Fibrillar collagens in connective tissues are organized into complex and diverse hierarchical networks. In dermis, bone, and tendon, one common phenomenon at the micrometer scale is the organization of fibrils into bundles. Previously, we have reported that collagen fibrils in these tissues exhibit a 10 nm width distribution of D-spacing values. This study expands the observation to a higher hierarchical level by examining fibril D-spacing distribution in relation to the bundle organization. We used atomic force microscopy imaging and two-dimensional fast Fourier transform analysis to investigate dermis, tendon, and bone tissues. We found that, in each tissue type, collagen fibril D-spacings within a single bundle were nearly identical and frequently differ by less than 1 nm. The full 10 nm range in D-spacing values arises from different values found in different bundles. The similarity in D-spacing was observed to persist for up to 40 μm in bundle length and width. A nested mixed model analysis of variance examining 107 bundles and 1710 fibrils from dermis, tendon, and bone indicated that fibril D-spacing differences arise primarily at the bundle level (∼76%), independent of species or tissue types.Keywords: 2D FFT; AFM; collagen bundle; fibril D-spacing; mixed model ANOVA

Nanoparticles conjugated with functional ligands are expected to have a major impact in medicine, photonics, sensing, and nanoarchitecture design. One major obstacle to realizing the promise of these materials, however, is the difficulty in controlling the ligand/nanoparticle ratio. This obstacle can be segmented into three key areas: First, many designs of these systems have failed to account for the true heterogeneity of ligand/nanoparticle ratios that compose each material. Second, studies in the field often use the mean ligand/nanoparticle ratio as the accepted level of characterization of these materials. This measure is insufficient because it does not provide information about the distribution of ligand/nanoparticle species within a sample or the number and relative amount of the different species that compose a material. Without these data, researchers do not have an accurate definition of material composition necessary both to understand the material–property relationships and to monitor the consistency of the material. Third, some synthetic approaches now in use may not produce consistent materials because of their sensitivity to reaction kinetics and to the synthetic history of the nanoparticle.In this Account, we describe recent advances that we have made in under standing the material composition of ligand–nanoparticle systems. Our work has been enabled by a model system using poly(amidoamine) dendrimers and two small molecule ligands. Using reverse phase high-pressure liquid chromatography (HPLC), we have successfully resolved and quantified the relative amounts and ratios of each ligand/dendrimer combination. This type of information is rare within the field of ligand–nanoparticle materials because most analytical techniques have been unable to identify the components in the distribution.Our experimental data indicate that the actual distribution of ligand–nanoparticle components is much more heterogeneous than is commonly assumed. The mean ligand/nanoparticle ratio that is typically the only information known about a material is insufficient because the mean does not provide information on the diversity of components in the material and often does not describe the most common component (the mode). Additionally, our experimental data has provided examples of material batches with the same mean ligand/nanoparticle ratio and very different distributions. This discrepancy indicates that the mean cannot be used as the sole metric to assess the reproducibility of a system. We further found that distribution profiles can be highly sensitive to the synthetic history of the starting material as well as slight changes in reaction conditions. We have incorporated the lessons from our experimental data into the design of new ligand–nanoparticle systems to provide improved control over these ratios.

A modular dendrimer-based drug delivery platform was designed to improve upon existing limitations in single dendrimer systems. Using this modular strategy, a biologically active platform containing receptor mediated targeting and fluorescence imaging modules was synthesized by coupling a folic acid (FA) conjugated dendrimer with a fluorescein isothiocyanate (FITC) conjugated dendrimer. The two different dendrimer modules were coupled via the 1,3-dipolar cycloaddition reaction (“click” chemistry) between an alkyne moiety on the surface of the first dendrimer and an azide moiety on the second dendrimer. Two simplified model systems were also synthesized to develop appropriate “click” reaction conditions and aid in spectroscopic assignments. Conjugates were characterized by 1H NMR spectroscopy and NOESY. The FA-FITC modular platform was evaluated in vitro with a human epithelial cancer cell line (KB) and found to specifically target the overexpressed folic acid receptor.

