The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:34-40 (2007)
© 2007 The Gerontological Society of America
Physicochemical Composition of Osteoporotic Bone in the Trichothiodystrophy Premature Aging Mouse Determined by Confocal Raman Microscopy
Aart A. van Apeldoorn,
Jan de Boer,
Harry van Steeg,
Jan H. J. Hoeijmakers,
Cees Otto and
Clemens A. van Blitterswijk
Departments of 1 Polymer Chemistry and Biomaterials and 4 Biophysical Techniques, Faculty of Technology and Sciences, University of Twente, Bilthoven, The Netherlands.
2 Department of Carcinogenesis, Mutagenesis and Aging, The National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
3 University Medical Center Rotterdam, Department of Genetics, The Netherlands.
Address correspondence to A. A. van Apeldoorn, PhD BioSc, Department of Polymer Chemistry and Biomaterials, Faculty of Technology and Sciences, University of Twente, P.O. Box 98, 3720 AB Bilthoven, The Netherlands. E-mail: a.a.vanapeldoorn{at}tnw.utwente.nl
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Abstract
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Although it has been established that premature aging trichothiodystrophy (TTD) mice display typical signs of osteoporosis, exact changes in physicochemical properties of these mice have not been elucidated. We used confocal Raman microscopy and histology to study femora of TTD mice. We measured femora isolated from xeroderma pigmentosum group A (XPA)/TTD double mutant mice to establish that Raman microscopy can be applied to measure differences in bone composition. Raman data from XPA/TTD mice showed remarkable changes in bone mineral composition. Moreover, we observed a severe form of osteoporosis, with strongly reduced cortical bone thickness. We used Raman microscopy to analyze bone composition in eight wild-type and eight TTD animals, and observed decreased levels of phosphate and carbonate in the cortex of femora isolated from TTD mice. In contrast, the bands representing the bone protein matrix were not affected in these mice.
TRICHOTHIODYSTROPHY (TTD) is a rare autosomal recessive disorder; patients have brittle hair, postnatal mental and growth retardation, peculiar face morphology, ichthyosis, photosensitivity, and skeletal disorders (1). Mice harboring the TTD-specific point mutation in the xeroderma pigmentosum group D gene (XPD) were found to show symptoms similar to those found in the human disease (2,3). Key features found in this mouse strain are skeletal abnormalities, such as kyphosis, osteosclerosis of the calvaria, and osteoporosis in the vertebrae and limbs (4). Osteoporosis is a condition of bone where decreased bone mineral density (BMD) and structural deterioration of bone tissue occurs (5,6), leading ultimately to fragility and an increased susceptibility to fractures. In humans, osteoporosis is found in older women and men, especially at later ages (>55 years). In The Netherlands alone about 430,000 people are thought to be positive for this bone defect (7). The TTD mouse seems to be an ideal candidate to investigate whether decreased BMD has an effect on the physicochemical bone composition, so we have used confocal Raman microscopy together with histology to study the femora of TTD mice.
Currently, Raman spectroscopy is more and more used to study biological samples, because there is little hindrance from water, and complex sample preparation is unnecessary. Several investigators have used Raman spectroscopy to study early mineralization (8,9), mechanical deformation of cortical bone (10), and fatigue-related microdamage of bovine bone samples (11). To eliminate the protein background fluorescence that frustrates the detection of the Raman spectra of bone, near-infrared excitation can be used (12). Frozen or embedded sample sections with thicknesses ranging from less than 1 µm to samples of intact tissues or cells can be analyzed by using a confocal setup. In hydroxyapatite, a major component of bone, the phosphate 
1 vibration is found at 960 cm–1. When hydroxyapatite becomes less perfect, for example, the mineral lattice has decreased crystalline-to-amorphous ratio, or vacancies and/or substitutions, the Raman band of phosphate can broaden or shift to lower wave numbers. The phosphate 1 vibration (960 cm–1) and monohydrogen phosphate 1 vibration (1003 cm–1) are considered accurate markers of substitution and structure of the mineral at hand (13,14). In addition, the Raman bands positioned around 1200–1300 (amide III), 1600–1700 (amide I), and 1400–1470 and 2800–3100 cm–1 (bending and stretching modes of CH groups, respectively) are markers for the organic protein component of bone matrix (mainly collagen type I).
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MATERIAL AND METHODS
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Animals
In this study, two mouse strains were examined by confocal Raman spectroscopy and histology. The first strain consisted of mice in a C57BL/6 background, which were either wild type (WT mice) or homozygous for the mutation XPDR722w (TTD mice). A group of eight TTD female mice were observed at 18 months of age, at which time the mice were killed and the femora removed for confocal Raman microscopy.
