NC-derived skeletogenic
system, namely the membrane bones in the maxillary process of the chick
embryo. The skeleton of the margin of the upper jaw comprises three membrane
bones and I shall consider the most posterior of these - the quadratojugal (QJ).
It is a slender bone comprising a shaft, and a hook at the posterior end where
it articulates with a groove in the quadrate. The QJ system was selected for
study, not for any major intrinsic properties but because a considerable body
of information concerning its developmental history has accumulated in recent
Skeletogenic potential of membrane bone periosteal cells
187
3
years (see Fig. 1). From experiments using grafts labelled with [ H]thymidine,
it is known that NC cells begin to leave the mesencephalic level of the developing
brain at approximately 36 h and migrate rostrally and ventrally (Johnston,
1966; Noden, 1975). The first of these cells, or their progeny, arrive in the
presumptive maxillary process at 52 h. There is subsequently a proliferation of
these 'ectomesenchyme' cells (Johnston, 1966) and at 7 days there is a condensation of the mesenchyme cells followed at 1\ days by the appearance of a calcified
matrix (Murray, 1963); note that this is bone formation directly not via a
cartilaginous model. Using the self-replicating marker of the heterospecific
quail-chick grafting system, it has been demonstrated that most, if not all, of
the bone and associated periosteum is derived from the NC (Le Lievre & Le
Douarin, 1975), On the twelfth day of incubation, two pads of cartilage appear
beneath the periosteum on the posterior hook. This constitutes the embryonic
articulating surface with the quadrate and, because it is cartilage deposited on
the surface of the bone by a periosteum, it has been termed 'secondary' or
'adventitious' cartilage (Murray, 1963). Analysis of the fate of quail mesencephalic grafts into chick embryo hosts has revealed that these chondrogenic
cells, like their osteogenic neighbours, are derived from the NC at the level of
the mid-brain (Le Lievre, 1978, personal communication).
It is now known that for NC-derived maxillary mesenchyme to become
osteogenic and subsequently form membrane bone an interaction with maxillary
epithelium must take place (Tyler, 1978; Hall, 1978). In vivo the cells from the
NC 'arrive' in the presumptive maxillary region at 2\ days and bone formation
starts at 1-1\ days (see Fig. 1). For isolated maxillary mesenchyme to form
bone in either organ culture (Hall, 1978) or when grafted onto the chorioallantoic membrane (Tyler, 1978), there is a dependency upon an epithelial
presence until 3-1—4 days of development. In the absence of that component,
the cultured or grafted mesenchyme gives rise only to fibrous connective tissue.
It has been proposed, that the causal mechanism underlying chondrogenesis is
simply the biomechanical effect of movement. The embryo starts to show a
dramatic increase in non-reflexogenic, spontaneous whole body movements
from about 8 days onwards (Hamburger, Balaban, Oppenheim & Wenger, 1965)
and it is believed that the local effects at embryonic articulations such as these,
are intermittent pressure and/or shearing forces. Murray and colleagues demonstrated that following in ovo paralysis by use of post-synaptic blocking agents
such as curare or decamethonium, membrane bone differentiated normally
but there was an absence of secondary cartilage associated with the articulations
between membrane bones (Murray & Drachman, 1967). The causal relationship
between movement and secondary chondrogenesis is better demonstrated by
grafting the joint to an ectopic site where the tissues are not exposed to such
forces (Murray & Smiles, 1965; Hall, 1972) or by organ culture of early QJ
rudiments in which the periosteal cells are not 'determined' for chondrogenesis.
By the simple expedient of applying gentle intermittent pressure to the joint
13
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54
188
P. THOROGOOD
surfaces early in the culture period, chondrogenesis can be stimulated (Hall,
1968).
The overall objective of this study and of work to be reported in subsequent
communications is to achieve an understanding of the developmental factors
which determine the spatial and temporal pattern in which NC-derived cells
differentiate into bone and cartilage during the formation of the head skeleton.
The initial objective was to analyse the relationship between embryonic movement and the subsequent differentiation of ectomesenchyme cells into cartilage.
The analysis was originally planned in two steps:
(i) Firstly, to investigate the temporal relationship between the commitment
of cells to cartilage formation and the duration of in ovo movement (i.e. when do
periosteal cells of the QJ hook become committed to chondrogenesis). To
establish this whole QJs from normal and paralysed embryos of various ages
were grown in organ culture and the incidence of in vitro chondrogenesis recorded: contralateral QJs were fixed at the outset of the culture period and used as
controls.
(ii) Having established the timing of commitment, was it then possible to
grow uncommitted periosteal cells in large numbers, and by subjecting the cells
to various in vitro manipulations, cause a chondrogenic differentiation? If such
an in vitro system of chondrogenesis could be established then it might provide
an insight into the acquisition of chondrogenic ability by the in vivo counterparts of these cells. Explants of 'uncommitted' periosteum were grown in order
to obtain the appropriate cells but unexpectedly the primary outgrowths from
such explants spontaneously became chondrogenic. Consequently the second
part of the investigation centred upon the acquisition of chondrogenic potential
by these cells and the factors controlling the expression of that potential.
