A Fine Structural Study of the Development of the Chick Flexor Digital Tendon: A Model for Synovial Sheathed Tendon Healing ' THEODORE K. GREENLEE, JR., CASSANDRA BECKHAM AND DIANA PIKE Department of Orthopaedics, School of Medicine, University of W a s h i n g t o n , Seattle, W a s h i n g t o n 98195
ABSTRACT The development of the synovial sheathed flexor digital tendon in the chick was studied by light and electron microscopy in 12-day embryos to 22-day post-hatched chickens. Areas of specialized connective tissue differentiation were identified in this complex structure consisting of a lubricated synovial sheath, elastic vincula and fibrocartilaginous adaptations on the surface of the tendon. The presence of some of these specialized adaptations may be related to the specific types of mechanical forces and stresses applied to the developing connective tissue system. This model system appears to be appropriate for the experimental study of tendon injuries related to the human hand.
Restoration of function to injured flexor digital tendons is a difficult medical problem (Kessler and Nissim, '69). Restoration is complicated by the complex structure of the organ, i.e., the synovial sheath in which the tendon glides, the tenuous blood supply available to the tendon within the sheath (Hollinshead, '58) and the pulleys (heavy fibrous condensations of the sheath) which keep the tendon closely applied to the bone during flexion. Any injury to the tendon within the sheath is usually followed by formation of scar tissue between the sheath and the tendon, thus limiting the excursion of the tendon. Even when the injury is treated by a specialist, some loss of function often occurs in the injured digit, and this functional loss is usually due to scarring. Our hypothesis is that insight into the healing mechanisms of this complex structure can be obtained by understanding the developmental processes. Developmental studies o n synovial sheathed tendons have been performed by Shields ('23), Fitton-Jackson ('56), and Greenlee and Ross ('67). In 1923 Shields used the light microscope to study synovial sheathed tendon development in the pig. In a n electron microscopic study of the metatarsal tendon of the chick, Sylvia Fitton-Jackson ('56) was concerned with AM. J. ANAT., 143: 303-314.
the process of collagen synthesis and secretion. The development of the hind flexor digital tendon of the rat was studied by Greenlee and Ross ('67) in a n attempt to develop a model system for the study of tendon healing. However, this model system was not considered to be optimum because of its small size and the lack of complexity of the tendon. The need for a model system by which to study synovial sheathed tendon healing is evident from the review studies of Mason and Shearon ('32), Salamon and Hamori ('66) and Verdan ('72). Tendons from many different animals have been used i n experimental studies on tendon healing: rats (Greenlee and Ross, '67), chicks (Lindsay and Thomson, '60), guinea pigs, rabbits, dogs, etc. (Mason and Shearon, '32). It is possible that some of the controversies arising from these studies result from the different sizes, anatomies and excursions of the tendons studied. The chicken flexor digital tendon is similar i n anatomy, size and excursion to that of the human hand (Lindsay and Thomson, '60) and is a good model by Accepted February 15, '75. 1This study was supported in part by U. S. Public Health Service Research Grant R01 AM 15762-03 and designated research funds from the Gainesville Veterans Administration Hospital, Gainesville, Florida. 2 Present address: 1201 Park Glen Drive, Knoxville, Tennessee 37919.
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which to study development and healing. Therefore, this developmental study of the chick flexor digital tendon is presented as a model system from which understanding of the mechanisms of the healing process may be derived. METHODS AND MATERIALS
Fertile white leghorn chicken eggs were obtained from a local hatchery and incubated in a humidified forced draft incubator a t 38°C. Chick embryos were killed at daily intervals from four days to hatching at 21 days. The embryos were not specifically staged and are designated only by their day of incubation. Post-hatched chickens were killed at 22 days. Specimens were obtained and fixed as described below: 1. Four-day limb buds through 21-day embryo digits were fixed in a 4% glutaraldehyde in phosphate buffer, then postfixed in 2% osmium tetroxide in phosphate buffer. 2. Post-hatched chick tendons were dissected from the digit and fixed in 2% osmium tetroxide in s-collidine buffer. The tissue was dehydrated in graded alcohols and embedded in epoxy resin according to Luft's method ('61). Specimens from each time interval were sectioned transversely for light and electron microscopy on a Porter-Blum MT I1 ultramicrotome. Thick sections were stained with azure TI methylene blue (Richardson et al., '60). Thin sections were stained with uranyl acetate and lead citrate (Reynolds, '63) and examined in a n Hitachi H U l l c electron microscope. Because of the large size of the digits and the desire to maintain the tendon and sheath relationship, certain fixation artifacts are present in the electron micrographs, These artifacts are limited to the mitochondria and when present are indicated in the illustrations. OBSERVATIONS
Overview of development The limb bud consists of mesDay 4 enchymal cells. Tendon cells are present, but Day 8 the sheath has not begun to form. Day 10 There is a condensation of
cells around the tendon to form the tendon sheath. Day 12 The tendon sheath is present, a synovial cleft has formed and the vincula are present. Day 13 Fasciculation of the tendon is obvious. Day 17 Large elastic fibers are present in the vincula. The fibro-cartilage area is present. There is a n increase in number and size of collagen fibrils. Microfilaments are present in septal cells. Day 22 Post-hatched. There is a great increase of microfilaments in the septal cells and a further increase in volume due to increase in size and number of collagen fibrils. Details of development Four to 12-day embryos. This period will not be discussed i n detail because during this time the chick flexor digital tendon (particularly cell function, cell contact within the tendon and formation of the tendon sheath) did not differ significantly from that of the rat (Greenlee and Ross, '67). Changes in cytoplasmic organelles in relation to collagen and other connective Fig. 1 This is a composite figure showing a longitudinal section and serial cross-sections of 12-day incubated chick embryo digits. The longitudinal section contains only part of the most proximal phalanx ( I ) . The general areas of the cross-sections are designated by the white lines o n the figure and lettered A to F. A is the most proximal cross-section along the longitudinal section. The longest, or deep tendon (dt), with its ultimate insertion into the distal phalanx (IV) is i n all of the sections. The middle tendon ( m t ) which splits and inserts into the base of the third phalanx (111) appears i n the longitudinal section and in cross-sections l A , 1B and 1C. The superficial tendon ( s t ) is not present on the longitudinal section as it has already split for insertion into the base of the second phalanx (IT). However, it is present i n figure l A , a section that it is a bit more proximal than is designated by the line on the longitudinal section. A synovial space ( s ) is present in the longitudinal and cross-sections, particularly in cross-sections l C , 1D and 1E. A synovial space is lacking at this early period i n cross-sections l A , 1B and IF. Even at this early stage of development certain specialized structures can be identified. They include the vincula ( v ) which are noted i n crosssections 1B and 1D and which are also identified in the longitudinal section. The pulleys ( p ) which keep the flexor tendons from bow-stringing are present in the longitudinal section. Figure 1 x 35.625, figures A-F x 71.25.
