Effects of TGF-β1 and IGF-I on the compressibility, biomechanics, and strain-dependent recovery behavior of single chondrocytes

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Abstract

The responses of articular chondrocytes to physicochemical stimuli are intimately linked to processes that can lead to both degenerative and regenerative processes. Toward understanding this link, we examined the biomechanical behavior of single chondrocytes in response to growth factors (IGF-I and TGF-β1) and a range of compressive strains. The results indicate that the growth factors alter the biomechanics of the cells in terms of their stiffness coefficient (∼two-fold increase over control) and compressibility, as measured by an apparent Poisson's ratio (∼two-fold increase over control also). Interestingly, the compressibility decreased significantly with respect to the applied strain. Moreover, we have again detected a critical strain threshold in chondrocytes at ∼30% strain in all treatments. Overall, these findings demonstrate that cellular biomechanics change in response to both biochemical and biomechanical perturbations. Understanding the underlying biomechanics of chondrocytes in response to such stimuli may be useful in understanding various aspects of cartilage, including the study of osteoarthritis and the development of tissue-engineering strategies.

Introduction

The biological responses of articular cartilage to mechanical forces play important roles in its normal physiology and disease (Buckwalter et al., 2006). This phenomenon, known as mechanotransduction, may have important ramifications toward understanding and treating debilitating musculoskeletal ailments, including osteoarthritis. For example, researchers have shown both beneficial and detrimental effects of mechanical forces on tissue explants and engineered tissues, using modalities such as direct compression (Aufderheide and Athanasiou, 2006; Ng et al., 2006; Lima et al., 2007), shear (Smith et al., 2004, Smith et al., 1995), and hydrostatic pressure (Smith et al., 2004; Hu and Athanasiou, 2006). Currently, the understanding of what elicits these different responses remains incomplete.

Particularly, vexing is the connection between the cellular biomechanics and biological processes. Toward understanding this vital interplay, it is important to characterize the biomechanics of the chondrocyte and how they change in response to physicochemical stimuli, such as growth factors and mechanical perturbations, as previously discussed (Guilak and Mow, 2000; Guilak et al., 2006, Guilak et al., 2002; Shieh and Athanasiou, 2002, Shieh and Athanasiou, 2005; Koay et al., 2003; Leipzig and Athanasiou, 2005; Leipzig et al., 2006; Shieh et al., 2006). Establishing a basic understanding of the mechanical nature of the chondrocyte aids theoretical models of cartilage that consider the cell (Guilak and Mow, 2000) and helps the development of new methodologies to study chondrocyte mechanobiology, such as a ‘single-cell’ approach (Shieh et al., 2006). This approach entails the use of instruments that can apply discreet forces to single adherent chondrocytes, such as the cytoindenter (Shin and Athanasiou, 1999; Koay et al., 2003), as well as the assessment of a cellular response, such as cartilage-relevant gene expression (Shieh and Athanasiou, 2006). Studies of single-cell biomechanics help to direct these efforts by providing information on the cellular characteristics of time constants for recovery, compressibility, and mechanical thresholds.

For example, when a possible change in chondrocyte mechanical behavior was identified between 25% and 30% compressive strain (termed the critical strain region), it was postulated that this represented a threshold akin to yield strain. The significance of this critical region remains unclear. However, it is possible that permanent damage may be incurred by the chondrocyte beyond this region. This damage may occur due to high strains or strain accumulation after repeated loading (Shieh et al., 2006), suggesting that this critical region would have important implications in directing appropriate in vitro mechanical stimulation regimens as well as for studies of mechanical damage to cartilage. While the cause of this critical region also remains uncertain, it is possible that cytoskeletal components, such as F-actin, rearrange or break down at high strains.

Considering that TGF-β1 and IGF-I increase F-actin levels and thereby increase cell stiffness to two-fold over control (Leipzig et al., 2006), in this study, we tested the hypothesis that these same growth factors would alter the critical strain region. We were also motivated to investigate how these growth factors and mechanical strains affected the compressibility of the cell, as measured by an apparent Poisson's ratio, since current computational models of cartilage assume this characteristic to be constant. Although we have previously observed that the apparent Poisson's ratio does not change with respect to increasing strains (Shieh et al., 2006), we hypothesized that the growth factors, due to their ability to reorganize the cytoskeleton (Gagelin et al., 1995; Boland et al., 1996; Berfield et al., 1997) and alter cellular mechanics (Leipzig et al., 2006), would increase the average apparent Poisson's ratio of the cells compared with control. We also expected that the growth factors would increase cell volume due to these effects on the cytoskeleton. Cell volume and shape are of interest since they are intimately tied to cell function (Urban et al., 1993; Bush and Hall, 2001; Guilak et al., 2002; Kerrigan et al., 2006). To test these hypotheses, single chondrocytes were exposed to IGF-I, TGF-β1, or no growth factors, and cells in each group were subjected to a range of compressive strains from 5% to 60% and their morphology, compressibility, stiffness, and recovery behavior were analyzed.

Section snippets

Methods

The basic protocols for this experiment were based on our previous work (Shieh et al., 2006). These methods and a few improvements are briefly explained below. Cell culture supplies were obtained from Invitrogen (Carlsbad, CA, USA) unless specified otherwise.

Results

In this section, treatments will be referred to as control, IGF-I, and TGF-β1. A total of 83 cells were tested and analyzed for this study (26 for control, 26 for IGF-I, and 31 for TGF-β1). Fig. 2 demonstrates how cells were analyzed.

Discussion

TGF-β1 and IGF-I are widely studied in cartilage research; understanding their effects on the biomechanics and recovery behavior of the chondrocytes can help to develop mechanical stimulation regimens of cartilage and may be relevant to the health and disease of cartilage. Additionally, it is important to understand how cellular biomechanics change with direct compressive strains, since this modality of mechanical stimulation influences cellular processes (Shieh et al., 2006). This study offers

Conflict of interest

None.

Acknowledgments

We gratefully acknowledge support from the NSF (traineeship for E.J. Koay, DGE-0114264), NIH (traineeship for G. Ofek, 5 T90 DK70121-03 and 5 R90 DK71504-03), and an Osteoarthritis Biomarkers Biomedical Science Grant from the Arthritis Foundation.

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