In this study we have investigated the potential of optical techniques to monitor changes in bone mineral density (BMD) via changes in scattering coefficient. For each of five bone samples, diffuse reflection and transmission coefficients were measured over the wavelength range 520-960 nm using an integrating sphere and CCD spectrometer. These were converted into optical absorption and scattering coefficients using a Monte Carlo inversion procedure. Measurements were made on samples immersed in formic acid solution for different lengths of time in order to investigate the effect of reduction in BMD on the optical properties. After full demineralization, the optical scattering coefficient fell by a factor 4. From the observed degree of fluctuation of the measurements, we estimate that BMD could be measured with an accuracy of 7% if optical scattering can be measured with an accuracy of 10%. We also report preliminary measurements of bone scattering using optical coherence tomography (OCT). An inter-side variability of 3% is obtained on dry samples with and without overlying periosteum. These results suggest that minimally invasive techniques for measuring optical scattering, such as OCT, may have a role in monitoring regional changes in BMD. This could be an important advance in our understanding of bone remodelling and its relationship to osteoarthritis. Both the integrating sphere and OCT measurements also suggest that light transport in bone is spatially anisotropic. OCT was used to assess probability of obtaining results in vivo.

General quantitative agreement was found between our method and histology in validation experiments. Qualitative results for cartilage suggest a complicated 3-D structure that warrants further study. There is potential to develop this approach into a tool that can provide depth-resolved information on collagen orientation in near real-time, non-destructively and in vivo.

Optical coherence tomography and polarization-sensitive optical coherence tomography images of equine articular cartilage are presented. Measurements were made on intact joint surfaces. Significant (e.g. × 2) variations in the intrinsic birefringence were found over spatial scales of a few millimetres, even on samples taken from young (18 month) animals that appeared visually homogeneous. A comparison of data obtained on a control tissue (equine flexor tendon) further suggests that significant variations in the orientation of the collagen fibres relative to the plane of the joint surface exist. Images of visually damaged cartilage tissue show characteristic features both in terms of the distribution of optical scatterers and of the birefringent components.

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Equine articular cartilage has been imaged using both polarization-sensitive optical coherence tomography (PS-OCT) and non-linear microscopy. PS-OCT has been used to spatially map the birefringence in the cartilage and we have found that in the vicinity of the lesion the images display a characteristic disruption in the regular birefringence bands shown by normal cartilage. We also note that significant (e.g. x2) variations in the apparent birefringence of samples taken from young (18 month) animals that otherwise appear visually homogeneous are found over spatial scales of a few millimeters. We have also imaged the cartilage using non-linear microscopy and compare the scans taken with second harmonic generation (SHG) light and the two photon fluorescence (TPF) light. SHG images collected using 800 nm excitation reveals the spatial distribution of collagen fibers, whilst TPF images clearly shows the distribution of intracellular and pericellular fluorophores.

Polarization-sensitive optical coherence tomography has been used to solve fast-axis fibre orientation in three dimension space. Previously we have demonstrated that the apparent variations in polar angle orientation of collagen fibers along sagittal ridge of equine third metacarpophalangeal joint exist. A quantitative method based on multiple angles of illumination has been proposed to determine the polar angle of the collagen fibers. This method however ignored the full 3D structure by assuming that the collagen fibers long-axis lay within the plane of incidence. A new quantitative method based on the theory of light propagation in uniaxial materials is described which avoids this assumption. To test this method we have performed control experiments on a sample of equine tendon (this tissue has well defined c-axis lying along the long-axis of the tendon). Several samples of tendon were cut to achieve a planar surface inclined at -20° to the long axis. Additional 30° rotation provided non-zero azimuthal angle. The surface was then imaged using incident beam angles -40°, -20°, 0, +20°, +40° in two orthogonal planes. Values for both the polar and azimuthal angles were then derived using a numerical optimisation procedure. Results agreed qualitatively with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence.