Three-dimensional structural interrelations between cells, extracellular matrix, and mineral in normally mineralizing avian leg tendon

Abstract

The basic mechanism by which vertebrate collagenous tissues are mineralized remains unclear, despite the importance of mineralization for skeletal formation and regeneration. The present study of normally mineralizing avian leg tendon, that presents structural, molecular biological, and biochemical features common to other collagenous vertebrate tissues, reveals previously unknown three-dimensional interrelationships between an extensive tendon lacuno-canalicular network, the extracellular matrix, and mineral deposits. These interrelationships suggest a mechanism where the lacuno-canalicular system facilitates the transport of mineral ions and possibly mineralization precursors. These ions and putative precursors are initially present in interfibrillar collagen spaces and subsequently translocated to neighboring collagen fibrils. Mineral particles then nucleate in association with collagen to form the well-known collagen–mineral composite material of the skeleton.The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.