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tissue fiber synthesis also paralleled those i n the rat. Changes in complexity began after the twelfth day; consequently, this paper specifically concentrates on the detailed development of the chick tendon from the 12-day embryo to post-hatching a t 22 days. Light microscopy 12-day embryo. The third and longest digit in the chick contains four phalanges. The relationship of the three tendons to this digit, the beginning of synovial cavity formation and the location of the pulleys and vincula can be seen in figure 1. The vincula provide the vasculature for the tendon (Brockis, ' 5 3 ) within the synovial sheath. 13-day embryo. By the thirteenth day fasciculi were quite obvious in the tendons. These appeared as bundles of collagen and cells cut in cross-section which were surrounded by relatively flat or longitudinally sectioned cells. These longitudinally sec-
tioned cells, or interfascicular cells, divided the tendon into many separate bundles or fascicles, 17-day embryo. The synovial sheath cavity was present (fig. 2 ) and extended proximally to include all three tendons. The volume of the tendon had also increased. By this time the specialized areas of the vincula and fibrocartilage were well differentiated. (See detailed description below.) No further developmental changes were observed with the light microscope other than an increase in volume of the tendon. Electron microscopy 13-day embryo. In cross-section the synovial cells appeared to pass between the fascicles on the surface of the tendon. Only a few longitudinally oriented collagen fibrils were associated with these interfascicular cells, while the tendon cells were surrounded by bundles of collagen. 17-day embryo. In cross-sectioned ten-
Fig. 2 This light micrograph demonstrates a n area similar to figure lB, from a 17-day embryo. It shows fasciculation ( f ) of the tendons and a well-defined synovial space ( s ) . X 270.
FINE STRUCTURE OF THE CHICK TENDON
don the fascicles were separated by longitudinally oriented interfascicular cells and increasing amounts of longitudinally oriented collagen fibrils. The synovial cells and the interfascicular cells differed in morphology from the cells of the fascicle (figs. 3 , 4 ) in that their cytoplasm had a lower concentration of rough endoplasmic reticulum. Intracellular microfilaments were also present in the interfascicular cells. The relationship of the interfascicular collagen fibrils to the tendon collagen fibrils is demonstrated in longitudinally sectioned tendon. A tangential section of a fascicle demonstrates longitudinally oriented collagen fibrils lying a t approximately right angles to each other (fig. 5). Comparison of multiple cross-sections with longitudinal sections suggests that the interfascicular collagen fibrils wrap around the fascicle a t approximately right angles to the bulk of the fascicle collagen. 21-day errthryo. The amount of collagen associated with the tendon had increased considerably. The interfascicular cells were more distinguishable from tendon cells than was observed in the 17-day embryo. In these older and larger specimens, slow diffusion of the fixative is believed to be responsible for the swelling of the mitochondria. 22-day, post-hatched. Aside from the large increase in the diameter of collagen fibrils, the major change here was in the appearance of interfascicular cells. The number of microfilaments in these cells had increased from the 17-day embryo to the 22-day post-hatched stage (fig. 6 ) . There was also a n increase in the amount of septa1 cell-associated collagen fibrils which varied considerably in size, as did the collagen fibrils in the tendon (400 A to 2300 A).
