A mandibular propulsive appliance modulates collagen-binding integrins distribution in the young rat condylar cartilage

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1 Biorheology 43 (2006) IOS Press A mandibular propulsive appliance modulates collagen-binding integrins distribution in the young rat condylar cartilage Mara Rúbia Marques, Denise Hajjar, Virgínia Oliveira Crema, Edna Teruko Kimura and Marinilce Fagundes Santos Cell and Developmental Biology Department, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil Abstract. We have previously shown that a mandibular propulsive appliance (MPA) stimulates cell proliferation and the synthesis of growth factors in the rat condylar cartilage. The aim of this study was to evaluate the effects of a MPA in the distribution of the integrin subunits α1 andα2 in this cartilage. Twenty eight days-old male Wistar rats were divided into treated (T) and age-matched control groups (C). Treated rats wore the appliance during 3, 5, 7, 9, 11, 15, 20, 30 and 35 days. The condyles were fixed, decalcified and paraffin-embedded. The distribution of α1 andα2 was studied by immunohistochemistry. Alpha1 distribution was uniform along the cartilage, increasing in 48 days-old rats (C20). Treated animals anticipated this increase to the age of 36 days (T9). The number of α2-positive cells was increased in C9 in the anterior condylar region, in C9 and C20 in the middle region and showed no differences in the posterior region. The MPA apparently abolished all variations, leading to a single increase at T30 in all regions. These results suggest that integrins containing the α1 andα2 subunits are modulated by forces promoted by the MPA, participating of the biological response to this therapy. Keywords: Functional orthopedics, condyle, alpha1, alpha2, integrin 1. Introduction The modulation of mandibular growth by the application of mechanical forces has been studied to improve the correction of discrepancies between the maxilla and the mandible, as well as dental malocclusions [19]. It is believed that the use of functional orthopedic appliances by young individuals generate tension in orofacial muscles, which are transmitted to the mandibular condyle, an important growth center of the mandible, generating adaptation. Clinical results obtained with this therapy are well characterized, although the molecular mechanisms underlying this response still remain unclear. The mandibular condyle is part of the temporomandibular joint, a bilateral synovial joint between the mandible and the temporal bones. The young condylar cartilage is a hyaline cartilage covered by a fibrous layer. Proliferative undifferentiated cells, located immediately below this layer, differentiate into chondroblasts, which produce extracellular matrix (ECM), mostly collagen type II and proteoglycans. * Address for correspondence: M.F. Santos, Cell and Developmental Biology Department, Av. Prof. Lineu Prestes 1524 sala 434, São Paulo, SP, Brazil, CEP X/06/$ IOS Press and the authors. All rights reserved

2 294 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage Later on, these cells become entrapped within this matrix, becoming chondrocytes. Hypertrophy of chondrocytes is followed by endochondral ossification. The proliferative compartment in the condylar cartilage is formed by undifferentiated cells and chondroblasts [19,24]. It is possible to modulate growth of the condylar cartilage by the application of forces [6]. Small load alterations such as those promoted by food hardness, for example, affect growth [1]. In cultured rat condylar cartilage hydrostatic compressive forces stimulated glycosaminoglycan (GAG) and DNA synthesis in a magnitude-dependent manner [22], and intermittent compressive loading increased collagen I and fibronectin synthesis [17]. We have previously observed that an intermittent indirect force generated by a mandibular propulsive orthopedic appliance was able to increase the expression of Insulin-like Growth Factors I and II (IGF-I and IGF-II), and cell proliferation in the young rat condylar cartilage [7]. Charlier et al. (1969) also observed an increase in the size of the undifferentiated layer after using this kind of appliance [2]. In our previous study, if this appliance was used for a short period of time (up to 9 days), the effect on mandibular repositioning was reversible. After 11 days of use, the mandibular repositioning was permanent, even after the appliance s withdrawn (unpublished observation). The transmission of forces applied to the condylar chondrocytes is probably mediated by integrins, heterodimeric transmembrane receptors for ECM proteins. Integrins contain two non-covalently associated subunits, α and β, which bind ECM proteins through their extracellular domains [5,9]. Through their cytoplasmic domains integrins usually interact with the cytoskeleton. Although integrins have no intrinsic enzymatic activity, they trigger signaling pathways upon binding to ECM, which might regulate several chondrocyte functions such as differentiation, matrix remodeling, gene expression, responses to mechanical stimulation and cell survival [13]. In normal human articular chondrocytes, integrin activation, consequent to mechanical stimulation in vitro, results in tyrosine phosphorylation of regulatory proteins and subsequent secretion of autocrine and paracrine factors [15]. The expression of integrins in human femoral condyles has been shown to be modulated by health condition and developmental stage [8,15,18,20]. During human growth, α1 and α2 integrin subunits were observed in the proliferative and hypertrophic layers [8], determined mostly by the ECM composition [11]. Condrossarcoma cells, as well as fetal chondrocytes, showed high levels of the collagen-binding α2β1 integrin [12]. We believe that an improved comprehension of how integrins mediate chondrocyte responses to mechanical stimulation, and how cross talk between integrins, ECM, and autocrine/paracrine signaling molecules (such as IGF-I and IGF-II) regulate mechanotransduction are important for further understanding how functional orthopedic appliances promote cartilage remodeling. 2. Materials and methods 2.1. Animals and treatment All experiments were conducted in accordance with the NIH guidelines, and the protocols were approved by the Biomedical Sciences Institute/USP Ethical Committee for Animal Research. Seventy-two male Wistar rats were used, beginning at 28 days of age. They were divided into treated (T) and control age-matched groups (C). Treated animals wore the appliance for 3, 5, 7, 9, 11, 15, 20, 30 and 35 days, from 8:00 a.m. to 6:00 p.m. The appliance was changed when necessary, according to the animal s development. Food and water were available at night, ad libitum. At the time of sacrifice, animals were

