# Determination of Collagen Fibers Arrangement in Bone Tissue by Using Transformations of Raman Spectra Maps

Authors: Tomasz Buchwald; Marek Kozielski; Mirosław Szybowicz
Journal: Spectroscopy: An International Journal
 (2012)

**Keywords:** Raman spectroscopy; bone tissue; collagen fibers arrangement


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## Abstract

The goal of this work was to evaluate the ability of Raman spectroscopy to identify molecular organization and chemical composition of extracellular matrix such as the collagen fibers arrangement, the level of mineralization, and the carbonate accumulation in mineral phase in spongy bone of the human head of the femur. Changes in composition and structure of the spongy bone tissue were illustrated using maps of polarized Raman spectra. In particular, the purpose of the present study was determination of arrangement of mineralized collagen on surface of trabecula by using transformations of Raman spectra maps. Transformations of Raman spectra maps were needed in order to remove impact of chemical composition on images of Raman spectra map, which display the collagen fibers orientation. These transformations allow to obtain simultaneously the distribution of constituents of bone and arrangement of collagen fibers on tissue surface. A method to indicate the collagen orientations is developed to understand the molecular organization in healthy and unhealthy bone at the microstructural level.


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## Body

## 1. Introduction

Bone tissue is a heterogeneous material composed of cells such as osteoblasts, osteocytes, osteoclasts, and an extracellular matrix, which consists primarily of collagen fibrils, mineral phase (hydroxyapatite and carbonate apatite crystals), and water [1–3]. The organic matrix is mainly made up of type I collagen fibers [4]. The collagen fibers are more elastic and resistant to fractures than the apatite crystals. Therefore, the bone structure strength is determined by the mineral components [5], whereas the elasticity of the tissue is determined by the organic components [6]. By combining the high toughness of collagen and the high stiffness of mineral, bone achieves its remarkable mechanical properties [7]. Bone in human and other mammal bodies is generally classified into two different forms: cortical bone and spongy bone. Cortical bone, also known as compact bone, is found mainly in the shaft of long bones and forms the outer shell around spongy bone. Spongy bone, also known as cancellous or trabecular bone, is found in the end of long bones, in vertebrae and in flat bones [8, 9]. Cortical bone is much denser and less porous than spongy bone. Both bone types have a hierarchical structure. The basic first level structure of cortical bone is osteon (osseous laminae arranged in a concentric pattern around Haversian canals), whereas spongy bone is formed by a network of trabeculae of different shape and thickness arranged along the directions of action of mechanical pressure forces [10, 11]. The mode of trabeculae arrangement allows to distribute and dissipate the energy from articular contact loads.Bone mass or bone mineral density is the most common parameter to clinically assess the bone quality. However, the content of mineral and organic constituents, the spongy bone microarchitecture, the orientation and cross-linking of collagen component, the accumulation of microdamage, and the bone remodeling activity or turnover also influence on the bone quality [3, 12–16]. In this way, assessment of the bone quality is complicated. One approach to address this issue is Raman microspectroscopy. This vibrational spectroscopy has been successfully applied to characterize tissues, especially bone tissue [17–20]. The use of Raman spectroscopy in biological investigation offers several advantages. This physical technique allows to determine the chemical composition and structure of tissue at the microstructural level [21, 22]. Raman spectroscopy offers micron-scale spatial resolution (0.5–1 μm) and enables analysis of biologically important sites such as individual cement lines, individual lamellae, regions around microcracks, and human dentin tubules [23, 24]. Spectroscopic data are obtained in a noninvasive manner, so the same sample can be examined by the various analytical methods. Raman spectra are not obscured by water, allowing analysis of fully hydrated samples, with minimal sample preparation. The visible or near-infrared optics, that are used by Raman instruments, are compatible with fiber-optic probes used in clinical instruments such as arthroscopes. In this way, Raman spectroscopy method could be used directly in arthroscopic surgery. Esmonde-White et al. showed the use of Raman spectroscopy for arthroscopy of human joint tissues using a custom-built fiber-optic probe [25]. Raman spectroscopy can provide detailed chemical information on the mineral and collagen matrix components in unprocessed bone preparations [20, 26]. Raman bands are sensitive to the molecular orientation; therefore the arrangement of collagen fibers and apatite crystals in osteons and trabeculae can be also determined [16, 27–29]. By using a motorized microscope stage, Raman spectroscopy technique allows to obtain the Raman spectra maps of the sample giving spatial information and showing the distribution and orientation of the chemical constituents on tissue surface. In earlier work, Raman spectral imaging has been used to study the complex organized systems such as bone tissue [24, 27, 28, 30].Polarization of excited and Raman scattered light provides the additional information on the structure and orientation of the molecular components of bone. Some of bands in Raman spectrum of bone tissue are quite sensitive to the molecular orientation [27–29]. Spectral parameters of phosphate ν1 and amide I Raman bands change more in different polarization direction of excited and scattered beam than spectral parameters of amide III, phosphate ν2, and phosphate ν4 Raman bands. The crystallographic C-axis of apatite crystals is along the collagen fibers and is associated with ν1 phosphate vibrations. Thus the phosphate ν1 band intensity has a maximum value when the polarization of incident light is parallel to the orientation of collagen fibers [31]. The collagen triple helix structure determines the positions of the amide bonds with respect to the backbone. The C=O bonds in amide I are perpendicular to the collagen molecular axis; therefore the amide I band, which is associated with the C=O stretching vibration, is more intense in the polarization direction perpendicular to the collagen fiber axis [32]. In this way, the phosphate ν1 and amide I bands are quite sensitive to the polarization direction of the incident and scattered light and have been related to the local orientation of collagen fibers and apatite crystals in the bone tissue. On the other hand, the bands such as those assigned to amide III, phosphate ν2, and phosphate ν4 are less sensitive to the orientation effects and indicate changes in mineral and organic composition of bone tissue [29]. In this study the linearly polarized incident and scattered light was used to obtain Raman spectra maps, which allow to show collagen fibers arrangement on trabecula surface.Raman spectra provide information on both mineral and organic matrix composition in bone tissue. Two of the most commonly reported measurements of Raman analysis of bone chemistry is the hydroxyapatite to collagen ratio and the carbonate apatite to hydroxyapatite ratio, obtained from the integrated areas of the phosphateν2 and ν4, carbonate ν1, and amide III bands. These parameters provide an indication of mineralization of bone tissue and describe the degree of carbonate substitution in the apatite crystal lattice. Raman spectroscopy also allows to indicate the bone tissue organization such as orientation of collagen fibers in osteons and trabeculae by the use of hydroxyapatite to collagen ratio parameter. The hydroxyapatite to collagen ratio is obtained from integrated area of the phosphate ν1 and amide I bands, which are dependent on tissue organization. In earlier studies the proposed parameters have been used to estimate the compositional and structural properties of bone tissue [16, 23, 24, 33–36]. Irregularity of surface of biological material influences intensity of bands in Raman spectrum. Due to potential variations in distance from the objective to the sample, the band area ratios were employed to describe composition and structure of bone tissue.The goal of this work was to evaluate the ability of Raman spectroscopy to identify molecular organization and chemical composition of extracellular matrix such as the collagen fibers arrangement, the level of mineralization, and the carbonate accumulation in mineral phase in spongy bone of the human head of the femur. Changes in composition and structure of the spongy bone tissue were illustrated using maps of polarized Raman spectra. In particular, the purpose of the present study was determination of arrangement of mineralized collagen on surface of trabecula by using transformations of Raman spectra maps. Transformations of Raman spectra maps, new application to determine the bone properties, were needed in order to remove impact of chemical composition on images of Raman spectra map, which display the collagen fibers orientation. These transformations allow to obtain simultaneously the distribution of constituents of bone and arrangement of collagen fibers on tissue surface. A method to indicate the collagen orientations is developed to understand the molecular organization in healthy and unhealthy bone at the microstructural level.