We have carried out stochastic boundary molecular dynamics simulations to estimate free energy changes for substitutions of Gly with Val, Arg and Trp residues in a collagen-like peptide. The relative free energy change differences of mutants containing a Val, an Arg and a Trp relative to the wild type are 5.7, 8.1 and 9.5 kcal/mol, respectively. The corresponding free energy change differences of mutants containing two mutated residues are on average 7.6, 10.5 and 14.7 kcal/mol, respectively. We show that the free energy change differences are correlated with the severity of OI from statistical analysis and mechanical properties of the individual tropocollagen molecules. This simulation result indicates an atomistic-level mechanistic understanding of the effect of OI mutations in terms of stability of the mutants relative to the wild type, which could eventually provide a new strategy for diagnosis and treatment of the disease.Research highlights► Changes in unfolding free energy for mutated collagen-like peptides were calculated. ► Larger ΔΔG values for G → V, R, W mutations were due to effects of size. ► Calculated ΔΔG values exhibit positive correlation with severity of OI. ► Calculated ΔΔG values are consistent with mechanical properties of collagen fibers.

The GM1/caveolin-1 lipid raft mediated endocytosis mechanism was explored for generation 5 and 7 poly(amidoamine) dendrimer polyplexes employing the Cos-7, 293A, C6, HeLa, KB, and HepG2 cell lines. Expression levels of GM1 and caveolin-1 were measured using dot blot and Western blot, respectively. The level of GM1 in the cell plasma membrane was adjusted by incubation with exogenous GM1 or ganglioside inhibitor PPMP, and the level of CAV-1 was adjusted by upregulation with the adenovirus vector expressed caveolin-1 (AdCav-1). Cholera toxin B subunit was employed as a positive control for uptake in all cases. No evidence was found for a GM1/caveolin-1 lipid raft mediated endocytosis mechanism for the generation 5 and 7 poly(amidoamine) dendrimer polyplexes.Keywords: caveolin-1; endocytosis; ganglioside GM1; PAMAM dendrimer; polyplexes; transfection;

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer−DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression.Keywords: cationic vectors; cellular uptake; flow cytometry; gene delivery; gene expression; lipoplexes; membrane permeability; Polyplexes;

Functional nanoparticles often contain ligands including targeting molecules, fluorophores, and/or active moieties such as drugs. Characterizing the number of these ligands bound to each particle and the distribution of nanoparticle−ligand species is important for understanding the nanomaterial’s function. In this study, the amide coupling methods commonly used to conjugate ligands to poly(amidoamine) (PAMAM) dendrimers were examined. A skewed Poisson distribution was observed and quantified using HPLC for two sets of dendrimer−ligand samples prepared using the amine-terminated form of the PAMAM dendrimer and a partially acetylated form of the PAMAM dendrimer that has been used for targeted in vivo drug delivery. The prepared samples had an average number of ligands per dendrimer ranging from 0.4 to 13. Distributions identified by HPLC are in excellent agreement with the mean ligand/dendrimer ratio, measured by 1H NMR, gel permeation chromatography (GPC), and potentiometric titration. These results provide insight into the heterogeneity of distributions that are obtained for many classes of nanomaterials to which ligands are conjugated and belie the use of simple cartoon models that present the “average” number of ligands bound as a physically meaningful representation for the material.Keywords: drug delivery; ligand distribution; nanoparticle characterization; nanotechnology; PAMAM dendrimer

The reaction of SnC(SiMe3)2CH2CH2C(SiMe3)2/ArI (Ar = Ph, 2,4,6-triisopropylphenyl) with cyclohexane, diethyl ether, tetrahydrofuran (THF), toluene, and mesitylene yields C−H activation products in which a new Sn−C bond is formed at the location of the weakest C−H bond. The regioselectivity of this reaction with trans-4-methyl-2-pentene and 4-methyl-2-pentyne was explored as a function of aryl halide for PhI, 2,4,6-trimethyliodobenzene, 2,4,6-triisopropyliodobenzene, 4-iodoanisole, 4-iodobenzonitrile, and 2,6-mesityliodobenzene. A degree of regiochemical control could be obtained as highlighted by increased amounts of primary activation. The use of 2,4,6-tert-butyliodobenzene resulted in C−H activation of the ortho tert-butyl groups in all solvents tried. The primary kinetic isotope effect for the reaction of SnC(SiMe3)2CH2CH2C(SiMe3)2/2,4,6-triisopropyliodobenzene with toluene/toluene-d7 was found to be 4.9 ± 0.5.

