INHAND: Non-proliferative and proliferative lesions of fish – Chapter 12. Fish skeletal and tooth

Introduction

The purpose of this publication is to provide a standardized nomenclature for classifying lesions in the skeletal system of laboratory fishes. Terms of systemic nonproliferative lesions that are not specific to the skeletal system are not included here. Neoplasms that affect skeletal tissues, but do not arise from the skeletal system, are described in other documents. The skeletal tissues described here are bones, fins, cartilage, teeth, notochord/vertebrae, and joints. Scales are described further in the integumentary system document, and intramembranous bones (pin bones) are included in the soft tissue system document. Application of nomenclature to other specialized elements of the exoskeleton (e.g., odontodes, denticles, dermal plates, scutes, etc.) are not specifically addressed; adaptation using bone, teeth, and scale terminology is advised.

The skeleton of fishes consists of two major categories: the endoskeleton and the dermal skeleton (exoskeleton)¹. The endoskeleton includes bony and cartilaginous elements that develop in the deeper tissues and are what we commonly refer to as the “skeleton” of an animal. The dermal skeleton comprises the hard tissues which form within the dermis without a cartilaginous template (e.g., dermal bone, teeth, odontodes, scales, scutes, fin spines, and fin rays)². The major categories of skeletal tissues (e.g., bone, cartilage, dentin, enamel) and their resident cell types (e.g., bone lining cells, osteoblasts/osteocytes/osteoclasts, chondroblasts/chondrocytes/chondroclasts, odontoblasts, and ameloblasts) are present in both teleosts and mammals. It is important to recognize that, unlike mammals, fish normally exhibit a range of intermediate tissue types as part of their mature skeleton. Among these is enameloid, an intermediate tissue sharing features of enamel and dentine, which coats the teeth. In addition, chondroid/hyaline-cell cartilage (tissue with features of both cartilage and connective tissue) and chondroid bone (tissue with features intermediate between cartilage and bone) are commonly present³ (Figs. 12.1 and 12.2). In some cases, the presence of intermediate tissue is pathological. At least 15 categories of cartilage have been described, some of which are not restricted to the endoskeleton, but develop within the dermal skeleton⁴. Skeletal regeneration is almost entirely restricted to the dermal skeleton (scales, fin rays, fin spines).

Fig. 12.1.

Zebrafish. Bone, chondroid. Hyomandibular bone, prootic. Example of normal chondroid bone. Decalcified section. H&E, 400×. Image courtesy Wes Baumgartner.

Fig. 12.2.

Zebrafish. Bone, chondroid. Ethmoid premaxilla. Normal chondroid bone. A joint is also present in the image. H&E, 400×. Image courtesy Wes Baumgartner.

Teleosts never stop growing, and thus all skeletal tissues continue to change throughout life whether by differentiation, mineralization, remodeling, or even metaplasia. Fluctuations in bone growth are typical and are often brought about by changes in temperature, light, diet, and other environmental cues. In some cases, bone growth may be delayed or even cease. These changes may leave defined ‘annual rings’, most evident in scales, opercula, and vertebrae⁵. The skeleton of fish is highly adaptive; altered feeding conditions and food types can alter cranial bone morphology, for example. Vertebral malformations, a relatively well-known occurrence, may develop from altered mechanical loads. In some species, remarkable changes in the skull, vertebrae, and scales can occur normally in association with migration/spawning. The ability of the skeleton to respond to mechanical changes in the environment, which in mammals is via the mechanosensory osteocyte and canalicular network, is significantly different in fishes. The canalicular network and connectivity of fish osteocytes are questionable; it is likely that osteoblasts, chondrocytes, and bone surface cells/osteocytes function as localized mechanosensory circuits⁶. Many of the characteristics of skeletal tissues are conserved among the vertebrates, and this includes cytokines and molecular factors familiar to those who work with mammals.

Scant information is available concerning joint specific pathology in fish and thus this topic will not be discussed in detail; lesions involving the joints are generally not seen in toxicologic studies. However, zebrafish knockout strains used for osteoarthritis modelling do exhibit changes similar to those in mammals^7-9. Intervertebral discs are described in the following notochord section, and for further comment on disease, see Deformation, Bone, Vertebra, Notochord, and Joint, Degenerative Joint Disease.

Necropsy and Trimming

The skeletal tissues are generally incorporated into the sectioning of fish tissues, particularly in small species where the entire fish is examined on a standard glass slide. Individual bones may be cleaned of soft tissue, fixed by immersion in neutral buffered formalin (10%), decalcified, embedded in paraffin, sectioned at 4–8 μm, and stained with hematoxylin and eosin (H&E). In young fish decalcification is often not necessary. Small laboratory fish may also be fixed in Davidson’s solution, which has mild decalcifying properties. Fish skeletal tissues are not as densely mineralized as mammalian bone, and decalcification with gentle acid solutions (such as Kristensen’s) or EDTA can be achieved relatively rapidly.

Specialized investigations may require examination of undecalcified sections; the use of H&E in addition to histochemical stains such as von Kossa and Goldner’s trichrome can be used. For decalcified sections, H&E, Masson’s trichrome, Toluidine blue, Alizarin red, and Alcian blue stains have been successfully used to delineate fine change in various fish skeletal tissue types^{4, 10, 11}.

An array of clinical and basic science tools has been used to examine fish skeletal tissues, including computed tomography, electron microscopy, and mechanical testing by various methods^12,13,14.

Morphology, Physiology, Nomenclature, Diagnostic Criteria, and Differential Diagnosis

1. Bone

In general, bone in fish is similar to that in mammals¹⁵. Specific terminology (described below) is used in the literature that the reader may find confusing as it applies to location, origin, mode of development and structural properties¹⁶. Endo- and exoskeleton refer to the location of the tissue; exoskeletal tissues form in contact with the epithelial ectoderm (scales, fin rays, teeth) while endoskeleton does not (endochondral bone). Dermal skeleton (exoskeleton) refers to teeth, scales, fin rays, and dermal bones that derive from the interaction of epidermis and mesenchyme. The dermal skeleton typically develops by membranous ossification, while endoskeleton is largely endochondral. Chondroid bone and cartilage in the dermal skeleton are secondary tissues. In a structural context, dermal skeletal and endoskeletal tissues may be woven or lamellar. Furthermore, bone may be compact or cancellous, and occasional osteons may be formed. Compact bone is characterized by tightly packed layers of mineralized matrix, while cancellous (spongy) bone is arranged in trabecular plates with intervening soft tissue cavities. Type I collagen is the major organic component; however, type II collagen can be present¹⁶. A recent bone tissue classification scheme has been published, which may prove useful in further harmonizing terminology among specialist groups¹⁷.

Unmineralized bone matrix (osteoid) is deposited by osteoblasts directly into connective tissue (dermal/membranous), or onto/within cartilage (perichondral/endochondral). Also, portions of tendons in the muscle bodies may mineralize, forming sesamoids or “pin” bones. In teleosts, woven bone develops first, and is subsequently remodeled into lamellar, and even osteonal bone. Like mammals, bone may contain encased osteocytes but, in many species, it is acellular (more properly termed “anosteocytic”), lacking obvious cell processes within the matrix (like acellular cementum in mammals and crocodiles, and first-generation teeth in teleosts), which distinguishes it from typical dentine^3,18. In some species, osteocytes remain at the surface but send cytoplasmic processes into the matrix (primary canaliculated, or tubular acellular bone)¹⁹. The classic paradigm that acellular bone is formed through polarized secretion of matrix without entrapment of osteocytes, has been recently challenged; authors propose that entrapped osteocytes undergo apoptosis and mineralization²⁰. Unlike mammals, laboratory fish may have a poorly developed osteocyte canalicular system (zebrafish-cellular bone), or none at all (medaka-acellular bone)²¹. Acellular bone, like cellular bone, is capable of remodeling and can be a source for calcium mobilization. Early cellular bone may initially be acellular or sparsely populated as to mimic acellular bone. Usually, the presence of cellular or acellular bone is uniform within the endoskeleton for any given fish, but endoskeletal and exoskeletal bone types may differ within a species. In adult zebrafish, bone is cellular and cancellous, while in the medaka, it is anosteocytic²². However, early stages of bone in zebrafish are anosteocytic²³.

Bone turnover estimates are lacking in teleosts, but reports in salmon indicate that the processes of resorption and remodeling are regular. In farming/laboratory conditions where growth is rapid compared to the wild, fast bone growth may lead to the production of abundant non-mineralized matrix, which in time mineralizes sufficiently²⁴. Calcium phosphate, in the form of hydroxyapatite, forms the mineral component. Mineralization occurs through inotropic calcification (crystal deposition along collagen fibrils). It is important to recognize that bone formation and bone mineralization are not the same; matrix is produced and mineralization occurs later. In zebrafish, mineralization of osteoid occurs within 6 hours of deposition, which is much faster than that reported in humans- several days²⁵. Evidence in fish suggests that while osteoblasts produce the organic matrix, they may not be actively involved in mineralization²⁶. In fish lacking osteocytes, other mesenchymal cells may assume osteocyte regulatory functions, similar to those of mammalian osteocytes²⁷.

Fish also have what is termed chondroid bone (CB), which is an intermediate tissue with characteristics of both cartilage and bone; it has a basophilic mineralized/bony collagenous matrix with nests of chondroid cells within lacunae, which may be immediately surrounded by cartilage matrix^{28, 29}. It may be highly cellular or poorly cellular, blends into other tissue types, and is found in joints, interfaces between cartilage and bone, and areas of increased stress. Formation of chondroid bone can have both type I and II collagen (typical of bone and cartilage, respectively) and can be identified with Trichrome staining, particularly in areas of growth^{30, 31}. It should be kept in mind, however, that other types of skeletal tissue, especially cartilage, may have both type I and II collagen³². Other histochemical stains may be used to better define the tissue and differentiate it from true cartilage³³.

Chondroid bone can be a stable tissue or appear in pathological processes such as metaplasia or neoplasia^{3, 34}. In zebrafish jawbone repair, chondrocyte-like cells produce both cartilage (Sox9a, Col2a1a) and bone (Runx2b, Col1a1a) associated proteins, which mineralize³⁵. These chondrocytic cells derive from the periosteum or osteoblasts. In sea bream gills, chondroid bone chondrocytes lose collagen II expression with maturity, but instead have collagen I and osteonectin expression³⁶. Thus, a population of mixed cartilage-bone cells can normally be present in a rapidly growing tissue, with or without subsequent differentiation into osteoblasts. As a result, bone repair in zebrafish can be a mixture of membranous and chondroid bone³⁵. Chondroid bone is to be distinguished from secondary cartilage which, in addition to lacking bone matrix features, is formed by periosteum/endosteum on membrane bone during normal development³⁷.

Two types of CB are described, according to different ontogenetic pathways. Type 1 CB develops in multipotential periostea; osteoblasts secrete osteoid which surrounds the cells and begins to calcify; acid carbohydrates then increase within the matrix, and cells then acquire a chondrocyte morphology, even losing cell contacts. Ultrastructurally, the cells have characteristics of chondrocytes and matrix has woven bone properties³⁸. In this way, young CB resembles cellular bone and is overlaid by a fibroblastic/mesenchymal layer. Type 2 CB is the equivalent of calcified hyaline cartilage or fibrocartilage^{4, 39, 40}.

Several characteristics of skeletal remodeling in teleosts distinguish it from that in mammals¹:

• Fish lack hematopoietic tissues in bone cavities; the dynamics between mammalian marrow and bone, which regulate osteoblast and osteoclast activities, are thus segregated in fish.

• Osteoclasts in many species are small, mononucleated cells that resorb bone in non-lacunar fashion.

• Teleosts obtain and release calcium through the gills (freshwater) or intestine (marine), and thus the endoskeleton is not usually a source or depot for calcium. Teleost bone metabolism is not primarily calcium driven, which is a common source of misinterpretation.

• If minerals are needed by the fish, mobilization occurs in the exoskeleton first (scales).

• The exoskeleton has a high capacity for regeneration, more than the endoskeleton.

• In teleosts, growth and remodeling are continuous throughout life, with the development of species-specific structures (the kype structure on the head of salmon) occurring well into adulthood.

Bone formation is typically perichondral, intramembranous, endochondral, or parachondral; the latter being characterized by the separation of osteogenesis from the cartilage by a few cell layers of connective tissue^{23, 41}. Perichondral bone is very common and predominates in young fish; osteoid is laid down in contact with the hyaline cartilage. This type of formation commonly persists as a thin layer of bone, without further ossification. As such, teleost long bones often present as a persistent rod of cartilage with a thin bony encasement, and cartilage condyles at both ends⁴².

As the first matrix to be laid down, compact woven bone fibers lack orientation but are remodeled into parallel-fibered and lamellar bone, both of which form the majority of teleost bone. In the case of lamellar bone, oriented fibrils are deposited on the inner aspect of lamellae that surround vascular canals (osteon formation in some species). Subsequent lamellae may not be concentric, but are arranged in piled “plywood stacks”, forming lamellated bone. Remodeling can occur repeatedly at the same site, in the form of localized resorption and filling that is seemingly unrelated to bone growth²². Sharpey’s fibers (collagen bundles connecting soft tissue to bone) may be present, particularly in growing intramembranous dermal bone. Due to the abundance of these fibers in some species, the term “Sharpey fiber bone” has been applied⁴³. Additionally, the presence of micron-sized tubules (termed “canals of Williamson”) within anosteocytic bone has led to some debate as to their content, whether cell processes (osteoblasts, cells of Williamson), hypomineralized collagen, or fluid^{19, 44}.