3 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage 295 anaesthetized with chloral hydrate (30 mg/100 g of body weight) and perfused with saline and with fixative solution (4% formaldehyde, Sigma, St. Louis, MO, USA). The mandibular condyles were dissected out, post-fixed in 4% formaldehyde at 4 C during 24 h, decalcified with 10% EDTA for 20 days and embedded in Paraplast R. Sagittal 5 µm-thick sections were placed on Poly-L-Lysine-coated slides (Sigma, St. Louis, MO, USA) Orthopedic appliance The appliance was designed as described by Hajjar et al. [7]. Briefly, it consisted of an inclined copper plane that raised the anterior displacement of the mandible every time the animals attempted to close their mouths at the normal position. The appliance was banded 90 at the anterior portion and a rubber tube was glued close, to fit in the upper incisor of the animal. All edges were rounded to avoid mucosal irritation. A soft leather collar was used to prevent the removal of the appliance; control animals wore the collar alone Immunohistochemistry Integrin subunits were investigated by immunohistochemistry, using the avidin biotin peroxidase technique. Rabbit polyclonal anti-α1 (Chemicon, AB1934, Temecula, CA, USA) and anti-α2 (Chemicon, AB1936, Temecula, CA, USA) were used at a 1 : 100 dilution in PBS, overnight at room temperature (RT). After washing in PBS, sections were incubated with the biotin-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA, USA) at a 1 : 250 dilution during 2 hours at RT. The sections were then revealed with 3,3 diamino-benzidine (DAB) in the presence of 0.02% H 2 O 2 for 30 min, mounted with Permount R (Fisher Chemicals) and examined on a Leitz microscope (Aristoplan). As a negative control the omission of the primary antibody was used, and no labeling was observed under these conditions. Pictures were taken using a CCD camera and the Scion Image software (NIH, Bethesda, USA) Counting of labeled cells and statistical analysis The quantification of labeled cells was performed by the evaluation of 900 cells in 3 randomly chosen microscopic fields at the anterior, central and posterior regions of the condylar cartilage per section. The number of labeled cells was expressed as a percentage of the total number of cells observed. Data were submitted to analysis of variance (ANOVA) followed by the Tukey s post-test for multiple comparisons. Main comparisons were made within the control group (between different ages) and between the treated groups and their age-matched controls. A P value of less than 0.05 was considered to be significant. Results were expressed as mean ± standard deviation (SD). 3. Results 3.1. The propulsor appliance modulates α1 integrin subunit distribution During normal development, α1 distribution was almost restricted to the hypertrophic layer in C3 (Fig. 1). From C5 on, α1 staining was distributed throughout the cartilage, including the proliferative

4 296 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage Fig. 1. Distribution of the α1 integrin subunit in the rat condylar cartilage during normal growth (C3 C30) and under therapy with a mandibular propulsive appliance (T3 T30). The α1 subunit was studied by immunohistochemistry using the avidin biotin-peroxidase technique in sagittal sections of the cartilage. T3, T9, T15, T20 and T30 correspond to young animals that wore the appliance for 3, 9, 15, 20 and 30 days, respectively. Each group was sacrificed with their age-matched control groups. In C3, all layers are shown: fibrous (F), undifferentiated (U), chondroblasts (C), mature chondrocytes (MC) and Hypertrophic (H). In detail, the negative control consisting of the omission of the primary antibody (CON). Magnification bar = 100 µm.