Two polymorphic structures of Cp*GeCl were determined by single-crystal X-ray diffraction analysis: (I) a dimer in the form of a parallelogram in solid state (yellow crystals, P21/n) and (II) an infinite ladder of alternating parallelograms (colorless crystals, C2/c). Melting experiments in X-ray capillary tubes revealed that the C2/c polymorph converts to the P21/n polymorphic structure. Variable-temperature and concentration-dependent 1H NMR spectroscopy suggest an equilibrium between a monomer and either a dimer or higher oligomer species. Variable-temperature 1H NMR (from 25 to −80 °C) of Cp*GeCl (P21/n) dissolved in toluene-d8 indicates that lower temperature favors the dimers and/or oligomers. Increasing concentration (from 0.05 to 0.65 M) also favors the dimer or oligomer structure. The addition of chloride ions (from 0.1 to 1.0 equiv) via the addition of tetrabutylammonium chloride to solutions prepared from Cp*GeCl resulted in the formation of Cp*2Ge. Addition of increasing amounts (from 5 to 42 mg) of Cp*GeCl to a sample of Cp*2Ge gave no evidence of exchange by 1H NMR spectroscopy. Similarly, the addition of one equivalent of methyl mesylate to various concentrations of Cp*GeCl (0.06, 0.09, and 1.8 M) did not result in the formation of methyl chloride, indicating that an equilibrium involving chloride ions is not operative.

This study demonstrates that collagen, the most abundant protein in animals, exists as a distribution of nanoscale morphologies in teeth, bones, and tendons. This fundamental characteristic of Type I collagen has not previously been reported and provides a new understanding of the nanoscale architecture of this ubiquitous and important biological nanomaterial. Dentin, bone, and tendon tissue samples were chosen for their differences in cellular origin and function, as well as to compare mineralized tissues with a tissue that lacks mineral in a normal physiological setting. A distribution of morphologies was present in all three tissues, confirming that this characteristic is fundamental to Type I collagen regardless of the presence of mineral, cellular origin of the collagen (osteoblast versus odontoblast versus fibroblast), anatomical location, or mechanical function of the tissue.

Partial acetylation of the amine-terminated poly(amidoamine) dendrimer has been used in the preparation of dendrimer particles conjugated with a wide variety of functional ligands including targeting moieties, therapeutic agents, and dye molecules. The effectiveness of mass transport during the partial acetylation reaction was found to have a major effect on subsequent distributions of dendrimer−ligand components and to be a major source of inconsistency between batches. This study has broad implications for a wide range of nanoparticle−ligand systems because it demonstrates that conjugates with the same mean ligand−particle ratios can have completely different distribution profiles.

Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by 1H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH2) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM1-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH2 dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH2 was not ganglioside GM1 dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM1 in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30−2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm2 in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1−100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making ∼3 nm holes in living cell membranes.

The reaction of silylene Si[N2(tBu)2C2H2] and Ph−X (X = I, Br) in alkane and ethereal solvents results in the formation of C−H activation product [C2H2(tBu2)N2]SiRI and an equivalent of benzene or oxidative-addition product [C2H2(tBu2)N2]SiPhI. The ratio of products obtained is dependent upon substrate and concentration. This class of reaction has been extended for Si[N2(tBu)2C2H2] and Ge[CH(SiMe3)2]2 to alkylamines. The primary kinetic isotope effect has been measured for the reaction of Si[N2(tBu)2C2H2] with Et2O and determined to be kH/kD = 5.1 ± 0.1. The reaction of Ge[CH(SiMe3)2]2 and Ph−Br with THF was determined to be second order. A large isotope effect ranging from 1.8 to 1.1 was measured for a variety of deuterated aryl halides, consistent with an initial electron transfer to the aryl halide.

