Growth regulation of the rat mandibular condyle and femoral head by transforming growth factor-{beta}1, fibroblast growth factor-2 and insulin-like growth factor-I

The mandibular condyle is a major growth site and is known to adapt to functional factors. Numerous studies have been performed on the effects of growth factors on the metabolism of primary cartilages, but only a few investigations have examined their action on primary and secondary cartilages. Therefore, the purpose of this study was to compare the effects of insulin-like growth factor-I (IGF-I), transforming growth factor-beta(1) (TGF-beta(1)), and fibroblast growth factor-2 (FGF-2) on the growth of secondary cartilage from the mandibular condyle and primary cartilage from the femoral head of new-born rats. In addition, synergy between these growth factors was investigated. The level of glycosaminoglycan (GAG) and DNA synthesis was analysed after 5 days in culture with the growth factors. The effects of TGF-beta(1) and FGF-2 on growth, tissue organization, and the GAG and collagen content were also evaluated.The stimulation of cell proliferation by the growth factors was higher in the mandibular condyles than in the femoral heads. The content of the matrix components was reduced more by FGF-2 in the mandibular condyles than in the femoral heads. Both TGF-beta(1) and FGF-2 antagonized the stimulatory effects of IGF-I on GAG synthesis in the two types of cartilage. In contrast, the total growth of mandibular condyles was not affected by TGF-beta(1) and FGF-2 while that of femoral heads was strongly reduced. This was mainly due to the inhibition of chondrocyte hypertrophy. These results show that in spite of the extensive effects of growth factors on the metabolism of mandibular condyles, their dimensional growth was not affected.     

Regulatory effects of FGF-2 on the growth of mandibular condyles and femoral heads from newborn rats

The secondary cartilage of the mandibular condyle is considered to be adaptive to functional factors. In the last decades, growth factors have also been shown to be potent regulators of cartilage metabolism. Moreover, it has been suggested that growth factors may differentially regulate the growth of primary and secondary cartilages. However, only a few studies have made a direct comparison of the effects of growth factors on both cartilages. Therefore, the aim here was to compare the effects of FGF-2 on secondary cartilage of the mandibular condyle and primary cartilage of the femoral head from 4-day-old rats in vitro. Cartilages were cultured for 1, 7 and 14 days with 0 and 100 ng/mL FGF-2. We evaluated the effects of FGF-2 on growth, tissue organisation, DNA and glycosaminoglycan (GAG) synthesis and GAG and collagen content. With FGF-2, the morphology of the mandibular condyles changed and the GAG and collagen contents were reduced. However, the growth of the mandibular condyles was not affected. On the contrary, the growth of the femoral heads was strongly reduced due to an inhibition of chondrocyte hypertrophy. In both cartilages, FGF-2 stimulated DNA synthesis in short-term cultures and reduced it in long-term cultures. In conclusion, FGF-2 had a larger effect on the metabolism of the mandibular condyles as compared to the femoral heads. However, the growth of the femoral heads was strongly reduced while that of the mandibular condyles was not affected.     

Growth stimulation of mandibular condyles and femoral heads of newborn rats by IGF-I

Primary and secondary cartilage differ in embryonic origin and are generally considered to have a different mode of growth. However, few experimental studies exist that directly compare the two types of cartilage and their growth regulation. The regulation of cartilage growth is a complex mechanism involving growth factors like insulin-like growth factor-I (IGF-I). The purpose of this study was to compare the growth of mandibular condyles of 4-day-old rats with that of femoral heads in vitro and to analyze the effects of IGF-I. Explants were cultured for up to 2 weeks with 0, 5, and 25 ng/ml IGF-1. Both, 5 and 25 ng/ml IGF-I significantly stimulated growth of the mandibular condyles while only 25 ng/ml IGF-I stimulated growth of the femoral heads. IGF-I increased glycosaminoglycan synthesis of both condylar and femoral cartilage. However, only the DNA synthesis of the mandibular condyles was significantly increased by IGF-I while that of the femoral heads was not affected. It is concluded that IGF-I stimulates growth of both secondary condylar cartilage and primary femoral cartilage. The mandibular condyle appears to be more sensitive to IGF-I than the femoral head, which may partly be due to the different developmental stage.     