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brils with a n occasional immature elastic fiber consisting of 100 A fibrils. Cartilage-like cells. Cells on the surface of the tendon adjacent to the volar sheath resembled cartilage cells (fig. 8). The collagen fibrils lying adjacent to the cells were randomly oriented. Ground substance extended over the surface of the cells, giving the appearance of fibrocartilage (fig. 8 A insert). DISCUSSION
Specialization in the synovial sheath tendon A synovial sheathed tendon has three distinct components: the tendon proper, consisting of longitudinally oriented collagen fibrils and cells, a visceral synovial layer on the tendon, which tends to be circumferentially oriented about the tendon, and a circumferentially oriented fibrous sheath associated with a parietal synovial layer (Greenlee and Ross, '67). When observing a n immature mesenchymal cell population in connective tissue, it is generally impossible to determine what the eventual function of a specific cell will be at maturity. Usually, this determination can be made only after the cell begins secreting its specialized connective tissue product. The four cell types of the chick tendon are: tendon cells, synovial and interfascicular cells, elastin-producing cells of the vincula and cartilaginous cells of the tendon surfaces. Their morphological and functional differences are discussed more fully below. Tendon fibroblast. The tendon fibroblast originates from a n immature mesenchymal cell containing large numbers of free ribosomes and scant rough endoplasmic reticulum (Greenlee and Ross, '67). These cells maintain contact with each other by means of junction sites (Ross and Greenlee, '66). As the cells mature they Specialized cellular adaptations develop a n extensive rough endoplasmic Vincula. I n the electron microscope the reticulum and large Golgi complexes. Howcells of the vincula resembled fibroblasts ever, the presence of endoplasmic reticualthough they produced chiefly elastic fi- lum and a well developed Golgi complex bers (fig. 7 ) . These cells were similar to is not diagnostic of a tendon fibroblast; those described in the ligamentum nuchae rather, i t is the association of a fibroblast of cattle (Greenlee et al., '66). However, with a n extracellular matrix of tightly in this same thin section, a fibroblast of packed, large collagen fibrils, and little else, the tendon was surrounded by collagen fi- which determines the diagnosis. Small,
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Fig. 3 This electron micrograph from a 17-day embryo shows the synovial space in the upper right hand corner. This is from the intermediate or middle tendon. The synovial cell (sc) contains some fine filaments and appears different morphologically from the tendon cells (tc). The cytoplasm of synovial cells is less dense than that of tendon cells. The tendon collagen (co) is cross-sectioned whereas the interfascicular collagen (arrow) is sectioned longitudinally. X 5,200. Fig. 4 Interfascicular cells (ic) with their longitudinally sectioned collagen (arrow) are similar to the synovial cells in figure 3. The tendon cells ( t c ) contain a rather dense rough endoplasmic reticulum (er). A mitochondrion ( m ) shows the artifacts of less than optimum fixation necessitated by the size of the specimens used for this study. x 7,400.
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Fig. 5 This is a longitudinal section of a tendon from a 17-day embryo. The collagen associated with the tendon proper ( t c ) is obliquely sectioned. The interfascicular collagen ( i c ) is also obliquely sectioned but at 90° to that of the tendon collagen. x 20,500.
poorly developed, elastic fibers are also present in the tendon, although in the adult tendon their concentration is quite low. During development the tendon cells become more spindle-shaped and elongated, and i t has been postulated that muscle tension and growth determine the extent of this elongation (Weiss, ’49). Biochemical studies indicate that the amounts of polysaccharides present in tendons are relatively low (Banga and Balo, ’60). Therefore, since the tendon fibroblast is the predominant cell and probably accounts for the bulk of the secreted material, it is assumed that tendon fibroblasts do not secrete large amounts of acid polysaccharides. Synovial a n d septa1 cells, Studies of joint synovial membranes usually mention two types of synovial cells, “ A and “B”, (Cherney et al., ’70; Barland et al., ’62). Type “ A ”is thought to be phngocytic, while type “B” is believed to be a synthetic cell responsible for the synthesis of lubricating polysaccharides. Henrikson and Cohen
( ’ 6 5 ) , in their study of the development of the chick interphalangeal joint, identified only one synovial cell type, as did studies of the rat flexor digital tendon (Greenlee and Ross, ’67). In the rat, morphologic differences between the tendon fibroblast and its associated synovial cells are differences of a quantitative rather than a qualitative nature. In the chick tendon there appears to be a more definable morphologic difference between these two cell types. Also, it appears that the synovial cells are continuous with the interfascicular cells. It is assumed that the main function of synovial cells is to secrete acid polysaccharides for lubrication, which may also be one function of the interfascicular cells. The fasciculation of the tendon might provide for a much more flexible structure than would occur if all the collagen fibrils in the tendon were bound together. ( A s an example, compare the flexibility of monofilament materials such as nylon with their woven polyfilament counterparts of similar size.)
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Fig. 6 This electron micrograph of a 22-day post-hatched chicken tendon shows that interfascicular cells have a distinct morphology of their own. These cells have a large number of perinuclear intracellular filaments ( f ) . Note the varying sizes of the interfascicular collagen (ic) and the tendon collagen (tc). x 12,400.
The interfascicular cells of the 22-day post-hatched chick have a unique morphologic appearance. They contain extremely large concentrations of intracellular filaments whose function is not known but, according to Wessels et al. ('71), these filaments probably have a contractile function in non-muscle cells. The functional significance of this increase in microfila-
ment concentration is not understood at this time. Elastic fiber producing cells of the vincula. The vincula which carry the major blood supply to the tendon secrete large elastic fibers (Beckham and Greenlee, '75). This elastic structure may prevent kinking of the blood vessels to this tendon; however, the exact function is unknown.
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Fig. 7 This is a n area of a vinculum from a 17-day chick embryo. The large elastic fibers (el), which are identified in niches of cells, are seen to consist of a n amorphous area with only occasional extracellular microfilaments ( f ) . The collagen fibrils (co) are oriented in a random fashion. One of the cells has a rather extensive rough endoplasmic reticulum ( e r ) and a well-developed Golgi apparatus (G), typical of fibroblasts. x 26,000.
