Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin.

BACKGROUND: Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS: Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION: Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

Functionalization of MgZnO nanorod films and characterization by FTIR microscopic imaging.

Metal organic chemical vapor deposition grown films consisting of MgxZn1-xO (4% < x < 5%) nanorod arrays (MgZnOnano) were functionalized with 11-azidoundecanoic acid (1). The MgZnOnano was used instead of pure ZnO to take advantage of the etching resistance of the MgZnOnano during the binding and subsequent sensing device fabrication processes of sensor devices, while the low Mg composition level ensures that selected ZnO properties useful for sensors development, such as piezoelectricity, are retained. Compound 1 was bound to the MgZnOnano surface through the carboxylic acid group, leaving the azido group available for click chemistry and as a convenient infrared spectroscopy (IR) probe. The progress of the functionalization with 1 was characterized by FTIR microscopic imaging as a function of binding time, solvents employed, and MgZnOnano morphology. Binding of 1 was most stable in solutions of 3-methoxypropionitrile (MPN), a non-protic polar solvent. This occurred first in µm-scale islands, then expanded to form a rather uniform layer after 22 h. Binding in alcohols resulted in less homogenous coverage, but the 1/MgZnOnano films prepared from MPN were stable upon treatment with alcohols at room temperature. The binding behavior was significantly dependent on the surface morphology of MgZnOnano. Graphical abstract The functionalization of MgZnO nanorod films with a click-ready linker and its dependence on bidning conditions and morphology has been studied by FTIR microscopic imaging using the azido group as the IR tag.

Graphene-Catalyzed Direct Friedel-Crafts Alkylation Reactions: Mechanism, Selectivity, and Synthetic Utility.

Transition-metal-catalyzed alkylation reactions of arenes have become a central transformation in organic synthesis. Herein, we report the first general strategy for alkylation of arenes with styrenes and alcohols catalyzed by carbon-based materials, exploiting the unique property of graphenes to produce valuable diarylalkane products in high yields and excellent regioselectivity. The protocol is characterized by a wide substrate scope and excellent functional group tolerance. Notably, this process constitutes the first general application of graphenes to promote direct C-C bond formation utilizing polar functional groups anchored on the GO surface, thus opening the door for an array of functional group alkylations using benign and readily available graphene materials. Mechanistic studies suggest that the reaction proceeds via a tandem catalysis mechanism in which both of the coupling partners are activated by interaction with the GO surface.

Microwave Enabled One-Pot, One-Step Fabrication and Nitrogen Doping of Holey Graphene Oxide for Catalytic Applications.

The unique properties of a holey graphene sheet, referred to as a graphene sheet with nanoholes in its basal plane, lead to wide range of applications that cannot be achieved by its nonporous counterpart. However, the large-scale solution-based production requires graphene oxide (GO) or reduced GO (rGO) as the starting materials, which take hours to days for fabrication. Here, an unexpected discovery that GO with or without holes can be controllably, directly, and rapidly (tens of seconds) fabricated from graphite powder via a one-step-one-pot microwave assisted reaction with a production yield of 120 wt% of graphite is reported. Furthermore, a fast and low temperature approach is developed for simultaneous nitrogen (N) doping and reduction of GO sheets. The N-doped holey rGO sheets demonstrate remarkable electrocatalytic capabilities for the electrochemical oxygen reduction reaction. The existence of the nanoholes provides a "short cut" for efficient mass transport and dramatically increases edges and surface area, therefore, creates more catalytic centers. The capability of rapid fabrication and N-doping as well as reduction of holey GO can lead to development of an efficient catalyst that can replace previous coin metals for energy generation and storage, such as fuel cells and metal-air batteries.

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function.

Plant-derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA-d34 ). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA-d34 and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose-dependent manner, with the highest increase from treatment with pure OA. OA-d34 penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA-d34 permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.

Infrared spectroscopic imaging tracks lateral distribution in human stratum corneum.

PURPOSE: To demonstrate the efficacy of infrared (IR) spectroscopic imaging for evaluation of lateral diffusion in stratum corneum (SC) and for elucidation of intermolecular interactions between exogenous agents and SC constituents. METHODS: In separate experiments, acyl chain perdeuterated oleic acid (OA-d) and deuterated dimethyl sulfoxide (DMSO-d) were applied to the surface of isolated human SC. The lateral distribution of permeant concentrations was monitored using the time-dependence of IR images. Diffusion coefficients (D) were estimated from Fick's second law. Interactions between the exogenous agents and the SC were tracked from changes in CD2 and Amide I stretching frequencies. RESULTS: Networked glyphs served as the major pathway for lateral distribution of OA-d. In glyph-poor regions, D values from 0.3-1 × 10(-8) cm(2)/s bracketed the OA-d data and apparently decreased with time. Although diffusion of DMSO-d is relatively fast compared to our experimental measurement time, the results suggest values of ~10(-7) cm(2)/s. OA-d spectral changes suggest penetration into the ordered lipids of the SC; DMSO-d penetration results in perturbation of SC keratin structure. CONCLUSIONS: IR imaging provides concentration profiles, diffusion coefficients, and unique molecular level information about structural changes in the endogenous SC constituents and exogenous agents upon their mutual interaction. Transport along glyphs is the dominant mode of distribution for OA-d.

