Measurement of local diffusion and composition in degraded articular cartilage reveals the unique role of surface structure in controlling macromolecular transport.

Developing effective therapeutics for osteoarthritis (OA) necessitates that such molecules can reach and target chondrocytes within articular cartilage. However, predicting how well very large therapeutic molecules diffuse through cartilage is often difficult, and the relationship between local transport mechanics for these molecules and tissue heterogeneities in the tissue is still unclear. In this study, a 150 kDa antibody diffused through the articular surface of healthy and enzymatically degraded cartilage, which enabled the calculation of local diffusion mechanics in tissue with large compositional variations. Local cartilage composition and structure was quantified with Fourier transform infrared (FTIR) spectroscopy and second harmonic generation (SHG) imaging techniques. Overall, both local concentrations of aggrecan and collagen were correlated to local diffusivities for both healthy and surface-degraded samples (0.3 > R2 < 0.9). However, samples that underwent surface degradation by collagenase exhibited stronger correlations (R2 > 0.75) compared to healthy samples (R2 < 0.46), suggesting that the highly aligned collagen at the surface of cartilage acts as a barrier to macromolecular transport.

The Effect of Charge and Mechanical Loading on Antibody Diffusion Through the Articular Surface of Cartilage.

Molecular transport of osteoarthritis (OA) therapeutics within articular cartilage is influenced by many factors, such as solute charge, that have yet to be fully understood. This study characterizes how solute charge influences local diffusion and convective transport of antibodies within the heterogeneous cartilage matrix. Three fluorescently tagged solutes of varying isoelectric point (pI) (4.7-5.9) were tested in either cyclic or passive cartilage loading conditions. In each case, local diffusivities were calculated based on local fluorescence in the cartilage sample, as observed by confocal microscopy. In agreement with past research, local solute diffusivities within the heterogeneous cartilage matrix were highest around 200-275 µm from the articular surface, but 3-4 times lower at the articular surface and in the deeper zones of the tissue. Transport of all 150 kDa solutes was significantly increased by the application of mechanical loading at 1 Hz, but local transport enhancement was not significantly affected by changes in solute isoelectric point. More positively charged solutes (higher pI) had significantly higher local diffusivities 200-275 µm from the tissue surface, but no other differences were observed. This implies that there are certain regions of cartilage that are more sensitive to changes in solute charge than others, which could be useful for future development of OA therapeutics.

Molecular transport in articular cartilage - what have we learned from the past 50 years?

Developing therapeutic molecules that target chondrocytes and locally produced inflammatory factors within arthritic cartilage is an active area of investigation. The extensive studies that have been conducted over the past 50 years have enabled the accurate prediction and reliable optimization of the transport of a wide variety of molecules into cartilage. In this Review, the factors that can be used to tune the transport kinetics of therapeutics are summarized. Overall, the most crucial factor when designing new therapeutic molecules is solute size. The diffusivity and partition coefficient of a solute both decrease with increasing solute size as indicated by molecular mass or by hydrodynamic radius. Surprisingly, despite having an effective pore size of ~6 nm, molecules of ~16 nm radius can diffuse through the cartilage matrix. Alteration of the shape or charge of a solute and the application of physiological loading to cartilage can be used to predictably improve solute transport kinetics, and this knowledge can be used to improve the development of therapeutic agents for osteoarthritis that target the cartilage.

The Effect of Antibody Size and Mechanical Loading on Solute Diffusion Through the Articular Surface of Cartilage.

Because of the heterogeneous nature of articular cartilage tissue, penetration of potential therapeutic molecules for osteoarthritis (OA) through the articular surface (AS) is complex, with many factors that affect transport of these solutes within the tissue. Therefore, the goal of this study is to investigate how the size of antibody (Ab) variants, as well as application of cyclic mechanical loading, affects solute transport within healthy cartilage tissue. Penetration of fluorescently tagged solutes was quantified using confocal microscopy. For all the solutes tested, fluorescence curves were obtained through the articular surface. On average, diffusivities for the solutes of sizes 200 kDa, 150 kDa, 50 kDa, and 25 kDa were 3.3, 3.4, 5.1, and 6.0 µm2/s from 0 to 100 µm from the articular surface. Diffusivities went up to a maximum of 16.5, 18.5, 20.5, and 23.4 µm2/s for the 200 kDa, 150 kDa, 50 kDa, and 25 kDa molecules, respectively, from 225 to 325 µm from the surface. Overall, the effect of loading was very significant, with maximal transport enhancement for each solute ranging from 2.2 to 3.4-fold near 275 µm. Ultimately, solutes of this size do not diffuse uniformly nor are convected uniformly, through the depth of the cartilage tissue. This research potentially holds great clinical significance to discover ways of further optimizing transport into cartilage and leads to effective antibody-based treatments for OA.

Cyclic Mechanical Loading Enhances Transport of Antibodies Into Articular Cartilage.

The goal of this study was to characterize antibody penetration through cartilage tissue under mechanical loading. Mechanical stimulation aids in the penetration of some proteins, but this effect has not characterized molecules such as antibodies (>100 kDa), which may hold some clinical value for treating osteoarthritis (OA). For each experiment, fresh articular cartilage plugs were obtained and exposed to fluorescently labeled antibodies while under cyclic mechanical load in unconfined compression for several hours. Penetration of these antibodies was quantified using confocal microscopy, and finite element (FE) simulations were conducted to predict fluid flow patterns within loaded samples. Transport enhancement followed a linear trend with strain amplitude (0.25-5%) and a nonlinear trend with frequency (0.25-2.60 Hz), with maximum enhancement found to be at 5% cyclic strain and 1 Hz, respectively. Regions of highest enhancement of transport within the tissue were associated with the regions of highest interstitial fluid velocity, as predicted from finite-element simulations. Overall, cyclic compression-enhanced antibody transport by twofold to threefold. To our knowledge, this is the first study to test how mechanical stimulation affects the diffusion of antibodies in cartilage and suggest further study into other important factors regarding macromolecular transport.