Cartilage-like cells. The cartilage-like cells are located on the surface of the tendon where the tendon is in direct contact with the sheath, where synovial cells are
normally observed, These cells are almost cuboid and are much larger than the associated tendon cells. An area of randomly oriented collagen fibrils is located on their
Fig. 8 This composite figure of the flexor tendon in the area of the pulley illustrates cell adaptations. Note in the light micrograph, insert B, the tendon ( t ) and the surface cells on the palmar aspect of the tendon which resemble cartilage cells ( c ) . These cells are directly opposed to the sheath ( s ) which is i n the area of the pulley. The electron micrograph demonstrates the difference between the cartilage cells (ca), with a matrix surrounding their free surface, and the typical visceral synovial cells ( v s ) with no matrix on their surface. Insert A is a higher magnification of an area between the fibro-cartilage cells ( c a ) which demonstrates that the matrix on the surface of the cells is composed of randomly oriented collagen fibrils (co) that come up to the potential space of the tendon sheath (sp). x 5,000. Insert A X 10,800, insert B x 340.
FINE STRUCTURE OF THE CHICK TENDON
surface, Another substance, probably a polysaccharide such as is found in cartilage, is also present. As is characteristic of cartilage, these cells and the matrix they are secreting presumably are better suited for accepting pressure than for bearing tension. The organelles of the cell are qualitatively the same as those in the tendon fibroblast. Functional adaptations. All of the cells constituting the synovial sheathed tendon apparatus appear to evolve from relatively similar mesenchymal cells during the early stages of embryonic development. About the twelfth day, these cells begin to assume individual characteristics for the performance of their specific functions. The tendon fibroblasts lay down large amounts of collagen to transmit the force of the muscle pull. Relatively frictionless motion of the tendon is provided by the synovial cells which secrete a buffer or lubricating fluid between the tendon and its sheath. In certain specialized areas, particularly where blood vessels enter the tendon, the cells surrounding the vessels change their function to secrete large elastic fibers. These fibers may be necessary for the protection and maintenance of the vessels which are subject to unusual stress because of the mobility of the tendon. In other areas where friction occurs between the tendon and its pulleys, the cells on the surface begin to assume the shape and characteristics of cartilage cells, thereby producing a necessary articular surf ace. The development of these structures in areas where they are essential for the functional development of the chick suggests that the forces applied to the cells might determine their ultimate state of differentiation. This study, however, does not prove this hypothesis or rule out other known controls of differentiation. LITERATURE CITED Banga, I., and J. Balo 1960 Isolation of neutral heteropolysaccharide containing mucoprotein from bovine Achilles tendon with the aid of collagenmucoproteinase. Biochem. J., 74: 388-393. Barland, P., A. B. Novikoff and D. Hamerman 1962 Electron microscopy of the human synovial membrane. J. Cell Biol., 1 4 : 207-220. Beckham, C., and T. K. Greenlee, Jr. 1975 The chick vincula - a n elastic structure. Accepted for publication by J. of Anat.
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Brockis, J. G. 1953 The blood supply of the flexor and extensor tendons of the fingers i n man. J. Bone & Joint Surg., 3543: 131-138. Cherney, D. D., W. D. Baxter and L. J. A. DiDio 1970 Synovial membrane of the rabbit knee during a n induced degenerative arthropathy. Acta Anat., 75: 225-247. Fitton-Jackson, S. 1956 The morphogenesis of avian tendon. Proc. R. SOC.(London) B., 144: 556-572. Greenlee, T. K. Jr., R. Ross and J. L. Hartman 1966 The fine structure of elastic fibers. J. Cell Biol., 30: 59-71. Greenlee, T. K., Jr., and R. Ross 1967 The development of the r a t flexor digital tendon, a fine structure study. J. Ultrastruct. Res., 18: 354-376. Henrikson, R. C., and A. S. Cohen 1965 Light and electron microscopic observations of the developing chick interphalangeal joint. J. Ultrastruct. Res., 13: 129-162. Hollinshead, W. H. 1958 Anatomy for Surgeons. Vol. 3. Chap. VI. The Back and Limbs. Paul B. Hoeber, Inc. New York, pp. 449-580. Kessler, I., and F. Nissim 1969 Primary repair without immobilization of flexor tendon division within the digital sheath. Acta Orthop. Scand., 40: 587-601. Lindsay, W. K., and H. G. Thomson 1960 Digital flexor tendons: an experimental study. part I. The significance of each component of the flexor mechanism in tendon healing. Brit. J. Plast. Surg., 12: 289-316. Luft, J. H. 1961 Improvements i n epoxy resin embedding methods. J. Biophy. Biochem. Cytol., 9: 409-414. Mason, M. L., and C. G. Shearon 1932 The process of tendon repair. A n experimental study of tendon suture and tendon graft. Arch. Surg., 25: 615-692. Reynolds, E. S. 1963 The u s e of lead citrate at high pH as a n electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-213. Richardson, K. C., L. Jarett and E. H. Finke 1960 Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol., 35: 313-323. Ross, R., and T. K. Greenlee, Jr. 1966 Electron microscopy: attachment sites between connective tissue cells. Science, 153: 997-999. Salamon, A., and J. Hamori 1966 Present state of tendon regeneration. Light and electron microscopic studies of the regenerating tendon of the rat. Acta Morphol. Acad. Sci. (Hungary), 14: 7-24. Shields, R. T. 1923 O n the development of tendon sheaths. Contrib. Embryol., 15: 55-62. Verdan, C. E. 1972 Half a century of flexortendon surgery. J. Bone Joint Surg., 54-A: 472491. Weiss, P. 1949 Differential growth. In: The Chemistry and Physiology of Growth. A. K. Parpart, ed., Princeton University Press, Princeton, N. J., 135-186. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Ludeunena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 1971 Microfilaments i n cellular and developmental processes. Science, 171: 135-143.
