Collagen fibril bundles: a branching assembly unit in tendon
morphogenesis
DAVID E. BIRK1*, JAMES F. SOUTHERN2, EMANUEL I. ZYCBAND1, JOHN T. FALLON2 and
ROBERT L. TRELSTAD1
1 Department of Pathology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane,Piscataway, New Jersey 08854, USA2Department of Pathology, Massachusetts General Hospital, Shriners Bums Institute and Harvard Medical School, 51 Blossom Street, Boston,Massachusetts 02114, USA
* To whom correspondence should be addressed
Summary
The assembly, deposition and organization of collagenfibril bundles and their composite fibrils were studiedduring morphogenesis of the chick embryo tendon usingelectron microscopy, serial sections and computer-as-sisted three-dimensional reconstruction techniques. The14-day chick embryo is a stage when tendon architectureis being established and rapid changes in the mechanicalproperties 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 ofthe developing tendon; and form arborizing networkswithin and among fascicles. The organization of discrete
fibril segments into bundles, during the establishment oftendon architecture and function, where the segmentalfibrillar components could interact with the interfibrillarmatrix as well as with adjacent fibrils would contributeto the stabilization of this structure. The observedgradual rotation of the bundles would serve to stabilizethe immature bundle through the physical twining of thecomposite fibrils while the extensive branching of thebundles observed at 14-days of development and theirintimate association with the cellular elements wouldprovide a higher order of structure stabilization.
Tendons transmit force from the muscular to theskeletal system and are composed primarily of fibro-blasts, type I collagen fibrils and a pro teogly can-richinterfibrillar matrix. Tendons are composed of highlyaligned collagen fibrils organized into bundles. Thefibril bundles (collagen fiber) together with the tendonfibroblasts are organized into fascicles, and the fasciclesare bound together in a connective tissue sheath to forma tendon (Elliot, 1965; Greenlee and Ross, 1967;Greenleeefa/. 1975; Kastelic et al. 1978; Davison, 1982;Squier and Magnes, 1983; Squier and Bausch, 1984;Parry and Craig, 1984; Baer et al. 1988). The systematicdevelopment of this hierarchy is required for structuralintegrity and normal function.
The mechanical properties of tendon are dependenton a number of factors including fibril and bundleorientation; 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 tendonshave been shown to be at least millimeters in lengthusing a statistical approach (PaiTy and Craig, 1984;Trotter and Wolfsy, 1989). We have proposed that fibrilsegments are precursors in collagen fibril formation inthe embryonic chick tendon and 'segmental' depositionand post-depositional rearrangements are importantsteps in the development of mechanical integrity.
Collagen fibrillogenesis is a multistep process involv-ing both intracellular and extracellular compartments(Trelstad, 1982; Birk and Trelstad, 1986). Collagensynthesis, molecular assembly and formation of supra-molecular aggregates occurs within a series of well-defined cytoplasmic compartments (Trelstad andHayashi, 1979). In tendon morphogenesis, a hierarchyof extracytoplasmic compartments establishes at leastthree different levels of matrix organization: collagenfibrils, bundles and tissue specific macroaggregates, e.g.the large bundles in tendon; the orthogonal bundles incornea or bone. The cellular control of local factors
438 D. E. Birk and others
within these highly partitioned environments is import-ant in the regulation of local functions within thevarious extracellular domains.
In 14-day chick embryos, fibril segments are as-sembled in narrow extracytoplasmic channels definedby the fibroblast (Birk and Trelstad, 1984; 1986; Birk etal. 1989a). The segments are then assimilated intobundles and the bundles are incorporated into thedeveloping matrix. Within the bundle the fibril seg-ments are coupled with other fibril segments into afunctionally continuous fibril (Birk et al. 19896). Theaddition of fibril segments to bundles and the post-depositional fusion, maturation and rearrangement ofthese fibril and bundle segments are important pro-cesses during tendon development and growth.
In the present study, the development and organiz-ation of collagen bundles and their constituent fibrilswere studied during morphogenesis of the chick embryotendon. The 14-day chick embryo is a stage whentendon micro- and macro-architecture are being estab-lished and rapid changes in mechanical propertiesoccur. This work describes how tendon matrix structuredevelops from discrete subunits and addresses therelationship between fibrillar architecture, bundlestructure and the tissue's mechanical properties.