MATERIALS AND METHODS
All embryos used in this study were from White Leghorn x Light Sussex
eggs obtained locally. The eggs were incubated at 37-0 ± 0-5 °C in a humidified
'Westernette' incubator and the embryos were staged according to the developmental table of Hamburger & Hamilton (1951). Embryos were used at stages
between 6 and 18 days of incubation; the appropriate tissues were removed and
rinsed briefly in sterile Dulbecco's phosphate buffered saline (PBS) pH 7-3. Any
fine dissections were carried out in aliquots of culture medium (see later).
Paralysis of embryos was induced by single doses of either 10 mg of D-tubocurarine chloride (Sigma Chemical Company) or 1 mg decamethonium iodine
(Koch-Light Laboratories) dissolved in 0-5 ml sterile PBS and injected into the
air space of eggs which had been incubated for 9 days (Hall, 1972). Embryos
were judged to be paralysed if they (i) showed a lack of movement on removal
from the egg and (ii) exhibited the 'baggy trouser' syndrome due to oedematous
hind limbs.
Skeletoge tic potential of membrane bone periostea! cells
189
Intact QJs were cultured individually using a standard 'Trowell-type' organ
culture system. The tissue was supported at the gas/medium interface on a
platform of Minimesh FDP (Expanded Metal Company, West Hartlepool,
U.K.) in 30 mm plastic tissue culture dishes (Sterilin) each containing 2 ml of
the a-modification of Eagle's Minimal Essential Medium with 10% foetal
calf serum and antibiotics. The 30 mm dishes were placed inside standard 9 cm
glass petri dishes which were humidified with damp filter paper and then incubated for 10 days at 37-5 °C at 5% CO2 in air in a Grant GC4 tissue culture
incubator fitted with a Gow Mac analytical CO2 monitor. In later experiments
(see Results) controlled pairs of QJs were cultured simultaneously in standard
and 'submerged' conditions. In these experiments the tissue was placed initially
on a 13 mm diameter Millipore filter, pore size 0-45 ju,m, and for standard organ
culture at the gas/medium interface this was placed on the Minimesh raft. For
'submerged' organ culture conditions in which the tissue was grown below the
interface the Millipore filter was 'sunk' by weighting down the filter with a
piece of Minimesh.
Three categories of tissue of various development ages were used for explant
cultures:
(i) periosteal cells from the tip of QJ hooks: this tissue is known to contain
cells which subsequently give rise to the cartilage pads associated with the
hook (Thorogood & Hall, 1976); removal of these periosteal cells prior to organ
culture of the QJ results in a complete absence of the cartilage pads (Thorogood,
unpublished observation).
(ii) segments of the mid-shaft and associated periosteum of the QJ; approximately 0-25-0-4 mm in length.
(iii) blocks of presumptive QJ tissue taken from the maxillary process and of
a size approximately equivalent to those used in (ii).
The explants were cultured in 2 ml of medium in 30 mm plastic tissue culture
dishes (Sterilin), both medium and incubation conditions being identical to
those described earlier for organ culture. Outgrowths were viewed in the living
state by inverted phase contrast microscopy. For cytological purposes some
explants were made on glass coverslips in order that cultures could be subsequently fixed and stained for morphological analysis (see later): no basic
difference in the nature of outgrowth from explants on glass or plastic substrates was observed.
Some tissue from organ culture was examined in whole mount preparations
by fixation in formol ethanol, staining in methylene blue and clearing in methyl
salicylate according to the Lundvall Technique (Hamburger, 1960). Other
material from organ culture was fixed in situ on the Millipore filter in 80 %
ethanol for histology. After decalcification in acid alcohol and paraffin wax
embedding, 7 /«n serial sections were cut and stained with Hansens haematoxylin,
alcian blue and chlorantine fast red (Lison, 1954). Explant cultures on glass
coverslips were gently rinsed in two changes of PBS, fixed in absolute methanol
13-2
190
P. THOROGOOD
Table 1. The incidence of cartilage formation in QJs grown
in organ culture
Developmental stage
day 0 culture
H.H.
H.H.
H.H.
H.H.
H.H.
st. 34
st. 35 (early)*
st. 35 (late)*
st. 36
st. 37
Incidence of
Cartilage in Cartilage in in vitro choncultured QJs
controls drogenesis (%)
1/12
3/12
6/11
10/10
6/6
0/12
0/12
0/11
0/11
2/6
8
25
55
100
-
* H.H. st. 35 was divided into two 'sub-stages'; 'early' at 9 days and 'late' at 9% days.
and stained with the May-Grumwald and Giemsa procedure (Paul, 1965). A
number of such explant cultures was subjected to enzymic digestion tests
to check the specificity of the metachromasia produced by the Giemsa stain.
Methanol-fixed material was incubated in a moist chamber at 37 °C for 2 h with
chondroitinase ABC (Seikagaku Kogyo Co. Ltd., Tokyo, Japan) according
to the method described by Katoh & Takayama (1977) and then stained with
May-Grumwald and Giemsa solutions: control material was incubated in PBS
before staining. Immunofluorescent techniques were used to investigate the
distribution of two principal collagen types - type I (skin, bone and tendon)
and type II (cartilage), by courtesy of Dr Klaus von der Mark, Max PlanckInstitut fur Biochemie, Munchen, West Germany. The antibodies used had
been raised in guinea-pigs against chick type-I and type-II collagens and purified
by immunoadsorption as described previously (von der Mark, von der Mark &
Gay, 1976). Explant cultures grown on 'Sterilin' 30 mm plastic tissue culture
dishes were washed twice in PBS fixed for 5 min in the vapour from an absolute
ethanol : glacial acetic 3:1 solution, then immersed into the fixative solution for
a further 20 min after which they were drained and air-dried. The procedure for
application of the guinea-pig collagen type-I or type-II antibodies and counterstaining with fluorescein-conjugated anti-guinea-pig y globulins has been
described elsewhere (von der Mark & von der Mark, 1977).
