Biomedical subjects

A Maroudas

Publications and source records attributed to A Maroudas.

At least 19 recordsLinked to original sources

PubMed2001

Age-related accumulation of the advanced glycation endproduct pentosidine in human articular cartilage aggrecan: the use of pentosidine levels as a quantitative measure of protein turnover.

During aging, non-enzymatic glycation results in the formation and accumulation of the advanced glycation endproduct pentosidine in long-lived proteins, such as articular cartilage collagen. In the present study, we investigated whether pentosidine accumulation also occurs in cartilage aggrecan. Furthermore, pentosidine levels in aggrecan subfractions of different residence time were used to explore pentosidine levels as a quantitative measure of aggrecan turnover. In order to compare protein turnover rates, protein residence time was measured as racemization of aspartic acid. As has previously been shown for collagen, pentosidine levels increase with age in cartilage aggrecan. Consistent with the faster turnover of aggrecan compared to collagen, the rate of pentosidine accumulation was threefold lower in aggrecan than in collagen. In the subfractions of aggrecan, pentosidine levels increased with protein residence time. These pentosidine levels were used to estimate the half-life of the globular hyaluronan-binding domain of aggrecan to be 19.5 years. This value is in good agreement with the half-life of 23.5 years that was estimated based on aspartic acid racemization. In aggrecan from osteoarthritic (OA) cartilage, decreased pentosidine levels were found compared with normal cartilage, which reflects increased aggrecan turnover during the OA disease process. In conclusion, we showed that pentosidine accumulates with age in aggrecan and that pentosidine levels can be used as a measure of turnover of long-lived proteins, both during normal aging and during disease.

Adolescent↗

PubMed2001

Depth-dependent compressive properties of normal aged human femoral head articular cartilage: relationship to fixed charge density.

OBJECTIVES: Determine the depth-varying confined and osmotic compression moduli of normal human articular cartilage from the femoral head, and test whether these moduli are dependent on fixed charge density. METHODS AND RESULTS: Using an automated instrument to allow epifluorescence microscopy analysis during confined compression testing on cartilage samples, the equilibrium confined compression modulus (H(A 0)) was found to vary markedly with depth (z=0-1500 microm) from the articular surface. H(A 0) increased from 1.16+/-0.20 MPa in the superficial (0-125 microm) layer to 7.75+/-1.45 MPa in the deepest (1250-1500 microm) layer tested, and was fit by the expression, H(A 0)(z) [MPa]=1.44 exp(0.0012.z [microm]). Also, in successive slices of cartilage extending from the articular surface to the middle-deep regions, the bulk modulus (K(0)) and fixed charge density (FCD) increased, consistent with previous findings. While H(A 0), K(0), and FCD each varied with depth from the articular surface, the dependence of H(A 0) and K(0) on depth did not appear to be completely related to variations in FCD. CONCLUSIONS: The confined compression modulus of normal aged human femoral head articular cartilage increases markedly with depth from the articular surface, a trend similar to that observed for articular cartilage from other joints in animals but with an absolute amplitude that is several-fold higher. The compressive properties were not simply related to FCD at different depths from the articular surface, suggesting that other as yet undefined factors also contribute to compressive properties.

Aged↗

PubMed2000

The increased swelling and instantaneous deformation of osteoarthritic cartilage is highly correlated with collagen degradation.

OBJECTIVE: To provide evidence for the hypothesis that the loss of tensile strength of osteoarthritic (OA) cartilage (resulting in swelling-the hallmark of OA) is due to an impaired collagen network and not to loss or degradation of proteoglycans. METHODS: The amount of degraded collagen molecules, the fixed charge density (FCD) on a dry-weight basis, the degree of swelling in saline, and the instantaneous deformation (ID; a test reflecting the tensile stiffness of the collagen network) were measured in full-depth OA femoral condyle samples. In addition, levels of the crosslink hydroxylysylpyridinoline (HP), the amount of degraded collagen molecules, and the degree of swelling were determined in the 3 zones (surface, middle, and deep) of OA cartilage. We also compared the ID of normal and OA cartilage. RESULTS: In full-depth OA cartilage, a close relationship was found between swelling and ID. Swelling and ID correlated strongly with the amount of degraded collagen molecules, and were not related to FCD. OA cartilage showed the same zonal pattern in HP levels as normal cartilage (i.e., an increase with depth). No relationship was found between collagen crosslinking and swelling of the surface, middle, and deep zones. In all 3 zones, swelling was proportional to the amount of degraded collagen molecules. Compared with that of normal cartilage, the change in ID of OA cartilage was most pronounced at the surface in a direction parallel to the direction of the collagen fibrils. CONCLUSION: The decreased stiffness of the OA collagen network (as measured by swelling and ID) is strongly related to the amount of degraded collagen molecules. The anisotropy in ID parallel and perpendicular to the direction of the fibrils revealed that the impairment of strength resides mainly in, and not between, the fibrils. Proteoglycans play only a minor role in the degeneration of the tensile stiffness of OA cartilage.

