Recent Studies of Rat Incisors, in Which the Dental Papilla Has Suffered

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Behavior of Dental and Surrounding Structures following Dental Papillary Injury A Histologic Study in Rats•\•\

Tetsuo OKAMOTO,* An Cesar Perri DE CARVALHO,* Acyr Lima DE CASTRO* and Ruy Dos Santos PINTO*

Recent studies of rat incisors, in which the dental papilla has suffered different intensities of injury, showed several pathological alterations of the continuously developing teeth. FERGUSON& LAWTON[1]verified a well localized reaction of the dental pulp and cessation of growth for 48 hours after injury of rats' lower incisors at the dental papilla level. After rats' upper incisors luxation[2], there were hyperemic vessels and quick recovery of the dental pulp. On the other hand, the tissue necrosis, enamel organ atrophy and tooth germs at the dental apical third could be seen after replanting and transplanting upper incisors of rats[3, 4]. These tooth germs, dental alveolus ankylosis and mineralized masses were related by DALLESTINI,OKA- MOTO,CASTRO and SANTOS-PINTO[5],following dental fractures in rats . The purpose of the present study is to verify histologically the behavior of the rat dental pulp and papilla, faced with a more intense injury in comparison with that cited by FERGUSON& LAWTON[1].Likewise, the clinical aspects of the gingival mucosa , dental crown, and growth of the same teeth are studied.

Material and Method For the present study, 24 male albino rats (Rattus norvegicus, Wistar strain) , weighing about 120 grams each were used. Before and during the entire experimental period, the animals received standard food and water ad libitum, except that 24 hours after the surgical procedures solid food was discontinued. The animals were anesthetized with sulfuric ether and following which, were sub- jected to a vertical incision measuring about 0.5 mm below the external edge of the left eye, above the mandible, to the osseous plane. After the visualization of the dental papilla under the cortical bone, a sterile root canal reamer (Hedstroem n.6) was pushed through the thin intervening bone and dentin into the dental papilla according to FERGUSON& LAWTON[1].A circular movement transmitted to the root canal reamer increased the injury to the dental papilla. The surgical wound was sutured with 4-0 Mersilenez.

* Department of Oral Surgery, Faculdade de Odontologia de Aracatuba, Aracatuba, Sao Paulo , Brasil. 127

For the measurement of the dental growth, a sulcus was made with a carborundum disc on the buccal surface of the lower right and left incisors at the free gingival level[2]. Four animals were killed after postoperative periods of 24 hours and 2, 5, 10, 20 and 30 days. After each rat's death the jaws were placed in 10 per cent formalin and, after fixation, decalcified in a formic acid-sodium citrate solution[6]. After this they were embedded in praffin for 15 minutes in a vacuum. The blocks thus obtained were cut with a rotary microtome into serial sections 6 microns thick. The tissues were stained with hematoxylin and eosin and Masson's trichrome for morhpologic study[7].

Results A. Clinical examination 1) No alteration of the gingival mucosa at the lower incisor level was seen. 2) There was no change in color of the incisor crowns. 3) It was not possible to verify the growth of the lower left before the postoperative day; following this time it grew 0.4 mm, daily. B. Histological examination In the 24-hour postoperative section blood clot and loss of the characteristic arrangement of the odontoblastic layer could be seen at the injured area (Fig. 1). There were also some polymorphonuclear neutrophils, histiocytes and several fibroblasts showing different degrees of degeneration. In the remainder of the dental pulp were seen hyperemic blood vessels. In the 2-day specimen there were hemorrhagic sites in the injured portion of the dental pulp with numerous histiocytes, some polymorphonuclear neutrophils, lym- phocytes and plasma cells, surrounding by a small number of young fibroblasts (Fig.

Fig. 1 Injured area. Loss of the characteristic arrangement of the odontoblastic layer (H & E stain ×240). 128

Fig. 2 Fibroblasts and hemorrhage at the injured site (H & E •~ 240).

Fig. 3 Bone-like tissue at the apical third of the tooth (arrows)-(H & E •~ 100) .

