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The macroscopic and microscopic structure of double-head antlers and pedicle bone of Cervidae (Mammalia, Artiodactyla)
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The macroscopic and microscopic structure of double-head antlers and pedicle bone of Cervidae (Mammalia, Artiodactyla) Uwe Kierdorf*, Horst Kierdorf* * and Michael Schultz***
* I. Zoo logi sches Instit ut de r Universitat Gotti nge n , Berliner StraBe 28 , D - 37073 G6ttingcn. ** Zoologisches lnstitut der Unive rsitat zu Kol n , Wey erta l 1[9, D - 50923 K61n, and ;'*·1' Zentru m Anatomic der Universitat Gottingen, Kreuzbergring 36, D - 37075 Gottingen, Germ any
Summary. Ortho- and heterotopicall y formed doubl e-h ead antlers were studied in red , fall ow and roe deer. The malform ation was the res ult of new antler grow th without pre viou s casting of the old antlers. Thus. two antler structures belonging to successive antl er ge nerations originated from one pedicle. These two structures were alw ays separated by a hori zont al groove. H istologicall y . sig ns of osteoclastic resorpt ion were observed in the inter ior and at the outer circumference of the distal parts of the pedicl es of the doubl e heads . The resorpti ve process had . ho wever. not been of an intensity necessary for subsequent antler casting . We also obse rved that the doubl e-h ead ' s seco nd antler gen eration had develope d as a per iosteal exostos is of the distal pedicle bone. Thu s, we assum e that in norm ogenesis form ation of the bony component of subsequent antlers is also probably dependent on cell s derived from pedicle periosteum . Finally . the process of antler regrowth in deer is compared with epimorphic rege nera tion occur ring in other vertebrates .
Key words: Antler regrowth - Bone reso rption - Deer Exostosis - Pedicle periosteum
Introduction The repl acem ent of antlers in deer is a developm ent al eve nt unp arallel ed in other mammals , since antlers are regarded as bein g the only mamm alian bon y appe ndages capable of
Correspond ence to : U . Kierdorf
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Ann Anat (1994) 176: 251-257 Gustav Fischer Verlag Jena
co mplete regeneration (Goss 1983, 1984 , 1992). At present, the origin of the cells forming the bony component of the regro wing velvet antler is still uncertain, as are the de velopmental conditions go vern ing subseq uent antler growth . According to both Nitsche (189 8) and Raesfeld (1919), the regro wing antler beam is buil t up ex clusively of cells originating from the perio steum of the pedicl e . Gruber (1937 " 195 2), who histologicall y studied antl er formation in the roe deer (Capreolus capreolus) , states that the proliferative zo ne of the seco ndary antler beam is form ed by cambial ce lls of the periosteum migrat ing onto the cas ting surface and by ce lls or iginating fro m the marr ow cav ities of the pedicle. In contras t to this, Wi slocki (1942) on the basis of light microscopical investigations in white-tailed deer (Odocoileus virgin ianu s), assumed that the oste ogenic germinal bed of the growing antler is made up of cells of the pedi cle ' s corium , as oppose d to its per iosteum . He therefore postulated "that the grow th of the entire antler is prob abl y dependent upon and co ntrolled by the formation of a germinal or periosteal bed derived from the skin" (Wislocki 1942 , 379 ). A similar view was later also expressed by Frank enberger ( 1954) . According to Goss et a!. ( 1992) , the cell s forming the new antler bud are derived from the connective tissues of the pedicle skin and from the tissu e present in and above the eroded pedicle bon e . If the views of the latter authors are cor rect , subsequent antler grow th wo uld not be a mere perio steal response , i.e. an activation of already osteogenic cell material (bone lining ce lls of the periosteum ), but wo uld co nstitute metapl asia of former co nnective tissue cells into chondro- and osteoblasts .
As already noticed by Olt (1921) and later stressed especially by Goss (1983, 1984, 1985, 1992), the healing process of the casting wound on top of the pedicle differs from that of other wounds in postnatal mammals in that no scar is formed. Rather, a mass of regenerative tissue gives rise to new bone growth. According to Goss (1983, 1984, 1992), the regrowing antler bud can be compared to regeneration blastemata found in other vertebrate taxa. More insight into the process of antler regrowth can be achieved by analyzing a particular antler malformation. the so-called "double head" (Altum 1896: Kierdorf and Kierdorf 1992 b), which results from new antler growth without previous casting of the old antler. Therefore. in the case of double-head antlers, either un i- or bilaterally two antler beams belonging to successive antler generations originate from one pedicle. Rarely, even tnple-head antlers, consisting of three consecutive antler generations, have been observed (Altum 1896: Raesteld 1920: Bubenik 1966).
Results Macroscopic observations In the fallow buck with bilateral double-head antlers, the bone tissue of the double head's second antler generation had on both sides formed a rim with a coronet-like surface structure, which was located around the distal part of the pedicles and beneath the spikes (Fig. I). The bases of the spikes, which represented the double-head's first antler generation, exhibited the knobby thickenings typical of first antlers in fallow deer. On the left side, the second antler generation was confined to the bony rim (Fig. I), whereas on the right side, an outgrowth about 24 ern in length had developed from its caudolateral portion.
Materials and methods The following specimens were at our disposal for macroscopical inspection: a) The bilateral double-head antlers of a fallow buck (Dam« aged 2.5 years. The double-head structure consisted of the undetached primary antlers (spikes) and the subsequently formed secondary antlers.
