Clinical and histochemical alterations of the periodontal ligament in gerbils after malocclusion induced/Alteraciones clinicas e histoquimicas del ligamento periodontal en gerbiles despues de maloclusion inducida.
KEY WORDS. Periodontal ligament; Histochemistry; Malocclusion, Gerbil; Meriones unguiculatus.
RESUMEN: El objetivo de este articulo es mostrar las alteraciones clinicas e histoquimicas del primer ligamento periodontal del lado derecho, despues de la extraccion del molar superior izquierdo en gerbiles (Meriones unguiculatus). Luego de dos meses, los ligamentos periodontales fueron retirados y procesados para el analisis histoquimico. Los resultados mostraron que la reaccion de TRAP es capaz de evidenciar la actividad osteoclastica en la hiperfuncion de la semimandibula derecha, explicando los cambios funcionales del ligamento periodontal despues de la extraccion dental, siendo observada una pequena recesion gingival y exposicion radicular de los dientes sin funcion, en los molares inferiores izquierdos.
PALABRAS CLAVE: Ligamento periodontal; Histoquimica; maloclusion; Gerbil; Meriones unguiculatus.
The periodontal ligament is the connective tissue localized between the root of the tooth and the alveolar bone, and is known to aid in relieving mechanical strain such as occlusal force and orthodontic force (Shuttleworth & Smalley, 1983; Kusters et al., 1991; Kuroiwa et al., 1992).
The periodontal ligament consists of cellular and intercellular components, which fills up the space around cells. Intercellular components are mainly composed of fibrous elements and the ground substance. The major fibrous elements are collagen fibers (type I and type III collagens), which play a role in resisting tensional force and holding teeth in the alveolar socket (Shuttleworth & Smalley; Kusters et al.).
Hypofunction such as a loss of the opposite tooth results in disuse atrophy, as observed in various organs. Although the periodontal principal fibers are disorganized at various degrees, disappear, and are replaced by a loose connective tissue under hypofunctional conditions, this atrophic phenomenon is reversed by recovery of the masticatory function (Cohn, 1965; Amemiya & Abe, 1980; Kinoshita et al., 1982; Koike, 1996).
Tartrate-sensitive acid phosphatase has been cytochemically localized in osteoblasts, osteocytes, and osteoclasts, whereas tartrate-resistant acid phosphatase (TRAP) is present in osteoclasts (Hammarstrom et al., 1983; Minkin, 1982). Osteoclasts have been identified, primarily by morphological criteria, as large, multi-nucleated cells which contact calcified bone matrix and exhibit ruffled borders surrounded by clear zones (Gothlin & Ericsson, 1976). Tartrate-resistant acid phosphatase as a histochemical and biochemical marker for the osteoclast would provide an additional experimental tool for studying osteoclastic differentiation and function. Baron et al., (1986) employed TRAP as such a marker for cytochemical identification of proposed osteoclast precursors at both the light and the electron microscopic level.
Thus, the objective of this study is to show the clinical and histochemical alterations of the periodontal ligament in the first molar, on the right side, after upper molar teeth extraction on the left side in gerbils.
MATERIAL AND METHOD
This study followed the requirements of the Ethics Committee on the Use of Animals in Experimentation at the University of Sao Paulo, Brazil.
It was used 10 adult male gerbils (Meriones unguiculatus), weighing 50g, maintained at the animal house under controlled temperature, with commercial ration and water available at libitum.
A half of these animals received the occlusal alteration induction by upper molars teeth extraction (Fig 1A), on the left side, experimental group, and the other half was considered as control, only submitted to surgical stress. These animals were used for histochemical study.
Induction of occlusal alteration. The animals were anesthetized with tribromoethanol (0.25g/kg of body weight) and submitted to teeth extraction of the upper molars in the left hemiarch, with animals receiving the antibiotic Pentabiotic (24000 IU for kg of body weight) as preoperative prophylaxis. After asepsis and disinfection of the surgical site, teeth extraction was performed with anatomical tweezers and hollenback 3S. The extraction area was compressed to stop bleeding (Chompret maneuver). After surgery, the animals received antiinflammatory and analgesic drugs (sodium diclofenac- 0.4mg/Kg of body weight) and were maintained in appropriate cages for two months.
Specimen preparation for histochemical analysis. After two months, the animals were anesthetized with urethane (1.5g/kg of body weight) and injected intracardially with 7% of body weight 0.9% physiological saline to wash the vessels, followed by perfusion with the same amount of 10% formalin in sodium phosphate buffer, pH 7.4. The hemimandibles with periodontal ligament were then removed and immersed in the same fixative solution. After fixation period, the specimens were washed in cacodilat solution and dryed and decalcified in EDTA 0.5M 10% solution with TRIS 0.2M (pH 7.4) for 7 days. After decalcified period, they were dehydratedin crescent alcohol sequence and immersed in a mixture of alcohol 95% + resin (1:1) during four hours and in pure resin "overnight". In the next day, it was included in historesin (Leica Historesin). Each historesin block was transversally sectioned in long orientation of the teeth with semi-seriated slices (3[micro]m) and submitted to histochemical reaction with TRAP (tartrate-resistant acid phosphatase).
