TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone

Bone 33 (2003) 270 –282

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TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone Matthew A. Rubin,a Iwona Jasiuk,a,* Jeannette Taylor,b Janet Rubin,c Timothy Ganey,d and Robert P. Apkarianb a

b

The G.W.W. School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0405, USA Integrated Microscopy and Microanalytical Facility, Department of Chemistry, Emory University, 1521 Pierce Drive, Atlanta, GA 30322, USA c Emory University School of Medicine, Veterans Affairs Medicine Center, 1670 Clairmont Road, Decatur, GA 30033, USA d Atlanta Medical Center, Department of Medical Education, 303 Parkway Drive NE, Atlanta, GA 30312, USA Received 25 January 2003; revised 12 May 2003; accepted 13 May 2003

Abstract Transmission electron microscopy (TEM) was used to investigate the crystal– collagen interactions in normal and osteoporotic human trabecular bone at the nanostructural level. More specifically, two-dimensional TEM observations were used to infer the three-dimensional information on the shape, the size, the orientation, and the alignment of apatite crystals in collagen fibrils in normal and osteoporotic bone. We found that crystals were of platelet shape with irregular edges and that there was no substantial difference in crystal length or crystal thickness between normal and osteoporotic trabecular bone. The crystal arrangement in cross-sectioned fibrils did not neatly conform to the parallel arrangement of crystals seen in longitudinally-sectioned fibrils. Instead, the crystal arrangement in both normal and osteoporotic trabecular bone took on more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns. The TEM imaging was done using bright fields only. Thus, the results presented are within the limitations of this approach. © 2003 Elsevier Inc. All rights reserved. Keywords: Nanostructure; Trabecular bone; Transmission electron microscopy; Osteoporotic Bone; Normal bone; Apatite crystals; Collagen fibrils

Introduction Well understood differences between normal and osteoporotic trabecular bone include lower bone mineral density (BMD) and thinner (or missing) trabecular struts in the osteoporotic bone. However, relatively few studies have analyzed the structure of normal compared with osteoporotic trabecular human bone at the nanoscale, leaving many unanswered questions concerning how a decrease in bone mineral density is affected by apatite crystal geometry and crystal organization. In fact, the exact crystal shape, size, and its three-dimensional (3D) relationship with collagen fibrils are still debated even for normal bone. Investigators have been divided between the plate-like or needle-like geometry of apatite crystals. Historically, Rob* Corresponding author. Fax: ⫹1-404-894-0186. E-mail address: [email protected] (I. Jasiuk). 8756-3282/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S8756-3282(03)00194-7

inson [1] proposed that apatite crystals in bone and other calcified tissue, such as the MTLT (mineralized turkey leg tendon), are plate-shaped. This observation was confirmed by Jackson et al. [2], Landis and Song [3], Landis et al. [4], Lees et al. [5], Prostak and Lees [6], Traub et al. [7], Weiner and Price [8], and others. Another group of researchers suggested that bone crystals are actually needle-like in shape, but the MTLT crystals are indeed plate-like [9,10]. A recent paper by Eppell et al. [11] summarizes developments in this area and conclusively shows that bone mineral crystals have a plate-like shape. Researchers still disagree about the apatite crystal size in both normal and osteoporotic bone. Transmission electron microscopy (TEM) studies [1,8,12] on normal bone and mineralized turkey tendon showed that crystals range in length from 15 to 150 nm, in width from 10 to 80 nm, and in thickness from 2 to 5 nm. Robinson [1] reported an average crystal size of 50 ⫻ 25 ⫻ 10 nm for normal human