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b) The unilateral (left side) double-head antler of another fallow buck, aged 3.5 years. This structure consisted of the proximal part of an undetached secondary antler. the distal part of which had been cut after velvet shedding. and the subsequently grown antler. c) The malformed antlers of a l3-year-old red deer stag tCcrvus elaphus). Detailed morphoiogical analysis had previously revealed that these antlers represented the second antler generation of a double head, the first antler generation of which had been belatedly cast (Kierdorf and Kierdorf I L)L) I ) d) Two atypically shaped double-head structures that had successively developed on the front leg of a roe buck. Heterotopic antler growth had been induced by autologous transplanting antlerogenic ("initial") periosteum to the metacarpus (Hartwig and Schrudde 1974). A pedicle had not formed at this site. In the years preceding double-head formation. the heterotopic antler structure had always been cast in due time For light microscopical observations. epoxy resin embedded. proximodistally oriented ground sections of 50 and 70 um thickness were prepared from segments of the three double-head structures of the fallow bucks (specimens A and 1:3: Il'It and right double head respectively, of the 2. 5-year-old buck: specimen C: left sided double head of the .\.5-year-old hue], I and from the right cranial appendage of the red deer stag I specimen f)) according to the method described by Schultz and Drommer ( IL)S3) and Schultz (1988). All segments included pedicle as well as antler bone. Sections were viewed and photographed in normal and polarized transmitted light.
Fig. I. Lett double-head antler of the 2.5-year-old fallow buck. First (asterisk) and second antler generation separated by a groove (arrow), P: pedicle. Fig. 2. Heterotopic double-head structure from the front leg of a roc buck First (asterisk) and second antler generation separated by a groove (arrow).
In the second fallow buck, bone tissue of the double head's second antler generation had also formed a coronetlike structure around the proximal part of the previous
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antler. From this coronet an anterior out growth (corrcspond ing to the brow tine ) 5 crn in len gth . and a lateral. terminally forked outgrowth 3X cm in length had de veloped. Len gth of the antl er beam on the right side was 46 em. In the red deer stag , the cas ting surfaces of the pedic les. formed by belated detachm en t of the double head ' s first antler ge neration, were surro unded by a slightly more proxima lly located rim of antler bone that had a coro net-like surface structure and gave rise to several outgrowths . T his antler bone co nstituted the seco nd ge neration of the for mer doubl e head . On the right side an ou tgrow th of 30 ern length or iginated from the med ial portion of the rim . Additio nally, two sma ll protubera nces had developed from its occ ipital and its occ ipitolatera l port ion On the left side , an outgrowth of 23 em length . corresponding to the brow tine, originated from the anter ior portion of the bony rim , and a larger out growth. 55 em in length. from its occ ipitolateral portion . In both heterotopic doubl e-h ead antlers of the roe buck . the seco nd antler generatio n was a lso represented by a ring-like mass of antler bone (Fig. 2). In these cases no outgrowths had sprouted from the rims. As ca n be dedu ced from the above descript ions . the mass of the bon e tissue form ing the dou ble-heads' seco nd antler generations was in each case lo wer than that for norm ally grow n subsequent antlers of the respective spec ies . Furthermore , in eac h of the complete dou bleheads analyzed , the antl er struct ures of the two generations were separated by a th in g roove (Figs . I . 2 ). The exi stence of such groove s. previously occ upied by velvet cove ring the seco nd antler ge nerat ion . is indica tive of do uble-heads and can therefore be used as a di scrimin ative criterion in differential diag nos is of antler- base malformations (Hartw ig 1981 ).
Microscopic observations A schematic illustration of the gro und sections is given in Figur es 3 a-d . Histolo gic ally . the pedicle s con sisted of dense os teo nic bone . Apart fro m a few typical Haversian sys tems , many modifi ed osteo ns, resembling the "FascrIilze' and " Faserfilz-Osteone" desribed by Kriese ct al. ( 1954) in human bone , were obse rved (F ig. 5 ). The bone formin g the proximal parts of the double heads ' first and seco nd antler ge neration (the antler regio ns analyzed in this study) also co nta ined both typical and modified Haversian systems, with primar y osteo ns dominating over seco nda ry ones (Fig. 4). In the basa l areas of the first antler ge neration. outer circumferential lamell ae were absent, the os teo ns co ntinuing up to the anatom ical surface of the antler beam (F ig. 6) . Th is is ev idence of a high degree of bone rem odel ing at these sites . In the compl ete doubl e heads analyzed . signs of osteo clastic resorption (i. e. Howships lacun ae ) wer e observed at various sites in the distal parts of the pedicles (Figs . 7. 8) . This resorption occurred both in the interior of the
pedicl e bone , leadin g to formation of large reso rptio n cav ities (Fig. 8), and in the bone lining the med ial and dorsal portions of the groove separating the two antler generations of the dou ble heads. In the latter location , signs of osteoclas ia were not only found on the pedicl e but also in the mediobasal area of the first antler genera tion dorsally bordering on the furrow . In co ntrast, no signs of resorpti on were observed in the bone formin g the proxim al borde r of the groove . In the areas of transition from pedi cle bone to that of the do uble heads' first and seco nd antler ge nera tion, respectively, no struc tural discontinuities were observed . Furthermore , at the interface between pedicl e and antler of the seco nd generation, no indica tion of a supraperiostea l nature of the antler bone was discernible. On the co ntrary, ped icle and antler bone clearl y formed a morphological entity . the latter being best characterized as an exostosis of the former . Due to a rather sharp change in the co urse followed by the bundles of co llage n fibers in the transition zone between pedicle and antler bone (of first and seco nd ge neration). the pedicl e-antl er boundary could , howe ver , be clearly demonstrated by polarized light microscop y (Fig . 9) . In the double-h ead structures from both fallow deers, surface appos ition of rather porous wove n bone with predo minantly radial orientatio n of co llagen fiber s was see n in the distal part of the ped icle as well as in the adjoining basa l area of the seco nd antler ge nera tion (Fig. 10). In the pedicle of the red deer spec imen, this surface layer of wove n bone had to a large ex tent alrea dy been repla ced by lamel lar bone. lea ving only traces of the former struc ture.
