The assembly, deposition and organization of collagen fibril bundles and their composite fibrils were studied during morphogenesis of the chick embryo tendon using electron microscopy, serial sections and computer-assisted three-dimensional reconstruction techniques. The 14-day chick embryo is a stage when tendon architecture is being established and rapid changes in the mechanical properties occur between days 14 and 17 of development. Tendon matrix structure develops from discrete sub-units, bundles of collagen fibrils. The bundles branch; undergo a gradual rotation over several micrometers; are intimately associated with the cellular elements of the developing tendon; and form arborizing networks within and among fascicles. The organization of discrete fibril segments into bundles, during the establishment of tendon architecture and function, where the segmental fibrillar components could interact with the interfibrillar matrix as well as with adjacent fibrils would contribute to the stabilization of this structure. The observed gradual rotation of the bundles would serve to stabilize the immature bundle through the physical twining of the composite fibrils while the extensive branching of the bundles observed at 14-days of development and their intimate association with the cellular elements would provide a higher order of structure stabilization.

Tendons transmit force from the muscular to the skeletal system and are composed primarily of fibro-blasts, type I collagen fibrils and a proteoglycan-rich interfibrillar matrix. Tendons are composed of highly aligned collagen fibrils organized into bundles. The fibril bundles (collagen fiber) together with the tendon fibroblasts are organized into fascicles, and the fascicles are bound together in a connective tissue sheath to form a tendon (Elliot, 1965; Greenlee and Ross, 1967; Greenlee et al. 1975; Kastelic et al. 1978; Davison, 1982; Squier and Magnes, 1983; Squier and Bausch, 1984; Parry and Craig, 1984; Baer et al. 1988). The systematic development of this hierarchy is required for structural integrity and normal function.

The mechanical properties of tendon are dependent on a number of factors including fibril and bundle orientation; fibril diameter; and fibril length. Recently, we have shown that in the 14-day chick embryo tendon, collagen fibrils are deposited as discrete fibril segments approximately 10 micrometers in length (Birk et al. 1989a). In contrast, collagen fibrils in mature tendons have been shown to be at least millimeters in length using a statistical approach (Parry and Craig, 1984; Trotter and Wolfsy, 1989). We have proposed that fibril segments are precursors in collagen fibril formation in the embryonic chick tendon and ‘segmental’ deposition and post-depositional rearrangements are important steps in the development of mechanical integrity.

Collagen fibrillogenesis is a multistep process involving both intracellular and extracellular compartments (Trelstad, 1982; Birk and Trelstad, 1986). Collagen synthesis, molecular assembly and formation of supra-molecular aggregates occurs within a series of well-defined cytoplasmic compartments (Trelstad and Hayashi, 1979). In tendon morphogenesis, a hierarchy of extracytoplasmic compartments establishes at least three different levels of matrix organization: collagen fibrils, bundles and tissue specific macroaggregates, e.g. the large bundles in tendon; the orthogonal bundles in cornea or bone. The cellular control of local factors within these highly partitioned environments is important in the regulation of local functions within the various extracellular domains.

In 14-day chick embryos, fibril segments are assembled in narrow extracytoplasmic channels defined by the fibroblast (Birk and Trelstad, 1984; 1986; Birk et al. 1989a). The segments are then assimilated into bundles and the bundles are incorporated into the developing matrix. Within the bundle the fibril segments are coupled with other fibril segments into a functionally continuous fibril (Birk et al. 1989b). The addition of fibril segments to bundles and the post-depositional fusion, maturation and rearrangement of these fibril and bundle segments are important processes during tendon development and growth.

In the present study, the development and organization of collagen bundles and their constituent fibrils were studied during morphogenesis of the chick embryo tendon. The 14-day chick embryo is a stage when tendon micro- and macro-architecture are being established and rapid changes in mechanical properties occur. This work describes how tendon matrix structure develops from discrete subunits and addresses the relationship between fibrillar architecture, bundle structure and the tissue’s mechanical properties.