## 2. Materials and Method

In this study, Raman microspectroscopy was used to estimate the chemical composition and orientation of collagen fibers of human spongy bone of femur heads collected from the five patients between 60 and 85 years of age, who underwent a high-energy femoral neck fracture. The treatment of choice in the above-mentioned patients was hip replacement surgery (total hip arthroplasty). The cross-sections of five millimeters in thickness were obtained from heads of femurs of these patients. In this work, the results of a study on one representative femur head from a 63-year-old woman are presented. The study has got the approval of the local Bioethical Commission at the Wielkopolska Medical Chamber in Poznań (no 14/2008 from August 27, 2008).All the spectroscopic measurements were performed on confocal Raman microspectroscope (inVia) made by Renishaw equipped with diode pumped laser emitting 785 nm infrared wavelength. The laser beam was tightly focused on the sample surface through a Leica 50x LWD microscope objective (LWD: long working distance) with numerical aperture (NA) equals 0.5, leading to a laser beam diameter about 2μm. The Raman scattering spectra were acquired from surface of the bone tissue in the back-scattering geometry in the spectral range of 200–1800 cm−1. During measurements, to minimize sample degradation or sample heating an incident power was dimmed to approximately 10 mW. The wavenumber and intensity were regularly calibrated with the use of 521 cm−1 band of a silicon internal sample. The position of the microscope objective with respect to the bone tissue was piezoelectrically controlled in the three axes during surface mapping. The measurements were made under a microscope in the high confocality mode with the depth resolution equals 2.2 μm. The incident light was linearly polarized optionally with a half-wave plate. The Raman scattered light was detected in the linear polarization (vertical and horizontal direction) using polarisers. The Raman spectra at different laser polarizations were obtained on the same position for given sample. The Raman spectra maps were achieved collecting single spectra, with an exposure time of 10 s for each spectrum without accumulation. The Raman images of surface of bone tissue were acquired in the rectangular areas of 100×100μm2 with steps of 10 μm at seven different sites of particular femur heads (thirty-five different sites of five femur heads). The analyses of arrangement of mineralized collagen on surface of trabecula by using transformations of these thirty-five Raman maps gave the same results. Therefore, the results of a study on one site of femur head are presented. The samples were measured at room temperature (296 K). Cosmic ray artefacts were removed and analyses of the spectra were performed in the WIRE 3.1 (Renishaw) software. Rayleigh scattering background was subtracted manually from each raw spectrum by using the polynomial curve. The integrated areas of Raman bands were calculated by curve-fitting or deconvolution in single spectra. The integrated area ratios of Raman bands were used to detect differences in the composition and structure of bone tissue. The images were generated by the OriginPro 8.0 software.


