Lesson 9 - Bone Notes PDF

_____________ LESSON 9 _____________ BONE I. Introduction Bone is a specialized form or subtype of connective tissue made up of cells, osteocytes, and calcified extracellular substance (bone matrix). However, despite being a hard and resistant material, bone is a living and dynamic material. The m...

_____________ LESSON 9 _____________ BONE I. Introduction Bone is a specialized form or subtype of connective tissue made up of cells, osteocytes, and calcified extracellular substance (bone matrix). However, despite being a hard and resistant material, bone is a living and dynamic material. The most important characteristic of this variety of connective tissue is the deposit of calcium salts in the extracellular substance, which gives it great hardness. These two elements (cells and extracellular matrix) are constant in bone tissue, what varies is their arrangement in bone, being observed macroscopically two varieties: compact bone and cancellous (spongy) bone (Figure 1). The first form the outer cortex of all the bones and the second is usually placed inside forming small trabeculae and between them the bone marrow islets are located, but no precise boundaries exist between the two varieties. B A Cells Compact bone Extracelular matrix Cancellous bone Figure 1. Detail of a bone showing compact and cancellous bone tissue (A). Diagram of a bone lamellae (B). Microscopically, cells and extracellular substance are arranged in bands or sheets of tissue called "bone lamellae" (Figure 1) which, in turn, are arranged two ways: 1) concentric layers to a canal constituting the morphological and functional unit of compact bone tissue called Osteon or Haversian System. 2) In the form of an anarchic three-dimensional network constituting the spongy bone. In the bone lamellae, cavities or lacunae called osteoplasts that house an osteocyte can be observed. The osteocytes of bone lamellae are in contact with each other and with the outside by means of cytoplasmic extensions that run 1 through ducts excavated in the bone matrix, called canaliculi and that communicate the different lacunae. The bone tissue is continually renewing itself through a reconstruction that lasts a lifetime. Thanks to this power of internal reconstruction, the bone can be modified by surgical procedures or modified by orthopedic prostheses. The bone tissue has a structure adapted to the functions of support and resistance to traction and compression. It is a plastic tissue that is sensitive to alterations in normal mechanical function, with low metabolism and a high content of inorganic substances. Lack of use produces atrophy and excess produces hypertrophy. II. FUNCTIONS 1) Mechanical, it is the skeletal support of the organism. 2) Protective, of vital organs: brain, heart, lung and bone marrow. 3) Storage, calcium, phosphate and other ions. III. COMPONENTS OF BONE TISSUE 1. Bone matrix The extracellular matrix of bone tissue is made up of: 1) Organic part (50%) consisting of collagen fibers type I and ground substance. The organic part of the extracellular matrix is synthesized by the osteoblast due to its great synthesis capacity, which is subsequently mineralized by the action of alkaline phosphatase. The bone matrix has a complex organization since collagen fibers are associated in thick parallel fascicles about 15 µm in diameter, joined by cementitious substance (ground substance), which are oriented within each bone lamella longitudinally, transversely or obliquely depending on the type of bone (compact or cancellous). The ground substance is rich in glycosaminoglycans, proteoglycans and adhesive glycoproteins (osteopontin, osteonectin and osteocalcin) that acts as cementitious substance of the collagen fibers, but without masking them and also as a substrate for the precipitation of calcium mineral salts in the form mainly of hydroxyapatite crystals. The non-calcified extracellular matrix is called osteoid matrix, but when mineral salts precipitate on it, it is called bone matrix. 2) Inorganic part (50%), consisting mainly of calcium and phosphorus, together with other components such as bicarbonate, citrate, magnesium, sodium and potassium. These minerals precipitate on the organic portion of the matrix in the form of hydroxyapatite crystals. 2 2. Cells of bone tissue There are four types: osteoprogenitor cells, osteoblasts, osteocytes and osteoclasts. 1) The osteoprogenitor or osteogenic cells originate from multipotent cells of the mesenchyme. They have a fusiform morphology and are located around the bone forming the periosteum (externally) and the endosteum (internally) and lining the Haversian canal. 2) The osteoblasts are bone tissue forming cells. The growing of the bone tissue is performed by apposition or layering; for this reason, the osteoblasts are placed on lamellae bone forming a cell line as a simple cuboidal epithelium, and with cytoplasmic processes that are introduced into the extracellular substance on which they rest. The osteoblasts are cuboidal cells with a central and vesicular nucleus and a basophilic cytoplasm. Using the electron microscopy osteoblasts have a structure resembling that of fibroblasts, since the function of both cells is to synthesize collagen fibers. For this reason, the cytoplasmic organoid most developed is the rough endoplasmic reticulum, occupying almost all the cytoplasm and presenting dilated cisterns with homogeneous content. Fibrils of protocollagen can be seen both inside cells and attached to the plasma membrane. 