Aged↗

PubMed1998

The effects of pH and ionic strength on intrafibrillar hydration in articular cartilage.

The hydration of articular cartilage is an essential determinant of its load bearing capacity. Here we have examined the dependence of the amount of intrafibrillar water, associated with the collagen molecules in both native and PG-depleted cartilage specimens, on the pH and ionic strength of the bathing solution, in the presence and absence of an externally applied pressure. We found that high ionic strength reduces the collagen intermolecular spacing over a large pH range: this is consistent with the electrostatic nature of the interactions between the charged groups within the intrafibrillar space. We also found that as the pH is lowered from neutral to approximately 3, there is, as expected, a gradual increase in the overall positive charge of the intrafibrillar compartment. However, surprisingly, this is not accompanied by an increase in the intrafibrillar hydration; only at pH 1.8 does the amount of intrafibrillar water increase markedly. We suggest that, rather than overall intrafibrillar charge, it is specific local axial and azimuthal relationships among collagen molecules in the fibril, and more particularly, among their charged amino acid residues, that determine the intermolecular collagen spacing, and hence intrafibrillar hydration.

Adult↗

PubMed1998

Mechanical properties of the collagen network in human articular cartilage as measured by osmotic stress technique.

We have used an isotropic osmotic stress technique to assess the swelling pressures of human articular cartilage over a wide range of hydrations in order to determine from these measurements, for the first time, the tensile stress in the collagen network, Pc, as a function of hydration. Osmotic stress was applied by means of calibrated solutions of polyethylene glycol. Calculations of osmotic stress were based on the balance, at equilibrium, between the applied stress, the collagen stress, and the proteoglycan osmotic pressure, piPG, acting within the extrafibrillar matrix compartment. Pc vs hydration was determined for several normal human samples, both native and trypsin-treated, and for cartilage from one osteoarthritic (OA) joint. We found that for normal cartilage the collagen network does not become "limp" until the volume of cartilage has decreased by 20-25% of its initial value and that its contribution to the balance of forces in cartilage therefore must be taken into account over a much wider range of hydrations than was previously thought. For normal cartilage, the Pc vs hydration curves exhibit a steep increase with increasing hydration; trypsin treatment does not change their slope, showing that PG concentration does not influence the inherent stiffness of the collagen network. By contrast, the curves for OA specimens are considerably shallower and displaced to higher hydrations. Our findings thus highlight the role of the stiffness of the collagen network in limiting hydration in normal cartilage and ensuring a high PG concentration in the matrix, which is essential for effective load-bearing and is lost in OA.

Cartilage, Articular↗

PubMed1998

Ageing and zonal variation in post-translational modification of collagen in normal human articular cartilage. The age-related increase in non-enzymatic glycation affects biomechanical properties of cartilage.

A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (>20 years), whereas in young cartilage (<10 years) the opposite was seen; highest levels were close to the bone. LP levels in cartilage sections at age 14 years are high at the surface and close to the bone, but they are low in the middle region. This indicates that maturation of cartilage in the second decade of life starts in the upper half of the tissue, and occurs last in the tissue close to the bone. The effect of NEG products on instantaneous deformation of cartilage was investigated as a functional of topographical variations in pentosidine levels in vivo and in relation to in vitro induced NEG. Consistently, higher pentosidine levels were associated with a stiffer collagen network. A stiffer and more crosslinked collagen network may become more brittle and more prone to fatigue.

