Morphological Changes in the Mandible of Male Mice Associated With Aging and Biomechanical Stimulus.код для вставкиСкачать
THE ANATOMICAL RECORD 292:431–438 (2009) Morphological Changes in the Mandible of Male Mice Associated With Aging and Biomechanical Stimulus MARIA LUISA TAGLIARO,1* RAQUEL MATTOS DE OLIVEIRA,1 DALVA MARIA PEREIRA PADILHA,2 SIDIA MARIA CALLEGARI-JACQUES,3 1 AND EMÍLIO ANTÔNIO JECKEL-NETO 1 Morphophysiological Sciences Department, Biosciences Faculty, Pontiﬁcal Catholic University of Rio Grande do Sul (PUCRS), Prédio 12, Av. Ipiranga, 6681, Porto Alegre, RS, Brazil 2 Hospital São Lucas, Instituto de Geriatria e Gerontologia, Pontiﬁcal Catholic University of Rio Grande do Sul (PUCRS), Av. Ipiranga, 6690, Porto Alegre, RS, Brazil 3 Statistics Department, Federal University of Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 9500, Porto Alegre, RS, Brazil ABSTRACT Degenerative changes in the temporomandibular joint (TMJ) associated with aging can affect mandibular shape and reduce growth potential when stimulated by functional appliance therapy. This study was designed to evaluate the morphological changes in the mandibles of male mice associated with aging and biomechanical stimulus. Every 3 days over the course of 1 month, the lower incisors were trimmed by 1 mm to induce mandibular advancement (MA) when the animal was feeding. The left mandibles of the 23 experimental and 27 control animals were subsequently dissected, and digital images were obtained to analyze nine linear/angular measurements. Because mandibular morphology depends on the maintenance of condylar cartilage, the surfaces of the condylar cartilage and the ascending ramus of the mandible were also analyzed by scanning electron microscopy (SEM). The linear measurements of the mandible showed changes according to age in the control group and a growth response in the mandibular condyle in 7- and 15-month-old mice after MA. Moreover, SEM analysis revealed depressions in the anterior region of the condylar cartilage and inclined vascular grooves in the ascending ramus in the 7- and 15-month-old experimental mice. Although the growth potential is reduced in mice after 6 months of age, the results showed that continuous growth of the mandible occurs after maturation, except in the condyle, and that biomechanical stimulus of the TMJ of male mice leads to condylar growth. These results suggest that mature and old individuals can favorably respond to maxillary functional orthopedic therapy. Anat Rec, 292:431–438, 2009. Ó 2009 Wiley-Liss, Inc. Key words: temporomandibular joint; mandible; functional maxillary orthopedics; condylar cartilage; condyle During the aging process, the mandible undergoes several morphological changes because it is a dynamic, cellular unit (Nomura et al., 2003). For example, the orientation of the osteons in the mandible adapts to the changes in the masticatory stimuli from the adjoining masticatory musculature (Nomura et al., 2003). Furthermore, the condylar cartilage, a growth center of the Ó 2009 WILEY-LISS, INC. *Correspondence to: Maria Luisa Tagliaro, PhD, Rua Garibaldi, 891/1002, Porto Alegre, RS, Brazil 90035-051. Fax: 15551-3286-1466. E-mail: email@example.com Received 20 November 2008; Accepted 14 October 2008 DOI 10.1002/ar.20861 Published online in Wiley InterScience (www.interscience.wiley. com). 432 TAGLIARO ET AL. mandible, becomes thin (Paulsen et al., 1999) with reduced growth potential due to biochemical changes (Blumenfeld et al., 1997; Blumenfeld and Livne, 1999) and diminished cellular activity (Paulsen et al., 1999). The mandibular condyle of young animals (6–8 weeks) is characterized by appositional growth, followed by endochondral ossiﬁcation. In male mice, till 6 months of age, both appositional and interstitial patterns of cellular growth occur, and after 6 or 7 months of age, degeneration of the condylar cartilage can be observed, accompanied by unremarkable insterstitial growth that might be associated with the repair process (Livne et al., 1990). However, mechanical stimuli in the mandible lead to muscle responses, neural activation, the enhancement of growth factors (Petrovic and