The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimer−phospholipid interactions were measured with isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and molecular dynamics (MD) simulations. Dendrimers of sixth-generation and smaller interacted with the lipids at an average stoichiometry and enthalpy proportional to the number of primary amines per dendrimers (4.5 ± 0.1 lipids/primary amine and 6.3 ± 0.3 kJ/mol of primary amines, respectively). Larger dendrimers, however, demonstrated a decreased number of bound lipids and heat release per primary amine, presumably due to the steric restriction of dendrimer deformation on the lipid bilayer. For example, eighth-generation PAMAM dendrimers bound to 44% fewer lipids per primary amine and released 63% less heat per primary amine as compared to the smaller dendrimers. These differences in binding stoichiometry support generation-dependent models for dendrimer−lipid complexation, which are consistent with previously observed generation-dependent differences in dendrimer-induced membrane disruption. Dendrimers of seventh-generation and larger bound to lipids with an average stoichiometry consistent with each dendrimer having been wrapped by a bilayer of lipids, whereas smaller dendrimers did not.Keywords: membrane disruption; membrane permeability; nanotoxicity; phospholipid bilayer; poly(amidoamine) dendrimer

Stochastic synthesis of a ligand coupled to a nanoparticle results in a distribution of populations with different numbers of ligands per nanoparticle. This distribution was resolved and quantified using HPLC and is in excellent agreement with the ligand/nanoparticle average measured by 1H NMR, gel permeation chromatography (GPC), and potentiometric titration, and yet significantly more disperse than commonly held perceptions of monodispersity. Two statistical models were employed to confirm that the observed heterogeneity is consistent with theoretical expectations.

The mixed reagent SnC(SiMe3)2CH2CH2C(SiMe3)2]/ArI reacts with alkynes to give primary and secondary propargylic C−H activation. Alkynes tested include 1-phenylpropyne, 1-phenylbutyne, 1-trimethylsily1hexyne, and 2-hexyne. Aryl halides tested include iodobenzene and 2,4,6-triisopropyliodobenzene. An X-ray crystal structure is reported for the product of the secondary propargylic activation of 2-hexyne.

The molecular structures and enthalpy release of poly(amidoamine) (PAMAM) dendrimers binding to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers were explored through atomistic molecular dynamics. Three PAMAM dendrimer terminations were examined: protonated primary amine, neutral acetamide, and deprotonated carboxylic acid. Fluid and gel lipid phases were examined to extract the effects of lipid tail mobility on the binding of generation-3 dendrimers, which are directly relevant to the nanoparticle interactions involving lipid rafts, endocytosis, lipid removal, and/or membrane pores. Upon binding to gel phase lipids, dendrimers remained spherical, had a constant radius of gyration, and approximately one-quarter of the terminal groups were in close proximity to the lipids. In contrast, upon binding to fluid phase bilayers, dendrimers flattened out with a large increase in their asphericity and radii of gyration. Although over twice as many dendrimer−lipid contacts were formed on fluid versus gel phase lipids, the dendrimer−lipid interaction energy was only 20% stronger. The greatest enthalpy release upon binding was between the charged dendrimers and the lipid bilayer. However, the stronger binding to fluid versus gel phase lipids was driven by the hydrophobic interactions between the inner dendrimer and lipid tails.

Third-generation (G3) poly(amidoamine) (PAMAM) dendrimers are simulated approaching 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers with fully atomistic molecular dynamics, which enables the calculation of a free energy profile along the approach coordinate. Three different dendrimer terminations are examined: protonated primary amine, uncharged acetamide, and deprotonated carboxylic acid. As the dendrimer and lipids become closer, their attractive force increases (up to 240 pN) and the dendrimer becomes deformed as it interacts with the lipids. The total energy release upon binding of a G3−NH3+, G3−Ac, or G3−COO− dendrimer to a DMPC bilayer is, respectively, 36, 26, or 47 kcal/mol or, equivalently, 5.2, 3.2, or 4.7 × 10−3 kcal/g. These results are analyzed in terms of the dendrimers’ size, shape, and atomic distributions as well as proximity of individual lipid molecules and particular lipid atoms to the dendrimer. For example, an area of 9.6, 8.2, or 7.9 nm2 is covered on the bilayer for the G3−NH3+, G3−Ac, or G3−COO− dendrimers, respectively, while interacting strongly with 18−13 individual lipid molecules.

