V. Pedrini

Age-Related Variations in Proteinpolysaccharides from Human Nucleus Pulposus, Annulus Fibrosus, and Costal Cartilage

Light fraction proteinpolysaccharide was prepared by water extraction of nucleus pulposus, annulus fibrosus, and costal cartilage obtained at autopsy from fifteen skeletally normal subjects ranging in age from four and one half to seventy-six years. Age dependent changes in glucosamine and galactosamine content of the crude tissue and of the light fraction proteinpolysaccharide were demonstrated. A decrease with aging in per cent of total hexosamine extractable in the light fraction proteinpolysaccharide and an increase in protein content was observed in all three tissues. Electrophoresis of the light fraction produced two main components differing in both polysaccharide and protein content.

Proteoglycans of human infant intervertebral disc. Electron microscopic and biochemical studies.

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 corresponding 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 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteoglycans collagen interactions in human costal cartilage

Abstract 1. 1. Adult human costal cartilage was repeatedly extracted using both dissociative and disruptive techniques. A considerable amount of proteoglycans could not be solubilized by these sequential extractions. 2. 2. This non-extractable proteoglycan fraction was solubilized by treating the cartilage residue with bacterial collagenase. In an attempt to define the interactions between the insoluble collagen matrix and the non-extractable proteoglycan, the collagenase-solubilized proteoglycans were extensively purified by gel filtration, carboxymethyl-cellulose chromatography, cesium chloride density gradient ultracentrifugation, electrophoresis and chromatography on cellulose columns in the presence of quaternary ammonium salts. 3. 3. Density gradient ultracentrifugation of collagenase-solubilized proteoglycans in 4.0 M guanidine·HCl yielded low-density and high-density fractions, both containing considerable amounts of hexuronate and hydroxyproline. 4. 4. Chromatography of the high-density fraction on a cellulose column equilibrated with cetyltrimethylammonium bromide yielded a purified proteoglycan fraction containing only a trace of hydroxyproline. The same treatment separated the low-density fraction into two fractions, one eluted with acetate buffer pH 4.0 and the other 1.25 M MgCl 2 . Both fractions contained polysaccharide material and hydroxyproline. These results are thought to indicate the presence of collagen peptides tightly bound to the proteoglycans. 5. 5. Digestion of the acetate-eluted fraction obtained from low-density proteoglycans with testicular hyaluronidase removed most of the chondroitin sulfate chains from the proteoglycan, but the hydroxyproline remained bound to the high molecular weight fraction which contained the protein core of the proteoglycan and keratan sulfate.

