The fine structure of elastic fibers.

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.

Electron microscopy: attachment sites between connective tissue cells.

Regions of attachment have been observed between connective tissue cells from four different structures: fibroblasts in embryonic and fetal tendons, fibroblasts in fetal ligamentum nuchae, odontoblasts, and osteoblasts. Morpho logically these sites appear to be focal and to consist of an approximation of the plasma membranes of adjacent cells to within approximately 200 A. In the region of approximation both the extracellular space and the cytoplasm adjacent to the plasmalemma are increased in density. We have postulated a role for these sites in the maintenance of structural integrity.

Improved three-year disease-free survival in osteogenic sarcoma.

Of 41 consecutive patients with newly diagnosed osteogenic sarcoma admitted to the Children's Orthopedic Hospital and Medical Center in Seattle, Washington, between 1952 and 1977, 19 treated before 1973 did not receive adjunctive chemotherapy (histological group) whereas after 1972 22 have been so treated (chemotherapy group). Chemotherapy consisted primarily of high doses of methotrexate and adriamycin for 16 months after surgical treatment. Patients in the historical group have been observed for a minimum of nine years (six patients) or until death (13 patients). The 13 surviving patients in the chemotherapy group have been followed for a minimum of three years (median five years) and all 12 disease-free patients have been off therapy for between one and a half and five and a half years (median three years). Overall, the chemotherapy group has had a significant increase in both survival (p = 0.03) and disease-free survival (P = 0.02) compared to the historical group. In 35 patients with localised disease at diagnosis, the three-year disease-free survival and the three-year survival rates were 18 per cent and 41 per cent respectively in the historical group, and 67 per cent and 78 per cent (life table estimates) respectively in the chemotherapy group. With adjunctive chemotherapy only one of the seven patients developing pulmonary metastases did so later than nine months after diagnosis. The superior results in the chemotherapy group could not be accounted for by differences in age, sex, presence of metastases at diagnosis, histopathology, location of primary tumour, type of initial or subsequent surgical treatment, or the use of standard or computerised lung tomography. Although the use of historical controls in this study does not exclude other changes as contributing to the observed improvement in outcome, our data support the contention that adjunctive chemotherapy improves both the disease-free survival and the overall survival of patients with osteosarcoma and rarely delays the onset of recurrent or metastatic disease.

Chick vincula: elastic structures with a check-rein mechanism.

From their mode of attachment and their elastic composition, it is clear that the vincula of the chick serve other functions besides that of carrying blood vessels to the digital flexor tendons within their synovial sheaths. Evidence is presented in support of the argument that elastic fibres bear the brunt of rapidly applied tensile forces and that the interweaving collagen fibres only become taut when the vincula are stretched to the limit and about to tear. Our hypothesis is that the collagen serves as a check-rein mechanism in an otherwise elastic structure.

The development of human digital tendons.

The development of human digital flexor and extensor tendons from 40 days to 112 days of gestation is described. The differentiation of the cell, the formation of collagen fibrils, and their organization into a relatively complex tendon organ are described. This word was supported by a grant from the Graduate Research Fund of the University of Washington. (Materials were obtained from the laboratory for human embryos which is supported by N.I.H. Grant no. HD00836.) We would like to thank Mrs Bea Watts for secretarial assistance.

A fine structural study of the development of the chick flexor digital tendon: a model for synovial sheathed tendon healing.

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.

The role of movement in the development of a digital flexor tendon.

D-tubocurarine was injected into the air sac of 8-day chick embryos to prevent movement of the digits of the hind limb. The embryos were paralyzed from the tenth to the eighteenth day, when the experiment was terminated. The immobilization of the flexor digitorum profundus tendons in the tarsus resulted in a loss of specialized structures around and on this tendon, as determined by light and electron microscopy. Specialized areas observed in the normal chick (synovial cavity, fibrocartilaginous area, and elastic vinculum) failed to form, as a result of the paralysis of the digit. Several authors have shown previously that movement is a requirement for the molding and maintenance of joint cavities in vivo, in ovo and in vitro (see text for references). We have shown that movement of the tendon is required to produce a functional tendon apparatus in the embryo and predict that movement is also required for regeneration after injury.

Collagen gene expression during development of avian synovial joints: transient expression of types II and XI collagen genes in the joint capsule.

