A model of unloaded human intervertebral disk based on NMR relaxation.

NMR relaxation rates were related to the composition of the nucleus pulposus from 11 and anulus fibrosus from six human intervertebral disks. Tissue water was proportional to glycosaminoglycan (GAG) and residue, the noncollagen, non-GAG portion of the dry weight (R2 = 0.74). The solid signal fraction depended on collagen and residue protons (R2 = 0.89). 1/T1 was proportional to collagen and residue (R2 = 0.97). T2 showed 2-4 components labeled A, B, C, and D, with means +/- standard deviations of 3.1 +/- 1.6, 17.5 +/- 9.5, 64 +/- 22, and 347 +/- 162 msec. Signal fractions of A and B depended on the collagen-associated water protons (R2 = 0.94 and 0.85), C on residue-associated water protons (R2 = 0.82), and D on GAG-associated water protons (R2 = 0.74). The data led to a model of disk architecture in which the collagen and residue were largely solid, forming distinct water compartments; the remaining water was present in a proteoglycan gel.

Non-proteoglycan forms of biglycan increase with age in human articular cartilage.

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.

The proteoglycans of the cartilaginous end-plate of the human intervertebral disc change after maturity.

Lumbar spines collected postmortem were assigned to one of two groups: group 1--three spines with healthy discs, or group 2--three spines with severely degenerated discs. The proteoglycans (PGs) of the cartilaginous end-plate (CEP) were extracted with 4 M guanidinium chloride containing protease inhibitors and were purified by caesium chloride density gradient ultracentrifugation. On Sepharose CL-2B chromatography, the most dense 20% of the gradient (the A1 fraction) showed two subfractions, one eluting near the void volume and one partitioned by the gel. Both fractions resembled those of the nucleus pulposus and the anulus fibrosus in the number of components seen on agarose-polyacrylamide gel electrophoresis. Both fractions changed with ageing/degeneration; the ratio of keratan sulphate to chondroitin sulphate, which was about 1 in group 1, increased to about 3 in group 2; the hydrodynamic volumes fell; the electrophoretically distinguishable component of lowest mobility disappeared while new, highly mobile components appeared; and the water content decreased slightly. Clearly, the PGs of the CEP of degenerated intervertebral discs differed from those of healthy discs; this supports the view that the CEP participates in the process of ageing/degeneration in the disc.

Magnetic resonance imaging reflects the chemical changes of aging degeneration in the human intervertebral disk.

T2 weighed spin echo magnetic resonance images (MRI) of the intervertebral disks of 4 lumbar spines were graded and the nuclei pulposi were analyzed for water, collagen and proteoglycan. The brightness of the nuclear image correlated directly with the proteoglycan concentration, but not with the water or collagen. The dark midnuclear cleft had a collagen concentration slightly higher and a water concentration slightly lower than the adjacent zones; no corresponding differences in proteoglycan were seen, although the relationship with MRI grade was confirmed.

Assay of keratan sulfate as anion-exchanger bound hexose.

An assay for keratan sulfate in papain digests of human intervertebral disc and other tissues has been developed. The digest is applied to the acetate form of a tertiary amine acrylic anion-exchange resin, the oligosaccharide hexose is removed by washing the resin with 0.2 M sodium acetate buffer pH 5.0, then the keratan sulfate is eluted quantitatively with 1.0 M pyridinium sulfate pH 2.5 and assayed for hexose by the anthrone reaction. The keratan sulfate content of human intervertebral disc tissues ranged from 7 to 78 mumole galactose equivalents/g fresh weight; the root mean square error was 2 mumole/g; 10-25 mg of tissue were required. The separation of oligosaccharides from keratan sulfate was confirmed by gel permeation chromatography, sugar composition, ester sulfate analysis, and nuclear magnetic resonance.

Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc.

A five-category grading scheme for assessing the gross morphology of midsagittal sections of the human lumbar intervetebral disc was developed. The ability of three observers to categorize a series of 68 discs with a wide spectrum of morphologies established the comprehensiveness of the classification. Three independent observers tested the reproducibility of the procedure by assignment of grades blindly to duplicate images of 68 discs taken from 15 spines. The intraobserver agreement ranged from 87 to 91%. The interobserver agreement was 61, 64, and 88% for the three pairs, the two low values being attributable to the bias of one observer. The agreement between the assigned and average grades was 85, 92, 68, 90, and 76% for Grades I through V, respectively. Except for Grade III, the disagreements were attributable mainly to the bias of one observer. Both the increase in the grade with age and the finding that all the discs within 14 of 15 spines had a narrow range of grades demonstrated the biologic credibility of the scheme.

Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc.

The link proteins of the human intervertebral disc were studied in tissue extracts by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, using a specific monoclonal antibody. Three link proteins were detected, corresponding in electrophoretic mobility to those present in articular cartilage. As with articular cartilage, the largest link protein predominates in the young, whereas in the adult the smallest link protein is equally abundant and internal fragmentation of the link proteins occurs. Only in the newborn is the quantity of extractable link protein comparable to that from articular cartilage. In the adult, the disc contains much less link protein than is present in autologous articular cartilage. Neither the amount nor heterogeneity of the link protein differs among different levels within the lumbar spine, although the proportions of the three proteins can differ between the anulus fibrosus and nucleus pulposus. The anulus always contained more extractable link protein relative to tissue wet weight than the nucleus, and the nuclear link protein, at least in adolescents, contained a greater proportion of the smallest link protein. Such changes in the quantity and structure of the disc link proteins may affect the properties of the proteoglycan aggregates and, thus, could influence disc function.

The heterogeneity of the non-aggregating proteoglycans of the human intervertebral disc.

Non-aggregating proteoglycans of differing average hydrodynamic volumes were prepared from nuclei pulposi and anuli fibrosi of three human lumbar spines and characterized by biochemical and immunochemical analyses. The hexose-to-hexuronate and protein-to-hexuronate ratios increased with decreasing hydrodynamic volume. Analysis by composite agarose/polyacrylamide-gel electrophoresis has demonstrated two aggregating subpopulations [McDevitt, Jahnke & Green (1982) Trans. Annu. Meet. Orthop. Res. Soc. 7, 50]. In the present study, electrophoresis of the non-aggregating pools has shown three additional subpopulations, here named bands III, IV and V. The two smallest proteoglycan pools from each tissue contained two and three components respectively. These components were isolated by preparative electrophoresis and analysed. Band III was a proteoglycan richer in keratan sulphate than in chondroitin sulphate; band IV was a proteoglycan richer in chondroitin sulphate than in keratan sulphate; band V contained only chondroitin sulphate. Unsaturated disaccharides prepared from the chondroitin sulphate of all bands were predominantly 6-sulphated, with only 5-15% 4-sulphated. The molecular masses of the chondroitin sulphate and keratan sulphate did not differ between the bands. The amino acid composition of band III differed from that of band IV. Thus three distinct subpopulations of non-aggregating proteoglycan were demonstrated in the human intervertebral disc.

Degeneration and the chemical composition of the human lumbar intervertebral disc.

Fifteen lumbar spines were collected postmortem. The intervertebral discs were assigned morphological grades and were analyzed for water, collagen, and proteoglycan. In order of increasing degeneration, five grade 0, four grade 1, 45 grade 2, 12 grade 3A, and nine grade 3B discs were identified. The proteoglycan concentration fell progressively with increasing grade, although the concentrations of each component overlapped extensively among the grades. Grade 2 discs showed no consistent differences from adjacent grade 3A or 3B discs in the same spine. All discs in seven of the eight spines with grade 3A or 3B discs and all discs in three spines with no grade 3A or 3B discs had proteoglycan concentrations below 52 mg/g fresh weight. Four of five spines with at least one disc of proteoglycan concentration above this value contained no grade 3A or 3B discs. These observations support the hypothesis that low proteoglycan concentrations in all the discs of a spine precede degeneration.

Concentration of plasma albumin in its accessible space in postmortem human dermis.

This study was designed to measure the effective concentration of plasma albumin in the interstitial space of human dermis. Discs of tissue taken postmortem from four donors have been separately analyzed for their content of plasma albumin and equilibrated with 125I-labeled monomeric plasma albumin in a specially designed cell which limited tissue swelling. The equilibrated discs and their surrounding fluid were assayed for radioactivity and the tissue space accessible to albumin was calculated after correction for swelling. The albumin content of serum was also measured. The concentration of albumin in the accessible space of the tissue ranged from 0.45 to 0.93 that in serum, averaging 0.68. The fraction of the total interstitial fluid accessible to albumin averaged, for three normal dermises, 0.35 and for an overhydrated specimen, 0.51. Thus, the effect of volume exclusion should be considered in measurements of the concentrations of plasma proteins in tissue.

