HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter 8 BONE FORMATION

Introduction
l Occurs in only one way - by the appositional or surface-depository action of osteoblasts; soon accompanied by the selective destructive action of osteoclasts in a remodelling process, continuously adapting the growing bone to developing soft tissues and dynamic mechanical forces, whilst meeting metabolic mineral demands.
2 Growth by remodelling is necessary because no interstitial growth is possible (except in growth cartilages).
3 Bone formed in the fetus is woven: only later is it mostly replaced by lamellar bone.
4 Dependent on whether bone is formed de novo in a soft tissue area, or in a site already taken by an established cartilaginous model, two situations of bone formation are noted - intramembranous and endochondral.

A INTRAMEMBRANOUS OSSIFICATION

l Seen in the skull vault, facial skeleton, and parts of the clavicle.
2 In one or more ossification centres for a given bone, mesenchymal cells become osteoblasts and start to lay skeletal claim to territory by forming branching trabeculae/struts of bone. The initial thin struts may be called spicules.
3 This trabecular bone becomes denser by widening of the trabeculae, and is then remodelled externally and internally, e.g., in the skull vault to two denser plates, tables, with spongy bone - diploë - between them.
4 The remodelling plates expand from their centres, but during growth remain separated by CT sutures for better adjustment to the enlarging brain, eyes, nasal cavities, etc. Skull bones grow by complex interactions and remodelling patterns that must cope also with, for instance, more teeth in the older child's jaws and the need for articulating cartilages on the mandible .

B PREPUBERTAL LONG BONE

l Diaphysis is the long tubular shaft containing marrow. The dense bone is the cortex, the marrow constitutes the medulla.
2 Epiphyses lie at each end of the bone. Each has:
... (a) a cap of hyaline articular cartilage over a
... (b) cushioning lattice of secondary-ossification-centre bone;
... (c) this bone on its deeper aspect is fused with an epiphyseal plate/growth disc of hyaline cartilage.
3 Metaphysis is a lattice of bone trabeculae (primary ossification bone) with cross-struts, that joins each end of the shaft to an epiphyseal plate.
4 Endosteum lines all internal bony surfaces.
5 Periosteum ensheaths the bone, except for a small circumferential perichondrium around the epiphyseal plates, and where tendons and ligaments fasten to the bone. The articulating surfaces are bare.
6 Longitudinal growth, while the bone is under the stresses of use, is provided for by the interstitial growth of cartilage in the growth plates.

C ENDOCHONDRAL/INTRACARTILAGINOUS OSSIFICATION

l Mesenchymal cells retracting their processes round up to become chondroblasts, which form a minute hyaline cartilage precursor having roughly the shape of the eventual bone, e.g., the femur. Other mesenchymal cells differentiate and make a perichondrium.

2 In the central, shaft, region of the cartilage:

(a) Cartilage cells hypertrophy.
(b) Matrix around them becomes basophilic, then calcifies.
(c) Perichondral cells close by become osteoblasts; i.e., the perichondrium becomes a periosteum.
(d) Periosteal bone deposition forms a subperiosteal bony collar or ring around the centre of the shaft.
(e) At the same time, through gaps in the bone, osteogenic/vascular buds invade the calcified cartilage matrix to form the primary ossification centre of bone forming in, and on the calcified walls of, spaces eroded in the matrix.

3  Cells and functions of an osteogenic bud are:
   (a) macrophages               |
   (b) chondroclasts             |- for selective cartilage erosion
   (c) endothelial cells?        |
   (d) progenitor cells of osteoblasts and osteoclasts/chondroclasts;
   (f) marrow cells - to populate intertrabecular spaces;
   (g) endothelial cells - to form capillaries and sinusoids.

4 Primary ossification zone establishes itself across the width of the shaft and starts extending in both directions towards the epiphyses, resulting in two transverse fronts of ossification across the diaphysis. At each front is the cartilaginous growth plate.

5 Epiphyseal plate. (This only becomes plate-like after secondary ossification has started within the epiphysis.) Starting farthest from the front, the zones are:

(a) Reserve/quiescent zone of hyaline cartilage with small cells and few cell divisions.
(b Proliferative zone where chondrocytes multiply and arrange themselves in ordered parallel columns (palisades) of disc-shaped cells. Growth, interstitial, in the long axis of the bone occurs mainly here.
(c) Hypertrophic/maturing zone has the chondrocytes no longer dividing, but enlarging at the expense of their matrix.
(d) Calcification zone, where the matrix stains basophilic and is impregnated by crystals of calcium salts. Mineralization may be triggered by the seeding action of calcium-rich matrix vesicles released from chondrocytes. Whether or not these chondrocytes then all die is disputed.
(e) Erosion zone encroaches into the calcification zone. Vascular bud elements destroy some cartilage, but leave longitudinal spicules as a scaffold on which bone is laid down. In HE preparations the calcified cartilage cores are blue, the superficial bone red.

6 Within the cartilage of the young epiphysis, a secondary ossification centre develops, again by processes of cartilage cell hypertrophy, matrix calcification, and its erosion by vascular elements penetrating from the perichondrium. However, orderly columns of chondrocytes and a defined marrow cavity are lacking.

7 The epiphyseal, secondary, ossification centre spreads to occupy much of the epiphysis and forms the bony border to the cartilaginous epiphyseal plate. The cartilage grows (thus lengthening the whole bone) keeping pace with the front of ossification invading it from the metaphyseal side, until puberty. Then resorption and ossification slowly overtake halting chondrocyte proliferation, until the primary ossification front fuses with the secondary epiphyseal bone - epiphyseal fusion/closure. The growth plate is obliterated, but an irregularity in the trabecular bone pattern marks its site.

