Bone Histology & Few Notes about Bone

BiomechanicsAssist. Professor Sadeq A. Al-Mukhtar

Prepared By Ali Abdul-Kareem Abbas

• Bone consists of extracellular bone matrix and bone cells.

• The composition of the bone matrix is responsible for the characteristics of bone.

• The bone cells produce the bone matrix, become entrapped within it, and break it down so that new matrix can replace the old matrix.

Bone Matrix

• By weight, mature bone matrix normally is approximately 35% organic and 65% inorganic material.

• Bone is formed by the hardening of this matrix entrapping the cells. When these cells become entrapped from osteoblasts they become osteocytes.

Molecular Structure

• Inorganic• The inorganic material primarily consists of a calcium phosphate

crystal called hydroxyapatite, which has the molecular formula [Ca10(PO4)6(OH)2].

• The matrix is initially laid down as un-mineralised osteoid (manufactured by osteoblasts).

• Mineralisation involves osteoblasts secreting vesicles contain in alkaline phosphatase. This cleaves the phosphate groups and acts as the foci for calcium and phosphate deposition.

• The vesicles then rupture and act as a centre for crystals to grow on.

• More particularly, bone mineral is formed from globular and plate structures, distributed among the collagen fibrils of bone and forming yet larger structure.

The Chemical Composition of Bone

Range in Literature for Healthy Adult Whole Bone (wt% of ash)

Calcium 32.6-39.5

Magnesium 0.32-0.78

Sodium 0.26-0.82

Potassium -

Stronium -

Phosphorus 13.1-18.0

Carbonate 3.2-13

Citrate 0.04-2.67

Chloride -

Fluoride 0.02-0.207

• Organic• The organic material primarily consists of

collagen (90%) and non-collagenous proteins (10%). The role of late unclear (osteocalcin, osteonectin, osteopontin and bone sialprotein).

• The collagen is synthesised intracellularly as tropocollagen and then exported, forming fibrils.

• One of the main things that distinguishes the matrix of a bone from that of another cell is that the matrix in bone is hard.

• The collagen and mineral components are responsible for the major functional characteristics of bone. Bone matrix might be said to resemble reinforced concrete.

• Collagen, like reinforcing steel bars, lends flexible strength to the matrix, whereas the mineral components, like concrete, give the matrix compression (weight-bearing) strength.

• If all the mineral is removed from a long bone, collagen becomes the primary constituent, and the bone becomes overly flexible. On the other hand, if the collagen is removed from the bone, the mineral component becomes the primary constituent, and the bone is very brittle.

Effects of Changing the Bone Matrix (a) Normal bone. (b) Demineralized bone, in which collagen is the primary remaining component, can be bent without breaking. (c) When collagen is removed, mineral is the primary remaining component, thus making the bone so brittle it’s easily shattered.

Bone Cells

Osteoblasts• Osteoblasts have an extensive endoplasmic reticulum and

numerous ribosomes. They produce collagen and proteoglycans, which are packaged into vesicles by the Golgi apparatus and released from the cell by exocytosis. Osteoblasts also form vesicles that accumulate calcium ions (Ca2+), phosphate ions (PO4=), and various enzymes. The contents of these vesicles are released from the cell by exocytosis and are used to form hydroxyapatite crystals. As a result of these processes, mineralized bone matrix is formed.

• Ossification, or osteogenesis, is the formation of bone by osteoblasts. Elongated cell processes from osteoblasts connect to cell processes of other osteoblasts through gap junctions. The osteoblasts then form an extracellular bony matrix that surrounds the cells and their processes.

• When the osteoblasts are not in the process of forming bone, they are flattened, elongated cells covering quiescent bone surfaces and are called resting osteoblasts or bone-lining cells.

Osteocytes• Oseocytes are the most abundant cells in mature bone with about

ten times more oseocytes than osteoblasts in normal human bone. • Once an osteoblast becomes surrounded by bone matrix, it is a

mature bone cell called an osteocyte. Osteocytes become relatively inactive compared to most osteoblasts, but it’s possible for them to produce components needed to maintain the bone matrix.

