Development of tooth germ heterotopically grafted within the ear skin. An histological study in the rat

MEZADRITJ, RIVERO-TAMES D, BOABAID F, ARMENGOL JA. DEVELOPMENT OF TOOTH GERM HETEROTOPICALLY GRAFTED WITHIN THE EAR SKIN. AN HISTOLOGICAL STUDY IN THE RAT. MED ORAL 2004;9:243-52.

SUMMARY

The main goal of this study was the analysis of the developmental potentiality of tooth germ from late bell stage on, after its heterotopic placement within the skin. Teeth germs of newborn rats were grafted within a skin pouch of the ear of adult rats. Seven to fourteen days after grafting, dental germs developed normal dental structures in which ameloblasts and odontoblasts were well differentiated. Twenty to forty-one days after graft, the inflammatory host reaction destroyed the dental developed tissues by cell infiltration. The dentin of the grafts was of osteoid characteristics, and its size increased dependinng on grafting time until the complete substitution of all dental tissues. This atypical dentin showed several degrees of polymerisation from collagen fibres smooth dentin devoid near the graft a to fibres rich dentin far from the dental germ. Present results suggest that this type of dental graft could be a valuable model to study the self-development of dental tissues and the reactive mechanisms taking place after dental injuries.

Key words: Dental germ, graft, odontoblast, dentin, osteoid tissue, odontoblastic reaction.

INTRODUCTION

Teeth germs have been grafted in clinical conditions to restore teeth loss or to correct defective dental occlusion. Clinical and radiological studies after embryonic dental grafts did not reporte periodontal pathologies. However, dental loss, pulp necrosis, increase of the periodontal space, ankylosis, and defective roots formation were systematically found (1-3).

Discrepancies remain open after different types of graft experiments. Thus, normal teeth were developed after the graft of dental germs transplanted before the end of the coronary stage (4-6). In contrast, grafts of dental germs between Mus musculus and Mus caroli mice strains showed the presence of multinucleated giant cells through the dentin pulp surface (7). Further, the initial development of dental matrices was followed by the appearance of epithelial and enamel ectopic foci within the dental follicle, metaplasy in the enamel organ, cysts, and loss of the anatomical integrity of the dental corona (8). In this way, the pulp and dentin of partially developed molar germs with attached periodontal ligament heterotopically grafted were substituted by fibrous connective tissue and osteoid dentin (9). However, other experiments described the normal development of postnatal dental germs isotopically transplanted within host rat pups, in which transplanted teeth were even able to eruptionate (10). Altogether, precedent data demonstrate that independently of final fate of the graft, the odontogenic competence acquired by dental ectomesenchyma before the graft plays a key role on its development .

Osteoid dentin is a tubule-less dentin observed in pulp areas devoid of odontoblasts, which were formerly replaced by neodifferentiated cells (11). This cell transformation was followed by papilla's cells of heteroropically grafted teeth germs (12). However, osteoid dentin formation was not exclusive of grafting experiments. Thus, several procedures as the subapical osteotomy (13), the systemic administration of cyclosporin-A (14), or antimitotic agents such as phosphamide (15), and the local application of calcium hydroxide (16) induced the formation of osteoid dentin.

In this paper, teeth germs in the bell stage were grafted within ear's skin pouches to analyse the development of isolated germ cells and their relationships with the ectopic environment.

MATERIALS AND METHODS

Twenty tooth germs from Wistar rats of one day of postnatal life were grafted within skin pouches performed in both ears of ten isogenic adult rats. Host rats were anaesthetised with Zoletil 50‚ [thietylamine chlorhydrate (125 mg) and zolazepan chlorhydrate (125 mg), 0.20 ml/kg ip]. An incision of 5 mm was transversally made in the back of the ear, and the skin was carefully separated from the ear cartilage by a watchmaker forceps.

Rat pups were deeply anaesthetised under ether vapours. The dental germs of superior first molars were carefully obtained, avoiding the extraction of adjacent periodontal tissues during tooth germ removal. Thereafter, dental germ was placed within the ear pouch.

A total of five grafts were collected at seven, fourteen, twenty-one and forty-two days after grafting (DAG). After hosts' anaesthesia ears were removed (right ear for 7 and 21 DAG, and left ear for 14 and 42 DAG), and immersed in 4% paraformaldehyde in phosphate buffer 0.1M (pH 7.4) for 24 hours. Blocks containing the graft and ear surrounding tissues were then dehydrated, cleared in xylol and paraffin embedded. Serial section 7µm thick were cut. Serial sections were used for Mallory's or haematoxylin-eosin staining, and to detect fragmented DNA using the Apoptotag® kit.

A series of 14 DAG transplants were used for electron microscopy. Blocks of tissue were fixed with 4% glutaraldehyde and 4% paraformaldehyde in cacodylate buffer 0.1M (pH 7.3) for 24 hours at 4 ºC. After 7% EDTA in the same buffer demineralisation for a week, the blocks were rinsed in buffer and postfixed for 1 hour with 1% osmium tetroxide and 5% saccharose in buffer. Thereafter, the blocks were dehydrated, cleared with propylene oxide and embedded in Araldite. Semithin sections 200 nm thick were made and counterstained with toluidine blue. Ultrathin sections 75 nm thick were stained with uranyl acetate and lead citrate, and viewed under a Zeiss EM 900 microscope.