5 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage 297 Fig. 2. Quantification of α1-labeled cells in different regions of the rat condylar cartilage: (A) anterior region; (B) central region; and (C) posterior region. The dotted line separates the early and late phases of the therapy. Data are presented as percentage of total cells counted, mean ± SD. # P < when compared to the previous group; P < when compared to the age-matched control group, according to ANOVA and Tukey s post-test. layer. Labeling intensity, however, was variable, mainly in the proliferative compartment and the chondrocyte layer (Fig. 1). The number of α1-positive cells was uniform among the anterior, central and posterior regions of the cartilage, varying from 50 70% (Fig. 2). In the control group, there was an increase in α1-positively labeled cells in the C20 group (100% labeled cells compared to 58.52% in C15). Labeling intensity was increased as well. Both the number of labeled cells and labeling intensity decreased afterwards, returning to initial levels (Figs 1 and 2).

6 298 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage Fig. 3. Distribution of the α2 integrin subunit in the rat condylar cartilage during normal growth (C3 C30) and under therapy with a mandibular propulsive appliance (T3 T30). The α2 subunit was studied by immunohistochemistry using the avidin biotin-peroxidase technique in sagittal sections of the cartilage. T3, T9, T15, T20 and T30 correspond to young animals that wore the appliance for 3, 9, 15, 20 and 30 days, respectively. Each group was sacrificed with their age-matched control groups. In detail, the negative control consisting of the omission of the primary antibody (CON). Magnification bar = 100 µm.

7 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage 299 The appliance promoted a slight increase in labeling intensity at T3, mostly in the hypertrophic layer (Fig. 1). At T9, however, both the number of positive cells and the intensity of the staining were enhanced, comparing to C9 (100% labeled cells vs % in C9) (Figs 1 and 2). From T9 on, the labeling was similar to that observed in the control group, except for T20, which did not show the increase in α1 observed in C The α2 integrin subunit distribution varies according to the cartilage region and is modulated by the propulsor appliance At C3, α2-positive cells were observed mostly in the hypertrophic layer (Fig. 3). At C9 there was a marked increase in the number of positive cells in the anterior and middle regions of the cartilage, but Fig. 4. Quantification of α2-labeled cells in different regions of the rat condylar cartilage: (A) anterior region; (B) central region; and (C) posterior region. The dotted line separates the early and late phases of the therapy. Data are presented as percentage of total cells counted, mean ± SD. # P < when compared to the previous group; P < when compared to the age-matched control group, according to ANOVA and Tukey s post-test.

8 300 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage not in the posterior region (100% labeled cells vs. 67% in C7, Fig. 4). We observed an enhanced labeling intensity as well, and the presence of α2 in all cartilage layers (Fig. 3). This increase in the number of labeled cells was observed at C20 again, but only in the central cartilage region (77% of labeled cells). From C20 on, the number of marked cells gradually returned to the initial levels, being mostly restricted to the hypertrophic layer (Figs 3 and 4). No variations were observed for α2 labeling in the posterior region (Fig. 4). As observed for α1, the staining in T3 was slightly more intense than that observed in the control group (Fig. 3). Curiously, the appliance abolished variations described above in the number of labeled cells, leading to a single increase in number and staining intensity at T30, in the anterior, central and posterior regions (Figs 3 and 4). This difference, observed mainly in the proliferative compartment, was not observed in T Discussion The modulation of the mandibular condylar cartilage growth and adaptation is considered an effective way to correct growth discrepancies between the mandible and maxilla. In ortodonthics, the application of mechanical forces through the use of orthopedic appliances promotes very good clinical results. In this study, we showed that a mandibular propulsor appliance changed the distribution of the integrin subunits α1 and α2, probably corresponding to the collagen-binding integrins α1β1 and α2β1, in the rat condylar cartilage. For both α1 and α2 there was variation in expression and distribution during growth. In control animals, the α1 subunit raised uniformly in all cartilage regions in C20, when the rats were 48 days-old, around puberty. The α2 subunit, on the other hand, was uniformly expressed along the condylar cartilage, but showed some differential distribution with growth. Specifically, there was an increase in C9, but only in the anterior and posterior regions of the cartilage, and another peak in C20, similar to α1, but observed only in the central region. An interesting idea is that while α1 is modulated by hormones and growth factors present in the cartilage during growth, α2 could be related to force, because its expression was higher close to the muscle insertion. However, maybe contrary to this hypothesis, the appliance, which we consider as an intermittent indirect force applied to the cartilage, anticipated the α1 peak (previously in C20) to 9 days of treatment (T9). On the other hand, all α2 variations were abolished by the appliance s use, and a peak of expression appeared at T30, only in the treated group. In general, expression peaks mean a broader distribution, with all layers marked, including the undifferentiated proliferative layer. It is tempting to think that α2 was being replaced by α1 in T9, and that there was a late effect of this therapy after 30 days of treatment, leading to an increase in α2. After 9 days of treatment, it is known that there is a marked increase in cell proliferation, as observed by PCNA (Proliferating Cell Nuclear Antigen) labeling [7]. However, we believe that in order to consolidate this effect on growth, the process of endochondral ossification must be continued, and the use of the appliance ensures that those new cells will differentiate and synthesize ECM proteins, such as collagen II and proteoglycans. If the appliance is removed after 9 days, the mandible does not return to its original position. Several studies showed that the condylar cartilage responds to mechanical stimuli by altering cell proliferation and ECM proteins expression. For example, intermittent compressive forces increased sulfated glycosaminoglycans and collagen synthesis, while continuous compressive forces reduced ECM synthesis [3,4]. Another study showed that compressive forces applied uniformly in vitro to the whole condyle using a pneumatic loading system increased collagen I and fibronectin [17]. One has to consider,