Discussion We believe that the present paper is the first one to give a detailed microscopical descri ption of the structures forming a double-head antler as well as of the adjacent pedicle bone . As has been dem on strated histologically, osteocl astic activity had occurred in the distal part of the pedi cle , both within the bone and at its o uter circumference . Th is is in full acco rdance with earl ier observations on the proces s of bone resorpt ion preced ing antler castin g (Ko llike r 1873 : Gruber 1937 : Wald o and Wislocki 195 1: Goss et al. 1992 ). Norma lly , detachm ent of the old antl ers by the action of mechani cal forces has already taken place before the last bon y trabecul ae co nnecting the rema ining part of the pedi cle with the antler bea m have been erod ed (Bertho ld 1831 : Kolliker 1873 ; Gruber 1937). Regardin g the com plete doubl e head s histolog icall y anal yzed in this study . it can be ass umed that osteoclastic activity during the " pre-c asting period" , although present , had not been of sufficient inten sity to allow for subseq uent det achm ent of the o ld antlers . As a result of this. no casting surface exi sted onto which cells of adja cent pedicle tissues could migrate . Instead of forming an antler bud on top of the pedicle , the
253
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b Fig. 3. Schematic illustrations (a-d) of ground sections, anterior view; the positionof the areas shown in Figures 4 to 10is indicated, a: specimen A, b: specimen B, c: specimen C. d: specimen D, stippled: pedicle, vertical hatching; first generation of double-head antler, horizontal hatching: second generation of double-head antler.
newly formed osteogenic tissue therefore established itself beneath the base of the undetached beams. As was seen in the fallow and red deer specimens examined, either uni- or bilaterally beam- or tine-like outgrowths emerged from this basal bulge of antler tissue. The fact that the growth of these beams or tines was rather limited in comparison with those of normal antlers can be attributed to a deficiency in the blood supply, due mainly to mechanical impairment of proper blood vessel development caused by the pressure of the basal part of the undetached beam on the adjacent growing velvet antler (Kierdorf and Kierdorf I991, 1992 b). The high incidence of modified osteons ("'faserfilze"
and "Faserfilz-Osteone'', Knese et al. 1954) in pedicle bone as well as in proximal antler bone is regarded as specific for these regions, since in a previous study (Kierdorf et al. 1993) it has been demonstrated that typical Haversian systems are found in the more distal regions of (fallow deer) antlers approximately 8 em above the coronet. "Faserfilz-Osteone" of the type found in pedicle bone have been observed both in normally structured bone (mandibular body, bony muscle attachments) as well as in bone tumors, especially solid, osteoblastic neoplasms (Schultz 1993). In the present study it has been further demonstrated histologically that the bone tissue forming the double-head's second antler generation develops as an
254
Fig. 4. Basal portion of a double head's second antler generation exhibiting modified as well as typical Haversian systems. Specimen A, ground section (50 urn), polarized light, x 57.
exostosis of the distal pedicle bone, the two structures thus forming a morphological entity. No observations were made that would point to a supraperiosteal origin of the bony tissue of the second antler generation. On the contrary, this antler bone has to be regarded as a product of the pedicle periosteum. Our results therefore corroborate the earlier findings of Nitsche (1898) who, on the basis of macroscopic inspection, stated that the double head' s second antler generation develops as a periosteal exostosis. This raises the question of whether formation of new antler bone (at least in this special case) can be regarded as a true epimorphic response, including generation of a blastema as well as cartilage and bone formation by cells of other than periosteal origin (Goss 19(2), or would be better characterized as the result of exaggerated tissue repair. A second case of periosteal osteogenesis. seen in the fallow deer (specimens A, B and C), is the apposition of woven bone onto the surface of the pedicle and the antler beam of the second generation. Regarding the pedicle. this apposition leads to thickening with age. a fact already described by Gruber (1937). As can be deduced from our results, in the (permanent) pedicles the initially formed porous, woven bone is during remodeling later replacement by lamellar bone of higher density. The fact that, in the case of double heads, new antler bone is derived from periosteal and not from suprape riosteal sources, suggested to Nitsche (1898) that also in norrnogenesis, i.e. in the case of antler bud formation on a casting surface, cells of periosteal origin are exclusively responsible for antler bone formation. On the basis of the available data, it is not yet possible to decide definitely whether the cells forming the osteogenic component of the regrowing velvet antler are derived solely from pedicle periosteum, or if and to what extent cells derived from the dermis or subcutis participate in the process of bone formation. However, taking into account the results of Nitsche (1898), Gruber 0937. IlJ52) and that of the present study, we conclude it to be unlikely that the cells
forming the new antler bone tissue are entirely of skin origin, as was assumed by Wislocki (1942). If the orthotopically developing antler bud really corresponds to a regeneration blastema, as has been proposed by Goss (1983, 1984, 1(92), we would expect the participation of cell material of other than periosteal origin in cartilage and bone formation. In order to decide whether the regrowing antler bud is a typical blastema, ultrastructural studies are necessary. Special regard should be given to the structure of the epitheliomesenchymal interface, because absence of the basal lamina seems to be an essential precondition for blastema formation and subsequent epimorphic regeneration (Neufeld 1(89). To us, the fact that, in the case of the double head's second generation, antler bone is formed in direct continuity with pedicle bone by periosteal activity, suggests that also in the process of normal antler regrowth, cells of periosteal origin are decisive for the process of antler bone formation, i.e. they carry the morphogenetic information necessary for renewed antler growth. This view is supported by the results of studies on experimental antler induction in deer. In a series of studies on red deer it has been shown that pedicle formation (but not subsequent antler growth) could be induced in males castrated before puberty (Jaczewski and Krzywinska 1(74) as well as in normal and ovariectomized females (Jaczewski 1976, 1977, 1981) by the administration of high doses of testosterone. Antler growth in these animals could be provoked by amputating the distal part of the pedicle. The procedure was. however, effective only when the wound involved bone and periosteum: wounding the pedicle skin alone was not followed by antler growth. With the same method, antler growth could also be induced in so-called hummels, i.e. fertile red deer stags possessing pedicles but lacking antlers (Lincoln and Fletcher 1(76), and in a white-tailed doe that had developed pedicles following postnatal blockade of the ovarian function (Bubenik 1(90). As has been already discussed earlier (Kierdorf and Kierdorf 1992a), the hypothesis that cells of the pedicle periosteum are determined for the growth of subsequent antler bone, irrespective of a possible contribution of other cell material to the antler bud, can be further based on the fact that this periosteum is a derivative of the antierogenic or initial periosteum responsible for the formation of primary cranial appendages, i.e. pedicles and first antlers (Hartwig and Schrudde 1974; Goss and Powel 1(85). It was therefore assumed that the pedicle periosteum may have retained the latter's state of determination, i.e. its antlerogenic tendencies, and is thus ultimately responsible for new antler growth in the orthotopic position. In the case of double-head structures developing on the front leg of the roe buck, it has been argued that growth of the second antler generation was triggered by cells derived from the heterotopically transplanted initial periosteum (Kierdorf and Kierdorf 1992a). Recently, Goss (1991) has produced experimental evicdence suggesting that the antlerogenic periosteum also carries morphogenetic information for the shape of primary and subsequent antlers.