White Leghorn chick embryos were incubated in a humidified atmosphere and staged according to Hamburger and Hamilton (1951). Chick embryo limbs were fixed in situ at stage40 (day 14) and stage 43 (day 17). Fixation was in 4% parafor-maldehyde, 2.5 % glutaraldehyde, in 0.1 M-sodium cacodylate pH7.4 with 8mM-CaCl2 for 15 min at room temperature followed by 40 min at 4°C. During fixation, the metatarsal tendons were dissected, and then washed in 0.1 M-cacodylate buffer pH 7.4 followed by post-fixation with 1% OsO4 in cacodylate buffer pH 7.4 for one hour at 4°C. The tissues were washed in buffer and dehydrated through a cold graded ethanol series followed by propylene oxide. The tendons were infiltrated, embedded in a fresh mixture of Polybed 812, nadie methyl anhydride, dodecenyl succinic anhydride and DMP-30 (Polysciences, Inc., Warrington, PA), polymerized and sectioned (Birk and Trelstad, 1984; 1986).

Serial sections (0.5 to 0.75 μm) of tendon were cut perpendicular to the tendon axis using a diamond knife. Sections were picked up onto formvar-coated 1×2mm slot grids and were stained with 2% aqueous uranyl acetate for 45 min, followed by 0.2 % lead citrate in 0.1 N-NaOH for 30min. The stained sections were stabilized by the evaporation of a thin layer of carbon, examined and photographed at 1000 kV using the AEI EM7 high voltage electron microscope at the New York State Department of Health Laboratories in Albany, New York (Birk and Trelstad, 1986). Additional thin sections (90 to 150 nm) were cut and stained with 2 % aqueous uranyl acetate for 10min, followed by 0.2% lead citrate in 0.1 N-NaOH for 5 min and examined using either a Philips 420 or JEOL 1200EX transmission electron microscope.

The serial thick sections were photographed as a montage containing 4 complete fascicles and portions of several others. Data from 2 different fascicles were collected for over 30 μm of tendon and portions of others were analyzed in detail. In addition, we have analyzed at least 10 other 14-day chick embryo metatarsal tendons in serial thick (0.5 to 0.75 μm) and thin (100 to 250 nm) sections over 5 to 100 μm.

Tendon fascicles were followed in 36 consecutive serial sections and computer-generated, graphic three-dimensional renderings were produced using MOVIE.BYU (Department of Civil Engineering, Brigham Young University, Provo, Utah). Areas of interest were identified in photographic prints and the appropriate profiles were digitized using DRAW.MGH (Department of Pathology, Massachusetts General Hospital, Boston, MA). The sections were aligned using a vertically trimmed edge of the section and a distinct knife mark as internal markers. The nuclei, cell outlines and bundles were contoured and the contours of interest were transferred to MOVIE.BYU and three-dimensional shaded renderings produced. The images were displayed on a Lexi-data LEX 90/35 high resolution graphics device interfaced with a DEC Micro VAX II computer for analysis. Photographs were taken using a Focus Graphics Imagerecorder.

Tendons are composed of discrete units called fascicles. Fascicles are composed of fibroblasts and their associated bundles of collagen fibrils. Bundles of collagen fibrils, not individual collagen fibrils, are the predominant extracellular structures observed. Fibril bundles (fibers) are discrete collections of collagen fibrils which form within specific fibroblast-defined compartments. The three-dimensional relationships among cells as well as the cells and collagen fibril bundles within a single fascicle of the 14-day and 17-day chick embryo tendon are illustrated in Fig. 1. At both stages of development, collagen bundles are the major extracellular structures. During development from 14 to 17 days, fascicles become better defined. At 14 days of development there is a poorly defined interfascicular matrix while by 17 days of development the fibroblasts of the endotendinium and their associated matrix, arranged roughly perpendicular to the fascicular fibroblasts, separate and define adjacent fascicles. Also as development proceeds from 14 to 17 days the cell-to-matrix ratio decreases and the fibroblasts become more attenuated. Concurrently, the long, slender processes separating bundles presumably retract and the small bundles coalesce to form larger ones characteristic of the mature tendon.

Fig. 1.