Fourier transform infrared spectroscopic imaging parameters describing acid phosphate substitution in biologic hydroxyapatite.

Acid phosphate substitution into mineralized tissues is an important determinant of their mechanical properties and their response to treatment. This study identifies and validates Fourier transform infrared spectroscopic imaging (FTIRI) spectral parameters that provide information on the acid phosphate (HPO4) substitution into hydroxyapatite in developing mineralized tissues. Curve fitting and Fourier self-deconvolution were used to identify subband positions in model compounds (with and without HPO4). The intensity of subbands at 1127 and 1110 cm(-1) correlated with the acid phosphate content in these models. Peak height ratios of these subbands to the ν3 vibration at 1096 cm(-1) found in stoichiometric apatite were evaluated in the model compounds and mixtures thereof. FTIRI spectra of bones and teeth at different developmental ages were analyzed using these spectral parameters. Factor analysis (a chemometric technique) was also conducted on the tissue samples and resulted in factor loadings with spectral features corresponding to the HPO4 vibrations described above. Images of both factor correlation coefficients and the peak height ratios 1127/1096 and 1112/1096 cm(-1) demonstrated higher acid phosphate content in younger vs. more mature regions in the same specimen. Maps of the distribution of acid phosphate content will be useful for characterizing the extent of new bone formation, the areas of potential decreased strength, and the effects of therapies such as those used in metabolic bone diseases (osteoporosis, chronic kidney disease) on mineral composition. Because of the wider range of values obtained with the 1127/1096 cm(-1) parameter compared to the 1110/1096 cm(-1) parameter and the smaller scatter in the slope, it is suggested that this ratio should be the parameter of choice.

Oleic acid disorders stratum corneum lipids in Langmuir monolayers.

Oleic acid (OA) is well-known to affect the function of the skin barrier. In this study, the molecular interactions between OA and model stratum corneum (SC) lipids consisting of ceramide, cholesterol, and palmitic acid (PA) were investigated with Langmuir monolayer and associated techniques. Mixtures with different OA/SC lipid compositions were spread at the air/water interface, and the phase behavior was tracked with surface pressure-molecular area (π-A) isotherms. With increasing OA levels in the monolayer, the films became more fluid and more compressible. The thermodynamic parameters derived from π-A isotherms indicated that there are preferential interactions between OA and SC lipids and that films of their mixtures were thermodynamically stable. The domain structure and lipid conformational order of the monolayers were studied through Brewster angle microscopy (BAM) and infrared reflection absorption spectroscopy (IRRAS), respectively. Results indicate that lower concentrations of OA preferentially mix with and disorder the ceramide-enriched domains, followed by perturbation of the PA-enriched domains and disruption of SC lipid domain separation at higher OA levels.

Microwave- and nitronium ion-enabled rapid and direct production of highly conductive low-oxygen graphene.

Currently the preferred method for large-scale production of solution-processable graphene is via a nonconductive graphene oxide (GO) pathway, which uncontrollably cuts sheets into small pieces and/or introduces nanometer-sized holes in the basal plane. These structural changes significantly decrease some of graphene's remarkable electrical and mechanical properties. Here, we report an unprecedented fast and scalable approach to avoid these problems and directly produce large, highly conductive graphene sheets. This approach intentionally excludes KMnO(4) from Hummers' methods and exploits aromatic oxidation by nitronium ions combined with the unique properties of microwave heating. This combination promotes rapid and simultaneous oxidation of multiple non-neighboring carbon atoms across an entire graphene sheet, thereby producing only a minimum concentration of oxygen moieties sufficient to enable the separation of graphene sheets. Thus, separated graphene sheets, which are referred to as microwave-enabled low-oxygen graphene, are thermally stable and highly conductive without requiring further reduction. Even in the absence of polymeric or surfactant stabilizers, concentrated dispersions of graphene with clean and well-separated graphene sheets can be obtained in both aqueous and organic solvents. This rapid and scalable approach produces high-quality graphene sheets of low oxygen content, enabling a broad spectrum of applications via low-cost solution processing.

Multiscale analysis of water uptake and erosion in biodegradable polyarylates.

