Type II collagen pro-alpha-chains containing a Gly574Ser mutation are not incorporated into the cartilage matrix of transgenic mice

The biochemical consequences of a type II procollagen mutation that contained a Gly574Ser amino acid substitution were analyzed in a transgenic mouse strain. The mutation correlated with one previously characterized in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al., 1992), and resulted in a similar shortlimbed phenotype. There were fewer collagen fibrils present in the transgenic cartilage and reduced immunofluorescence of cartilage matrix using a type II collagen antibody. Pepsin-extracted collagen from transgenic mouse embryo cartilage was analyzed electrophoretically and indicated less type II as well as type XI collagen compared to their wild-type littermates. A pulse-chase experiment was performed to evaluate the biosynthesis and fate of type II collagen. Chondrocytes isolated from transgenic tissue synthesized fewer stable molecules, resulting in decreased secretion of the procollagen chains. By amino acid sequence analysis of the type II collagen peptides from cartilage of transgenic mouse embryos, serine was not detected at residue 574, the site mutated in the transgene. Based on sequence data, we believe that the molecules incorporated into collagen fibrils of the extracellular matrix, while fewer in number, were composed of normal alpha 1(II) chains.     

Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene

PURPOSE: To characterize the morphological changes in the eyes of transgenic mice harboring different mutations in type II collagen gene to elucidate the function of this collagen in the eye, and to find out whether these animals could function as models for the human arthro-ophthalmopathies of the Kniest, Stickler and Wagner types. METHODS: Three genetically engineered mouse lines representing two types of mutations in the triple-helical domain of type II collagen and their nontransgenic littermates used as controls were analyzed on day 18.5 embryonic development. After genotyping by polymerase chain reaction (PCR) and Southern hybridization the embryos were prepared for routine histology. Polarization microscopy was done on hyaluronidase-treated sections. RESULTS: Histological analysis revealed several genotype-dependent abnormalities in the eyes of the transgenic mice. Most striking changes were observed in the vitreous architecture; in one line of mice the vitreous was tightly packed in the posterior region of the vitreous space with thick fibrils, empty cavities and dense membrane-like material. The other mutation resulted in reduced filament density of the vitreous. In the most severely affected phenotype the internal limiting membrane was detached from the retinal layers and was markedly thickened, and the posterior lens capsule was thickened. The anterior chamber was shallow or absent in all transgenic lines but was well formed in the normal animals. Changes were also observed in the lens, corneal and scleral structures. CONCLUSIONS: The ocular changes observed in transgenic mice harboring mutations in type II collagen gene show similarities to the human ocular findings in Kniest dysplasia, and in Stickler and Wagner syndromes. We therefore propose that these animals could serve as models for systematic analysis of vitreoretinal degeneration and other abnormalities, as seen in these syndromes.     

Collagen II is essential for the removal of the notochord and the formation of intervertebral discs

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.     

Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.     

Ulcerative colitis--late functional results of ileoanal pouch anastomosis

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.     

Immunohistochemical findings type I and type II collagen in prenatal mouse mandibular condylar cartilage compared with the tibial anlage

In growing animals the mandibular condylar cartilage serves not only as an articular but also as a growth cartilage, yet, condylar cartilage has some characteristic features that are not found in growth cartilage. For example, some reports suggest that type I collagen, which is not seen in the growth plate cartilage of long bones, is present in the extracellular matrix of condylar cartilage postnatally. Here, the condylar and limb bud cartilage of fetal mice was examined. The distribution of type I and type II collagen in condylar cartilage was already different from that in the limb bud at the first appearance of the cartilage. Type I collagen was demonstrated in the extracellular matrix of the condylar cartilage that first appeared on day 15 of gestation. However, the reaction for type II collagen was much weaker than that for type I collagen. On day 18 of gestation, type I collagen was still found throughout the cell layers but became gradually weaker with depth. Type II collagen was limited exclusively to the deeper layers at this stage. These findings are different from those in the limb bud cartilage, indicating a characteristic feature of the cells in the condylar cartilage present from the prenatal period.     

