Histometric Analysis of Rat Alveolar Wound Healing

 
Teresa Lúcia LAMANO CARVALHO
Karina Fittipaldi BOMBONATO
Luiz Guilherme BRENTEGANI
 
Departamento de Estomatologia, Laboratório de Patologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

Braz Dent J (1997) 8(1): 9-12 ISSN 0103-6440

| Introduction | Material and Methods | Results and Discussion | Acknowledgments | References |

The chronology of alveolar wound healing was analyzed by a standard stereological method (point-counting volumetry) in normal rats. The upper right incisors were extracted and the animals were killed 1, 2, 3 and 6 weeks postoperatively. A light camera was used to determine the volume fraction of histologic components in the apical, middle and cervical thirds of the alveolus. Progressive bone neoformation was quantified, in parallel to a decrease in percent volume of connective tissue. A significantly smaller volume fraction of bone trabeculae in the cervical third of the socket was observed at all periods except the 6th week. The present histometric data show that bone neoformation continued to proceed beyond the 3rd postoperative week.

Key Words: alveolar wound healing, histometry.

Introduction

Alveolar healing following tooth extraction has been extensively investigated in humans and in several animal species under normal and experimental conditions that might interfere with the time course of healing. Most of these studies were qualitative reports limited to histologic examination of tissue repair (Boyne, 1966; Carlsson et al., 1967; Astrand and Carlsson, 1969; Simpson, 1969; Carvalho and Okamoto, 1985; Alves and Okamoto, 1989; Santos Jr. and Melhado, 1990). Some histochemical (Amler et al., 1960; Okamoto and Russo, 1973) and radiographic (Guglielmotti et al., 1985, 1986) analyses have also been published.

Few studies deal with quantitative histometric evaluation of the healing of dental extraction wounds. Histometric analysis of alveolar healing in normal, uranyl nitrate-intoxicated and X-irradiated rats was carried out by Guglielmotti and Cabrini (1985) and Guglielmotti et al. (1985, 1986) with the use of an image analyzing system. Histometric studies of dental healing around implants have recently been performed in dogs (Parr et al., 1993) and rabbits (Matsui et al., 1994) using a computer system for image analysis also.

The purpose of the present study was to quantify the time course of dental wound healing in the apical, middle and cervical thirds of the alveolus of normal rats, using a simple standard stereological method.

Material and Methods

Male albino Wistar rats weighing 190-200 g were anesthetized with an intraperitoneal injection of 2,2,2,-tribromoethanol (Aldrich, Milwaukee, WI, USA) at a dose of 25 mg/100 g body weight. The upper right incisors were extracted with a forceps after disconnection of the surrounding gingiva and luxation with an enamel hatchet with a cutting edge. Immediately after surgery the gingival tissues were sutured with monofilament and a single intramuscular dose of antibiotic (Pentabiótico Veterinário, Wyeth, São Bernardo do Campo, SP, Brazil, 0.2 ml/rat) was given. The animals were killed by decapitation 1, 2, 3 and 6 weeks postoperatively (N = 5-7 per group), their mandibles were removed and their heads immersed in 10% formalin for 48 h. After fixation, the maxilla was dissected and divided along the median sagittal plane. The right halves were cut tangentially to the distal surface of the molars, decalcified and processed for paraffin embedding. Longitudinal 6-mm thick sections, cut at intervals of 60 mm, were stained with hematoxylin and eosin.

A light camera (Zeiss, Jena) was used to measure the volume fraction of alveolar components by a differential point-counting method (Weibel et al., 1966). Microscopic images of the alveolus, at a final magnification of 420X, were superimposed on sheets of paper with a grid containing 100 equidistant points. Six thousand points lying on clot and blood vessels, connective tissue and bone trabeculae were counted in the apical, middle and cervical thirds of each alveolus.

Data were analyzed for statistical significance by a non-parametric test (Kruskall-Wallis, ANOVA).

Results and Discussion

The different phases of alveolar healing were observed by histologic examination 1 to 6 weeks after dental extraction. At the end of the 1st week, delicate bone trabeculae lined with osteoblasts were observed mainly in the apical and middle thirds of the internal surfaces of the buccal walls; connective tissue was abundant and neoformed capillaries were still present; remnants of blood clots were observed in the central regions. By the 2nd week, with progressive bone neoformation, the socket was equally occupied by mature connective tissue and bone trabeculae. At the 3rd week, the entire socket appeared to be filled with thick trabecular bone, as also observed at the 6th week. The intra-alveolar healing process in the present study closely resembled data regarding white rats (Astrand and Carlsson, 1969; Okamoto and Russo, 1973; Carvalho and Okamoto, 1985).

