Arrhs orul Bid. Vol. 24, pp. 283 to 291 Petgm~on Press Ltd 1979. Printed in Great Britain
SUTURE
DEVELOPMENT AND BONY FUSION THE FETAL RABBIT PALATE
IN
M. PERSSON* and W. ROY Department of Anatomy, University of Virginia, Charlottesville, VA 22901, U.S.A. SummaryYThe intermaxillary and the interpalatine regions of the fetal rabbit palate are contiguous areas of suture development and bony fusion, respectively. As these regions must differ regarding the factors responsible for the formation of a suture, the palate was selected as a model for the study of the mechanism of suture development. Palates from 21-25-day rabbit fetuses were classified into developmental stages using cleared and alizarin-stained specimens. The developmental stages of the two areas were compared in paraffin and Epon sections. PAS-staining and autoradiography following [3H]-proline injection were used. The difference in development of the two areas resulting in suture formation and fusion, respectively, could not be explained by morphologic differences in the interzonal tissue, but by analysing the osteogenic pattern along the bony rims adjacent to the maxillary and the palatine processes, it was concluded that widening of the bony palate in the maxillary area occurred by displacement and in the palatine area mainly by cortical drift. A lack of separation of the palatine ossification centres is considered to be a possible regulating factor for the fusion of the palatine bones and the continuous spatial movement of the maxillary ossification centres a factor in the formation of the intermaxillary suture.
INTRODUCTION Some of the ossification centres that appear in the mammalian skull during fetal development fuse completely early in development, for instance those of the human parietal bone; others remain separated by fibrous connective tissue till long after birth (Bruce, 1941; Noback, 1944; Noback and Robertson, 1951). The factors responsible for fusion between some ossification centres and the development of fibrous sutures between others remain unknown. For an understanding of premature closure of sutures, which results in a skull deformity (craniostenosis), knowledge of the factors controlling maintenance of the sutures is essential. Studies performed on suture development have suggested three factors possibly playing a role in this type of joint formation. Pritchard, Scott and Girgis (1956) drew attention to the morphological similarity between developing sutures and diarthrodial joints. From observations on paralysed chick embryos, Drachmann and Sokoloff (1966) concluded that skeletal muscle contractions are essential in embryonic development of diarthrodial joints, a conclusion consistent with an older concept that movement between adjacent bones is responsible for maintaining the patency of sutures (Loeschcke and Weinnoldt, 1922; Sitsen, 1935). Buckland-Wright (1972) has shown that minor movements between cranial bones occur normally, so that movements may play an important role in postnatal maintenance of the suture. Markens (1975a) found that the presumptive coronal suture of fetal rats remained patent when transplanted to the dura mater of older animals, where
no movement could occur. He suggested that a biochemical factor, an osteogenesis-inhibiting factor, WAS responsible for suture development and preventing fusion of the bones. Cell death is a normal feature in the formation of a number of tissues (Gliicksmann, 1951) and was also observed electron microscopically by Ten Cate, Freeman and Dickinson (1977) during cranial suture development in the rat. The developing secondary palate of the fetal rabbit provides a suitable model to study suture formation and bony fusion between ossification centres. The growing palatine bones meet in the midline and fuse, forming a single unpaired bone, but immediately anterior to the fusion area the palatal processes from the maxillary bones remain separated by an intermaxiliary suture (Bruce, 1941). Hence, in contiguous areas, widely-different factors controlling suture formation must operate.
MATERIAL AND METHODS New Zealand White rabbits, weighing 3.54.5 kg, were mated for two l-h periods with a 12-h interval. The day of mating was designated as day 1 of gestation. Fetuses were removed by Caesarian section at 24-h intervals starting on day 20. By staining the skulls with alizarin and clearing them according to Staples and Schnell (1964), a series of developmental stages of the fetal bony palates was obtained. Using the data from the developmental series, fetuses ranging in age from day 21-25 were selected for further study. The secondary palates were fixed in alcoholic formalin (three parts 95 per cent ethanol: one part formalin) for 2 days and demineralized in a mixture of formic acid and sodium formate (45 per
* Present address: Department of Orthodontics, Faculty of Dentistry, University of Gothenburg, S-400 33 Gothenburg. Sweden. 283
2x4
M. Persson and W. Roy
cent and 15 per cent, equal parts) for 7-14 days. Following embedding in Paraplast (Sherwood Medical Industries, St Louis. Miss.), serial 6+m sections in the horizontal plane were cut through the maxillarypalatine area. Serial transverse sections through the same area were made in other specimens. Every fourth section was stained with Harris haematoxylineosin and adjacent sections were stained with Heidenhain azan for collagen. The remaining sections were stained with the periodic acid-Schiff (PAS) method (Lillie, 1954) to study differentiation of osteogenic ceils by demonstrating intracellular glycogen (Pritchard, 1952). To test the specificity of the PAS-staining reaction, consecutive sections were treated with diastase (diastase of malt, Fischer Sci. Co, Pittsburg, Penn.) for extraction of glycogen before staining (Lillie, t 954). Some palates were fixed in 3 per cent glutaraldehyde buffered in 0.1 M sodium cacodylate buffer and postfixed in 1 per cent osmium tetroxide. Before postfixation, some palates were demineralized in 10 per cent EDTA. The tissue was routinely processed and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, Penn.). Sections 2 Ltmthick were cut and stained with Toluidine blue in 1 per cent sodium borate. Although C3H]-proline is not a specific indicator of the synthesis of collagen, there is reason to consider that the bulk of the uptake in autoradiographs represents the uptake in collagen (Carneiro, 1965). 4 /G/g body wt tritiated proline (L-proline-[3H], specific activity 29 Ci/mmol; New England Nuclear, Boston, Mass.) was administered intra-peritoneally to fetuses on days 21 and 22 through an opening in the uterine wall. The does were kept under pentobarbital anaesthesia and the fetuses removed 30min, 2 h or 4 h after injection. After fixation in alcoholic formalin, embedding in paraffin and sectioning (61(m), autoradiographs were prepared according to the dipping technique of Kopriwa and Leblond (19623, using Kodak NTB2 emulsion.
RESULTS
Bo11.vpalate derefopment specimens
in dizarin-stained
and cleared
Alizarin-stained specimens showed that the bony processes of the maxilla became distinguishable on day 20-21. During day 21-22, the bony processes approached each other rapidly and on day 22-23 reached the stable distance that constitutes the suture (Figs. 1 and 2). The bony processes of the palatine bones became visible approximately 1 day later than the maxillary ones, and fused with each other on day 23-24, establishing a single unpaired palatine bone across the midtine (Fig. 2). Ossification detected in the alizarinstained and cleared specimens was a little less advanced than in paraffin and plastic-embedded sections. Because of the difference in chronology of development of maxillary and palatine bony processes, we shall describe comparable developmental stages rather than chronological stages. The terms defined by Pritchard et al. (1956) as “stage of approaching bone territories” and “stage of meeting bone territories” will be used.
