The distribution of calcific deposits in intervertebral discs of the lumbosacral spine.

The incidence of intervertebral disc calcifications (IVDCs) was examined in 52 lumbosacral spines obtained sequentially at autopsy. The presence of calcific deposits was detected by fine-grain roentgenograms. The nature of these deposits was determined by wide-angle x-ray diffraction, and histologic observations were made. A high prevalence of IVDC, 18 spines of 52, some with multiple deposits, was noted. Calcium pyrophosphate dihydrate (CPPD) deposits were found in 3% of the spines and accounted for 29% of the 42 deposits analyzed. The CPPD deposits occurred at multiple disc levels (an average of four per spine), were diffuse, and involved a major portion of the disc (nucleus pulposus, annulus fibrosus, and endplate) but were not generally associated with histologic disc degeneration. Hydroxyapatite (HA) deposits occurred in 12% of the spines, most often in the nucleus pulposus and endplate. The HA deposits appeared as small punctate radiodensities. Roentgenographic evidence of degenerative changes, i.e., disc space narrowing, endplate disruption, desiccation, and osteophyte formation, were present in all but one of the spines containing HA deposits. An additional 19% of the spines had deposits that could not be characterized by x-ray diffraction but were very similar in roentgenographic appearance to HA deposits. No conclusions could be drawn on the relationship between the presence of HA or CPPD and collagen or hexosamine content.

Keratan sulphate: a functional substitute for chondroitin sulphate in O2 deficient tissues?

Keratan sulphate and chondroitin sulphate can each fill space and exert swelling pressure in collagenous fibrillar matrices, but whereas the former is synthesised from glucose precursor without consuming NAD, the latter converts 2 mols of NAD for each uronate residue in the polymer chain. We suggest that the observed distribution of keratan sulphate and chondroitin sulphate in cartilage, cornea and intervertebral disc are determined by the ambient oxygen tension, and that keratan sulphate is preferentially synthesised in conditions of oxygen lack. The implications of this hypothesis in the physiology of contact lenses, cartilage degeneration, corneal scar repair and ageing are discussed.

Fibronectin content of the annulus fibrosus in diabetic and non-diabetic sand rats.

Annulus fibrosus of intervertebral discs from diabetic and non-diabetic sand rats were examined by microspectrophotometry for fibronectin content. This was higher in the diabetic animals both in the dorsal and ventral parts and in the outer and inner lamellae of the annulus. It is suggested that diabetes-related changes in fibronectin are similar to changes in annular collagen observed in species other than sand rats.

Age-related changes in fibronectin in annulus fibrosus of the sand rat (Psammomys obesus).

Fibronectin stains were carried out and evaluated by microspectrophotometric techniques in annuli fibrosi of vertebral discs of sand rats (Psammomys obesus) of both sexes and two age groups, 13-18 months and over 2 years of age. The distribution of fibronectin in the annulus shows a centripetal gradient from the outer to the inner laminae. Fibronectin was significantly more abundant in the annuli of old than of young animals of corresponding sex. Sex differences were not significant. The dorsal segment contained more fibronectin than the ventral, but the difference was statistically significant only in the aged females. The outer laminae of the annuli appeared consistently higher in fibronectin content than the inner laminae.

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.

Metaplastic proliferative fibrocartilage as an alternative concept to herniated intervertebral disc.

It is hypothesized on the basis of experimental intervertebral disc degeneration that herniated disc is actually newly synthesized proliferative metaplastic fibrocartilage and not herniation of pre-existing disc tissue, particularly that of the nucleus pulposus. Human material in selected surgical tissues was examined to test this concept. Histology revealed evidence for proliferative fronts of fibroblastic cells in herniated discs with hypocellular interiors. Hydroxypyridinium cross-link assay was used to determine the maturity of the collagen. Results indicated, with statistical significance, that herniated disc is a younger tissue than in situ annulus fibrosis, and that herniated disc is not from the nucleus pulposus, thus supporting the hypothesis.

Cervical spine anatomy and pathologic processes. Applications of new MR imaging techniques.

Important information regarding the MR appearance of the cervical spine can be obtained using spin-echo and gradient-pulse sequences with motion reduction techniques and intravenous gadolinium-DTPA. In this article the normal MR appearance of the cervical spine is presented, using these newer techniques. Some artifacts and pathologic conditions that may alter the normal appearance of the cervical spine are illustrated.

Collagen stability and cross-linking in normal and kyphoscoliotic mouse intervertebral discs.

Intervertebral discs of the cervical-thoracic region of the spine of BDL mice which are homozygous for the ky gene mutation undergo degeneration. Discs from these mice have a normal collagen content and undergo normal collagen cross linking prior to the appearance of degenerative changes. The major reducible collagen cross-link formed in discs of these mice and in normal CBA strain mice is hydroxylysino-5-ketonorleucine. These results and other previous results indicate that the discs in the ky mouse develop degenerative disease due to an extrinsic factor rather than to an intrinsic abnormality of their extracellular matrix. The extrinsic factor has been identified as spinal muscle atrophy.

