by Robert C. Mellors, M.D., Ph.D.


  1. Bone Formation
  2. The process of bone formation (osteogenesis) involves three main steps: 
    • production of the extracellular organic matrix (osteoid); 
    • mineralization of the matrix to form bone; 
    • and bone remodeling by resorption and reformation. 
    The cellular activities of osteoblasts, osteocytes, and osteoclasts are essential to the process. Osteoblasts synthesize the collagenous precursors of bone matrix and also regulate its mineralization. As the process of bone formation progresses, the osteoblasts come to lie in tiny spaces (lacunae) within the surrounding mineralized matrix and are then called osteocytes. The cell processes of osteocytes occupy minute canals (canaliculi) which permit the circulation of tissue fluids. To meet the requirements of skeletal growth and mechanical function, bone undergoes dynamic remodeling by a coupled process of bone resorption by osteoclasts and reformation by osteoblasts.
  3. Osteoblasts and Bone Matrix
  4. Osteoblasts are derived from mesenchymal stem cells of the bone marrow stroma. They possess a single nucleus, have a shape that varies from flat to plump, reflecting their level of cellular activity, and in later stages of maturity line up along bone-forming surfaces. Osteoblasts synthesize and lay down precursors of collagen 1, which comprises 90-95% of the organic matrix of bone. Osteoblasts also produce osteocalcin -the most abundant non-collagenous protein of bone matrix- and the proteoglycans of ground substance and are rich in alkaline phosphatase, an organic phosphate-splitting enzyme.Osteoblasts have receptors for parathyroid hormone and apparently for estrogen.Hormones, growth factors, physical activity, and other stimuli act mainly through oteoblasts to bring about their effects on bone.

    The collagen 1 formed by osteoblasts is typically deposited in parallel or concentric layers to produce mature (lamellar) bone. But when bone is rapidly formed, as in the fetus or certain pathological conditions (fracture callus, fibrous dysplasia, hyperparathyroidism), the collagen is not deposited in a parallel array but in a basket-like weave and is called woven, immature, or primitive bone.

    In fully decalcified bone sections, the extracellular matrix stains pink with H+E, similar to collagen elsewhere but with a more homogeneous than fibrillar structure which latter is easily observed by polarizing microscopy.

  5. Bone Mineralization
  6. The main mineral component of bone is an imperfectly crystalline hydroxyapatite [Ca10(PO4)6(OH)2] which comprises about 1/4 the volume and 1/2 the mass of normal adult bone. The mineral crystals, as shown by electron microscopy, are deposited along, and in close relation to, the bone collagen fibrils. Calcium and phosphorus (Pi, inorganic phosphate) are, of course, derived from the blood plasma and ultimately from nutritional sources. Vitamin D metabolites and parathormone (PTH) are important mediators of calcium regulation, and lack of the former or excess of the latter leads to bone mineral depletion. 

    Undecalcified bone sections, such as those stained with the von Kossa stain, are best used for the histological study of bone mineral distribution. The extracellular matrix of bone is mineralized soon after its deposition, but a very thin layer of unmineralized matrix is seen on the bone surface, and this is called the osteoid layer or osteoid seam. In some pathological conditions, the thickness and extent of the osteoid layer may be increased (hyperosteoidosis) or decreased. Hyperosteoidosis may be caused by conditions of delayed bone mineralization (as in osteomalacia/rickets resulting from vitamin D deficiency) or of increased bone formation (as in fracture callus, Paget's disease of bone, etc.). 

    Mineralization dynamics can be studied in undecalcified bone sections if two time-spaced doses of tetracycline (which binds to actively mineralizing surfaces and is autofluorescent) are given to a patient before a bone biopsy is performed. A biopsy of normal bone will show two discrete and separated layers of fluorescent label uptake marking successive mineralization fronts. If mineralization is blocked, there is no uptake of the labels, and if mineralization is deficient and delayed, as in osteomalacia/rickets caused by vitamin D deficiency, the labels are often smudgy in appearance or in some cases there is no uptake at all. 