The interzone rerritories
during
the stage of approaching
bone
When the maxillary and the palatine bony processes started to approach.each other, the interzone between the processes consisted of 3 different cell zones (Fig. 3): (a) a narrow, cell-rich midline zone. (b) a wide zone with undifferentiated mesenchyme on each side of the midline, (c) a differentiating osteogenic zone bilaterally as parts of the approaching bone territories. The midline zone consisted of a narrow band with irregular cell density. It extended antero-posteriorly through the future bony palate, but decreased in density and disappeared in the area of the future soft palate. In transverse sections the zone extended between the oral and nasal epithelial linings, but was most pronounced in the oral part. In some regions, the cells formed small aggregates, in others the zone was less apparent. The majority of the cells had a small, clearly distinguishable, nucleus with one or two prominent nucleoli (Fig. 4). Some cells showed deeply staining nucleus and cytoplasm; in others the nucleus had broken into fragments, indicating cell degeneration (Fig. 5). In PAS-stained sections of the midline zone, remnants of a basement membrane could be seen (Fig. 6). We believe that the midline zone contains remnants of the epithe~al seam which was formed when the epithelial linings of the palatal shelves fused. The undifferentiated mesenchyme cells on each side of the midline zone were widely separated and without any specific orientation (Fig. 4). They were irregular in shape and their nuclei stained less intensely than those of the midline zone. Scattered among the mesenchyme cells were some spindle-shaped cells characterized by a basophilic cytoplasm. Laterally the zone of undifferentiated mesenchyme gradually gave way to a zone of denseiy-packed cells with the features of differentiating osteogenic cells (Fig. 7). Close to the undifferentiat~ mesenchyme, most of the cells had a moderately stained nucleus with prominent nucleoli; they also stained intensely for glycogen and were therefore thought to be preosteoblasts. More laterally there were cells with the features of osteoblasts, that is a large, marked basophilic cytoplasm and an eccentric hypochromatic nucleus (Fig. 7). Mitosis was absent in the osteoblast population and intracellular glycogen was rare. No differences were found between the interzones of the maxillary and palatine processes. In sections of both maxillary and palatine areas stained with azan, osteogenic fibre bundles were seen to emerge from the bone trabeculae and to radiate between the pre-osteoblasts. More medially, the number of fibres was low, with only a few scattered fibres without specific orientation outside the osteogenic zones. In autoradiographs prepared 30 min and 4 h after injection, the maxillary interzone was more heavily labelled than the palatine one (Figs. 8 and 9). No difference in intensity, however, was evident when comparable stages of development were examined. The midline zone showed the weakest labelling but towards the osteogenic tones it gradually increased. Osteoblasts were most intensely labelled. In 4 h
Suture development autoradiographs, there was a large number of grains outside the osteoblasts, indicating that extracellular proteins had been deposited. This phenomenon was less evident in other parts of the interzone. The site of presumptive suture ligament formation thus could not be distinguished at this stage from the site of later fusion either by the number and direction of the collagenous fibres, or by the distribution of, or rate of, accumulation of labelled proteins. Through continuous expansion of the bony processes, at the expense of the undifferentiated mesenthyme, the interzone of both sites became gradually narrow. The midline zone persisted until it was encroached upon by the osteogenic zones, when it could no longer be distinguished. The fusing osteogenic zones, characterized by marked cellular density, gradually made up the interzone between the bony processes. The first indication of a possible difference between the maxillary and the palatine site was in the osteogenic zone. In the maxillary area, a zone with a PASreaction positive for glycogen remained separated from the bone by a zone of weak staining, but in the palatine area it was found immediately adjacent to the surface of the bone (Figs. 10 and 11). Hence, the difference in staining for glycogen in cells close to the bone was the only observation indicating a different .development between the two sites during this stage. The interzone during stage of meeting bone territories Following meeting of the osteogenic zones, markedly different changes occurred in the maxillary and the palatine sites. When the bone trabeculae of the maxillary processes approached each other closely, a new central zone was formed characterized by cells with a large round to oval nucleus and a darklystained vacuolated cytoplasm (Fig. 12). In paraffin sections, these cells showed an intensely-positive PAS reaction (Fig. 13) that was missing in adjacent sections treated with diastase. As cells with similar characteristics were present in the adjacent periosteum, we believe the central zone represents the fusion of two outer periosteal cambial layers. The bony processes from this stage remained at a constant distance from each other, and a suture separating the processes was thus established. When the bone trabeculae of the palatine bony processes approached each other closely, no central zone developed as seen between the maxillary ones (cf. Figs. 12 and 14) and the interzone appeared to be compressed. Some of the cells in the merging osteogenic zones (Fig. 14) assumed an elongated irregular shape with their longitudinal axes oriented parallel to the bony surface and an increased stainability, others had a large pale nucleus and a prominent nucleolus and appeared similar to pre-osteoblasts. The osteoblast layer became less conspicuous and mitoses were rare. Some of the cells continued to stain positively for glycogen (Fig. 15). Following partial fusion of the processes, clusters and narrow strips of cells temporarily remained in the midline (Figs. 16 and 17). These cells, probably rests of the earlier interzone tissue, were of bizarre shape and variable in size and staining intensity, and many were probably degener-
285
and bony fusion
ating (Fig. 17). Thus, whereas the maxillary bony processes remained separated by the joined bilateral osteogenic zones, the palatine processes became fused. Osteogenic pattern of adjacent bony margins The anterior extensions of the maxillary palate, forming the lateral walls of the incisive foramen, consisted of dense bone with fairly smooth surface (Figs 18 and 21). These surfaces were covered by a single layer of mostly cuboidal or slightly flattened cells with conspicuous cytoplasm. No osteoclasts were observed. The surfaces retained these cellular appearances even after a definitive suture had formed. The posterior extensions of the maxillary palate, along the palato-maxillary suture, had mainly a .trabecular appearance and were completely covered by large active osteoblasts (Fig. 19). The structure of the palatine bone margins at the palato-maxillary sutures was similar (Fig. 19). Before and after fusion they were lined by osteoblasts, and there was no osteoclastic activity. In contrast, the surfaces of the palatine bone, forming the lateral wall of the oronasal cavity, consisted of dense bone with a non-trabecular structure and scalloped surface (Fig. 20). The walls were mainly covered by small cells, interspersed with large multinuclear osteoclasts. These appearances persisted after the palatine bony processes had fused. DISCUSSION
In the rabbit, elevation and fusion of the palatal shelves is completed on prenatal day 17 (Walker, 1971). Formation of the bony palate began on prenatal day 20 and was completed on day 24 (Bruce, 1941). We found remnants of the epithelial linings of the shelves still identifiable while the bony processes approached each other, whereas in the rat the epithelial seam breaks down rapidly and the basement membrane disappears even before degeneration of the epithelial seam (Hughes, Furstman and B:rnick, 1967). In man also a distinct basement membrane lines the fused epithelial lamina (Barry, 1961) in which epithelial remnants are frequently found postnatally (Wood and Kraus, 1962). These epithelial remnants apparently do not influence subsequent suture formation or bony fusion. The cellular aggregation persisting from the original line of fusion between the shelves was morphologically quite different from the blastema consisting of thin collagenous fibres interspersed between mesenchymal cells and fibroblasts described by Markens (1975b) in cranial suture areas in the rat one day before the suture was established. We observed a blastema only when the bone territories met; hence, we agree with Pritchard et al. (1956) that no specific morphological features indicate where a suture will develop. The bony processes of the maxillary and palatine bones approach each other by penetrating a loose mesenchyme tissue. The low uptake of labelled proline and the few collagenous fibres in this intervening tissue indicate that non-osteogenic collagen production is a relatively late feature of suture formation in the palate. Ten Cate, Freeman and Dickinson (1977) suggest that the development and structure of