RESULTS
(a) Organ culture experiments
To establish the temporal relationship between the commitment of cells to a
chondrogenic fate and in ovo movement, intact QJs of various developmental
ages were grown in organ culture: from each embryo the contralateral QJ was
fixed at the outset of the experiment as a control. Cartilage was never observed
in any control until stage 37 - the 12th day of development (Table 1). In contrast
the incidence of cartilage formation during a 10-day in vitro period rose with
Skeletogenic potential of membrane bone periosteal cells
191
tissue taken from progressively older embryos, increasing from 8 % in cultures
of stage-34 QJs to 100 % in stage-36 tissue (Table 1). Chondrogenesis in cultures
of stage-37 tissue was discounted because cartilage formation has apparently
already commenced in vivo by this time, judging by the incidence of cartilage
in some of the controls at this stage. In the Lundvall whole mount preparations
of normal QJs at stage 39, two discrete pads of cartilage can be identified
within the hook (Fig. 2); the narrow space between the two pads is occupied by
the spicule of bone projecting into the hook from the shaft. In those cultured
QJs in which cartilage was found in the hook, the two pads were generally less
well defined and the final form ranged from two chondrogenic loci apparently
fused together (Fig. 3) to an ill-defined mass of cartilage at the tip of the hook.
In sections of equivalent QJs such areas possessed a characteristic cartilage
phenotype comprising an extensive alcian-blue-positive matrix in which the
cells were located within distinct lacunae (Fig. 4). In order that the effect of in
ovo movement on subsequent chondrogenesis might be studied, QJs were cultured
from embryos paralyzed by a single in ovo injection of curare. At a developmental
stage at which high incidences of in vitro chondrogenesis had been observed in
normal QJs, tissue from paralysed embryos failed to exhibit cartilage formation
in vitro (Table 2). In two cultures of tissue taken from stage-36 paralysed embryos
limited cartilage formation was observed: it had been noted at the outset of
the experiment that these two embryos alone, although exhibiting the oedematous 'baggy trouser' syndrome, had been incompletely paralysed and had shown
some slight limb movement on removal from the egg.
(b) Explant culture experiments
The results from the previous experiments were interpreted as demonstrating
that cells in the periosteum overlying the QJ hook were committed to chondrogenesis by stage 36 and that in ovo movement prior to culturing the tissue
was involved (see Discussion). To provide the large numbers of uncommitted
periosteal cells needed to examine this relationship further using in vitro
techniques (see Introduction), explant culture technique was employed. Periosteal tissue was dissected away from the QJ hook and used as a primary explant.
However, it was found that periosteal tissue taken from embryos of an age at
which cells were not committed to forming cartilage, as judged by the previous
organ culture results, produced outgrowths which contained an abundance of
chondrogenic cells.
Irrespective of the developmental stage used as a source of explant tissue, the
incidence of cartilage formation in the outgrowths was always 100 % (Table 3).
Also the rate of outgrowth and the timing of the appearance of various cell
phenotypes within the outgrowth were independent of developmental stage.
The explant usually became attached to the substrate within the first 24 h of
culture and shortly afterwards cells with a fibroblast-like phenotype grew out in
an apparently random fashion.
192
P. THOROGOOD
Skeletogenic
potential
of membrane
bone periosteal
cells
193
Table 2. The incidence of cartilage formation in organ culture of
QJs taken from embryos paralysed in ovo at 9 days
Developmental stage
dayO culture
Incidence of
Cartilage in Cartilage in in vitro choncultured QJs controls drogenesis (%)
H.H. st. 35 (late)
0/10
0/10
0
H.H. st. 36
2/10*
0/10
20
* Incomplete paralysis in ovo noted at outset - see text.
Table 3. The incidence of chondrogenesis in explant culture of
periosteum isolated from the QJ hook
Incidence of
Developmental stage
day 0 culture
H.H.
H.H.
H.H.
H.H.
H.H.
H.H.
H.H.
st. 34
st. 35 (early)
st. 35 (late)
st. 36
st. 37
st. 38
st. 43
Cartilage in
outgrowth
in vitro
chondrogenesis (%)
17/17
15/15
17/17
11/11
6/6
9/9
100
100
100
100
100
100
0/7 (N.A.) *
* 'N.A.' refers to non-attachment'. The incidence of attachment in samples of older
periosteum was very low (see Table 4 also) due to the tissue curling up on release from the
bone surface. In the absence of attachment there was no outgrowth.
FIGURES 2-5
Fig. 2. A normal stage-29 quadratojugal (QJ). Cartilage is limited to two discrete
pads within the hook (arrows). Lundvall preparation. Bar represents 1 mm.
Fig. 3. A normal QJ fixed at stage-36 (A) and its contralateral partner grown in
organ culture for 10 days (B). Note the absence of cartilage in the control and the
partially-fused pads of cartilage in the cultured specimen. Lundvall preparation.
