13C NMR relaxation studies on cartilage and cartilage components

We have investigated the molecular motions of polysaccharides of bovine nasal and pig articular cartilage by measuring the 13C NMR relaxation times (T1 and T2). Both types of cartilage differ significantly towards their collagen/glycosaminoglycan ratio, leading to different NMR spectra. As chondroitin sulfate is the main constituent of cartilage, aqueous solutions of related poly- and monosaccharides (N-acetylglucosamine and glucuronic acid) were also investigated. Although there are only slight differences in T1 relaxation of the mono- and the polysaccharides, T2 decreases about one order of magnitude, when glucuronic acid or N-acetylglucosamine and chondroitin sulfate are compared. It is concluded that the ring carbons are motion-restricted primarily by the embedment in the rigid pyranose structure and, thus, additional limitations of mobility do not more show a major effect. Significant differences were observed between bovine nasal and pig articular cartilage, resulting in a considerable line-broadening and a lower signal to noise ratio in the spectra of pig articular cartilage. This is most likely caused by the higher collagen content of articular cartilage in comparison to the polysaccharide-rich bovine nasal cartilage.     

Prediction of biomechanical properties of articular cartilage with quantitative magnetic resonance imaging

Quantitative magnetic resonance imaging (MRI) is the most potential non-invasive means for revealing the structure, composition and pathology of articular cartilage. Here we hypothesize that cartilage mechanical properties as determined by the macromolecular framework and their interactions can be accessed by quantitative MRI. To test this, adjacent cartilage disk pairs (n=32) were prepared from bovine proximal humerus and patellofemoral surfaces. For one sample, the tissue Young's modulus, aggregate modulus, dynamic modulus and Poisson's ratio were determined in unconfined compression. The adjacent disk was studied at 9.4T to determine the tissue T(2) relaxation time, sensitive to the integrity of the collagen network, and T(1) relaxation time in the presence of Gd-DTPA, a technique developed for the estimation of cartilage proteoglycan (PG) content. Quantitative MRI parameters were able to explain up to 87% of the variations in certain biomechanical parameters. Correlations were further improved when data from the proximal humerus was assessed separately. MRI parameters revealed a topographical variation similar to that of mechanical parameters. Linear regression analysis revealed that Young's modulus of cartilage may be characterized more completely by combining both collagen- and PG-sensitive MRI parameters. The present results suggest that quantitative MRI can provide important information on the mechanical properties of articular cartilage. The results are encouraging with respect to functional imaging of cartilage, although in vivo applicability may be limited by the inferior resolution of clinical MRI instruments.     

Biomechanical properties of knee articular cartilage

Structure and properties of knee articular cartilage are adapted to stresses exposed on it during physiological activities. In this study, we describe site- and depth-dependence of the biomechanical properties of bovine knee articular cartilage. We also investigate the effects of tissue structure and composition on the biomechanical parameters as well as characterize experimentally and numerically the compression-tension nonlinearity of the cartilage matrix. In vitro mechano-optical measurements of articular cartilage in unconfined compression geometry are conducted to obtain material parameters, such as thickness, Young's and aggregate modulus or Poisson's ratio of the tissue. The experimental results revealed significant site- and depth-dependent variations in recorded parameters. After enzymatic modification of matrix collagen or proteoglycans our results show that collagen primarily controls the dynamic tissue response while proteoglycans affect more the static properties. Experimental measurements in compression and tension suggest a nonlinear compression-tension behavior of articular cartilage in the direction perpendicular to articular surface. Fibril reinforced poroelastic finite element model was used to capture the experimentally found compression-tension nonlinearity of articular cartilage.     

Contribution of tissue composition and structure to mechanical response of articular cartilage under different loading geometries and strain rates

Mechanical function of articular cartilage in joints between articulating bones is dependent on the composition and structure of the tissue. The mechanical properties of articular cartilage are traditionally tested in compression using one of the three loading geometries, i.e., confined compression, unconfined compression or indentation. The aim of this study was to utilize a composition-based finite element model in combination with a fractional factorial design to determine the importance of different cartilage constituents in the mechanical response of the tissue, and to compare the importance of the tissue constituents with different loading geometries and loading rates. The evaluated parameters included water and collagen fraction as well as fixed charge density on cartilage surface and their slope over the tissue thickness. The thicknesses of superficial and middle zones, as based on the collagen orientation, were also included in the evaluated parameters. A three-level resolution V fractional factorial design was used. The model results showed that inhomogeneous composition plays only a minor role in indentation, though that role becomes more significant in confined compression and unconfined compression. In contrast, the collagen architecture and content had a more profound role in indentation than with two other loading geometries. These differences in the mechanical role of composition and structure between the loading geometries were emphasized at higher loading rates. These findings highlight how the results from mechanical tests of articular cartilage under different loading conditions are dependent upon tissue composition and structure.     

