Behaviour of cells of condylar cartilage of foetal mouse mandible maintained in vitro

ArchsoralBid. Vol. 16,pp. 1379-1391, 1971.Pergamon Press.Printedin GreatBritain.

BEHAVIOUR OF CELLS OF CONDYLAR CARTILAGE OF FOETAL MOUSE MANDIBLE MAINTAINED IN VITRO A. H. MELCHER Faculty of Dentistry, University of Toronto, Toronto 101, Ontario, Canada Summary-Mandibles of 18 day in utero mouse foetuses were maintained for periods up to 14 days on a chemically defined medium with or without hydrocortisone, and in atmospheres comprising differing oxygen tensions. Under these conditions the organization of the mandibular condyle could not be maintained, nor was there development comparable with that seen in mandibles of the same chronological age in duo. At the end of the culture period the condylar cartilage was found to be populated wholly by hypertrophic cells, and some of these, as well as many of the perichondral cells, were found to have incorporated [3H]-thymidine. The hypertrophic cells were also found to have secreted [3H]-proline into the surrounding matrix. An osteoid-like material was found to have been deposited on the periphery of the condyle and also, possibly, within some areas of the condylar cartilage. The behaviour of the cells of the condylar cartilage was different from those of Meckel’s cartilage in the same explanted mandible. There is a surprising morphological resemblance between the changes in the condylar cartilage maintained in vitro in this investigation, and the changes reported by other workers in condylar cartilages transplanted in uiuo. INTRODUCTION

There have been many reports of culture in vitro of cartilage of developing long bones, and of the effect of hormones, vitamins and oxygen tension upon this tissue (see, for example, BIGGERS,1965; LE DOUARIN,1970; KIENY, 1970). In contrast to this, there appear to have been few attempts to examine the behaviour in vitro of the cartilages of the developing mandible. Meckel’s cartilage, and the condylar and angular cartilages, have been shown to differentiate in explants that were dissected from 11 and 13 day foetal mice and maintained in vitro (GLASSTONE,1967, 1968). Mandibles of older mouse foetuses have been maintained in vitro on a chemically defined medium, but the report contains only a passing reference to chondrocytes (MELCHERand HODGES, 1968). The resected condyle and Meckel’s cartilage of 5, 10 and 15 day post-par&m rats have been cultured in vitro by CHARLIERand PETROVIC(1967). However, these investigators found that the organization and growth of the mandibular condyle was not maintained. The purpose of the present investigation was to determine whether the organization and development of the cartilage of the condyle can be maintained when near-term foetal mouse mandible is cultured in vitro on a chemically defined medium; and, in addition, to examine the effect of different oxygen tensions and hydrocortisone on maintenance of the cartilage. MATERIALS

AND METHODS

Explants Pregnant Connaught-strain mice were killed by cervical dislocation when their foetuses were 18 days old. The uteri were removed aseptically and placed in sterile WAYMOUTH’S(1959) MB752/1 1379

