Short-wave infrared light imaging measures tissue moisture and distinguishes superficial from deep burns.

Existing clinical approaches and tools to measure burn tissue destruction are limited resulting in misdiagnosis of injury depth in over 40% of cases. Thus, our objective in this study was to characterize the ability of short-wave infrared (SWIR) imaging to detect moisture levels as a surrogate for tissue viability with resolution to differentiate between burns of various depths. To accomplish our aim, we constructed an imaging system consisting of a broad-band Tungsten light source; 1,200-, 1,650-, 1,940-, and 2,250-nm wavelength filters; and a specialized SWIR camera. We initially used agar slabs to provide a baseline spectrum for SWIR light imaging and demonstrated the differential absorbance at the multiple wavelengths, with 1,940 nm being the highest absorbed wavelength. These spectral bands were then demonstrated to detect levels of moisture in inorganic and in vivo mice models. The multiwavelength SWIR imaging approach was used to diagnose depth of burns using an in vivo porcine burn model. Healthy and injured skin regions were imaged 72 hours after short (20 seconds) and long (60 seconds) burn application, and biopsies were extracted from those regions for histologic analysis. Burn depth analysis based on collagen coagulation histology confirmed the formation of superficial and deep burns. SWIR multispectral reflectance imaging showed enhanced intensity levels in long burned regions, which correlated with histology and distinguished between superficial and deep burns. This SWIR imaging method represents a novel, real-time method to objectively distinguishing superficial from deep burns.

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition.

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.

Combined reflectance and Raman spectroscopy to assess degree of in vivo angiogenesis after tissue injury.

BACKGROUND: Angiogenesis, 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 METHODS: To 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. RESULTS: Our 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. CONCLUSIONS: A 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.

Design and microfabrication of a miniature fiber optic probe with integrated lenses and mirrors for Raman and fluorescence measurements.

Fiber optics coupled to components such as lenses and mirrors have seen extensive use as probes for Raman and fluorescence measurements. Probes can be placed directly on or into a sample to allow for simplified and remote application of these optical techniques. The size and complexity of such probes however limits their application. We have used microfabrication in polydimethylsiloxane (PDMS) to create compact probes that are 0.5 mm thick by 1 mm wide. The miniature probes incorporate pre-aligned mirrors, lenses, and two fiber optic guides to allow separate input and output optical paths suitable for Raman and fluorescence spectroscopy measurements. The fabricated probe has 70 % unidirectional optical throughput and generates no spectral artifacts in the wavelength range of 200 to 800 nm. The probe is demonstrated for measurement of fluorescence within microfluidic devices and collection of Raman spectra from a pharmaceutical tablet. The fluorescence limit of detection was 6 nM when using the probe to measure resorufin inside a 150-µm inner diameter glass capillary, 100 nM for resorufin in a 60-µm-deep × 100-µm-wide PDMS channel, and 11 nM for fluorescein in a 25-µm-deep × 80-µm-wide glass channel. It is demonstrated that the same probe can be used on different sample types, e.g., microfluidic chips and tablets. Compared to existing Raman and fluorescence probes, the microfabricated probes enable measurement in smaller spaces and have lower fabrication cost. Graphical abstract A microfabricated spectroscopic probe with integrated optics was developed for chemical detection in small spaces and in remote applications.

Selective detection and complete identification of triglycerides in cortical bone by high-resolution (1)H MAS NMR spectroscopy.

Using (1)H-based magic angle spinning solid-state NMR spectroscopy, we report an atomistic-level characterization of triglycerides in compact cortical bone. By suppressing contributions from immobile molecules present in bone, we show that a (1)H-based constant-time uniform-sign cross-peak (CTUC) two-dimensional COSY-type experiment that correlates the chemical shifts of protons can selectively detect a mobile triglyceride layer as the main component of small lipid droplets embedded on the surface of collagen fibrils. High sensitivity and resolution afforded by this NMR approach could be potentially utilized to investigate the origin of triglycerides and their pathological roles associated with bone fractures, diseases, and aging.

Raman spectroscopy delineates radiation-induced injury and partial rescue by amifostine in bone: a murine mandibular model.