9 M.R. Marques et al. / Orthopedic therapy modulates integrins in condylar cartilage 301 however, the force direction in these experiments, since it appears to be determinant for the effects on cartilage. Opposite forces such as those exerted by a protrusive appliance compress the anterior portion of the condyle against the temporal articular eminence, decreasing cartilage thickness and aggrecan immunostaining in rats [23]. However, in the same anterior region there are considerable stretching forces compared to the rest of the condyle, due to the muscle intermittent activity. Apparently, ECM composition defines the expression of different integrins in chondrocytes. For example, in vitro, α1 and α2 expression were upregulated by collagens I and II, respectively, in porcine knee articular cartilage [11]. It is possible that, in our study, the increase in α2 after 30 days of treatment was related to a higher amount of collagen type II produced by chondrocytes, after cell proliferation and differentiation. In monkeys and humans, the distribution and expression of collagens I and II vary with age [10,16], suggesting that the normal variation of α1 and α2 in rats could also be associated with ECM composition variations. Corroborating this hypothesis, stretching strain in vitro upregulated cartilaginous collagen II and aggrecan expression, as well as α2 and α5 (fibronectin-binding) integrin subunits [14]. Similar observation was made for the β1 integrin subunit, in vivo [21]. We have shown previously that this mandibular propulsive appliance stimulates the synthesis of IGF-I and IGF-II in the condylar cartilage, closely related to an increase in PCNA labeling [7]. After 9 days of treatment, for example, the expression of IGF-I and IGF-II was increased in the cartilage, which is coincident with the increased expression of α1 observed in the present study. Even in control animals, the expression of IGFs was slightly higher during puberty [7]. It is possible that IGFs affected α1 expression in the condylar cartilage in our study, and this hypothesis deserves further investigation. Alternatively, α1-containing integrins, functioning as mechanoreceptors, might activate signal transduction pathways that regulate gene expression, leading to an increase in IGFs production. In human articular chondrocytes, for example, the fibronectin receptor α5β1 functions as a mechanoreceptor. After mechanical stimulation, there was activation of a signal cascade involving stretch-activated ion channels, the actin cytoskeleton and the tyrosine phosphorylation of several components of focal adhesions. Subsequently, there was secretion of interleukin-4, which acted in an autocrine manner to regulate several cell functions and gene expression [20]. In summary, we have shown that integrin subunits α1 and α2 are expressed in the rat condylar cartilage during normal growth, with varying distribution according to the animal s age. A mandibular propulsive appliance, probably acting as an intermittent force, altered their expression and distribution, suggesting that this modulation may be part of the appliance s effect on a molecular level. The understanding of how forces exerted by orthopedic appliances modulate chondrocyte function is very important to improve our knowledge about this widely employed therapy. Acknowledgements The authors are grateful to Emília Ribeiro for the excellent technical support. This study was supported by the São Paulo State Research Foundation (FAPESP), grants # 97/ and 01/ Mara R. Marques is the recipient of a scholarship from FAPESP (02/ ). References [1] M. Bouvier and M.L. Zimny, Effects of loads on surface morphology of the condylar cartilage of the mandible in rats, Acta Anat. 129 (1997),

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