286
M. Persson and W. Roy
cranial vault sutures can be described “in terms of the functional activity of two cell populations, namely, the osteocytic and fibrocytic series”. Besides collagen production, they found removal of fibrous tissue to be an activity of the fibrocytic series. Although their stages of development are not clearly defined, the differences between our observations and theirs probably arise because in the cranial vault the bone components are connected from the beginning by the more densely fibrous ectomeninx whereas, in the palate, the bones approach each other in a loose, mesenchymatous tissue (Pritchard et al., 1956). Osteoblasts in fetal tissue are characterized by a marked glycogen storage (Harris, 1932; Pritchard, 1952). Ultrastructural studies in the rat suggest that the glycogen content is maximal in pre-osteoblasts, in which it may occupy two-thirds of the cell volume (Scott and Glimcher, 1971). Using glycogen storage as a criterion for distinguishing fibrocytic from osteogenic cell populations, our observations indicate that the majority of the cells between the approaching bony processes are differentiating into osteogenic cells, in both the suture-forming and the fusing areas. Meeting of bone territories in the palate initially consisted of the simple coalescence of two PAS-positive osteogenic zones in both the suture-forming area and the fusing area. In the developing suture, however, a new central layer subsequently was recognized. Pritchard et al. (1956) stated that a loose cellular layer in the middle of the suture is provided by remnants of the “inter-territorial” mesenchymal tissue, separating the bilateral fibrous and cambial layers of the osteogenic zones. This conclusion was reinforced by conspicuous glycogen storage and alkaline phosphatase activity in the cambial osteogenic layers, but not in the intervening fibrous and loose cellular middle zone of the suture. In our experiments, however, the central layer of the developing palatal suture was characterized by a high glycogen content which would indicate that it is made up mainly by what Scott and Glimcher (1971) called “poorly differentiated” osteogenic cells. Sutural tissue growth is characterized by a high proliferative activity of “suture” cells, collagen synthesis, and production of glycosaminoglycans in the extracellular substance (Persson, 1973). The accumulation of glycogen in the suture cells may be specifically related to one of these activities, especially as glycogen has been implicated as a source material for the intracellular synthesis of mucosubstances in the fetal osteoblast (Scott and Glimcher, 1971). The glycogen content of matrix-producing osteoblasts is low (Nilsen, 1977). It is therefore possible that glycogen accumulation in the central layer of the suture indicates that some differentiating pre-osteoblasts are by chance trapped temporarily in this layer, when other cells differentiate further into osteoblasts. Although we cannot classify these cells in the new central layer of the suture or explain their high glycogen content, they cannot be remnants of the mesenchymatous tissue. Furthermore, as no difference was found with regard to the presence of a fibrous layer between the suture-forming and fusing areas, a remnant of the interzone cannot be essential in preventing fusion of the bones at a suture, as suggested by Troitsky (1932) and Pritchard et al. (1956).
The ultimate fate of the tissue between the fusing bone processes could not be conclusively determined, but many cells showed signs of degeneration. Fusion of ossification centres during early skeletal development appears, therefore, to be morphologically distinct from the fusion process observed in the mature suture (Moss, 1958; Persson, Magnusson and Thilander, 1978). We cannot support the suggestion by Ten Cate et al. (1977) that cell death is significant in the development of a suture. Our observations indicate that cellular degeneration in the interzone is more related to the fusion process between bony components. In agreement with this concept is the observation by Piischmann (1975) that extensive necrosis of the interzone in explanted synovial joints is followed by fusion of the cartilaginous anlage. We looked for an explanation of the different patterns of development in suture formation and fusion outside the interzonal tissue. In the maxillary area, the bone deposition along the margins of the incisive foramen, bilaterally in the palato-maxillary suture and at the mid-palatal suture means that there is a simultaneous separation of the two maxillary bones (Fig. 21). In the palatine area, on the other hand, the extensive resorption along the surfaces facing the oronasal cavity, together with osteoblastic activity on the opposite surface at the palato-maxillary suture indicates that the vertical plates of the palatine bones move laterally by remodelling. Hence, whereas widening of the maxillary bony palate occurs by lateral movements of the maxillary bones in roro, the palatine area widens by so-called cortical drift. This has been interpreted by Hoyte (1976) as moving the palatine bones postnatally pari pussu with the maxillary bones, when separation of the two palatine bones is made impossible by the lack of an intervening suture. Our observations indicate that this lateral displacement of the palatine bones is minimal or absent even before the two sides have fused. A lack of such movement by the expanding palatine ossification centres may, therefore, be the primary factor responsible for the cellular packing and necrosis of the interzone and the ultimate fusion of the bony palatine processes. The contrasting spatial separation of the maxillary ossification centres during their expansion is thus a possible determining maxillary suture.
factor
in the formation
of the
inter-
Acknowledgements-This study was performed under grant Nos. S-T32-DE-07037 and 5-ROl-04402 from the United States National Institutes of Health. We are indebted to Dr. J. Langman for his support and helpful suggestions and to Mrs Mildred Marshall for technical assistance.
REFERENCES
Barry A. 1961. Development
of the branchial region of human embryos with special reference to the fate of epithelia. In: Congeniral Anomalies of the Face and Associated Structures (Edited by Pruzansky S.), pp. 46-62. Charles C. Thomas, Springfield, Ill. Bruce J. 1941. Time and order of appearance of ossification centers and their development in the skull of the rabbit. Am. J. Anat.
68, 41-67.
Buckland-Wright J. D. 1972. The shock-absorbing effect of cranial sutures in certain mammals. J. dent. Res. 51, 1251 abstract.
Suture
development and bony fusion
Noback C. R. and Robertson G. G. 1951. Sequences of appearance of ossification centers in the human skeleton during the first five prenatal months. Am. J. Anar. 89, l-28. Persson M. 1973. Structure and growth of facial sutures. Histologic, microangiographic and autoradiographic studies in rats and a histologic study in man. Odont. Revy 24, Suppi. 26. Persson M., Magnusson B. and Thilander B. 1978. Suturai closure in rabbit and man: a morphological and histochemical study. J. Anat., Lond. 123, 313-322. Pritchard J. J. 1952. A cytological and histochemicai study of bone and cartilage formation in the rat. 1. Anat., Lond. 86. 259-277. Pritchard J: J., Scott J. H. and Girgis F. G. 1956, Structure and development of cranial and facial sutures. J. Anat., Lond. 90, 73-89. Piischmann H. 1975. Comparison of joint formation in oivo and in vitro. In: New Approaches to rhe Evaluation of Abnormal Embryonic Development (Edited by Neubert D. and Merker H. J.), pp. 227-239. George Thieme, Stuttgart. Scott B. L. and Glimcher M. J. 1971. Distribution of giycogen in osteobiasts of the fetal rat. J. Ultrastruct. Res.
Carneiro J. 1965. Synthesis and turnover of collagen in periodontal tissuds. In: The Use of Radioautography in Investigating Protein Synthesis (Edited by Leblond C. P. and Warren K. B.), pp. . 247-257. Academic Press, New York. Drachmann D. B. and Sokoioff L. 1966. The role of movement in embryonic joint development. Deul. Biol. 14, 401-420. Giiicksmann A. 1951. Ceil death in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. Harris H. A. 1932. Glycogen in cartilage. Nature, Land. 130, 99&997. Hoyte D. A. N. 1976. Growth of the face and the jaws compared with the rest of the skeleton. In: The ~~uRt~on and .~cclusjon of Teeth (Edited by Poole D. F. G: and Stack M. V.), pp. 31-51. Butterworths, London. Hughes L. V., Furstman L. and Bernick S. 1967. Prenatal development of the rat palate. J. dent. Res. 46, 373-379. Kopriwa B. M. and Lebiond C. P. 1962. Improvements in the coating technique of radioautography. J. Histothem. Cytochem. IO, 269-284. Liiiie R. D. 1954. Hisropathologic Technic and Practical Histochemistry, pp. 274-276. Biakinston, New York. Loeschcke H. and Weinnoldt H. 1922. ifber den Einfluss von Druck und Entspannung auf das Knochenwachstum des Hirnschldeis. Beifr. Parhof. Atzuf. 70, 406-439. Markens I. S. 1975a. Transplantation of the future coronal suture on the dura mater of 3 to 4-months-old rats. Acta Anat.