Stutzmann, 1999), and vascular ﬂow (Rabie et al., 2002). Condylar cartilage and mandibular bone growth (Petrovic and Stutzmann, 1999; Rabie et al., 2002), and repair (Tagliaro et al., 2006) are the results of this stimulation. Furthermore, the growth response is regulated by the orchestrated inﬂuences of many growth and regulatory factors expressed by cellular components in the mandibular condyle (Rabie and Hagg, 2002). Functional Maxillary Orthopaedics is based on the principle of delivering neural and mechanical stimuli to the craniofacial complex through bimaxillary appliances (Simões, 1985), leading to growth responses of condylar cartilage (Fanghanel and Miehe, 1994; Fuentes et al., 2003; Rabie et al., 2003a,c; Tang et al., 2004), and the mandible of young and adult animals (Xiong et al., 2004, 2005). The pterygoid muscle forms a stabilizing system with the mandibular condyle and the articular capsule in the temporomandibular joint (TMJ) during mastication. The stimulus of mandibular advancement (MA) provokes a contraction of the lateral pterygoid muscle, which changes the functional and biomechanical environment of the TMJ (Takahashi et al., 1995). In 16month-old female mice, application of the MA stimulus for 30 days results in the enhancement of the length of the mandible, condylar process, and the mandibular condyle as compared to both a control group of females of the same age and to 2- and 7-month-old MA-stimulated female mice (Tagliaro et al., 2006). In male mice, however, the natural tendencies of mandible growth in the aging process are unclear, and the analysis of probable growth response and condylar cartilage regeneration after mechanical stimuli at all ages is important. Thus, the present study compared the morphological changes of the mandible of male mice of different ages during natural growth and after the application of MA stimuli. Fig. 1. The lower incisors were bilaterally trimmed by 1 mm in the incisal third to induce mandibular advancement during feeding. chow ad libitum. The mice were randomly assigned to either the experimental (N 5 23) or the control (N 5 27) group. The 2-month-old group was divided into nine experimental individuals and seven controls, the 7-monthold mice into eight experimentals and nine controls and the 15-month group into 10 experimentals and seven controls. In the experimental group, the lower incisors were trimmed bilaterally by 1 mm at the incisal third every 3 days, eight times in total, to induce an increase in MA when the animals were feeding (Fig. 1). The experimental animals were subsequently examined to conﬁrm that the contact of the posterior upper teeth and lower teeth was preserved, weighed to verify whether they ate less during the experimental period, and examined for any signs of illness. The animals were euthanized 4 days after the last trim, the left mandibles were dissected, and the tissues were ﬁxed in 10% paraformaldehyde solution for 24 hr. Trimming procedures and cervical dislocation were carried out in anesthetized animals (10% ketamine and 2% xylazine, 2:1, 0.1 ml/100 g). MATERIALS AND METHODS Animals and Experimental Protocol This experiment was approved by the Research Ethics Committee of the Pontifı́cal Catholic University of Rio Grande do Sul (PUCRS), Brazil. Fifty male Mus domesticus domesticus CF1 mice (FEPPS, Fundação Estadual de Produção e Pesquisa em Saúde do Rio Grande do Sul, Brazil) that were 2, 7, and 15 months of age, were studied. The animals were housed under standard conditions with a 12-hr light:12-hr dark cycle and supplied with tap water and standard rodent Scanning Electron Microscopy The left mandibular condyles were separated from the mandible, dehydrated, gold-coated in a Balzers Bal-Tec SCD-005 sputter coater, and examined using a Philips XL-30 scanning electron microscope. The descriptive analysis looked at images of the surface of the mandibular condyle and the ascending ramus. The characteristics observed were signs of condylar cartilage degeneration, the orientation of the vascular grooves in the 433 MORPHOLOGICAL CHANGES IN MANDIBLE Fig. 2. The mandibular measurements and growth tendencies related to aging and