Determination of the Orientation of Collagen Fibers in Human Bone

Transcription

1 THE ANATOMICAL RECORD 266: (2002) DOI /ar Determination of the Orientation of Collagen Fibers in Human Bone SHIGEYOSHI OSAKI, 1 *SETSUKO TOHNO, 2 YOSHIYUKI TOHNO, 2 KAZUO OHUCHI, 3 AND YOSHINORI TAKAKURA 4 1 Department of Chemistry, Nara Medical University, Nara, Japan 2 Department of Anatomy, Nara Medical University, Nara, Japan 3 Department of Orthopedics, Fukushima Medical University, Fukushima, Japan 4 Department of Orthopedics, Nara Medical University, Nara, Japan ABSTRACT Ahuman calcaneus bone, consisting of hydroxyapatite and collagen fibers, was successively sliced into samples in adirection perpendicular to the long axis of the bone and parallel to the long axis of the human lower limb. The transmitted microwave intensities of 12 GHz, reflecting the dielectric property, were measured for the slice samples using Osaki s microwave method (Tappi J., 1987; 70: ). The complex dielectric constant of 12 GHz of the collagen fiber film was much greater than that of hydroxyapatite disc, which demonstrated that the dielectric anisotropy observed for the sliced bone was mainly affected by the collagen fibers. The angular dependence of the transmitted microwave intensity gives the orientation angle reflecting the direction of the collagen-fiber orientation, and the degree of orientation reflecting the anisotropic property of collagen fibers. The orientation angle and the degree of orientation for the slice sampleschangedwithchangingpositionalongthelongaxisofthecalcaneus bone. The direction of orientation deviated to the lateral side at the heel part of the left calcaneus, and to the medial side at the middle part. The degree of orientation is relatively high at the heel part and low at the middle. Such results give atwo-dimensional distribution of collagen-fiber orientation in the left calcaneus, and suggest that the direction and degree of orientation are closely related to the direction and magnitude of the stress applied to the bone, respectively. Anat Rec 266: , Wiley-Liss, Inc. Key words: human bone; calcaneus; slice sample; collagen fibers; orientation Determination of the orientation of collagen fibers in human tissues, such as bones, is indispensable when studying the relationship between the physical properties and the structures. From the viewpoint of orthopedic biomechanics, such adetermination will be useful when estimating the mechanical stress acting on human bones consisting of collagen fibers and hydroxyapatite crystals. Mechanical compression, ultrasound velocity, and X-ray methods (Gierse, 1976; Greenfield et al., 1981; Wahner and Fogelman, 1996; Yettram and Camilleri, 1993) have been used to study the mechanical and structural anisotropies of bulk bones. However, it has been impossible to estimate the orientation of collagen fibers using such methods because of the difficulty in separating only the effects of the fibers in bulk bones. Although amethod using neutron diffraction (Bacon et al., 1984) was developed for measuring the orientation of the hydroxyapatite crystals in bones, it was not possible to apply this to the determination of the collagen-fiber orientation in bones. Thus, no methods have been available to determine the orientation of collagen fibers in human bones because it was very difficult to isolate the physical properties of the collagen fibers. *Correspondence to: Shigeyoshi Osaki, Nara Medical University, Kashihara, Nara , Japan. s-osaki@naramed-u.ac.jp Received 8August 2001; Accepted 7November 2001 Published online 00 Month WILEY-LISS, INC.