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36, 565-586.
Sitsen A. E. 193.5.ijber die Ursachen der Verknacherung der Schldeinghte. Frankfurt. Z. Pathoi. 48. 499-525. Staples R. and Schneli V.-L. 1964. Refinements in rapid clearing technic in the KOH-alizarin-Red-S method for fetal bone. Stain Techno~. 39, 61-63. Ten Cate A. R., Freeman E. and Dickinson J. B. 1977. Suturai development: Structure and its response to rapid expansion. Am. J. Orthodonr. 71. 622-636. Troiisky W. 1932. Zur Frage der Formbiidung der Schldeidaches. 2. Morph. Anthrop. 30, 504-532. Walker B. E. 1971. Palate morphogenesis in the rabbit.
93, 29-44.
Markens I. S. 1975b. Embryonic development of the coronai suture in man and rat. Acta Anar. 93, 257-273. Moss M. L. 1958. Fusion of the frontal suture in the rat. Am. J. Anat. 102, 141-166. Nilsen R. 1977. Electron microscopy of induced heterotopic bone formation in guinea pigs. Archs oral Biol. 22. 485493. Noback C. R. 1944. The developmental anatomy of the human osseous skeleton during embryonic, fetal and circumnatai periods. Anar. Rec. 88, 91-i 17.
Archs
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Wood P. J. and Kraus B. S. 1962. Prenatal development of the human palate and some histological observations. Archs oral Viol. 7, 137-150.
Plate 1 overleaf. Fig. 1. Cleared and aiizarin-stained plate of a 22-day-old rabbit fetus from the oral side. The bilateral processes of the maxillary and the palatine bones approach the midline to form the bony palate. The processes of the maxillary bones (M) almost touch but those of the palatine bones (P) are further apart. IF, incisive foramen; NC, posterior nasal choana. Fig. 2. Similar specimen to Fig. 1, but from a 25-day-old rabbit fetus. A suture has been established between the maxillary processes (M), but the processes of the palatine bones (P) have completely fused. Fig. 3. Parapiast section of the secondary palate region of a 21-day fetus. The interzone between the approaching maxillary (M) and palatine (P) bony processes consists of different layers: (a) a narrow ceil-rich midline zone; (b) on each side of the midline, a zone with undifferentiated mesenchyme ceils; (c) differentiating osteogenic zones bilaterally just in front of the approaching bone processes. The midline
zone is distinguishable
in both
maxillary
and palatine
areas
(arrows).
x40
Fig. 4. Epon section of the interzone on day 21. The midline zone (a) is characterized of ceils of varying
by an aggregation morphology. The zone is considered to be a remnant of the epitheiiai seam resulting from the fusion of the paiatai shelves. x 400
Fig. 5. Epon section of the interzone, day 21. Many ceils in the midline zone close to the oral surface show characteristics of ceil degeneration, such as density of nucleus (vertical arrows) and fragmentation (horizontal arrows). x 600
Fig. 6. Transverse paraplast section of the palate on day 21 and PAS-stained. Remnants of the basement membranes persisting from the fusion of the palatai shelves are still visible in the midzone (arrows), although
most of the epitheiiai
ceils have disappeared.
oe, invagination
of the oral epitheiium.
x 200
Fig. 7. Epon section of the differentiating osteogenic zone of the maxillary area on day 21. The osteogenic zone is wide and cells of the interzone gradually (left to right) acquire the characteristics of pre-osteobiasts (pob) and osteoblasts (ob). Some of the densely packed ceils show degenerative changes (arrow). ost, osteoid of the maxillary process. x 400
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M. Persson
and W. Roy
Plate 2. Figs. 8 and 9. Autoradiograph of the on day 22. There are slightly more the palatine area (Fig. 9). Grains are towards the maxillary
middle part of the interzone grains in the interzone of fewer in the midline zone (M) and palatine (P) bones
4 h after idection with [3H]-proline the maxillary area (Fig. 8) than in of both areas, but gradually increase beyond the field. x400
Figs. IO and I I. Paraplast section of the interzone on day 22, and PAS-stained. In the maxillary area (Fig. IO), the intense PAS-positive reaction of the interzone is separated from the bony surface (M) by a zone of weak staining corresponding to the osteoblast layer (+-). In the palatine area (Fig. ll), the intense reaction is in cells adjacent to the palatine bone (P). x400 Fig.
12. Epon section of the maxillary by cells with a large oval nucleus
suture on day 24. There is a new central zone characterized and darkly-stained vacuolated cytoplasm (arrows). x 400
Fig. 13. Paraplast section of the same stage and area as Fig. 12, PAS-stained. Cells of the newly-formed central zone retain the intense PAS-positive reaction for intracellular glycogen shown in earlier stages by cells of the merging bone territories. The zone of weak staining reaction adjacent to the bony maxillary process (M) is now distinct (cf. Fig. 10). x 200 Fig. 14. Epon section of the palatine area on day 23. The palatine processes are now close to each other and the interzone appears compressed. Some interzone cells are elongated or irregular in shape and show varying nuclear and cytoplasmic characteristics (vertical arrows). Others appear similar to pre-osteoblasts (horizontal arrows), but the earlier welldefined osteogenic zones have disappeared. x400 Fig. 15. Paraplast section contiguous bony palatine
of the same area as in Fig. I4 but on day 24, PAS-stained. Between closely processes (P), many cells continue to show a positive reaction for glycogen. x200 Plate
3.
Fig. 16. Paraplast section of the palatine area on day 24, haematoxylin-eosin processes have partially fused. Clusters of cells enclosed by meeting bony remnants of the earlier interzone tissue. x 100
stained. trabeculae
The palatine are probably
Fig. 17. Epon section of the same stage and area as Fig. 16. The cells remaining in the midline after partial fusion of the processes show a bizarre shape and some appear to be pyknotic and disintegrating. x 400 Figs. 18-20. Epon sections of maxillary and palatine bone surfaces prior to fusion of the palatine processes on day 23 (the surface areas in the figures are outlined in Fig. 21). The surface of the maxillary bone (M), forming the lateral wall of the incisive foramen (Fig. 18) and the maxillary bone surface (M) at the palate-maxillary suture (Fig. 19) are lined by active osteoblasts. The opposite margin of the palato-maxillary suture is formed by the lateral wall of the vertical lamina of the palatine bone (P). Its surface is covered by active qsteoblasts; the opposite surface of the lamina, facing the oronasal cavity (Fig. 20). shows several osteoclasts (arrows) and only a few active osteoblasts. x 100 Fig. 21. Cleared and alizarin-stained palate of a 22-day old fetus. Osteoblastic activity is marked +, osteoclastic activity is marked -. The depository and resorptive osteogenic patterns in Figs. 18-20 and at the approaching bony processes are outlined. Lateral displacement is indicated by arrows. IF, incisive forarnen; M. maxillary bone: P. palatine bone: pm, palato-maxillary suture.
Suture development
and bony fusion
Plate 1.