Materials and methods
White Leghorn chick embryos were incubated in a humidifiedatmosphere and staged according to Hamburger and Hamil-ton (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 cacodylatepH7.4 with 8mM-CaCl2 for 15min at room temperaturefollowed by 40min at 4°C. During fixation, the metatarsaltendons were dissected, and then washed in O.lM-cacodylatebuffer pH7.4 followed by post-fixation with 1% OsO4 incacodylate buffer pH 7.4 for one hour at 4°C. The tissues werewashed in buffer and dehydrated through a cold gradedethanol series followed by propylene oxide. The tendons wereinfiltrated, embedded in a fresh mixture of Polybed 812, nadicmethyl anhydride, dodecenyl succinic anhydride and DMP-30(Polysciences, Inc., Warrington, PA), polymerized and sec-tioned (Birk and Trelstad, 1984; 1986).
Serial sections (0.5 to 0.75 /an) of tendon were cut perpen-dicular to the tendon axis using a diamond knife. Sectionswere picked up onto formvar-coated 1x2mm slot grids andwere stained with 2% aqueous uranyl acetate for 45min,followed by 0.2 % lead citrate in 0.1 N-NaOH for 30min. Thestained sections were stabilized by the evaporation of a thinlayer of carbon, examined and photographed at 1000 kV usingthe AEI EM7 high voltage electron microscope at the NewYork State Department of Health Laboratories in Albany,New York (Birk and Trelstad, 1986). Additional thin sections(90 to 150 run) were cut and stained with 2 % aqueous uranylacetate for lOmin, followed by 0.2% lead citrate in 0.1 N-NaOH for 5 min and examined using either a Philips 420 orJEOL 1200EX transmission electron microscope.
The serial thick sections were photographed as a montagecontaining 4 complete fascicles and portions of several others.Data from 2 different fascicles were collected for over 30 /anof tendon and portions of others were analyzed in detail. Inaddition, we have analyzed at least 10 other 14-day chick
embryo metatarsal tendons in serial thick (0.5 to 0.75 /an) andthin (100 to 250 run) sections over 5 to 100 /an.
Tendon fascicles were followed in 36 consecutive serialsections and computer-generated, graphic three-dimensionalrenderings were produced using MOVIE.BYU (Departmentof Civil Engineering, Brigham Young University, Provo,Utah). Areas of interest were identified in photographicprints and the appropriate profiles were digitized usingDRAW.MGH (Department of Pathology, MassachusettsGeneral Hospital, Boston, MA). The sections were alignedusing a vertically trimmed edge of the section and a distinctknife mark as internal markers. The nuclei, cell outlines andbundles were contoured and the contours of interest weretransferred to MOVIE.BYU and three-dimensional shadedrenderings produced. The images were displayed on a Lexi-data LEX 90/35 high resolution graphics device interfacedwith a DEC Micro VAX II computer for analysis. Photographswere taken using a Focus Graphics Imagerecorder.
Results
Tendons are composed of discrete units called fascicles.Fascicles are composed of fibroblasts and their associ-ated bundles of collagen fibrils. Bundles of collagenfibrils, not individual collagen fibrils, are the predomi-nant extracellular structures observed. Fibril bundles(fibers) are discrete collections of collagen fibrils whichform within specific fibroblast-defined compartments.The three-dimensional relationships among cells as wellas the cells and collagen fibril bundles within a singlefascicle of the 14-day and 17-day chick embryo tendonare illustrated in Fig. 1. At both stages of development,collagen bundles are the major extracellular structures.During development from 14 to 17 days, fasciclesbecome better defined. At 14 days of developmentthere is a poorly defined interfascicular matrix while by17 days of development the fibroblasts of the endotendi-nium and their associated matrix, arranged roughlyperpendicular to the fascicular fibroblasts, separate anddefine adjacent fascicles. Also as development proceedsfrom 14 to 17 days the cell-to-matrix ratio decreases andthe fibroblasts become more attenuated. Concurrently,the long, slender processes separating bundles presum-ably retract and the small bundles coalesce to formlarger ones characteristic of the mature tendon.
