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Farnum CE, Williams RW, and Donnelly E. (2009) Analyzing primary cilia by multiphoton microscopy. Method Cell Biol 94: 117-135.

DOI

Abstract

In this chapter, a technique is outlined for the use of immunohistochemistry (IHC) followed by multiphoton microscopy (MPM) for the analysis of incidence, length, and 3D orientation of the axoneme of the primary cilium. Although the application presented specifically emphasizes localizations in tenocytes and chondrocytes, the technique is applicable to cells in a wide range of connective tissues. The primary advantages of utilizing MPM as opposed to TEM for these kinds of ciliary analyses are the rapidity of the technique for preparation of the samples and the ability to collect data from multiple cells simultaneously. Using MPM, the axoneme, basal body, and associated centriole can be visualized by specific IHC with localizing antibodies. However, the resolution achieved through TEM analyses allows the complex morphology of the primary cilium to be visualized, and this remains the primary advantage of TEM versus MPM. SHG, which occurs only with MPM, allows visualization of collagen fibrils and is particularly advantageous for localizing primary cilia associated with cells in connective tissues. This, and the deep penetration with less photobleaching, are the primary advantages of MPM compared to confocal microscopy. As with any microscopical technique, the protocol needs to be optimized for any given tissue. In particular, additional antigen retrieval techniques to enhance the unmasking of specific epitopes for antibody binding may be required for adaptation of this approach to other dense connective tissues with complex spatial organizations such as intervertebral disc or meniscus.

Keywords

Donnelly E, Ascenzi M-G, and Farnum CE. (2010) Primary cilia are highly oriented with respect to collagen direction and long axis of extensor tendon. J Orthop Res 28: 77-82.

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Abstract

Skeletal tissues adapt to their mechanical environments by modulating gene expression, cell metabolism, and extracellular matrix (ECM) architecture; however, the mechanosensory mechanisms for these processes are incompletely understood. Primary cilia have emerged as critical components of the cellular mechanosensory apparatus and have been hypothesized to participate in establishment of cellular and ECM orientation, but their function in skeletal tissues is just beginning to be examined. Here we focused on tendon, a tissue with an oriented matrix that is ideal for analysis of spatial relationships between primary cilia and the ECM. The objective of this study was to characterize the incidence and orientation of tenocyte primary cilia in their native ECM. Primary cilia, nuclei, and collagen were analyzed three-dimensionally in immunofluorescently labeled rat extensor tendon using multiphoton microscopy and semiautomated morphometry. Primary cilia were observed in 64% of tenocytes. The cilia were highly oriented with respect to the ECM: cilia were aligned parallel to the collagen fibers and the long axis of the tendon. This study represents the first quantification of the in situ incidence and orientation of primary cilia in tendon. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:77–82, 2010

Keywords

primary cilia; tendon; tenocyte; collagen; multiphoton microscopy

Donnelly E, Boskey AL, Baker SP, and van der Meulen MCH. (2010) Effects of tissue age on bone tissue material composition and nanomechanical properties in the rat cortex. J Biomed Mater Res 92A: 1048-1056.

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Abstract

Although osteoporosis is known to alter bone tissue composition, the effects of such compositional changes on tissue material properties have not yet been examined. The natural gradient in tissue mineral content arising from skeletal appositional growth provides a basic model for investigation of relationships between tissue composition and mechanical properties. The purpose of this study was to examine the effects of tissue age on bone tissue composition and nanomechanical properties. The nanomechanical properties and composition of regions of differing tissue age were characterized in the femoral cortices of growing rats using nanoindentation and Raman spectroscopy. In addition, spatial maps of the properties of periosteal tissue were examined to investigate in detail the spatial gradients in the properties of newly formed tissue. Newly formed tissue (0–4 days) was 84% less stiff and had 79% lower mineral:matrix ratio than older intracortical (15–70 days) tissue. Tissue modulus, hardness, mineral:matrix ratio, and carbonate:phosphate ratio increased sharply with distance from the periosteum and attained the properties of intracortical tissue within 4 days of formation. The mineral: matrix ratio explained 54% and 62% of the variation in tissue indentation modulus and hardness, respectively. Our data demonstrate significant variations in tissue mechanical properties with tissue age and relate mechanical properties to composition at the microscale. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010

Keywords

bone mineralization; mechanical properties; nanoindentation; Raman spectroscopy; rat

Donnelly E, Williams RW, and Farnum CE. (2008) The primary cilium of connective tissue cells: Imaging by multiphoton microscopy. Anat Rec 291: 1062-1073.

