Raman spectroscopy is widely used to characterize pathogenic alterations in bone during disease or treatment. The intensity of Raman scattering provides information on the relative content of key molecular components in bone, including hydroxyapatite, carbonate, collagen, and collagen crosslinks. The spectral parameters of composition are related to bone's mechanical functions,1 and have been established as important indicators of bone quality. Recently, spatially offset Raman spectroscopy (SORS) has enabled differential detection of buried substances from the surface of the body, by introducing a distance between the laser illumination and the collection paths: see Figure 1(A).2,3 Conducting Raman measurements with different offsets between the source and the collection allows for extraction of surface and subsurface spectra. Noninvasive transcutaneous probing of bone signals thus becomes feasible in live subjects, which allows for potential translation of this technique to clinical settings.4
Figure 1. (A) The spatially offset Raman spectroscopy (SORS) experimental setup for in situ measurement of a rat model of fracture healing. Raman scattering from callus and bone are detectable with the increasing offset (d) between laser and collection fibers. (B) Radiography of the rat model with healing femur and contralateral control. (C) Extracted spectral factors of bone (i, gray line), and soft tissue (ii, gray line), showed good agreement with the Raman spectra from bare bone (i, black line) and pure soft tissue (ii, black line). (D) Extracted bone factors from contralateral control (i) and healing bone (ii).
The essential component of SORS is the fiber optic probe that introduces offsets between illumination and collection paths. Previous explorations of probe configurations include point, global, or ring illumination, where the aim was to minimize thermal damage and maximize signal recovery from subsurface objects. Most SORS probes adopted a design with a predetermined source/collection distance. Now, however, we have developed a handheld fiber probe for SORS with adjustable offsets, which enables more data collection at different depths with varying offsets.5 This probe setup is especially helpful for large subjects or deeper samples. Furthermore, it could provide a set of spectra with more variability in source/detector offsets, which is a prerequisite for successful separation of mixed bone/soft tissue spectra into individual spectral components during data processing.
We explored the application of our SORS device for bone fracture healing assessment. We hypothesized that we might acquire bone signals from healing bones in situ through our SORS setup, and that the spectral parameters obtained from extracted bone factors are related to the composition and material properties of the repaired bone. The goal was to investigate the potential of SORS for non-destructive assessment of the quality of healing, and predicting resistance to re-fracture.
Assessment of the healing progress is important in evaluating fracture repairs with time, and comparing therapeutic outcomes in fracture-healing studies. During the healing process, callus forms around the fracture site, and progressively ossifies until the physiology and mechanical strength of the healing bone is restored. Figure 1(B) shows the callus formation around a fractured rat femur at four weeks post-fracture, and the contralateral control of fracture healing.
We positioned laser illumination on the skin above the fractured point, and moved the position of the collection fibers in millimeter steps after each collection, enabling us to collect Raman spectra, as shown in Figure 1(A). The spectral contribution from bone increased with incrementing offsets, as expected. From previous experience, we knew that increased variation in the relative contribution of bone to soft tissue in the data set could improve efficacy and accuracy in bone signal recovery. We recovered spectral factors arising from soft tissue and the underlying bone, which were in good agreement with those collected from excised tissue immediately after SORS measurements: see Figure 1(C). The mean bone factors, shown in Figure 1(D), clearly show the differences in the peak height of hydroxyapatite, proline, and carbonate, which are related to the relative content of mineral and collagen in the surrogated region.6
The quality of healing is mainly determined by the extent of restoration in bone mechanical properties. Consequently, we tested the excised femurs after SORS measurements using reference point indentation and tissue mechanical testing at the area of callus. The bone material strength at four weeks post-fracture was higher than at two weeks, showing longitudinal improvement in mechanical properties during the fracture healing course. Our results in compositional parameters show a concurrent trend of variation with bone mechanics, indicating a potential correlation of the two measures.
In summary, we have demonstrated the potential of SORS in detecting the composition of callus in situ. We used a laboratory-developed SORS probe with adjustable source/detection offsets that provided data sets with more variation in the relative bone/soft tissue signals, and thus led to a successful recovery of bone factors closely matching the spectra from corresponding bare bones. In future work we will include more animals in the study to gain enough power for a statistical correlation test between spectral parameters and mechanical properties. This will enable evaluation of the potential of the compositional indicators to predict callus quality during fracture healing.
Hao Ding, Guijin Lu, Christopher West, Gloria Gogola, James Kellam, Catherine Ambrose, Xiaohong Bi
University of Texas Health Science Center at Houston
Xiaohong Bi is an assistant professor of nanomedicine and biomedical engineering. Her focus is on developing the application of optical spectroscopy and imaging techniques for disease detection, especially for musculoskeletal and cancer research.
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11(6), p. 060502, 2006. doi:10.1117/1.2400233
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5. Z. Wang, H. Ding, G. Lu, X. Bi, Use of a mechanical iris-based fiber optic probe for spatially offset Raman spectroscopy, Opt. Lett. 39(13), p. 3790-3793, 2014.
6. H. Ding, G. Lu, C. West, G. Gogola, J. Kellam, C. Ambrose, X. Bi, Noninvasive assessment of fracture healing using spatially offset Raman spectroscopy. Presented at SPIE Photonics West 2016.