A blind spot in confocal reflection microscopy: the dependence of fiber brightness on fiber orientation in imaging biopolymer networks.

We investigate the dependence of fiber brightness on three-dimensional fiber orientation when imaging biopolymer networks with confocal reflection microscopy (CRM) and confocal fluorescence microscopy (CFM). We compare image data of fluorescently labeled type I collagen networks concurrently acquired using each imaging modality. For CRM, fiber brightness decreases for more vertically oriented fibers, leaving fibers above approximately 50 degrees from the imaging plane entirely undetected. As a result, the three-dimensional network structure appears aligned with the imaging plane. In contrast, CFM data exhibit little variation of fiber brightness with fiber angle, thus revealing an isotropic collagen network. Consequently, we find that CFM detects almost twice as many fibers as are visible with CRM, thereby yielding more complete structural information for three-dimensional fiber networks. We offer a simple explanation that predicts the detected fiber brightness as a function of fiber orientation in CRM.

Preventing distal pullout of posterior spine instrumentation in thoracic hyperkyphosis: a biomechanical analysis.

STUDY DESIGN: An in vitro biomechanical study. OBJECTIVE: Compare the mechanical behavior of 5 different constructs used to terminate dual-rod posterior spinal instrumentation in resisting forward flexion moment. SUMMARY OF BACKGROUND DATA: Failure of the distal fixation construct can be a significant problem for patients undergoing surgical treatment for thoracic hyperkyphosis. We hypothesize that augmenting distal pedicle screws with infralaminar hooks or sublaminar cables significantly increases the strength and stiffness of these constructs. METHODS: Thirty-seven thoracolumbar (T12 to L2) calf spines were implanted with 5 configurations of distal constructs: (1) infralaminar hooks, (2) sublaminar cables, (3) pedicle screws, (4) pedicle screws+infralaminar hooks, and (5) pedicle screws+sublaminar cables. Progressive bending moment was applied to each construct until failure. The mode of failure was noted and the construct's stiffness and failure load determined from the load-displacement curves. RESULTS: Bone density and vertebral dimensions were equivalent among the groups (F=0.1 to 0.9, P>0.05). One-way analysis of covariance (adjusted for differences in density and vertebral dimension) demonstrated that all of the screw-constructs (screw, screw+hook, and screw+cable) exhibited significantly higher stiffness and ultimate failure loads compared with either sublaminar hook or cable alone (P<0.05). The screw+hook constructs (109+/-11 Nm/mm) were significantly stiffer than either screws alone (88+/-17 Nm/mm) or screw+cable (98+/-13 Nm/mm) constructs, P<0.05. Screw+cable construct exhibited significantly higher failure load (1336+/-328 N) compared with screw constructs (1102+/-256 N, P<0.05), whereas not statistically different from the screw+hook construct (1220+/-75 N). The cable and hook constructs failed by laminar fracture, screw construct failed in uniaxial shear (pullout), whereas the screws+(hooks or wires) failed by fracture of caudal vertebral body. CONCLUSIONS: Posterior dual rod constructs fixed distally using pedicle screws were stiffer and stronger in resisting forward flexion compared with cables or hooks alone. Augmenting these screws with either infralaminar hooks or sublaminar cables provided additional resistance to failure.

Robust pore size analysis of filamentous networks from three-dimensional confocal microscopy.

We describe a robust method for determining morphological properties of filamentous biopolymer networks, such as collagen or other connective tissue matrices, from confocal microscopy image stacks. Morphological properties including pore size distributions and percolation thresholds are important for transport processes, e.g., particle diffusion or cell migration through the extracellular matrix. The method is applied to fluorescently labeled fiber networks prepared from rat-tail tendon and calf-skin collagen, at concentrations of 1.2, 1.6, and 2.4 mg/ml. The collagen fibers form an entangled and branched network. The medial axes, or skeletons, representing the collagen fibers are extracted from the image stack by threshold intensity segmentation and distance-ordered homotopic thinning. The size of the fluid pores as defined by the radii of largest spheres that fit into the cavities between the collagen fibers is derived from Euclidean distance maps and maximal covering radius transforms of the fluid phase. The size of the largest sphere that can traverse the fluid phase between the collagen fibers across the entire probe, called the percolation threshold, was computed for both horizontal and vertical directions. We demonstrate that by representing the fibers as the medial axis the derived morphological network properties are both robust against changes of the value of the segmentation threshold intensity and robust to problems associated with the point-spread function of the imaging system. We also provide empirical support for a recent claim that the percolation threshold of a fiber network is close to the fiber diameter for which the Euler index of the networks becomes zero.

An algorithm for extracting the network geometry of three-dimensional collagen gels.

