The effect of sliding velocity on chondrocytes activity in 3D scaffolds

Abstract

Sliding motion and shear are important mediators for the synthesis of cartilage matrix and surface molecules. This study investigated the effects of velocity magnitude and motion path on the response of bovine chondrocytes cultured in polyurethane scaffolds and subjected to oscillation against a ceramic ball. In order to vary velocity magnitude, the ball oscillated ±25° at 0.01, 0.1, and 1 Hz to generate 0.28, 2.8, and 28 mm/s, respectively. The median velocity of these ‘open’ motion trajectories was tested against ‘closed’ motion trajectories in that the scaffold oscillated ±20° against the ball at 1 Hz, reaching 2.8 mm/s. Constructs were loaded twice a day for 1 h over 5 days. Gene expression of cartilage oligomeric matrix protein (COMP), proteoglycan 4 (PRG4, lubricin), and hyaluronan synthase 1 (HAS1) and release of COMP, PRG4, and hyaluronan (HA) were analyzed.

Velocity magnitude determined both gene expression and release of target molecules. Using regression analysis, there was a positive and significant relationship with all outcome variables. However, only COMP reacted significantly at 0.28 mm/s, while all other measured variables were considerably up-regulated at 28 mm/s. Motion path characteristics affected COMP, but not PRG4 and HAS1/HA.

To conclude, velocity magnitude is a critical determinant for cellular responses in tissue engineered cartilage constructs. The motion type also plays a role. However, different molecules are affected in different ways. A molecule specific velocity threshold appears necessary to induce a significant response. This should be considered in further studies investigating the effects of continuous or intermittent motion.

Introduction

The synthesis of several molecules which are important for the development and maintenance of healthy articular cartilage is regulated through mechanical interaction. Important parameters include the type, duration and magnitude of the mechanical stimulus. For example, it has been shown that bovine and human chondrocytes seeded in polyurethane open-celled matrices increased the expression of proteoglycan 4 (PRG4, also known as lubricin or superficial zone protein) when surface motion was applied to the matrices, but not when axial compression was applied in isolation (Grad et al., 2005; Candrian et al., 2008). It was suggested that the release of this lubricating protein is tied to surface shear and fluid film transport within the joint space. Similar results were found with native cartilage, where whole bovine stifle joints were maintained in a bioreactor and submitted to continuous passive motion for 24 h. Compared with non-contacting regions, the synthesis of PRG4 was higher in femoral cartilage regions, where sliding against the meniscus or the tibial cartilage occurred (Nugent-Derfus et al., 2007). Other investigations identified PRG4 expression in various canine musculoskeletal tissues, whereby the distribution of PRG4 variants appeared to be related to the mechanical functions of the connective tissues, with certain variants expressed predominantly in tissues subjected to significant shear and frictional forces (Sun et al., 2006). Thus, sliding motion and shear have widely been recognized as important mechanical mediators of PRG4. The specific contribution of the corresponding sliding velocity vector, however, has not been systematically addressed.

Sliding velocity is an important parameter to determine the lubrication state of articulating surfaces in technical applications (Wimmer and Fischer, 2007). This relationship is important for cartilage and regenerated cartilage tissue in vitro alike. The relative speed between two opposing surfaces determines the entraining velocity of the interfacial fluid and, thus, governs its film thickness. Relative speed may also play a central role for cell signaling and therefore for cartilage development. It has been shown in recent bioreactor experiments that hydrodynamic shear through fluid flow increases collagen deposition (Gemmiti and Guldberg, 2006) as well as newly synthesized proteoglycans (Raimondi et al., 2006). In one of our own studies we observed that oscillation (±25°, 0.1 Hz) of a ∅32 mm ball against a tissue engineered construct had a stronger effect on chondrocytic gene expression patterns than axial oscillation (±7.5°, 0.1 Hz) of the ∅8 mm cylindrical construct against the ball (Grad et al., 2006). Although the same shear frequency was applied, sliding velocities were dissimilar. Oscillation of the ball generated a significantly higher average sliding velocity (2.8 mm/s) than axial rotation of the construct (0.1 mm/s). In addition, the shape of the motion trajectories was different. The ‘linear open’ velocity vectors at the ball allowed fluid drag into the contact zone, while the ‘curvilinear closed’ velocity vectors at the scaffold surface prevented direct fluid ingress. Because of these dissimilarities in magnitude and velocity profile, we speculated that either sliding velocity magnitude or motion trajectory shape (or both) play a crucial role for cell stimulation.

The aims of the present study were (a) to evaluate the influence of varying velocity magnitudes and (b) to study the effect of different motion paths. We hypothesized that both sliding velocity magnitude and motion path affect the chondrocytic response to articular motion. In order to control and limit variables, the influence of varying velocity magnitude was studied using identical motion paths and the influence of motion trajectory was studied keeping the average velocity constant. The analysis of gene expression and release of molecules was limited to those that are known to be responsive to mechanical stimuli simulating joint articulation (Grad et al., 2005, Grad et al., 2006).