After two months, it was found in this work a little gingival recession and radicular exposure of teeth without function, inferior molars of the left side.
The stained histological glasses by tartrate-resistant acid phosphatase (TRAP) showed positive cells by red stain in cytoplasm (Fig 1B). These cells may be multinucleated placed at Howship lacunae or mononucleated (Figs. 2A, B). It was observed a great number of TRAP positive cells in the cervical and apical regions of the periodontal ligament, in the inferior first molars. These cells were not different between the sides of the control group and between the control and experimental groups on the right side, hyperfunction region, (Figs. 2C, D).
[FIGURE 1 OMITTED]
The periodontal ligament tissue is thought to play an important part in the regeneration of the periodontium, but the precise function of the ligament tissue and cells is not yet clearly understood. Here we have attempted to clarify the histochemical alterations of the periodontal ligament in first molars, on the right side, after upper molars teeth extraction on the left side in gerbils.
After two months, it was found in this work a little gingival recession and radicular exposure of the teeth without function, inferior left molars, according to Burkland et al., 1976 and Schropp et al., 2003 works. This situation is explained by the fact that the opposite teeth extraction, permitted the continuous eruption of these teeth ahead of occlusal plane, probably by the cementary neoformation, although this process has been observed only in the apical region of the hypofunction side. These results, however, diverged to the data obtained in mice, after a long period of time in observation of antagonist teeth, it was not observed cement hyperplasic tissue or tooth supra-eruption, being that found intense resorption of remanescent alveolar bone and a little quantity of periodontal ligament in the interdental region, due to the lack of periodontal stimulation of the periodontal tissues by occlusal contact (Cohn), because the lack of function causes involution of the soft periodontal tissues, according to that was observed in humans and monkeys (Pihlstrom & Ramfjord, 1971).
[FIGURE 2 OMITTED]
The histological results of this study showed thick vessels in the periodontal ligament, principally in the apical region of the hemimandible submitted to hyperfunction activity, which is in agreement with Palcanis (1973) and Kvinnsland et al., (1992) studies. They showed that an occlusal trauma can alter the vascular function and that blood pressure, even though the histological tissue presents a normal appearance. The numbers suggest an increase of the vessels in the periodontal ligament of the first molar, on the right hemimandible of the experimental group.
According to Cole & Walters (1987), tartrate-resistant acid phosphatase (TRAP) is able to show the osteoclasts presence. Thus, it has been used as a histochemical marker for osteoclasts in many studies (Brudvik & Rygh, 1993, 1994; Rody et al., 2001; Kawarizadeh et al., 2003, 2004; Kawasaki et al., 2004; Fukushima et al., 2005). It was observed in this study a high intensity staining for TRAP reaction in the hyperfunction hemimandible, which is explained by the intense bone remodeling process, represented by the increase of osteoclasts cells.
It was possible to conclude that the TRAP reaction was able to show high intensity of osteoclastic activity in the hyperfunction hemimandible, explaining the functional changes in the periodontal ligament after teeth extraction.
Amemiya, A. & Abe, S. An electron microscopic study on the effects of extraction of opposed teeth on the periodontal ligament in rats. Jpn. J. Oral. Biol., 22:72-83, 1980.
Baron, R.; Neff, L.; Tran Van, P.; Nefussi, J. R. & Vingnery, A. Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am. J. Pathol., 122:363-78, 1986.
Brudvik, P. & Rygh, P. The initial phase of orthodontic root resorption incident to local compression of the periodontal ligament. Eur. J. Orthod., 15:249-63, 1993.
Brudvik, P. & Rygh, P. Multi-nucleated cells remove the main hyalinized tissue and start resorption of adjacent root surfaces. Eur. J. Orthod., 16:265-73, 1994.
Burkland, G.A.; Heeley, J. D. & Irving, J. T. A histological study of regeneration of the completely disrupted periodontal ligament in the rat. Arch. Oral. Biol., 21:349-54, 1976.
Cohn, S. A. Disuse atrophy of the periodontium in mice. Arch. Oral. Biol., 10:909-19, 1965.
Cole, A. A. & Walters, L.M. Tartrate-resistant acid phosphatase in bone and cartilage following decalcification and cold embedding in plastic. J. Histochem. Cytochem., 35:203-6, 1987.
Fukushima, H.; Jimi, E.; Okamoto, F.; Motokawa, W. & Okabe, K. IL-1-induced receptor activator of NF-kappa B ligand in human periodontal ligament cells involves ERK-dependent PGE2 production. Bone, 36:267-75, 2005.