Bone morphogenetic protein 3 expression pattern in rat condylar cartilage, femoral cartilage and mandibular fracture callus

Mandibular condylar cartilage differs from primary cartilage in morphological organization of the chondrocytes and in responses to biomechanical stress and humoral factors. For the first time, we describe the expression of Bmp3 mRNA in relation to types I, II and X collagen mRNA (as determined by in situ hybridization) in chondrocytes of the rat mandibular condylar cartilage, femoral articular cartilage, femoral growth plate cartilage, and temporal cartilage, which transiently appeared in the reparative response stage of mandibular ramus fracture healing. In all cartilages evaluated, Bmp3 was expressed in proliferating chondrocytes that expressed type I collagen in condylar cartilage, articular cartilage, and temporal cartilage appearing during fracture healing. Bmp3 was also found in hypertrophic chondrocytes that expressed type X collagen mRNA in all cartilages evaluated. Furthermore, in remodeling bone, Bmp3 mRNA was strongly expressed in active osteoblast cells in periosteal reaction layers formed after fracture. These findings suggest that Bmp3 expression in a special layer of typical articular cartilage may be regulated by mechanical stress stimulation. We also found that Bmp3 was expressed in the periosteal layers of the bone segments near the fracture site during fracture healing.     

The effects of elevated fibroblast growth factor 23 on mandibular growth in rats

ObjectiveThe aim of this study is to elucidate the local effects of fibroblast growth factor 23 (FGF23) in on mandibular condylar growth in growing rats.DesignGrowing Sprague–Dawley rats received intra-temporomandibular joint injections of phosphate buffer solution (PBS), adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-mediated fibroblast growth factor 23 (Ad-FGF23), which were marked as groups A, B, and C, respectively. In vitro, we treated rat mandibular cartilage chondrocytes with PBS, Ad-GFP, and Ad-FGF23.ResultsThe mandibular condyles in group C grew smaller sizes than those in the other control groups due to significant differences among the experimental and control groups with the value of C–D, Q–R (P ≤ 0.05), accompanied by diminished bone mass of sub-cartilage condyles via micro CT analysis. Histologically, the length of the hypertrophic zone was diminished and was associated with decreasing chondrocyte proliferation in group C. Quantitative real-time PCR indicated significant decreases in the expression of chondrogenesis marker genes, including Type X collagen (Col X) and SRY-type box 9 (Sox 9). Moreover, elevated Ad-FGF23 suppressed chondrocyte proliferation and the expression of the chondrogenic differentiation markers Col X and Sox 9 of in vitro.ConclusionsLocal injection of FGF23 suppressed the development and decreased the bone mass of condyles through the decreasing the formation of condylar cartilage, specifically by regulating condylar cartilage cell viability and chondrogenesis expression.     

Forward mandibular positioning enhances condylar adaptation in adult rats

The aim of this investigation was to assess quantitatively the adaptive changes in the condyles of adult rats to forward mandibular positioning. The level of types II and X collagen expressed in the condyles of adult rats was compared with that formed in response to forward mandibular positioning and the levels of expression were correlated to the amount of bone formed in response to mandibular advancement. Seventy-eight 120-day-old female Sprague-Dawley rats were included in this study. The rats were randomly allocated to six groups. Each group consisted of nine rats with bite-jumping devices and four untreated controls. The animals in each group were sacrificed on days 3, 7, 14, 21, 30, and 60. Immunostaining was used for the detection of types II and X collagen, while Alcian blue-PAS was used to observe the extracellular matrix and new bone formation. The results showed that new cartilage was formed in the posterior condyle. The highest level of expression of types II and X collagen were present on day 21, the amount of increase was 247.99 and 540.08 per cent, respectively. The highest level of new bone formation was measured at day 30 of advancement when the amount of increase in new bone formation was 318.91 per cent. These findings indicate that forward mandibular positioning causes changes in the biophysical environment of the temporomandibular joint (TMJ) of adult rats that leads to condylar adaptation.     