Relationship among fibroblasts and bundles in a tendon fascicle. Transmission electron micrographs of sections (150 to 200 nm) cut perpendicular to the axis of 14-day (A–B) and 17-day (C–D) chick embryo tendons are presented. At 14 days of tendon development the fibroblasts have become organized into fascicles (A), however, the boundaries between adjacent fascicles are not well defined. By 17 days of development, the boundaries of the fascicle are readily identifiable (C), with fibroblasts and matrix oriented perpendicular to the tendon axis separating adjacent fascicles. At both stages of development, the compartmentalization of the extracellular space is readily apparent and bundles of collagen fibrils within clearly defined compartments are the major extracellular structure. Fibril forming channels (arrows) containing single or small groups of fibrils and bundle forming compartments (B) containing fibril bundles are numerous and easily identifiable at both stages of development. The fibroblast-to-cell ratio decreases as development proceeds (B vs. D). Presumably, the slender cytoplasmic processes separating bundle-forming compartments retract (curved arrows) and the bundles coalesce to form large bundles characteristic of the mature tendon. Bar, 1.0μm.

Fig. 1.

Relationship among fibroblasts and bundles in a tendon fascicle. Transmission electron micrographs of sections (150 to 200 nm) cut perpendicular to the axis of 14-day (A–B) and 17-day (C–D) chick embryo tendons are presented. At 14 days of tendon development the fibroblasts have become organized into fascicles (A), however, the boundaries between adjacent fascicles are not well defined. By 17 days of development, the boundaries of the fascicle are readily identifiable (C), with fibroblasts and matrix oriented perpendicular to the tendon axis separating adjacent fascicles. At both stages of development, the compartmentalization of the extracellular space is readily apparent and bundles of collagen fibrils within clearly defined compartments are the major extracellular structure. Fibril forming channels (arrows) containing single or small groups of fibrils and bundle forming compartments (B) containing fibril bundles are numerous and easily identifiable at both stages of development. The fibroblast-to-cell ratio decreases as development proceeds (B vs. D). Presumably, the slender cytoplasmic processes separating bundle-forming compartments retract (curved arrows) and the bundles coalesce to form large bundles characteristic of the mature tendon. Bar, 1.0μm.

The fibroblast–bundle relationship in the 14-day chick embryo tendon is complex. Collagen bundles branch to form an anastomosing bundle network within and among fascicles. In Fig. 2, a single bundle is followed in a series of high voltage electron micro-graphs from thick sections (0.5 to 0.75 μm) cut perpendicular to the axis of the tendon. In this series of consecutive micrographs, the branching of bundles within a tendon fascicle is illustrated with a small bundle separating from a larger bundle to join an entirely different bundle.

Fig. 2.

Bundle branching. A single bundle is followed in this series of serial high-voltage electron micrographs. Thick sections (0.5 to 0.75 μm) cut perpendicular to the axis of 14-day chick embryo tendons are presented as a series of consecutive micrographs. A collagen bundle (arrowhead) is followed from its initial appearance as part of a larger bundle (section no. 1) to its incorporation into an entirely different bundle (section no. 29). In sections 4 through 25 this small bundle is distinct. However, in following its course, it is clear that this is a branch connecting two distinct bundles. When the fibril content of this small bundle was determined in section numbers 12–15 and 21–22, where the fibrils were easily counted, the bundles were found to be discontinuous in fibril number from section to section, indicating that some of the collagen fibrils terminated within this portion of the bundle. Also, the bundles were found to rotate, the small bundle indicated rotated approximately 180° over a 10 to 12 μm distance. Bar, 500 nm.

Fig. 2.

Bundle branching. A single bundle is followed in this series of serial high-voltage electron micrographs. Thick sections (0.5 to 0.75 μm) cut perpendicular to the axis of 14-day chick embryo tendons are presented as a series of consecutive micrographs. A collagen bundle (arrowhead) is followed from its initial appearance as part of a larger bundle (section no. 1) to its incorporation into an entirely different bundle (section no. 29). In sections 4 through 25 this small bundle is distinct. However, in following its course, it is clear that this is a branch connecting two distinct bundles. When the fibril content of this small bundle was determined in section numbers 12–15 and 21–22, where the fibrils were easily counted, the bundles were found to be discontinuous in fibril number from section to section, indicating that some of the collagen fibrils terminated within this portion of the bundle. Also, the bundles were found to rotate, the small bundle indicated rotated approximately 180° over a 10 to 12 μm distance. Bar, 500 nm.

