Bone has three main functions. Its earliest function, on an evolutionary scale, was to protect the brains of the first bony fish. It later developed in these bony fish as a flexible skeleton of rigid components against which swimming muscles could work with great efficiency. Finally, it provided a store and source of calcium, whose homeostasis was important. There is little further that can be added to the role of bone in mammals and man. Teeth and bones share a common property. They are both hard and are resistant to dissolution by acids. This is because part of their structure is made of apatite crystals, which are only slightly soluble. The bones and teeth of a mammal may remain intact for hundreds of years after it has died. When we handle such old relics, it is hard to realize that they were once dynamic, reactive, and constantly changing components of a living body. Living bone is constantly being turned over, resorbed, and remodeled in response to changing demands. One of the challenges to astronauts, who spend any prolonged period in the zero gravity of space, is the rapid resorption of bone which is no longer weight bearing. Maintaining the bone around teeth and preventing its resorption if the teeth are lost provide one of the great challenges of dentistry. The more we understand of the dynamics of bone resorption and remodeling, the better we will manage the clinical problem of bone loss.
Calcium and phosphate have a strong affinity for each other and therefore form stable compounds, which have structural and physiological importance in many living organisms. Collectively, they are referred to as biological apatites. The name is derived from the Greek “apatite” to deceive. Apatite is a very hard salt and almost insoluble in water. Crystals of apatite form the bulk of the mineralized part of hard tissues in bones and teeth. The crystals are not pure apatite; they include carbonate, citrate, sodium, and magnesium, in amounts of about 1% each. There are small amounts of fluoride and traces of heavy metals.
The proportions of calcium and phosphates in the crystals of apatite are not consistent; the most common form, however, is hydroxyapatite (Ca10(PO4)6OH2), but there are other apatites in which the calcium and phosphate parts are in different ratios. The ratio of Ca: PO4 in hydroxyapatite is 1.67, the highest of all the apatites and the most insoluble.
When crystals of an almost insoluble salt like apatite are placed in water, both calcium and phosphate ions enter the water. The process continues until the solution around the crystals is saturated and can hold no more ions. At this point, the salt and the solution are in equilibrium. If, at this point, either Ca+ or PO4− is added to the solution, the reaction would go into reverse, and ions would precipitate onto the crystal. So, the reaction may move in either direction, dissolution or precipitation. The principles of saturated solutions may be summarized by the following relationship:
The concentration of ions in such a saturated solution will be depend on the solubility of the apatite and will have a constant value known as the solubility product (Ksp). The Ksp will be highest for the most soluble apatites.
Before precipitation of ions can occur, their concentrations must reach certain levels for a given set of conditions. The different ions, calcium, and phosphate, in this case, do not have to be in the same proportions, but the product of their concentrations, [Ca+] × [HPO4−], must reach the solubility product (Ksp). In test tube experiments (in vitro), this product must reach 4.3 mmol2 before nuclei of hydroxyapatite will form. If the pH does not change, this solubility product is constant. The solubility for any apatite is decreased if the solution is alkali. Hence, precipitation of salt would be assisted in an alkaline medium.
There are three recognizable stages in the production of hydroxyapatite crystals (▶ Fig. 7.1). Firstly, Ca+ and PO4− must accumulate in such concentration as to exceed the solubility of an apatite salt and to precipitate. Secondly, the ions must precipitate in a specific pattern which will allow other ions to spontaneously arrange themselves in the proper orientation for the third stage, which is crystal growth.
Fig. 7.1 A diagrammatic representation of the stages in the production of hydroxyapatite crystals. 1. The first stage in crystal formation is the increase is salt concentration until the solution is saturated. Saturation is reached sooner in an alkali medium. 2. Crystals begin to form in a specific pattern determined by the template offered by existing crystals. 3. Once the crystals first form, they grow rapidly provided the solution remains saturated.
The presence of existing crystal nuclei alters the conditions very significantly. Existing crystals reduce the concentrations required for precipitation to as low as 0.83 mmol2, compared to the concentration of salts required to allow precipitation in a test tube of 4.3 mmol2. The concentration product of [Ca+] × [HPO4−] ions in tissue fluid is around 1.76 mmol2. This means that hydroxyapatite cannot precipitate without a local catalyst or template, but once nucleation has occurred, crystals can grow rapidly in body fluids.