Organic-coated superparamagnetic iron oxide nanoparticles (OC-SPIONs) were synthesized and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. OC-SPIONs were transferred from organic media into water using poly(amidoamine) dendrimers modified with 6-TAMRA fluorescent dye and folic acid molecules. The saturation magnetization of the resulting dendrimer-coated SPIONs (DC-SPIONs) was determined, using a superconducting quantum interference device, to be 60 emu/g Fe versus 90 emu/g Fe for bulk magnetite. Selective targeting of the DC-SPIONs to KB cancer cells in vitro was demonstrated and quantified using two distinct and complementary imaging modalities: UV–visible and X-ray fluorescence; confocal microscopy confirmed internalization. The results were consistent between the uptake distribution quantified by flow cytometry using 6-TAMRA UV–visible fluorescence intensity and the cellular iron content determined using X-ray fluorescence microscopy.Keywords: cancer; dendrimers; magnetic nanoparticles; phase transfer; superparamagnetism; targeted MRI contrast agents; X-ray fluorescence microscopy;

The interaction of generation 5 (G5) and 7 (G7) poly(amidoamine) (PAMAM) dendrimers with mica-supported Survanta bilayers is studied with atomic force microscopy (AFM). In these experiments, Survanta forms distinct gel and fluid domains with differing lipid composition. Nanoscale defects are induced by the PAMAM dendrimers. The positively charged dendrimers remove lipid from the fluid domains at a significantly greater rate than for the gel domains. Dendrimer accumulation on lipid edges and terraces preceding lipid removal has been directly imaged. Immediately following lipid removal, the mica surface is clean, indicating that lipid defects are not induced by dendrimers binding to the mica substrate and displacing the lipid.

Polycationic organic nanoparticles are shown to disrupt model biological membranes and living cell membranes at nanomolar concentrations. The degree of disruption is shown to be related to nanoparticle size and charge, as well as to the phase–fluid, liquid crystalline, or gel–of the biological membrane. Disruption events on model membranes have been directly imaged using scanning probe microsopy, whereas disruption events on living cells have been analyzed using cytosolic enzyme leakage assays, dye diffusion assays, and fluorescence microscopy.

Dendrimer-based anticancer nanotherapeutics containing ∼5 folate molecules have shown in vitro and in vivo efficacy in cancer cell targeting. Multivalent interactions have been inferred from observed targeting efficacy, but have not been experimentally proven. This study provides quantitative and systematic evidence for multivalent interactions between these nanodevices and folate-binding protein (FBP). A series of the nanodevices were synthesized by conjugation with different amounts of folate. Dissociation constants (KD) between the nanodevices and FBP measured by SPR are dramatically enhanced through multivalency (∼2,500- to 170,000-fold). Qualitative evidence is also provided for a multivalent targeting effect to KB cells using flow cytometry. These data support the hypothesis that multivalent enhancement of KD, not an enhanced rate of endocytosis, is the key factor resulting in the improved biological targeting by these drug delivery platforms.

Atomic force microscopy (AFM) is employed to observe the effect of poly(amidoamine) (PAMAM) dendrimers on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers. Aqueous solutions of generation 7 PAMAM dendrimers cause the formation of holes 15–40 nm in diameter in previously intact bilayers. This effect is observed for two different branch end-groups—amine and carboxyl. In contrast, carboxyl-terminated core-shell tectodendrimer clusters do not create holes in the lipid membrane but instead show a strong affinity to adsorb to the edges of existing bilayer defects. A possible mechanism for the formation of holes in the lipid bilayer is proposed. The dendrimers remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules. Dynamic light scattering (DLS) measurements as well as 31P NMR data support this explanation. The fact that tectodendrimers behave differently suggests that their cluster-like architecture plays an important role in their interaction with the lipid bilayer.