289
290
M. Persson
and W. Roy
Plate 2.
Suture development
and bony fusion
Plate 3.
291
SUTURE
DEVELOPMENT AND BONY FUSION THE FETAL RABBIT PALATE
IN
M. PERSSON* and W. ROY Department of Anatomy, University of Virginia, Charlottesville, VA 22901, U.S.A. SummaryYThe intermaxillary and the interpalatine regions of the fetal rabbit palate are contiguous areas of suture development and bony fusion, respectively. As these regions must differ regarding the factors responsible for the formation of a suture, the palate was selected as a model for the study of the mechanism of suture development. Palates from 21-25-day rabbit fetuses were classified into developmental stages using cleared and alizarin-stained specimens. The developmental stages of the two areas were compared in paraffin and Epon sections. PAS-staining and autoradiography following [3H]-proline injection were used. The difference in development of the two areas resulting in suture formation and fusion, respectively, could not be explained by morphologic differences in the interzonal tissue, but by analysing the osteogenic pattern along the bony rims adjacent to the maxillary and the palatine processes, it was concluded that widening of the bony palate in the maxillary area occurred by displacement and in the palatine area mainly by cortical drift. A lack of separation of the palatine ossification centres is considered to be a possible regulating factor for the fusion of the palatine bones and the continuous spatial movement of the maxillary ossification centres a factor in the formation of the intermaxillary suture.
INTRODUCTION Some of the ossification centres that appear in the mammalian skull during fetal development fuse completely early in development, for instance those of the human parietal bone; others remain separated by fibrous connective tissue till long after birth (Bruce, 1941; Noback, 1944; Noback and Robertson, 1951). The factors responsible for fusion between some ossification centres and the development of fibrous sutures between others remain unknown. For an understanding of premature closure of sutures, which results in a skull deformity (craniostenosis), knowledge of the factors controlling maintenance of the sutures is essential. Studies performed on suture development have suggested three factors possibly playing a role in this type of joint formation. Pritchard, Scott and Girgis (1956) drew attention to the morphological similarity between developing sutures and diarthrodial joints. From observations on paralysed chick embryos, Drachmann and Sokoloff (1966) concluded that skeletal muscle contractions are essential in embryonic development of diarthrodial joints, a conclusion consistent with an older concept that movement between adjacent bones is responsible for maintaining the patency of sutures (Loeschcke and Weinnoldt, 1922; Sitsen, 1935). Buckland-Wright (1972) has shown that minor movements between cranial bones occur normally, so that movements may play an important role in postnatal maintenance of the suture. Markens (1975a) found that the presumptive coronal suture of fetal rats remained patent when transplanted to the dura mater of older animals, where
no movement could occur. He suggested that a biochemical factor, an osteogenesis-inhibiting factor, WAS responsible for suture development and preventing fusion of the bones. Cell death is a normal feature in the formation of a number of tissues (Gliicksmann, 1951) and was also observed electron microscopically by Ten Cate, Freeman and Dickinson (1977) during cranial suture development in the rat. The developing secondary palate of the fetal rabbit provides a suitable model to study suture formation and bony fusion between ossification centres. The growing palatine bones meet in the midline and fuse, forming a single unpaired bone, but immediately anterior to the fusion area the palatal processes from the maxillary bones remain separated by an intermaxiliary suture (Bruce, 1941). Hence, in contiguous areas, widely-different factors controlling suture formation must operate.
MATERIAL AND METHODS New Zealand White rabbits, weighing 3.54.5 kg, were mated for two l-h periods with a 12-h interval. The day of mating was designated as day 1 of gestation. Fetuses were removed by Caesarian section at 24-h intervals starting on day 20. By staining the skulls with alizarin and clearing them according to Staples and Schnell (1964), a series of developmental stages of the fetal bony palates was obtained. Using the data from the developmental series, fetuses ranging in age from day 21-25 were selected for further study. The secondary palates were fixed in alcoholic formalin (three parts 95 per cent ethanol: one part formalin) for 2 days and demineralized in a mixture of formic acid and sodium formate (45 per
* Present address: Department of Orthodontics, Faculty of Dentistry, University of Gothenburg, S-400 33 Gothenburg. Sweden. 283
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M. Persson and W. Roy
cent and 15 per cent, equal parts) for 7-14 days. Following embedding in Paraplast (Sherwood Medical Industries, St Louis. Miss.), serial 6+m sections in the horizontal plane were cut through the maxillarypalatine area. Serial transverse sections through the same area were made in other specimens. Every fourth section was stained with Harris haematoxylineosin and adjacent sections were stained with Heidenhain azan for collagen. The remaining sections were stained with the periodic acid-Schiff (PAS) method (Lillie, 1954) to study differentiation of osteogenic ceils by demonstrating intracellular glycogen (Pritchard, 1952). To test the specificity of the PAS-staining reaction, consecutive sections were treated with diastase (diastase of malt, Fischer Sci. Co, Pittsburg, Penn.) for extraction of glycogen before staining (Lillie, t 954). Some palates were fixed in 3 per cent glutaraldehyde buffered in 0.1 M sodium cacodylate buffer and postfixed in 1 per cent osmium tetroxide. Before postfixation, some palates were demineralized in 10 per cent EDTA. The tissue was routinely processed and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, Penn.). Sections 2 Ltmthick were cut and stained with Toluidine blue in 1 per cent sodium borate. Although C3H]-proline is not a specific indicator of the synthesis of collagen, there is reason to consider that the bulk of the uptake in autoradiographs represents the uptake in collagen (Carneiro, 1965). 4 /G/g body wt tritiated proline (L-proline-[3H], specific activity 29 Ci/mmol; New England Nuclear, Boston, Mass.) was administered intra-peritoneally to fetuses on days 21 and 22 through an opening in the uterine wall. The does were kept under pentobarbital anaesthesia and the fetuses removed 30min, 2 h or 4 h after injection. After fixation in alcoholic formalin, embedding in paraffin and sectioning (61(m), autoradiographs were prepared according to the dipping technique of Kopriwa and Leblond (19623, using Kodak NTB2 emulsion.
RESULTS
Bo11.vpalate derefopment specimens
in dizarin-stained
and cleared
Alizarin-stained specimens showed that the bony processes of the maxilla became distinguishable on day 20-21. During day 21-22, the bony processes approached each other rapidly and on day 22-23 reached the stable distance that constitutes the suture (Figs. 1 and 2). The bony processes of the palatine bones became visible approximately 1 day later than the maxillary ones, and fused with each other on day 23-24, establishing a single unpaired palatine bone across the midtine (Fig. 2). Ossification detected in the alizarinstained and cleared specimens was a little less advanced than in paraffin and plastic-embedded sections. Because of the difference in chronology of development of maxillary and palatine bony processes, we shall describe comparable developmental stages rather than chronological stages. The terms defined by Pritchard et al. (1956) as “stage of approaching bone territories” and “stage of meeting bone territories” will be used.