3) The osteocytes are the adult cells of the bone tissue that are cloistered in the lacuna or osteoplast, interacting with neighbouring cells and surface osteoblasts through thin cytoplasmic processes located in the bone canaliculi. Osteocytes are ovoid in shape, with a small central nucleus and a basophilic cytoplasm with paraplasmic inclusions. The thin cytoplasmic processes of one cell contact those of others, but no modes of attachment between them have been observed. 4) The fourth cellular element is the osteoclast whose function is to disintegrate bone tissue that, in a first phase, has been formed indiscriminately, to enter, in a second phase, in a remodelling process. This disintegrating function is carried out thanks to its high lysosomal content. Morphologically, it is a large multinucleated cell, approximately 100 µm in diameter, and is found embedded in bony cavities: the Howship lacunae, which appear to be formed by the osteoclasts themselves. With the light microscope, the osteoclast has a smooth surface outside the lacuna and an anfractuous surface in contact with the bone matrix called striated edge. The very numerous nuclei are ovoid and vesicular and are located at the opposite pole to the bone matrix. Electron microscopy shows that the striated edge corresponds to the presence of abundant microvilli in contact with the bone matrix. At this level, the cytoplasm contains abundant lysosomes with different shape and size rich in hydrolytic enzymes (alkaline phosphatase, principally). Osteoclasts originate 3 from coalescence of mononuclear cells from, according to some authors, the bone matrix, and according to others, the blood (possibly monocytes). A B Figure 2. Diagrams of an osteoblast (A); an osteocyte (B); and an osteoclast (C). I V. TYPES OF BONE TISSUE Macroscopically, bones can be long, short, flat, irregular and sesamoid. In a longitudinal section, cancellous bone can be identified inside the epiphyses and the compact bone surrounding the entire bone (Figure 1). Bone is surrounded externally by the periosteum and internally by the endosteum. Microscopically, we can classify bone tissue into: 1. Immature or primary bone tissue, is the first bone to develop in the foetus and during repairs. It is constituted by abundant osteocytes and irregular bundles of collagen. 2. Secondary, mature or laminar bone tissue, is made up of mature bone lamellae. These bone lamellae can be arranged in a honeycomb shape forming cancellous bone tissue or concentrically to a channel forming compact bone. ▪ Cancellous (spongy) or trabecular bone It is located inside the flat bones and in the medullary cavity of the long bones, mainly in the epiphysis. Morphologically it has a very simple architecture since it is formed by bone lamellae and trabeculae associated with each other as a threedimensional network, more numerous in the lines that correspond to those of maximum pressure or tension, leaving cavities occupied by bone marrow between them. These lamellae contain osteoplasts containing osteocytes, whose canaliculi are directly related to the medullary cavity. 4 C ▪ Compact bone This type of tissue constitutes the cortical zone of the bones. It is covered externally by the periosteum and internally, when it is not continued with spongy bone tissue, by the endosteum. Bony lamellae, in compact bone, are organized mainly in osteons or Haversian system and to a lesser extent in laminar systems: 1) external limiting system, 2) internal limiting system, 3) interstitial system. The osteon or Haversian system is the morphological and functional unit of compact bone. This system has a central duct called "Haversian canal" in which vessels (arterial and venous) and nerves are housed. Surrounding this canal there is a series of concentric bone lamellae, between 5 and 12, and the last, the outermost, is called the “EBNER limiting lamella” or cementitious lamina. In each lamella there is a row of oval-shaped osteoplasts with their corresponding osteocytes and their major axis oriented perpendicular to the radius of the osteon. The osteoplasts of the innermost bone lamellae of each osteon communicate, on the one hand, with the central canal and, on the other, with the osteoplasts of the immediate lamellae through the canaliculi. The intermediate lamellae are related to each other by said ducts, and the last or limiting of Ebner, is related to the previous one by the canaliculi of its internal face, but the canaliculi of its external face bend finishing in a “cul-de-sac”. The fascicles of the collagen fibers are arranged parallel to the lamella itself and perpendicular in relation to adjacent lamellae. The periosteum is formed by two layers: an outer, fibrous, rich in not calcified collagen fibers whose function is to distribute blood circulation and nerve supply to the bone, and an internal layer, called "cambium" which has osteoprogenitor