THE ANATOMICAL RECORD 295:1421–1429 (2012)
Morphometry and Patterns of Lamellar Bone in Human Haversian Systems UGO E. PAZZAGLIA,1* TERENZIO CONGIU,2 MARCELLA MARCHESE,1 FRANCESCO SPAGNUOLO,1 AND DANIELA QUACCI2 1 Clinica Ortopedica dell’Universit a degli Studi di Brescia, II Divisione di Ortopedia e Traumatologia, Spedali Civili di Brescia, Brescia, Italy 2 Dipartimento di Scienze Chirurgiche e Morfologiche, Universit a dell’Insubria, Varese, Italy
ABSTRACT The lamellar architecture of secondary osteons (Haversian systems) has been studied with scanning electron microscopy (SEM) in transverse sections of human cortical bone. Na3PO4 etching was used to improve the resolution of the interface between neighboring lamellae and the precision of measurements. These technical improvements permitted testing of earlier morphometry assumptions concerning lamellar thickness while revealing the existence of different lamellar patterns. The mean lamellar thickness was 9.0 6 2.13 lm, thicker and with a wider range of variation with respect to earlier measurements. The number of lamellae showed a direct correlation with the lamellar bone area, and their thickness had a random distribution for osteonal size classes. The circular, concentrical pattern was the more frequently observed, but spiral and crescent-moonshaped lamellae were also documented. Selected osteons were examined by either SEM or SEM combined with polarized light microscopy allowing comparisons of corresponding sectors of the osteon. The bright bands observed with polarized light corresponded to the grooves observed in etched sections by SEM. The dark bands corresponded to the lamellar surface with the cut fibrils oriented approximately longitudinally along the central canal axis. However, lamellae with large and blurred bright bands could be observed, which did not correspond to a groove observed by SEM. These findings are in contrast with the assumption that all the fibril layers within a lamella are oriented along a constant and unchangeable angle. The different lamellar patterns may be explained by the synchronous or staggered recruitment and activation of osteoblasts committed to the osteon’s completion. Anat C 2012 Wiley Periodicals, Inc. Rec, 295:1421–1429, 2012. V
Key words: human; cortical bone; lamellae; scanning electron microscopy
The concentric laminar structures found in secondary bone were first recognized by Havers in 1691 (thus, ‘‘Haversian’’ systems and canals). A mature Haversian system structure in bone is also called an ‘‘osteon.’’ The structure of Haversian systems was subsequently confirmed and extended by many subsequent studies. However, these lead to two contrasting models on how the collagen fibrils were arranged within the individual lamellae (Gebhardt, 1906; Ruth, 1947; Rouiller et al., 1952; Engstrom and Engfeld, 1953; Frank et al., 1955; Smith, 1960; Ascenzi and Bonucci, 1968; Boyde and Hordell, 1969; Reid, 1986; Giraud-Guille, 1988; Carando et al., 1989; Boyde and Riggs, 1990; Marotti, 1993; C 2012 WILEY PERIODICALS, INC. V
Grant sponsor: Dipartimento di Specialit a Chirurgiche, Scienze Radiologiche e Medico-Forensi, Brescia University. *Correspondence to: Ugo E. Pazzaglia, M.D., Clinica Ortopedica dell’Universit a degli Studi di Brescia, II Divisione di Ortopedia e Traumatologia, Spedali Civili di Brescia, Brescia, Italy. E-mail:
[email protected] Received 27 April 2011; Accepted 21 June 2012. DOI 10.1002/ar.22535 Published online 16 July 2012 in Wiley Online Library (wileyonlinelibrary.com).
