The present study, for the first time, provides a detailed description of the microscopic structure of both pedicle and hard antler bone in a cervid species, the European roe deer. Our results demonstrate pronounced seasonal changes in pedicle structure that can be related to the seasonal antler cycle. These findings corroborate and extend those of von Raesfeld (1919), who first described the seasonal variation in pedicle porosity based on macroscopic observations on longitudinally sectioned pedicles of roe deer.
Around antler casting, pedicle porosity is very high due to extensive osteoclastic resorption that has occurred in the pedicle bone. In addition to bone erosion within the pedicle, there is also extensive sub-periosteal resorption along the pedicle surface. Osteoclastic resorption is especially pronounced in the distal pedicle, where it causes the formation of extended, frequently confluent resorption spaces that were termed resorption sinuses by Kölliker (1873). During the pre-casting period, huge numbers of osteoclasts have been reported to occur first at the pedicle periphery and subsequently in progressively more central locations (Goss et al. 1992). In fallow bucks, in which premature antler casting was induced by castration, first osteoclasts were observed 3 days after castration along the sides of the pedicle, and 2 weeks after castration osteoclasts and associated resorption spaces were present along the full width of the pedicle bone (Goss et al. 1992). The weakening of the bony connections within the distal pedicle eventually leads to antler casting. Following antler casting, osteoclast numbers decline (Goss et al. 1992), but bone erosion continues for some time, causing a smoothing of the pedicle's separation plane (Gruber, 1937; Kierdorf et al. 2003).
Some apposition of new bone onto the resorption surfaces shortly after casting was observed in the present study. However, basically pedicle porosity remains high during the period of intense antler growth, and only during late antler growth the porosity becomes progressively reduced (von Raesfeld, 1919). As a consequence, pedicle bone is very compact during the hard antler phase. It may be hypothesized that maximum compactness is reached during the rutting period (mid-July to mid-August in Germany; Stubbe, 1997), when the antlers are used for fighting and the pedicles are therefore subjected to the strongest mechanical impacts within the seasonal cycle.
The seasonal variation in pedicle porosity is considered an adaptation to the varying functional demands on the pedicle during the antler cycle. During antler growth, pedicle vessels play a role in antler circulation in that the venous drainage of the antlers occurs internally through veins in the pedicle (Waldo et al. 1949). Also, arterial communications between pedicle and antler have been observed in this period, although at all stages of their growth the main blood supply of the antlers is through external arteries that ascend in the vascular layer of the velvet. As a result of the progressive reduction in antler porosity, venous drainage in the later stages of antler growth occurs largely via veins in the velvet (Waldo et al. 1949).
During the hard antler stage, especially the rutting period, the pedicles have to withstand the same forces as the antlers, and from a biomechanical point of view hard antlers and pedicles must be considered a functional unit. Previous studies demonstrated that the dry hard antler bone is very well suited for its function in sparring and fighting, as it exhibits a very high impact resistance and work to fracture (Currey et al. 2009). In contrast to the functional (dry) antlers, the pedicles consist of living, wet bone. It is presumed that, regardless of this difference, the mechanical properties of the pedicles during the hard antler period at least equal those of the antlers. It might even be hypothesized that the mechanical properties of the pedicles exceed those of the hard antlers during the rutting period. If this was the case, fractures would more likely occur in the antler than the pedicle. This would be selectively advantageous, as a fractured antler will be replaced by a normal one in the next growth phase, while a pedicle fracture will very probably cause a permanent malformation of the cranial appendage and may even result in the death of the animal. Thus far, studies on the mechanical properties of pedicles are lacking. Such studies have to take into account the strong seasonal variation in pedicle structure documented in the present and previous studies that will certainly be reflected in a corresponding variation of mechanical properties.
