Histological studies of bone formation during pedicle restoration and early antler regeneration in roe deer and fallow deer.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 273A:741–751 (2003) Histological Studies of Bone Formation During Pedicle Restoration and Early Antler Regeneration in Roe Deer and Fallow Deer UWE KIERDORF,1* ELKE STOFFELS,2 DIETER STOFFELS,2 HORST KIERDORF,3 THOMAS SZUWART,4 AND GÜNTER CLEMEN2 1 Institute of General and Systematic Zoology, Justus Liebig University of Giessen, Giessen, Germany 2 Institute of Animal Evolution and Ecology, University of Münster, Münster, Germany 3 Department of Biology, University of Hildesheim, Hildesheim, Germany 4 Clinical Anatomy, Anatomical Institute, University of Münster, Münster, Germany ABSTRACT The purpose of the present study was to examine the process of bone formation in the regenerating cranial appendages of roe deer (Capreolus capreolus) and fallow deer (Dama dama) during the early postcasting period. After the antlers are cast, osteoclastic and osteoblastic activities lead to a smoothing of the pedicle’s separation surface, a strengthening of the pedicle bone, and a partial restoration of the distal pedicle portion that was lost along with the cast antler. Initially, bone formation occurs by intramembranous ossiﬁcation, but early during the regeneration process cartilage is formed at the tips of the cranial appendages, and is subsequently replaced by bone in a process of endochodral ossiﬁcation. Shortly after the antlers are cast, the cambium layer of the periosteum in the distal pedicle is markedly enlarged, which suggests that the periosteum serves as a cell source for the bone-forming tissue covering the exposed pedicle bone. The histological ﬁndings of our study are consistent with the view that the bony component of the regenerating cranial appendages of deer is largely derived from the pedicle periosteum. Based on ﬁndings in other bone systems, we speculate that stem cells that can undergo both osteogenic and chondrogenic differentiation are present in the pedicle periosteum. The early onset of chondrogenesis in the regeneration process is regarded as an adaptation to the necessity of producing a huge volume of bone within a short period. This parallels the situation in other cases of chondrogenesis in membrane bones. Anat Rec Part A 273A:741–751, 2003. © 2003 Wiley-Liss, Inc. Key words: deer; antler; pedicle; regeneration; periosteum; intramembranous ossiﬁcation; endochondral ossiﬁcation; cartilage Antlers are periodically cast and regrown cephalic appendages of deer. Since the replacement of antlers constitutes the only case of regeneration of a complete bony appendage in mammals (Goss, 1984, 1992; Bubenik, 2001), this developmental process deserves special attention. Antlers do not grow directly from the top of the deer skull; rather, they are cast and regenerate from the apices of permanent protuberances of the frontal bones, the pedicles. There is considerable evidence supporting the notion that the timing of the antler cycle is closely linked to seasonal ﬂuctuations in concentrations of circulating sex steroids—particularly testosterone (Wislocki et al., 1947; Goss, 1968; Suttie et al., 1995). Thus, premature casting of hard antlers can be induced by castration (Wislocki et al., 1947; Lincoln, 1971; Goss et al., 1992; Kierdorf et al., © 2003 WILEY-LISS, INC. 1995), as well as by the administration of compounds that inhibit LH and thus testosterone release (medroxyprogesterone acetate), or interfere with both the release of testosterone and its action at the receptor level (cyproterone acetate) (Muir et al., 1982; Bubenik et al., 1987; Kierdorf *Correspondence to: Uwe Kierdorf, Institute of General and Systematic Zoology, Justus Liebig University of Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany. E-mail: email@example.com Received 4 November 2002; Accepted 5 May 2003 DOI 10.1002/ar.a.10082 742 KIERDORF ET AL. et al., 1993; Suttie et al., 1995). On the other hand, antler casting can be postponed by administration of testosterone or estradiol to deer carrying hard antlers (Goss, 1968). These ﬁndings suggest that antler casting is triggered by a decrease in testosterone levels. Antler casting itself is preceded by the action of numerous