The fibroblast-bundle relationship in the 14-daychick embryo tendon is complex. Collagen bundlesbranch to form an anastomosing bundle network withinand among fascicles. In Fig. 2, a single bundle isfollowed in a series of high voltage electron micro-graphs from thick sections (0.5 to 0.75 /an) cut perpen-dicular to the axis of the tendon. In this series ofconsecutive micrographs, the branching of bundleswithin a tendon fascicle is illustrated with a small bundleseparating from a larger bundle to join an entirelydifferent bundle.
The collagen fibrils within these branching bundleshave discrete lengths. When the fibril content of thesmall bundle in Fig. 2 was determined in sections no.12-15 and no. 21-22, where the fibrils were easilycounted, the bundles were found to be discontinuous infibril number from section to section, indicating that it is
Collagen fibril bundles 439
Fig. 1. Relationship among fibroblasts and bundles in a tendon fascicle. Transmission electron micrographs of sections (150to 200 nm) cut perpendicular to the axis of 14-daj (A-B) and 17-day (C-D) chick embryo tendons are presented. At 14 daysof tendon development the fibroblasts have become organized into fascicles (A), however, the boundaries between adjacentfascicles are not well defined. By 17 days of development, the boundaries of the fascicle are readily identifiable (C), withfibroblasts and matrix oriented perpendicular to the tendon axis separating adjacent fascicles. At both stages ofdevelopment, the compartmentalization of the extracellular space is readily apparent and bundles of collagen fibrils withinclearly defined compartments are the major extracellular structure. Fibril forming channels (arrows) containing single orsmall groups of fibrils and bundle forming compartments (B) containing fibril bundles are numerous and easily identifiable atboth stages of development. The fibroblast-to-cell ratio decreases as development proceeds (B vs. D). Presumably, theslender cytoplasmic processes separating bundle-forming compartments retract (curved arrows) and the bundles coalesce toform large bundles characteristic of the mature tendon. Bar, 1.0/an.
440 D. E. Birk and others
Fig. 2. Bundle branching. A single bundle is followed in this series of serial high-voltage electron micrographs. Thicksections (0.5 to 0.75 im\) cut perpendicular to the axis of 14-day chick embryo tendons are presented as a series ofconsecutive 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 smallbundle is distinct. However, in following its course, it is clear that this is a branch connecting two distinct bundles. When thefibril 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 collagenfibrils terminated within this portion of the bundle. Also, the bundles were found to rotate, the small bundle indicatedrotated approximately 180° over a 10 to Yljjm distance. Bar, 500 nm.
Fig. 4. Three-dimensional reconstructions of a 14-day chick embryo tendon fascicle. The data described in Fig. 3 werereconstructed using 36 serial sections with the graphics routines available with MOVIE.BYU. Shaded renderings of thereconstructed (A) nuclei (red); the (B) bundles (green); and (C) cells (blue) were reconstructed from sections cutperpendicular to the tendon axis and are presented with a longitudinal orientation. The nuclei are labelled as in Fig. 3. Thesection shown in Fig. 3 is at the level indicated by the arrow. In D, the three parts were rebuilt and displayed with the sameorientation and colors as in A-C. However, the blue cells were made partially transparent to permit the viewing of somedeeper structures. The section shown in Fig. 3 is at the level indicated by the arrow and the position of the * corresponds tothat in panel B. Bars, 2.
Collagen fibril bundles 441
I!
Fig. 3. Collagen bundles were studied within a single fascicle from a 14-day chick embryo tendon using computer-assistedthree-dimensional reconstructions. Serial 0.5 /im thick sections cut perpendicular to the tendon axis were examined andphotographed using the high-voltage electron microscope. In A, one of these micrographs is presented. The nuclei of threefibroblasts are indicated (I,II,III). The cells, nuclei and bundles (not individual fibrils) were hand digitized for each sectionthrough 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 bothpanels A and B. Bar, 2.0/an.
composed of collagen fibrils that terminate at differentlevels. Also, the bundles were found to rotate approxi-mately 180° over a 10 to 12/mi distance relative tointernal markers.