mandibular advancement. Although the condyle length (C–D) and width (R–Q) decreased in older control animals, the length of the condylar process (B–F), and mandible (A–F) and the distance from the point of intersection between the lingual surface of the lower incisors and its lingual alveolar bone to middle point of the mandibular foramen (A–B) were enhanced. After mandibular advancement, the C–D length was increased (white arrow) in the oldest group. ascending ramus, and morphological changes induced by MA in both structures. Mandibular Measurements All measurements were made according to the procedure described by Xiong et al. (2004). The width and length of the condylar head were directly measured using a Vernier caliper. The linear measurements were made by digital imaging of the mandible medial side (Fig. 2) and consisted of: the length of the condylar process (B–F), mandibular length (A–F), the distance from the point of intersection between the lingual surface of the lower incisors and its lingual alveolar bone to the middle point of the mandibular foramen (A–B), condylar length (C–D), condylar width (Q–R), the distance from the most anterior point of the condyle to the mandibular plane (C–GH), the distance from the intersection point of the (B–E) extension line and the outer contour of the condyle to the mandibular plane (F–GH), and the distance from the most inferior and posterior points of the condyle to the mandibular plane (D–GH). The angle of the condylar process axis to the mandibular plane (BF/ GH) was also measured. The images were obtained at a 90-degree angle using a Sony DSC-W1 digital camera at a ﬁxed distance and a 90-drgree angle and analyzed by Image Pro-plus1 4.1 software. Fig. 3. A: Weight of individuals in the experimental group (2, 7, and 15 months) at each treatment (events 1–8) and on the day the animals were euthanized (event e). B: Final weights in the experimental (EG) and control (CG) groups. Student’s t-test. SPSSÓ v. 8 software was used for the statistical analyses. RESULTS Body Weight Statistical Analysis The changes in linear/angular measurements produced by experimental MA in mice of different ages were evaluated by multivariate analysis of variance (MANOVA) using a 2-way model with ﬁxed effects and interaction. The means of treated and untreated individuals in each age group were compared using the least signiﬁcant difference (LSD) criterion and conﬁrmed by Student-Newman-Keuls (SNK) or Tamhane’s test, according to the acceptance of homogeneity or heterogeneity of the group variances. The values of the weight differences among the groups were evaluated using the Weight loss was observed from the ﬁrst to the second trim in 7- (5.9%) and 15- (2.5%) month-old MA mice. In the 2-month-old experimental group, the weight loss occurred after trim 3 (2.0%). Over the course of the experiment, the weight of 7-month-old MA mice was reduced by 6%, 1.0% (15 months), and the weight of 2-month-old mice increased by 2.5% (Fig. 3A). The ﬁnal weights of the 2- and 7-month-old experimental mice were 0.8% and 2.0%, respectively, less than that of the control mice, whereas 15-month-old MA mice weighed 3.7% more than control mice of the same age (Fig. 3B). 434 TAGLIARO ET AL. The weight differences among the groups were not statistically signiﬁcant and no sign of illness was detected. Scanning Electron Microscopy The mandibular ascending ramus in the experimental group was characterized by inclined vascular grooves, most likely a result of the MA stimulus, whereas the grooves were less inclined in control groups of the same age (Fig. 4A–F). In the posterior region of the mandibular condyles, an elongation of the condylar surface in 7and 15-month-old experimental animals was observed compared with controls (Fig. 5A–F). Depressions in the anterior region of the condylar cartilage were accentuated in 7- and 15-month-old experimental mice (Fig. 6A,B). Linear and Angular Measurements The mean and standard deviations of the eight linear and one angular mandibular in individuals of different ages, in both the control and experimental groups, are presented in Table 1. Age had a signiﬁcant effect on all linear