An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.     

Histochemical localisation of versican, aggrecan and hyaluronan in the developing condylar cartilage of the fetal rat mandible

We investigated the histochemical localisation of versican, aggrecan and hyaluronan in the developing condylar cartilage of the fetal rat mandible at d 15–17 of gestation. At d 15 of gestation, immunostaining for versican was detected in the anlage of the future condylar process (condylar anlage), although the staining intensity showed a considerable regional variation. At d 16 of gestation, a metachromatically stained matrix firstly appeared in the condylar anlage. Aggrecan, hyaluronan and versican were simultaneously detected in this newly formed condylar cartilage. At d 17 of gestation, immunostaining for versican became restricted to the perichondrium and was barely detected in the cartilage. Colocalisation of versican and aggrecan was also seen in the cranial base cartilage at d 14 of gestation. These results indicate that although versican is replaced by aggrecan during the transition from prechondrogenic tissue to cartilage, both molecules were temporally colocalised in the newly formed cartilage. A hyaluronan-rich, low-versican area was identified in the posterior end of the condylar anlage during d 15–17 of gestation. The existence of this area is a unique structural feature of the developing condylar cartilage.     

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

Immunohistochemistry of collagen types II and X, and enzyme-histochemistry of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible

We investigated the immunohistochemical localisation of types II and X collagen as well as the cytochemical localisation of alkaline phosphatase in the developing condylar cartilage of the fetal mouse mandible on d 14–16 of pregnancy. On d 14 of pregnancy, although no immunostaining for types II and X collagen was observed, alkaline phosphatase activity was detected in all cells in the anlage of the future condylar process. On d 15 of pregnancy, immunostaining for both collagen types was simultaneously detected in the primarily formed condylar cartilage. Alkaline phosphatase activity was also detected in chondrocytes at this stage. By d 16 of pregnancy, the hypertrophic cell zone rapidly increased in size. These findings strongly support a periosteal origin for the condylar cartilage of the fetal mouse mandible, and show that progenitor cells for condylar cartilage rapidly or directly differentiate into hypertrophic chondrocytes.     

Vitality of chondrocytes in the mandibular condyle as revealed by collagen formation. An autoradiographic study with 3H-proline

Histological and autoradiographic studies using ³H-proline indicate that cartilaginous tissue in the mandibular condyle maintains morphologic and metabolic characteristics of an embryonic type of tissue. Cartilage cells in the condyle lack the specific arrangement and cellular homogeneity characteristic of more differentiated endochondral growth sites. Through dedifferentiation many chondrocytes in the mandibular condyle appear to outlive the hypoxic conditions that are reported to prevail within the mineralizing zone. Chondrocytes in this zone reveal only a minimal amount of ³H-proline uptake in comparison with the cells in the chondroblastic and premineralizing zones. The dedifferentiated chondrocytes appear to redifferentiate into more specialized cells, possibly osteoprogenitor cells, as they reveal a significant increase in ³H-proline incorporation in the vicinity of the ossifying front. These observations on proline metabolism support the concept that calcification in the condylar cartilage is not necessarily accompanied by degeneration and death of the chondrocytes.     

Tissue engineering the mandibular condyle

Tissue engineering provides the revolutionary possibility for curing temporomandibular joint (TMJ) disorders. Although characterization of the mandibular condyle has been extensively studied, tissue engineering of the mandibular condyle is still in an inchoate stage. The purpose of this review is to provide a summary of advances relevant to tissue engineering of mandibular cartilage and bone, and to serve as a reference for future research in this field. A concise anatomical overview of the mandibular condyle is provided, and the structure and function of the mandibular condyle are reviewed, including the cell types, extracellular matrix (ECM) composition, and biomechanical properties. Collagens and proteoglycans are distributed heterogeneously (topographically and zonally). The complexity of collagen types (including types I, II, III, and X) and cell types (including fibroblast-like cells, mesenchymal cells, and differentiated chondrocytes) indicates that mandibular cartilage is an intermediate between fibrocartilage and hyaline cartilage. The fibrocartilaginous fibrous zone at the surface is separated from hyaline-like mature and hypertrophic zones below by a thin and highly cellular proliferative zone. Mechanically, the mandibular condylar cartilage is anisotropic under tension (stiffer anteroposteriorly) and heterogeneous under compression (anterior region stiffer than posterior). Tissue engineering of mandibular condylar cartilage and bone is reviewed, consisting of cell culture, growth factors, scaffolds, and bioreactors. Ideal engineered constructs for mandibular condyle regeneration must involve two distinct yet integrated stratified layers in a single osteochondral construct to meet the different demands for the regeneration of cartilage and bone tissues. We conclude this review with a brief discussion of tissue engineering strategies, along with future directions for tissue engineering the mandibular condyle.     