In endochondral ossification, cartilage may be resorbed along a complete front, often without a spongiosa (zebrafish and medaka); adipose tissue fills the space^{23, 30, 45}. In long bones, epiphysis-like maturation gradients may exist in the cartilage at either end, as well as in the diaphysis, where a bi-directional gradient can occur^{46, 47}. The removal of the cartilage may be incomplete, and cartilage may persist. Enlargement of bone cavities occurs by endosteal resorption and periosteal deposition. In bones that do not develop a central cavity, the cartilage model proceeds through a transition stage, where the matrix loses histochemical features of cartilage and assumes bone morphology and staining. The chondrocytes themselves also exhibit alterations with loss of collagen II production and upregulation of collagen X, osteonectin, and collagen I³⁶.

Osteoblasts can assume different morphologies depending on their level of activity. They may be pyriform, spindled, or cuboidal and can be indistinguishable from mononuclear osteoclasts³⁰. Actively secreting osteoblasts exhibit polarization with increased basophilia and are positive for alkaline phosphatase⁴².

Osteoclasts in fish may either be large multinucleated cells, such as those seen in mammals, or small mononuclear cells; both can, but do not necessarily have, ruffled borders. In medaka, osteoclasts in the early stages of resorption are mononucleated, with multinucleated forms appearing secondarily⁴⁸. In zebrafish, the appearance of osteoclast types depends on age and the bone studied²². Therefore, proper visualization of remodelling activity may require staining of tissues for tartrate-resistant acid phosphatase (TRAP).

Preosteoclasts also express TRAP, while osteoblasts express alkaline phosphatase. Osteoclasts are able to resorb calcified cartilage (chondroclasts) and uncalcified tissues associated with bone resorption areas^{49, 50}. Mononuclear osteoclasts are often flat/squamous, similar to other bone lining cells. In this manner, such osteoclasts form a smooth, non-lacunar type of bone resorption, and such cells may predominate in the scales. Aside from osteoclasts, bone mineral resorption may occur by osteocytic osteolysis and halastasis (removal of calcium phosphate without matrix lysis)^{1, 51,52,53}.

The regulation of calcium in teleosts is primarily through the gill, secondarily in the intestine, and a minor component in the opercular epithelium. The kidneys maintain internal calcium levels through excretion of excess. Hormones involved in calcium homeostasis include stanniocalcin, calcitonin, estrogen, melatonin, calcitriol, retinoic acid, cortisol, growth hormone, prolactin, parathyroid hormone and parathyroid hormone related protein (PTHrP). Fibroblast growth factor 23 in fish may function in a similar manner to mammals^{1, 51,52,53,54,55,56,57,58}. Stress, circadian rhythms, vitellogenesis, smolting (the physiological and anatomical metamorphic adaptations observed with changes in environmental salinity), and osmoregulation may lead to changes in these hormones, effectively suppressing osteoblast or osteoclast activity^{1, 59}.

Calcium deficiency is uncommon in teleosts, and the role of parathormone (PTH) in fish is uncertain. A PTHrP is present in some species which may function as a hypercalcemic factor, counteracting the effects of stanniocalcin. In teleosts, dietary phosphorus is a requirement; thus, inadequate phosphorus intake or starvation may lead to insufficient bone mineralization or resorption⁶⁰. Phosphorus uptake in fish, like mammals, is vitamin D dependent. Vitamin D is stored in the liver of fish.

Vertebrae form around the notochord, which is a mesodermally derived rod of large cartilage-like cells, constrained by a thick sheath, situated between the spinal cord and coelom; it serves as the major skeletal element for locomotion as well as for developmental fate information⁶¹. In elasmonbranchs the notochord persists throughout life. In teleosts it is retained in a reduced form, largely between vertebra, and functions like the mammalian nucleus pulposus. A single layer of chordoblasts, in a stack-of-coins formation, divides into chordocytes which then enlarge and become vacuolated⁶². Vacuolar content is constrained by thick basal laminar sheaths, which allow cells to exert pressure on the tissue walls⁶³. Notochord cells throughout are joined together by junctional complexes, contain keratin, and secrete a sparse chondroid matrix⁴. The developed notochord is delimited by a complex sheath rich in type II collagen, proteoglycans and elastin. Along the inner aspect of this sheath, a single layer of non- vacuolated chordoblasts (notochord epithelium) surrounds a core of vacuolated (physaliforous) chordocytes (Figs. 12.3, 12.4, 12.5, and 12.6). The innermost layer of the sheath is the basal lamina, which is surrounded by a thick fibrous collagen sheath, then a thin external elastin layer, and finally an outer connective tissue sheath with fibroblasts (Fig. 12.6)⁶⁴. The notochordal sheath is the first structure to mineralize in developing fish.

Fig. 12.3.

Channel catfish, juvenile. Vertebra, notochord. Normal. Black arrow: chordacentral bone. Blue arrow: notochord. Green arrow: intervertebral region filled with vacuolated chordocytes, some of which lose their vacuoles, developing into intervertebral septa and central notochord strands. H&E, 100×. Image courtesy Wes Baumgartner.

Fig. 12.4.

Largemouth bass, juvenile. Vertebra, notochord. Normal. Green arrow: notochord lacuna that forms as a result of chordocyte loss; it is filled with fluid and is lined by unvacuolated chordocytes. This cavity atrophies with maturation. Black arrow: Fibrous sheath, adjacent to the layer of cuboidal chordoblasts. H&E, 100×. Image courtesy Wes Baumgartner.

Fig. 12.5.

Largemouth bass, juvenile. Vertebrae. Normal endplate, sagittal section. Green arrow: endplate bone. Black arrow: osteogenic zone. Blue arrow: Intervertebral ligament with an outer pink internal periosteal ligament and inner (dark pink) elastic membrane. H&E, 200×. Image courtesy Wes Baumgartner.

Fig. 12.6.

Largemouth bass, juvenile. Vertebra, notochord margin, transverse. Normal. Blue arrow: cuboidal layer of chordoblasts, adjacent vacuolated chordocytes. Black arrow: fibrous sheath, which is sandwiched between the internal elastic membrane at the junction of the chordoblasts, and external elastic membrane where the sheath meets pink bone (membranes are inapparent with H&E stain). Green arrow: osteogenic layer. H&E, 400×. Image courtesy Wes Baumgartner.

Three mesenchymal components converge to produce the vertebral body in teleosts; the chordacentra, arcocentra, and autocentra (Fig. 12.7; detailed below). In the initial phase of development, the intervertebral regions thicken and the notochord fibrous sheath that becomes the vertebrae mineralizes and expands circumferentially, forming a series of rings (chordal centers, or chordacentra) along the notochord (Figs. 12.8 and 12.9). The cells responsible for sheath mineralization, and therefore chordacentra formation, are not clearly recognized. The chordoblasts in Atlantic salmon express alkaline phosphatase (ALP) at the time of sheath mineralization, similar to pre-osteoblasts and osteoblasts, suggesting that chordacentra formation is related to chordoblast function⁶⁵. However, in the medaka and zebrafish, ALP expression was found only in the perichondral osteoblasts, suggesting a different method of chordacentra formation⁶⁶. In some species it is reported that the chordacentra are transitory structures that degenerate during development⁶⁷. The intervertebral portions of the sheath then thicken and remain unmineralized, forming the internal intervertebral ligaments (notochord sheath and outer elastic membrane) which are lined internally by the internal elastic membrane (in some species) and chordoblasts. Subsequently, a continuous layer of collagen (external collagenous ligament) is formed by the fibroblasts surrounding the sheath⁶⁸. Thus, the growth plate zone is composed of several layers, from internal to external: chordoblasts, intervertebral ligament/notochord sheath, an external elastic membrane, an external intervertebral ligament (with an external layer of fibroblasts) delimited by the osteogenic zone/bone rims, a layer of osteoblasts abutting the bone, and an investment of loose vascular connective tissue⁶⁹.

Fig. 12.7.

Diagram types of centrum formation in actinopterygians. A: Cartilage elements: arcualia (no centra). B: Dorsal and ventral arcocentra. C: Dorsal and ventral arcocentra and holocentrum including chordacentrum. D: Dorsal and ventral arcocentra and chordacentrum . E: Dorsal plus ventral arcocentrum and chordacentrum. F: Dorsal and ventral arcocentra, chordacentrum, and thin autocentrum. G: Dorsal and ventral arcocentra, chordacentrum, and well developed autocentrum. H: Dorsal and ventral arcocentra plus autocentrum. Abbreviations: AUC: autocentrum; BD: basidorsal arcuale; BV: basiventral arcuale; CHC: chordacentrum; DARC: dorsal arcocentrum; DARC+AUC+VARC: fused dorsal arcocentra, autocentrum, and ventral arcocentra; DARC+VARC: fused dorsal and ventral arcocentra; FSH: fibrous sheath of notochord; NC.C: canal for spinal cord; NO: notochord; NS: neural spine; VARC: ventral arcocentrum. Used by permission, from Arratia et.al, 2001, Wiley⁷¹.

Fig. 12.8.

Development of the larger structures of the notochord and the vertebral joints. Schematic summary of mid‐sagittal sections, drawn to different scales. (A) Early embryos (up to ca. 170 d°) have disc‐shaped chordocytes, with each cell spanning the diameter of the notochord. (B) In late embryos, up to 700 d°, the chordocytes become spherical, with no single cell spanning the notochord. (C) Newly hatched alevin have a segmented notochord with vacuolated chordocytes. (D) Parr have a notochord with prominent extracellular spaces, lacunae, and mainly dense chordocytes forming a longitudinal notochordal strand and transverse septa. (E) Smolt have a prominent longitudinal strand and transverse septa, and early formation of large vacuolated chordocytes close to the intervertebral ligaments. (F) Adults have a notochord consisting mainly of large vacuolated cells, while the strand, septa, and lacunae are insignificant. The notochord in Atlantic salmon (Salmo salar L.) undergoes profound morphological and mechanical changes during development. Used by permission from Wiley⁶³.

Fig. 12.9.

Schematic views of different stages in the development of the notochord and the vertebral bodies. The chordoblast layer of the notochord is in green, while the segmental zones that develop within the chordoblast layer are shown in yellow and orange. The chordocyte core is shown in light green. The notochordal sheath is in light blue, and the chordacentra that develop within the sheath are shown in red. The sclerotomal bone is in dark blue and the sclerotomal part of the intervertebral ligament is coloured brown. The drawings show the chordoblast layer, the notochordal sheath, neural and haemal arches and sclerotomal bone. A. Unsegmented notochord. B Segmentation of the chordoblast layer showing a dorsad-moving wedge-shaped wave of chordoblasts with a new arrangement. Yellow zone indicates chordoblasts with an orientation perpendicular to the cranio-caudal axis. The orange zone indicate bands of cuboidal chordoblasts at the center of the vertebral segments. Chordacentra (red) develop in the notochordal sheath adjacent to the rearranged chordoblasts. C Fully differentiated notochord with complete segmentation of the chordoblast layer and fully developed chordacentra. D Later stage with bone of sclerotomal origin (dark blue) external to and deposited around the chordacentra. E Lateral view of the vertebral column with its main segmental components. Vertical lines indicate how the primary segmental elements of the notochord relate to the definite vertebral and intervertebral regions, respectively. With permission, from Grotmol et al, 2003, Springer¹⁹⁵.

At the same time, paired cartilages (arcualia), both dorsal and ventral to the notochord, fuse to one another and to the vertebra, forming the neural and hemal arches of the spine, respectively. The basal portions of the arcualia rest on the vertebra or are incorporated into it (arch centers, or archocentra). The arcualia undergo endochondral ossification. The third component (auto centers, or autocentra) is membranous bone that develops outside the collagenous investment of the chordacentra^{26, 70}. Studies in medaka have reported conflicting results^{66, 71, 72}. Osteoblasts deposit osteoid, which is modelled into lamellar bone along the anterior and posterior endplate rims⁶⁸. When combined with the remnants of the arcualia, the autocentra may developed elaborate ornamentations (maltese cross appearance), particularly when viewed in cross section.

Vertebral growth occurs at the anterior and posterior endplates in progressively wider rings, forming an hourglass/biconoid shape (amphicoelous) with notochord tissue between. The outer rim of the autocentra is the growth zone; chordacentra do not grow appreciably and are subsequently incarcerated within the vertebra^{67, 73}. Within each vertebra, the two cones are composed of compact bone while the remaining autocentra bony tissues are cancellous. Osteoclasts are not necessary for vertebral development since mineralization occurs through the notochord sheath and intervertebral ligament. As such, remnants of the notochord are retained between vertebral bodies.

Within the notochord tissue of salmon there may be remarkable reorganization as the fish develop, with the formation of large toroid chambers filled with clear content within the central axis, which are interrupted by central strands and intervertebral septa⁶². In this process, the cells lose their vacuoles and accumulate intermediate filaments. Remarkably, at sexual maturity these changes disappear and the chordocytes become secondarily vacuolated, much as before. In this process, chordocytes progress through various morphologies, including squamous, acanthocytic, finely vacuolated, and large vacuolated forms. Such changes can be seen in other fish species; however, they have not been investigated.

Alterations in the vertebrae are commonly seen; exercise can increase bone formation and vertebral length significantly⁷³. In aging fish, vertebral body resorption may be increased. Spinal deformities are commonly encountered in fish, particularly salmon, and are due to varied causes⁷⁴. Vertebrae may be compressed, enlarged, fused, or dysplastic due to a combination of increased or decreased bone, collagen, cartilage, and mineral. Changes are also evident in the intervertebral discs. For further comment and references, see ‘Deformation, Bone and Notochord’ and ‘Joint, Degenerative Joint Disease’, ‘Bone, decreased’, ‘Bone, increased’, and ‘Metaplasia’ sections. Under pathological conditions, the vertebral bone or notochord tissues may exhibit cartilage metaplasia^{26, 75}.

