BackgroundAngiogenesis, the formation of blood vessels, is a critical aspect of wound healing. Disorders of wound healing are often characterized by lack of angiogenesis, a condition frequently observed in aging and diabetic patients. Current techniques for assessing blood at injury sites are limited to contrast-imaging, including angiography. However, these techniques do not directly observe oxygenation of blood and are not amenable to serial evaluation. A multimodal noninvasive reflectance and Raman spectrometer have been proposed to help clinicians as a point-of-care tool to interrogate local angiogenesis and tissue architecture, respectively. The spectrometer system is a rapid, noninvasive, and label-free technology well-suited for the clinical environment.Materials and methodsTo demonstrate feasibility, the spectrometer system was used to interrogate angiogenesis serially over 9 wk as a result of heterotopic ossification (HO) development in a validated murine model. End-stage HO was confirmed by micro-computed tomography.ResultsOur preliminary results suggest that reflectance spectroscopy can be used to delineate vessel formation and that pathologic wounds may be characterized by unique spectra. In our model, HO formed at sites 1-3, whereas sites 4 and 5 did not have radiographic evidence of HO.ConclusionsA point-of-care system like that demonstrated here shows potential as a noninvasive tool to assess local angiogenesis and tissue architecture that may allow for timely intervention in a clinical setting.

Raman spectroscopy provides information on bone chemical composition and structure via widely used metrics including mineral to matrix ratio, mineral crystallinity and carbonate content, collagen cross-linking ratio and depolarization ratios. These metrics are correlated with bone material properties, such as hardness, plasticity and Young's modulus. We review application of Raman spectroscopy to two important irradiated animal models: the mouse tibia, a model for damage to cortical bone sites including the rib (breast cancer) and to healthy tissue adjacent to extremity sarcomas, and the rat mandible, a model for radiation damage in head and neck cancer radiotherapy. Longitudinal studies of irradiated mouse tibia demonstrate that radiation-induced matrix abnormalities can persist even 26 weeks post-radiation. Polarized Raman spectroscopy shows formation of more ordered orientation of both mineral and collagen. At 8 weeks post-radiation, irradiated rat hemimandible exhibits transient hypermineralization, increased collagen cross-linking and decreased depolarization ratios of mineral and collagen. A standard radioprotectant, amifostine, mitigates rat mandible radiation damage, with none remaining detectable 18 weeks post-radiation. Already a powerful tool to monitor radiation damage, Raman spectroscopy may be important in development of new radiotherapy protocols and radioprotective agents. Further in vivo studies of radiation effects on the rodent models are underway, as are development of methodologies for eventual use in human subjects.Raman spectroscopy can monitor radiation-induced changes in both bone chemical composition and structural disorder in animal models at cancer therapy radiation doses. Spectroscopy can be used to evaluate the extent of damage and the effectiveness of chemical agents designed to minimize damage or speed repair.

This review describes new technologies for the diagnosis and treatment, including fracture risk prediction, of postmenopausal osteoporosis. Four promising technologies and their potential for clinical translation and basic science studies are discussed. These include reference point indentation (RPI), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and magnetic resonance imaging (MRI). While each modality exploits different physical principles, the commonality is that none of them require use of ionizing radiation. To provide context for the new developments, brief summaries are provided for the current state of biomarker assays, fracture risk assessment (FRAX), and other fracture risk prediction algorithms and quantitative ultrasound (QUS) measurements.

Advances in fiber optic probe design are moving Raman spectroscopy into the clinic, although there remain important practical problems. While much effort has been devoted to minimizing Raman and fluorescence background from fibers, less attention has been given to the need to generate reference Raman signals that can correct for variations in tissue albedo, which is important in quantifying changes in tissue composition. To address this shortcoming, we have developed a fiber optic probe that incorporates a fluorinated ethylene-propylene copolymer (FEP) cap at the end of each excitation fiber. Transmission of laser light through the transparent cap generates a 732 cm−1 Raman band whose intensity scales linearly with the laser power delivered to the tissue of interest. In our first design, the FEP cap functions as a waveguide with only a small insertion loss (∼5%). Laser transmission through 1 mm of the polymer is sufficient to generate a usable reference Raman signal. We show the application of the probe to quantitative non-invasive Raman spectroscopy of animal tissues using rat leg phantoms as models. Ex-vivo Raman spectroscopy of excised rat tibia supports the use of the probe for spectroscopy of various tissues. These results provide proof of principle that the Raman probe can be used in multiple spectroscopic applications.

To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating the human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.