Bar represents 1 mm.
Fig. 4. L.S. through the hook of a stage-36 QJ grown in organ culture for 10 days.
A thin spicule of bone (Bo) is surrounded on both sides by the chondrocytes of the
two cartilage pads (C). Alcian blue: chlorantine fast red. Bar represents 50/tm.
Fig. 5. Fibroblastic cells at the growing edge of the outgrowth from a 7-day explant
of stage-36 QJ hook periosteum. Inverted phase contrast microscopy. Bar represents
100/tin.
194
P. THOROGOOD
Skeletogenic potential of membrane bone periostea! cells
195
Table 4. The incidence of chondrogenesis in explant culture of
segments of periostea! tissue taken from the QJ shaft
Developmental stage
day 0 culture
H.H.
H.H.
H.H.
H.H.
st. 34
st. 35 (early)
st. 37
st. 43
Cartilage in
outgrowth
10/10
10/10
9/10
3/10
(7/10 N.A.)
Incidence of
in vitro chondrogenesis (%)
100
100
90
100
3/3 outgrowths)
As more cells appeared the outgrowth became consolidated and grew in a
centrifugal fashion, always with fibroblast cells at the growing circumference
(Fig. 5). Behind this growing edge but within the fibroblastic outgrowth a second
cell phenotype arose on the 4th day. These cells had a flattened epithelioid
morphology and each cell was surrounded by a refractile extracellular space;
this phenotype initially arose in small monolayer loci around the original
explant and these loci subsequently expanded and coalesced (Fig. 6). This cell
phenotype was interpreted as chondrogenic on the basis of its characteristic
in vitro chondrocyte morphology (e.g. Solursh, Meier & Vaerwyck, 1973) and
because of the composition of the extracellular matrix produced by the cells.
Initially these chondrogenic cells existed in monolayers but these subsequently
grew into multilayers composed of more rounded cells. After 10-12 days
secondary loci of chondrogenesis arose away from the primary outgrowth: these
were assumed to be established by floaters from the primary outgrowth which
settle, attach and divide. Such secondary loci exhibited the same morphological
FIGURES
6-9
Fig. 6. A monolayer of epithelioid chondrogenic cells and refractile extracellular
matrix within the outgrowth from a 12-day explant of a stage-35 (early) QJ hook
periosteum. Inverted phase contrast microscopy. Bar represents 100 /*m.
Fig. 7. Fixed and stained preparation of outgrowth from 14-day explant of stage-34
QJ hook periosteum. Note the metachromatic matrix organized into distinct lacunae
(arrows), each occupied by a cartilage cell. May-Grunwald and Giemsa. Bar
represents 40/tm.
Fig. 8. Immunofluorescent staining of the outgrowth from a 14-day explant of
stage-35 (early) QJ hook periosteum; fixed in absolute ethanol: glacial acetic,
air-dried and incubated with antibody to type-II collagen. The extracellular material
around each lacunae stains positively, indicating the presence of the cartilagespecific collagen type in the matrix. Bar represents 100/*m.
Fig. 9. Myotubes (arrows) overlying a loose monolayer of fibroblastic cells in the
outgrowth of an 8-day explant of stage-29 presumptive maxillary mesenchyme. A
number of dark cells withfineelongate processes (arrowheads) abutting onto the myotubes are also present and these were interpreted as nerve cells. Inverted phase
contrast microscopy. Bar represents 100/*m.
196
P. THOROGOOD
Table 5. The incidence of chondrogenesis and myogenesis in explant cultures of
tissue taken from the presumptive maxillary process and from the early QJ
rudiment
Developmental stage
day 0 culture
Maxillary mesenchyme at
H.H. st. 29
QJ condensation at
H.H. st. 31
QJ rudiment at H.H. st. 33
Cartilage in
outgrowth
Incidence
Myotubes in chondrogenesis
outgrowth
(%)
Incidence
myogenesis
(%)
4/11
11/11
4/8
5/8
50
62-5
22/22
0/22
100
0
36-4
100
sequence of epithelioid monolayered cells, followed by cells in multilayers which
subsequently grew to produce small nodules of cartilage after several weeks of
culture.
The chondrogenic nature of these cells was confirmed by a number of other
criteria. Firstly, in the outgrowths of explant cultures grown on glass coverslips
and stained by the May-Grumwald and Giemsa technique, there were extensive
areas of metachromatic extracellular matrix (Fig. 7). The matrix had a lacunar
organization and resembled the characteristic morphology of sectioned cartilage.
The metachromasia was abolished by preincubation in chondroitinase ABC
prior to staining whereas control preparations preincubated in PBS retain the
metachromatic staining property. Secondly, the ultrastructural appearance of
these cells was characteristic of chondroblasts and chondrocytes - a scalloped
cell profile and a cytoplasm packed with rough E.R. and surrounded by a matrix
containing granular and fibrillar components (unpublished observations).
Lastly, the immunofluorescent analysis of collagen types revealed that the
extracellular matrix was strongly positive for type-II collagen - the collagen
species characteristic of cartilage, but contained very little type I (Fig. 8). Type I
was found sparsely distributed among the fibroblasts at growing edge but more
strongly located in a border around the nodules of cartilage formed in older
cultures.