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bone. Recent measurements by atomic force microscopy (AFM) [11] give smaller values of 12 ⫻ 10 ⫻ 1 nm for normal bone. There is also a disagreement on the difference in size between normal and osteoporotic bone. Three distinct views on the size of osteoporotic crystals have been presented in literature: that the osteoporotic crystals are either smaller [13], of same size [14], or larger than those in normal bone [15–17]. Several experimental techniques, which include X-ray diffraction (XRD) [14,18 –22], Fourier transform-infrared (FTIR) technique [23–29], infrared spectrophotometry [30,31], small angle X-ray scattering (SAXS) [9,10,32,33], back scattering electron imaging (BSEI), phosphorus-31 solid state nuclear magnetic resonance (NMR) [23,34], Xray pole analysis [35,36] as well as electron microscopy: scanning electron microscopy (SEM), transmission electron microscopy (TEM) [6,37], and AFM [11] have been used to investigate the crystal shape, size, and/or chemical composition. All of the above techniques require some processing of tissue and thus each technique is subject to different limitations [16]. In addition the measurements are difficult because of the very small size of crystals. Most of these studies focused on normal bone. XRD, FTIR, SAXS, and BSEI address site-to-site variation in mineral quality and can provide information about crystallinity (crystal size, perfection, and maturation) of bone mineral. Electron microscopy images provide information on the crystal shape, size, and location with respect to collagen fibrils. Regardless of the crystal shape and size, there have been proposed several different models describing the 3D crystal– collagen interaction at the nanoscale level. The first model states that the plate-like crystals are arranged in parallel layers transversing the fibril and that the layered motif of the crystals in one collagen fibril is aligned with neighboring fibrils [3,7,37– 40]. This model, however, is based only on the observations of the crystal– collagen interaction within fibrils of the MTLT and the longitudinallysectioned fibrils from nonhuman osteonal bone (rat and chicken). In addition, this model suggests that the majority of the plate-like crystals reside within the collagen fibrils and not between them. The second model proposes a completely different crystal– collagen relationship based on the investigation of longitudinally sectioned and cross-sectioned fibrils from the MTLT and from human osteonal bone [5,6,41]. This model suggests that plate-like crystals are situated both within and outside the fibrils, but the majority of crystals lie outside the collagen fibrils. The crystals outside the fibrils tangentially surround the fibril, while the plate-like crystals within the collagen are inclined at an acute angle to the longitudinal axis of the fibril. The third model proposes that needle-like apatite crystals in bone and the plate-like apatite crystals in the MTLT, which lie predominately inside the collagen, are aligned with the long axis of the fibril [9,10]. The fourth model [42] suggests that gap-filling crystals are needle-like with the c-axis par-

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allel to collagen fibril, while the crystals that fill pores and interfibril spaces are plate-like and they have their c-axes perpendicular to the fibril direction. The fifth model [43] assumes that apatite crystals are located mainly outside collagen fibrils and that they form a rigid random network. In this paper we use TEM to determine the ultrastructure of normal and osteoporotic human trabecular bone at the nanostructural level. In particular, our two-dimensional TEM observations are used to infer the 3D information on the geometry, size, and orientation of apatite crystals within longitudinally sectioned and cross-sectioned collagen fibrils in normal and osteoporotic trabecular bone. The TEM results presented in this paper complement several other TEM reports on the crystal structure of human and animal bones, as well as mineralized tendons and cartilage. Those TEM studies include the investigations of Arsenault [44,45], Glimcher [46 – 49], Glimcher and Krane [50], Lee and Glimcher [51], Prostak and Lees [6], and Lees et al. [5], among others. The main motivation for our study was to characterize the crystal/collagen fibril interaction in normal and osteoporotic bone, the shape and size of crystals and their arrangement with respect to collagen fibrils, so we could use this information as an input in the mechanics modeling of normal and osteoporotic bone. Bone can be considered as a composite material with a complex hierarchical structure. At the nanoscale level (collagen fibril/apatite crystal level) it consists of two main phases: collagen and apatite. Stiff apatite crystals give bone its stiffness and strength while the collagen gives bone its fracture toughness. Mechanical properties of composite materials are strongly influenced by the shape, size and arrangement of the reinforcing phase [52]. Thus, the detailed knowledge of these factors in both normal and osteoporotic bone is crucial in the accurate predictions of bone’s mechanical properties. The additional important parameters needed for modeling include the properties of constituents (collagen and apatite crystals), bonding between collagen and crystals, and how these two phases are influenced by osteoporosis. However, this information cannot be obtained from TEM images, and thus was not addressed in this paper. The studies focusing on the prediction of mechanical properties of bone based in the crystal structure and organization include those of Landis [53], Sasaki et al. [54], Wagner and Weiner [55], Gilmore and Katz [56], and Currey [57], among others.

Methods and materials Trabecular (cancellous) bone material was extracted from a fibula and a tibia of three normal human males ranging from 20 to 30 years of age and from a femur of three osteoporotic human males and females ranging from 79 to 91 years of age. Samples were collected at the Atlanta Medical Center in Atlanta, Georgia. The normal bone tissue

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Table 1 Bone mineral density measurements Bone sample

Bone type

Bone mineral density (mg/mm3)

