Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation
Introduction
The articulating ends of joint surfaces are covered by articular cartilage, which acts as a load-bearing surface that transmits applied forces to the underlying bone. Damage to this tissue from disease or trauma is of particular concern due to the limited ability of articular cartilage to self-repair. Attempts to treat damaged cartilage have met with limited success (Darling and Athanasiou, 2003, Hunziker, 2002). Repair of joint defects using in vitro-formed cartilage tissue is an alternative treatment, but the physical properties of this tissue are a fraction of native cartilage, which may affect its ability to successfully resurface a large joint defect in the long term. We and others have developed methods of applying compressive forces that mechanically stimulate chondrocytes and result in increased matrix accumulation and improved tissue formation (Lima et al., 2004, Mauck et al., 2003, Waldman et al., 2003, Waldman et al., 2005). However, the molecular mechanism regulating this physical improvement has not been determined.
One possible explanation is that mechanical stimulation is inhibiting matrix metalloprotease (MMP) expression, resulting in increased matrix accumulation. Transcriptional regulation of most MMPs is predominately controlled by DNA binding activity at the AP-1 consensus sequence. AP-1 sites are found in most MMPs, except for MMP-2 and MMP-10 (Crawford and Matrisian, 1996)and MT-MMPs (Westermarck and Kahari, 1999). The AP-1 transcription factor is a protein composed of Jun family homodimers (cJUN, JunB, JunD) or Jun/Fos (cFOS, FRA-1, FRA-2, FOS-b) heterodimers, that bind to the AP-1 site (Hai and Curran, 1991, Smeal et al., 1989), thus upregulating the expression of MMP. Activation of cJUN is through phosphorylation by SAPK/JNK whereas cFOS is activated by phosphorylation by the ERK1/2 pathway (Monje et al., 2003). In rat bladder smooth muscle cells, mechanical stretch has been shown to upregulate AP-1 binding activity and alter MMP expression which correlates with the development of bladder hypertrophy (Park et al., 1999). Alternatively, mechanical loading may increase chondrocyte gene expression of the matrix molecules, aggrecan and collagen. AP-1 sites have not been identified in the promoter regions of either type II collagen or aggrecan so regulation of these macromolecules is quite different than the MMPs. For example, type II collagen and aggrecan expression is regulated by several transcription factors including zinc finger Sp-1/Sp-3 (Chadjichristos et al., 2003, Tan et al., 2003), sox 5,6 and 9 (Lefebvre et al., 1998) and the Snail family (Seki et al., 2003). In other studies, mechanical stimulation has been shown to alter expression of these different genes and their corresponding protein but the ultimate response appears to be influenced by the type and duration of the mechanical force applied (Benjamin and Hillen, 2003).
The aim of this study was to determine the mechanism(s) by which cyclic compressive loading leads to increased matrix accumulation by chondrocytes. The profiles of metalloproteases (MMP 1, 2, 3, and 13) which have been implicated in cartilage degradation and collagen type II and aggrecan gene expression were examined post-stimulation and the signal transduction pathway activated by this mechanical loading was assessed. Understanding the mechanism(s) resulting in increased matrix accumulation may identify a way to further improve the properties of in vitro-formed cartilage. In addition it may allow a better understanding of the mechanism(s) regulating matrix production by chondrocytes, a poorly understood process.
Section snippets
Effect of mechanical stimulation on matrix and metalloprotease gene expression
As the increase in matrix accumulation with stimulation could be due to increased expression of matrix genes, and/or a decrease in expression of metalloproteases, the chondrocytes were examined for changes in levels of type II collagen, aggrecan and metalloproteases (1, 2, 3, and 13) mRNA at various times post-stimulation. Cyclic compression of in vitro grown cartilage tissue on a porous calcium polyphosphate (CPP) construct (Fig. 1) induced a sequential change in catabolic and anabolic genes.
Discussion
In this study, a single application of uniaxial, confined cyclic compression of chondrocytes within the first three days of culture resulted in activation of the ERK1/2 and JNK MAPKs, increased AP-1 binding, followed by a sequential increase in catabolic and anabolic gene sets. There was a transient upregulation of MMP-3 and MMP-13 gene expression by 2 h, which corresponded with an increase in active MMP-13 protein and evidence of matrix degradation as shown by increased release of matrix
Mechanical stimulation
Chondrocytes were isolated by sequential enzymatic digestion from bovine (6 to 9 month old animal) metacarpal-phalangeal joints as described previously (Boyle et al., 1995). Cells for each experiment were obtained from one to two animals and pooled to obtain sufficient cells for the experiment. Chondrocytes were seeded in Ham's F-12 supplemented with 25 mM HEPES and 5% fetal bovine serum (SIGMA) at a density of 160,000 cells/mm2 onto the surfaces of calcium polyphosphate (CPP) substrates. The
Acknowledgements
The authors wish to acknowledge Ryding-Regency Meat Packers and Mr. H. Bojarski for providing tissue samples, Dr. Jason Hong for manufacturing CPP and Dr. Jian Wang for the mechanical stimulation. This work was supported by CIHR, The Arthritis Society, NSERC and a CIHR training grant to JNAD.
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