The os penis of the rat. I. Phosphomonoesterases in its proximal growth cartilage

The distribution of reaction for acid and alkaline phosphatases in the proximal cartilage of the os penis and the mandibular condylar cartilage has been compared. The distribution of acid phosphatase in the two structures seems to be identical, whereas the distribution of alkaline phosphatase in the os penis cartilage seems to differ from that in the mandibular condylar cartilage and, by this, from all other studied growth cartilages.     

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.     

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.     

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.     

Histogenesis of the Os Penis and Os Clitoridis in Rats

The development of the os penis in normal male rats and the os clitoridis in the females treated with testosterone were studied histologically and histochemically. Alcian blue-positive materials, alkaline phosphatase activities and calcium deposit were detected. The os penis of the rats was composed of a proximal segment and a distal one. The proximal segment of the os penis was formed by the fusion of a membrane bone in its distal half and an ossifying hyaline cartilage in its proximal half. The distal segment of the os penis developed as a fibrocartilage bone.
Two steps were demonstrated in the development of the os penis: the formation of the rudiments, and the differentiation of the proximal and distal segments. The treatment of newborn females with testosterone induced an os clitoridis homologous to the proximal segment of the os penis. The development of the os clitoridis was compared with that of the os penis.     

Immunohistochemical studies of the extracellular matrix in the condylar cartilage of the human fetal mandible: collagens and noncollagenous proteins.

This study provides data concerning the cells and their extracellular matrix in prenatal human mandibular condylar cartilage. The latter cartilage represents a secondary type of cartilage since it develops late in the morphogenesis of the craniofacial skeleton. The cartilage of the mandibular condyle is actively involved in endochondral ossification, thus showing all the phases of cartilage growth, maturation, and mineralization that precedes de novo bone formation. The present study focused on the localization and distribution of the major macromolecules that are normally encountered in cartilage and bone, including collagens, proteoglycans, fibronectin, osteonectin, osteocalcin, alkaline phosphatase, and anchorin CII. It became clear that the mineralized zone of the cartilage already contained bone-specific antigens; thus the above zone might serve as an essential propagative predecessor in the ossification process.     

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.     

Spatio-temporal expression patterns of Wnt signaling pathway during the development of temporomandibular condylar cartilage

There is a growing body of evidence supporting the involvement of the Wnt signaling pathway in various aspects of skeletal and joint development; however, it is unclear whether it is involved in the process of temporomandibular joint development. In order to clarify this issue, we examined the spatio-temporal distribution of mRNAs and proteins of the Wnt family during the formation of the mandibular condylar cartilage at the prenatal and postnatal stages. An in situ hybridization test revealed no mRNAs of β-catenin and Axin2 during early mesenchymal condensation; the ligands surveyed in this study (including Wnt-4, 5a, and 9a) were clearly detected at various ranges of expression, mainly in the condylar blastema and later distinct cartilaginous layers. Apart from β-catenin and Axin2, the Wnt family members surveyed in this study, including Lef-1, were found to be immunopositive during early chondrogenesis in the condylar cartilage at E14.5. After distinct chondrocyte layers were identified within the cartilage at E16.5, the expression of the Wnt signaling members was different and mainly restricted to proliferating cells and mineralized hypertrophic chondrocytes. In the adult mandibular condylar cartilage, the Wnt-4 mRNA, as well as the Wnt-4 and Wnt-9a proteins, was not observed. Our findings demonstrated that the Wnt signaling pathway was associated with the development of mandibular condylar cartilage.     

Alkaline phosphatase and phosphoamino acid phosphatases in normal and cancerous tissues of the human larynx

The activities of alkaline phosphatase and phosphoamino acid phosphatases were measured in normal and cancerous regions of the human larynx. For each larynx, alkaline phosphatase and phosphotyrosine phosphatase activities were higher in the tumor than in the corresponding normal tissue. Phosphothreonine and phosphoserine phosphatase activities were relatively low and there were no consistent trends. The increased alkaline phosphatase activity in the tumors supports histological observations that ossification of cartilage seems to occur at the site of invasion; the phosphatase acting on phosphotyrosine could serve as a regulator of cell differentiation during tumorigenesis.     

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.     