An immunohistochemical study on the localization of type II collagen in the developing mouse mandibular condyle

The present chronological investigation assessed the distribution of type II collagen expression in the developing mouse mandibular condyle using immunohistochemical staining with respect to the anatomy of the anlage of the mandibular condyle, the histological characteristics of which were disclosed in our previous investigation. We analyzed fetuses, obtained by cross breeding of ICR strain mice, between 14.0 and 19.0 days post-conception (dpc) and pups on 1, 3, and 5 days post-natal (dpn) using immunohistochemical staining with 2 anti-type II collagen antibodies. The expression of type II collagen was first detected at 15.0 dpc in the lower part of the hypertrophic chondrocyte zone; thereafter, this type II collagen-positive layer was expanded and intensified (P1 layer). At 17.0 dpc, we identified a type II collagen-negative layer (N layer) around the P1 layer and we also identified another newly formed type II collagen-positive layer (P, layer) on the outer surface of the N layer. The most typical and conspicuous 3-layered distribution was observed at 1 dpn; thereafter, there was a reduction in the intensity of expression, and with it, the demarcation between the layers was weakened by 5 dpn. The P1 layer was derived from the central region of the core cell aggregate of the anlage of the mandibular condyle and participated in endochondral bone formation. The N layer was derived from the fringe of the core cell aggregate of the anlage, formed the bone collar at the side of the condyle by intramembranous bone formation, and showed a high level of proliferative activity at the vault. The P2 layer was formed from the outgrowth of the N layer, and could be considered as the secondary cartilage. The intensive expression of type II collagen from 17.0 dpc to 3 dpn was detected in the fibrous sheath covering the condylar head, which is derived from the peripheral cell aggregate of the anlage. Since its expression in the fibrous sheath was not detected in the neighboring section in the absence of hyaluronidase digestion, some changes in the extracellular matrix of the fibrous sheath appear to participate in the generation of the lower joint space. The results of the present investigation indicate that further studies are required to fully characterize the development of the mouse mandibular condyle.     

Presence of chondroid bone on rat mandibular condylar cartilage

Summary Immunohistochemical techniques were used to examine the locations of type I and type II collagens in the the most anterior and the posterosuperior regions of the mandibular condylar cartilages of young and adult rats. Large ovoid and polygonal cells, which were morphologically different from any of the neighboring cells, e.g., mature or hypertrophied chondrocytes, osteoblasts, or fibroblasts, were observed at the most anterior margin of the young and adult condylar cartilages. In the extracellular matrix (ECM) of this area, an eosinophilic staining pattern similar to that in bone matrix was observed, while the peripheral ECM showed basophilic staining and very weak reactivity to Alcian blue. Immunohistochemical examination showed that the ECM was stained heavily and diffusely for type I collagen, while a staining for type II collagen was faint and limited to the peripheral ECM. Two different staining patterns for type II collagen could be recognized in the ECM: one pattern revealed a very faint and diffuse reaction while the other showed a weak rim-like reaction. These staining patterns were markedly different from those in the cartilaginous cell layer in the posterosuperior area of the condylar secondary cartilage, which showed faint staining for type I collagen and a much more intense staining for type II collagen. These observations reveal the presence of chondroid bone, a tissue intermediate between bone and cartilage tissues, in the mandibular condylar cartilage, and suggest the possibility of osteogenic transdifferentiation of mature chondrocytes.     

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.     

Mechanical stimulation of TMJ condylar chondrocytes encapsulated in PEG hydrogels

Temporomandibular joint (TMJ) disorders are most commonly associated with TMJ disc dislocation and osteoarthritis, which can cause erosion of the articular cartilage on the head of the mandibular condyle. There has been little attention focused on treating the damaged condylar cartilage. Therefore, the overall goal of this research is to create a tissue engineering therapy for resurfacing the damaged cartilage of the condylar process with healthy living tissue. Initially, bovine condylar cartilage explants were studied to understand the tissue structure, composition, and gene expression of the native tissue. The cell response of isolated condylar chondrocytes encapsulated in photopolymerized poly(ethylene glycol) hydrogels as a tissue engineering scaffold was examined in the presence and absence of dynamic loading for up to three days of culture. Condylar chondrocyte viability was maintained within the PEG hydrogel constructs over the culture period and loading conditions. Cell response was examined through real-time RTPCR for collagen types I and II and aggrecan, nitric oxide production, cell proliferation, proteoglycan (PG) synthesis, and spatial distribution of extracellular matrix through histology. This study demonstrates that PEG hydrogel constructs are suitable for condylar chondrocyte encapsulation in the absence of loading. However, dynamic compressive strains resulted in inhibition of gene expression, cell proliferation, and PG synthesis.     