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Fig. 5 . Dense osteonic pedicle bone consisting of a few typical Haversian systems and of modified osteons. Specimen B, ground section (50 um) , polarized light using a quart z (I), X 57. Fig, 6. Basal area of a double head's first antler genera tion. Osteonic bone reaching up to the anatomica l surface of the antler beam (upper right comer of figure). Specimen A , ground section (50 urn), polarized light using a quartz (I), X 57. Fig. 7. Howship's lacunae (arrows) in the peripheral area of the pedicle bone. Specimen A, ground section (50 urn), polarized light using a quartz (I) , X 57 . Fig, 8. Resorption cavities formed by osteoclastic activity in the interior of the pedicle, arrows : Howship's lacunae. Spec imen A , ground section (50 urn), polarized light using a quartz (I) , X 57. Fig . 9 , Zone of transition between pedicle bone (lower left part of figure) and bone of the double head' s second antler generation. Specimen A , ground section (50 urn), polarized light using a quartz (1), X 57 . Fig . 10. Spicules of woven hone with radially oriented collagen fibers at the surface of tl1e double head 's second antler generation. Specimen B, ground section (50 urn), polarized light using a quartz (I ), X 57 .
Acknowledgements. The expert technical assistance of Mr.
References
M, Brandt is gratefully acknowledged . We are very much indebted to the Verein der Freunde und Forderer der Universitat ZlI K61n e. V. for their generous assistance in financing the colour reproductions.
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Wiedererzeugung der Hirschgeweihe. Beitr zur Anatomie, Zootomie, Physiologie 5: 39-96 Bubenik AB (1966) Das Geweih. Parey, Hamburg Berlin Bubenik GA (1990) The role of the nervous system in the growth of antlers. In: Bubenik GA, Bubenik AB (eds) Horns, pronghorns, and antlers. Springer, New York, pp 339- 358 Frankenberger Z (1954) Pucnice a paroh. Ceskoslov Morfol 2: 89-95 Goss RJ (1983) Deer antlers. Academic Press, New York Goss RJ (1984) Epimorphic regeneration in mammals. In: Hunt RB, Heppenstall RB, Pines E, Rovee D (eds) Soft and hard tissue repair. Praeger, New York, pp 554- 573 Goss RJ (1985) Tissue differentiation in regenerating antlers. In: Fennessy PF, Drew KR (eds) Biology of deer production. Royal Soc New Zealand Bull 22: 229-238 Goss RJ (1991) Induction of deer antlers by transplanted periosteum: III. Orientation. J Exp Zool 259: 246-251 Goss RJ (1992) Regeneration versus repair. In: Cohen IK, Diegelmann RF, Lindblad WJ (eds) Wound healing - Biochemical and clinical aspects. Saunders, Philadelphia, pp 20- 39 Goss RJ, Powei RS (1985) Induction of deer antlers by transplanted periosteum I. Graft size and shape. J Exp ZooI 235: 359-373 Goss RJ, van Praagh A, Brewer P (1992) The mechanism of antler casting in the fallow deer. J Exp Zool 264: 429-436 Gruber GB (1937) Morphobiologische Untersuchungen am Cerviden-Geweih, Nachr Ges Wiss Gottingen, Math-Physik KI NF 3: 9-63 Gruber GB (1952) Uber das Wesen der Cervidengeweihe. Dtsch Tierarzt Wschr 59: 225-228 and 241-243 Hartwig H (1981) RosenmiBbildung bei einem Rehgehorn. Z Jagdwiss 27: 287-291 Hartwig H, Schrudde J (1974) ExperimenteJle Untersuchungen zur Bildung der primaren Stirnauswiichse beim Reh (Capreolus capreolus L.). Z Jagdwiss 20: 1-13 Jaczewski Z (1976) The induction of antler growth in female deer. Bull Acad Polon Sci Cl II 24: 61- 65 Jaczewski Z (1977) The artificial induction of antler cycles in female red deer. Deer 4: 83-86 Jaczewski Z (1981) Further observations on the induction of antler growth in red deer females. Folia BioI 29: 131-140 Jaczewski Z, Krzywinska K (1974) The induction of antler growth in a red deer male castrated before puberty by traumatization of the pedicle. Bull Acad Polon Sci CI V 22: 67-72 Kierdorf H, Kierdorf U (1992a) State of determination of the antlerogenic tissues with special reference to double-head formation. In: Brown RD (ed) Biology of deer. Springer, New York, pp 525-531
Kierdorf U, Kierdorf H (1991) Abnortnes Rothirschgeweih als Folge einer Doppelkopfbildung. Nieders Jager 36: 564-567 Kierdorf U, Kierdorf H (1992b) Der Doppelkopf, ein fur das Verstandnis der Folgegeweih-Bildung aufschluBreiches Naturexperiment. Z Jagdwiss 38: 244-251 Kierdorf U, Schultz M, Fischer K (1993) Effects of antiandrogen treatment on the antler cycle of male fallow deer (Dama dama L.). J Exp Zool 266: 195-205 Knese KH, Voges D, Ritschl I (1954) Untersuchungen iiber die Osteon- und Lamellenfortnen im Extremitatenskelett des Erwachsenen. Z Zellforsch 40: 323-360 Kolliker A (1873) Die normale Resorption des Knochengewebes und ihre Bedeutung fur die Entstehung der typischen Knochenformen. Vogel, Leipzig Lincoln GA, Fletcher TJ (1976) Induction of antler growth in a congenitally polled scottish red deer stag. J Exp Zool 195: 247-251 Neufeld DA (1989) Epidermis, basement membrane, and connective-tissue healing after amputation in mouse digits: implications for mammalian appendage regeneration. Anat Rec 223: 425-432 Nitsche H (1898) Studien tiber Hirsche. Engelmann, Leipzig Olt A (1921) Uber den innersekretorischen EinfluB der Hoden auf die Entwicklung des Cervidengeweihes. Dtsch Jager-Zeitung 76: 417-420 and 433-436 Raesfeld Fv (1919) Das Rehwild. 