Muscle Intro PDF

CH APTER 16 Bones, Joints, Tendons, and Ligamentsa Erik J. Olson, Jaclyn A. Dykstra, Alexandra R. Armstrong, and Cathy S. Carlson Key Readings Index Structure and Function, 1037 Dysfunction/Responses to Injury, 1048 Portals of Entry/Pathways of Spread, 1055 Defense Mechanisms/Barrier Systems, 1056 Diseases Affecting Multiple Species of Domestic Animals, 1056 Diseases of Horses, 1091 Diseases of Ruminants (Cattle, Sheep, and Goats), 1091 The skeleton consists of bones and joints and their supporting structures (tendons, ligaments, connective tissue [e.g., joint capsules], and fasciae) and is responsible for supporting the body and enabling movement initiated by the nervous system and facilitated by muscles. The skeleton can be divided into the axial skeleton (head, vertebrae, ribs, and sternum) and the appendicular skeleton (thoracic and pelvic limbs). Information on the methods for postmortem examination and evaluation of the skeleton is available in E-Appendix 16.1, which can be found at www.expertconsult.com. Structure and Function Bone Development of Bone The development of bone and thus the skeletal system occurs concurrently and interdependently with that of the muscular and nervous systems during the embryonic period of intrauterine development. As a result, developmental processes that define the structure of the skeleton are closely aligned with the development of the muscular and nervous systems. Together these processes unite the three systems into one functional unit that facilitates movement. This successful outcome results from a stepwise chronologic sequence of signaling events among and between cells and tissues within the three emerging systems during embryonic development. Clinically and anatomically, the adult skeletal system has welldefined symmetry and planar orientation and positions, such as cranial, caudal, dorsal, and ventral, as examples, that are used to characterize and diagnose musculoskeletal and nervous system diseases. It is an amazing process when one considers how these anatomic planes and positions arise from a starting point of a single cell (zygote) resulting from the fusion of the ovum and sperm. The zygote then proceeds via cell divisions through morulation, blastulation, and gastrulation to eventually become an embryo and then a fetus with a well-defined head-tail axis. It appears that the head-tail axis of the embryo is established early in development; however, the mesenchymal cell aggregates (also called bone matrix) that form the eventual structural components of the skeleton along this axis are not identifiable until later stages in the development of the embryo. aFor a glossary of abbreviations and terms used in this chapter, see E-Glossary 16.1. Diseases of Pigs, 1091 Diseases of Dogs, 1091 Diseases of Cats, 1094 The process of going from a single cell to an adult animal with a head-tail axis and the establishment of anatomic planes requires the development of “planar polarity” in the developing cells. It is believed this polarity is established very early in development, likely before gastrulation, possibly as early as in the 2-, 4-, or 8-cell stages or during morulation or blastulation. Once polarity is established, the sequence of events that leads to the development of the skeleton and its integration with muscles and nerves occurs chronologically. Developmentally, bone arises during the embryonic period of intrauterine development from mesenchymeb that is distributed bilaterally, symmetrically, and segmentally along the developing neural tube (E-Fig. 16.1; also see E-Figs. 14.1 and 14.2). The genetic profile of individual mesenchymal cell aggregates determines whether these cells differentiate into cranial neural crest cells, somites (paraxial mesoderm), or lateral plate mesoderm and hence the eventual structure and function of each bone and whether it becomes part of the axial or appendicular skeleton. Collections of these cells within somites also differentiate into accompanying muscle groups. Innervation arises from adjoining cells that form the spinal ganglia (see E-Fig. 16.1). In the axial skeleton, the structure of the bones of the head arises from cranial neural crest cells, whereas structure of the bones of the vertebrae, ribs, and sternum arises from somites (paraxial mesoderm). The structure of the bones of the thoracic and pelvic limbs (known as the appendicular skeleton) arises from lateral plate mesoderm within limb bud primordia (see E-Fig. 16.1). Once the mesenchyme templates for the bones of the appendicular and axial skeletons are determined and positioned within the developing embryo, they grow in size and shape during the fetal period of intrauterine development. The processes of structural development of bone matrix begin through intramembranous and endochondral ossification (also see the sections on Bone at the Organic Matrix and Mineral Level and also Bone Growth). These processes involve the transformation of mesenchyme (bone matrix) into bone via the deposition of osteoid followed by mineralization of the osteoid through deposition of hydroxyapatite.c Intramembranous ossification occurs when mesenchyme differentiates and matures bLoosely arranged aggregates of mesenchymal stem cells embedded within abundant extracellular matrix (ECM). cInorganic mixture made up of calcium, phosphate, and hydroxide, deposited in bones and teeth in a crystallized lattice-like form to give these structures rigidity. 1037 CHAPTER 16 Bones, Joints, Tendons, and Ligaments 1037.e1 E-Glossary 16.1 Glossary of Abbreviations and Terms ADAM AECC AGEs Ankylosis Appendicular skeleton Arthritis Arthrogryposis Arthropathy Axial skeleton Bone dust BMP Brachygnathia inferior (mandibular brachygnathism) Brachygnathia superior (maxillary brachygnathism) BTF Callus Canaliculi Cancellous bone Cartilage canals Cement lines Chondrocyte clones Chondromalacia A disintegrin and metalloprotease; a family of peptidase proteins Articular-epiphyseal cartilage complex Advanced glycation end products Fusion of a joint; can be iatrogenic (surgical) or associated with natural disease processes (e.g., spondylosis of vertebrae or degenerative joint disease) The bones and cartilage that support the appendages of vertebrates (thoracic and pelvic limbs and supporting pectoral and pelvic girdles) Inflammation of a joint; a general term that includes degenerative, immune-mediated, and infectious causes The congenital contracture of one or more joints (often occurs with bilateral symmetry) Any joint disease The part of the skeleton that includes the head, vertebrae, ribs, and sternum (those bones not included in the appendicular skeleton) A common processing artifact in histologic sections of bone that have been cut with saws; composed of debris Bone morphogenetic proteins Relative shortening of the mandibles CHS CLAD CPDD Diaphysis Diarthrodial joint Diskospondylitis DJD DLX5 Eburnation ECF Endochondral ossification Relative shortening of the maxillae Bone tissue fluid A disorganized meshwork of woven bone that forms after a fracture Microscopic tunnels/canals within bone through which osteocytes make contact with osteoblasts and other osteocytes by means of long cytoplasmic processes Synonymous with trabecular bone or spongy bone; typically found at the ends of long bones and is less dense than cortical bone Minute tunnels in the epiphyseal/ growth cartilage of the fetal skeleton that contain blood vessels and possibly nerves and lymphatic vessels Collagen-poor, proteoglycanrich seams between adjacent remodeling and/or modeling units; appear as basophilic lines in H&E sections Clusters of chondrocytes that represent histological evidence of local chondrocyte replication in response to injury; also referred to as “complex chondron” Softening of articular cartilage; caused by a number of different underlying conditions (e.g., trauma and inflammation) Enthesophyte Epiphysis Exostosis FOD H&E HHM HO/HPO Howship’s lacuna Hydroxyapatite Calvarial hyperostosis syndrome Canine leukocyte adhesion deficiency Calcium pyrophosphate deposition disease The midsection or main shaft of a long bone. (See also epiphysis, metaphysis, and physis.) A movable joint in the axial or appendicular skeleton; also known as a synovial joint; characterized by the presence of a fibrocartilage layer or hyaline cartilage that lines the opposing bony surfaces as well as a lubricating synovial fluid within the synovial cavity Inflammation of the vertebral body, centered on or originating from the intervertebral disk Degenerative joint disease (synonyms are osteoarthritis and osteoarthrosis) Distal-less homeobox 5 gene A degenerative process of bone commonly associated with osteoarthritis; ulceration and loss of articular cartilage results in sclerosis (thickened subchondral bone), and the exposed bone surface develops a smooth polished (ivory-like) appearance Extracellular fluid One of two essential processes that result in bone formation during fetal development of the mammalian skeletal system; relies on a cartilage model and the replacement of cartilage by bone Abnormal bony projections at the attachment of a tendon or ligament The end of a long bone; at the level of the joint, the epiphysis is covered with articular cartilage A nodular, benign bony growth projecting outward from the surface of a bone Fibrous osteodystrophy Hematoxylin and eosin stain Humoral hypercalcemia of malignancy (also known as pseudohyperparathyroidism); a paraneoplastic syndrome Hypertrophic osteopathy (hypertrophic pulmonary osteopathy) A groove or cavity usually containing osteoclasts that occurs in bone, which is undergoing resorption (also known as erosion or resorption lacunae) Inorganic mixture comprised of calcium, phosphate, and hydroxide, deposited in bones and teeth in a crystallized latticelike form to give these structures rigidity Continued 1037.e2 SECTION II Pathology of Organ Systems E-Glossary 16.1 Glossary of Abbreviations and Terms—cont’d Excessive growth of bone; the diameter of the bone is increased, and this term implies more uniform thickening on the periosteal surface versus osteophyte or exostosis Intramembranous ossification Formation of bone on, or in, fibrous connective tissue (e.g., flat bones of the skull) Lamellar bone Mature bone in which the collagen fibers are arranged in parallel lamellae Luxation Complete dislocation of a joint MCSF Macrophage colony-stimulating factor Metaphysis The wide portion of a long bone between the epiphysis and the narrower diaphysis Modeling Change of the shape or contour of a bone in response to normal growth, altered mechanical use, or disease. The bone surfaces can go directly from resting to either formation or resorption, depending on the stimulus. Oligoarthritis Arthritis affecting a small number of joints OPG Osteoprotegerin Osteitis Inflammation of bone. See also osteomyelitis and periostitis. Osteoarthrosis A condition of chronic arthritis, usually mechanical and with minimal inflammation Osteochondrosis A heterogeneous group of lesions involving the growth cartilage of young animals characterized by focal or multifocal failure (or delay) of endochondral ossification; characterized by cartilage necrosis in the AECC and retained hypertrophic chondrocytes in the physis Osteochondrosis dissecans Osteochondrosis at the AECC that results in formation of clefts in the necrotic cartilage with subsequent fracture of the overlying articular cartilage Osteochondrosis latens A stage of osteochondrosis in