Chondroclasts are mature osteoclasts which are capable of cartilage matrix resorption

Multinucleated cells termed chondroclasts have been observed on the deep surface of resorbed hyaline cartilage but the relationship of these cells to macrophages and osteoclasts and their role in rheumatoid arthritis (RA) and other arthritic conditions is uncertain. Multinucleated cells in RA and other arthritic conditions showing evidence of cartilage resorption were characterised immunohistochemically for expression of macrophage/osteoclast markers. Mature human osteoclasts formed from circulating monocytes and tissue macrophages were cultured for up to 4?days on slices of human cartilage and glycosaminoglycan (GAG) release was measured. Multinucleated cells resorbing unmineralised cartilage were seen in osteoarthritis, RA, septic arthritis, avascular necrosis and in four cases of giant cell tumour of bone that had extended through the subchondral bone plate. Chondroclasts expressed an osteoclast-like phenotype (TRAP+, cathepsin K+, MMP9+, CD14-, HLA-DR-, CD45+, CD51+ and CD68+). Both macrophages and osteoclasts cultured on cartilage released GAG. These findings indicate that chondroclasts have an osteoclast-like phenotype and that mature human osteoclasts are capable of cartilage matrix resorption. Resorption of unmineralised subchondral cartilage by chondroclasts and macrophages can be a feature of joint destruction in inflammatory and non-inflammatory arthropathies as well as inflammatory and neoplastic subchondral bone lesions.     

An ultrastructural study of cartilage resorption at the site of initial endochondral bone formation in the fetal mouse mandibular condyle

An ultrastructural study was undertaken on cartilage resorption at the site of initial endochondral bone formation in the mouse mandibular condyle on d 16 of pregnancy. After resorbing the bone collar, the osteoclasts extended their cell processes into the cartilage matrix and made contact with hypertrophic chondrocytes. By means of cell processes or vacuolar structures, these osteoclasts entrapped the calcified cartilage matrices, cell debris, and the degraded uncalcified cartilage matrices. In particular, since the calcified cartilage matrices were sometimes seen to be disrupted within the osteoclastic vacuolar structures, they were probably disposed of by the osteoclasts. Invading endothelial cells giving rise to capillaries also directly surrounded the degraded uncalcified cartilage matrices and small deposits of cell debris. In addition, hypertrophic chondrocytes that had attached to or were in the process of attaching to the invading osteoclasts often enclosed the small calcified cartilage matrices. Other cell types that have often been reported in other regions of cartilage resorption were not seen at the site of initial endochondral bone formation in this study. Our findings in relation to cartilage resorption may therefore represent unique features of the site of initial endochondral bone formation site. We consider that the manner of cartilage resorption is likely to vary by site, age, and species.     

Chondroclasts and endothelial cells collaborate in the process of cartilage resorption.

The condylar cartilage of the young rat is a major growth center of the craniofacial complex. Differences between the mechanism that results in bone formation from growth centers in the epiphyseal plates of long bones are dictated primarily by the different character of the mineralization of the cartilage. In this ultrastructural study we demonstrate that the terminal hypertrophic chondrocytes undergo apoptosis and disintegration while simultaneously chondroclasts dissolve gaps in the calcified cartilage that engulfs them. The latter are also phagocytizing debris of the chondrocytes. The chondroclasts are intimately followed by tube-forming endothelial cells that most probably coalesce to create extensions of the invading capillaries into the evacuated lacunae. The chondroclasts have ultrastructural features similar to osteoclasts. They are multinucleate, are rich in mitochondria and vacuoles, form clear zones that adhere to the spicules of the calcified cartilage, and also form a sort of ruffled border. The latter is not as elaborate and orderly arranged as is known from osteoclasts. The capillaries that follow orient the stroma cells to the evacuated lacunae and, together with the calcified cartilaginous scaffold, supply the adequate environmental conditions for the stroma cells to differentiate into osteoblasts and to build up trabecular bone.     

Chondroclasts and endothelial cells collaborate in the process of cartilage resorption

The condylar cartilage of the young rat is a major growth center of the craniofacial complex. Differences between the mechanism that results in bone formation from growth centers in the epiphyseal plates of long bones are dictated primarily by the different character of the mineralization of the cartilage. In this ultrastructural study we demonstrate that the terminal hypertrophic chondrocytes undergo apoptosis and disintegration while simultaneously chondroclasts dissolve gaps in the calcified cartilage that engulfs them. The latter are also phagocytizing debris of the chondrocytes. The chondroclasts are intimately followed by tube-forming endothelial cells that most probably coalesce to create extensions of the invading capillaries into the evacuated lacunae. The chondroclasts have ultrastructural features similar to osteoclasts. They are multinucleate, are rich in mitochondria and vacuoles, form clear zones that adhere to the spicules of the calcified cartilage, and also form a sort of ruffled border. The latter is not as elaborate and orderly arranged as is known from osteoclasts. The capillaries that follow orient the stroma cells to the evacuated lacunae and, together with the calcified cartilaginous scaffold, supply the adequate environmental conditions for the stroma cells to differentiate into osteoblasts and to build up trabecular bone. © 1992 Wiley-Liss, Inc.     

