The problem of bone lamellation: An attempt to explain different

JOURNAL OF MORPHOLOGY 274:543–550 (2013)

The Problem of Bone Lamellation: An Attempt to Explain Different Proposed Models Gastone Marotti, Marzia Ferretti, and Carla Palumbo* Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Sezione di Morfologia umana, Universita` di Modena e Reggio Emilia, Italy ABSTRACT Collagen texture and osteocyte distribution were analyzed in human woven- and lamellar-bone using scanning and transmission electron microscopy. We provide data substantiating the concept that lamellar bone is made up of an alternation of dense-acellular lamellae and loose-cellular lamellae, all exhibiting an interwoven texture of collagen fibers. An attempt is also made to explain how the present findings might conform to those of authors whose models propose orderly, geometric arrangements of collagen fibers inside bony lamellae. Such a comparison is possible because the present investigation analyzes split loose lamellae and tangentially-sectioned dense lamellae. It emerged that only loose lamellae can be dissected, revealing a loose interwoven collagen texture and halved osteocyte lacunae. Dense lamellae cannot be split because of their compactness. The analysis of tangentially sectioned dense lamellae demonstrates that they consist of a network of interwoven collagen fiber bundles. Inside each bundle, collagen fibers run parallel to each other but change direction where they enter adjacent bundles, at angles as described by other authors whose TEM investigations were performed at a much higher magnification than those of the present study. Consequently, what these authors consider to be a lamella are, instead, bundles of collagen fibers inside a lamella. There is discussion of the role played by the manner of osteocyterecruitment in the deposition of lamellar- and wovenbone and how the presence of these cells is crucial for collagen spatial arrangement in bone tissues. J. Morphol. 274:543–550, 2013. Ó 2013 Wiley Periodicals, Inc. KEY WORDS: dense acellular lamellae; loose cellular lamellae; fiber arrays; osteocyte lacunae

The first hypothesis is known as the Gebhardt’s (1906) model and is currently accepted by the vast majority of authors (Ascenzi and Bonucci, 1967; Ascenzi et al., 1973, 1983, 1987, 2003, 2008; Ascenzi and Benvenuti, 1986; Rho et al., 1999; Weiner et al., 1999; Bromage et al., 2003; GiraudGuille et al., 2003, 2008; Xu et al., 2003; Ascenzi and Lomovtsev, 2006; Hofmann et al., 2006; Wagermaier et al., 2006; Reisinger et al., 2011; Yamamoto et al., 2012). It is cited in almost all histology textbooks as an established fact. Gebhardt’s model was refined by the Giraud-Guille group (Giraud-Guille, 1988, 1989, 1992, 1994, 1996; Besseau and Giraud-Guille, 1995), and the Weiner group (Weiner et al., 1991, 1997; Ziv et al., 1996a,b). Using transmission electron microscopy (TEM), these two research groups identified offset bony lamellae (viz, plywood-units) made up of parallel arrays of fibers of varying orientation in each lamella. In orthogonal plywood-units, the angle between consecutive lamellae is about 908, while in twisting plywood-units the fibrillar directions progress from one lamella to the next at intermediate angles, giving rise to a cholesteric-type structure. It was proposed that each individual lamellar unit (two adjacent lamellae with the interposed transition zone) is composed of five arrays of parallel collagen fibers, each offset by 308 (Weiner et al., 1997). In recent years, various authors have analyzed osteon lamellar organization in relation to mechanical properties (Ascenzi and Lomovtsev 2006; Hofmann et al., 2006) or to cellular content (Ascenzi et al. 2008; Yamamoto et al., 2012), considering bone tissue organization as a precise geometric scheme often also implying synchronized

INTRODUCTION Despite many recent studies, the structure of lamellar bone remains a subject of debate. Currently, two different hypotheses are discussed. In the first, bony lamellae are considered homogeneous in structure, all with the same collagen density and containing orderly arranged collagen fibers; the difference between successive lamellae consists in the orientation of the fibers. In the second, the lamellar pattern of the bone is thought to depend on an alternation of layers of heterogeneous structure in which the collagen fibers are interwoven. Ó 2013 WILEY PERIODICALS, INC.