measurements (P < 0.001 for six variables except C–D and Q–R in which P 5 0.020); no age effect was observed on angle BF-GH (P 5 0.885). The differences in all linear measurements of the untreated groups of all ages were statistically signiﬁcant although angle BFGH was not. The smallest differences between ages are: B–F (P < 0.014), A–F (P < 0.003), A–B (P < 0.014), C–D (P < 0.002), Q–R (P < 0.042), C-GH (P < 0.008), F-GH (P < 0.023), and D-GH (P < 0.021). The effects of treatment and of age as well as the interaction between them were tested using a whole set of nine variables by MANOVA; all the results were found to be statistically signiﬁcant (P 5 0.04; P < 0.001 and P < 0.025, respectively). As for the treatment effect, MA produced lower averages for D-GH (P 5 0.015), independent of age group (no interaction effect); the C–D measurement, however, also presented a signiﬁcant effect of MA (P 5 0.001) and a signiﬁcant interaction among the treatment and age group (P 5 0.003). For the 7- and 15-month old groups, the average mandibular condyle lengths (C–D) were signiﬁcantly (P < 0.05) greater than those of controls. The average measurements from treated and control 7-month old mice are 2.26 and 1.91, respectively; for 15-month mice they are 2.04 and 1.79, respectively. There were no statistically signiﬁcant differences between the experimental and control groups for any other variable. DISCUSSION The microstructure, morphology, biochemical environment, and the cellular and molecular composition of bone and cartilage tissues change through the life of an animal (Weiss et al., 1986; Kloss and Gassner, 2006). Endogenous and exogenous factors affect the articular cartilage through mechanical forces (Yamamoto et al., 2005), and the effects of these factors differ depending on age for the condylar cartilage of the mandible, the ﬁbrocartilage of lumbar intervertebral disc, the elastic cartilage of external ear and the hyaline cartilage of the xiphoid process. All types of cartilage tend to present some potential for regeneration in the early stages of life, followed by a gradual and continuous decline in metabolic activities (Weiss et al., 1986). Also important is the distinction between the effects of aging in males and females. Sex hormones regulate bone metabolism. For example, in C57BL/6J mice orchiectomized or ovariectomized on the 5th day after birth, both males and females showed suppressed craniofacial growth, and these differences suggest an important role for sex hormones in growth control (Fujita et al., 2004). In addition, in humans, biochemical ﬁndings (bone loss markers) and histomorphometric analysis at different skeletal sites suggests that osteoclast function is preserved during aging in men (Kloss and Gassner, 2006). This study evaluated the morphological changes of the mandible and mandibular condyle associated with aging and the effect of MA, a biomechanical stimulus, in male mice. In agreement with our previous ﬁndings in mice (Tagliaro et al., 2006), a cephalometric longitudinal study of humans was performed, and researchers observed natural and continuous craniofacial bone growth over a period of 66 years and detected different patterns of growth for men and women (Behrents, 1993). These results suggest the potential for growth after mechanical stimulus. In the present study, many linear measurements of the mandible were enhanced in 7- and 15-month-old male mice compared with controls. However, in the condyle, an important growth center of the mandible, different effects of age were observed. The length C–D was diminished in the oldest animals and the width Q–R was reduced in 7-month-old mice. By contrast, in 15-month-old mice, the width Q–R was increased to similar values of the 2-month-old animals (Table 1). A decrease in the levels of several growth factors and an increase in the levels of pro-inﬂammatory cytokines occur in the TMJ. These changes lead to the reduced growth response and the reduced regenerative ability of the condylar cartilage of rodents (Livne, 1994; Livne et al., 1997; Blumenfeld and Livne, 1999; Gepstein et al., 2002). The enhancement of the linear measurements of the