RESULTS

Seven days after grafting (DAG) the dental germ developed three areas: (i) a proliferating region with early odontoblasts, which resembled the Hertwig's root sheath, (ii) an odontoblastic layer placed under a dentin matrix layer, and (iii) a region characterized by an osteoid dentin which continued the normal one. The dental pulp was occupied by hypertrophied blood vessels (Figure 1A,C). Odontoblasts were arranged in "palisade" without specific intercellular unions (Fig.1H), and possessed a cytoplasm rich in rough endoplasmic reticule (Fig, 1G), Golgi apparatus and transport vesicles (Figure 1H).

Fourteen DAG, odontoblasts keep the same morphological arrangement and secrete the matrix from the apical pole (Fig. 1D). The continuous genesis of dentin and enamel increased their size (Figure 1E). From this date on, the formation of osteoid dentin became most evident. Disorganized odontoblasts secreted this dentin without polarization, being finally embedded within the osteoid matrix (Figure 2A-D).

The ameloblasts were always in intimate contact with the enamel, preserving their polarized cylindrical cell morphology (Figure 2B-D). In contrast, disorganised odontoblast cells layer was devoid of pre-dentin, and coincided with a pulp invaded by a connective tissue rich in hyperaemic blood vessels and lymphocytes. Cells embedded within the osteoid dentin conserved the morphology of secreting cells rich. However, the loss of cell polarity signs, as the presence of the cell nucleus at the cell's centre rather than within their basal pole, were clearly detected (Figure 2E).

The osteoid dentin, whose structure resembles the primary osseous tissue, was the most abundant tissue from fourteen DAG on. Odontoblast-like cells embedded within the osteoid dentin possessed abundant endoplasmic reticule cisterns, endo- and exocytic vesicles (Figure 2A-D). Osteoid dentin matrix occupied by cell prolongations (Figure 3A-D), showed two different morphologies that could explain their differential staining. The matrix near the cell prolongations was granular and devoid of collagen fibres; while, distant osteoid dentin was very rich in collagen fibres with few cellular rest or inclusions (Figures 2F, 3A-B). The cisterns of rough endoplasmic reticule were anomalously dilated, index of an anomalous and fast protein synthesis (Figure 3C-D). However, it should be stated here that decalcification procedures used during the post-fixation could also explain the presence of altered mitochondria and artefactual vesicular dilatations. The large extracellular space is traversed by cell prolongations of several sizes, which did not establish any type of intercellular contact (Figure 3C-D). The cell surface possessed numerous exocytic pits and endocytic vesicles (Figures 2G, 3A-B), which fused on primary endosomes through the nearing cell cytoplasm (Figure 3B). The granular and electrodense content of exocityc vesicles resembled to the extracellular matrix directly apposed to cell prolongations (Figure 3A-D).

Multinucleated giant cells spreaded throughout the surface of the osteoid dentin, the dentin and the enamel were occasionaly form fron 14 DAG (Figure 3E). These osteoclast-like cells possessed several nuclei eccentrically located within a vacuolated cytoplasm (Figure 3F), were most abundant from 21 DAG on. Lytic areas defined by osteoclast-like cells, increased between 21 to 42 DAG. Date in which few rest of osteoid dentin remained within a reactive connective tissue filled by lymphocytes and hyperaemic blood vessels (Figure 4A).

Images of fragmented DNA labelled cells, indicative of apoptosis, were detected within the graft. Dying cells were randomly distributed throughout the dental pulp (Figure 4B), and under the dentin within the odontoblast cells layer (Figure 4C-F).

DISCUSSION

The first molar is the model of choice for the experimental analyses of odontogenesis (17). Dental germs used here were in the late bell stage in which the genesis of both enamel and dentin was started. From 7 DAG on, dental germs developed three clear odontogenic regions: (i) an undifferentiated region resembling the Hertwig's root sheath, (ii) a region, presumably induced by the former, of differentiated odontoblasts that secrete the pre-dentin lying under the ameloblastic layer, and (iii) an osteoid dentin area different from normal dental tissues. This area together with the tissue resulting from the host's inflammatory reaction will be the final fate of the transplant.