  7. Osteoclasts and Bone Resorption
  8. Osteoclasts are derived from hematopoietic stem cells that also give rise to monocytes and macrophages.Typically multinucleated, osteoclasts adhere to the surface of bone undergoing resorption and lie in depressions termed Howship's lacunae or resorption bays. The boundary between the old and new bone is distinguished in an H+E section by a blue (basophilic) line called a cement line or reversal line. Several metabolic bone diseases (such as hyperparathyroidism, Paget's disease, and others) are characterized by increased modeling and increased osteoclastic activity. Osteoclasts are apparently activated by "signals" from osteoblasts. For example, osteoblasts have receptors for PTH whereas osteoclasts do not, and PTH-induced osteoclastic bone resorption is said not to occur in the absence of osteoblasts.
  9. Bone Development
  10. At an early stage of human embryonic development, a cartilage model of much of the skeleton ( of extremities, trunk, and base of the skull) is formed from the mesenchyme. In the further fetal development of long bones, a rim of primitive bone is first laid down in layers over the middle of the shaft by osteoblasts arising from the overlying periosteum, and subperiosteal bone formed in this way soon extends up and down the shaft (diaphysis). The process by which bone tissue replaces membranous fibrous tissue is called intramembranous ossification 

    3042: Intramembraneous ossification, femur of 4 month fetus, H&E. 

    This is the process by which the diaphysis increases in width throughout postnatal growth. Some bones, such as the flat bones of the calvarium, are formed entirely, or in great part, by intramembranous ossification.

    The cartilage cells of the core of the fetal shaft degenerate upon contact with penetrating buds of periosteal osteoblasts, the cartilage matrix becomes mineralized and resorbed, and the resulting surfaces and spaces are lined by osteoblasts which lay down woven bone and form primitive bone trabeculae. The process by which bone tissue replaces cartilage is called endochondral ossification 

    3043: Endochondral ossification, femur of term fetus, H&E. 

    and begins in the femur at about the ninth week of fetal life. Some of the trabeculae fuse with the subperiosteal new bone while others are resorbed to form a medullary cavity which will be occupied by hematopoietic tissue. Thus, the primitive bone shaft is formed 

    3044: Primitive bone shaft and cartilage models of the skeleton, lower extremity of 4 month fetus, H&E.

    and lies between the cartilaginous ends which become the epiphyses. In the later months of fetal life, the woven bone of the diaphysis will be replaced by lamellar bone of mature type. Over time, the bone cortex is thickened and remodeled to serve mechanical functions and is permeated by haversian systems (bone-forming units) of longitudinal, vascularized canals bounded by concentric lamellae of bone, culminating in the typical appearance of compact cortical bone as seen in the adult. 

    3045: Compact cortical bone and haversian systems (bone-forming units),femur of 31 year old male, polished bone block. 

    3046: Compact cortical bone, PAMS (periodic acid methenamine silver, rabbit femur.

    The longitudinal growth of the long bones occurs in the epiphysial (epiphyseal) growth plate as cartilage cells, arising from reserve cells, undergo mitosis and proliferate in orderly longitudinal columns. 

    3047: Epiphysial growth plate, femur of term fetus, H&E. 

    The proliferated cartilage cells vacuolate as they move toward the cartilage-bone junction (metaphysis), the cartilage matrix becomes mineralized, and buds of osteoblasts emerging from the metaphysis replace the mineralized cartilage with bone. This process of cartilage cell proliferation and endochondral ossification is repeated over and over, and the bones become longer and larger. Meanwhile, at age-related intervals, secondary centers of endochondral ossification ("radiologic epiphysis") begin to form on the articular side of the growth plate. 

    3048: Secondary center of ossification, femur of term fetus, H&E. 

    When skeletal maturity is reached, the cartilage cells of the growth plate cease to proliferate, the growth plate becomes thinner, is replaced by bone and disappears, and the epiphysis is "closed" or fused with the shaft.