There is experimental evidence that casting of the dry hard antlers is prevented by higher levels of circulating androgens (Suttie et al. 1984, 1995; Goss et al. 1992; Lincoln, 1992; Kierdorf et al. 1993, 1995b). This suggests that higher androgen levels inhibit osteoclast formation in the pedicle, which would otherwise occur as a consequence of the death of the antlers due to insufficient blood supply and the attempt of the organism to sequester the dead bone (Goss, 1983). When androgen levels drop sharply after the rutting period, or experimentally following castration, this enables massive osteoclastogenesis and rapid bone resorption to take place in the pedicle (Goss et al. 1992). An inhibitory effect of androgens on osteoclastogenesis and osteoclast activity has been demonstrated experimentally in other mammals (Wiren, 2008).
The view that hard antlers are dead structures has been challenged by some authors, who claim that antlers maintain a functional blood supply through the pedicle after velvet shedding. In consequence, these authors posit that hard antlers survive until shortly before casting and that there is even some ongoing bone formation in them (Brocksted-Rasmussen et al. 1987; Rolf & Enderle, 1999; Rolf et al. 2001). Results from more recent studies, however, showed that hard antlers effectively dry out (being in equilibrium with the moisture content of the ambient air) and that there is no further mineralization of antler bone after velvet shedding (Currey et al. 2009; Gomez et al. 2013), thereby supporting the traditional view of hard antlers as dead bone. Moreover, the hypothesis that hard antlers are living structures cannot explain why premature casting of hard antlers, but not of antlers in velvet, can be induced by castration or treatment with anti-androgens (Kierdorf & Kierdorf, 2011).
It has been known for a long time that pedicles increase in diameter and shorten in length with age (Stubbe, 1997). The progressive shortening of the pedicles is due to the fact that the separation plane is located in the pedicle, and that with each casting event a portion of the distal pedicle is lost along with the cast antler (Berthold, 1831; Gruber, 1937; Goss et al. 1992; Kierdorf & Kierdorf, 2012; Li, 2013). There is some bone restoration in the distal pedicle, but this is insufficient to compensate for the loss at casting (Kierdorf et al. 2003; Li, 2013). A central biological function of pedicles is probably to ensure that the massive osteoclastic activity leading to antler casting occurs at a ‘safe distance’ from the skull roof. Moreover, pedicles provide a connection with a defined diameter between antler and skull that can effectively be eroded within the seasonal period of low androgen levels (Kierdorf et al. 2004b).
The increase in pedicle diameter due to periosteal bone apposition ensures that the pedicle can serve as a firm base for the regeneration of successively larger and heavier antlers, and as a firm connection between antler and skull when the antlers are used for fighting. In the yearling roe buck, the outer pedicle zone can be described as fibrolamellar bone, composed of mostly circumferentially oriented sheets of woven bone and of lamellar (primary osteonal) bone that fills in the elongate vascular compartments between these sheets. A similar structure of the outer pedicle zone (termed plexiform in that study) has previously been reported from yearling red deer stags, although in the red deer this zone was relatively broader than in the roe deer (Kierdorf et al. 2000). In the older roe bucks, the outer pedicle zone also consisted predominantly of fibrolamellar bone, with some areas showing a woven bone structure. Only in roe buck no. 5 was an outermost zone of lamellar bone present.
The formation of woven and fibrolamellar bone occurs at sites of rapid growth, while lamellar bone is laid down more slowly (Amprino, 1947; Currey, 2002; Huttenlocker et al. 2013; Lee et al. 2013). The fact that the structure of periosteal bone tissue reflects its rate of deposition is known as Amprino's rule (Lee et al. 2013). The finding that the outer pedicle zone of roe bucks (this study) and of young red deer stags (Kierdorf et al. 2000) consists mainly of fibrolamellar bone can thus be seen as indicative of a rapid seasonal growth process that is responsible for the increase in pedicle diameter. The observation of an outermost pedicle bone layer with a lamellar structure in roe buck no. 5 suggests that during the later stages of growth the rate of periosteal bone deposition onto the pedicle surface becomes considerably reduced. The results of the present study thus lead us to hypothesize that the seasonal pedicle growth can be divided into an early phase of rapid bone formation and a later phase of slower growth. To test this hypothesis, labeling studies on forming pedicle bone, similar to those recently performed to analyze bone formation in the antler cortex (Gomez et al. 2013), are encouraged.