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medium (Microbiological Associates, Bethesda, Md., U.S.A.), where the foetuses were delivered surgically. Each foetus was then decapitated, and the two mandibles dissected in fresh medium, the mandibular condyle being separated from the cranial part of the temporomandibular joint. The explants were placed on rafts of Millipore filter (pore size l-2 pm), and the two together on expanded steel grids located in plastic Trowell-type culture dishes (No. 3010, Falcon Plastics, Los Angeles, California). Each dish contained a single grid (Falcon No. 3014) spanning a well having a capacity of 1 ml, and the interior of the dish was kept humid by placing 3 ml of sterile, triple-distilled water in a moat surrounding the well. A total of 96 mandibles were cultured and examined. Medium and gases The culture medium comprised antibiotiofree Waymouth’s MB752/1 medium supplemented with 0*45 pg/ml ferrous sulphate and 300 pg/rnl ascorbic acid (WFeA). Ferrous sulphate and ascorbic acid have been shown to be necessary for collagen synthesis (see, for example, HUTTONet al., 1967), and ascorbic acid has, in addition, been found to prevent waterlogging of cartilage explants maintained on a chemically defined medium (REYNOLDS,1966a). In some experiments, WFeA was further supplemented by 1-O rg/ml hydrocortisone-21-sodium succinate (Sigma Chemical Co., St. Louis, MO.), (WFeAHc). Hydrocortisone was added to the medium as this substance has been shown to protect the extracellular substance of cartilage from the resorption that occurs in the presence of high tensions of oxygen (SLEDGEand DINGLE,1965). and the dosage was selected on the basis of the morphological appearance of mandibles cultured in earlier experiments. The media were constituted under normal laboratory conditions and were then sterilized by passage through Millipore filter (pore size O-22 pm). Two explants were placed in each dish, and medium was pipetted into the well until it just reached the undersurface of the grid. The dishes were placed in gas-tight Plexiglass boxes having a capacity of 12.5 1, the interiors of which were kept humid by containers of triple-distilled water. The boxes were filled with an appropriate gas mixture, sealed, and incubated in a water-jacketed incubator at 38°C. The gas-mixtures used were 95 per cent O2 + 5 per cent COz, 40 per cent O2 + 5 per cent CO2 + 55 per cent Nz, and air + 5 per cent COz (Union Carbide Canada Ltd.). The oxygen tension in each Plexiglass box was checked using a Servomex Oxygen Analyser (Servomex Controls Ltd., Crowborough, Sussex, England). In some experiments, the oxygen tension in the box at the end of the culture period was again checked and was found not to have shown a measurable decrease. In each experiment, comparison was made between two different sets of culture conditions. The two explants from each foetus were maintained either on two different media but in the same gaseous environment, or on the same medium but in two different gaseous environments. The cultures were generally maintained for 14 days, but a few were harvested after 7 days. Except where otherwise stated, the results described refer to observations on 14day cultures. The medium and gas were changed three times a week. HistoIogy At the end of the culture period most of the explants were tied in Bouin’s fluid and processed without further demineralixation for the preparation of paraffin sections. Mandibles from the younger intact mice were processed similarly, but tissues from mice older than 5 days post-partum were demineralized in equal volumes of 20 per cent sodium citrate and 45 per cent formic acid. Cultures and in vivo mandibles from which sections were to be stained by the Von Kossa method were fixed in buffered formalin, pH 7.4. All explants were sectioned serially in the longitudinal plane. Most of the sections were stained with haematoxylin and eosin, but appropriate sections were stained by Van Gieson’s picro-fuchsin, Gomori’s trichrome using light green, the Von Kossa method for mineral salts, or with alcian blue at pH 0.5 (LEV and SPICER,1964). Some explants and some in viuo mandibles younger than 5 days post-partum were not fixed, but were frozen. These were then sectioned in a cryostat, and the sections mounted on microscope slides. The sections were fixed for 30 set in citratebuffered acetone, pH 4.5, and processed by the method of Burstone (PEARSE,1968) for acid phosphatase, omitting MnCl,, and using red-violet LB diazonium salt. Some sections were incubated without the Naphthol-AS/BI-phosphate to provide controls for the reaction. Radioautography One microcurie per millilitre [3H]-thymidine or 5-O &/ml [3H]-proline(Amersham Searle, Toronto Canada) were added on the 9th to 12th day to the medium of some explants cultured on WFeA or WFeAHc for 14 days in 40 per cent O2 or 95 per cent Oz. In one-week cultures, the thymidine

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was added to the medium on the third to fifth day. In a number of the thymidine experiments, 0.2 mg/ml colchicine (Sigma Chemical Co., St. Louis, MO.) was added to the medium for the last 18 hours of culture. Observations were also made on material from another investigation in which explants were maintained on WFeA and in 95 per cent Oz for 7 days, and in which 5.0 &i/ml [3H]-proline (Amersham Searle, Toronto, Canada) was added to the medium on the first to fifth days. In all instances sections were dipped in NTB2 nuclear track emulsion (Kodak, Rochester N.Y.), exposed in light-tight boxes for 7 or 14 days, and developed in Dektol: distilled water (1 :l) (Kodak, Rochester, N.Y.) for 2 min at 13°C. The sections were stained through the emulsion with haematoxylin and eosin. RESULTS In-vivo

mandibles

The development of the mandibular joint in viuo has been described in Swiss mice before birth (FROMMER,1964) and in C57 mice after birth (LEVY, 1948). Observations have shown that, in general, the development in vivo up to 11 days post-partum of the condyle of the Connaught strain mouse used in the present investigation is not dissimilar to these descriptions. At the time of explant the condyle is elongated proximo-distally (Figs. la and b). A large proportion of the cartilage comprises hypertrophic cells, and distally it is being eroded and replaced. The cells in the proximal quarter of the condyle are not hypertrophic. This area of the cartilage is noticeably cellular, and the perichondrium is also thick and cellular (Fig. 2). The distal part of the external aspect of the cartilage is surrounded by a perichondral collar com-

FIG. la. A drawing of a mature mouse mandible for orientation. mandibular condyle is encircled.