The collagen fibrils within these branching bundles have discrete lengths. When the fibril content of the small bundle in Fig. 2 was determined in sections no. 12–15 and no. 21–22, where the fibrils were easily counted, the bundles were found to be discontinuous in fibril number from section to section, indicating that it is composed of collagen fibrils that terminate at different levels. Also, the bundles were found to rotate approximately 180° over a 10 to 12 μm distance relative to internal markers.

The relationship of bundles to one another and to the fibroblasts within a fascicle were studied further from a 14-day chick embryo tendon using computer-assisted three-dimensional reconstructions. Serial 0.5 μm sections were cut perpendicular to the tendon axis and photographed using the high-voltage electron micro-scope. The profiles of the nucleus, major portions of the cell and bundles were digitized for two complete fibroblasts and portions of two others within a fascicle. In Fig. 3A, one of these micrographs is presented and three nuclei (I, II, III) are indicated. These cells and their associated fibril bundles were chosen for reconstruction. In Fig. 3B the profiles of the nuclei, cells and bundles are shown with the same orientation as in Fig. 3A.

Fig. 3.

Collagen bundles were studied within a single fascicle from a 14-day chick embryo tendon using computer-assisted three-dimensional reconstructions. Serial 0.5 μm thick sections cut perpendicular to the tendon axis were examined and photographed using the high-voltage electron microscope. In A, one of these micrographs is presented. The nuclei of three fibroblasts are indicated (I,II,III). The cells, nuclei and bundles (not individual fibrils) were hand digitized for each section through 18 consecutive micrometers (36 sections). The sections were oriented using a vertically trimmed edge (not shown) and a distinct knife mark (seen across the bottom of A). In B, the profiles from A are presented, the nuclei are labelled (I,II,III), the cells are clear and the bundles are stippled. The same bundle (B) and portion of a cell (C) is labelled in both panels A and B. Bar, 2.0 μm.

Fig. 3.

Collagen bundles were studied within a single fascicle from a 14-day chick embryo tendon using computer-assisted three-dimensional reconstructions. Serial 0.5 μm thick sections cut perpendicular to the tendon axis were examined and photographed using the high-voltage electron microscope. In A, one of these micrographs is presented. The nuclei of three fibroblasts are indicated (I,II,III). The cells, nuclei and bundles (not individual fibrils) were hand digitized for each section through 18 consecutive micrometers (36 sections). The sections were oriented using a vertically trimmed edge (not shown) and a distinct knife mark (seen across the bottom of A). In B, the profiles from A are presented, the nuclei are labelled (I,II,III), the cells are clear and the bundles are stippled. The same bundle (B) and portion of a cell (C) is labelled in both panels A and B. Bar, 2.0 μm.

Approximately 18 μm of tendon from 36 consecutive, aligned sections, digitized as in Fig. 3B, were reconstructed into a three-dimensional image for analysis of bundle and cell relationships using computer-assisted reconstruction techniques. Fig. 4AC is a presentation of the component parts of this reconstruction. Three-dimensional renderings were produced of the nuclei, cells and bundles. This reconstruction is presented with a perpendicular axial orientation (it is a longitudinal representation of the tendon built from sections cut perpendicular to the tendon axis), but has the same rotational orientation as in Fig. 3. The nuclei (A), fibril bundles (B), and cells (C) are reconstructed from over 18 μm of tissue and in Fig. 4D the entire reconstruction is presented with the same orientation as in A–C, showing the three-dimensional relationships of the nuclei (red), bundles (green), and cells (blue, partially transparent) within a single tendon fascicle throughout 18 μm of tissue.

Fig. 4.

Three-dimensional reconstructions of a 14-day chick embryo tendon fascicle. The data described in Fig. 3 were reconstructed using 36 serial sections with the graphics routines available with MOVIE.BYU. Shaded renderings of the reconstructed (A) nuclei (red); the (B) bundles (green); and (C) cells (blue) were reconstructed from sections cut perpendicular to the tendon axis and are presented with a longitudinal orientation. The nuclei are labelled as in Fig. 3. The section shown in Fig. 3 is at the level indicated by the arrow. In D, the three parts were rebuilt and displayed with the same orientation and colors as in A–C. However, the blue cells were made partially transparent to permit the viewing of some deeper structures. The section shown in Fig. 3 is at the level indicated by the arrow and the position of the * corresponds to that in panel B. Bars, 2.0 μm.