• The spaces occupied by the osteocyte cell bodies are called lacunae, and the spaces occupied by the osteocyte cell processes are called canaliculi. In a sense, the cells and their processes form a “mold” around which the matrix is formed.

• Functions: (1) stabilize bone minerals by maintaing approperiate local ionic milieu. (2) detect microdamage, and (3) respond to the amount and distribution of strain within bone tissue (modeling-remodelling)/reconstruction.

Ossification

(a) Osteoblasts on a preexisting surface, such as cartilage or bone. The cell processes of different osteoblasts join together. (b) Osteoblasts have produced bone matrix. The osteoblasts are now osteocytes. (c) Photomicrograph of an osteocyte in a lacuna with cell processes in the canaliculi.

Osteoclasts• Osteoclasts are large cells with several nuclei and care responsible

for the resorption, or breakdown, of bone. Where the plasma membrane of osteoclasts contacts bone matrix, it forms many projections called a ruffled border.

• Hydrogen ions are pumped across the ruffled border and produce an acid environment that causes decalcification of the bone matrix. The osteoclasts also release enzymes that digest the protein components of the matrix. Through the process of endocytosis, some of the breakdown products of bone resorption are taken into the osteoclast.

• Osteoclasts break down bone best when they are in direct contact with mineralized bone matrix. Osteoblasts assist in the resorption of bone by osteoclasts by producing enzymes that break down the thin layer of unmineralized organic matrix normally covering bone. Removal of this layer by osteoblasts enables the osteoclasts to come into contact with the mineralized bone.

Origin of Bone Cells• Connective tissue develops embryologically from

mesenchymal cells. • Some of the mesenchymal cells become stem cells,

which have the ability to replicate and give rise to more specialized cell types.

• Osteochondral progenitor cells are stem cells that have the ability to become osteoblasts or chondroblasts. Osteochondral progenitor cells are located in the inner layer of the perichondrium, the inner layer of the periosteum, and in the endosteum. From these locations, they can be a potential source of new osteoblasts or chondroblasts.

• Osteoblasts are derived from osteochondral progenitor cells, and osteocytes are derived from osteoblasts. Whether or not osteocytes freed from their surrounding bone matrix by resorption can revert to active osteoblasts is a debated issue.

• Osteoclasts are not derived from osteochondral progenitor cells but are derived instead from stem cells in red bone marrow.

• The bone marrow stem cells that give rise to a type of white blood cell, called a monocyte, also are the source of osteoclasts. The multinucleated osteoclasts probably result from the fusion of many stem cell descendants.

Woven and Lamellar Bone• Bone tissue is classified as either woven or lamellar bone according

to the organization of collagen fibers within the bone matrix. In woven bone, the collagen fibers are randomly oriented in many directions.

• Woven bone is first formed during fetal development or during the repair of a fracture. After its formation, osteoclasts break down the woven bone and osteoblasts build new matrix. This process of removing old bone and adding new bone is called remodeling. Woven bone is remodeled to form lamellar bone.

• Lamellar bone is mature bone that is organized into thin sheets or layers approximately 3–7 micrometers (μm) thick called lamellae. In general, the collagen fibers of one lamella lie parallel to one another but at an angle to the collagen fibers in the adjacent lamellae. Osteocytes, within their lacunae, are arranged in layers sandwiched between lamellae.

• In human long bones, woven bone is replaced by lamellar bone at age 2 and 3 years.

• The lamellae of adult cortical bone appear in three major pattern;

• (1) circular ring of lamellae (concentric).• (2) circuferential lamellae: extended

uninterrupted.• (3) angular (interstitial) that filling the gap

between Haversian system.

Cancellous and Compact Bone• Bone, whether woven or lamellar, can be classified

according to the amount of bone matrix relative to the amount of space present within the bone. Cancellous bone has less bone matrix and more space than compact bone, which has more bone matrix and less space than cancellous bone.

