We show the application of near-infrared Raman Spectroscopy to in-vitro monitoring of the viability of tissue constructs
(EVPOMEs). During their two week production period EVPOME may encounter thermal, chemical or biochemical
stresses that could cause development to cease, rendering the affected constructs useless. We discuss the development of
a Raman spectroscopic technique to study EVPOMEs noninvasively, with the ultimate goal of applying it in-vivo. We
identify Raman spectroscopic failure indicators for EVPOMEs, which are stressed by temperature, and discuss the
implications of varying calcium concentration and pre-treatment of the human keratinocytes with Rapamycin. In
particular, Raman spectra show correlation of the peak height ratios of CH<sub>2</sub> deformation to phenylalanine ring breathing,
providing a Raman metric to distinguish between viable and nonviable constructs. We also show the results of singular
value decomposition analysis, demonstrating the applicability of Raman spectroscopic technique to both distinguish
between stressed and non-stressed EVPOME constructs, as well as between EVPOMEs and bare AlloDerm® substrates,
on which the oral keratinocytes have been cultured. We also discuss complications arising from non-uniform thickness
of the AlloDerm® substrate and the cultured constructs, as well as sampling protocols used to detect local stress and
other problems that may be encountered in the constructs.
Polarized Raman spectroscopy allows measurement of molecular orientation and composition and is widely used in the study of polymer systems. Here, we extend the technique to the extraction of quantitative orientation information from bone tissue, which is optically thick and highly turbid. We discuss multiple scattering effects in tissue and show that repeated measurements using a series of objectives of differing numerical apertures can be employed to assess the contributions of sample turbidity and depth of field on polarized Raman measurements. A high numerical aperture objective minimizes the systematic errors introduced by multiple scattering. We test and validate the use of polarized Raman spectroscopy using wild-type and genetically modified (oim/oim model of osteogenesis imperfecta) murine bones. Mineral orientation distribution functions show that mineral crystallites are not as well aligned (p<0.05) in <i>oim/oim</i> bones (28±3 deg) compared to wild-type bones (22±3 deg), in agreement with small-angle X-ray scattering results. In wild-type mice, backbone carbonyl orientation is 76±2 deg and in <i>oim/oim</i> mice, it is 72±4 deg (p>0.05). We provide evidence that simultaneous quantitative measurements of mineral and collagen orientations on intact bone specimens are possible using polarized Raman spectroscopy.
Polarized Raman spectroscopy is widely used in the study of molecular composition and orientation in synthetic and
natural polymer systems. Here, we describe the use of Raman spectroscopy to extract quantitative orientation
information from bone tissue. Bone tissue poses special challenges to the use of polarized Raman spectroscopy for
measurement of orientation distribution functions because the tissue is turbid and birefringent. Multiple scattering in
turbid media depolarizes light and is potentially a source of error.
Using a Raman microprobe, we show that repeating the measurements with a series of objectives of differing numerical
apertures can be used to assess the contributions of sample turbidity and depth of field to the calculated orientation
distribution functions. With this test, an optic can be chosen to minimize the systematic errors introduced by multiple
scattering events. With adequate knowledge of the optical properties of these bone tissues, we can determine if elastic
light scattering affects the polarized Raman measurements.
Raman spectroscopic studies have shown that the properties of the organic matrix and the orientation of the mineral and
matrix components of bone have a large influence on its properties. We employ polarized Raman microspectroscopy to
monitor the changes in the orientation of mineral crystallites during tensile loading of bovine femora in the elastic
regime. We load tissue in a custom-built dynamic mechanical tester that fits on the stage of a Raman microprobe and can
accept hydrated tissue specimens. Parallel and perpendicular polarization components of the Raman spectra along the
long axis of the diaphysis are obtained. We propose that the orientation and structure of mineral crystallites change on
deformation of bone tissue by tensile loading.