Our characterization of the primary bone in the outer pedicle of the roe bucks as fibrolamellar and of the lamellar systems forming within the vascular compartments as primary osteons is in line with the use of these terms by other authors. Thus, Currey (2002) describes fibrolamellar bone as being composed of a thin scaffolding of woven (sometimes parallel-fibered) bone and lamellar bone that fills in the cavities (vascular compartments) within the scaffold, thereby forming primary osteons (Currey, 2002). Similarly, Huttenlocker et al. (2013, p. 21) characterize fibrolamellar bone as a ‘bone complex composed of a woven-fibered scaffolding and intervening primary osteons of varying orientations’. The initially large cavities (that may contain one or more blood vessels) between the woven bone layers are referred to as primary Haversian or osteonal cavities by these authors.
The primary nature of the circular lamellar systems forming within the vascular compartments of fibrolamellar bone was already emphasized by Enlow & Brown (1956, 1957, 1958) in their seminal study of fossil and recent bone tissues. However, in these papers, the authors use the term ‘primary osteons’ for primary vascular canals, while applying the term ‘protohaversian system’ to concentric primary lamellar systems deposited in primary vascular and marrow spaces. In a later paper, Enlow (1962b, p. 278) characterizes the primary osteon as ‘a variety of the Haversian system [..] formed by the deposition of concentric lamellae within tubular, anastomosing spaces located in surface deposits of fine-cancellous, non-lamellar bone’, and distinguishes between primary and secondary osteonal systems on the one hand and (‘non-Haversian’) primary vascular bone on the other. This is more in line with the way the term ‘primary osteon’ is used in the present paper and the works of other authors (Currey, 2002; Locke, 2004; Huttenlocker et al. 2013).
Stover et al. (1992), in their study of cortical bone growth in the metacarpal diaphysis of the horse, describe the formation of circumferential trabeculae of more highly mineralized woven bone by periosteal apposition and subsequent deposition of lamellar bone on the inner trabecular surfaces, resulting in the formation of what they describe as rows of primary osteons in the vascular compartments between woven-bone trabeculae. Likewise, when characterizing the growth in diameter of mammalian long bones, Locke (2004, p. 559) states that the predominantly axially oriented vessels present in the vascular compartments of the cortical diaphysis during later development become surrounded by cylindrical lamellae, which developmentally ‘are primary osteons in the sense that laminar bone is primary’. Note that this author applies the term ‘laminar bone’ to the primary bone formed during the growth in width of the long bone diaphysis. While Currey (2002) treats the terms fibrolamellar, plexiform and laminar bone as broadly synonymous, other authors distinguish between different sub-types of fibrolamellar bone based on vessel orientation in the vascular compartment or use terms like ‘laminar’ only for the orientation of the vascular system (Huttenlocker et al. 2013).
Secondary osteons in different stages of development were observed in the outer pedicle zone, where they replaced the primary (fibrolamellar or woven) bone. This indicates that erosion of the newly formed peripheral bone and its replacement by secondary osteonal bone laid down around one or more blood vessels occur rapidly. Occasionally different generations of secondary osteons were observed, likewise demonstrating the rapidity of the remodeling process. Replacement of laminar bone by secondary osteons also occurs in the shafts of mammalian long bones (Locke, 2004).
