Three-dimensional culture of human meniscal cells: extracellular matrix and proteoglycan production

Abstract

Background: The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-beta (TGF-beta).

Methods: Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-beta. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen.

Result and conclusion: Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-beta (2.48 microg/ml +/- 1.00, mean +/- S.D., vs control levels of 1.58 +/- 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology.

Figure 1

Low confluence human meniscus cells are spindle-shaped in monolayer culture. Note long processes which extend from the cells. (Hoffman modulation contrast image; bar = 10 μm).

Figure 2

Histologic images of two micromass cultures formed by human meniscus cells. Note the presence of ECM rich in proteoglycans as indicated by the pink stain. (Paraffin-embedded micromass nodule, stained with toluidine blue; bar = 50 μm).

Figure 3

Histologic images of human meniscus cells cultured under control conditions in the 3D collagen sponge. A. Arrows show good attachment of cells to the sponge surface. B. * denotes concentration of ECM. C. Margin shows a tight layer of cells along the perimeter of the sponge. (Paraffin embedded toluidine-blue stained specimens; for all images, bar = 50 μm).

Figure 4

Histologic features of human meniscal cells cultured in 3D collagen sponge in the presence of TGF-β. A, note the high cell density; B, note accumulations of ECM marked by *. (Paraffin embedded sections stained with toluidine blue; for both images, bar = 50 μm).

Figure 5

Immunolocalization of major ECM components in control and TGF-β-treated meniscal cells in 3D culture. The top row presents images from control cells; the second row presents images for TGF-β-treated cells. Chondroitin sulfate (CS): A: control cells show modest CS content in the ECM. B: ECM formed by TGF-β-treated cells shows more abundant CS compared to controls. Type I collagen (Col type I) is abundant in both ECM formed by control (C) and TGF-β-treated cells (D). Type II collagen (Col type II): Control cells produced less type II collagen (E) when compared to ECM from TGF-β-treated cells (F). Figure 5G presents a representative negative control specimen. (For all images, bar in G = 50 μm).