Picrotoxin, a gamma-aminobutyric acid-receptor antagonist, retards craniofacial development in the weaning rat: I. Effect on mandibular bone growth

The in vivo effects of elevated doses of picrotoxin, a gamma-aminobutyric acid (GABA) A-receptor antagonist, were studied in the skulls of weaning rats. Twenty-one-day-old male rats were treated daily with 2 mg/kg of pictroxin for a period of 3 weeks. This study revealed that chronic administration of the agent caused a reduction in bone formation in various growth sites in the skull along with a significant decrease in the calcium content and alkaline phosphatase activity in the mandible. Serum levels of calcium were unchanged, but the activity of alkaline phosphatase decreased. The decrease in bone alkaline phosphatase was accompanied by structural changes in the developing mandible. The latter was manifested by qualitative changes in the structure of ossification sites, in the appearance of the osteoblasts, and in the pattern of bone mineralization. These findings indicate that picrotoxin affects the normal growth of the craniofacial skeleton in an intact growing animal, probably because of central changes in GABA-ergic control on motor function along with possible alteration in corticosteroid secretion.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

Time-Dependent Effects of a Functional'-Type Orthopedic Appliance on the Rat Mandible Growth

The modus operandi and the time-dependent variations in the effects of the LSU-activator, an orthopedic appliance currently used in human orthodontic therapy, was experimentally analyzed in growing rats. This appliance causes a forward positioning of the lower jaw and a restriction of mandibular motility. After a 4-week treatment, the following changes were observed:

(i) the growth rate of the condylar cartilage was accelerated, this growth-promoting effect being more pronounced when the LSU-activator was worn during the animal's rest span.

(ii) the direction of condylar growth became more backward-oriented; no significant difference between day and night treatment, i.e. during the rest and activity spans could be detected;

(iii) the supplementary lengthening of the mandible was greater in rats treated during rest than in rats treated during waking and

(iv) the number of serial sarcomeres in the lateral pterygoid muscle was smaller. This growth retardation of the muscle was greater in rest-time than in waking-time treated individuals.

The LSU-type activator's action implies a two-step effect: during the time of wearing the appliance, the more forward positioning of the mandible causes a reduced growth of the lateral pterygoid muscle; during the time the LSU-type activator is not worn, the mandible is functioning in a more forward position in such a way that it stimulates the growth rate of the condylar cartilage and the subperiosteal ossification of the posterior border of the ramus. It is therefore essential, for a few hours every day, that the mandible be allowed to move freely from the appliance in a more forward position.     

Load application induces changes in the expression levels of Sox-9, FGFR-3 and VEGF in condylar chondrocytes

Experimental and clinical observations have proven the modulatory effects of mechanical loading on the development and maintenance of cartilage architecture. Here we examined the involvement of Sox-9, FGFR-3 and VEGF (pivotal factors controlling cartilage development and growth) in the mechano-transduction pathway of mandibular condylar cartilage by changing the dynamics of the transmitted load via changes in food hardness. To this end, condyle cartilage tissue of rats fed with hard or soft food was analyzed immunohistochemically at various time points. Our findings demonstrate that different mechanical loading conditions in condylar chondrocytes trigger differentiation-/maturation-related processes by affecting the expression levels of these factors, ultimately influencing condylar cartilage growth.     

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

The effect of dietary consistency on gross and histologic morphology in the craniofacial region of young rats

Three groups of weanling rats and three groups of juvenile rats were fed diets which differed in physical consistency for periods of 5 and 8 weeks, respectively. In both the weanling and juvenile rats, one group was fed a soft diet, a second group was fed a hard diet, and a third group was initially fed the soft diet and then was switched to the hard diet for the remainder of the experimental period. The effects of these differences in dietary consistency on gross and histologic morphology of the craniofacial region were examined. Significant differences were found in the dimensions and morphology of the condyle and condylar cartilage as a result of the differences in dietary consistency in both the weanling and juvenile groups. Soft-diet rats generally had smaller condyles and a thinner layer of condylar cartilage than either hard-diet or soft/hard-diet rats. Little change, however, was found in the overall dimensions of the mandible and maxilla in any of the groups of rats.     

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.     

