PROTEOGLYCANS OF HUMAN INFANT INTERVERTEBRAL DISC

The ground substance of the intervertebral disc consists primarily of proteoglycans, which give the tissue its stiffness to compression and its resiliency. To investigate the structure and composition of these molecules, we extracted them from human infant nucleus pulposus under associative conditions and from human infant annulus fibrosus and cartilage end-plate under dissociative conditions. We examined the degree of aggregation, the composition, the electron microscopic appearance, and the dimensions of the proteoglycans of the intervertebral disc and compared their structure and dimensions with those of the proteoglycans from bovine hyaline cartilage. Aggregates represented 52 per cent of the proteoglycans of the nucleus pulposus between the ages of one and ten days but only 28 per cent between the ages of six and eight months. Preparations from the correspondlug annuli contained 59 per cent aggregates at one to ten days and 47 per cent at six months. The corresponding cartilage end-plate preparations contained 45 and 40 per cent aggregates The proteoglycans of the annulus fibrosus and cartilage end-plate contained more protein and less hexosamine than did those of the nucleus pulposus. Electron microscopy showed that approximately two-thirds of the aggregates from nucleus pulposus consisted of very short hyaluronate filaments with closely packed monomers. The other third had longer hyaluronate filaments and wider distances between monomers, and closely resembled the aggregates from the annulus fibrosus and cartilage end-plate. Aggregated monomers consisted of two segments: a thin segment connecting directly to the hyaluronic acid filament and a thick segment extending peripherally from the thin segment. The thin segment formed about 12 per cent of the total monomer length in the samples from all three disc tissues. The lower proportion of aggregated monomers, the lower protein content, and the smaller aggregates with closely packed monomers suggest that the nucleus pulposus may contain less link protein than do the annulus fibrosus and cartilage end-plate. Compared with proteoglycan aggregates from bovine hyaline cartilage, proteoglycan aggregates from human intervertebral disc were shorter and had fewer monomers and wider spacing between monomers. The * Department of Orthopaedics, University of Iowa College of Medicine, Iowa City, Iowa 52242. aggregated monomers from the three components of the intervertebral disc had an average length of 209 ± 90 nanometers, compared with 210 ± 114 nanometers for monomers from hyaline cartilage of skeletally mature cows, 250 ± 116 nanometers for monomers from hyaline cartilage of skeletally immature calves, and 288 ± 108 nanometers for monomers from fetal animals. The monomer length in the intervertebral disc varied less than that in mature and immature cartilage but more than that in fetal cartilage. The average length of the thin segment of disc monomers was significantly shorter than the average length of the thin segment in any population ofproteoglycan monomers from bovine cartilage, perhaps reflecting the greater concentration of keratan sulphate in monomers in intervertebral disc. CLINICAL RELEVANCE: The mechanical function of the intervertebral disc depends primarily on its extracellular matrix. Abnormalities of disc function stem from or are reflected in alterations of the composition and organization ofthe matrix. Since the upright posture of humans imposes particular demands on the discs and may result in particular disorders, the examination of human discs provides information that is not obtainable from animal tissue. This study demonstrated that the proteoglycans in the discs of human infants share their basic structure with proteoglycans from hyaline cartilage, but differ in their dimensions. Furthermore, the proteoglycan population in the annulus fibrosus and cartilage end-plate differs from the proteoglycan population in the nucleus pulposus. This documentation of the proteoglycan structure of the disc of the human infant forms the basis for future examination of changes with age in disc proteoglycans that may produce or accompany disorders of human intervertebral discs. The mobility and stability of the human spine depend to a large extent on the intervertebral discs. In early life, the discs consist of two distinct components: the peripheral fibrous annulus fibrosus and the central gelatinous nucleus pulposus4”6’2527. On the cranial and caudal surfaces, a cartilage plate caps the disc and covers the ends of each vertebra. With increasing age, fibrocartilage gradually replaces the nucleus pulposus, ultimately transforming the intervertebral disc into a fibrocartilaginous plate4’626. Differentiation of these age-related changes from disc degeneration or disease has proved difficult’ , but regardless of their etiology these changes alter the mechanical properties PROTEOGLYCANS OF HUMAN INFANT INTERVERTEBRAL DISC 285 VOL. 67-A, NO. 2, FEBRUARY 1985 of the spine and may produce clinical syndromes. Thus, knowledge of the basic structure and composition of the disc forms the foundation for progress in understanding many problems of the spine. The tissues of the disc consist of relatively few cells and an abundant extracellular matrix. Collagen and proteoglycan form the macromolecular framework of the disc matrix and largely determine its mechanical properties4-’2-23-28. In hyaline cartilage, proteoglycans exist on two levels of organization: subunits or monomers composed of protein core filaments with multiple covalently bound chondroitin and keratan sulphate chains; and aggregates large molecules composed of multiple subunits, non-covalently associated filaments of hyaluronic acid, and link proteins. Previous studies of proteoglycans from the nucleus pulposus suggested that they consist of the same components as do proteoglycans from hyaline cartilage’-2’2-’4’7, and that proteoglycan aggregates exist as part of the disc. However, gel-chromatography and sedimentation-velocity studies have indicated that the proteoglycan monomers and aggregates from the nucleus pulposus are smaller than analogous molecular species from hyaline cartilage214.23.24 Furthermore, monomers from the nucleus pulposus have more keratan sulphate, longer keratan sulphate chains, less chondroitin sulphate, and shorter chondroitin sulphate chains than do monomers from hyaline cartilage23-24. These previous studies demonstrated important chemical and physicochemical differences between the proteoglycans of the nucleus pulposus and those of hyaline cartilage, but they did not demonstrate the structural basis for these differences. However, electron microscopic monolayer methods make it possible to study the form and dimensions of individual proteoglycan molecules6 10.2 I .22 and elucidate the structural differences among proteoglycan populations from different tissues. In this investigation we examined the composition, degree of aggregation, and molecular architecture of proteoglycans from human infant nucleus pulposus, annulus fibrosus, and cartilage end-plate. This allowed comparison of proteoglycans from the nucleus pulposus with those from the annulus and cartilage end-plate and with previously studied proteoglycans of hyaline cartilage. Furthermore, our study forms the basis for future study of age and diseaserelated changes in proteoglycans from the disc.