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Oct 3, 2002 - assisted histometry (Adobe Photoshop software). Since long bones of the adult mallard are mainly made of primary osteonal tissue ('fibro- ...

J. Anat. (2002) 201, pp521–526


Blackwell Science, Ltd

Laminar bone as an adaptation to torsional loads in flapping flight Emmanuel de Margerie CNRS UMR 8570, 2, place Jussieu, case 7077, 75251 Paris Cedex 05, France

Abstract Using a new histometric method, the orientation of primary osteons was measured in the main long bones of adult mallards (Anas platyrhynchos). In the light of previous biomechanical and ontogenetic studies, a functional hypothesis is proposed, explaining the histological differences observed between long bones; laminar bone tissue, mainly found in the wing bones, may be a biomechanical adaptation to torsional loads caused by flapping flight. Key words Anas platyrhynchos; bone histology; bone microstructure; primary osteon; shear; torsion.

Introduction A large part of the microstructural variability of bone as a tissue remains to be functionally interpreted. Classically, periosteal primary bone microstructure is known to reflect the rate of osteogenesis (Amprino, 1947; de Ricqlès et al. 1991; Castanet et al. 1996, 2000). However, a recent quantitative work on the mallard (Anas platyrhynchos) has shown that the orientation of primary osteons, when present, is quite independent of growth rate, and thus it remains to be functionally interpreted (de Margerie et al. 2002). The aim of the present work was to quantify carefully this primary osteonal orientation in the long bones of the mallard, and to devise a functional hypothesis to explain the pattern of variation observed (i.e. anatomical segregation of tissue types).

Materials and methods This study is based on a sample of 40 bones: eight long bones (humerus, radius, ulna, carpometacarpus, femur, tibiotarsus, tarsometatarsus, second phalanx of toe III) of five hunt-killed adult mallards, 557–931 days old

Correspondence Emmanuel de Margerie, CNRS UMR 8570, 2, place Jussieu, case 7077, 75251 Paris Cedex 05, France. E-mail: [email protected] Accepted for publication 3 October 2002 © Anatomical Society of Great Britain and Ireland 2002

(tagged animals). Decalcified mid-diaphyseal (smallest diameter of bone shaft) cryogenic cross-sections were stained with Ehrlich’s haematoxylin, and observed by natural light microscopy. High-resolution (19 000 dpi, or 0.75 pixels µm−1) digital pictures of the 40 sections (taken with an Olympus C3030 camera mounted on the microscope) were used for the following computerassisted histometry (Adobe Photoshop software). Since long bones of the adult mallard are mainly made of primary osteonal tissue (‘fibro-lamellar complex’ and ‘lamellar bone with primary osteons’, after de Ricqlès et al. 1991), osteonal orientation was estimated through the orientation of the vascular network. Primary vascular canals were classified into four categories, according to their individual orientation in the cortex (Fig. 1): 1 Circular canals (C): canals lying in the plane of section, approximately parallel (0 ± 22.5°) to the bone wall. 2 Radial canals (R): canals lying in the plane of section, approximately orthogonal (90 ± 22.5°) to the bone wall. 3 Oblique canals (O): other canals lying in the plane of section. 4 Longitudinal canals (L): canals going through the plane of section. To be considered as ‘lying in the plane of section’, a canal should exhibit a length/width ratio greater than 3. Otherwise, it was considered to be a longitudinal (L) canal. Anastomosed canals were unconnected and submitted to the same analysis. The true three-dimensional

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bone’ (de Ricqlès et al. 1991). One value of laminarity was thus determined per analysed bone, resulting from the analysis of all primary canals of each section (150– 1500 per section). The effect of the long bone category on laminarity was first tested globally using within-subjects nonparametric analysis of variance (Friedman’s ANOVA). Then differences between pairs of long bone categories were tested by non-parametric multiple comparisons, using the method given by Hollander & Wolfe (1973; p. 151).

Results Analysis of variance demonstrates a strong effect of long bone category on laminarity (P < 0.0002, n = 40). Most notably, multiple comparisons detect significant differences between some wing bones and some leg bones (Fig. 2). A dichotomy near Laminarity = 0.5 differentiates long bones exhibiting ‘laminar bone’ (LAM > 0.5: ulna, humerus, carpometacarpus and femur) from long bones lacking such tissue (toe phalanx, tibiotarsus, tarsometatarsus and radius: primary osteons mostly longitudinal; LAM < 0.5). Note that the first group includes all wing bones except the radius, the second all leg bones except the femur.

