Biochemical anatomy of human bone: comparative study of compact and spongy bone in femur, rib and iliac crest

Using EDTA extraction procedure, compact and spongy bone from human femur, rib and iliac crest were compared in terms of their content in collagen, sialoprotein, proteoglycan and carbohydrate. The bone matrix sizes displayed significant variations, the femur having the smallest size and iliac crest the largest one. No significant difference in the matrix size has been found between the spongy and compact bone. The EDTA extractability of the spongy bone was higher than that of the compact bone, with femur showing the lowest extractability. The collagen content of the 3 bones studied was similar although the femur had slightly lower values. The sialic and uronic acids and hexose contents were higher in the femur than in the rib and iliac crest. The collagen/hexose, collagen/sialic acid and collagen/uronic acid ratios in the bone matrix were highest in the iliac crest and lowest in the femur, suggesting that alterations in the amounts of bone matrix can affect the mechanical properties of different parts of the bony skeleton and vice versa.     

A comparison of bone matrix and tendon with particular reference to glycoprotein content.

Bone matrix and tendon are compared in terms of their carbohydrate and non-collagenous protein composition. The collagen content of both tissues was similar (90-91%), but bone matrix had at least three times as much sialic acid (0.28%) as tendon (0.08%). Smaller differences were found in the analysis of hexoses and hexosamines. After digestion with bacterial collagenase, about 9% of the total protein from both tissues was non-diffusible on dialysis, and this contained only 0.15% (bone) and 0.7% (tendon) of the original hydroxyproline; recovery of sialic acid was 86-87%. The collagenase-resistant soluble material amounted to about 9% (bone matrix) and 5% (tendon); the insoluble residues were 1 and 4% respectively. There were clear differences in the carbohydrate contents of the digests, but the amino acid compositions were similar. When the soluble digests were chromatographed on DEAE-cellulose, the elution profiles indicated the presence in each tissue of a variety of glycoproteins and a proteoglycan fraction, and showed clearly that an acidic glycoprotein corresponding to bone sialoprotein was not present in tendon.     

Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen

Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.     

Collagen and non-collagenous proteins in different mineralization stages of human femur

Mineralized tissues exhibit varying degrees of mineralization in different areas within the same bone. Using the technique of density gradient fractionation, bone powder from the diaphysis of human femur has been separated in different fractions corresponding to the degree of mineralization. Isolated bone fractions were analysed for their content in collagen and non-collagenous proteins. The results showed marked differences between compact and spongy bones, this latter containing higher proportions of little mineralized bone particles than the former (p less than 0.01). The ethylenediaminetetra-acetic acid (EDTA) extractability and the bone matrix size decreased relative to the decrease in specific gravity of bone particles. Among the matrix components of different fractions, sialoprotein consistently increased with the increase in specific gravity while proteoglycan decreased in reverse manner to the increase in collagen. However, in the most mineralized fraction (specific gravity: 2.33 g/cm3), the proteoglycan amount increased while collagen decreased. In conclusion, this study of bone maturation in human femur confirms the suitability of the technique of density gradient fractionation in the studies of bone matrix-mineral interactions. Apart from the fraction with the highest specific gravity, the analytical results obtained in fractions are similar to those observed in age-related bone changes, suggesting that the increase in mineralization degree of bone particles may be related to their age.     

Sialic acids in bone demineralised in organic solvents

The sialic acid content of bovine shin bone, demineralised by a new process in ethanol-trimetrylammonium EDTA, was measured and compared with that of the original whole bone. New variants of the direct Ehrlich procedure, as well as the periodate-thiobarbituric acid assay, were employed. About 75% of the sialic acid remains in the bone matrix from ethanolic demineralisation, and the PAS reaction is correspondingly much more intense, compared with matrix from aqueous EDTA demineralisation.     

The composition and protein metabolism in the immature rabbit intervertebral disc

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.     

