Deformation Thresholds for Chondrocyte Death and the Protective Effect of the Pericellular Matrix

Abstract

In cartilage tissue engineering studies, the stimulatory effect of mechanical perturbation declines after the first 2 weeks of culture. Similarly, it is known that chondrocyte-agarose constructs should not be loaded within the first days after seeding, to prevent considerable cell death, suggesting a mechanical threshold. This study aims to establish a relationship between chondrocyte deformation and death, and to evaluate the protective effect of the pericellular matrix (PCM) that is formed in 3D cultures. Chondrocyte viability was monitored every hour for 24 h after applying a strain range of 0% to 25% to agarose constructs containing chondrocytes, cultured for 1, 3, 5, 7, or 10 days. At these culture time points, the PCM thickness and chondrocyte deformation were assessed by means of histology and assayed for biochemical contents. Inverse finite element (FE) simulations were used to evaluate the change of mechanical properties of the chondrocyte and PCM over the 10-day culture duration. Chondrocyte death was demonstrated to be dependent on both the magnitude and duration of straining. The highest cell death was observed at day 1 (43%), reducing over culture duration (15% at day 3 and 2.5% at day 10). Cell deformation at 25% compression decreased significantly over culture duration (aspect ratio of 2.24±0.67 at day 1 and 1.45±0.24 at day 3) and with increased matrix production. Inverse FE simulations showed an increasing PCM Young's modulus of 45 kPa at day 3 to 162 kPa at day 10. The current results provide evidence for a mechanical threshold for chondrocyte death and for the protective effect of the PCM. As such, these insights may help in establishing mechanical loading protocols for cartilage tissue engineering studies.

The Influence of Cell-Matrix Attachment and Matrix Development on the Micromechanical Environment of the Chondrocyte in Tissue-Engineered Cartilage

“…28,29 Cell deformation indexes, defined as the size of the cell in the direction of compression divided by the perpendicular length, compare well when constructs are subjected to 5-20% strain (Fig. 7).…”

“…The deformation index at the presence of PCM was predicted to be in the range of 0.94-0.97 (depending on the PCM maturation) which is in agreement with the experimental findings. 28 Besides these agreements, it is also worth mentioning that the constitutive material model for native ECM has previously been validated and used for studies at both the macroscopic tissue 21,22,30 and the microscopic cell level. 24,[31][32][33][34][35][36] The different macroscopic and microscopic depthdependent strain fields between native cartilage and TE constructs may have implications for the mechano-response of the chondrocytes to mechanical stimulation.…”

“…This may then partly explain why loading during TE should be started few days after cell seeding, in order to have an anabolic effect. 6,28 Numerical predictions further indicate that with PCM maturation, the externally applied compression is more effectively transmitted to the cell (i.e., strain magnification), while at the same time, PCM protects the cell from excessive tensile strains in the direction perpendicular to the loading direction. These effects are independent of cell size.…”

“…7). Further, a recent study by De Vries et al 28 showed that during culture, PCM becomes protective for cell deformation, so the cell deformation in the direction perpendicular to the loading direction becomes less if the matrix around the cell becomes more mature. This is consistent with our finding (Fig.…”

Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage

“…A change from interterritorial to pericellular localisation was also observed in the other zones of the growth plate and in the articular cartilage; however, the time‐point of the shift varied between proteins. Increasing thickness of the pericellular matrix over time has been observed in cultured chondrocytes and is associated with increased protection against mechanical deformation (de Vries et al ). The spatial change with increased pericellular staining of the more developed cartilage in our study may indicate an increase in production of the specific molecule.…”