Anatomic location

Age

Sex

1 2 3 4 5 6

Normal Normal Normal Osteoporotic Osteoporotic Osteoporotic

0.576 0.593 0.597 0.227 0.232 0.273

Fibula Radius Tibia Femur Femur Femur

20 20 30 79 86 91

Male Male Male Female Female Male

was obtained from a traumatic amputation or from an accident victim, while the osteoporotic tissue was collected from hip orthoplasty procedures. We used the tissue from different anatomic locations for normal and osteoporotic samples due to bone availability. The bone mineral density (BMD) and the anatomic locations for the normal and osteoporotic subjects are shown in Table 1. The subjects had no known bone diseases except for osteoporosis (osteoporotic patients). The BMD measurements were obtained by dual excitation absorptiometry (DEXA) using cubes measuring 1 cm3. The tissue was stored in 90% ethanol solution. Prior to the collection of tissue, the experimental protocol was submitted to the IRB at the Atlanta Medical Center and given approval. In line with Food and Drug Administration (FDA) and Office for the Protection of Research Risks (OPRR) guidelines, no separate informed consent was required for collecting the tissue. TEM preparation Bone specimens were postfixed in 1% osmium tetroxide, dehydrated in an acetone series (30, 50, 70, 80, 90, and 100%), infiltrated with a graded series of acetone and three changes of Spurr resin, and finally, embedded in fresh Spurr resin in labeled Beem capsules. Ultrathin sections (90 –100 nm) of human trabecular bone were cut with a diamond knife on a RMC MT 7000 Ultramicrotome and picked up on 300-mesh copper and Formvar-coated, single slot, copper grids. Up to four blocks were selected for thin sections from each specimen. Several sections were taken from each block and collected onto three grids for each block. A JEOL JEM-1210 Analytical TEM operated at 80 and 90 kV was used to record images of the calcified human trabecular bone. Concurrently to cutting ultrathin sections for TEM, thick sections were also cut. These were viewed using a light microscope to determine the orientations of TEM thin sections with respect to a trabecular structure. Light microscopy analysis revealed that, in most instances, the cuts were made in longitudinal directions of trabeculae. We took record of locations from which samples were procured but this information was difficult to track when preparing TEM specimens. TEM images were photographed at low (2,000⫻), inter-

mediate (20,000⫻), and high (80,000⫻) magnifications (low-dose imaging) to best observe nanostructural features of bone. The negatives were then scanned with an Agfa T-2500 scanner into a computer to generate high resolution, 45-megabyte image files. These images provided monitor magnification 10-fold greater than the recorded magnification for detail recognition. Adobe Photoshop 6.0 was then used to adjust the black, white, and gray tonal ranges of the images for better visualization and detail recognition of the bone crystals. An unpaired two-sample t test was used to analyze the statistical significance of apatite crystal dimensions between normal and osteoporotic human bone. Most TEM images included both longitudinal and cross-sectioned fibrils in the same plane. However, in order to study the apatite crystal arrangement in longitudinally- and crosssectioned fibrils, we zoomed in on these two perpendicular fibril directions separately and illustrated them in separate figures.

Results Nanostructure of normal bone We observed both distinct plate-like and tablet-like apatite crystals within the mineralized collagen fibrils (Figs. 1–2). Plate-like crystals had irregular edges, with no welldefined profile and were rather low in density. Tablet-like crystals, however, had distinct boundaries, with a welldefined, oblong profile and were much denser than platelike crystals. The abundance of dense mineral in the bone matrix as well as the merging and overlapping of crystal profiles made it difficult to isolate the less dense plate-like crystals. The widthwise dimension of plate-like crystals observed in this study was oriented perpendicular to the collagen long axis, while the lengthwise dimension, which corresponded to the crystallographic c-axis of the plate-like crystals [3], was generally aligned parallel to the long axis of the fibril. Additionally, the plate-like crystals observed in this study were found only in longitudinally-sectioned fibrils and not in cross-sectioned fibrils. Tablet-like crystals were also aligned lengthwise with the long axis of the fibril. This type of crystal geometry was observed in micrographs containing both longitudinally-sectioned and cross-sectioned mineralized fibrils. To provide a more accurate description of their threedimensional structure, crystals were measured in both longitudinally-sectioned and cross-sectioned fibrils. Plate-like crystals were almost impossible to isolate within the TEM micrographs, making measurements of their size difficult. However, a few favorable micrographs enabled the measurement of a small number of single plate-like crystals. The acquired dimensions reflect the observed plate-like crystals in a very small population (n ⫽ 6) and should be considered carefully. On the other hand, tablet-like crystals were much

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Fig. 1. TEM micrographs of longitudinally-sectioned mineralized collagen fibrils in human normal trabecular bone. (A) Distinct individual apatite crystals are seen within the longitudinally-sectioned mineralized collagen fibrils as plate-like (arrows) and tablet-like (plates on edge) (dotted arrows) shapes. Tablet-like crystals are generally stacked in parallel layers that are aligned with the fibril’s long axis. This arrangement could be seen extending over several adjacent fibrils. The overlapping of adjacent plate-like crystals is seen to form larger parallel units of mineral (white arrow heads). (B) Distinct tablet-like apatite crystals (arrows), within the longitudinally-sectioned mineralized collagen fibrils, are seen stacked in parallel layers (white arrows) that are aligned with the fibril’s long axis.

