Non-calcified Cartilage Research Articles

Growth plate abnormalities in a new dwarf mouse model: tich

Growth plate cartilage calcification has been examined in a recently described mouse mutant, tich, which is co-isogenic with the A.TL strain. Long bones were studied from 1-day-old and 1-month-old mice which carried a homozygous recessive gene mutation making them short limbed and dumpy. Specimens were studied by routine histology, scanning electron microscopy and radiography. In 1-day-old tich mice the front of calcified cartilage was recessed behind the advancing periosteum and bone. No similar recess was seen in control mice. At 1 month of age, a number of the long bone growth plates were irregularly thickened, particularly in the central area. This produced a central tongue of non-calcified cartilage (particularly prominent in the proximal tibia) which gave rise to a corresponding pit in the calcified cartilage layer, in macerated specimens. This was accompanied by poor resorption of calcified cartilage. At both ages the presence of the respective defects was radiographically confirmed. At present it is not known whether this is primarily a defect of calcification or resorption but its presence, apparently from a single mutation in a genetically defined mouse strain, makes it a potentially valuable model.

Cartilage: The ‘F’-factor fallacy

It remains common practice to treat skin lesions overlying cartilage with electrons, apparently because of an assumption that radiation absorption qualities of non-calcified cartilage are similar to those of bone. We present data based on tissue composition and calculated mass absorption coefficients, supporting the view that such lesions may safely be treated with superficial X-rays.

Constitutive myc expression impairs hypertrophy and calcification in cartilage

The myc oncogene is expressed by proliferating quail embryo chondrocytes (QEC) grown as adherent cells and is repressed in QEC maintained in suspension culture. To investigate the interference of myc expression during chondrocyte differentiation, QEC were infected with a retrovirus carrying the v-myc oncogene (QEC-v-myc). Uninfected or helper virus-infected QEC were used as control. In adherent culture, QEC-v-myc displayed a chondrocytic phenotype and synthesized type II collagen and Ch21 protein, while control chondrocytes synthesized type I and type II collagen with no Ch21 protein detected as long as the attachment to the plastic was kept. In suspension culture, QEC-v-myc readily aggregated and within 1 week the cell aggregates released small single cells; still they secreted only type II collagen and Ch21 protein. In the same conditions control cell aggregates released hypertrophic chondrocytes producing type II and type X collagens and Ch21 protein. In the appropriate culture conditions, QEC-v-myc reconstituted a tissue defined as nonhypertrophic, noncalcifying cartilage by the high cellularity, the low levels of alkaline phosphatase enzymatic activity, and the absence of type X collagen synthesis and of calcium deposition. We conclude that the constitutive expression of the v-myc oncogene keeps chondrocytes in stage I (active proliferation and synthesis of type II collagen) and prevents these cells from reconstituting hypertrophic calcifying cartilage.

Metabolic activity in the calcified zone of cartilage: Observations on tetracycline labelled articular cartilage in human osteoarthritic hips

The tidemark is a metabolically active zone in the calcified layer of the cartilage in which it is possible to show that there is calcifying activity even over a short period of time using a tetracycline labelling technique. The tidemark slowly advances in the direction of the non-calcified cartilage and analysis of double-tetracycline labelled cartilage shows that this is not an appositional phenomenon like that occurring in bone, but that, where present, several tidemarks can be labelled at the same time. Each tidemark may therefore be metabolically active and it is not just the tidemark adjacent to hyaline cartilage which incorporates calcium.

Cytosolic ionized calcium concentration in isolated chondrocytes from each zone of the growth plate.