*Correspondence to: Carla Palumbo, Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, Sezione di Morfologia umana – Istituti Anatomici, Universita` di Modena e Reggio Emilia, Via del Pozzo 71 (area Policlinico), I-41125 Modena, Italy. E-mail: [email protected] Received 18 May 2012; Revised 24 October 2012; Accepted 4 November 2012 Published online 5 January 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmor.20114

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movements of osteoblastic laminae during preosseus matrix deposition. The second hypothesis has less supporters than the first. While several authors have suggested in the past, in relation to diverse issues, that the lamellar aspect of bone depends on an alternation of layers of heterogeneous structure (Ranvier, 1889; Ruth, 1947; Rouiller et al., 1952; Frank et al., 1955; Rouiller, 1956; Mjo¨r, 1969), it was only in 1988 that the present research group used scanning electron microscopy (SEM) to analyze the structure of the same lamellae in both cross- and longitudinal-section, providing clear evidence that lamellar bone is made up of alternating collagenrich (dense lamellae) and collagen-poor (loose lamellae) layers, all with interwoven fibers (Marotti and Muglia, 1988). In a subsequent series of comparative polarized light, SEM and TEM studies, conducted in the 1990s, the authors further demonstrated that: a) lamellar bone can also be considered as a structure in which cellular lamellae alternate with acellular lamellae, given that osteocytes were only found in loose lamellae; b) loose lamellae are thicker than dense lamellae and their thickness closely corresponds to the minor transverse axis of the almond-like shaped osteocyte lacunae; c) loose cellular lamellae are less resistant than dense acellular lamellae, with experimentallyinduced microfractures occurring only in the former (Marotti, 1993, 1996; Marotti et al., 1994a,b). The present paper is based on SEM and TEM observations of human lamellar bone. Besides providing additional findings in support of the concept of lamellar bone structure, an attempt is made to explain how published observations and findings, in particular of the Girauld-Guille and Weiner groups, can conform to the model proposed here. MATERIALS AND METHODS The present study was carried out on human woven- and lamellar-bone (mainly on Haversian systems) of the cortex at the mid-shaft level of the tibiae removed from nine cadavers in six male subjects aged 9, 19, 23, 45, 59, and 70 years. In none of the subjects were recognized pathological skeletal changes detectable at autopsy. All withdrawals were performed according to the Bioethical Committee of the Italian National Institute of Health.

Scanning Electron Microscopy Analyses were performed on cross- and longitudinal sections (50 6 10 lm thick) as well as on cubic or rectangular-prismatic samples (side 5 5–10 mm, edge of angles 5 908 6 108) in order to observe each lamella sectioned both transversely and longitudinally, as previously reported (Marotti, 1993). The samples were fixed in 4% buffered paraformaldehyde and then polished with emery paper and fresh water. Before being gold palladium sputtered (K550, Emitech Ltd, United 224 Kingdom), both sections and prismatic samples were etched with 0.1 N HCl for 90 seconds, treated with trypsin (80 U/ml; pH 7.4 for 4 hours at 378C) alcohol dehydrated and air dried at 378. Same specimens were also treated according to the technique used by Reid (1986), namely, they were fixed in 3% glutaraldehyde buffered

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with 0.15 mol l21 cacodylate, treated with 0.25% trypsin-EDTA at 378C for 2 hours, and critical-point dried (CPD030, Bal-Tec, Liechtenstein). No significant differences were observed among specimens prepared by either method. SEM observations were performed under XL-30-FEI Company (Heidhoven, NL). To test the mechanical resistance of bony lamellae, we submitted to rolling friction (volvent forces) cylindrical samples (about 5 mm), isolated from human compact bone, containing cross-sectioned lamellar osteons.

Transmission Electron Microscopy After fixation in 4% buffered paraformaldehyde, the specimens were decalcified with 10% EDTA for one week, post-fixed in 1% osmium tetroxide in 0.13 M phosphate buffer for about 2 hours, and embedded in epoxy resin (Durcupan ACM). Crossand longitudino-tangential sections (70–80 nm thick) of secondary Haversian systems were obtained with an ultramicrotome (Ultracut Reichert-Jung) and then stained with uranyl acetate and lead citrate before the ultrastructural observation. TEM observations were performed under EM 109 Carl Zeiss AG (Oberkochen, Germany).

