Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage

Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidin-peroxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.     

Tensile properties of the mandibular condylar cartilage

Mandibular condylar cartilage plays a crucial role in temporomandibular joint (TMJ) function, which includes facilitating articulation with the temporomandibular joint disc and reducing loads on the underlying bone. The cartilage experiences considerable tensile forces due to direct compression and shear. However, only scarce information is available about its tensile properties. The present study aims to quantify the biomechanical characteristics of the mandibular condylar cartilage to aid future three-dimensional finite element modeling and tissue engineering studies. Porcine condylar cartilage was tested under uniaxial tension in two directions, anteroposterior and mediolateral, with three regions per direction. Stress relaxation behavior was modeled using the Kelvin model and a second-order generalized Kelvin model, and collagen fiber orientation was determined by polarized light microscopy. The stress relaxation behavior of the tissue was biexponential in nature. The tissue exhibited greater stiffness in the anteroposterior direction than in the mediolateral direction as reflected by higher Young's (2.4 times), instantaneous (1.9 times), and relaxed (1.9 times) moduli. No significant differences were observed among the regional properties in either direction. The predominantly anteroposterior macroscopic fiber orientation in the fibrous zone of condylar cartilage correlated well with the biomechanical findings. The condylar cartilage appears to be less stiff and less anisotropic under tension than the anatomically and functionally related TMJ disc. The anisotropy of the condylar cartilage, as evidenced by tensile behavior and collagen fiber orientation, suggests that the shear environment of the TMJ exposes the condylar cartilage to predominantly but not exclusively anteroposterior loading.     

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.     

The significance of a differential distribution of alkaline phosphatase in the perichondrium of the mandibular condylar cartilage in the wistar albino rat

Summary The aim of the present investigation has been to further study an incidentally observed rare distribution of alkaline phosphatase in the covering of the mandibular condyle. It was felt that this phenomenon might be related to the necessary interaction between the bony and the cartilaginous condylar head during the transformative growth movements of the condylar process.The study has been based on histomorphological and histochemical observations on frontal and sagittal sections of mandibular condyles from rats between 10 and 21 days of age. As regards the bony condylar head which is oval with its long axis in the antero-posterior direction the observations showed that this structure during growth is transformed in a superior, posterior and medial direction. This involves differential resorption on the surfaces in the anterior part and differential apposition on the surfaces in the posterior part.As regards the cartilaginous condylar head, the observations showed that its shape in the frontal plane changes from triangular in the anterior part to rectangular in the posterior part. Alkaline phosphatase reaction in its perichondrium always reaches a higher level medially than laterally.General observations of perichondrial alkaline phosphatase reaction were applied to the distribution of the enzyme in the perichondrium of the mandibular condyle. These data suggest that as the condylar cartilage grows medially, it becomes narrower anteriorly and broader posteriorly.     

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.     

Dynamic changes in acid mucopolysaccharides during mineralization of the mandibular condylar cartilage

Summary Sequential histochemical changes related to acid mucopolysaccharides (AMPS) were studied in the calcifying cartilage of the mandibular condyle. Non-decalcified, 1 Eponembedded sections were subjected to a variety of histochemical procedures. The results indicate that AMPS are synthesized and secreted mainly by hypertrophic chondrocytes in the premineralizing zone. Within the matrix at the mineralization front the AMPS complexes are apparently degraded by lysosomal enzymes to yield a highly anionic fraction which is maintained in the matrix. This fraction could function as the site for mineralization and cationic dye reaction which allows for histochemical visualization.Supported in part by Grant DE 00163 from the National Institute of Dental Research, U.S.P.H.S.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Mechanical stress promotes matrix synthesis of mandibular condylar cartilage via the RKIP-ERK pathway

Mandibular hypoplasia is a common jaw deformity that affects breathing, occlusal function and facial aesthetics. Stimulating mandibular condylar growing with functional appliances is an ordinary but controversial treatment method in orthodontics. Therefore, it is vital to clarify how functional appliances affect condylar growing. Raf-1 kinase inhibitor protein (RKIP), as an endogenous inhibitory molecule of the ERK signaling, is postulated to involve in stress-induced response to articular cartilage. This study was to reveal the role of RKIP in regulating cartilage matrix synthesis with functional appliance treatment. Here, position rat mandibular forward simulating functional appliance effect to examine the stress-induced modification of mandibular condylar in vivo, meanwhile rat mandibular condylar chondrocytes (Mccs) were subjected to cyclic tensile stress (CTS, 16%, 1 HZ). The results showed that mandibular forward therapy enhanced condylar cartilage growth. The thicknesses of all layers of condylar cartilage were increased significantly. RKIP expression was also increased in the mature cartilage layer. In addition, CTS could enhance extracellular matrix formation and cartilage marker expression (aggrecan and collagen II), which shared a similar expression pattern with RKIP in Mccs. However, CTS induced up-regulation of collagen II and aggrecan was blocked by RKIP knockdown. Nuclear p-ERK, targeting downstream of RKIP, showed a decrease after CTS,which was disappeared in RKIP-knockdown Mccs. Taken together, physiological mechanical stimulation promotes cartilage growth modification by up-regulating RKIP through inhibiting ERK signaling pathway.     