cells. The periosteum is attached to the bone by strong collagen fiber bundles introduced on the "external limiting system", forming the so-called "perforating fibers" or " Sharpey fibers". This external or "fundamental " limiting system is constituted by a series of bone lamellae concentric to the total bone axis and similar to those that constitute the Haversian system. In close contact with the limiting system, abundant osteons are located surrounded by a basophilic material that forms the "cementing lines" or "Ebner lines" and separated from each other by the "interstitial systems" or "intercalary systems", which are remnants of Haversian systems formed by portions of bone lamellae. The different osteons communicate with each other, with the periosteum and with the medullary canal of the bone through "perforating ducts " or " Volkmann canals ", whose difference from Haversian is that they are not concentrically surrounded by bony lamellae. Larger vessels circulate within these ducts than in Haversian canals, generally accompanied by more connective tissue. The endosteum, which is made up of a fibrous layer, appears covering the "internal limiting system" which is similar in its constitution to the external fundamental system, and also perforated by the Volkmann canals. 5 V. TYPES OF OSSIFICATION The process by which bone tissue is formed is known by the name of ossification. This process can originate either on primitive connective tissue or on a pre-existing cartilaginous model. In the first case, ossification is "intramembranous", and occurs normally in flat bones, and in the second "endochondral", which is typical of long bones. The ossification process requires two phases: 1) an osteoid matrix appears on which the primary bone tissue is formed, a task carried out by the osteoblasts; and 2) there is a remodelling of the bone by the osteoclasts in its two varieties: compact and cancellous. Figure 3. Diaphysis wall diagram of long bones. There are three types of lamellar bone tissue: the Haversian systems and the external and internal limiting systems. The Havers system is represented in three dimensions in the upper left and shows the orientation of the collagen fibers in the lamellae. The projecting Haversian system, on the left, shows the direction of the collagen fibers in each lamellae. On the right is an isolated Haversian system with a central blood vessel (there are also nerves in this central canal, which are not shown in the diagram) and numerous osteocytes with their processes. Image taken from Junqueira y Carnero. Basic histology. 2005. V.1. Intramembranous ossification This type of ossification is typical of the flat bones of the skull, called “membranous bones”. They are constituted, initially, by a mesenchymal tissue or connective membrane with abundant ground substance rich in 6 mucopolysaccharides, in which mesenchymal stellate cells can be seen, with a similar structure to fibroblasts and with numerous cytoplasmic processes through which they are related to each other; there is a vascular network that nourishes this mesenchymal tissue. The ossification process develops from this tissue, thanks to its pluripotent cells, with the ability to differentiate into bone matrixforming cells. The ossification process in a flat or membranous bone that, initially, is constituted by a mesenchymal tissue or connective membrane, begins in different areas of this membrane due to a greater blood supply, which causes certain groups of mesenchymal cells to begin to differentiate into spherical cells or osteoblasts, capable of synthesizing collagen fibers and acid phosphatase around them, constituting a "matrix or osteoid tissue", formed by a group of osteoblasts immersed in a matrix rich in collagen fibers and acid phosphatase. Towards this focus of osteoid tissue there is a greater blood supply that initiates a precipitation of calcium salts in the form of hydroxyapatite, cloistering the osteoblasts that become osteocytes, thus obtaining a "calcified osteoid matrix" or "primary focus of ossification". This primary focus of ossification induces the pluripotent mesenchymal cells to arrange surrounding it as a cuboidal epithelium and to differentiate into osteoblasts capable of synthesizing collagen fibers and acid phosphatase, which are deposited in its vicinity, giving rise to new osteoid tissue that, later, calcifies, cloistering the new osteocytes and producing growth by apposition of the initial primary focus of ossification. The different primary foci of ossification that have appeared in the membranous bone grow by apposition until they unite with each other and constitute a "primary bone tissue", on which the osteoclasts subsequently act for their remodelling, giving rise to an adult bone tissue. 