Adult↗

PubMed1998

Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age.

Aggrecan is a key component of the cartilage matrix. During aging, many changes occur in its composition and structure; in particular, there is an increase in the proportion of lower molecular weight monomers and of the "free" binding region. An important question has been whether these changes represent alterations in biosynthesis or whether they are due to the accumulation with age of the partially degraded fragments of the originally synthesized large monomer. In the present work we have used an independent tool, viz., the extent of racemization of aspartic acid to study the molecular "age" of different buoyant density fractions of the aggrecan of human articular cartilage, as well as of isolated free binding region and link protein. By measuring the D/LAsp ratio of the different aggrecan species, we were able to establish directly the relative residence times of these molecules in the cartilage matrix and, in combination with compositional and structural analyses, to define their "history" and calculate some of the kinetics constants characterizing their turnover. The value of the turnover constant for the large monomer in fraction A1D1 is 0.206 per year, which corresponds to a half-life of 3.4 years, while the turnover constant for the free binding region is 0.027 per year, which corresponds to a half-life of 25 years. It is thus clear that the rate of formation and turnover of the large monomer is much more rapid than the final degradation of the free binding region fragments, which explains the accumulation of the latter in cartilage during aging.

Adult↗

PubMed1997

A simplified measurement of degraded collagen in tissues: application in healthy, fibrillated and osteoarthritic cartilage.

Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simplified method for the quantification of the amount of degraded collagen in the collagen network of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by alpha-chymotrypsin, hydrolysis in 6 M HCl of the released fragments as well as the residual tissue, and then measurement of the amount of hydroxyproline in both pools. Since the digestion of degraded collagen by alpha-chymotrypsin and measurement of hydroxyproline is not restricted to a specific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investigations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy cartilage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in osteoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.

Adult↗

PubMed1995

Concentration and size distribution of insulin-like growth factor-I in human normal and osteoarthritic synovial fluid and cartilage.

The concentration of free insulin-like growth Factor-I (IGF-I) and its complexes was determined in human normal and osteoarthritic synovial fluids, using ultrafiltration through 20- and 100-kDa membranes, followed by a radioimmunoassay of each fraction. In addition, freshly obtained samples of normal and osteoarthritic cartilage were incubated for several days, at both 4 and 37 degrees C. The incubation media (desorbates) were analyzed the same way as the synovial fluid samples to yield the concentration of IGF-I in cartilage in situ. Our findings are (i) Free IGF-I content is extremely low in both human serum and synovial fluid and there is no significant difference between the two; (ii) The concentration of total IGF-I in normal human synovial fluid is an order of magnitude lower than that in serum due mainly to the decrease in the concentration of the large complex; (iii) Preliminary results show that the total IGF-I in osteoarthritic synovial fluids is twice as high as in normal fluids; (iv) In normal human cartilage the levels of IGF-I in all its forms are very low and are consistent with the expected exclusion of large molecules by the extracellular matrix; (v) By contrast, in osteoarthritic cartilage, the concentrations of all forms of IGF-I are high, probably due to increased permeability of the matrix and binding; (vi) The levels of IGF-I found in normal human cartilage are more than an order of magnitude lower than those which stimulate proteoglycan synthesis in human cartilage in culture, while the IGF-I levels in osteoarthritic cartilage lie in the range in which stimulation does occur.

Adult↗

PubMed1995

Insulin-like growth factor-I and its complexes in normal human articular cartilage: studies of partition and diffusion.

Insulin-like growth factor-I (IGF-I) plays a major role in cartilage homeostasis. Our objective was to study the penetration of IGF-I, both alone and bound to serum proteins, into the different zones of normal human cartilage using radioactively labeled IGF-I. The uptake of free IGF-I was higher than that predicted on the basis of excluded volume calculations and showed concentration dependence: we attributed this to reversible binding of the hormone to the tissue. Since the extent of binding was much higher than that calculated for binding to cell receptors, we concluded that IGF-I binds to matrix components. The kinetics of desorption of IGF-I from cartilage confirmed our conclusions regarding binding. The degree of uptake of IGF-I protein complexes prepared by labeling human serum with [125I]IGF-I showed that such complexes are largely excluded from normal cartilage and that the amounts present in the tissue are too low to affect proteoglycan metabolism.