The area of the

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prising trabeculae of bone and periosteum, but this does not extend proximally (Fig. 1). Two days post-partum there is evidence that the hypertrophic zone is narrower and that it is being replaced distally by bone and cellular soft connective tissue. There is no evidence that bone or osteoid is present in the proximal aspect of the condyle. By 11 days post-partum, the proximo-distal dimension of the condyle is markedly reduced (Fig. 3). The hypertrophic zone is quite narrow and there is a narrow, but clear, zone of cartilage proximal to it. No bone or osteoid is present on the proximal aspect of the condyle. Replacement of the cartilage distally does not occur in a manner similar to endochondral ossification, with formation of a primary spongiosa. Instead, the mineralized extracellular substance is removed and replaced by bone and cellular soft connective tissue; multinucleated giant cells appear to be associated with this process. This observation is similar to that which has been made by DURKIN et al. (1969 a, b) on the mandibular condyle of the guinea pig. Histochemical examination for acid phosphatase showed that there was more reaction-product over the hypertrophic cells than other cartilage cells at all the time-periods examined. In vitro mandibles None of the explants cultured for 14 days in this study showed development towards the morphological configuration seen at the corresponding chronological period of development in vivo (11 days post-partum); and, furthermore, in no instance was the organization pertaining in the condyle at the time of explant maintained. Replacement of the distal aspect of the cartilage by bone and soft connective tissue had not progressed greatly (Fig. 4). Most of the chondrocytes of the cartilage were hypertrophic and the intervening extracellular substance attenuated (Fig. 5). Histochemical examination for acid phosphatase showed the presence of impressive amounts of reaction product over these cells. Neither addition of 1 *Opg/ml hydrocortisone to the medium, nor culturing in an atmosphere of air and 5 per cent CO2 rather than in 95 per cent O2 and 5 per cent COZ, prevented hypertrophy of the chondrocytes. In contrast to this finding, the organization of the distal portion of Meckel’s cartilage in the same explants, where the cartilage was being replaced by bone in a process akin to endochondral ossification, was relatively well maintained, especially in WFeAHc and in an atmosphere of 40 per cent 0, (Fig. 6). Sections of condyle that had been processed by the Von Kossa method showed that little mineral could be demonstrated in the extracellular substance of the distal part of the condylar cartilage, and radioautographs revealed that little [3H]-proline was incorporated at the bone face distal to the condylar cartilage, even during the first 5 days of culture. The former observation suggested that the normal process of calcification that occurs in the developing condylar cartilage did not take place during the period of culture. In occasional explants maintained for 7 days, some vestige of the organization of the condyle at the time of explant could be recognized. In most of the explants, even those maintained for only 7 days, varying lengths of the periphery of the cartilage, including its proximal aspect, were embraced by osteoid-like material. This material was present not only on the circumference of the cartilage but appeared also to have extended into the substance of the cartilage in

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some sites (Figs. 4 and 5). The belief that some of the substance of the cartilage was occupied by osteoid-like material was supported by reconstructions that were made from assembled tracings of each tenth section. When examined in polarized light, this material was seen to contain orientated birefringent fibres (Fig. 5), and it was stained by the acid fuchsin in the van Gieson solution and by light green, but was less reactive to alcian blue than was the adjacent cartilage. The material was not stained by the Von Kossa method, but osteoid and predentine known to have been deposited during the period of culture were not either. The lacunae of the osteoid-like material were large. Radioautography of explants maintained on medium to which [3H]-thymidine had been added on the ninth to twelfth day of culture showed nuclei in many of the cells of the perichondrium or “periosteum”, and in occasional chondrocytes (Fig. 7), to be marked by the presence of silver grains. Mitotic figures were also seen, particularly in the explants maintained on medium to which colchicine had been added during the last 18 hr of culture. Radioautographs of explants maintained for 7 days on medium to which [3H]-proline had been added from the first to fifth day showed a heavy deposit of silver grains in the osteoid-like material and in the extracellular substance of much of the cartilage (Fig. 8), and a lesser deposit in the extracellular substance of the distal part of the cartilage. Many of the hypertrophic chondrocytes were also labelled lightly. Labelling of similar distribution, but of diminished intensity, was seen in the 14 day cultures. DISCUSSION