The developmental sequence of the embryonic joint has been well studied morphologically. There are, however, no definitive studies of cell function during joint development. In order to begin to understand the differentiation events that contribute to joint formation, we examined the expression of collagen mRNAs encoding types I, IIA, IIB, and XI. In situ hybridization was performed on chicken embryo hind limb buds and digits from day 7 to day 18 (Hamburger and Hamilton stages 31-44). In the day 7 (stage 31) limb bud, there was a condensation of mesenchyme forming the primitive tarsal and metatarsal bones that showed abundant expression of type IIA procollagen message, but no type IIB or type alpha 1(XI) message. By day 8 (stage 33), co-expression of types IIA, and type XI procollagen mRNAs was observed in the condensations, with expression of IIB restricted to early chondrocytes with metachromatically staining matrix. At this stage, DNA fragmentation characteristic of apoptosis was observed in cells near the midline of the interzone region between the developing anlagen, and in areas between and around the individual digits of the paddle. The presumptive apoptotic cells were more numerous at day 9 (stage 35), and were not found in the developing joint at subsequent time points, including the initiation of spatial cavitation of the joint. From days 11-18, type IIA procollagen mRNA was expressed in flattened cells at the surface of the anlagen, and in the perichondrium and in the developing joint capsule; type IIB mRNA message was found only in chondrocytes. Type XI mRNA was expressed by all type II-expressing cells. Alpha 1(I) mRNA was expressed early by cells of the interzone and capsule, but as cavitation progressed, the type I expressing cells of the interzone merged with the superficial layer of the articular surface. Thus, at the time of joint cavitation, there was a distinct pattern of expression of procollagen messages at the articular surface, with type I being outermost, followed by morphologically similar cells expressing type IIA, then chondrocytes expressing type IIB. The progenitor cells expressing type IIA message define a new population of cells. These cell populations contribute to the molecular heterogeneity of the articular cartilage, and these same populations likely exist in the developing joints of other species. The transient transcription of type II and type XI collagen genes, characteristic of chondrocytes, by cells in the joint capsule demonstrates that these cells may have chondrogenic potential.

A low-serum medium for tendon cells: effects of growth factors on tendon cell growth and collagen production.

Primary avian tendon cells maintain a higher percentage of net collagen synthesis when cultured in low serum concentrations than in high. However, under these conditions the cells grow slowly and can only be subcultured one or two times. We therefore examined various growth factors for their effects on tendon cell multiplication in order to develop a new medium for these cells. Of the growth factors tested, epidermal growth factor, insulin, transferrin, and selenium each stimulated tendon cell division. We also investigated how these factors affected collagen synthesis, and found that they all increased both collagen and noncollagen protein synthesis equally, thus leaving the percentage of protein synthesis devoted to collagen the same. When combined with 0.2% fetal bovine serum in Ham's F12 medium, epidermal growth factor and insulin together stimulated cell multiplication to a level comparable to that of cells grown in Ham's F12 plus 10% serum. Cells could also be successfully subcultured in this medium. Thus, by using selected growth factors we have reduced significantly the serum requirements of cultured tendon cells without affecting population doubling time or subculture capability. This low-serum medium should prove useful for the study of the regulation of collagen biosynthesis.

Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells.

Fibroblasts of the synovium of sheathed tendons were isolated, and their biochemical properties were compared with those of the fibroblasts of the remaining tendon. The synovial cells had a lower attachment efficiency than did the tendon cells. On the day of cell isolation the synovial cells synthesized collagen as 10% of their total protein, whereas the tendon cells synthesized 30% collagen. After growth in fetal bovine serum (FBS), the percentage of collagen synthesized by both populations decreased; however, the synovial cells still made less collagen than did the tendon cells (5 versus 11%). On the basis of cyanogen bromide peptide analysis, the synovial cells were found to synthesize Types I and III collagen in primary culture, whereas the tendon cells synthesized only Type I. The synovial cells also synthesized two to three times less sulfated glycosaminoglycans in culture than did the tendon cells. Thus, the two cell populations differed in attachment efficiency and in their biosynthesis of collagen and sulfated glycosaminoglycans. These differences reflect extracellular matrix differences that have been observed in the tendon in vivo. In addition, the results augment existing data showing that not all fibroblasts have identical phenotypes.

Magnetic resonance imaging of soft tissue masses.

Twenty-nine soft tissue masses were studied with magnetic resonance imaging (MRI) which proved to be useful in the preoperative evaluation of these lesions. Other imaging modalities employed had significant limitations. Plain films were of little value because of the intrinsically low contrast of soft tissues. Angiography was not necessary unless MRI suggested a vascular lesion or proximity to major blood vessels. Computed tomography (CT) and MRI both readily identified fatty lesions and their relationship to adjacent structures. Some soft tissue tumors could not be delineated from normal muscle with CT, but were easily seen with MRI. MRI is ideally suited for the study of suspected soft tissue tumors because of its excellent soft tissue contrast and its ability to image directly in any plane. Optimum evaluation required imaging in at least two planes with spin echo sequences chosen to bring out both T1 and T2 features.