Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs in the body. Bone health is an integral part of overall health, but our lack of understanding of the ultrastructure of healthy bone precludes us from knowing how disease may impact nanoscale properties in this biological material. Here, we show that quantitative assessments of a distribution of Type I collagen fibril morphologies can be made using atomic force microscopy (AFM). We demonstrate that normal bone contains a distribution of collagen fibril morphologies and that changes in this distribution can be directly related to disease state. Specifically, by monitoring changes in the collagen fibril distribution of sham-operated and estrogen-depleted sheep, we have shown the ability to detect estrogen-deficiency-induced changes in Type I collagen in bone. This discovery provides new insight into the ultrastructure of bone as a tissue and the role of material structure in bone disease. The observation offers the possibility of a much-needed in vitro procedure to complement the current methods used to diagnose osteoporosis and other bone disease.
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 which 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.
This paper details a quantitative method to measure the D-periodic spacing of Type I collagen fibrils using Atomic Force Microscopy coupled with analysis using a 2D Fast Fourier Transform approach. Instrument calibration, data sampling and data analysis are all discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of Type I collagen morphology in disease models of estrogen depletion and Osteogenesis Imperfecta are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage Osteoporosis and Osteogenesis Imperfecta. The ability to quantitatively characterize nanoscale morphological features of Type I collagen fibrils will provide important structural information regarding Type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knock out studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments.
The success of high-speed atomic force microscopy in imaging molecular motors 1 , enzymes 2 and microbes 3 in liquid environments suggests that the technique could be of significant value in a variety of areas of nanotechnology. However, the majority of atomic force microscopy experiments are performed in air, and the tapping-mode detection speed of current highspeed cantilevers is an order of magnitude lower in air than in liquids. Traditional approaches to increasing the imaging rate of atomic force microscopy have involved reducing the size of the cantilever 4,5 , but further reductions in size will require a fundamental change in the detection method of the microscope [6][7][8] . Here, we show that high-speed imaging in air can instead be achieved by changing the cantilever material. We use cantilevers fabricated from polymers, which can mimic the high damping environment of liquids. With this approach, SU-8 polymer cantilevers are developed that have an imaging-in-air detection bandwidth that is 19 times faster than those of conventional cantilevers of similar size, resonance frequency and spring constant.A primary research goal in atomic force microscopy (AFM) is to increase the imaging speed, improve its ease of use and expand its potential range of applications 9 . In the most widely used AFM mode (a.c. mode or tapping mode) the detection speed (mechanical bandwidth, BW) of the AFM cantilever fundamentally limits the imaging speed. The bandwidth is a measure of the maximum rate of topography change the cantilever can accurately detect. It is related to the cantilever resonance as BW ∝ f 0 /Q, where the cantilever resonance frequency f 0 is primarily determined by the cantilever mass and elastic modulus, and the quality factor Q is determined by the cantilever damping 10 . When the oscillating cantilever experiences a change in boundary condition (that is, topography), it requires several cycles to reach a new steady-state amplitude (Fig. 1a). A cantilever with higher resonance frequency runs through the required number of cycles more quickly, thereby enabling faster imaging (Fig. 1b). The number of required oscillatory cycles is determined by the damping of the cantilever, characterized by Q. A cantilever with low resonance frequency and low Q can therefore be equally as fast as a cantilever with high resonance frequency and high Q (compare Fig. 1b and c). From a detection bandwidth perspective, the ideal combination is a high resonance frequency and low quality factor (Fig. 1d).The development of current high-speed AFM (HS-AFM) technology was enabled by the miniaturization of silicon and silicon nitride (SiN) cantilevers to x-y dimensions below 10 µm (the approach taken in Fig. 1b), resulting in cantilevers with megahertz resonance frequencies 4,5 . Modelling and an improved understanding of cantilever behaviour in fluids has greatly benefited this geometric optimization. For nearly all cantilevers, viscous damping in the surrounding medium determines Q (ref. 11). In liquid, viscous damping yields Q ≈ 2...
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