Proteoglycans of human scoliotic intervertebral disc

To determine the effects of scoliosis, irrespective of its etiology, on the intervertebral disc, we isolated the proteoglycans from the nucleus pulposus, annulus fibrosus, and cartilaginous end-plates of patients with idiopathic scoliosis, paralytic scoliosis, and cerebral palsy. The chemical composition of the proteoglycans, the proportions of aggregates and monomers, the sizes of the monomers, and the limiting viscosity numbers of the purified preparations were determined. The results showed that: (1) The scoliotic nucleus pulposus contained normal amounts of proteoglycan aggregates and monomers. The molecules that were isolated from the nucleus pulposus of a sixyear-old patient had a normal composition, but those from two adolescents with scoliosis contained more protein and less carbohydrate than the proteoglycans of normal tissue. (2) The disc level or location of the tissue in the scoliotic curve (concave or convex) did not affect either the amount or the molecular-weight distribution of proteoglycan aggregates from the fibrocartilaginous portion of the annulus fibrosus. (3) The convex portion of the end-plates appeared histologically normal, but in all specimens studied the tissue contained only traces or greatly reduced amounts of proteoglycan aggregates. The monomers were smaller than normal. Premature closure of the growth plate on the concave side of the curve was observed histologically in the only specimen that was available for study (from an eleven-year-old child with a 70-degree lumbar curve) . The proteoglycans extracted from the remaining hyaline cartilage, however, behaved as do their counterparts from normal tissue. CLINICAL RELEVANCE: Proteoglycans have important roles in determining the shock-absorbing ability of the nucleus pulposus, the elastic behavior of the annulus fibrosus, and the resilience to compression of the cartilaginous end-plates. In addition, proteoglycans play a role in the process of endochondral ossification occurring in the growth-plate portion of the end-plate. S Supported by a grant from the Hearst Foundation. t Read in part at the Annual Meeting of the Orthopaedic Research Society, New Orleans, Louisiana, January 19, 1982. Department of Orthopaedic Surgery, Biochemistry Research Laboratory, 180 Medical Laboratories, University of Iowa, Iowa City, Iowa 52242. The results of this study indicate that the scoliotic curve, irrespective of its etiology, produces secondary changes in the proteoglycans of the intervertebral disc that could alter the properties of the tissues and might be important in the progression of the disease. Scoliosis can occur in patients with generalized connective-tissue disorders (Marfan’s disease , osteogenesis imperfecta , Ehler-Danlos syndrome , and others) or in association with paralysis of the trunk, spina bifida, cerebral palsy, or congenital hemivertebra. Most often, however, scoliosis occurs in otherwise normal individuals and it is called “idiopathic”29”2. The intervertebral disc, a major load-carrying component of the spine, is formed by three distinct structures: the nucleus pulposus, the annulus fibrosus, and the cartilaginous end-plates. In a normal spine the nucleus pulposus is located centrally or slightly posteriorly (lumbar region), and in the young it is a gelatinous structure formed by a loose network of collagen fibers embedded in a gel of proteoglycans. Its water content, ranging from 70 to 90 per cent, is highest at birth and decreases with age’3. When a compressive load is applied to the spine, a fluid pressure develops within the nucleus and pushes the annulus fibrosus radially outward and the centers of the end-plates away from each other’3. The mechanical behavior of the intervertebral disc is largely dictated by its two major macromolecular components: proteoglycans and collagen. While the negatively charged groups of the glycosaminoglycan chains are largely responsible for the fluid pressure in the nucleus pulposus”, the collagenous structure of the annulus fibrosus is ideally suited to accommodate the complex stresses caused by compression of the nucleus From a mechanical point of view, the cartilaginous end-plates may be considered the weakest structure of the intervertebral disc. They have been shown to fracture under compressive loads that leave both the nucleus and the annulus intact5’27. Together with the periphery of the annulus they represent, however, the main route for the nutrition of the discs20”4, the largest avascular structures of the body. The end-plates also provide for growth and ossification of the vertebral bodies and are the sites of insertion of the fibers of the annulus fibrosus. During the 816 ANGIOLA PEDRINI-MILLE ET AL. THE JOURNAL OF BONE AND JOINT SURGERY growth years the end-plates are formed by a layer of hyaline cartilage overlying a growth plate. Endochondral growth and ossification occur uniformly across the entire vertebral body until ossification centers appear in the outer regions of the end-plates at around the age of twelve yearsl7. The resulting epiphyseal ring is totally ossified and joined to the body of the vertebra by the age of seventeen to twenty-five years. This coincides with the cessation ofgrowth, although hyaline cartilage persists in the central portions of the end-plates8”0. Normal endochrondral growth i nvolves proliferation, maturation, hypertrophy, and degeneration of chondrocytes leading to calcification of the intercellular matrix. The synthesis of proteoglycans, collagen, and possibly non-collagenous matrix proteins changes in a characteristic fashion at different stages of the process23. In order for these events to proceed normally, an optimum pressure is also required. In 1862 Heuter and Volkmann noted an inverse relationship between compressive forces parallel to the axis of epiphyseal growth and the rate of growth of the cartilage, and suggested that a decrease in compression would lead to an acceleration of growth i:t, This was proved by Hert1’, who also demonstrated that a decrease of the compressive forces beyond a point at which the epiphysis could effectively be considered to be under traction leads to dedifferentiation of the chondrocytes or dosure of the growth plate. Irrespective of its etiology, the scoliotic curve severely affects the mechanical properties of the spine. Structures on the concave side of the curve are subject to much greater loads than are their counterparts on the convex side. In 1973 we reported that the nucleus pulposus of children with idiopathic scoliosis contained more collagen and less glycosaminoglycans than normal tissue, while the composition of the corresponding annulus fibrosus was normal2’. This was confirmed by Zaleske et al. 16, who found the same abnormalities in tissues of patients with scoliosis secondary to myelomeningocele. Normal nucleus pulposus contains only type-Il collagen whereas annulus fibrosus contains both type-I and type-Il collagen, their proportions changing in a gradient-like fashion from the outer zone, where type I predominates, to the inner region, where 70 per cent of the collagen is type II I0#{149} The relative amounts of the two types of collagen have recently been found to be severely altered in scoliotic annuli taken from the convex side of the curve, although a somewhat abnormal pattern of distribution was also seen in the outer and middle regions of the annulus fIbrosus from the concave side2t. The present study of the proteoglycans of the nucleus pulposus, annulus fibrosus, and end-plates of the scoliotic spine was motivated by the involvement of such molecules in determining the mechanical properties of a tissue and by their role in endochondral ossification. Our main purpose was to see whether the uneven distribution of forces caused by the scoliotic curve, irrespective of its etiology, would affect the proteoglycans of the intervertebral disc. Materials and Methods Intervertebral discs were obtained at autopsy, within twelve hours of death, from skeletally normal individuals and at surgery from eight patients. Five patients had idiopathic scoliosis: one girl, six years and six months old, with a 90-degree curve and three adolescent girls and one boy, fifteen to eighteen years old, with curves measuring between 45 and 50 degrees. One patient had paraplegia: a fifteen-year-old boy who had been completely paralyzed below the sixth thoracic vertebra since the age of five years. The thoracolumbar curve. first noted when he was seven years old, remained rather mild and non-progressive up to the age of thirteen years and then increased approximately 50 degrees in the sixteen months preceding surgery, when it measured 90 degrees. Two girls had cerebral palsy: one fifteen-year-old girl with a right lumbar curve of 70 degrees and one eleven-yearold girl with a rapidly progressing curve of 74 degrees.

Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus

Human nucleus pulposus and annulus fibrosus, obtained at autopsy from patients 7-30 years of age, were extracted with 2 M guanidine-HCl (pH 5.82) to remove proteoglycans, then stirred with pepsin in 0.5 M acetic acid, followed by three 24-h extractions with 1 M NaCl (pH 7.5) and one 24-h extraction with 2 M KSCN (potassium thiocyanate) (pH 7.2). Pepsin and NaCl solubilized an average of about 30% of nucleus pulposus collagen and 18% of annulus fibrosus collagen. KSCN extracted a further 34% of nucleus pulposus collagen and only 4% of annulus fibrosus collagen. CM-cellulose chromatography of nucleus and annulus collagen purified from the pepsin, NaCl and KSCN supernatants consistently revealed only one peak, always appearing slightly ahead of the alpha1 position for rat tail tendon type I collagen. Polyacrylamide and SDS-gel electrophoresis consistently revealed only one band with the mobility of alpha1 chains. Amino acid composition of collagen from nucleus and annulus is comparable to those of mammalian and avian cartilage type II collagen, and distinctly different from those of rat tail tendonand guinea pig skin type I collagens. Periodate oxidation of nucleus and annulus collagens showed that 81% and 67%, respectively, of the hydroxylysine residues survive treatment, compared to 71% for bovine articular cartilage collagen and 17% for guinea pig skin collagen. Total hexose analysis revealed 1.8 muM and 2.0 muM hexose per muM periodate-stable hydroxylysine in nucleus and annulus collagens, respectively. Ion exchange chromatography showed the presence of glucose and galactose in a ratio of 0.92:1 in nucleas collagen and 1.07:1 in annulus collagen. Pepsin-solubilized, NaCl-extracted collagen from nucleus and annulus formed native-type fibrils in vitro. The banding patterns of ATP-induced segment-long-spacing precipitates of nucleus and annulus collagens were identical to each other and indistinguishable from those of cartilage (type II) collagen, but distinctly different from those of rat tail tendon (type I) collagen. These data suggest that the collagen which can be extracted after limited pepsin attack of human nucleus and annulus is of the form [alpha1 (II)]3.

