Immunolocalization of matrix proteins in different human cartilage subtypes

Cartilage exerts many functions in different tissues and parts of the body. Specific requirements presumably also account for a specific biochemical composition. In this study, we investigated the presence and distribution pattern of matrix components, in particular collagen types in the major human cartilages (hyaline, fibrous, and elastic cartilage) by histochemical and immunohistochemical means. Macroscopically normal articular cartilages, menisci, disci (lumbar spine), epiglottal, and tracheal tissues were obtained from donors at autopsy. Aurical and nasal cartilages were part of routine biopsy samples from tumor resection specimens. Conventional histology and immunohistochemical stainings with collagen types I, II, III, IV, V, VI, and X and S-100 protein antibodies were performed on paraformaldehyde-fixed and paraffin-embedded specimens. The extracellular matrix is the functional component of all cartilages as indicated by the low cell densities. In particular major scaffold forming collagen types I (in fibrous cartilage) and II (in hyaline and elastic cartilages) as well as collagen type X (in the calcified layer of articular cartilages, the inner part of tracheal clips, and epiglottis cartilage) showed a specific distribution. In contrast, the "minor" collagen types III, V, and VI were found in all, collagen type IV in none of the cartilage subtypes. In this study, we present a biochemical profile of the major cartilage types of the human body which is important for understanding the physiology and the pathophysiology of cartilages.     

Developmental origins and evolution of jaws: new interpretation of "maxillary" and "mandibular"

Cartilage of the vertebrate jaw is derived from cranial neural crest cells that migrate to the first pharyngeal arch and form a dorsal "maxillary" and a ventral "mandibular" condensation. It has been assumed that the former gives rise to palatoquadrate and the latter to Meckel's (mandibular) cartilage. In anamniotes, these condensations were thought to form the framework for the bones of the adult jaw and, in amniotes, appear to prefigure the maxillary and mandibular facial prominences. Here, we directly test the contributions of these neural crest condensations in axolotl and chick embryos, as representatives of anamniote and amniote vertebrate groups, using molecular and morphological markers in combination with vital dye labeling of late-migrating cranial neural crest cells. Surprisingly, we find that both palatoquadrate and Meckel's cartilage derive solely from the ventral "mandibular" condensation. In contrast, the dorsal "maxillary" condensation contributes to trabecular cartilage of the neurocranium and forms part of the frontonasal process but does not contribute to jaw joints as previously assumed. These studies reveal the morphogenetic processes by which cranial neural crest cells within the first arch build the primordia for jaw cartilages and anterior cranium.     

Gross morphology and topographical relationships of the hyobranchial apparatus and laryngeal cartilages in the ostrich (Struthio camelus)

The ostrich hyobranchial apparatus consists of the centrally positioned paraglossalia and basiurohyale and paired caudo‐lateral elements (horns), each consisting of the ceratobranchiale and epibranchiale. The paraglossalia lie within the tongue parenchyma and consist of paired, flat, caudo‐laterally directed cartilages joined rostrally. The basiurohyale forms a single dorso‐ventrally flattened unit composed of an octagonal‐shaped body from which extend rostral (the rostral process) and caudal (the urohyale) projections. The laryngeal skeleton consists of cricoid, procricoid and paired arytenoid cartilages. The large ring‐shaped cricoid cartilage displays a body and paired wings which articulate with each other and with the procricoid. The blunt, ossified, rostral projection of the cricoid and the scalloped nature of the arytenoid cartilages are unique to the ostrich. The procricoid is a single structure which links the paired arytenoids and wings of the cricoid. The hyobranchial apparatus is firmly attached to the tongue parenchyma and to the larynx and proximal trachea. In contrast to previous reports in this species, the horns of the hyobranchial apparatus are not related to the skull. Ossification of the body of the basihyale, the ceratobranchials and the rostral process and body of the cricoid cartilage of the larynx lends stability to these structures.     

Assessment of cartilage invasion in case of laryngeal cancer by means of longitudinal sectioning for histopathology – Clinical implications

