Ultrastructural study of the three-dimensional tenocyte network in newly hatched chick Achilles tendons using serial block face-scanning electron microscopy

Abstract

The lateral cytoplasmic processes of tenocytes extend to form three-dimensional network surrounding collagen fibers. It is unknown whether connections between two cytoplasmic processes involve overlapping of the processes or merely surface contact. In this study, the two-dimensional and three-dimensional structure of tenocytes in the Achilles tendons of the newly hatched chicks were studied using transmission electron microscopy and serial block face-scanning electron microscopy. Observation of the two-dimensional structures revealed various forms of cellular connections, including connections between the cytoplasmic processes of adjacent tenocytes and between the cytoplasmic process of tenocytes and fibroblasts. Three-dimensional observation showed spike-like cytoplasmic processes extending from one tenocyte that interlocked with cytoplasmic processes from other tenocytes. Cytoplasmic processes from each tenocyte within the chick tendons interlocked to ensure a tight cell-to-cell connection around growing collagen fibers. A cellular network formed by these cytoplasmic processes surrounds each collagen fiber. Cell-cell junctions, which were suggested to be gap junctions, observed at sites of cytoplasmic process overlap most likely represent the major route for communication between tenocytes associated with fibroblasts, enabling vital signals important for maintaining the cell and tendon integrity to be transmitted.

Keywords: Achilles tendon; chick; serial block face-scanning electron microscope; tenocyte; three-dimensional.

Fig. 1.

Diagram showing the organization of a collagen fiber bundle. Tenocyte cytoplasmic processes extend around a group of collagen fibrils forming a collagen fiber. A collagen fiber bundle comprises a group of collagen fibers surrounded by the endotendineum, which is a sheath of connective tissue embedded with fibroblasts.

Fig. 2.

Transmission electron microscope images of the tendon. A: Tenocytes (blue) are present within each collagen fiber bundle, which is surrounded by the endotendineum (yellow dotted line), but fibroblasts (red) remain on the outside of the bundle. B: The cytoplasmic processes of most tenocytes stretch to form networks surrounding collagen fibers (white arrowheads). Some tenocyte cytoplasmic processes extend to connect with fibroblasts (white arrow). C: Most collagen fibers (*) are surrounded by a network of cytoplasmic processes from three tenocytes (Tc). D: Magnified image of the box in C. Electron density is intense at sites of overlapping processes (black arrow).

Fig. 3.

Topographical images of collagen fiber bundles at the beginning (A), middle (B) and end (C) of the cut tendon. A group of collagen fibers is gathered together to form a collagen fiber bundle, which is enclosed within the endotendineum (yellow dotted line). The cytoplasmic processes of the tenocytes (†) arrayed longitudinally along the collagen fibers extend to form a three-dimensional network around the collagen fibers. Fibroblasts located outside the collagen fiber bundle are also arrayed along the length of the collagen fiber bundle. D: A connection between the cytoplasmic process of tenocyte (blue dotted line) and a fibroblast (red dotted line) can be seen in the magnified box in C.

Fig. 4.

Three-dimensional reconstruction of tenocytes delineated with different colors. Networks formed by the cytoplasmic processes of all tenocytes surrounding each collagen fiber were clearly observed on a horizontal plane (A). Tenocytes with lateral non-continuous spike-like cytoplasmic processes were arranged in tandem along the length of collagen fibers (B, C). The vertical slice section of C shows gaps between the ends of tenocytes (arrows, D). Differently sized and shaped processes extended to fill the recesses left by the processes extending from the adjacent tenocyte, to form an interlocking structure (E). Primary processes (arrows) and secondary processes (arrowheads) can be seen in the magnified box in E (F). However, these processes were not observed at the terminal ends of these cells, where instead intercellular gaps were clearly seen (D).