Collagen cross-linking, a major post-translational modification of collagen, plays important roles in the biological and biomechanical features of bone. Collagen cross-links can be divided into lysyl hydroxylase and lysyl oxidase-mediated enzymatic immature divalent cross-links, mature trivalent pyridinoline and pyrrole cross-links, and glycation- or oxidation-induced non-enzymatic cross-links (advanced glycation end products) such as glucosepane and pentosidine. These types of cross-links differ in the mechanism of formation and in function. Material properties of newly synthesized collagen matrix may differ in tissue maturity and senescence from older matrix in terms of cross-link formation. Additionally, newly synthesized matrix in osteoporotic patients or diabetic patients may not necessarily be as well-made as age-matched healthy subjects. Data have accumulated that collagen cross-link formation affects not only the mineralization process but also microdamage formation. Consequently, collagen cross-linking is thought to affect the mechanical properties of bone. Furthermore, recent basic and clinical investigations of collagen cross-links seem to face a new era. For instance, serum or urine pentosidine levels are now being used to estimate future fracture risk in osteoporosis and diabetes. In this review, we describe age-related changes in collagen cross-links in bone and abnormalities of cross-links in osteoporosis and diabetes that have been reported in the literature.

Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus

Glycosaminoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGs can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGs is organized macroscopically by segregation into alternating blocks of specific sulfation patterns and microscopically by formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.

Enzymatic degradation of glycosaminoglycans.

Agarose-based biomaterials for tissue engineering.

To determine whether subchondral bone in osteoarthritis differs from that seen in normal human aging, osteoarthritic femoral heads removed for total hip arthroplasty were compared with normal age-matched and young autopsy controls. Standardized, 1-cm deep, weight-bearing and nonweight-bearing subchondral bone blocks, as well as cancellous core bone, 2–4 cm deep to the articular surface, were examined in each femoral head. Mineralization was assessed using density fractionation and chemical analysis, and compared to histomorphometry. In osteoarthritis, both weight-bearing and nonweight-bearing surface subchondral bone showed a lower degree of mineralization than age-matched and young controls. Histomorphometric analysis showed that subchondral bone thickness, as well as all osteoid parameters and eroded surfaces, were increased in osteoarthritic samples versus controls. Mineralization in the deep cancellous core bone increased with normal aging but underwent less change with osteoarthritis. Histomorphometry of the cancellous core showed that osteoid parameters, but not bone volume, were increased in osteoarthritis versus controls. In conclusion, osteoarthritis is associated with a thickening of the subchondral bone with an abnormally low mineralization pattern.

Subchondral bone in osteoarthritis

The Biochemistry of Bone

Binding of calcium to glycosaminoglycans: an equilibrium dialysis study.

The relation between bone mineralization and glycosaminoglycans (GAG) was investigated in newborn and mature rabbit diaphyseal bone. Using the density fractionation technique, the bone was separated into fractions of increasing density from 1.4 to 2.3 grams/ml. Each fraction was analyzed by X-ray diffraction to determine the average crystal size. The GAG content of each fraction and of the unfractionated bone was determined by direct extraction and on a few fractions by sequential extraction in guanidine hydrochloride and guanidine/EDTA. There was a decrease in the GAG content with animal age and with increasing fraction density in the newborn rabbit. In one overlapping fraction (2.0-2.1 grams/ml), the GAG content was twice as high in the newborn as in the mature animal. Finally, the crystal size substantially increased from newborn to mature rabbits. Therefore, calcification and maturation of bone is associated with a decrease in the proteoglycan content of the organic matrix.

Bone mineral and glycosaminoglycans in newborn and mature rabbits.

The provisional calcification of epiphyseal cartilage involves deposition of hydroxyapatite (calcium phosphate) crystals in an extracellular matrix consisting principally of Type II collagen and cartilage proteoglycan. A mechanism is now proposed to explain how epiphyseal cartilage calcification is initiated. Calcium exists at high concentration in cartilage, but is mainly bound to the anionic groups of proteoglycans, and thus is unavailable for precipitation. A local increase in phosphate concentration displaces calcium ions from proteoglycan by an ion-exchange effect, raising the Ca X PO4 product above the threshold for precipitation of hydroxyapatite. Evidence for this hypothesis has been derived from studies of the effect of phosphate on the binding of calcium to cartilage proteoglycan, and on hydroxyapatite formation in the presence of chondroitin sulfate.

An ion-exchange mechanism of cartilage calcification.

Collagen has long been suspected to be a promoter of hydroxyapatite (HA) deposition in bone. This theory was tested by comparison of HA formation in gels composed of collagen, gelatin or agarose. Collagen gels supported substantially more HA precipitation than gelatin gels, but slightly less than agarose gels. Analysis of the relative diffusion rates for calcium in these matrices indicated that, in this system, amount of HA formation is dependent upon the rate of diffusion. Under conditions in which the diffusion rates for collagen and agarose gels were comparable, similar amounts of HA were formed. This suggests that fibrillar collagen is not per se a promoter of HA deposition. Extracellular matrix macromolecules may influence calcification by restricting ionic diffusion through connective tissues.

Hydroxyapatite formation in collagen, gelatin, and agarose gels.

Formation of calcium pyrophosphate dihydrate (CPPD) crystals in native collagen gels represent an in vitro model system for the study of pathological cartilage calcification. The conditions under which CPPD forms in collagen gels have been determined. At low Ca X pyrophosphate product, CPPD forms directly. At high Ca X pyrophosphate product, CPPD forms via the amorphous intermediate calcium magnesium pyrophosphate (CMPP). Chondroitin sulfate (CS) inhibits formation of CPPD by both pathways, but apparently by different mechanisms. Direct CPPD formation is inhibited with low potency by CS, apparently by binding of Ca2+ ions. Indirect formation of CPPD is inhibited with high potency by CS, apparently by stabilization of the CMPP intermediate. Comparison of the inhibition of direct CPPD formation by the two glycosaminoglycan species occurring in cartilage proteoglycan showed that CS is a more potent inhibitor than keratan sulfate (KS), in agreement with the greater Ca2+-binding affinity of CS. The increase in KS/CS ration which occurs in human hyaline cartilage with aging may facilitate deposition of CPPD crystals by decreasing the exclusion of pyrophosphate anions.

Graeme K. Hunter

Papers

Binding of calcium to glycosaminoglycans: an equilibrium dialysis study.

Bone mineral and glycosaminoglycans in newborn and mature rabbits.

An ion-exchange mechanism of cartilage calcification.

Hydroxyapatite formation in collagen, gelatin, and agarose gels.

Effect of glycosaminoglycans on calcium pyrophosphate crystal formation in collagen gels.