Effects of TGF-β1 and IGF-I on the compressibility, biomechanics, and strain-dependent recovery behavior of single chondrocytes
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.
References (40)
- et al.
Rapid phenotypic changes in passaged articular chondrocyte subpopulations
Journal of Orthopaedic Research
(2005) - et al.
The mechanical environment of the chondrocyte: a biphasic finite element model of cell–matrix interactions in articular cartilage
Journal of Biomechanics
(2000) - et al.
The effects of osmotic stress on the viscoelastic and physical properties of articular chondrocytes
Biophysical Journal
(2002) - et al.
Biosynthetic response and mechanical properties of articular cartilage after injurious compression
Journal of Orthopaedic Research
(2001) - et al.
Unconfined creep compression of chondrocytes
Journal of Biomechanics
(2005) - et al.
The effects of TGF-beta1 and IGF-I on the biomechanics and cytoskeleton of single chondrocytes
Osteoarthritis and Cartilage
(2006) - et al.
The beneficial effect of delayed compressive loading on tissue-engineered cartilage constructs cultured with TGF-beta3
Osteoarthritis and Cartilage
(2007) - et al.
Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis
Archives of Biochemistry and Biophysics
(2000) - et al.
Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration
Journal of Biomechanics
(2006) - et al.
In vitro measurement of articular cartilage deformations in the intact human hip joint under load
Journal of Bone and Joint Surgery America
(1979)
Comparative study of the intrinsic mechanical properties of the human acetabular and femoral head cartilage
Journal of Orthopaedic Research
Biomechanical properties of hip cartilage in experimental animal models
Clinical Orthopaedics and Related Research
Biomechanical topography of human articular cartilage in the first metatarsophalangeal joint
Clinical Orthopaedics and Related Research
A direct compression stimulator for articular cartilage and meniscal explants
Annals of Biomedical Engineering
Insulin-like growth factor (IGF-I) induces unique effects in the cytoskeleton of cultured rat glomerular mesangial cells
Journal of Histochemistry and Cytochemistry
TGF beta 1 promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture
Journal of Cell Science
Perspectives on chondrocyte mechanobiology and osteoarthritis
Biorheology
Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes
Journal of Cellular Physiology
Transforming growth factor-beta-induced mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA
Molecular Biology of the Cell
Chondrocyte cells respond mechanically to compressive loads
Journal of Orthopaedic Research
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