An Immunohistochemical Study of Matrix Components in Early‐Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15–16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle‐shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin‐C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin‐C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate‐resistant acid phosphatase (TRAP)‐positive cells were present. Meanwhile, TRAP‐positive, multinucleated chondroclasts and flattened, osteoblast‐like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles. Anat Rec, 298:1560–1571, 2015. © 2015 Wiley Periodicals, Inc.     

Transmission and scanning electron microscope studies of calcified cartilage resorption

The authors' previous report (Savostin-Asling and Asling, 1973) demonstrated that Meckel's cartilage is a favorable site for study of calcified cartilage resorption. In the present study the ultrastructural features at this resorption front have been examined by transmission and scanning electron microscopes (19-day rat fetus). Multinucleated giant cells (chondroclasts) dominated the erosion front. The many features which they showed in common with osteoclasts included abundant mitochondria, vacuolation, lysosomes, sparsity of roughsurfaced endoplasmic reticulum, and deep infoldings at loci of contact with calcified matrix. Crumbling of matrix (with mineral crystals penetrating between these foldings) and fragmentation of collagen fibrils were also seen. The propensity of chondroclasts for spanning several opened lacunae provided special opportunity to demonstrate cell surface modifications in presence or absence of matrix contact. Ameboid processes extending into lacunae were seen by both transmission and scanning procedures; they were sometimes tipped with a veil of filamentous processes as small as 0.3 μm in diameter. Most hypertrophic chondrocytes, when released from lacunae, appeared to be disintegrating. However, in accord with previous evidence of their possible merger with chondroclasts (in light microscopic studies) there was also evidence for breakdown of cell walls between a chondroclast and a chondrocyte in intimate contact, with possibility of cytoplasmic continuity.     

Transmission and scanning electron microscope studies of calcified cartilage resorption.

The authors' previous report (Savostin-Asling and Asling, '73) demonstrated that Meckel's carilage is a favorable site for study of calcified cartilage resorption. In the present study the ultrastructural features at this resorption front have been examined by transmission and scanning electron microscopes (19-day rat retus). Multinucleated giant cells chondroclasts) dominated the erosion front. The many features which they showed in common with osteoclasts included abundant mitochondria, vacuolation, lysonsomes, sparsity of rough-sufaced endoplasmic reticulum, and deep infoldings at loci of contact with calcified matrix. Crumbling of matrix (with mineral crystals penetrating between these foldings) and fragmentation of collagen fibrils were also seen. The propensity of chondroclasts for spanning several opened lacunae provided special opportunity to demonstrate cell surface modifications in presence or absence of matrix contact. Amebiod processes extending into lacunae were seen by both transmission and scanning procedures; they were sometimes tipped with a veil of filamentous processes as small as 0.3 mum in diameter. Most hypertrophic chondrocytes. when released from lacunae, appeared to be disintegrating. However, in accord with previous evidence of their possible merger with chondroclasts (in light microscopic studies) there was also evidence for breakdown of cell walls between a chondroclast and a chondrocyte in intimate contact, with possibility of cytoplasmic continuity.     

The lead line in bone---a lesion apparently due to chondroclastic indigestion.

The metaphyseal line of increased radiodensity which occurs in lead poisoning was studied in children and young monkeys with lead encephalopathy and in guinea pigs. The histologic lesion consists of impaired resorption of calcified metaphyseal cartilage, depressed bone deposition on cartilaginous surfaces, and the accumulation of numerous multinucleate giant cells, some containing lead inclusions. By electron microscopy, the giant cells appear to be osteoclasts and chondroclasts containing large amounts of mineralized cartilage matrix. We interpret the lead line to be the result of a lead-induced inability of cartilage-resorbing cells to degrade mineralized matrix, with a resultant impairment of metaphyseal cartilage resorption. The radiodensity of the lead line would thus be due to persistent mineralized metaphyseal cartilage and not to a primary osseous change. Some observations on lead inclusions in these cells suggest that the fibrillar component forms before the amorphous part.     

Mandibular coronoid process in parathyroid hormone-related protein-deficient mice shows ectopic cartilage formation accompanied by abnormal bone modeling

Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller, this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller, supporting previous immunohistochemical results.     