Progress in the diagnosis and prediction of fragility fractures depends on improvements to the understating of the compositional contributors of bone quality to mechanical competence. Raman spectroscopy has been used to evaluate alterations to bone composition associated with aging, disease, or injury.In this survey we will (1) review the use of Raman-based compositional measures of bone quality, including mineral-to-matrix ratio, carbonate-to-phosphate ratio, collagen quality, and crystallinity; (2) review literature correlating Raman spectra with biomechanical and other physiochemical measurements and with bone health; and (3) discuss prospects for ex vivo and in vivo human subject measurements.ISI Web of Science was searched for references to bone Raman spectroscopy in peer-reviewed journals. Papers from other topics have been excluded from this review, including those on pharmaceutical topics, dental tissue, tissue engineering, stem cells, and implant integration.Raman spectra have been reported for human and animal bone as a function of age, biomechanical status, pathology, and other quality parameters. Current literature supports the use of mineral-to-matrix ratio, carbonate-to-phosphate ratio, and mineral crystallinity as measures of bone quality. Discrepancies between reports arise from the use of band intensity ratios rather than true composition ratios, primarily as a result of differing collagen band selections.Raman spectroscopy shows promise for evaluating the compositional contributors of bone quality in ex vivo specimens, although further validation is still needed. Methodology for noninvasive in vivo assessments is still under development.

The use of bone structural allografts for reconstruction following tumor resection is widespread, although successful incorporation and regeneration remain uncertain. There are few non-invasive methods to fully assess the progress of graft incorporation. Computed tomography and MRI provide information on the morphology of the graft/host interface. Limited information is also available from DXA and ultrasound. Only few techniques can provide information on the metabolic status of the graft, such as the mineral and matrix composition of the regenerated tissue that may provide early indications of graft success or failure. To address this challenge, we discuss here the implementation of Raman spectroscopy for in vivo assessment of allograft implantation in a rat model. An array of optical fibers was developed to allow excitation and collection of Raman spectra through the skin of rat at various positions around the rat's tibia. The system is calibrated against locally constructed phantoms that mimic the morphology, optics and spectroscopy of the rat. The system was evaluated by carrying out transcutaneous Raman measurement on rat. Bone mineral and matrix Raman bands are successfully recovered. This new technology provides a non-invasive method for in vivo monitoring of bone graft osseointegration.

•We irradiated murine tibiae with 4 × 5 Gy doses, approximating dosage for human cancer radiotherapy.•Using Raman, we observed both bone mineral and collagen abnormalities induced by radiation.•Initial increases in collagen cross-linking return to near-normal values over 26 weeks. Collagen and mineral orientation along diaphysis followed same pattern.•Other Raman parameters (mineral/matrix, crystallinity, carbonate content) also showed initial deviation, with return to normal over 26 weeks.•Results are consistent with initial radiation damage to mineral, matrix and slow remodeling of damaged tissue.IntroductionRadiotherapy to the appendicular skeleton can cause an increased risk of developing catastrophic fractures with delayed bone healing or non-union, and may subsequently require multiple procedures and amputation. Biomechanical studies suggest that irradiated bone is more brittle, but the cause is unclear and cannot be explained by changes to bone structure or quantity, suggesting that there are crucial changes in irradiated bone material properties. Raman spectroscopy provides a means to assess the chemical properties of the mineral and matrix constituents of bone, which could help explain post-radiation embrittlement. In this study we use a murine tibial model with focal irradiation and perform Raman spectroscopy to test the hypothesis that changes in bone chemistry following irradiation is consistent with reduced bone quality and persists in the long term after irradiation.MethodsFemale BALB/F mice aged 12 weeks were subjected to unilateral, localized hindlimb irradiation in 4 daily 5 Gy fractions (4 × 5 Gy) totaling 20 Gy, and were euthanized at 1, 4, 8, 12, and 26 weeks post-irradiation (n = 6/group). The irradiated (right) and non-irradiated contralateral control (left) tibiae were explanted and assessed by non-polarized and polarized Raman spectroscopy over the proximal cortical bone surface. Raman parameters used included the mineral/matrix ratio, mineral crystallinity, carbonate/phosphate ratio, collagen cross-link ratio, and depolarization ratio.ResultsSignificantly increased collagen cross-link ratio and decreased depolarization ratio of matrix were evident at 1 week after irradiation and this persisted through 26 weeks. A similar significant decrease was observed for depolarization ratio of mineral at all time points except 8 and 26 weeks. At 4 weeks after irradiation there was a significantly increased mineral/matrix ratio, increased mineral crystallinity, and decreased carbonate/phosphate ratio compared to controls. However, at 12 weeks after irradiation these parameters had moved in the opposite direction, resulting in a significantly decreased mineral/matrix ratio, decreased crystallinity and increased carbonate/phosphate ratio compared to controls. At 26 weeks, mineral/matrix, crystallinity and carbonate/phosphate ratios had returned to normal.DiscussionIn this mouse model, Raman spectroscopy reports both bone mineral and collagen cross-link radiation-induced abnormalities that are evident as early as one week after irradiation and persists for 26 weeks. The picture is one of extensive damage, after which there is an attempt at remodeling. We hypothesize that pathological cross-links formed by radiation damage to collagen are poorly resorbed during the altered remodeling process, so that new tissue is formed on a defective scaffold, resulting in increased bone brittleness.