Although there are several cell types that contribute to the overall health and function of the skeletal system, the main cellular components of bone are osteoblasts, osteoclasts, and osteocytes (Figure 1). These cell types reside directly within bone and regulate the synthesis and degradation of the acellular matrix components of bone.¹ Bone resorption and formation are predominantly carried out by osteoblasts and osteoclasts. Osteocytes, the most numerous of the bone cells, are important mechanosensors that translate mechanical stimuli to biochemical processes. A fourth cell type, referred to as bone-lining cells, represents terminally differentiated
osteoblasts that adhere tightly to the surface of bone and play a regulatory role in bone remodeling.

Osteoblasts are the main bone-forming cells and are identified histologically based on the expression of several components critical to their function and regulation including alkaline phosphatase and parathyroid hormone receptor. Osteoblasts are efficient synthesizers of extracellular matrix (ECM). The primary function of this cell type is secretion of osteoid (unmineralized bone), which is predominantly composed of type 1 collagen, osteocalcin, osteonectin, and osteopontin. On a molecular level, osteoblasts can be identified by expression of the lineage-specifying transcription factors Runx2.² Osteoblasts possess a very organized cellular composition that allows them to sense environmental regulators at their apical surface and secrete molecules important for new bone formation at the basal surface. Metabolically, as described in a 2021 study, osteoblasts use predominantly glycolytic programs that allow for shunting of important metabolic byproducts into biosynthesis pathways for the production of various ECM components.³ Osteoblasts are responsive to a variety of hormonal stimuli. Estrogen promotes bone formation by restricting osteoblast homeostasis, whereas chronic glucocorticoid use can decrease bone mineral density partially through augmentation of osteoblast and osteocyte apoptotic rate.

Osteoclasts are the main mediators of bone resorption. Histologically, osteoclasts are identified as multinucleated giant cells with abundant expression of the cell surface molecules receptor activator of nuclear
factor kappa B (RANK, an activator of the osteoclast resorption program) and the integrin receptor αvβ3 (cell surface adhesion molecule critical for osteoclast adhesion to bone surfaces).⁴ The basal surface is identified by a characteristic ruffled border, which creates a sealing zone where resorption of mineralized bone matrix takes place. Bone resorption is stimulated by signaling through the RANK pathway. There are several sources of RANK ligand (RANKL) that can induce osteoclast-mediated bone resorption, including osteoblasts, T cells, stromal fibroblasts and adipocytes, and cancer cells. Osteoclast activity is also tightly orchestrated by several hormonal stimuli. Estrogen has been shown to limit c-Jun activity, thus restricting RANKL-induced osteoclast differentiation. Histologically, osteoclasts are defined by multiple nuclei and abundant cytoplasm containing a high density of lysosomes and mitochondria to support their immense and diverse energy demands. Osteoclasts have dynamic and tightly regulated metabolic programs. Preliminary studies suggest that glycolysis is the predominant mechanism of energy generation in osteoclasts actively engaged in bone resorption, whereas oxidative phosphorylation is the main bioenergetic source for osteoclast formation. Metabolic regulation of osteoclast formation and function remains an active area of ongoing research and potential therapeutic intervention.⁵

Osteocytes are terminally differentiated cells that originate from osteoblasts, reside in characteristic lacunae embedded by bone matrix, and function as the main mechanosensors in bone.⁶ The mechanisms that regulate osteoblast differentiation to mature osteocytes remain incompletely elucidated, but involve a selective downregulation of canonical osteoblast genes and upregulation of selective osteocyte-associated genes: dentin matrix protein 1, fibroblast growth factor 23 (FGF23), ECM phosphoglycoprotein, and phosphate-regulating endopeptidase homolog X-linked. Osteocytes are characterized by extensive dendritic cytoplasmic projections that tunnel through mineralized bone matrix to establish the lacunar-canalicular arrangement in bone.⁶ These interconnections coordinate intercellular communication within bone and transfer mechanical stimuli into biochemical processes that influence bone resorption and formation.⁷ In response to mechanical signals such as shear stress or mechanical loading, osteocytes increase RANKL secretion (activates osteoclasts) and decrease sclerostin (an inhibitor of osteoblast anabolism) production.⁸ Collectively, mechanical stresses on bone activate the remodeling system mediated by osteocyte mechanotransduction of osteoblast and osteoclast-regulating molecules.

One of the main physiologic functions of bone is to provide a supportive environment for bone marrow stem cell self-renewal and differentiation. Far from simply serving as structural support, bone cells and the proteins they secrete have important influence on the fate commitment of various stem cell precursors. For example, in response to mechanical stress, osteocytes coordinate commitment of hematopoietic stem cells to the osteoclast lineage, thus promoting skeletal remodeling in a guided manner directly in response to mechanical stimuli.