mandibles in control mice observed in this study can be explained by variations in the calciﬁcation of cranial and facial sutures in young adults and by extrinsic factors such as functional and biomechanical stimuli (Behrents, 1993). There were no changes in the direction of condylar growth in the control group. This observation is conﬁrmed by the measurement averages of angle BF-GH, which were maintained in the three age groups and by the signiﬁcant enhancement from 2 to 15 months of age in mice in all three linear distances from condylar points C, F, and D to the mandibular plane (GH). In addition all measurements were consistent with no modiﬁcation of the axial rotation (Table 1). However, in the experimental group, the C–D averages were enhanced in the 7- and 15-month-old mice, and the distance D–GH diminished independently of age group. These results conﬁrm the presence of posterior condylar growth, due to the posterior axial rotation of the mandibular condyle when the mandible is advanced to incisal contact. Stuzmann and Petrovic (1999) demonstrated that in young untreated rats, the angle and direction of condylar growth is in the same direction as the trabecular bone. However, in mice stimulated by MA (postural hyperpropulsor) or growth hormone (STH), a modiﬁcation of rotational growth, a different angular direction (BF-GH), MORPHOLOGICAL CHANGES IN MANDIBLE 435 Fig. 4. Scanning electron microscopy of the mandibular ascending ramus. In 2- (A), 7- (C), and 15- (E) month-old mandibular advancement mice an elongation characterized by inclined vascular grooves was observed while the grooves were less inclined in control mice of the same age (B, D, and F). condylar growth, and new trabecular bone were observed (Stutzmann and Petrovic, 1999). In the present study, the condyle of control male mice decreased in length from 2 to 15 months of age over the course of natural development. The width was less in 7-month-old animals compared with 2-month-old animals, and it was greater in 15-month-old animals compared with 7month-old animals (Table 1). These data are compatible 436 TAGLIARO ET AL. Fig. 5. Scanning electron microscopy of the condyles. An elongated form was observed in condyles of the 7- (C) and 15- (E) month-old experimental groups than in 7- (D) and 15- (F) control group. No difference was detected between the 2-month-old treated (A) and control mice (B). with the fact that condylar cartilage becomes thin in the oldest mice and that cellular activity is involved in the repair of cartilage (Livne et al., 1990); however, it does not explain why the width was increased in 15-month- old animals. According to Blumenfeld et al. (1997), the degenerative changes in the condylar cartilage remain stable in female mice beginning at 12 months of age, suggesting that 15-month-old mice are metabolically 437 MORPHOLOGICAL CHANGES IN MANDIBLE (Kobayashi et al., 2002), it could affect the condylar length but not the width in mice. The results also showed a signiﬁcant enhancement in the length of mandibular condyle in 7- and 15-month-old mice after MA (Table 1). This protrusive movement modiﬁes the biophysical and biochemical environment in the TMJ and increases vascular endothelial growth factor expression (Rabie et al., 2002). As a result, blood-vessels proliferate; mesenchymal cells migrate, chondrogenesis is induced, cartilaginous matrix and type II collagen synthesis are enhanced, which together promote the formation of new cartilage and bone (Rabie et al., 2003a,b, 2004). By contrast, a morphological study in 7-month-old female mice showed no condylar growth after 30 days of MA, although the regeneration of cartilage was observed by SEM (Tagliaro et al., 2006). In 2-month-old male mice, MA produced lower, but not signiﬁcantly different values, in distance D–GH and in angle BF-GH (Table 1). This result suggests that the stimulus changed the activity of the lateral pterygoid muscle modifying the direction of condylar growth, causing it to become horizontally oriented as justiﬁed by the posterior rotation of the mandible (Stutzmann and Petrovic, 1999). In this study, the SEM analyses of the ascending ramus showed a different