ABSTRACT The development of the synovial sheathed flexor digital tendon in the chick was studied by light and electron microscopy in 12-day embryos to 22-day post-hatched chickens. Areas of specialized connective tissue differentiation were identified in this complex structure consisting of a lubricated synovial sheath, elastic vincula and fibrocartilaginous adaptations on the surface of the tendon. The presence of some of these specialized adaptations may be related to the specific types of mechanical forces and stresses applied to the developing connective tissue system. This model system appears to be appropriate for the experimental study of tendon injuries related to the human hand.
Restoration of function to injured flexor digital tendons is a difficult medical problem (Kessler and Nissim, '69). Restoration is complicated by the complex structure of the organ, i.e., the synovial sheath in which the tendon glides, the tenuous blood supply available to the tendon within the sheath (Hollinshead, '58) and the pulleys (heavy fibrous condensations of the sheath) which keep the tendon closely applied to the bone during flexion. Any injury to the tendon within the sheath is usually followed by formation of scar tissue between the sheath and the tendon, thus limiting the excursion of the tendon. Even when the injury is treated by a specialist, some loss of function often occurs in the injured digit, and this functional loss is usually due to scarring. Our hypothesis is that insight into the healing mechanisms of this complex structure can be obtained by understanding the developmental processes. Developmental studies o n synovial sheathed tendons have been performed by Shields ('23), Fitton-Jackson ('56), and Greenlee and Ross ('67). In 1923 Shields used the light microscope to study synovial sheathed tendon development in the pig. In a n electron microscopic study of the metatarsal tendon of the chick, Sylvia Fitton-Jackson ('56) was concerned with AM. J. ANAT., 143: 303-314.
the process of collagen synthesis and secretion. The development of the hind flexor digital tendon of the rat was studied by Greenlee and Ross ('67) in a n attempt to develop a model system for the study of tendon healing. However, this model system was not considered to be optimum because of its small size and the lack of complexity of the tendon. The need for a model system by which to study synovial sheathed tendon healing is evident from the review studies of Mason and Shearon ('32), Salamon and Hamori ('66) and Verdan ('72). Tendons from many different animals have been used i n experimental studies on tendon healing: rats (Greenlee and Ross, '67), chicks (Lindsay and Thomson, '60), guinea pigs, rabbits, dogs, etc. (Mason and Shearon, '32). It is possible that some of the controversies arising from these studies result from the different sizes, anatomies and excursions of the tendons studied. The chicken flexor digital tendon is similar i n anatomy, size and excursion to that of the human hand (Lindsay and Thomson, '60) and is a good model by Accepted February 15, '75. 1This study was supported in part by U. S. Public Health Service Research Grant R01 AM 15762-03 and designated research funds from the Gainesville Veterans Administration Hospital, Gainesville, Florida. 2 Present address: 1201 Park Glen Drive, Knoxville, Tennessee 37919.
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which to study development and healing. Therefore, this developmental study of the chick flexor digital tendon is presented as a model system from which understanding of the mechanisms of the healing process may be derived. METHODS AND MATERIALS
Fertile white leghorn chicken eggs were obtained from a local hatchery and incubated in a humidified forced draft incubator a t 38°C. Chick embryos were killed at daily intervals from four days to hatching at 21 days. The embryos were not specifically staged and are designated only by their day of incubation. Post-hatched chickens were killed at 22 days. Specimens were obtained and fixed as described below: 1. Four-day limb buds through 21-day embryo digits were fixed in a 4% glutaraldehyde in phosphate buffer, then postfixed in 2% osmium tetroxide in phosphate buffer. 2. Post-hatched chick tendons were dissected from the digit and fixed in 2% osmium tetroxide in s-collidine buffer. The tissue was dehydrated in graded alcohols and embedded in epoxy resin according to Luft's method ('61). Specimens from each time interval were sectioned transversely for light and electron microscopy on a Porter-Blum MT I1 ultramicrotome. Thick sections were stained with azure TI methylene blue (Richardson et al., '60). Thin sections were stained with uranyl acetate and lead citrate (Reynolds, '63) and examined in a n Hitachi H U l l c electron microscope. Because of the large size of the digits and the desire to maintain the tendon and sheath relationship, certain fixation artifacts are present in the electron micrographs, These artifacts are limited to the mitochondria and when present are indicated in the illustrations. OBSERVATIONS
Overview of development The limb bud consists of mesDay 4 enchymal cells. Tendon cells are present, but Day 8 the sheath has not begun to form. Day 10 There is a condensation of
cells around the tendon to form the tendon sheath. Day 12 The tendon sheath is present, a synovial cleft has formed and the vincula are present. Day 13 Fasciculation of the tendon is obvious. Day 17 Large elastic fibers are present in the vincula. The fibro-cartilage area is present. There is a n increase in number and size of collagen fibrils. Microfilaments are present in septal cells. Day 22 Post-hatched. There is a great increase of microfilaments in the septal cells and a further increase in volume due to increase in size and number of collagen fibrils. Details of development Four to 12-day embryos. This period will not be discussed i n detail because during this time the chick flexor digital tendon (particularly cell function, cell contact within the tendon and formation of the tendon sheath) did not differ significantly from that of the rat (Greenlee and Ross, '67). Changes in cytoplasmic organelles in relation to collagen and other connective Fig. 1 This is a composite figure showing a longitudinal section and serial cross-sections of 12-day incubated chick embryo digits. The longitudinal section contains only part of the most proximal phalanx ( I ) . The general areas of the cross-sections are designated by the white lines o n the figure and lettered A to F. A is the most proximal cross-section along the longitudinal section. The longest, or deep tendon (dt), with its ultimate insertion into the distal phalanx (IV) is i n all of the sections. The middle tendon ( m t ) which splits and inserts into the base of the third phalanx (111) appears i n the longitudinal section and in cross-sections l A , 1B and 1C. The superficial tendon ( s t ) is not present on the longitudinal section as it has already split for insertion into the base of the second phalanx (IT). However, it is present i n figure l A , a section that it is a bit more proximal than is designated by the line on the longitudinal section. A synovial space ( s ) is present in the longitudinal and cross-sections, particularly in cross-sections l C , 1D and 1E. A synovial space is lacking at this early period i n cross-sections l A , 1B and IF. Even at this early stage of development certain specialized structures can be identified. They include the vincula ( v ) which are noted i n crosssections 1B and 1D and which are also identified in the longitudinal section. The pulleys ( p ) which keep the flexor tendons from bow-stringing are present in the longitudinal section. Figure 1 x 35.625, figures A-F x 71.25.
