Intramembranous or endochondral ossification generates bone tissue.
Bones consist of a dense cortical shell and sponge-like trabecular network.
Bone formation depends on metabolically active osteoblasts synthesizing matrix proteins.
Bone marrow fat increases with age at the expense of osteoblasts.
Resorption of bone is mediated by multinucleated osteoclasts.
The most abundant cell type in bone is the osteocyte.
Bone is continuously rebuilt, a process known as bone remodeling.
The immune system, in particular T lymphocytes, influences bone remodeling.
Neuroendocrine loops exert systemic control on bone remodeling.
Structure and Composition of Bone
Bone is a specialized connective tissue that assists in (1) locomotion, by providing the insertion site of the muscles; (2) protection of the internal organs and the bone marrow; and (3) metabolic function, such as storage and provision of calcium to the body. Bone consists of cells and the extra-cellular matrix, which is composed of type I collagen fibers and a number of noncollagenous proteins. The specific composition of the bone matrix allows its mineralization, which is a specific feature of bone.
The two major types of bones are flat bones, which are built by intramembranous ossification, and long bones, which emerge from endochondral ossification. Intramembranous bone formation is based on the condensation of mesenchymal stem cells, which directly differentiate into bone-forming osteoblasts. In contrast, during endochondral ossification of the long bones, the mesenchymal stem cells first differentiate into chondrocytes that will later be replaced by osteoblasts. Long bones consist of the following: (1) epiphyses, which are protrusions at the ends of the long bones; (2) diaphysis, constituting the bone’s shaft; and (3) metaphyses, which are located between the epiphysis and the diaphysis ( Fig. 4.1 ). The metaphysis is separated from the epiphysis by the growth plate, a proliferative cartilage layer that is essential for the longitudinal growth of bones. After the growth is completed, this cartilage layer is entirely remodeled into bone.
The external shape of bones is formed by a dense cortical shell (cortical or compact bone), which is particularly strong along the diaphysis, where the bone marrow is located. The cortical bone shell becomes progressively thinner toward the metaphyses and epiphyses, where most of the trabecular bone is located. Trabecular bone (also called cancellous bone ) is a sponge-like network consisting of myriad highly interconnected bony trabeculae. The outer and the inner surfaces of cortical bone are covered by layers of osteogenic cells, termed the periosteum and the endosteum, which are involved in the growth of width by bone apposition at the periosteal sites and bone resorption at the endosteal sites.
Although cortical and trabecular bone is composed of the same cells and the same matrix components, there is a substantial difference between these two forms of skeletal tissue. Cortical bone almost exclusively consists of mineralized tissue (up to 90%), allowing it to fulfill its mechanical requirements. In contrast, only 20% of trabecular bone is mineralized tissue, with the bone marrow, blood vessels, and a network of mesenchymal stem cells constituting the remainder of the bone. As a consequence, trabecular bone shares a vast surface with the nonmineralized tissue, which is the basis for the metabolic function of bone, necessitating a high level of communication between the bone surface and the nonmineralized tissue.
Bone Vasculature
The metabolic function of the bone is dependent on being supplied with nutrients, oxygen, hormones, growth factors, and neurotransmitters. Bone marrow resident cells and mesenchymal stem cells respond to bone mediators but can also produce metabolic factors that are relevant for body homeostasis. The exchange of such mediators into and out of the bone is mediated by a complex vascular system.
Oxygen is supplied by nutrient arteries; these enter the bone mainly at the metaphysis, run longitudinally along the bone shaft, and ramify towards the inner surface of the cortical bone, the endosteum. At this interface, the arteries form loops and interconnect with sinusoidal capillaries. This fenestrated venous vessel type allows a fast exchange of metabolites between the blood circulation and the bone marrow tissue. The sinusoids form a dense, irregular network that converges into a large central sinus in the middle of the bone marrow tissue. This central sinus exits the bone shaft and connects the bone marrow with the general blood circulation.