In newly formed bone, about 70% of the mineral is amorphous calcium phosphate (ACP), but in older bone, the proportions of ACP and hydroxyapatite are reversed. ACP may form first and then become crystallized into hydroxyapatite as this has been observed in in vitro studies. In vivo, a local increase in pH would encourage this change from ACP to hydroxyapatite.
Mineralization is the deposition of apatites and other salts in an extracellular matrix of fibers and ground substance. As calcium is not the only mineral involved, it is incorrect to refer to the process as calcification. Mineralization goes on throughout life as bone is continually being deposited and removed from the skeleton. The mineralization of the teeth is for the most part confined to their period of development. There is no completely satisfactory explanation for the apparent control of the process of mineralization. It is not clear how it is initiated in embryonic development, and it is not certain how the process, once started, is restrained. There are at least two possible processes which could be involved in the control of mineralization. The first results directly from cellular activity via the production of vesicles high in calcium content; the second depends on the presence of matricellular proteins including collagen.
The matrix in which mineralization takes place contains collagen, proteoglycans, citrates, lipids, and plasma constituents. The cells concerned in the mineralization process all contain large quantities of the enzyme alkaline phosphatase. There is some evidence for the involvement of each of these organic components in mineralization.
Crystal growth may be initiated by the presence of a nucleus in a saturated solution. This nucleus may be an impurity or a previously formed crystal introduced into the solution, which acts like a template against which ions can form up in order. This process is referred to as epitaxy. Support for the epitaxy theory has come from electron microscope studies which have shown that hydroxyapatite crystals are initiated first at the light bands of 64.0-nm repeating collagen, and that the crystals orientate along the long axis of the collagen fiber. It is also suggested that for crystal formation to occur within the fiber, the phosphate and calcium ions must pass through the gaps between the tropocollagen molecules. For type I collagen, the gap is 0.6 nm (large enough for the phosphate molecule), but for the type II collagen, (soft tissue) the gap (0.3 nm) is too small, and the collagen does not calcify.
A calcium-phospholipid-phosphate complex has been isolated from young bone and found to induce crystallization. Acidic phospholipids from dentin have also been found to have the same potential. Phospholipids are also associated with the matrix vesicles (see Chapter 7.2.2 Control of Mineralization by Cells) in calcifying cartilage. Glycosaminoglycans (GAGs) in cartilage seem to have an inhibitory effect on mineral nucleation. Their removal may be necessary to make room for mineral crystals.
All the components of supportive connective tissue, including the matrix of fibers and ground substance and its mineralization with apatite, are formed as a result of bone-forming (osteoblast) and bone-removing (osteoclast) cells. The activity of osteoblast and osteoclast is influenced by systemic hormones such estrogen, parathyroid, calcitonin, and growth hormone. At a local level, the activity of these cells is also influenced by a wide range of local chemical mediators, and the matrix molecules around the cells, which are both supportive and inductive. The provision of high concentrations of calcium at levels which are highly toxic to cells would seem to require mechanisms which take place outside the cell, although it is possible that mineralizing cells could accumulate and store high levels of calcium safely in their mitochondria.
The final concentration, however, is not achieved in the cell but in an extracellular vesicle. Membrane-bound vesicles about 100 µm in diameter have been found within the organic matrix destined to become bone, and within predentin, and in the precalcifying zone of cartilage. The contents of these matrix vesicles are rich in acid phospholipids, which have a strong affinity for calcium. One of these, phosphatidylserine, not only binds calcium but also inorganic phosphate as well. Matrix vesicles also contain high concentrations of phosphatases, enzymes which generate phosphates. Matrix vesicles are able to concentrate both phosphate and calcium ions until the threshold for the precipitation of hydroxyapatite is reached.