2). Some newly formed capillaries and odontoblastic layer in degeneration were also seen. In some cases was present little zone of mineralized bone-like tissue on theapical third of the tooth (Fig. 3). At the "buccal plate" the vascularization of the dental pulp was deficient and some fibroblasts and odontoblasts were degenerating. On the other hand, the dental pulp was well vascularized near the "lingual plate", with a great number of fibroblasts. By 5 postoperative days the apical third of the tooth presented the mineralized bone-like tissue with a small number of osteoclast-like cells at its periphery; at this time mineralized dentin-like tissue was seen in other areas (Fig. 4). The dental pulp showing deficient vascularization at the "buccal plate" presented degenerated fibroblasts and odontoblasts (Fig. 5). At the "lingual plate" the dental pulp was well vascularized with a great number of fibroblasts. Characteristic embryonic tissue at the apical third of the dental pulp could also be seen. 129

Fig. 4 Mineralized tissue with characteristic dentin (H & E •~ 100).

Fig. 5 Degenerating fibroblasts and odontoblasts at the "buccal plate" (H & E •~ 100).

Fig. 6 Mineralized tissue with bone and dentin characteristic (H & E •~ 100). 130

In the 10-day specimen the dental pulp showed a comparable characteristic to the previous postoperative period. The mineralized tissue formation increased sometimes showing dentin or bone tissue characteristics (Fig. 6). At the "lingual plate" the dental pulp showed a small quantity of hyaline degeneration. In the 20-day specimen the dental pulp exhibited embryonic tissue at the apical third. On approximately the entire portion of the pulp chamber there was mineralized tissue with dentin (Fig. 7) and characteristic bone tissue. In some areas were present dystrophic mineralized masses with nuclear fragments (Fig. 8) and erythrocytes within. A small portion of the dental pulp was degenerating. After 30 days, there were embryonic structures at the apical level (Fig. 9) and mineralized tissue on the pulp chamber with the same characteristic of that of the an- terior period. There was an increase of dystrophic mineralized masses.

Fig. 7 Mineralized tissue within the pulp chamber with characteristic dentin (H & E •~ 100) .

Fig. 8 Dystrophic mineralized masses with nuclear fragments (H & E •~ 240). 131

Fig. 9 Embryonic structures at the apical level (H & E •~ 240).

Discussion In this paper, we have observed dental papilla at the 5th postoperative day. Until this time, the dental growth could be seen clinically. This fact suggests that during this period, the dental papilla seems to have reacted to the injury, having regenerated itself. After this, it initiated its histodifferentiation function. This fact seems to be true, that between the 10th and 30th postoperative day, the dental growth was similar to that cited by ADDISON& APPLETON[8],SHADLE, WAGNER & JACOBS[9]and MICHAELI& WEINREB[10],that is, 2.8 mm, 2.93 mm and 3.0 Mm per week, respectively. The dental growth clinically observed from the 5th postoperative day was based in the histological sections of the most advanced stages when a whole dental papilla in continuity with a small differentiated dental pulp portion is observed. Mineralized masses present within the pulp chamber after different degrees of trauma have been described by RAmALllo[4], CALLESTINIet al[5], KRoNFELD[11], Blackwood[12], ARwILL[13], ULMANSKY& SHAPIRo[14],and EDLuND[15],but there was not a definition about their nature. In the present paper, we could notice appearance of mineralized formations with osseous trabeculae aspect beside an atypical dentin without solution of continuity with the bone tissue. This mineralized structures seem to be produced by osteoblasts and odontoblasts originating from mesenchymal cells and/or fibroblasts. This particular behavior of the dental papilla or the dental pulp at the apical level was probably due to a metabolic alteration following the traumatic injury. Such structures are similar to that described by CALLESTINIet al[5], at the apical dental level. Different ones ocurring at the apical dental level, in the mineralized masses of the pulp chamber seem to arise from the necrotic pulp. The pulp necrosis originated initially from the injury to dental papilla and is favored by the progressive increase of calcified masses that obstruct the blood supply. Hyaline masses also contribute for this calcification. According to POLICARD& 132

COLLET[16],these hyaline masses suffering disintegration in periphery would have affinity to the calcium salts originating from mineralized and dystrophic masses.

Conclusions Following dental papilla injury, there was a temporary cessation of the tooth growth. Mineralized masses of different origins were seen within the pulp chamber and at the apical dental third. Other efforts must be made to verify the behavior of several dental structures after injury.

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