RESULTS No significant differences in the structure of bony lamellae were observed in relation to the age of subjects or to the radius of curvature of the microscopic specimens examined (i.e., Haversian systems and outer/inner circumferential systems). Low magnification SEM observation of prismatic bone samples allowed analysis of the same lamellae in both cross- and longitudinal-section (Fig. 1). Each lamella clearly has the same appearance, regardless of the plane of the section, a finding at odds with Gebhardt’s model and other authors who suggest an orderly arrangement of fibers in the bony lamellae. At higher magnification, SEM and TEM both show that lamellar bone is made up of alternating collagen-rich (dense lamellae) and collagen-poor (loose lamellae) layers, all with a highly interwoven arrangement of fibers (Fig. 2). It also appears from both cross and longitudinal sections that the two types of lamellae have different thickness, the loose lamellae being thicker (3.3–4.2 lm) than the dense ones (1.8–2.2 lm) in the majority of samples examined. Additionally, osteocyte lacunae are only located inside loose lamellae and their size (i.e., the minor axis of the triaxial ellipsoid) closely matches the thickness of the loose lamellae (Fig. 3 A,B). Thus, lamellar bone can also be considered as a structure in which cellular lamellae alternate with acellular lamellae. In contrast, osteocyte lacunae in woven-bone exhibit a disorderly distribution and are surrounded by irregularly arranged bundles of collagen fibers (Fig. 3 C). Osteocyte lacunae in both woven and lamellar bone are encircled by a layer of looselyarranged collagen fibers, with a structure resembling that of loose lamellae (Fig. 4). Cylindrical samples of lamellar bone were submitted to volvent forces, making it possible to analyze tangentially-split loose lamellae under SEM

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The dissection of loose lamellae (Fig. 5), besides revealing a dissociation of their interwoven poor collagen content, enabled observation of the surface (see Fig. 6) of the two adjacent dense lamellae containing the splitted loose one. Dissected osteocyte lacunae can also be seen together with the numerous small holes of the osteocyte canaliculi crossing the lamellae. The tangentially sectioned dense lamellae viewed in SEM (Fig. 6) and TEM (Figs. 7 and 8) show three crucial observations: a) dense lamellae are made up of a network of thick interwoven collagen fiber bundles arranged not only along the lamellar plane, but also running perpendicular to the lamellae; b) inside each bundle, collagen fibers run parallel to each other, but than change direction when they enter adjacent bundles, at various angles (frequently of 308); c) collagen fiber bundles curve around the osteocyte canaliculi and cross, at right angle, the fibers running inside the canaliculi

Fig. 1. Scanning electron micrographs of a cross-sectioned secondary lamellar osteons (A) and of a prismatic sample of the same type of osteon (B); the arrows point to the edge of the prismatic sample. Note in B that dense lamellae are thinner than loose lamellae and that each lamella looks the same in both the cross- and longitudinal-sectional surfaces of the osteon. Scale bars: A 5 10 lm , B 5 15 lm.

(Fig. 5) and tangentially-sectioned dense lamellae under SEM and TEM (Figs. 6–8). Only loose lamellae were found to be split, while dense lamellae were not.

Fig. 2. Electron micrographs (A 5 SEM; B 5 TEM) of human lamellar bone tissue, showing the highly intermingled texture of collagen fibers inside both dense (white arrow heads) and loose lamellae. Scale bars: A, B 5 2 lm.

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Fig. 3. Scanning electron micrographs of a cross- (A) and longitudinal-sectioned (B) lamellar bone, and (C) woven bone. Note that osteocyte lacunae are located in lamellae displaying the same loose aspect in both cross- and longitudinal section. Note also the strict correspondence between the minor axis of the lacunae and that of the loose lamellae. Osteocyte lacunae in woven bone (C) are randomly distributed and surrounded by perilacunar matrices (osteocytic collagen) of loosely arranged collagen fibers. Scale bars: A, B, C 5 8 lm.