Loss of organization of the cartilage

The in vitro system used in this investigation did not support continued development of the mandibular condyle, nor did it maintain the cellular organization of the cartilage that pertained at the time of explant. For development to have continued in vitro in a manner comparable with that seen in vivo, a number of processes would have had to have been supported. These processes would have included: (a) Proliferation of the cells of the perichondrium; (b) Differentiation of many of these cells into chondrocytes, and secretion by them of extracellular substance of cartilage; (c) Hypertrophy of chondrocytes distally; (d) Calcification of extracellular substance of cartilage more distally; (e) Removal of the extracellular substance from the distal aspect of the cartilage at a rate in excess of cartilage development proximally; (f) Replacement of the resorbed cartilage by soft connective tissue and bone. Of all these processes, only one was supported adequately by the in-vitro system, and that was proliferation of the cells of the perichondrium. Evidence that this took place was provided by the fact that these cells incorporated [3H]-thymidine, and by the recognition of mitotic figures in the cells. The progeny of these cells, however, did not differentiate into typical chondrocytes, although they were able to secrete a prolinecontaining extracellular substance. This topic will be discussed further below.

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Why the distal aspect of the cartilage was not resorbed and replaced by new bone and soft connective tissue is not known. Osteoid and predentine were deposited abundantly elsewhere in the same explant (see also, MELCHERand HODGES,1968). Furthermore, BRIGHTONet al. (1969) have found that the zone of hypertrophic cells disappears from the epiphyseal cartilage of explants of ribs from young rats maintained in 90 per cent 02, and on synthetic medium supplemented with foetal calf serum. It is conceivable that this defect may have been due to the fact that the extracellular substance of the distal aspect of the condylar cartilage was not demonstrably calcified ilz vitro. This could be of particular importance were, as seems possible from the in-vivo observations, multinucleated giant cells responsible for removal of the cartilage (see, for example, WEINMANN and SICHER,1955, p. 285; IRVINGand HANDELMAN,1963). On the other hand, CHARLIERand PETROVIC(1967), on the basis of radioautographic observations on uptake of 45Ca, have reported light calcification of extracellular substance in their resected condyles maintained in vitro, but did not find that cartilage was replaced by bone. Perhaps the extent of calcification could be important in this context. Irrespective of the oxygen tension or the presence or absence of hydrocortisone in the culture medium, the chondrocytes that were present in the condyle when the explants were harvested were found to be hypertrophic. This suggests firstly that many of the hypertrophic chondrocytes present at the time of explant could be maintained in the culture system, and the conclusion is supported by the fact that some of the cells were shown to be able to incorporate [3H]-thymidine and [3H]-proline. Secondly, it is apparent that the chondrocytes in the proximal part of the condyle became hypertrophic during the period of culture. In this regard, the observations of SLEDGEand DINGLE(1965) and SLEDGE(1968) are of interest. These investigators have found that exposure of chick limb-bud rudiments cultured in vitro to elevated tensions of oxygen increases production and release of acid phosphatase, but that excessive secretion of the enzyme is prevented by addition of cortisol to the medium. Similarly, REYNOLDS(1966b) has found that, when developing long bones of chick are cultured on a chemically defined medium, addition of hydrocortisone reduces the hypertrophy of diaphyseal chondrocytes. In the 2-3 day post-partum animals and the explants examined in the present investigation, the hypertrophic cells were found to be reactive for acid phosphatase. The former observation is similar to that made by GREENSPAN and BLACKWOOD (1966) on mandibular condyles of rats. Thus, unlike what has been reported to occur in chick long bones, decreasing the oxygen tension in the environment and adding hydrocortisone to the medium did not prevent hypertrophy of cartilage cells in the mandibular condyles cultured in this investigation. However, it is not possible to say from the observations made what the effect of these parameters was on release of hydrolytic enzymes by the hypertrophic chondrocytes. In strong contrast to the behaviour of the cells of the cultured condyle, it was possible, in the same explanted mandible, to maintain reasonably well the organization in the distal part of Meckel’s cartilage where bone was being replaced by an endochondral-like process. This was particularly true in the presence of hydrocortisone. It has also been found possible to maintain reasonably well the organization of the