2. Fins

Fish fins are skin folds supported by endoskeletal endochondral bone, and rays that derive from the dermis. Most fish have two sets of paired fins, the pectoral and pelvic, and three median fins⁷⁶. The paired fins are attached to the pectoral and pelvic girdles, which are endoskeletal and do not regenerate. Fin rays (exoskeleton) are composed of either segmented (soft rays) or fused (spines) bone. Each ray, or lepidotrich, is composed of two symmetrical bony halves (hemitrichs), which are fused to varying degrees by ligaments; they are present in all bony fish and are derived from modified scales.

Soft ray hemitrichs are composed of multiple uniform bony segments with intervening fibrous joints, allowing flexibility. Between each hemitrich pair is a layer of connective tissue, vessels, and nerves. When injured, scleroblasts associated with the epithelium synthesize the osteoid for bone production. Lepidotrichia regenerate from proximal to distal, successively adding new segments. Actinotrichia are fine cords of connective tissue that attach to the hemitrichs, generally at the distal tips; they are made of collagen-like elastoidin⁷⁷. For many years the scales and fin rays of fish have been regarded as neural crest derivatives. Recently however it has been demonstrated that these tissues have a mesodermal origin in the medaka and zebrafish⁷⁸. Methods for studying fin and scale regeneration are published⁷⁹.

In broader context, the homology between skeletal elements (bone, cartilage, fins, and scales) and teeth in fish may have bearing on the interpretation of chondro-osseous elements found in cases of chronic injury, as well as in sarcomas of the jaw, fins, and skin.

Table 1 shows nomenclature, diagnostic criteria and differential diagnosis for bone and notochord.

Table 1. Nomenclature, diagnostic criteria and differential diagnosis of bone and notochord.

Bone & Notochord			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Congenital non-proliferative
	*Ectopic cartilage			X
	*Malformation		X
	Non-proliferative
		Apoptosis		X
		Cyst, bone				X
		*Bone, decreased		X
		*Bone, increased		X
		*Degeneration	X
		*Deformation	X
		*Eroded surface, increased	X
		Fibro-osseous lesion				X
		Fibrous osteodystrophy				X
		*Fracture		X
		*Fungus		X
		Infiltrate [insert predominant cell type]^	X
		*Inflammation	X
		*Metaplasia		X
		*Necrosis		X
		*Osteoblastic surface, increased	X
		*Osteoclasts, increased		X
		*Osteoid, increased	X
		*Parasite		X
Proliferative
	Non-neoplastic
		*Hyperplasia		X
	Neoplastic
		*Chordoma		X
		*Myxosarcoma		X
		Osteoblastoma				X
		Osteofibroma				X
		Osteogenic fibrosarcoma				X
		*Osteoma		X
		*Osteochondroma		X
		*Osteosarcoma		X
		*Teratoma, extragonadal, bone		X

*Terminology with diagnostic criteria or comment described in the main text.

^ See hematopoietic chapter for detailed description.

Bone – Congenital/Developmental Lesions

Congenital lesions involving piscine skeletal tissues are not frequently observed in small fish species used in toxicological studies but are well known in salmonids. However, zebrafish exposed to TCDD developed craniofacial deformities, in addition to other system deformities⁸⁰. Malformations occur with low prevalence and are attributed to genetic factors, hypoxic episodes, nutritional imbalances, rearing density, infections, trauma, and many more^{16, 81}. Mortality in deformed fry is high. In general, skeletal anomalies are conspicuous; vertebral anomalies are well described. Vertebral malformations have been observed in zebrafish⁸².

Ectopic cartilage, bone

Ectopic cartilage can be a feature of growth abnormalities in vertebrae, and depending on the cause, may be found in the intervertebral space/notochord/growth ring, or in the spongy bone that forms the body of the centrum^{26, 69}.

Malformation, bone

(Fig. 12.10)

Fig. 12.10.

Channel catfish. Vertebra (transverse section). Vertebral malformation/dysplasia. Notochord tissues are fibrotic, ragged, poorly cellular, and vacuolated (circle). There is necrosis of chordoblasts and chordocytes. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

Developmental alterations in the shape of bones have been observed in numerous species due to genetic disorders (spontaneous or modified genetic strains), early nutritional deficiencies or excesses (e.g. vitamin E deficiency in catfish), infections, toxins, xenobiotics or other causes. Ectopic cartilage may be a feature of growth abnormalities in vertebrae, and depending on the cause, may be found in the intervertebral space/notochord/growth ring, or in the spongy bone that forms the body of the centrum ^26,69. The persistence of cartilage in the vertebral endplates may lead to restricted elongation, and subsequent fusion. Caution should be applied in the interpretation of either deformation or malformation as chordocytes may exhibit marked variation in morphology as a consequence of normal development⁶². Additional references are also available for those wishing to delve more deeply into the subject^{11, 83,84,85,86,87,88}.

Congenital or developmental bone malformations should be carefully distinguished from induced findings by appropriate clinical history and signalment.

Bone – Non-Proliferative

Bone, decreased – Similar to rodent

(Fig. 12.11)

Fig. 12.11.

Species unknown. Cranium (transverse section). Bone, decreased, affecting both cortex and trabeculae. Note there is generalized loss of bone thickness. Decalcified section. H&E, 400×. Image courtesy Wes Baumgartner.

Comments

Decreased bone results from imbalance in formation and resorption, with a relative increase in resorption. The terms osteopenia and osteoporosis are not recommended as qualitative diagnoses; these terms should be reserved for quantitative assessments. Morphometric/densitometric data may be required to detect subtle decreases in bone mass. A variety of zebrafish mutants germane to osteoporosis have been published⁷⁹. In addition, restricted mobility has been reported to induce osteopenia in zebrafish⁸⁹.

Interestingly, in studies using post smolt salmon to generate a primary dietary phosphorus deficiency, it was found that while bone and scale mineral decreased by 50% (homogeneous osteomalacia), no appreciable changes were seen histologically. Bone matrix formation was uninterrupted, and bone/scale resorption were not evident despite the lack of mineral. Thus, bone growth and mineralization in fish may not be linked, at least in some species^26,60. Spawning migration in Atlantic salmon is associated with halastasy (diffuse demineralization without degradation of bone matrix), which may be more common than previously appreciated⁵².

Calcium deprivation and repletion in tilapia has been associated with decreased bone⁹⁰. As a function of delayed mineralization, decreased bone was seen in haddock subjected to phosphorus deficiency⁹¹. Zebrafish typically exhibit reduced vertebral bone mass as a result of aging⁹².

Bone, increased

(Fig. 12.12)

Fig. 12.12.

Gizzard shad. Operculum. Bone, increased, nodular. Characterized by few osteocytes. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Modifiers/Locator

• Trabecular

• Cortex

• Nodular (Fig. 12.12). Increased nodular bone may be appropriate where focal lesions are observed macroscopically or microscopically

• Locator modifiers can be applied to reflect the extent and/or pattern of this finding, which may variably affect all bones, ligaments, tendons, soft tissues around joints, muscle

Other Terms

• Nodular hyperostosis, ossicles, osteophyte (nodular bone growth at diarthrodial joint margins), enthesophyte (ossification of tendon/ligament), exostosis (nodular bone growth from bone surfaces), ectopic bone, heterotopic bone

Pathogenesis/Cell of Origin

• Non-neoplastic focal or multifocal augmentation of bone mass, intraskeletal or extraskeletal

• Increased bone matrix production by well differentiated osteoblasts

• Due to a modeling-based process (formation independent of resorption)

Diagnostic Features

• Increased bone matrix/surfaces in the periosteum, within the bone, joint cartilage, or within soft tissues

• Increased, well differentiated osteoblasts in single layers on bone surfaces with lack of atypia and may exhibit evidence of remodeling

• A general regular pattern to bone trabeculae. May exhibit growth. Various bone forms are observed

○ Fine cancellous spongy bone

○ Very dense to osteonal bone

○ Hollow ossicles/spheres of chondroid and lamellar bone (parasite-induced)

Differential Diagnoses

• Fracture/callus

○ Focal area of bone discontinuity may be evident

○ Large fibroblasts in collagenous stroma

○ Mineralization or ossification of injured stroma or inflammation may be present

• Fibroma

○ Fibroblast rich neoplasm

○ May have abundant secondary bone production (not derived directly from neoplastic cells) or mineralization

• Osteoma

○ Tumorous mass of mature bone produced by neoplastic cells

○ Progressive growth

○ No cellular atypia, low mitotic rate

• Osteosarcoma

○ Densely cellular, atypical cells that produce osteoid

○ Immature bone matrix

Comments

As in mammals, the formative and resorptive aspects of bone metabolism are coupled in fish. Situations that cause increased mineral demands, and therefore bone metabolism (acidosis for example), may lead to increased osteoblast and osteoclast activity. As such, the stimulation of bone resorption may in the end paradoxically lead to increased bone volume²⁴. Increased bone in fish, nodular or otherwise, can be associated with various etiologies to include trauma and parasites.

It is helpful to differentiate diffuse increased bone from focal, nodular increased bone as there can be different causes of each manifestation.

Bony growths may arise from bone surfaces for various reasons, as a result of disruption of the periosteum. Trauma or infection over time may lead to bone production within a robust fibrotic inflammatory response; such cases are best termed “fracture” (above), “fibrosis”, or “fibroplasia” (see Soft Tissue document) when appropriate. Within such areas there is often a general regularity to the bone trabeculae, with an observable progression or variation in osteogenic maturity that suggests a reactive condition. Inflammation, whether an inciting cause or secondary, complicates diagnosis. Within any bony mass, diligent search for a primary cause is necessary. A fungal infection was reported in association with such growths⁹³.

Bony protuberances composed of mature bone (hyperostosis, exostosis) predictably affecting specific neural and hemal spines are common and well known, affecting many wild species⁹⁴. Hyperostotic nodules are often densely mineralized and can replace/expand existing bone, rather than emanating from surfaces⁹⁵. The etiology is obscure, and various hypotheses have been given^{96, 97}. They may be a physiological response; however, they are not seen in small laboratory species.

Log perch (Perca caprodes) infected with the myxozoan Myxobolus scleroperca develop plasmodia within the cartilages of the skull, which become incarcerated by bone nodules⁹⁸. Yellow perch (Perca flavescens) infected with Apophallus brevis metacerariae develop intramuscular/soft tissue bony ossicles around the parasite, as a part of its life cycle^{99, 100}.

Degeneration, bone – Similar to rodents

(Figs. 12.10 and 12.13)

Fig. 12.13.

Zebrafish. Vertebra. Degeneration, notochord. Note the hyperplastic chordoblasts with vacuolation along the inner bone surface (black arrow). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

May affect any bone or the notochord in growth or development.

Deformation, bone

(Fig. 12.14)

Fig. 12.14.

Killifish. Vertebra. Deformation. Decalcified section. H&E. Image courtesy of Rani Sellers.

Modifier/Locator

Vertebra, bone, notochord

Other Terms

• Scoliosis, kyphosis, lordosis, vertebral fusion, ankylosis, humpback, hunchback, short fish

Pathogenesis/Cell of Origin

• Alteration of bone or vertebral bodies with abnormal notochordal and bony elements, in excess or deficiency from normal

Pathogenesis

• Varied stressors and age-related changes lead to abnormal alterations in growth plate, bone, or notochord tissues that result in complex deformations

Diagnostic Features

• Vertebrae may be short, enlarged, fused, irregular, or displaced with irregular or flattened endplates

• A combination of increased or decreased bone, collagen, cartilage, notochord, and mineral in vertebral bodies and/or joints

• Trabecular bone remodeling and osteophytic changes can be marked

• Vertebral joints may be irregular, compressed, mineralized, ossified, fibrotic, or exhibit chondroid metaplasia

• Osteogenic zone orientation changes

• The ligaments of the joint become irregular, split, or are lost

• Notochord tissues (chordocytes) may exhibit chondroid metaplasia, fibrosis, mineralization, ossification, or extrusion through joint spaces

• Chordoblasts may become hyperplastic, vacuolated or necrotic

• Vertebral trabecular cartilage/chondroid bone matrix becomes altered and chondrocyte numbers change, often with loss and necrosis

• Inflammation may be evident in the perivertebral stroma

• Non-vertebral bones that are deformed share many of the features described for vertebrae

Differential Diagnosis

• Dysplasia, bone. This term, which may be observed in published literature can have many similar diagnostic features but should be avoided due to potential connotations of pre-neoplastic proliferation

• Malformation, bone. This term should be applied when alterations in normal bone morphology are associated with congenital or developmental events, in contrast to deformation that may reflect varied stressors and age-related changes as well as infection, trauma and/or inflammation

Comments

Vertebral deformation is a commonly encountered entity and is well described in salmonids, Senegalese sole, and zebrafish^{101, 102}. Increased or decreased bone, in association with vertebral abnormalities (scoliosis, lordosis, kyphosis), has been described in association with aging, parasitic infections, bacterial infections, low pH, abnormal temperatures, viruses, dietary imbalances (especially phosphorus and vitamin C), heavy metal toxicosis, trifluralin, dibutylphthalate, toxaphene, polychlorinated biphenyls (PCBs), malathion, chlorpyrifos, and trichlorfon exposure, among others^{74, 103,104,105,106,107}. Often the pathologic features in dysplastic vertebrae affect the growth plate rims/articular surfaces, but this is not always the case; phytic acid dietary supplementation led to a proliferation of bone trabeculae and cartilage in the central part of the vertebral bodies^{108, 109}. Adaptive responses in vertebral disease are often associated with chondroid bone formation, which is further elaborated upon under ‘metaplasia’.