The interzone rerritories
during
the stage of approaching
bone
When the maxillary and the palatine bony processes started to approach.each other, the interzone between the processes consisted of 3 different cell zones (Fig. 3): (a) a narrow, cell-rich midline zone. (b) a wide zone with undifferentiated mesenchyme on each side of the midline, (c) a differentiating osteogenic zone bilaterally as parts of the approaching bone territories. The midline zone consisted of a narrow band with irregular cell density. It extended antero-posteriorly through the future bony palate, but decreased in density and disappeared in the area of the future soft palate. In transverse sections the zone extended between the oral and nasal epithelial linings, but was most pronounced in the oral part. In some regions, the cells formed small aggregates, in others the zone was less apparent. The majority of the cells had a small, clearly distinguishable, nucleus with one or two prominent nucleoli (Fig. 4). Some cells showed deeply staining nucleus and cytoplasm; in others the nucleus had broken into fragments, indicating cell degeneration (Fig. 5). In PAS-stained sections of the midline zone, remnants of a basement membrane could be seen (Fig. 6). We believe that the midline zone contains remnants of the epithe~al seam which was formed when the epithelial linings of the palatal shelves fused. The undifferentiated mesenchyme cells on each side of the midline zone were widely separated and without any specific orientation (Fig. 4). They were irregular in shape and their nuclei stained less intensely than those of the midline zone. Scattered among the mesenchyme cells were some spindle-shaped cells characterized by a basophilic cytoplasm. Laterally the zone of undifferentiated mesenchyme gradually gave way to a zone of denseiy-packed cells with the features of differentiating osteogenic cells (Fig. 7). Close to the undifferentiat~ mesenchyme, most of the cells had a moderately stained nucleus with prominent nucleoli; they also stained intensely for glycogen and were therefore thought to be preosteoblasts. More laterally there were cells with the features of osteoblasts, that is a large, marked basophilic cytoplasm and an eccentric hypochromatic nucleus (Fig. 7). Mitosis was absent in the osteoblast population and intracellular glycogen was rare. No differences were found between the interzones of the maxillary and palatine processes. In sections of both maxillary and palatine areas stained with azan, osteogenic fibre bundles were seen to emerge from the bone trabeculae and to radiate between the pre-osteoblasts. More medially, the number of fibres was low, with only a few scattered fibres without specific orientation outside the osteogenic zones. In autoradiographs prepared 30 min and 4 h after injection, the maxillary interzone was more heavily labelled than the palatine one (Figs. 8 and 9). No difference in intensity, however, was evident when comparable stages of development were examined. The midline zone showed the weakest labelling but towards the osteogenic tones it gradually increased. Osteoblasts were most intensely labelled. In 4 h
Suture development autoradiographs, there was a large number of grains outside the osteoblasts, indicating that extracellular proteins had been deposited. This phenomenon was less evident in other parts of the interzone. The site of presumptive suture ligament formation thus could not be distinguished at this stage from the site of later fusion either by the number and direction of the collagenous fibres, or by the distribution of, or rate of, accumulation of labelled proteins. Through continuous expansion of the bony processes, at the expense of the undifferentiated mesenthyme, the interzone of both sites became gradually narrow. The midline zone persisted until it was encroached upon by the osteogenic zones, when it could no longer be distinguished. The fusing osteogenic zones, characterized by marked cellular density, gradually made up the interzone between the bony processes. The first indication of a possible difference between the maxillary and the palatine site was in the osteogenic zone. In the maxillary area, a zone with a PASreaction positive for glycogen remained separated from the bone by a zone of weak staining, but in the palatine area it was found immediately adjacent to the surface of the bone (Figs. 10 and 11). Hence, the difference in staining for glycogen in cells close to the bone was the only observation indicating a different .development between the two sites during this stage. The interzone during stage of meeting bone territories Following meeting of the osteogenic zones, markedly different changes occurred in the maxillary and the palatine sites. When the bone trabeculae of the maxillary processes approached each other closely, a new central zone was formed characterized by cells with a large round to oval nucleus and a darklystained vacuolated cytoplasm (Fig. 12). In paraffin sections, these cells showed an intensely-positive PAS reaction (Fig. 13) that was missing in adjacent sections treated with diastase. As cells with similar characteristics were present in the adjacent periosteum, we believe the central zone represents the fusion of two outer periosteal cambial layers. The bony processes from this stage remained at a constant distance from each other, and a suture separating the processes was thus established. When the bone trabeculae of the palatine bony processes approached each other closely, no central zone developed as seen between the maxillary ones (cf. Figs. 12 and 14) and the interzone appeared to be compressed. Some of the cells in the merging osteogenic zones (Fig. 14) assumed an elongated irregular shape with their longitudinal axes oriented parallel to the bony surface and an increased stainability, others had a large pale nucleus and a prominent nucleolus and appeared similar to pre-osteoblasts. The osteoblast layer became less conspicuous and mitoses were rare. Some of the cells continued to stain positively for glycogen (Fig. 15). Following partial fusion of the processes, clusters and narrow strips of cells temporarily remained in the midline (Figs. 16 and 17). These cells, probably rests of the earlier interzone tissue, were of bizarre shape and variable in size and staining intensity, and many were probably degener-
285
and bony fusion
ating (Fig. 17). Thus, whereas the maxillary bony processes remained separated by the joined bilateral osteogenic zones, the palatine processes became fused. Osteogenic pattern of adjacent bony margins The anterior extensions of the maxillary palate, forming the lateral walls of the incisive foramen, consisted of dense bone with fairly smooth surface (Figs 18 and 21). These surfaces were covered by a single layer of mostly cuboidal or slightly flattened cells with conspicuous cytoplasm. No osteoclasts were observed. The surfaces retained these cellular appearances even after a definitive suture had formed. The posterior extensions of the maxillary palate, along the palato-maxillary suture, had mainly a .trabecular appearance and were completely covered by large active osteoblasts (Fig. 19). The structure of the palatine bone margins at the palato-maxillary sutures was similar (Fig. 19). Before and after fusion they were lined by osteoblasts, and there was no osteoclastic activity. In contrast, the surfaces of the palatine bone, forming the lateral wall of the oronasal cavity, consisted of dense bone with a non-trabecular structure and scalloped surface (Fig. 20). The walls were mainly covered by small cells, interspersed with large multinuclear osteoclasts. These appearances persisted after the palatine bony processes had fused. DISCUSSION
In the rabbit, elevation and fusion of the palatal shelves is completed on prenatal day 17 (Walker, 1971). Formation of the bony palate began on prenatal day 20 and was completed on day 24 (Bruce, 1941). We found remnants of the epithelial linings of the shelves still identifiable while the bony processes approached each other, whereas in the rat the epithelial seam breaks down rapidly and the basement membrane disappears even before degeneration of the epithelial seam (Hughes, Furstman and B:rnick, 1967). In man also a distinct basement membrane lines the fused epithelial lamina (Barry, 1961) in which epithelial remnants are frequently found postnatally (Wood and Kraus, 1962). These epithelial remnants apparently do not influence subsequent suture formation or bony fusion. The cellular aggregation persisting from the original line of fusion between the shelves was morphologically quite different from the blastema consisting of thin collagenous fibres interspersed between mesenchymal cells and fibroblasts described by Markens (1975b) in cranial suture areas in the rat one day before the suture was established. We observed a blastema only when the bone territories met; hence, we agree with Pritchard et al. (1956) that no specific morphological features indicate where a suture will develop. The bony processes of the maxillary and palatine bones approach each other by penetrating a loose mesenchyme tissue. The low uptake of labelled proline and the few collagenous fibres in this intervening tissue indicate that non-osteogenic collagen production is a relatively late feature of suture formation in the palate. Ten Cate, Freeman and Dickinson (1977) suggest that the development and structure of