2. Aufl, Parey, Berlin Raesfeld Fv (1920) Das Rotwild. 3. Aufl, Parey, Berlin Schultz M (1988) Methoden der Licht- und Elektronenmikroskopie. In: Knussmann R (ed) Anthropologie, Vol I, 1, Fischer, Stuttgart, pp 698-730 Schultz M (1993) Initial stages of systemic bone disease. In: Grupe G, Garland AN (eds) Histology of ancient human bone. Springer, Berlin, pp 185-203 Schultz M, Drommer R (1983) Moglichkeiten der Praparateherstellung aus dem Gesichtsschadelbereich fur die makroskopische und mikroskopische Untersuchung unter Verwendung neuer Kunststofftechniken. In: Hoppe W (ed) Experimentelle Mund-Kiefer-Gesichtschirurgie. Mikrochirurgische Eingriffe. Thieme, Stuttgart, pp 95-97 Waldo CM, Wislocki GB (1951) Observations on the shedding of the antlers of Virginia deer (Odocoileus virginianus borealis). Am J Anat 88: 351-396 Wislocki GB (1942) Studies on the growth of deer antlers. I. On the structure and histogenesis of the antlers of the virginia deer (Odocoileus virginian us borealis). Am J Anat 71: 371-415
Accepted June 14, 1993
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The macroscopic and microscopic structure of double-head antlers and pedicle bone of Cervidae (Mammalia, Artiodactyla) Uwe Kierdorf*, Horst Kierdorf* * and Michael Schultz***
* I. Zoo logi sches Instit ut de r Universitat Gotti nge n , Berliner StraBe 28 , D - 37073 G6ttingcn. ** Zoologisches lnstitut der Unive rsitat zu Kol n , Wey erta l 1[9, D - 50923 K61n, and ;'*·1' Zentru m Anatomic der Universitat Gottingen, Kreuzbergring 36, D - 37075 Gottingen, Germ any
Summary. Ortho- and heterotopicall y formed doubl e-h ead antlers were studied in red , fall ow and roe deer. The malform ation was the res ult of new antler grow th without pre viou s casting of the old antlers. Thus. two antler structures belonging to successive antl er ge nerations originated from one pedicle. These two structures were alw ays separated by a hori zont al groove. H istologicall y . sig ns of osteoclastic resorpt ion were observed in the inter ior and at the outer circumference of the distal parts of the pedicl es of the doubl e heads . The resorpti ve process had . ho wever. not been of an intensity necessary for subsequent antler casting . We also obse rved that the doubl e-h ead ' s seco nd antler gen eration had develope d as a per iosteal exostos is of the distal pedicle bone. Thu s, we assum e that in norm ogenesis form ation of the bony component of subsequent antlers is also probably dependent on cell s derived from pedicle periosteum . Finally . the process of antler regrowth in deer is compared with epimorphic rege nera tion occur ring in other vertebrates .
Key words: Antler regrowth - Bone reso rption - Deer Exostosis - Pedicle periosteum
Introduction The repl acem ent of antlers in deer is a developm ent al eve nt unp arallel ed in other mammals , since antlers are regarded as bein g the only mamm alian bon y appe ndages capable of
Correspond ence to : U . Kierdorf
~I
Ann Anat (1994) 176: 251-257 Gustav Fischer Verlag Jena
co mplete regeneration (Goss 1983, 1984 , 1992). At present, the origin of the cells forming the bony component of the regro wing velvet antler is still uncertain, as are the de velopmental conditions go vern ing subseq uent antler growth . According to both Nitsche (189 8) and Raesfeld (1919), the regro wing antler beam is buil t up ex clusively of cells originating from the perio steum of the pedicl e . Gruber (1937 " 195 2), who histologicall y studied antl er formation in the roe deer (Capreolus capreolus) , states that the proliferative zo ne of the seco ndary antler beam is form ed by cambial ce lls of the periosteum migrat ing onto the cas ting surface and by ce lls or iginating fro m the marr ow cav ities of the pedicle. In contras t to this, Wi slocki (1942) on the basis of light microscopical investigations in white-tailed deer (Odocoileus virgin ianu s), assumed that the oste ogenic germinal bed of the growing antler is made up of cells of the pedi cle ' s corium , as oppose d to its per iosteum . He therefore postulated "that the grow th of the entire antler is prob abl y dependent upon and co ntrolled by the formation of a germinal or periosteal bed derived from the skin" (Wislocki 1942 , 379 ). A similar view was later also expressed by Frank enberger ( 1954) . According to Goss et a!. ( 1992) , the cell s forming the new antler bud are derived from the connective tissues of the pedicle skin and from the tissu e present in and above the eroded pedicle bon e . If the views of the latter authors are cor rect , subsequent antler grow th wo uld not be a mere perio steal response , i.e. an activation of already osteogenic cell material (bone lining ce lls of the periosteum ), but wo uld co nstitute metapl asia of former co nnective tissue cells into chondro- and osteoblasts .