The tissues which form the skeleton of vertebrates are cartilage and bone. The early fish all had cartilaginous skeletons, and some, like sharks and rays still have not progressed beyond using cartilage for their backbones and fins. Many millions of years ago, some fish developed a modification to cartilage. These first truly bony fish actually still had a backbone of cartilage, but they grew protective sheets of bone under the skin around the head, improving the protection of the brain and eyes. These bony plates are still part of our skeletons and are known as the dermal (skin) bones of the skull. Bone had many other advantages over cartilage, particularly where a rigid thin structure was required. The flesh of fishes-like mackerel and trout is well supported with “bones” to support the action of strong swimming muscles. Land animals do not have the support water gives to the body and require the skeleton to prevent them from collapsing under their weight. Cartilage would not support the weight of a horse, but bone, with its mineral crystals, toughened by a network of collagen fibers is able to withstand heavy compression. Bone is, however, brittle; it does not withstand tension well. If the horse should fall badly, a leg bone might snap under the tension. Parts of the mammal skeleton which must be flexible, like the ears, respiratory passages, and the ends of long bones, are made of cartilage.
Cartilage has a very simple structure when viewed under a microscope. The only cells visible in mature cartilage are chondrocytes. The matrix surrounding the cells usually has a glassy appearance and is given the name hyaline cartilage (Greek hyalos = glass). Hyaline cartilage can be found keeping the airway open, from the nose to the bronchi and covering the articular surface of joint surfaces. The matrix is not in fact featureless, as it contains a fine network of collagen fibers embedded in an extracellular matrix. In some cartilage, such as that found between the intervertebral disks, the fibers are visible, as they are more densely packed. This, so-called fibrous cartilage, is stronger but less flexible than hyaline cartilage. When prepared for histological section, the matrix of cartilage takes up basic dies (basophilic) because it contains acid GAGs. These GAGs are huge molecules which account for some of the important physical properties of cartilage (see Chapter 6.1 Glycosaminoglycans). GAGs retain large amount of water which is attracted to the molecule and trapped within its large domain. It is difficult to squeeze this water out of the GAG molecule, so cartilage is resistant and to compression. One of its great disadvantages is that if it is torn or damaged, it has little ability to repair itself because it does not have a blood supply. Nutrients reach the cartilage cells by diffusion from the perichondrium, a surrounding layer of dense connective tissue. Cartilage is not a biologically active tissue.
Cartilage is a useful skeletal material in the developing embryo. At this time and during infancy, the skeleton needs to grow fast. Cartilage is flexible and can grow by expansion from within (interstitial growth) and so increase in size rapidly. Bone, being rigid, cannot grow except by addition of new bone to the surface, so expansion is gradual. With the exception of the dermal bones of the skull, the bones of the skeleton develop first in cartilage, which is later replaced with bone. Cartilage also has a crucial role in the interface between skeletal elements. Not only it is a growth zone in young bones, but also it later remains to provide the joint surfaces. The combination of GAGs and a network of collagen gives it a high resistance to compression, flexibility, and a slippery surface. This makes it an ideal material for joint surfaces which have to sustain high impact, such as those of the lower limbs. There are some exceptions, however. The temporomandibular joint surfaces and the disk in between are not cartilage but dense fibrous tissue. The reason for this may be that cartilage provides a suitable surface in a joint as long as it is stable and well fitting when loaded (like the knee joint). The temporomandibular joint rotates and slides while under load in a grinding manner, and the joint surfaces are not well fitting. If the lining surface were incompressible cartilage, there would be concentrations of pressure to the supporting bone. Fibrous tissue, on the other hand, allows for some distortion under load thereby conforming to the constantly changing points of contact between the joint surfaces. The intervening disk, also of dense fibrous tissue, ensures smooth movement of the joint under load (see Chapter 9.2 The Structure of the Temporomandibular Joint).
The first bones to form in the embryo are those which surround the brain. These skull bones form in between the outer membrane of the brain and the skin, hence the name, intramembranous. The cells which form bone are osteoblasts. These are specialized cells which are differentiated from stem cells in bone marrow and the periosteum. The osteoblasts secrete a matrix around themselves which is rich in collagen fibers and ground substance known as osteoid. The ground substance consists of the GAG chondroitin sulphate and osteocalcin. The osteoblasts then start depositing calcium and other mineral salts into the osteoid, where the salts precipitate as crystals. These first spicules of bone appear as an ossification center, which grows progressively in size to fuse with other ossification centers (▶ Fig. 7.2). This first formed bone is called embryonic, or woven bone, because it is not well organized. Bone which first forms during repair of fractures, or in tooth sockets after extraction, is also irregular, woven bone.
Fig. 7.2 A diagrammatic representation of the stages of embryonic or woven bone formation. Mesenchymal stem cells differentiate into preosteoblasts, which further differentiate into osteoblasts. Osteoblasts lay down a matrix of osteoid, which contains collagen fibers in a ground substance of the glycosaminoglycan, chondroitin sulfate. Mature osteoblasts secrete calcium and phosphate salts, which form crystals of hydroxyapatite around the collagen fibers. The bone formed may trap osteoblasts, which become osteocytes.
The organization of this bone soon begins by its removal by osteoclasts. These are multinucleated giant cells, whose origins are from monocytes. Osteocytes emerge from blood vessels and remove a tunnel of bone around the blood vessel, which in the two dimensions of a histological section appear as a circular ring of bone removal (▶ Fig. 7.3). The osteoclasts’s work is done. Osteoblasts begin to form bone against the inner walls of the tunnel in layers of concentric plates called lamellae. The layers are distinguished by the regular orientation of collagen fibers. The tunnel is not completely filled up, as a central bundle of blood vessels remains. As the lamellae are formed, some osteoblasts remain trapped in between them. They are in this situation, called osteocytes, as they no longer form bone. They retain contact via long cellular projections, with osteoblasts, and contribute greatly to control bone metabolism from their inner caverns.
Fig. 7.3 A diagrammatic representation of the formation of lamella (compact) bone. Embryonic bone surrounding a blood vessel is remodeled, forming a tunnel. Against the walls of the tunnel, new osteoid, containing ordered collagen fibers, is laid down then mineralized forming concentric plates of lamella bone. The blood vessel and surrounding plates of lamella bone are called an osteon.
The concentric lamellae of bone and central blood vessels have been called a haversian system. The haversian system in three dimensions forms the basic structural unit of bone and is called an osteon. The concentric tubes of plated bone resemble the stem of a leek, but of course they are microscopic in size. The process of cutting a tunnel and lining it again is in order to align the collagen fibers, before they are surrounded by crystals of bone salts. This alignment gives the osteon great strength, as not only are the fibers arranged in a number of useful directions, but their direction is reversed in each adjacent layer of the osteon. Fibers in each osteon are formed as either long spirals, transverse circles, or longitudinal rods (▶ Fig. 7.4). Osteons have greater density than embryonic bone and are the reason this bone has a compact appearance, even when it is not magnified. If the leg bone of any mammal is sectioned through the shaft, it will be seen to consist of a thick dense tube called the cortex and an inner softer center called the medulla. The medulla in the center of long bones may be filled with red bone marrow in the young and fatty tissue in older subjects. If the bone is sectioned at either end of the shaft, the medulla is seen to be filled with spongy-looking bone.
The dense bone of the cortex is compact lamella bone, and this can be confirmed by examining a section using a microscope. The spongy inner core appears to lack any pattern of arrangement. When a section is examined microscopically, it appears even less structured, as all that can be detected is isolated spicules of bone. However, if a radiograph is made, a distinct orientation of spicules may be seen. These spicules have been called trabeculae, and so the bone is called trabecular bone (▶ Fig. 7.5) (see Appendix F.1 Stress and Trabecular Orientation).
Fig. 7.5 The structural appearance of compact and trabecular bone. (a) A sagittal section through the mandible, with a few remaining anterior teeth, shows the compact cortical bone (C), thickest at the lower border of the mandible. Most of the posterior teeth have been lost for some time before death. Note, the absence of any cortical bone on the residual alveolar ridge. One of the premolar teeth has been removed postmortem, and the cortical bone lining the tooth socket is visible. The mental foramen is visible (mf). The central spongy bone has supportive trabeculae (T) which are not readily recognizable. (b) The radiograph of the same sample reveals the direction of trabeculae, which are more radiopaque, and therefore slightly lighter than the surrounding spongy bone.