The inner pedicle zone of the yearling roe buck consisted of a trabecular framework of woven bone filled with primary osteons and secondary osteons. The latter were mostly small, with a single vascular canal. Formation of such typical secondary osteons can be ascribed to the action of basic multicellular units or bone remodeling units (Currey, 2002; Stout & Crowder, 2012). In this process, a relatively small volume of bone is first eroded by osteoclasts, and the resulting cavity (cylindrical in compact bone) is then refilled with new bone that is deposited in concentric lamellae. This sequence occurs in a spatially and temporally tightly coordinated manner, and normally without significantly affecting overall bone mass or markedly impairing the biomechanical properties of the bone undergoing remodeling.
The seasonal variation in bone structure observed in the pedicles of the older bucks deviates in a number of aspects from the above picture. Our findings indicate a strong seasonal rhythm of bone formation and resorption, with formation probably considerably lagging behind resorption. In consequence, it may take some months until the bone regains a dense structure. The large cavities resulting from the extensive resorption activity are filled in with complex secondary lamellar systems that possess multiple vascular canals. These systems are referred to as complex secondary osteons to indicate their difference from smaller and more typically formed secondary osteons. The seasonal changes in the roe deer pedicle may be classified as an unusual case of remodeling, thereby corroborating the statement by Hall (2005) that regional bone remodeling can occur in very different ways.
In the yearling roe buck, no remnants of mineralized cartilage were observed in the inner pedicle zone. Cartilage remnants were also absent from the pedicles of the older bucks, although in these cases it could be argued that they had been removed in the course of the repeated cycles of extensive resorption and infilling. The increase in pedicle diameter occurred via sub-periosteal bone apposition, i.e. by direct (intramembranous) ossification. Our findings are thus consistent with the view expressed by Gruber (1937) that pedicle formation in roe deer occurs via direct (intramembranous) ossification. It should, however, be mentioned that in other species (fallow deer, red deer) initial pedicle growth (not secondary growth in thickness) has been reported to involve endochondral ossification (Kierdorf et al. 1994; Li & Suttie, 1994).
Antler bone formation in roe deer can be summarized as follows. Initially a trabecular framework of woven bone with more or less cylindrical pores is formed that, in a process of endochondral ossification, largely replaces the preexisting trabeculae of mineralized cartilage. Resorption of the cartilaginous trabeculae is performed by chondroclasts, whose occurrence and activity has been demonstrated in previous studies (Banks, 1974; Kierdorf et al. 1995a; Szuwart et al. 2002). In the process of cartilage replacement, woven bone is initially laid down onto a scaffold of mineralized cartilage (Banks, 1974; Kierdorf et al. 1995a). The presence of remnants of this mineralized cartilage within the woven bone trabeculae of hard antlers of roe deer demonstrates that the replacement of cartilage by bone during antler growth is incomplete, as was previously shown also to be the case in red deer (Landete-Castillejos et al. 2012a). The above description pertains to the formation of the main cortex and the cancellous core of the roe deer antler. In contrast to these antler zones, the outer cortex lacks remnants of mineralized cartilage, thereby indicating that it is not formed by endochondral ossification, but by sub-periosteal direct (intramembranous) ossification.
The soft tissue spaces lined by the trabecular scaffold of woven bone are filled by endosteal deposition of lamellar bone in the form of primary osteons oriented in the long axis of the antler. In this paper, the entire tubular lamellar system formed within an intertrabecular cavity is regarded as one primary osteon. Such primary osteons may contain one or several vascular canals, and are often morphologically more variable than secondary osteons. Our use of the term primary osteon in antler bone corresponds to that of Krauss et al. (2011) in their study of red deer antlers. This concept of the primary osteon is the basis for the latter authors' statement that the number of lumina within the trabecular framework of developing antlers corresponds to the number of primary osteons present in mature antler bone.
