14. Bone biochemistry

Bone tissue is a special type of connective tissue. Bone tissue is a major component of bone. It forms a bony plate. Depending on the density and arrangement of plates we distinguish compact and spongy bone substance. In bodies of long (tubular) bones primarily compact bone substance is contained. In the epiphysis of long bones, as well as short and wide bones the spongy bone substance is dominated. Cellular elements of bone tissue are osteoblasts, osteocytes and osteoclasts.

Osteoblast is a cell of bone tissue, involved in the formation of intercellular substance. A distinctive feature of osteoblasts is the presence of highly developed endoplasmic reticulum and the powerful apparatus of protein synthesis. Procollagen is synthesized in osteoblasts, as well as glycosaminoglycans, protein components of proteoglycans, enzymes and other compounds, many of which are then quickly transferred into the intercellular substance.

Osteocyte is a mature dendritic cell of the bone tissue that produces the components of the intercellular matrix and usually walled in it. Osteocytes are derived from osteoblasts during bone formation.

Osteoclast is a giant multinucleated cell of bone tissue, which is capable to resorb calcified cartilage and the intercellular matrix of bone tissue in the process of the development and reconstruction of bone. This is the main function of osteoclast. Osteoclasts, as well as osteoblasts, synthesize RNA, proteins. However, in osteoclast, this process is less intense because they have poorly developed endoplasmic reticulum and there is a small number of ribosomes, but they contain many lysosomes and mitochondria.

The chemical composition of bone tissue. The content and composition of the organic matrix are subject to significant changes depending on the degree of mineralization of bone tissue.

Intercellular organic matrix of compact bone is about 20%, inorganic substances are 70% and water is 10%. Organic components are dominated in the spongy bone; they make up more than 50%, while inorganic compounds have 33-40%. The amount of water stored in the same range as in compact bone.

Due to the differences in the relative specific weight of organic and inorganic components insoluble minerals account half of bone mass.

The inorganic composition of bone tissue.

Hydroxyapatite crystals Са₁₀(РО₄)₆(ОН)₂ are part of the mineral phase of bone tissue, have the form of plates or sticks. The other part consists of an amorphous calcium phosphate Са₃(РО₄)₂. Amorphous calcium phosphate prevails at an early age; in mature bone becomes crystalline hydroxyapatite predominate. Typically, amorphous calcium phosphate is considered as labile pool of Са²⁺ ions and phosphate.

The composition of the mineral phase of bone consists of sodium, magnesium, potassium, chlorine, etc. In the crystal lattice of hydroxyapatite Са²⁺ ions can be replaced by other divalent cations, whereas anions other than phosphate and hydroxyl ions, are adsorbed on the surface of crystals, or dissolved in the hydration shell of crystal lattice.

The organic matrix of bone. About 95% of organic matrix accounts for collagen. Collagen is the main factor determining the mechanical properties of bone. Collagen fibrils of bone matrix are formed by collagen type 1. This type of collagen is also part of the tendons and skin, but bone collagen has some peculiarities. Bone collagen has few more hydroxyproline than the collagen of tendons and skin. Bone collagen is characterized by a high content of free amino groups of lysine and oxylysine residues. Another feature of bone collagen is high content of phosphate compared with collagen of other tissues. Much of this phosphate is associated with the serine residues.

The dry demineralized bone matrix contains about 17% of non-collagenic proteins, among which are protein components of proteoglycans. In tote an amount of proteoglycans in the formed dense bone is small.

The composition of the organic matrix of bone tissue consists of glycosaminoglycans, the main representative of which is chondroitin-4-sulfphate. Chondroitin-6-sulphate, keratan sulphate and hyaluronic acid are presented in small quantities.

Ossification accompanied by a change of glycosaminoglycans: sulphurized compounds give a way to non-sulphurized. Bone matrix contains lipids, which are a direct component of bone tissue, and are not mixed due to the insufficient removal of lipid-rich bone marrow. Lipids may play a significant role in the formation of nuclei of crystallization in bone mineralization.

Osteoblasts are rich in RNA. The high content of RNA in bone cells reflects their activity and constant biosynthetic feature. Feature of the bone matrix is high concentration of citrate: 90% of the total amount of citrate in the body accounts for the bone tissue. Citrate is necessary for bone mineralization. It forms complexes with salts of calcium and phosphorus, providing an opportunity to increase their concentration in the tissue to a level at which the crystallization and mineralization can start. In addition to citrate, succinate, fumarate, malate, lactate and other organic acids are detected in bone tissue.