Absorption↗

PubMed1995

Age-related changes in collagen packing of human articular cartilage.

We have used X-ray scattering techniques to determine if the lateral packing of collagen molecules in the fibrils of human articular cartilage changes with age. Such changes would affect the available intrafibrillar volume and consequently the amount of intrafibrillar water. Measurements were made both in the presence and absence of compression on samples from donors aged 20 to 90 years. We find a weak though statistically significant tendency towards less dense collagen packing in native tissue as a function of age. However, the increase in packing density in response to pressure does not change with age, and the packing density in articular cartilage from which the proteoglycan molecules have been removed is similarly not age-dependent. The small increase in intrafibrillar water indicated by our data is insufficient to explain the reported increase in fibril diameter in samples from aged donors.

Adult↗

PubMed1993

Human facet cartilage: swelling and some physicochemical characteristics as a function of age. Part 2: Age changes in some biophysical parameters of human facet joint cartilage.

This study was aimed at investigating, in relating to aging, some of the biochemical and biophysical characteristics of the facet cartilage that determine the functional behavior of this tissue. In addition, facets and discs from the same segment were graded according to their macroscopic appearance. The proportion of severely degenerate discs was low in young subjects and increased with age; by contrast, the proportion of coarsely fibrillated and/or ulcerated facets was high in spines from young adults and remained constant throughout adulthood. Unlike discs, facets do not show an age-related loss of proteoglycans or a consequent decrease in the resistance to a compressive load. However, even in relatively young age groups (30-50 years) a high hydration was observed more often in facet joints than in cartilage from other joints studied. These characteristics are known to accompany damage of the collagen network and cartilage degeneration. Unlike normal femoral head cartilage, facet cartilage does not show a rise in fixed charge density with age. The cartilage from the superior processes (concave) is thicker than that from the inferior processes (convex) and has a higher fixed charge density. At the same time it has a higher water content, which indicates that damage occurs more frequently.

Adolescent↗

PubMed1992

Physicochemical properties of the aging and diabetic sand rat intervertebral disc.

Hydration, fixed charge density, (FCD) and hydration under various osmotic pressures were compared in young, old, and young diabetic sand rats. This rat is a desert animal that may develop diabetes when fed a regular diet; it is also known to have radiographic and histologic evidence of intervertebral disc (IVD) disease. Forty-five rats and 180 IVD were used in this study; they were divided into three equal groups: young healthy, old healthy, and young diabetics. IVD, cancellous bone, and muscle were sampled from distal lumbar spines. The young diabetic rats (YD) were considerably heavier than the age-matched controls, had higher insulin and glucose levels, and all YD had cataracts. The discs of the young diabetic animals demonstrated decreased hydration, FCD and ability to resist compression under osmotic pressures as compared with the young and healthy discs and were more similar to the discs from old rats. The IVD is the most affected musculoskeletal connective tissue in sand rats with aging and diabetes. The aged and diabetic discs in the sand rat demonstrated changes similar to human changes with regard to lower hydration, FCD, and ability to resist osmotic pressure. Therefore, the sand rat may be a suitable animal model for studying the pathogenesis of disc degeneration.

Aging↗

PubMed1992

Racemization of aspartic acid in human articular cartilage.

The rate of racemization of aspartic acid was measured in young and aged human femoral head cartilage. Normal femoral heads were obtained at postmortem, osteoarthritic specimens at operations for total hip replacement. In order to distinguish between the aspartic acid racemization in collagen from that in proteoglycan (PG), in addition to native tissue, we tested cartilage specimens from which PG had been enzymatically removed. Preliminary results indicate that there is only a very slow collagen turnover in normal adult cartilage. The same is true of residual cartilage from osteoarthritic femoral heads, indicating no rapid repair except where osteophytes are formed. Native, PG-containing cartilage, whether normal or osteoarthritic was found to have unexpectedly high racemization rates.

Adult↗

PubMed1992

Human facet cartilage: swelling and some physico-chemical characteristics as a function of age. Part 1: Swelling of human facet joint cartilage.