Demonstration of link protein in proteoglycan aggregates from human intervertebral disc.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

Target tissue models: the proteoglycans and degeneration of the human intervertebral disc.

Seventy-five intervertebral discs from 15 spines were analyzed after morphologic grading. With degeneration, the water and proteoglycan (PG) contents decreased and the collagen content increased. The PG was isolated from 20 discs by single-stage dissociative density gradient ultracentrifugation. Neither the chondroitin 6-sulphate nor the protein contents of the PG changed with degeneration although the keratan sulphate fell slightly.

Elastic fibers in human skin: quantitation of elastic fibers by computerized digital image analyses and determination of elastin by radioimmunoassay of desmosine.

The elastic fibers in the skin and other organs can be affected in several disease processes. In this study, we have developed morphometric techniques that allow accurate quantitation of the elastic fibers in punch biopsy specimens of skin. In this procedure, the elastic fibers, visualized by elastin-specific stains, are examined through a camera unit attached to the microscope. The black and white images sensing various gray levels are then converted to binary images after selecting a threshold with an analog threshold selection device. The binary images are digitized and the data analyzed by a computer program designed to express the properties of the image, thus allowing determination of the volume fraction occupied by the elastic fibers. As an independent measure of the elastic fibers, alternate tissue sections were used for assay of desmosine, an elastin-specific cross-link compound, by a radioimmunoassay. The clinical applicability of the computerized morphometric analyses was tested by examining the elastic fibers in the skin of five patients with pseudoxanthoma elasticum or Buschke-Ollendorff syndrome. In the skin of 10 healthy control subjects, the elastic fibers occupied 2.1 +/- 1.1% (mean +/- SD) of the dermis. The volume fractions occupied by the elastic fibers in the lesions of pseudoxanthoma elasticum or Buschke-Ollendorff syndrome were increased as much as 6-fold, whereas the values in the unaffected areas of the skin in the same patients were within normal limits. A significant correlation between the volume fraction of elastic fibers, determined by computerized morphometric analyses, and the concentration of desmosine, quantitated by radioimmunoassay, was noted in the total material. These results demonstrate that computerized morphometric techniques are helpful in characterizing disease processes affecting skin. This methodology should also be applicable to other tissues that contain elastic fibers and that are affected in various heritable and acquired diseases.

The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.

Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma.

Characterization of collagenous meshworks by volume exclusion of dextrans.

The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754--1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.

Distribution of glycosaminoglycans across the normal and the scoliotic disc.

This paper reports on our investigations of the glycosaminoglycans (GAGs) (hyaluronic acid, chondroitin-6-sulfate, and keratan sulfate) of 13 adjacent zones of normal and scoliotic discs obtained at necropsy. The isolation and separation of these GAGs on the microgram scale was achieved using the different solubilities of the calcium and cetyl pyridinium chloride complexes. In normal discs the distribution of these GAGs is symmetric about the nucleus pulposus where their concentration is highest, and lowest in the outer annulus regions. A shift in this distribution profile was observed for the GAGs of the scoliotic discs, particularly at the apex of the curve. On the concave side of this disc the chondroitin-6-sulfate was elevated relative to discs at a lower level. In those annulus zones on the convex side of the curve, the keratan sulfate and hyaluronic acid levels were elevated and chondroitin sulfate depressed. These results suggest the presence in these respective regions of at least two different species of proteoglycans accompanied by elevated hyaluronate levels.

Exclusion of dextrans by meshworks of collagenous fibres.

Insoluble collagen from human dermis was equilibrated in a physiological medium with mixtures of 3H2O and fluorescein-conjugated dextrans of different molecular weights. Dextrans of mol.wts. greater than 10(5) were excluded from a volume of 3.82+/-0.87 ml(S.D.) per g of collagen; dextrans of lower molecular weight occupied a larger volume. The apparent excluded volume was proportional to the weight of the collagen. Dansylated albumin behaved similarly to dextran; the polymeric collagen from rat skin exhibited a much larger excluded volume than the insoluble collagen. These results indicated that the volume available to the plasma proteins in human dermis was limited by insoluble collagen as well as by the glycosaminoglycans of the tissue.