AimThe aim of the study was to assess the accuracy of radiological diagnosis of laryngeal cartilage infiltration by histopathological examination of laryngeal specimen after total laryngectomy.BackgroundDespite the development of new medical technologies and significant clinical advances allowing early diagnosis and treatment of laryngeal cancer, mortality is still on the rise. Neoplastic infiltration of the laryngeal cartilages is the most common source of error in the assessment of cancer staging. Furthermore, cartilage invasion is listed as a contraindication to partial surgical techniques as well as radiotherapy.Materials and methodsThe study was carried out on 21 larynges following total laryngectomy. Before taking the decision to perform surgery, high-resolution CT scans were performed in all cases. An extended histopathological examination was conducted using a unique vertical cross-section of the whole larynx.ResultsPathology reported 2 cases of arytenoid cartilage invasion, 5 cases of cricoid cartilage invasion, 12 cases of thyroid cartilage penetration, 1 case of internal cortex invasion and 9 cases of extra-laryngeal spread. CT imaging identified 8 of 13 cases (61.5%) of pathologically proven invasion of thyroid cartilage and only 2 cases (2/9, 22%) of extra-laryngeal spread. According to CT results, arytenoid cartilage invasion was correctly identified in 2 cases, cricoid cartilage invasion in 4 (4/5, 80%). The positive predictive values for thyroid, cricoid and arytenoid cartilage invasion and penetration were 80%, 66.7% and 50%, respectively. In case of pre-laryngeal spread the positive predictive value was 100%.ConclusionDespite increasingly advanced methods involved in the diagnosis of laryngeal cancer, many discrepancies may be observed between the radiological and histopathological assessments. CT imaging has limitations especially in thyroid cartilage penetration and extra-laryngeal spread assessment in advanced laryngeal cancer cases. An extended histopathological examination, involving vertical cross-sections of the whole larynx is a very precise study that allows a precise determination of local cancer staging (T).     

Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl- epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.     

Three‐dimensional reconstruction of the odontophoral cartilages of Caenogastropoda (Mollusca: Gastropoda) using micro‐CT: Morphology and phylogenetic significance

Odontophoral cartilages are located in the molluscan buccal mass and support the movement of the radula during feeding. The structural diversity of odontophoral cartilages is currently known only from limited taxa, but this information is important for interpreting phylogeny and for understanding the biomechanical operation of the buccal mass. Caenogastropods exhibit a wide variety of feeding strategies, but there is little comparative information on cartilage morphology within this group. The morphology of caenogastropod odontophoral cartilages is currently known only from dissection and histology, although preliminary results suggest that they may be structurally diverse. A comparative morphological survey of 18 caenogastropods and three noncaenogastropods has been conducted, sampling most major caenogastropod superfamilies. Three‐dimensional models of the odontophoral cartilages were generated using X‐ray microscopy (micro‐CT) and reconstruction by image segmentation. Considerable morphological diversity of the odontophoral cartilages was found within Caenogastropoda, including the presence of thin cartilaginous appendages, asymmetrically overlapping cartilages, and reflexed cartilage margins. Many basal caenogastropod taxa possess previously unidentified cartilaginous support structures below the radula (subradular cartilages), which may be homologous to the dorsal cartilages of other gastropods. As subradular cartilages were absent in carnivorous caenogastropods, adaptation to trophic specialization is likely. However, incongruence with specific feeding strategies or body size suggests that the morphology of odontophoral cartilages is constrained by phylogeny, representing a new source of morphological characters to improve the phylogenetic resolution of this group. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)-dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of "ghost" cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.     

A unique sound production mechanism in the pipid anuran Xenopus borealis

The totally aquatic pipid frog Xenopus borealis produces long trains of click-like sound at high sound pressure levels (> 105 dB SPL) underwater at night. While X. borealis retains an essentially terrestrial respiratory tract, the larynx is highly modified in two ways. First, the cricoid cartilage is greatly expanded posteriorly to form a large 'box'. Portions of this cricoid box are composed of an unusual elastic cartilage. Second, portions of the arytenoid cartilages are elaborated into calcified rods with disc-like enlargements at their posterior ends. These discs are the only freely moveable components within the larynx–there are no vocal cords. Artificial stimulation of a pair of muscles controlling the discs and discrete lesions that impair their movement demonstrate that motion of the discs is both necessary and sufficient for click production. Unlike all other anurans, X borealis does not use a moving air column in sound production. A possible mechanism of click production involves two steps: (1) at first, the discs are held tighdy apposed in the midline by fluid adhesive forces, and contraction of bipennate muscles is isometric; (2) when the muscle tension exceeds the adhesive force, the discs separate with very high acceleration leaving a vacuum between them. Air rushing into the space at high speed (an implosion) produces the click. The cricoid box shapes the frequency spectrum of the clicks, and opening the box broadens the power spectrum. The power spectrum of clicks produced by males after breathing helium is unchanged.     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

Vertebral column and associated elements in dipnoans and comparison with other fishes: development and homology.