The Isolation of Soluble Proteins,Glycoproteins, and Proteoglycans from Bone

Procedures are described for the isolation from bone of fractions containing proteins, glycoproteins and proteoglycans. Extraction of powdered bone with solutions of the sodium salts of ethylendiaminetetra-acetic acid (EDTA) at pH 7.5 solubilised about 7% of the organic material. These extracts contained about 1.8% of the total collagen and at least 60% of the total non-collagenous protein of bone. The extracts were dialysed against water to remove EDTA and then against a pH 5 buffer. At this stage a precipitate (Cl) formed which was removed by centrifugation. The supernatant was applied to a column of the carboxylio ion-exchange resin, Amberlite CG-50. The effluent at pH 5 contained the proteoglycans and more-acidic glycoproteins and was therefore named the Acidic Fraction (AF). The material adsorbed to the resin (Fraction G2) was eluted by equilibration to pH 8. AF was further fractionated by cetylpyridinium chloride (CPC) precipitation into three relatively pure components:(i) CP-S, a glycoprotein soluble in CPC, (ii) bone sialoprotein (BSP) which formed a CPC precipitate soluble in 0.2M-MgCl₂; and (iii) a proteoglycan fraction which formed a CPC precipitate insoluble in 0.2M-MgCl₂. The G2 fraction contained most of the soluble collagen together with glycoproteins and other non-collagenous proteins. These were fractionated by chromatography on DEAE-cellulose at pH 7.2 using stepwise elution with increasing concentrations of NaCl. Some resolution of the mixture was obtained, though most of the fractions contained more than one component. These procedures have been used on an analytical scale to assess the yields and recoveries of total protein, hydroxyproline and sialic acid in the fractions described above. This has been compared with the large scale procedure for the preparation of the fractions, which have been studied in previous work.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Insoluble non-collagenous cartilage glycoproteins with aggregating sub-units.

Two insoluble non-collagenous glycoprotein fractions (A and G) have been separated from puppy rib cartilage, following extraction of most of the proteoglycan and digestion of the insoluble residue with purified collagenase. After reduction, alkylation and extraction with sodium dodecylsulfate most of each protein is solubilized. Gel electrophoresis of solubilized A or G shows the presence of either one or two bands and gel chromatography shows both high and low molecular weight peaks. The production of a low molecular weight electrophoresis band from the high molecular weight Sephadex fraction indicates that there is aggregation and disaggregation of sub-units in sodium dodecylsulfate. Both A and G are high in aspartate plus glutamate and have a low hydroxyproline content. The insoluble A and G both contain hexose, uronic acid, galactosamine, glucosamine and a small amount of sialic acid, but they differ in their contents of hexose and six amino acids. They both form single bands in CsCl gradients but they differ in density. Electron microscopy shows that both insoluble glycoprotein fractions stain with lead, ruthenium red, or alcian blue plus phosphotungstate and that G contains many fine filaments. Material with the same appearance and staining properties was found to occur on the surface of collagen fibres in the undigested cartilage residue.     

The acid mucopolysaccharides of cattle retina

1. Two polysaccharides were isolated from the interstitial matrix surrounding the photoreceptor cells of cattle retina. They were liberated from this region of the tissue in a soluble form after agitation of whole retinas in 0.9% sodium chloride. One, which comprises two-thirds of the polysaccharides present, is a hyaluronidase-sensitive ;half-sulphated' chondroitin sulphate containing uronic acid, galactosamine and sulphate in the molar proportions 1.27:1.0:0.54. The other is a hyaluronidase-resistant non-sulphated heteropolysaccharide for which the name sialoglycan is proposed. It contains galactose, glucosamine and sialic acid in the molar proportions 2.4:1.0:0.4. Both polysaccharides contain only small amounts of nitrogen in excess of the amount calculated from their amino sugar and sialic acid content. 2. A similar combination of mucopolysaccharides is associated with the pigment epithelial-cell layer but in quantities only one-fifth of those present in the adjacent matrix area. 3. The ease with which they are released into aqueous media is consistent with the assumption that they are present in the extracellular spaces in both of these tissue layers. 4. The retinal residue left after removal of the two soluble polysaccharides is rich in amino sugar- and sialic acid-containing polymers, which appear to be firmly bound to the tissue fragments. 5. About one-third of the sialic acid and one-tenth of the amino sugar could be extracted with chloroform-methanol. The components in this fraction were tentatively identified as gangliosides. 6. Digestion of the chloroform-methanol-insoluble residue with Pronase yielded as the principal product a heteropolysaccharide containing 16.5% of glucosamine, 24.3% of neutral sugar (galactose plus fucose) and 18.1% of sialic acid. This substance has been classified as a sialoglycan of composition similar to (but not identical with) that of the soluble one isolated from the matrix area of the tissue.     