As already noticed by Olt (1921) and later stressed especially by Goss (1983, 1984, 1985, 1992), the healing process of the casting wound on top of the pedicle differs from that of other wounds in postnatal mammals in that no scar is formed. Rather, a mass of regenerative tissue gives rise to new bone growth. According to Goss (1983, 1984, 1992), the regrowing antler bud can be compared to regeneration blastemata found in other vertebrate taxa. More insight into the process of antler regrowth can be achieved by analyzing a particular antler malformation. the so-called "double head" (Altum 1896: Kierdorf and Kierdorf 1992 b), which results from new antler growth without previous casting of the old antler. Therefore. in the case of double-head antlers, either un i- or bilaterally two antler beams belonging to successive antler generations originate from one pedicle. Rarely, even tnple-head antlers, consisting of three consecutive antler generations, have been observed (Altum 1896: Raesteld 1920: Bubenik 1966).
Results Macroscopic observations In the fallow buck with bilateral double-head antlers, the bone tissue of the double head's second antler generation had on both sides formed a rim with a coronet-like surface structure, which was located around the distal part of the pedicles and beneath the spikes (Fig. I). The bases of the spikes, which represented the double-head's first antler generation, exhibited the knobby thickenings typical of first antlers in fallow deer. On the left side, the second antler generation was confined to the bony rim (Fig. I), whereas on the right side, an outgrowth about 24 ern in length had developed from its caudolateral portion.
Materials and methods The following specimens were at our disposal for macroscopical inspection: a) The bilateral double-head antlers of a fallow buck (Dam« aged 2.5 years. The double-head structure consisted of the undetached primary antlers (spikes) and the subsequently formed secondary antlers.
dania),
b) The unilateral (left side) double-head antler of another fallow buck, aged 3.5 years. This structure consisted of the proximal part of an undetached secondary antler. the distal part of which had been cut after velvet shedding. and the subsequently grown antler. c) The malformed antlers of a l3-year-old red deer stag tCcrvus elaphus). Detailed morphoiogical analysis had previously revealed that these antlers represented the second antler generation of a double head, the first antler generation of which had been belatedly cast (Kierdorf and Kierdorf I L)L) I ) d) Two atypically shaped double-head structures that had successively developed on the front leg of a roe buck. Heterotopic antler growth had been induced by autologous transplanting antlerogenic ("initial") periosteum to the metacarpus (Hartwig and Schrudde 1974). A pedicle had not formed at this site. In the years preceding double-head formation. the heterotopic antler structure had always been cast in due time For light microscopical observations. epoxy resin embedded. proximodistally oriented ground sections of 50 and 70 um thickness were prepared from segments of the three double-head structures of the fallow bucks (specimens A and 1:3: Il'It and right double head respectively, of the 2. 5-year-old buck: specimen C: left sided double head of the .\.5-year-old hue], I and from the right cranial appendage of the red deer stag I specimen f)) according to the method described by Schultz and Drommer ( IL)S3) and Schultz (1988). All segments included pedicle as well as antler bone. Sections were viewed and photographed in normal and polarized transmitted light.
Fig. I. Lett double-head antler of the 2.5-year-old fallow buck. First (asterisk) and second antler generation separated by a groove (arrow), P: pedicle. Fig. 2. Heterotopic double-head structure from the front leg of a roc buck First (asterisk) and second antler generation separated by a groove (arrow).
In the second fallow buck, bone tissue of the double head's second antler generation had also formed a coronetlike structure around the proximal part of the previous
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antler. From this coronet an anterior out growth (corrcspond ing to the brow tine ) 5 crn in len gth . and a lateral. terminally forked outgrowth 3X cm in length had de veloped. Len gth of the antl er beam on the right side was 46 em. In the red deer stag , the cas ting surfaces of the pedic les. formed by belated detachm en t of the double head ' s first antler ge neration, were surro unded by a slightly more proxima lly located rim of antler bone that had a coro net-like surface structure and gave rise to several outgrowths . T his antler bone co nstituted the seco nd ge neration of the for mer doubl e head . On the right side an ou tgrow th of 30 ern length or iginated from the med ial portion of the rim . Additio nally, two sma ll protubera nces had developed from its occ ipital and its occ ipitolatera l port ion On the left side , an outgrowth of 23 em length . corresponding to the brow tine, originated from the anter ior portion of the bony rim , and a larger out growth. 55 em in length. from its occ ipitolateral portion . In both heterotopic doubl e-h ead antlers of the roe buck . the seco nd antler generatio n was a lso represented by a ring-like mass of antler bone (Fig. 2). In these cases no outgrowths had sprouted from the rims. As ca n be dedu ced from the above descript ions . the mass of the bon e tissue form ing the dou ble-heads' seco nd antler generations was in each case lo wer than that for norm ally grow n subsequent antlers of the respective spec ies . Furthermore , in eac h of the complete dou bleheads analyzed , the antl er struct ures of the two generations were separated by a th in g roove (Figs . I . 2 ). The exi stence of such groove s. previously occ upied by velvet cove ring the seco nd antler ge nerat ion . is indica tive of do uble-heads and can therefore be used as a di scrimin ative criterion in differential diag nos is of antler- base malformations (Hartw ig 1981 ).