In isolated chondrocytes from the growth plate, there is an increase in both the cytosolic ionized calcium concentration and in total cell calcium content as the cells approach the mineralization front. The reserve zone chondrocytes have a cytosolic ionized calcium concentration, [Ca2+]i, of 124 +/- 23 nM and a total cell calcium content, CaT, of 12.8 +/- 6.8 nmol/mg cell protein. Proliferative zone chondrocytes have a [Ca2+]i of 172 +/- 29 nM and a CaT of 16.1 +/- 11.8 nmol/mg cell protein. Hypertrophic zone chondrocytes have a [Ca2+]i of 273 +/- 49 nM and a CaT of 35.8 +/- 16 nmol/mg protein. Chondrocytes isolated from the entire growth plate have a [Ca2+]i of 132 +/- 29 nM and a CaT of 13.2 +/- 2.5 nmol/mg cell protein. Chondrocytes isolated from noncalcifying hyaline cartilage have a [Ca2+]i of 85 +/- 10 nM and a CaT of 11.5 +/- 1.4 nmol/mg cell protein. There appears to be a correlation between intracellular calcium accumulation, an increase in the intracellular ionized calcium concentration, and the process of cartilage matrix mineralization.

A mathematical modelling study of epiphyseal cartilage calcification

Recent studies in this laboratory have suggested that proteoglycan may function as a Ca ion-exchanger in the calcification of epiphyseal growth plate cartilage. Specifically, it has been proposed that phosphate liberated from hypertrophic chondrocytes may displace calcium ions bound to the anionic groups of proteoglycans, thereby raising the Ca x PO4 activity product above the threshold for precipitation of hydroxyapatite. In order to determine whether this mechanism is quantitatively feasible, a mathematical model of the interaction between Ca, Na, proteoglycan and phosphate has now been developed. This model is based on a general binding theory, and utilizes previously-determined values for the binding constants of the Ca-proteoglycan interaction, inhibition constants for the effect of Na and phosphate on this interaction, and literature values for the concentrations of proteoglycan, Na and Ca in epiphyseal cartilage. Using this approach, it was predicted that the free Ca concentration in epiphyseal cartilage in the absence of phosphate will be 1.55 mM. At 0.7 mM phosphate, the approximate concentration in non-calcified cartilage matrix, the free Ca concentration will be 2.40 mM, corresponding to a Ca x PO4 product of 1.68 (mM)2. In order to achieve a Ca x PO4 product sufficient for spontaneous precipitation of hydroxyapatite [approximately 4.3 (mM)2], a phosphate concentration of approximately 1.40 mM is required. Therefore, calcification of epiphyseal cartilage matrix by the mechanism described above will require an approximate doubling of the phosphate concentration in the pre-calcifying zones, indicating that the release of a fraction of the intracellular phosphate could trigger the calcification process.

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro.

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.

Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA).

Recent immunofluorescent and histochemical data did not detect changes in the concentration of proteoglycans between noncalcified and calcified cartilage in fetal bovine growth plate or metaphyseal bone. These findings were constant, regardless of prior fixation before demineralization with disodium ethylenediaminetetraacetate (EDTA) or prior demineralization before fixation. Previous experience has shown that EDTA can extract proteoglycans from calcified cartilage. With this in mind, we determined the amount of proteoglycan extracted from calcified cartilage in metaphyseal bone and uncalcified growth plate cartilages during decalcification of unfixed fresh tissues with EDTA. To this end, fresh growth plate cartilages and metaphyses were decalcified at 5 degrees C for 48 hours in a buffered solution of EDTA to which several protease inhibitors were added. Under these conditions 20-25% of the total proteoglycan (measured as uronic acid and hexosamine) was extracted from mineralized cartilage but only about 1% from the uncalcified (growth plate) cartilages. Thus, histochemical and immunohistochemical studies appear to be insensitive measures of proteoglycan concentrations in histological sections of mineralized tissue and may not give quantitative information.

C-propeptide of type ii procollagen; a protein associated with cartilage mineralization

A matrix protein has been identified in both calcifying and noncalcifying cartilage during development, and named chondrocalcin. The concentration of this protein is greatly increased in calcifying cartilage and it appears in the matrix when and where mineralization occurs. Amino acid sequencing has recently shown that chondrocalcin is identical to the C-propeptide of type II procollagen. Type II procollagen is a high molecular weight precursor of type II collagen and it is characterized by amino (NH2) and carboxy (C) propeptide extensions. These nonhelical extensions are normally cleaved extracellularly by proteinases to give the collagen molecule. To investigate the synthesis, secretion and matrix distribution of the C-propeptide, particularly during mineralization, this protein has been localized at the EM level with immunogold techniques.