osteoclasts that erode the bony connection between the antler and pedicle along the future separation plane (Goss et al., 1992). The histogenesis of antlers during the more advanced stages of antler regeneration has been investigated in considerable detail. These studies demonstrated that antler bone is formed by both endochondral and intramembranous ossiﬁcation (Gruber, 1937; Banks, 1974; Banks and Newbrey, 1983a, b; Price et al., 1996). Antler elongation occurs in distal growth zones, where mesenchymal cells sequentially proliferate and differentiate into chondroblasts and chondrocytes. The highly vascularized antler cartilage is then mineralized, eroded by chondro-/osteoclasts, and replaced by bone. Thus, bone is formed by an endochondral process. However, at the periphery of the antler, a sleeve of bone is formed via direct, intramembranous ossiﬁcation by osteoblasts derived from the osteogenic (cambium) layer of the periosteum. So far, less attention has been given to the histogenetic processes that occur shortly after antlers are cast, i.e., during the closure of the casting wound at the apex of the pedicle and the earliest stages of antler regrowth (Gruber, 1937; Wislocki, 1942; Waldo and Wislocki, 1951; Goss et al., 1992). However, a detailed knowledge of these events is crucial to obtain a better understanding of antler regeneration, and to compare it with other restorative or regenerative processes in vertebrates. Therefore, the purpose of the present study was to characterize the process of bone formation during pedicle restoration and early antler regeneration, using light and electron microscopy. MATERIALS AND METHODS Animals and Tissue Sampling The tissues studied originated from four adult roe bucks (Capreolus capreolus; specimens R1–R4, used only for light microscopy), and one juvenile fallow buck (Dama dama; specimen F, used for light and electron microscopy) that were killed quite shortly after their antlers were cast (but at different developmental stages; for details, see the Results section). The four roe bucks were wild animals that were shot during hunting operations outside the normal hunting season (normally, roe bucks are shot only during the hard antler stage). Their heads were cut off and kept frozen until further processing for light microscopy. The fallow buck was a farm animal that was killed by a shot in the head and subsequent exsanguination after its ﬁrst (spike) antlers were cast. The tissues from this buck were sampled and ﬁxed immediately after death for both light and electron microscopy. In the roe bucks, the pedicles and a portion of the adjacent frontal region were removed from the heads with a bone saw, and ﬁxed by immersion in either 3.5% buffered formalin or Bouin’s solution. In two of the animals, the complete pedicles were cut in the median-sagittal plane. In the other two specimens, the pedicles were longitudinally split into four segments by two sections oriented in a median-sagittal and a coronal plane, respectively, using an electric saw equipped with a water-cooled diamond blade. Afterwards, the pedicle segments were removed from the skull by a horizontal section. In order not to dislodge the overlying soft tissues from the pedicle bone during sampling, incisions down to the bone were made with a scalpel prior to sawing. These incisions were kept open during sampling, and care was taken to ensure that the saw cuts exactly followed the incision lines. Tissue samples from the fallow buck were obtained in a similar way as described for the roe deer. Sectioning was performed under constant rinsing with Sørensen’s buffer (pH 7.4). For light microscopy, a medial and a lateral pedicle half were ﬁxed in Bouin’s solution. The remaining two halves from the contralateral side, designated for electron microscopy, were each further sectioned into smaller pieces and ﬁxed in a cold (4°C), phosphate-buffered (pH 7.4) paraformaldehyde-glutaraldehyde-osmium mixture (Hatae et al., 1986). Light Microscopy The tissue samples were decalciﬁed in a saturated aqueous EDTA solution for up to 8 weeks, followed by thorough rinsing ﬁrst in tap water and then in distilled water. Part of the material was shock-frozen (–15°C) and sectioned at 8 –10 m with a frozen-section microtome (type 