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epiphyseal cartilage of 18 day in utero mouse humerus cultured for 14 days on WFeAHc and in 95 per cent O2 (MELCHER,1971). While it is likely that the gaseous environment and the nutrients provided for the explant were not adequate for maintenance and development of the condylar cartilage in vitro, the medium for example did not contain sulphation factor (MCCONAGHEY and SLEDGE,1970), it is striking that there are reports that the organization of the condyle may not be maintained after transplantation in vivo. OSTERGREN(1958) transplanted mandibles under the skin of isologous mice, and has reported failure of normal chondrogenesis in the mandibular condyle less than 15 days post-operatively. Similarly, R~~NNINGand KOSKI (1969) transplanted rat mandibular condyles, with or without the adjacent articular disc, to the brains of inbred littermates, and found that even 5 days post-operatively the thickness of the zone of proliferative cells had been reduced “. . . so as to bring the hypertrophic cells closer to the upper surface of the cartilage.” Epiphyseal cartilages of long bones transplanted in the same experiment were maintained, whereas the adjacent articular cartilage in the same long bones disintegrated. These, and other findings, have been interpreted as suggesting that the condylar cartilage is more like an articular cartilage than a growth cartilage (DURKIN et al., 1969a). They also raise the possibility that the failure to maintain the condylar cartilage in vitro in the present investigation may, at least in part, have been due to lack of function. Potentials of the cells of the condyle

It has been found in this laboratory in a combined histological and biochemical study using [3H]-proline, that cells of mouse mandibles maintained in vitro on WFeA in 95 per cent O2 for one week under the conditions described here, can synthesize collagen (MELCHER,BURGESSand KUCEY, in preparation). However, although it is evident from the radioautographs in the present investigation that [3H]-proline was secreted into the extracellular substance of the condyle during the period of culture, and that hypertrophic cells were involved in the process, the identity of the molecules into which the amino acid was incorporated is not known. Peripherally, including the proximal aspect of the condyle where bone and osteoid were never seen in vivo, and in some areas more centrally, the radioactively labelled proline was associated with a material which exhibited histological characteristics that were more consistent with those possessed by osteoid than extracellular substance of cartilage. These characteristics include birefringence, staining by acid fuchsin and light green, and decreased reactivity with alcian blue. It is of course, possible that the material could be fibrocartilage, but the decreased staining by alcian blue does not favour this. Furthermore, the premise that the material deposited subperiosteally could be osteoid-like receives some support from the observations of SHAWand BASSETT(1967). These investigators have reported subperiosteal osteogenesis in chick tibia1 cartilage rudiments cultured in plasma clots. Thus, the cells of the perichondrium of the condylar cartilage of the mandible, like cells of perichondrium and periosteum elsewhere, appear to have a dual potential (see HALL, 1970). Unfortunately, the osteoid-like material was deposited under all the conditions of culture used. Consequently, no conclusions can be drawn in this investigation about the factors that govern the nature of the extracellular

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substance that is secreted by the perichondral cells and perhaps the chondrocytes. It remains to be seen whether the same changes would occur if the temporomandibular joint was kept intact at the time of dissection. In contrast to these observations, CHARLIERand PETROVIC(1967) have not reported the presence of this osteoid-like material in their cultures of isolated condyles. Apart from the osteoid-like material that was deposited on the periphery of the condyle by cells of the perichondrium, it is conceivable that chondrocytes may have synthesized and secreted similar material more centrally in the cartilaginous part of the condyle. HALL (1970) has recently discussed extensively the potential of chondrocytes to become bone-secreting cells. He has pointed out that the studies of CRELIN (1967), CRELIN and KOCH (1965, 1967) and HOLTROP(1966, 1967) provide experimental evidence “. . . that the hypertrophic chondrocytes of the cartilage model are able to dedifferentiate to an ‘osteoprogenitor cell type’ and then redifferentiate as osteoblasts.” Furthermore, Hall maintains that, despite assertions to the contrary, the bulk of available evidence appears to favour the possibility that cartilage can be transformed into bone. WHEELERHAINES(1968) and BOHATIRCHUK (1969) appear to support this view on the basis of their work on dogs and humans and on rats and rabbits respectively. Despite these claims, it is very difficult to see how chondrocytes or their progeny can transform the highly distinctive extracellular substance of cartilage directly into that of bone or osteoid, which is so different. The observations made here suggest a mechanism whereby the changes seen in the condylar cartilage of the explants could have been wrought. The chondrocytes, which at the end of the culture period were hypertrophic, were found to have been able to secrete a proline-containing substance, and some of them had been able to synthesize DNA. In addition, the extracellular substance between the cells was attenuated. These observations could be interpreted as suggesting that, in the more central areas of the condyle, chondrocytes tist removed extracellular substance surrounding them, then occasional of these cells divided, thus returning to the progenitor state. The resulting daughter cells subsequently could have differentiated and assumed the capacity to secrete and surround themselves with the osteoid-like material. That cells in and around bone can return to the progenitor state and differentiate into alternative functional types has been disdiscussed by OWEN (1970). Thus, if osteoid-like material was deposited in an area of the condyle that was formerly occupied by cartilage, it seems much more likely that this occurred as a result of replacement of the cartilage after removal of its extracellular substance by chondrocytes, than as a result of the direct transformation of its extracellular substance into a demonstrably fibrous material. Of considerable relevance to the findings made in this investigation is the claim by R~~NNING(1966) that mandibular condyles transplanted to the brains of isologous rats sometimes exhibited “. . . direct transformation of cartilage into bone . . .“. Furthermore, FELTS (1961), in his Fig. 12, illustrates a section of a mouse mandible transplanted for 40 days under the skin of an isologous host, in which it is claimed that “large-cell cartilage is partially replaced by primitive bone . . .“. Indeed, the resemblance of this photomicrograph of a condyle transplanted in vivo to the condyles maintained in vitro in the present experiment is quite remarkable. The relationship