easier to isolate and measure, permitting a larger population size (n ⫽ 45). All crystals that had a well-defined profile were carefully measured in each figure. They were a sample of the total number of tablet-like crystals within the images. Crystals that appeared to be part of a cluster were not chosen. In longitudinally-sectioned bone collagen fibrils, the displayed length and width dimensions of observable plate-like crystals were in the range of 57.0 ⫾ 6.7 and 27.3 ⫾ 3.5 nm, respectively. Tablet-like crystals had a length of 50.7 ⫾ 9.1 and width of 7.7 ⫾ 1.5 nm. In mineralized regions containing solely cross-sectioned fibrils, the tablet-like crystals had a length of 27.2 ⫾ 3.0 and a width of 7.7 ⫾ 5.6 nm. The above data give the mean and the mean standard deviation. The measurements showed that the tablet-like crystals, in cross-sectioned fibrils were, on average, half the length of tablet-like crystals seen in longitudinal sectioned fibrils, but widths were consistent regardless of sectioning. This suggests that the tablet-like crystals in cross-sectioned fibrils are actually plate-like crystals cut widthwise, instead of lengthwise as found in longitudinally-sectioned fibrils (Fig. 3). Note that since tablet-like crystals are just plates on edge, the width of the tablet-like crystals is actually the thickness of the plates. Thus, to make the description more clear, we use only the terms length and width in both longitudinal- and cross-sections.

Plate-like and tablet-like crystals were readily apparent in regions containing longitudinally-sectioned fibrils (Figs. 1A and B). Tablet-like crystals were stacked in parallel layers that were aligned with the fibril’s long axis. This arrangement could be seen extending over several adjacent fibrils. The distance of separation between adjacent parallel crystals was not discernable in these micrographs; however, they appeared to traverse the entire diameter of the fibrils. Organization of individual plate-shaped crystals within the fibrils was much more difficult to discern because of their low density and tendency to merge with other plate-like crystals. The layering of parallel-aligned crystals traversing the fibril’s width, observed in longitudinal-sections, was not seen within cross-sectioned fibrils. Crystal organization in cross-sectioned fibrils displayed more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns of only tablet-like crystals (Figs. 2A and B). The light regions in Fig. 2B reflect regions deficient in mineral. The amount of crystal rotation was dependent on location, since it was unusual to find two nearby regions harboring the same sequence of crystal orientations. A revolution of the crystal arrangement was seen within approximately a 100 –200-nm diameter; collagen fibrils are about 100 nm in diameter (Fig. 4). Thus, the rotation of fibrils about their long axis throughout the region

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Fig. 2. TEM micrographs of cross-sectioned mineralized collagen fibrils in human normal trabecular bone. (A) Distinct individual apatite crystals are seen within the cross-sectioned mineralized collagen fibrils as tablet-like (plates on edge) shapes (arrows). Crystal organization appears to have more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns (black bars indicate orientation of the crystal at that particular location). The orientation of the tablet-like crystals distinctively changes within a 100 –200-nm-diam area. (B) Lower magnification of the cross-sectioned mineralized region in A (represented by box). Crystal organization in cross-sectioned fibrils displayed more of a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns of only tablet-like crystals.

would account for the undulated and circular appearance of the crystal organization (see Fig. 8B). Nanostructure of osteoporotic bone Crystal– collagen composite Individual apatite crystals were evident throughout the mineralized regions as plate-like and tablet-like shapes, as similarly seen in normal bone (Figs. 5A– 6B). Crystal appearance, geometry, and alignment, for both plate-like and tablet-like crystals, were also identical to those observed in normal bone. Again, the overlapping crystal pattern, also seen in the normal bone, made it difficult to isolate single, plate-like crystals. The crystallographic c-axis of the platelike crystals was generally aligned parallel to the long axis of the fibril, and the widthwise dimension of plate-like crystals was oriented perpendicular to the collagen long axis. Additionally, plate-like crystals were found only in longitudinally-sectioned fibrils and not in cross-sectioned fibrils in human bone. Tablet-like crystals were much denser, and thus more readily observable, than plate-like crystals and were also aligned with the long axis of the fibril. This type of crystal geometry was observed in micrographs that contained both longitudinally-sectioned and cross-sectioned mineralized fibrils. Crystals were measured in both longitudinally-sectioned and cross-sectioned fibrils to provide a more accurate measurement of their 3D structure. As in normal bone, plate-like crystals in osteoporotic bone were difficult to isolate with