Gothlin, G. & Ericsson, J. L. E. The osteoclast: review of ultrastructure, origin, structure-function relationship. Clin. Orthop. Relat. Res., 120:201-31, 1976.
Hammarstrom, L. E.; Anderson, T. R.; Marks, S. C. Jr. & Toverud, S.U. Inhibition by dithionite and reactivation by iron of the tartrate-resistant acid phosphatase in bone of osteopetrotic rats. J. Histochem. Cytochem., 31:1167-174, 1983.
Kawarizadeh, A.; Bourauel, C. & Jager, A. Experimental and numerical determination of initial tooth mobility and material properties of the periodontal ligament in rat molar specimens. Eur. J. Orthod., 25:569-78, 2003.
Kawarizadeh, A.; Bourauel, C.; Zhang, D.; Gotz, W.; Jager, A. Correlation of stress and strain profiles and the distribution of osteoclastic cells induced by orthodontic loading in rat. Eur. J. Oral. Sci., 112:140-7, 2004.
Kawasaki, N.; Hamamoto, Y.; Nakajima, T.; Irie, K. & Ozawa, H. Periodontal regeneration of transplanted rat molars after cryopreservation. Arch. Oral. Biol., 49:59-69, 2004.
Kinoshita, Y.; Tonooka, K. & Chiba, M. The effect of hypofunction on the mechanical properties of the periodontium in the rat mandibular first molar. Arch. Oral. Biol., 27:881-5, 1982.
Koike, K. The effects of loss and restoration of occlusal function on the periodontal tissues of rat molar teeth: histopathological and histometrical investigation. J. Jpn. Soc. Periodont., 38:1-19, 1996.
Kuroiwa, M.; Kodaka, T. & Higashi, S. Scanning electron microscopic observations of the periodontal ligament fibers and cells in rat molar teeth. Acta Anat., 67:200-6, 1992.
Kusters, S.T.; Kuijpers-Jagtman, A. M. & Maltha, J.C. An experimental study in dogs of transseptal fiber arrangement between teeth which have emerged in rotated or non-rotated positions. J. Dent. Res., 70:192-7, 1991.
Kvinnsland, S.; Kristiansen, A.B.; Kvinnsland, I. & Heyeraas, K. J. Effect of experimental traumatic occlusion on periodontal and pulpal blood flow. Acta. Odontol. Scand., 50:211-9, 1992.
Minkin, C. Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif. Tissue. Int., 34:285-90, 1982.
Palcanis, K.G. Effect of occlusal trauma on interstitial pressure in the periodontal ligament. J. Dent. Res., 52: 903-10, 1973.
Pihlstrom, B. L.; Ramfjord, S.P. Periodontal effect of nonfunction in monkeys. J. Periodontol., 42: 748-56, 1971.
Rody, W. J. Jr.; King, G. J. & Gu, G. Osteoclast recruitment to sites of compression in orthodontic tooth movement. Am. J. Orthod. Dentofacial. Orthop., 120:477-89, 2001.
Schropp, L.; Wenzel, A.; Kostopoulos, L. & Karring, T. Bone healing and soft tissue contour changes following single-tooth extraction: a clinical and radiographic 12-month prospective study. Int. J. Periodontics. Restorative. Dent., 23:313-23, 2003.
Shuttleworth, C. A. & Smalley, J.W. Periodontal ligament. Int. Rev. Connect. Tissue. Res., 10:211-47, 1983.
Dr. Joao Paulo Mardegan Issa
Faculdade de Odontologia de Ribeirao Preto - USP
Departamento de Morfologia, Estomatologia e Fisiologia
CEP: 14040-904 Av. Cafe S/N, Ribeirao Preto
Phone: +55-16-36024095 Fax: +55-16-36330999
* Leandro Moura Leite Naves; * Joao Paulo Mardegan Issa; ** Dimitrius Leonardo Pitol; *** Sandra Yasuyo Fukada; **** Miguel Angel Sala di Matteo & **** Mamie Mizusaki Iyomasa
* Graduate student, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
** Graduate student, Biosciences Institute- Molecular and Cellular Biology (UNESP), Rio Claro, Sao Paulo, Brazil.
*** Graduate student, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
**** Professor, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
NAVES, L. M. L.; ISSA, J. P. M.; PITOL, D. L.; FUKADA, S. Y.; DI MATTEO, M. A. S. & IYOMASA, M. M. Clinical and histochemical alterations of the periodontal ligament in gerbils after malocclusion induced. Int. J. Morphol., 25(4):907-910, 2007.
NAVES, L. M. L.; ISSA, J. P. M.; PITOL, D. L.; FUKADA, S. Y.; DI MATTEO, M. A. S. & IYOMASA, M. M. Alteraciones clinicas e histoquimicas del ligamento peridontal en gerbiles despues de maloclusion inducida. Int. J. Morphol., 25(4):907-910, 2007.