Transdifferentiation of bone or notochord into chondroid bone or cartilage has been described in fish, both in normal development and as part of pathologic processes, particularly vertebral dysplasia^{43, 75, 76, 101, 109}. Abnormal compression loads, instability or impaired vascularization can produce cartilage within bone. Ectopic cartilage is a feature of growth abnormalities in vertebrae, and depending on the cause, may be found in the intervertebral space/notochord/growth ring, or in the spongy bone that forms the body of the centrum^{26, 69}. The persistence of cartilage in the vertebral endplates may lead to restricted elongation, and subsequent fusion. Finally, it should be recognized that chordocytes may exhibit marked variation in morphology as a consequence of normal development⁶². The following papers, in addition to those listed above, are useful references^{11, 83,84,85,86,87,88}.

Eroded surface, increased, bone – Similar to rodent

(Fig. 12.15)

Fig. 12.15.

Koi. Operculum. Eroded surface, increased, bone. Note irregular depressions on the bone surfaces with plump osteoblasts. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

As in mammals, the formative and resorptive aspects of bone metabolism are coupled in fish. Situations that cause increased mineral demands, and therefore bone metabolism (acidosis for example), lead to increased osteoblast and osteoclast activity. Calcium deprivation in tilapia has been associated with increased eroded surfaces¹¹⁰.

Fracture, bone – Similar to rodent

(Figs. 12.16, 12.17, 12.18, and 12.19)

Fig. 12.16.

Channel catfish. Gill. Fracture, gill filament with displacement, cartilage necrosis, and inflammation in association with myxozoan infection. An example of Proliferative Gill Disease (PGD). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Fig. 12.17.

Species unknown. Vertebra. Fracture with callus formation (black star). Note as well bone remodeling with decreased bone (black arrows). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Fig. 12.18.

Channel catfish. Gill. Fracture, gill filament, with callus formation following previous cartilage necrosis (myxozoan infection) with epithelial inflammation in association with myxozoan infection. An example of Proliferative Gill Disease (PGD). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Fig. 12.19.

Channel catfish. Gill. Fracture, gill filament, with increased chondrocytes, osteoid, and inflammation associated with myxozoan infection. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

Tissue response to endoskeletal bone fracture in fish is similar to that in mammals. The features of fractures are influenced by the mechanical and metabolic factors placed on the tissue, and species studied. The presence of secondary cartilage (chondrocytes derived from the periosteal cells of bone) as a feature in fracture healing may vary, particularly in membranous or dermal bone^{28, 111}. Depending on the temporal proximity to fracture occurrence, a diagnosis of ‘fracture/callus’ may be used.

Comparison of fractures in an anosteocytic cichlid (Sarotherodon melanotheron) and a cyprinid (Carassius auratus) with cellular bone found that, under calcium deficient conditions the cyprinid (cellular bone) could mineralize the callus, while the anosteocytic cichlid could not¹¹². Under normal conditions, the cyprinid formed a larger callus in a shorter amount of time compared to the cichlid. Fractured fin rays have a high capacity for regeneration.

An obvious focal distortion in the bone is the hallmark of a historical fracture. Fracture disrupts the blood supply, and bone necrosis is frequently present. Fractures in housed fish may be rather common, particularly on fins and the snout/jaw. A complication is a nonunion, where the tissues fail to ossify. Such lesions are characterized by an irregular growth of otherwise reactive bone, sometimes in a multilobular and seemingly haphazard pattern, all in a dense irregular fibrous stroma. It may be difficult if not impossible to discriminate this from true neoplasia. This is compounded by the fact that fish normally display a remarkable diversity in cartilage and bone tissue elements, resulting in non-neoplastic lesions that exhibit a wide range of skeletal tissue morphologies⁴.

Bone deformity and fracture have been reported in fish with vitamin C deficiency, heavy metal toxicosis, exposure to oxytetracycline antimicrobials, electrocution, and other etiologies^{74, 113, 114}. See sections for ‘Bone, increased, and Bone, decreased’ for further comment.

In broader context, the homology between skeletal elements (bone, cartilage, fins, and scales) and teeth in fish may have bearing on the interpretation of chondro-osseous elements found in cases of chronic injury, as well as in sarcomas of the jaw, fins, and skin.

Fungus, bone

(Fig. 12.20)

Fig. 12.20.

Zebrafish. Vertebra. Fungus, Pseudoloma neurophila infection of the endplate which is subsequently distorted. Decalcified section. H&E, 400×. Image courtesy Jan Spitsbergen.

Other Terms

• Infestation or infection by a variety fungal agents (e.g. microsporidian agents)

Diagnostic Features

• Presence of encysted or intraosseus fungi of various species, such as Pseudoloma neurophilia

• Irregular expansion and/or proliferation of the bone in a localized or nodular pattern

Differential Diagnosis

• Osteoma or osteosarcoma – neoplastic expansion or an invasive neoplasm without the presence of parasites

• Nodular bone formation (increased bone) increases in bone matrix in the absence of pathogens

Comments

A wide variety of organisms infect/infest fish bone inducing varying degrees of inflammation, and osteoid formation. Additionally, fungi can induce nodular expansion of the bone mimicking neoplasia or nodular bone formation. In zebrafish, this finding may be associated with Pseudoloma neurophilia spp. infection.

Inflammation, bone

Comments

Detailed discussion can be found in the hematopoietic system chapter. The term ‘osteomyelitis’ is not appropriate in fishes, since the hematopoietic tissues do not reside within bone, but within the kidneys. While in diagnostic and environmental studies ‘osteitis’ has been used, it is most appropriate to use ‘inflammation, bone’ in toxicity studies.

Metaplasia, bone

Other Terms

• Chondroid bone, fibrocartilaginous tissue

Pathogenesis/Cell of Origin

• A differentiated cell type transforms into another, with or without cell division, or dedifferentiation.

Diagnostic Features

• The presence of an alternative connective tissue compared to controls

• Intermediate forms, such as chondroid bone, may occur prominently

• Cells with osteoblast features may develop into chondrocytes, with territorial arrangements, cartilage matrix, lack cell processes, and exhibit features of cartilage matrix mineralization

• Fibrocartilage in the intervertebral spaces, notochord tissue, and growth rings of dysplastic vertebrae

• A mixture of chondroid bone and Sharpey fiber bone may develop

Differential Diagnoses

• Osteosarcoma: Abundant pleomorphic large mesenchymal cells producing osteoid

• Chondrosarcoma: Abundant pleomorphic chondrocytes producing cartilage matrix, and not osteoid

Comments

Precursor cells in intramembranous bone have the capacity to develop into osteogenic or chondrogenic cells^{115, 116}. Depending on the factors involved in bone development or reaction to injury, chondrocytic cells may develop from the osteoprogenitor cells within a bone matrix (chondroid bone- see Introduction section).

Transdifferentiation of bone or notochord into chondroid bone or cartilage have been described in fish, both in normal development and as part of pathologic processes, particularly vertebral dysplasia^{43, 75, 76, 101, 109}.

Abnormal compression loads, instability or impaired vascularization can produce cartilage within bone. Metaplastic (ectopic) cartilage is a feature of growth abnormalities in salmonid vertebrae, and depending on the cause, may be found in the intervertebral space/notochord/growth ring, or in the spongy bone that forms the body of the centrum²⁶. The persistence of cartilage in the vertebral endplates may lead to restricted elongation, and subsequent fusion.

Necrosis, bone – Similar to rodent

Comments

Bones in fish are normally acellular to poorly cellular, therefore use of empty lacunae as a criterion for necrosis can be challenging due to their scarcity. Necrosis occurs in conditions where blood supply to the bone is impaired. Trauma (fracture), inflammation (osteitis) and neoplasia are typical causes. Dead bone is brittle, predisposing it to fracture. The primary diagnosis in cases where lesions include necrosis, inflammation, and fracture should focus on the pre-eminent or initiating lesion.

Apoptosis (or any other cell death pathway term) should not be used as a diagnostic term unless definitive techniques can confirm this process (TEM, IHC). The term apoptosis/necrosis may be used if morphologic features of both necrosis and apoptosis are present in the same lesion¹¹⁷.

Osteoblastic surface, increased, bone – Similar to rodent

(Fig. 12.21)

Fig. 12.21.

Channel catfish. Gill. Osteoblastic surface, increased, gill arch. Note a thin layer of hyperplastic osteoblasts (black arrows). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

An increase in the osteoblast surface interpreted to be producing matrix can occur as a consequence of increased remodeling or modeling-based bone formation. Increased osteoblast surface area without a change in bone mass may occur in the acute response to skeletal loading. As in mammals, the formative and resorptive aspects of bone metabolism are coupled in fish. Situations that cause increased mineral demands, and therefore bone metabolism (acidosis for example), lead to increased osteoblast and osteoclast activity. Calcium deprivation and repletion in tilapia have been associated with increased osteoblast numbers⁹⁰.

Osteoclasts, increased, bone

(Fig. 12.22)

Fig. 12.22.

Brook trout. Mandible. Osteoclasts, increased. Multinucleate osteoclasts (black arrowheads) in irregular depressions (also called Howship’s lacunae) in mineralized cartilage lacunae. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Diagnostic Features

• Apparent increase in osteoclast numbers (hyperplasia)

• An irregular bone surface, with surface depressions representing osteoclast lacunae

• Mononuclear osteoclasts may be abundantly present, but may not produce resorption lacunae

Special Diagnostic Methods

• Typically biomarkers for osteoclasts are required to adequately detect them, particularly in those species with mononuclear osteoclasts. Many, but not all, fish have both mononuclear and multinuclear osteoclasts¹¹⁸

• Procedures to highlight osteoclasts in tissue sections include tartrate-resistant acid phosphatase (TRAP)

Comments

Osteoclasts are essential for bone modeling and healing. Osteoclasts in fish can be small mononuclear cells, indistinguishable from osteoblasts in some cases.

Increased osteoclasts can be associated with a variety of pathologic processes, including metabolic/hormonal disturbances, neoplasia, and prolonged osteoclast lifespan. The presence of increased osteoclasts does not necessarily indicate increased bone resorption.

As in mammals, the formative and resorptive aspects of bone metabolism are coupled in fish. Situations that cause increased mineral demands, and therefore bone metabolism (acidosis for example), lead to increased osteoblast and osteoclast activity. Zebrafish typically exhibit reduced vertebral bone mass as a result of aging⁹².

Increased osteoclasts have been seen in studies of phosphorus and calcium deficiency^{91, 110}.

Bone mineral resorption is not only mediated by osteoclasts; evidence for osteocyte resorption (referred to as osteocytic osteolysis) and generalized demineralization without matrix resorption (halastasis) have been reported-see introduction.

Osteoid, increased, bone – Similar to rodent

(Fig. 12.23)

Fig. 12.23.

Channel catfish. Gill. Osteoid increased, gill arch, with osteoblastic surface, increased. Undecalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

Osteoid is the unmineralized organic precursor matrix of bone composed of collagen, non-collagenous proteins (osteocalcin, osteonectin, osteopontin, others), and proteoglycans¹¹⁹. Numerous staining methods have been used to identify it in decalcified tissues, and none are fully satisfactory. In fish, bone (and presumably osteoid) may be composed of both type I and II collagens³².

Increased osteoid can be seen in metabolic diseases, especially those that lead to vertebral deformations (see sections for Bone, increased and Bone, decreased), increased mechanical stress, and fractures. Calcium deprivation and repletion in tilapia has been associated with increased osteoid⁹⁰. As a function of delayed mineralization, increased osteoid was seen in haddock subjected to phosphorus deficiency⁹¹.

In routine studies, a provisional diagnosis of increased osteoid can be made from decalcified H&E sections based on the presence of clearly demarcated zones of pale staining bone matrix associated with plump osteoblasts. Osteoid is finely fibrillar/amorphous, is intimately associated with or entraps osteoblasts, and becomes mineralized; features which differentiate it from fibrous stromal collagen. With trichrome stains in decalcified sections, osteoid seams will stain pale green or blue (when compared to to the red, more mature and presumably well mineralized bony tissues), adjoined by regimented osteoblasts situated between fine collagen bundles.

Osteoid is composed of collagen and is therefore birefringent (unlike fibrin). The use of picrosirius red collagen staining has been reported as a method to discriminate osteoid from bone by the relative quality of birefringence and collagen type¹²⁰. However, the ability of picrosirius red to differentiate types of collagen is disputed^{121, 122}.

Histochemical staining generally does not allow reliable differentiation of osteoid from fibrous stromal collagen when evaluating intercellular matrices in osteogenic neoplasia¹²³. However, methods have been published using human tissues that make a strong case for their discriminatory ability¹²⁴. Nevertheless, validation of such mammalian methods must be done for any fish species in question, since the staining characteristics of the various skeletal tissues are likely to be significantly different. In areas of active membranous bone formation, the detection of osteoid may be straightforward⁴³. Studies with undecalcified sections would be required to confirm the presence of osteoid. It is important to realize that fish, depending on circumstances, can have severe hypomineralization of bone without identifiable changes histologically²⁶. As such, the changes inherent to bone maturation that allow histochemical discrimination may not be present or may be altered in the population studied. Minor variations in the tinctorial qualities of the matrix in standard H&E sections (decalcified) should not be interpreted as definitive evidence of variations in osteoid.

