Immunohistochemical localization of fibroblast growth factor receptors in the rat mandibular condylar cartilage and tibial cartilage

The fibroblast growth factor receptors (FGFRs), members of the tyrosine-kinase receptor family, are known to play a crucial role in the growth and development of cartilaginous tissues. The mandibular condylar cartilage has been suggested to have a characteristic growth pattern compared with the tibial growth plate cartilage, e.g., cell alignment, mode of proliferation and differentiation, and response to humoral and mechanical factors. To examine the mRNA expression and localization of fibroblast growth factor receptor (FGFR)-1, -2, and -3 in the condylar and tibial growth plate cartilages, reversed transcribed polymerase chain reaction (RT-PCR) assay and immunohistochemistry were carried out using growing rats. The enzymatically isolated rat condylar and tibial chondrocytes expressed mRNA of aggrecan and type II collagen, which are together known as the major cartilaginous extracellular matrices. Both types of cells expressed mRNA of FGFR-1, -2, and -3 by RT-PCR. In the neonatal rat, immunolocalization of FGFR-1, -2, and -3 was found in the middle of the condylar cartilage, mainly in the hypertrophic zone of the tibial cartilage. At 3 weeks old, the three FGFRs were broadly observed in both cartilages. At 8 weeks old, localization of FGFR-3 was absent in the hypertrophic cell layer of the condyle, whereas it was still broadly observed in the tibial growth plate cartilage. In the same stage, FGFR-1 and FGFR-2 showed similar localization in both cartilages to that at 3 weeks of age. All these observations suggest that FGFRs play an important role in the differential growth pattern of the condylar cartilage. Received: Jan. 14, 1999 / Accepted: March 3, 1999     

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.     

Increased mandibular condylar growth in mice with estrogen receptor beta deficiency

Temporomandibular joint (TMJ) disorders predominantly afflict women of childbearing age, suggesting a role for female hormones in the disease process. In long bones, estrogen acting via estrogen receptor beta (ERβ) inhibits axial skeletal growth in female mice. However, the role of ERβ in the mandibular condyle is largely unknown. We hypothesize that female ERβ‐deficient mice will have increased mandibular condylar growth compared to wild‐type (WT) female mice. This study examined female 7‐day‐old, 49‐day‐old, and 120‐day‐old WT and ERβ knockout (KO) mice. There was a significant increase in mandibular condylar cartilage thickness as a result of an increased number of cells, in the 49‐day‐old and 120‐day‐old female ERβ KO compared with WT controls. Analysis in 49‐day‐old female ERβ KO mice revealed a significant increase in collagen type X, parathyroid hormone–related protein (Pthrp), and osteoprotegerin gene expression and a significant decrease in receptor activator for nuclear factor κ B ligand (Rankl) and Indian hedgehog (Ihh) gene expression, compared with WT controls. Subchondral bone analysis revealed a significant increase in total condylar volume and a decrease in the number of osteoclasts in the 49‐day‐old ERβ KO compared with WT female mice. There was no difference in cell proliferation in condylar cartilage between the genotypes. However, there were differences in the expression of proteins that regulate the cell cycle; we found a decrease in the expression of Tieg1 and p57 in the mandibular condylar cartilage from ERβ KO mice compared with WT mice. Taken together, our results suggest that ERβ deficiency increases condylar growth in female mice by inhibiting the turnover of fibrocartilage. © 2013 American Society for Bone and Mineral Research.     

Biomechanical and biochemical characteristics of the mandibular condylar cartilage

The human masticatory system consists of a mandible which is able to move with respect to the skull at its bilateral temporomandibular joint (TMJ) through contractions of the masticatory muscles. Like other synovial joints, the TMJ is loaded mechanically during function. The articular surface of the mandibular condyle is covered with cartilage that is composed mainly of collagen fibers and proteoglycans. This construction results in a viscoelastic response to loading and enables the cartilage to play an important role as a stress absorber during function. To understand its mechanical functions properly, and to assess its limitations, detailed information about the viscoelastic behavior of the mandibular condylar cartilage is required. The purpose of this paper is to review the fundamental concepts of the biomechanical behavior of the mandibular condylar cartilage. This review consists of four parts. Part 1 is a brief introduction of the structure and function of the mandibular condylar cartilage. In Part 2, the biochemical composition of the mandibular condylar cartilage is summarized. Part 3 explores the biomechanical properties of the mandibular condylar cartilage. Finally, Part 4 relates this behavior to the breakdown mechanism of the mandibular condylar cartilage which is associated with the progression of osteoarthritis in the TMJ.     