## 3. Results and Discussion

Figure1 gives a general view of the Raman spectra from spongy bone sample showing the major bands corresponding to the mineral and organic phases. The position of the main vibrational bands in the observed Raman spectra of bone tissue and their respective assignments are listed in Table 1. The mineral bands of the spectrum are dominated by the ν1 phosphate (PO43-) internal mode at 961 cm−1. The other markers of the mineral component connected with the ν2 and ν4 phosphate vibrations are detected at 431 cm−1 and 589 cm−1, respectively [20, 27]. The internal modes of the carbonate group (CO32-) are detected at 1072 cm−1 (ν1 mode of B-type carbonate) and 1103 cm−1 (mode of A-type carbonate) [16]. However, the weakness of the A-type carbonate band does not permit getting information about the composition of bone tissue; therefore B-type mode only is used. The bands assigned to the organic components (collagen and noncollagen moieties) are found in regions: ~1200–1305 cm−1 (amide III), ~1599–1701 cm−1 (amide I), ~1405–1490 cm−1, and ~2800–3100 cm−1 (bending and stretching modes of C–H groups, resp.) [20, 28]. The Raman signal depends not only on the composition but also on the local orientation of collagen fibers or apatite crystals. Therefore, to indicate changes in chemical composition in bone tissue bands, which are less sensitive to the orientation effects were used. The mineral and organic composition were determined from the following Raman bands: the amide III (integrated area ~1200–1305 cm−1), phosphate ν2 (integrated area ~370–486 cm−1), phosphate ν4 (integrated area ~511–632 cm−1), and carbonate ν1 (integrated area ~1041–1107 cm−1). Dependency of amide I and phosphate ν2 Raman bands intensity on structural organization allows to determine arrangement of collagen fibers in bone tissue. The orientation of collagen fibers was indicated using the integrated area ~1599–1701 cm−1 of amide I band and ~932–989 cm−1 of phosphate ν2 band.Table 1
Position of the main vibrational bands in the observed Raman spectra of bone tissue and their respective assignments.
Wavenumber (cm−1)Assignment431ν2 (PO43-)589ν4 (PO43-)961ν1 (PO43-)1072ν1 (CO32-) B type1103ν (CO32-) A type1200–1305Amide III1405–1490C–H bending1599–1701Amide I2800–3100C–H stretchingFigure 1
Representative Raman spectrum collected from spongy bone tissue showing the major bands and the corresponding compounds. Background signal has been removed.The ratios of integrated area of the appropriate bands in the Raman spectra were employed to obtain the biochemical properties of bone tissue. As markers of chemical composition the ratios ofν2PO43-/amide III, ν4PO43-/amide III, and ν1CO32-/ν2PO43- were used. The phosphate to collagen ratio identified differences in mineralization of bone tissue, whereas the carbonate-to-phosphate ratio gave information about the degree of carbonate accumulation in the apatite crystal lattice. The ratio of ν1PO43-/amide I was calculated and used to investigate orientation of collagen fibers. The compositional and structural changes in spongy bone were illustrated using maps of polarized Raman spectra. Figure 2 presents images of the ratios of ν2PO43-/amide III (Figures 2(a), 2(d), and 2(g)), ν1CO32-/ν2PO43- (Figures 2(b), 2(e), and 2(h)) and ν1PO43-/amide I (Figures 2(c), 2(f), and 2(i)). The maps of Raman spectra were generated in the vertical and horizontal polarization of incident and scattered light. The arrows in the figures indicate these polarization directions. Each image displays the same area of spongy bone surface. In the scale bars, the maximum values of the ratios are specified and the minimum value is 0 for all images. The bright contrast corresponds to the maximum ratio, while dark to the lowest one.Figure 2
The contrast images based on the ratios ofν2PO43-/amide III ((a), (b), and (g)), ν1CO32-/ν2PO43- ((b), (e), and (h)), and ν1PO43-/amide I ((c), (f), (i)). Arrows indicate polarization of laser incident and scattered beams, and the color bar displays the maximum ratio for each image.The bands used in the ratios ofν2PO43-/amide III and ν1CO32-/ν2PO43- are less sensitive to orientation effect, so the maps based on these ratios are similar and do not show considerable changes in the contrast for different polarizations of incident and scattered light. In this way, the ν2PO43-/amide III and ν1CO32-/ν2PO43- ratio images are used as an indicator of distribution of chemical components in spongy bone tissue. Conclusions on the degree of mineralization of spongy bone can be drawn from Figure 2 showing the ratios of ν2PO43-/amide III obtained for the vertical polarization of laser light (V polarization) (Figure 2(a)), the vertical polarization of laser incident and scattered beams (VV polarization) (Figure 2(d)), and the horizontal polarization of laser incident and scattered beams (HH polarization) (Figure 2(g)). These images present very similar contrast changes despite the fact that the Raman maps were obtained in different polarization directions. Therefore, contrast images based on the ratio of ν2PO43-/amide III are a good indicator of local variations in the content of hydroxyapatite crystals with respect to that of collagen on surface of trabecula. The brighter and darker areas in these images correspond to higher and lower ratios of mineral to organic content. Figure 2 displays images for the ratios of ν1CO32-/ν2PO43- obtained for the vertical polarization of laser light (Figure 2(b)), the vertical polarization of laser incident and scattered beams (Figure 2(e)), and the horizontal polarization of laser incident and scattered beams (Figure 2(h)). These images