The second strain consisted of mice with a mixed C57BL/6:129 background all with a homozygous mutation in the xeroderma pigmentosum group A gene (XPA) (15). XPA encodes a DNA binding zinc-finger protein that recognizes DNA damage caused by ultraviolet radiation. As such, the XPA protein participates in the initial step of the process of nucleotide excision repair. These mice were either XPA mice or XPA mice also homozygous for the mutation in XPDR722w (XPA-TTD mice). The four female XPA-TTD mice used to evaluate mineral content were previously described by de Boer and colleagues (4). Because XPA-TTD mice have a very low neonatal survival, and fail to thrive at 3 weeks of age, at which point most animals die, the number of animals observed in this group was low (n = 4) because of their limited survival. Therefore, the data obtained from the XPA-TTD mice can only be used to describe the phenotype in a qualitative manner. The femora from these 3-week-old mice were used for confocal Raman microscopy and histology in the same manner as those from the TTD mice. The control groups consisted of wild-type strain counterparts of the same age as each of the two different mutant mice strain groups.
Femora
After removal, the femora were fixed in a 4% paraformaldehyde solution for at least 24 hours, after which time surrounding tissue, skin, connective tissue, and muscle were removed for Raman measurements or left intact for histology. Fixation protocols have been proven to have no or very little influence on bone or soft tissue composition, although a minor shift of amide bonds can sometimes be observed, which does not affect the intensity of Raman bands (16,17). The samples were then stepwise dehydrated in an increasing ethanol series starting at 70% and finishing at 100%, after which they were critical point dried using a BAL-TEC CPD 030 Critical Point Dryer machine (Bal-Tec AG, Witten, Germany). In the femora obtained from XPA-TTD mice, the left leg was used for histological evaluation and the right leg was used for critical point drying and Raman spectroscopy.
Confocal Raman Microscopy
We used a confocal Raman microscope extensively described elsewhere (18–21) to measure the isolated femora at the bone surface. In short, the excitation wavelength used was the 647.1 nm line from a Kr ion laser. The field of view is approximately 58 µm in diameter when a 63 Zeiss Plan Neofluar (NA = 1.2) water immersion objective (Carl Zeiss BV, Sliedrecht, The Netherlands) is used. A blazed holographic grating with 600 grates/mm (Jobin-Yvon, Paris, France) was used for dispersion. The lateral resolution is limited by the diffraction, and is 550 nm for this system. The definition of the resolution is based on the diameter of the laser beam waist at which the beam intensity has fallen to 1/e2 of its peak value. In all cases the specimens were measured with 30 mW for 45 seconds to not damage the specimens by overexposure, and to ensure a good signal-to-noise ratio. The charged coupled device (CCD) is connected with a computer for data collection and analysis using WinSpec (Roper Scientific BV, Vianen, The Netherlands) and Microcal Origin (OriginLab Corporation, Northampton, MA) data analysis software. All measurements (n = 10) per femora (n = 8) were performed in a randomized manner at the cortex surface, to obtain a total of 80 spectra. All 80 spectra were then baseline-corrected, and the average spectrum was calculated by using Microcal Origin; this method allows one to compare the data obtained from TTD mice with the data obtained from the control group. After data correction for background fluorescence, the intensity of specific bone mineral and bone matrix Raman bands were determined. (Because the raw data were not exactly horizontal presented when plotted, but on a straight slope, caused by background fluorescence, we corrected for this effect by subtracting the same straight slope from all spectra to ensure the spectra are horizontally aligned with X = 0). Based on data published by de Boer and colleagues (4), we expect the bone mineral content in TTD mice to be lower than that in the wild-type mice. The average intensities of both wild-type and TTD mouse strain bone mineral bands (phosphate and carbonate) were compared using a one-tailed Student t test with homoscedastic sampling. The intensities of bone matrix specific bands, mostly related to collagen type I, were compared using a two-tailed distribution (no data on the bone matrix content of these bones were known beforehand).
Histology
To study its histology, we fixed the complete left hind leg of XPA-TTD mice and control animals (XPA mice) in 4% paraformaldehyde for at least 24 hours. After fixation, the samples were dehydrated in an increasing ethanol series (70%–100%) after which they were immersed in a methyl methacrylate (MMA) solution. Vacuum was applied for 2 hours to ensure that air bubbles were removed before placing the samples in a water bath at 37°C for 2 days to polymerize the MMA. After embedding, serial sections were made on a Leica SP 1600 innerlock diamond saw (Leica Microsystems BV, Rijswijk, The Netherlands). The sections were stained using methylene blue (Sigma, Zwijndrecht, The Netherlands) and basic fuchsine (Sigma) for light microscopic examination.
