Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a three-dimensional extracellular matrix synthesized by human diploid fibroblasts in vitro.

Development and maturation of an extracellular matrix, synthesized by human gingival fibroblasts, have been studied microscopically. Pairs of demineralized, fibronectin-coated slices of human tooth root, 300 micron thick, were placed on confluent cell layers, defining a 0.5 mm wide space. The cultures were grown under standard conditions with ascorbic acid (50 micrograms ml-1) added daily. At various times up to 13 weeks, the cultures were fixed and the samples prepared for light and electron microscopy. Cells from the monolayer became attached to, and migrated up, the vertical root surface and, during the time studied, completely filled the space between the root slices with an extracellular matrix. A close association was seen between the cell membrane and collagen fibres in the demineralized surface initially. A thin layer of fibrillar material was deposited between the cell and the vertical surface, and eventually an extracellular matrix surrounding the cells and attaching to the root surface was present. Samples fixed in the presence of Ruthenium Red showed intense staining of the fibrillar material, indicating the presence of anionic molecules. Additional cells migrated onto the newly synthesized matrix and up the root surface. Growth of the fibrillar networks on either side, horizontally and vertically, continued and, eventually, an extracellular matrix attaching to the vertical surfaces completely filled the previously empty space. Immunocytochemical staining showed that the matrix contained hyaluronic acid, chondroitin sulphate, dermatan sulphate and fibronectin at this time. Collagen fibres were observed at 6 weeks, and at later times collagen types I, III and V were the primary matrix components. The fibroblasts attaching to the root slice and those present at the edge of the matrix had an elongated, polar form. The cells within the matrix frequently showed a stellate appearance with numerous extended processes, in contact with fibrillar material or collagen fibres. Fibroblast processes were at later times seen to enclose bundles of collagen fibres and to mediate cell-to-cell contact, occasionally via desmosome-like structures. The structure and composition of the matrix and the appearance and apparent behaviour of the cells were similar to that observed in the healing wound. This system thus could provide a model for studying various aspects of regeneration of extracellular matrix.

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system.

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1 beta and transforming growth factor-beta.

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1 beta and TGF-beta increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1 beta; 1 ng/ml TGF-beta) and time-dependent (IL-1 beta 12-hr peak; TGF-beta 24-hr peak). Effect of IL-1 beta and TGF-beta on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with 125I-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1 beta (4.7 x 10(6)/cell) and TGF-beta (3.9 x 10(6)/cell)-treated cells, compared to control (3.0 x 10(6)/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1 beta and TGF-beta have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q.