Abstract. The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.T o suit their functions, connective tissues must have distinct biomechanical properties that critically depend on the molecular structure of the components of the extracellular matrix as well as their supramolecular assembly. As the principal tensile elements, fibrils play a key role in structural stabilization. The distribution of the diameters and the organization of the fibrils into meshworks, fiber bundles, or highly ordered layers are characteristics of different tissues. For the stabilization of the tissue structure, fibrils require unique surface properties enabling them to specifically interact with themselves as well as other matrix components, such as proteoglycans and matrix glycoproteins. The morphological diversity of connective tissues is paralleled by a variety of the major molecular fibril components, the collagens (25). Recent reports have established the existence of heterotypic fibrils assembled from more than one collagen type (2,12,17,18,20,23,37,38). The interaction of different collagens during fibrillogenesis may well be crucial in the regulation of the fibril architecture and the modulation of the fibril surface properties.Cartilage is unique in that it contains a tissue-specific set of collagens (25); i.e., collagens II, IX, and XI. Collagen X is predominantly found in cartilage but also occurs at low levels in intramembranous bone (31). In cartilage, however, collagen X is restricted to hypertrophic zones and, therefore, may play a role in the transition of cartilage to bone (32). Collagen II is the major fibril component and is similar to collagens I and HI of other tissues in that the molecule essentially consists of a single uninterrupted helical domain 300 nm in length. Collagen II comprises three identical cd(II)-chains. Collagen XI probably is the structural analogue in cartilage to collagen V because, in the tissue form, both proteins contain a large amino-terminal noncollagenous domain in addition to a 300-nm triple helix (4, 26). Collagen XI is a heterotrimer (26). The od(XI)-and tx2(XI)-chains are structurally similar to the or(V)-and ot2(V)-chains (6, 11). Curiously, the ot3(XI)-chain is similar if not identical with an overglycosylated form of the txl(...

Abstract. It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril. major question in cell biology is how individual macromolecules combine to form the often large and complex supramolecular structures of the extracellular matrix. Approaches to this problem include the direct microscopic visualization of tissue sections aided by chemical stains and immunological tools, reconstitution and binding studies with purified components in solution followed by analysis of the resulting products, and, where possible, direct x-ray analysis of highly ordered arrays in situ.This strategy is particularly well exemplified by the many detailed studies of collagen fibrils and fibrillogenesis. This work has resulted in an understanding of the interactions that govern lateral association of fibrillar collagens (for recent reviews see references 3, 7, and 19). The quarter stagger model that arose from these studies has been considerably refined since its introduction, but still remains as the cornerstone of current models as it explains how fibrils formed in vitro from purified collagen molecules give rise to the characteristic staining patterns observed in the electron microscope. Although the fibril staining patterns produced in vitro match those seen in vivo, the diameter regulation of collagen fibrils in vivo and their complexity, including association with other components, especially proteoglycans, are features not reproduced by mixing solutions of collagens in vitro. In summary, two critical questions remain: how is the construction of fibrils regulated in vivo and what role might other molecules play in these processes?This problem is particularly well illustrated in cartilage. The morphology of fibrils reconstituted from purified type II collagen in solution is vastly different from that of cartilage fibrils in situ. Large tactoidal aggregates with d-periodic staining patterns are formed under appropriate reconstitution conditions (21). In contrast, fibrils from chicken embry...

Abstract. Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification.At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability.Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.