1206; Reichert-Jung, Nussloch, Germany). The remaining samples were dehydrated in a graded series of isopropanol, transferred into butanol, and embedded in paraplast wax or ﬁbrowax (Plano Co., Wetzlar, Germany). The embedded tissue blocks were sectioned at 6 –10 m, and the sections stained with either hemalum-eosin (HE), Heidenhain’s azan, or alcian blue (pH 2.5)-PAS. Photographs of the stained sections were taken with a photomicroscope (Axioskop 2, Zeiss, Jena, Germany). Electron Microscopy Tissue samples for transmission electron microscopy (TEM) were thoroughly rinsed with Sørensen’s buffer, dehydrated in a graded series of ethanol, transferred into acetone, and embedded in Durcupan (Fluka Chemie, Deisenhofen, Germany). Semithin sections (1–2 m) were cut with an ultramicrotome (Reichert-Jung Ultracut), stained with a 1% toluidine blue-borax solution, and studied by light microscopy. Ultrathin sections were cut with diamond knives, stained with lead citrate (5 min) and uranyl acetate (30 min), and studied with a Zeiss EM 900 transmission electron microscope, operated at an accelerating voltage of 50 keV. RESULTS Specimens R1 and R2 (Largely Unhealed Casting Wound) Since the ﬁndings in these two specimens were similar, they are described together. Healing of the casting wounds had just started in the peripheral portions of the pedicles in both bucks; however, most of the top of the bony pedicle was still a wound area covered by a scab (Fig. 1a). Between the scab and the pedicle bone, a ﬁbrocellular tissue (granulation tissue) was present. This tissue layer was continuous with the connective tissue in the marrow spaces between the spicules of pedicle bone. Numerous leukocytes were present in the scab as well as in the granulation tissue, especially its distal portion. The skin bordering on the casting wound formed a tumescent rim. Starting from this rim, the epidermis had just begun to reepithelialize the wound area (Fig. 1a). At the leading edge, a thin epidermal sheet was migrating beneath the PEDICLE RESTORATION AND ANTLER REGENERATION Fig. 1. Pedicles of roe bucks shortly after antler casting. a: Separation surface of pedicle with numerous, often pointed bone spicules (asterisks). A layer of granulation tissue (G) is present between the scab (S, partially lost during histological processing) and the pedicle bone. The epidermis (E) has just begun to reepithelialize the wound area. C, cambium layer of pedicle periosteum; D, dermis; arrow, sebaceous gland; arrowhead, epidermal papilla. Specimen R1: HE (⫻26, bar ⫽ 500 743 m). b: Peripheral area of pedicle bone. Bundles of collagen ﬁbers (Sharpey’s ﬁbers, arrows) connect periosteum and pedicle bone. C, cambium layer of pedicle periosteum; F, ﬁbrous layer of pedicle periosteum; P, pedicle bone. Specimen R2: HE (⫻105, bar ⫽ 100 m). c: Resorption of pedicle bone (asterisks) by osteoclasts (arrowheads). Specimen R1: HE (⫻83, bar ⫽ 100 m). 744 KIERDORF ET AL. scab. In this region, the epidermis did not possess papillae; however, closely behind the migration front, epidermal papillae were already present that projected into the underlying tissue (Fig. 1a). Also, new hair follicles and sebaceous glands were formed. As is typical of antler velvet, no arrector pili muscles were associated with the hair follicles. The separation surfaces of the bony pedicles were uneven, with numerous projecting osseous spicules (Fig. 1a). The pedicle stumps consisted of lamellar bone (Fig. 1b). In the distal pedicle portion, numerous multinucleated osteoclasts were observed that were situated in Howship’s lacunae and resorbing the pedicle bone (Fig. 1c). The marrow spaces in the distal pedicle were enlarged. No distinct boundary existed between the connective tissue of the marrow spaces deeper within the pedicle and that located in the distal pedicle portion. The thickness of the periosteum lining the pedicle bone (especially that of the inner cambium layer) was markedly increased in the distal compared to the proximal pedicle portion (Fig. 1a and b). In more proximal pedicle portions, bundles of collagen ﬁbers (Sharpey’s ﬁbers) connected periosteum and bone (Fig. 1b). Bone resorption was observed also along the pedicle periphery, especially in specimen R2. Specimen R3 (Early Pedicle Restoration) In this specimen, the bony