Discussion Fig. 1 Measurement of vascular orientation. Bone tissue laminarity was defined as the ratio of the area of the circular canals to total vascular area. C, circular vascular orientation; O, oblique vascular orientation; R, radial vascular orientation; L, longitudinal vascular orientation. See text for further details.

organization of the vascular network is complex (Currey, 1960). The artificial segregation employed here is somewhat simplified, but provides a convenient starting point for a first quantitative histometric analysis of vascular orientation. Secondary canals (Haversian or endosteal) were excluded from analysis. The cumulative vascular area of each of the four categories of canals was calculated for the whole crosssection. A synthetic index of orientation was defined as the ratio of cumulative area of circular canals to the total vascular area. This dimensionless histometric index was called ‘laminarity’ because primary bone with a majority of circular primary osteons is called ‘laminar

Studies of bird and bat wing mechanics (Pennycuick, 1967; Kirkpatrick, 1993) and in vivo strain measurements (Swartz et al. 1992; Biewener & Dial, 1995) have shown that the wing skeleton is subjected to considerable torsional loading during flight, shearing the bony material. Swartz et al. (1992) wrote that ‘torsion and shear are unique and crucial features of skeletal biomechanics during flight’, and that ‘the evolution of skeletal design in bats and other flying vertebrates may be driven by the need to resist these loads’. As shown by the present results, histological differences exist between the wing and leg bones of the mallard. In congruence with the above-mentioned biomechanical findings, the following hypothesis is proposed: within fast-growing bone tissues, primary osteonal orientation may reflect preferential loading mode. Hence laminar bone may be an adaptation (sensu lato) to torsional loads. Whether this correspondence between structure and function results from adaptation sensu stricto (i.e. exclusively from natural selection), from epigenetic © Anatomical Society of Great Britain and Ireland 2002

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Fig. 2 Diagram demonstrating variable laminarity in different long bone categories. Circles: individual values. ANOVA detects a strong effect of bone category on laminarity (P < 0.0002, n = 40). Stars: significant differences revealed by multiple comparisons (5% experimentwise error rate). H, humerus; R, radius; U, ulna; CMC, carpometacarpus; F, femur; TT, tibiotarsus; TMT, tarsometatarsus; P, phalanx 2 of foot digit III.

accommodation during ontogeny (i.e. ‘functional adaptation’ sensu Carter et al. 1991) or both is beyond the scope of the present paper (see De Ricqlès, 1991, for a detailed discussion of such concepts). This putative adaptation must have an ultrastructural basis, perhaps through particular collagen fibre orientation in the primary osteons of laminar bone. However, quantitative investigation of collagen fibre orientation in varied primary osteonal bone tissues remains to be done.

‘Anomalous’ cases The femur, a leg bone with high laminarity, could be considered ‘anomalous’. However, modelling and © Anatomical Society of Great Britain and Ireland 2002

experiments on bipedalism of extant and extinct dinosaurs (Carrano, 1998; Carrano & Biewener, 1999) show that ‘torsion is maximal in horizontal femora’, as is found in birds. Thus the femur, though it is a leg bone, probably experiences torsional loading during terrestrial locomotion, deserving laminar microstructure. The radius, a wing bone with low laminarity, could be considered anomalous, too. But this bone probably endures little torsion: its known function is to act as a ‘pushrod’ between humerus and carpus, coupling movement of the wrist with movement of the elbow (Hildebrand, 1982: p. 556). Moreover, flight loads transmitted through flight feathers affect the ulna, not the radius (Fig. 2). Finally, an examination of the shape of the joints between the wing bones also suggests

524 Bone microstructure in the mallard, E. de Margerie

Fig. 3 The ulna has the highest laminarity in the study. According to the proposed hypothesis, such microstructure might be an adaptation to the torsional loads this bone bares during flapping flight. Its particular shape (high circularity, thin walls), optimal for torsion, supports such an interpretation. As a reference, tibiotarsus exhibits quite opposite characteristics at both structural levels (histology and cross-sectional shape).

that the ulna is the main supporting bone of the zeugopodium, bearing the main part of loads transmitted from stylo- and autopodium (incidentally, the ulna has the highest laminarity of all bones; see Fig. 3). Longitudinally organized bone tissue of the radius and of some other long bones (tibiotarsus, tarsometatarsus, toe phalanx) may be better adapted to longitudinal stresses caused by compression, tension or bending loads. Mechanical tests on the long bones of birds (Cubo & Casinos, 2000), which revealed that the bone material’s bending strength and stiffness are maximal in the radius and tibiotarsus, support this idea.