Changes in cartilage proteoglycans associated with calcification.

The purposes of these experiments were to study the biosynthetic and postbiosynthetic relationships between proteoglycans in noncalcified growth cartilage and calcified cartilage in metaphysis from the costochondral junctions of immature rabbits. Based on in vivo experiments in which 35 S-sodium sulfate was injected into rabbits, it is shown that proteoglycans from the hypertrophic region becomes part of the calcified cartilage matrix which is to be incorporated into the metaphysis. The proteoglycan aggregates in the growth apparatus undergo partial disaggregation and degradation. There is approximately a 25% decrease in aggregation from regions of the rib distal to the metaphyseal-growth plate junction (69%) to the region proximal to it (50%). In contrast, in their final state in calcified cartilage, the proteoglycans are more completely disaggregated and the proteoglycans subunits are smaller, as adjudged from gel chromatography. Control experiments indicate that although some artifactual disaggregation is produced by the extraction process, it is not of the same magnitude as that seen in the actual isolation experiments nor are the subunits reduced in size.

Comparative Chondro-Osseous Development and Growth of Marine Turtles

Longitudinal skeletal growth in appendicular long bones of Caretta caretta resembles the patterns in Pseudemys scripta and Carettochelys insculpta. A metaphyseal cone of cartilage becomes transiently isolated through the formation of a subphyseal plate of calcified cartilage which undergoes early peripheral vascular irruption and ossification. The noncalcified epiphyseal cartilage remains thin and avascular. Skeletal growth in Dermochelys coriacea is remarkably divergent from other living chelonians. Primary vascular irruption occurs into the noncalcified hypertrophied cartilage of the metaphysis with rapid extension of vascularity also into the non-hypertrophied epiphyseal cartilage. Endochondral ossification of the metaphysis advances contiguously from the diaphysis, a subphyseal calcified cartilage plate does not form, and there is no isolation of a metaphyseal cartilage cone. The noncalcified epiphyseal cartilage remains thick with transphyseal as well as perichondral vascularization through cartilage canals. The pattern in Dermochelys may be due to very rapid skeletal growth to a large body size. The giant Cretaceous Archelon ischyros has a similar skeletal vascular pattern, whereas the giant Tertiary Stupendemys geographicus resembles normal chelonians. In many respects, skeletal growth of Dermochelys resembles marine mammalian patterns.

Ultrastructural cytochemistry of proteoglycans associated with calcification of shark cartilage.

Proteoglycans (PGs) as well as sulfated glycosaminoglycans (GAGs) are closely associated with cartilage calcification. An inner zone of endoskeletal tesserae of sharks is composed of a unique calcified hyaline cartilage. Initial calcification can be seen in the cartilage close to the inner zone. We have ultrastructurally examined shark, Triakis scyllia, noncalcifying, calcifying, and calcified cartilage using the tannic acid-ferric chloride (TA-Fe), the high iron diamine (HID), and the HID-thiocarbohydrazide-silver proteinate (HID-TCH-SP) methods for localization of sulfated complex carbohydrates. In noncalcifying cartilage, TA-Fe and HID strongly stained matrix granules which were round, ovoid, elongated, or irregularly shaped and presumably represented PG monomers. The size and staining intensity of the reactive matrix granules progressively decreased in calcifying cartilage toward the calcification front of the calcified cartilage. Similarly, a progressive decrease in the size of the HID-TCH-SP stain deposits in the matrix granules was observed in the calcifying cartilage close to the calcification front and was interpreted as a decrease in length of sulfate containing GAG chains. In the calcified cartilage, the highly calcified areas were often localized in the calcification front and contained few or no small HID-TCH-SP stain deposits, whereas the weakly calcified regions contained more stain deposits. These results indicate that partial and complete degradation of sulfated GAGs and/or PGs may be a requisite for calcification of shark cartilage.