The role of hydration in degradation and erosion of materials, especially biomaterials used in scaffolds and implants, was investigated by studying the distribution of water at length scales from 0.1 nm to 0.1 mm using Raman spectroscopy, small-angle neutron scattering (SANS), Raman confocal imaging, and scanning electron microscopy (SEM). The measurements were demonstrated using L-tyrosine derived polyarylates. Bound- and free- water were characterized using their respective signatures in the Raman spectra. In the presence of deuterium oxide (D(2)O), H-D exchange occurred at the amide carbonyl but was not detected at the ester carbonyl. Water appeared to be present in the polymer even in regions where there was little evidence for N-H to N-D exchange. SANS showed that water is not uniformly dispersed in the polymer matrix. The distribution of water can be described as mass fractals in polymers with low water content (~5 wt%), and surface fractals in polymers with larger water content (15 to 60 wt%). These fluctuations in the density of water distribution are presumed to be the precursors of the ~ 20 µm water pockets seen by Raman confocal imaging, and also give rise to 10-50 µm porous network seen in SEM. The surfaces of these polymers appeared to resist erosion while the core of the films continued to erode to form a porous structure. This could be due to differences in either the density of the polymer or the solvent environment in the bulk vs. the surface, or a combination of these two factors. There was no correlation between the rate of degradation and the amount of water uptake in these polymers, and this suggests that it is the bound-water and not the total amount of water that contributes to hydrolytic degradation.

Infrared reflection-absorption spectroscopy: principles and applications to lipid-protein interaction in Langmuir films.

Infrared reflection-absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.

Infrared assessment of bone quality: a review.

BACKGROUND: Bone strength depends on both bone quantity and quality. The former is routinely estimated in clinical settings through bone mineral density measurements but not the latter. Bone quality encompasses the structural and material properties of bone. Although its importance is appreciated, its contribution in determining bone strength has been difficult to precisely quantify partly because it is multifactorial and requires investigation of all bone hierarchical levels. Fourier transform infrared spectroscopy provides one way to explore these levels. QUESTIONS/PURPOSES: The purposes of our review were to (1) provide a brief overview of Fourier transform infrared spectroscopy as a way to establish bone quality, (2) review the major bone material parameters determined from Fourier transform infrared spectroscopy, and (3) review the role of Fourier transform infrared microspectroscopic analysis in establishing bone quality. METHODS: We used the ISI Web of Knowledge database initially to identify articles containing the Boolean term "infrared" AND "bone." We then focused on articles on infrared spectroscopy in bone-related journals. RESULTS: Infrared spectroscopy provides information on bone material properties. Their microspectroscopic versions allow one to establish these properties as a function of anatomic location, mineralization extent, and bone metabolic activity. It provides answers pertaining to the contribution of mineral to matrix ratio, mineral maturity, mineral carbonate substitution, and collagen crosslinks to bone strength. Alterations of bone material properties have been identified in disease (especially osteoporosis) not attainable by other techniques. CONCLUSIONS: Infrared spectroscopic analysis is a powerful tool for establishing the important material properties contributing to bone strength and thus has helped better understand changes in fragile bone.

IPr# - highly hindered, broadly applicable N-heterocyclic carbenes.

IPr (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) represents the most important NHC (NHC = N-heterocyclic carbene) ligand throughout the field of homogeneous catalysis. Herein, we report the synthesis, catalytic activity, and full structural and electronic characterization of novel, sterically-bulky, easily-accessible NHC ligands based on the hash peralkylation concept, including IPr#, Np# and BIAN-IPr#. The new ligands have been commercialized in collaboration with Millipore Sigma: IPr#HCl, 915653; Np#HCl; 915912; BIAN-IPr#HCl, 916420, enabling broad access of the academic and industrial researchers to new ligands for reaction optimization and screening. In particular, the synthesis of IPr# hinges upon cost-effective, modular alkylation of aniline, an industrial chemical that is available in bulk. The generality of this approach in ligand design is demonstrated through facile synthesis of BIAN-IPr# and Np#, two ligands that differ in steric properties and N-wingtip arrangement. The broad activity in various cross-coupling reactions in an array of N-C, O-C, C-Cl, C-Br, C-S and C-H bond cross-couplings is demonstrated. The evaluation of steric, electron-donating and π-accepting properties as well as coordination chemistry to Au(i), Rh(i) and Pd(ii) is presented. Given the tremendous importance of NHC ligands in homogenous catalysis, we expect that this new class of NHCs will find rapid and widespread application.

FT-IR investigation of Terbinafine interaction with stratum corneum constituents.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm-1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm-1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial ß-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

Visualization of Epidermal Reservoir Formation from Topical Diclofenac Gels by Raman Spectroscopy.

