1.

Introduction

Arthritis and other rheumatic conditions represent a public health burden that is both significant and growing. In 2008, it was estimated that more than 21% of U.S. adults (46.4 million persons) were diagnosed with some type of arthritis, with rheumatoid arthritis affecting 1.3 million patients.¹

Glucocorticoids are among the most extensively used anti-inflammatory agents in the management of the painful symptoms of rheumatoid arthritis. However, since the glucocorticoid (GC) receptor is expressed in almost every cell in the body, glucocorticoids have many effects, including potentially harmful ones on bone metabolism. These adverse effects include inhibition of bone formation and an enhancement of bone resorption, often leading to osteoporosis, and ultimately an increased risk of fracture. Currently, extended exposure to glucocorticoids is the leading cause of physician-induced osteoporosis, leaving patients susceptible to fractures at reported rates of 30% to 50%.² It has also been shown that GC excess in mice affects bone metabolism in a similar fashion, leading to significant osteoporosis that mimics clinical symptoms in human patients.^{2, 3, 4}

To reverse GC-induced osteoporosis, several classes of drugs have been developed, including antiresorptive drugs of the bisphosphonate family^{5, 6, 7} and anti-tumor necrosis factor (TNF) drugs.⁸ Clinical trials of these drugs typically report increases in bone mineral density (BMD), as measured by dual energy x-ray absorptiometry (DXA) or computed tomography, as the primary outcome of drug efficacy. However, this increase in BMD is not predictive of fracture risk in the nearly 10% of treated patients that experience a fracture.^{9, 10} Therefore, new diagnostic and monitoring technologies for detection of bone changes that better correlate with fracture risk could potentially be more definitive than BMD in establishing the efficacy of new generations of these drugs.

Vibrational spectroscopic techniques, including Raman spectroscopy, have been used extensively to study bone and other mineralized tissue.^{11, 12} In particular, the bone diseases postmenopausal osteoporosis,¹³ osteogenesis imperfecta,¹⁴ and early-onset osteoarthritis¹⁵ all induce perturbations to cortical bone that can be detected by Raman spectroscopy. In this communication, we report the results of the first Raman spectroscopy study of cortical bone in a transgenic mouse model of rheumatoid arthritis that constitutively overexpresses the inflammatory molecule human tumor necrosis factor-alpha (hTNF-α), which was developed by Keffer ¹⁶ Because of its chronic progressive nature, 100% incidence, and simple etiology, the hTNF-α transgenic (hTNF-Tg) mouse is an excellent model to investigate the pathogenesis and treatment of rheumatoid arthritis and side effects such as GC-induced osteoporosis.

2.

Materials and Methods

3.

Results

The average Raman spectra acquired from each of the 15 mice are overlaid in Fig. 1. Major Raman bands associated with mineral ( [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ and [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm CO}_3^{2-}$\end{document} ${\mathrm{CO}}_3^{2-}$ ) and protein matrix (amide III, CH₂, and amide I) content are labeled. Band area ratios frequently studied in vibrational spectroscopic analysis of diseased cortical bone include the mineral-to-matrix ratio (MTMR; [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}/$\end{document} ${\mathrm{PO}}_4^{3-}/$ CH₂ or [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}/$\end{document} ${\mathrm{PO}}_4^{3-}/$ amide I), which describes the degree of phosphate mineralization, and the phosphate-to-carbonate ratio (PTCR; [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ / [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm CO}_3^{2-}$\end{document} ${\mathrm{CO}}_3^{2-}$ ), which describes the amount of carbonate substitution in the apatite crystal lattice.^{13, 15} Since all spectra have been normalized to the area under the amide I peak, variation in the MTMR is represented by the variation in the [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ peak near 960 cm⁻¹. The spectral insets in Fig. 1 highlight this variation, showing a decrease in the MTMR dependent upon treatment regimen and mouse genotype.

Fig. 1

Mean Raman spectra from each of the tibiae. The major Raman bands are labeled along with their peak positions (in parentheses) in relative wavenumber shift. The spectral insets highlight the variation in the mineral-to-matrix ratio (specifically, the band area ratio of [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ to amide I) between the four groups of mice. (Color online only.)