The hydration of cartilage from human facet joints was measured after the joints had been subjected to different treatments. One group of facets was opened and directly exposed to physiologic saline solution before extraction of cartilage plugs. The plugs were weighed, re-equilibrated in fluid, and weighed again. The swelling results obtained under these conditions were compared with those when similar plugs of cartilage were excised from joints that had not been exposed to solution or had been exposed to solution while still closed. It was found that swelling was least (and similar in value to hip cartilage) for joints that had been exposed open to saline solution, highest for joints that had not been exposed to solution, and intermediate for joints that had been exposed to solution while still closed. The same trends were observed whether the cartilage on the joint was intact or fibrillated, although in each group the swelling and the final hydration were higher for fibrillated than for intact tissue. It was concluded that facet cartilage, unlike human hip or knee cartilage, is underhydrated when excised from the joint. This underhydration is thought to reflect the permanent presence of stresses in vivo on some part of the facet joints, the position of the loaded site changing with time. The authors attempted to distinguish between the swelling caused by this underhydration and that from disruption of the collagen network in the case of fibrillated specimens.

Aging↗

PubMed1991

The effect of osmotic and mechanical pressures on water partitioning in articular cartilage.

X-ray diffraction measurements on native and proteoglycan-free articular cartilage have been made in order to test the dependence of the lateral packing of the collagen molecules on the osmotic pressure gradient, either naturally occurring or externally applied, between the intra- and extrafibrillar compartments. From the information on collagen packing we have been able to calculate, albeit with several assumptions, the amount of intrafibrillar water as a function of pressure. In parallel with the above measurements, we have quantitated, using serum albumin partitioning, the intrafibrillar water in proteoglycan-free cartilage, as a function of mechanically applied pressure. The results of both sets of experiments lead to the conclusion that the molecular packing density, and hence the intrafibrillar water content, are a function of the osmotic pressure difference between the extrafibrillar and intrafibrillar spaces or the equivalent mechanically applied pressure. The determination of intrafibrillar water has enabled us to calculate, from measured values of fixed charge density, the internal osmotic pressure of cartilage specimens, both in compressed and uncompressed states.

Adult↗

PubMed1990

Influence of cyclic loading on the nutrition of articular cartilage.

Articular cartilage is avascular. Nutrients are transported to the cells mainly by diffusion from the synovial fluid. Nutrient transport is also sometimes thought to be assisted by movement of fluid in and out of cartilage in response to cyclic loading of the tissue ('pumping'). The influence of pumping on transport of solutes through cartilage was measured by subjecting plugs of human femoral head cartilage immersed in medium containing radioactive solutes to a simulated walking cycle of 2.8 MPa at 1 Hz. The rate of absorption or desorption of tracers from the cycled plugs was compared with that of unloaded control plugs. For small solutes (urea, NaI) fluid transport did not affect the rate of solute transport significantly. Most major nutrients, such as glucose and oxygen, are small solutes and thus nutrition should not be affected by pumping. The rate of desorption of a large solute (serum albumin), however, was increased by 30-100% in plugs subjected to cyclic loading.

Biological Transport↗

PubMed1989

Some biochemical and biophysical parameters for the study of the pathogenesis of osteoarthritis: a comparison between the processes of ageing and degeneration in human hip cartilage.

We have investigated the changes in some of the biochemical and biophysical properties of human femoral head cartilage on the one hand during ageing and on the other hand in osteoarthritis. Topographical variations were also investigated. The parameters studied were those relevant to cartilage function, viz., proteoglycan concentration (as expressed by the concentration of negatively charged groups), the rate of glycosaminoglycan synthesis, water content, osmotic pressure and fluid loss during compression. During ageing the fixed charge density was found to increase at all sites of the femoral head provided fibrillation was absent: osmotic pressure increased accordingly whilst loss of fluid under the effect of externally applied compression diminished. In cartilage from osteoarthritic joints the opposite changes were found. The rate of GAG synthesis varied considerably with site on the femoral head. It decreased somewhat with age on the superior surface, but increased on the inferior surface. When the same sites were compared, the rate of GAG synthesis in cartilage from osteoarthritic heads was either the same as or lower than in cartilage form normal heads in the same group.

Adolescent↗