A case of metastatic leptomeningeal carcinomatosis from early gastric carcinoma

Intramuscular myxoma is a rare benign soft tissue tumor which may be mistaken for other benign and low-grade malignant myxoid neoplasms. We present the case of a 63-year-old woman with an asymptomatic intramuscular myxoma discovered incidentally on a whole-body F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography. PET images showed a mild FDG uptake (maximum standardized uptake value, 1.78) in the left gluteus maximus. Subsequent magnetic resonance (MR) imaging revealed a well-defined ovoid mass with homogenous low signal intensity on T1-weighted sequences and markedly high signal intensity on T2-weighted sequences. Contrast-enhanced MR images showed heterogeneous enhancement throughout the mass. The diagnosis of intramuscular myxoma was confirmed on histopathology after surgical excision of the tumor. The patient had no local recurrence at one year follow-up. Our case suggests that intramuscular myxoma should be considered in the differential diagnosis of an oval-shaped intramuscular soft tissue mass with a mild FDG uptake.     

Collagen in tissue-engineered cartilage: Types,structure, and crosslinks

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/ water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale. J. Cell. Biochem. 71:313–327, 1998. © 1998 Wiley-Liss, Inc.     

Depth-dependent Compressive Equilibrium Properties of Articular Cartilage Explained by its Composition

For this study, we hypothesized that the depth-dependent compressive equilibrium properties of articular cartilage are the inherent consequence of its depth-dependent composition, and not the result of depth-dependent material properties. To test this hypothesis, our recently developed fibril-reinforced poroviscoelastic swelling model was expanded to include the influence of intra- and extra-fibrillar water content, and the influence of the solid fraction on the compressive properties of the tissue. With this model, the depth-dependent compressive equilibrium properties of articular cartilage were determined, and compared with experimental data from the literature. The typical depth-dependent behavior of articular cartilage was predicted by this model. The effective aggregate modulus was highly strain-dependent. It decreased with increasing strain for low strains, and increases with increasing strain for high strains. This effect was more pronounced with increasing distance from the articular surface. The main insight from this study is that the depth-dependent material behavior of articular cartilage can be obtained from its depth-dependent composition only. This eliminates the need for the assumption that the material properties of the different constituents themselves vary with depth. Such insights are important for understanding cartilage mechanical behavior, cartilage damage mechanisms and tissue engineering studies.     

The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

Nonlinear tensile properties of bovine articular cartilage and their variation with age and depth

Tensile stiffness of articular cartilage is much greater than its compressive stiffness and plays an essential role even in compressive properties by increasing transient fluid pressures during physiological loading. Recent studies of nonlinear properties of articular cartilage in compression revealed several physiologically pertinent nonlinear behaviors, all of which required that cartilage tensile stiffness increase significantly with stretch. We therefore performed sequences of uniaxial tension tests on fresh bovine articular cartilage slices using a protocol that allowed several hours to attain equilibrium and measured longitudinal and transverse tissue strain. By testing bovine cartilage from different ages (6 months to 6 years) we found that equilibrium and transient tensile modulus increased significantly with maturation and age, from 0 to 15 MPa at equilibrium and from 10 to 28 MPa transiently. Our results indicate that cartilage stiffens with age in a manner similar to other highly hydrated connective tissues, possibly due to age-dependent content of enzymatic and nonenzymatic collagen cross links. The long relaxation period used in our tests (5-10 hours) was necessary in order to attain equilibrium and avoid a very significant overestimation of equilibrium modulus that occurs when much shorter times are used (15-30 minutes). We also found that equilibrium and transient tensile modulus increased nonlinearly when cartilage is stretched from 0 to 10% strain without any previous tare load. Although our results estimate a nonlinear increase in tensile stiffness with stretch that is an order of magnitude lower than that required to predict nonlinear properties in compression, they are in agreement with previous results from other uniaxial tension tests of collagenous materials. We therefore speculate that biaxial tensile moduli may be much higher and thereby more compatible with observed nonlinear compressive properties.     

Species-common antigen of connective tissues.

Collagen-free extracts were prepared from bovine, porcine and canine hyaline, elastic and fibrous cartilages, articular capsule, tendon, aorta, cortical bone and regenerating articular surfaces. The extracts were investigated with antisera to bovine nasal septal cartilage, dog articular cartilage and non-collagenous protein fraction of bovine cortical bone. Immunodiffusion, immunoelectrophoresis, and immunohistochemical methods were used. In the different supporting tissues of the three animal species a common antigen, probably of proteoglycan origin, was demonstrated. The finer differences in antigenicity between the different tissues are probably due to the variations in proteoglycan composition of the given supporting tissues. Owing to the wide-spread occurrence of the antigen, the authors suggest the term "species-common connective tissue antigen" instead of the "species-common cartilage antigen" used so far.     