TEM, making measurements of their size and shape difficult. The problem of isolating plate-like crystals made it challenging to distinguish any shape differences in platelike crystals between osteoporotic and normal bone. Again, a few favorable micrographs enabled the measurement of a small number (n ⫽ 5) of single, plate-like crystals. The acquired dimensions reflected the observed plate-like crystals in a very small population, as in the normal bone. Tablet-like crystals, however, were much easier to isolate and measure, permitting a larger population size (n ⫽ 45). The length and width of observable plate-like crystals in longitudinally-sectioned collagen fibrils were 47.7 ⫾ 4.6 and 27.5 ⫾ 4.8 nm, respectively. Tablet-like crystals had a length of 49.8 ⫾ 10.4 and a width of 7.1 ⫾ 1.7 nm. In cross-sectioned fibrillar regions, tablet-like crystals were 26.9 ⫾ 3.8 nm in length and 6.6 ⫾ 1.1 nm in width. In regions containing longitudinally-sectioned fibrils, both plate-like and tablet-like crystals were readily seen. Within the collagen, groups of individual tablet-like crystals were either stacked in parallel layers or aligned in a colinear fashion along the axis of the collagen fibrils (Figs. 5A and B). In mineralized areas of only cross-sectioned fibrils, tablet-like crystals were predominately observed (Figs. 6A and B) (Note: The region depicted in Fig. 6B is to the left of the region shown in Fig. 6A). The neat and orderly arrangement of crystals in parallel stacks, apparent in longitudinal-sections and presumed in cross-sections, was not seen within regions of fibrils in cross-section. As observed in normal bone, the crystal organization in cross-sectioned fibrils dis-

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Fig. 3. Schematic illustration of the length- and widthwise dimensions and c-axis of the apatite crystals (not drawn to scale). (A and B) The relationship between the crystal cut planes to the proposed final shape of the plate-like and tablet-like crystals seen in longitudinally-sectioned collagen fibrils. Likewise, C shows the relationship between the crystal cut plane to the proposed final shape of the tablet-like crystals seen in cross-sectioned collagen fibrils. In cross-sectioned fibrils (C), crystals were half the length (L) of tablet-like crystals seen in longitudinally-sectioned fibrils (A and B), but the widths (w) and the thickness (t) were consistent regardless of sectioning.

played more of a random undulated arrangement, with certain localized areas demonstrating circular oriented patterns of only tablet-like crystals (Figs. 6A and B). The orientation of the tablet-like crystals distinctively changed within a 100 –200-nm diameter area (Figs. 3 and 6A). In addition to the above described circular patterns, we also observed larger semicircular crystal patterns, with spans of approximately 500 nm in the mineralized fibrils (Fig. 6C). At the arc junctions, crystals could be seen converging (or diverging) at relatively similar angles to each other. These junc-

tions also appeared to be denser than the surrounding crystal areas. Thus, the undulated and circular appearance of the crystal organization suggests the fibrils are rotated differently about their long axis throughout the region (see Fig. 8B) and might suggest why tablet-like and plate-like crystals were seen together in longitudinally-sectioned fibrils. In regions where there was a successive transition from longitudinal, oblique, cross-sectioned and back to longitudinally sectioned fibrils, another motif of crystal orientation was discerned (Figs. 7A and B). Orderly groups of tablet-

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Fig. 4. Illustration of circular dispersion of crystals within a 100 –200-nm region in normal and osteoporotic human bone (not drawn to scale). (A) The dotted black rings depict rotations of crystals within a 100-nm-diam area. (B) The solid black ring shows rotation of crystals within a 200-nm region

like crystals were seen rotating from an oblique to a roughly parallel orientation with respect to the longitudinally-sectioned fibrils, as fibril orientation progressed through the sequence mentioned above. However, various areas throughout Fig. 7B showed clusters of crystals at different stages of rotation than others, indicating that

crystal rotation was not uniform throughout the fibril progression. This resulted in a crystal organization that was undulated in appearance and was more evident in regions consisting of cross-sectioned fibrils. Nonetheless, the rotation and undulated appearance of crystals returned to a more parallel arrangement of the crystals

Fig. 5. TEM micrographs of longitudinally-sectioned mineralized collagen fibrils in human osteoporotic trabecular bone. (A) Distinct individual apatite crystals are seen within the longitudinally-sectioned mineralized collagen fibril as plate-like (arrows) and tablet-like (plates on edge) shapes (dotted arrows). Tablet-like crystals are generally stacked in parallel layers that are aligned with the fibril’s long axis. This arrangement could be seen extending over several adjacent fibrils. Crystals were either stacked in parallel layers or aligned in a colinear fashion along the axis of the collagen fibrils (white arrows). (B) Distinct individual apatite crystals are seen within the longitudinally-sectioned mineralized collagen fibril as plate-like (white arrows) and tablet-like (plates on edge) shapes (white dotted arrows). Within the collagen, groups of individual tablet-like crystals are either stacked in parallel layers or aligned in a linear fashion along the axis of the collagen fibrils (arrows).