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Weiner et al., 1997, 1999; Ascenzi et al., 2004; Pazzaglia et al., 2011). In the first model, the lamellar arrangement has been explained by separate, concentrical layers with a parallel orientation of the collagen fibrils within the layer, but forming a different angle with the vascular canal axis in the next lamella; whereas in the second by an alternating sequence of dense and loose fibrils aggregation. It is generally not questioned, if all the lamellar systems of secondary osteons have always had in their development a regular concentric pattern, perhaps because their initial osteonal design has been partially hidden by ongoing bone remodeling. Thus, most attention has been focused on the orientation of the collagen fibrils, whereas other parameters of lamellar organization (like thickness, completeness, or patterns) have generated less interest. Marotti (1993) observed that no appreciable differences in cortical bone structure were related to the age of the subject or to the radius of curvature of the lamellae within the osteon. In some studies, the thickness of the lamellae was measured (Reid, 1986; Weiner et al., 1999) with values reported in a range from 1.8 to 5 lm. Weiner et al. (1997, 1999) examined vitrified sections cut approximately in the plane of the lamellar boundary distinguished in each lamella five layers and measured the plywood-like structure of each sublayer. These authors assigned different thicknesses to each sublayer, and this reflected what was observed in baboon tibiae (Liu et al., 1999). It was assumed that the lamellae in the osteon were regular with minor deviances from a standard thickness and that they generally developed on the whole circumference and concentrically. By applying a short and controlled etching with Na3PO4 to transverse sections of human cortical bone prepared for scanning electron microscopy (SEM) observation, it was possible to improve the resolution of the interface between neighboring lamellae and thus, the precision of measurements (Congiu and Pazzaglia, 2011). Therefore, we could test using morphometric methods previous assumptions concerning the patterns and the geometry of lamellar systems in osteons. The application to the study of lamellar organization of different methods (from histology to electron microscopy and diffractometry) has expanded our understanding of the osteonal lamellar system. However, there are few studies that attempted to correlate SEM morphology with the images obtained by polarized light microscopy (Reid, 1986). These observations, however, were carried out on different osteons. In this article, we were able to examine the same osteon using both SEM and polarized microscopy. As the fibrillar geometry of the lamellae is the result of a complex process that develops within a tunnel dug by the cutting cone and implies not only collagen synthesis and extrusion by isolated osteoblasts but rather coordination of the activity of a pool of osteogenic cells (Pazzaglia et al., 2011), this morphometric approach to characterize lamellar geometry provided indications on the synchronization, cycles, and behaviors of the osteoblast pool in general.
MATERIALS AND METHODS Four tibias were obtained from male patients between 25 and 52 years of age who underwent an amputation
below the knee because of severe leg traumatic injuries. All patients gave consent that a segment of the amputated tibia would be used for scientific purposes, and the protocol was approved by the Ethics Committee of Brescia Spedali Civili. Two segments of the tibia about 1 cm of thickness were obtained between the one-third distal and the twothird proximal portion of the bone. These cylinders of diaphysis were cleaned of soft tissues and fixed in neutral formaldehyde (10%) for a week. One of the samples was then placed in hydrogen peroxide (40%) at room temperature for 4 weeks to remove all remaining soft tissues without physical manipulation. Three parallelepipeds were cut from the annular segment using a lowspeed bone saw with diamond-coated wafering blades (Remet, Casalecchio di Reno, Bologna, Italy), corresponding, respectively, to the antero-medial, lateral, and posterior sector of the tibia. Four slices of about 1 mm in thickness cut in a plane perpendicular to the long axis of the tibia were obtained from each of the blocks. The sections were ground to about 400 lm in thickness with an automated grinder (Remet, Casalecchio di Reno, Bologna, Italy) and further reduced by manual grinding to about 300 lm. The sections were then polished, washed repeatedly in distilled water, and further cleaned in an ultrasonic bath. To enhance the lamellar pattern of the osteons, the cut surface of the slices was exposed for 1 min to a 6% Na3PO4 solution (pH ¼ 9.1) at room temperature. The sections were then dehydrated in ascending concentrations of ethanol and critical-point dried in CO2. The sections were secured on stubs with conducting tape, coated with a thin layer of gold in a vacuum sputter-coater (Emitech) and examined with a Philips XL 30 scanning electron microscope in back-scattered electron imaging (BEI) and direct modes. The bone specimens of the second ring (one for each tibia) were ground to about 40 lm as described earlier. The sections were then stored in 2% formaldehyde until further processing. These slices were observed wet with an Olympus BX51 microscope under polarized light and 10 osteons from each slice that could be easily traced by their position on the map of the specimen were photographed. The same specimens were then subjected to Na3PO4 etching and prepared for SEM as described earlier. The same osteons that were identified and photographed under polarizing light were also observed by SEM in both the BEI and the direct modes.
Morphometry SEM digital images of the transverse surfaces etched with Na3PO4 were acquired in the direct mode with the electron beam perpendicular to the plane of the section, at 20 kV and 10 mm of working distance. The same magnification of 350 was used for each of all examined sections. We selected for morphometry all the osteons satisfying the following conditions: (a) a central canal perpendicular to the section or forming an angle =70 degrees; this angle was calculated measuring the short cathetus of the right triangle (central canal axis) with the perpendicular (long cathetus ¼ section thickness) (Fig. 1); (b) a complete lamellar pattern without marginal notching; (c) a central canal perimeter