2 104 OSAKI ET AL. Fig. 1. a: Side and back views of left calcaneus bone. b: A slice sample (ca. 1.5 mm thick) used for the microwave measurements. The slice was cut in the direction perpendicular to the long axis of the calcaneus and parallel to the long axis of the human lower limb, and then was cut on both sides parallel to the SD. The SD corresponds to the direction parallel to the long axis of the human lower limb. It is expected that if the orientation of collagen fibers in bones could be determined, information could be obtained regarding the distribution of stress applied to the bone, and on the growth of bones. In previous studies, Osaki (1987, 1990a, 1997) applied a microwave method to determine the molecular and fiber orientations of paper sheets (Osaki, 1987), nonwoven fabrics (Osaki, 1989), and calf leather (Osaki, 1990b, 1999; Osaki et al., 1993), and also found a new method (Osaki, 2001) of using hair pores to determine the orientation of collagen fibers in leather sheets prepared from calf skin. These studies showed that the orientation, as determined by the microwave and hairpore methods, reflected the mechanical anisotropy. In the present study we overcame the difficulty in successively cutting human calcaneus bone into slice samples, and applied the microwave method to determine the orientation of the collagen fibers. It was found that the orientations of the collagen fibers could be determined for slice samples at different positions along the long axis of the human calcaneus, providing a two-dimensional distribution of collagen-fiber orientation that reflected the mechanical anisotropy. MATERIALS AND METHODS Preparation of Slice Samples From Calcaneus Bone The bone used in this study was the human left calcaneus (ca. 8 cm long) (Fig. 1a), which was fixed in a solution of 36% ethanol, 13% glycerin, 6% phenol, and 6% formaline (Tohno et al., 1985), and prepared for anatomical experiments by medical students. The calcaneus was roughly similar in shape to a cylindrical rod, with a diameter of ca cm from the heel end (the anterior part up to ca. 5 cm facing the toe part was not cylindrical). The left calcaneus bone was cut into slices at a thickness of ca. 1.5 mm, in the direction perpendicular to the long axis of the calcaneus and parallel to the long axis of the human lower limb, by using the cutter MC120 (Maruto Co., Kyoto, Japan). The slices were dried in an air flow at a temperature of 50 C for 1 day, and then at room temperature for more than 5 days. Both sides of the slices were then cut in the direction parallel to the standard direction (SD) and subjected to the microwave measurements (Fig. 1b). The SD corresponds to the direction parallel to the long axis of the human lower limb. A plus value of orientation angle means that the direction of orientation deviated to the lateral side (Fig. 1a). The samples were designated as specimens 3-1 to 3-17, changing position from the heel to the middle of the long axis of the calcaneus bone. Minerals were chemically removed from the calcaneus slices by chelate extraction with EDTA-3Na and the chelate-extracted slices were also subjected to the microwave measurements. The chemical content of the calcaneus bone, excluding water and lipids, was determined to be about 35% organic materials, such as collagen fibers, and about 65% inorganic hydroxyapatite, consisting of calcium phosphate, calcium carbonate, and magnesium phosphate. Hydroxyapatite discs were prepared from the powders according to the compression method, which is similar to that used in infrared measurements (Tadokoro, 1979).

3 Microwave Measurements Each slice sample was inserted into the narrow gap between a pair of waveguides constituting the cavity resonator system and rotated around the central axis normal to the sample plane. The microwaves were irradiated to this plane and the transmitted microwave intensity was measured at different rotation angles (Osaki, 1990a, 1997, 1999). The angular dependence of transmitted microwave intensity, called the orientation pattern (Osaki, 1990a), was measured at 12 GHz. The orientation pattern gives the orientation angle and the degree of fiber orientation (DFO). The direction in which the transmitted microwave intensities are at a minimum is designated as the orientation angle corresponding to the angle between the main axis of the collagen-fiber chains and the SD (Osaki, 1990a, 1999). The maximal-to-minimal ratio of transmitted microwave intensity is defined as the DFO reflecting