Identification of IgG in the canine intervertebral disc.

Nucleus pulposus was taken at necropsy from five mongrel dogs and tested in an enzyme linked immunoabsorbent assay (ELISA) system designed to indicate the presence of IgG and IgM. This assay positively identified IgG. Agarose-bound Protein A was used to treat the nucleus pulposus material to extract the IgG. Repeating the ELISA using Protein A-treated nucleus pulposus showed that the ELISA was greatly diminished in activity, confirming the presence of whole molecules of IgG in nucleus pulposus. Investigators have postulated an immunologic basis for spinal pain syndromes. None to date has demonstrated IgG in the nucleus pulposus. This IgG may be a mediator by which an inflammatory response is activated which contributes to the clinical picture of chronic back and radicular pain.

The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-beta-D-galactosidase digestion.

The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-beta-D-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.

Localization of different alcian blue-proteoglycan particles in the intervertebral disc.

A histochemical investigation was carried out on proteoglycans of bovine intervertebral disc. Samples obtained from the "annulus fibrosus" (A.F.) and "nucleus pulposus" (N.P.) were treated with Alcian blue (AB) diluted in solutions of MgCl2 at critical electrolyte concentrations (CEC); some samples were incubated in testicular hyaluronidase before AB treatment. At least four types of elongated AB-proteoglycan particles were recognized: a) in A.F. lamellae and N.P., 1 nm rod-like particles were arranged orthogonally to the collagen fibrils and spaced at a distance equivalent to the fibril D-period (Figs. 1-5); b) within the A.F. lamellae, other 16-20 nm particles formed a close network among the collagen fibrils (Figs. 1,2,3,5); c) in the A.F. interlamellar crevices, 30-50 nm leaf-like particles were present (Fig. 6); d) in the N.P.Z., 20-30 nm leaf-like particles formed a wide-mesh (Fig. 4). The alcianophylic particle sizes suggest they may correspond to proteoglycan monomers in the A.F. lamellae and mostly proteoglycan aggregates in A.F. interlamellar crevices and N.P.. Both alcianophylia degrees at MgCl2 CEC solutions and enzymatic susceptibility indicate the presence of chondroitin sulphate and keratan sulphate and that the large particles in the A.F. interlamellar crevices are the keratan sulphate richest proteoglycans. The features of the observed AB-proteoglycan particles are consistent with previous morphological data reported for other tissues as well as some biochemical data for the intervertebral disc and may be correlated to the composite mechanical properties of this tissue.

Universal spondylodiscitis in a patient with erosive peripheral arthritis and apatite crystal deposition.

A patient with erosive peripheral arthritis in whom vasculitis and monoclonal IgG kappa paraprotein were associated with sacroiliitis and widespread destruction of intervertebral discs is reported. Crystals resembling apatite were identified in intervertebral disc material, and we postulate that the discitis was accelerated by apatite deposition. Our case illustrates a unique example of axial involvement in rapidly progressive joint disease.

Measure-set computed tomographic analysis of internal architectures of lumbar disc. Clinical and histologic studies.

Measure-set computed tomography (MSCT) scan was performed in 30 adult subjects (17 men and 13 women, mean age 42 years) and eight cadavers (five men and three women, mean age 42 years) to establish prospectively a normal CT pattern of the internal architectures of the lumbar disc. Based on the uniformity or nonuniformity of MSCT composition of the nucleus pulposus (NP) and annulus fibrosus (AF), and also on how well NP was discerned from the surrounding AF, the internal architecture could be categorized into well-defined (WD), intermediate (IM), and ill-defined (ID) types. An age and prevalence study of the categorized disc showed that, with age, the disc undergoes an architectural transformation from WD through IM to ID. In addition, 72.5% of WD-type discs and 72.1% of ID-type discs occurred in the third and fourth decades and in the sixth and seventh decades, respectively, suggesting that the former is a young form with mild degeneration and the latter an old form with advanced degeneration. Fifty-eight percent of WD-type discs and 60.4% of ID-type discs occurred at L1-3 and L4-S1 levels, respectively, regardless of age. This finding suggests the old form tends to occur at the lumbosacral junction, which is the site of maximum weight bearing in humans. The correlation of water content and histologic findings of degeneration with the three categorized disc types in cadavers showed that WD, IM, and ID discs were associated with the largest, intermediary, and least amount of water of NP, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine proteinase inhibitors of the canine intervertebral disc.

Several species of cysteine proteinase inhibitors have been demonstrated in the greyhound intervertebral disc which were resolved into four species (Mr 15,800, 16,600, 17,200 and 17,800) by gelatin-SDS-polyacrylamide gel electrophoresis. Reductive alkylation did not affect their inhibitory capability nor their electrophoretic migration on gelatin-SDS-polyacrylamide gel electrophoresis. The cysteine proteinase inhibitors from the nucleus pulposus and annulus fibrosus were identical as assessed by the aforementioned criteria, although the level in the nucleus was found to be higher than that in the annulus. Ion-exchange chromatography demonstrated distinct acidic and basic forms of the disc cysteine proteinase inhibitor. The latter species was the most abundant and its Mr was determined to be 16,900 by gelatin-SDS-polyacrylamide gel electrophoresis. Both forms were shown to be strongly inhibitory against the cysteine proteinases, papain and ficin, but were less strongly inhibitory against cathepsin B (EC 3.4.22.1). Presumably these disc cysteine proteinase inhibitors play a regulatory role in the metabolism of proteoglycans and collagen by endogenous cysteine proteinases.