A new method for investigating osteoarthritis using Fast Field-Cycling nuclear magnetic resonance

Osteoarthritis in synovial joints remains a major cause of long-term disability worldwide, with symptoms produced by the progressive deterioration of the articular cartilage. The earliest cartilage changes are thought to be alteration in its main protein components, namely proteoglycan and collagen. Loss of proteoglycans bound in the collagen matrix which maintain hydration and stiffness of the structure is followed by collagen degradation and loss. The development of new treatments for early osteoarthritis is limited by the lack of accurate biomarkers to assess the loss of proteoglycan. One potential biomarker is magnetic resonance imaging (MRI). We present the results of a novel MRI methodology, Fast Field-Cycling (FFC), to assess changes in critical proteins by demonstrating clear quantifiable differences in signal from normal and osteoarthritic human cartilage for in vitro measurements. We further tested proteoglycan extracted cartilage and the key components individually. Three clear signals were identified, two of which are related predominantly to the collagen component of cartilage and the third, a unique very short-lived signal, is directly related to proteoglycan content; we have not seen this in any other tissue type. In addition, we present the first volunteer human scan from our whole-body FFC scanner where articular cartilage measurements are in keeping with those we have shown in tissue samples. This new clinical imaging modality offers the prospect of non-invasive monitoring of human cartilage in vivo and hence the assessment of potential treatments for osteoarthritis. Keywords: Fast Field-Cycling NMR; human hyaline cartilage; Osteoarthritis; T1 dispersion; quadrupolar peaks; protein interactions     

T1-Weighted Sodium MRI of the Articulator Cartilage in Osteoarthritis: A Cross Sectional and Longitudinal Study

Structural magnetic resonance imaging (MRI) has shown great utility in diagnosing soft tissue burden in osteoarthritis (OA), though MRI measures of cartilage integrity have proven more elusive. Sodium MRI can reflect the proteoglycan content of cartilage; however, it requires specialized hardware, acquisition sequences, and long imaging times. This study was designed to assess the potential of a clinically feasible sodium MRI acquisition to detect differences in the knee cartilage of subjects with OA versus healthy controls (HC), and to determine whether longitudinal changes in sodium content are observed at 3 and 6 months. 28 subjects with primary knee OA and 19 HC subjects age and gender matched were enrolled in this ethically-approved study. At baseline, 3 and 6 months subjects underwent structural MRI and a 0.4ms echo time 3D T1-weighted sodium scan as well as the knee injury and osteoarthritis outcome score (KOOS) and knee pain by visual analogue score (VAS). A standing radiograph of the knee was taken for Kellgren-Lawrence (K-L) scoring. A blinded reader outlined the cartilage on the structural images which was used to determine median T1-weighted sodium concentrations in each region of interest on the co-registered sodium scans. VAS, K-L, and KOOS all significantly separated the OA and HC groups. OA subjects had higher T1-weighted sodium concentrations, most strongly observed in the lateral tibial, lateral femoral and medial patella ROIs. There were no significant changes in cartilage volume or sodium concentration over 6 months. This study has shown that a clinically-feasible sodium MRI at a moderate 3T field strength and imaging time with fluid attenuation by T1 weighting significantly separated HCs from OA subjects.     

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.     

Acoustic properties of articular cartilage under mechanical stress

Mechano-acoustic and elastographic techniques may provide quantitative means for the in vivo diagnostics of articular cartilage. These techniques assume that sound speed does not change during tissue loading. As articular cartilage shows volumetric changes during compression, acoustic properties of cartilage may change affecting the validity of mechano-acoustic measurements. In this study, we examined the ultrasound propagation through human, bovine and porcine articular cartilage during stress-relaxation in unconfined compression. The time of flight (TOF) technique with known cartilage thickness (true sound speed) as well as in situ calibration method [Suh, Youn, Fu, J. Biomech. 34 (2001), 1347-1353] were used for the determination of sound speed. Ultrasound speed and attenuation decreased in articular cartilage during ramp compression, but returned towards the level of original values during relaxation. Variations in ultrasound speed induced an error in strain and compressive moduli provided that constant ultrasound speed and time-of-flight data was used to determine the tissue thickness. Highest errors in strain (-11.8 +/- 12.0%) and dynamic modulus (15.4 +/- 17.9%) were recorded in bovine cartilage. TOF and in situ calibration methods yielded different results for changes in sound speed during compression. We speculate that the variations in acoustic properties in loaded cartilage are related to rearrangement of the interstitial matrix, especially to that of collagen fibers. In human cartilage the changes, are, however relatively small and, according to the numerical simulations, mechano-acoustic techniques that assume constant acoustic properties for the cartilage will not be significantly impaired by this phenomenon.     