Resident bone marrow stem cells are localized within trabecular spaces of the skeleton. Within these bone marrow niches reside a variety of precursor cell types, including stem cells of mesenchymal and hematopoietic origin that give rise to osteoblasts and osteoclasts, respectively. Stem cells capable of forming osteoblasts and other mesenchymal tissues were historically referred to as mesenchymal stem cells, a term that encompasses a heterogenous population of undifferentiated cells capable of producing cells of osteogenic and chondrogenic origin. Recent studies have identified a more pure skeletal stem cell characterized by distinct cell surface markers and differentiation patterns.⁹ These cells are particularly enriched in hypertrophic zones of the epiphyseal plate, but can also be isolated from other regions of bone including the femoral head, from bone morphogenetic protein 2 treated adipose cells, and from induced pluripotent stem cells. As discussed in a 2021 study, skeletal stem cells proliferate in the setting of bone injury and demonstrate age-associated phenotypic changes that predispose to diseases such as osteoporosis.¹⁰

Osteoclasts are a distinct bone cell in that they are derived of hematopoietic rather than mesenchymal origin. As such, they share many similarities to monocytes and macrophages histologically, genetically, and functionally. Differentiation of hematopoietic stem cells to the osteoclast lineage requires intimate interaction with bone stromal elements. Commitment to the osteoclast lineage is coordinated by stromal derived factors including RANKL and colony-stimulating factor 1. RANKL activates RANK receptors on hematopoietic stem cells, which in turn stimulates tumor necrosis factor receptor-associated cytoplasmic factors (TRAF), including TRAF6, to localize to the cytoplasmic portion of the RANK receptor. TRAF6 activates signal transduction pathways that support osteoclastogenesis by inducing expression of osteoclast-specific genes.¹¹

Despite a multitude of specialized cell types residing within and contributing to the many functions of bone, the physical mass of bone tissue is predominantly composed of acellular ECM. The ECM of bone can be largely classified into two components: organic and mineralized. The organic ECM components are formed predominantly by osteoblasts and are secreted in an unmineralized form called osteoid. Osteoid is generally found over bone surfaces and in areas of new bone formation. Mineralization of osteoid is a hallmark in the formation of new bone. It is ultimately the mineralized bone matrix that comprises a large majority of overall bone mass and largely defines bone’s material properties.

The organic component of ECM is 90% type 1 collagen. Other important organic components of ECM include osteocalcin, fibronectin, proteoglycans, bone morphogenetic proteins, and growth factors. In order for collagen to be secreted by osteoblasts, it must be modified posttranslationally. These modifications include hydroxylation of proline groups, which mechanistically explains why hydroxyproline can be used as a clinical marker of collagen breakdown. Secreted collagen fibrils exist in a helical structure and are held stable by intrinsic interactions between helices as well as interchain covalent cross-links. This specific collagen arrangement provides the tensile strength of bone, and modification to the level of collagen interaction can produce bone tissue of various tensile strengths.¹²

At any given time, approximately 70% of the skeletal ECM is mineralized. Mineralization of organic matrix refers to the inorganic ionic components that are demoisted in close association with collagen fibrils. The mineral crystals found in bone are a calcium phosphate compound called hydroxyapatite. In a process termed nucleation, calcium and phosphate ions are arranged into a crystalline hydroxyapatite molecule, which is then deposited in pockets of collagen fibrils under the promotion of supportive proteins such as biglycan and bone sialoprotein. After deposition of an initial hydroxyapatite crystal, mineralization of the organic ECM becomes an efficient process with multidirection secondary nucleation ensuing from the surface of the initially deposited crystal.¹

Through mineralization and demineralization of ECM tissue, bone plays an important role in systemic ion homeostasis, predominantly calcium balance. As such bone is exquisitely responsive to hormonal regulators of calcium balance. Parathyroid hormone can activate osteoblasts to secrete RANKL, which in turn activates osteoclasts. This leads to increased bone resorption, demineralization of existing bone matrix, and elevation of serum calcium levels. In the setting of hypercalcemia, calcitonin is produced by cells of the thyroid gland and leads to decreased serum calcium through dual effects in activating osteoblast and inhibiting osteoclast function. Vitamin D is a critical component of calcium ion homeostasis as it is required for dietary calcium absorption. Recent studies have also demonstrated a direct role for vitamin D in stimulating the proliferation and differentiation of human osteoblasts.¹³

Despite significant variability in the shape and size of bones in the human skeleton, the functional elements that support distinct physiologic roles in bone are conserved. Mature bone structure is characterized by its functional organization, either trabecular/cancellous or cortical. Trabecular bone is characterized by a loose organization of bony struts with interspersed bone marrow and stromal elements. The bony matrix components in trabecular bone are organized parallel to the direction of the trabecular strut and form discrete packets of bone tissue where bone remodeling cycles take place. Cortical bone is densely packed compact bone arranged in an organized concentric lamellar structure. The functional unit of cortical bone is called an osteon or haversian system, which are concentric rings of osteocytes and organized mineralized matrix with a central haversian canal. Each osteon is separated from other osteons by a cement line, but interosteon communication is facilitated by Volkmann canals, which run approximately perpendicular to the haversian system.

