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Visualizing molecular polar order in tissues via electromechanical coupling
2012-12, Denning, Denise, Alilat, Sofiane, Habelitz, S., Fertala, A., Rodriguez, Brian J.
Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides.
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Piezoelectric properties of aligned collagen membranes
2014-02, Denning, Denise, Paukshto, Michael V., Habelitz, S., Rodriguez, Brian J.
Electromechanical coupling, a phenomenon present in collagenous materials, may influence cell–extracellular matrix interactions. Here, electromechanical coupling has been investigated via piezoresponse force microscopy in transparent and opaque membranes consisting of helical-like arrays of aligned type I collagen fibrils self-assembled from acidic solution. Using atomic force microscopy, the transparent membrane was determined to contain fibrils having an average diameter of 76 ± 14 nm, whereas the opaque membrane comprised fibrils with an average diameter of 391 ± 99 nm. As the acidity of the membranes must be neutralized before they can serve as cell culture substrates, the structure and piezoelectric properties of the membranes were measured under ambient conditions before and after the neutralization process. A crimp structure (1.59 ± 0.37 µm in width) perpendicular to the fibril alignment became apparent in the transparent membrane when the pH was adjusted from acidic (pH = 2.5) to neutral (pH = 7) conditions. In addition, a 1.35-fold increase was observed in the amplitude of the shear piezoelectricity of the transparent membrane. The structure and piezoelectric properties of the opaque membrane were not significantly affected by the neutralization process. The results highlight the presence of an additional translational order in the transparent membrane in the direction perpendicular to the fibril alignment. The piezoelectric response of both membrane types was found to be an order of magnitude lower than that of collagen fibrils in rat tail tendon. This reduced response is attributed to less-ordered molecular assembly than is present in D-periodic collagen fibrils, as evidenced by the absence of D-periodicity in the membranes. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.
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Electromechanical properties of dried tendon and iso-electrically focused collagen hydrogels
2012-08, Denning, Denise, Abu-Rub, M. T., Zeugolis, Dimitrios I., Habelitz, S., Pandit, A., Fertala, A., Rodriguez, Brian J.
Assembling artificial collagenous tissues with structural, functional, and mechanical properties which mimic natural tissues is of vital importance for many tissue engineering applications. While the electro-mechanical properties of collagen are thought to play a role in, for example, bone formation and remodeling, this functional property has not been adequately addressed in engineered tissues. Here the electro-mechanical properties of rat tail tendon are compared with those of dried isoelectrically focused collagen hydrogels using piezoresponse force microscopy under ambient conditions. In both the natural tissue and the engineered hydrogel D-periodic type I collagen fibrils are observed, which exhibit shear piezoelectricity. While both tissues also exhibit fibrils with parallel orientations, Fourier transform analysis has revealed that the degree of parallel alignment of the fibrils in the tendon is three times that of the dried hydrogel. The results obtained demonstrate that isoelectrically focused collagen has similar structural and electro-mechanical properties to that of tendon, which is relevant for tissue engineering applications.