Early detection and treatment of wear particle-induced inflammation and bone loss in a mouse calvarial osteolysis model using HPMA copolymer conjugates

Abstract

Wear particle-induced inflammation is considered to be the major cause of aseptic implant loosening and clinical failure after total joint replacement. Due to the frequent absence of symptoms, early detection and intervention prior to implant failure presents a significant challenge. To address this issue, a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based optical imaging contrast agent (P-IRDye) was developed and used for the detection of wear particle-induced inflammation employing a murine calvaria osteolysis model. The particle-induced osteolysis of calvaria was evaluated by H&E, tartrate-resistant acid phosphatase (TRAP) staining and μ-CT after necropsy. One-day post particle implantation, P-IRDye was administrated to the mice via tail vein injection. Live imaging of the animals 6 days after implantation revealed the preferential distribution and sustained retention of the macromolecular contrast agent at the site of particle implantation. Immunohistochemical staining and FACS analyses of the calvaria-associated soft tissue revealed extensive uptake of the HPMA copolymer by F4/80, Ly-6G (Gr1) and CD11c positive cells, which accounts for the retention of the macromolecular probes at the inflammatory sites. To test the potential of the system for therapeutic intervention, an acid-labile HPMA copolymer-dexamethasone conjugate (P-Dex) was prepared and shown to prevent the particle-induced inflammation and bone damage in the calvaria osteolysis model.

Figure 1

The syntheses of P-IRDye and P-Alexa.

Figure 2

Chemical structure of P-Dex.

Figure 3

Representative images of undecalcified calvaria after TRAP-staining (A and B) and H&E stained decalcified calvaria tissue sections (C and D). Panels A and C are from mouse calvaria injected with PBS, while B and D are from animals implanted with PMMA particles. TRAP-staining of the undecalcified calvarial tissue shows the presence of abundant TRAP-positive cells from the PMMA particle implanted animal (panel B). The arrows in panel D indicates the region of focal bone resorption.

Figure 4

Live optical imaging after PBS or PMMA implantation. P-IRDye was given via tail vein injection the following day after PBS or PMMA implantation. The mice were imaged prior and each day after the administration of the optical imaging agent for the following 6 days (n = 3). A. The upper panel shows the images from the mice with calvarial injection of PBS. The lower panel shows the images from the mice with calvarial PMMA-particle implantation. Compared to the PBS group, PMMA particle implanted animals demonstrated more intense and longer lasting NIR signals in the calvarial region where the PMMA particles were implanted; B. The NIR signal intensity was measured from a consistent region of interest (red circle) in the calvaria site for all the mice. The signal intensity differences in the two groups were statistically significant (p < 0.05).

Figure 5

Representative confocal images of anti-Ly-6G (Gr-1, Gr1), anti-F4/80, anti-CD11c and anti-P4HB antibody stained frozen sections of calvaria and adjacent soft tissue from PMMA particle implanted mice (treated with P-Alexa). Each panel was composed of four sub-images: antibody red staining, P-Alexa green fluorescence, DIC image and the co-localization of the three. The co-localization of red and green color in both panels yields a yellow color, which confirms the internalization of the HPMA copolymer conjugate by Ly-6G (Gr-1, Gr1), F4/80 or CD11c positive cells at the sites of inflammation. The spherical structures are PMMA particles. Bar = 50 μm.

Figure 6

Representative data from fluorescence-activated cell scanning (FACS) analysis of cells isolated from sites of PMMA particle-induced inflammation 24 h after systemic administration of P-Alexa. The histogram plots show the intensity of staining with the specific antibodies designated on the x-axis (orange fills) with isotype control antibodies (blue lines) on the same plots. The percentages represent the percent of antibody positive cells among P-Alexa positive cells. (A) 25.12 % P-Alexa positive cells were F4/80 positive; (B) 35.15 % P-Alexa positive cells were Ly-6G positive; (C) 9.73 % P-Alexa positive cells were CD11c positive; (D) < 1 % P-Alexa positive cells were P4HB positive. Abundant spherical structures present in the tissue sections are PMMA particles.

Figure 7

Representative images of TRAP-stained undecalcified calvaria from different treatment groups. (A) Sham control group; (B) UHMWPE + Dex group; (C) UHMWPE + P-Dex group; (D) UHMWPE + PHPMA group; (E) UHMWPE + PBS group. TRAP-positive areas (arrow) are abundant on calvaria surfaces from the PHPMA and PBS groups (midline region, magnifications are at 45×). Panel F focuses on a particular TRAP positive region of a calvarium from the PHPMA group. Bone resorption pits were visible surrounded by TRAP positive cells. Bar = 250 μm.

Figure 8

Representative micro-CT reconstructed images of the mouse calvaria after different treatments. The whole calvarium micro-CT image from a healthy (sham operated) mouse is shown in panel A. The red circle represents the spherical region of interest (ROI) of 2 mm in diameter in the center of calvarium used for quantitative analysis of BV/TV (%). Panels B to F show enlarged images of the selected blue square regions represented in panel A. (B) Sham control group (no particle); (C) UHMWPE + PBS group; (D) UHMWPE + PHPMA group; (E) UHMWPE + Dex group; (F) UHMWPE + P-Dex group. There was significant bone resorption in PBS and PHPMA treatment groups, while the UHMWPE-induced bone resorption was greatly attenuated in Dex and P-Dex treatment groups.

Figure 9

Quantitative analysis results of BV/TV (%) from micro-CT evaluation. Implantation of UHMWPE particles in vehicle (PBS) resulted in statistically significant reduction in BV/TV (%) values compared to sham control (no particles) (p < 0.05). Polymer alone (PHPMA) did not alter the UHMWPE-induced reduction in BV/TV. However, both Dex and P-Dex treatment groups significantly (p < 0.05) reversed the UHMWPE-induced reduction in BV/TV to levels comparable to sham mice. ns = not significant.