A vertebral column consisting of a persistent notochord and ossified arcocentra is the primitive condition for Gnathostomata; it still persists in primitive actinopterygians and sarcopterygians. Advanced actinopterygians and sarcopterygians develop numerous types of centra that include, among others, the presence of holocentrum, chordacentrum, and autocentrum. The chordacentrum, a mineralization or calcification of the fibrous sheath of the notochord, is only found in actinopterygians, whereas an autocentrum is a synapomorphy of teleosts above Leptolepis coryphaenoides. The chordacentrum, formed by migration of cartilaginous cells from the arches into the fibrous sheath of the notochord and usually covered by a thin calcification, is a unique feature of chondrichthyans. The actinopterygian chordacentrum and the chondrichthyan chordacentrum are not homologous. The postcaudal cartilaginous centrum is only known in postcaudal vertebrae of living dipnoans. The holocentrum is present in certain fossil dipnoans and actinopterygians, where it has been independently acquired. It is formed by proliferation of cartilage cells around the elastica externa of the notochord. These cells later ossify, forming a compact centrum. A vertebral column formed by a persistent notochord without vertebral centra is the primitive pattern for all vertebrates. The formation of centra, which is not homologous among vertebrate groups, is acquired independently in some lineages of placoderms, most advanced actinopterygians, and some dipnoans and rhipidistians. Several series of structures are associated with the vertebral column such as the supraneurals, interhaemals, radials, and ribs. In living dipnoans median neural spine, "supraneural," and dorsal radial result from growth and distal differentiation of one median cartilage into two or three median bones during ontogeny. The median neural spine articulates with the neural arch and fuses with it in the caudal vertebrae early in ontogeny. Two bones differentiate in the anterior abdominal vertebrae, i.e., the proximal neural spine and the distal "supraneural." Three bones differentiate in front of the dorsal fin, i.e., the proximal neural spine, the middle "supraneural", and the distal radial; the same pattern is observed in front of the anal fin (the proximal haemal spine, the middle interhaemal, and the distal radial). Considering that the three dorsal (and also the three ventral) bones originate from growth of only one cartilage, they cannot be serial homologs of the neural spines, or "supraneural." They are linear homologs of the median neural cartilage in living dipnoans. The development of these elements differs within osteichthyans from sarcopterygians to actinopterygians, in which the neural spine originates as a continuation of the basidorsal arcualia and in which the supraneural and radial originate from independent cartilages that appear at different times during early ontogeny. The ribs of living dipnoans are unique in that they are not articulated with parapophyses, like in primitive fossil dipnoans, but a remnant of the ventral arcuale surrounded by a small arcocentrum remains at its base. A true caudal fin is absent in living dipnoans. The postcaudal cartilages extend to the caudal tip of the body separating dorsal and ventral rays (or the camptotrichia). Actinotrichia are present in young dipnoans. They are also known in extant actinistians and actinopterygians. They probably represent the primitive state for teleostomes. In contrast, the camptotrichia are unique for extant dipnoans (and probably Carboniferous and younger dipnoans). Lepidotrichia apparently developed many times among osteichthyans.     

Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage

Introduction

The intra-helical cleavage of type II collagen by proteases, including collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of collagen cleavage generated by collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared.

Methods

Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in type II collagen by the action of cathepsin K and possibly by other proteases, but not by any collagenases studied to date.

Results

In most cases, the staining patterns for collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix.

Conclusions

Cleavage of collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of type II collagen.     

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.     

The true hanging columella: simplified diagnosis and treatment using a modified direct approach

An imbalance between the alar rim and the columella border can be a disturbing aesthetic deformity. If the cause is a pseudohanging columella, the therapy should be directed to the alar rims. When the deformity is a true hanging columella with unusually wide medial crural cartilages, balance can be restored by excising a C-shaped crescent of cartilage from the cranial border of the medial crura of the alar cartilages in a direct approach. This condition was present in approximately 15 percent of the patients reviewed. The treatment of a true hanging columella adds a subtle beneficial enhancement to the results of a rhinoplasty. The authors describe a simplified diagnostic method and present their experience treating the true hanging columella using a modified "direct approach" through a closed endonasal rhinoplasty.     

