Atomic force microscopy imaging for nanoscale and microscale assessments of extracellular matrix in intervertebral disc and degeneration.

Degeneration of the intervertebral disc (IVD) is a condition that is often associated with debilitating back pain. There are no disease-modifying treatments available to halt the progression of this ubiquitous disorder. This is partly due to a lack of understanding of extracellular matrix (ECM) changes that occur at the micro- and nanometer size scales as the disease progresses. Over the past decade, atomic force microscopy (AFM) has been utilized as a tool to investigate the impact of disease on nanoscale structure of ECM in bone, skin, tendon, and dentin. We have expanded this methodology to include the IVD and report the first quantitative analysis of ECM structure at submicron size scales in a murine model for progressive IVD degeneration. Collagen D-spacing, a metric of nanoscale structure at the fibril level, was observed as a distribution of values with an overall average value of 62.5 ± 2.5 nm. In degenerative discs, the fibril D-spacing distribution shifted towards higher values in both the annulus fibrosus and nucleus pulposus (NP) (P < .05). A novel microstructural feature, collagen toroids, defined by a topographical pit enclosed by fibril-forming matrix was observed in the NP. With degeneration, these microstructures became more numerous and the morphology was altered from circular (aspect ratio 1.0 ± 0.1) to oval (aspect ratio 1.5 ± 0.4), P < .005. These analyses provide ECM structural details of the IVD at size scales that have historically been missing in studies of disc degeneration. Knowledge gained from these insights may aid the development of novel disease-modifying therapeutics.

Microstructure dependent binding of pigment epithelium derived factor (PEDF) to type I collagen fibrils.

Pigment epithelium derived factor (PEDF) is a multifunctional extracellular protein. In addition to its known anti-angiogenic and neurotrophic roles in collagen rich tissues, PEDF is thought to be involved in collagen fibril assembly due to its sequence specific binding to the collagen fibril and high expression in regions of active bone formation. In order to image the presence of the protein on the fibrils, PEDF was recombinantly made with a strep tag (strep-PEDF) and then gold nanoparticles conjugated to streptavidin (AuNP) were used as a secondary tag. The gold nanoparticles were detected using phase imaging in tapping mode AFM to image where exogenous PEDF bound in rabbit femur. These findings demonstrate that PEDF binds heterogeneously in cortical rabbit femur. Exogenous PEDF binding was concentrated at areas between microstructures with highly aligned collagen fibrils. Binding was not observed on or within the collagen fibrils themselves.

In vivo targeting of metastatic breast cancer via tumor vasculature-specific nano-graphene oxide.

Angiogenesis, i.e. the formation of neovasculatures, is a critical process during cancer initiation, progression, and metastasis. Targeting of angiogenic markers on the tumor vasculature can result in more efficient delivery of nanomaterials into tumor since no extravasation is required. Herein we demonstrated efficient targeting of breast cancer metastasis in an experimental murine model with nano-graphene oxide (GO), which was conjugated to a monoclonal antibody (mAb) against follicle-stimulating hormone receptor (FSHR). FSHR has been confirmed to be a highly selective tumor vasculature marker, which is abundant in both primary and metastatic tumors. These functionalized GO nano-conjugates had diameters of ∼120 nm based on atomic force microscopy (AFM), TEM, and dynamic laser scattering (DLS) measurement. (64)Cu was incorporated as a radiolabel which enabled the visualization of these GO conjugates by positron emission tomography (PET) imaging. Breast cancer lung metastasis model was established by intravenous injection of click beetle green luciferase-transfected MDA-MB-231 (denoted as cbgLuc-MDA-MB-231) breast cancer cells into female nude mice and the tumor growth was monitored by bioluminescence imaging (BLI). Systematic in vitro and in vivo studies have been performed to investigate the stability, targeting efficacy and specificity, and tissue distribution of GO conjugates. Flow cytometry and fluorescence microscopy examination confirmed the targeting specificity of FSHR-mAb attached GO conjugates against cellular FSHR. More potent and persistent uptake of (64)Cu-NOTA-GO-FSHR-mAb in cbgLuc-MDA-MB-231 nodules inside the lung was witnessed when compared with that of non-targeted GO conjugates ((64)Cu-NOTA-GO). Histology evaluation also confirmed the vasculature accumulation of GO-FSHR-mAb conjugates in tumor at early time points while they were non-specifically captured in liver and spleen. In addition, these GO conjugates can serve as good drug carriers with satisfactory drug loading capacity (e.g. for doxorubicin [DOX], 756 mg/g). Enhanced drug delivery efficiency in cbgLuc-MDA-MB-231 metastatic sites was demonstrated in DOX-loaded GO-FSHR-mAb by fluorescence imaging. This FSHR-targeted, GO-based nanoplatform can serve as a useful tool for early metastasis detection and targeted delivery of therapeutics.

Estrogen depletion and drug treatment alter the microstructure of type I collagen in bone.

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.

High-resolution NMR characterization of low abundance oligomers of amyloid-ß without purification.

Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-ß (Aß). The aggregation of Aß leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aß oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling (1)H-(1)H NMR experiments to overcome many of these limitations. Using (1)H-(1)H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aß1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time (1)H-(1)H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aß1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-ß-sheet fibrils.

Alteration of Type I collagen microstructure induced by estrogen depletion can be prevented with drug treatment.

Two independent biological replicates of estrogen depletion were employed with differing drug treatment conditions. Data Set I consisted of 9-month-old New Zealand white female rabbits treated as follows: sham-operated (n=11), ovariectomized (OVX; n=12), OVX+200 µg kg(-1) alendronate (ALN), 3 × a week for 27 weeks (n=12) and OVX+10 mg kg(-1) Cathepsin-K inhibitor (CatKI) daily for 27 weeks. Data Set II consisted of 6-month-old New Zealand white female rabbits that were sham-operated (n=12), OVX (n=12) or OVX+0.05 mg kg(-1) 17ß-estradiol (ERT) 3 × a week for 13 weeks (n=12). Samples from the cortical femur were polished and demineralized to make them suitable for atomic force microscopy (AFM) imaging. Type I collagen fibrils present in bundles or sheets, running parallel to each other, were combined into a class termed Parallel. Fibrils present outside of such structures, typically in images with an angular range of non-parallel fibrils, were combined into a class termed Oblique. The percentage of fibrils coded as Parallel for Sham animals in Data Sets I and II was 52% and 53%, respectively. The percentage of fibrils coded as Parallel for OVX animals in Data Sets I and II was 35% in both cases. ALN and ERT drug treatments reduced the change from 18 to 12%, whereas CatKI treatment reduced the change to 5%.