Whereas in the center of the antler shaft the lamellar infilling is incomplete, leading to the formation of the cancellous antler core, in more peripheral areas the intertrabecular spaces are completely filled in, causing a compaction of the bone and the formation of a dense antler cortex. This process somewhat resembles the so-called lamellar compaction of fine cancellous bone occurring in the metaphysis of long bones, which was described as involving ‘endosteal lamellar deposition on the surface of pre-existing endochondral trabeculae [..] containing spicules of calcified cartilage matrix’ (Enlow, 1962a, p. 82). Contrary to the situation in antlers, the appearance of the lamellar bone formed in the long bone metaphyses is rather irregular (Enlow, 1962a). This can be attributed to the fact that in the antlers the distinctly tubular nature of the trabecular framework determines the shape of the lamellar bone filling in the intertrabecular cavities.
Our findings indicate that in the main cortex and the cancellous antler core, the infilling of the intertrabecular voids by primary osteons was in places apparently preceded by bone resorption on the trabecular scaffold. A corresponding observation was previously reported by Gomez et al. (2013) for red deer in a study using fluochrome labeling of forming antler bone. Using BSE imaging, we observed the presence of bright (relatively hypermineralized) thin scalloped lines around primary osteons. On the basis of their morphological characteristics and the observation of signs of resorption on the trabecular scaffold, these lines were interpreted as cement lines formed at reversal sites. Skedros et al. (1995) previously reported the occurrence of highly mineralized seams around primary osteons in mature antler bone. They termed these structures hypermineralized lamellae and assumed that they function as analogs of cement lines in secondary bone. Krauss et al. (2011) likewise observed hypermineralized lines around primary osteons in mature antler bone of red deer, which they interpreted as resting lines that were laid down before the start of osteon formation. Based on the findings of the present and our previous study (Gomez et al. 2013), we suppose that the hypermineralized lamellae of Skedros et al. (1995) and the resting lines described by Krauss et al. (2011) may likewise represent cement lines formed at reversal sites following resorption on the trabecular scaffold. Previous studies on antler growth demonstrated early osteoclastic resorption of the bone that had been laid down on the scaffold of the mineralized cartilage, so that chondroclastic, osteoblastic and osteoclastic processes could be observed side by side in a single section (Banks, 1974; Kierdorf et al. 1995a). This means that right from the beginning of the formation of the main cortex and the cancellous core, bone resorption occurs in the antler growth process.
The finding that not only secondary osteons but, according to our interpretation, sometimes also primary osteons were delimited by cement lines can complicate the distinction between them. Our study also suggests that secondary osteons in antler bone, like those in pedicle bone, may not always conform to the typical morphological picture of relatively small circular lamellar systems with a single central vascular canal. A recent fluorochrome labeling study of cortical bone formation in red deer antlers confirmed the formation of secondary osteons during antler osteogenesis also in this species (Gomez et al. 2013). It was demonstrated that the formative period of secondary osteons overlapped with that of primary ones. As these secondary osteons were formed in bone that was still undergoing modeling, they were referred to as modeling osteons (Gomez et al. 2013).
Our findings corroborate the view that secondary osteons are scarce in antlers and that most of the osteons present in antler bone are primary ones (Chen et al. 2009; Launey et al. 2010; Krauss et al. 2011; Gomez et al. 2013). In consequence, the forming antler can be said to show very limited remodeling activity during its short lifespan. Signs of ongoing (limited) remodeling activity were observed in the permanently viable antlers of castrated fallow bucks (Kierdorf et al. 2004a).
The present study confirmed the observation previously made for red deer antlers (Gomez et al. 2013) that during antler growth bone resorption at trabecular endosteal surfaces is followed by bone formation at the same sites. Generally, modeling in a specific bone location is distinguished from remodeling in that osteoblastic bone formation is not preceded by osteoclastic bone resorption at modeling sites (Erben, 1996; Seeman, 2008). Recently, however, Maggiano (2012) suggested that the term modeling should be applied to all processes occurring at periosteal or endosteal surfaces during bone development and growth, including those where bone deposition follows resorption at a given site. As shown, primary osteon formation in growing antlers may involve resorption on the endosteal trabecular surface prior to lamellar infilling of the intertrabecular cavities. Nevertheless, the process can be characterized as a case of bone modeling in the sense defined by Maggiano (2012).