illustrating relative distribution of mineral components on the surface of trabecula present also similar contrast changes as those for the ratios of ν2PO43-/amide III despite the fact that the Raman maps were obtained in different polarization directions. The brighter and darker areas in these images correspond to higher and lower ratios of carbonate apatite to hydroxyapatite content. The higher ratio of carbonate-to-phosphate crystals in Figures 2(b), 2(e), and 2(h) occurs in the same area as the lower ratio of hydroxyapatite to collagen content in Figures 2(a), 2(d), and 2(g). It is possible that the increased carbonate substitution in the apatite crystal lattice occurred in response to deficient mineralization.Figure2 displays ν1PO43-/amide I ratio contrast images of the bands quite sensitive to the collagen fibers orientation obtained for the vertical polarization of laser light (Figure 2(c)), the vertical polarization of laser incident and scattered beams (Figure 2(f)), and the horizontal polarization of laser incident and scattered beams (Figure 2(i)). The ν1PO43-/amide I ratio contrast images should determine the collagen fibers arrangement in trabecula; however these images do not show a pronounced structural effect. Figures 2(c), 2(f), and 2(i) based on the ν1PO43-/amide I ratio reveal slight contrast changes in comparison with those in the other ratio images referring to the chemical composition in trabecula (Figures 2(a), 2(d), and 2(g)). The similarity in the character of the maps referring to the collagen orientation and chemical composition is probably related to the distribution of bone tissue constituents. This means that differences in contrast in the maps referring to chemical composition are too large with respect to these referring to collagen fibers arrangement, so the orientation effect is undetectable. Hence, Figures 2(c), 2(f), and 2(i) present very similar contrast images to Figures 2(a), 2(d), and 2(g). Moreover, the images in Figures 2(f) and 2(i) illustrating collagen orientation on surface of trabecula are almost identical and their contrasts do not change much for different laser incident and scattered light polarizations. Therefore, it is difficult to conclude if the highest contrast corresponds to the orientation of collagen fibers or to the changes in chemical composition. To remove impact of chemical composition on ν1PO43-/amide I ratio contrast images obtained for different laser incident and scattered light polarizations, transformations of these contrast images were used. Transformations permit to display on images the orientation of collagen fibers without chemical composition.Figure3 displays contrast images obtained by using transformations of Raman spectra maps. In the scale bars, the maximum values of the ratios are specified and the minimum value is 0 for all images. The bright contrast corresponds to the maximum values, while dark to the lowest one. Figures 3(a), and 3(b) display images obtained by using the first transformation of Raman spectra maps. Figure 3(a) illustrates image with different values between ν1PO43-/amide I ratio values obtained for the horizontal polarization of laser incident and scattered beams (HH polarization) and ν1PO43-/amide I ratio values obtained only for the horizontal polarization of laser light (H polarization). Figure 3(b) illustrates image with difference values between ν1PO43-/amide I ratio values obtained for the vertical polarization of laser incident and scattered beams (VV polarization) and ν1PO43-/amide I ratio values obtained only for the vertical polarization of laser light (V polarization). This first transformation of Raman spectra maps allows to display collagen fibers orientation, which is parallel to the horizontal direction (Figure 3(a)) and parallel to the vertical direction (Figure 3(b)). If the orientation of collagen fibers is not parallel to the horizontal or vertical polarization of laser light, so if the fibers are skewed, then the changes in polarizability of collagen molecule are detected for both light polarizations. Therefore, this collagen fibers arrangement is also visible simultaneously in Figures 3(a) and 3(b). The arrows in Figures 3(a) and 3(b) indicate collagen fibers arrangement on surface of trabecula.Figure 3
The contrast images obtained by using transformations of Raman spectra maps. The contrast images with difference values betweenν1PO43-/amide I ratio values obtained for the HH polarization and H polarization [HH−H] (a), for the VV polarization and V polarization [VV−V] (b). The contrast images with the total values [(VV−V) + (HH−H)], which is the sum of [VV−V] values and [HH−H] values from the first transformation (c). Arrows indicate collagen fibers arrangement on surface of trabecula, and the color bar displays the maximum value for each image.Figure3(c) displays contrast image obtained by using the second transformation of Raman spectra maps. Figure 3(c) illustrates image with the total values [(VV−V) + (HH−H)] which is the sum of [VV−V] values and [HH−H] values from the first transformation. This second transformation allows to display collagen fibers arrangement on surface of trabecula in each direction. Circles in Figure 3(c) indicate areas with the maximum values. Areas with the maximum values are found in the parts of trabecula surface where the arrows are crossed. It could mean that collagen fibers overlap each other in these parts of trabecula. The results of Raman maps transformations show that this method to analysis of collagen fibers arrangement on surface of trabecula could be very useful to determine properties of bone tissue at microstructural level.