The relationship of bundles to one another and to thefibroblasts within a fascicle were studied further from a14-day chick embryo tendon using computer-assistedthree-dimensional reconstructions. Serial 0.5/xm sec-tions were cut perpendicular to the tendon axis andphotographed using the high-voltage electron micro-scope. The profiles of the nucleus, major portions of thecell and bundles were digitized for two completefibroblasts and portions of two others within a fascicle.In Fig. 3A, one of these micrographs is presented and
Fig. 5. Bundle branching. Shaded renderings of thereconstructed (A-D) nuclei (red); the (A-D) bundles(green), and (A,C) cells (transparent blue) are presentedafter being manipulated using the computer. In A and B,the fascicle was sectioned at 30°, rotated -105° from theposition in Fig. 4, and the top half removed. Note thebranching (arrow) and fusion of the bundles as well as theirtwisting. In C and D, the fascicle was sectioned at 30°,rotated 80° from the position in Fig. 4, and the top halfremoved. Again, note the branching (arrow) and fusion ofthe bundles as well as their twisting. In panels A and C thenuclei are labelled as in Figs 3 and 4. Bars, 2.0/an.Fig. 6. Anastomosis of bundles at 14 days of tendondevelopment. A longitudinal section of the bundlereconstruction demonstrating branching (arrow) ispresented. This reconstruction is from the data presented inFigs 3-5 and is similar to that presented in Fig. 2. Bar,2.0//m.
three nuclei (I, II, III) are indicated. These cells andtheir associated fibril bundles were chosen for recon-struction. In Fig. 3B the profiles of the nuclei, cells andbundles are shown with the same orientation as inFig. 3A.
Approximately 18 /xm of tendon from 36 consecutive,aligned sections, digitized as in Fig. 3B, were recon-structed into a three-dimensional image for analysis ofbundle and cell relationships using computer-assistedreconstruction techniques. Fig. 4A-C is a presentationof the component parts of this reconstruction. Three-dimensional renderings were produced of the nuclei,cells and bundles. This reconstruction is presented witha perpendicular axial orientation (it is a longitudinalrepresentation of the tendon built from sections cutperpendicular to the tendon axis), but has the samerotational orientation as in Fig. 3. The nuclei (A), fibrilbundles (B), and cells (C) are reconstructed from over18 /xm of tissue and in Fig. 4D the entire reconstructionis presented with the same orientation as in A-C,showing the three-dimensional relationships of thenuclei (red), bundles (green), and cells (blue, partiallytransparent) within a single tendon fascicle throughout18/OTI of tissue.
The reconstructed tendon fascicle shown in Fig. 4was studied further after being manipulated using thegraphics routines available with MOVIE.BYU. InFig. 5, the reconstruction is sectioned and the top halfremoved to reveal internal detail of the fascicle. Thesegraphic reconstructions demonstrate the complexity ofthe bundle architecture within a fascicle. Bundles areseen to branch, bifurcate and rotate about their long
442 D. E. Birk and others
axes. The bundles arborize with branching of bundles,subbranches connecting adjacent branches and fusionof branches with other bundles.
In Fig. 6, a longitudinal section of the reconstructionpresented in Figs 4 and 5 is presented. This internalaspect of the reconstruction clearly demonstrates thebranching and connection of 2 adjacent bundles. Thisreconstruction presents data similar to that presented in30 consecutive 0.5 /xm thick sections in Fig. 2.
Discussion
The assembly, deposition and organization of collagenfibril bundles and their composite fibrils were studiedduring morphogenesis of the chick embryo tendon. The14-day chick embryo is a stage when tendon architec-ture is being established (Birk and Trelstad, 1986;McBride et al. 1985) and rapid changes in the mechan-ical properties occur between days 14 and 17 of devel-opment (McBride et al. 1988). This work describes howtendon matrix structure develops from discrete sub-units, bundles of collagen fibrils and addresses therelationship between fibrillar architecture, higher orderstructure and the tissue's mechanical properties.