The geometric structure of a biopolymer network impacts its mechanical and biological properties. In this paper, we develop an algorithm for extracting the network architecture of three-dimensional (3d) fluorescently labeled collagen gels, building on the initial work of Wu et al., (2003). Using artificially generated images, the network extraction algorithm is then validated for its ability to reconstruct the correct bulk properties of the network, including fiber length, persistence length, cross-link density, and shear modulus.

Using magnetic resonance microscopy to study the growth dynamics of a glioma spheroid in collagen I: A case study.

BACKGROUND: Highly malignant gliomas are characterized by rapid growth, extensive local tissue infiltration and the resulting overall dismal clinical outcome. Gaining any additional insights into the complex interaction between this aggressive brain tumor and its microenvironment is therefore critical. Currently, the standard imaging modalities to investigate the crucial interface between tumor growth and invasion in vitro are light and confocal laser scanning microscopy. While immensely useful in cell culture, integrating these modalities with this cancer's clinical imaging method of choice, i.e. MRI, is a non-trivial endeavour. However, this integration is necessary, should advanced computational modeling be able to utilize these in vitro data to eventually predict growth behaviour in vivo. We therefore argue that employing the same imaging modality for both the experimental setting and the clinical situation it represents should have significant value from a data integration perspective. In this case study, we have investigated the feasibility of using a specific form of MRI, i.e. magnetic resonance microscopy or MRM, to study the expansion dynamics of a multicellular tumor spheroid in a collagen type I gel. METHODS: An U87mEGFR human giloblastoma multicellular spheroid (MTS) containing approximately 4.103 cells was generated and pipetted into a collagen I gel. The sample was then imaged using a T2-weighted 3D spoiled gradient echo pulse sequence on a 14T MRI scanner over a period of 12 hours with a temporal resolution of 3 hours at room temperature. Standard histopathology was performed on the MRM sample, as well as on control samples. RESULTS: We were able to acquire three-dimensional MR images with a spatial resolution of 24 x 24 x 24 microm3. Our MRM data successfully documented the volumetric growth dynamics of an MTS in a collagen I gel over the 12-hour period. The histopathology results confirmed cell viability in the MRM sample, yet displayed distinct patterns of cell proliferation and invasion as compared to control. CONCLUSION: In this study, we demonstrate that a specific form of MRI, i.e. magnetic resonance microscopy or MRM, can be used to study the dynamic growth of a multicellular tumor spheroid (MTS) with a single cell scale spatial resolution that approaches the level of light microscopy. We argue that MRM can be employed as a complementary non-invasive tool to characterize microscopic MTS expansion, and thus, together with integrative computational modeling, may allow bridging of the experimental and clinical scales more readily.

The degenerative state of the intervertebral disk independently predicts the failure of human lumbar spine to high rate loading: an experimental study.

BACKGROUND: In the elderly, 30%-50% of patients report a fall event to precede the onset of vertebral fractures. The dynamic characteristics of the spine determine the peak forces on the vertebrae in a fall. However, we know little about the effect of intervertebral disk degeneration on the failure of human spines under the high loading rates associated with such falls. We hypothesized that MR estimates of disk hydration and viscoelastic properties will provide better estimates of failure strength than bone density alone. METHODS: Seventeen L1-L3 human spine segments were imaged (magnetic resonance imaging, dual-energy X-ray absorptiometry), their dynamic responses quantified using pendulum based impact, and the spines tested to failure under high rate loading simulating a fall event. The spines' stiffness and damping constants were computed (Kelvin-Voigt model) with disk hydration and geometry assessed from T2 and proton density images. FINDINGS: Under impact, the spines exhibited a second-order underdamped response with stiffness and damping ranging (17.9-754.5) kN/m and (133.6-905.3) Ns/m respectively. Damping, but not stiffness, was negatively correlated with higher ultimate strength (P<0.05). Higher bone mineral density and MR-estimated disk hydration correlated with higher ultimate strength (P<0.01 for both). No such correlations were observed for the T2 values. Adding disk hydration yielded a 20% increase in the model's association with failure load compared to bone density alone (MANOVA, P<0.001). INTERPRETATION: The strong correlation between disk viscoelastic properties and MR-estimated hydration with the spine segments' ultimate strength clearly demonstrates the need to include disk degeneration as part of fracture risk assessment in the elderly spine.

Biopolymer network geometries: characterization, regeneration, and elastic properties.

We study the geometry of biopolymer networks and effects of the geometry on bulk mechanical properties. It is shown numerically that the physical network geometry can be quantified statistically and regenerated from its statistical description, so that the regenerated network exhibits the same network mechanics as the physical network in the elastic regime. A collagen-I biopolymer network is used for validation. The method enables parametric studies of the network geometry, whose parameters are often difficult to vary independently in experiments.

Strain-induced alignment in collagen gels.

Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.