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tissue fiber synthesis also paralleled those i n the rat. Changes in complexity began after the twelfth day; consequently, this paper specifically concentrates on the detailed development of the chick tendon from the 12-day embryo to post-hatching a t 22 days. Light microscopy 12-day embryo. The third and longest digit in the chick contains four phalanges. The relationship of the three tendons to this digit, the beginning of synovial cavity formation and the location of the pulleys and vincula can be seen in figure 1. The vincula provide the vasculature for the tendon (Brockis, ' 5 3 ) within the synovial sheath. 13-day embryo. By the thirteenth day fasciculi were quite obvious in the tendons. These appeared as bundles of collagen and cells cut in cross-section which were surrounded by relatively flat or longitudinally sectioned cells. These longitudinally sec-
tioned cells, or interfascicular cells, divided the tendon into many separate bundles or fascicles, 17-day embryo. The synovial sheath cavity was present (fig. 2 ) and extended proximally to include all three tendons. The volume of the tendon had also increased. By this time the specialized areas of the vincula and fibrocartilage were well differentiated. (See detailed description below.) No further developmental changes were observed with the light microscope other than an increase in volume of the tendon. Electron microscopy 13-day embryo. In cross-section the synovial cells appeared to pass between the fascicles on the surface of the tendon. Only a few longitudinally oriented collagen fibrils were associated with these interfascicular cells, while the tendon cells were surrounded by bundles of collagen. 17-day embryo. In cross-sectioned ten-
Fig. 2 This light micrograph demonstrates a n area similar to figure lB, from a 17-day embryo. It shows fasciculation ( f ) of the tendons and a well-defined synovial space ( s ) . X 270.
FINE STRUCTURE OF THE CHICK TENDON
don the fascicles were separated by longitudinally oriented interfascicular cells and increasing amounts of longitudinally oriented collagen fibrils. The synovial cells and the interfascicular cells differed in morphology from the cells of the fascicle (figs. 3 , 4 ) in that their cytoplasm had a lower concentration of rough endoplasmic reticulum. Intracellular microfilaments were also present in the interfascicular cells. The relationship of the interfascicular collagen fibrils to the tendon collagen fibrils is demonstrated in longitudinally sectioned tendon. A tangential section of a fascicle demonstrates longitudinally oriented collagen fibrils lying a t approximately right angles to each other (fig. 5). Comparison of multiple cross-sections with longitudinal sections suggests that the interfascicular collagen fibrils wrap around the fascicle a t approximately right angles to the bulk of the fascicle collagen. 21-day errthryo. The amount of collagen associated with the tendon had increased considerably. The interfascicular cells were more distinguishable from tendon cells than was observed in the 17-day embryo. In these older and larger specimens, slow diffusion of the fixative is believed to be responsible for the swelling of the mitochondria. 22-day, post-hatched. Aside from the large increase in the diameter of collagen fibrils, the major change here was in the appearance of interfascicular cells. The number of microfilaments in these cells had increased from the 17-day embryo to the 22-day post-hatched stage (fig. 6 ) . There was also a n increase in the amount of septa1 cell-associated collagen fibrils which varied considerably in size, as did the collagen fibrils in the tendon (400 A to 2300 A).