Histometric analysis permitted us to quantify progressive bone neoformation in parallel to a decrease in the volume fraction of connective tissue up to the 6th week after dental extraction and showed a significantly smaller volume fraction of bone trabeculae in the cervical third of the socket at all time points except the 6th week (Table 1, Figure 1). It has been assumed that, in rats, healing following dental extraction is fully complete by the 21st postoperative day (Okamoto and Russo, 1973; Santos Jr. and Melhado, 1990). However, results from the present study showed that bone neoformation continued to proceed beyond this time.

Figure 1- Volume fraction of connective tissue and bone trabeculae in the apical (Api), middle (Mid) and cervical (Cer) thirds of the alveolus 1 to 6 weeks after tooth extraction (mean ± SEM for 5-7 rats per group).

Using an image analyzing system, Guglielmotti and Cabrini (1985) quantified alveolar wound healing in Wistar rats on the basis of histometric parameters including bone volume density. Measurements, however, were confined to a rectangle outlined on the apical third of the alveolus. Although no statistical analysis was applied, maximal bone volume density was apparently reached by the 30th postoperative day. The authors reported that maximal bone formation and maximal alveolar volume occurred on the 14th day after tooth extraction. The marked differences in methodology rendered unviable comparisons between our results and those reported by Guglielmotti and Cabrini (1985).

Even though the method presented here is simple and does not require sophisticated equipment, it appears to be precise and might be useful in comparative studies of alveolar wound healing under different types of experimental conditions.

Acknowledgments

The authors are indebted to Albertina A. Teixeira, Antonio de Campos and Edna A.S. Moraes for technical assistance.

References

Alves MCR, Okamoto T: Influência do stress no processo de reparo em feridas de extração dental. Estudo histológico em ratos. Rev Odont UNESP 18: 119-130, 1989

Amler MH, Johnson PL, Salman I: Histological and histochemical investigation of human alveolar socket healing in undisturbed extraction wounds. J Am Dent Assoc 61: 32-44, 1960

Astrand P, Carlsson GE: Changes in the alveolar process after extractions in the white rat. A histologic and fluorescent microscopic study. Acta Odont Scand 27: 113-127, 1969

Boyne PJ: Osseous repair of the postextraction alveolus in man. Oral Surg Oral Med Oral Pathol 21: 805-813, 1966

Carlsson GEH, Thilander H, Hedegard B: Histologic changes in the upper alveolar process after extractions with or without insertion of an immediate full denture. Acta Odont Scand 25: 21, 1967

Carvalho AC, Okamoto T: Interferências sistêmicas sobre o processo de reparo em feridas de extração dental. Rev Odont UNESP 14: 27-33, 1985

Guglielmotti MB, Cabrini RL: Alveolar wound healing and ridge remodeling after tooth extraction in the rat: a histologic, radiographic and histometric study. J Oral Maxillofac Surg 43: 359-364, 1985

Guglielmotti MB, Ubios AM, Cabrini RL: Alveolar wound healing alteration under uranyl nitrate intoxication. J Oral Pathol 14: 565-572, 1985

Guglielmotti MB, Ubios AM, Cabrini RL: Alveolar wound healing after X-irradiation. J Oral Maxillofac Surg 44: 972-976, 1986

Matsui Y, Ohno K, Michi K, Tachikawa T: Histomorphometric examination of healing around hydroxylapatite implants in 60Co-irradiated bone. J Oral Maxillofac Surg 52: 167-172, 1994

Okamoto T, Russo MC: Wound healing following tooth extraction. Histochemical study in rats. Rev Fac Odont Araçatuba 2: 153-164, 1973

Parr GR, Steflik DE, Sisk AL: Histomorphometric and histologic observations of bone healing around immediate implants in dogs. Int J Oral Maxillofac Implants 8: 534-540, 1993

Santos Jr PV, Melhado RM: Efeitos da estimulação ultra-sônica sobre o processo de reparo em ferida de extração dental: estudo histológico em ratos. Rev Odont UNESP 19: 291-299, 1990

Simpson HE: The healing of extraction wounds. Brit Dent J 126: 550-557, 1969

Weibel ER, Kistler GS, Scherle WF: Practical stereological methods for morphometric cytology. J Cell Biol 30: 23-38, 1966

Correspondence: Teresa Lúcia Lamano Carvalho, Departamento de Estomatologia, Laboratório de Patologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.

Accepted January 3, 1997
Electronic publication: September, 1997

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