The outer antler cortex, which was referred to as the sub-velvet zone by Rolf & Enderle (1999), has thus far received little attention. We show that where it is thin, the outer cortex consists of woven bone. In areas where the antler exhibits protuberances (pearls) and the outer cortex is broader, the woven bone forms a trabecular framework whose voids are incompletely filled with primary osteons. These observations suggest a less mature nature of the outer compared with the main cortex. In contrast to the main cortex, resorption on the trabecular scaffold does not occur prior to the infilling of the intertrabecular spaces.
It has been shown that at sites of bone mineralization, proteoglycans of the osteoid are partially degraded and lost, and that there is a decrease in the glycosaminoglycan content of bone with tissue age (Pugliarello et al. 1970; Baylink et al. 1972; Grynpas & Hunter, 1988). The more intense autofluorescence observed in the outer cortex of the roe deer antlers is therefore seen as indicative of a higher proteoglycan content and thus a younger age of this zone compared with the main cortex. In line with our findings, a higher degree of autofluorescence of the bone matrix of newly formed trabecular excrescences compared with the established (older) matrix was recently reported by Taylor et al. (2012). The above view of a younger age of the antler's outer cortex compared with the main cortex is in line with the results of a recent fluorochrome labeling study in red deer (Gomez et al. 2013).
On the basis of our findings we conclude that the outer cortex is formed late during antler growth by intramembranous ossification as a consequence of periosteal activity occurring in the more proximal antler portions. This is in line with the view of Bubenik (1966), who stated that the pearls of the antler develop secondarily during late antler growth. Late formation by intramembranous ossification explains the lack of mineralized cartilage remnants and signs of bone resorption in the outer cortex that are both prominent features of the endochondrally forming central antler portions.
The antlers of castrated deer tend to develop conspicuous bony protuberances (von Raesfeld, 1919; Bubenik, 1966; Goss, 1983; Kierdorf et al. 1995b). Histological investigations in castrated fallow bucks have shown that these protuberances, which develop in proximal and distal antler portions, are formed by periosteal bone apposition onto the original antler surface (Kierdorf et al. 2004a). These protuberances can therefore be characterized as a hypertrophic form of the structures that form during normal antler growth, due to (seasonally renewed) activation of the antler periosteum in the castrates.
During the short lifespan of the antlers, a large amount of bone has to be mineralized. Thus, in adult red deer stags (≥ 3 years old), dry antler weight accounted for 28% of total skeletal weight (Gomez et al. 2012). The resulting high calcium and phosphorus demand for antler mineralization is partly met by mobilization from the skeleton (Meister, 1956; Banks et al. 1968; Baxter et al. 1999). The more recently formed bone in the transition zone and the cancellous core of the roe deer antlers showed signs of hypomineralization. This could indicate that during peak mineralizing activity the mineral demands of the forming antler bone could not be met in full. If this was the case, the findings would indicate an impairment of the mineralization process caused by physiological exhaustion, especially regarding calcium supply (Landete-Castillejos et al. 2012b). An alternative explanation would be that velvet shedding, which is caused by a rise in circulating androgens levels, sets an abrupt end to the antler mineralization process regardless of the stage of antler maturation. This was observed in an experimental situation in which, due to anti-androgen treatment, out-of-season antler casting had been induced that was followed by regeneration of a new set of antlers. These antlers were cleaned of velvet at a very immature stage of bone development after a short period of growth (Kierdorf et al. 1993). In the roe deer, the most recently formed antler portions, namely the outer cortex and the youngest bone formed in the cancellous and transition zones, showed signs of incomplete development and hypomineralization. This suggests that also during normal antler development, velvet shedding can be triggered before full maturation of the antler bone has been accomplished.