## 4. Conclusion

In this study the linearly polarized incident and scattered light was used to obtain the Raman spectra maps to show changes in the structure of trabecula. The Raman spectra maps based on the ratios ofν2PO43-/amide III and ν1CO32-/ν2PO43- do not show considerable changes for different polarizations of incident and scattered light, so these images were taken as an indicator of spongy bone composition, that is, give the information about relative amount of hydroxyapatite to collagen and carbonate apatite to hydroxyapatite. The Raman spectra maps based on the ratio of ν1PO43-/amide I should show the collagen fibers arrangement in trabecula, because bands in this ratio are quite sensitive to the collagen orientation. However, this image does not show distinct changes for different polarizations of incident and scattered light. It could mean that differences in contrast in the maps referring to chemical composition are too large with respect to those referring to collagen fibers orientation so the orientation effect is undetectable. These results suggest to use transformations of Raman spectra maps, which allow to remove impact of chemical composition on contrast images of these maps. In this study, we use two transformations of Raman spectra maps. The first transformation removed impact of chemical composition on contrast images and indicated collagen fibers arrangement on surface of trabecula in parallel to the horizontal and vertical direction. The second transformation allowed to display collagen fibers arrangement on surface of trabecula in each direction. The results allow concluding that ν1PO43-/amide I ratios permit to determine the orientations of collagen fibers in spongy bone tissue; however if we would like to say more about collagen fibers arrangement, we could use transformations proposed in this work.Results of investigations demonstrate the possibility of the Raman spectroscopy application for microanalysis of composition and collagen fibers arrangement in the human bone tissue. The method, which allows to distinguish between orientation and compositional contribution, is crucial as bone is comprised of mineralized collagen fibers with alternating orientation in osteons and trabeculae. Understanding the bone tissue organization at the microstructural level can help finding the origins of bone diseases such as osteoporosis or osteoarthritis. Determination of the mode of collagen fibers arrangement permits evaluation of the biomechanical properties of bone, so provides information of susceptibility of bone to fracture.



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*Source: 261487-2012-05-28.xml*