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cranial vault sutures can be described “in terms of the functional activity of two cell populations, namely, the osteocytic and fibrocytic series”. Besides collagen production, they found removal of fibrous tissue to be an activity of the fibrocytic series. Although their stages of development are not clearly defined, the differences between our observations and theirs probably arise because in the cranial vault the bone components are connected from the beginning by the more densely fibrous ectomeninx whereas, in the palate, the bones approach each other in a loose, mesenchymatous tissue (Pritchard et al., 1956). Osteoblasts in fetal tissue are characterized by a marked glycogen storage (Harris, 1932; Pritchard, 1952). Ultrastructural studies in the rat suggest that the glycogen content is maximal in pre-osteoblasts, in which it may occupy two-thirds of the cell volume (Scott and Glimcher, 1971). Using glycogen storage as a criterion for distinguishing fibrocytic from osteogenic cell populations, our observations indicate that the majority of the cells between the approaching bony processes are differentiating into osteogenic cells, in both the suture-forming and the fusing areas. Meeting of bone territories in the palate initially consisted of the simple coalescence of two PAS-positive osteogenic zones in both the suture-forming area and the fusing area. In the developing suture, however, a new central layer subsequently was recognized. Pritchard et al. (1956) stated that a loose cellular layer in the middle of the suture is provided by remnants of the “inter-territorial” mesenchymal tissue, separating the bilateral fibrous and cambial layers of the osteogenic zones. This conclusion was reinforced by conspicuous glycogen storage and alkaline phosphatase activity in the cambial osteogenic layers, but not in the intervening fibrous and loose cellular middle zone of the suture. In our experiments, however, the central layer of the developing palatal suture was characterized by a high glycogen content which would indicate that it is made up mainly by what Scott and Glimcher (1971) called “poorly differentiated” osteogenic cells. Sutural tissue growth is characterized by a high proliferative activity of “suture” cells, collagen synthesis, and production of glycosaminoglycans in the extracellular substance (Persson, 1973). The accumulation of glycogen in the suture cells may be specifically related to one of these activities, especially as glycogen has been implicated as a source material for the intracellular synthesis of mucosubstances in the fetal osteoblast (Scott and Glimcher, 1971). The glycogen content of matrix-producing osteoblasts is low (Nilsen, 1977). It is therefore possible that glycogen accumulation in the central layer of the suture indicates that some differentiating pre-osteoblasts are by chance trapped temporarily in this layer, when other cells differentiate further into osteoblasts. Although we cannot classify these cells in the new central layer of the suture or explain their high glycogen content, they cannot be remnants of the mesenchymatous tissue. Furthermore, as no difference was found with regard to the presence of a fibrous layer between the suture-forming and fusing areas, a remnant of the interzone cannot be essential in preventing fusion of the bones at a suture, as suggested by Troitsky (1932) and Pritchard et al. (1956).
The ultimate fate of the tissue between the fusing bone processes could not be conclusively determined, but many cells showed signs of degeneration. Fusion of ossification centres during early skeletal development appears, therefore, to be morphologically distinct from the fusion process observed in the mature suture (Moss, 1958; Persson, Magnusson and Thilander, 1978). We cannot support the suggestion by Ten Cate et al. (1977) that cell death is significant in the development of a suture. Our observations indicate that cellular degeneration in the interzone is more related to the fusion process between bony components. In agreement with this concept is the observation by Piischmann (1975) that extensive necrosis of the interzone in explanted synovial joints is followed by fusion of the cartilaginous anlage. We looked for an explanation of the different patterns of development in suture formation and fusion outside the interzonal tissue. In the maxillary area, the bone deposition along the margins of the incisive foramen, bilaterally in the palato-maxillary suture and at the mid-palatal suture means that there is a simultaneous separation of the two maxillary bones (Fig. 21). In the palatine area, on the other hand, the extensive resorption along the surfaces facing the oronasal cavity, together with osteoblastic activity on the opposite surface at the palato-maxillary suture indicates that the vertical plates of the palatine bones move laterally by remodelling. Hence, whereas widening of the maxillary bony palate occurs by lateral movements of the maxillary bones in roro, the palatine area widens by so-called cortical drift. This has been interpreted by Hoyte (1976) as moving the palatine bones postnatally pari pussu with the maxillary bones, when separation of the two palatine bones is made impossible by the lack of an intervening suture. Our observations indicate that this lateral displacement of the palatine bones is minimal or absent even before the two sides have fused. A lack of such movement by the expanding palatine ossification centres may, therefore, be the primary factor responsible for the cellular packing and necrosis of the interzone and the ultimate fusion of the bony palatine processes. The contrasting spatial separation of the maxillary ossification centres during their expansion is thus a possible determining maxillary suture.
factor
in the formation
of the
inter-
Acknowledgements-This study was performed under grant Nos. S-T32-DE-07037 and 5-ROl-04402 from the United States National Institutes of Health. We are indebted to Dr. J. Langman for his support and helpful suggestions and to Mrs Mildred Marshall for technical assistance.
REFERENCES
Barry A. 1961. Development
of the branchial region of human embryos with special reference to the fate of epithelia. In: Congeniral Anomalies of the Face and Associated Structures (Edited by Pruzansky S.), pp. 46-62. Charles C. Thomas, Springfield, Ill. Bruce J. 1941. Time and order of appearance of ossification centers and their development in the skull of the rabbit. Am. J. Anat.
68, 41-67.
Buckland-Wright J. D. 1972. The shock-absorbing effect of cranial sutures in certain mammals. J. dent. Res. 51, 1251 abstract.
Suture
development and bony fusion
Noback C. R. and Robertson G. G. 1951. Sequences of appearance of ossification centers in the human skeleton during the first five prenatal months. Am. J. Anar. 89, l-28. Persson M. 1973. Structure and growth of facial sutures. Histologic, microangiographic and autoradiographic studies in rats and a histologic study in man. Odont. Revy 24, Suppi. 26. Persson M., Magnusson B. and Thilander B. 1978. Suturai closure in rabbit and man: a morphological and histochemical study. J. Anat., Lond. 123, 313-322. Pritchard J. J. 1952. A cytological and histochemicai study of bone and cartilage formation in the rat. 1. Anat., Lond. 86. 259-277. Pritchard J: J., Scott J. H. and Girgis F. G. 1956, Structure and development of cranial and facial sutures. J. Anat., Lond. 90, 73-89. Piischmann H. 1975. Comparison of joint formation in oivo and in vitro. In: New Approaches to rhe Evaluation of Abnormal Embryonic Development (Edited by Neubert D. and Merker H. J.), pp. 227-239. George Thieme, Stuttgart. Scott B. L. and Glimcher M. J. 1971. Distribution of giycogen in osteobiasts of the fetal rat. J. Ultrastruct. Res.
Carneiro J. 1965. Synthesis and turnover of collagen in periodontal tissuds. In: The Use of Radioautography in Investigating Protein Synthesis (Edited by Leblond C. P. and Warren K. B.), pp. . 247-257. Academic Press, New York. Drachmann D. B. and Sokoioff L. 1966. The role of movement in embryonic joint development. Deul. Biol. 14, 401-420. Giiicksmann A. 1951. Ceil death in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. Harris H. A. 1932. Glycogen in cartilage. Nature, Land. 130, 99&997. Hoyte D. A. N. 1976. Growth of the face and the jaws compared with the rest of the skeleton. In: The ~~uRt~on and .~cclusjon of Teeth (Edited by Poole D. F. G: and Stack M. V.), pp. 31-51. Butterworths, London. Hughes L. V., Furstman L. and Bernick S. 1967. Prenatal development of the rat palate. J. dent. Res. 46, 373-379. Kopriwa B. M. and Lebiond C. P. 1962. Improvements in the coating technique of radioautography. J. Histothem. Cytochem. IO, 269-284. Liiiie R. D. 1954. Hisropathologic Technic and Practical Histochemistry, pp. 274-276. Biakinston, New York. Loeschcke H. and Weinnoldt H. 1922. ifber den Einfluss von Druck und Entspannung auf das Knochenwachstum des Hirnschldeis. Beifr. Parhof. Atzuf. 70, 406-439. Markens I. S. 1975a. Transplantation of the future coronal suture on the dura mater of 3 to 4-months-old rats. Acta Anat.
