Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C
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.
Section snippets
Polymer nomenclature
The different formulations of this copolymer system are indicated as follows: a-PEG b:c, where a is the molecular weight of PEG (g/mol), b is the weight percentage of PEGT, and c is the weight percentage of PBT. For example, the polymer 1000-PEG 70:30 has PEG molecular weight of 1000 g/mol and a PEGT–PBT ratio of 70:30.
Processing of polymer culture substrates
Polymer particles were dissolved in chloroform (Sigma, Uithoorn, The Netherlands) and 60–100 μm thick dense films were cast on a glass plate. These substrates were vacuum dried
Inhibition of RhoA expression and modulation of cell shape and actin stress fiber organization by tenascin-C
Cellular actin distribution was observed by binding to fluorescently labeled phalloidin. There was a time and dose-dependent inhibition of cell spreading by soluble Tenascin-C (Fig. 1). When cultured on TCPS for 5 h, p3 chondrocytes were spread in −TN medium, thinly elongated in 35 nM TN conditions, and completely rounded with diffuse actin that was concentrated towards the cell membrane in 70 nM TN (Figs. 1a–c). By 24 h, cells had spread further in the control (TN-free) and 30 nM TN conditions
Discussion
There have been previous reports that examined the effects of substrate chemistry on chondrocyte behavior [27], [42], [43], [44], [45], [46]. We have recently shown that chondrocyte attachment and phenotypic gene expression did not always correlate with substrate wettability, but with differential protein adsorption from serum [47]. Specifically, the unique affinities of vitronectin and fibronectin for different substrates influenced the modalities of cell attachment and downstream gene
Conclusions
Chondrocyte phenotype can be modified by its extracellular environment via differential receptor binding and cytoskeleton organization. Analysis of matrix and focal adhesion components of chondrocytes grown on polymers with different chemistries demonstrated that cell adhesion and its subsequent signaling cascades are responsible for maintenance or loss of the chondrogenic phenotype, an understanding of which is a requisite for engineering substrate-induced cellular response.
Acknowledgment
We would like to thank Dr. J. de Boer for critical review of this paper.
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