which the lesion of cartilage necrosis is confined to the epiphyseal cartilage and is only visible microscopically Osteochondrosis manifesta A stage of osteochondrosis in which the lesion (retained necrotic epiphyseal cartilage) results in focal failure of endochondral ossification and is visible grossly and radiographically Osteoclasis Breaking down or absorption of osseous tissue Osteoid The unmineralized organic portion of the bone matrix that forms prior to the maturation of bone tissue Osteomyelitis Inflammation of the bone and medullary cavity/bone marrow Hyperostosis A condition in which bone mineral density is lower than normal and/ or the amount of bone present is less than normal Osteophyte Bony projection that forms along joint margins Osteoporosis A disease in which bone fractures occur secondary to a reduction in bone density or mass (osteopenia) Osx Osterix, a protein that plays a major role in driving the differentiation of mesenchymal precursor cells into osteoblasts and, eventually, osteocytes Pannus A fibrovascular and histiocytic tissue (inflammatory granulation tissue) that arises from the synovial membrane and spreads as a membrane over articular cartilage Pathologic fracture Abnormal bone that fractures with minimal trauma Perichondral ring The periosteum covering the physis; adds new cartilage to the periphery of the physis, enabling it to expand in width as the skeleton grows Periostitis Inflammation of the periosteum Physis A hyaline cartilage plate located between the epiphysis and the metaphysis at each end of a long bone; the tissue that is responsible for longitudinal growth (also known as growth plate) Polyostotic A process that affects several bones Primary center of ossification Site in mid-diaphysis of a long bone where endochondral ossification first occurs Primary spongiosa The mineralized cartilage (and accompanying bone matrix) in the developing metaphysis Pseudoarthrosis A false joint formed as a pocket of fibrous tissue and cartilage PTH Parathyroid hormone PTHrP Parathyroid hormone-related peptide/protein RANK Receptor activator of nuclear factor κB RANKL (ODF = osteoclast Receptor activator for nuclear differentiation factor) factor κB ligand Remodeling A lifelong process in which mature bone is removed from the skeleton and new bone tissue is formed; in remodeling, resorption must precede formation to keep bone mass and shape constant Resting lines Cement lines that occur when osteoblast formation ceases and subsequently resumes; usually smooth and follow the contour of the overlying surface Reversal lines Scalloped basophilic lines indicating where bone resorption stopped and was followed by formation Runx2 Runt-related transcription factor 2 Osteopenia Continued CHAPTER 16 Bones, Joints, Tendons, and Ligaments 1037.e3 E-Glossary 16.1 Glossary of Abbreviations and Terms—cont’d Sclerosis Sclerostin Secondary centers of ossification Secondary spongiosa Subchondral bone Subluxation Synovial fossa Synovitis TEM Tidemark Increased bone per unit of area A protein that inhibits bone formation by osteoblasts Sites of endochondral ossification located in the epiphyses of growing bones during normal development The second stage of mineralization of bony trabeculae; comprises the enlarged, mineralized bony trabeculae immediately subjacent to the primary spongiosa The area of bone located immediately subjacent to, and providing support for, the articular cartilage Partial dislocation of a joint Normal depression on non–weightbearing articular cartilage surfaces that develop bilaterally in the larger appendicular joints, particularly in the horse, pig, and ruminant Inflammation of the synovium Transmission electron microscopy A line observed histologically that delimits the boundary between the uncalcified articular cartilage and the subjacent calcified cartilage E-Appendix 16.1 Postmortem Examination and Evaluation of Bones The entire skeleton is rarely examined at necropsy; instead, a focused examination usually is dictated by the clinical history. Antemortem clinical and radiographic findings are valuable and should be in hand before the postmortem examination is begun, especially for cases suspected of having relatively small, localized lesions. Because of the variability in bone and joint tissues among sites, species, and ages of individuals, it is extremely useful to compare suspected lesions with age-matched control tissue, although this is not always possible. In addition, it should be remembered that a lesion responsible for lameness may involve the skeletal, muscular, or nervous systems. Regardless of whether lesions specific to the skeletal system are suspected, certain areas of the skeleton should be examined in every necropsy for completeness and to provide familiarity with normal osseous structures. This includes examination of marrow for fat cell stores and the presence and number of hematopoietic cells; thickness of cortical bone; amount and distribution of cancellous bone; and thickness and uniformity of metaphyseal growth plates, articular surfaces, and tendon insertions in at least one long bone that is cut longitudinally. In small animals, testing bone strength by breaking a rib can be informative, although these results are relative because of the marked variation in size among species and breeds. Also, determining the degree to which the rib bends before breaking is important because increased pliability may indicate the presence of a fibrous osteodystrophy lesion. Bony tissues are more readily visualized if bone marrow contents are flushed out with a jet of water, and some lesions can be best visualized radiographically. Postmortem radiographs of slabs of bones or entire bones with much of the soft tissue removed can provide information that may not be visible in routine radiographs that are taken in vivo. Postmortem autolytic TIMPs TRAP Traumatic fracture TRIC Valgus Varus Wnt Woven bone Tissue inhibitors of metalloproteinases Tartrate-resistant acid phosphatase; a histochemical stain used to identify osteoclasts A type of bone fracture occurring secondary to trauma Trimeric intracellular cation channels Angular limb deformity in which the limb distal to the lesion deviates laterally Angular limb deformity in which the limb distal to the lesion deviates medially Signal transduction pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning, and organogenesis during embryonic development Immature bone in which the collagen fibers of the matrix are arranged irregularly in the form of interlacing networks; normally present in growing animals but considered pathologic in adults changes do not usually pose major problems in the evaluation of the skeleton at necropsy because postmortem bacterial invasion is less rapid than in most other tissue. Bone marrow cultures taken postmortem can be useful in detecting bacteremia (e.g., salmonellosis). Sometimes, bones are fractured at euthanasia and/or by postmortem transport and handling, and these fractures are distinguished from those occurring in vivo by the absence of hemorrhage in bone or adjacent soft tissue. Fracture of bones during euthanasia could be difficult to distinguish from very recent fracture, especially if recent antemortem trauma was reported in the history. However, as in a postmortem bone fracture, less blood from hard and soft tissue is expected in agonal fracturing of bones. Bone dust (microscopic fragments of mineralized debris) is a common artifact occurring when bones are sawed before fixation (see Fig. 16.44, B and C). Specimens for histopathologic evaluation should be fixed in 10% neutral-buffered formalin. A formalin-to-tissue volume ratio of 10 to 1 or greater is recommended but may not be practical. Preparation of cleaned osteologic specimens can be very helpful when examining skeletal lesions. A number of different techniques may be used to remove the soft tissues from bone, including cold or warm water maceration and the use of dermestid beetles. Ancillary information regarding bone density can be obtained through bone ash analysis or densitometry scans. For detailed evaluation of bone turnover, bone labels such as tetracycline or calcein can be administered before euthanasia for dynamic histomorphometry studies on undecalcified bone. In addition to conventional radiographs, advanced imaging modalities such as computed tomography (CT) scans, magnetic resonance imaging, and micro-computed tomography (micro-CT) may provide additional information. Decalcification of bone may be accomplished by either immersion of samples in ethylenediaminetetraacetic acid (EDTA), which is a chelating agent, or use of an acid decalcification method (e.g., by 1037.e4 SECTION II Pathology of Organ Systems immersion in formic or hydrochloric acid). The former method takes considerably longer but avoids some of the artifacts caused by acid decalcification and allows future immunohistochemistry studies. Postmortem Examination and Evaluation of Joints The routine necropsy examination also should include opening and examining several large synovial joints, such as the shoulder and hip, to become familiar with the expected age-related changes in joint tissues. In cases in which septicemic joint disease is suspected, many joints should be examined, including the carpal and tarsal joints, because the larger joints may not be affected. Joints should be disarticulated so that articular surfaces, synovial fluid, and all associated structures are clearly visible. Consideration should be given to aspirating synovial fluid before disarticulation to obtain a sample free of contamination that is suitable for culture and analysis that includes viscosity (mucin precipitation), cell count, and cytology. The best time to retrieve a sample of synovial membrane is while the joint is being opened because this tissue retracts rapidly. If lesions are mild, it may be difficult to locate a membrane in an opened joint. Articular cartilage also should be examined as soon as the joint is opened because dehydration of cartilage occurs rapidly on exposure to air. If the site of interest cannot be immersed in fixative immediately, it should be kept covered with a moist paper towel. Fine finger-like proliferations of synovium (villous hypertrophy/hyperplasia) are best evaluated grossly when the specimen is submerged in water, saline, or formalin. Microscopic examination of synovium is required to further characterize the lesions. Specimens for histopathologic evaluation should be fixed in 10% neutral-buffered formalin. A formalin-to-tissue volume ratio of 10 to 1 or greater is recommended but may not be practical. CHAPTER 16 Bones, Joints, Tendons, and Ligaments 1037.e5 Cranial neural crest cells (eventual bones of the calvarium, maxilla, and mandible) Calvarium, maxilla, and mandible Epidermis (skin) Lateral plate mesoderm Cervical vertebrae (eventual bones of the thoracic limb) Forelimb Neural tube Spinal ganglion (eventual CNS) (eventual PNS) Somite paraxial mesoderm Lateral plate mesoderm (eventual vertebrae, ribs, and sternum precusors) Forelimb (eventual bones of the limbs) Limb precursors Limb precursors Notochord Somite - paraxial mesoderm (eventual bones of the vertebrae, ribs, and sternum) Direction of growth Thoracic vertebrae Spinal ganglion (eventual PNS) Epidermis (skin) Somite - paraxial mesoderm Neural tube (eventual CNS) Spinal ganglion (eventual vertebrae, ribs, and sternum precursors) Lumbar vertebrae (eventual PNS) Neural tube (eventual CNS) Hindlimb Lateral plate mesoderm (eventual bones of the pelvic limb) Notochord Hindlimb Sacral vertebrae E-Figure 16.1 Development of Bone. Mesenchymal cell aggregates (also known as bone matrix) differentiate into cranial neural crest cells, somites (paraxial mesoderm), or lateral plate mesoderm. In the axial skeleton, the structure of the bones of the head arise from cranial neural crest cells, whereas structure of the bones of the vertebrae, ribs, and sternum arise from somites (paraxial mesoderm). The structure of the bones of the thoracic and pelvic limbs, the appendicular skeleton, arise from lateral plate mesoderm within limb bud primordia. