Gurjit S. Mandair

Raman Assessment of Bone Quality

BackgroundProgress 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/purposesIn 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.MethodsISI 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.ResultsRaman 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.ConclusionsRaman 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.

Contributions of Raman spectroscopy to the understanding of bone strength

Raman spectroscopy is increasingly commonly used to understand how changes in bone composition and structure influence tissue-level bone mechanical properties. The spectroscopic technique provides information on bone mineral and matrix collagen components and on the effects of various matrix proteins on bone material properties as well. The Raman spectrum of bone not only contains information on bone mineral crystallinity that is related to bone hardness but also provides information on the orientation of mineral crystallites with respect to the collagen fibril axis. Indirect information on collagen cross-links is also available and will be discussed. After a short introduction to bone Raman spectroscopic parameters and collection methodologies, advances in in vivo Raman spectroscopic measurements for animal and human subject studies will be reviewed. A discussion on the effects of aging, osteogenesis imperfecta, osteoporosis and therapeutic agents on bone composition and mechanical properties will be highlighted, including genetic mouse models in which structure-function and exercise effects are explored. Similarly, extracellular matrix proteins, proteases and transcriptional proteins implicated in the regulation of bone material properties will be reviewed.

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.

Potential of Raman Spectroscopy for Evaluation of Bone Quality in Osteoporosis Patients. Results of a Prospective Study

As part of our ongoing assessment of bone tissue composition and structure, we report the first experimental protocols of a prospective study to investigate the potential of using Raman spectroscopy to diagnose and predict skeletal fragility in postmenopausal osteoporosis patients. This multi-center study will assess several potential spectroscopic and X-ray based diagnostic techniques. One hundred and twenty participants will be enrolled in this five year study and the investigators will be blinded to information concerning patient history and status. Iliac crest bone biopsy specimens are provided with no identifying information except a patient study number. Our team will use micro-computed tomography (micro-CT) to identify regions of interest in both cortical and cancellous bone from specimens delivered to us. Raman mapping will be performed using a line-focused 785 nm laser in order to obtain local and averaged values on several spectroscopic metrics of bone quality. These metrics include carbonate/phosphate and phosphate/matrix ratios. Results from an initial set of biopsies will be presented. Protocols for obtaining measurements are discussed, with emphasis on the challenges presented by the use of fixed and polymer embedded specimens. These protocols are illustrated will data from a biopsy specimen.

Hox11 Function Is Required for Region-Specific Fracture Repair

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα+/CD51+/LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss‐of‐function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton.

Detection of potential osteoarthritis biomarkers using surface enhanced Raman spectroscopy in the near-infrared

A novel application of surface-enhanced Raman spectroscopy (SERS) for in-vitro osteoarthritis (OA) biomarker detection is described. Hyaluronic acid (HA) is a potential OA biomarker and synovial fluid levels of HA have been correlated with progression of joint space narrowing. However, current immunoassay and chromatographic methods that identify HA in synovial fluid are cumbersome and often require sophisticated instrumentation. Raman spectroscopy may be an alternative to these analytical methods, providing rapid identification of HA using characteristic Raman bands. Yet, previous reports of un-enhanced Raman spectroscopy for hyaluronic acid are in aqueous solutions exceeding 1000X in-vivo concentrations because HA is a weakly scattering polysaccharide. Surface-enhanced Raman spectroscopy can improve detection limits by 100-1000 times and we present, to our best knowledge, the first surface-enhanced Raman spectra of hyaluronic acid. Moreover, the recent commercial availability of stable SERS gold substrates has enabled rapid SERS detection of this biomarker at concentrations diluted by more than an order of magnitude, compared to previous literature reports. Preliminary results of easily and rapidly observing hyaluronic acid at low concentrations in aqueous solutions supported further studies in synthetic models of biofluids, such as artificial synovial fluid, that contain HA at low concentrations. These complex fluids contain proteins that compete for the SERS-active sites on the substrate, and the resulting spectra are dominated by protein Raman bands. We apply a simple and validated protein precipitation protocol to artificial synovial fluid prior to deposition onto the SERS substrate. We find that HA is easily detected in these fluids after protein removal treatment.