Fig. 4.

Three-dimensional reconstructions of a 14-day chick embryo tendon fascicle. The data described in Fig. 3 were reconstructed using 36 serial sections with the graphics routines available with MOVIE.BYU. Shaded renderings of the reconstructed (A) nuclei (red); the (B) bundles (green); and (C) cells (blue) were reconstructed from sections cut perpendicular to the tendon axis and are presented with a longitudinal orientation. The nuclei are labelled as in Fig. 3. The section shown in Fig. 3 is at the level indicated by the arrow. In D, the three parts were rebuilt and displayed with the same orientation and colors as in A–C. However, the blue cells were made partially transparent to permit the viewing of some deeper structures. The section shown in Fig. 3 is at the level indicated by the arrow and the position of the * corresponds to that in panel B. Bars, 2.0 μm.

The reconstructed tendon fascicle shown in Fig. 4 was studied further after being manipulated using the graphics routines available with MOVIE.BYU. In Fig. 5, the reconstruction is sectioned and the top half removed to reveal internal detail of the fascicle. These graphic reconstructions demonstrate the complexity of the bundle architecture within a fascicle. Bundles are seen to branch, bifurcate and rotate about their long axes. The bundles arborize with branching of bundles, subbranches connecting adjacent branches and fusion of branches with other bundles.

Fig. 5.

Bundle branching. Shaded renderings of the reconstructed (A–D) nuclei (red); the (A–D) bundles (green), and (A,C) cells (transparent blue) are presented after being manipulated using the computer. In A and B, the fascicle was sectioned at 30°, rotated −105° from the position in Fig. 4, and the top half removed. Note the branching (arrow) and fusion of the bundles as well as their twisting. In C and D, the fascicle was sectioned at 30°, rotated 80° from the position in Fig. 4, and the top half removed. Again, note the branching (arrow) and fusion of the bundles as well as their twisting. In panels A and C the nuclei are labelled as in Figs 3 and 4. Bars, 2.0μm.

Fig. 5.

Bundle branching. Shaded renderings of the reconstructed (A–D) nuclei (red); the (A–D) bundles (green), and (A,C) cells (transparent blue) are presented after being manipulated using the computer. In A and B, the fascicle was sectioned at 30°, rotated −105° from the position in Fig. 4, and the top half removed. Note the branching (arrow) and fusion of the bundles as well as their twisting. In C and D, the fascicle was sectioned at 30°, rotated 80° from the position in Fig. 4, and the top half removed. Again, note the branching (arrow) and fusion of the bundles as well as their twisting. In panels A and C the nuclei are labelled as in Figs 3 and 4. Bars, 2.0μm.

In Fig. 6, a longitudinal section of the reconstruction presented in Figs 4 and 5 is presented. This internal aspect of the reconstruction clearly demonstrates the branching and connection of 2 adjacent bundles. This reconstruction presents data similar to that presented in 30 consecutive 0.5 μm thick sections in Fig. 2.

Fig. 6.

Anastomosis of bundles at 14 days of tendon development. A longitudinal section of the bundle reconstruction demonstrating branching (arrow) is presented. This reconstruction is from the data presented in Figs 3–5 and is similar to that presented in Fig. 2. Bar, 2.0 μm.

Fig. 6.

Anastomosis of bundles at 14 days of tendon development. A longitudinal section of the bundle reconstruction demonstrating branching (arrow) is presented. This reconstruction is from the data presented in Figs 3–5 and is similar to that presented in Fig. 2. Bar, 2.0 μm.

The assembly, deposition and organization of collagen fibril bundles and their composite fibrils were studied during morphogenesis of the chick embryo tendon. The 14-day chick embryo is a stage when tendon architecture is being established (Birk and Trelstad, 1986; McBride et al. 1985) and rapid changes in the mechanical properties occur between days 14 and 17 of development (McBride et al. 1988). This work describes how tendon matrix structure develops from discrete subunits, bundles of collagen fibrils and addresses the relationship between fibrillar architecture, higher order structure and the tissue’s mechanical properties.