pedicle was already fully covered by newly formed velvet skin, which consisted of a multilayered epidermis and a dermis (Fig. 2a). However, overt antler regrowth had not yet started. Beneath the regenerated skin at the pedicle top was a cell- and ﬁberrich mesenchymal tissue, which was thicker in peripheral areas than in more central ones, and was peripherally continuous with the pedicle periosteum (Fig. 2a and b). At the separation surface of the pedicle, the osseous projections were much shorter and less pointed than in specimens R1 and R2 (Fig. 2a). This smoothing of the bone surface had apparently been brought about by combined osteoclastic and osteoblastic activities. A few active osteoclasts were still present in the distal pedicle bone. However, the remaining bone surface was covered by osteoblasts, which had laid down a thin layer of new bone onto the surface of the pedicle bone. Evidence for this consisted of a seam of osteoid and the occurrence of osteocytes, which had apparently just become incorporated into the bone matrix (Fig. 2b). A few places also showed the formation of new, slender, osseous trabeculae extending from the much thicker trabeculae of the pedicle stump (Fig. 2b). As in specimens R1 and R2, large marrow spaces were present in the distal pedicle bone (Fig. 2a). Some bone formation was also observed along the sides of the pedicle. In all cases, the new bone was formed by intramembranous ossiﬁcation, and no signs of chondrogenesis were observed. Specimen F (Advanced Stage of Pedicle Restoration) Wound healing in this individual was almost complete. Except for a scab-covered central area (approximately 5 mm in diameter), the pedicle top was covered by newly formed velvet skin. The former separation surface was slightly concave (Fig. 3a). In the distal pedicle bone, the marrow spaces between the osseous trabeculae were enlarged. The relatively thick osseous trabeculae of the pedicle stump were continuous with much thinner, newly formed trabeculae (Fig. 3a and b). The osteoid secreted by the osteoblasts lining these newly formed trabeculae (Fig. 3c) was undergoing rapid mineralization, and cells were observed that had just become entrapped in the collagenrich bone matrix as osteocytes (Fig. 3d). By this appositional bone formation, the trabeculae increased in thickness. Occasionally, multinucleated osteoclasts eroding the newly formed bone were also observed. Thus, osteoblastic and osteoclastic activities occurred simultaneously. The intertrabecular mesenchymal tissue contained bundles of collagen ﬁbers oriented parallel to the slender trabeculae, as well as numerous capillaries (Fig. 3c and e). The newly formed osseous trabeculae were mainly oriented in a vertical direction (Fig. 3b and c). Electron microscopy showed that the osteoblasts lining the newly formed osseous trabeculae possessed an acentric nucleus consisting mainly of euchromatin, a well developed rough endoplasmic reticulum, Golgi vesicles and secondary lysosomes, numerous mitochondria, and a meshwork of intermediary ﬁlaments (Fig. 4a). The cells were in contact with each other by long, slender processes. The extracellular matrix contained high numbers of collagen ﬁbers, and numerous mineralization foci were observed (Fig. 4a). The osteocytes were completely surrounded by mineralized matrix (Fig. 4b). The cells were connected by cell processes lying in canaliculi, and possessed fewer mitochondria and a less developed rough endoplasmic reticulum than the osteoblasts. Distally, the zone of newly formed, slender osseous trabeculae merged into a cell-rich, ﬁbrous, and highly vascularized mesenchymal tissue (Fig. 3a and e). Within this tissue, cellular condensations associated with reticular bundles of collagen ﬁbers were present (Fig. 3e). The cells within these condensations contained numerous mitochondria and a prominent rough endoplasmic reticulum (Fig. 5). In the distal direction, the bundles of collagen ﬁbers became less prominent. The extracellular matrix of the mesenchymal tissue did not exhibit the alcianophilia, which histochemically characterizes the presence of acid mucosubstances of the cartilage matrix (not shown). This lack of alcianophilia suggested that chondrogenesis had not yet started in this specimen. Peripherally, the mesenchymal