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between these in vivo observations and those made here in vitro raises once again the possibility that the changes seen in vitro may in part be due to lack of function. Acknowledgement-I

wish to acknowledge my indebtedness to Mrs. WILMA for her competent technical assistance. This investigation was supported by Grant No. MA-3803 awarded by the Medical Research Council of Canada. HIDDLESTONE

Rt%um&--Les mandibules de foetus de souris, ages de 18 jours in utero, sont cultivees pendant 14 jours sur un milieu chimiquement defini avec ou sans hydrocortisone et sous diverses tensions d’exygene. Dans ces conditions, la croissance normale du condyle mandibulaire n’a pu etre maintenue: il en est de meme du developpement mandibulaire compare avec des echantillons d’bges identiques in vivo. A la fin de la pbiode de culture, le cartilage condylien ne contient que des cellules hypertrophiques, dont certaines incorporent de la jH-thymidine, ainsi que de nombreuses cellules perichondrales. Les cellules hypertrophiques secretent de la 3H-proline dans Ia matrice. Un materiel d’aspect osteoide est depose a la p&ipherie du condyle et, sans doute aussi, dans certaines regions du cartilage condylien. Le comportement des cellules du cartilage condylien est different de celui du cartilage de Meckel dans la meme mandibule explant&,. 11 existe une ressemblance morphologique etonnante entre les changements du cartilage condylien cultive in vitro, et les changements observes par d’autres auteurs sur des cartilages condyliens implant&s in vivo.

Zusammenfassung-Unterkiefer von Mausefeten im Foetalalter von 18 Tagen wurden bis zu 14 Tage lang auf einem chemisch definieten Medium mit oder ohne Hydrokortison und bei unterschiedlicher Sauerstoffspannung gehalten. Unter diesen Bedingungen konnte weder die Organisation des mandibularen Kondylus erreicht werden, noch war die Entwicklung mit der vergleichbar, die bei Unterkiefern derselben Altersstufe in vivo zu beobachten ist. Am SchluB der Kulturzeit war der Gelenkknorpel tiber und tiber mit hypertrophischen Zellen besiedelt; einige von diesen wie such viele perichondrale Zellen hatten 3H-Thymidin inkorporiert. Die hypertrophischen Zellen hatten such 3H-Prolin in die umgebende Matrix sezemiert. Ein osteoid-ahnliches Material war in der Peripherie des Kondylus abgelagert and mijglicherweise such innerhalb einiger Bezirke des Kondylenknorpels. Die Zellen des Kondylenknorpels verhielten sich anders als die des Meckel’schen Knorpels in derselben explantierten Mandibula. Uberraschend morphologisch ahnlich waren die in dieser Untersuchung festgestellten Verlnderungen im Kondylenknorpel in vitro und die von anderen Untersuchern beobachten Veranderungen des in vivo transplantierten Kondylenknorpels.

REFERENCES BIGGERS,J. D. 1965. Cartilage and bone. In: CelIs and Tissues in Culture, pp. 197-260 (Edited by WILLMER,E. N.). Academic Press, New York. BOHATIRCHUK,F. P. 1969. Metaplasia of cartilage into bone-A study by stain historadiography Am. J. Amt. 126,243-254. BRIGHTON,C. T., RAY, R. D., SOBLE,L. W. and KUETTNER,K. E. 1969. In vitro epiphyseal-plate growth in various oxygen tensions. J. Bone Jr Surg. %A, 1383-1396. CHARLIER.J.-P. and PETRO~IC.A. 1967. Recherches sur la mandibule de rat en culture d’oraanes: le cartilage condylien a-t-il’un potential de croissance independant ? L’Orthod. Franc. 38,16?-175. CRELINE. S. 1967. An autoradiographic study of endochondral ossification in vitro. Amt. Rec. 157, 354. CRELIN, E. S. and KOCH, W. E. 1965. Development of mouse pubic joint in vivo following initial differentiation in vitro. Anat. Rec. 153, 161-171. CRELIN,E. S. and KOCH, W. E. 1967. An autoradiographic study of chondrocyte transformation into chondroclasts and osteocytes during bone formation in vitro. Anat. Rec. 158,473-483.