that perpendicularly pass through the lamellae. These findings indicate that each lamella, independently of its dense or loose micro-architecture, is not only made up of a network of interwoven collagen fibers running in the lamellar plane, but it is also intersected by fibers running both outside and inside the osteocyte canaliculi. DISCUSSION The main difference between the present investigations on lamellar bone and those performed by previous authors consists in the fact that the present study took into account the existence of osteo-

cytes inside the bone matrix. We suggest that disregarding the location and distribution of osteocyte hinders, a priori, the possibility of establishing the true collagen texture of bone tissue. These findings and observations were exhaustively reported in previous papers (Marotti and Muglia, 1988; Marotti, 1993, 1996; Marotti et al., 1994a,b) along with the results on split loose lamellae and tangentially sectioned dense lamellae described here, strongly confirming that lamellar bone is made up of alternating collagen-rich layers without osteocyte cell bodies (dense acellular lamellae) and collagen-poor layers containing osteocytes (loose cellular lamellae), all with a

Fig. 4. Electron micrographs (A 5 SEM; B 5 TEM) of tangentially sectioned osteocyte lacunae in human bone. Note the loose collagen fiber texture in the perilacunar matrix (osteocytic collagen). Scale bars: A, B 5 1 lm.

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Fig. 5. Scanning electron micrograph of a tangentially split loose lamella. The loosely arranged collagen fibers forming the lamella appears dissociated, whereas the upper and lower surfaces bordering the lamella display the compact and interwoven arrangement of the fibers in the two adjacent dense lamellae. Note numerous holes of the osteocyte canaliculi running across the lamellae and, to the right, an osteocyte lacuna cut in half. Scale bar 5 4 lm.

highly interwoven arrangement of fibers. Thus, lamellar bone should be considered a variety of woven bone. Some authors, while accepting the view that collagen fibers do not follow a parallel array in each lamella, assume that significant variations in average fiber orientation between adjacent lamellae have been overlooked. They are convinced that a preferential orientation of collagen fibers must exist inside each lamella (Carando et al., 1989, 1991; Boyde and Riggs, 1990; Riggs et al., 1993a,b; Martin et al., 1996; Ascenzi and Lomovtsev 2006 Ascenzi et al. 2008; Hofmann et al., 2006; Yamamoto et al., 2012). The fact that: a) each lamella always displays the same aspect in both cross- and longitudinal-section, and b) osteocyte lacunae always appear to be located inside lamellae displaying the same loose aspect in both cross- and longitudinal-section, do not fit with a preferential orientation of fibers inside each lamella. If lamellae were comprised as suggested by Gebhardt and the authors supporting his view, their so-called longitudinal lamellae (considered by them to consist of fibers running parallel to the Haversian canal), which appear stippled in cross-section, would appear striped in longitudinal-section, and vice versa. The same applies to the osteocyte lacunae, which always appear located in lamellae of the same loose appearance in both cross- and longitudinal-section. The orderly distribution of osteocytes in lamellar bone (inside loose lamellae only), compared with the random arrangement of osteocyte in woven bone, indicates that the difference in texture between the two bony tissues depends on the distribution of osteocytes in the bone matrix, that is to say, on the

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manner of recruitment of the osteocyte-differentiating osteoblasts from the osteogenic laminae. Since all osteocyte lacunae in both woven- and lamellarbone are surrounded by a layer of loosely-arranged collagen fibers (osteocytic collagen, as named by Boyde 1972), with structure resembling that of loose lamellae, it is likely that loose lamellae can form as a result of the alignment and fusion of the osteocytic collagen of the osteocytes they contain which, in turn, depends on an orderly manner of osteocyte recruitment. On the contrary, in woven-bone, the osteoblasts that differentiate into osteocytes are recruited haphazardly and ‘‘enter’’ the bone in a random fashion. Consequently, woven bone consists of an irregular distribution of osteocyte-rich areas where the collagen is loosely arranged, since it corresponds to that of the osteocytic collagen, interposed with other acellular areas, similar to the structure of dense lamellae (for more exhaustive information, see Marotti 1996). The importance of osteocyte lacunae in conditioning collagen arrangement also emerges from the observations of Ascenzi et al. (2008), when collagen fibers were found to be running with variable (smaller or larger) angles relative to the osteon axis, depending on the location of the fibers,

Fig. 6. Scanning electron micrograph of a tangentiallyviewed dense lamella. This micrograph virtually corresponds to an enlargement of the dense lamella bordering the lower surface of the dissected loose lamella in Figure 5. It is evident that dense lamellae are made up of a network of thick interwoven collagen fiber bundles. Each bundle is made up of an array of parallel collagen fibers, which branch by an angle of about 308 in the transition zones, when they enter adjacent bundles. The holes encircled by the bundles correspond to cross-sectioned osteocyte canaliculi running across the lamellae. Scale bar 5 0.3 lm.