DOI

Abstract

Although the role of the primary cilium as a sensory organelle in epithelial cells has been elucidated significantly over the past decade, the function of primary cilia in connective tissue cells has been studied less extensively. Primary cilia have been implicated as mechanotransducers in connective tissues, but the mechanisms by which the cells sense loads and convert them to biochemical signals for tissue formation and adaptation are poorly understood. Before hypotheses regarding the function of the primary cilium in connective tissue cells can be tested, methods for quantitation of incidence as well as three-dimensional visualization of primary cilia with respect to the extracellular matrix (ECM) are needed. The objective of this study was to develop a rapid method for visualizing primary cilia in their native ECM in a wide range of connective tissues. Whole-mount immunohistochemical and multiphoton microscopy techniques were developed to simultaneously image primary cilia, cell nuclei, and collagen and their relationships to each other in situ. Axonemes of primary cilia projecting into the ECM were successfully visualized in thick sections of growth plate cartilage, tendon, ligament, meniscus, intervertebral disc, and perichondrium. These methodologies will allow analysis of the incidence and three-dimensional orientation of primary cilia and enable investigation of the role of primary cilia in normal and pathological growth and adaptation in a variety of musculoskeletal tissues. Anat Rec, 291:1062–1073, 2008. © 2008 Wiley-Liss, Inc.

Keywords

Small cracks; Biaxial stress; Fatigue crack growth

Fig 7 from The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology
Volume 291, Issue 9, pages 1062-1073, 25 AUG 2008 DOI: 10.1002/ar.20665
http://onlinelibrary.wiley.com/doi/10.1002/ar.20665/full#fig7

Donnelly E, Williams RW, Downs SA, Dickinson ME, Baker SP, and van der Meulen MCH. (2006) Quasistatic and dynamic nanomechanical properties of cancellous bone tissue relate to collagen content and organization. J Mater Res 21: 2106-2117.

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Abstract

Cancellous bone plays a crucial structural role in the skeleton, yet little is known about the microstructure-mechanical property relationships of the tissue at the microscale. Cancellous tissue is characterized by a microstructure consisting of layers interspaced with transition zones with different proportions of collagen and mineral. In this study, the quasistatic and dynamic mechanical properties of lamellar and interlamellar tissue in human vertebrae were assessed with nanoindentation, and the collagen content and organization were characterized with second harmonic generation microscopy. Lamellar tissue was 35% stiffer, 25% harder, and had a 13% lower loss tangent relative to interlamellar tissue. The stiff, hard lamellae corresponded to areas of highly ordered, collagen-rich material, with a relatively low loss tangent, whereas the compliant, soft interlamellar regions corresponded to areas of disordered or collagen-poor material. These data suggest an important role for collagen in the tissue-level mechanical properties of bone.

Keywords

Bone; Hardness; Microstructure

Donnelly E, Baker SP, Boskey AL, and van der Meulen MCH. (2006) Effects of surface roughness and maximum load on the mechanical properties of cancellous bone measured by nanoindentation. J Biomed Mater Res 77A: 426-435.

DOI

Abstract

The effects of two key experimental parameters on the measured nanomechanical properties of lamellar and interlamellar tissue were examined in dehydrated rabbit cancellous bone. An anhydrous sample preparation protocol was developed to maintain surface integrity and produce RMS surface roughnesses ∼10 nm (5 × 5-μm2 area). The effects of surface roughness and maximum nanoindentation load on the measured mechanical properties were examined in two samples of differing surface roughness using maximum loads ranging from 250 to 3000 μN. As the ratio of indentation depth to surface roughness decreased below ∼3:1, the variability in material properties increased substantially. At low loads, the indentation modulus of the lamellar bone was ∼20% greater than that of the interlamellar bone, while at high loads the measured properties of both layers converged to an intermediate value. Relatively shallow indentations made on smooth surfaces revealed significant differences in the properties of lamellar and interlamellar bone that support microstructural observations that lamellar bone is more mineralized than interlamellar bone. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

Keywords

cancellous bone; nanoindentation; mechanical properties; surface roughness; sample preparation

Donnelly E and Nelson D. (2002) A study of small crack growth in aluminum alloy 7075-T6 Int J Fatigue 24: 1175-1189.

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Abstract

Previous studies of the growth of small fatigue cracks in aluminum alloy 7075-T6 are reviewed to compare observed behavior and explanations for that behavior. The results of small crack growth tests conducted in this work are then reported. Crack growth was monitored periodically between initial crack sizes of approximately 10 μm to final sizes of approximately 0.5 mm in rotating bending and plate specimens subjected to fully-reversed cycling. Both longitudinal and transverse stress components were present in the central region of the plate specimens, while rotating bending specimens experienced nearly uniaxial cyclic stress. Specimens were taken from rolled 7075-T651 with a pancake microstructure. Tests were conducted at a number of different strain amplitudes. Small cracks grew faster than large cracks for the same stress intensity range ΔK, as expected from previous research by others. When compared on the basis of ΔK, growth rates in the plate specimens varied from being little different than those in rotating bending specimens to approximately four times higher, depending on strain amplitude. The growth rates of cracks at different locations around the circumference of the rotating bending specimens varied little, in spite of the different microstructural orientations of the different locations. In both plate and rotating bending tests, some cracks exhibited marked decelerations and accelerations in growth rate, while others did not. For those cracks that did not exhibit such oscillations in growth rate plus those cracks that had grown large enough for such oscillations to have subsided, growth rates were found to be proportional to crack size and to amplitude of maximum shear strain to a power of approximately 4.5. Growth rate data from both rotating bending and plate specimens were well correlated by that strain parameter.

Keywords

Small cracks; Biaxial stress; Fatigue crack growth