Despite its therapeutic role in head and neck cancer, radiation administration degrades the biomechanical properties of bone and can lead to pathologic fracture and osteoradionecrosis. Our laboratories have previously demonstrated that prophylactic amifostine administration preserves the biomechanical properties of irradiated bone and that Raman spectroscopy accurately evaluates bone composition ex vivo. As such, we hypothesize that Raman spectroscopy can offer insight into the temporal and mechanical effects of both irradiation and amifostine administration on bone to potentially predict and even prevent radiation-induced injury. Male Sprague-Dawley rats (350-400 g) were randomized into control, radiation exposure (XRT), and amifostine pre-treatment/radiation exposure groups (AMF-XRT). Irradiated animals received fractionated 70 Gy radiation to the left hemi-mandible, while AMF-XRT animals received amifostine just prior to radiation. Hemi-mandibles were harvested at 18 weeks after radiation, analyzed via Raman spectroscopy, and compared with specimens previously harvested at 8 weeks after radiation. Mineral (ρ958) and collagen (ρ1665) depolarization ratios were significantly lower in XRT specimens than in AMF-XRT and control specimens at both 8 and 18 weeks. amifostine administration resulted in a full return of mineral and collagen depolarization ratios to normal levels at 18 weeks. Raman spectroscopy demonstrates radiation-induced damage to the chemical composition and ultrastructure of bone while amifostine prophylaxis results in a recovery towards normal, native mineral and collagen composition and orientation. These findings have the potential to impact on clinical evaluations and interventions by preventing or detecting radiation-induced injury in patients requiring radiotherapy as part of a treatment regimen.

Tissue phantoms to compare spatial and temporal offset modes of deep Raman spectroscopy.

Time-resolved and spatially offset Raman spectroscopies have previously been demonstrated for depth analysis through strongly scattering, non-transparent materials. In this study, several series of tissue phantoms were created with varied compositions and thicknesses to compare the potential of these different Raman techniques for biomedical applications. Polydimethylsiloxane (PDMS) phantoms were made with TiO2 particles suspended as a scattering agent, mimicking the scattering properties of biological tissues. The phantom layers contained embedded biomineral simulating inclusions (sphere or layer-shaped) with varied carbonate to phosphate ratios. The tissue phantoms were studied using Time Resolved Raman Spectroscopy (TRRS), Spatially Offset Raman Spectroscopy (SORS), and their combination, using a single instrumental setup with picosecond pulsed excitation at 720 nm and two different detectors. A comparison is made of the efficiency of these techniques to resolve chemical information from these heterogeneous scattering phantom samples. Measurements with continuous wave detection were found to offer a better signal-to-noise ratio than with TRRS, and in SORS measurements ratios of target to matrix signal were found to vary depending on the structural geometry and optical properties of the phantoms. Anomalous SORS behaviour, in which the relative contribution from the target decreases with offset, was observed in cases where the target was highly scattering and the top layer was relatively transparent. Time gating with an intensified charge-coupled device (ICCD) detector can yield more direct information on the depth of the hidden material.

Aged male rats regenerate cortical bone with reduced osteocyte density and reduced secretion of nitric oxide after mechanical stimulation.

Mechanical loading is integral to the repair of bone damage. Osteocytes are mechanosensors in bone and participate in signaling through gap junction channels, which are primarily comprised of connexin 43 (Cx43). Nitric oxide (NO) and prostaglandin E2 (PGE2) have anabolic and catabolic effects on bone, and the secretion of these molecules occurs after mechanical stimulation. The effect of age on the repair of bone tissue after damage and on the ability of regenerated bone to transduce mechanical stimulation into a cellular response is unexplored. The goal of this study was to examine (1) osteocytes and their mineralized matrix within regenerated bone from aged and mature animals and (2) the ability of regenerated bone explants from aged and mature animals to transduce cyclic mechanical loading into a cellular response through NO and PGE2 secretion. Bilateral cortical defects were created in the diaphysis of aged (21-month-old) or mature (6-month-old) male rats, and new bone tissue was allowed to grow into a custom implant of controlled geometry. Mineralization and mineral-to-matrix ratio were significantly higher in regenerated bone from aged animals, while lacunar and osteocyte density and phosphorylated (pCx43) and total Cx43 protein were significantly lower, relative to mature animals. Regenerated bone from mature rats had increased pCx43 protein and PGE2 secretion with loading and greater NO secretion relative to aged animals. Reduced osteocyte density and Cx43 in regenerated bone in aged animals could limit the establishment of gap junctions as well as NO and PGE2 secretion after loading, thereby altering bone formation and resorption in vivo.

Characterization of biofluids prepared by sessile drop formation.