Microscopic observations A schematic illustration of the gro und sections is given in Figur es 3 a-d . Histolo gic ally . the pedicle s con sisted of dense os teo nic bone . Apart fro m a few typical Haversian sys tems , many modifi ed osteo ns, resembling the "FascrIilze' and " Faserfilz-Osteone" desribed by Kriese ct al. ( 1954) in human bone , were obse rved (F ig. 5 ). The bone formin g the proximal parts of the double heads ' first and seco nd antler ge neration (the antler regio ns analyzed in this study) also co nta ined both typical and modified Haversian systems, with primar y osteo ns dominating over seco nda ry ones (Fig. 4). In the basa l areas of the first antler ge neration. outer circumferential lamell ae were absent, the os teo ns co ntinuing up to the anatom ical surface of the antler beam (F ig. 6) . Th is is ev idence of a high degree of bone rem odel ing at these sites . In the compl ete doubl e heads analyzed . signs of osteo clastic resorption (i. e. Howships lacun ae ) wer e observed at various sites in the distal parts of the pedicles (Figs . 7. 8) . This resorption occurred both in the interior of the
pedicl e bone , leadin g to formation of large reso rptio n cav ities (Fig. 8), and in the bone lining the med ial and dorsal portions of the groove separating the two antler generations of the dou ble heads. In the latter location , signs of osteoclas ia were not only found on the pedicl e but also in the mediobasal area of the first antler genera tion dorsally bordering on the furrow . In co ntrast, no signs of resorpti on were observed in the bone formin g the proxim al borde r of the groove . In the areas of transition from pedi cle bone to that of the do uble heads' first and seco nd antler ge nera tion, respectively, no struc tural discontinuities were observed . Furthermore , at the interface between pedicl e and antler of the seco nd generation, no indica tion of a supraperiostea l nature of the antler bone was discernible. On the co ntrary, ped icle and antler bone clearl y formed a morphological entity . the latter being best characterized as an exostosis of the former . Due to a rather sharp change in the co urse followed by the bundles of co llage n fibers in the transition zone between pedicle and antler bone (of first and seco nd ge neration). the pedicl e-antl er boundary could , howe ver , be clearly demonstrated by polarized light microscop y (Fig . 9) . In the double-h ead structures from both fallow deers, surface appos ition of rather porous wove n bone with predo minantly radial orientatio n of co llagen fiber s was see n in the distal part of the ped icle as well as in the adjoining basa l area of the seco nd antler ge nera tion (Fig. 10). In the pedicle of the red deer spec imen, this surface layer of wove n bone had to a large ex tent alrea dy been repla ced by lamel lar bone. lea ving only traces of the former struc ture.
Discussion We believe that the present paper is the first one to give a detailed microscopical descri ption of the structures forming a double-head antler as well as of the adjacent pedicle bone . As has been dem on strated histologically, osteocl astic activity had occurred in the distal part of the pedi cle , both within the bone and at its o uter circumference . Th is is in full acco rdance with earl ier observations on the proces s of bone resorpt ion preced ing antler castin g (Ko llike r 1873 : Gruber 1937 : Wald o and Wislocki 195 1: Goss et al. 1992 ). Norma lly , detachm ent of the old antl ers by the action of mechani cal forces has already taken place before the last bon y trabecul ae co nnecting the rema ining part of the pedi cle with the antler bea m have been erod ed (Bertho ld 1831 : Kolliker 1873 ; Gruber 1937). Regardin g the com plete doubl e head s histolog icall y anal yzed in this study . it can be ass umed that osteoclastic activity during the " pre-c asting period" , although present , had not been of sufficient inten sity to allow for subseq uent det achm ent of the o ld antlers . As a result of this. no casting surface exi sted onto which cells of adja cent pedicle tissues could migrate . Instead of forming an antler bud on top of the pedicle , the
253
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b Fig. 3. Schematic illustrations (a-d) of ground sections, anterior view; the positionof the areas shown in Figures 4 to 10is indicated, a: specimen A, b: specimen B, c: specimen C. d: specimen D, stippled: pedicle, vertical hatching; first generation of double-head antler, horizontal hatching: second generation of double-head antler.
newly formed osteogenic tissue therefore established itself beneath the base of the undetached beams. As was seen in the fallow and red deer specimens examined, either uni- or bilaterally beam- or tine-like outgrowths emerged from this basal bulge of antler tissue. The fact that the growth of these beams or tines was rather limited in comparison with those of normal antlers can be attributed to a deficiency in the blood supply, due mainly to mechanical impairment of proper blood vessel development caused by the pressure of the basal part of the undetached beam on the adjacent growing velvet antler (Kierdorf and Kierdorf I991, 1992 b). The high incidence of modified osteons ("'faserfilze"
and "Faserfilz-Osteone'', Knese et al. 1954) in pedicle bone as well as in proximal antler bone is regarded as specific for these regions, since in a previous study (Kierdorf et al. 1993) it has been demonstrated that typical Haversian systems are found in the more distal regions of (fallow deer) antlers approximately 8 em above the coronet. "Faserfilz-Osteone" of the type found in pedicle bone have been observed both in normally structured bone (mandibular body, bony muscle attachments) as well as in bone tumors, especially solid, osteoblastic neoplasms (Schultz 1993). In the present study it has been further demonstrated histologically that the bone tissue forming the double-head's second antler generation develops as an
254
Fig. 4. Basal portion of a double head's second antler generation exhibiting modified as well as typical Haversian systems. Specimen A, ground section (50 urn), polarized light, x 57.
