Experiments in vitro on the Role of Movement in
the Development of Joints
by G. LELKES 1
From the Department of Anatomy, Histology and Embryology, Medical University of Debrecen
WITH ONE PLATE
I T has been pointed out by Fell & Canti (1934) as a result of their experiments
in vitro concerning the early formation of the avian limb skeleton and kneejoint, that the appearance of the articular rudiment is independent of the bloodand nerve-supply as well as of mechanical influences. These authors believe that
the formation of articular surfaces occurs in consequence of the differential
growth of the scleroblastema (the 'Anlage' of the limb skeleton, skeletal rudiment) the essential factor in joint formation being the association of undifferentiated tissue with the rapidly growing chondrification centres. They emphasize,
however, that only the earlier stages of joint formation can be obtained in vitro',
the conditions of cultivation are not adequate for the further development of the
joints. The articular rudiment disappears by secondary fusion of the cartilages
of the limb skeleton. Fell & Canti mention the possibility that though mechanical
movement is not necessary for the appearance of the joint, it may be an important and possibly an essential factor in its subsequent development.
Giindisch (1943), examining cultures of chick-limb scleroblastemas, came to
the conclusion that the growth observed in vitro is, for the most part, erroneously
interpreted as organotypical, since the development in vitro of the organ anlage
represents a histotypical proliferation of tissues. According to his work only the
cartilage cells continue to differentiate in the explanted limb skeletal blastema,
multiplying and producing ground substance. Thus cartilage centres are produced which should be considered as separate cultures of cartilage tissue in an
organ culture undergoing dedifferentiation. In consequence of the histotypical
growth of these cartilage centres the loose tissue between the articular surfaces
becomes chondrified. The more developed the joints are at explantation, that is,
the smaller the undifferentiated tissue between the articular ends is, the later the
chondrification takes place. Giindisch mentions that movement inhibited to
some extent the fusion of the cartilaginous surfaces.
In the present experiments the influence of mechanical movement on the
1
Author's address: Department of Anatomy, Histology and Embryology, Medical University
of Debrecen, Debrecen 12, Hungary.
[J. Embryol. exp. Morph. Vol. 6, Part 2, pp. 183-186, June 1958]
5584.6
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G. L E L K E S — E F F E C T OF M O V E M E N T ON
development in vitro of the knee-joint of 6-7-day-old chick-embryos was studied.
At this stage the joint has already appeared. No consideration has been given to
the earlier stages of its development.
MATERIAL AND METHOD
Knee-joints of 6-7-day-old chick-embryos were cleaned from soft tissues, care
being taken that the perichondrium should remain intact. The joints were cultivated, some according to Fell's watch-glass technique, some in Carrel flasks.
Always the two limbs of the same embryo were explanted in the same watchglass or flask, one limb being subjected to movement., the other one serving as
control. The limbs were moved with a thin glass rod, five times a day. A mixture
of fowl blood-plasma and fowl embryonic extract (2:1) was employed as
medium. The explants cultivated on watch-glasses were transferred to new
medium every 48 hours. The liquid medium in the Carrel flasks, composed of
Tyrode solution and embryonic extract (5 : 1), was changed every two days.
Forty joints were explanted. After cultivation for different periods of time the
cultures were fixed in Susa mixture, embedded in paraffin, and cut in serial sections. The sections were stained with haemalum-eosin, toluidine blue, or Azan.
RESULTS
It was observed that both the unmoved controls and the joints subjected to
movement presented some regressive alterations which, especially in certain
portions of the epiphyseal cartilage, were manifested in a pale staining of the
nuclei of the cartilage cells and in disintegration of the ground substance. Besides
this regression, however, there were to be found tissue differentiations too, such
as hypertrophy of cartilage cells in the diaphysis and formation of a perichondral bony sheath, as well as proliferation of cartilage tissue originating from the
epiphyseal cartilage. In explants which had not been moved this proliferation
of the cartilage tissue often forms a continuous cartilage bridge between the
femur and tibia by chondrifying the undifferentiated interarticular tissue (Plate,
fig. 1). There is no remainder of the interarticular tissue, the site of fusion of the
cartilage-anlage being marked only by the elongated cartilage cells that correspond to the previously free surface (Plate, fig. 2). There is a little basophil
ground substance between the cells which completely masks the collagen fibres.
In other cases there is an entirely irregular histotypical proliferation of epiphyseal cartilage which tends to fusion.
In explants that were moved the cartilaginous fusion of the parts of the limb
skeleton does not take place at all. Between the articular ends there is to be seen
a loose mesenchymal tissue with its cells situated along the articular surfaces
(Plate, fig. 3). The flat cells are arranged in 4-5 rows with collagen fibres between
them. Beginning from the 6th row the fibres are masked by the ground substance
and here the cells assume a more rounded form. The superficial layers show the
character of procartilage (Plate, fig. 4). More striking still is the separation of
J O I N T D E V E L O P M E N T IN VITRO
185
the articular surfaces with the formation of an articular cavity in the explant
shown in Plate, fig. 5 (knee-joint of a 7-day-old chick-embryo cultivated for 6
days, moved five times a day). The upper part of the picture is particularly interesting in that the proliferating young cartilage there shows a definite articular
surface. The cells of the articular surfaces are flattened and show the character
of a procartilage. Collagen fibres are to be found only between the cells of the
most superficial 2-3 rows. The amount of ground substance rapidly increases in
the deeper layers. The arrangement of the flattened cells is parallel to the articular
surface. There is no remainder of tissue between the articular surfaces opposite
to each other (Plate, fig. 6).