The ensemble-averaged ultimate torque, BMD, cortical thickness, MTMR, and PTCR for each group of mice are presented in Fig. 2. Trends are readily visible for all five parameters: they decrease with GC treatment and also in transgenic mice. For ultimate torque and the Raman-based parameters, both of these trends were significant (as determined by two-way ANOVA); for the microCT parameters, only the mouse genotype was found to be significant. The magnitudes of the standardized ultimate torque effect sizes (along with 95% confidence intervals) were 1.20 [0.07,2.34], 2.25 [1.11,3.39], and 0.09 [0,1.22] for the treatment, genotype, and interaction effects, respectively. Only the main effects of treatment regimen and mouse genotype were included in the final ANOVA models since the interaction effects were not statistically significant.

Fig. 2

Bar plots of the mean (a) ultimate torque, (b) BMD, (c) cortical thickness, (d) MTMR, and (e) PTCR of each group. The error bars are plotted as the standard error of the mean. The gray bars represent PL-treated groups and the white bars represent GC-treated groups. [TeX:] \documentclass[12pt]{minimal}\begin{document}$\textsuperscript {*} p \le 0.05$\end{document} $*p\le 0.05$ as determined by two-way ANOVA.

Figure 3 provides scatter plots of ultimate torque as predicted by regression against BMD alone (a), multiple microCT parameters (b), and the full Raman spectrum (c). All of the plots show significant correlation between the measured and predicted values of ultimate torque, with the BMD plot being noticeably noisier. The accuracy of each prediction model is quantified in Table 1.

Fig. 3

Scatter plots of the ultimate torque prediction for each tibia versus its measured value for the (a) BMD, (b) microCT PLSR, and (c) Raman PLSR prediction models. The RMSECV of each prediction model is presented in Table 1. (Color online only.)

Table 1

Accuracy of ultimate torque prediction.

In order to isolate the Raman spectral features most associated with compromised bone strength, a Raman peak-based prediction algorithm was also sought. The Raman spectral features that were most predictive of ultimate torque were identified by analyzing the PLSR coefficients as described by Beier ²⁹ Briefly, the Hadamard, or entrywise, product of the mean-centered spectrum of the tibia with the largest ultimate torque, [TeX:] \documentclass[12pt]{minimal}\begin{document}$S_{\tilde{\nu }}$\end{document} $S_{\tilde{\nu }}$ , and the regression coefficients generated by PLSR, [TeX:] \documentclass[12pt]{minimal}\begin{document}$b_{\tilde{\nu }}$\end{document} $b_{\tilde{\nu }}$ , was calculated. By definition, this vector ( [TeX:] \documentclass[12pt]{minimal}\begin{document}$b_{\tilde{\nu }}\,\circ \,S_{\tilde{\nu }}$\end{document} $b_{\tilde{\nu }}\circ S_{\tilde{\nu }}$ ) assigns large absolute values to spectral regions with high predictive power. Analysis of the [TeX:] \documentclass[12pt]{minimal}\begin{document}$b_{\tilde{\nu }}\,\circ \,S_{\tilde{\nu }}$\end{document} $b_{\tilde{\nu }}\circ S_{\tilde{\nu }}$ spectrum revealed that the [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ , [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm CO}_3^{2-}$\end{document} ${\mathrm{CO}}_3^{2-}$ , and CH₂ peaks were strongly leveraged in predicting ultimate torque. A separate peak-based prediction model was built using the areas under the [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ , [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm CO}_3^{2-}$\end{document} ${\mathrm{CO}}_3^{2-}$ , and CH₂ peaks, as well as the first moment of the [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ peak, which is related to the peak's spectral location. Using a LOOCV approach, these four Raman parameters were used to predict the ultimate torque of each bone. The prediction accuracy of this model is included in Table 1.

4.

Discussion

The trend in the ensemble-averaged measurements of the microCT-derived BMD and Raman spectroscopy derived MTMR closely matches the biomechanical ultimate torque measurements as seen in Fig. 2. The BMD and MTMR measurements suggest that both the total mineral content and phosphate mineral content relative to the protein matrix are reduced in the bones of GC-treated and hTNF-Tg mice. Hypomineralization is expected in each of these cases since both GC-treatment and arthritic inflammation decrease bone formation and increase bone resorption.³⁰ The concomitant decrease in the PTCR implies that the degree of carbonate substitution in the apatite crystal lattice is larger in the bones of GC-treated and hTNF-Tg mice compared to the wild-type controls. Others have reported a similar decrease in the PTCR in women who had experienced an osteoporotic fracture.¹³