In addition to the system of bone marrow vascularization, the mineralized bone also exhibits a dense vascular system, which is termed the Haversian-Volkmann System . This vascular network contains both arterial and venous vessels that ensure nutrient supply and metabolic exchange of the calcified bone tissue. Its structural organization is very complex because it can contain trans-cortical vessels (TCVs), which directly connect the bone marrow vascularization with the periosteum and the general circulation. Next to this, TCVs can show bifurcated or complex rope ladder–like morphologies ( Fig. 4.2 ). Although their diameters are much smaller than those of nutrient arteries and central sinus exits, numbers of TCVs are drastically higher. Based on their multitude, they facilitate the major blood transport through the bone.
At the endosteum, the vessels feed into the sinusoidal-arterial transition zones. These so-called type H vessels are also formed at the vascular transition zones of the metaphysis and exhibit specific metabolic profiles. Based on differences in tissue oxygenation and metabolic activity, they influence the growth potential and metabolism of hematopoietic stem cells as well as bone remodeling cells. Thus, the vascular supply of nutrients and oxygen is not only relevant for bone marrow metabolism but also for the formation and destruction of the mineralized bone matrix.
Bone Matrix
The key protein component of bone is type I collagen. Collagen fibers follow specific directions, forming the basis for the lamellar structure of bone. This lamellar structure, which can be visualized when examining bone in the polarized light, allows dense packaging, resulting in optimal resistance to mechanical load. The lamellar collagen structures can be assembled in parallel (e.g., along the cortical bone surfaces and inside the bony trabeculae) or concentrically around blood vessels embedded in the Haversian channels of the cortical bone. Upon rapid deposition of new bone, such as during fracture healing, this lamellar structure is missing, and the bone is then called woven bone. Woven bone is consecutively remodeled into lamellar bone, which is also considered “mature” bone. The composition of the collagen backbone also assists in the deposition of spindle- or plate-shaped hydroxyapatite crystals, which contain calcium phosphate, thus allowing the calcification of the bone matrix.
In addition to type I collagen, other so-called noncollagenous proteins also exist in bone. Some of them, such as osteocalcin, osteopontin, and fetuin, are mineralization inhibitors, which serve to balance the degree of mineralization of the skeletal tissue. Aside from their intrinsic function in bone, noncollagenous proteins also exert important metabolic functions, such as the control of energy metabolism by osteocalcin.
Bone Cells: Osteoblasts
Osteoblasts are the bone-forming cells that derive from the mesenchymal stem cells of the bone marrow. Mesenchymal stem cells also form chondrocytes, myocytes, and adipocytes. Osteoblasts are cuboid-shaped cells that form clusters covering the bone surface. They are metabolically highly active, synthesizing the collagenous and noncollagenous bone matrix proteins that are excreted and then deposited between the osteoblasts and the bone surface. This newly built matrix, which is not yet calcified, is termed the osteoid. The lag phase between osteoid deposition and its mineralization is approximately 10 days. Osteoblast differentiation depends on the expression of two key transcription factors: runt domain transcription factor 2 (Runx2) and its target Osterix-1. The transcription factors confer the differentiation of mesenchymal cells into osteoblasts in response to external stimuli. Prostaglandin E 2 (PGE 2 ), insulin-like growth factor (IGF)-1, parathyroid hormone (PTH), bone morphogenic proteins (BMPs), and Wingless and Int-1 (Wnt) proteins are key stimuli for osteoblast differentiation. For instance, PGE 2 is an important anabolic factor for bone and induces the expression of bone sialoprotein and alkaline phosphatase in mesenchymal cells. BMPs and transforming growth factor (TGF)-β, which shares structural similarities with BMPs, foster osteoblast differentiation by activating intra-cellular Smad proteins. Finally, Wnt proteins, a family of highly conserved signaling molecules, are potent stimulators of osteoblast differentiation. Wnt proteins bind to surface receptors on mesenchymal cells, such as frizzled and LRP5, thereby eliciting activation and nuclear translocation of the transcription factor β-catenin, which induces the transcription of genes involved in osteoblast differentiation. Wnt proteins, thereby, act not only in close synergy with BMPs but also cross talk to the receptor activator of nuclear factor-κB ligand (RANKL)–osteoprotegerin (OPG) system, which is involved in the differentiation and function of bone-resorbing osteoclasts.
