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Antibody Charge Affects Depth-Dependent Diffusion in Healthy Articular Cartilage
DiDomenico, Chris1; Goodearl, Andrew2; Yarilina, Anna2; Sun, Victor2; Mitra, Soumya2; Schwartz Sterman, Annette2; Bonassar, Lawrence1
1
Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States.; 2AbbVie, Worcester, MA
Disclosures: C. DiDomenico: 6; AbbVie. A. Goodearl: 3A; AbbVie. 4; AbbVie; Abbott. A. Yarilina: 3A; AbbVie. 4; AbbVie; Abbott. V. Sun: 3A;
AbbVie. 4; AbbVie; Abbott. S. Mitra: 3A; AbbVie. 4; AbbVie; Abbott. A. Schwartz Sterman: 3A; AbbVie. 4; AbbVie; Abbott. L. Bonassar: 5; AbbVie.
INTRODUCTION
In the past decade, breakthroughs for treatment of rheumatoid arthritis (RA), which rely on anti-inflammatory antibodies to hinder systemic symptoms and
joint degradation, have been developed [1,2]. Antibody-based therapy for osteoarthritis (OA) is actively being investigated as well, but this has several
unaddressed issues and necessitates antibody penetration into the dense, avascular cartilage matrix to reach locally-produced inflammatory cytokines [3–5].
The ability of large molecules like antibodies to diffuse through cartilage is limited, with potential need for optimization, as synovial clearance times are on
the order of hours [3,6]. Due to the depth-dependent mechanical properties and varying negative charge density of cartilage, diffusion of drugs through the
articular surface is complex and there are many factors that may affect transport, such as solute size and charge [7,8]. Previous work has indicated that
antibodies have spatially-dependent diffusive properties within cartilage that is influenced by molecular size [10]. Therefore, the goals of this study are to
investigate how charged solutes diffuse through the negatively charged tissue matrix and how charge affects diffusion behavior.
METHODS
Fresh, full-thickness (2 mm) articular cartilage plugs (4 mm diameter) were harvested from the patellofemoral groove of 1-3 day old bovids. These plugs
were bisected axially, and one 2x4x1.15 mm slice of tissue was obtained from each bisected half, for a total of 20-24 slices for each experiment. Then, one of
three fluorescently labeled (Alexa Fluor 633) full-sized (150 kDa) antibody variants (AbbVie, Worcester, MA) were added at a concentration of ~1 μM in PBS
to randomized wells in a 24-well plate. These structurally similar antibodies had three different average isoelectric points: 4.7, 5.4, and 5.9. Cartilage slices
were then randomly placed in wells such that the articular surface and deep zones were exposed to the antibody solution on the lateral faces. An impermeable
platen array was placed on top of the well plate, applying a 15% strain offset to all samples to limit fluid contact to the lateral faces only (Figure 1 inset). All
samples were placed in an incubator at 37°C for three hours during testing. Before assessment using confocal microscopy, samples were rinsed and cut to
image fluorescence perpendicular to the articular surface. Fluorescence data was used to determine local diffusivities at discrete depths from the articular
surface using a multi-layer transient diffusivity model [9].
RESULTS
Overall, fluorescence curves and local diffusivities were heterogeneous through the depth of the tissue, with three distinct depth-dependent sections for each
solute (Figure 1 and 2). On average, diffusivities for all solutes were about 4 μm2/s within 0-100 μm from the articular surface and did not vary significantly
between isoelectric points (repeated-measures two-way ANOVA, p > 0.05). Diffusivities increased to a maximum of 14.9, 16.9, and 19.0, μm2/s for the solutes
with an isoelectric point of 4.7, 5.4, and 5.9, respectively, between 200-275 μm (p < 0.05). In this region, negative charge inhibited antibody diffusion, with
the pI 4.7 antibody having a diffusivity 20-30% lower than that of the pI 5.9 antibody (p < 0.05). Deeper in the tissue, 400-800 μm below the articular surface,
diffusivities were similar to those found in the surface region and had no significant dependence on isoelectric point (p > 0.05).
DISCUSSION
Negatively-charged human monoclonal antibodies penetrated ~800 μm into healthy cartilage in three hours, which is on the time scale of the residence time
of similarly-sized molecules within the joint space in vivo [11]. Overall, the depth-wise composition of this cartilage tissue greatly affected the diffusion of
these antibodies. It has been shown that this diffusive behavior is highly related to tissue matrix density and collagen fiber orientation, with highest diffusivities
occurring near a combination of low collagen fiber alignment and low aggrecan content (immediately past the articular surface) [10]. In agreement with past
research, local diffusivities for all solutes were highest around 200 μm from the articular surface, but 300-400% lower near the articular surface and in the
deeper zones [10]. Differences in isoelectric point caused local differences in diffusivities within the range 200-300 μm, but no other differences were observed.
Within this range of isoelectric points, its seems that charge interactions between matrix and solute were only significant at certain aggrecan concentrations
that were present in this region. The time scale of these experiments and limited range of isoelectric points could have masked differences deeper within the
tissue, where charge was expected to have a greater effect due to increasing tissue charge density. These data support that isoelectric point represents another
tunable property that could be changed to optimize macromolecular transport within cartilage.
SIGNIFICANCE
Negatively charged, full-sized antibody molecules were able to penetrate through the articular surface of cartilage and have spatially-dependent diffusivities
through the tissue depth. This research provides further insight on how macromolecular transport occurs within cartilage and how different factors may affect
the development of OA antibody therapy.
30
Rinse in PBS
Diffusiviity (μm2/s)
Articular
surface
0.8
0.4
pI 4.7
pI 5.4
pI 5.9
0.2
AS
0.6
DZ
Fluorescence Intensity
1
*
25
20
15
10
5
0
0
0
200
400
600
Depth from articular surface (μm)
800
Figure 1: Average (n =6-8) fluorescence intensity of confocal images for
different antibody isoelectric points. Higher isoelectric points tended to have
higher fluorescence between 200-400 μm. Inset: Depiction of cutting
procedure to generate slices and experimental setup showing the
PBS/Antibody bath having contact only with the deep zone (DZ) and
articular surface (AS) faces. Only fluorescence on the AS side was analyzed
for this study.
0
200
400
600
Depth from articular surface (μm)
800
Figure 2: Average (n =6-8) local diffusivities for each antibody isoelectric
point. Higher isoelectric point variants had significantly increased
diffusivities compared to other solutes between 200-350 μm (*: p < 0.05),
but no other differences between groups were found. Curves were analyzed
using a repeated-measures two-way ANOVA. Inset: Confocal image
showing fluorescence gradient perpendicular to the surface of the cartilage,
with the analyzed region highlighted in red (middle 50% of sample).
Ref: [1] Bang+, A Rheum., 2008. [2] Feldmann+, Annu. Rev. Immunol., 1996. [3] Goldring+, Rheum. Dis., 2001. [4] Allen+, Tissue Eng., 2010. [5] Moos+,
J. Rheum., 1999 [6] Martel-Pelletier, Osteo Cart., 2004. [7] Mow+, J Biomech., 1984. [8] Poole+, Clin. Orthop. Relat. Res., 2001. [9] Carr+, Appl. Math.
Model., 2016. [10] DiDomenico+, [Abstract] ORS, 2016. [11] Owen+, Clin. Pharmacol. 1994.
ORS 2017 Annual Meeting Poster No.0476