• Cancellous bone consists of interconnecting rods or plates of bone called trabeculae. Between the trabeculae are spaces that in life are filled with bone marrow and blood vessels. Cancellous bone is sometimes called spongy bone because of its porous appearance.

• Most trabeculae are thin (50–400 μm) and consist of several lamellae with osteocytes located between the lamellae. Each osteocyte is associated with other osteocytes through canaliculi.

• Usually no blood vessels penetrate the trabeculae, so osteocytes must obtain nutrients through their canaliculi. The surfaces of trabeculae are covered with a single layer of cells consisting mostly of osteoblasts with a few osteoclasts.

• Trabeculae are oriented along the lines of stress within a bone. If the direction of weight-bearing stress is changed slightly (e.g., because of a fracture that heals improperly), the trabecular pattern realigns with the new lines of stress.

Cancellous Bone(a) Beams of bone, the trabeculae, surround spaces in the bone. In life, the spaces are filled with red or yellow bone marrow and with blood vessels. (b) Transverse section of a trabecula.

Lines of StressThe proximal end of a long bone (femur) showing trabeculae oriented along lines of stress (arrows).

• Compact bone is denser and has fewer spaces than cancellous bone. Blood vessels enter the substance of the bone itself, and the lamellae of compact bone are primarily oriented around those blood vessels. Vessels that run parallel to the long axis of the bone are contained within central, or haversian (haver shan), canals.

• Central canals are lined with endosteum and contain blood vessels, nerves, and loose connective tissue. Concentric lamellae are circular layers of bone matrix that surround a common center, the central canal.

• An osteon, or haversian system, consists of a single central canal, its contents, and associated concentric lamellae and osteocytes. In cross section, an osteon resembles a circular target; the “bull’s-eye” of the target is the central canal, and 4–20 concentric lamellae form the rings.

• Osteocytes are located in lacunae between the lamellar rings, and canaliculi radiate between lacunae across the lamellae, producing the appearance of minute cracks across the rings of the target. The outer surfaces of compact bone are formed by circumferential lamellae, which are flat plates that extend around the bone. In some bones, such as certain bones of the

• face, the layer of compact bone can be so thin that no osteons exist, and the compact bone is composed of only circumferential lamellae.

• In between the osteons are interstitial lamellae, which are 174 Part 2 Support and Movement remnants of concentric or circumferential lamellae that were partially removed during bone remodeling.

• Osteocytes receive nutrients and eliminate waste products through the canal system within compact bone.

• Blood vessels from the periosteum or medullary cavity enter the bone through perforating, or Volkmann’s, canals, which run perpendicular to the long axis of the bone.

• Perforating canals are not surrounded by concentric lamellae but pass through the concentric lamellae of osteons.

• The central canals receive blood vessels from perforating canals. • Nutrients in the blood vessels enter the central canals, pass into the

canaliculi, and move through the cytoplasm of the osteocytes that occupy the canaliculi and lacunae to the most peripheral cells within each osteon. Waste products are removed in the reverse direction.

Bone Development• During fetal development, bone formation occurs in two patterns

called intramembranous and endochondral ossification. • The terms describe the tissues in which bone formation takes place:

intramembranous ossification in connective tissue membranes and endochondral ossification in cartilage.

• Both methods of ossification initially produce woven bone that is then remodeled.

• After remodeling, bone formed by intramembranous ossification cannot be distinguished from bone formed by endochondral ossification.

• Bone remodeling produces and maintains bone that is biomechanically and metabolically competent.

This table compares intramembranous and endochondral ossification.

Bone surfaces • Bone restricted to growth appositionally, because of non-

expandable nature of mineralized bone tissue; thus all bone activities occur at bone surfaces.

• Bone tissue has 2 major surfaces, perosteal and endosteal (subdivieded to intracortical –osteonal-, endocortical and cancellous or trabecular surfaces).

• Cacellous bone surface contributes >61% of the total bone surface because the mean trabecular surface-to-volume ratio is 8 times greater in cancelous (20mm2/mm3) than in cortical bone (2.5mm2/mm3).