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota and Dr. J.F. Zachary, College of Veterinary Medicine, University of Illinois.) 1038 SECTION II Pathology of Organ Systems directly into bone. Endochondral ossification, an indirect process, occurs when mesenchyme first differentiates into cartilaginous facsimiles of bone, which subsequently differentiate and mature into bone. Last, developing bone establishes functional linkages with the muscular system through tendons (“origins and insertions”) and ligaments, and with the nervous system through cranial and spinal nerves. Intramembranous Ossification. Intramembranous ossification occurs primarily in mesenchyme derived from cranial neural crest cells that ultimately develop into the bones of the calvaria. Once the location and shape of these bones are determined in the embryonic period of intrauterine development, a unique population of cells in the mesenchyme differentiates structurally and functionally into osteoblasts that secrete osteoid into the existing mesenchyme. Osteoid is an extracellular matrix protein mixture consisting of collagen and proteoglycans, which is able to bind calcium. Once osteoblasts are surrounded by osteoid that fills the existing mesenchyme, they functionally differentiate into osteocytes, which act to mineralize the existing osteoid via calcium and phosphate deposition in the form of hydroxyapatite. Endochondral Ossification. Endochondral ossification occurs primarily in mesenchyme derived from somites (paraxial mesoderm) and lateral plate mesoderm that ultimately develop into the bones of the vertebrae, ribs, and sternum and the bones of the thoracic and pelvic limbs, respectively. Once the location and shape of these bones are determined in the embryonic period of intrauterine development, the mesenchyme differentiates into cartilaginous facsimiles of bone, which are later replaced by mineralized bone through a complicated process that is discussed in other sections of this chapter (see the section on Bone at the Organic Matrix and Mineral Level; also see the section on Bone Growth) and in other veterinary textbooks.d,e Endochondral ossification is a process that occurs predominantly in the vertebral column, ribs, pelvis, and limbs. Mineralization of osteoid occurs via calcium and phosphate deposition in the form of hydroxyapatite. Bone at the Cellular Level Structure and function can be discussed at the organ, tissue, and cellular levels. In this section, normal structure and function are briefly reviewed, beginning at the cellular level and including bone matrix and mineral. Cells directly involved with the structural integrity of bone include osteoblasts, osteocytes, and osteoclasts (Box 16.1). Osteoblasts are cells on any bone surface (periosteal, endosteal, trabecular, and intracortical) that produce bone matrix (osteoid), initiate the mineralization of this matrix (deposition of hydroxyapatite), and seemingly paradoxically initiate the resorption of this matrix by osteoclasts. Osteoblasts are derived from mesenchymal stem cells, with differentiation driven largely by the following transcription factors: runt-related transcription factor 2 (RUNX2), Osterix (OSX), and distal-less homeobox 5 gene (DLX5). The canonical Wnt signaling pathway and expression of various bone morphogenetic proteins (BMPs) also play key roles in bone metabolism and differentiation. Although these signaling cascades are complex, important events in Wnt signaling in the osteoblast include the binding of Wnt ligands to a receptor complex composed of a dKierszenbaum AL, Tres LL: Histology and cell biology, an introduction to pathology, ed 4, 2015, Mosby. eHyttel P, Sinowatz F, Vejlsted M, et al: Essentials of domestic animal embryology, 2010, Saunders. Box 16.1 Bone Cell Function Osteoblasts: Cells of stromal stem cell origin that reside on the surface of bone and form bone matrix, initiate bone mineralization, and initiate bone resorption (by signaling to osteoclasts) in response to physiologic stimuli Osteocytes: Osteoblasts that have become encased in bone matrix. These cells detect changes in stress (force applied to the bone) and strain (structural deformation in response to the force) in the bone and signal these changes to osteoblasts to either form bone or initiate resorption; may mobilize calcium from the bone through osteocytic osteolysis Osteoclasts: Cells formed by the fusion of cells of the monocytemacrophage cell line; function is to resorb mineralized bone matrix o o Figure 16.1 Osteoblasts and Osteoid, Long Bone, Cynomolgus Macaque. Prominent cuboidal (active) osteoblasts (arrows) line the trabecular surfaces. Mineralized trabecular bone is black; unmineralized bone matrix (osteoid [o]) is present (light blue) between mineralized bone and osteoblasts. Hematopoietic marrow is present between trabeculae. Von Kossa tetrachrome stain. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota.) member of the Frizzled family of receptors and a coreceptor (either Lrp5 or Lrp6), facilitating movement of β-catenin into the nucleus to regulate gene expression. Active osteoblasts are plump cuboidal cells (Figs. 16.1 and 16.2; E-Fig. 16.2), with abundant basophilic cytoplasm that is rich in rough endoplasmic reticulum and contains a prominent Golgi apparatus and numerous mitochondria (E-Fig. 16.3). Inactive osteoblasts are disk-shaped cells with little cytoplasm because fewer organelles are needed for matrix synthesis and secretion (E-Fig. 16.4). Osteoblasts likely interact with osteocytes to assist in fine control of calcium homeostasis and detection of mechanical use and microscopic damage to bone, as discussed later. Indirect measurements of osteoblast activity are reflected in blood concentrations of the bone-specific isoform of alkaline phosphatase, an enzyme on the cell membrane of osteoblasts, and the noncollagenous protein, osteocalcin, that is secreted by osteoblasts and is present in bone matrix. Both are thought to have roles in mineralization and calcium ion homeostasis, with recent work identifying an additional role for the osteoblast in the endocrine system, largely mediated through the effects of osteoblast-derived osteocalcin on adipose tissue, male gonadal tissue, and neurons. Osteocytes are osteoblasts that have been surrounded by mineralized bone matrix (E-Fig. 16.5; also see Fig. 16.2) and are the most numerous and longest living of the bone cell types. They occupy small spaces in the bone called lacunae (singular: lacuna) and make CHAPTER 16 Bones, Joints, Tendons, and Ligaments E-Figure 16.2 Osteoblasts and Osteocytes, Long Bone, Young Dog. Prominent cuboidal osteoblasts (arrows) line the endosteal surface. Hematopoietic marrow is present (above the endosteal surface). More mature (older) osteocytes are present deeper in the cortex (below the endosteal surface) and are recognizable as small elliptical cells (arrowheads) residing in lacunae. The more recently embedded (younger) osteocytes are in larger lacunae closer to the endosteal surface. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) 1038.e1 A B ER E-Figure 16.3 Osteoblast, Bone, Tibia, Rodent. Osteoblast (below) with abundant endoplasmic reticulum (ER) on an actively mineralizing surface. Only a portion of the cell, not including the nucleus, is shown. Cell processes (arrows) of the osteoblasts extend out into the osteoid (above). Mineralization (black spicules) is initiated within matrix vesicles (arrowheads) and then extends onto the adjacent collagen. Transmission electron microscopy (TEM). Uranyl acetate and lead citrate stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) E-Figure 16.4 Quiescent Osteoblasts. A, Vertebra, adult dog. Quiescent osteoblasts (arrows) on the endosteal surface of adult bone are fusiform with little cytoplasm and form an inconspicuous layer between the cortical bone (below the endosteal surface) and active hematopoietic marrow (above the endosteal surface) of the adult axial skeleton. Hematoxylin and eosin (H&E) stain. B, Inactive osteoblast, bone, tibia, rodent. Quiescent osteoblast has reduced cytoplasmic volume due to the less-developed or less-active organelles, which are necessary for collagen synthesis. The narrow rim of unmineralized (light gray) bone matrix between the osteoblast and the mineralized bone (black) is termed the lamina limitans. Transmission electron microscopy (TEM). Uranyl acetate and lead citrate stain. (A and B courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) 1038.e2 SECTION II Pathology of Organ Systems E-Figure 16.5 Osteocyte, Bone, Tibia, Rodent. A recently embedded osteocyte still has residual rough endoplasmic reticulum and Golgi apparatus that were used during its osteoblastic period. Cell processes extend into the mineralized matrix (black) through tunnels called canaliculi (arrows). Transmission electron microscopy (TEM). Uranyl acetate and lead citrate stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) CHAPTER 16 Bones, Joints, Tendons, and Ligaments * cc 1039 cc * cc Figure 16.2 Osteoblasts, Osteocytes, and Osteoclasts, Femoral Head, Immature Pig. Active osteoblasts (arrows) on the trabecular surface; osteocytes are embedded in bone matrix (asterisks); multinucleated osteoclasts (arrowheads). Areas of retained calcified cartilage (cc) are present within and hematopoietic marrow is present between bone trabeculae in this young animal. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota.) contact with osteoblasts and other osteocytes by means of long cytoplasmic processes (also referred to as dendrites) that pass through thin tunnels in the bone called canaliculi (singular: canaliculus). This arrangement may be referred to as the osteocyte lacunar-canalicular system (LCS). The osteocyte cell body and cytoplasmic processes are surrounded by pericellular fluid, the composition of which has not been well defined. Osteocytes communicate with one another directly through gap junctions and indirectly by paracrine signaling, with recently identified effects farther afield, including an endocrine role in systemic phosphate metabolism. Under conditions of extreme stress to calcium homeostasis, osteocytes might have the ability to resorb perilacunar mineral and matrix, thus enlarging the lacuna (osteocytic osteolysis). This process apparently is rare and likely does not contribute significantly to development of osseous lesions. Osteocytes also retain a limited capacity to form bone and are exquisitely sensitive to mechanical strain in the form of shear stress. Other functions of osteocytes are somewhat speculative and are presented later in osteoblast-osteocyte interactions. Osteoclasts are multinucleated cells that are derived from hematopoietic stem cells of the monocyte-macrophage series and are responsible for bone resorption (Fig. 16.3). Several cytokines play a role in osteoclast differentiation and survival, with receptor activator for nuclear factor κB ligand (RANKL) and macrophage colonystimulating factor (MCSF) having the most essential roles (see later discussion). Osteoclasts have abundant eosinophilic cytoplasm and a specialized brush border along the margin of the cell that is adjacent to the bone surface that is being resorbed (E-Figs. 16.6 and 16.7). For osteoclasts to resorb bone, they must gain access to the bone surface that is usually covered by osteoblasts, and they must attach to the mineralized surface by transmembrane receptors in their sealing zones. The osteoclast transmembrane integrin receptors bind to noncollagenous matrix proteins; however, osteoclasts are not able to bind to unmineralized bone matrix, even though it contains these same ligands. Once bound to the matrix, the osteoclast resorbs bone in two stages. First, the mineral is dissolved by secretion of hydrogen ions through a proton pump located in the brush border. These Figure 16.3 Osteoclast and Quiescent Osteoblast, Long Bone, Cynomolgus Macaque. A large multinucleated cell (osteoclast) (arrow) is present within a scalloped cavity of resorbed bone known as a Howship’s (or erosion/ resorption) lacuna. Quiescent osteoblast (arrowhead). Von Kossa tetrachrome stain. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota.) hydrogen ions are derived from carbonic acid produced within the osteoclast from water and carbon dioxide by the enzyme carbonic anhydrase. Second, the collagen of the matrix is cleaved into polypeptide fragments by cysteine proteinases, metalloproteinases, and cathepsins, particularly cathepsin K, released from the numerous lysosomes in the osteoclast and secreted through the brush border. The concavity in the bone created by the resorbed bone matrix is called a Howship’s lacuna, or a resorption lacuna. Physiologically, osteoclast activation is controlled by osteoblasts and bone marrow stromal cells (see later discussion of interactions). Calcitonin is a systemic inhibitor of osteoclasts. Osteoclasts have receptors for calcitonin and respond to this hormone by involuting their brush border and detaching from the bone surface. The activity of osteoclasts can be indirectly measured by determining serum concentrations of collagen degradation products (pyridinoline and deoxypyridinoline cross-links) or tartrate-resistant acid phosphatase (TRAP) activity. TRAP is a glycosylated monomeric metalloenzyme that is highly expressed by osteoclasts. TRAP staining also may be used as a histochemical marker of osteoclasts in histologic sections (E-Fig. 16.8). Osteoblast-osteocyte interactions are apparent from their connections to each other by thin, tortuous, cytoplasmic processes. This network of osteoblasts and osteocytes forms a functional membrane that separates the extracellular fluid (ECF) bathing bone surfaces from the general ECF and can regulate the flow of calcium and phosphate ions to and from the bone fluid compartment (E-Fig. 16.9). Because of the large surface area of perilacunar and canalicular bone available for rapid ion exchange, significant amounts of calcium can be shifted from the bone fluid compartment to the ECF compartment without structural changes within the bone. In addition, this network allows osteocytes to detect alterations in the fluid flow within the bone ECF compartment. It is thought that such flow contributes to electric currents called streaming potentials. Changes in these streaming potentials caused by altered stress and strain on the bone or disruption of these potentials by microcracks (minuscule fractures within the bone visible only microscopically) may be detected by osteocytes with subsequent signaling to the overlying osteoblasts to initiate bone formation or resorption. Osteocytes also are capable of secreting sclerostin, a protein that inhibits bone formation by osteoblasts. During bone modeling (change of shape in response to normal growth, altered mechanical use, or disease), sclerostin may keep bone-lining cells in a quiescent state and may therefore prevent activation of osteoblasts and bone formation. Sclerostin also CHAPTER 16 Bones, Joints, Tendons, and Ligaments E-Figure 16.6 Osteoclasts, Long Bone, Young Dog. Several multinucleated cells (osteoclasts) (arrows) are resorbing endocortical bone and creating a scalloped endosteal surface. These scalloped cavities of resorbed bone are called Howship’s (or erosion) lacunae. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) BB E-Figure 16.7 Osteoclasts, Brush Border, Bone, Tibia, Rodent. Spicules of hydroxyapatite (arrows) are apparent between the projections of the osteoclast brush border (BB). The crystals have been separated from the matrix and are in the process of being dissolved by acid and enzyme secretions across the brush border. Transmission electron microscopy (TEM). Uranyl acetate and lead citrate stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) 1039.e1 E-Figure 16.8 Osteoclast, Bone, Pig. Tartrate-resistant acid phosphatasepositive osteoclast (multinucleated cell with red cytoplasm) on the surface of mineralized bone in an area of active remodeling. Plump, cuboidal osteoblasts line the remainder of the bone surface. Marrow space is above, and bone is below. TRAP stain. (Courtesy Dr. C.S. Carlson, College of Veterinary Medicine, University of Minnesota.) 1039.e2 SECTION II Pathology of Organ Systems Capillary Osteoclasts ECF Osteoblasts BTF BTF Osteocytes Bone matrix E-Figure 16.9 Movement of Calcium. The hypothesized intracellular and extracellular movement of calcium (arrows) in bone and the relationships of osteoblasts (blue, single nuclei), osteocytes (yellow), and osteoclasts (blue, multiple nuclei) to blood vessels, extracellular fluid (ECF), and bone tissue fluid (BTF) compartment is shown. (Redrawn from Matthews JL, Vander Wiel C, Talmage RV: Bone lining cells and the bone fluid compartment, an ultrastructural study, Adv Exp Med Biol 103:456, 1978.) 1040 SECTION II Pathology of Organ Systems has been shown to decrease the life span of osteoblasts by stimulating apoptosis. In modeling, bone surfaces (periosteal, endosteal, intracortical, and trabecular) can go directly from resting to either formation or resorption, depending on the stimulus. In modeling, bone resorption and formation occur independently on separate surfaces/anatomic locations (i.e., they are not directly coupled). This process allows the shape or size of bone to change, enables the medullary cavity to enlarge, and allows the overall shape of the bone to be maintained while it is growing. Modeling is in contrast to remodeling, in which resorption must precede formation to keep bone mass and shape constant (see later discussion). Osteoclast-osteoblast/stromal cell interactions are required for physiologic resorption of bone. The bone surface is protected from osteoclastic resorption by a continuous layer of osteoblasts and by a very thin layer of unmineralized bone matrix normally present beneath resting osteoblasts (lamina limitans) (see E-Fig. 16.2). For parathyroid hormone (PTH) to initiate bone resorption, receptors on the osteoblast bind PTH. The binding of PTH to osteoblasts signals them to retract and secrete collagenases, which erode the unmineralized layer of matrix and allow osteoclasts access to a mineralized bone surface. More recently, it has been determined that human osteoclasts express PTH and can respond directly to PTH. In addition, osteoblasts and bone marrow stromal cells that are activated by binding PTH and in response to a variety of other bone-resorbing stimuli (1,25-dihydroxyvitamin D3; interleukin [IL]-1, IL-6, and IL-11; tumor necrosis factor-α [TNF-α]; prostaglandin E2 [PGE2]; and glucocorticoids) express or secrete RANKL, also called osteoclast differentiation factor (ODF). RANKL binds to the RANK receptor on osteoclasts and activates the resorption process. Osteocytes also express RANKL and have been shown to regulate osteoclasts during bone remodeling. Osteoblasts and bone marrow stromal cells also can secrete osteoprotegerin (OPG), a RANK homologue that works by binding to RANKL, thus blocking the RANK-RANKL interaction and inhibiting the differentiation of osteoclast precursors into mature osteoclasts. OPG expression can be stimulated by transforming growth factor-β (TGF-β). Therefore, osteoblasts and stromal cells have the ability to both up- and downregulate osteoclastic bone resorption (Fig. 16.4). In conditions of inflammation and necrosis, inflammatory mediators, such as IL-1 and TNF-α, can stimulate osteoclasts directly, causing bone resorption independent of the presence of viable osteoblasts. Bone at the Organic Matrix and Mineral Level The mineralized matrix of bone provides the organ’s strength. Bone organic matrix consists of type I collagen and “ground substance” (the noncollagenous extracellular matrix that includes water, proteoglycans, glycosaminoglycans, noncollagenous proteins, and lipids). Type I collagen polymers are secreted by osteoblasts and assembled into fibrils that are embedded in the ground substance and then mineralized. The fundamental unit of the type I collagen molecule (known as tropocollagen) is composed of three intertwined amino acid chains, unique to which is the hydroxylated form of the amino acid proline (hydroxyproline). Type I collagen molecules have extensive cross-linkages among the amino acid chains within the molecule and between adjacent molecules. Collagen molecules are deposited in rows with a gap between each molecule and with the rows staggered so that the molecules overlap by one-quarter of their length. This specific packing of the collagen molecules and the cross-linkages contributes to the strength and insolubility of the fibrous component of the bone matrix. Other than in rapidly deposited bone (i.e., woven bone found in the embryonic skeleton or in pathologic conditions such as fracture repair, in which the collagen RANK ligand Stromal cell / Osteoblast Osteoclast precursor RANK NFκB Differentiation M-CSF receptor Osteoclast M-CSF PTH receptor Bone Osteoprotegerin (blocks RANK-RANK ligand interaction) Figure 16.4 Interaction between Stromal Cells/Osteoblasts and Osteoclast/Osteoclast Precursors. MCSF produced by stromal cells/osteoblasts binds to a receptor on osteoclast precursors to enhance their differentiation to mature osteoclasts. Stromal cells/osteoblasts are also involved in the activation of osteoclasts through production of RANK ligand, which binds to the RANK receptor on the osteoclast and its precursors. This process results in the differentiation of osteoclast precursors to mature osteoclasts and the activation of mature osteoclasts, allowing them to dissolve and resorb bone tissue (osteoclasis). Conversely, osteoblasts and stromal cells can inhibit the activation of osteoclasts by secreting osteoprotegerin, which can bind to RANK ligand and block its binding to RANK receptor. PTH receptors on osteoblasts (not shown) bind PTH to initiate and signal these cells to retract from the bone surface and secrete collagenases. Osteoclasts also have been shown to express PTH receptors, allowing them to respond directly to PTH. MCSF, Macrophage colony-stimulating factor; NF-κB, nuclear factor κB; PTH, parathyroid hormone. fibers are arranged haphazardly), collagen fibers in bone are arranged in parallel lamellae (singular: lamella) and the tissue is called lamellar bone. In cortical (compact) bone, lamellae are arranged concentrically (E-Fig. 16.10). In trabecular