Braz Dent J (2000) 11(2): 117-126 ISSN 0103-6440

This study was undertaken to examine the normal and abnormal epithelial alterations of secondary palate in rats. Control and dexamethasone-treated embryos and fetuses of Wistar rats were evaluated by macroscopic and scanning electron microscopic analysis prior to, during, and after fusion of palatal processes. Normal alterations of the surface topography included growth and disorganization of medial edge epithelial cells followed by fusion and posterior migration to both the oral and nasal aspects of the palate. No evidence of epithelial cell death or transformation was observed. Dexamethasone-treated fetuses showed epithelial cells increased in size with a large amount of desquamation, followed by deposition of a disorganized cell layer with keratin-like characteristics. This allowed no fusion of palatal processes.

Key Words: dexamethasone, epithelial cells, secondary palate.

Introduction

Development of the secondary palate is a complex and critical event and its formation is a result of fusion of palatal processes during fetus evolution. Initially, the palatal processes grow vertically down the side of the tongue, then elevate to a horizontal position above the dorsum of the tongue and fuse with each other to form an intact palate. Although palate embryogenesis has been the target of much research, some aspects related to the mechanism of fusion remain ill-defined.

Noting that fusion occurs between specific epithelial surfaces at specific times during normal development suggests that localized, prerequisite cellular changes may occur in areas of presumptive fusion prior to their contact (Waterman and Meller, 1973; Hehn et al., 1998; Miettinen et al., 1999). Midline epithelial cells manifest morphological signs of necrosis, probably involved with a programmed lysosomal-mediated autolysis (Greene and Pratt, 1978). It has been recently suggested that seam degeneration occurs by transformation of basal medial edge epithelial cells to mesenchyme (Fitchett and Hay, 1989). Another hypothesis is that there is no cell death or transformation. Instead, epithelial cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate (Carette and Ferguson, 1992).

The mechanism of secondary palate formation also has to be evaluated when related to cleft palate. Congenital malformations initially require clinical recognition and documentation but should then be complemented by laboratory research which cannot be carried out in the clinic. These aspects are important because the directly associated and derived effects of facial clefts are profound and lead to severe clinical consequences. The complexity of facial structures and their functions are reflected clearly in the degree and frequency of derived difficulties associated with clefts (Lin et al., 1999). Therefore, knowledge about normal development and alterations that result in abnormal secondary palate formation remains important.

Information obtained from animal models usually cannot be directly extrapolated to humans (Navia and Narkates, 1980); however, the answers obtained from animal models to properly formulated research questions are extremely useful and valid information. The answers could either validate the formulated hypothesis or not, yet they will help improve the understanding of the biology of health and the mechanisms of disease in man. Therefore, a comprehensive study of palatogenesis in the Wistar rat was made, combining macroscopic and microscopic observations of the secondary palate of both normal fetuses and those with experimentally induced cleft palate.

Material and Methods

Immature Wistar rats, 36 males and 72 females, 35 days of age, obtained from Oswaldo Cruz Foundation Rodent Department, were kept for 55 days under controlled conditions of temperature (23 ± 1^oC), humidity (50 ± 5%), and an alternating light-dark cycle (12:12), with 10-15 changes of room air per hour. All procedures fulfilled ethical and legal recommendations for animal experimentation (CCAC, 1980; CBEA, 1991). The rodent cages were made of semi-opaque polypropylene, with stainless steel tops, built-in hoppers and water bottle fixings, with 3 animals per cage. To assure that rats could use their behavioral heat regulatory mechanism, bedding of wood origin was selected.

Males and females were maintained in different places in the room and when they were 90 days of age, females were introduced to cages previously occupied by males in order to enable the Whitten effect (Whitten et al., 1968). On the appropriate day of the estrous cycle (after 4 days), the animals were mated overnight and the presence of spermatozoa in the vaginal smear on the following morning was taken as evidence of copulation. The conception time was arbitrarily calculated from the midpoint (9 AM) of the fertilization period and was designated as the onset of time 0.

Procedures were carried out with half of the animals, in which normal palatogenesis was studied (control). Another group formed by the other half of the animals was used in the experiments where the drug-altered palatogenesis was analyzed (test group). Intramuscular injections of dexamethasone acetate (3 mg/kg; Merck, Whitehouse Station, NJ, USA) were given to the thigh of each pregnant animal of the test group. Half of the rats were submitted to this treatment from day 11 to 14 of gestation and the other half from day 14 to 17 of gestation.

Control and test animals were sacrificed with chloroform at 24-h intervals from day 11 to 21 of gestation. Laparotomy was performed and the uteruses were maintained in RPMI-1640 media. The fetuses were removed and subsequently used for macro- and microscopic analysis.

For macroscopic analysis, the heads of fetuses were fixed for at least 16 h in 4% paraformaldehyde buffered with 0.1 M sodium phosphate (pH 7.4). The lower portion of face and tongue were removed to facilitate the study of the developing palate. Earlier fetuses were dissected and analyzed using a gross dissecting microscope.