Pseudoachondroplasia: biochemical and histochemical studies of cartilage.

Cartilage from patients with pseudoachondroplasia is characterized by unique inclusions in the cisternae of the endoplasmic reticulum and proteoglycan abnormalities have been suggested in this form of dwarfism. To elucidate the nature of the proteoglycan defect, we determined the amount of the individual glycosaminoglycans present in iliac-crest cartilage of three patients and extracted the proteoglycan monomers from one of the samples. Sections of iliac-crest cartilage and proximal fibular growth plates were examined by electron microscopy and also stained with hematoxylin and eosin, safranin O-fast green, and alcian blue in the presence of increasing concentrations of magnesium chloride (zero to one molar). The chondrocytes of the iliac crest and fibular physes were arranged in clusters more than in columns and contained characteristic endoplasmic reticulum inclusions, which were particularly large in the hypertrophic cells. The cartilage stained very poorly with hematoxylin and eosin and with safranin O-fast green. The alcian-blue stain was abolished from perilacunar areas and from longitudinal septa by magnesium chloride concentrations that were lower than those required by normal tissue. The proteoglycans of iliac-crest cartilage were found to be significantly enriched in keratan sulphate and had a below-normal ratio of chondroitin-4-sulphate to chondroitin-6-sulphate, although the amount of the two isomeric chondroitin sulphates combined was within normal limits. The urinary excretion of glycosaminoglycan by the three patients was normal. Pseudoachondroplasia appears to be a generalized cartilage disorder involving abnormalities of proteoglycans, probably related to the core protein or to enzymes that are responsible for the formation of the glycosaminoglycan chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease of enzymatic synthesis of hexosamine in epiphyseal plates of aminoacetonitrile-treated rabbits.

Summary Enzymatic synthesis of hexosamine from glutamine and glucose-6-phosphate in epiphyseal plates of normal and lathyric rabbits has been studied. In the cartilage from experimental animals a decrease of 75.92% of this synthesis was found. AAN added in vitro did not have any inhibitory effect upon formation of hexosamine.

Multiple Epiphyseal Dysplasia in Two Siblings: HISTOLOGICAL AND BIOCHEMICAL ANALYSES OF EPIPHYSEAL PLATE CARTILAGE IN ONE

Two siblings with multiple epiphyseal dysplasia and slipped capital femoral epiphysis are reported. In one an osteosarcoma developed in the femur necessitating amputation.The tibial growth plates of the amputated extremity were profoundly disorganized, with disordered cartilage-cell columns, clefts,

Mucopolysaccharide Defect in Experimental Lathyrism.

Summary The hexosamine content of bone callus of aminoacetonitrile-treated rats has been studied 10 and 17 days after fracture and has been found to be decreased by about 50% as compared to controls. The decrease has been found to be due wholly to galactosamine, while glucosamine content remains the same. On the basis of these and of previously available data it is concluded that only Ch-4-SO4 and Ch-6-SO4 are affected by the AAN intoxication. It has also been confirmed that the solubility of collagen in the lathyritic bone callus is greatly increased.

Chemical studies on the ground substance of human epiphyseal-plate cartilage.

Six samples of human epiphyseal cartilage obtained from skeletally normal children, from one day to thirteen years old, were studied. The collagen and mucopolysaccharide content was rather constant in epiphyseal-plate cartilage of older children, but in the cartilage from the new born infant the amount of galactosamine was significantly increased. Light protein polysaccharides were extracted from three of the samples. In each case, both chondroitin sulphate and keratosulphate were present in the light protein-polysaccharide complex. The ratio of these substances seemed to be age-dependent. The amino acid composition was very similar to that of other light protein-polysaccharide preparations described in the literature. By electrophoresis, the light protein-polysaccharide fraction did not behave as an homogenous compound. Prior to any enzymatic digestion or alkaline degradation, the light protein-polysaccharide complex in each case could be resolved in two well defined bands by electrophoresis. The heavy protein-polysaccharides complex had a very low amino sugar content, which increased slightly in the samples obtained from older children.