Development of more effective diagnostic and therapeutic solutions is vital to tackling the growing challenge of bone diseases and disorders in aging societies. Spatially offset Raman spectroscopy (SORS) enables the chemical specificity of conventional Raman spectroscopy to be combined with sub-surface probing. SORS has successfully been applied to transcutaneous investigations of underlying bone and shows great potential to become an in vivo tool for non-invasive diagnosis of various bone conditions.

The volume within the complex hierarchical bone tissue probed by SORS depends on the specimen's optical properties. Understanding the actual sampling depth is important to correctly assign detected chemical changes to specific areas in the bone. This study explores the hypothesis that the effective Raman signal recovery from certain depths requires different spatial offsets depending on the bone mineralisation.

SORS depth investigations were conducted on three bones with significantly different mineralisation levels. Thin slices (0.6 – 1.0 mm thickness) were cut from deer antler, horse metacarpal and whale tympanic bulla and stacked together (4 – 7 layers; 4.1 – 4.7 mm total thickness). A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as reference sample and was inserted in between the layers of stacked bone slices. Raman spectra were acquired at 30 s using 830 nm excitation.

A quantitative relation between the SORS offset and the primarily interrogated depth inside the bone was established. Maximum accessible depths at small offset strongly depend on the mineralisation level. Using large spatial offsets of 7 – 9 mm PTFE signal recovery depths of 4.4 – 4.6 mm through cortical bone can be realized with only minor dependence on the bone mineralisation.

These findings highlight the potential of SORS for medical diagnostics by enabling the non-invasive detection of bone conditions characterised by chemical alterations several millimetres inside compact bone tissue (e.g. infections, tumours, etc.).

Osteoarthritis (OA) is a common, debilitating joint disease involving degeneration of cartilage and bone. It has been suggested that subtle changes in the molecular structure of subchondral bone may precede cartilaginous changes in the osteoarthritic joint. To explore these changes Raman spectroscopy was employed as a diagnostic tool. Raman spectroscopy measures inelastic scattered laser light produced when photons interact with chemical materials. Resultant changes in wavelength form spectra relative to the chemical composition of the given sample: with bone this includes the mineral and matrix components, unlike conventional X-rays. The aim of our study is to explore the hypothesis: Changes in matrix composition of osteoarthritic subchondral bone can be detected with Raman spectroscopy. pQCT and Raman spectroscopy were employed to determine the bone mineral density (BMD) and bone quality, respectively. Ten medial compartment OA and five control (non-OA) tibial plateaus were interrogated and analysis performed to compare OA to control, and medial to lateral compartments. The subchondral bone of the medial OA compartments had higher BMD (p=0.05) and thickness compared to lateral and control samples. Spectral analysis revealed there is no difference between the medial and lateral compartments within either cohort. However, there is a statistically significant (p=0.02) spectral difference between the OA and control specimens. The detection of bone matrix changes in osteoarthritis using Raman spectroscopy contributes to the understanding of the biochemical signature of subchondral bone across diseased and control tibial plateaus. This technique has potential to shed light on the role of bone in osteoarthritis.

In this study we explore the hypothesis that there is a correlation between the ratio of the intensities of specific peaks of the Raman spectrum of bone tissue and the material properties of that particular type of bone. Raman spectroscopy is a powerful analytical technique capable of providing rich chemical information on the composition of skeletal tissue matrices and it has been used extensively to interrogate bone in the past. Spectra are presented of a selection of animal bones, each having greatly differing material properties, the differences having been produced by evolution in response to their greatly differing functions. The main examples described are deer antler (a bone naturally selected for toughness), tympanic bulla from a fin whale (naturally selected for stiffness) and the intermediate ‘standard’ bone from adult mammalian limbs which must be both tough enough to resist fracture and stiff enough to resist deformation during physiological loading (from an ovine femur in our case). In order to illustrate the specific relationship between material properties and Raman spectra additional mineralized tissues also with differing functions and of known Young's moduli are also introduced. The results show that a strong correlation exists between the mineral to collagen ratio of these different bone tissues as measured with Raman spectroscopy and their (previously published) Young's moduli. Raman spectra have been retrieved through skin and tissue in other studies in the past, an amalgamation of refined versions of those in vivo techniques with the work introduced here paves the way for the emergence of novel systems for assessing the material properties of bone tissue at specific anatomical sites in vivo in the future.

Introduction: The ‘gold standard’ currently used to assess bone quality is bone mineral density (BMD) measured by Dual Energy X-ray Absorptiometry (DEXA). However BMD accounts for no more than 60 – 70% of bone strength. X-rays are affected primarily by the mineral phase of bone; the organic phase remains essentially invisible. Yet it is known that the material strength and toughness of bone is critically dependent on its organic phase. A Raman spectroscopic technique was used that permitted visualisation of both phases of bone deep to unbroken skin by successfully removing spectral information from the overlying tissues.

Hypothesis: Spectral features of both the mineral and organic phases of bone from different murine genotypes can be measured objectively through the unbroken skin using time-resolved Raman spectroscopy.

Methods: We used an 800 nm probe laser (1 kHz, 1 ps pulses, focussed to 1 mm diameter) with a synchronised 4 ps optical Kerr gate that had a variable picosecond delay that effectively shuttered out photons from the overlying tissues. We measured bone spectra at a point 2mm above the carpus from two mouse genotypes: wildtype and oim/oim (matched for age, sex and weight) at a typical depth 1.1mm. We then repeated the measurements once the overlying tissues had been carefully removed to expose the bones directly. Oim/oim mice produce only homotrimeric collagen I, (á1(I)3), associated with this change in collagen is a poor mineralisation of the bone tissue, making it an ideal model for a this study.

Results: We recorded the main spectral features in both phases of bone and showed that the ratios of spectral bands from the two phases were similar within each genotype, whether measured through the skin or directly from exposed bone. However, there was a significant difference in the same ratios between genotypes associated with a reduced mineralisation in the oim/oim mice; a significant difference that was apparent both directly from bone and through skin. The band associated with CH2 wag of collagen (organic phase) showed a frequency shift between the genotypes.

Discussion: Measurements of the spectra and their analysis were similar whether made directly on bone or transcutaneously. We were able to detect changes in mineralisation between genotypes and, unlike measurements of BMD, we showed also changes in collagen. Since the material strength of bone is critically dependent on collagen, this indicates an appreciable advantage of this technique over DEXA.

Conclusions: This novel technique allowed objective transcutaneous spectral measurements of bone tissue and was able to distinguish between normal and unhealthy bone tissue. With a laser focussed to 1 mm diameter that was readily moveable, these measurements were specific to that site (2 mm proximal to the carpus). After further optimisation, this technology is likely to improve fracture risk assessments in comparison to the use of DEXA alone, opening opportunities for screening in anticipation of the predicted increase in fragility fractures.