For scanning electron microscopic (SEM) analysis, the heads were fixed in 2.5% glutaraldehyde. After initial hardening of the tissue, specimens were dissected to obtain an occlusal view of the head and remained in this primary fixative for approximately 72 h. They were then washed in phosphate buffered saline (pH 7.2) and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 30 min at room temperature. Osmicated specimens were washed in phosphate buffered saline and dehydrated in a graded ethanol series at room temperature. They were then dried with liquid CO₂ by the critical point method, affixed to aluminum slugs with silver conductive paint and coated with gold palladium on an omnirotating stage in an FL 9496 vacuum evaporator at a minimum vacuum of 5 x 10^-5 torr. Observations and electromicrographs were made with a Jeol JSM 840-A scanning electron microscope.
 

Results

A total of 143 fetuses constituted the control group for analysis of normal development, and 140 fetuses constituted the test group. No gross malformations were detected in any of the control fetuses and by day 16 the palatal processes were either fused or, at least, in contact. Older fetuses showed a gradual increase in the formation of palatal structures.

All dexamethasone-treated fetuses exhibited clefts of the secondary palate. Eighteen-day fetuses displayed the palatal processes at a horizontal position, which remained unaltered until day 21 when fetuses presented a great cleft of the whole extension of the palate.

Control fetuses on day 11 presented formed, but not fused, first and second branquial arches. Fusion of the first branquial arch occurred on day 13, which contributed to limit the inferior portion of the primitive mouth. Fetuses on day 14 presented fusion of median nasal processes and subsequent proliferation of mesenchymal tissue on its inferior region to form the median portion of the upper lip. These aspects can be better evaluated in Figure 1. Test group fetuses did not present any disturbance of upper lip formation or structures of primary palate.

Observation of palatal processes was possible after its complete formation in 14-day fetuses. In the control group, the medial edge epithelial cells were round and presented different sizes, which indicates great mitotic activity, without any evidence of cellular desquamation (Figure 2a). On day 15 epithelial cells showed an increase in size, probably because of an increase in the amount of cytoplasmic material (Figure 2b). Fetuses on day 16 showed great disorganization of medial edge epithelial cells, which presented a lot of free spaces between them and a large quantity of extracellular matrix (Figure 2c). Fusion of palatal processes was complete in 17-day fetuses, which presented an increase in the amount of epithelial cell desquamation (Figure 2d). Older fetuses also exhibited this trend and it was possible to observe the gradative formation of polygonal shape keratin structures covering oral epithelium (Figure 2e-h).

In test group fetuses, on day 14, the medial edge epithelial cells of palatal processes were very irregular, disorganized and with a large amount of extracellular matrix, besides the large cellular volume indicating an increase in cytoplasmic material (Figure 3a). On day 15, epithelial cells presented a large degree of desquamation (Figure 3b). This rate of disorganization and desquamation of medial edge epithelial cells increased in fetuses on days 16 and 17 (Figure 3c-d). Older fetuses showed the progressive formation of a disorganized and keratin-like layer of cells covering both palatal processes extending from the oral epithelium to the nasal limit of the medial edge (Figure 3e-h).

Figure 1 - Frontal view of scanning electromicrographs of control fetuses. a) 11 days of age, showing frontonasal (F) and maxillary (M) processes above first and second branquial arches; b) 12 days, median (MN) and lateral (LN) nasal processes; c) 13 days, union of mandibular processes (Md) with each other and maxillary process with median and lateral nasal processes have occurred; d) 14 days, presenting union of median nasal processes with each other; e), f) and g) 15 to 17 days, gradative formation of middle portion of upper lip (Magnification: X80).

Figure 2 - Scanning electromicrographs of epithelial cells of control fetuses from 14 to 21 days of age. a), b) and c) 14, 15, and 16 days of age, respectively: medial edge region of palatal processes, showing the progressive increase in size and in amount of extracellular matrix. d), e), f), g) and h) 17-21 days of age, respectively: palatal fusion zone where it is possible to observe the gradative epithelial keratinization (Magnification: X1,800).

Figure 3 - Scanning electromicrographs of medial edge epithelial cells of dexamethasone-treated fetuses from 14 to 21 days of age. a), b), c) and d) 14-17 days of age, respectively: a large rate of cellular desquamation and disorganization. e), f), g) and h) 18-21 days of age, respectively: deposition of a keratin-like layer presenting sharp irregularity (Magnification: X1,800).

Discussion

Development of an individual is a complex and critical event and requires the integration of many mechanisms to allow the correct formation of all structures. During facial arrangement most embryonic tissue union does not occur by fusion. On the contrary, cleft elimination is a result of mesenchymal merging within these components. This is true when related to maxillary processes. Simultaneously with their growth toward midline, tissue proliferation below the just-bound median nasal processes can be observed and this activity is responsible for upper lip and anterior portion of maxillary bone formation.

It has been reported (Moore, 1982; Ten Cate, 1994) that median nasal processes are responsible for formation of these structures. However, it can be verified that this union occurs by the mesenchymal merging which occurs below them and not by a true fusion. The same mechanism is present in maxillary and mandibular processes union to form the lip corner which limits the oral cavity.