Electron microscopical x-ray microanalysis of mineralization nodules and several other tissue compartments in fetal bone

Several chemical components are involved in bone and cartilage mineralization. Acid glycosaminoglycans (GAG) form one class of these components. In this study, the possible presence of sulfated GAG in areas of beginning bone mineralization was investigated. We analyzed areas containing acid groups, which were made visible with positive colloidal THO 2 by means of x-ray microanalysis. Four tissue compartments were investigated: osteoid, mineralization nodules, mineralized internodular matrix, and noncalcified cartilage matrix. Sulfur proved to be present in a higher concentration in the mineralization nodules than in the osteoid, whereas no significant difference could be demonstrated between the sulfur concentrations in mineralized internodular matrix and osteoid. The ratio of the sulfur content between the mineralization nodules and the cartilage matrix is significantly lower than the corresponding thorium ratio. This difference suggests that acid groups different from sulfate groups occur in a relatively larger amount in mineralization nodules than in cartilage matrix.

Freeze-fracture images of matrix vesicles of epiphyseal cartilage and non-calcified tracheal cartilage.

Freeze-fracture images of matrix vesicles of epiphyseal cartilage and non-calcified tracheal cartilage.

Properties of nucleating systems

A brief review of the various theories of bone mineralization is presented. It is not clear at present whether the many mineralization events which have been proposed are redundant or cooperative. A short description is given of homogeneous and heterogeneous nucleation of solids from solution and how these mechanisms affect hydroxyapatite formation, in vitro and in vivo. The remainder of the paper deals with specific experiments by the authors on (a) the stabilization of amorphous calcium phosphate and (b) the inhibitory role of proteoglycans in preventing cartilage mineralization. Stable amorphous calcium phosphate is found in the mitochondria of cells involved in tissue mineralization. In addition, it is believed that in bone mineral deposition an unstable amorphous calcium phosphate is formed first before transforming to bone apatite. A number of chemical species have been shown to act as stabilizers of amorphous calcium phosphate. The example given here in detail is the stabilization of the amorphous granules found in mitochondria by a mixture of Mg and adenosine triphosphate. The effect of proteoglycan aggregates and subunits from calcifying and non-calcifying cartilage was observed on the following two hydroxyapatite formation systems: (a) transformation of amorphous calcium phosphate to hydroxyapatite; (b) direct precipitation of hydroxyapatite from supersaturated calcium phosphate solutions. The aggregates were more effective inhibitors of apatite formation in both test systems, than equal weight solutions of subunits, but the subunit also delayed apatite formation. This suggests that in the epipryseal growth plate even the proteoglycan subunit must be removed by diffusion or enzyme cleavage for cartilage calcification to take place.

Resistant proteoglycans in epiphyseal plate cartilage. Variations in their distribution in relationship to age and species.

The localizations of resistant proteoglycans (RPGs) in the epiphyseal plates of rats, dogs, and humans are similar. In the epiphysial plates from young rats, dogs, and humans, the RPGs form a stratum at the junction of the zones of resting and proliferating cells. Non-calcified cartilage RPGs are associated with cells which have the potential for proliferation or column organization. As the individuals age, RPGs are found in intercolumnar regions or at times are even absent. There is also a type of RPGs in calcified cartilage, including the calcified cartilage subjacent to the articular surface, in all species. In human epiphyseal plates, looser fibrillar RPGs change abruptly to a more condensed type in the zone of provisional calcification. Calcified cartilage RPGs stain more intensely with toluidine blue and may represent a different type of RPGs.

The effect of varus stress on the moving rabbit knee joint.

Unicompartmental osteoarthritis was produced by applying varus stress to moving rabbit knee joints. Degenerative changes were confined to the medial tibial and the medial femoral articular surfaces. Within the range of varus stress used, duration appears to be more important than magnitude of varus stress in determining the severity of cartilage damage. The calcified zone remained histologically unchanged despite advanced changes in the noncalcified zone superficial to the tidemark. Intrachondral degenerative cysts were frequently found in the basilar layers of the noncalcified cartilage adjacent to the tidemark where shear stresses were likely to be highest and diffusible nutrients least available. Highly cellular cartilaginous tissue was noted in the subchondral marrow spaces in the specimens with advanced cartilage degeneration. These areas appeared to be continuous with the overlying degenerated cartilage through gaps in the calcified cartilage. Subchondral bone did not show remarkable trabecular thickening despite advanced degenerative changes in the articular cartilage.