The formation, deposition and organization of collagen fibrils into a tissue-specific pattern are important processes in tendon morphogenesis. However, it is not the individual fibril, but groups of fibrils organized as bundles, that are organized, during morphogenesis, into well-defined patterns characteristic of different tissues. Tendon fibroblasts and their associated fibrillar matrix are organized as fascicles. The fascicle is easily recognized by 14 days of chick tendon development and the tendon becomes increasingly fascicular with continued development. We have shown that fasciculation is initially associated with an increased complexity of the fibroblast surface and a concurrent development of well-defined extracytoplasmic compartments containing bundles of collagen fibrils (Birk and Trelstad, 1986; Yang and Birk, 1988; Birk et al. 1989b). With development, the fibroblasts become increasingly attenuated and the large bundles characteristic of the mature tendon are compartmentalized within extracellular chambers defined by two or more adjacent fibroblasts within a fascicle.

The growing ends of collagen fibrils have been identified intimately associated with the fibroblast within narrow extracytoplasmic channels (Birk and Trelstad, 1986). The production of a continuous matrix requires the incorporation of newly formed fibrils into the developing stroma. The analysis of serial sections has demonstrated that the tendon fibroblast produces fibrils as segments. The 14-day chicken embryo tendon is composed of discrete fibril segments, 10 μm in length (Birk et al. 1989a). These segments are then assimilated into bundles and the bundles are incorporated into the developing matrix. When the fibril content of bundles was determined in serial sections, the bundles were found to be discontinuous in fibril number from section to section. The bundles also were found to rotate approximately 180° over a 10 to 12μm distance. This gradual rotation would serve to twine the composite fibrils.

The mechanical properties of a developing tendon composed of discontinuous fibril segments are dependent on non-covalent interactions at the surfaces of the discontinuous segments. In the tendon, it is likely that proteoglycans are integrated into the type I collagen fibril structure (Scott, 1984; 1988; Scott et al. 1981) as well as other collagen species (Birk et al. 1988; Keene et al. 1987; Mendler et al. 1989). In addition, other macromolecules such as type VI collagen may interact with collagen fibrils as well as the cellular elements (Bruns et al. 1986). Some of these additional macromolecules may interact to form the non-covalent fibril-fibril interactions, which are necessary for mechanical integrity. As development proceeds and the fibril segments exceed a critical length, estimated to be 30 micrometers for tendon fibrils, the composite has tensile properties equal to that of the covalent strength of the contributing monomers (McBride et al. 1989). The organization of fibril segments into bundles, during the establishment of tendon architecture and function, where the segmental fibrillar components could interact with the interfibrillar matrix as well as with adjacent fibrils, would contribute to the stabilization of this structure. Also, the observed gradual rotation of the bundles would serve to stabilize the immature bundle through the physical twining of the composite components. This arrangement also may contribute to the elasticity of the tendon. In addition, the extensive branching of the bundles observed at 14 days of development and their intimate association with the cellular elements would provide a higher order of structure stabilization.

The finding of fibril ends within a bundle at 14 days of development indicates that fibril segments are maintained for a period of time. The post-depositional fusion, maturation and rearrangement of these fibril and bundle segments are important processes which presumably occur within the largest extracytoplasmic compartment. At 14 days of development tendon structure is presumably stabilized by the collection of fibril segments into bundles, the close association of bundles with the tendon fibroblasts and the branching of bundles with the formation of an anastomosing bundle network within and among tendon fascicles.

We would like to gratefully acknowledge the assistance of Patrick Lardieri with the three-dimensional reconstructions. This work was supported by National Institutes of Health grant AR37003 and the Shriners Burns Institute. DEB is supported by a Research Career Development Award (EY00254). Portions of this work were carried out using the High Voltage Electron Microscope at the New York State Department of Health Laboratories, assisted by NIH Grant RR 01219 supporting the New York State High Voltage Microscope as a National Biotechnology Resource.

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