Distribution and characterization of proliferative cells in the rat mandibular condyle during growth

Distribution of proliferative cells and localization of types I and II collagen were examined in the rat mandibular condylar cartilage of 36 long-Evans/Turku strain rats during normal postnatal growth using an immunohistochemical method combined with histomorphometry. There were considerable differences in the thickness of the proliferative cell layer in the condylar head, with most mitoses occurring in the postero-superior area. It was found that the extracellular matrix of the proliferative cells does not stain for type II collagen in 20-day-old and older rats, and that besides the subchondral bone, the strongest intensity for type I collagen stain was always localized in the articular surface of the condylar head. Statistically significant overlapping of the proliferative cell layer and the one secreting type II collagen occurred during the earlier stages of development, particularly in the postero-superior area of the condylar head. As type II collagen is considered to be a marker for identification of typical cartilage cells, the findings indicate that, in addition to undifferentiated cells, a portion of the proliferative cells can be characterized as chondroblasts during the early postnatal period in rats, but not in the later stages of development. The developmental phase of the condylar cartilage should therefore be taken into consideration when the effect of various biomechanical and humoral/hormonal factors on growth of the condylar cartilage is examined.     

Osteoarthritis of the temporomandibular joint: a light microscopic and scanning electron microscopic study of the articular cartilage of the mandibular condyle

The aim of this study was to investigate the organization of collagen fibrils and the histopathologic alterations as well as the morphologic aspects of osteoarthritic articular cartilage of the human mandibular condyle. Nine osteoarthritic condyles, three obtained at necropsy and the other six during surgery, were examined by light microscopy and scanning electron microscopy. Light microscopic observations revealed features of progressive and regressive remodeling and the presence of clefts. Scanning electron microscopic observations showed the presence of thick, coiled fibrils at the joint surface and numerous osmiophilic lipid globules scattered between the collagen fibrils. The collagen fibrils were disordered. It was concluded that collagen fiber network disintegration and fatty degeneration comprise the osteoarthritic changes of the articular cartilage of the human mandibular condyle.     

Expression of extracellular matrix in human mandibular condyle

ObjectivesThe age-related expression of the extracellular matrices in human mandibular condyle was examined.Study designThe distribution patterns of types I to V collagens, laminin, fibronectin, fibronectin receptor, and transforming growth factor β in 34 human mandibular condyles dissected from autopsy specimens were studied by immunohistochemical procedure with special attention on the age-related changes.ResultsType I collagen was detected in the full layer of the condylar cartilage, and a stronger immunoreaction was delineated in the articular and cartilage zone. Types II and III collagen were mainly localized in the fibrocartilage zone. Type IV collagen and laminin were detected not only in the basement membrane of the blood vessels but also in the degenerated lesion where the expression of transforming growth factor β was also detected. Immunostaining of type V collagen and fibronectin was noted in the perichondrocytic area, whereas that of fibronectin receptor was seen in the chondrocytes. In materials from younger cadavers types I, II, IV, and V collagens, fibronectin, its receptor, and laminin showed stronger expression in the degenerative lesions than in the normal portions. In the sections from cadavers over the seventh decade, the immunoreaction of extracellular matrices was weak compared with the younger materials, and no increased reaction of extracellular matrices in the degenerative lesions was detected. In addition, severe osteoarthrosis was frequently seen in the older materials in macroscopic findings.ConclusionsThese results suggest that the expression of extracellular matrices thus seems to be closely related to agingand degenerative changes in the condyle.     

Effects of estrogen on the condylar cartilage of the rat mandible in organ culture.

PURPOSE: The effects of estrogen on bone have been well documented. However, very little is known about the regulatory role of estrogen on cartilage and, in particular, the secondary cartilage of the mandibular condyle. The aims of this study were to determine whether estrogen receptors are present in the condylar cartilage of the rat mandible and to assess the effect of varying 17beta-estradiol (E2) concentrations on the proteoglycan content of this tissue. MATERIALS AND METHODS: Mandibular condyles of 16 female Sprague-Dawley rats were resected. Eighteen of these condyles were divided into three groups and the condylar cartilage was removed and placed in organ culture for 4 days with media containing different concentrations of estrogen: 10(-11) mol/L, 10(-8) mol/L, and 10(-6) mol/L. The cartilage then was analyzed for proteoglycan content along with six specimens not passed through the organ culture. Six intact mandibular condyles also were resected and placed in organ culture with the same varying E2 concentrations, and the condylar cartilage was analyzed for estrogen receptors along with two condyles not passed through the culture system. RESULTS: Estrogen receptors were evenly distributed within the chondroblastic and hypertrophic zones in the control group and the group with 10(-11) mol/L E2. With E2 concentrations of 10(-8) mol/L and 10(-6) mol/L, there was a qualitative decrease in hypertrophic chondroblasts, thickness of the condylar cartilage, and a significant decrease in proteoglycan content. CONCLUSIONS: This study shows the presence of estrogen receptors in the secondary cartilage of the rat mandibular condyle. Estrogen has the potential to cause a decrease in extracellular matrix and thickness of this cartilage.     