Sessile drop formation, also called drop deposition, has been studied as a potential medical diagnostic, but the effects of complex biofluid rheology on the final deposition pattern are not well understood. We studied two model biofluids, blood plasma and synovial fluid, when deposited onto slightly hydrophilic substrates forming a contact angle of 50-90°. Drops were imaged during the evaporation process and geometric properties of the drop, such as contact angle and drop height, were calculated from the images. The resulting dried biofluid drops were then examined using light microscopy and Raman spectroscopy to assess morphological and chemical composition of the dried drop. The effect of substrate contact angle (surface wetting) and fluid concentration was examined. We found that when biofluids are deposited onto slightly hydrophilic surfaces, with a contact angle of 50-90°, a ring-shaped deposit was formed. Analysis of the drying drop's geometric properties indicates that biofluid dynamics follow the piling model of drop formation, as proposed by Deegan et al. The final deposition pattern varied with substrate surface and concentration, as shown by light microscopy photos of dried drops. The chemical composition of the outer ring was minimally affected by substrate surface, but the spatial heterogeneity of protein distribution within the ring varied with concentration. These results indicate that biofluid drop deposition produces ring-shaped deposits which can be examined by multiple analytical techniques.

Novel assessment tools for osteoporosis diagnosis and treatment.

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.

Effortful Control, Behavior Problems and Peer Relations: What Predicts Academic Adjustment in Kindergarteners from Low-income Families?

This study examined the role of effortful control, behavior problems, and peer relations in the academic adjustment of 74 kindergarten children from primarily low-income families using a short-term longitudinal design. Teachers completed standardized measures of children's effortful control, internalizing and externalizing problems, school readiness, and academic skills. Children participated in a sociometric interview to assess peer relations. Research Findings: Correlational analyses indicate that children's effortful control, behavior problems in school, and peer relations are associated with academic adjustment variables at the end of the school year, including school readiness, reading skills, and math skills. Results of regression analyses indicate that household income and children's effortful control primarily account for variation in children's academic adjustment. The associations between children's effortful control and academic adjustment did not vary across sex of the child or ethnicity. Mediational analyses indicate an indirect effect of effortful control on school readiness, through children's internalizing problems. Practice or Policy: Effortful control emerged as a strong predictor of academic adjustment among kindergarten children from low-income families. Strategies for enhancing effortful control and school readiness among low-income children are discussed.

Manual lesion segmentations for traumatic brain injury characterization.

Traumatic brain injury (TBI) often results in heterogenous lesions that can be visualized through various neuroimaging techniques, such as magnetic resonance imaging (MRI). However, injury burden varies greatly between patients and structural deformations often impact usability of available analytic algorithms. Therefore, it is difficult to segment lesions automatically and accurately in TBI cohorts. Mislabeled lesions will ultimately lead to inaccurate findings regarding imaging biomarkers. Therefore, manual segmentation is currently considered the gold standard as this produces more accurate masks than existing automated algorithms. These masks can provide important lesion phenotype data including location, volume, and intensity, among others. There has been a recent push to investigate the correlation between these characteristics and the onset of post traumatic epilepsy (PTE), a disabling consequence of TBI. One motivation of the Epilepsy Bioinformatics Study for Antiepileptogenic Therapy (EpiBioS4Rx) is to identify reliable imaging biomarkers of PTE. Here, we report the protocol and importance of our manual segmentation process in patients with moderate-severe TBI enrolled in EpiBioS4Rx. Through these methods, we have generated a dataset of 127 validated lesion segmentation masks for TBI patients. These ground-truths can be used for robust PTE biomarker analyses, including optimization of multimodal MRI analysis via inclusion of lesioned tissue labels. Moreover, our protocol allows for analysis of the refinement process. Though tedious, the methods reported in this work are necessary to create reliable data for effective training of future machine-learning based lesion segmentation methods in TBI patients and subsequent PTE analyses.

Polymer-capped fiber-optic Raman probe for non-invasive Raman spectroscopy.

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.

Fiber-optic Raman spectroscopy of joint tissues.

In this study, we report adaptation of Raman spectroscopy for arthroscopy of joint tissues using a custom-built fiber-optic probe. Differentiation of healthy and damaged tissue or examination of subsurface tissue, such as subchondral bone, is a challenge in arthroscopy because visual inspection may not provide sufficient contrast. Discrimination of healthy versus damaged tissue may be improved by incorporating point spectroscopy or hyperspectral imaging into arthroscopy where the contrast is based on the molecular structure or chemical composition. Articular joint surfaces of knee cadaveric human tissue and tissue phantoms were examined using a custom-designed Raman fiber-optic probe. Fiber-optic Raman spectra were compared against reference spectra of cartilage, subchondral bone and cancellous bone collected using Raman microspectroscopy. In fiber-optic Raman spectra of the articular surface, there was an effect of cartilage thickness on recovery of signal from subchondral bone. At sites with intact cartilage, the bone mineralization ratio decreased but there was a minimal effect in the bone mineral chemistry ratios. Tissue phantoms were prepared as experimental models of the osteochondral interface. Raman spectra of tissue phantoms suggested that optical scattering of cartilage has a large effect on the relative cartilage and bone signal. Finite element analysis modeling of light fluence in the osteochondral interface confirmed experimental findings in human cadaveric tissue and tissue phantoms. These first studies demonstrate the proof of principle for Raman arthroscopic measurement of joint tissues and provide a basis for future clinical or animal model studies.