tissue was continuous with the cambium layer of the periosteum (Fig. 3a). Specimen R4 (Early Antler Regeneration) In this specimen, the antlers had already regenerated to a length of approximately 4 cm and were covered by velvet skin. The pedicle bone and the proximal portion of the regenerated antler were lined by a periosteum/perichondrium that was divided into an outer ﬁbrous and an inner cambium/cellular layer (Fig. 6a). In the distal direction, both layers increased in thickness, and the layering itself became less distinct. The tip of the regenerating antler was capped by a highly vascularized hyperplastic perichondrium (not shown). Beneath this hyperplastic perichondrium, mesenchymal cells sequentially differentiated into chondroblasts and chondrocytes. The chondrocytes were arranged in mostly vertically oriented columnar structures that were separated by a highly vascularized mesenchymal tissue (Fig. 6b). PEDICLE RESTORATION AND ANTLER REGENERATION 745 Fig. 2. Early pedicle restoration in a roe buck. a: The former casting wound is completely covered by newly formed skin. Note the smoothing of the pedicle bone’s separation surface compared to Figure 1. The square indicates the region shown in b. C, cambium layer of pedicle periosteum; F, ﬁbrous layer of pedicle periosteum; asterisks, pedicle bone. Specimen R3: HE (⫻22, bar ⫽ 500 m). b: Higher magniﬁcation of the pedicle region indicated by the square in a. Beginning bone formation on the surface of the pedicle bone (asterisk). Black arrows, thin layer of osteoid; white arrows, osteocytes that were recently incorporated into the osteoid; arrowhead, slender bony outgrowth extending from the pedicle bone; Ob, seam of osteoblasts; P, pedicle periosteum. Specimen R3: HE (⫻180, bar ⫽ 50 m). Further proximally, the chondrocytes underwent hypertrophic changes, and the cartilage matrix became mineralized. The calciﬁed cartilage was then eroded by multinucleated chondro-/osteoclasts, and woven bone was laid down on the remnants of the cartilaginous trabeculae (Fig. 6c). These combined chondroclastic and osteoblastic activities were responsible for the formation of a primary spongiosa, composed of bone and calciﬁed cartilage. Further proximally, the primary spongiosa, whose trabeculae no longer exhibited a preferential orientation, was converted into a secondary spongiosa consisting of lamellar bone. Along the periphery of the growing antler, a sleeve of bone was formed by intramembranous ossiﬁcation from the periosteum. The present investigation provides histological information on pedicle restoration and early antler regeneration in roe and fallow deer. Since the studied specimens represent different stages of this developmental process, we combined the results to hypothetically reconstruct the basic histogenetic events that occur during this period of cephalic appendage regeneration in deer. Shortly after the antlers were cast, ongoing osteoclastic bone resorption was observed in the distal pedicle. Active osteoclasts were also present along the pedicle periphery. The bone resorption leading to antler casting occurs both deep within the pedicle and along the pedicle periphery DISCUSSION 746 KIERDORF ET AL. (Gruber, 1937; Waldo and Wislocki, 1951; Wislocki and Waldo, 1953; Goss et al., 1992). Osteoclastic activity is most intense in a rather narrow layer, which corresponds to the future separation plane. However, some bone resorption also occurs in other areas of the pedicle surface, as was observed in the present study and in previous studies by Gruber (1937) and Kierdorf U et al. (1994). In accordance with the ﬁndings of Gruber (1937), the present investigation showed that bone erosion in the distal pedicle continues for some time after antler casting, leading to a smoothing of the uneven separation surface. However, osteoclastic activity is soon reduced, and the histological picture is then dominated by bone formation. In this way, the pedicle bone is strengthened, and a base for the regenerating antler is formed. While initially osteoblastic activity is limited to a thickening of the preexisting osseous trabeculae of the pedicle stump, later new, slender, osseous trabeculae extending from the separation plane are also formed. The formation of a narrow zone of delicate