The Mandibular Cartilage Metabolism is Altered by Damaged Subchondral Bone from Traumatic Impact Loading

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a degenerative disease caused by excessive external loading. Recently, it was reported that the damage in the mineralized subchondral bone caused by traumatic impact-loading is responsible for the initiation and progression of cartilage degeneration. Thus far, we have hypothesized that cytokines released from damaged subchondral bone from impact-loading affect the cartilage catabolism under pathological conditions. An impactor of 200 gw was dropped onto the top of a porcine mandibular condyle. After organ culture for 2 days, we investigated the association between the subchondral bone and cartilage using histological and biochemical experiments. The impact-loading induced the expression of IL-1β immunohistochemically and prominently up-regulated IL-1α and IL-1β mRNA levels in subchondral bone. We confirmed a significant decrease in type II collagen and aggrecan mRNA expressions in chondrocytes by co-culture with osteoblasts after impact-loading, and significant increase in mRNA and protein expressions of IL-1β in subchondral osteoblasts from impact-loaded subchondral bone. The mRNA expressions of type II collagen, aggrecan, and type X collagen in chondrocytes were decreased significantly by the co-culture with osteoblasts pre-treated by IL-1β, -6, and TNF-α. Among them, osteoblasts pre-treated by IL-1β affected chondrocytes most strongly. It was also shown that IL-1β-treated osteoblasts enhanced the MMP-1 mRNA level most markedly in chondrocytes among the four cytokines. These results suggest that the TMJ subjected to impact-loading can increase directly IL-1β synthesis in the subchondral region, subsequently altering the metabolism of adjacent cartilage and may eventually resulting in the onset and progression of TMJ-OA.     

Postnatal Development of Type II Collagen and Aggrecan mRNA Expression in a Rabbit Craniomandibular Joint

The aim of this study was to investigate the postnatal growth changes in the condyle and disc of the rabbit craniomandibular joint (CMJ). Forty‐eight rabbits from newborn to an age of 120 days were divided into eight groups, and chondrocytic differentiation and function were evaluated within the CMJ by in situ hybridization of type II collagen and aggrecan mRNA. The morphology of the posterior band and the bilaminar zone were similar in the newborn group and were composed primarily of mesenchymal cells and capillaries. After weaning, mastication loading induced the differentiation of mesenchymal cells, which was accompanied by structural differentiation between the posterior band and the bilaminar zone. Aggrecan first appeared in the posterior band of the disc at 30 days postnatally, when the rabbits began to masticate solid food. Type II collagen emerged in the disc at the age of 45 days. Both genes coexpressed in the deeper half of the proliferative layer, the whole hypertrophic layer, and the mineralized layer of the condylar cartilage and staining intensity increased with age. The coexpression of aggrecan and Type II collagen indicates the maturation of chondrocyte differentiation in the disc and condyle, which contributes to the biomechanical characteristics of the CMJ that resist functional stimulation. Anat Rec 293:1574–1580, 2010. © 2010 Wiley‐Liss, Inc.     

Chondrocyte terminal differentiation, apoptosis, and type X collagen expression are downregulated by parathyroid hormone

Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis through the endocrine system. Parathyroid hormone-related peptide (PTHrP) is a heterogeneous polypeptide with sequence homology to PTH in its first 13 amino acid residues. Both bind and activate a common receptor, the type 1 PTH/PTHrP receptor (PTH1R). Activation of this G-protein-coupled receptor by PTHrP has been shown to regulate chondrogenesis in a manner that attenuates chondrocyte hypertrophy. Here, we report the dose-response (10(-7) to 10(-15) M) effects of PTH on chondrogenesis using an avian sternal organ culture model. PTH increased cartilaginous tissue length and downregulated the deposition of type X collagen and its mRNA expression. In addition, PTH increased chondrocyte cell diameter in prehypertrophic and proliferative regions while decreasing chondrocyte apoptosis in the hypertrophic zone. In conclusion, these experiments demonstrate that PTH regulates cartilage growth, chondrocytic apoptosis, deposition of type X collagen protein, and expression of type X collagen mRNA. Type X collagen mRNA expression was downregulated by PTH in this organ culture model, but cell size, another marker for terminal differentiation, increased.     

Mandibular growth retardation in corticosteroid-treated juvenile mice.

Juvenile mice were treated for up to eight weeks with weekly doses of a synthetic analogue of cortisol:triamcinolone hexacetonide. The mandibular condylar cartilage was studied histologically and histochemically at regular intervals. Morphometric measurements were performed along the mandibular posterior vertical dimension (condylar process and ramus). By the second injection significant morphological changes were noted in the condylar cartilage, followed by retardation of bone growth. The most distinctive feature in the cartilage of triamcinolone-treated mice was a marked increase in the dimension of its mineralized zone concomitant with a significant increase in the number of hypertrophic chondrocytes. The role of condylar cartilage in mandibular growth is discussed.