The cast of clasts: catabolism and vascular invasion during bone growth,repair, and disease by osteoclasts,chondroclasts, and septoclasts

Three named cell types degrade and remove skeletal tissues during growth, repair, or disease: osteoclasts, chondroclasts, and septoclasts. A fourth type, unnamed and less understood, removes nonmineralized cartilage during development of secondary ossification centers. “Osteoclasts,” best known and studied, are polykaryons formed by fusion of monocyte precursors under the influence of colony stimulating factor 1 (CSF)-1 (M-CSF) and RANKL. They resorb bone during growth, remodeling, repair, and disease. “Chondroclasts,” originally described as highly similar in cytological detail to osteoclasts, reside on and degrade mineralized cartilage. They may be identical to osteoclasts since to date there are no distinguishing markers for them. Because osteoclasts also consume cartilage cores along with bone during growth, the term “chondroclast” might best be reserved for cells attached only to cartilage. “Septoclasts” are less studied and appreciated. They are mononuclear perivascular cells rich in cathepsin B. They extend a cytoplasmic projection with a ruffled membrane and degrade the last transverse septum of hypertrophic cartilage in the growth plate, permitting capillaries to bud into it. To do this, antiangiogenic signals in cartilage must give way to vascular trophic factors, mainly vascular endothelial growth factor (VEGF). The final cell type excavates cartilage canals for vascular invasion of articular cartilage during development of secondary ossification centers. The “clasts” are considered in the context of fracture repair and diseases such as arthritis and tumor metastasis. Many observations support an essential role for hypertrophic chondrocytes in recruiting septoclasts and osteoclasts/chondroclasts by supplying VEGF and RANKL. The intimate relationship between blood vessels and skeletal turnover and repair is also examined.     

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.     

Regenerative Potential of Mandibular Condyle Cartilage and Bone Cells Compared to Costal Cartilage Cells When Seeded in Novel Gelatin Based Hydrogels

The field of temporomandibular joint (TMJ) condyle regeneration is hampered by a limited understanding of the phenotype and regeneration potential of cells in mandibular condyle cartilage. It has been shown that chondrocytes derived from hyaline and costal cartilage exhibit a greater chondro-regenerative potential in vitro than those from mandibular condylar cartilage. However, our recent in vivo studies suggest that mandibular condyle cartilage cells do have the potential for cartilage regeneration in osteochondral defects, but that bone regeneration is inadequate. The objective of this study was to determine the regeneration potential of cartilage and bone cells from goat mandibular condyles in two different photocrosslinkable hydrogel systems, PGH and methacrylated gelatin, compared to the well-studied costal chondrocytes. PGH is composed of methacrylated poly(ethylene glycol), gelatin, and heparin. Histology, biochemistry and unconfined compression testing was performed after 4 weeks of culture. For bone derived cells, histology showed that PGH inhibited mineralization, while gelatin supported it. For chondrocytes, costal chondrocytes had robust glycosaminoglycan (GAG) deposition in both PGH and gelatin, and compression properties on par with native condylar cartilage in gelatin. However, they showed signs of hypertrophy in gelatin but not PGH. Conversely, mandibular condyle cartilage chondrocytes only had high GAG deposition in gelatin but not in PGH. These appeared to remain dormant in PGH. These results show that mandibular condyle cartilage cells do have innate regeneration potential but that they are more sensitive to hydrogel material than costal cartilage cells.

     

Partial inhibition of bone resorption by disodium cromoglycata in a synchronized model of bone remodeling

In a previous study we observed that mast cell degranulation might be associated to bone resorption. To verify this assumption, the efficiency of cromoglycate was assessed on a synchronized sequence of bone remodeling induced in male Wistar rats along the mandibular cortex. After 4 days (time of osteoclastic peak in this model), cromoglycate (15/mg/kg and 30 mg/kg/d per os) decreased the number of degranulating mast cells, especially in the population adjacent to the bone surface. Concomitantly the extent of resorption surface was decreased vs untreated animals. Active osteoclasts were lower whereas the total number of osteoclasts (both active and inactive) was not statistically modified. The mean osteoclast-bone interface was not modified. No dose effect was found. These data indicate that mast cell degranulation is involved in the events leading to osteoclast resorption, presumably in the mechanisms providing osteoclast access to bone surface.     

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.     

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.     

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.     

Immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in Meckel's cartilage compared with developing endochondral bones in mice

We examined the immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14-16 (E14-16) in Meckel's cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckel's cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckel's cartilage, tartrate-resistant acid phosphatase (TRAP)-positive cells appeared on the lateral side of the cartilage. Furthermore, the dental follicle showed moderate immunoreactivity for RANKL and OPG, whereas osteoblasts derived from perichondral cells were immunonegative for RANKL and OPG in that area. On E16, cartilage resorption by TRAP-positive cells had progressed at the differential position, and intensely immunoreactive products of RANKL were overlapped on and found to exist next to TRAP-positive cells in the resorption area. In developing metatarsal tissue, OPG immunoreactivity was intense in periosteal osteoblasts, whereas RANKL was only faintly seen in some of the periosteal cells. In epiphyseal chondrocytes of the developing femur, RANKL immunoreactivity was moderate, and OPG scarcely detected. These results indicate a peculiarity of RANKL and OPG immunolocalization in resorption of Meckel's cartilage. Growth of the incisor teeth may be involved in the time- and position-specific resorption of Meckel's cartilage through local regulation of the RANKL/OPG system in dental follicular cells and periosteal osteoblasts, whereas RANKL and OPG in chondrocytes seem to contribute to resorption through regulation of the chondroclast function.     

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.