bone, the lamellae usually are arranged parallel with the bone surface. The collagen content of bone and its lamellar arrangement give bone its strength and flexibility. The ground substance of bone, which also is synthesized by osteoblasts, consists of noncollagenous proteins, proteoglycans, and lipids. Many of the noncollagenous proteins are cytokines that are capable of influencing bone cell activity and may play pivotal roles in controlling the extent of bone formation and resorption in normal remodeling and in disease (Fig. 16.5). Also, among the noncollagenous proteins are enzymes that can function in the degradation of collagen (e.g., matrix metalloproteinases) and can destroy inhibitors of mineralization (e.g., pyrophosphates). Other noncollagenous proteins in the matrix can function as adhesion molecules and help bind cells to cells, cells to matrix, and mineral to matrix. Examples of these are osteonectin and osteocalcin. The major role of proteoglycans in bone matrix is uncertain, although recent work suggests an important role for proteoglycans in tethers that attach osteocyte processes to the canalicular walls as part of the mechanical signaling between osteocyte and matrix, along with roles in cell-signaling pathways and matrix structural integrity. There is also evidence that proteoglycans influence bone cell differentiation and proliferative activity. Lipids may assist in binding calcium to cell membranes and in promoting calcification. Bone mineral is in the form of a crystal called hydroxyapatite. Mineralized bone makes up approximately 65% of the bone by weight and consists in part of calcium, phosphorus, carbonate, magnesium, sodium, manganese, zinc, copper, and fluoride. The mineral content gives bone its hardness. The production of osteoid (unmineralized organic matrix) by osteoblasts is followed by a period of maturation, after which mineral is deposited in exchange for water. Mineralization in woven bone is initiated within cytoplasmic blebs (matrix CHAPTER 16 Bones, Joints, Tendons, and Ligaments E-Figure 16.10 Osteonal Remodeling, Cortical Bone. Endocortical surface of bone has undergone extensive osteonal remodeling. Collagen fibers are birefringent when viewed in appropriate plane with polarized light; alternate lamellae polarize when viewed in the same plane. This alternating pattern of birefringence demonstrates the parallel arrangement of collagen layers in lamellar bone. Much of the cortex has been remodeled into osteonal bone (concentric layers with central channel for vessels and nerves), but there are a few small areas of cortex that remain unosteonized (unremodeled). Unstained and fully mineralized. Polarized light micrograph. (Courtesy Dr. L.P. Krook, College of Veterinary Medicine, Cornell University.) 1040.e1 CHAPTER 16 Bones, Joints, Tendons, and Ligaments Microdamage Osteoclast Mediators of osteoclastogenesis Osteoclast precursor Liberated matrix bound growth factors Surface osteoblasts Osteocyte Mechanical factors Hormones Cytokines inorganic pyrophosphates and, in doing so, destroy these inhibitors. Once the gaps are filled with mineral and the inhibitors of mineralization are destroyed, the process continues so that eventually the surfaces of collagen fibers, as well as spaces between collagen fibers, are mineralized. Initiation of mineralization in lamellar bone may not require matrix vesicles because glycoproteins, such as bone sialoprotein and osteonectin, can act as the nidus for the mineralization process. Bone as a Tissue Proliferation Osteoprogenitor cells Proliferation and maturation Active osteoblasts Runx2 Wnt BMP LRP5/6 β-catenin Figure 16.5 Relationship between Osteoclasts, Osteoblasts, and Growth Factors. Osteoclasts are able to liberate and activate growth factors from the matrix that are stimulatory to osteoblast progenitor cells, allowing them to proliferate and differentiate to mature osteoblasts, which may stimulate osteoclast differentiation and activation as described in Fig. 16.4. The result is a “coupling” of the process of osteoclastic bone lysis with subsequent bone formation. vesicles) of osteoblasts in the osteoid (see E-Fig. 16.2). Initiation of mineralization involves concentrating calcium, phosphorus, and other elements in these matrix vesicles to a level that causes precipitation of the mineral in the form of amorphous (not yet crystalline) hydroxyapatite. Matrix vesicles contain phospholipids and enzymes such as alkaline phosphatase and adenosine triphosphatase in their membranes. It is speculated that the membrane phospholipids attract calcium and phosphorus to the surface of the vesicle and that the alkaline phosphatase and adenosine triphosphatase enzymes may function in the pumping of these ions into the cell against a concentration gradient. On reaching a critical mass, the amorphous mineral becomes crystalline. The crystalline hydroxyapatite pierces the lipid membrane of the matrix vesicle and extends to the gaps (holes) between collagen molecules. It is within these holes that the mineral crystals are first deposited in collagen. For the mineralization to spread beyond these gaps between the collagen molecules, it is necessary for naturally occurring inhibitors of mineralization, such as inorganic pyrophosphate, in the matrix to be destroyed. Certain molecules promote matrix mineralization, including dentin matrix protein 1 (DMP1) and bone sialoprotein. Inorganic pyrophosphates are normal by-products of cellular metabolism and are deposited in unmineralized matrix by osteoblasts. The phosphatase enzymes described previously on the matrix vesicles have the ability to cleave these 1041 In the cortex and subjacent to articular cartilage (subchondral bone), bone is organized into osteons (also called Haversian systems), which are cylinders of concentric layers of lamellae that are oriented parallel to the longitudinal axis of the bone and contain centrally located vessels and nerves (Fig. 16.6). The bone between the osteons is called interstitial lamellae. Layers of bone oriented parallel to the internal and external circumference of the bone (beneath the endosteal and periosteal surfaces) are called circumferential lamellae. The osteonal system provides channels for the vascular supply to the cortex and acts as tightly bound cables, giving the cortical bone strength and limited flexibility. This osteonal system also may be important in limiting propagation of microcracks in bone by diverting cracks along cement lines, which are collagen-poor, proteoglycan-rich seams between adjacent remodeling and/or modeling units (see later discussion). In hematoxylin and eosin (H&E)-stained histologic sections of decalcified bone, cement lines appear as basophilic lines. In contrast to the dense compact bone of the cortex and the subchondral bone plates, the bone in the medullary cavity is in the form of anastomosing plates or rods and is termed cancellous, trabecular, or spongy bone (see Figs. 16.6 and 16.8). The orientation of the trabeculae usually reflects adaptation (modeling) to mechanical stresses applied to the bone. This is readily apparent when examining the trabeculae in the femoral neck, which are oriented in lines and arcs that are perpendicular to the stress applied and are thicker and more numerous on the ventral aspect (side of compression) compared with the dorsal aspect (side of tension). The lamellae within a trabecula usually are arranged parallel to the surface of the trabecula and are not arranged into tubes or osteons, as they are in cortical bone. In most species, bone undergoes a low but constant process called remodeling, in which old bone is resorbed and replaced by new bone. Remodeling is a tightly coordinated event that includes synchronized activities of multiple cellular participants to ensure that bone resorption and formation occur sequentially at the same anatomic location to preserve bone mass. The basal level of this remodeling activity (number of sites in the skeleton being remodeled at any one time) is likely “programmed” for each species. The number of active remodeling sites, however, can be markedly increased or decreased in response to altered mechanical use (see later discussion). This turnover of old bone to new bone allows for the repair of accumulated microscopic injury in the bone (microcracks/microfractures). In normal bone remodeling, slightly less bone is replaced than is removed, leaving a small negative net change in bone mass with each remodeling cycle and explaining in part the reduced bone mass in aged animals. Furthermore, in disease states, such as hyperparathyroidism, resorption is often increased and formation decreased, leaving a significant net negative bone balance. Interestingly, in small, short-lived animals, such as the mouse and rat, cortical bone is not remodeled. Bone formation may be greater than bone resorption at the periosteal surface, leading to small increases in bone diameter with age. The remodeling unit of cortical bone is called the osteon, whereas the remodeling unit of trabecular bone is called the basic structural unit. The shape of the osteon is cylindrical; however, the basic 1042 SECTION II Pathology of Organ Systems Osteons (Haversian systems) Periosteum Endosteum Inner layer Outer layer Trabeculae Compact bone Haversian canals (vertical) Cancellous (spongy) bone A Volkmann canals (horizontal) Medullary marrow cavity Osteon (Haversian system) Circumferential lamellae Figure 16.7 Remodeling in Compact Subchondral Bone Adjacent to a Joint with Bacterial Infection, Bone, Horse. Resting cement lines appear as basophilic smooth lines indicating where formation has temporarily stopped (arrowhead). Reversal lines are scalloped basophilic lines (arrow) indicating where bone resorption stopped and was followed by formation. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) Blood vessels within Haversian or central canal Lacunae containing osteocytes Interstitial lamellae B Periosteum Blood vessel within Volkmann or perforating canal Concentric lamellae resorption stopped and the process was reversed by formation) (Fig. 16.7). Cement lines also can occur when osteoblast formation ceases and subsequently resumes; these are called “resting lines” and are usually smooth, following the contour of the overlying surface. Bone as an Organ Compact bone Cancellous bone Trabeculae Lacunae Central Mineralized matrix canal Individual bones of the skeleton vary in their manner of formation, growth, structure, and function. Flat bones of the skull develop by the process of intramembranous ossification, in which mesenchymal cells differentiate into osteoblasts and produce bone directly, in the absence of a preexisting cartilage model. In contrast, most bones develop from cartilaginous models by the process of endochondral ossification, in which cartilage is invaded by blood vessels, undergoes mineralization, and forms primary (diaphyseal) and secondary (epiphyseal) centers of ossification. Bones forming by endochondral ossification, such as the long bones of the appendicular skeleton and the vertebral bodies, are divided anatomically into epiphyses, metaphyseal growth plates (physes), metaphyses, and diaphyses (Fig. 16.8). Blood Supply to Bone C Figure 16.6 Structure of Compact and Cancellous Bone. A, Longitudinal section of a long bone showing both cancellous and compact bone. B, Magnified view of compact bone. C, Section of a flat bone. Outer layers of compact bone surround centrally located cancellous bone. Fine structure of compact and cancellous bone is shown at a higher magnification. (From Thibodeau GA, Patton KT: Anatomy and physiology, ed 6, St. Louis, 2007, Mosby.) structural unit has the contour of a shallow trench filled with parallel lamellae. The time required from initiation to completion of these remodeling units (from initiation of bone removal by osteoclasts to complete filling of the defect by osteoblasts), regardless of the type of bone, is estimated to be 3 to 4 months in human beings. Basophilic cement lines that mark the limit of previous resorptive activity are usually somewhat scalloped, following the contours of the Howship’s lacunae created by osteoclasts, and are called reversal lines (where Developmentally, long bones are first apparent as a mesenchymal condensation that then differentiates to hyaline cartilage (Fig. 16.9). Osteogenic cells (stem cells) differentiate into osteoblasts, which secrete bone matrix onto the periphery of the diaphysis, forming a bony periosteal collar. The central area of the shaft then becomes vascularized and mineralizes, forming the primary center of ossification. At a later stage of development, the ends of the growing long bones become vascularized centrally and become mineralized, forming the secondary centers of ossification. The growth plate (physis) forms between the primary and secondary centers of ossification and is composed of epiphyseal (growth) cartilage that is responsible for longitudinal growth. Epiphyseal cartilage also is present at the ends of the growing long bones, subjacent to the articular cartilage, and is responsible for forming the shape of these structures. Arterial blood from the systemic circulation enters bones through nutrient, metaphyseal, periosteal, and epiphyseal arteries (see Fig. 16.9). Nutrient arteries penetrate the diaphyseal cortex through a nutrient foramen covered by strong, protective CHAPTER 16 Bones, Joints, Tendons, and Ligaments Trabecular bone Physis (growth plate) Articular cartilage Epiphyseal cartilage Trabecular bone Epiphysis Physis (growth plate) Metaphysis Cortical bone Cortical bone Endosteum Medullary cavity/bone marrow Endosteum Diaphysis Medullary cavity/bone marrow Periosteum Physis (growth plate) 1043 Periosteum Metaphysis Physis (growth plate) Epiphysis A B Figure 16.8 Longitudinal Section of Long Bone (Tibia) Showing Trabecular (Cancellous) and Compact (Cortical) Bone and Names of the Regions of Bone. A, Schematic diagram. B, Long bone (tibia), macerated specimen. The marrow has been flushed from this specimen. (B courtesy Dr. E.J. Olson and Dr. J.A. Dykstra, College of Veterinary Medicine, University of Minnesota.) fascial attachments; once within the medulla, these arteries divide into proximal and distal intramedullary branches. Other arteries penetrating the cortex include the proximal and distal metaphyseal arteries, which are smaller and more numerous than the nutrient arteries. They penetrate the cortex and anastomose with the terminal branches of the nutrient arteries in the medullary cavity, protecting against infarction in case of obstruction of a nutrient artery. Small periosteal arteries also pass through the diaphyseal cortex at sites of fascial attachment and can supply one-quarter to onethird of the outer cortex. The remainder of the cortex is supplied by the nutrient artery and its anastomotic branches. This blood flow is centrifugal (from medulla to periosteum) because of greater pressures in the intramedullary vessels. The chondrocytes of the physis nearest the epiphysis are supplied by epiphyseal arteries, whereas the chondrocytes of the physis nearest the metaphysis are supplied by branches of metaphyseal and nutrient arteries. As capillaries from these vessels approach the metaphyseal side of the physis, they make abrupt turns (loops), which are sites of predisposition to bacterial embolization in neonatal sepsis, sometimes resulting in osteomyelitis. Bone Growth Bone grows in length by interstitial growth within the metaphyseal growth plates (physes) (Fig. 16.10). The calcified longitudinal septa of the growth plates serve as struts on which bone is deposited, a process called endochondral ossification. The metaphyseal growth plate is divided into a reserve or resting zone, a proliferative zone, a hypertrophic zone, and a calcifying zone (see Fig. 16.10). The resting or reserve zone serves as a source of cells for the proliferating zone in which cells multiply, accumulate glycogen, produce matrix, and become arranged in longitudinal columns. In the hypertrophic zone, the chondrocyte volume expands and the chondrocytes secrete macromolecules that modify the matrix to allow capillary invasion and initiate matrix mineralization. The overall lengthening of the bone is due to both chondrocyte proliferation and hypertrophy; recent experimental evidence indicates that the latter is more important to this process. Calcification begins in the longitudinal septa of cartilaginous matrix between columns of chondrocytes. Although the majority of the terminal hypertrophic/calcifying chondrocytes appear to undergo apoptosis, these cells also are capable of undergoing transformation to osteoblasts. Matrix vesicles derived from chondrocytes (analogous to those described previously for mineralization of bone) form in the calcifying zone and initiate the mineralization process. The processes of mineralization and vascular invasion of the growth plate are codependent events. To supply salts for the mineralization, a nearby blood supply is necessary. Vascular invasion, a critical step in endochondral ossification, does not take place in mammalian growth plates unless there is mineralization of the longitudinal septum. Blood vessels from the metaphysis invade into the advancing growth plate, providing an entryway for osteoblasts that form bone on the cartilage spicules (Fig. 16.11). The chondro-osseous junction in the metaphysis is a fragile lattice of bone-covered spicules of calcified cartilage (primary spongiosa). As the growth plate advances and elongates the metaphysis, the more mature trabeculae deeper in the metaphysis become fewer and thicker (secondary spongiosa) and are composed primarily of bone with only residual fragments of cartilage. Growth plates (physes) are thickest when growth is most rapid; as growth slows, the growth plate becomes thin and “closes” (becomes replaced by bone) at skeletal maturity. The age at which a growth plate closes varies, depending on site, species, and sex. For example, the physes of vertebrae usually remain open longer than the physes of the long bones. Androgens and estrogens play a major role in determining the time of growth plate closure, and early castration results in delayed growth plate closure with subsequent increased length of bones compared with intact individuals. Growth of the epiphysis contributes both to the overall length of the bone and to the shape of the ends of the bone. This is 1044 SECTION II Pathology of Organ Systems R P A B C D H C Figure 16.10 Growth Plate (Physis), Long Bone, Immature Pig. Resting (R), proliferating (P), hypertrophic (H), and calcifying (C) zones of the growth plate are visible. Apoptotic chondrocytes are released from their lacunae by invading vessels and chondroclasts, leaving only the longitudinal septa (arrow) as a template on which bone will be deposited to form a primary trabecula. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. A.R. Armstrong, College of Veterinary Medicine, University of Minnesota.) E F Mesenchyme G Cartilage matrix Calcified cartilage Bone Figure 16.9 Correlation of Long Bone Development and Vascularization. The primitive mesenchyme that makes up the skeletal primordia contains no blood vessels (A). This mesenchyme condenses and undergoes central mineralization; a bony collar forms in the periosteum of the diaphysis (B and C). The nutrient artery enters the mineralized cartilaginous tissue in the diaphysis, bringing osteogenic and osteoclast precursors and enabling endochondral ossification to occur (primary center of ossification) (D). Similarly, the epiphyseal arteries bring these cells to the secondary centers of ossification in the epiphyses (located at the ends of the growing long bones) (E). Extensive anastomoses develop as the bone continues to develop and as the subarticular growth cartilage is replaced by bone and the growth plates close (F and G). Cartilage canal vessels, arising from the perichondrium and entering the epiphyseal cartilage present at the ends of the growing long bones, are not shown in this diagram but are present before the development of the secondary centers of ossification and persist for a variable time frame afterward, depending on site and species. (Redrawn from Banks WJ: Applied veterinary histology, ed 3, St. Louis, 1993, Mosby.) accomplished by endochondral ossification at the articular-epiphyseal cartilage complex (AECC). The AECC is composed of permanent articular cartilage, as well as a subjacent temporary growth/ epiphyseal cartilage that is vascularized and contains the same zones as the growth plate (Fig. 16.12). In the mature individual, endochondral ossification no longer occurs at the AECC. Although the articular cartilage remains throughout life, the epiphyseal cartilage is completely replaced by bone once growth has ceased (Fig. 16.13). Bone grows in width by intramembranous bone formation. Except for articular surfaces (including the ends of the vertebral bodies), the surfaces of bones are covered by periosteum. This covering is a thin membrane that is loosely attached to underlying bone, except at heavy fascial attachments on bony prominences and at tendon insertions, where its attachments are strong and are associated with large vessels penetrating the underlying bone. Microscopically, the periosteum is composed of an outer fibrous layer that provides structural support and an inner osteogenic or cambium layer that is capable of forming normal lamellar appositional bone on the cortex of growing bones (Fig. 16.14). The cambium layer also is capable of Epiphyseal vessel Endplate Resting chondrocytes Proliferative chondrocytes Hypertrophic chondrocytes Calcified cartilage Woven bone on calcified cartilage spicule Capillary loop Cartilage matrix Calcified cartilage Woven bone Figure 16.11 Major Blood Supply to the Physis. Branches of the epiphyseal artery supply the resting zones of the growth plate. Branches of the metaphyseal artery form capillary loops at the metaphyseal side of the physis where endochondral ossification is occurring. (Redrawn from Banks WJ: Applied veterinary histology, ed 3, St. Louis, 1993, Mosby.) CHAPTER 16 Bones, Joints, Tendons, and Ligaments 1045 AC f EC o Figure 16.14 Periosteum, Bone, Dog. The outer fibrous layer (f) and inner osteogenic layer (o) line the periosteal surface. The osteogenic layer is able to rapidly deposit woven bone as a nonspecific response to injury. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) Figure 16.12 Articular-Epiphyseal Cartilage Complex (AECC), Long Bone, Immature Pig. The AECC is composed of a layer of articular cartilage (AC) and a subjacent layer of growth (epiphyseal) cartilage (EC). The growth cartilage is present only in immature individuals, and it often contains cartilage canal vessels (not shown); its structure and function are similar to those of the physis. Compare with Fig. 16.13. Toluidine blue stain. (Courtesy Dr. C.S. Carlson, College of Veterinary Medicine, University of Minnesota.) SZ MZ AC DZ TM CC Figure 16.13 Articular Cartilage, Long Bone, Adult Dog. The articular cartilage is present throughout life and contains superficial, middle, and deep zones; however, the epiphyseal cartilage is absent in adults. In adults, endochondral ossification has ceased and the tidemark (TM) delimits the boundary between the uncalcified articular cartilage (AC) and the calcified cartilage (CC). Compare with Fig. 16.12. DZ, Deep zone; MZ, middle zone; SZ, superficial zone; Toluidine blue stain. (Courtesy Dr. C.S. Carlson, College of Veterinary Medicine, University of Minnesota.) forming woven bone, known as periosteal new bone, in response to injury. The periosteum is well supplied with lymph vessels and with fine myelinated and nonmyelinated nerve fibers that account for the intense pain that occurs with periosteal injury. The periosteum covering the physis is called the perichondral ring. The perichondral ring adds new cartilage to the periphery of the physis, enabling it to expand in width as the animal grows. The metaphyseal cortex immediately adjacent to the perichondral ring is normally very thin in the growing bone because its surfaces are the sites of very active osteoclastic bone resorption. Structurally, this area is the weakest part of the bone. Joints Joints (articulations) join skeletal structures, provide for movement, and in some cases have shock-absorbing functions. This section of the chapter is primarily devoted to synovial joints, also called movable or diarthrodial joints. Synovial joints occur in both the axial and the appendicular skeleton. These joints allow for a variable degree of movement and, anatomically, they are composed of two bone ends joined together by a fibrous capsule and ligaments. The inner surface of the articular capsule is lined by a synovial membrane, and the bone ends are covered by articular cartilage. The joint space contains synovial fluid; fibrocartilaginous menisci or disks are present at some sites (e.g., femorotibial and temporomandibular joints). Synovial joints operate with very low coefficients of friction and are self-lubricating. Articular cartilage serves as the bearing substance and subchondral bone as the supporting material. Articular cartilage functions to minimize friction created by movement to transmit mechanical forces to underlying bone and to maximize the contact area of the joint under load. Joints receive and absorb energy of impact. Both articular cartilage and subchondral bone deform under pressure, but it is the subchondral bone that has the most significant force-attenuating properties. Articular Cartilage Articular (hyaline) cartilage is normally a white to blue-white material with a smooth, moist surface. Cartilage thickness is greatest in the young and at sites of maximal weight bearing. Thinning and yellow discoloration occur in old age. At its margins, articular cartilage merges with a periosteal surface that is lined by fibrous tissue contiguous with the synovial membrane. Synovial fossae are normal cartilage-free depressions on non–weight-bearing articular cartilage surfaces (Fig. 16.15) that develop bilaterally in the larger appendicular joints of the horse, pig, and ruminant. These fossae are usually not present at birth but are fully formed by skeletal maturity. The function of synovial fossae is not known; however, they are significant because they are often mistaken for lesions. Adult articular cartilage contains no nerves, blood, nor lymph vessels; its nutrients are obtained by diffusion from synovial fluid and to a lesser 1046 SECTION II Pathology of Organ Systems A B Figure 16.15 Synovial Fossa, Joint, Radius and Ulna, Bone, Proximal End, Adult Horse. A, Synovial fossae are depressions in the cartilage on the non–weight-bearing surfaces of the sagittal ridge of the radius and in the semilunar notch of the ulna (arrows). The parallel linear grooves apparent on the weight-bearing surface (articular cartilage) of the radius are the result of degenerative joint disease. B, Histologically, the surface of the synovial fossa is covered by a thin fibrous membrane (arrow) rather than articular cartilage. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) extent from subchondral vessels. In the immature skeleton, articular cartilage overlies the temporary growth cartilage of the epiphysis (epiphyseal cartilage) (see Fig. 16.12). The epiphyseal cartilage is highly dependent on a network of blood vessels that originate from the perichondrium and the subchondral bone. Similar to the physis, it undergoes endochondral ossification and thereby contributes to the growth/development of the epiphysis. At skeletal maturity, the epiphyseal cartilage has been entirely replaced by bone, and the only joint cartilage remaining is the adult articular cartilage, which overlies a thin subjacent zone of calcified cartilage. In skeletally mature individuals, the junction between the noncalcified articular cartilage and the deeper calcified cartilage forms a thin basophilic line in H&E-stained sections called the tidemark (see Fig. 16.13). As animals age, multiple tidemarks can be formed, indicating an advance (thickening) of the calcified layer of articular cartilage and thinning of the overlying noncalcified articular cartilage. The calcified cartilage serves to anchor articular cartilage to subchondral bone and limits the diffusion of substances between bone and cartilage. Articular cartilage is 70% to 80% water by weight. It is a viscoelastic, hydrated fiber-reinforced gel that contains chondrocytes, collagen fibers (mostly type II), noncollagenous proteins, and proteoglycan aggregates. The largest proteoglycan in articular cartilage is aggrecan, which is composed of the immensely long, nonpolysulfated glycosaminoglycan, hyaluronan, to which core proteins are attached in a perpendicular arrangement like bristles on a brush. Similarly, smaller, negatively charged polysulfated glycosaminoglycans (chondroitin sulfate and keratan sulfate) are attached to the core proteins. The large content of negatively charged polysaccharide chains in the aggrecan aggregate is responsible for the extremely high osmotic swelling pressure of cartilage. The swelling pressure provided by aggrecan is counteracted by the resistance of the intact type II collagen fibers, giving cartilage its characteristic properties of being able to resist compressive forces and having a high tensile strength. Collagen fibers are arranged in arcades so that the tops of the arcades are parallel to the articular surface, and the sides are perpendicular to the surface and parallel with the radial or intermediate zone of chondrocytes. Functionally, the superficial zone of articular cartilage resists shearing forces, the middle zone functions in shock absorption, and the calcified deep zone of cartilage serves to attach articular cartilage to the subchondral bone by its irregular (and therefore interlocking) interfaces. Scanning electron microscopy reveals that the surface of articular cartilage is not smooth but, rather, contains numerous depressions that can serve as reservoirs for synovial fluid. Articular cartilage contains a single population of cells called chondrocytes that are responsible for the production, maintenance, and turnover of intercellular substances. In routine histologic sections, chondrocytes appear to be present within lacunae. However, although the lacunae within which osteocytes reside contain fluid spaces, the apparent lacunae of chondrocytes have been clearly demonstrated to be the result of shrinkage artifact. In fact, the chondrocyte cell membrane is in direct contact with the pericellular matrix and contains surface receptors for matrix components (e.g., hyaluronan). Normal matrix turnover is enzymatic, and this process is balanced by enzyme inhibitors. Proteases capable of degrading aggrecans are called aggrecanases and are members of the ADAM (a disintegrin and metalloprotease) protein family, whereas proteases capable of breaking the peptide bonds in collagen are called collagenases. Damage to the matrix occurs if there is increased destruction or decreased synthesis of matrix components. It is important to remember that, compared with bone that normally renews itself by remodeling, articular cartilage has extremely poor regenerative capabilities; damaged articular cartilage repairs through the replacement of hyaline cartilage with fibrocartilage. Although there is evidence for proteoglycan synthesis in normal articular cartilage, the turnover of cells and type II collagen occurs at an extremely low rate. Correspondingly, the cellularity of articular cartilage declines with age, and the life span of individual chondrocytes is thought to be long. Articular Capsule/Synovium/Synovial Fluid The articular capsule consists of outer fibrous and inner synovial tissue layers. The outer layer is a heavy sheath that contributes to joint stability and, at its insertion, attaches to bone at the margins of the joint, thereby enclosing a segment of bone of variable length within the joint cavity. It is well supplied with blood vessels and nerve endings. The inner synovial tissue layer is called the synovial membrane and covers all the inner surfaces of the joints except for the surface of the articular cartilage. The synovial membrane is normally very thin, lacks a basement membrane, and is barely visible grossly. In fact, collection of normal synovial membrane for histologic evaluation is best done immediately after opening the joint because it quickly retracts and becomes difficult to locate. The inner surface of the synovial membrane may be flat or may contain tiny projections (villi). Synovial intimal or lining cells, one to four cells thick, form a discontinuous surface layer and include two cell types: (1) A cells, which are macrophages that are responsible for the removal of microbes and the debris resulting from normal wear and tear in the joint (Fig. 16.16; E-Fig. 16.11), and (2) B cells, which are fibroblastlike cells that produce synovial fluid. These cell types are not distinguishable from each other in routine histologic sections. Normal synovial fluid contains hyaluronic acid, lubricin (a water-soluble CHAPTER 16 Bones, Joints, Tendons, and Ligaments E-Figure 16.11 Synovial Membrane, Joint, Dog. The normal synovial membrane (arrows) consists of an incomplete layer of histiocytes (phagocytic cells) and fibrocytes with subjacent loose fibrous and/or fibrofatty tissue. The joint lumen is at the top of the figure. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) 1046.e1 CHAPTER 16 Bones, Joints, Tendons, and Ligaments 1047 js gp ac gc js bm Figure 16.16 Synovial Membrane, Stifle Joint, Mouse. The normal synovial membrane (arrows) encloses an optically clear joint space (js) and consists of an incomplete layer of histiocytes (phagocytic cells) and fibrocytes with subjacent loose fibrous and/or fibrofatty tissue. bm, Bone marrow. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota.) glycoprotein), proteinases, and collagenase and is clear, colorless to pale yellow, and viscous. In addition to lubricating the joint surfaces, it supplies oxygen and nutrients to, and removes carbon dioxide and metabolic wastes from, the chondrocytes in articular cartilage. The synovial subintima can be classified according to the type of tissue that predominates (areolar, adipose, or fibrous), and it contains blood and lymph vessels that supply and drain the intraarticular structures. Adipose tissue sometimes accumulates in the deep layers of the synovium, forming fat pads that serve as soft cushions in joint cavities (e.g., the infrapatellar fat body of the stifle joint). Joint lubrication depends on the microscopic roughness, elasticity, and hydration of articular cartilage and on the presence of hyaluronic acid and lubricin in synovial fluid. The lubricating properties of lubricin depend on its ability to bind to articular cartilage, where it retains a protective layer of water molecules. In contrast, hyaluronic acid, the molecule that makes synovial fluid viscous, has largely been excluded as a lubricant of the cartilage-on-cartilage bearing and instead lubricates the site of surface contact between synovium and cartilage. When pressure is applied to the joint, as in weight bearing, the articular surface is supplied with pressurized fluid that carries most of the load. Synovial fluid is combined with water that is released from the underlying cartilage when pressure is applied during weight bearing. When the load is removed, the water returns to the cartilage because of the hydrophilic properties of proteoglycans. This flushing of fluid in and out of the articular cartilage enables nutrients to enter cartilage from the synovial fluid and waste products to be removed. Subchondral Bone The subchondral bone has a variable morphologic appearance depending on the maturity of the individual. In immature individuals, it is composed of fine, interconnecting trabeculae that contain a large, calcified cartilage component and are the result of replacement Figure 16.17 Shoulder Joint, Rat. Glenoid cavity (gc), articular cartilage (ac) of humeral head (dashed-line ellipsoid), secondary center of ossification lies between the articular cartilage and the growth plate (gp). js, Joint space. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. C.S. Carlson and Dr. E.J. Olson, College of Veterinary Medicine, University of Minnesota.) of epiphyseal cartilage by bone through the process of endochondral ossification (see Fig. 16.12). In mature individuals, it is composed of a thin layer of cortical bone (cortical end plate) and underlying subchondral bone trabeculae (see Fig. 16.13). In both cases, it acts to support the overlying cartilage and dissipate concussive forces to the peripheral cortical bone (Fig. 16.17; E-Fig. 16.12). The thickness of the subchondral bone plate varies in proportion to the degree of weight bearing. Increased subchondral bone thickness is a common pathologic finding in osteoarthritis. In larger animals (nonrodent species), subchondral bone often is composed of compact osteonal bone rather than trabecular bone. Tendons and Ligaments A tendon is a unit of musculoskeletal tissue that transmits force from muscle to bone. Morphologically, tendons are similar to ligaments and fascia; however, ligaments join bone to bone, and fascia connects muscle to muscle. The musculotendinous unit is composed predominantly of parallel arrays of closely packed collagen fibers and rod- or spindle-shaped fibroblast-like cells (tenocytes) within a well-ordered extracellular matrix (Fig. 16.18). Collagen fibrils are bundled into large fibers that are evident throughout the tendon and have a crimped or a sinusoidal pattern when visualized with light microscopy that facilitates a 1% to 3% elongation of the tendon. This elongation of the individual fibers serves to buffer the tendon from sudden mechanical loading. Water represents approximately 55% of the weight of tendon, is present mainly in the extracellular matrix, and is believed to reduce friction, facilitating the gliding of fibrils in response to mechanical loading. The major fibrillar component of tendon is type I collagen, which constitutes approximately 80% of the dry weight, whereas type III collagen is present in the endotenon (connective tissue binding together groups of collagen fibers into fascicles) and epitenon (exterior sheath of connective tissue that surrounds groups of fascicles). The remainder of the tendon components include elastin, proteoglycans, and inorganic components. Collagen is synthesized by the tenocytes and constitutes the CHAPTER 16 Bones, Joints, Tendons, and Ligaments a b E-Figure 16.12 Rat. Compact bone (b) supports the articular cartilage (a) in the adult animal when there is no longer endochondral ossification occurring at the articular-epiphyseal cartilage complex. Hematoxylin and eosin (H&E) stain. (Courtesy Dr. S.E. Weisbrode, College of Veterinary Medicine, The Ohio State University.) 1047.e1 1048 SECTION II Pathology of Organ Systems t c A t ts B Figure 16.18 Tendon, Joint, Mouse. A, Gastrocnemius tendon (t). The tendon is composed of parallel arrays of closely packed collagen fibers and low numbers of fibroblast-like cells called tenocytes (see higher magnification in B; arrowheads). The tendon is covered by flattened synoviocytes (see higher magnification in B), so it may glide smoothly over the synoviocytes of the tendon sheath. c, Calcaneus insertion. Hematoxylin and eosin (H&E) stain. B, Higher magnification of tendon sheath. The enveloping structure enclosing the tendon is the tendon sheath (ts), and it is lined by synoviocytes (arrows). t, Gastrocnemius tendon. H&E stain. (Courtesy Dr. J.F. Zachary, College of Veterinary Medicine, University of Illinois.) basic structural unit of tendon. The collagen polypeptides form a triple helix, which self-assembles into collagen fibrils with intermolecular cross-links that form between adjacent helices. The collagen polypeptides and the ensuing triple helix are synthesized inside the cell, secreted into the extracellular matrix, and assembled into the microfibrillar units that constitute the collagen fibers. This step is promoted by a specialized enzyme called lysyl oxidase, which promotes cross-link formation—a process involving placement of stable cross-links within and between the molecules. Cross-link formation is the critical step that gives collagen fibers their strength. Tendon is stronger per unit area than muscle, and its tensile strength equals that of bone, although it is flexible and slightly extensible. The parallel arrangement of tendon collagen fibers resists tension so that contractile energy is not lost during transmission from muscle to bone. The mechanical properties of tendons depend on the collagen fiber diameter and orientation; however, the proteoglycan components of tendons also are important to the mechanical properties. Whereas the collagen fibrils allow tendons to resist tensile stress, the proteoglycans allow them to resist compressive stress. Skeletal ligaments are defined as dense bands of collagenous tissue that span a joint and then become anchored to the bone at either end. They vary in size, shape, orientation, and location. Their bony attachments are called insertions. Biochemically, ligaments are approximately two-thirds water and one-third solid. Type I collagen accounts for 85% of the collagen (other collagen types include III, V, VI, XI, and XIV), and the collagens account for approximately 75% of the dry weight, with the balance being made up by proteoglycans, elastin, and other proteins. While the ligament appears as a single structure, during joint movement some fibers appear to tighten or loosen depending on the bone positions and the forces that are applied, confirming the complexity of these structures. Histologically, ligaments are composed of fibroblasts that are surrounded by matrix. The fibroblasts are responsible for matrix synthesis and are relatively few in number, representing a small percentage of the total ligament volume. Recent studies have indicated that normal ligament cells may communicate by means of prominent cytoplasmic extensions that extend for long distances and connect to cytoplasmic extensions from adjacent cells, thus forming an elaborate three-dimensional architecture. Gap junctions have also been detected in association with these cell connections, raising the possibility of cell-to-cell communication and the potential to coordinate cellular and metabolic responses throughout the tissue. Ligament microstructure reveals collagen bundles aligned along the long axis of the ligament and displaying an underlying “waviness” or crimp along the length, similar to that present in tendon. Crimp is thought to play a biomechanical role, possibly relating to the ligament’s loading state with increased loading likely resulting in some areas of the ligament uncrimping, allowing the ligament to elongate without sustaining damage. One of the main functions of ligaments is mechanical because they passively stabilize joints and help guide those joints through their normal range of motion when a tensile load is applied. Another function of ligaments relates to their viscoelastic behavior in helping provide joint homeostasis. Ligaments “load relax,” which means that loads/stresses decrease within the ligament if they are pulled to constant deformations. A third function of ligaments is their role in joint proprioception. In joints such as the stifle, proprioception is provided principally by joint, muscle, and cutaneous receptors. When ligaments are strained, they invoke neurologic feedback signals that then activate muscular contraction, which appears to play a role in joint position sense. Tendons and ligaments attach to bone in a similar manner, through osteotendinous or osteoligamentous junctions, respectively. These junctions are known as entheses and are distinguished as fibrous and fibrocartilaginous according to the type of tissue present at the attachment site. At fibrous entheses, the tendon or ligament attaches either directly to the bone or indirectly to it via the periosteum. Fibrocartilaginous entheses are sites in which chondrogenesis has occurred and commonly contain four tissue types: dense fibrous connective tissue, uncalcified fibrocartilage, calcified fibrocartilage, and bone. Dysfunction/Responses to Injury Bone Mechanical forces that can affect bone are both internal and external. Internal forces associated with extremes in work or exercise can influence modeling or remodeling (see previous discussion) and occasionally cause fracture (failure of the bone). External forces (trauma) are more commonly associated with damage to the periosteal surface or fracture of the cortex and/or trabecular bone. Hormonal agents, particularly calcitriol and PTH, enter the bone by the bloodstream as described previously. Infectious agents are discussed later in the section on Bone, Inflammation. Defense against mechanical forces includes the structure of the bone and the bone’s ability to model and remodel to adapt to chronic changes in forces applied to it. The dense bone of the cortex enables the bone to resist most external forces. The formation of osteons and cement lines in the cortex (primary remodeling) facilitates dissipation of microcracks and bending of the bone in response to stress. Defense against hormonal agents that are capable of resorbing bone include the protective covering on mineralized surfaces by the lamina limitans and osteoblasts. In addition, hormonal signaling to resorb bone is done through the osteoblast, which has the ability to modulate the resorption signal. Defense against infectious agents is discussed later in the section on Diseases Affecting Multiple Species of Domestic Animals, Bone, Inflammation. The hard tissue of bone can be likened to the rings in a tree that leave clues to the history

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