The molecular interactions guiding the development of the corona need the anatomical integrity between the epithelium of the dental germ and the surrounding mesenchyma (18). Discrepancies have been raised from previous studies on dental germ transplants. Thus, dental germs grafted before the beginning of dental tissues formation seem to develop dental and periodontal normal structures (4-6,19). However, dental germs grafted after the initial development of the enamel and dentin, like present experiments, evolved in normal dental tissues (10-12,20,21), or developed aberrant structures (8-9). Our results agree with the last finding in the sense that the final evolution of grafted dental germs is the degeneration and later absorption of transplanted tissues. This degeneration seems not be lied to the absence of some of the embryonic dental tissues or by defective grafting procedures. Thus, the graft Hertwig's root sheath presence, and the beginning of the root development, suggests that the source of inductive relationships regulating the epithelium-mesenchyma interactions were already preserved. Hence, its possible that early hox genes, expressed at the beginning of the intercellular relationships between the epithelial cells and the ectomesenchyma (22,23), were also expressed during the former steps of graft development. The heterotopic placement of dental germs could be a good model, not only to define the role of the dental papillae and/or the dental epithelium in the inductive phenomena allowing the dental development (24,25); but, to study the inductive role of embryonic hox genes on the non-odontogenic adult host mesenchyma (26).

The osteoid dentin, an anomalous dental tissue found in several experimental conditions (13-16,27), was the most developed dental tissue observed in our study. Atypical secreting cells whose morphology resembled to the odontoblasts were embedded within matrix of the osteoid dentin. However, no data are available on the structure of the osteoid matrix (28,29). Two different types of matrix were observed in our grafts: (i) a clear matrix, which lacks of collagen fibres was located in the boundaries of the atypical odontoblasts, and (ii) other rich in collagen fibres that was systematically placed far from secreting cells. Furthermore, the distance of rich fibres matrix from the surface of the cells embedded through the osteoid dentin was preserved, irrespective of the size and location of the cell branches or the cell bodies. This gradient of the richness degree on collagen fibres indicates that the polymerisation of tropocollagen fibres takes place far from they was secreted. This constant relationship between both matrix types and their cell/matrix gradient has not been previously described. However, methods used here do not allow us to elucidate why and how this process occurs.

Cell prolongations of osteoid dentin secreting cells resembled the prolongations of the odontoblasts. However, the number of exocytic vesicles in these prolongations was higher than that found in normal odontoblasts, which were almost inexistent (28). This abundance on exocytic vesicles indicates the active synthesis and secretion of the matrix by these cells, and could support the great size attained by the osteoid dentin. Other alternative explanation to the matrix-cell gradient could be based on the existence of a fibrinolityc phenomenon at the interface between the cell surface and the matrix, which depolymerises the collagen fibres nearing to the cell surface. Nevertheless, the almost complete absence of phagocytic vacuoles together with the scarce presence of lysosomes within the cell cytoplasm seems to deny this possibility. Present pioneer observations in our experimental model open new insights on the study of synthesis and secretion of both collagen and non-collagen proteins in reactive matrices in which polymerisation process takes place far from the cell surface.

Osteoid dentin genesis has been related to the odontoblast cell loss that is replaced by the neodifferentiated pulp cells that form a tubule-less tissue resembling the osseous tissue (11). In the same way that young mesenchyme cells secrete and mineralise an osteoid dentin-like matrix before to differentiate in odontoblats (12). Our results suggests that, in an early stage, the osteoid dentin precedes the normal dentin, and could induce the ameloblasts differentiation and enamel formation. In addition, heterotopically grafted adult dentin induces the formation of osteoid tissue from the host (30,31). Hence, the great size attained by the osteoid dentin -although cell differentiation of odontoblasts in osteoid dentin secreting cells is evident in our grafts- will also be explained by the existence of graft factors inducing the synthesis of osteoid dentin by the host.

Regressive phenomena such programmed cell death play a key role in late developmental stages (for a review, see 32-34). Apoptosis has been described in several odontogenic stages (35-37). In our grafts, fragmented DNA labelled cells were identified at 14 DAG, a critical stage of the graft similar to the advanced late bell stage in which the withdrawal of dental tissues began. This coincidental stage induces us to suggest that apoptotic cells within the graft followed their developmental fate, rather than represent a necrotic cell death induced by the host's inflammatory reaction. Analysis now in progress by using the immunohistochemical detection of active 3-caspase, enzyme linked to the final apoptotic cascade (38,39), will solve this question.

On the way of regressive phenomena, the synthesis of osteoid dentin declines from 21 DAG on. Activity that was replaced by the increase on lytic activity characterised by the presence of multinucleated giant cells throughout the osteoid dentin, dentin and enamel. This observation differs from those described after the intraosseous grafts of dental germs. In these cases, the abnormal development of mineralised tissues, together with enamel metaplasia, enamel ectopies, cysts formation, and absence of periodontal ligament, continues without reabsorptive activity (8,9). Lityc activity could be related with the host's tissue reaction rather than to the graft. Thus, in anisogenic dental grafts multinucleated cell similar to those found here have been described. In clinical trials this reaction has been suggested as explanation for the unsuccessful of dental germs auto-grafts (3). In our grafts, the inflammatory reaction of the host was found from the first analysed date. However, its maximum was reached from 21 DAG on, dates that are coincidental with the increase of lityc activity. Therefore, it can be assumed that reactive response of host is directly involved in graft rejection.

Grafting procedures have attained a great importance in biological studies from the past decade on. However, the present study is the first devoted to the analysis of the development of dental germ heterotopically grafted into the adult skin, and represents an useful model to elucidate the mechanisms of formation and destruction of dental tissues.

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