Parasite, bone

Other Terms

• Infestation or infection by a variety of agents (i.e. crustaceans, nematodes, cestodes, digenea, myxozoans)

Diagnostic Features

• Presence of encysted or intraosseus parasites

• Irregular expansion and/or proliferation of the bone in a localized or nodular pattern

Differential Diagnosis

• Osteoma or osteosarcoma – neoplastic expansion or an invasive neoplasm without the presence of parasites

• Nodular bone formation (increased bone) – increases in bone matrix in the absence of parasites

Comments

A wide variety of organisms infect/infest fish bone inducing varying degrees of inflammation, and osteoid formation. Often parasites in bone elicit remarkably little inflammation until excystment or rupture of parasite-induced lesions; at that point it may be difficult to discriminate parasitism from other causes of inflammation, as the parasites may not be evident. Additionally, parasites can induce nodular expansion of the bone mimicking neoplasia or nodular bone formation. In zebrafish, this finding may be associated with microsporidia (Pseudoloma neurophilia spp.) infection which is recommended to record under Fungus. Whirling disease is associated with Myxozoan spp. infection in the calvaria of salmonids and a variety of other parasites in other species.

Bone – Proliferative Non-Neoplastic

Hyperplasia, osteoblast/chordoblast/chordocyte

(Fig. 12.13)

Modifer/Locator

• Osteoblast

• Chordoblast

• Chordocyte (Fig. 12.13)

Pathogenesis/Cell of Origin

• Non-neoplastic proliferation of osteoblasts, chordoblasts along intervertebral joints or chordocytes in areas of degeneration or repair

• Non-neoplastic proliferation of chordocytes in the intervertebral space

Diagnostic Features

Osteoblast

• Single or multiple proliferations of well differentiated plump osteoblasts along bone surfaces

• Occurs concurrently in bones with increased bone matrix

• Often occurs with evidence of osteoid production

• May fill inter-trabecular spaces, but does not disrupt architecture

• Hyperplastic osteoblasts may be admixed with fibroblasts/mesenchymal cells in collagenous matrix as part of a healing response

Chordoblast

• Increased numbers of chordoblasts along the basal lamina

• Chordoblasts may contain clear vacuoles

Chordocyte

• Increased numbers of cells in notochord tissues

• Cells may adopt various morphologies including fibroblastic, spindled, microvacuolated, and acanthocytic

Differential Diagnoses

Osteoblast

• Soft tissue fibroplasia: Well differentiated fibroblasts in collagenous stroma, within and around bone and an active repair process

• Osteoblastic surface, increased – a diffuse increase in the surface extent of osteoblasts

• Osteosarcoma: abundant pleomorphic large neoplastic mesenchymal cells producing osteoid with tissue disruption

Chordocyte

• Vertebral malformation/dysplasia – Chondroid metaplasia of notochord tissue; fibrosis and/or ossification of notochord tissue

Comments

Chordoblast or chordocyte hyperplasia of the bone may be observed in association with vertebral malformation/dysplasia (Fig. 12.13).

Chordocyte hyperplasia may be seen as an age-related change in zebrafish or as a consequence of vertebral deformities. Chordocytes can adopt a wide variety of morphologies ranging from fibroblastic, spindyloid, and acanthocytic throughout normal vertebral development⁶².

Bone – Proliferative Neoplastic

Chordoma, bone (M)

Pathogenesis/Cell of Origin

• Notochord mesenchymal cells

• Axial skeleton (vertebra), intestine, pancreas

Diagnostic Features

• Lobulated and well demarcated tumor with or without a fibrous capsule

• Irregular cells with distinct borders, single to many clear vacuoles, centralized nuclei (physaliforous or physaliferous cells)

• Signet ring cells- peripheralized nuclei with homogeneous cytoplasm- may be present

• Mucin matrix may be present

• Few if any mitoses

• Necrosis and bone spicules may be evident

• Can be invasive

• May occur in the intestine as well (see Gastrointestinal chapter for relevant images)

Special Diagnostic Techniques

• Chordomas can be distinguished by histochemistry or immunohistochemistry from other tumors. They are lipid negative (Oil-red-O, Sudan black), glycogen positive (Periodic acid-Schiff), keratin and vimentin positive, and S100 positive. Alcian blue and Toluidine blue demonstrate mucin.

Differential Diagnoses

• Hyperplasia, chordocyte: Small round cells that cluster within degenerate chordoid tissues (age related change), often associated with mineralization

• Notochord hamartoma: Presence of excess chordocytes in normal locations – may be categorized as Malformation

• Parachordoma: A myoepithelial tumor/soft tissue neoplasm not reported in fish

• Chondroma: Well differentiated chondrocyte neoplasm

• Liposarcoma: Mesenchymal neoplasm with abundant intracellular lipid vacuoles

• Myxosarcoma: Mesenchymal neoplasm with abundant extracellular mucin

Comments

Chordomas are rare neoplasms in vertebrates and have recently been described in zebrafish^{125, 126}. Medaka exposed to aminoproprionitrile developed spinal chordomas¹²⁷. They may occur in the vertebral column or in the intestine and tend to be broadly invasive but not metastatic, although involvement of the pancreas from intestinal occurrences has been reported.

Myxosarcoma, (M) bone

(Fig. 12.24)

Fig. 12.24.

Zebrafish. Vertebra. Myxosarcoma, malignant. Induced by 7,12-Dimethylbenz[a]anthracene (DMBA). Decalcified section. Alcian Blue stain. Image courtesy of Jan Spitsbergen.

Diagnostic Features

• Densely cellular mass

• Stellate mesenchymal cells

• Poorly demarcated, infiltrative

• Increased atypia and mitoses

• Abundant amorphous blue/myxomatous matrix in a network of collagen

Differential Diagnoses

• Myxoma: No cellular atypia, low mitotic rate

• Fibrosarcoma: Minimal to no myxomatous matrix

Comments

Uncommon tumor arising in bone. Reported in zebrafish¹²⁶.

Osteoma, (B) bone

(Fig. 12.25)

Fig. 12.25.

Species unknown. Gill. Osteoma (B). Decalcified section. H&E.

Other Terms

None

Pathogenesis/Cell of Origin

Benign neoplastic proliferation of mesenchymal cells with bone differentiation generally arising from the periosteal surface of bone

Differential Diagnoses

• Bone, increased, nodular: Reactive/physiological bone nodules distinguishable by the absence of neoplastic character, and location (depending on species)

• Osteochondroma: A mass of well differentiated bone and cartilage, with significant abundance and admixture of both elements

• Osteosarcoma: Densely cellular, atypical cells and immature bone matrix

Comments

The term osteoma has been used to describe many bone lesions that are not true neoplasms, particularly osseous growths that result from infection or trauma as well as heavily ossified dysplastic lesions. Osteomas are to be differentiated from Bone, increased, nodular (see above), which may not be possible in many cases. Diligent search of lesions for evidence of non-neoplastic etiology is necessary before diagnosis of osteoma can be made. They have been diagnosed in zebrafish, among others^{128, 129}.

Osteochondroma, bone

Pathogenesis/Cell of Origin

• Typical structures affected include skull, eye (sclera), gill cartilage

• A benign mixed mesenchymal tumor with prominent cartilaginous and osseous components

Diagnostic Features

• Lobulate well demarcated tumor with or without a fibrous capsule

• Few if any mitoses

• Tissue elements are well differentiated and appear hyperplastic

• Cartilage and bone are admixed

Differential Diagnoses

Bone, increased, nodular

• Reactive/physiological bone nodules distinguishable by the absence of neoplastic character, and location (depending on species)

Osteoma

• Tumorous mass of mature bone produced by neoplastic cells

• Progressive growth

• No cellular atypia, low mitotic rate

• Cartilage is a minor component and may be reactive, if present

Chondroma

• Tumorous mass of cartilage produced by neoplastic cells

• No pleomorphism, no features of malignancy

• Excessive disorganization

• Mineralized cartilage may be present

Osteosarcoma

• Neoplastic cells producing osteoid is an essential feature

• Abundant cartilage or chondroid bone (chondrocytic cells embedded in mineralized bony matrix)

Comments

The term ‘osteochondroma’ has been used in a few reports, denoting a tumorous mixture of cartilage and bone elements^{128, 130, 131}. This type of neoplasm has also been identified rarely as a spontaneous occurrence in zebrafish populations¹³². The term is problematic, as it is also a diagnosis in mammals that refers specifically to a hamartomatous proliferation of bone and cartilage that arises from bone surfaces, and forms by endochondral ossification. Given the plasticity of skeletal elements in fish, the presence of mixed tumors in fish is not surprising.

Osteosarcoma, (M) bone

Other Terms

• Osteogenic sarcoma

Affected Structures

• May arise from any bone (including scales- lepidosarcoma) or extra-skeletal sites

Pathogenesis/Cell of Origin

• A malignant primary bone tumor

Histogenesis

• Pluripotential mesenchymal cells, osteoblasts, osteocytes

Diagnostic Features

• Formation of osteoid by neoplastic mesenchymal cells is an essential feature

• Cells have increased mitotic figures

• Neoplastic cells fill in the spaces between bone

• Immature woven bone often formed

• Variation/mixture of collagen, cartilage, bone, myxomatous stroma

• Necrosis of associated normal bone

Differential Diagnoses

Osteoma

• No cellular atypia, low mitotic rate

Osteochondroma

• A mass of well differentiated bone and cartilage, with significant abundance and admixture of both elements

Bone, increased, nodular

• Mature well differentiated bone

Soft tissue fibroplasia/fibrosis

• Reactive bone formation as a result of injury to soft tissues, persistent inflammation

Mixed mesenchymal tumor

• Prominent admixture of more than one connective tissue type (see soft tissue document)

Chondrosarcoma

• Malignant cells do not form osteoid, only cartilage

• Bone is produced by endochondral ossification of tumor cartilage

Melanoma/Chromatophoroma

• Cells contain pigment or birefringent material consistent with pigmented/reflective neural crest cells.

Sarcoma, undifferentiated

• Pleomorphic cell type without differentiation

Lepidosarcoma

• Polygonal to spindled mesenchymal cells arranged in fascicles or storiform arrays to resemble scales. Small spicules may be present. Hard scale tissue may or may not be abundant (see Integument chapter)

Comments

The determination of osteoid being produced by the neoplastic cell population is the definitive feature of this neoplasm (see Comment under the Osteoid, increased section in this document). By convention, any sarcomatous neoplasm that produces osteoid/bone, regardless of other stromal types (cartilage, collagen, mucin, etc.) produced, is an osteosarcoma. Melanomas in mammals, on rare occasions, may produce bone. Non-neoplastic bone (woven or lamellar) may form around or within neoplasms of various types as well as inflammatory lesions, and this must be differentiated. Reactive bone trabeculae are interconnected, have a general pattern, and are lined by single layers of osteoblasts¹²³.

Various osteosarcoma subtypes are recognized in mammals, and cartilage or fibrous tissues are a common component (chondroblastic, fibroblastic osteosarcoma); however, subtyping of these tumors is not conducted in toxicity studies. The various presentations are not surprising, as the progenitor cells of bone can produce bone, cartilage, and fibrous tissue as a reparative response. In addition, the commonality of chondroid bone or mixed bone types in fish may lead to additional osteosarcoma subtypes specific to fish.

Bone tumors of scales (lepidocytomas/lepidosarcomas) are described in the Integument chapter. Reported tumors are single, lack metastasis, and do not arise from osteomas^{128, 133, 134}.

Teratoma, extragonadal, (B/M) bone

Comments

Extragonadal teratomas have been observed in bone of guppies and caudal fin of zebrafish¹³⁵.

3. Cartilage

Cartilaginous tissues figure prominently in the skeleton, not only during development, but as persistent tissues in the adult. The blastema that becomes cartilage begins as a condensation of mesenchymal cells that develop into chondroblasts and eventually become separated by secretion of extracellular matrix (differentiation). These blastemas may be very small and have very little matrix; this may be problematic, as fully differentiated cartilages in fish may look similar. Hyaline cartilages are generally similar to that seen in mammals and often merge with other cartilage types. A simplified classification system highlights the common hyaline cartilage modifications seen, which generally fall into the following: addition of matrix fibers (collagen, elastin), calcified cartilage, chondroid bone, and transformation/resorption (replacement/infiltration by adipose, endochondral bone formation, connective tissue metaplasia)^4,136. These tissue types can be recognized and discriminated from bone using H&E, trichrome, toluidine blue, PAS, Alcian blue, safranin O/fast green, and elastin stains^{4, 31}.

The term ‘chondroid’ has historically been used to describe tissue types which have properties of both of cartilage and fibrous tissue, but has also been more specifically used to describe hyaline-cell cartilage, which is composed of large ovoid cells separated by scant matrix (as defined in the classification system cited above; it is distinct from matrix-rich hyaline cartilage which is typical of mammalian hyaline cartilage)^{3, 5, 137}. The use of the term ‘chondroid’ as a tissue category should be avoided, as it is often used non-specifically as a descriptive term and more precise terms are available.

In some cases, hyaline cartilage exhibits a maturation gradient, producing a physis with resting, proliferating, and hypertrophied zones. Cartilage growth can occur at the periphery (perichondrium) as well as interstitially. Resorption of cartilage, developmentally, does not primarily occur by macrophage ingestion, but rather may involve transdifferentiation/metaplasia of cartilage into other connective tissues¹³⁸. The major collagen in fish is type II. Chondroid bone is described further in the Bone section of this chapter.