orientation of vascular grooves in the experimental groups of all ages in comparison with controls. The grooves are more prominent and follow the direction of the protrusive movement (Fig. 4). The MA provokes the contraction of the pterygoid lateral muscle, which pulls the mandibular condyle to a more forward and lower position (Petrovic and Stutzmann, 1999) and could cause a tension remodelling of the vascular grooves in the ascending ramus. In the 7- and 15-month-old MA groups, the condyle is elongated in the posterior region, conﬁrming the signiﬁcant enhancement of condyle length in the linear measurements after MA (Fig. 5). The biomechanical stimulus and the consequent condylar cartilage enhancement mostly occurred in the middle to posterior region. As a result, the depressions in the anterior region of the condylar cartilage became pronounced and deep. It was observed that in 7- and 15-month-old experimental male mice the growth response occurred in the condyle length and that no signiﬁcant differences occurred in the remainder of the mandible (Table 1). These patterns of growth suggest that the MA stimulus in 7- and 15-month-old male mice can activate the growth potential of the condylar cartilage. This step stable. Therefore, the 15-month-old group could repair naturally the cartilage and return to the original dimensions of the condyle. Because postero-anterior movements are predominant during the mastication Fig. 6. Scanning electron microscopy of the anterior region of a representative condylar cartilage from the 7-month-old group. The depressions (white arrows) were more accentuated in the experimental mice (A) than in the control mice (B). TABLE 1. Mean 6 standard deviation for nine linear (in mm) and angular (in degrees) measurements in the mandibles of no treated male mice of different ages Linear (mm) and angular (8) variables B–F A–F A–B C–D Q–R C–GH F–GH D–GH BF/GH (8) 2-month-old control (N 5 9) 2.91 12.73 10.25 2.07 0.84 6.00 5.88 4.71 40.22 6 6 6 6 6 6 6 6 6 0.22a 0.40a 0.34a 0.18a 0.10a 0.25a 0.24a 0.22a 3.12a 2-month-old experimental (N 5 7) 2.94 12.88 10.28 1.99 0.84 5.89 5.65 4.40 36.85 6 6 6 6 6 6 6 6 6 0.18a 0.51a 0.32a 0.13a 0.00a 0.21a 0.19a 0.15a 2.54a 7-month-old control (N 5 8) 3.12 13.44 10.80 1.91 0.74 6.12 5.97 4.82 39.06 6 6 6 6 6 6 6 6 6 0.07b 0.19b 0.19b 0.19a,b 0.05b 0.25a 0.31a 0.25a 2.67a 7-month-old experimental (N 5 9) 3.03 13.39 10.84 2.26 0.78 6.08 5.85 4.67 38.83 6 6 6 6 6 6 6 6 6 0.20b 0.18b 0.18b 0.20c 0.11b 0.18a 0.21a 1.80a 4.67a 15-month-old control (N 5 10) 3.39 13.76 10.81 1.79 0.81 6.50 6.33 5.12 38.10 6 6 6 6 6 6 6 6 6 0.15c 0.60b,c 0.60b 0.15b 0.06a 0.32b 0.38b 0.28b 2.87a 15-month-old experimental (N 5 7 3.36 13.84 10.86 2.04 0.83 6.50 6.34 5.04 38.79 6 6 6 6 6 6 6 6 6 Means indicated by the same letter do not differ signiﬁcantly at 0.05 level in each linear and angular measurement. 0.18c 0.41c 0.26b 0.19a 0.00a 0.31b 0.32b 0.32b 1.20a 438 TAGLIARO ET AL. precedes bone formation as a morphologic guide for blood invasion (Rabie and Hagg, 2002), although it may be necessary to prolong the stimulus to obtain mandibular bone growth. These data suggest that some mechanisms of growth exist in the mandibles of male mice during aging. The linear and angular measurements revealed a continuous growth of the mandible during the aging process, except in the condyle, which showed the potential for growth when induced by a biomechanical stimulus. In addition, MA induced growth of the mandibular condyle. Thus, the functional maxillary orthopaedics could be an option for treating mature and old individuals when mandibular growth and regeneration of the condylar cartilage are necessary. ACKNOWLEDGMENTS We are very grateful to Luisa Maria Gomes de Macedo Braga, PhD and Patricia Sesterheim MSc for mouse husbandry at FEPPS (Fundação Estadual de Produção e Pesquisa em Saúde), and to Clı́via Pazin Miwa, Deise Forneck Flores and Marcus Rodrigo Guidoti Soares, undergraduate students of PUCRS, for laboratory assistance. LITERATURE CITED Behrents R. 1993. Crescimento facial adulto. 3rd ed. Porto Alegre. Editora Artes Médicas Ltda. 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