During aging, bone marrow adipocytes (BMAs) derived from bone marrow mesenchymal stem cells (BMSCs) accumulate, which correlates with osteoporosis. Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ) promotes adipocyte differentiation. However, activation of the Wnt/β-catenin signaling pathways stimulate BMSCs to differentiate into osteoblasts and inhibit adipogenesis. BMAs are metabolically active cells that play an active role in energy storage, endocrine function, and bone metabolism.
Bone Cells: Osteocytes
Osteocytes are by far the most abundant cell type within bone. One cubic millimeter of bone contains up to 25,000 osteocytes that are well connected with each other and the bone surface by small tubes (canaliculi). This large and dense communication network inside the bone shares similarities to the nervous system. The surface of this network of lacunae contains the osteocytes and canaliculi, which consist of interconnecting filaments of the osteocytes. The network covers an area of 1000 to 4000 square meters. Osteocytes are derived from osteoblasts, which are subsequently entrapped in the bone matrix. Osteocytes, however, also start to express genes that are specific for these cells and are not found in other cells, such as osteoblasts. One of the most interesting products of the osteocyte is sclerostin, a secreted molecule that binds lipoprotein receptor–related proteins (LRPs) and blocks Wnt-stimulated bone formation. Consistent with its function as an inhibitor of bone formation, overexpression of sclerostin leads to low bone mass, whereas deletion of sclerostin leads to increased bone density and strength. This effect has recently been successfully used as a therapeutic strategy to increase bone mass by inhibiting sclerostin by a specific antibody. Loss-of-function mutations in the human SOST gene that encodes sclerostin entail increased bone mass, a disease termed sclerosteosis. Several local and systemic factors have been suggested as possible regulators of sclerostin expression by osteocytes. For instance, intermittent administration of PTH, which is associated with strong anabolic effects on the bone, potently inhibits sclerostin expression.
Recent genetic studies in mice have revealed that osteocytes provide the majority of the RANKL that controls osteoclast formation in cancellous bone. Of note, osteocyte death is most strongly linked to the pathogenesis of osteonecrosis, a disease that occurs when excessive death of bone tissue and absence of bone regeneration result in the collapse of necrotic bone.
Bone Cells: Osteoclasts
Osteoclasts are multinucleated cells that contain up to 20 nuclei and are unique in their ability to resorb bone. They are directly attached to the bone surface and build resorption lacunae (Howship lacunae). Apart from their multiple nuclei, another characteristic of the osteoclast is the ruffled border, a highly folded plasma membrane facing the bone matrix and designed to secrete and resorb proteins and ions into the space between the osteoclast and bone surface ( Fig. 4.3 ). The space between this ruffled border and the bone surface is the place where bone resorption occurs. It is sealed by a ring of contractible proteins and tight junctions because it represents one of the few regions of the human body where a highly acidic milieu is found. Bone degradation by osteoclasts consists of two major steps: first, demineralization of inorganic bone components, and second, removal of organic bone matrix. To demineralize bone, osteoclasts secrete hydrochloric acid through proton pumps into the resorption lacunae. This proton pump requires energy that is provided by an adenosine triphosphatase (ATPase), allowing the enrichment of protons in the resorption compartment, which, in fact, represents an extra-cellular lysosome. In addition to protons and chloride, osteoclasts release matrix-degrading enzymes, including tartrate-resistant acid phosphatase (TRAP), lysosomal cathepsin K, and other cathepsins. Cathepsin K can effectively degrade collagens and other bone matrix proteins. Consequently, inhibitors of cathepsin K block osteoclast function and slow down bone resorption.
