Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone

Abstract

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal "ghost" in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.

How interface size, density, and viscosity affect creep and relaxation functions of matrix-interface composites: a micromechanical study

“…which is consistent with the interface viscosity of η i = 1.83 × 10 12 GPa s m −1 as given in [22], and an interface radius of 50 nm, consistent with electron microscopic images [30]. In order to study interaction among two interface families exhibiting different interface sizes and viscosities, the corresponding product a 2 η i,2 , referring to the second interface family, is considered to be a multiple of the amount given in (108),…”

“…This expectation is supported by the theoretical derivation of Ponte Castañeda and Willis [28] that the Mori-Tanaka scheme actually considers spatial distributions of inhomogeneities following the same ellipsoidal shape as the inhomogeneities themselves. For material systems targeted at by our approach, including calcium silicate hydrates in concrete [29], extrafibrillar spaces in bone [30], or montmorillonite interlayers in clay [31], these spatial distributions appear as natural and appropriate. The remaining two state equations establish relations between dislocation vectors of the two interface families,…”

“…To this end, we consider the example of creep of bone stemming from viscous interfaces between hydroxyapatite crystals, slightly extending the discussion given in [14]. In this reference, needle-type bulk viscoelastic mineral phases were introduced as part of the porous polycrystal forming the extrafibrillar space of bone [30]-for further details on the mechanics of such porous polycrystals, we refer to [33]. Herein, we regard the viscoelasticity of these aforementioned mineral phases stemming from viscous interfaces being embedded, at a yet smaller scale, within the bulk crystal phases.…”

Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: Experimentally supported micromechanical explanation of bone strength

“…Such micromechanical models predict, on the basis of mechanical properties of bone elementary constituents (hydroxyapatite, collagen, water), the (poro-)elasticity tensors at the different hierarchical levels of the material, from tissue-specific composition data, such as porosities and mineral/collagen content. There-the explicit consideration of the extrafibrillar mineral crystals whose existence was evidenced earlier [Lees et al, 1984a, 1994, Prostak and Lees, 1996, Pidaparti et al, 1996, Benezra Rosen et al, 2002, and further confirmed by the kinetics of recent demineralization experiments [Balooch et al, 2008]. In this sense, the challenge of micromechanics-supported, consistently upscaled microstructure-property relationships for poroelasticity in bone has been met quite reasonably.…”

The Ultrastructure of Bone and Its Relevance to Mechanical Properties

“…Many previous TEM studies using microtome-cut sections of bone concluded that much of the mineral in bone resided in the gap zone between collinear triple helices of collagen (e.g., [27][28][29][30][31]). While there is a wide consensus that a large part of the mineral of bone must also lie outside the fibrils ( [32][33][34][35][36][37][38][39][40][41][42][43][44][45]), the detailed structure of this extrabrillar component was poorly known before the use of ion milling for preparation of TEM samples.…”