These aspects refer to primary palate formation and no disturbance was observed in dexamethasone-treated fetuses. Absence of glucocorticoid receptors in this region or a delay of drug administration correlated with primary palate formation period can explain this fact.

Secondary palate development starts on day 14 when the palatal processes can be perceived as outgrowths of maxillary processes. Although there have been some attempts to determine when palatal processes elevation occurs, controversy exists regarding the sequence of events of shelf elevation. Some authors report that the shelves start to elevate at the back (Miettinen et al., 1999) while others maintain that they start at the front portion (Wragg et al., 1972). After elevation, the palatal shelves grow and approach each other towards midline, showing some superficial cells undergoing degeneration and basal cells in the process of mitotic division at the same time.

They fuse themselves and fuse with the free margin of the nasal septum as well as with the primary palate. This mechanism further obeys an antero-posterior gradient of closure in humans and epithelial fusion along the entire length of the shelves forming the secondary palate (Moore, 1982). In rats, it was possible to observe that initial fusion occurs at the middle portion of secondary palate and spreads anteriorly and posteriorly at the same time. Epithelial fusion is a normal morphogenetic event which can be defined as a type of tissue interaction involving adhesion between localized regions of epithelial surfaces (Waterman and Meller, 1973) and posterior migration of mesenchyme cells, thus establishing the formation of the secondary palate (Singh et al., 1997; Degitz et al., 1998).
 

Prior to contact, the superficial cells of the palatal shelves show varying degrees of sharp degenerative changes and occasionally disappear (Kaartinen et al., 1997). Fusion occurs between the deeper cells of the opposing epithelia (Ferguson, 1988; Gibbins et al., 1999). The resulting epithelial seam is then two to three cell layers thick (Greene and Pratt, 1978) and is bound on each side by an uninterrupted basal lamina (Kaartinen et al., 1997).

In this investigation it was possible to observe that changes in the appearance of the epithelial surface over the presumptive medial edge occurred prior to contact between the shelves. These alterations were bilateral and correlated to changes in the histological organization of the underlying epithelium. Surface alterations were seen only over areas of the shelves normally involved in fusion and showed few signs of cell desquamation; this mechanism may reflect the specificity of fusion. It was also noted that surface cells of the medial edge presented signs of migration, which can indicate a mechanism that may aid in the rapid thinning of the lamina during its dissolution, as suggested by Jacobsson in 1997. Waterman and Meller (1973) reported that migration of surface cells away from the medial edge might expose the surfaces of deeper cells which are more capable of fusing; however, this possibility cannot be determined by means of SEM alone. The epithelial seam is lost in 12 h in normal rat development (Gibbins et al., 1999), whereas it persists for many weeks in normal human development.

Dexamethasone-treated fetuses presented another pattern of alterations of medial edge epithelial cells. At the beginning, these cells were increased in size with much disorganization. They also showed a large amount of extracellular matrix and later, much desquamation. This situation remained the same during the gestation period, with the only alterations restricted to the formation of a keratin-like cell layer, especially on the medial edge.

Normal and dexamethasone-treated mechanisms of secondary palate formation suggest that prefusion alterations in the palatal shelf epithelium may also result from a mesenchymal-epithelial interaction. One event can influence or be influenced by the other and this sequence of events can either form a normal or abnormal secondary palate.

Acknowledgments

The authors wish to thank Dr. Marcos Farina for his assistance with SEM specimens manipulations and Dr. Jayme Mendes for his help with electromicrographs. This investigation was supported in part by Research National Council (CNPq).

Resumo

Bittencourt MAV, Bolognese AM: Alterações epiteliais durante a formação do palato secundário. Braz Dent J 11(2): 117-126, 2000

Este trabalho foi realizado com o objetivo de avaliar as alterações epiteliais normais e anormais durante a formação do palato secundário em ratos Wistar. Animais controle e animais tratados por dexametasona foram avaliados macroscopicamente e através da microscopia eletrônica de varredura antes, durante e após a fusão dos processos palatinos. As alterações normais na superfície dos processos incluiram o crescimento e a desorganização das células epiteliais da crista medial, seguida por fusão e posterior migração celular para as porções oral e nasal do palato. Nenhuma evidência de morte ou transformação celular foi observada. Os fetos tratados por dexametasona apresentaram células epiteliais aumentadas, com uma grande quantidade de descamação, seguida pela deposição de uma camada celular desorganizada com características de queratinização. Isto impediu a fusão dos processos palatinos.

Unitermos: dexametasona, células epiteliais, palato secundário.

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Correspondence: Prof. Marcos Alan Vieira Bittencourt, Av. Antônio Carlos Magalhães, 429 - Sala 510, Centro Emp. Itaigara Sul, Itaigara, 41825-000 Salvador, BA, Brasil. Tel: +55-71-353-9862. E-mail: ortoufba@cdl.com.br

Accepted April 26, 2000
Eletronic Publication october, 2000