Alternative hypotheses Previous authors have viewed the function of laminar bone as increasing blood supply, crack-stopping or modulating electrical signals (Currey, 1960, 1984; Macginitie et al. 1997). These hypotheses result from comparisons of laminar bone with Haversian (i.e. secondary) bone. The functional hypothesis presented

here is based on more ‘homologous’ comparisons, within the scope of primary bone tissues. Another hypothesis is to see laminar bone as the result of a faster osteogenesis. This could explain the occurrence of laminar bone in long bones with larger diameters (e.g. humerus, ulna or femur), resulting from faster diametral growth. However, a previous study (de Margerie et al. 2002) has shown that laminar bone does not grow faster than bone tissues with other osteonal orientations (e.g. ‘reticular’ or ‘longitudinal’). The occurrence of laminar bone in long bones with larger diameters is better explained by the new hypothesis: an histological optimization for torsion (i.e. high laminarity) probably goes with a shape optimization for torsion, resulting in large-diameter, thin-walled cross-sections (Alexander, 1968; Currey, 1984; Swartz et al. 1992).

Laminar bone occurrence and loading mode Traditionally, laminar bone is known to be present in bovine bone (an important model in biomedical osteology) © Anatomical Society of Great Britain and Ireland 2002

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and, more generally, in species of terrestrial mammals as large as or larger than dogs (Amprino & Godina, 1947; Amprino, 1947; Enlow & Brown, 1958; Stover et al. 1992). The new hypothesis is supported by numerous in vivo strain measurements that have now demonstrated significant torsion in the limb bones of dogs (Carter et al. 1980), horses (Schneider et al. 1982; Hartman et al. 1984; Gross et al. 1992) and sheep (Gautier et al. 2000), emphasizing the fact that limb (parasagittal) posture and kinematics alone are poor predictors of the real complexity of bone loading (Carter et al. 1980; Demes et al. 1998). Torsional loading (or ‘twisting’) of the entire bone shaft causes shear stresses at the bone tissue level, in the transverse plane as well as in the longitudinal plane (Drucker, 1967). Thus it is less restrictive to say that laminar bone would be an adaptation to shear stress. Indeed, shear stresses in those planes need not be caused by torsional loads. Any loading mode causing principal stresses to lie at some angle to the shaft’s axis would cause shear stresses in the same planes (Drucker, 1967), and thus might also be better resisted by laminar bone. Dynamic variation of the stress orientation during the locomotor cycle (considerable in the bird’s humerus: Biewener & Dial, 1995) is also an important parameter of bone loading mode. Due to the lack of comparative data, it still cannot be said whether laminar bone is an adaptation to static torsional loading, dynamic torsional loading or both.

Conclusion and perspectives After this comparative structural study of the mallard’s long bones, it appears that laminar bone tissue may be a better resistant organization to torsional loading of the bone shaft, as occurs in vertebrates’ wings during flapping flight. This hypothesis needs to be tested in other species, as well as to be given an ultrastructural basis. Mechanical torsional testing of long bones could also give direct evidence for the proposed interpretation. Osteonal orientation in rapidly growing primary bone tissues could become a useful tool for investigating the skeletal biomechanics of extant and extinct tetrapods. It would be of similar significance as crosssectional geometry, but potentially more precise (bone histological structure can differ within a single crosssection; De Ricqlès, 1976), and is more practical to use in fossil crushed and/or fragmentary bones. © Anatomical Society of Great Britain and Ireland 2002

Acknowledgments I would like to thank V. de Buffrénil, J. Castanet, J. Cubo, M. Laurin, J.-Y. Sire and A. de Ricqlès for their useful comments on previous versions of the manuscript, and M.-M. Loth for technical help.

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© Anatomical Society of Great Britain and Ireland 2002

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