Primary correction of the unilateral cleft nasal deformity

An 18-year experience with the management of the unilateral cleft nasal deformity in 1200 patients is presented. A primary cleft nasal correction was performed at the time of lip repair in infancy; a secondary rhinoplasty was done in adolescence after nasal growth was complete. The technical details of the authors' primary cleft nasal correction are described. Exposure was obtained through the incisions of the rotation-advancement design. The cartilaginous framework was widely undermined from the skin envelope. The nasal lining was released from the piriform aperture, and a new maxillary platform was created on the cleft side by rotating a "muscular roll" underneath the cleft nasal ala. The alar web was then managed by using a mattress suture running from the web cartilage to the facial musculature. In 60 percent of cases, these maneuvers were sufficient to produce symmetrical dome projection and nostril symmetry. In the other 40 percent, characterized by more severe hypoplasia of the cleft lower lateral cartilage, an inverted U infracartilaginous incision and an alar dome supporting suture (Tajima) to the contralateral upper cartilage were used. Residual dorsal hooding of the lower lateral cartilage was most effectively managed with this suture. This primary approach to the cleft nasal deformity permits more balanced growth and development of the ala and domal complex. Some of the psychological trauma of the early school years may be avoided. Also, because of the early repositioning of the cleft nasal cartilages, the deformity addressed at the time of the adult rhinoplasty is less severe and more amenable to an optimal final result.     

A cadaveric analysis of the ideal costal cartilage graft for Asian rhinoplasty

Augmentation rhinoplasty of the Asian nose may be effectively accomplished with alloplastic materials. However, certain circumstances mandate the use of autologous grafts (e.g., dorsal augmentation that exceeds 8 mm and patient intolerance of alloplastic implants). Septal and auricular cartilages are inadequate for dorsal augmentation of the Asian nose. The use of costal cartilage for autologous augmentation in select Asian patients has proven to be a reliable method in more than 500 operative cases during a 10-year period. This study was designed to evaluate the ideal costal cartilage graft for augmentation rhinoplasty. Forty-two preserved cadavers were studied for the relationship of the individual rib cartilages to the surrounding tissue and for the length and caliber of each costal cartilage. The seventh rib was found to be the ideal rib graft by virtue of its safe location and overall size for grafting. The seventh rib is situated over the abdominal cavity, so the risk of pneumothorax is insignificant. The internal thoracic artery and vein descend in close apposition behind the first to sixth ribs but begin a course medial to the ribs inferior to this point, and therefore vascular injury during seventh-rib harvesting is unknown. The seventh rib also provides the greatest overall available length (90.7 mm, right; 89.6 mm, left) and thickness (17.6 mm, right; 17.5 mm, left). Despite the more conspicuous location of the incision required to harvest the seventh rib, the limited 3-cm incision that is used has healed favorably in almost all cases. The other major drawback for seventh-rib harvesting is the dissection required through the overlying rectus abdominis muscle, but little technical difficulty or postoperative morbidity is added with muscle dissection. The seventh rib is advocated as the ideal choice for augmentation rhinoplasty and potentially other recipient sites.     

Stress relaxation behaviors of articular cartilages in porcine temporomandibular joint

In this study, we tested the compressive stress relaxation behaviors of the mandibular condylar and temporal cartilages in the porcine temporomandibular joint (TMJ). The aim was to determine the quantitative and qualitative similarities and differences of compressive stress relaxation behaviors between the two cartilages. Ten porcine TMJs were used; the articular surface was divided into 5 regions: anterior, central, posterior, lateral and medial. Compressive relaxation test was carried out at a strain level of 5% in each region of the two cartilages. The stress relaxation was monitored over a period of 5 min. In all the regions of the two cartilages, the time-dependent stress relaxation curves showed a marked drop in stress within the initial 10 s, which can be fitted by a standard linear viscoelastic model. The instantaneous moduli in the temporal cartilage were dominantly larger than those in the condylar cartilage, while the condylar cartilage had slightly larger relaxation moduli than the temporal cartilage except for the medial region. The both cartilages showed the regional differences in the compressive stress relaxation behavior, and in the temporal cartilage the lateral and medial regions revealed the largest values for the instantaneous and relaxation moduli. The present results demonstrate that the viscoelastic properties of compressive stress relaxation in both cartilages are region-specific, which might have an important implication for stress distribution and transmission along with the TMJ disc.     

Gene and protein expressions of type I collagen are regulated tissue-specifically in rat hyaline cartilages in vivo

The present study was designed to investigate how rat hyaline cartilages at various sites in vivo express the gene and protein of type I collagen using in situ hybridization and immunohistochemistry. The gene of pro alpha 1(I) collagen was expressed by chondrocytes in articular cartilage, and the protein of type I collagen was identified in the cartilage matrix. In contrast, growth plate cartilage expressed the gene of pro alpha 1(I) collagen, but no protein of type I collagen. Neither gene nor protein of type I collagen was expressed in cartilages of trachea and nasal septum. The present study suggested that expression of type I collagen in hyaline cartilages may be regulated tissue-specifically at gene and/or protein levels.