Endochondral ossification, in the mammalian sense, exists in teleosts (salmon, carp) but is generally absent from smaller species such as zebrafish and medaka. In these species the replacement of cartilage by bone occurs by the perichondral deposition of a bony collar. Chondrocytes can undergo hypertrophy, but the cartilage is removed from the diaphyseal body and is not replaced by bone. The resulting space (sometimes referred to as marrow spaces, although fish do not deposit hematopoietic marrow in bone, but rather in the kidneys) is filled with adipose. Physeal structures are encountered in various locations in some species (such as carp), resembling epiphyseal growth plates in mammals³¹. Recent investigations in experimental osteoarthritis in the jaw of zebrafish elucidate the regenerative capacity of cartilage^{8, 9}.

Calcified cartilage is a permanent feature in the skeleton of many species; hydroxyapatite is identified by the presence of globules of spheritic calcification (described in the Teeth section) with the matrix, forming concentric rings of Liesegang; groups may be further surrounded by waves of Liesegang¹³⁹. The calcified globules exhibit metachromasia with histochemical staining³⁷.

Mucochondroid is a special cartilage-type tissue in the skull that contains fibroblasts or hyaline cells in a mucinous matrix (mucous connective tissue)¹⁴⁰; it is described further in the Soft Tissues manuscript.

Table 2 shows nomenclature, diagnostic criteria and differential diagnosis of cartilage.

Table 2. Nomenclature, diagnostic criteria and differential diagnosis of cartilage.

Cartilage			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	*Altered matrix, cartilage			X
	Apoptosis			X
	*Cartilage, decreased			X
	*Cartilage, increased			X
	*Fracture			X
	Growth plate closed				X
	^Infiltrate [insert predominant cell type]			X
	^Inflammation			X
	*Necrosis			X
	Physis, decreased thickness				X
	Physis, increased thickness				X
Proliferative
	Non-neoplastic
		*Hyperplasia		X
	Neoplastic
		*Chondroma		X
		*Chondrosarcoma		X

Uncommon as a background finding; commonly induced.

*Terminology with diagnostic criteria or Comment described in the main text.

^ Detailed discussion can be found in the hematopoietic system chapter.

Cartilage – Non-Proliferative

Altered matrix, cartilage

(Fig. 12.26)

Fig. 12.26.

Channel catfish. Gill. Altered matrix, cartilage and increased, cartilage. Numerous disorganized chondrocytes are present in a pale amphophilic matrix. Decalcified section, H&E. Image courtesy Wes Baumgartner.

Other Terms

• Chondromucinous degeneration, chondroid metaplasia, fibrocartilaginous metaplasia, degenerative change, chondroid mineralization

• Degenerative/reactive altered matrix

• Metaplasia

Pathogenesis/Cell of Origin

• Non-neoplastic abnormal alterations in matrix fibers and/or ground substance, encompassing degenerative, reactive/regenerative, or metaplastic change

• Biochemical or physical change to the extracellular matrix as a result of chondrocyte dysfunction, modulation, or necrosis

Diagnostic Features

• Change in matrix fiber type or ground substance quality (loss of basophilia in H&E-stained sections, metachromasia in toluidine blue stained sections, or elastin fiber increase/decrease in aldehyde-fuchsin stained sections for instance)

• Matrix calcification may be reduced (when a normal condition) or increased (when not normally present)

• Chondrocytes may be disorganized, increased or decreased in number

• Extracellular matrix may be disorganized, increased, or decreased in amount

• Peripheral chondroblasts may appear in chondrones (proliferation)

Comments

Altered cartilage matrix may be used in situations with primary biochemical changes in the ground substance or altered chondrocyte function that lead to changes in the normal maintenance or development of the cartilage matrix. Such alterations can be associated with cartilage necrosis; however, changes may not be associated with necrosis and may develop for reasons unrelated to those that induce necrosis.

Connective tissues in fishes exhibit phenotypic continuity, and metaplastic change involving cartilage includes transdifferentiation into bone and fibrous tissue⁷⁶.

May be seen in conjunction with altered chondrocyte numbers (increased/decreased)¹⁴¹. Mineralization of matrix is not necessarily an abnormal change, depending on species/strain. Histochemical staining will be required to identify fine alterations in the matrix.

Cartilage, decreased, cartilage

Other Terms

• Cartilage atrophy

Pathogenesis/Cell of Origin

• Non-neoplastic reduction in cartilage mass

• Decreased cartilage matrix formation and/or increased cartilage resorption

Diagnostic Features

• Reduced cartilage matrix

Differential Diagnoses

• Altered matrix: Non-neoplastic change in matrix fiber type or ground substance quality

Comments

May be seen in conjunction with reduced chondrocyte numbers¹⁴¹. Histochemical staining may be required to identify fine alterations in the matrix.

Cartilage, increased, cartilage

(Fig. 12.27)

Fig. 12.27.

Channel catfish. Gill. Cartilage, increased, gill filament. Note large and poorly organized chondrocytes in abundant matrix. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Pathogenesis/Cell of Origin

• Non-neoplastic augmentation of cartilage mass

Pathogenesis

• Increased matrix production by chondroblasts and/or decreased resorption by chondroclasts

Diagnostic Features

• Increased cartilage matrix

• Chondrocytes may be hypertrophied

• Matrix calcification may be reduced (when normal) or increased (when not normally present)

• Chondrocytes may be disorganized, and increased or decreased in number

Differential Diagnoses

• Chondroma: Neoplastic mass of well differentiated chondrocytes producing chondroid matrix

• Chondrosarcoma: Abundant pleomorphic large mesenchymal cells producing cartilage

• Altered matrix, cartilage: A change to cartilage matrix characterized by abnormal matrix and chondrocyte appearance and disorganization

Comments

Cartilage retains a remarkable ability to proliferate and alter matrix characteristics, depending on circumstances. Cartilage may proliferate in a hyperplastic or dysplastic manner and can occur as a metaplastic response in various tissues, in some cases due to parasitism^142,143,144. Histochemical staining may be required to identify fine alterations in the matrix.

Fracture, cartilage

(Figs. 12.16, 12.17, and 12.18)

Pathogenesis/Cell of Origin

• Localized disruption of structure resulting in discontinuity

• Fractures develop secondary to trauma. Predisposing factors that lead to fracture susceptibility include defective matrix production, altered cartilage matrix with reduced material properties, neoplasia, or inflammation

Diagnostic Features

• Early stages are dominated by hemorrhage (from surrounding tissues), edema, fibrin, and necrosis

• Intermediate stages are characterized by increased mesenchymal cells with fibrous tissue, fibrocartilage, or dysplastic cartilage

• Late stages may exhibit varying degrees of hyperplasia and fibrosis, depending on species and cartilage type

Differential Diagnoses

• Chondroma: Neoplastic mass of well differentiated chondrocytes producing chondroid matrix

• Chondrosarcoma: Abundant pleomorphic large mesenchymal cells producing cartilage

• Altered matrix, cartilage: A change to cartilage matrix characterized by abnormal appearance, such as disorderly arrangement of matrix and/or cells

Comments

The features of fractures are influenced by the mechanical and metabolic factors placed on the tissue. An obvious focal distortion in the cartilage is the hallmark of a fracture. Fracture disrupts the blood supply to the area, and necrosis is frequently present. Healing of cartilage fractures in fish is not well understood. Developing cartilage seems to be much more amenable to repair; repair in adult poikilotherms may be poor or show minimal chondrogenic differentiation from the perichondrium^{111, 145}.

Fish normally display a remarkable diversity in cartilage and bone tissue elements, resulting in non-neoplastic lesions that exhibit a wide range of skeletal tissue morphologies⁴.

Necrosis, cartilage – Similar to rodent

(Fig. 12.28)

Fig. 12.28.

Channel catfish. Gill. Necrosis, cartilage, gill filament. Note this example of Proliferative Gill Disease (PGD) with inflammation in adjacent tissue. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Comments

Necrosis most often occurs in conditions where blood supply to cartilage is impaired. Trauma (fracture), inflammation (chondritis) and neoplasia are typical causes. Dead cartilage is brittle, predisposing it to fracture. The primary diagnosis in cases where lesions include necrosis, inflammation, and fracture should focus on the pre-eminent or initiating lesion.

Use of the term ‘necrosis’ or ‘apoptosis/necrosis’ can be applied in accordance with Elmore et al.¹¹⁷

Cartilage – Proliferative Non-Neoplastic

Hyperplasia, chondrocyte, cartilage

(Fig. 12.29)

Fig. 12.29.

Channel catfish. Gill. Hyperplasia, chondrocyte, gill filament. Finding characterized by large irregular chondrocytes with variable matrix staining. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Differential Diagnoses

Chondroma

• Excessive disorganization

• Increased cellular atypia

Comments

Chondrocyte hyperplasia commonly occurs in association with inflammation and trauma (healing responses).

Cartilage –Proliferative Neoplastic

Chondroma, cartilage (B)

Pathogenesis/Cell of Origin

• Benign primary cartilage tumor

• Chondroblasts, chondrocytes

Diagnostic Features

• Well circumscribed, expansile tumor of irregular mature cartilage

• Chondrocytes are disorderly, but well differentiated and arranged individually or in small groups within lacunae

• Minimal cellular and nuclear pleomorphism

• Mitoses are rare

• The matrix is amorphous, hyaline, and pale blue

• Occasional binucleate cells are present, but not multinucleate

• Matrix mineralization or secondary ossification may be present

• Ossification may be present, but changes are metaplastic and not convincingly formed from neoplastic osteoblasts

Differential Diagnoses

Chondrocyte hyperplasia

• Well differentiated chondrocytes

Chondrosarcoma

• Pleomorphic, binucleated, or multinucleated cells.

• Infiltrative growth

• Mitoses may be present

Fracture

• Hyperplastic chondrocytes, osteoblasts, or scleroblasts (bone-forming cells of the scale)

• Chondro-osseous matrix admixture

• Mineralized matrix, osteoid, and bone are present

Chordoma

• Vacuolated tumor cells with distinct cell borders and centralized nuclei (physaliferous cells)

Comments

Chondromas do not invade surrounding tissues. Mineralization or ossification of cartilage matrix as a secondary change should not be confused with osteoid production by neoplastic osteoblasts (osteosarcoma), but this is admittedly problematic in fish due to plasticity of skeletal tissue types. Chondromas have been associated with carcinogen exposure in zebrafish¹²⁹.

Chondrosarcoma, cartilage (M)

Pathogenesis/Cell of Origin

• A malignant primary cartilage tumor

• Pluripotential mesenchymal stem cells, chondroblasts, chondrocytes

Diagnostic Features

• Highly cellular, lobulated mass composed of cartilage

• Often well differentiated, with large cells and nuclei

• Occasional pleomorphic, binucleated, and multinucleated cells

• Increased mitoses

• Poorly differentiated tumors may have abundant spindle cells

• Matrix mineralization may be present

• Ossification may be present, but changes are metaplastic and not convincingly formed from neoplastic osteoblasts

Differential Diagnoses

Chondroma

• No pleomorphism, no features of malignancy

Osteosarcoma

• Neoplastic cells producing osteoid is an essential feature

• Abundant cartilage or chondroid bone (chondrocytic cells embedded in mineralized bony matrix)

Chordoma

• Vacuolated tumor cells with distinct cell borders and centralized nuclei (physaliferous cells)

Comments

The cartilage matrix within the mass may mineralize, and even secondarily ossify, particularly in areas of previous necrosis. Scrutiny of the mass for neoplastic production of osteoid or tumor bone is important; by convention such change warrants a diagnosis of osteosarcoma¹⁴⁶.

4. Joint

The prevailing assumption that ray-finned fish lack true synovial joints has recently been questioned, with support for their presence in the jaw and pectoral girdle of zebrafish. Indeed, fish species are in possession of any variety of joint types throughout the body. In many species of fish, cartilaginous joints consistent with that of typical synovial joints may be found, complete with stratified articular cartilage and synovial membranes⁷ (Fig. 12.2). In addition, fish have fibrous joints (syndesmoses) that are most apparent in the sutures of the cranium and remain patent throughout life¹⁴⁷.

Table 3 shows nomenclature, diagnostic criteria and differential diagnosis of joint. For description of vertebral joint degeneration, see Degeneration, bone in the Bone section above.

Table 3. Nomenclature, diagnostic criteria and differential diagnosis of joint.

Joint			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	Degeneration, chrondromucinous					X
	Degenerative joint disease			X
	Inflammation			X
Proliferative
	Non-neoplastic
		Hyperplasia		X
	Neoplastic
		Sarcoma, synovial			X

5. Tooth

Aside from edentulous species, fish characteristically have continuous life-long formation and replacement of teeth. They may be present throughout the mouth, on the branchial arches, and/or in the caudal pharynx¹. Teeth are present in a broad spectrum of forms, and bear similarities to integumentary structures (dermal skeleton) such as placoid scales and denticles (odontodes)². In most cases, teeth are peg-like or conical. Cypriniforms, including the zebrafish, lack oral teeth and have only pharyngeal teeth. The medaka has both an oral and pharyngeal dentition, with as many as 1000 erupted pharyngeal teeth alone¹⁴⁸. Oral and pharyngeal teeth also develop differently, in terms of germ layer contribution; oral teeth derive from the ectoderm, while pharyngeal teeth derive from endoderm¹⁴⁹.