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brils with a n occasional immature elastic fiber consisting of 100 A fibrils. Cartilage-like cells. Cells on the surface of the tendon adjacent to the volar sheath resembled cartilage cells (fig. 8). The collagen fibrils lying adjacent to the cells were randomly oriented. Ground substance extended over the surface of the cells, giving the appearance of fibrocartilage (fig. 8 A insert). DISCUSSION
Specialization in the synovial sheath tendon A synovial sheathed tendon has three distinct components: the tendon proper, consisting of longitudinally oriented collagen fibrils and cells, a visceral synovial layer on the tendon, which tends to be circumferentially oriented about the tendon, and a circumferentially oriented fibrous sheath associated with a parietal synovial layer (Greenlee and Ross, '67). When observing a n immature mesenchymal cell population in connective tissue, it is generally impossible to determine what the eventual function of a specific cell will be at maturity. Usually, this determination can be made only after the cell begins secreting its specialized connective tissue product. The four cell types of the chick tendon are: tendon cells, synovial and interfascicular cells, elastin-producing cells of the vincula and cartilaginous cells of the tendon surfaces. Their morphological and functional differences are discussed more fully below. Tendon fibroblast. The tendon fibroblast originates from a n immature mesenchymal cell containing large numbers of free ribosomes and scant rough endoplasmic reticulum (Greenlee and Ross, '67). These cells maintain contact with each other by means of junction sites (Ross and Greenlee, '66). As the cells mature they Specialized cellular adaptations develop a n extensive rough endoplasmic Vincula. I n the electron microscope the reticulum and large Golgi complexes. Howcells of the vincula resembled fibroblasts ever, the presence of endoplasmic reticualthough they produced chiefly elastic fi- lum and a well developed Golgi complex bers (fig. 7 ) . These cells were similar to is not diagnostic of a tendon fibroblast; those described in the ligamentum nuchae rather, i t is the association of a fibroblast of cattle (Greenlee et al., '66). However, with a n extracellular matrix of tightly in this same thin section, a fibroblast of packed, large collagen fibrils, and little else, the tendon was surrounded by collagen fi- which determines the diagnosis. Small,
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T. K. GREENLEE, JR., C. BECKHAM AND D. PIKE
Fig. 3 This electron micrograph from a 17-day embryo shows the synovial space in the upper right hand corner. This is from the intermediate or middle tendon. The synovial cell (sc) contains some fine filaments and appears different morphologically from the tendon cells (tc). The cytoplasm of synovial cells is less dense than that of tendon cells. The tendon collagen (co) is cross-sectioned whereas the interfascicular collagen (arrow) is sectioned longitudinally. X 5,200. Fig. 4 Interfascicular cells (ic) with their longitudinally sectioned collagen (arrow) are similar to the synovial cells in figure 3. The tendon cells ( t c ) contain a rather dense rough endoplasmic reticulum (er). A mitochondrion ( m ) shows the artifacts of less than optimum fixation necessitated by the size of the specimens used for this study. x 7,400.
FINE STRUCTURE OF T H E CHICK TENDON
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Fig. 5 This is a longitudinal section of a tendon from a 17-day embryo. The collagen associated with the tendon proper ( t c ) is obliquely sectioned. The interfascicular collagen ( i c ) is also obliquely sectioned but at 90° to that of the tendon collagen. x 20,500.
poorly developed, elastic fibers are also present in the tendon, although in the adult tendon their concentration is quite low. During development the tendon cells become more spindle-shaped and elongated, and i t has been postulated that muscle tension and growth determine the extent of this elongation (Weiss, ’49). Biochemical studies indicate that the amounts of polysaccharides present in tendons are relatively low (Banga and Balo, ’60). Therefore, since the tendon fibroblast is the predominant cell and probably accounts for the bulk of the secreted material, it is assumed that tendon fibroblasts do not secrete large amounts of acid polysaccharides. Synovial a n d septa1 cells, Studies of joint synovial membranes usually mention two types of synovial cells, “ A and “B”, (Cherney et al., ’70; Barland et al., ’62). Type “ A ”is thought to be phngocytic, while type “B” is believed to be a synthetic cell responsible for the synthesis of lubricating polysaccharides. Henrikson and Cohen
( ’ 6 5 ) , in their study of the development of the chick interphalangeal joint, identified only one synovial cell type, as did studies of the rat flexor digital tendon (Greenlee and Ross, ’67). In the rat, morphologic differences between the tendon fibroblast and its associated synovial cells are differences of a quantitative rather than a qualitative nature. In the chick tendon there appears to be a more definable morphologic difference between these two cell types. Also, it appears that the synovial cells are continuous with the interfascicular cells. It is assumed that the main function of synovial cells is to secrete acid polysaccharides for lubrication, which may also be one function of the interfascicular cells. The fasciculation of the tendon might provide for a much more flexible structure than would occur if all the collagen fibrils in the tendon were bound together. ( A s an example, compare the flexibility of monofilament materials such as nylon with their woven polyfilament counterparts of similar size.)
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T. K. GREENLEE, JR., C. BECKHAM AND D. PIKE
Fig. 6 This electron micrograph of a 22-day post-hatched chicken tendon shows that interfascicular cells have a distinct morphology of their own. These cells have a large number of perinuclear intracellular filaments ( f ) . Note the varying sizes of the interfascicular collagen (ic) and the tendon collagen (tc). x 12,400.
The interfascicular cells of the 22-day post-hatched chick have a unique morphologic appearance. They contain extremely large concentrations of intracellular filaments whose function is not known but, according to Wessels et al. ('71), these filaments probably have a contractile function in non-muscle cells. The functional significance of this increase in microfila-
ment concentration is not understood at this time. Elastic fiber producing cells of the vincula. The vincula which carry the major blood supply to the tendon secrete large elastic fibers (Beckham and Greenlee, '75). This elastic structure may prevent kinking of the blood vessels to this tendon; however, the exact function is unknown.
FINE STRUCTURE OF THE CHICK TENDON
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Fig. 7 This is a n area of a vinculum from a 17-day chick embryo. The large elastic fibers (el), which are identified in niches of cells, are seen to consist of a n amorphous area with only occasional extracellular microfilaments ( f ) . The collagen fibrils (co) are oriented in a random fashion. One of the cells has a rather extensive rough endoplasmic reticulum ( e r ) and a well-developed Golgi apparatus (G), typical of fibroblasts. x 26,000.