exostosis of the distal pedicle bone, the two structures thus forming a morphological entity. No observations were made that would point to a supraperiosteal origin of the bony tissue of the second antler generation. On the contrary, this antler bone has to be regarded as a product of the pedicle periosteum. Our results therefore corroborate the earlier findings of Nitsche (1898) who, on the basis of macroscopic inspection, stated that the double head' s second antler generation develops as a periosteal exostosis. This raises the question of whether formation of new antler bone (at least in this special case) can be regarded as a true epimorphic response, including generation of a blastema as well as cartilage and bone formation by cells of other than periosteal origin (Goss 19(2), or would be better characterized as the result of exaggerated tissue repair. A second case of periosteal osteogenesis. seen in the fallow deer (specimens A, B and C), is the apposition of woven bone onto the surface of the pedicle and the antler beam of the second generation. Regarding the pedicle. this apposition leads to thickening with age. a fact already described by Gruber (1937). As can be deduced from our results, in the (permanent) pedicles the initially formed porous, woven bone is during remodeling later replacement by lamellar bone of higher density. The fact that, in the case of double heads, new antler bone is derived from periosteal and not from suprape riosteal sources, suggested to Nitsche (1898) that also in norrnogenesis, i.e. in the case of antler bud formation on a casting surface, cells of periosteal origin are exclusively responsible for antler bone formation. On the basis of the available data, it is not yet possible to decide definitely whether the cells forming the osteogenic component of the regrowing velvet antler are derived solely from pedicle periosteum, or if and to what extent cells derived from the dermis or subcutis participate in the process of bone formation. However, taking into account the results of Nitsche (1898), Gruber 0937. IlJ52) and that of the present study, we conclude it to be unlikely that the cells
forming the new antler bone tissue are entirely of skin origin, as was assumed by Wislocki (1942). If the orthotopically developing antler bud really corresponds to a regeneration blastema, as has been proposed by Goss (1983, 1984, 1(92), we would expect the participation of cell material of other than periosteal origin in cartilage and bone formation. In order to decide whether the regrowing antler bud is a typical blastema, ultrastructural studies are necessary. Special regard should be given to the structure of the epitheliomesenchymal interface, because absence of the basal lamina seems to be an essential precondition for blastema formation and subsequent epimorphic regeneration (Neufeld 1(89). To us, the fact that, in the case of the double head's second generation, antler bone is formed in direct continuity with pedicle bone by periosteal activity, suggests that also in the process of normal antler regrowth, cells of periosteal origin are decisive for the process of antler bone formation, i.e. they carry the morphogenetic information necessary for renewed antler growth. This view is supported by the results of studies on experimental antler induction in deer. In a series of studies on red deer it has been shown that pedicle formation (but not subsequent antler growth) could be induced in males castrated before puberty (Jaczewski and Krzywinska 1(74) as well as in normal and ovariectomized females (Jaczewski 1976, 1977, 1981) by the administration of high doses of testosterone. Antler growth in these animals could be provoked by amputating the distal part of the pedicle. The procedure was. however, effective only when the wound involved bone and periosteum: wounding the pedicle skin alone was not followed by antler growth. With the same method, antler growth could also be induced in so-called hummels, i.e. fertile red deer stags possessing pedicles but lacking antlers (Lincoln and Fletcher 1(76), and in a white-tailed doe that had developed pedicles following postnatal blockade of the ovarian function (Bubenik 1(90). As has been already discussed earlier (Kierdorf and Kierdorf 1992a), the hypothesis that cells of the pedicle periosteum are determined for the growth of subsequent antler bone, irrespective of a possible contribution of other cell material to the antler bud, can be further based on the fact that this periosteum is a derivative of the antierogenic or initial periosteum responsible for the formation of primary cranial appendages, i.e. pedicles and first antlers (Hartwig and Schrudde 1974; Goss and Powel 1(85). It was therefore assumed that the pedicle periosteum may have retained the latter's state of determination, i.e. its antlerogenic tendencies, and is thus ultimately responsible for new antler growth in the orthotopic position. In the case of double-head structures developing on the front leg of the roe buck, it has been argued that growth of the second antler generation was triggered by cells derived from the heterotopically transplanted initial periosteum (Kierdorf and Kierdorf 1992a). Recently, Goss (1991) has produced experimental evicdence suggesting that the antlerogenic periosteum also carries morphogenetic information for the shape of primary and subsequent antlers.
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Fig. 5 . Dense osteonic pedicle bone consisting of a few typical Haversian systems and of modified osteons. Specimen B, ground section (50 um) , polarized light using a quart z (I), X 57. Fig, 6. Basal area of a double head's first antler genera tion. Osteonic bone reaching up to the anatomica l surface of the antler beam (upper right comer of figure). Specimen A , ground section (50 urn), polarized light using a quartz (I), X 57. Fig. 7. Howship's lacunae (arrows) in the peripheral area of the pedicle bone. Specimen A, ground section (50 urn), polarized light using a quartz (I) , X 57 . Fig, 8. Resorption cavities formed by osteoclastic activity in the interior of the pedicle, arrows : Howship's lacunae. Spec imen A , ground section (50 urn), polarized light using a quartz (I) , X 57. Fig . 9 , Zone of transition between pedicle bone (lower left part of figure) and bone of the double head' s second antler generation. Specimen A , ground section (50 urn), polarized light using a quartz (1), X 57 . Fig . 10. Spicules of woven hone with radially oriented collagen fibers at the surface of tl1e double head 's second antler generation. Specimen B, ground section (50 urn), polarized light using a quartz (I ), X 57 .
Acknowledgements. The expert technical assistance of Mr.
References
M, Brandt is gratefully acknowledged . We are very much indebted to the Verein der Freunde und Forderer der Universitat ZlI K61n e. V. for their generous assistance in financing the colour reproductions.
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Accepted June 14, 1993
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