The capacity of pig articular cartilage in organ culture to regenerate after breakdown induced by complement-sufficient antiserum to pig erythrocytes.

Explants of pig articular cartilage including invading marrow and subchondral bone (together='invasion zone') were cultivated for 10 or 14 days in complement-sufficient rabbit antiserum to pig erythrocytes (AS+C'), and then transferred to heat-inactivated normal rabbit serum (NRS) for a period of recovery. In AS+C' the cartilage matrix lost first proteoglycan and then collagen, but the cells remained viable. The degradation of collagen was accompanied by a fibroblastic transformation of the chodrocytes, first seen in the region immediately above the invasion zone. Immunohistochemical studies showed that after cultivation in AS+C', IgG antibodies entered areas in which the matrix was depleted of proteoglycan and reacted strongly with the majority of chondrocytes; those that had under-gone fibroblastic transformation exhibited little or no reaction. The degree of recovery in NRS depended on the extent to which the matrix had broken down in AS+C'. If degradation of collagen was confined to the region immediately above the invasion zone, and elsewhere only proteoglycan had been lost, new metachromatic material was regenerated in the non-calcified cartilage, and the fibroblast-like chondrocytes resumed their normal appearance and regained their reactivity with the IgG antibodies of AS; new cartilage and chondroid tissue appeared in the cavities of the invasion zone. If degradation of collagen and fibroblastic transformation of chondrocytes had spread throughout the cartilage, breakdown continued in NRS and cartilage disappeared completely above the invasion zone; new cartilage waw sometimes formed in the cavities of the invasione zone.

The Ultrastructure and Biomechanical Significance of the Tidemark of Articular Cartilage

Thirty specimens of human articular cartilage obtained at surgery were examined by scanning electron microscopy to determine the ultrastructure of the tidemark, the junction of the non-calcified and calcified portions of mature articular cartilage. Three distinct variations of the collagen framework of the tidemark were observed: (1) A band of randomly oriented compacted fibrils that appeared to be continuous with those of the non-calcified and calcified zones. (2) A band of flattened fibrils paralleling the undulating surface of the calcified cartilage. (3) A band of perpendicularly oriented fibrils having a distinct continuous transition between the non-calcified and calcified zones, the amount of calcified material applied about the fibrils rapidly increasing as the fibrils entered the calcified zone. The tidemark may serve to provide a tethering mechanism for the relatively flexible and perpendicularly oriented collagen fibrils of the deepest portion of the non-calcified articular cartilage and may prevent them from being sheared at their point of anchorage to the calcified zone. The undulating pattern of the tidemark affords a strong geometric pattern in providing resistance to the shearing action of articulation. Small gaps present in the tidemark may provide pathways for the passage of nutrients into the deep non-calcified zone of articular cartilage from the subchondral bone.

Intracartilaginous defects in adult sheep

Standardized, intracartilaginous articular cartilage defects were studied in the knee joints of adult sheep at 3, 4 and 6 months. The histologic and autoradiographic examinations of the articular cartilage in the margins of the defects showed superficial fibrillation and retention or increase of sulphate uptake over chondrocytes close to areas without labelled cells. These changes were mainly confined to the defect area. No progress with time could be ascertained. Desintegrated and dead chondrocytes as well as matrix alterations were ultrastructurally found in the margins of the defects. Beneath the defects in the deeper parts of the remaining non-calcified cartilage, there were matrix alterations that were not apparent with the light microscope. Microradiographic and fluorescence microscopic (after Tetracycline) examination showed loss of mineral from calcified cartilage and bone in the immediate vicinity of the defect and increased fluorescence in mineralized structures at 3 and 4 months. Moreover, at 6 months subchondral osteophytes were seen within and outside the defect area. Thus, in this experimental system superficial articular cartilage defects evoke matrix alterations in the deeper regions of the remaining cartilage and reactive changes in the mineralized structures.