Biomedical tissue phantoms with controlled geometric and optical properties for Raman spectroscopy and tomography.

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.

Raman assessment of bone quality.

BACKGROUND: 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. QUESTIONS/PURPOSES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Altered collagen chemical compositional structure in osteopenic women with past fractures: A case-control Raman spectroscopic study.

Incidences of low-trauma fractures among osteopenic women may be related to changes in bone quality. In this blinded, prospective-controlled study, compositional and heterogeneity contributors of bone quality to fracture risk were examined. We hypothesize that Raman spectroscopy can differentiate between osteopenic women with one or more fractures (cases) from women without fractures (controls). This study involved the Raman spectroscopic analysis of cortical and cancellous bone composition using iliac crest biopsies obtained from 59-cases and 59-controls, matched for age (62.0 ± 7.5 and 61.7 ± 7.3 years, respectively, p = 0.38) and hip bone mineral density (BMD, 0.827 ± 0.083 and 0.823 ± 0.072 g/cm3, respectively, p = 0.57). Based on aggregate univariate case-control and odds ratio based logistic regression analyses, we discovered two Raman ratiometric parameters that were predictive of past fracture risk. Specifically, 1244/1268 and 1044/959 cm-1 ratios, were identified as the most differential aspects of bone quality in cortical cases with odds ratios of 0.617 (0.406-0.938 95% CI, p = 0.024) and 1.656 (1.083-2.534 95% CI, p = 0.020), respectively. Both 1244/1268 and 1044/959 cm-1 ratios exhibited moderate sensitivity (59.3-64.4%) but low specificity (49.2-52.5%). These results suggest that the organization of mineralized collagen fibrils were significantly altered in cortical cases compared to controls. In contrast, compositional and heterogeneity parameters related to mineral/matrix ratios, B-type carbonate substitutions, and mineral crystallinity, were not significantly different between cases and controls. In conclusion, a key outcome of this study is the significant odds ratios obtained for two Raman parameters (1244/1268 and 1044/959 cm-1 ratios), which from a diagnostic perspective, may assist in the screening of osteopenic women with suspected low-trauma fractures. One important implication of these findings includes considering the possibility that changes in the organization of collagen compositional structure plays a far greater role in postmenopausal women with osteopenic fractures.

Natural-abundance 43Ca solid-state NMR spectroscopy of bone.

Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Development of non-invasive Raman spectroscopy for in vivo evaluation of bone graft osseointegration in a rat model.

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.

Radiation-induced changes to bone composition extend beyond periosteal bone.

BACKGROUND: Cancer patients receiving radiotherapy for soft tissue sarcomas are often at risk of post-irradiation (post-RTx) bone fragility fractures, but our understanding of factors controlling radiation-induced bone injury is limited. Previous studies have evaluated post-RTx changes to cortical bone composition in the periosteum of irradiated tibiae, but have not evaluated effects of irradiation in deeper tissues, such as endosteal or mid-cortical bone, and whether there are differential spatial effects of irradiation. In this study, we hypothesize that post-RTx changes to cortical bone composition are greater in endosteal compared to mid-cortical or periosteal bone. METHODS: A pre-clinical mouse model of limited field hindlimb irradiation was used to evaluate spatial and temporal post-RTx changes to the metaphyseal cortex of irradiated tibiae. Irradiation was delivered unilaterally to the hindlimbs of 12-wk old female BALB/cJ mice as 4 consecutive daily doses of 5 Gy each. RTx and non-RTx tibiae were obtained at 0, 2, 4, 8, and 12 wks post-RTx (n = 9 mice/group/time). Raman spectroscopy was used to evaluate spatial and temporal post-RTx changes to cortical bone composition in age-matched RTx and non-RTx groups. RESULTS: Significant early spatial differences in mineral/matrix and collagen crosslink ratios were found between endosteal and periosteal or mid-cortical bone at 2-wks post-RTx. Although spatial differences were transient, mineral/matrix ratios significantly decreased and collagen crosslink ratios significantly increased with post-RTx time throughout the entire tibial metaphyseal cortex. CONCLUSIONS: Irradiation negatively impacts the composition of cortical bone in a spatially-dependent manner starting as early as 2-wks post-RTx. Long-term progressive post-RTx changes across all cortical bone sites may eventually contribute to the increased risk of post-RTx bone fragility fractures.