Cartilage-like cells. The cartilage-like cells are located on the surface of the tendon where the tendon is in direct contact with the sheath, where synovial cells are
normally observed, These cells are almost cuboid and are much larger than the associated tendon cells. An area of randomly oriented collagen fibrils is located on their
Fig. 8 This composite figure of the flexor tendon in the area of the pulley illustrates cell adaptations. Note in the light micrograph, insert B, the tendon ( t ) and the surface cells on the palmar aspect of the tendon which resemble cartilage cells ( c ) . These cells are directly opposed to the sheath ( s ) which is i n the area of the pulley. The electron micrograph demonstrates the difference between the cartilage cells (ca), with a matrix surrounding their free surface, and the typical visceral synovial cells ( v s ) with no matrix on their surface. Insert A is a higher magnification of an area between the fibro-cartilage cells ( c a ) which demonstrates that the matrix on the surface of the cells is composed of randomly oriented collagen fibrils (co) that come up to the potential space of the tendon sheath (sp). x 5,000. Insert A X 10,800, insert B x 340.
FINE STRUCTURE OF THE CHICK TENDON
surface, Another substance, probably a polysaccharide such as is found in cartilage, is also present. As is characteristic of cartilage, these cells and the matrix they are secreting presumably are better suited for accepting pressure than for bearing tension. The organelles of the cell are qualitatively the same as those in the tendon fibroblast. Functional adaptations. All of the cells constituting the synovial sheathed tendon apparatus appear to evolve from relatively similar mesenchymal cells during the early stages of embryonic development. About the twelfth day, these cells begin to assume individual characteristics for the performance of their specific functions. The tendon fibroblasts lay down large amounts of collagen to transmit the force of the muscle pull. Relatively frictionless motion of the tendon is provided by the synovial cells which secrete a buffer or lubricating fluid between the tendon and its sheath. In certain specialized areas, particularly where blood vessels enter the tendon, the cells surrounding the vessels change their function to secrete large elastic fibers. These fibers may be necessary for the protection and maintenance of the vessels which are subject to unusual stress because of the mobility of the tendon. In other areas where friction occurs between the tendon and its pulleys, the cells on the surface begin to assume the shape and characteristics of cartilage cells, thereby producing a necessary articular surf ace. The development of these structures in areas where they are essential for the functional development of the chick suggests that the forces applied to the cells might determine their ultimate state of differentiation. This study, however, does not prove this hypothesis or rule out other known controls of differentiation. LITERATURE CITED Banga, I., and J. Balo 1960 Isolation of neutral heteropolysaccharide containing mucoprotein from bovine Achilles tendon with the aid of collagenmucoproteinase. Biochem. J., 74: 388-393. Barland, P., A. B. Novikoff and D. Hamerman 1962 Electron microscopy of the human synovial membrane. J. Cell Biol., 1 4 : 207-220. Beckham, C., and T. K. Greenlee, Jr. 1975 The chick vincula - a n elastic structure. Accepted for publication by J. of Anat.
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Brockis, J. G. 1953 The blood supply of the flexor and extensor tendons of the fingers i n man. J. Bone & Joint Surg., 3543: 131-138. Cherney, D. D., W. D. Baxter and L. J. A. DiDio 1970 Synovial membrane of the rabbit knee during a n induced degenerative arthropathy. Acta Anat., 75: 225-247. Fitton-Jackson, S. 1956 The morphogenesis of avian tendon. Proc. R. SOC.(London) B., 144: 556-572. Greenlee, T. K. Jr., R. Ross and J. L. Hartman 1966 The fine structure of elastic fibers. J. Cell Biol., 30: 59-71. Greenlee, T. K., Jr., and R. Ross 1967 The development of the r a t flexor digital tendon, a fine structure study. J. Ultrastruct. Res., 18: 354-376. Henrikson, R. C., and A. S. Cohen 1965 Light and electron microscopic observations of the developing chick interphalangeal joint. J. Ultrastruct. Res., 13: 129-162. Hollinshead, W. H. 1958 Anatomy for Surgeons. Vol. 3. Chap. VI. The Back and Limbs. Paul B. Hoeber, Inc. New York, pp. 449-580. Kessler, I., and F. Nissim 1969 Primary repair without immobilization of flexor tendon division within the digital sheath. Acta Orthop. Scand., 40: 587-601. Lindsay, W. K., and H. G. Thomson 1960 Digital flexor tendons: an experimental study. part I. The significance of each component of the flexor mechanism in tendon healing. Brit. J. Plast. Surg., 12: 289-316. Luft, J. H. 1961 Improvements i n epoxy resin embedding methods. J. Biophy. Biochem. Cytol., 9: 409-414. Mason, M. L., and C. G. Shearon 1932 The process of tendon repair. A n experimental study of tendon suture and tendon graft. Arch. Surg., 25: 615-692. Reynolds, E. S. 1963 The u s e of lead citrate at high pH as a n electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-213. Richardson, K. C., L. Jarett and E. H. Finke 1960 Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol., 35: 313-323. Ross, R., and T. K. Greenlee, Jr. 1966 Electron microscopy: attachment sites between connective tissue cells. Science, 153: 997-999. Salamon, A., and J. Hamori 1966 Present state of tendon regeneration. Light and electron microscopic studies of the regenerating tendon of the rat. Acta Morphol. Acad. Sci. (Hungary), 14: 7-24. Shields, R. T. 1923 O n the development of tendon sheaths. Contrib. Embryol., 15: 55-62. Verdan, C. E. 1972 Half a century of flexortendon surgery. J. Bone Joint Surg., 54-A: 472491. Weiss, P. 1949 Differential growth. In: The Chemistry and Physiology of Growth. A. K. Parpart, ed., Princeton University Press, Princeton, N. J., 135-186. Wessells, N. K., B. S. Spooner, J. F. Ash, M. 0. Bradley, M. A. Ludeunena, E. L. Taylor, J. T. Wrenn and K. M. Yamada 1971 Microfilaments i n cellular and developmental processes. Science, 171: 135-143.