PURPOSE: This work investigated whether topical pain relief diclofenac gels can form a diclofenac reservoir in the epidermal and dermal layers of human skin. METHODS: Excised human skin samples were treated with three topical diclofenac gels ex vivo and examined using Raman microscopy of transversally microtomed sections. The relative diclofenac concentration in the skin layers was calculated as the ratio of the integrated areas of bands characteristic of diclofenac (~445 cm-1) and skin (Amide I). A customized masking algorithm ensured that only diclofenac-specific signal was mapped in the resulting Raman images. RESULTS: A heterogenous spatial distribution of diclofenac was clearly visible in both the epidermis and the dermis in all samples, with a markedly higher diclofenac relative content and number of pixels above the detection limit in the epidermis compared to the dermis. CONCLUSION: The Raman images evidenced that the studied topical gels deliver diclofenac through the stratum corneum skin barrier and form a drug depot localized in the epidermis. The data are in line with earlier clinical findings that this depot acts like a true reservoir and enables sustained drug release.

Complex N-glycan and metabolic control in tumor cells.

Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched complex N-glycans on cell surface glycoproteins that bind to galectins and promote surface residency of glycoproteins, including cytokine receptors. Carcinoma cells from polyomavirus middle T (PyMT) transgenic mice on a Mgat5-/- background have reduced surface levels of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) receptors and are less sensitive to acute stimulation by cytokines in vitro compared with PyMT Mgat5+/+ tumor cells but are nonetheless tumorigenic when injected into mice. Here, we report that PyMT Mgat5-/- cells are reduced in size, checkpoint impaired, and following serum withdrawal, fail to down-regulate glucose transport, protein synthesis, reactive oxygen species (ROS), and activation of Akt and extracellular signal-regulated kinase. To further characterize Mgat5+/+ and Mgat5-/- tumor cells, a screen of pharmacologically active compounds was done. Mgat5-/- tumor cells were comparatively hypersensitive to the ROS inducer 2,3-dimethoxy-1,4-naphthoquinone, hyposensitive to tyrosine kinase inhibitors, to Golgi disruption by brefeldin A, and to mitotic arrest by colcemid, hydroxyurea, and camptothecin. Finally, regulation of ROS, glucose uptake, and sensitivities to EGF and TGF-beta were rescued by Mgat5 expression or by hexosamine supplementation to complex N-glycan biosynthesis in Mgat5-/- cells. Our results suggest that complex N-glycans sensitize tumor cells to growth factors, and Mgat5 is required to balance responsiveness to growth and arrest cues downstream of metabolic flux.

A unique gel matrix moisturizer delivers deep hydration resulting in significant clinical improvement in radiance and texture.

Introduction: As skin ages, it loses its ability to retain moisture and becomes rough and dry. This results in a clinically dull appearance with a loss of radiance, firmness, and suppleness. Symptoms can be improved with use of a moisturizer that builds and maintains skin hydration over time; however, most moisturizers that occlude the skin surface are perceived as heavy and greasy and are not consumer preferred. Methods: A unique, consumer-preferred gel matrix formula was developed by combining liquid crystal structures, which mimic skin barrier lipid assembly, with specific emulsifiers that deliver water deep into skin. Ex vivo studies were conducted to investigate the superior hydrating effects of the gel matrix formula. Confocal Raman microscopy studies assessed the spatial distribution of water in ex vivo skin after application of the gel matrix formula. To determine the effects of the gel matrix formula on dry facial skin, a 12-week clinical study was conducted with subjects with self-perceived skin dryness and dullness. Results: The formulation significantly increased the relative water content throughout epidermal regions, which was not observed with the application of a competitive gel formula. Instrumental measurements assessed improvements in skin surface moisturization and barrier function. Clinical grading showed significant improvements in hydration-related endpoints including radiance, clarity, and texture. Subject self-agree assessment demonstrated that subjects observed improvements in the appearance of their facial skin. Conclusion: These studies demonstrated that the gel matrix formula increased skin water content in deeper layers, and resulted in significant clinical improvements in hydration, barrier function, and clinical appearance of radiance.

Graphene oxide catalyzed ketone α-alkylation with alkenes: enhancement of graphene oxide activity by hydrogen bonding.

Direct α-alkylation of carbonyl compounds represents a fundamental bond forming transformation in organic synthesis. We report the first ketone-alkylation using olefins and alcohols as simple alkylating agents catalyzed by graphene oxide. Extensive studies of the graphene surface suggest a pathway involving dual activation of both coupling partners. Notably, we show that polar functional groups have a stabilizing effect on the GO surface, which results in a net enhancement of the catalytic activity. The method represents the first alkylation of carbonyl compounds using graphenes, which opens the door for the development of an array of protocols for ketone functionalization employing common carbonyl building blocks and readily available graphenes.

Interaction of recombinant surfactant protein D with lipopolysaccharide: conformation and orientation of bound protein by IRRAS and simulations.

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.

Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy.

Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.