osseous trabeculae, which leads to a partial restoration of the distal pedicle portion lost along with the cast antler, was described by Gruber (1937) in the roe deer and by Wislocki (1942) in the white-tailed deer (Odocoileus virginianus). Corroborating and extending previous observations by Gruber (1937), the present study demonstrated by both light and electron microscopy that this narrow bone zone is formed by intramembranous ossiﬁcation. During the later stages of cranial appendage regeneration, the ossiﬁcation process in the distal portion of the developing outgrowth is clearly an endochondral one, in that cartilage is formed ﬁrst and is subsequently replaced by bone. This mode of bone formation corresponds to the situation during more advanced stages of antler regrowth (Gruber, 1937; Banks, 1974; Banks and Newbrey, 1983a, b; Price et al., 1996). There appears to be a parallel between the sequence of events occurring in cranial appendage regeneration in deer and in secondary bone repair in fractured mammalian long bones. For example, osteogenesis in the fracture callus is likewise reported to include initial intramembranous bone formation mediated by cells undergoing primary osteoblastic differentiation, and secondary endochondral bone formation with chondrocytic differentiation of progenitor cells and subsequent replacement of cartilage by bone (Yoo and Johnstone, 1998). The origin of the cells forming the bony component of the regenerating antler has been a matter of controversy (Goss, 1985, 1995; Kierdorf and Kierdorf, 1992, 2001; Li and Suttie, 2001). Based on histological studies in whitetailed deer, Wislocki (1942, p. 379) stated that, “one gains Fig. 3. Advanced stage of pedicle restoration in a fallow buck. a: Overview of regenerated cranial appendage. Newly formed slender osseous trabeculae (T) distal to the pedicle stump (asterisk). The zone of the slender trabeculae is capped by mesenchymal tissue (M). D, dermis; E, epidermis. The rectangles indicate the regions shown in b, c, and e. Specimen F: Heidenhain’s azan (⫻6.4, bar ⫽ 1.25 mm). b: Higher magniﬁcation of the transition zone between the pedicle stump (asterisk) and the newly formed osseous trabeculae (T). The mesenchymal tissue (M) overlying the trabecular bone is seen in the upper left corner of the ﬁgure. Specimen F, Heidenhain’s azan (⫻13.2, bar ⫽ 600 m). c: Newly formed, mostly vertically oriented osseous trabeculae (T) lined by osteoblasts (arrow). Note the presence of numerous capillaries (asterisk) in the intertrabecular mesenchymal tissue. Specimen F: Heidenhain’s azan (⫻53, bar ⫽ 150 m). d: Higher magniﬁcation of a newly formed osseous trabecula with some recently incorporated cells (osteocytes, black arrow). White arrows, osteoblasts; asterisk, mineralized bone matrix. Specimen F: semithin section, toluidine blue-borax (⫻211, bar ⫽ 38 m). e: Cellular condensations and accompanying reticular bundles of collagen ﬁbers (arrow) in the richly vascularized mesenchymal tissue overlying the zone of the newly formed osseous trabeculae. Asterisk, vascular space. Specimen F: Heidenhain’s azan (⫻53, bar ⫽ 150 m). PEDICLE RESTORATION AND ANTLER REGENERATION Fig. 4. Cells from the newly formed osseous trabeculae of the fallow deer pedicle. a: Osteoblast from the periphery of a newly formed osseous trabecula. Note the richness of collagen ﬁbers (cut at different angles) in the extracellular matrix, and the occurrence of numerous foci of mineralization (arrows) in that matrix. ER, rough endoplasmic reticulum; N, nucleus; arrowhead, cell process of osteoblast; asterisks, min- 747 eralized bone matrix. Transmission electron micrograph (⫻4,540, bar ⫽ 2 m). b: Osteocyte from a newly formed osseous trabecula. The cell is embedded in mineralized bone matrix. M, mitochondrion; N, nucleus; arrowhead, cell process of osteocyte. Transmission electron micrograph (⫻6,600, bar ⫽ 2 m). 