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Fig. 6. TEM micrographs of cross-sectioned mineralized collagen fibrils in human osteoporotic trabecular bone. (A) Distinct individual hydroxyapatite crystals are seen within the cross-sectioned mineralized collagen fibril as tablet-like (plates on edge) shapes (arrows). Crystal organization in cross-sectioned fibrils displayed more of a random undulated arrangement, with certain localized areas demonstrating circular oriented patterns of only tablet-like crystals patterns (bars indicate orientation of the crystal at that particular location). The orientation of the tablet-like crystals distinctively changes within a 100 –200-nm-diam area. (B) Lower magnification of the cross-sectioned mineralized region in A (represented by box). (C) Distinct individual apatite crystals are seen within the cross-sectioned mineralized collagen fibril as tablet-like (plates on edge) shapes (arrows). Continuous arcing patterns (dotted lines) with spans of approximately 500 nm are evident among the crystal arrangement.

along the axis of the collagen once the fibrils were longitudinally-sectioned.

Discussion At the nanostructural level, this study demonstrated that both normal and osteoporotic bone crystals were indeed plate-like (or tablet-like) in shape rather than needle-like. This explicit demonstration, showing that crystals were thin plate-like crystals with irregular edges, supported the results

of earlier studies on mineral shape in bone [1,2,8,11]. Crystal dimensions, which we obtained for normal human bone (57 ⫾ 6.7 ⫻ 27 ⫾ 3.0 ⫻ 7.7 ⫾ 3.5 nm), were consistent with other electron microscopy investigations [1,8] on normal human bone (45–50 ⫻ 25 ⫻ 10 nm). However, our results were higher by about a factor of three than those obtained by AFM [11] and several TEM studies [44,58]. The differences between AFM and TEM measurements were reported by Eppel et al. [11]. The reasons for discrepancy may include different preparation protocoles and techniques: crushing of bone to isolate crystals versus in situ

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Fig. 7. TEM micrographs of osteoporotic bone. (A) TEM micrograph of the characteristic arcing pattern of unmineralized twisted or rotated plywood motif in osteoporotic bone. A successive transition of longitudinal (L), oblique (O) and cross-sectioned fibrils (C) is apparent. Uc, unmineralized collagen; M, mineral zone. (B) Crystals can be seen at different stages of rotation, indicating that crystal rotation was not uniform throughout the fibril progression. This resulted in a crystal organization that was undulated and circular in appearance.

observations, bright versus dark field imaging, and other factors. Our TEM images included only bright field images. As pointed our by a reviewer, the dark field images would ensure that crystals observed were single crystals and not clumps of crystals. This subject is beyond of the scope of this paper. For a discussion on the advantages of dark field imaging and its application to the analysis of apatite crystals see Jackson et al. [2], for example. In this paper we provided detailed 3D measurements and geometry comparisons of apatite crystals between normal and osteoporotic bone by means of TEM. However, the method we used could not provide information on whether the majority of crystals lie inside or outside the fibrils. We were unable to identify individual mineralized collagen fibril profiles as well as spaces between the mineralized collagen fibrils in the TEM micrographs. Thus, the undulated and circular crystal patterns seen in the mineralized cross-sectioned areas did not differentiate between crystals inside or outside the fibrils, rather they showed the overall area encompassed by the mineralized fibrils and the crystals residing in it. In other words, we were not able to identify what aspect of the undulated or circular crystal arrangement was influenced by crystals inside or outside the fibril by means of TEM. It is quite possible that the crystals on the surface and between the fibrils contribute to the circular patterns or that they have another orientation that was undetectable in the micrographs. We determined there was no significant difference in crystal length and morphology of tablet-like crystals between normal and osteoporotic bone. However, the mea-

sured length difference of about 10 nm (normal 57.0 ⫾ 6.7; osteoporotic 47.7 ⫾ 4.6 nm) we observed for plate-like crystals in longitudinally-sectioned fibrils in normal and osteoporotic bone was not accurate, since the lengths we observed for tablet-like crystals in both longitudinally-sectioned (normal 50.7 ⫾ 9.1 nm; osteoporotic 49.8 ⫾ 10.4 nm) and cross-sectioned fibrils (normal 27.2 ⫾ 3.0 nm; osteoporotic 26.9 ⫾ 3.8 nm) for both osteoporotic and normal bone measured in this experiment were not significantly different. Another reason for the discrepancy in crystal length could be due to the very small population of observable plate-like crystals in both normal and osteoporotic bone in this study. Despite this, the results of our study were similar to results of Simmonds et al. [14] showing that osteoporotic crystals do not differ in size from those in normal bone. Also, recent reports, which provided twodimensional structural information on osteoporotic bone crystals from the effects of chemical treatment [23,33,59], suggested that the differences in structure and size of crystals were not statistically significant between normal and osteoporotic bone. We have also demonstrated in this study that no significant difference in 3D structure of crystals between normal and osteoporotic bone was found by means of TEM. The comparison of crystal characteristics between normal and osteoporotic bone reported in this paper is summarized in Table 2. Our TEM characterization of the 3D crystal arrangement in normal and osteoporotic cross-sectioned collagen fibrils contrasts with the first model, mentioned in the Introduction, which suggests that the parallel layering of plate-like