慢性睡眠限制状态下大鼠髁突软骨MMP-1,MMP-13表达的变化

目的:探讨MMP-1,MMP-13在慢性睡眠限制引起大鼠髁突软骨结构变化中的表达变化及作用。方法:180只雄性Wistar大鼠随机分为3组(n=60):慢性睡眠限制组(CSR)、大平台组(LC)、笼养组(CON)。每组根据试验时间不同分别分为3个亚组(n=20):7天(7D)、14天(14D)、21天(21D)组。参考改良多平台法(MMPM)建立大鼠的慢性睡眠限制模型。通过HE染色观察大鼠髁突软骨的结构变化。通过免疫组化和实时定量PCR分别检测MMP-1和MMP-13的蛋白水平及m RNA水平的表达变化。结果:HE染色和扫描电镜结果显示,CSR组的大鼠髁突软骨出现了病理性的改变。与CON和LC组比较,CSR组MMP-1和MMP-13的m RNA转录和蛋白表达水平明显升高(P0.05)。结论:慢性睡眠限制能够引起大鼠颞下颌关节髁突软骨的病理性变化。MMP-1和MMP-13的表达水平的变化可能在大鼠髁突软骨病理性改变中起关键作用。     

Effects of different concentrations of serum on cartilage growth in an organ culture system

Summary The purpose of the present study was to examine the effects of various concentrations of serum on the behavior of neonatal condylar cartilage when cultured in an organ culture system. Mandibular condylar cartilages were obtained from newborn ICR mice, of which the zone of undifferentiated chondroprogenitor cells along with a few layers of young cartilage cells were cultivated at the medium-air interface. The incubation medium included fetal bovine serum at concentrations ranging from 0 to 10%, and the explants were kept in vitro up to 10 d. The serum-free medium maintained the chondrogenic expression, and the overall size of the cartilagenous protion of the explants increased with the decrease of the concentrations of serum in the medium. When explants were labeled with [³H]thymidine and were then processed for autoradiography, the peak of labeling was noticed at 48 h, a feature that recapitulated itself in all cultures (73, 140, 175, 201, and 129 labeled cells per chondroprogenitor zone in explants grown in 0, 1, 2.5, 5, and 10%, respectively). It can be concluded that serum-free medium maintains the chondrogenic phenotype of condylar cartilage in vitro. This study was supported in part by a research grant from the Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig-Stockheim, Federal Republic of Germany.     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Os penis of the rat. IV. The proximal growth cartilage

Histomorphological and histochemical aspects of the proximal cartilage of os penis and its surrounding perichondrium in 60 rats aged between 1 and 100 days are described. Comparisons at 11-14 days with the mandibular condylar cartilage reveal a slight difference in their general morphological composition. The developmental changes which take place in the os penis cartilage reveal histomorphologic events, some of which may be brought into agreement with previous observations of patterns of transformations of the bone. Observations on an age-dependent morphological appearance of the area adjacent to the proximal surface of the cartilage suggest certain agreements between the mandibular angular cartilage and the os penis cartilage. The study of phosphomonoesterases in the os penis cartilage and its perichondrium reveals significant, unexplained differences in the distribution of alkaline phosphatase between this cartilage and the mandibular condylar cartilage.     

Light- and electron-microscopic observations on the mandibular condylar cartilages in growing rats on a low-calcium diet.

In order to obtain more insight into the physiologic mechanism of endochondral ossification, histological changes occurring in the mandibular condylar cartilage of growing rats fed on a low-calcium diet were investigated by light and electron microscopy. Twenty-three-day-old rats were fed on a normal diet or a low-calcium diet for 8 weeks. For the histological observations the mandibular condyles were dissected from each animal at 1, 2, 4, 5 and 8 weeks after the initiation of the experiment. Histological changes occurring in the mandibular condylar cartilages of the rats fed on a low-calcium diet were as follows: (1) narrow proliferative and mature cell zones and a wide hypertrophic cell zone, (2) inhibition of development of cell organelles in the mature chondrocytes, (3) decrease in dead cells in the proliferative zone, (4) decrease in glycogen accumulation in the chondrocytes and (5) inhibition of calcification in the extracellular matrix of the hypertrophic cell zone. Additionally at the end of the experimental period, the following findings were observed: (1) appearance of small light cells in the mature cell zone and the hypertrophic cell zone and (2) decrease in proteoglycan granules and appearance of large collagen fibrils in the pericellular region of the hypertrophic cell zone.     

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.     