It had been previously noticed that in organ culture of intact QJs small
diffuse patches of cartilage occasionally occurred at loci away from QJ hook
and therefore it was felt necessary to test the chondrogenic potential of periosteum from other regions in explant culture. Segments of QJ shaft and associated
periosteum were removed from embryos ranging from stage 34 to stage 43 and
grown as explants. Like the previous cultures of QJ hook periosteum, the present
set of cultures exhibited virtually a 100% incidence of chondrogenesis which
again was independent of the developmental stage used as a source of tissue
(Table 4). The manner of outgrowth, range of cell phenotypes and differentiation
Skeletogenic potential of membrane bone periosteal cells
197
Table 6. The incidence of chondrogenesis in explant culture of QJ hook periosteum
taken from embryos paralysed in ovo at 9 days
Developmental stage
day 0 culture
H.H. st. 36
Cartilage in
outgrowth
Incidence of
in vitro chondrogenesis (%)
14/14
100
of cartilage were all identical to those described for the previous explant cultures.
As all tissues grown as explants had exhibited chondrogenesis in vitro,
including the earliest tissue- taken from stage-34 embryos, it was necessary to
establish how early this chondrogenic potential was acquired by the cells
concerned. Tissue for explant culture was taken from embryos of progressively
earlier developmental stages (see Fig. 1):
(i) QJ rudiments at stage 33: that is, soon after membrane bone formation
commences.
(ii) The QJ condensation at stage 31 when it consists of a mass of tightly
packed cells and uncalcified matrix.
(iii) Samples of maxillary mesenchyme at stage 29, that is approximately 12 h
before condensation, were selected as 'presumptive' QJ tissue.
Cultures of stage-33 QJ rudiments grew in a manner identical to that described
previously for periosteum explant cultures and a 100% incidence of chondrogenesis was recorded (Table 5). In contrast, in cultures of tissue taken from the
developmentally earliest source, that is stage-29 maxillary mesenchyme, the
incidence of cartilage formation was far lower -36-4% (Table 5) and the
chondrogenic loci were much smaller, grew slowly and generally failed to
coalesce, apparently being overgrown by vigorous proliferation of fibroblastic
cells. Myotubes were always observed in maxillary mesenchyme cultures by the
6th day of culture and were regularly found lying over a layer of fibroblastic
cells (Fig. 9). Irregularly shaped cells with slender, elongated processes were
frequently found in association with the myotubes and at the growing edge of
the outgrowth these were tentatively interpreted as nerve cells. By approximately
12 days of culture both the myogenic and neurogenic phenotypes could no
longer be identified and the outgrowths were composed largely of rapidly
proliferating fibroblastic cells and the occasional locus of chondrogenic cells.
Explants of stage-31 QJ condensations grew in a similar fashion to the maxillary
mesenchyme cultures although the incidence of chondrogenesis was higher at
50% and the incidence of myogenesis lower at 62-5% (Table 5). It should be
noted that myogenesis was confined to these cultures of younger tissues and
that myotubes were never seen in outgrowths of explant cultures of tissues taken
from embryos older than stage 31. Their occurrence was interpreted as the result
of inadvertent inclusion of cells which in vivo would have contributed to
198
P. THOROGOOD
Table 7. The incidence of cartilage formation in 'submerged' organ culture ofQJs
taken from stage-36 embryos paralysed in o vo at 9 days
Culture conditions
Standard organ
culture
'Submerged' organ
culture
Number of cultures
containing cartilage
3/19*
18/19
Incidence of in vitro
chondrogenesis (%)
15-8
945
* The three cultures recorded as containing cartilage each contained a single small nodule
of chondrogenic tissue which in all cases occurred at the interface between the bone and the
filter surface.
facial muscle but could not be excluded from the presumptive maxillary
ectomesenchyme, or from the ill-defined stage-31 QJ condensation, during
explantation.
Tissue removed from in ovo paralysed embryos had been previously found
to lack the ability to form cartilage in organ culture, even at a stage when QJs
from normal embryos are apparently committed to chondrogenesis (see earlier Tables 1 and 2). The elimination of in ovo movement by paralysis had been
thought to block the event leading to chondrogenic commitment of periosteal
cells (see later - Discussion). Explant culture had been found to be 'permissive'
to chondrogenesis by this same population of cells irrespective of developmental
stages and in order to find out if the permissiveness of explant conditions would
overcome the in ovo block, explants of QJ periosteum from paralysed embryos
were set up. Embryos were paralysed by a single in ovo injection of curare or
decamethonium at stage 35, and the QJ hook periosteum removed at stage 36
and cultured (Table 6). All explants exhibited a pattern of outgrowth and chondrogenic differentiation identical to that shown by previous explant cultures
of stage-33 and older tissues.
(c) ' Submerged' organ culture experiments
Previous results indicated that using organ culture techniques the commitment of periosteal cells to a chondrogenic fate was stage-dependent, and that
the dependency was apparently based upon the existence and duration of
in ovo movement prior to culture. In contrast any tissue grown in explant
culture failed to show a stage-dependent commitment to a chondrogenic fate
and explant culture conditions appeared to be 'permissive' in that cells of
virtually any age, from any region of the periosteum, always formed cartilage.