748 KIERDORF ET AL. Fig. 5. Cells from the cellular condensations in the ﬁbrous mesenchymal tissue of the fallow deer pedicle distal to the newly formed osseous trabeculae. The cells contain numerous mitochondria (M) and a prominent rough endoplasmic reticulum (ER). N, nucleus; asterisks, bundles of collagen ﬁbers in the extracellular matrix cut at different angles (⫻4,800, bar ⫽ 2 m). the impression that it is the proliferating deeper portion of the corium, rather than adjacent periosteal tissue, which restores the surface of the pedicle and gives rise to the osteogenic germinal bed.” However, based largely on the morphological analysis of so-called double-head antlers, other authors have proposed that the pedicle periosteum is the (main) cell source for the osteogenic component of the regenerating antler (Nitsche, 1898; Kierdorf and Kierdorf, 1992, 2001; Kierdorf U et al., 1994). Gruber (1937) suggested that in addition to periosteal cells, cells originating from the marrow spaces of the pedicle also contribute to the osteogenic tissue that covers the separation surface of the pedicle. Results of genetic labeling of cells from the antlerogenic periosteum (i.e., the specialized periosteum overlying the prospective pedicle region (Hartwig and Schrudde, 1974; Goss and Powel, 1985)) indicate that the bony component of pedicles and ﬁrst antlers is derived from this periosteum (Li and Suttie, 2001). As yet, corresponding data for the regenerating antler are not available. The histological ﬁndings of the present study are consistent with the view that the bony component of the regenerating cranial appendages of deer is, at least to a large extent, derived from the pedicle periosteum. Shortly after casting, the periosteum of the distal pedicle portion (especially its cambium layer) is markedly increased in thickness, which suggests that it serves as the major source of osteogenic cells. However, some contribution of endosteal cells or of cells originating from the marrow spaces of the pedicle to the osteogenic tissue can not be excluded. Evidence from in vivo and in vitro studies on other skeletal structures indicates that periosteal cells can un- dergo both osteogenic and chondrogenic differentiation, and that both osteoblast and chondroblast precursors are located in the cambium layer of the periosteum (Fang and Hall, 1997; Yoo and Johnstone, 1998; Ito et al., 2001). Moreover, there is evidence to support the notion that a common precursor population of both chondrogenic and osteogenic cells exists in the periosteum (Fang and Hall, 1997; Ito et al., 2001). It may thus be speculated that also in the case of cranial appendage regeneration in deer, the pedicle periosteum is a source of both osteoblast and chondroblast progenitors, which originate from a common stem cell population. The histological ﬁndings of the present study indicate that early postcasting bone is formed by intramembranous ossiﬁcation; however, the process of bone formation at the tip of the cranial appendage soon changes to an endochondral one. This could be taken as indicating that osteoblasts are initially recruited from the (hypothetical) stem cell population residing in the periosteum, and that later a chondrogenic pathway is followed. Further studies are necessary to address this question of a potential sequential recruitment of osteoblast and chondroblast precursors from the periosteum in the case of cranial appendage regeneration in deer. Chondrogenesis in membrane bones occurs wherever and whenever rapid skeletal growth is required, and is considered to be an adaptive response to local microenvironmental stimulation. Fang and Hall (1997) noted that cartilage is the only skeletal tissue that has active cell division, and chondrogenic differentiation therefore achieves a larger volume of skeletal tissue within a shorter period compared to osteogenesis. These authors convincingly argued that the chondrogenic potential of PEDICLE RESTORATION AND ANTLER REGENERATION Fig. 6. Early regenerating antler of a roe buck. a: Middle portion of regenerated antler. The cartilage (asterisks) is lined by a perichondrium consisting of an inner cellular (C) and an outer ﬁbrous layer (F). D, dermis; E, epidermis; H, hair follicle; S, sebaceous gland; arrows, vascular spaces. Specimen R4: Heidenhain’s azan (⫻26, bar ⫽ 500 m). b: Vertical columns of cartilage (C) separated by highly vascularized mes- 749 enchymal tissue. Arrowheads, vascular spaces. Specimen R4: Heidenhain’s azan (⫻80, bar ⫽ 100 m). c: Zone of cartilage resorption and replacement by bone. Asterisk, cartilage; arrows, newly formed bone; arrowheads, multinucleated chondro-/osteoclasts; I, intertrabecular tissue. Specimen R4: Heidenhain’s azan (⫻160, bar ⫽ 50 m). 