In summary, of 20 moved explants, 7 showed complete separation of femur
and tibia with formation of an articular cavity; and 13 had well-formed articular
surfaces but with loose tissue between them, though no cartilaginous fusion. Of
20 control (unmoved) explants, 9 showed complete cartilaginous fusion between
femur and tibia, and 11 showed entirely irregular histotypical proliferation of
the epiphyses.
DISCUSSION
Szekely (1943) demonstrated with limbs of human embryos that muscular
activity may be presumed—judging on morphological grounds—to occur as
early as the cartilaginous skeletal rudiments appear. At first the muscular
activity, by forming the tendon structure, produces only a very slight movement. Later, with the strengthening of muscles and tendons, a movement with
greater amplitude of flexion and extension becomes possible, which promotes
the formation of the articular surfaces and capsule and prevents at the same
time the fusion of the articular ends.
Hamburger (1929) denied the role of movement in the formation of joints. He
extirpated in frogs—in the neural-plate stage—the lumbosacral part of the spinal
cord. After the operation the morphogenesis of the reduced limbs was stated by
him to be normal. As a counter-argument it may, however, be mentioned that
since the paralysed limbs were floating in water the articulations had some
possibility of movement which might have provided the minimal stimulus necessary for the development of a joint. The articular cavity actually obtained was
in fact reduced.
The experiments recorded here on the role of movement in the development
of joints suggest the following points:
1. In explanted joints which are not moved during cultivation cartilaginous
fusion across the joint occurs, as observed by several authors (Giindisch, 1943,
in vitro; Pellegrini, 1934, in chorio-allantoic grafts) and in my own control
preparations. This fusion is prevented if movement, resembling the normal
movement at the joint, is produced during cultivation.
2. Not only is fusion prevented by movement, but the formation of an articular cavity is induced by movement.
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G. L E L K E S — J O I N T D E V E L O P M E N T IN
VITRO
3. Movement also exerts a formative effect on the shape and structure of the
articular surfaces.
It may therefore be suggested that proper differentiation of a joint depends on
its undergoing movement.
SUMMARY
1. The effect on joint differentiation of movement at the knee-joint of leg
rudiments of 6-7-day-old chick-embryos cultivated in vitro has been studied.
2. In control explanations, where no movement was applied, cartilaginous
fusion across the joint of the skeletal parts was generally found. Movement
prevents this fusion. An articular cavity was sometimes formed in the moved
explants but not in the controls.
3. Movement exerts a formative effect on the shape and structure of the
articular surfaces, and on the histotypical proliferation of cartilage tissue.
ACKNOWLEDGEMENT
I am indebted to Prof. Dr. St. Krompecher, director of the Department of
Anatomy, Histology and Embryology, for stimulating interest during this work
and for criticism of the manuscript.
REFERENCES
FELL, H. B., & CANTI, R. G. (1934). Experiments on the development in vitro of the avian kneejoint. Proc. roy. Soc. 116, 316-51.
GUNDISCH, M. (1943). The mechanism of development of skeleton and joints of limbs. Experiments in vitro on embryonic limbs of chicken. Erdelyi Muzeum Egyesiilet, Orv. En. 54,
33-44 (in Hungarian).
HAMBURGER, W. (1929). Die Entwicklung experimentell erzeugter nervenloser und schwach
innervierter Extremitaten von Anuren. Roux Arch. EntwMech. Organ. 114, 272-363.
PELLEGRINI, O. (1934). Lo sviluppo di abbozzi di articolazioni impiantati nelle membrane corioallantoidee. Atti Soc. med.-chir. Padova, 11, 927-41.
SZEKELY, K. (1943). Causal histogenetic studies on human embryonic limbs. Erdelyi Muzeum
Egyesiilet, Orv. Ert. 54, 18-32 (in Hungarian).
EXPLANATION OF PLATE
All microphotographs were taken of sections stained with Azan (Heidenhain).
FIG. 1. Knee-joint of 7-day-old chick-embryo cultivated for 6 days, not moved. Cartilaginous
fusion of femur and tibia, x 40.
FIG. 2. Cartilage bridge between femur and tibia, from the preparation seen infig.1; higher
magnification, x 400.
FIG. 3. Knee-joint of 7-day-old chick-embryo cultivated for 6 days, moved five times a day.
Definite articular surfaces with loosened tissue between them, x 40.
FIG. 4. Area between articular surfaces from the preparation seen infig.3; at greater magnification. x200.
FIG. 5. Knee-joint of 7-day-old chick-embryo, cultivated for 6 days, moved five times a day.
Articular surfaces well developed, articular cavity present, x 40.
FIG. 6. Articular surfaces from the preparation seen infig.5 with greater magnification, x 400.
(Manuscript received 27: v: 57)
J. Embryol. exp. Morph.
Vol. 6, Part 2
G. LELKES