Treatment of cartilage defects by Low-intensity pulsed ultrasound in a sheep model

Aim of this study was to evaluate effects of Low-intensity pulsed ultrasound on repair of articular cartilage defects. Low-intensity pulsed ultrasound (Lipus) can induce the differentiation and activation of chondrocytes. This study was designed to evaluate the effect of Lipus on articular cartilage defects in a sheep. Eight sheep were divided in to two groups. The animals received bilateraly, articular cartilage defects 4 mm in diameter and 2 mm in deep on the patellar groove and experimental groups were treated with intensity 200 mW/cm², 20 min/day with low-intensity pulsed ultrasound for 2 month. Then both knee joints underwent surgery for remove of formed tissue sample from defects.The samples were evaluated by Quantitative real-time polymerase chain reaction (qRT-PCR), Safranin-o staining, Immunofluorescence Staining and Morphological characterization. The best and worst sample per group according to Macroscopic and micriscopic scoring were icentified. The results showed that the operated groups with-Lipus-treatment and without-Lipus treatment had considered statistically significant. Gross photography revealed that the defects in experimental groups were filled with proliferative tissue, while in control groups, a thin layer of proliferative tissue was formed in defects. qRT-PCR results showed the expression of coll2, sox9, aggrecan and Osteocalcin in experimental groups. Intense safranin-O staining show the formation cartilage tissue in ultrasound treated group, while loose safranin-o-staining were observed at the control groups. Immunofluorescence staining showed the type 2 Collagen protein expression. We suggest that low-intensity pulsed ultrasound provide the mechanistic basis force for articular cartilage repair and effective treatment modality for improving of articular cartilage defects.

     

Is Biochemical Assessment of Pheochromocytoma Necessary in Adrenal Incidentalomas with Magnetic Resonance Imaging Features not Suggestive of Pheochromocytoma?

Objective: Currently, it is unclear whether pheochromocytomas can be ruled out based on low intensity on T2-weighted sequences and signal loss on out-of-phase magnetic resonance imaging (MRI) sequences. Hence, in this study, we investigated whether biochemical screening for pheochromocytoma in patients with adrenal incidentalomas (AIs) showing MRI features not suggesting pheochromocytoma would prove beneficial.Methods: We performed MRI for 300 AIs in 278 consecutive patients. All patients were screened for pheochromocytoma with plasma metanephrine and normetanephrine. Patients with high plasma levels of metanephrine and/or normetanephrine were also assessed for pheochromocytoma by urinary metanephrines.Results: Hyperintensity was detected on T2-weighted MRI sequences in 28 (9.3%) of the 300 AIs. Among these 28 incidentalomas, pheochromocytoma was diagnosed in 13 (46.4%) of the cases by histopathologic analysis. Hyperintensity on T2-weighted MRI was significantly higher in pheochromocytomas compared to the remaining AIs (P<.001). All 13 pheochromocytomas were characterized by hyperintensity on T2-weighted sequences and the absence of signal loss on out-of-phase MRI sequences. Pheochromocytoma was not detected in any of the 272 AIs that appeared hypointense or isointense on T2-weighted MRI sequences or in the 250 cases with signal loss on out-of-phase sequences.Conclusion: The results of this study suggest that AIs that appear hypointense or isointense on T2-weighted MRI sequences and those with signal loss on out-of-phase sequences may not require routine biochemical screening for pheochromocytoma. Further studies including a higher number of pheochromocytomas are required to confirm our results.Abbreviations:AI = adrenal incidentalomaCT = computed tomographyMRI = magnetic resonance imaging     

Identification of a high-molecular-weight (> 400 000) protein in hyaline cartilage

A high-molecular-weight (> 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2–4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the > 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.     

Identification of a high-molecular-weight (greater than 400 000) protein in hyaline cartilage

A high-molecular-weight (greater than 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2-4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the greater than 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Transient solute diffusion in articular cartilage

The one-dimensional transient diffusion of glucose, inulin and dextran into adult bovine knee articular cartilage was determined for transport times of 1, 5, 15 and 60 min, and 4, 12, 24 and 48 h. The apparent diffusion coefficient and apparent interface partition coefficient were calculated from the concentration-depth profiles within the tissue using a theoretical model for non-steady state solute diffusion. The diffusion coefficient was found to decrease with both solute size and transport time. The partition coefficient also decreased with solute size but increased with transport time. Neither coefficient was dependent on normal tissue fluid or proteoglycan content variations.