Chondrocyte-specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specific enhancer

Type XI collagen and type II collagen are coexpressed in all cartilage, and both are essential for normal cartilage differentiation and skeletal morphogenesis. This laboratory has recently identified a 48-base pair (bp) enhancer element in the type II collagen gene Col2a1 that contains several HMG-type protein-binding sites and that can direct chondrocyte-specific expression in transient transfection and in transgenic mice. The present study has identified two short chondrocyte-specific enhancer elements within a region in the 5' portion of the type XI collagen gene Col11a2 that has previously been shown to influence chondrocyte-specific expression in transgenic mice. These Col11a2 enhancer elements, like the Col2a1 enhancer, contain several sites with homology to the high mobility group (HMG) protein-binding consensus sequence. In electrophoretic mobility shift assays, the Col11a2 elements formed a DNA-protein complex that was dependent on the presence of the HMG-like sites. It had the same mobility as the complex formed with the Col2a1 48-bp enhancer and appeared to contain the same or similar proteins, including SOX9. The Col11a2 elements directed gene expression in transient transfections of chondrocytes but not fibroblasts, and their activity was abolished by mutation of the HMG-like sites. Ectopically expressed SOX9 activated these enhancers in non-chondrocytic cells, as it also activates the Col2a1 enhancer. Finally, the Col11a2 enhancer elements both directed transgene expression to cartilage in developing mouse embryos. Overall, our results indicate that the two Col11a2 chondrocyte-specific enhancer elements share many similarities with the Col2a1 48-bp enhancer. These similarities suggest the existence of a genetic program designed to coordinately regulate the expression of these and perhaps other genes involved in the chondrocyte differentiation pathway.     

Proteoglycan and collagen of "achondroplastic" (cn/cn) neonatal mouse cartilage

Proteoglycans and collagen were analyzed in epiphyseal cartilage of 5 day old mice homozygous for the recessive gene, achondroplasia, and their normal littermates. No significant differences were observed in either hydroxyproline or hexosamine content. Extractable proteoglycans from control and mutant tissue were found to contain a similar distribution of aggregate and monomer. The component glycosaminoglycans were similar in size and composition. Collagen from both was identified as type II collagen. In addition, the collagen content of hind limb bones of 28 day dwarf mice was found to be similar to control amounts. These findings suggest that dwarfism in the cn/cn mouse is not associated with a defect in any of the major matrix constituents.     

Cartilage fibrils of mammals are biochemically heterogeneous: differential distribution of decorin and collagen IX

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents-decorin and collagen IX-in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.     

The role of type X collagen in endochondral ossification as deduced by Fourier transform infrared microscopy analysis

Type X collagen has been implicated in the morphogenetic events of endochondral ossification (EO), including the calcification of hypertrophic cartilage and trabeculae prior to their replacement by bone and marrow. Recently, transgenic mice which expressed a truncated collagen X protein were reported to exhibit morphologic alterations in all tissues arising through EO. Fourier Transform InfraRed (FTIR) spectroscopy has previously been shown to provide quantitative and qualitative information about the relative amount of mineral and carbonate present, mineral composition, and crystal perfection. To determine the role of collagen X in mineralization, the "quality" of mineral crystals was analyzed in thin sections of calcified cartilage from tibia obtained from several independent transgenic mouse lines showing varying degrees of the mutant phenotype and mice without type X collagen expression, by means of Fourier Transform InfraRed microscopy (FTIRM). In the present paper, the term "mineral quality" is employed to describe crystallinity/crystal maturation, and acid phosphate content. The results indicate significant differences between normal and transgenic mice bone mineral, both in the amount present and the "quality" of the crystals. In contrast, the analysis of the mineral in mice without type X collagen expression was not different from their age/sex-matched controls.     

Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.     

Administration of interleukin 12 in combination with type II collagen induces severe arthritis in DBA/1 mice

The induction of arthritis in DBA/1 mice usually requires immunization with the antigen type II collagen emulsified with Mycobacterium tuberculosis in oil. Here we describe that interleukin 12 (IL-12) can replace mycobacteria and cause severe arthritis of DBA/1 mice when administered in combination with type II collagen. Immunization of DBA/1 mice with type II collagen emulsified in oil alone resulted in a weak immune response, and only a few animals (10-30%) developed arthritis. Administration of IL-12 for 5 days simultaneously with each immunization strongly enhanced the anti-type II collagen immune response. Collagen-specific interferon gamma (IFN-gamma) synthesis by ex vivo activated spleen cells was enhanced 3- to 10-fold. IFN-gamma was almost completely produced by CD4+ T cells. Furthermore, the production of collagen-specific IgG2a and IgG2b antibodies was upregulated 10- to 100-fold. As a consequence, the incidence of arthritis in the group of mice immunized with collagen plus IL-12 was very high (80-100%). The developing arthritis was severe, involving approximately 50% of all limbs with strongly increased footpad thickness in most cases. Furthermore, histological examination revealed massive, mainly polymorphonuclear cell infiltration, synovial hyperplasia, cartilage and bone destruction, as well as new bone formation. In many cases, this resulted in the complete loss of joint structure. Neutralization of IFN-gamma in vivo prevented the development of arthritis in collagen-immunized and IL-12-treated mice. In conclusion, our data show that in vivo administered IL-12 can profoundly upregulate a T helper I-type autoimmune response, resulting in severe joint disease in DBA/1 mice.     

Brain natriuretic peptide enhances the endothelium-independent cAMP and vasorelaxant responses of calcitonin gene-related peptide in rat aorta

The localization of cathepsin K protein in mouse osteoclasts was examined by immunolight and immunoelectron microscopy using the avidin-biotin-peroxidase complex method with anti-cathepsin K (mouse) antibody. With light microscopy, a strong immunoreaction for cathepsin K was found extracellularly along the bone and cartilage resorption lacunae and detected intracellularly in vesicles, granules, and vacuoles throughout the cytoplasm of multinuclear osteoclasts and chondroclasts attached to the surface of the bone or cartilage. Mononuclear cells, probably preosteoclasts, some distance from the bone also contained a few cathepsin K-positive vesicles and granules. Cathepsin K was sometimes found in the cisternal spaces of the rough endoplasmic reticulum and vesicles of the Golgi apparatus with electron microscopy of the basolateral region of the osteoclasts. Cathepsin K-positive vesicles and granules as lysosomal compartments were present in various stages of fusion with vacuoles as endosomal compartments that contained fragmented cathepsin K-negative fibril-like structures. Some of the vacuoles (endolysosomes), which seemed to be formed by this process of fusion, contained cathepsin K-positive vesicles and fibril-like structures that did not show the regular cross striation of type I collagen fibrils. In the apical region of the osteoclasts, cathepsin K-positive vesicles and pits had already fused with or were in the process of fusing with the ampullar extracellular spaces. There were large deposits of cathepsin K on fragmented fibril-like structures without regular cross striation in the extracellular spaces, as well as on and between the cytoplasmic processes of the ruffled border. There were also extensive deposits of cathepsin K on the type I collagen fibrils with cross striation in the bone resorption lacunae. Osteoblasts and osteocytes were negative for cathepsin K. In the immunocytochemical controls, no immunoreaction was found in the osteoclasts or preosteoclasts, or on the collagen fibrils in the resorption lacunae. The results indicate that cathepsin K is produced in mature osteoclasts attached to the bone and secreted into the bone resorption lacunae. The findings suggest that cathepsin K participates in the extracellular degradation of collagen fibrils in the resorption lacunae and in the subsequent degradation of the fragmented fibrils in the endolysosomes. It is also suggested that cathepsin K degrades the organic cartilage matrix.     

Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model

The objective of our study was to evaluate reparative tissues formed in chondral defects in an adult canine model implanted with cultured autologous articular chondrocytes seeded in type I and II collagen GAG matrices. Two defects were produced in the trochlea grooves of the knees of 21 dogs, with cartilage removed down to the tidemark. This study includes the evaluation of 36 defects distributed among five treatment groups: Group A, type II collagen matrix seeded with autologous chondrocytes under a sutured type II collagen flap; Group B, type I collagen matrices seeded with chondrocytes under a sutured fascia flap; Group C, unseeded type I collagen matrix implanted under a sutured fascia flap; Group D, fascia lata flap alone; and Group E, untreated defects. All animals were killed 15 weeks after implantation. Six other defects were created at the time of death and evaluated immediately after production as 'acute defect controls'. In three additional defects, unseeded matrices were sutured to the defect and the knee closed and reopened after 30 min to determine if early displacement of the graft was occurring; these defects served as 'acute implant controls'. The areal percentages of four tissue types in the chondral zone of the original defect were determined histomorphometrically: fibrous tissue (FT); hyaline cartilage (HC); transitional tissue (TT, including fibrocartilage); and articular cartilage (AC). New tissue formed in the remodeling subchondral bone underlying certain defects was also assessed. Bonding of the repair tissue to the subchondral plate and adjacent cartilage, and degradation of the adjacent tissues were evaluated. There were no significant differences in the tissues filling the original defect area of the sites treated with chondrocyte-seeded type I and type II matrices. Most of the tissue in the area of the original defect in all of the groups was FT and TT. The areal percentage of HC plus AC was highest in group E, with little such tissue in the cell-seeded groups, and none in groups C and D. The greatest total amount of reparative tissue, however, was found in the cell-seeded type II matrix group. Moreover, examination of the reparative tissue formed in the subchondral region of defects treated with the chondrocyte-seeded collagen matrices (Groups A and B) demonstrated that the majority of the tissue was positive for type II collagen and stained with safranin O. These results indicate an influence of the exogenous chondrocytes on the process of chondrogenesis in this site. In all groups with implants (A-D), 30(50% of the FT and TT was bonded to the adjacent cartilage. Little of this tissue (6-22%) was attached to the subchondral plate, which was only about 50% intact. Remarkable suture damage was found in sections from each group in which sutures were used. Harvest sites showed no regeneration of normal articular cartilage, 18 weeks after the biopsy procedure. Future studies need to investigate other matrix characteristics, and the effects of cell density and incubation of the seeded sponges prior to implantation on the regenerative response.     

A scanning electron microscopic study of the degenerative cartilage in patellar chondropathy

Patellar chondropathy as cartilage degeneration localized in patellar cartilage in young persons is characterized by cartilaginous changes, such as softening, swelling, and fissuring. With a view to structural characterization of early cartilaginous degeneration before erosion, the morphology of affected cartilage was studied under a scanning electron microscope. The surface network of cartilage constituting fibrils had an edematous change, presenting with fibrillation on the medial facet, whereas many fibrils of the central ridge had a collagen bundle, and fissuring of varying size was observed. It appeared that a mechanical force (shearing) acting on the site of the central ridge was associated with the formation of a collagen bundle and its destruction. On the lateral facet, fibrils were arranged perpendicular to the joint surface; the superficial layer of fibrils was worn by hyper-pressure acting on the lateral facet. On the fractured surface, the coarseness of collagen fibrils showed changes that varied with the site and stage of cartilage degeneration. Frequent changes were signs of fibril loosening (coarsening), such as reduction in fibril density (i.e., edematous change), collagen fibril aggregation, and fissuring, and longitudinal restructuring of fibrils. The patellar cartilage in the patients of this series showed a structure adapted to the mechanical force. The initial structural changes of cartilage consisted of collagen fibril aggregation and reduction in fibril density. These changes give rise to matrix rarefaction, which in turn causes cartilage degeneration to progress. These changes were concurrent in both the superficial and middle layers and were not localized as basal degeneration.     

Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage

We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite.     

Tumours aneurysms

Ultrastructural and cytochemical studies on the remodelling of the rat tracheal cartilage have been carried out. The thickness of the tracheal cartilage was constant, during the observation periods (1 to 54 days after birth). The external perichondrium of the tracheal cartilage consisted of active fibroblasts and intercellular fibrils. The inner part of this perichondrium was a chondrogenic layer, where appositional growth was taking place. On the other hand, the internal perichondrium contained fibroblast-like cells, which were nearly twice as large as the external perichondrial fibroblasts in size and were arranged in three or four layers. The cells had well developed organella and large vacuoles which contained numerous fragments of fibrils and/or glycosaminoglycan. Many cytoplasmic processes protruded to the cartilage matrix, where the intercellular fibrils were particularly irregular in arrangement. Some vacuoles included collagen fibrils. Based on an intense acid phosphatase activity in these vacuoles and other findings, the fibrils were thought to be phagocytosed collagen of the cartilage matrix. An extensive alkaline phosphatase activity was demonstrated on the plasma membrane of fibroblasts and chondroblasts in the external perichondrium. The present investigation revealed distinct functional difference between the external and internal perichondrium of the tracheal cartilage. It is resorbed at the internal perichondrium, while it appositionally grows at the external perichondrium. The fibroblast-like cells of the internal perichondrium play an essential role in resorption of the matrix in cartilage remodelling.     

Differences in submicroscopic structure of the extracellular matrix of canine femoral and tibial condylar articular cartilages as revealed by polarization microscopical analysis

The submicroscopic orientation patterns of sulfated glycosaminoglycan side chains of proteoglycan molecules and collagen fibrils were compared in different extracellular matrix areas of femoral and tibial articular cartilages of young adult beagle dogs using qualitative and quantitative polarization microscopic analytical methods. Paraffin sections were cut perpendicularly to the articular surfaces from the femoral and tibial condyles and stained. Picrosirius red F38 staining combined with an antecedent digestion with testicular hyaluronidase was used to enhance the optical anisotropy of collagen. Birefringence of sulfated glycosaminoglycan molecules was selectively amplified by a combination of carboxymethylation with CH3I and a subsequent staining with toluidine blue. The specimens were analysed in a polarization microscope equipped with compensator plates, and retardation values of birefringence were determined in territorial and interterritorial matrix areas of different zones using monochromatic plane polarized light. It was found that besides some similarities there were significant differences in the submicroscopic organization of extracellular matrix between femoral and tibial articular cartilages. Common structural features of the femoral and tibial cartilages were the sulfated glycosaminoglycans and collagen fibrils which were circularly oriented in the territorial matrix, and these components were longitudinally arranged within the trabeculae of the interterritorial matrix. Furthermore, the territorial matrix was a more densely packed structure than the interterritorial matrix. Our results revealed the following major differences between the two cartilages: The degree of orientation of sulfated glycosaminoglycans was higher in the femoral cartilage matrix areas as compared to the identical structures of the tibial cartilage; the collagen structure was more densely packed in the interterritorial matrix of the superficial and mineralization zones of the femoral cartilage than in the tibial cartilage, and except for the zone of mineralization, the degree of collagen orientation was higher in the territorial matrix of the femoral than the tibial cartilage. These findings suggest that the extracellular matrix of femoral condylar cartilage has a more densely packed molecular structure than the softer tibial cartilage matrix. This structural difference may have an influence on the pathogenesis of diseases involving articular cartilage.     

The primary calcification in bones follows removal of decorin and fusion of collagen fibrils

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.