Scanning tunneling microscopy (STM), in conjunction with X-ray photoemission (XPS) and reflection-absorption infrared (RAIRS) spectroscopy, has been used to investigate the reaction of octahydridosilsesquioxane clusters (H8Si8O12) on the Si(1 0 0)-2×1 and Si(1 1 1)-7×7 surfaces. The clusters exhibit a markedly different reactivity upon exposure to the two clean silicon surfaces. STM data is presented that, in conjunction with XPS and RAIRS data, provides numerous constraints upon possible geometries for the chemisorbed clusters. The sum of the data is consistent with a dissociative reaction mechanism on Si(1 0 0)-2×1, resulting in cluster attachment to the surface via a single vertex. Conversely, data of Si(1 1 1)-7×7 subject to a saturation exposure of H8Si8O12 is presented that is highly suggestive of cluster decomposition on the surface.

Tri-(tert-butoxy)silanol (tBOS) rapidly forms a self-limited ∼10-Å-thick silicon oxide film upon exposure to a Si(100)-2×1 surface at 300 K. The majority of hydrocarbon spontaneously desorbs at this temperature. Heating to ∼700 K removes the remaining tert-butoxy groups. The films were characterized by conventional X-ray photoelectron spectroscopy (XPS), synchrotron XPS of the Si 2p core-level and valence band regions, and reflection absorption infrared spectroscopy (RAIRS).

Tissue cryo-sectioning combined with atomic force microscopy imaging reveals that the nanoscale morphology of dermal collagen fibrils, quantified using the metric of D-periodic spacing, changes under the condition of estrogen depletion. Specifically, a new subpopulation of fibrils with D-spacings in the region between 56 and 59 nm is present 2 years following ovariectomy in ovine dermal samples. In addition, the overall width of the distribution, both values above and below the mean, was found to be increased. The change in width due to an increase in lower values of D-spacings was previously reported for ovine bone; however, this report demonstrates that the effect is also present in non-mineralized collagen fibrils. A nonparametric Kolmogorov–Smirnov test of the cumulative density function indicates a statistical difference in the sham and OVX D-spacing distributions (P<0.01).

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype–phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen–ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.

•Estrogen depletion altered collagen fibril structure for cortical and trabecular bone in rabbits.•In rabbit cortical femur, estrogen depletion altered the formation of collagen bundles and sheets.•In trabecular lumbar vertebrae, estrogen depletion altered collagen fibril microstructure across a multimicron scale.The impact of estrogen depletion and drug treatment on type I collagen fibril nanomorphology and collagen fibril packing (microstructure) was evaluated by atomic force microscopy (AFM) using an ovariectomized (OVX) rabbit model of estrogen deficiency induced bone loss. Nine month-old New Zealand white female rabbits were treated as follows: sham-operated (Sham; n = 11), OVX + vehicle (OVX + Veh; n = 12), OVX + alendronate (ALN, 600 μg/kg/wk., s.c.; n = 12), and OVX + cathepsin-K inhibitor L-235 (CatKI, 10 mg/kg, daily, p.o.; n = 13) in prevention mode for 27 weeks. Samples from the cortical femur and trabecular lumbar vertebrae were polished, demineralized, and imaged using AFM. Auto-correlation of image patches was used to generate a vector field for each image that mathematically approximated the collagen fibril alignment. This vector field was used to compute an information-theoretic entropy that was employed as a quantitative fibril alignment parameter (FAP) to allow image-to-image and sample-to-sample comparison. For all samples, no change was observed in the average FAP values; however significant differences in the distribution of FAP values were observed. In particular, OVX + Veh lumbar vertebrae samples contained a tail of lower FAP values representing regions of greater fibril alignment. OVX + ALN treatment resulted in a FAP distribution with a tail indicating greater alignment for cortical femur and less alignment for trabecular lumbar vertebrae. OVX + CatKI treatment gave a distribution of FAP values with a tail indicating less alignment for cortical femur and no change for trabecular lumbar vertebrae. Fibril alignment was also evaluated by considering when a fibril was part of discrete bundles or sheets (classified as parallel) or not (classified as oblique). For this analysis, the percentage of parallel fibrils in cortical femur for the OVX group was 17% lower than the Sham group. OVX + ALN treatment partially prevented the proportion of parallel fibrils from decreasing and OVX + CatKI treatment completely prevented a change. In trabecular lumbar vertebrae, there was no difference in the percentage of parallel fibrils between Sham and any of the other treatment groups.