Biomechanical properties of the mandibular condylar cartilage and their relevance to the TMJ disc

Mandibular condylar cartilage plays a crucial role in temporomandibular joint (TMJ) function, which includes facilitating articulation with the TMJ disc, reducing loads on the underlying bone, and contributing to bone remodeling. To improve our understanding of the TMJ function in normal and pathological situations, accurate and validated three-dimensional (3-D) finite element models (FEMs) of the human TMJ may serve as valuable diagnostic tools as well as predictors of thresholds for tissue damage resulting from parafunctional activities and trauma. In this context, development of reliable biomechanical standards for condylar cartilage is crucial. Moreover, biomechanical characteristics of the native tissue are important design parameters for creating functional tissue-engineered replacements. Towards these goals, biomechanical characteristics of the condylar cartilage have been reviewed here, highlighting the structure–function correlations. Structurally, condylar cartilage, like the TMJ disc, exhibits zonal and topographical heterogeneity. Early structural investigations of the condylar cartilage have suggested that the tissue possesses a somewhat transversely isotropic orientation of collagen fibers in the fibrous zone. However, recent tensile and shear evaluations have reported a higher stiffness of the tissue in the anteroposterior direction than in the mediolateral direction, corresponding to an anisotropic fiber orientation comparable to the TMJ disc. In a few investigations, condylar cartilage under compression was found to be stiffer anteriorly than posteriorly. As with the TMJ disc, further compressive characterization is warranted. To draw inferences for human tissue using animal models, establishing stiffness–thickness correlations and regional evaluation of proteoglycan/glycosaminoglycan content may be essential. Efforts directed from the biomechanics community for the characterization of TMJ tissues will facilitate the development of reliable and accurate 3-D FEMs of the human TMJ.     

Picrotoxin, a gamma-aminobutyric acid-receptor antagonist, retards craniofacial development in the weaning rat: II. Effect on mandibular condylar cartilage

The in vivo effects of picrotoxin, a gamma-aminobutyric acid (GABA)-receptor antagonist, were studied in the mandibular condyles of weaning rats. Male rats 21 days old were treated daily with 2 mg/kg of picrotoxin for a period of 3 weeks. This study revealed that chronic administration of the agent caused a reduction in bone formation in various sites in the mandible, along with significant changes in the structure of the condylar cartilage and its ossification front. The length of the chondroblastic zone increased, yet the length of the hypertrophic zone was reduced. The latter phenomenon was manifested by qualitative changes in the overall structure of various cellular zones, in the appearance of the osteoblasts, and in the pattern of cartilage mineralization. The changes in the condylar cartilage cannot be attributed to a direct effect of picrotoxin; in vitro studies indicated no significant change in the incorporation of 3H-thymidine and 35S-sulfate in picrotoxin-treated cultures. These findings indicate that picrotoxin affects the normal growth of the mandible in an intact, growing animal, probably through an indirect route involving neurons in the central nervous system.     

3H]thymidine uptake in cells of rat condylar cartilage

Weanling rats were injected intraperitoneally with [3H]thymidine and sacrificed from 5 min to 20 days later. Their mandibular condylar cartilages were examined histologically, by thin-layer autoradiography, and by using liquid scintillation and microscopic counting methods. Labeled DNA appeared in some of the chondrocytes of the resting zone as early as 10 min postinjection, and reached the proliferative zone by 24 hr and the hypertrophic zone by 4 days. The labeling pattern in the last zone was more disperse, being oriented toward the periphery of the cells as they became hypertrophic. The maximum number of labeled chondrocytes was reached by 2 hr postinjection. These amounted to approximately 11% of the total chondrocyte population, the majority of which were located in the resting zone (73%). It is concluded that, over this period, the mitotic index for these cells is 50-60 per thousand resulting in approximately 100 labeled chondrocytes. In addition, some of the chondroclasts at the erosion front contained labeled DNA as early as 5 min after [3H]thymidine administration. By 10 min, 65% of these cells exhibited one or more labeled nuclei, and the ratio of labeled cells remained high through 20 days. Chondroclasts were seen to contain a diffuse label within their cytoplasm after 5 days. This label was similar to that seen in hypertrophic chondrocytes that had reached the erosion front by that time. Clearly, chondroclasts exhibit nuclear division and do not form from fusion of hypertrophic chondrocytes, although which specific mononuclear cells may act as chondroclast progenitors is not clear. In addition, these multinucleate resorbing cells are capable of ingesting or phagocytizing nuclear remnants from hypertrophic chondrocytes at the eroding face of cartilage.     

