Imaging articular cartilage in osteoarthritis using targeted peptide radiocontrast agents

Abstract

Background: Established MRI and emerging X-ray contrast agents for non-invasive imaging of articular cartilage rely on non-selective electrostatic interactions with negatively charged proteoglycans. These contrast agents have limited prognostic utility in diseases such as osteoarthritis (OA) due to the characteristic high turnover of proteoglycans. To overcome this limitation, we developed a radiocontrast agent that targets the type II collagen macromolecule in cartilage and used it to monitor disease progression in a murine model of OA.

Methods: To confer radiopacity to cartilage contrast agents, the naturally occurring tyrosine derivative 3,5-diiodo-L-tyrosine (DIT) was introduced into a selective peptide for type II collagen. Synthetic DIT peptide derivatives were synthesised by Fmoc-based solid-phase peptide synthesis and binding to ex vivo mouse tibial cartilage evaluated by high-resolution micro-CT. Di-Iodotyrosinated Peptide Imaging of Cartilage (DIPIC) was performed ex vivo and in vivo 4, 8 and 12 weeks in mice after induction of OA by destabilisation of the medial meniscus (DMM). Finally, human osteochondral plugs were imaged ex vivo using DIPIC.

Results: Fifteen DIT peptides were synthesised and tested, yielding seven leads with varying cartilage binding strengths. DIPIC visualised ex vivo murine articular cartilage comparably to the ex vivo contrast agent phosphotungstic acid. Intra-articular injection of contrast agent followed by in vivo DIPIC enabled delineation of damaged murine articular cartilage. Finally, the translational potential of the contrast agent was confirmed by visualisation of ex vivo human cartilage explants.

Conclusion: DIPIC has reduction and refinement implications in OA animal research and potential clinical translation to imaging human disease.

Fig 1. Design of targeted peptide radiocontrast agents through the insertion of DIT into the WYRGRL hexamer.

Strategies include N-terminal placement of DIT (A), an integrated design (B) replacing Tyr with DIT (red) and containing Arg or Lys amino acids (circled), and an N/C-terminal PEG-based linker (AEEA, blue) approach to separate signalling (DIT) and targeting (WYRGRL) entities (C, D). Histology of ex vivo murine articular cartilage 24 h after intra-articular injection of Ac-YRLGRW-DOTAM-Cy5.5 (E) or Ac-WYRGRL-DOTAM-Cy5.5 (F) into mouse knee joints. Only targeted probe (F) showed binding to cartilage (Cy5.5, red) with the pericellular proteoglycan perlecan (Alexa Fluor 488, green) revealing the location of chondrocytes.

Fig 2. Ex vivo imaging of healthy murine articular cartilage using DIPIC and other radiocontrast agents.

(A) Representative micro-CT coronal views of mouse tibiae incubated with W'Y'KGKL, 'Y'-AEEA-WYKGKL and 'Y'WGKKL upon 1 h and 24 h of washing in saline. The colour-coded maps of absorption (HU) for the lateral tibial plateau (red dotted box) highlight the loss of contrast enhancement as probes are cleared from cartilage over time. The dotted white lines delineate cartilage from bone. (B) Representative micro-CT coronal views of mouse tibiae after overnight incubation with iopamidol, 'Y'-AEEA-WYKGKL (both at 20 mg iodine/ml), and PTA (1% PTA in 70% ethanol). X-ray absorption profiles for each contrast agent were generated by drawing a line across cartilage from probe to bone. (C) X-ray fluorescence images for ex vivo murine articular cartilage after incubation with 'Y'-AEEA-WYKGKL showing calcium, phosphorous, sulphur and iodine channels (500 nm/pixel). (D) Elemental fluorescence profiles were generated by drawing a line down the middle of each image from (C).

Fig 3. Ex vivo and in vivo imaging of murine osteoarthritis using DIPIC.

(A) Representative micro-CT coronal views of contralateral tibia (negative control) and operated tibiae at 4 weeks and 12 weeks post-surgery imaged using DIPIC and subsequently PTA-CT. Despite PTA-CT providing higher contrast enhancement of cartilage than DIPIC (see colour-coded maps of absorption), both methods revealed the progressive degradation of this tissue in the medial tibial plateau (dotted red boxes). Dotted white lines delineate cartilage from bone. The presence of lesions in DMM mice was validated by histopathology (haematoxylin & eosin and safranin-O staining in the far-right panel). (B) Average articular cartilage thickness in DMM-operated and contralateral tibiae in weeks post-DMM surgery. Mean ± SEM, *p < 0.05, obtained by one-way ANOVA with Dunnett’s multiple comparison post-test for differences between 2 weeks vs. subsequent time points in the medial DMM tibial plateau and ^#p < 0.05 for the same test in the medial contralateral tibial plateau. Parametric correlations between articular cartilage thickness measurements obtained from histological sections and by DIPIC (C) (n = 32) and PTA-CT (D) (n = 17); Pearson correlation coefficients (r) and p-values are indicated in the graph. (E) Representative micro-CT coronal views of DMM mouse knee joints and corresponding colour-coded maps of absorption of the medial aspect of the joint after injection of either 'Y'-AEEA-WYKGKL or saline into the synovial space. Progressive cartilage degradation is highlighted by DIPIC, but not by saline, owing to the loss of contrast enhancement.

Fig 4. Ex vivo imaging of human articular cartilage with the DIT peptide 'Y'-AEEA-WYKGKL.

(A) Representative micro-CT views of a human osteochondral plug at different time points upon incubation with probe and corresponding colour-coded maps of absorption. (B) X-ray absorption profiles (dashed coloured lines in micro-CT images) obtained to evaluate probe penetration into tissue over time. (C) Representative micro-CT views of the same osteochondral plug at different time points upon washing in saline and corresponding colour-coded maps of absorption. (D) X-ray absorption profiles (dashed coloured lines in micro-CT images) obtained to evaluate probe washing from tissue over time. (E) Representative views of a 3D reconstruction of the osteochondral plug showing articular cartilage imaged by the DIT peptide (green) and underlying subchondral bone.