• At any specific time the bone surfaces may be in one of the three functional states: forming, resorbing, or quiescent, according to the cell type present at that time.

Bone structural unit • In cortical bone: Osteon or Haversian system (2/3). • In cancellous or trabecular bone: trabecular packet or

hemiosteon (shallow crescent).

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Strength of Trabecular Bone • Strength: stress at which the specimen fails, by an offset yield point

or at maximal load carrying capacity, i.e. ‘’the ultimate point‘’. • Uniaxial Properties: the strength of trabecular bone depends on

volume fraction, architecture, and the tissue material properties, in that order of importance. E.g. tensile, compressive strengths.

• Strength-Density Relations:

• Where ult is ultimate stress (in MPa), p is apparent density (in g/cm3), and is strain rate (in s-1).

• Multiaxial Behavior: it is important clinically since multiaxial stresses can occur during falls, trauma, and at the bone-implant interface. Formation of a multiaxial failure criteration for trabecular bone particularly challenging since it is necessary to account for interspecimen variations in volume fraction and architecture (Tsai-Wu Criterion).

Skeletal Mass and It’s Changes • Mechanical regulation of bone biology begins at about 5-7 weeks of prenatal life

when most of the adult skeletal elements and soft tissue has formed. • The mechanostat best explained by Frost’s hypothesis. Frost proposed the

mechanostat as a name for the biologic mechanisms that skeletal mass and architecture to the needs of one’s normal physical activities. It is well recognized that bone mass adapts to mechanical usage (MU) in special way: the MU makes it biologic mechanisms correct errors between the mass and its MU.

• MU bone mechanostat bone mass effect • The mechanostat has been proposed to consist of at least these tissue–level

biologic mechansims: growth, modeling and remodeling mechanisms (location and rates) + mechanical transducers.

• The mechanostat concept is based on the idea that exists an effective strain, set point (range of strain at which no response), with a mechanical overload, a response will occur that increases muscle and bone mass and strength.

• > 1500-3000 microstrain will evoke a positive response while <200 micostrain trigger a response that cause bone loss. So remodelling at 2 ends.

• Non-mechanical agents can evoke changes or errors in the set point range.

• The Utah paradigm of skeletal physiology: Health/Disease --------- Mechanical Feedback Loop Muscle Loads Bone Signals Biologic Mechanisms Mechanical & non-mechanical agents 4 conditions: (determine, time & space, after birth neuromotor

physiology & anatomy>biologic mechanisms, and non-mechanical help or hinder the mechanical control)

Mechanical Testing Methods

• There are numbers of biomechanic parameters that can be used to characterize the integrity of bone.

• The key relationship is that between load applied to a structure and displacement in response to the load.

• The slop of the elastic region of the load-displacement curve represents the extrinsic stiffness or rigidity of the structure.

• Besides stiffness, several other biomechanical properties can be derived, including ultimate load (force at failure), work to failure (area under the load displacement curve) and ultimate displacement.

The load-displacement curve, illustrating the four key bio mechanical parameters for the bone specimen: ultimate load, extrinsic stiffness or rigidity (S), work to failure (U), and ultimate displacement.

Tensile Test

Compressive Test

Bending Test

Torsion Test

Site-Specific Tests

Mechanobiological Models of Fracture Healing

• Pauwel’s theory: regulative effect of mechanical forces on tissue differentiation, using simple experimental models of fracture callus.

• Shearing stress change in shape, whereas hydrostatic pressure change in volume.

• 1. Shear, which causes a change in cell shape, stimulates mesenchymal cell differentiation into fibroblasts, and

• 2. Hydrostatic compression, which causes a change in volume without a change in shape, stimulates mesenchymal cell differentiation into chondocytes.

• If both differentiation of fibrocartilage (e.g. in menisci). • So primary bone formation requires a stable mechanical

enviroment so that endochondral bone formation will proceed only if the soft tissues create this low strain enviorment.

Thank you