We have noted that intramembranous bone formation occurs in the skull bone to protect the brain. It is clear that as soon as some bone has formed, it cannot increase in size from within, but only by the apposition of new layers of bone on the surface. The shape may be controlled by both appositional growth on the outside and resorption on the inside. Where two cranial bone surfaces meet, for example, the parietal and frontal bones, a fibrous suture separates the two membranous bones until growth ceases (▶ Fig. 7.6). Bone growth by apposition and resorption is a relatively slow process; it serves well enough for the bones of the skull which are in infancy, disproportionately large, but grow slowly, and must at all times provide a rigid protective shield for the brain.
Fig. 7.6 A diagrammatic representation of the difference between interstitial and appositional bone growth. (a) A growing skull bone increases in size slowly by apposition (+ +) and changes shape by a balance of apposition (+ +) and resorption (– –). (b) Long bones increase in length rapidly, as growth occurs first, by interstitial growth of cartilage before bone formation occurs.
Growth of the axial skeleton must be more rapid in order to achieve adult size during the years of childhood. The axial skeleton also has to grow while bearing the weight of the child. Bone-forming cells at sutures would have to be capable of withstanding considerable compression due to weight and at the same time provide a high growth rate. Subperiosteal appositional bone formation would not meet the demands made for growth of the axial skeleton.
The axial skeleton of the embryo is therefore first formed in cartilage. Cells forming cartilage (chondrocytes) are flattened parallel to the surface and several layers deep. The rubbery consistency of the cartilage surrounding each chondrocyte protects it from compression due to the weight of the child. It has been noted that the matrix of chondroitin sulfate and type II collagen fibers provide cartilage with resistance to compression and a slippery joint surface. Nests of chondrocytes are able to form cartilage rapidly. Unlike the rigid matrix surrounding bone cells, cartilage can increase in size by expansion from within, that is, by interstitial growth. The chondrocytes increase in size and their extracellular matrix increases in mass. Then they begin to calcify their surrounding matrix with hydroxyapatite crystals. Their mission is suicidal. As soon as they have completed this task of growth and calcification, they die by a process known as apoptosis (programmed cell death). The calcified matrix becomes the framework against which osteoblasts start to form bone (▶ Fig. 7.7).
Fig. 7.7 A diagrammatic representation of the conversion of a cartilaginous limb into bone. Blood vessels invade the cartilage and begin to vascularize it. As this occurs, some cartilage cells calcify their surrounding matrix and die, leaving channels for blood vessels to follow. Osteoblast lays down bone against the calcified cartilage and gradually replaces it, except for two plates, which remaining at the epiphysis, and allows continued growth of cartilage until adulthood.
Before new bone can replace the calcifying cartilage, a blood supply (angiogenesis) must be established. Blood vessels penetrate the cartilage in the center of the shaft, the diaphysis and at each end, the epiphysis. The new blood vessels move into the spaces created by the dying cartilage cells and establish a highly active connective tissue capable of supporting the principal bone-forming cells the osteoblasts. Having a calcified matrix to work against the replacement of the calcified cartilage by bone proceeds swiftly. This type of bone formation by replacement of cartilage is called endochondral ossification. Bone is also being formed on the outside of the cartilage in the perichondrium. This periosteal bone contributes to thickening the long bones, but the major growth in length is still achieved by cartilage growth. The cartilage is gradually replaced with bone along the shaft of the long bones and at the ends (epiphysis) of the bone where it forms a joint. In between the shaft and the epiphysis is a plate of cartilage which remains the active growing site until the adult dimensions of the bone are reached. The epiphyseal plate then ossifies, fusing the shaft and epiphysis, and growth in length of the bone stops. The time of fusion of the epiphyseal plate of bones of the wrist is a useful indicator of the biological age of an adolescent (see Chapter 184.108.40.206 Measuring Growth).
As we have seen, cartilage may become calcified, so mineralization is not always a useful distinction between cartilage and bone. There are, however, some essential differences between bone and calcified cartilage, which are related to their internal structure.
Firstly, in bone, the collagen fibers are arranged in layers and organized into longitudinal, circular, and spiral patterns, which are all united into a macroscopic structural unit, the osteon. This orientation gives bone structural strength which cartilage lacks. The fibers in cartilage are arranged in an interlocking mesh, and there is no macroscopic structural unit.