Many species of fish (including zebrafish) have pharyngeal jaws which have robust musculature³. Pharyngeal teeth are arranged in rows that are constantly replaced, though not necessarily in synchronous fashion. The frequency of replacement depends on relative attrition, diet, and time of year (which may all be related)⁵. The first generation of teeth in teleosts forms outside the jaw bones, within the dermis (extramedullary), while replacement teeth may develop in similar fashion (as in zebrafish), or within the jaw (intramedullary). The ability of fish to have multiple replacement cycles allows for phenotypic plasticity, which may occur in single or successive generations¹⁵⁰. Variation in tooth shape, number, and tooth rows are associated with pathology, diet, attrition, intraspecific variation, and development^{3, 151}. In some species, dental formulae are asymmetric¹⁵².

1) Tooth formation (Figs. 12.30, 12.31, 12.32 and 12.33)

Fig. 12.30.

Schematic representation of replacement tooth formation in A: extramedullary or B: intramedullary situation, as observed in teleost fishes. In both schemes, successive stages of development of the tooth germ are shown from left to right. The predecessor (functional tooth) is only represented once (left of each figure), to show its relationship to the successor (replacement tooth). The zebrafish conforms to the upper scheme; other teleosts, e.g. cichlid fish, to the lower. Bone, pink; epithelium, purple; tooth matrix, red. Abbreviations: ab, attachment bone; d, dentine; db, dentigerous bone; ep, buccal or pharyngeal epithelium; en, enameloid; eo, enamel organ; m, mesenchyme; mc, medullary cavity. Used by permission, Wiley & Sons¹⁹⁶.

Fig. 12.31.

Largemouth bass, adult. Tooth. Normal with newly forming “bell” stage teeth (black arrowheads). Decalcified section. H&E. Image courtesy Wes Baumgartner.

Fig. 12.32.

Channel catfish, adult. Tooth. Normal. Pulp cavity with the base of the tooth, ligamentous attachments, and jaw bone. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Fig. 12.33.

Channel catfish. Tooth. Normal. Tooth histology in normal adult fish characterized by continuous eruption with little protrusion. Decalcified section. H&E. Image courtesy Wes Baumgartner.

Similar to mammals, tooth formation is a complex interaction of oral epithelium and mesenchyme, with a dental organ, dental papilla and in some cases a recognizable dental sac or follicle (which may only be evident in developing teeth)¹⁵³. Teeth are formed and replaced continuously (polyphyodont), and in the more commonly examined species do not exhibit growth after formation (as opposed to rodents). Tooth development in fish can be remarkably fast, in as little as two days¹⁵⁴. In medaka, pharyngeal teeth may develop in 3 weeks, with a functional lifespan of only 1 week¹⁴⁸. Fish teeth are generally acrodont (sitting atop the bone) with few thecodont (a tooth base in a deep pocket within alveolar bone) examples¹⁵⁵. Acrodont teeth are attached to the skull bone proper by bony ankylosis or by fibrous ligaments between dentin and bone. An intervening collar of mineralized tissue between the dentin or ligament and bone has been termed ‘bone of attachment’ or ‘pedicel’¹⁵⁶. Ankylosis occurs when the opposing outgrowths of tooth and bone form a composite tissue that is mineralized. In the pharyngeal teeth of zebrafish, the tissue has features of both dentine and bone, termed ‘dentinous bone’¹⁵⁶. In other species it remains to be determined whether or not the pedicel is truly bone, dentine, predentine/collagen, or an intermediate type^{157, 158}. The teeth and bony elements may be fused to one another or otherwise held in position through various ligaments and rings of collagenous tissue (annular ligament of Tomes)^{159, 160}. Thus, the teeth may be fixed or achieve varying degrees of mobility⁵. Attachment type can vary among teeth within single fish, and even along the same bone¹⁵⁴. Additionally, attachment type may be sexually dimorphic, as seen in the medaka¹⁶¹.

Initiation/Formation: In fish the teeth may originate directly from the oral epithelium, via a dental lamina, or from epithelial layers associated with predecessor teeth¹⁶². Dental laminae can be characterized as (1) continuous or discontinuous, and (2) permanent or non-permanent¹⁶³. The origin location of the tooth germ can be independent of development location (i.e., extramedullary or intramedullary)^{158, 164}. The formation of a dental lamina in fish is less obvious than in mammals, and in some cases can be arguably absent; small regions of cells may be arranged in isolated units within the mesenchyme, rather than in a continuous fashion. In fish with intramedullary dentition, epithelial downgrowths must pass through replacement pores within the bone, which may be re-established many times for the replacement teeth³. In the zebrafish, all functional teeth are associated from the beginning with a non-permanent, discontinuous epithelial downgrowth (successional laminae). The enamel organ of first-generation teeth develops directly from the oropharyngeal epithelium, without a lamina¹⁶⁵. The first indication of tooth formation is a focal thickening of the epithelium (basal layer or entire thickness), with eventual accumulation of mesenchymal cells in the subjacent stroma. The invagination of the oral epithelium into the mesenchyme results in a dental organ and papilla². In salmon the opposite occurs; it is the mesenchyme that evaginates into the epithelium¹⁶⁶.

Differentiation: The proximal end of the dental lamina proceeds through an infolding around the papilla (bell stage) (Fig. 12.31), such that the dental organ is composed of two layers; an inner dental epithelium (IDE) next to the papilla, and an outer dental epithelium (ODE). The proximal-most limit of the dental organ is the cervical loop. Dental organs in fishes do not, in most cases, have as complicated a structure as seen in mammals. A stellate reticulum is often inapparent and, if present, may only briefly exist in developing teeth. The IDE differentiates into ameloblasts of similar character to those in other animals.

Mesenchymal cells subjacent to the IDE differentiate into pre-odontoblasts; immediately prior to enameloid formation they mature into odontoblasts. Enameloid is very similar to enamel, but with a less orderly crystalline arrangement; the collagen component is deposited by odontoblasts with contribution of matrix by ameloblasts¹⁶⁷. The first deposits of enameloid are in the matrix surrounding odontoblast processes, which themselves may extend to the basal lamina of the ameloblast layer. The basal lamina between the ameloblasts and enameloid is subsequently removed, and mineralization begins in the enameloid. At this point, pre-dentine is already present and mineralization begins at the tooth tip. The dentine-enameloid junction, which may be irregular, marks the original location for the basal lamina⁵. Centripetal mineralization of the pre-dentine, toward the odontoblast layer (tip-down, outward-in), marks the formation of dentine. Odontoblast processes are not generally observed, or are relatively short, in first generation teeth. The dental papilla in first generation teeth is often composed entirely of odontoblasts or even a single odontoblast, lacking capillaries and nerves. Subsequent dentitions, and in particular larger teeth, may have larger and more developed pulp¹⁵⁸. Dentine deposition can continue after tooth eruption¹⁶⁸.

Enameloid maturation begins with increased mineralization and progressive loss of organic matrix, at a time just prior to tooth attachment. The bone of attachment forms at the point of dentinal proximity, with or without an underlying supporting bone. The dentin links to the bone by ligaments that may or may not mineralize². The cells producing the collagen, dentin, and attachment bone are often difficult to discriminate from one another, as they derive from the same mesenchymal population^{168, 169}. In zebrafish, fusion of dentine with attachment bone occurs (in decalcified sections toluidine blue staining can differentiate the tissues)¹⁷⁰. Hertwig’s root sheath does not disintegrate in fishes^{171, 172}.

Replacement: New teeth form beneath or next to the older teeth, being arranged in generational rows connected via an epidermal lamina that buds from the previous tooth. In zebrafish and most species studied, functional teeth are associated with epithelial downgrowths termed ‘successional laminae’, from which replacement teeth develop. Such laminae may remain quiescent, only to develop upon stimulation^{165, 173}. Replacement teeth may differ significantly from first generation teeth in terms of size, shape, skeletal support, and epithelial connections¹⁵⁴. As the dentin grows, constriction of the pulp vessels and nerves may lead to pulp atrophy. Shedding is an active process, not a result of wear or damage, and proceeds in most cases from base to apex¹⁵⁸. Osteoclasts/odontoclasts remove the bony attachments, and teeth are lost or resorbed^{5, 174}.

Eruption: In fish with intramedullary tooth development, the resorption of dentigerous bone occurs in conjunction with tooth movement and eventual eruption, where the tooth migrates through bone and attaches to its surface¹⁶⁶ (Figs. 12.32 and 12.33). In zebrafish, tooth movement does not occur, but rather epithelial remodeling and retraction leads to dental exposure¹⁶⁵.

2) Tissue types

Cementum is a “bone-like mineralized tissue secreted by cementoblasts on the surface of root dentine or, in some animals, crown enamel”¹⁷¹. Acellular and cellular cementum (primary and secondary, respectively) are characterized by laminations, a lower degree of mineralization compared to bone and dentine, and many coarse fibers (Sharpey’s fibers) running through it¹⁷⁵. The organic matrix of cementum is similar to bone and dentine, but differs in that it lacks innervation and vascularization¹⁶⁷. Cementum is conspicuously present in thecodont vertebrates (typical of mammals), which have a discrete periodontium, as opposed to most fishes³. In some species, particularly with robust dentitions (Anarhichas, wolffish), tissue resembling cementum is evident, but in most species a clearly defined cementum is absent^{5, 172, 175}. The “bone of attachment” in many teleost teeth is derived from odontogenic cells and is a type of dentine rather than cementum or true bone^{3, 167}. In bony fish, teeth are generally attached to the jaw by ligaments.

Dentine is a mineralized collagenous tissue surrounding a mesenchymal papilla which, at some stage in development, possesses cells with long processes that penetrate the tissue. Generally, dentine lacks cell bodies enclosed within the matrix; however, occasional cells can become entrapped within complex teeth. Calcification of the matrix may occur in two ways: inotropic and spheritic calcification. Inotropic, or homogeneous calcification develops from seeding centers associated with collagen bundles. Spheritic calcification, on the other hand, leads to the formation of globular lime bodies where hydroxyapatite crystallites radiate from the center, without regard to collagen. Spheritic calcification is also present in scales (corpuscles of Mandl), calcified cartilage, and bone in some cases¹³⁹.

More than one type of dentine may be present, depending on tooth type, however orthodentine is predominantly present, as seen in mammals. The dentine of fishes is of an intermediate type, which contains regular tubules running from the pulp (containing odontoblast processes- Tomes fibers) orthogonally across the entire thickness of the dentin, and well into the enameloid¹⁷⁶. The first-generation teeth (and sometimes adult teeth, as well as vasodentine) may lack dentinal tubules (and thus, Tomes fibers), forming an acellular, atubular tissue that is not readily distinguishable from acellular bone^{3, 166}. However, even without tubules, dentine should not contain Sharpey’s fibers (thick collagenous fiber bundles that extend from mesenchymal tissue to bone/cementum)¹⁷⁶. The structural markings identified in mammalian (particularly human) orthodentine, such as Schreger lines and incremental lines, have been identified in fishes^176,177,178. Other types of dentine, including vasodentine (Gadidae), osteodentine (pike), and plicidentine (gar) are remarkably different from typical orthodentine, and may be encountered in some species, particularly in larger teeth^{5, 139, 176}.

Enameloid is a hypermineralized, non-prismatic, fibrous tissue that coats the teeth (cap and collar enameloid) and is composed of fluoridated carbonate-apatites¹⁷⁹. It forms by the combined action of the ameloblasts and odontoblasts, unlike enamel³⁴. Enameloid appears prior to dentine development, contains many collagen fibers, and lacks growth lines since mineralization occurs in one event¹⁶³. Odontoblast processes are often present in enameloid, and thus it may be considered a form of modified dentine¹⁷⁹. Unlike enamel, enameloid forms in a centripetal manner; the external shape of the tooth is therefore similar to the boundary between the IDE and dental papilla at the initiation of matrix deposition. In some species, the first-generation teeth enameloid may not be distinguishable, or it may be entirely lacking¹⁵⁸. In demineralized sections, enameloid usually lacks any apparent matrix, but in some species the Tomes fibers from the dentinal tubules may persist¹⁷⁵. Amelogenin is the major matrix protein.

Table 4 shows nomenclature, diagnostic criteria and differential diagnosis for teeth.

Table 4. Nomenclature, diagnostic criteria and differential diagnosis of teeth.

Teeth			Common	Uncommon	Not observed, but potentially relevant	Not applicable
Non-proliferative
	Alteration, dentin matrix				X
	Apoptosis				X
	Cyst (s)					X
	*Degeneration			X
	Denticle (s)					X
	Dentin, decreased				X
	Dentin niches				X
	Dentin matrix alteration				X
	*Fracture			X
	Necrosis			X
	Periodontal pocket					X
	Pulp concretion					X
	Malformation/dysplasia				X
	*Resorption			X
	Thrombus					X
Proliferative
	Non-neoplastic
		*Hyperplasia, fibrous connective tissue		X
	Neoplastic
		*Ameloblastoma		X
		*Fibroma, odontogenic		X
		*Odontoma		X
		Tumor, odontogenic, benign		X
		Tumor, odontogenic, malignant		X

Uncommon as a background finding; commonly induced.

*Terminology with diagnostic criteria or Comment described in the main text.

Tooth–Non-proliferative

Degeneration, tooth – Similar to rodent

Comments

Enameloid may be difficult to discriminate from dentin and is often absent in decalcified sections since it is largely mineral. Any injury to the formative cells may result in irregularities in the enameloid. Degenerative changes in enameloid and increased apoptosis of ameloblasts has been seen in zebrafish following excessive fluoride exposure¹⁸⁰.