The role of mandibular condylar cartilage in articular cartilage repair.

The articular hyaline cartilage of synovial joints has a very limited capacity for repair after injury. In contrast, the mandibular condylar cartilage of the temporomandibular joint possesses as intrinsic potential for regeneration. This study aimed to test the hypothesis that cultured allografts of mandibular condylar cartilage could be used to promote biological repair of injured orthotopic joint surfaces. Using a primate animal model, cultures of mandibular condylar cartilage cells were grafted into surgically created defects in a recipient hyaline cartilage joint surface. Articular wound healing was assessed macroscopically and histologically over a postoperative period of 52 weeks. Mandibular condylar cartilage cells scheduled for allogenous transplantation were initially characterised in vitro. Expansion of primary colonies in organ culture provided the allogenic cellular material for in vivo grafting. Grafting of osteochondral articular wounds with 5-week cultures of mandibular cartilage cells led to wound regeneration with complete reconstitution of articular surface continuity by 52 weeks. There was novel synthesis of cartilage collagens and sulphated glycosaminoglycans within the repair tissue and no evidence of immunological rejection. Healing of grafted defects was thought to occur by a combination of donor cell proliferation and ingress of host mesenchymal cells. In contrast, grafted control wounds underwent largely fibrous repair with incomplete articular regeneration. In conclusion, transplanted allografts of cultured mandibular condylar cartilage appeared to have the ability, in this primate model, to promote cartilaginous repair and regeneration of orthotopic articular wounds.     

Expression of superficial zone protein in mandibular condyle cartilage

OBJECTIVE: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. METHODS: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage. RESULTS: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta. CONCLUSIONS: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.     

Effect of compressive forces on extracellular matrix in rat mandibular condylar cartilage

To reveal the effect of compressive force on the mandibular condylar cartilage, an appliance was set on 8-week-old Wistar rats to load continuous compressive force. Immunohistochemical and histochemical analyses were performed using toluidine blue, antibodies, and probes for aggrecan, hyaluronan, type II collagen, type X collagen, and 5-bromo-2-deoxyuridine (BrdU). Histomorphometry and statistical analyses were also performed for aggrecan and BrdU immunostaining. In toluidine blue staining, tissue metachromasia was observed in the transitional zone and the hypertrophic zone of the mandibular condylar cartilage. In histomorphometry and statistical analysis, thickness of the cartilage decreased significantly in all regions in the 3-day experimental group. However, the thickness of the cartilage in the anterior region showed recovery while it decreased continuously in the posterior region. Distributional changes of aggrecan, hyaluronan, type II collagen, and type X collagen in the experimental groups were similar to those for toluidine blue staining. The immunostained area of all these molecules decreased as a result of the decrement of the cartilage area. However, enhanced immunostaining for aggrecan in the proliferative zone was observed only in the 1-day experimental group. BrdU-positive cells, observed in the proliferating zone and the transitional zone, decreased significantly in the experimental group 3 days after force was applied. These results demonstrate that continuous compressive forces on the mandibular condylar cartilage decrease the proliferation of chondrocytes and the amount of extracellular matrices.     

Matrix synthesis in mandibular condylar cartilage of aging mice

Osteoarthritic (OA) lesions often develop along the articular surface of mandibular condylar cartilage of aging mice. Metabolic activities of chondrocytes in condylar cartilage of newborn to 18-month-old mice were evaluated morphologically and biochemically in vivo and in vitro. In the period between birth and 3 months of age, marked age-dependent reductions in the number of cells per unit area (-56.43%), in DNA (-64.38%) and in glycosaminoglycan (GAG) (-46.32%) contents were observed. In the same time period, protein content remained almost constant. Reduced rates in the incorporation of [3H]thymidine, [3H]leucine and [35S]sulfate between birth and 3 months of age (-81.60%, -25.98% and -67.75%, respectively) were also observed in vitro. Collagen synthesis was similarly reduced, from 38.70% in newborns to 27.86% in 18-month-old animals. Morphology and autoradiography in mandibular cartilage revealed that the reductions of cellularity and of sulfated macromolecular synthesis appeared to be more pronounced along the articular surface. In this region OA lesions were often observed. It may thus be that the combination of reduced sulfated GAGs and the decreased number of cells in this region could result in tissue with architecture that is less suited to withstanding stress and thus more prone to the development of the typical OA lesions that are seen along the articular surfaces of mandibular cartilage in aged mice.     

Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.     

Key points in surgical management of mandibular condylar fractures

Mandibular condylar fractures are among the most common facial fractures and some of the most difficult to manage.Opinions about the management of mandibular condylar fractures differ among surgeons.With the implementation of new technology,an increased understanding of fracture man-agement,and better functional and morphological outcomes reported in the literature,open reduction and internal fixation is becoming many surgeons preferred choice for the treatment of condylar fractures.Because surgical treatment of such fractures is complex,certain factors must be considered to achieve satisfactory outcomes.In this article,we summarise six key points in the management of mandibular condylar fractures:virtual evaluation of condylar fracture,a suitable surgical approach,good reduction,stable internal fixation,repair of the articular disc,and restoration of the mandibular arch width.We believe that these points will help to improve the prognosis of mandibular condyle fractures.     

The mandibular condylar growth center: Separation and characterization of the cellular elements

The developing mandibular condylar growth center consists of a number of histologically distinct cell types. There is an increase in cell volume that takes place from the condylar surface layer through the center of ossification, resulting in a disorganized, irregular cellular pattern. Consequently, the isolation and separation of the different cells from this tissue is difficult using standard methodologies. Countercurrent centrifugal elutriation, whereby cells are separated on the basis of size, was applied to bovine mandibular condylar growth center cells. The cell volume, alkaline phosphatase content, proteoglycan synthesis, and type X collagen synthesis all showed a positive correlation with increasing cell size. The largest cells had characteristics that are consistent with hypertrophic chondrocytes; the smallest cells, on the other hand, had many fibroblastic characteristics.     

Basic fibroblast growth factor stimulates articular cartilage enlargement in young rats in vivo

Basic fibroblast growth factor is a potent mitogen for chondrocytes and influences the protein synthesis of their extracellular matrix in vitro. To investigate its effect on normal developing articular cartilage in vivo, we injected basic fibroblast growth factor once into the knee joints of 4-week-old rats. Phosphate buffered saline was similarly injected into the contralateral knee joints as controls. A histological analysis showed that an injection of basic fibroblast growth factor induced enlargement of the articular cartilage area, especially in the condylar ridge region on day 7 after the injection. The extent of the enlargement was dose-dependent. The localization and amount of proliferating cells in the articular cartilage were analyzed immunohistochemically by the detection of proliferating cell nuclear antigen. On day 1 after the injection, the number of cells positive for proliferating cell nuclear antigen increased significantly in the joints that were injected compared with the controls, and Northern blot analysis showed that the level of messenger RNA for α(II) procollagen was lower in these joints than in the controls. The message in the joints that had been injected increased on day 7, and it was greater than that in the controls. This suggests that proliferating chondrocytes in developing articular cartilage respond to basic fibroblast growth factor with a resulting proliferation of chondrocytes followed by enlargement of cartilage.     

培养软骨、关节软骨、生长板和半月板的超微结构研究

目的 探讨离心管培养软骨作为移植材料的可能性。方法 3周龄兔关节软骨细胞经离心管培养2周形成软骨,另取6周龄兔肱骨头关节软骨、肱骨近端生长板和半月板,并对4种软骨进行透射电镜观察,比较其软骨细胞和细胞外基质结构。结果 离心管培养软骨具有独特的超微结构,与关节软骨和生长板有一定的相似性,而与半月板有明显区别。4种软骨都形成了各自不同的细胞和细胞外基质特征,离心管培养软骨呈现典型软骨细胞凋亡,生长板表现“黑暗”软骨细胞,关节软骨和半月板未见细胞凋亡。结论 离心管培养软骨具有独特的超微结构,可作为关节软骨和生长板的移植物。     