1 2 3 4 5 Figure 4. Primary nuclei of intramembranous ossification. V.2. Endochondral ossification This type of ossification occurs in long bones consisting primarily of a cartilaginous matrix. Long bones have two ends or "epiphysis" and a central area or "diaphysis". They have a transversal axis of symmetry located in the latter. It is from this axis of symmetry where the ossification process begins that leads to the development 7 of the bone towards both ends, thus growing in length. The growth in thickness is made by apposition from the periosteum. Initially, in the cartilaginous model of the long bone, the cells are arranged in an orderly manner and in strata or areas that, from the epiphysis to the diaphysis, are: o "Germinal zone", located in the outermost part of the epiphysis and made up of small cells, with nonspecific morphology and a high mitotic index; if this area exists, the bone is growing. o "Serial zone", below the previous one, and in which the chondrocytes, still small, are arranged in rows; it is considered as a transition zone. o "Columnar zone", after the previous one and already in the diaphysis. Seriation becomes clearer and chondrocytes are larger, laying on top of each other as thick vertical cords; cells in this area have lost their ability to divide. o " Hypertrophic zone", the cells hypertrophy, the chondroplasts increase in volume and the columnar arrangement is maintained. o "Necrosis zone" or "calcified cartilage zone", present in those bones in which the ossification process has begun and in the innermost zone and close to the axis of symmetry of the diaphysis. Hypertrophic chondrocytes show clear degenerative signs. The cartilaginous model of bone is surrounded by a highly developed and active perichondrium, especially at the level of the diaphysis, made up of numerous pluripotent stellate cells immersed in a mesenchymal matrix. The onset of ossification corresponds to an increase in blood flow in the perichondrium of the diaphysis and, as in intramembranous ossification, "primary foci of ossification" appear in a thin band in this area, which develop to form "primary bone tissue". When this change of mesenchymal tissue to bone occurs in the perichondrium, in the hypertrophic zone of the cartilaginous bone model, the chondrocytes enter in degenerative processes that end with cell death; when the chondrocytes die, the chondroplasts are free, open and remain a disintegrating cartilaginous matrix. Simultaneously with this process there is an increase in blood flow, the blood vessels provide calcium salts and are accompanied by connective tissue with pluripotent mesenchymal cells that differentiate into osteoblasts. On the disintegrated cartilaginous matrix, the precipitation of calcium salts occurs in the form of hydroxyapatite, giving rise to a "provisional or preliminary calcification zone". In turn, the blood vessels, accompanied by connective tissue, penetrate between the open chondroplasts, originating channels whose walls are constituted by the calcified cartilaginous matrix. The cells of the perivascular connective tissue, in contact with the cartilaginous matrix, differentiate into osteoblasts that are arranged as a cuboidal epithelium on the matrix, and begin to produce collagen fibers and ground substance, on which calcium salts precipitate, forming a primary focus of ossification: first in the central area or diaphysis of the bone and later it extends towards both ends or epiphysis in an identical process to the one previously 8 exposed. In such a way, if there is a germinal zone in the epiphysis in the cartilaginous bone model, which by division originates the rest of the zones, the ossification process will take place, since there will be a hypertrophic zone where vascular phenomena and deposition of calcium will take place. Around the diaphysis and starting from the perichondrium, and simultaneously with endochondral ossification, a bone sheath is formed and the bone thickness growth is carried out by apposition from the surrounding periosteum. Figure 5. Photomicrograph of the epiphyseal plate in which the five zones, the alterations that take place in the cartilage and the formation of bone tissue are observed. Pararosaniline and toluidine blue stain. Image taken from the book of Basic Histology Junqueira y Carnero, 2005. VI. BONE GROWTH AND REMODELLING Growth involves a net increase in skeletal mass that occurs prior to closure of the epiphysis plate. Modelling is the process responsible for maintaining the characteristic morphology of growing bone. Both growth and modelling require bone formation (osteoblastic activity) and bone resorption (osteoclastic activity) to occur at anatomically separate locations. As we have indicated previously, bone, a dynamic organ, is composed of an inorganic extracellular matrix, and its microarchitecture is constantly being modified by two groups of bone cells with hormonal responses: osteoblasts and osteoclasts. The conjugated activity of osteoblasts and osteoclasts, called coupling or rotation, is the basis for bone turnover or remodelling. 9 Bone remodelling serves to: o Guarantee the function of the skeleton as a mineral reserve. o Help maintain functions and fluid exchange in cortical bone tissue. o Allow repair, rearrangement and reorganization in trabecular bone tissue. The factors involved in remodelling are: o Endogenous (hormonal): PTH stimulates resorption and calcitonin inhibits resorption. o Environmental: nutrition and exercise inhibit resorption. Immobility increases resorption. Bone remodelling does not end with the replacement of primary by secondary bone but continues throughout life. 10