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DURIUN, J. F., IRVING,J. T. and HEELY,J. D. 1969a. A comparison of the circulatory and calcification patterns in the mandibular condyle in the guinea pig with those found in the tibia1 epiphyseal and articular cartilages. A&s oral Biol. 14, 1365-1371. DURKIN, J. F., IRVING, J. T. and HEELY,J. D. 1969b. A comparison of circulatory and calcification changes induced in the mandibular condyle, tibia1 epiphyseal and articular cartilages of the guinea pig by the onset and healing of scurvy. Archs oral Biol. 14, 1373-1382. FELIS, W. J. L. 1961. In viva implantation in skeletal biology. In: Znt. Rev. Cyfol., pp. 243-302 (Edited by BOURNE,G. H. and DANIELLI,J. F.). Academic Press, New York. FROMMER,J. 1964. Prenatal development of the mandibular joint in mice. hat. Rec. 150,449-462. GLASSTONE,S. 1967. Morphodifferentiation of teeth in embryonic mandibular segments in tissue culture. J. dent. Res. 46, 611-614. GLASSTONE,S. 1968. Tissue culture of mandible and mandibular joint of mouse embryos. Nature 220, 705-706. GREENSPAN,J. S. and BLACKWOOD,H. J. J. 1966. Histochemical studies of chondrocyte function in the cartilage of the mandibular condyle of the rat. J. Anat. 100, 615-626. HALL, B. K. 1970. Cellular differentiation in skeletal tissues. Biol. Rev. 45, 455-484. HOLTROP, M. E. 1966. The origin of bone cells in endochondral ossification. In: Third European Symposium on Calcified Tissues pp. 32-36. (Edited by FLEISCH,H., BLACKWOOD,H. J. J. and OWEN M.). Springer-Verlag, Berlin. HOL~ROP, M. E. 1967. The potencies of the epiphyseal cartilage in endochondral ossification. Proc. ned. Akad. Wet. (C) Biol. Med. SC. 70,21-28. HUTTON,J. J. JR., TAPPEL, A. L. and UDENFRIEND,S. 1967. Cofactor and substrate requirements of collagen proline hydroxylase. Archs. biochem. Biophys. 118, 231-240. IRVING,J. T. and HANDELMAN,C. S. 1963. Bone destruction by multinucleated giant cells. In: Mechanisms of Hard Tissue Destruction, pp. 515-530. (Edited by SOGNNAES,R. F.), Am. Ass. Adv. Sci. Washington, D.C. KIENY, M. 1970. Culture of embryo organs in synthetic media. In: Organ Culture, pp. 95-100, pp. 103-115. (Edited by ANDRE THOMAS,J.), Academic Press, New York. LE DOUARIN,G. 1970. Differentiation of organs in natural media. In: Organ Culture, pp. 15-18, pp. 53-54 (Edited by ANDRE THOMAS,J.). Academic Press, New York. LEV, R. and SPICER, S. S. 1964. Specific staining of sulphate groups with alcian blue at low pH. J. Histochem. Cytochem. 12,309. LEVY, B. M. 1948. Growth of mandibular joint in normal mice,. J. Am. dent. Ass. 36,177-182. MCCONAGHEY,P. and SLEDGE,C. B. 1970. Production of “sulphation factor” by the perfused liver. Nature 225, 1249-1250. MELCHER,A. H. 1971. Role of chondrocytes and hydrocortisone in resorption of proximal fragment of Meckel’s cartilage. An in vitro and in vivo study. Anat. Rec. In press. MELCHER,A. H. and HODGES,G. M. 1968. In vitro culture of an organ containing mixed epithelial and connective tissues on a chemically defined medium. Nature (Land.) 219,301-302. OSTERGREN,C. D. 1958. The development of the embryonic mouse mandible as an isologous subcutaneous transplant, and as a chorio-allantoic graft. M.S.D. Thesis, University of Minnesota, Minneapolis, Minnesota: Quoted by, FELTS, W. J. L. (1961). OWEN, M. 1970. The origin of bone cells. In: Znt. Rev. Cyto. Vol. 28, pp. 213-238. (Edited by BOURNE,G. H. and DANIELLI,J. F.). Academic Press, New York. PEARSE,A. G. E. 1968. Histochemistry, Theoretical and Applied, 3rd edn, p. 731. Churchill, London. REYNOLDS,J. J. 1966a. The effect of ascorbic acid on the growth of chick bone rudiments in chemically defined medium. Exp. Cell Res. 42, 178-188. REYNOLDS,J. J. 1966b. The effect of hydrocortisone on the growth of chick bone rudiments in chemically defined medium. Exp. Cell Res. 41, 174-189. RUNNING, 0. 1966. Observations on the intracerebral transplantation of the mandibular condyle. Acta odont. stand. 24, 443-457. RBNNING,0. and Kosxr, K. 1969. The effect of the articular disc on the growth of condylar cartilage transplants. Trans. Eur. Orthodont. Sot. 45, 99-108. SHAW, J. L. and BASSETT,C. A. L. 1967. The effects of varying oxygen concentrations on osteogenesis and embryonic cartilage in vitro. J. Bone Jt Surg. 49A 73-80. SLEDGE,C. B. 1968. Biochemical events in the epiphyseal plate and their physiologic control. Clin. Orthopaed. 61, 3747. SLEDGE,C. B. and DINGLE, J. T. 1965. Oxygen induced resorption of cartilage in organ culture. Nature 205, 140-141.