Growth and growth pressure of mandibular condylar and some primary cartilages of the rat in vitro

To compare the in vitro development of the secondary cartilage of the mandibular condyle with that of primary cartilages, several cartilaginous explants derived from 4-day-old rats were cultured in a serum-free culture system. The following cartilages were used: the mandibular condylar cartilage, the distal epiphyseal cartilage (including the growth plate) of the third metatarsal, a fragment of costal cartilage (including the osteochondral junction) of the fourth rib, the spheno-occipital synchondrosis and the chondroepiphysis of the femoral head. In addition, with a specially designed, in vitro pressure registration system, the maximal growth pressures for each of the explants, except the femoral head, were determined. The results show an independent growth potential for the primary cartilages of the epiphyseal and costal growth plates with a maximal growth pressure of 9.5 and 7.8 g/mm2, respectively. The primary cartilage of the spheno-occipital synchondrosis, on the other hand, although it possesses an independent growth potential, could exert a maximum growth pressure of only 1.5 g/mm2. The secondary cartilage of the mandibular condyle showed a limited intrinsic growth potential, as well as a low maximal growth pressure (2.6 g/mm2). If calculated per dividing and/or matrix synthesizing cell (cells mainly responsible for the cartilage growth), the cells of the condylar cartilage showed the least growth potency (0.08 mg/cell in comparison to 1.9, 1.5 and 0.3 for epiphyseal, costal, and synchondroseal cartilages, respectively.     

负荷改变对髁突软骨细胞合成蛋白多糖的影响

目的：研究负荷变化对髁突软骨细胞蛋白多糖合成的影响。方法：拔除成年家兔左下磨牙,分别于术后2周、1月和3月取双侧颞颌关节,进行阿辛兰和甲苯胺兰等组织化学染色和免疫组化染色。结果：酸性粘多糖和蛋白多糖术后量均降低,以非拔牙侧更显著,随着缺牙时间的延长这些变化而更显著。结论：异常负荷可导致软骨细胞蛋白多糖合成的减少及其构成的变化,从而使髁突纤维软骨的粘弹性降低。     

Distribution of insulin-like growth factor-I mRNA in the mandibular condyle and rib cartilage of the rat during growth

This study makes a molecular biological comparison of primary and secondary cartilage at an early phase of postnatal development. The distribution of insulin-like growth factor-I (IGF-I) mRNA expression in the mandibular condyle and rib cartilage of 1-28-day-old rats was examined after in situ hybridisation using an oligo probe cocktail for IGF-I mRNA. In the condyle, expression was localised to a narrow strip under the articular layer where the cells are undifferentiated. Essentially, no differences were found in IGF-I synthesis within three samples from the same age group or between different age groups. In rib cartilage, IGF-I mRNA was localised within the germinative, proliferative and early hypertrophic cell layers in 1-28-day-old rats. Again, there were no differences in expression among animals of the same age or as a function of age. This pattern of IGF-I mRNA expression indicates that IGF-I synthesis during growth of the mandibular condylar cartilage is different from that of costal cartilage. The findings shed light on the problem of overgrowth often associated with the use of costochondral grafts to replace defective mandibular condyles.     

Spatial arrangement of collagen fibrils in the articular cartilage of the mandibular condyle: A light microscopic and scanning electron microscopic study

To determine the spatial arrangement of collagen fibrils in articular cartilage of the human mandibular condyle, ten healthy condyles obtained at necropsy were examined by light microscopy and scanning electron microscopy (SEM). Observations using light microscopy showed the existence of four different zones. The organization and alignment of the collagen fibrils were different in every zone and varied from layers to bundles of fibrils running parallel, obliquely, or radially to the articular surface. Observations using scanning electron microscopy revealed a thin, surface layer of disorganized small fibrils with a cotton-wool appearance and a well-organized architecture of collagen fibrils in every zone. It was concluded that the organization of collagen fibrils in articular cartilage shows a three-dimensional network with a special system in every zone.     