This dramatic difference in the in vitro expression of chondrogenic potential was
investigated using a third culture technique. A principal difference between
tissue grown in organ culture and tissue cultured as an explant is that the
former is maintained at the gas phase/medium interface whereas the latter
Skeletogenic potential of membrane bone periostea! cells
199
becomes attached to the floor of the culture vessel and is submerged beneath the
medium. In order to test whether or not the factor of submersion was important
intact QJs were grown in organ culture conditions but submerged beneath the
gas phase/medium interface (see Materials and Methods). All QJs were taken
from stage-36 embryos previously paralysed by in ovo injection of curare or
decamethonium at stage 35 and consequently the tissue lacked cells committed
to chondrogenesis (see earlier - Table 2). From each embryo the QJ from one
side was grown in conditions of submerged organ culture and the contralateral QJ grown in standard organ culture as a control. In all other respects
conditions were identical to the organ culture experiments described previously.
On examination of the control tissue a 15-8% incidence of in vitro chondrogenesis was observed and in each of these positive cases the chondrogenesis
was limited to single small nodules of cartilage (Table 7). The controls in this
experiment are identical to the experimental cultures recorded in Table 2 and
the results are essentially the same, that is a low incidence of limited chondrogenesis. However, the submerged QJs whose control partners produced little
if any cartilage in standard organ culture, were found to regularly contain
abundant cartilage (Table 7 and Fig. 10). The positive staining for cartilage was
generally heavy and much more widespread than the cartilage found in
previous experiments: compare Fig. 10 with Fig. 3.
Histological examination of the tissues from the experiment revealed that
control QJs contained healthy membrane bone surrounded by osteoblastic
and fibroblastic cells whereas submerged tissue contained, in addition to these
components, extensive cartilage which not only covered and invested the
periosteal surfaces but also filled the spaces within the membrane bone (Figs.
11 and 12). The alcian blue-positive matrix was abundant and in some instances
could also be found within osteocytic lacunae and surrounding the osteocytes.
DISCUSSION
(a) Commitment and chondrogenesis
It is evident from the organ culture experiments described in section (a) of the
Results that commitment of periosteal cells to chondrogenesis is normally a
time or stage-dependent phenomenon. The incidence of in vitro chondrogenesis
rose progressively with the stage of embryo used as a tissue source until stage 36 the 11th day development, when all cultured membrane bones subsequently
exhibited cartilage formation. Evidently cells in the QJ periosteum have become
committed to chondrogenesis at least 24 h before secondary cartilage is histologically identifiable, as cartilage in vivo is first seen at stage 37. In the light
of extensive work demonstrating a causal relationship between the biochemical
effects of movement and secondary chondrogenesis (see Introduction) it is
possible that the pattern of commitment revealed in this experiment reflects a
quantitative dependence upon the amount of in ovo movement and intermittent
200
P. THOROGOOD
Skeletogenic potential of membrane bone periostea! cells
201
pressure to which the tissue is exposed prior to culture. If this is the case then
by stage 36 a threshold has been passed and a state of commitment attained by
the cells. When analysing the non-reflexogenic spontaneous activity of chick
embryos Hamburger et al. (1965) found that although motility is exhibited as
early as 2>\ days of development the proportion of overall time occupied by such
activity is low and only starts to increase sharply at stage 32 when it rises from
25 % to 70 % at stage 37 and subsequently peaks at 80% by stage 39. Furthermore, the actual phases of activity start to lengthen dramatically from stage 34
and by this time spontaneous motility is exhibited not only by the neck, trunk
and limbs but also by the mouth, tongue and eyelids. Thus if periosteal cells
at articulations between membrane bones are exposed to intermittent pressure
during embryonic movement it is appropriate that the incidence and duration
of embryonic motility increases shortly before the time at which periosteal cells
become committed to forming secondary cartilage. That movement is involved
in establishing commitment is clearly demonstrated by the culture of QJsfrom
paralysed embryos (Table 2) where elimination of in ovo movement from 9
days of incubation onwards, results in the elimination of an ability to form
cartilage in vitro, presumably as a consequence of blocking the events leading to
commitment. Precisely how intermittent pressure and/or shearing forces affect
the periosteum at the cellular and subcellular levels is not known (see discussion
in Hall, 1978).
In connexion with this causal relationship between movement and secondary
chondrogenesis it should be noted that the present system is very different from
the chondrogenesis occurring in 'mesodermal' mesenchyme. If one eliminates
movement from a system of developing diarthrodial joints (such as in the limb)
by paralysis or by ectopic grafting, cartilage formation is actually enhanced to
the extent that secondary fusion of the articulating surfaces occurs (e.g. Drach-
FIGURES
10-12
Fig. 10. Intact QJs from a single stage 36 embryo paralysed at stage 35 with decamethonium iodide and cultured for 10 days ; fixed and processed by the Lundvall
technique as in Figs. 2 and 3. The lower specimen was grown in 'submerged£
organ culture and the upper specimen was the contralateral control grown in
standard organ culture. The submerged specimen contains extensive cartilage associated with the hook (arrow) and along the greater part of the shaft; cartilage is
absent in the control. Bar represents. 2 mm.
Fig. 11. L.S. through the shaft of a QJ from a stage-36 embryo paralysed at stage 35
and grown in submerged organ culture for 10 days. The darker staining bone (Bo)
is surrounded by, and invested with, cartilage which has a lighter staining matrix (c).