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     

Collagen I and II mRNA distribution in the rat temporomandibular joint region during growth

The distribution of type I and II collagen synthesis in the temporomandibular joint (TMJ) area of 1- to 28-day-old rats was studied after hybridization with probes to pro alpha1(I) and pro alpha1(II) collagen mRNA, and stain intensity through the various cartilaginous zones of the mandibular condyle and other areas of TMJ was assessed. The pro alpha(I) collagen mRNA was detected in the perichondrium/periosteum, in the fibrous and undifferentiated cell layers of the mandibular condyle, in the articular disc, and in all bone structures and muscles. The pro alpha1(II) collagen mRNA was found in the condylar cartilage and the articular fossa. Intensity in the condyle was highest in the chondroblastic layer and decreased towards the lower hypertrophic layer. In the condylar cartilage of the 21- to 28-day-old rats the chondroblastic cell zone was relatively narrow compared with the younger animals, whereas the reverse seems to be the case in the cartilage of the articular fossa. Changes in the pro alpha1(II) collagen mRNA were observed in the osseochondral junction area of the primary spongiosa, in that at the age of 5 days intense staining was found, whereas no staining was observed by 14 days. In the mineralizing zone, however, the majority of osteoblastic cells gave a positive signal with the pro alpha1(I) collagen probe. In conclusion, type II collagen synthesis of the mandibular condyle is restricted to its upper area. This differs from the long bone epiphyseal plate, where this type of collagen is produced virtually throughout the cartilage. Type II collagen synthesis of the fossal cartilage seems to increase as a function of age.     

Histomorphology of the mandibular condylar cartilage in greater galagos (Otolemur spp.)

The functional morphology of the primate craniomandibular complex and temporomandibular joint (TMJ) components is frequently discussed in terms of gross skeletal structure. At the histomorphologic level, however, the TMJ has only been studied in Old World anthropoids. The present study is designed to describe the microanatomy of the condylar cartilage of the TMJ in two closely related species of greater galago: the exudativorous Otolemur crassicaudatus and the frugivorous O. garnettii. TMJs with intact joint capsules were harvested from adult, cadaveric specimens of these species (four O. crassicaudatus and five O. garnettii). The samples were decalcified, processed for paraffin sectioning, and sectioned at 10-18 microm in the coronal plane. The samples were then stained with hematoxylin/eosin, Gomori trichrome, and Alcian blue, and examined with a photomicroscope. Generally, condylar cartilage in O. crassicaudatus was thickest both laterally and centrally, while O. garnettii had the relatively thickest cartilage laterally. Both species displayed a superficial articular zone, a middle proliferative zone, and a deeply located hypertrophic zone in the condylar cartilage. O. crassicaudatus typically had the greatest cell density in each of these zones. In addition, O. crassicaudatus had focal concentrations of Alcian blue laterally and centrally, while O. garnettii had the greatest reactivity in the central portion only. These results suggest that O. crassicaudatus may be specialized to resist greater compressive force at the TMJ condylar cartilage in specific regions of the mandibular condyle.     

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.     

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.     

Phosphomonoesterases in growth cartilages of the rat

Summary Epiphyseal plate cartilage, epiphyseal cartilage, synchondroseal cartilage and mandibular condylar cartilage were studied morphologically and histochemically in 14 days old rats. Ordinary decalcified paraffin sections were stained with hematoxylin & eosin, van Giesons connective tissue stain, or toluidine blue, and used for morphological studies of the different cartilaginous structures. Undecalcified cryostat sections were used for demonstration of acid and alkaline phosphatase. The enzyme activity was tested for at regular intervals during incubation from 15 sec to 120 min.The morphologic study revealed that a marked similarity of construction exists between epiphyseal plate cartilage and synchrondroseal cartilage. The construction of epiphyseal and condylar cartilage differ from that of the other two structures and also differ mutually.With small variations the reaction for both alkaline and acid phosphatase was found to be identical in the zones of erosion, hypertrophy and maturation of the four structures. Intercellularly, acid phosphatase is present in all zones in the synchondroseal and the epiphyseal plate cartilage, while in the epiphyseal and condylar cartilages it is only present in the zones of erosion, hypertrophy and maturation.The identical reaction for acid phosphatase in the epiphyseal and the condylar cartilage is thought, in all likelihood, to be accidental. When kinetic conditions are taken into account, epiphyseal cartilage seems to react like epiphyseal plate and synchondroseal cartilage, while the condylar cartilage takes up an exceptional position among growth cartilages.