750 KIERDORF ET AL. membrane bone is retained for circumstances in which such a rapid increase of bone volume is demanded—for example, in the case of fracture repair. The pedicles of deer are outgrowths from the frontals and as such represent membrane bones. Moreover, the rapidity of antler growth, which leads to the formation of a huge volume of bone within a seasonally ﬁxed period of only a few months, is without parallel in other skeletal structures in vertebrates. Thus it comes as no surprise that cartilage is formed very early in the process of cranial appendage regeneration, and that the pedicle periosteum is apparently capable of producing not only osteoblast precursors but also chondroblast precursors. There are certain parallels between antler regrowth and the formation of the primary cranial appendages of deer (i.e., the pedicles and ﬁrst antlers) (Kierdorf and Kierdorf, 2002). Studies in fallow deer and red deer (Cervus elaphus) have demonstrated that in these species only the very early stages of pedicle growth occur by intramembranous ossiﬁcation, whereas most of the elongation is brought about by proliferation of cartilage, which is subsequently replaced by bone in a process of endochondral ossiﬁcation (Kierdorf H et al., 1994; Li and Suttie, 1994; Szuwart et al., 1994). Compared to fallow and red deer, in the roe deer the onset of chondrogenesis occurs much later, when the cranial outgrowths have already reached a considerable height (Gruber, 1937). Since roe deer are smaller than both fallow deer and red deer, and their pedicles contain a much smaller volume of bone compared to that in the other two species, this interspecies difference with regard to the start of cartilage formation during cranial appendage development correlates with the view that chondrogenesis by membrane bones is a speciﬁc growth response when rapid formation of large volumes of bone is required. Based on studies in red deer, Li and Suttie (2000) proposed that the stimulus for the onset of cartilage formation during pedicle growth may be a mechanical pressure on the underlying tissues caused by the stretched pedicle skin (which results from the subdermal expansion of the bony component of the pedicle). It seems unlikely that this “pressure hypothesis” can be extended to also cover the onset of chondrogenesis in the case of cranial appendage regeneration in deer. In pedicle formation, a full-thickness scalp skin is initially stretched by a subcutaneously expanding tissue mass, and only during later stages of development is this stretch released by neogenesis of skin (Li and Suttie, 2000). In contrast, during cranial appendage regeneration, growth of the bony component and rapid new (velvet) skin formation occur simultaneously and in a coordinated manner. It is therefore unlikely that the newly formed velvet skin exerts any signiﬁcant pressure on the underlying tissues. In conclusion, the present investigation revealed that after antlers are cast, osteoclastic and osteoblastic activities lead to a smoothing of the separation surface, a strengthening of the pedicle bone, and a partial restoration of the distal pedicle, thereby forming the base for subsequent antler regeneration. Bone formation during early cranial appendage regeneration initially occurs by intramembranous ossiﬁcation, while antler elongation is later brought about by the proliferation of cartilage, which is subsequently replaced by bone. The histological ﬁndings of our study are consistent with the view that the bony component of the regenerating antler is largely derived from the cells of the pedicle periosteum. It is further suggested that the cells originating from the pedicle periosteum can undergo both osteogenic and chondrogenic differentiation, and that the early onset of chondrogenesis is a reaction to the necessity of producing a huge volume of bone within a short period. LITERATURE CITED Banks WJ. 1974. The ossiﬁcation process of the developing antler in the white-tailed deer (Odocoileus virginianus). Calcif Tissue Res 14:257–274. Banks WJ, Newbrey JW. 1983a. Light microscopic studies of the ossiﬁcation process in developing antlers. In: Brown RD, editor. 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