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RESULTS
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The Raman spectra obtained from one of the four XPA-TTD mice observed in this study showed remarkable differences with respect to the control group. Although the number of animals did not allow for a sound statistical analysis and the differences in the other animals observed were not as extreme, the differences in these spectra are quite noteworthy. In Figure 1, an example of a spectrum in which the differences were most extreme from a XPA-TTD mouse is shown in comparison to spectra obtained from a control group animal. The Raman spectrum of this mouse showed severe decreased intensity of bone mineral specific bands when compared to the control group. The intensities of bone matrix specific bands, in contrast, were similar to those found in the control group. Interestingly, during isolation of these femora we noticed that the XPA-TTD mice seemed to have thinner femora than the wild-type animals. This finding might be related to the above-mentioned spectral data indicating a low mineral content. Based upon these results it was decided to study these samples in more detail by histology to check for altered bone morphology. Several serial cross-sections from the femora of XPA-TTD mice were observed using light microscopy. In Figure 2, a typical light micrograph from an overview cross-section of the femur of XPA-TTD mouse shown in Figure 1 compared to a control can be seen.
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Figure 1. Raman spectrum showing the differences between a wild-type (WT) and a xeroderma pigmentosum group A–trichothiodystrophy (XPA-TTD) mouse in which the lowest amount of bone mineral was observed
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Figure 2. Top and middle: Morphology of wild-type (WT) (left panel) and xeroderma pigmentosum group A–trichothiodystrophy (XPA-TTD) (right panel) mice. Bottom: Morphology of WT (left) and TTD mice (right). Scale bars are 400 µm (top) and 200 µm (bottom). B = bone, BM = bone marrow, SK = skin region, M = muscle region
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In both XPA-TTD and XPA mice, a similar appearance of bone marrow and osteocytes can be found, which is shown in the high magnification light micrographs in Figure 2 (middle). However, in the case of the XPA-TTD mouse, the thickness of the cortical bone was at least 2-fold less than that observed in the controls. These results relate to what was observed initially during explantation of the femora, where we observed that XPA-TTD mouse femora were frailer than wild-type and TTD femora.
In the case of TTD mice, average Raman spectra were used for determination of peak intensities. Raman spectra of both the controls and TTD mice showed specific bone mineral and bone matrix Raman bands (see Figure 3, A and B). The bone mineral bands observed were the phosphate (
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1 symmetric stretch at 960 cm–1 and the B-type carbonate (
) 1 symmetric stretch at 1070 cm–1. The bone matrix band markers for the collagen backbone—amide I at 1655 cm–1, amide III at 1250 cm–1, methylene (CH2)-wag at 1450 cm–1, and phenylalanine at 1004 cm–1—could all clearly be observed in both groups. The average spectra of TTD mice showed a slightly lower intensity of the main phosphate band with respect to the control mice (see Figure 4). In Figure 5, A and B, the average intensities of the most important Raman bands, as shown in Table 1, were plotted, showing the variation in mineral content of the individual mice within the group of animals investigated. A summary of the average intensities of these different Raman bands can be found in Table 1. The data suggest that mineral content in non-TTD mice is slightly higher than that of TTD mice. Results after statistical analysis showed that the average mineral content in TTD mice was lower than that found in the wild-type group. The band for the 1 symmetrical stretch (960 cm–1) and (1070 cm–1) were both lower in the TTD mice than in the wild-type mice (p =.029 and p =.033, respectively), indicating a change in physicochemical content. The bone matrix specific bands, which can be found around 1250, 1450, and 1650 cm–1 do not seem to be affected in the TTD mice as the intensities of these bands did not show any differences with respect to the control group. Interesting was the fact that no clear histological differences could be observed between TTD mice and their wild-type counterparts (Figure 2, bottom).