the mechanical anisotropy (Osaki et al., 1993). For example, a DFO of 1.0 reflects random orientation. The transmitted microwave intensity, I, at a fixed measuring frequency, f m, after insertion of a sample in the cavity resonator system (Osaki, 1990a, 1997) is expressed as a function of rotation angle,, as I( ) I 20 ( )/{1 Q 2 2 ( )[f 20 ( )/f m f m /f 20 ( )] 2 } [1] Here, the I at the f m higher than a resonance frequency f 20 is just half of the peak intensity I 20 at f 20 (Osaki, 1990a), and Q 2 is the Q-value corresponding to the ratio of the f 20 to the half-width f in the resonance curve, all at. The subscript 2 indicates the values after the insertion of the sample. The errors in the values observed for the orientation angle and the degree of orientation measured by Osaki s microwave method were within 1 and 5%, respectively. An accuracy in the values is due to variations in the chemical components and the density, uneven sample thickness, and adjustment of the sample axis to the SD. The dielectric constant and dielectric loss were determined from the values of the resonance frequency and the Q-value. When the sample is a sheet material and its size is larger than that of the opening of each waveguide, the and are given by (Osaki, 1997): 1 A(c/t)[(f 10 f 20 )/f 20 ] [2] (Bc/2t)[1/Q 2 1/Q 1 ] [3] Here, c is a parameter related to the depth of the rectangular waveguides, and A and B are constants associated with the instrument. The subscripts 1 and 2 indicate the values before and after the insertion of the sample. The and for the 1-mm-thick hydroxyapatite disc and the 20- m-thick collagen film were measured at 12 GHz and at a temperature of 25 C using the microwave method (Osaki, 1997). RESULTS Orientation Pattern of Calcaneus Bone Using the Microwave Method Figure 2 shows the angular dependence of transmitted microwave intensity at 12 GHz for three different slice COLLAGEN-FIBER ORIENTATION IN HUMAN CALCANEUS 105 Fig. 2. The angular dependence of transmitted microwave intensity at 12.0 GHz for slice specimens (specimens 3-1, 3-9, and 3-17) at three different positions from the heel to the middle along the long axis of the left calcaneus. The orientation pattern differs with changing position up to ca. 5 cm from the heel end on the long axis. The orientation angle and the DFO change from 13 to 22 and from to for specimens 3-1 to 3-17, respectively. The SD corresponds to the direction parallel to the long axis of the human lower limb. specimens cut in the direction perpendicular to the long axis of the left calcaneus and parallel to the long axis of the human lower limb. The angular dependence differs at three different positions on the long axis of the calcaneus. That is, the orientation angle and the DFO change with position along the long axis. Figure 3 shows the orientation angle of slice specimens (ca. 1.5 mm thick) at 17 different positions on the long axis of the left calcaneus. The orientation angle changes continuously and gives plus to minus values from the heel to the middle part along the long axis. In particular, the direction of orientation is deviated to the lateral side at the heel part of the left calcaneus, and to the medial side at the middle part. Such a result suggests that the direction of orientation corresponds to that of the stress applied to the calcaneus. Figure 4 shows the DFO for slice samples at different positions on the long axis. The DFO is relatively large in the heel part, but is small in the middle part. Although the variation in the data is large, the DFO decreases, on average, from the heel to the middle. The small value of DFO in the middle means that the fiber orientation is almost random around the sample plane. Thus, the orientation determined by the microwave method may reflect the orientational distribution of stress applied to the calcaneus bone. Complex Dielectric Constant of Hydroxyapatite and Collagens In order to study the contribution of collagen fibers to calcaneus bones, dielectric measurements were carried out for a hydroxyapatite disc and a collagen-fiber film. The hydroxyapatite crystals showed a dielectric constant of 2.65 and dielectric loss of 0.19 at 12 GHz, while the collagen film showed a dielectric constant of 4.30 and dielectric loss of The complex dielectric constant of the collagen film was much larger than that of the hydroxyapatite. This means that the orientation pattern reflecting the dielectric anisotropy of the slice calcaneus may be mainly affected by the collagen fibers.

