Evaluation of protein undernourishment on the condylar process of the Wistar rat mandible correlation with insulin receptor expression.

UNLABELLED: The mandible condylar process cartilage (CP) of Wistar rats is a secondary cartilage and acts as a mandibular growth site. This phenomenon depends on adequate proteins intake and hormone actions, including insulin. OBJECTIVES: The present study evaluated the morphological aspects and the expression of the insulin receptor (IR) in the cartilage of the condylar process (CP) of rats subjected to protein undernourishment. MATERIAL AND METHODS: The nourished group received a 20% casein diet, while the undernourished group (U) received a 5% casein diet. The re-nourished groups, R and RR, were used to assess the effects of re-nutrition during puberty and adulthood, respectively. CPs were processed and stained with picro-sirius red, safranin-O and azocarmine. Scanning electron microscopy and immunohistochemistry were also performed. RESULTS: The area of the CP cartilage and the number of cells in the chondroblastic layer decreased in the U group, as did the thickness of the CP layer in the joint and hypertrophic layer. Renourishment during the pubertal stage, but not during the adult phase, restored these parameters. The cell number was restored when re-nutrition occurred in the pubertal stage, but not in the adult phase. The extracellular matrix also decreased in the U group, but was restored by re-nutrition during the pubertal stage and further increased in the adult phase. IR expression was observed in all CPs, being higher in the chondroblastic and hypertrophic cartilage layers. The lowest expression was found in the U and RR groups. CONCLUSIONS: Protein malnutrition altered the cellularity, the area, and the fibrous cartilage complex, as well as the expression of the IRs.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats.

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Age-dependent physiological changes in the histoarchitecture of the articular cartilage of the rabbit mandibular condyle: a morphological and morphometric study.

Mandibular condyle articular cartilage participates in condylar postnatal growth and is responsible for adaptations to anatomical and/or biomechanical alterations throughout life. In a preliminary study in rabbits, differences were observed in the thickness of the layers of articular cartilage in control animals at 5 and 6 months (generally considered adults for this purpose). This study aimed to describe sagittally sectioned condylar cartilages stained with Picrosirius-hematoxylin in rabbits at 40 days and 5, 6, 8, 13, and 18 months to determine when histological maturity is reached. At 40 days, 5 layers were seen: fibrous, proliferative, transition, maturation, and hypertrophic. Older animals (5-18 months) lacked the transition layer. Fibrous, proliferative, and hypertrophic regions were considered for morphometric analysis. The thickness of the fibrous region did not change during the analyzed period (p = 0.1899). When proliferative and hypertrophic regions and the total thickness of the cartilage were compared, a difference was detected (p < 0.001). The thickness of the proliferative region was greatest at 40 days and decreased at 5 months; however, it increased at 6 months, when it was significantly thicker than at 5, 8, 13, and 18 months. Both the hypertrophic region and the total thickness were thickest at 40 days, intermediate at 5, 6, and 8 months, and thinnest at 13 and 18 months. In summary, our data suggest a physiological period of increased cartilage growth at 6 months. Additionally, rabbits at this age should be avoided in experiments involving condylar cartilage. Finally, 13-month-old rabbits have reached histological maturity of the condylar cartilage.

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

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 alpha1 and alpha2 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 alpha1 and alpha2 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 alpha2-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 alpha1 and alpha2 subunits are modulated by forces promoted by the MPA, participating of the biological response to this therapy.

Mandibular repositioning modulates IGFBP-3, -4, -5 and -6 expression in the mandibular condylar cartilage of young rats.

Functional orthopedic appliances correct dental malocclusion partially by exerting indirect mechanical stimulus on the condylar cartilage, modulating growth and the adaptation of orofacial structures. However, the exact nature of the biological responses to this therapy is not well understood. Insulin-like growth factors I and II (IGF-I and II) are important local factors during growth and differentiation in the condylar cartilage [D. Hajjar, M.F. Santos and E.T. Kimura, Propulsive appliance stimulates the synthesis of insulin-like growth factors I and II in the mandibular condylar cartilage of young rats, Arch. Oral Biol. 48 (2003), 635-642]. The bioefficacy of IGFs at the cellular level is modulated by IGF binding proteins (IGFBP). The aim of this study was to verify the mRNA and protein expression of IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 in the condylar cartilage of young male Wistar rats that used a mandibular propulsive appliance for 3, 9, 15, 20, 30 or 35 days. For this purpose, sagittal sections of decalcified and paraffin-embedded condyles were submitted to immunohistochemistry and the condylar cartilage to RT-PCR. The control group showed a gradual increase in the protein expression of all IGFBPs, except IGFBP-4. Following use of the appliance, IGFBP-3 and IGFBP-6 expression decreased in the early stage of the treatment. At 20 days of treatment there was a decline in the IGFs and IGFBP-3, IGFBP-4 and IGFBP-5 expression and at 30 days there was a peak in the IGFs and all IGFBPs expression except for IGFBP-3 where the peak was observed in the control animals. The expression patterns of all IGFBPs in the condylar cartilage were similar. The modulation of IGFBP-3, -4, -5 and -6 expression in the condylar cartilage in response to the propulsive appliance suggests that those peptides are involved in the mandibular adaptation during this therapy.

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat.

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.

[The mandibular condyle. The prenatal evolutionary process]. / Condyle mandibulaire. Processus évolutif prénatal.

Fourteen prenatal maxillary condyles were studied from 66 to 305 mm. vertex-coxa, making slides with different orientation and using several staining techniques. The most significative characteristics were described in the different ages. The lining connective tissue increased its thickness but not change appreciably its structure. The mesenchymal layer, generating cells, maintained its character, but its columnar invaginations observed early were getting more tense as its age increased. The cartilage without typical isogeneic groups and few intercellular substance, decreased as substituted by osteoid trabeculae observing few osteoclasts. In the subchondral area, the difference between the columnar mesenchyma and haematogenous narrow was outstanding.

Relative growth of the condylar cartilage of the rat mandibular head during postnatal life.

In a study using morphometric techniques, the changes in area undergone by the various constituting layers of the condylar cartilage of the mandibular head of normal rats were examined, the cell/cartilage matrix ratios were determined, and the relative growth of the various cartilage layers was analyzed during postnatal life. The approximate 15% decrease of the cell contingent, the approximate 48% increase in cartilage matrix, the abrupt changes in the descriptive curve around the 15th to 20th d of postnatal life and the analysis of the relative growth pattern of the fibrous, embryogenic, cartilage, and hypertrophic cartilage layers were related to the functional adaptations which regulate the growth of the mandible condyle by a direct or indirect action on the temporal-mandibular articulation system.