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Table 2 Crystal observations Fibril section

Visible crystal shape Crystal size (nm) Length Width Crystal orientation

a

Normal bone

Osteoporotic bone

Long-section fibrils

Cross-section fibrils

Long-sect fibrils

Cross-section fibrils

Plate-like crystals and tablet-like crystals

Tablet-like crystals

Plate-like crystals and tabletlike crystals

50.7 ⫾ 9.1a 7.7 ⫾ 1.5a

27.2 ⫾ 3.0 7.7 ⫾ 5.6

49.8 ⫾ 10.4 7.1 ⫾ 1.7

26.9 ⫾ 3.8 6.6 ⫾ 1.1

Long axis aligned with c-axis of fibril

Circular oriented patterns in localized areas. Crystals rotate in about 100–200-nm diam area.

Long axis aligned with c-axis of fibril

Circular oriented patterns in localized areas. Crystals rotate in about 100–200nm-diam area.

Measurements are for tablet-like crystals.

crystals in one collagen fibril is aligned with the crystal layers in neighboring fibrils [3,7,38 – 40]. The first model is limited by the fact that the crystal– collagen interaction was only investigated in longitudinally-sectioned fibrils of the

MTLT, as opposed to investigating crystal organization in cross-sectioned fibrils in bone itself, which was performed in our study. Thus, we have demonstrated that, in crosssectioned collagen bone fibrils, crystals exhibited more of a

Fig. 8. Schematic illustrations of collagen fibrils in cross-section and in longitudinal-section. (A) Accepted model: I and II represent collagen fibrils in cross-section and in longitudinal-section, respectively (not drawn to scale). The white, plate-like objects within the collagen fibrils denote apatite crystals (not drawn to scale). Plate-like crystals are arranged in parallel layers that traverse the fibril. The layered motif of the crystals in one collagen fibril is aligned with neighboring fibrils [3,7,38,40]. (B) Proposed model: I and II represent collagen fibrils in cross-section and in longitudinal-section, respectively, as observed by us experimentally (not drawn to scale). The plate-like objects within the collagen fibrils denote HA crystals (not drawn to scale). The white faces on the crystals in I and II depict the exposed crystal surface due to sectioning. Note too that the crystal layers in each fibril are rotated differently to give a random, undulated arrangement, with certain localized areas demonstrating circular oriented patterns of crystals in fibril cross-section. The undulated crystal organization suggests that the fibrils are rotated differently about their long axis. As a result, crystals are seen as both tablet-like (plates on edge) and plate-like shapes in longitudinal-section, while crystals appear only as tablets in cross-section. The extent of crystal rotation shown between individual fibrils is arbitrary. III shows a different perspective of the fibril sections. Axis X and Axis Y illustrate the different rotation of the fibrils about their long axis.

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random, undulated arrangement, with certain localized areas of circular oriented patterns rather than in neat and orderly alignment of crystal layers of neighboring collagen fibrils (Figs. 2, 3, 4, and 6). Such crystal arrangement resembled those seen in other TEM studies [46 –51]. A physical explanation for the undulated and circularly oriented patterns observed in cross-sectioned collagen fibrils is provided by the different rotation of adjacent collagen fibrils about their long axis, but not the rotation of crystal layers. In other words, this unexpected crystal appearance was a consequence of numerous collagen fibrils rotated arbitrarily about their axis, with little or no alignment of crystals layers from collagen fibril to neighboring collagen fibrils. Weiner and Traub[40] and Ziv et al. [60] have shown through SEM that mineralized fibrils do indeed rotate around their axes in different lamellar sublayers in rat and human bone, such that the layers of crystals in these successive sublayers are also rotated with the fibrils to form the rotated, plywood structure. This rotation about the collagen axis gave the appearance of crystal layers rotated about each other. Thus, this may explain why the groups of crystal layers we observed with the TEM appeared at an angle to each other and rotated about each other in crosssectioned fibrils. If the crystal layers were aligned with neighboring crystal layers in other fibrils, the cross-sectional view of the crystal layers would appear as uniform layered region of parallel, tablet-like structures, which was not the case. To our knowledge the semicircular pattern we observed (Fig. 6C) is reported here for the first time. A possible physical explanation for the semicircular pattern observed in cross-sectioned collagen fibrils could be due to a gradual rotation of adjacent collagen fibrils about their long axis, giving rise to the arc shape. Nonetheless, this pattern still suggests that the fibrils are rotated about their long axis, resulting in crystal patterns that are not from the alignment of neighboring crystal layers in adjacent fibrils. Crystal arrangement, seen for example in Fig. 2A (normal bone) and Fig. 6A (osteoporotic bone), demonstrates how the orientation of groups of tablet-like crystals rotates within a 100 –200-nm-diam area. The undulated and circular orientation indicates that the crystal layers in collagen are not all axially aligned and explains why both tablet-like and plate-like crystals are seen in longitudinally-sectioned fibrils. The varying crystal widths and thicknesses seen in longitudinally-sectioned collagen fibrils arise from apatite crystals lying oblique to the section plane. Thus, the type of crystal geometry seen in longitudinally-sectioned collagen fibrils is dependent on how the crystal layers are sectioned. A comparison between crystal orientation as observed by us experimentally and by Weiner and Traub [40] is better visualized in the schematic illustrations of Figs. 8A and B, respectively. Fig. 8A illustrates Weiner and Traub’s [40] observations of a uniform, parallel layered region of crystals, while Fig. 8B shows the rotation of apatite crystals in collagen fibrils in cross-section and in longitudinal-section.