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C. 1959. Rapid proliferation of sublines of NCTC Clone 929 (Strain L) mouse cell in a simple chemically defined medium. (MB 752/l). J. Natn. Cancer Inst. 22, 1003-1015. WEINMANN, J. P. and SIGHER,H. 1955. Bone and Bones, 2nd Edn, p. 285, Kimpton, London. WHEELER HNNES, R. and MOHUIDDIN, A. 1968. Metaplastic bone, J. Anat. 103,527-538. WAYMOUTH,

PLATEal AND 2 OVERLEAF

1390

A. H. MELCHER

PLATE 1 FIG. lb. Mandibular perichondrium;

condyle of 18 day in utero mousefoetus. H-hypertrophiccells; PArrows-perichondral collar. Haematoxylin and eosin. x 60

FIG. 2. A higher magnification of an area in the proximal part of the condyle illustrated in Fig. 1. P-perichondrium; C-chondrogenic cells. Haematoxylin and eosin. x 750 FIG. 3. Mandibular condyle of 11 day post-purtutn mouse. Note that the cartilaginous part of the structure (between the arrows) is no longer elongated proximo-distally. Haematoxylin and eosin. x 60 FIG. 4. Mandibular condyle of 18 day in utero foetal mouse after culture for 14 days on WFeAHc and in 40 per cent Oz. The cartilage (C) comprises hypertrophic chondrocytes. Note the osteoid-like material (0). M-Meckel’s cartilage. The architecture of the condyle should be compared with that illustrated in Fig. 3. Haematoxylin and eosin. x 75

CELLS OF CONDYLAR CARTILAGE itt U&I?

I *c

PLATE 1 A.O.B.

F.p.1390

A.H.MELCHER

PLATE 2

CELLS OF CONDYLAR CARTILAGE in Vitro

1391

PLATE 2 FIG. 5.

A higher magnification of a portion of the osteoid-like material (0) illustrated in Fig. 4. Note the hypertrophic chondrocytes in the adjacent cartilage (C). Haematoxylin and eosin. x 300 Inset: An area of the osteoid-like

material

photographed

in polarized

light. x300

FIG. 6. Distal portion of Meckel’s cartilage in an 18 day in utero foetal mouse mandible after culture for 14 days on WFeAHc and in 40 per cent OZ. C-cartilage; H-Hypertrophic cells; S-soft connective tissue and osteoid. Haematoxylin and eosin. x 300 FIG.

7. Radioautograph to show incorporation of t3H]-thymidine into cells of the perichondrium covering a part of the condyle adjacent to that illustrated in Fig. 4. x 750 Inset: Chondrocytes in the depth of the cartilage in the same radioautograph labelled by silver grains. x 750

FIG. 8. Radioautograph of part of a condyle of an 18 day in utero foetal mouse mandible after culture for 7 days on WFeAHc and in 40 per cent OZ. [3H]-proline was added to the medium from the iirst to fifth days. An intense band of silver grains is present over newly-deposited osteoid (0), but not over old osteoid of the perichondral collar (arrowed). Grains are also present over the extra-cellular substance of the cartilage (C) and some chondrocytes. x 750