Odontoblasts are mesenchymal cells filling the dental papilla or lining the pulp perimeter. Tubules may not be identifiable normally, depending on species. Degenerative changes in the odontoblasts may lead to abnormalities in dentine and enameloid, as well as tooth attachment.

The functional lifespan of fish teeth may be on the order of days, and the developmental lifespan a matter of a few weeks; transient injury may therefore be identifiable within a relatively narrow timeframe. Additionally, teeth in fish retain developmental plasticity over their lifetime, allowing for alterations in shape and number^{150, 152}. Indirect (secondary) effects in successional teeth, beyond the lifespan of the tooth generations directly affected in the study, should be considered.

Fracture, tooth

Other Terms

• None

Pathogenesis/Cell of Origin

• Disruption of tooth integrity

Pathogenesis

• Discontinuity or “break” in the tooth, typically from trauma

Diagnostic Features

• Histologic appearance is influenced by amount of time since fracture occurrence

• Osteoclast (mononucleate or multinucleate) activity may be increased

• Associated epithelium may show evidence of injury (necrosis)

• Formative cells in successional teeth may exhibit hyperplasia

• May see inflammation

Differential Diagnoses

• Dental dysplasia – aberrant development without a fracture

• Odontogenic neoplasia – masses of cells (+/– matrix production) with one or more odontogenic cell lineages

Comments

Fractures may occur as a result of trauma, dietary or otherwise, and infection. The high turnover rate of fine teeth in many species suggests that attrition and fracture are an expected outcome that has been compensated for in many species¹⁴⁸. With this in mind, the normal dental wear or fracture rate must be carefully examined in the control population before a determination of significance is applied to this diagnosis.

Resorption, tooth – Similar to rodent

Comments

Resorption of dental hard tissues may be initiated by trauma, inflammation, or possibly fungal organisms^{181, 182}. Shedding and replacement of teeth is a normal condition in fishes, which involves increased clastic activity. The diagnosis rests on the determination of what constitutes abnormal removal of tissues.

Tooth –Proliferative Non-Neoplastic

Hyperplasia, fibrous connective tissue, tooth

Other Terms

• Reactive hyperplasia

Pathogenesis

• Epithelial cells (oral mucosa and dental organ) and mesenchymal cells proliferate in response to persistent irritation

Diagnostic Features

• Well circumscribed, concentric growth, symmetrical; may arise on both upper and lower jaw (kissing lesions)

• Loose, bland, hyperplastic mesenchyme with fine bony spicules (metaplasia) dispersed near the attachment site

• Surface epithelium is hyperplastic and well differentiated, with deep clefts and cords extending well into the stroma

• Well differentiated enamel organs and formed teeth, if present, radiate from the epithelial ingrowths

• Inflammation may be present

Differential Diagnoses

• Fibroma – A neoplastic mass of soft tissue origin, composed of fibroblasts, metaplastic bone, and entrapped denticles in some cases (common in angelfish). See Soft Tissues chapter

• Ameloblastoma – an odontogenic epithelial neoplasm that does not produce dental hard tissues

• Odontoma (complex or compound) – A mixed tumor largely composed of differentiated but abnormal mineralized dental hard tissues

• Fibroma, odontogenic – a neoplasm composed of dental follicle-like mesenchyme

• Fibroma – A mass of non-odontogenic neoplastic fibrous connective tissue with dysplastic mineralization or entrapped teeth. See Soft Tissues chapter

Comments

Hyperplastic lesions are less cellular than true neoplasms and atypia is not a feature. Edema, inflammation, metaplasia, and hyperplasia are frequently evident¹⁸³. Although numerous denticles can be present in association with epithelial hyperplasia, careful examination may be required to reveal hard tissue elements within the stroma.

Tooth development requires both epithelium and mesenchyme, and hyperplasia secondary to trauma/irritation can produce nodular lesions that contain dental elements. Fish maintained in tanks may impact the tank sides or other fish, resulting in focal or multifocal hyperplastic lesions of the mouth and lips that may contain one or more elements of tooth or bone, including complete teeth. Hyperplastic oral epithelium is continuous with the formative cells of enamel organs; thus, the presence of hyperplastic surface epithelium in association with hyperplastic enamel organs and teeth would be expected. The dental mesenchyme in fish can produce dentin, bone, and collagenous tissue. A distinct periodontal ligament tissue is generally lacking in fishes. Tissue strain at the base of the mass may induce hemorrhage and necrosis, with subsequent metaplastic bone formation.

See comments under ‘Soft Tissues - Proliferative Neoplastic’ in the Soft Tissues chapter.

Tooth –Proliferative Neoplastic

Ameloblastoma (M), tooth

(Fig. 12.34)

Fig. 12.34.

Chinook salmon. Tooth. Ameloblastoma. Neoplastic tissue is contiguous with enamel organ cells (lower left), and also forms a separate nodular mass (upper right). Note the acantholysis and a tendency to form clefts, features which further help to distinguish this from squamous cell carcinoma. Decalcified section. Image courtesy of Jeffrey Wolf. H&E.

Other Terms

• None

Pathogenesis/Cell of Origin

• Arising from cells of the enamel organ and basal layer of the oral mucosa, this is a locally aggressive neoplasm that is not known to metastasize

Diagnostic Features

• May be multiple, even bilaterally symmetrical. Although most are found within the mouth, these can also originate in extra-oral locations

• Composed of islands, nests, or anastomosing strands of odontogenic epithelial cells with enamel organ features, in collagenous stroma

• Epithelium may consist of a peripheral layer of tall columnar cells resembling the inner dental epithelium; they may be pseudostratified, polygonal, or even goblet cells

• Intercellular bridges and desmosomal attachments, or loosely arranged cells similar to stellate reticulum, may be evident

• Epithelial islands may be solid (solid type) or may show degenerative (acantholysis, clefting) changes resulting in cyst formation (cystic type)

• Dental hard tissues (enamel, dentin, attachment bone) are not produced, or are minimally present

• Underlying bone may be unaffected (extramedullary dentition species)

Differential Diagnoses

• Squamous/basal cell carcinoma – an epithelial neoplasm that is not associated with the dental laminae and does not form clefts/acantholytic spaces

• Odontoma (complex or compound) – A mixed tumor largely composed of differentiated but abnormal mineralized dental hard tissues

• Fibroma – may incorporate developing teeth (common in angelfish)- see Soft Tissues chapter

• Fibroma, odontogenic – a neoplasm composed of dental follicle-like mesenchyme

Comments

Ameloblastomas are uncommon neoplasms in fish but, surprisingly, can be multifocal and have occurred as epizootics^{184, 185}. The etiology of these tumors is unknown, however environmental contamination and viral infection are suspected. A wide variety of histologic patterns have been described¹⁸⁶. Some ameloblastomas in Chinook salmon (Oncorhynchus tshawytscha) appeared to originate in extra-oral locations such as the gill arch and fin¹⁸⁴.

A final note concerning tumors composed of a mixture of odontogenic epithelium and mesenchyme (ameloblastic fibroma/fibro-odontoma). In domestic animals such neoplasms present along a continuum of inductive differentiation, spanning from ameloblastic fibroma (least differentiated) to compound odontoma (highly differentiated)¹⁸⁷. Typically, mesenchymal components comprise the bulk of the lesion.

Consensus regarding nomenclature of mixed dental tumors in animal species has not been reached; it can hardly be done for fish, where few cases are available to examine. The term ameloblastic odontoma is not favored^{186, 187}. Currently there are too few cases available to address the diagnosis of odontoameloblastoma in fishes at this time¹⁸⁸.

Ameloblastic fibromas and fibro-odontomas are described as exophytic, often densely cellular masses; although numerous denticles can be present, careful examination may be required to reveal hard tissue elements¹⁸⁹.

The dental mesenchyme in fish is capable of producing dentin, bone, and collagenous tissue. A distinct periodontal ligament tissue is generally lacking in fishes. Dentin within neoplastic tissue may lack tubules/Tomes fibers, making discrimination from bone difficult. Sharpey’s fibers should not be present in dentin, but can be present in bone (and cementum, see introductory Comment).

Fibroma, odontogenic (B), tooth

Other Terms

• None

Behavior

• Benign neoplasm

Pathogenesis

• Mesenchymal cells of the dental papilla or follicle

Diagnostic Features

• Well circumscribed expansile tumor

• Composed of primitive-appearing follicle mesenchyme with areas of collagen formation

• Strands or islands of non-neoplastic, mostly undifferentiated small epithelial cells are scattered throughout, but are a minor component

• Irregular foci of bony tissue may be present in the mass, but enameloid does not form

Differential Diagnoses

• Hyperplasia, fibrous connective tissue, tooth – a non-neoplastic nodule of mesenchyme associated with surface epithelial hyperplasia

• Fibroma – A mass of non-odontogenic neoplastic fibrous connective tissue with dysplastic mineralization throughout. May incorporate developing teeth. (see Soft Tissues chapter, Fibroma)

• Ameloblastoma – an odontogenic epithelial neoplasm that does not produce dental hard tissues

• Odontoma (complex or compound) – A mixed tumor largely composed of differentiated but abnormal mineralized dental hard tissues

Comments

Odontogenic fibromas have been reported in gilthead seabream¹⁹⁰. A distinct periodontal ligament tissue, the compelling feature of odontogenic fibromas in mammals, is generally lacking in fishes. The dental mesenchyme in fish is capable of producing bone or dentin, which may be abundant. Dentin within neoplastic tissue may lack tubules/Tomes fibers, making discrimination from bone difficult. Sharpey’s fibers should not be present in dentin, but can be present in bone (and cementum, see introductory Comment). As these tumors may contain bone elements, they must be thoroughly examined to rule out odontoma or reactive bone metaplasia within an area of Hyperplasia, fibrous connective tissue, tooth. They appear similar to ossified fibromas in soft tissues, and may be impossible to discriminate¹⁹¹.

Odontoma (B), tooth

(Figs. 12.35, 12.36, 12.37, and 12.38)

Fig. 12.35.

Zebrafish, adult. Tooth. Odontoma. Induced by exposure to DMBA. Decalcified section. H&E. Image courtesy Jan Spitsbergen.

Fig. 12.36.

Carp. Tooth. Odontoma. Note that mass is characterized by an island of cells forming stellate reticulum surrounded by an ameloblastic layer. Decalcified section. H&E. Image courtesy Heike Schmidt-Posthaus.

Fig. 12.37.

Carp. Tooth. Odontoma. Note that the mass is characterized by a cluster of epithelium resembling the odontogenic cap and dental lamina. Decalcified section. H&E. Image courtesy Heike Schmidt-Posthaus.

Fig. 12.38.

Carp. Tooth. Odontoma. Decalcified section. H&E.

Other Terms

• None

Pathogenesis/Cell of Origin

• A benign mixed epithelial-mesenchymal tumor with differentiated dental components

Diagnostic Features

• Dental hard tissues are abundant with varying degrees of differentiation (enameloid, dentin, bone)

• The contribution of ameloblastic epithelium is minor

• Hard tissues exhibit haphazard and/or primitive features

• Multiple tumors may be present, in various places throughout the oral cavity

Differential Diagnoses

• Ameloblastoma – an odontogenic epithelial neoplasm that does not produce dental hard tissues

• Fibroma, odontogenic – a neoplasm composed of dental follicle-like mesenchyme

• Osteoma – a neoplasm of well differentiated bone trabeculae, lacking abnormal tooth elements within the mass (associated denticles may be entrapped at the periphery)

• Hyperplasia, fibrous connective tissue, tooth – a non-neoplastic nodule of mesenchyme associated with surface epithelial hyperplasia, which may contain entrapped well differentiated teeth

• Fibroma – a benign fibroblast neoplasm forming irregular fascicles in loose stroma, which may contain entrapped, well-differentiated denticles- see Soft Tissues, Proliferative Neoplastic section

Comments

The nature of dental formation in fish (continuous production) complicates the interpretation of masses that contain dental hard tissues; reactive hyperplasia or fibromas may incorporate normal teeth that would otherwise arise in this area. Hamartomatous malformations are also possible, which would be present early in development, however injury to developing teeth in adult fish could also lead to malformed teeth that may present as grossly evident nodules. The occurrence of a true neoplasm that produces dental hard tissues therefore requires unequivocal evidence of neoplastic tissue; haphazard odontogenic mesenchyme and tooth-like elements with some primitive features^{183, 192}.

Few cases are described in fishes; they are often considered to be malformations (hamartomas) rather than true neoplasms, however local bone invasion has been reported^{126, 189, 193, 194}. Angelfish (Pterophyllum scalare) are particularly well known to produce lip nodules (fibromas or fibrous hyperplasia) that contain denticles. Typically, they are pedunculated lip masses composed of spongy fibrous tissue containing bone and well-formed teeth in various stages of development, all radiating from the point of attachment and oriented perpendicular to the epithelial surface; such features suggest reactive hyperplasia rather than neoplasia¹⁸³. Depending on the cell of origin, these may be diagnosed as foci of Fibroplasia, soft tissues, or Hyperplasia, fibrous connective tissue, tooth.

Significant bone may be produced within an odontogenic mass (possibly a result of repeated trauma), obscuring the presence of abnormal tooth elements; careful examination of the tumor is necessary to differentiate this from osteoma¹⁹².

The dental mesenchyme in fish is capable of producing dentine, bone, and collagenous tissue. A distinct periodontal ligament tissue is generally lacking in fishes. Dentine within neoplastic tissue may lack tubules/Tomes fibers, making discrimination from bone difficult. Sharpey’s fibers should not be present in dentine, but can be present in bone (and cementum, see introductory Comment).