Adaptation of bone and tendon to prolonged hindlimb suspension in rats

The rat hindlimb suspension model was used to ascertain the importance of ground reaction forces in maintaining bone and tendon homeostasis. Young female Sprague-Dawley rats were randomly assigned to either a suspended or a nonsuspended group. After 28 days, femur bones and patellar tendons were obtained for morphological and biochemical analyses. Prolonged suspension induced a significant change in the geometric configuration of the femur middiaphysis by increasing the minimum diameter (12%) without any significant alterations in cortical area, density, mineral, and collagen concentrations. Femur wet weight, length, DNA, and uronic acid concentrations of suspended animals were not significantly different from bones of nonsuspended rats. However, the collagen and proteoglycan concentrations in patellar tendons of suspended rats were 28% lower than the concentrations of matrix proteins in tissues obtained from nonsuspended animals. These data suggest that elimination of ground reaction forces induces alterations in tendon composition and femur diaphyseal shape by changing regional rates in bone remodeling and localized tendon strain. Therefore it appears that ground reaction forces are an important factor in the maintenance of cortical bone and patellar tendon homeostasis during weight-bearing conditions.     

Non-collagenous proteins of predentine from dentinogenically active bovine teeth.

Predentin(e) was dissected out from unerupted permanent bovine teeth. The non-collagenous proteins were extracted at -13 degrees C by 4 M-guanidinium chloride containing proteinase inhibitors and separated by DEAE-Sepharose and Sephadex G-100 chromatography. In addition to a few minor constituents, the only major non-collagenous components that could be demonstrated were albumin and proteoglycan. The localization of the former, demonstrated by optical-microscopical immunochemistry, was such that it was concluded that albumin is not a constituent of predentin matrix. Very low amounts of phosphoprotein were found in predentin matrix. This was of two types, high- and low-phosphorylated. Larger amounts of phosphoprotein were not present until the dissection was carried deeper into newly formed dentin(e). On the basis of the present results and previously obtained morphological data the conclusion was drawn that predentin matrix, containing virtually only collagen type I and proteoglycan, is similar in composition to that of loose connective tissue and primarily aimed at the production and maturation of collagen fibres. Only immediately before the mineralization front are the non-collagenous protein components secreted that initiate and govern calcium-phosphate mineral formation.     

Collagenase and proteinase induction in implants of bone matrix

Enzymes capable of digesting collagen and non-collagenous proteins are present in implants of bone matrix. In the early stages of bone morphogenesis, implants produce relatively large amounts of trypsin-labile proteins and have high non-collagenolytic neutral proteinase and low collagenase activities. Enzymatic activity is maximal three weeks after implantation. The results indicate that increased synthesis of non-collagenous proteins and non-collagenolytic proteinases precedes the induction of significant amounts of collagenases. The importance of these findings in bone morphogenesis is discussed.     

Effects of endosulfan on intestinal functions in protein-malnourished rats

In rats fed 18% protein diet, administration of endosulfan (2mg/kg body weight daily for 7 days) significantly decreased the brush border sialic acid and increased the hexoses contents. The intestinal uptake of glucose was increased while that of glycine and calcium was reduced. Brush border enzymes and lipids were not affected. However, in protein malnourished rats (fed 8% protein) exposed to endosulfan, brush border sucrase and peptidase activities were enhanced, while alkaline phosphatase activity was decreased compared to untreated malnourished animals. Membrane sialic acid content was low while fucose and cholesterol levels were augmented in endosulfan fed malnourished animals. The uptake of glucose and glycine was elevated under these conditions. These results Suggest that the nutritional status of the animals has an important bearing on thc susceptibility of intestinal tissue to endosulfan toxicity in rats.     

Pig vitreous gel: macromolecular composition with particular reference to hyaluronan-binding proteoglycans

The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction. As estimated by gel chromatography on Sepharose CL-2B, hyaluronan presents a polydisperse hydrodynamic behavior with a lower molecular mass (M(r)) value of 220 kDa. The existence of low amounts of a hyaluronan-binding proteoglycan population with structural and immunological characteristics similar to a member of the hyalectan family, versican, has also been demonstrated. The concentration of this versican-like proteoglycan in whole vitreous accounts for 50 microg proteoglycan protein per ml of vitreous gel and represents a minor proportion (about 7%) of the total protein content. The proteoglycan has an average M(r) of 360 kDa and is substituted by chondroitin sulphate (CS) side chains. Study of the CS sulphation pattern showed that the chains were composed of both type 4- and 6-sulphated disaccharide units.     

Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon)

The aim of this study was to provide basic information to allow improved scientific assessment of velvet antlers’ quality by investigating the change of chemical composition depending on antler growth period in sika deer (Cervus nippon). Twelve antlers harvested from sika deer stags (aged 4–5 years) by antler growth periods of 40 days (FDG) and 60 days (SDG) after the casting of antler buttons from the previous set were analysed to compare the chemical composition, such as crude protein, ash, ether extract, amino acid, collagen, glycosaminoglycans (GAGs), uronic acid and sialic acid. The weight of velvet antler in FDG was lower than that of the SDG (P<0.05). SDG had a higher (P<0.05) content of crude ash than FDG. FDG had a higher (P<0.05) content of crude protein than SDG. Amino acid composition was also higher in FDG than in SDG for all sections. The content of collagen was higher in SDG than in FDG for the middle and base (P<0.05) sections. However, collagen levels were exceptionally higher (P<0.05) in FDG than in SDG for the upper section. While the content of collagen was significantly higher (P<0.05) for the upper section than for the middle and base sections in FDG, this was significantly increased (P<0.05) downward from the upper to the base section in SDG. Uronic acid content was higher in FDG than SDG for all three sections but there was no significant difference between groups in the middle and base sections. The content of GAGs was significantly higher (P<0.05) in FDG than SDG for all three sections. The content of sialic acid was the same as that of GAGs. Consequently, in this study, velvet antler production was increased with the extension of antler growth period, but the contents of protein, total amino acid composition, GAGs, uronic acid and sialic acid were decreased and those of ash and collagen were increased. Therefore, it is expected that the quality of velvet antler may be decreased greatly by extension of antler growth period.     

Composition en glucides conjugués de l'hémolymphe de Blaberus craniifer: Variations au moment de l'ecdysis imaginale

Analysis of macromolecular carbohydrates performed by gas liquid chromatography on Gregarina blaberae haemolymph showed the presence of hexoses (galactose, mannose, glucose), methyl-pentose (fucose), and hexosamines (N-acetylgalactosamine, N-acetylglucosamine). No trace of pentose or sialic acid or uronic acid was found. Mannose was the main neutral sugar. A change in the molar ratio of mannose consisting of an enrichment of female haemolymph occurred during larval-adult ecdysis. There was a parallel increase in glycoprotein staining with periodate-fuchsin after cellulose acetate electrophoresis of female haemolymph.     

The removal of non-collagen components from newborn calf dermis with magnesium chloride solution.

1. Non-collagenous substances in newborn calf dermis were extracted with solutions of various concentrations of MgCl2. The total protein and hydroxyproline contents in MgCl2 extracts increased with increase in the concentration of MgCl2 in the solutions. In particular, steep increases of their contents were observed at concentrations of MgCl2 from 0.5 to 1.0 M. Total amounts of hydroxyproline in 1.0, 2.0, and 3.0 M MgCl2 extracts were equivalent to 40-50% of the hydroxyproline content in the whole connective tissue. Hexose and hexosamine contents of MgCl2 extracts increased with increase of the MgCl2 concentration. Hexuronic acid was hardly present in the residues after extractions with 0.5, 1.0, 2.0, and 3.0 M MgCl2. 2. Plasma proteins, hyaluronic acid, and dermatan sulfate were extracted at low concentrations of MgCl2. A non-collagenous protein and MgCl2-soluble collagen were extracted with 1.0, 2.0, and 3.0 M MgCl2 solutions. The disperson of collagen fibrils was observed in the residue extracted with 1.0 M MgCl2 solution by electron microscopy; the fibril structure of collagen was disordered by extraction with 2.0 and 3.0 M MgCl2. The results suggest that the dispersion and disorder of collagen fibrils lead to the release of a non-collagenous protein. Furthermore, it is suggested that the removal of hyaluronic acid and dermatan sulfate was not very effective for the solubilization of a large amount of collagen, but was suitable as a pretreatment to the extraction of a non-collagenous protein accompanied by the solubilization of a large amount of collagen. 3. The non-collagenous protein was purified by DEAE-cellulose column chromatography. Polyacrylamide gel electrophoresis of this protein at pH 8.5 showed a single band moving to the cathode. The non-collagenous protein contained 3.7% hexose, 1.8% hexosamine, and no hexuronic acid. This protein is rich in glycine, glutamic acid, and alanine, and contains neither hydroxyproline nor hydroxylysine. Sedimentation analysis showed a single peak with 1.8 S and the molecular weight was approx. 43,000 as determided by SDS polyacrylamide gel electrophoresis.     

The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and non-collagen components.

Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.     

Encapsulation of foreign particles in vitro by separated blood cells from crayfish,Astacus leptodactylus

Summary Bone matrix consists of type-I collagen and noncollagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin — a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils — is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.