The fact that statistically significant interaction effects were not observed in this study may be due to the relatively small sample size (15 total specimens). Potentially relevant ultimate torque effect sizes are those greater than about 2.6 N·mm (the prediction accuracy of the full-spectrum Raman PLSR model), which corresponds to a standardized effect size of about 0.6. Further study of the interaction between glucocorticoid treatment and mouse genotype is warranted since this effect size, as well as an effect size of zero, is included in the 95% confidence interval for the magnitude of the ultimate torque interaction effect [0,1.22].

The regression models for prediction presented in Fig. 3 and Table 1 suggest that Raman signatures from bone, if obtained transcutaneously, may be capable of screening for fracture risk more reliably than the conventional measurement of BMD via DXA or computed tomography. DXA is the current standard for diagnosing osteoporosis since it is the most extensively validated technology for clinical prediction of fracture risk.³¹ Computed tomography can also be used to measure BMD and provides additional quantitative information about bone structure. However, computed tomography requires a higher radiation dose and remains more expensive and less accessible than DXA. The scatter plots in Fig. 3, along with Table 1, provide estimates of the fracture risk prediction accuracy of DXA [Fig. 3a], computed tomography [Fig. 3b], and Raman spectroscopy [Fig. 3c]. Among these technologies, Raman spectroscopy produced the smallest prediction error. Raman spectroscopy could also provide a potential cost and space benefit over clinical DXA and computed tomography scanners, and it is arguably safer as it does not require the use of ionizing radiation. While transcutaneous acquisition of bone Raman signatures will inevitably reduce the signal strength, the ability to acquire such spectra in mice,^{32, 33} rats,³⁴ chickens,³⁴ canines,³⁵ and humans³⁶ has recently been demonstrated.

Previous Raman spectroscopy studies of osteoporotic bone have primarily focused on changes to individual spectral features associated with specific biochemical properties of the bone.¹¹ Recent studies have also revealed correlations between various Raman spectral features and a number of parameters related to the bone's biomechanical strength.^{37, 38, 39} Specifically, correlations between the ultimate stress (maximum applied stress prior to fracture) and the Raman-derived MTMR and crystallinity (which is related to the inverse of the [TeX:] \documentclass[12pt]{minimal}\begin{document}${\rm PO}_4^{3-}$\end{document} ${\mathrm{PO}}_4^{3-}$ ν₁ peak width) have been observed in the femurs of rats and mice. The present results emphasize that full-spectrum regression methods such as PLSR can often provide stronger correlations with biomechanical properties. For comparison to PLSR, simple univariate models for predicting ultimate torque were built based upon single Raman band area ratios (MTMR and PTCR). While these ratios have shown potential for diagnosing osteoporosis,¹¹ the MTMR and PTCR both proved to be poor univariate predictors of ultimate torque (RMSECV = 4.5 and 4.6 N·mm, respectively), compared to the more advanced PLSR modeling (Table 1). This underscores the importance of using the full plethora of spectral data, rather than a single spectral feature, for predicting the biomechanical properties of a bone.

This study is the first to evaluate bone changes using a variety of minimally invasive imaging and spectroscopic techniques and to establish predictive correlations with the biomechanical properties of cortical bone in a mouse model of rheumatoid arthritis. The data presented here examined mice at only a single GC-exposure time point (6 weeks). Interesting future directions include examining mice at earlier time points to evaluate whether Raman spectroscopy can predict early, subtle chemical changes to the bone before later changes in BMD and deterioration of biomechanical properties are manifest. Toward clinical translation of this diagnostic technology, longitudinal in vivo studies in mice utilizing transcutaneous detection will also be pursued.

5.

Conclusion

Raman spectroscopy has been used to study the chemical perturbations to cortical bone associated with both glucocorticoid-induced osteoporosis and rheumatoid arthritis. Each disease was associated with statistically significant changes in the degree of phosphate mineralization and overall mineral composition in the tibial cortical bone of mice. Raman spectroscopy and microcomputed tomography were also separately used to predict tibial fracture risk as quantified by a biomechanical measurement of ultimate torque (maximum applied torque prior to fracture). Predictions based upon the measured Raman spectrum were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density.