Bone is the main support and protection tissue of vertebrates. Besides acting as a scaffold, it has other functions such as storage and metabolic control of elements such as calcium and phosphorus, as well as being a place for production of new blood cells by a process referred as hematopoiesis. Hematopoietic cells are housed in the bone marrow. The most characteristic component of bone tissue is the mineralized extracellular matrix containing hydroxyapatite minerals (crystallized calcium phosphate is up to 65% of the bone extracellular matrix). The remainder of the extracellular matrix is composed of organic molecules, which consist of highly abundant collagen fibers (mainly Type I, which can be up to 95% of the organic part) and smaller amounts of glycosaminoglycans. Extracellular matrix allows the bone tissue to show great consistency, hardness, compressive strength, and some elasticity. Bone remodeling is a permanent process driven by bone cells. The cells that resorb bone are known as osteoclasts, while new bone tissue is synthesized by osteoblasts. Osteoblasts are locked in cavities of extracellular matrix, and they eventually become osteocytes, the cells constituting the mature bone tissue. Unlike cartilage, bone tissue is heavily irrigated by the blood system.
Depending on the density of extracellular matrix, two types of bone tissue can be distinguished: a) cancellous or trabecular bone, when the bone tissue has large spaces that give a loose or spongy appearance; b) compact or cortical bone, when the extracellular matrix is very dense and empty cavities are not found.
At light microscopy, there are three categories of bone tissue depending on the collagen fibers; laminar, with parallel fibers forming bundles; osteonic or concentric laminar, with parallel collagen fibers but arranged in concentric structures.
Trabecular or spongy bone has large spaces known as vascular cavities. These cavities are occupied by blood vessels and hematopoietic cells. Bone trabeculae are the walls of vascular cavities and may contain intertwined collagen fibers (woven trabecular bone) or collagen fibers arranged in lamellae (lamellar trabecular bone). Generally, during the formation of bone, i.e. osteogenesis, the woven trabecular bone is first formed, which is called primary bone. It is subsequently replaced by a secondary bone, laminar trabecular bone, which is usually found in the shaft and head of long bones, and it is always surrounded by compact bone.
Compact or cortical bone has no vascular cavities, and its extracellular matrix is organized into bone lamellae, which can be arranged in parallel (lamellar compact bone) or concentrically around a canal (osteonic compact bone). This canal is known as Haversian canal. The Haversian system is composed of Haversian canal, concentric bone lamellae, and osteocytes located among the lamellae. Haversian system is also known as osteon. Haversian canals of neighbour osteons are connected by transverse ducts referred as Volkmann canals. Usually, 4 to 20 bone lamellae are arranged around a Haversian channel. Osteocytes are located in small cavities, known as bone lacunae, within bone lamellae. From bone lacunae, small ducts, also known as canaliculi, are extended through the bone lamella. Osteocyte cellular processes are inside these small channels. Canaliculi make a net of channels that connect every osteocyte with the Haversian canal. In this way, osteocytes may be fed by blood vessels.
The inner surfaces of compact bone and vascular cavities of spongy bone are covered by connective tissue, the endosteum, which contains osteogenic cells, osteoblasts, and osteoclasts. Externally, the bone is covered by periosteum, which is formed by an external layer of fibrous connective tissue and a deeper layer containing osteogenic material, where osteoblasts are found. Periosteum is attached to the bone by collagen bundles embedded in the calcified bone matrix.
Osteogeneis is the process of bone formation. It should be discerned between the origin of bone cells and the way the bone is formed. There are three cell lineages in the embryo that can differentiate in bone cells: paraxial mesoderm provides vertebrae and part of the skull and face bones, lateral mesoderm cells differentiate into the bones of the limbs, and neural crest cells, derived from ectoderm, differentiate into some bones of the skull and face. There are two modes of bone formation from mesenchymal cells (coming from some of the three lineages mentioned before): intramembranous and endochondral ossification. Intramembranous ossification is the formation of bone directly from mesenchymal cells, whereas during endochondral ossification there is a first differentiation of mesenchymal cells into cartilage, which is later substituted by bone tissue. As it can be noticed in both modes, bone tissue replaces a previous one. The first signal of the appearing of bone tissue is the synthesis of a net of trabeculae, which are progressively remodeled as bone tissue matures.
