Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C

Abstract

Chondrocyte ‘dedifferentiation’ involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and intracellular proteins that are involved in signal transduction pathways, including those associated with cell shape and actin microfilament organization. The objective of this study was to examine the modulation of chondrocyte phenotype by cultivation on polymer substrates containing poly(ethylene glycol) (PEG). We observed differential arrangement of actin organization in articular chondrocytes, depending on PEG length. When cultivated on 300 g/mol PEG substrates at day 19, chondrocytes had lost intracellular markers characteristic of the differentiated phenotype, including type II collagen and protein kinase C (PKC). On these surfaces, chondrocytes also expressed focal adhesion and signaling proteins indicative of cell attachment, spreading, and FA turnover, including RhoA, focal adhesion kinase, and vinculin. The switch to a dedifferentiated chondrocyte phenotype correlated with integrin expression. Conversely, the expression of CD44 receptors coincided with chondrogenic characteristics, suggesting that binding via these receptors could play a role in maintaining the differentiated phenotype on such substrates. These effects can be similar to those of compounds that interfere in intracellular signaling pathways and can be utilized to engineer cellular response.

Introduction

The characteristic phenotype of differentiated chondrocytes is that of rounded cells that secrete extracellular matrix proteins (specifically collagen II and aggrecan) and with a diffuse actin microfilament network [1], [2]. Upon attachment to substrates in two dimensions however, chondrocytes have frequently been observed to attain a spread morphology with a reorganization of filamentous actin into distinct stress fibers [3], [4], [5]. During this dedifferentiation towards a more fibroblastic phenotype, type II collagen production is reduced and eventually replaced with type I collagen, with concomitant reduction or cessation of aggrecan synthesis [3], [4].

A number of researchers have investigated techniques to reexpress the chondrogenic phenotype during chondrocyte expansion in monolayer culture by growing cells on microcarriers [6] using growth factors, such as basic fibroblast growth factor (bFGF-2) [7], [8] or incorporating cytoskeleton modifying drugs such as cytochalasin D or dihydrocytochalasin B [5], [9], [10], [11], [12], [13], [14], [15], [16]. And although there have been reports discussing cell behavior on different substrates [17], [18], [19], [20], [21], there has been little in the way of mechanistically addressing the influence of materials on the events that regulate cellular phenotype. Understanding such behavior has become increasingly relevant, not only for investigative cell science but because of the possibilities for cell transplantation and tissue engineering using polymers as cell delivery devices [22].

Given the importance of understanding cell response to biomaterials, we have attempted to answer the following question: How do substrate properties influence cell behavior? In addition, what substrates elicit the expression of intracellular signaling proteins that are involved in the maintenance of the differentiated chondrogenic phenotype?

To that end, we used block copolymers of poly(ethylene glycol) terephthalate (PEGT) and poly(butylene terephthalate) (PBT) as model substrates for cell attachment and growth. The overall copolymer properties are determined by its two components—the PEG segment is hydrated, whereas PBT provides hydrophobic, protein binding domains, and stiffness. These copolymers can be synthesized with different fractions of soft and hard components, as well as with PEG chains of different molecular weights. These were shown to be biocompatible [23], [24], [25] and have been studied extensively for tissue engineering applications [25], [26], [27], [28]. The ability to synthesize polymers with controllable properties makes them useful model substrates for investigating cell behavior.

Focal adhesions (FAs) are the primary sites by which cells detect their substrate. They are plasma membrane clusters of receptors, cytoskeletal proteins, and other proteins that link the transmembrane receptors to the cytoskeleton [29], [30]. They form the foci of signal transduction from the external substrate-dependent microenvironment to cells (outside–in signaling), as well as of feedback signals transmitted by cells to their external milieu (inside–out) [31], [32], [33]. It has also been suggested that FAs are sites where mechanical stress is converted to biochemical signals [34], [35]. Thus, the signaling mechanisms in play at, or due to FAs, are crucial in determining cell fate, especially as it relates to differentiation or proliferation. Given the complexities of intracellular signaling and cross-talk between pathways, we have used a candidate approach to examine the expression of selected proteins that are known to be involved in general FA formation and turnover, as well as those associated with the maintenance of differentiated chondrogenic characteristics.

We also studied the effects of blocking the activation of RhoA by tenascin-C (TN). RhoA is involved in cytoskeletal regulation [36], [37], a feature we exploited to probe how interference in RhoA signaling by using soluble TN (an exogenous matrix protein) or varying substrate chemistry can affect stress fiber assembly and FA formation, thereby modulating chondrocyte differentiation.

The objective of this study was to examine the differential expression of FA receptors and intracellular signaling proteins involved in preserving the chondrogenic phenotype, after varying polymer substrate composition and by using an exogenous compound to interfere in cell signaling.