8 Hyaline cartilage remains as a thin cap over the epiphysis to be the articular surface.

9 Growth in width of the shaft is by a periosteal deposition on the outside surface, coordinated with an osteoclastic resorption on the inner, marrow, aspect. These patterns may be reversed at sites of change in shape or drift. At the same time, shaft bone is remodelled internally to be more lamellar and have the layers of Chapter 7.D.

D OSTEOID

l The osteoid seam is a very poorly mineralized, narrow zone of organic matrix seen sometimes with LM between the true bone and active osteoblasts.
2 It results from a definite lag between the formation of collagen fibrils and the later deposition of mineral crystals.
3 The presence of osteoid can be determined for sure by methods, e.g., von Kossa's silver, microradiography, EM, which are able to show the absence of mineral, but certain stains for decalcified sections are reliable.
4 The seam widens significantly in osteomalacia and rickets when too little Ca²⁺ is available, e.g., in kidney disease.

E PHYSIOLOGICAL FACTORS AFFECTING CONNECTIVE TISSUES

l Hormones

Growth: for matrix synthesis, particularly in epiphyseal cartilage; lack causes dwarfism, excess gigantism, or in adult-onset, acromegaly.
Parathyroid: acts on osteoblasts to cause osteoclasts to resorb bone, thus raising blood Ca²⁺; lack of hormone results in death by tetany; large eroded spaces from an excess fill with fibrous CT in 'osteitis fibrosa' - hyperparathyroidism. (There is also the paradox that small doses of PTH stimulate bone formation.)
Calcitonin: acts on the 'clast to block bone resorption and lower blood Ca²⁺, but its role seems to be minor (except in treating Paget's disease - uncoordinated 'clasts and 'blasts).
Thyroid: acts indirectly on all cell activities by controlling metabolic rate; lack thus slows growth; an excess favours resorption.
Sex: affect genital tract CT, e.g., endometrial stroma; also the timing of secondary ossifications and epiphyseal closure (premature fusion and dwarfing follow an excess of gonadal hormone in childhood).
Glucocorticoid: excess impairs bone and CT matrix synthesis; used clinically to reduce inflammation, perhaps by reducing prostaglandins.
Insulin, Norepinephrine, etc: control fat cell metabolism.
Parathyroid hormone-related protein/PTHrP is a PTH-like peptide released from various tumours that causes a cancer-linked destruction of bone, and hypercalcaemia. (PTHrP acts more in a local - paracrine - manner than the endocrine PTH.)

2 Agents
l Vitamin D: in its active form is needed for Ca²⁺ to be absorbed in the gut; low blood Ca²⁺ from a lack of D prevents mineralization of growth cartilage matrix, resulting in rickets, and causes the failure of osteoid to mineralize in osteomalacia; excess D may raise blood Ca²⁺ to the point where soft tissue calcifications occur.
2 Prostaglandins: stimulate osteoclastic bone resorption
3 Peptides: although thought of originally in the contexts of immunity and haemopoiesis, the cytokines (see F below) influence matrix formation and destruction, and the numbers and activities of all connective tissue cells, e.g., macrophages stimulate fibrogenesis, and osteoblasts interact with osteoclasts, etc.

3 Diet
l Calcium, phosphorus: see 2.l above for Ca²⁺ deficiency.
2 Vitamin A: excess and deficiency disturb ossification and remodelling in different ways.
3 Vitamin C: deficiency (scurvy) impairs collagen synthesis in all CTs.
4 Copper: needed for making elastin.
5 Toxic elements, e.g., Pb, ⁹⁰Sr, F, may substitute for the natural elements and ions in the mineral crystals of bone and teeth.

4 Use
CTs respond to more use by making a matrix better able to withstand the greater forces, e.g., osteoblasts build more and wider bone trabeculae; conversely, disuse leads to the few thin and frail trabeculae of osteoporosis.

F CYTOKINES

1 The name does not indicate the only source or action; all cell types use cytokines for signalling, including neural and epithelial cells.
2 The materials are protein or glycoprotein and express sub-types, e.g., acidic and basic fibroblast growth factors (aFGF & bFGF), and control cells by binding to receptors, but comprise a system separate from hormones, neurotransmitters, and the eicosanoids (derivatives of arachidonic acid, e.g., prostaglandins and leukotrienes).
3 Cytokines' actions are diverse, and not consistently stimulatory or inhibitory, but depend on the target cell type and the action of other agents.
4 Factors that could qualify, but are already known as hormones, such as erythropoietin & insulin, are not listed.
5 Cytokines are important for the control of renewing cell populations, in inflammation and healing (wound and fracture), and the immune responses, and are used clinically to influence disease or tardiness in these processes.
6 A few cytokines and their actions are listed below, but which cytokine does what can wait.

Epidermal growth factor  EGF            Insulin-like growth factors IGFs            
Platelet-derived growth factor PDGF     Fibroblast growth factors  FGFs             
Transforming growth factors  TGF-1      Interleukins 1,2,3-- IL-1,2, --IL-10   
Tumor necrosis factor-alpha TNF-a      Colony-stimulating factors CSFs
Interferons  IFs                        Stem-cell factor  SCF

Help to start and/or complete cell proliferation;
Promote or inhibit differentiation;
Activate white blood cells, osteoclasts, etc.;
Raise or lower the rate of synthesis of ECM;
Alter the release of proteases or their inhibitors;
Induce chemotaxis, motility & change of cell shape;
Change sensitivity to other cytokines or hormones;
Cause fever;
Cause vasoconstriction

William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159