Finally, MTLT has been used extensively in literature to analyze the 3D crystal arrangement in mineralized tissues [3,7,38,40]. This is mainly because of local parallelism of the collagen fibrils, as opposed to the more complex fibril orientation in bone [16]. The parallelism could be seen in the schematic illustration of a cross-section through collagen fibrils from a MTLT (as seen in Weiner and Traub [40]) that showed how the parallel layers of crystals in adjacent fibrils could be aligned in 3D. This parallel structure of collagen fibrils might be irrelevant in some parts of bone, especially in regions consisting of predominately crosssectioned fibrils (i.e., in orthogonal plywood motifs), since cross-sectioned, mineralized fibril regions in our investigation did not show a parallel alignment of tablet-like crystals between neighboring fibrils. In summary, the TEM images of crystal patterns reported in this paper resemble closely those found in other TEM studies of bone structure due to Arsenault [44,45], Glimcher [46 – 49], Glimcher and Krane [50], Lee and Glimcher [51], Prostak and Lees [6], and Lees [6], and Lees et al. [5], among others. These studies are very comprehensive and provide a wealth of information on crystal– collagen level in normal bone. Our main interest was in identifying the differences between normal and osteoporotic bone. To our knowledge our investigation is one of the few studies focusing on crystal shape, size, and its arrangement with respect to collagen fibrils for normal compared with osteoporotic bone using the TEM. We concluded that there were no significant structural differences between these two bone types at the crystal– collagen level. The results reported in this paper are based on three sources for normal bone and three sources for osteoporotic bone. Since the results for normal and osteoporotic bone were very similar and consistent we did not proceed with a larger number of sources. Second, we used tissue from different anatomic locations: femur for osteoporotic samples and long bones (fibula and tibia) for normal samples because of availability. Again, since the results for these two bone types were similar, we did not broaden this study to include the same anatomic locations.

Conclusions In summary, in this study we demonstrated using TEM that normal and osteoporotic human bone crystals are platelike in shape and that the tablet-like appearance was only the plates viewed on edge. Second, we determined that there was no significant variation in length or thickness between normal and osteoporotic bone crystals. We also showed that crystal arrangement appeared differently when the fibrils were viewed in longitudinal-sections and cross-sections. Last, we observed that more of a random, undulated crystal arrangement, with areas of circularly oriented patterns of apatite crystals, were characteristic in cross-sectioned

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fibrils, while apatite crystals in longitudinal sections were arranged in parallel, ordered layers. These TEM images involved bright field images only. Thus, the results presented are within the limitations of such an approach. Since our results at the nanostructural scale showed no significant differences in crystal size or arrangement between normal and osteoporotic bone, these observations might suggest that the chief mechanism underlying the lower BMD is a reduction in trabecular structure, rather than a deterioration of the bone crystals at the nanoscale. The other hypothesis can be whether a reduced content of calcium is guiding the BMD reduction in trabecular bone. Presumably an inability to form plates in a normal collagen bone matrix might predispose the bone to resorption (because of less mineral crystal, different degree of crystallinity, and different mechanical properties) and increase the lability of the matrix. Noting that the alignment of the crystals with the long axis of collagen and equal size of the crystal has been confirmed by our study, the chief difference in BMD likely relies on the amount of trabecular bone that was seen in the samples. Without doing detailed morphometric comparison of the two sets of tissues, it would be impossible to validate the raw values of BMD with the mean trabecular volume of the tissue. However, the reduction in BMD and the common values of the size and orientation of the crystals themselves would argue that it is not a failure of the osteoblasts to produce an appropriate matrix, but an imbalance in metabolic turnover that results in the osteoporosis. It is an attractive consideration to validate that crystals outside the fibrils may be more labile and that interstitial acidification that accompanies aging may tip the metabolic balance toward catabolic change, but we do not have data to support the connection.

Acknowledgments The support of the Emory/Georgia Tech Seed Grant Program 1999 –2000 (to I.J. and J.R.) and the support of the National Science Foundation (Grant CMS-0085137) (to I.J.) is acknowledged.

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