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36, 565-586.
Sitsen A. E. 193.5.ijber die Ursachen der Verknacherung der Schldeinghte. Frankfurt. Z. Pathoi. 48. 499-525. Staples R. and Schneli V.-L. 1964. Refinements in rapid clearing technic in the KOH-alizarin-Red-S method for fetal bone. Stain Techno~. 39, 61-63. Ten Cate A. R., Freeman E. and Dickinson J. B. 1977. Suturai development: Structure and its response to rapid expansion. Am. J. Orthodonr. 71. 622-636. Troiisky W. 1932. Zur Frage der Formbiidung der Schldeidaches. 2. Morph. Anthrop. 30, 504-532. Walker B. E. 1971. Palate morphogenesis in the rabbit.
93, 29-44.
Markens I. S. 1975b. Embryonic development of the coronai suture in man and rat. Acta Anar. 93, 257-273. Moss M. L. 1958. Fusion of the frontal suture in the rat. Am. J. Anat. 102, 141-166. Nilsen R. 1977. Electron microscopy of induced heterotopic bone formation in guinea pigs. Archs oral Biol. 22. 485493. Noback C. R. 1944. The developmental anatomy of the human osseous skeleton during embryonic, fetal and circumnatai periods. Anar. Rec. 88, 91-i 17.
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Wood P. J. and Kraus B. S. 1962. Prenatal development of the human palate and some histological observations. Archs oral Viol. 7, 137-150.
Plate 1 overleaf. Fig. 1. Cleared and aiizarin-stained plate of a 22-day-old rabbit fetus from the oral side. The bilateral processes of the maxillary and the palatine bones approach the midline to form the bony palate. The processes of the maxillary bones (M) almost touch but those of the palatine bones (P) are further apart. IF, incisive foramen; NC, posterior nasal choana. Fig. 2. Similar specimen to Fig. 1, but from a 25-day-old rabbit fetus. A suture has been established between the maxillary processes (M), but the processes of the palatine bones (P) have completely fused. Fig. 3. Parapiast section of the secondary palate region of a 21-day fetus. The interzone between the approaching maxillary (M) and palatine (P) bony processes consists of different layers: (a) a narrow ceil-rich midline zone; (b) on each side of the midline, a zone with undifferentiated mesenchyme ceils; (c) differentiating osteogenic zones bilaterally just in front of the approaching bone processes. The midline
zone is distinguishable
in both
maxillary
and palatine
areas
(arrows).
x40
Fig. 4. Epon section of the interzone on day 21. The midline zone (a) is characterized of ceils of varying
by an aggregation morphology. The zone is considered to be a remnant of the epitheiiai seam resulting from the fusion of the paiatai shelves. x 400
Fig. 5. Epon section of the interzone, day 21. Many ceils in the midline zone close to the oral surface show characteristics of ceil degeneration, such as density of nucleus (vertical arrows) and fragmentation (horizontal arrows). x 600
Fig. 6. Transverse paraplast section of the palate on day 21 and PAS-stained. Remnants of the basement membranes persisting from the fusion of the palatai shelves are still visible in the midzone (arrows), although
most of the epitheiiai
ceils have disappeared.
oe, invagination
of the oral epitheiium.
x 200
Fig. 7. Epon section of the differentiating osteogenic zone of the maxillary area on day 21. The osteogenic zone is wide and cells of the interzone gradually (left to right) acquire the characteristics of pre-osteobiasts (pob) and osteoblasts (ob). Some of the densely packed ceils show degenerative changes (arrow). ost, osteoid of the maxillary process. x 400
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and W. Roy
Plate 2. Figs. 8 and 9. Autoradiograph of the on day 22. There are slightly more the palatine area (Fig. 9). Grains are towards the maxillary
middle part of the interzone grains in the interzone of fewer in the midline zone (M) and palatine (P) bones
4 h after idection with [3H]-proline the maxillary area (Fig. 8) than in of both areas, but gradually increase beyond the field. x400
Figs. IO and I I. Paraplast section of the interzone on day 22, and PAS-stained. In the maxillary area (Fig. IO), the intense PAS-positive reaction of the interzone is separated from the bony surface (M) by a zone of weak staining corresponding to the osteoblast layer (+-). In the palatine area (Fig. ll), the intense reaction is in cells adjacent to the palatine bone (P). x400 Fig.
12. Epon section of the maxillary by cells with a large oval nucleus
suture on day 24. There is a new central zone characterized and darkly-stained vacuolated cytoplasm (arrows). x 400
Fig. 13. Paraplast section of the same stage and area as Fig. 12, PAS-stained. Cells of the newly-formed central zone retain the intense PAS-positive reaction for intracellular glycogen shown in earlier stages by cells of the merging bone territories. The zone of weak staining reaction adjacent to the bony maxillary process (M) is now distinct (cf. Fig. 10). x 200 Fig. 14. Epon section of the palatine area on day 23. The palatine processes are now close to each other and the interzone appears compressed. Some interzone cells are elongated or irregular in shape and show varying nuclear and cytoplasmic characteristics (vertical arrows). Others appear similar to pre-osteoblasts (horizontal arrows), but the earlier welldefined osteogenic zones have disappeared. x400 Fig. 15. Paraplast section contiguous bony palatine
of the same area as in Fig. I4 but on day 24, PAS-stained. Between closely processes (P), many cells continue to show a positive reaction for glycogen. x200 Plate
3.
Fig. 16. Paraplast section of the palatine area on day 24, haematoxylin-eosin processes have partially fused. Clusters of cells enclosed by meeting bony remnants of the earlier interzone tissue. x 100
stained. trabeculae
The palatine are probably
Fig. 17. Epon section of the same stage and area as Fig. 16. The cells remaining in the midline after partial fusion of the processes show a bizarre shape and some appear to be pyknotic and disintegrating. x 400 Figs. 18-20. Epon sections of maxillary and palatine bone surfaces prior to fusion of the palatine processes on day 23 (the surface areas in the figures are outlined in Fig. 21). The surface of the maxillary bone (M), forming the lateral wall of the incisive foramen (Fig. 18) and the maxillary bone surface (M) at the palate-maxillary suture (Fig. 19) are lined by active osteoblasts. The opposite margin of the palato-maxillary suture is formed by the lateral wall of the vertical lamina of the palatine bone (P). Its surface is covered by active qsteoblasts; the opposite surface of the lamina, facing the oronasal cavity (Fig. 20). shows several osteoclasts (arrows) and only a few active osteoblasts. x 100 Fig. 21. Cleared and alizarin-stained palate of a 22-day old fetus. Osteoblastic activity is marked +, osteoclastic activity is marked -. The depository and resorptive osteogenic patterns in Figs. 18-20 and at the approaching bony processes are outlined. Lateral displacement is indicated by arrows. IF, incisive forarnen; M. maxillary bone: P. palatine bone: pm, palato-maxillary suture.
Suture development
and bony fusion
Plate 1.
289
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M. Persson
and W. Roy
Plate 2.
Suture development
and bony fusion
Plate 3.
291