4 106 OSAKI ET AL. The complex dielectric constant for the hydroxyapatite disc and collagen film suggests that the contribution of the hydroxyapatite crystals to the orientation pattern obtained for the slice clacaneus is relatively small. The chelate extraction of slice samples indicates that the collagen fibers are, on average, oriented in a preferred direction. These results show that the orientation pattern measured for sliced calcaneus consisting of hydroxyapatite crystals and collagen fibers accurately reflects the orientation of collagen fibers in human bones. Fig. 3. The orientation angle determined by the microwave method is plotted against the position of the slice specimens (specimens 3-1 to 3-17) up to ca. 5 cm from the heel end on the long axis of the calcaneus. The direction of orientation for the left calcaneus deviates to the lateral side at the heel of the left calcaneus and to the medial side at the middle. Here, a plus sign of angle means the deviation to the lateral side. Fig. 4. The DFO determined by the microwave method is plotted against the position of the slice specimens (specimens 3-1 to 3-17) up to ca. 5 cm from the heel end on the long axis of the left calcaneus. The degree of orientation is large in the heel and small in the middle along the long axis of the left calcaneus. Fiber Orientation After Chelate Extraction The angular dependence of transmitted microwave intensity was measured for the slice samples from which minerals (such as Na and Ca) were removed, using the microwave method (Osaki, 1999). The direction of orientation obtained for the slices with less minerals was almost the same as that obtained for the untreated slice samples, while the degree of orientation changed after the chelate extraction with EDTA-3Na. The change in the degree of orientation for samples treated by chelate extraction may be due to the change in the content of collagen fibers oriented in a preferred direction. This supports the notion that the direction of orientation measured for the sliced bones with geometrical defects induced by chelate extraction corresponds to that in which the collagen fibers are, on average, aligned. DISCUSSION Orientation of Collagen Fibers for Estimating Mechanical Stress In the present study it was found that the microwave method is useful for determining the orientation of collagen fibers in human bones. A previous study (Osaki, 1999) showed that the orientation direction of collagen fibers in calf leather corresponds to the direction of highest mechanical strength. Similarly, the orientation direction of collagen fibers in a calcaneus slice will correspond to the direction in which the mechanical compression strength is high. The DFO will also reflect that of the mechanical anisotropy. In particular, the direction of collagen-fiber orientation in the calcaneus bone may correspond to the direction in which mechanical stress is applied, and areas with a high degree of orientation in the calcaneus bone may correspond to the areas where the stress is highest. In the present study, the orientation of the collagen fibers obtained in the left calcaneus indicates that mechanical stress is applied to the lateral side at the heel part of the calcaneus and to the medial side at the middle, and that the mechanical stress is relatively high at the heel and low at the middle of the calcaneus. Such results will provide useful information for estimating the distribution map of mechanical stress applied to the calcaneus bone. CONCLUSIONS The orientation of collagen fibers offers useful information concerning the mechanical functions of the calcaneus and other bones. If slice samples are prepared by cutting the bone in three different perpendicular directions, the microwave method will give a three-dimensional distribution of collagen-fiber orientation in human bones. It then provides important information regarding the growth of bones and differences in fiber orientation between races and walking styles, and will also clarify the various functions of human bones from the clinical point of view. In the near future we will report on the three-dimensional distribution of collagen-fiber orientation in human bones and discuss the relationship between collagen-fiber orientation and mechanical stress, and compare our findings with the fine structures observed by optical and electron microscopic methods. LITERATURE CITED Bacon GE, Bacon PJ, Griffiths RK A neutron diffraction study of the bones of the foot. J Anat 139: Gierse H The cancellous structure in the calcaneus and its relation to mechanical stressing. Anat Embryol 150:63 68.

5 Greenfield MA, Graven JD, Huddleston A, Kehler ML, Wishko D, Stern R Measurement of the velocity of ultrasound in human cortical bone (in vivo). Radiology 138: Osaki S Microwaves quickly determine the fiber orientation of paper. Tappi J 70: Osaki S Dielectric anisotropy of nonwoven fabrics by using the microwave method. Tappi J 72: Osaki S. 1990a. Explanation of orientation patterns determined for sheet materials by means of microwaves. J Appl Phys 67: Osaki S. 1990b. Orientation test. Nature 347:132. Osaki S A new microwave cavity resonator for determining molecular orientation and dielectric anisotropy of sheet materials. Rev Sci Instrum 68: Osaki S Distribution map of collagen fiber orientation in a whole calf leather. Anat Rec 254: COLLAGEN-FIBER ORIENTATION IN HUMAN CALCANEUS 107 Osaki S Use of hair pores to determine the orientation of collagen fibers in skin. Anat Rec 263: Osaki S, Yamada M-O, Takakusu A, Murakami K A new approach to collagen fiber orientation in cow skin by the microwave method. Cell Mol Biol 39: Tadokoro H Structure of crystalline polymers. New York: John Wiley & Sons. p Tohno Y, Tohno S, Matsumoto H, Naito K A trial of introducing soft X-ray apparatus into dissection practice for students [Japanese]. J Nara Med Assoc 36: Wahner HW, Fogelman I The evaluation of osteoporesis: dual energy X-ray absorptiometry in clinical practice. London: Martin Duntiz. Yettram AL, Camilleri NN The force acting on the human calcaneus. J Biomed Eng 15:46 50.