An In Situ Hybridization Study of Perlecan,DMP1, and MEPE in Developing Condylar Cartilage of the Fetal Mouse Mandible and Limb Bud Cartilage

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.Key words: Mandibular condylar cartilage, perlecan, DMP1; MEPE, in situ hybridization     

Structural characterization of the mandibular condyle in human fetuses: light and electron microscopy studies.

Mandibular condyles from 18- to 20-week-old human fetuses were examined in the light and electron microscope with particular attention to intratissue organization and extracellular matrix. In the human fetus the condyle has been divided into five layers: (1) the most superficial, articular layer, (2) chondroprogenitor cell layer, (3) condroblast cell layer, (4) nonmineralized hypertrophic cell layer, and (5) mineralized hypertrophic cell layer. The articular layer is rich in collagen fibers (mostly of the type I collagen), but the cells seldom divide. By contrast, in the chondroprogenitor cell layer and upper part of the chondroblastic cell layer mitosis gives rise to new cells. The matrix in the latter layer is composed of thick banded 'lucent' fibrils in a loose feltwork of granules representing cartilage proteoglycans. The daughter cells in the progenitor cell layer undergo differentiation which is apparently completed along the lower border of the mineralized hypertrophic cell layer--the ossification front. The matrix in the hypertrophic cell layer reveals distinct matrix vesicles that undergo mineralization and subsequently coalesce to form larger sheets of mineralized extracellular matrix. Mineralized cartilage serves as a backbone for new bone formation as marrow-derived osteoblasts and osteoclasts attach to remnants of mineralized cartilage, which enables the turning on of the remodeling cycles involved in new bone formation. It can be concluded that the process of endochondral ossification as has been reported in lower animals is recapitulated in the human fetus, thus the dynamics associated with condylar morphogenesis is maintained through phylogeny.     

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.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.     

A comparison of the immunohistochemical localization of type I and type II collagens in craniofacial cartilages of the rat.

The immunohistochemical localization of types I and II collagen was examined in the following 4 cartilaginous tissues of the rat craniofacial region: the nasal septal cartilage and the spheno-occipital synchondrosis (primary cartilages), and the mandibular condylar cartilage and the cartilage at the intermaxillary suture (secondary cartilages). In both primary cartilages, type II collagen was present in the extracellular matrix (ECM) of the whole cartilaginous area, but type I collagen was completely absent from the ECM. In the secondary cartilages, type I collagen was present throughout the cartilaginous cell layers, and type II collagen was restricted to the ECM of the mature and hypertrophic cell layers. These observations indicate differences in the ECM components between primary and secondary craniofacial cartilages, and that these differences may contribute to their modes of chondrogenesis.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Summary Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.     

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies,including comparisons with developing endochondral bones

The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

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.     

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.     

Matrix metalloproteinases are not essential for aggrecan turnover during normal skeletal growth and development

The growth plate is a transitional region of cartilage and highly diversified chondrocytes that controls long bone formation. The composition of growth plate cartilage changes markedly from the epiphysis to the metaphysis, notably with the loss of type II collagen, concomitant with an increase in MMP-13; type X collagen; and the C-propeptide of type II collagen. In contrast, the fate of aggrecan in the growth plate is not clear: there is biosynthesis and loss of aggrecan from hypertrophic cartilage, but the mechanism of loss is unknown. All matrix metalloproteinases (MMPs) cleave aggrecan between amino acids N341 and F342 in the proteinase-sensitive interglobular domain (IGD), and MMPs in the growth plate are thought to have a role in aggrecanolysis. We have generated mice with aggrecan resistant to proteolysis by MMPs in the IGD and found that the mice develop normally with no skeletal deformities. The mutant mice do not accumulate aggrecan, and there is no significant compensatory proteolysis occurring at alternate sites in the IGD. Our studies reveal that MMP cleavage in this key region is not a predominant mechanism for removing aggrecan from growth plate cartilage.     

Perlecan,the multidomain HS-proteoglycan of basement membranes,is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.     

Studies on hormonal regulation of the growth of the craniofacial skeleton: III. Effects of 24,25 (OH)2D3 on cartilage growth in the mandibular condyle of suckling mice

This study examined the influence of 24,25 (OH)2D3, an active metabolite of vitamin D, on the growth and development of cartilage cells in condylar cartilage of suckling mice. It became evident that when the hormone was administered even at high doses, it did not significantly affect the incorporation of [3H]thymidine, but led to a marked decrease in the number of both chondroblasts and hypertrophic chondrocytes. At the same time, condyle of hormone-treated mice reached an increase in the number of mesenchymelike cells within the chondroprogenitor zone. High values of correlation were noted between the overall dimensions of the condylar cartilage and those of the chondroblastic and hypertrophic zones. The hormone also significantly reduced the degree of matrix metachromasia (indicative of proteoglycan content) and concomitantly altered the mineralization pattern of the cartilaginous matrix. This study indicates that in young animals increased doses of 24,25(OH)2D3 do not affect the proliferative activity of chondroprogenitor cells yet possess an inhibitory effect upon the capacity of the latter cells to differentiate into chondroblasts. The hormone also seems to affect the already differentiated cells--chondroblasts and hypertrophic chondrocytes--both structurally as well as metabolically. In so doing, this metabolite of vitamin D affects the normal process of endochondral bone formation in one of the mandible's main growth sites. Thus, in the developing animal, elevated concentrations of 24,25(OH)2D3 may impair the growing mandible's ability to achieve its normal size and shape.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Expression of collagen type I,II, X and Ki-67 in osteochondroma compared to human growth plate cartilage

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.     

Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.