The collagen composition of the mandibular joint of the foetal calf

The distribution of genetically distinct collagen molecules in the mandibular joint was investigated. Type II collagen was found only in the articular cartilage. Both the fibrous layer covering the articular cartilage and the articular disc were composed of type I collagen and no type II collagen was detected. The histological appearance of these tissues was consistent with the biochemical findings. Small amounts of type III collagen were associated with the fibrous layer covering the articular cartilage; its function is unknown.     

血管内皮生长因子及其受体在大鼠下颌骨髁突软骨细胞中的表达和意义

目的：研究VEGF及其受体(Fh—1和Flk—1)在大鼠下颌骨髁突软骨细胞中的表达，探讨其对大鼠下颌骨髁突软骨生长发育的影响。方法：用免疫组化方法，对VEGF及其受体(Fh—1和Flk—1)在大鼠下颌骨髁突软骨细胞中的表达进行检测。结果：大鼠下颌骨髁突软骨的增殖层、肥大层、矿化和钙化层均有表达，而纤维层没有表达。结论：大鼠下颌骨髁突软骨的增殖层、肥大层及矿化和钙化层软骨细胞可产生并分泌VEGF及其受体(Fh—1和Flk—1)，VEGF可能在大鼠下颌骨髁突软骨生长发育中发挥作用。     

Bone characteristics in condylar hyperplasia of the temporomandibular joint: a microcomputed tomography,histology, and Raman microspectrometry study

Unilateral condylar hyperplasia (UCH) of the temporomandibular joint is a progressive deformation of the mandibular condyle of unknown origin. UCH is characterized by excessive growth of the condylar head and neck, leading to an increase in size and volume. The aim of this study was to investigate the characteristics of the bone in patients with UCH using microcomputed tomography (micro-CT), histology, and Raman microspectroscopy. The mandibular condyles of six patients with UCH were analysed using micro-CT, histology, and Raman microspectrometry and imaging, and the results were compared with those obtained for a normal control subject. Three-dimensional micro-CT models revealed focal abnormalities of the bone microarchitecture, with foci of osteosclerosis. Histological sections showed that these foci included islands of calcified cartilage matrix with live chondrocytes. Raman analysis revealed that the cartilage matrix was more heavily calcified than the bone matrix and that the cartilage could be identified by the phenylalanine (PHE) band of its matrix, as well as by its glycosaminoglycan (GAG) content. The persistence of foci of live and active chondrocytes within the bone matrix is intriguing and appears to be pathognomonic of UCH. These new findings on UCH could help to determine its pathophysiology and thus prevent this disease, which can lead to major facial deformity.     

Mandibular functional positioning only in vertical dimension contributes to condylar adaptation evidenced by concomitant expressions of L-Sox5 and type II collagen

OBJECTIVE: Concerted expressions of L-Sox5 and type II collagen play an important part in osteogenic transition in epiphyseal cartilage. This study was designed to elucidate the role of mandibular vertical functional positioning in condylar adaptive remodelling by examining L-Sox5 and type II collagen expressions in condylar cartilage. DESIGN: 40 female Sprague-Dawley rats at age of 5 weeks were randomly divided into the experimental (n=20) and control groups (n=20). Bite plates were fitted on the upper posterior teeth of the experimental animals to induce functional repositioning of mandible in vertical dimension. The animals in both experimental and matched control groups were sacrificed on days 3, 6, 9 and 12, respectively. Tissue sections were cut in the sagittal plane through the mandibular condyles and processed with histomorphological examination for cellular response and immunohistochemical test for expressions of L-Sox5 and type II collagen. Quantitative assessment was conducted with computer-assisted imaging system to reveal the correlation between these two factors. RESULTS: (1) Both L-Sox5 and type II collagen were expressed in prechondroblastic cells and chondroblastic cells. (2) When mandible was downward positioned, the amount of L-Sox5 expression was significantly higher by 16.1% (day 9) and 24.2% (day 12) than that of the control (P<0.05); Similarly, type II collagen expression in the experimental group was also significantly stronger by 9.3% (day 9) and 12.3% (day 12) than control group (P<0.05), indicating an enhanced osteogenic transition occurring in condylar cartilage. (3) There was a similarity in temporospatial patterns between the expressions of these two factors, indicating their integral functions in facilitating condylar adaptation. CONCLUSIONS: It is suggested that L-Sox5 plays a key role in adaptive remodelling of condylar cartilage resulting from downward positioning of the mandible. Integration with type II collagen enables L-Sox5 to induce osteogenic transition and consequently to encourage endochondral ossification.