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Figure 3. Raman spectra from femora of trichothiodystrophy (TTD) (A) and wild-type (WT) (B) mice showing both the molecular assignment and band position of bone mineral and matrix-specific Raman bands
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Figure 5. Comparison of the variation of the intensities of the main Raman bands observed in the trichothiodystrophy (TTD) mouse and the wild-type (WT) cohort
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DISCUSSION
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TTD mice were shown to have many symptoms of an early aging phenotype, including a decreased mineral density in vertebrae and limbs leading to osteoporosis, as suggested by previous studies (2–4). TTD-XPA mutant mice exhibited an even faster rate of aging compared to TTD mice (4), resulting in a severely shortened life span (3 weeks) and spinal kyphosis, which is indicative for osteoporosis. The primary indication for osteoporosis is loss of bone, which can be measured by decreased BMD. Raman spectroscopy and/or imaging have been used in several studies to evaluate bone mineral and bone matrix composition of bone samples (9,11,22–26). In this study, we have used confocal Raman microscopy to investigate the physicochemical composition of femoral bone from TTD and XPA-TTD mice.
XPA-TTD Mouse Femora
Raman measurements of one of the XPA-TTD mice used in this study showed remarkable differences in bone mineral composition in them compared to that in wild-type animals. Although the data presented here do not allow for a statistical analysis because of the small number of animals studied, the amount of bone mineral seems to be reduced in the femora from these animals. The XPA-TTD mouse showing the lowest amount of bone mineral appeared not only to have less bone mineral, but less bone material as well. The femora of these animals seemed to be more fragile, based on physical examination during explantation, than TTD or WT femora, in concurrence with these observations. In addition, histology revealed that the thickness of cortical bone in these mice was about 2-fold less than that found in the control mice. However, the morphology of the bone (apart from the thickness) was comparable to that in the control group. Because we used a high resolution confocal Raman microscope, allowing us to measure a volume of approximately 0.5 x 0.5 x 1.5 µm (simplified ellipsoid laser spot volume), the observation of decreased bone mineral content must be a property of these bones, which might in turn influence their cortical thickness.
TTD Mouse Femora
Raman spectroscopy showed a decreased level of phosphate and carbonate content present in cortical bone of femora isolated from TTD mice compared to WT animals. In contrast, the bone matrix content was not affected, leading to the conclusion that collagen type I content is not changed in these mice. The variation in bone mineral content per femora among TTD mice was similar to what was observed for TTD mice when x-ray radiographs were used by de Boer et al., to determine bone density (4). However, the method used gives limited results, so we decided to use confocal Raman microscopy, which can provide more detailed information on the molecular composition of bone. Although the differences in bone mineral content in TTD and WT mice found in this study are not large, it might explain the decreased bone density found in the study by de Boer and colleagues. A study in Sprague-Dawley rats has shown that bone stiffness increased with increase of degree of mineralization, carbonate content, and crystallinity of bone mineral (27). Although no crystallinity and mechanical measurements were done in our study, the differences in bone composition of TTD and XPA-TTD mice compared to the control animals could imply that bone stiffness in these animals is lower than that of the wild-type animals, comparable to the aforementioned study. Therefore, thorough mechanical testing is needed in future studies to gain more insight into the biomechanic properties of bone obtained from these mice.
Bone formation is highly dependent on the presence of sufficient osteoblasts, which actively deposit bone extracellular matrix. However, the number of cells with osteogenic potential depends on the individual's age and physiological state. Research done on the effect of donor age and the capacity of mesenchymal stem cells (MSCs) isolated from patients shows a significant decrease in osteogenic potential of these cell populations at age 50 years and higher (28). The decrease in osteogenicity was related to proliferative capacity of the MSCs in culture, especially at older ages. The ability to form so-called colony-forming-units, or CFUs, from human bone marrow was also found to be negatively influenced at later ages (29). Furthermore, studies performed in animals (30) and humans (31,32) show that the capacity to express alkaline phosphatase, an osteoblast-specific marker, also decreases during aging in these cells. For future experiments it would be interesting to isolate MSCs from TTD and XPA-TTD mice to gain insight into their proliferative and differentiation capacity. Because XPA-TTD mice show fast and premature aging, MSCs isolated from even young animals could very well be hampered in their ability to form bone, like MSCs from humans.
In this study, we concentrated on the use of confocal Raman microscopy to analyze bone physicochemical composition derived from the premature aging TTD mouse model. The data found by Raman spectroscopy presented in this article add to results published by us on these mouse models; bone mineral content in TTD mice was found to be lower, whereas bone matrix protein content was similar to that found in WT animals.
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Acknowledgments
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This research was funded by the Dutch Technology Foundation (STW).
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Footnotes
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Decision Editor: Huber R. Warner, PhD
Received October 27, 2005
Accepted July 31, 2006
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Abstract
Material and Methods
, indicating a lower mineral content in TTD mice