Optimized multi-axial loading to enhance chondrogenesis and limit proteoglycan loss in cartilage explants during bioreactor experimentation

Abstract

Purpose: Osteoarthritis (OA) is a disease of the whole joint, with associated changes in the subchondral bone, synovium, bone marrow, muscles and ligaments, but with cartilage degeneration being the hallmark of OA. Mechanical loading is a key control signal in the regulation of the metabolic activity (i.e. homeostasis) of the articular chondrocytes, the unique cell population found in cartilage, embedded in their self-produced extra-cellular matrix (ECM). To study the effects of mechanical loading on the biological response of cartilage, explants can be loaded in bioreactors. Multi-axial loading bioreactors that combine compressive with torsional loading can mimic the loading of articular cartilage. This study aims at informing bioreactor protocol development to define mechanical parameters that optimize the biological response of cartilage explants during multi-directional loading experiments. Indeed, the local biological response may vary from chondrogenesis in some parts to proteoglycan loss in others depending on the mechanical parameters. This study uses a finite element method to investigate the effects of multi-axial loading characteristics on the local mechanical environment (stresses and strains) of the explants while accounting for material properties of the non-fibrillar matrix of cartilage explants. The mechanical environment is then related to chondrogenesis (based on maximum compressive principal strain (MCPS) - Zahedmanesh et al., 2014) and proteoglycan loss (based on the fluid velocity (FV) - Orozco et al., 2018). Methods: The cartilage explant was modelled as a 3D cylinder of 8mm diameter and equal height in Abaqus. A fiber-reinforced poroelastic (FRPE) constitutive model (Ebrahimi et al., 2019) was used to define the material, in which the non-fibrillar matrix of the cartilage explant was modelled as a porous hyperelastic (Neohookean) material saturated with fluid (water) and the collagen fiber network was modelled as nonlinear elastic material that can only withstand tensile loading. Four families of organized collagen fibrils (primary fibrils) and 13 families of randomly oriented fibrils (secondary fibrils) formed the collagen network. Ratio of density of the primary to the secondary fibrils was set to 12.16. The bottom surface of the explant was fully constrained and sinusoidal (frequency of 1Hz) unconfined compression and torsion loading were applied to the top surface. Free fluid flow was allowed through the lateral surface. The initial and strain-dependent fibril network moduli were respectively set to 0.41 and 15.42 MPa and the constant for displacement dependence of permeability in the non-fibrillar matrix was set to 3.36 (Ebrahimi et al., 2019). The sensitivity of the maximum values of FV and MCPS was studied at 9 key locations (see figure a), whereby the material parameters of the non-fibrillar matrix and loading range were varied. This resulted in five input parameters: elastic modulus (E=[0.15,0.6]MPa), Poisson’s ratio (nu=[0.1,0.45]), initial permeability (k0=[0.4,1.6]×10-15m4/Ns), compressive strain (Comp=[5,20]%) and torsion loading (Tor=[5,20]°). A full-factorial design of experiment method was used and a first-order polynomial surface including the interactions fitted the responses. Results: MCPS varied between 8.4% and 36.5% and is independent of the material properties of non-fibrillar matrix (E, nu and k0) but has a high and moderate dependency on Compressive (Comp) and torsion loading (Tor) magnitudes, respectively (figure b). The maximum value of MCPS always occurs at the bottom edge, i.e. at the distal cartilage bone interface (point 7 in figure a). FV varies between 1.2×10-7 mm/sec and 4.8×10-5 mm/sec and dominantly depends on initial permeability (k0) and compressive loading (Comp), while being independent of Poisson’s ratio (nu) and slightly dependent on elastic modulus (E) and torsion loading (Tor) (figure c). The maximum value of FV always occurs at the center of the side surface (point 4 in figure a). Conclusions: The parameter sensitivity analysis Results show that by increasing compression and torsion loading within the bioreactor the chondrogenesis (which depends on MCPS) may increase, independent of the material properties studied. Therefore, within the selected ranges studied here, a decrease in material stiffness (softening of the cartilage) due to OA won’t affect the chondrogenesis in cartilage explants. Based on these results, if enough high-quality chondrocyte cells are provided in a cell-seeded implant-cartilage unit, chondrogenesis will increase by increasing the compression and torsion loadings applied within the bioreactor up to at least 20% and 20°, respectively. On the other hand, increasing compression loading will increase FV, thereby degenerating the extracellular matrix through proteoglycan loss. Also, increasing initial permeability (k0) will increase FV under loading, while FV is independent of elastic modulus (E), Poisson’s ration (nu) and torsion loading (Tor). Therefore, to limit proteoglycan loss and enhance chondrogenesis in cartilage explants during bioreactor experiments, compression loading might be decreased (to control FV ) but torsion loading increased (to increase MCPS). In future, follow-up biological studies are needed to test the added value of these model-informed bioreactor protocols.