Superficial zone protein affects boundary lubrication on the surface of mandibular condylar cartilage

We examined the localization and boundary lubricating function of superficial zone protein (SZP) on the surface of mandibular condylar cartilage. Chondrocytes were separated from the surface layer of mandibular condylar cartilage of 6- to 9-month-old female pigs. A cyclic tensile strain of 7% or 21% cell elongation was applied to the cultured chondrocytes. Gene expression levels of cartilage matrix proteins and secretory phospholipase A₂ (sPLA₂) were quantified by real-time polymerase chain reaction analysis. The friction coefficient of the mandibular condylar surface was measured by a friction tester before and after treatment with 0.1 U/ml sPLA₂. Significantly higher mRNA levels of SZP and type I collagen were found in chondrocytes from the superficial layer than in those in the other layers. The SZP mRNA level was up-regulated by cyclic tensile strain of 7% and 21% cell elongation. Cyclic tensile strain of 21% cell elongation up-regulated the sPLA₂ mRNA level. The friction coefficient of the condylar surface was increased significantly by treatment with sPLA₂. The removal of SZP from the surface layer of mandibular condylar cartilage by sPLA₂ resulted in a significant increase in the friction coefficient on the surface of articular cartilage.     

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.     

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick

Summary The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.The proliferating osteogenic and chondrogenic cells of the chick embryo have been classified and compared histochemically. Collagen synthesis was found to be high in the osteogenic cells and acid mucopolysaccharide and mucoprotein synthesis high in the chondrogenic cells.It has been postulated that the morphogenetic switch from osteogenesis to adventitious chondrogenesis most probably involves a change in the rate of collagen and acid mucopolysaccharide synthesis by the germinal cells of the membrane bones.     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

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.     

Abnormal development of the os penis in male mice treated neonatally with tamoxifen

The os penis of male C57BL/Tw mice given 5 daily injections of 100 micrograms tamoxifen (Tx) starting on the day of birth (day 0) was examined at ages of 5-60 days; the bones of males given Tx injections for 5 days starting at 0-10 days and of those given neonatal injections of 100 micrograms clomiphene or nafoxidine were examined at 60 days. In the control males given the vehicle alone, the proximal segment of the os penis, composed of a compact cell mass found at day 0, developed at 5 days into the membrane bone with bone marrow and hyaline cartilage; the distal segment, composed of mesenchymatous cells until 10 days, developed at 30 days into fibrocartilage characterized by a distribution of type I collagen. By contrast, in Tx mice, fibrocartilage in the distal segment, and hyaline cartilage characterized by a distribution of type II collagen, and bone marrow in the proximal segment disappeared by 30 days. The maximum area of the proximal and distal segments gradually increased with age in control mice, whereas the proximal segment area remained unchanged in Tx mice. In clomiphene and nafoxidine mice at 60 days, the proximal segment was composed of hyaline cartilage; however, the distal segment lacked fibrocartilage. Hyaline cartilage in the proximal segment and fibrocartilage in the distal segment disappeared in all 60-day-old mice given Tx starting within 5 days. Neonatal castration did not suppress the formation of bone marrow and fibrocartilage in the os penis, though the bone size was smaller than in the intact controls. Formation of spines on the glans penis skin was suppressed by Tx given within 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

Ultrahistochemistry of matrix vesicles in elastic cartilage.

Matrix vesicles in the elastic cartilage of epiglottis were negative for acid phosphatase, alkaline phosphatase, and ATPase. This is in agreement with the very rare occurrence of mineralization of elastic cartilage. Only the lysosomes of the chondrocytes showed a positive reaction for acid phosphatase, and a positive reaction for alkaline phosphatase and ATPase was found in relation to the cells of the perichondrium.     

FGFR2功能增强对小鼠下颌骨髁突发育影响

成纤维细胞生长因子受体2(Fibroblast growth factor receptor, FGFR2)是参与调控骨骼发育的重要分子,在调控软骨内成骨过程中发挥着重要作用。为了探讨FGFR2功能增强对小鼠下颌骨髁突生长发育的影响,文章以FGFR2功能增强型点突变(Fgfr2^+/S252W)小鼠为研究对象,采用番红固绿染色研究Fgfr2^+/S252W小鼠下颌骨髁突不同生长发育阶段的组织形态;利用免疫细胞化学染色和实时荧光定量PCR方法检测X型胶原(Col X)在3周龄小鼠髁突肥大软骨细胞中的表达。结果显示,1周龄、3周龄和6周龄突变型小鼠下颌骨髁突的软骨细胞层宽度都比同窝野生型窄,钙化软骨细胞层退化时间早,骨小梁钙化绿染程度深;Col X在突变型小鼠下颌骨髁突的表达高于同窝野生型小鼠(P<0.001)。结果表明,FGFR2功能增强可导致小鼠下颌骨髁突软骨层组织形态异常,抑制髁突软骨内成骨,从而导致下颌骨髁突发育畸形。