Raman spectroscopy of murine bone in response to simulated spaceflight conditions

Astronauts exposed to spaceflight conditions can lose 1-2% of their bone mineral density per month from the weight-bearing portions of the skeletal system. Low bone mineral density, termed osteopenia, is the result of decreased bone formation and/or increased bone resorption. In this study, Raman spectroscopy is used to examine if the physicochemical composition of murine femurs is altered in response to simulated spaceflight conditions (hindlimb suspension). Female C57BL/6J mice, aged 53 days, were divided into ground control and simulated spaceflight groups for a period of 12 days, modeling the experiment profile of mice flown on Space Shuttle flight STS-108. After the study, the mice were sacrificed and femur specimens harvested. Mid-diaphysis sections were probed using near-infrared Raman microscopy. Spectra were collected at various anatomical sites (anterior, lateral, medial, and posterior quadrants) and/or cortical locations (periosteal, midosteal, and endosteal). Chemometric recovery of spectra was employed to reduce signal contributions from the epoxy embedding agent. Mean values for mineralization, carbonation, crystallinity, and other parameters associated with the matrix were estimated. Correlations between mineralization and carbonation were observed, despite the small absolute changes between the two groups. We present more detailed analysis of this data and comment on the prospects for Raman spectroscopic evaluation of bone quality in hindlimb suspended (HLS) specimens.

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.

Raman spectroscopic evidence of crystalline phosphate precursor to bone apatitic mineral

Bone is a highly specialized connective tissue comprised of cross-linked collagen fibers interspersed with apatitic mineral crystallites of various sizes, shapes, orientation, and composition. However, the nucleation, growth, and propagation of mineral crystallite into the collagenous matrix are not clearly understood. By using a research grade inverted microscope fitted with a line-shaped 830 nm laser and spectrograph, we show that the Raman scatter from mineralizing cell cultures in an incubation chamber can be collected and monitored directly through the bottom of the well-plates over a period of 24 hours. In our studies, murine-derived MC3T3 cells were incubated at 37°C in the presence of 5% CO2 and 85% humidity. Results show a gradual shift in the phosphate ν1 apatitic band center (955-957 cm-1) during the first hour of mineralization. The phosphate ν1 apatitic band width also narrowed during this time. To quantify the amount of crystal growth in vivo, we used a calibration curve derived from X-ray powder diffraction and Raman studies performed on a series of synthetic carbonated apatites and deproteinated mouse femoral specimens. Mineralization in neonatal mouse calvarial culture was observed along the lambdoid suture. Deposition proceeded in a stepwise fashion over the course of ~30 h.

Osteoarthritis screening using Raman spectroscopy of dried human synovial fluid drops

We describe the use of Raman spectroscopy to investigate synovial fluid drops deposited onto fused silica microscope slides. This spectral information can be used to identify chemical changes in synovial fluid associated with osteoarthritis (OA) damage to knee joints. The chemical composition of synovial fluid is predominately proteins (enzymes, cytokines, or collagen fragments), glycosaminoglycans, and a mixture of minor components such as inorganic phosphate crystals. During osteoarthritis, the chemical, viscoelastic and biological properties of synovial fluid are altered. A pilot study was conducted to determine if Raman spectra of synovial fluid correlated with radiological scoring of knee joint damage. After informed consent, synovial fluid was drawn and x-rays were collected from the knee joints of 40 patients. Raman spectra and microscope images were obtained from the dried synovial fluid drops using a Raman microprobe and indicate a coarse separation of synovial fluid components. Individual protein signatures could not be identified; Raman spectra were useful as a general marker of overall protein content and secondary structure. Band intensity ratios used to describe protein and glycosaminoglycan structure were used in synovial fluid spectra. Band intensity ratios of Raman spectra indicate that there is less ordered protein secondary structure in synovial fluid from the damage group. Combination of drop deposition with Raman spectroscopy is a powerful approach to examining synovial fluid for the purposes of assessing osteoarthritis damage.