The lacunar organization of the cartilage is more clearly seen in the upper part of the
figure (arrows). Alcian blue and chlorantine fast red. Bar represents 100/tm.
Fig. 12. L. S. through the shaft of the contralateral partner of the specimen in Fig.
11, grown in standard organ culture. The bone (Bo) is surrounded by fibroblastic
cells and no cartilage is present. Filter (f). Alcian blue and chlorantine fast red.
Bar represents 100 /.cm.
202
P. THOROGOOD
mann & Sokoloff, 1966). In the present system of NC-derived cells the absence
of movement results in a lack of chondrogenesis - a point which emphasizes
the difference in developmental histories and underlying developmental mechanisms of tissues which are phenotypically alike, but which are, in an operational
sense, 'non-equivalent' (Lewis & Wolpert, 1976).
(b) Chondrogenesis in explant culture
The original objective of using the explant culture technique described in
Section (b) of the Results was to grow large numbers of uncommitted and nonchondrogenic periosteal cells. It was intended that such cells should be subjected
to various in vitro manipulations in an attempt to cause a chondrogenic differentiation thereby establishing an in vitro system which might provide some insight
into the acquisition of chondrogenic ability by these cells in vivo. However, this
had to be revised as a result of the unpredicted finding that virtually all periosteal
explant cultures spontaneously formed cartilage in the primary outgrowths.
Irrespective of age of embryo providing the source of tissue (Tables 3 and 5) or
the precise site from which the periosteal tissue was taken (Tables 4 and 5) all
cultures showed a similar pattern of differentiation. The temporal sequence of
cell phenotypes appearing in the outgrowths and the type of extracellular
matrix produced was virtually identical in all types of explant (with the exception
of the incidence of myogenesis and overgrowth by fibroblastic cells observed
in cultures of presumptive QJ tissue - see Table 5). Even the timing of cartilage
formation in explant culture is not stage-dependent as the onset of chondrogenesis was the same in all categories of the experiment, that is at 4 days after
explantation. These findings are in marked contrast to the earlier conclusion
made from the organ culture results. For the same tissue now grown in explant
culture the concept of temporal 'commitment' seems inappropriate in that the
age of tissue, or stage of embryo from which the tissue is removed prior to
explantation, is not related to cartilage formation. Periosteal tissue of any
stage-even from the presumptive tissue of the early maxillary process, is
capable of expressing a chondrogenic potential in explant culture. In some way
explant culture conditions appear to mimic or simulate the in ovo events which
lead to a chondrogenic commitment and the subsequent formation of secondary
cartilage in vivo or in organ culture. Indeed explant culture can apparently
reverse the effects of blocking these in ovo events: periosteal tissue from paralysed
embryos, which fails to form cartilage in organ culture, regularly produces
cartilage in explant culture. The explant culture environment may therefore be
regarded as 'permissive' in the sense that a simple change in culture conditions
and one lacking any element of specific informational content, results in a
specific change in the fate of the cells concerned and in their pattern of gene
expression. The ability to form cartilage, which in the QJ system in vivo is
ostensibly restricted to a precise region of the hook, and both in vivo and in
organ culture is only expressed in a stage-dependent fashion, is now expressed
Skeletogenic potential of membrane bone periosteal cells
203
by cells from any part of the QJ and without relationship to the age of tissue
concerned.
The results shown in Table 4 and to a lesser extent those in Table 5, indicate
that latent chondrogenic potential is widespread throughout the periosteum and
not restricted to the periosteum overlying the hook. In the intact embryo this
potential to form secondary cartilage is normally expressed only by the periosteum overlying the hook but it can be concluded from the present results that
ectomesenchyme cells with a latent chondrogenic potential are widely distributed
in association with membrane bone. One interpretation of the present results
is that only those cells which are finally located at the sites of presumptive
joints subsequently express this potential and proceed to form secondary
cartilage. Cells with equivalent potential and located elsewhere do not do so but
when placed in explant culture will form secondary cartilage. Some evidence in
support of this interpretation of widespread but unexpressed chondrogenic
potential is provided by the observation that during the repair of fractured
membrane bones cartilage is formed in spite of membrane bones not going
through a cartilaginous phase during their development (Girgis & Pritchard,
1958). One source of such cartilage is apparently the periosteum and if the
periosteum is removed prior to experimental fractures being created, this
'periosteal' cartilage does not form (Hall & Jacobson, 1975).
(c) Chondrogenesis and tissue submersion
It is not possible to decide if explant culture conditions promote chondrogenesis by stimulating the microenvironment created by movement and intermittent pressure at a developing joint or mimic this effect by some other
mechanism. However, the fact remains that cartilage does form in explant
culture when it would not do so in vivo or in organ culture. By considering the
fundamental differences between the two culture techniques can we learn why
explant conditions promote chondrogenesis so readily? In standard organ culture
tissue is grown at the gas medium interface on a filter supported by a stainless
steel mesh platform and skeletal tissues tend to grow as a solid tissue rather
than spread out. In contrast tissue in explant culture is allowed to sink, attach
to the floor of the culture vessel and the cells grow out, initially using the
floor of the vessel as a substrate; growth is essentially two-dimensional in
nature and centrifugal in direction. In addition to the fact that there is a size
discrepancy between the two techniques - larger tissue samples were grown in
organ culture, the two principle differences are therefore the mode of growth and
the degree of submersion. In explant culture the growing tissue is submerged
with the medium whereas in organ culture it is maintained at the surface of the
medium. It was the second parameter - submersion, which was tested experimentally and described in Section (c) of the Results. Tissue from paralysed
embryos, which in standard organ culture generally does not form cartilage,
was grown in an organ culture apparatus, identical in all respects to the standard
14
EMB 54
204
P. THOROGOOD
pattern except that the tissue was submerged. Whereas the contralateral controls
in standard organ cultures showed virtually no cartilage formation the submerged
organ cultures exhibited a high incidence of extensive in vitro chondrogenesis.