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Fig. 7. Transmission electron micrograph of a tangentially sectioned dense lamella; B corresponds to an enlargement of the squared area in A. Under TEM, too, dense lamellae appear to be made up of a network of interwoven bundles of collagen fibers, which run parallel to each other inside the bundles and change direction where they branch to join the adjacent bundles. In B, the arrows point to cross-sectioned fibers running perpendicular to the plane of the lamella; the arrow head indicates the fibers running inside an osteocyte canaliculus. Hence, dense lamellae do not only contain bundles of fibers irregularly interwoven in the lamellar plane but also fibers running perpendicular to the lamellar plane, both inside and outside the canaliculi. Scale bars: A, B 5 0.6 lm.

respectively, in extint lamellae (perilacunar zone) or in bright lamellae. The analysis of split lamellae revealed, once again, their loose and interwoven arrangement of

collagen fibers. It is important to note that only loose lamellae can be split due to their poor and loosely-arranged collagen content, while dense lamellae cannot be split because of their compact-

Fig. 8. Transmission electron micrograph of a tangentially sectioned dense lamella; B corresponds to an enlargement of the squared area in A. Dense lamellae appear to be made up of a network of interwoven bundles of collagen fibers, which run parallel to each other inside the bundles and change direction where they branch to join the adjacent bundles. Arrows point to cross-sectioned bundles of collagen fibers running at 908 with respect to the lamellar plane. Scale bars: A, B 5 0.3 lm.

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ness. Another interesting point is the evidence for microfracture location only inside cellular splitted loose lamellae, in line with the observations of Ascenzi et al. (2008) that stress and strain are clearly affected by collagen fiber orientation in the equatorial perilacunar region (i.e., the peripheral zone inside loose lamellae, in view of the present authors). Hence, contrary to the proposals of Gebhardt and the authors supporting his view, all lamellae do not share the same collagen density or mechanical resistance (Ascenzi and Lomovtsev 2006; Hofmann et al., 2006), the loose cellular lamellae being weaker than dense acellular one as previously shown (Marotti et al., 1994). Scanning electron microscopy and TEM analyses of tangentially sectioned dense lamellae confirm their highly interwoven and compact collagen texture, and also provide an explanation for the results reported in literature, and in particular the above cited papers of Giraud-Guille and Weiner can be seen to conform to the observations of the present study. The TEM micrographs published by the above mentioned authors were taken at a magnification incomparably higher than those of the present authors’ study. If their micrographs are compared with those reported here in Figures 6–8, it emerges that what they interpret as a lamella is instead a bundle of collagen fibers within a lamella. In Figures 6–8, numerous bundles comprising five or more arrays of parallel collagen fibers are clearly recognizable, as well as transition zones where they offset by 308. Again, it must be emphasized that these arrays are not lamellae. They are bundles making up a single lamella, and the transition zones represent the sites where they branch by about 308 to join adjacent bundles. These findings are in agreement with those of Hofmann et al. (2006), who observe collagen bundles between adjacent lamellae tilting progressively, layer by layer, from a transverse to a longitudinal direction. We conclude that the TEM images published by Giraud-Guille and Weiner are reliable, but suggest a different interpretation. In conclusion, we suggest that investigations into collagen texture in bone tissues should be performed under low magnification and, above all, taking into account the location and distribution of osteocyte cell bodies and dendrites, the presence of which, per se, will undoubtedly disrupt any perfect and orderly/geometric arrangement of collagen.

ACKNOWLEDGMENTS This study was supported by funds of Fondazione of Vignola and Banca Popolare of Emilia Romagna. The authors wish to thank Professor M.A. Muglia, now retired, for her past SEM inves-

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