The carboxypropeptide trimer of type II collagen is a prominent component of immature cartilages and intervertebral-disc tissue.

Immature bovine cartilages and intervertebral-disc tissue all revealed a prominent protein, not present in the adult tissues, in non-denaturing extracts made with chondroitin ABC lyase (EC 4.2.2.4), Streptomyces hyaluronidase (EC 4.2.2.1) or 1 M NaCl. The protein ran on SDS-polyacrylamide electrophoresis, before disulphide reduction, as a close doublet of bands of apparent molecular weight 110,000 and 105,000. After reduction, they dissociated respectively into two protein bands at 37,000 and 35,000, indicating that the initial molecules were disulphide-bonded trimers. Amino-terminal sequence analysis established the identity of both proteins (Mr 110,000 and Mr 105,000) as forms of the carboxypropeptide of type II collagen. The larger molecule appeared to be the trimer of intact alpha 1(II) carboxypropeptides and the smaller, a version composed of chains that were ten residues shorter at their amino-terminal ends. The material appears to be identical to chondrocalcin, a protein previously found to be enriched in fetal growth plate and named on the basis that it may play a role in cartilage calcification. The present findings, however, indicate that the protein is equally abundant in all type II collagen-synthesizing young cartilages, including nucleus pulposus of the intervertebral disc and other cartilages that never calcify.

A pathological study of experimental chemonucleolysis with collagenase.

This study was undertaken to examine and quantitate the chemical injury and repair of the dog nucleus pulposus in collagenase-induced chemonucleolysis. Thirty three adult mongrel dogs were used into which the purified collagenase was injected at a rate of 200 units. The dogs were sacrificed after roentgenography. Disc specimens were stained primarily with safranin-O for microscopic study, and fresh tissues were used for electron microscopic study. For immunohistochemical analysis, paraffin sections were reacted with anti-bovine types I, II and III collagen antibodies. Two weeks after the injection, the height of the injected disc was reduced by about 50% and there was no sign of recovery for 52 weeks. There was a profound loss of safranin-O stainability in the nucleus pulposus, annulus fibrosus and cartilagenous endplate. By 8 weeks, regeneration of the nucleus pulposus began and chondrocytes appeared from the junctional area. The regenerated nucleus pulposus was stained well with safranin-O and consisting largely of type II collagen immunohistochemically. In the regenerated nucleus pulposus, collagen fibers and proteoglycans were reconstructed in a net-work with hyaluronic acids.

Water content in human intervertebral discs. Part I. Measurement by magnetic resonance imaging.

Tension-relaxation experiments were performed on human disc lamellae specimens. The water content was found to affect the viscoelastic behavior and a master relaxation curve was constructed from the experimental data. The water content of disc phantoms is measured by magnetic resonance imaging (MRI) techniques. MRI was used to compare the discs of patients of different ages. The possibility of obtaining cross-sectional water distribution in human intervertebral disc material using MRI techniques and its relation to the disc's mechanical properties was explored, with the goal of constructing a realistic mathematical model of the disc which takes into account the water content of the disc.

Water content in human intervertebral discs. Part II. Viscoelastic behavior.

Water content of intervertebral discs is a significant aspect of both viscoelastic behavior and age-related degenerative changes. Using water content as a dependent variable, stress-relaxation was measured using standardized anulus fibrosus specimens strained at various levels of strain. Synthesis of experimental data into a master relaxation curve allows prediction of specimen response over time intervals not readily accessible experimentally. A quantitative understanding of the role of water content may have important clinical application, since magnetic resonance imaging is a tool which should allow water content determination in vivo.

Low molecular weight serine proteinase inhibitors of the human intervertebral disc.

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.

[A histochemical study of acid glycosaminoglycans on normal and pathological cartilage, ligaments and several other connective tissues].

Histochemical estimation of acid glycosaminoglycan was critically re-evaluated, using the meniscus, intervertebral disc, ossified yellow ligament, ganglion, Dupuytren's fascia and several other tissues. Each tissue was stained with toluidine blue, alcian blue, high iron diamine, low iron diamine, aldehyde fuchsin and dialyzed iron-ferrocyanide. Digestion techniques for GAG were used in these staining methods, and the effects of protease inhibitors (PI) on digestion were also examined. In this study, the optimal temperature for digestion with Streptomyces hyaluronidase was between 37 degrees C and 43 degrees C, which varied according to the tissue examined. The addition of PI seemed necessary because the enzymatic treatment without PI resulted in an excessive decrease of staining. Protease-free chondroitinase ABC, which did not excessively decrease staining results, was found to be more useful than chondroitinase ABC without PI.