Shear and Compression Differentially Regulate Clusters of Functionally Related Temporal Transcription Patterns in Cartilage Tissue

Abstract

Chondrocytes are subjected to a variety of biophysical forces and flows during physiological joint loading, including mechanical deformation, fluid flow, hydrostatic pressure, and streaming potentials; however, the role of these physical stimuli in regulating chondrocyte behavior is still being elucidated. To isolate the effects of these forces, we subjected intact cartilage explants to 1-24 h of continuous dynamic compression or dynamic shear loading at 0.1 Hz. We then measured the transcription levels of 25 genes known to be involved in cartilage homeostasis using real-time PCR and compared the gene expression profiles obtained from dynamic compression, dynamic shear, and our recent results on static compression amplitude and duration. Using clustering analysis, we determined that transcripts for proteins with similar function had correlated responses to loading. However, the temporal expression patterns were strongly dependent on the type of loading applied. Most matrix proteins were up-regulated by 24 h of dynamic compression or dynamic shear, but down-regulated by 24 h of 50% static compression, suggesting that cyclic matrix deformation is a key stimulator of matrix protein expression. Most matrix proteases were up-regulated by 24 h under all loading types. Transcription factors c-Fos and c-Jun maximally responded within 1 h to all loading types. Pre-incubating cartilage explants with either a chelator of intracellular calcium or an inhibitor of the cyclic AMP pathway demonstrated the involvement of both pathways in transcription induced by dynamic loading.Mechanical forces within synovial joints are known to influence the metabolic behavior of chondrocytes, the sole cell type of cartilage (1-3). Chondrocytes are responsible for the maintenance and turnover of the cartilage extracellular matrix, in particular the biosynthesis of type II collagen and proteoglycan (aggrecan), which in combination provide the tensile, shear, and compressive stiffness of cartilage. Cartilage thickness and proteoglycan content were found to be enhanced in load-bearing areas in vivo (4). Joint inactivity (1, 2) and injury (5) have been found to promote degradation of the extracellular matrix and to reduce cartilage load bearing capacity. The exact mechanisms by which mechanical forces influence the biological activity of chondrocytes are under intense study. In addition, the precise component(s) of the physical stimuli that induce such biological changes following joint movement are not known.The effects of mechanical loading on cartilage in vivo have been studied using intact human and animal cartilage explants in vitro to determine the precise biophysical forces and physicochemical changes that result (for reviews, see Refs. 6 and 7

An integrated microfluidic device for characterizing chondrocyte metabolism in response to distinct levels of fluid flow stimulus

“…Of note, the extremely low shear stress comparable to interstitial flow level showed remarkable effects on chondrocytes than static condition, suggesting that interstitial flow has an important regulatory role in chondrocyte metabolism. In healthy cartilage, interstitial flow is able to transmit the state of cell surroundings and is beneficial for stability of extracellular microenvironments and matrix production (Fitzgerald et al 2006). Nevertheless, in current condition, mass transport effects cannot be separated from the mechanical effects of fluid flow.…”

Regulation of Chondrocytic Gene Expression by Biomechanical Signals

“…14 Dynamic compression has been shown to upregulate expression of anabolic genes such as ACAN, COL2 α1 (COL2A1), and tissue inhibitor of metalloproteinase 3 (TIMP3) 15 while downregulating specific genes of the matrix metalloproteinase (MMP) family. 4,9,15,16 More importantly, compressive forces at low magnitudes have been shown to be antiinflammatory in nature. This is evidenced by the findings that when chondrocytes seeded into agarose hydrogel scaffolds were stimulated by exogenous interleukin-1β (IL-1β), sinusoidal form compression at 15% strain suppressed aggrecanase 1 (a disintegrin-like and metalloprotease domain (reprolysin-type) with thrombospondin type I motifs 4 [ADAMTS4]) and aggrecanase 2 (ADAMTS5) but not MMP3 gene expression.…”

“…16,24 Contrary to dynamic loading, static compression induces an initial accumulation of interstitial hydrostatic pressure within the cartilage; however, this pressure reaches an equilibrium stasis due to stress relaxation of the tissue. 3 Static compression invariably downregulates anabolic gene expression 4,12,16 and upregulates catabolic (MMP3, MMP9, MMP13, and ADAMTS4) 4 and inflammatory (tumor necrosis factor-α [TNF-α; TNFA], COX2/PTGS2, iNOS/NOS2A). 4,6 gene expression.…”

“…3 Static compression invariably downregulates anabolic gene expression 4,12,16 and upregulates catabolic (MMP3, MMP9, MMP13, and ADAMTS4) 4 and inflammatory (tumor necrosis factor-α [TNF-α; TNFA], COX2/PTGS2, iNOS/NOS2A). 4,6 gene expression. Murata et al 6 have shown that static compression activates the IL-1 signaling pathway by upregulating IL-1α, IL-1β, and NOS2A gene expression in the presence and absence of IL-1 receptor antagonist (Fig.…”

“…Additionally, the biomechanical signals received by the viscoelastic ECM are transduced and propagated to the resident chondrocytes. The molecular effects of compressive forces on gene expression in chondrocytes has been studied using several different models, largely ex vivo, including cartilage explants [3][4][5][6][7][8] and in vitro systems such as agarose, 9-11 alginate, 5 and other polymeric scaffolds. [12][13][14] Although the cartilage explant tissue models provide exceptional physiologic simulation of in vivo biomechanics, the tissue-level environment and signaling complexity creates challenges for dissecting the individual molecular responses to the applied compressive forces.…”