In this type of ossification, mesenchymal cells are differentiated directly into bone cells. First, there is a condensation of mesenchymal cells in the place where the bone is going to be formed. These groups of cells are well irrigated by blood vessels. The first signals of bone tissue are the appearance of strips of extracellular matrix located at equidistant points of nearby blood vessels, so that they show a reticular morphology. Some mesenchymal cells gather around these new strips and start to differentiate into osteoblasts by increasing in size and getting a cuboidal to columnar morphology. Osteblasts start to synthesize extracellular matrix with abundant proteoglycans and collagen, which is organized into fibrils. Chondroitin sulphate and keratan sulphate facilitate the precipitation of calcium phosphate that form the bone calcium mineral hydroxyapatite, part of the bone extracellular matrix. A nucleation zone of ossification is generated, and the process of maturation of mesenchymal cells into osteoblasts is repeated at the border, leading to the growth of the trabeculae. The extracellular matrix is secreted around osteoblasts, which become completely surrounded by bone matrix. Then, osteblasts differentiate into osteocytes. Periosteum is formed at the external surfaces of this trabecular system, whereas endosteum appears at the surfaces of the internal cavities. Bone marrow develops in the internal space of these cavities.
The organization of the initial bone matrix is referred as immature or reticular. By constant remodeling of the bone matrix, the reticular organization is transformed into a layered organization. Bones originated by intramembranous ossification do not develop typical osteones. They have compact bone in the periphery and cancellous bone in the inner parts.
In this type of ossification, mesenchymal cells differentiate first into chondrocytes, which are later replaced by bone cells. First, mesenchymal cells express molecular markers that are typically found in precursors of cartilaginous cells. At the same time, they express specific cadherins, adhesion proteins, that make cells to join between each other to form compact groups, known as nodules. Nodules develop into hyaline cartilage, whereas mesenchymal cells located at the periphery of these nodules start the expression of genes for differentiating into bone cells.
Chondrocytes proliferate to form cartilage, which will be the scaffold to build the bone. This cartilage has chondrocytes secreting extracellular matrix typically found in hyaline cartilage, as well as perichondrium forming the external layer.
At this time of development, some chondrocytes stop proliferation. They grow in size and become hypertrophic chondrocites. This stage is important because it determines the final size of the bone, particularly in long bones, by affecting the growing rate and total length of the bone, which are different in every particular bone. Hypertrophic chondrocytes synthesize now a type of extracellular matrix containing molecules that favour the formation of minerals. They also synthesize type X cartilage and fibronectin. Among the new molecules released to the extracellular matrix, there are two that are important for the formation of bone: an angiogenic factor that attract blood vessels toward the interior of the cartilage, and a molecule that makes the peripheral mesenchymal cells to differentiate into osteoblasts.
Hypertrophic chondrocytes die by apoptosis and the space they leave is invaded by blood vessels. Osteblasts enter the interior of the ossification area thanks to this blood vessels invasion and, as soon as they are inside, start to synthesize bone matrix. Meanwhile, osteoblasts that were in the old perichondrium also produce bone matrix that surround the degenerating cartilage. Periosteum progressively appears in this peripheral location. Internally, the area of dead chondrocytes expands along the cartilage structure, at the same time that it is being occupied by blood vessels that bring cells of the bone marrow, as well as osteoclasts. Osteoclasts differentiate from blood cells.
Some of the cartilage remains alive at the ends of the long bones, that allows the growth in length during the juvenile stage of the animal. This cartilage disappears and ossifies when the animal enters the adult stage. These remaining cartilage areas in young bones are known as physis or growth plates. They have several layers: basal, proliferation, maturation and ossification, which are a summary of the ossification process, so that the basal layer is chondrocyte source and in the ossification layer dead chondrocytes are being substituted by osteoblasts, bone matrix and bone marrow. There is a perichondral ring wrapping the growth plate, which allows the support and increase in diameter of this plate.
Long bones also increase in diameter, at the same time than they do in length. The increase in diameter is possible by the work of the periosteum, which produces bone by intramembranous ossification toward the inner part, that pushes itself outward.