Ultrastructural localization of acid phosphatase in cartilage of young mandibular condyles

Summary The fine structural localization of acid phosphatase was studied in cartilage of mandibular condyles of the mouse. Although the final product was found to be deposited within most chondroblasts and chondrocytes, the most abundant precipitate was observed within the hypertrophic chondrocytes in the vicinity of the mineralization front. In these cells, lead phosphate precipitates were noted along the rough endoplasmic reticulum and within lysosome-like bodies. Positive reaction to acid phosphatase was also noticed within vacuoles which were located in the matrix close to the centers of mineralization. It is conceivable that this enzyme is involved in matrix production at one stage of chondrogenesis and in the mineralization process at a later stage.This investigation was supported in part by Grant No. DE 00163 from the National Institute of Dental Research, U.S.P.H.S., and in part by Tufts University Cancer Research Center Institutional Grant IN-23-0.     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

Immunolocalization of vascular endothelial growth factor in rat condylar cartilage during postnatal development

It is well known that angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. To address angiogenesis of endochondral ossification in the condyle, we examined the appearance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in condylar cartilage of the growing rat. The early expression of VEGF at various sites during condylar cartilage development indicates that VEGF plays a role in the regulation of angiogenesis at each site of bone formation. From the findings of Flt-1 immunoreactivity, the VEGF produced by the chondrocytes of the hypertrophic zone should contribute to the promotion of endothelial cell proliferation and to stimulate migration and activation of osteoclasts in condylar cartilage, resulting in the invasion of these cells into the mineralized zone.Junko Aoyama and Eiji Tanaka contributed equally to this work     

Death and proliferation of chondrocytes in the degraded mandibular condylar cartilage of rats induced by experimentally created disordered occlusion

Objective  To investigate the effect of experimentally created disordered occlusion (ECDO) on cell death and proliferation in rat mandibular condylar cartilage. Methods  Sprague–Dawley rats were randomly assigned to experimental and control groups. In the experimental groups, ECDO was created by the dental orthodontic method. By means of histological evaluation, immunohistochemistry and TUNEL staining, we studied the histomorphological changes, the death and proliferation of chondrocytes. Results  Time- and sex-related progressive histologic degradation was observed in the condylar cartilage of ECDO rats, accompanied with diminished chondrocyte proliferation in the female 12-week ECDO subgroup (P < 0.05). An increase in the number of apoptotic chondrocytes was seen in both the female 8- and 12-week ECDO subgroups and in the male ECDO 12-week subgroup (all P < 0.05), but not in the male ECDO 8-week subgroup (P > 0.05). Conclusion  ECDO induces degradation in the rat condylar cartilage accompanied by an increase in chondrocyte death.     

Electron microscopic observations on the fate of hypertrophic chondrocytes in condylar cartilage of rat mandible

The present study focused on the hypertrophic cell zone and the adjacent region of primary spongiosa in the mandibular condylar cartilage in growing rats (3 to 7 weeks old). In this cartilage, chondrocytes were not arranged in columns, and there was no clear distinction between longitudinal and transverse septum. The hypertrophic chondrocytes were not surrounded entirely by calcified matrix, and capillaries were in close contact with cartilage cells. The staining intensity of the pericellular matrix decreased in the lower hypertrophic cell zone in comparison with that in the upper part of the hypertrophic cell zone. Electron microscopic examinations indicated that the lowest hypertrophic cells contained lysosomes and pinocytotic vesicles. Some hypertrophic chondrocytes appeared to have been released from their lacunae and were observed in the region of the primary spongiosa. Hence it is suggested that the lowest hypertrophic chondrocytes in the rat mandibular condyle do not die but are released from their lacunae into the bone marrow. Further study is needed to determine whether or not these cells do indeed become osteoblasts and/or chondroclasts.     

Immunohistochemical localization of phosphatidylcholine in rat mandibular condylar surface and lower joint cavity by cryotechniques

The immunolocalization of phospholipids has not yet been clearly demonstrated in temporomandibular joints (TMJs). We have examined the distribution of one of phospholipids, phosphatidyl-choline (PC), in the rat mandibular condylar surface and lower joint cavity. Some fresh resected TMJs with their disks attached were immediately plunged into isopentane-propane cryogen (-193 degrees C). Cryostat sections were cut, mounted on NH3+-coated slides, and fixed with paraformaldehyde (PF). Cryosections were first immunostained with anti-mouse PC antibody (JE-1). Subsequently, they were labeled with immunogold particles following silver enhancing for light microscopic analyses. Some cryosections were subjected to double immunofluoresecence labeling with anti-fibronectin antibody or hyaluronic acid-binding protein in combination with the anti-PC antibody. As an immunocontrol, other cryosections were pretreated with phospholipase A2 before such immunofluorescence labeling. We have confirmed the presence of PC in the lower joint cavity of rat TMJs as well as on the mandibular condylar surface layer, which was colocalized with hyaluronic acid and fibronectin respectively. However, by treatment with phospholipase A2, such immunolabeling for PC was clearly decreased, showing that the PC is a component in the rat in vivo TMJ. These findings suggest that PC, hyaluronic acid and fibronectin may interact each other in the TMJ articular surface areas to play a functional role for lubrication in TMJ.