Bone formation. Intercellular matrix formation and mineralization of bone tissue are the result of osteoblasts activities, which, as the bone formation, are immured in the intercellular substance and become osteocytes. Bone tissue is the main depot of calcium in the body and is actively involved in calcium metabolism. Calcium release is achieved by breaking down (resorption) of bone tissue, and its binding – through the formation of bone tissue. A process of constant adjustment of bone tissue is related to this, continuing throughout the life of the organism. At the same time there are changes in the shape of bone, respectively changing mechanical loads. Bone of the human skeleton is almost completely rebuilt every 10 years.

Ossification process is possible only if there are strictly oriented collagen fibers. Structural feature of the collagen fiber is that arranged in a row tropocollagen molecules are not related to the type of end to end. Between the end of one molecule and the beginning of the next is an available space. It is likely that the intervals along a series of tropocollagen molecules are the original centers of deposition of mineral components of bone tissue. The formed crystals in the collagen zone then, in turn, become the nuclei of mineralization, where hydroxyapatite is deposited in the space between collagen fibers.

During the formation of bone in the area of calcification, there is the degradation of proteoglycans with the participation of lysosomal proteases. As the mineralization of bone hydroxyapatite crystals displace not only proteoglycans, but water. Thick, fully mineralized bone is almost dehydrated. Under these conditions, collagen is about 20% by weight and 40% of the volume of bone tissue; the rest is accounted for mineral components.

Not all collagen tissues in the body are subject to ossification.

Apparently, there are specific inhibitors of calcification. A number of researchers believe that prevents the constant presence of proteoglycans in these tissues the process of mineralization of collagen in the skin, tendons, vascular wall. There is also the view that inorganic pyrophosphate can be an inhibitor of calcification. By mineralization of tissue the inhibitory effect of pyrophosphate is removed by pyrophosphatase, which, inter alia, is found in bone tissue. In general, the biochemical mechanisms of bone mineralization require further study.

Catabolism of bone matrix is a complex problem too. Both in physiological and in pathological conditions, bone resorption occurs, in which almost simultaneously have resolution of both mineral and organic structures of bone tissue. In the removal of mineral salts a particular role belongs to an increasing production of organic acids, including lactate, during osteolysis,. It is known that the tissue pH shift in the acidic side helps to dissolve minerals, thus facilitating their removal.

Resorption of organic matrix requires the presence and action of the appropriate enzymes. These include lysosomal acidic hydrolases, which range in bone tissue is quite wide. They are involved in intracellular digestion of fragments of resorbable structures.

Therefore, might occur an intracellular hydrolysis, you need beforehand to pre-expose structures of organic matrix an action, as a result of which fragments of polymers would have formed. Thus, the resorption of collagen fibers requires prior exposure to collagenolytic enzymes.

Factors affecting bone tissue metabolism, primarily include hormones, enzymes and vitamins.

Mineral components of bone are almost in a state of chemical equilibrium with the ions of calcium and phosphate in blood serum. Flux, deposit and excretion of calcium and phosphate levels are regulated by very complex system in which, among other factors important role belongs to parathyroid hormone and calcitonin. When the concentration of Са² ions in blood serum is decreased secretion of parathyroid hormone is increased. Directly under the influence of this hormone in bone tissue cell systems involved in bone resorption (increased number of osteoclasts and their metabolic activity) are activated, i.e. osteoclasts contribute to an increased dissolution of mineral compounds contained in bones. Parathyroid hormone increases the reabsorption of Са²⁺ ions in renal tubules. The cumulative effect is to increase the level of calcium in the blood serum. With increasing content of Са²⁺ ions in serum hormone calcitonin is secreted, the effect of which is to decrease the concentration of Са²⁺ ions due to its deposition in bone tissue. It increases bone mineralization and decreases the number of osteoclasts in the zone of action, i.e., inhibits bone resorption process. All this increases the rate of bone formation.

In the regulation of content of Са²⁺ ions the important role belongs to the vitamin D, which is involved in the biosynthesis of Са²⁺-binding proteins. These proteins are necessary for the absorption of Са²⁺ ions in the intestine, their reabsorption in the kidneys and their mobilization of calcium from the bones. Optimal intake of vitamin D is essential for normal processes of calcification of bone tissue. With deficiency of vitamin D, these processes are disturbed. Admission of excessive amounts of vitamin D to the long-term leads to bone demineralization. Cessation of bone growth is an early manifestation of vitamin A deficiency. It is believed that this fact is due to violation of the synthesis of chondroitin sulfate. When administered to animals high doses of vitamin A in excess of physiological need and causing the development of hypervitaminosis A, there is bone resorption, which can lead to fractures.

For the normal development of bone tissue vitamin C is also necessary. The influence of vitamin C on bone metabolism is due primarily to the influence on biosynthesis of collagen. Ascorbic acid is essential for the reaction of hydroxylation of proline and lysine. A deficiency of vitamin C also causes changes in the synthesis of glycosaminoglycans: hyaluronic acid content in the bone tissue is increased by several times, whereas the biosynthesis of chondroitin sulfates slows.