Not only was there a virtual 100 % incidence of chondrogenesis but the cartilage
which did form was not confined to the 'normal' loci found in vivo or in the
earlier standard organ culture experiments. The cartilage covered a much larger
surface area of the bone and examination of sectioned material revealed that
extensive chondrogenesis had occurred within the membrane bone itself. Evidently the parameter of submersion resolves the difference in results from the
two techniques but to what extent factors of pH, oxygen and carbon dioxide
concentrations and ionic gradients may be involved is not known. Given the
proposed causal relationship between anaerobiosis and chondrogenesis in some
systems (e.g. Bassett & Hermann, 1961; Pawalek, 1969), the possibility that
submersion generates relatively anaerobic conditions and thereby promotes
chondrogenesis, may provide a link with events in vivo, if one effect of movement at a developing joint is to generate an anaerobic environment. In fact it
has recently been demonstrated that preceding the formation of secondary
cartilage in the QJ in vivo there is an increase in the specific activity of lactate
dehydrogenase (LDH) - a key enzyme in anaerobic glycolysis, within the
periosteal tissue of the hook and that this increase fails to occur in the absence
of movement (Thorogood & Hall, 1976). Furthermore, the maintained increase
in LDH activity occurring during chondrogenesis is associated with a proportionate increase in the 'anaerobic' isozymic forms of LDH, both in this system
(Coffin & Hall, 1974) and during limb chondrogenesis in the mouse (Thorogood
& Law, 1979). However, this isozyme transition probably reflects an adaptation
by chondrocytes to the anaerobic environment created by the subsequent
accumulation of extracellular matrix and is unlikely to be characteristic of early
chondrogenesis. To what extent the microenvironment created by submersion
in vitro simulates or mimics the microenvironment created by movement and
pressure at a developing joint in vivo remains to be investigated. Nevertheless
this simple manipulation in culture does provide a means of causing large
numbers of periosteal cells to become chondrogenic and to differentiate in a
way in which they would not have done otherwise.
(d) The neural crest and chondrogenesis
The subject of cell lineages and the relationships between various cell types
in the periosteum has not been investigated in this work. The question remains
as to whether cartilage and bone formation in this system is the result of modulation in synthetic activity by a single skeletal progenitor population or is due
to the selective amplification and repression of the activities of two discrete
progenitor populations, one chondrogenic and the other osteogenic. Hall and
his colleagues have presented considerable circumstantial evidence for the
former interpretation (reviewed Hall, 1978) but in the absence of a clonal
Skeletogenic potential of membrane bone periosteal cells
205
analysis the second interpretation remains equally plausible. However, the
cells forming cartilage in this system, like their osteogenic counterparts forming
the membrane bone, are ectomesenchyme cells derived from the cranial N.C.
or more precisely, from the mesencephalic level of the cranial N.C. (Le Lievre,
1978, personal communication). Thus the cells from this level of the crest
differentiate into three principal skeletal tissues - the primary cartilage of the
sclera and of the quadrate, the membrane bones of the upper jaw and the
secondary cartilages associated with these bones (Le Lievre, 1974,1978; Noden,
1978). In the differentiation of the first two of these tissue types, epitheliomesenchymal interactions have been shown to be involved (Newsome, 1972,
1976; Tyler, 1978) although precisely what the role these interactions play in the
progressive restriction of the cells, their commitment and the subsequent
expression of the differentiated state, is unclear at present. For the third tissue
type—secondary cartilage, tissue interactions are apparently not involved in
commitment or expression. It has been proposed in the present work that the
differentiation of this particular type of cartilage is controlled in an epigenetic
fashion as it is merely the spatial position of the cell after migration which
determines whether or not it will be permitted to express its chondrogenic
potential. It has been previously suggested that N.C. cells differentiate, not
according to their point of origin along the anterioposterior axis but according
to morphogenetic factors encountered along the migration pathways and/or
at the site where migration is arrested (Le Douarin & Teillet, 1974, Morris &
Thorogood, 1978). If that generalization is correct then this mechanism controlling secondary cartilage formation provides a clear example of morphogenetic factors at the final site controlling the development of a N.C.-derived
tissue.
1 express thanks to my former colleagues in the Zoology Department, Oxford, for helpful
discussion during the course of the work, and to Jim Bee for carrying out the immunofluorescent analysis for me using facilities very kindly provided by Klaus von der Mark at
the Max Planck-Institut fur Biocheme, Munich. The work was supported by a Scientific
Investigations Grant from the Royal Society.
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{Received 19 March 1979, revised 26 July 1979)