Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts.
Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.
Schlumberger
W
W
Institut für Arterioskleroseforschung an der Universität Münster/Bundesrepublik Deutschland.
Thie
M
M
Rauterberg
J
J
Kresse
H
H
Robenek
H
H
D000464:Alginates; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000648:ultrastructure*; D005109:Extracellular Matrix / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure*; D005347:Fibroblasts; D005782:Gels; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D011509:Proteoglycans / Q000032:analysis* / Q000096:biosynthesis / Q000648:ultrastructure
D000464:Alginates; D005782:Gels; D011509:Proteoglycans; D003094:Collagen