All bone surfaces are covered by specialized tissues. The outermost surface of bone is called the periosteal membrane, a highly innervated and well-vascularized structure consisting of fibrous connective tissue and an inner osteogenic layer of progenitor cells that can mature in response to various cues to stimulate the formation of new bone. Periosteal vessels supply the outer one-third of cortical bone. The inner surface of bone is referred to as the endosteal surface, is typically very thin, and consists of terminally differentiated osteoblasts referred to in this context as bone-lining cells. Vascular supply to the inner two-thirds of cortical bone is provided by nutrient arteries that pass through cortical foramen and travel through the haversian and Volkmann canals. Blood supply to trabecular bone is mediated by diffusion from adjacent bone marrow elements.

The effect of nutritional status on bone health and bone mineral density is well established. Deficiencies
in vitamin D lead to poor intestinal calcium absorption and subsequent decreased bone mineralization. As such, vitamin D and calcium supplementation have been long-standing interventions for conditions characterized by low bone mineral density such as osteoporosis. A rapidly developing area of study and clinical intervention is the influence of the gut microbiome on bone health.¹⁴ The human microbiome refers to the trillions of microbes that inhabit the human body and the products they secrete. Alterations in the microbiome have been associated with several pathologies, including osteoporosis. A healthy intestinal microbiome maintains gut epithelial health to aid in calcium absorption. Moreover, short chain fatty acids that are produced from bacterial digestion of dietary fiber can limit osteoclast differentiation and function without compromising bone formation.¹⁵ Recent studies demonstrate that reconstitution of healthy gut microbiome with a specialized diet consisting of supplemental sialylated milk oligosaccharides leads to increased femoral trabecular bone volume and cortical thickness in preclinical murine models and increased serum markers of osteoblastic differentiation in malnourished children¹⁶,¹⁷ (Figure 2). An understanding of the specific microbial species and their metabolic byproducts that influence overall bone health is an exciting area of ongoing research and represents immense clinical opportunity for low-cost intervention for a variety of skeletal pathologies.

Osteogenesis imperfecta, also known as brittle bone disease, is a genetic skeletal disorder estimated to affect approximately 1 in 13,500 to 15,000 births.¹⁸ The disease results from autosomal dominant mutations in the genes encoding type 1 collagen (COL1A1/2). Four types of osteogenesis imperfecta have been described:¹⁹ type I corresponds to the mildest form, type II to the perinatal lethal form, type III to the most severe form compatible with life, and type IV is an intermediate group. Over the past 10 years, the classification, which was initially phenotypically based, has expanded to currently include more than 16 different types as this number is constantly increasing with novel gene discovery.¹⁸

Heterozygous mutations in the collagen genes COL1A1 and COL1A2 are the most common cause of osteogenesis imperfecta. The large size of these genes explains the numerous known mutations and part of the heterogeneity of the clinical symptoms. Two different pathophysiologies can be described: Loss-of-function mutations such as stop mutations lead to haploinsufficiency. Patients have a reduced amount of collagen, but this is of normal quality. In contrast, other mutations (mostly glycine substitutions) lead to qualitative alterations of the ECM, because the collagen molecules and later fibrils cannot assemble properly. Because of the reduced bone stability and the stimulated degradation, osteoblasts produce as much osteoid as possible, but this is of lower quality. This results in high-turnover osteoporosis. Additionally, mutations in several other genes can result in the clinical picture of osteogenesis imperfecta. Moreover, mutation in several genes can result in the clinical picture of osteogenesis imperfecta without changing the collagen sequences but rather affecting the biosynthetic pathway and secretion of collagens. These include P4HB, P3HI, CRTAP, PPIB, and several others.

The symptoms of the disease can be divided into skeletal and extraskeletal findings. Skeletal symptoms are decreased bone mass leading to reduced bone stability. This results in an increased fracture rate of the long bones after minor trauma, as well as deformities of vertebrae. Scoliosis is an additional problem that develops frequently during puberty in more severely affected patients and can lead to impairment of pulmonary function. Short stature is present in almost all patients and extremities can be disproportioned. As a collagen disorder, additional extraskeletal symptoms can
include hypermobility of ligaments and increased fragility of vessels. An effect on heart valves has also been described as well as an early loss of hearing. An obvious but not always persistent finding is a blue-gray discoloration of the sclera in approximately 50% of patients with osteogenesis imperfecta. Because of the close biochemical relationship between collagen and dentin, the teeth are affected in some patients, leading to dentinogenesis imperfecta with amber-colored appearance and increased brittleness.²⁰

Histologic changes include quantitative deficiencies in ECM production and mineralization. In addition, the persistence of cartilage within bone trabeculae of the diaphysis, and the absence of lamellar bone and mature haversian systems indicate abnormalities in the bone maturation process by which cartilaginous precursor structures are replaced by woven bone, and subsequently by mature lamellar bone (Figure 3, A).

Medical treatments with diphosphonates are currently used as standard therapy in patients with a moderate or severe course of the disease in childhood and adolescence. These drugs effectively reduce bone resorption and thereby increase bone mass. In addition, the strengthening of muscles induces an osteoanabolic stimulus, which leads to an increase in the synthesis of ECM by osteoblasts. Although the function of osteoblasts can be impaired in osteogenesis imperfecta, using the muscles is still the best way to stimulate bone formation.²¹