Gender differences in expression of androgen receptor in tibial growth plate and metaphyseal bone of the rat

In this study, we investigate the expression of the androgen receptor (AR) in the tibial growth plate and metaphyseal bone of male and female rats at the mRNA and protein level. Using in situ hybridization and immunohistochemistry, AR mRNA and protein were demonstrated in proliferating and early hypertrophic chondrocytes in the growth plate of 1-, 4-, and 7-week-old male and female rats. Immunostaining for AR was observed both in the nucleus and the cytoplasm. After sexual maturation at 12 and 16 weeks of age, AR expression decreased in both genders and was confined to a small rim of prehypertrophic chondrocytes. In female rats of 40 weeks of age, this expression pattern was still visible. In most age groups there was a tendency toward an increased AR mRNA expression in male vs. female rats except in the 7-week-old animals. At the protein level, sexually maturing 7-week-old male rats demonstrated a higher staining intensity compared to their female counterparts. At this stage, AR staining in the males was mainly confined to the nucleus, whereas in females staining was predominantly found in the cytoplasm. In the tibial metaphysis, AR mRNA was detected in lining cells, osteoblasts, osteocytes, and osteoclasts at all stages of development. At the protein level, a similar expression pattern was observed, except for an absence of immunostaining in the lining cells. The staining was both nuclear and cytoplasmic. In most age groups, mRNA and protein signals were higher in males compared with females. We have demonstrated the presence of AR mRNA and protein in the tibial growth plate and the underlying metaphyseal bone during development of the rat. In male rats, the presence of higher messenger and protein staining intensities, as well as preferential nuclear staining during sexual maturation, suggests that direct actions of androgens in chondrocytes and in bone forming cells may be involved in establishing the gender differences in the skeleton.     

Expression of parathyroid hormone-related protein in rat articular cartilage

Expression and localization of parathyroid hormone-related protein (PTHrP) in rat articular cartilage during fetal and postnatal periods were investigated by immunohistochemistry and in situ hybridization. PHTrP displayed distinct distribution and intensity of staining at different ages. In fetal (18-day-old) and young (3-week-old) rats, articular chondrocytes expressed abundant PTHrP throughout the entire thickness of cartilage. In contrast, in 60-week-old rats, PTHrP was expressed in a few articular chondrocytes of superficial and middle layers. Regulation of PTHrP and PTH/PTHrP receptor mRNA was also studied in cultured rat articular chondrocytes. Northern blot analysis revealed that both transforming growth factor- (TGF-), an important stimulator for chondrocyte proliferation and differentiation, and 10% fetal bovine serum (FBS) stimulated the expression of PTHrP mRNA with down-regulation of its receptor mRNA. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulated the expression of receptor without changes of PTHrP mRNA level. These results suggest that the changes in abundance and localization of PTHrP and its receptor may be directly involved in the cell growth and differentiation of articular cartilage.     

The distribution of S-100 protein positive chondrocytes in the human articular cartilages under aging or diseased conditions]

There have been few reports on the localization of S-100 protein positive chondrocytes in the human articular cartilages. We studied 59 articular cartilages of the aged subjects, 65 osteoarthritic (OA) and 39 rheumatoid arthritic (RA) articular cartilages, to detect the histological localization of S-100 protein using immunoperoxidase method (ABC). The results obtained from normal cartilages demonstrated strongly positive cells representing hypertrophic chondrocytes in the perivascular areas of the neonatal articular cartilage and in the deep zone of the infant articular cartilage. The moderately positive cells were found in the intermediate zone of infant and adult articular cartilages. In mild OA, there were many positive chondrocytes in the intermediate zone with erosion of the surface layer, while in moderate or severe OA many strongly positive cells were found in clusters. The hypertrophic cells in the metaplastic cartilage arising from bone marrow in subjects with severe OA, or from pannus after RA were also positive. It is therefore, suggested that S-100 protein may be correlated with the metabolic activity of the cartilage matrix such as collagen and proteoglycan, as reported in the literature. S-100 protein further, appears to be useful for evaluating histologically the activity of cartilage repair in the pathologic human articular cartilages.