The formation, deposition and organization of col-lagen fibrils into a tissue-specific pattern are importantprocesses in tendon morphogenesis. However, it is notthe individual fibril, but groups of fibrils organized asbundles, that are organized, during morphogenesis,into well-defined patterns characteristic of differenttissues. Tendon fibroblasts and their associated fibrillarmatrix are organized as fascicles. The fascicle is easilyrecognized by 14 days of chick tendon development andthe tendon becomes increasingly fasdcular with con-tinued development. We have shown that fasciculationis initially associated with an increased complexity ofthe fibroblast surface and a concurrent development ofwell-defined extracytoplasmic compartments contain-ing bundles of collagen fibrils (Birk and Trelstad, 1986;Yang and Birk, 1988; Birk et al. 1989b). With develop-ment, the fibroblasts become increasingly attenuatedand the large bundles characteristic of the maturetendon are compartmentalized within extracellularchambers defined by two or more adjacent fibroblastswithin a fascicle.
The growing ends of collagen fibrils have beenidentified intimately associated with the fibroblastwithin narrow extracytoplasmic channels (Birk andTrelstad, 1986). The production of a continuous matrixrequires the incorporation of newly formed fibrils intothe developing stroma. The analysis of serial sectionshas demonstrated that the tendon fibroblast producesfibrils as segments. The 14-day chicken embryo tendonis composed of discrete fibril segments, 10/mi in length(Birk etal. 1989a). These segments are then assimilatedinto bundles and the bundles are incorporated into thedeveloping matrix. When the fibril content of bundleswas determined in serial sections, the bundles werefound to be discontinuous in fibril number from sectionto section. The bundles also were found to rotate
approximately 180° over a 10 to 12pan distance. Thisgradual rotation would serve to twine the compositefibrils.
The mechanical properties of a developing tendoncomposed of discontinuous fibril segments are depen-dent on non-covalent interactions at the surfaces of thediscontinuous segments. In the tendon, it is likely thatproteoglycans are integrated into the type I collagenfibril structure (Scott, 1984; 1988; Scott et al. 1981) aswell as other collagen species (Birk etal. 1988; Keene etal. 1987; Mendler et al. 1989). In addition, othermacromolecules such as type VI collagen may interactwith collagen fibrils as well as the cellular elements(Bruns et al. 1986). Some of these additional macromol-ecules may interact to form the non-covalent fibril-fibril interactions, which are necessary for mechanicalintegrity. As development proceeds and the fibril seg-ments exceed a critical length, estimated to be 30 mi-crometers for tendon fibrils, the composite has tensileproperties equal to that of the covalent strength of thecontributing monomers (McBride et al. 1989). Theorganization of fibril segments into bundles, during theestablishment of tendon architecture and function,where the segmental fibrillar components could interactwith the interfibrillar matrix as well as with adjacentfibrils, would contribute to the stabilization of thisstructure. Also, the observed gradual rotation of thebundles would serve to stabilize the immature bundlethrough the physical twining of the composite com-ponents. This arrangement also may contribute to theelasticity of the tendon. In addition, the extensivebranching of the bundles observed at 14 days ofdevelopment and their intimate association with thecellular elements would provide a higher order ofstructure stabilization.
The finding of fibril ends within a bundle at 14 days ofdevelopment indicates that fibril segments are main-tained for a period of time. The post-depositionalfusion, maturation and rearrangement of these fibriland bundle segments are important processes whichpresumably occur within the largest extracytoplasmiccompartment. At 14 days of development tendon struc-ture is presumably stabilized by the collection of fibrilsegments into bundles, the close association of bundleswith the tendon fibroblasts and the branching ofbundles with the formation of an anastomosing bundlenetwork within and among tendon fascicles.
We would like to gratefully acknowledge the assistance ofPatrick Lardieri with the three-dimensional reconstructions.This work was supported by National Institutes of Healthgrant AR37003 and the Shriners Burns Institute. DEB issupported by a Research Career Development Award(EY00254). Portions of this work were carried out using theHigh Voltage Electron Microscope at the New York StateDepartment of Health Laboratories, assisted by N1H GrantRR 01219 supporting the New York State High VoltageMicroscope as a National Biotechnology Resource.
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