Impact of PEG Sensitization on the Efficacy of PEG-Hydrogel Mediated Delivery of Bone Morphogenetic Protein 2

Introduction:: Poly(ethylene glycol) (PEG)-based hydrogels have been used extensively in tissue engineering and clinical applications due to their being regarded as bioinert and having highly tunable physicochemical properties. However, a growing body of evidence suggests an increasing prevalence of anti-PEG antibodies in the general public. Anti-PEG antibodies have been documented to contribute to adverse health outcomes, with cases of anaphylaxis being reported following administration of PEG-conjugated therapeutics. Furthermore, anti-PEG antibodies have been found to contribute to accelerated blood clearance of PEGylated pharmaceuticals. Given these concerns, we sought to investigate the implications of PEG immunogenicity for tissue engineering. To achieve this goal, we immunized male and female C57BL/6 mice against PEG and treated a critical-sized bone defect with PEG-based microporous annealed particle (MAP) hydrogels loaded with bone morphogenetic protein 2 (BMP-2) to promote bone regeneration. Bone formation at the defect site was then quantitatively analyzed to evaluate differences between naïve and PEG-sensitized mice. Bone morphology and the host response were also evaluated via histology.

Materials and Methods:: PEG-based microgels were fabricated by electrospraying a 0.75:1 thiol-norbornene precursor solution of 4-arm, 20kDa PEG-amide-norbornene (10wt%), lithium acylphosphinate (10mM), cell adhesive peptide CGRGDS (1mM), and dithiol matrix metalloproteinase-degradable peptide crosslinker KCGPQGIWGQCK. To evaluate the impact of PEG immunogenicity on the efficacy of PEG-based hydrogels in tissue engineering applications, male and female C57BL/6 mice were sensitized to PEG via subcutaneous administration of multi-PEGylated keyhole limpet hemocyanin (PEG-KLH, 2mg/kg of body weight) for 4 weeks. Naïve controls were administered saline for the same duration. Calvarial defects were created on the right parietal bone 2 weeks after the final PEG-KLH administration, and the defects were filled with PEG microgels, loaded with or without 500ng of BMP-2, and a PEG-di-tetrazine linker (2mM). Defects of negative control groups were left empty. Mice were euthanized 3 or 6 weeks post-implantation. Tissue was collected, fixed in 10% neutral buffered formalin for 48 hours, and subsequently immersed in Carson’s fixative. Effects of PEG sensitization on bone formation were quantified via microcomputed tomography (μCT), where samples were scanned in 0.5° increments over a total 360° at a voltage, current, and pixel resolution of 30kV, 200μA, and 18μm, respectively. Tissue samples were decalcified in 0.5M EDTA (pH ~7.2) prior to embedding in paraffin and sectioning at a 6μm thickness. The host responses following the administered treatment types were assessed through hematoxylin and eosin (H&E) and Masson’s trichrome staining. Histology samples were imaged using bright-field microscopy at 4x and 10x magnifications.

Results, Conclusions, and Discussions:: μCT analysis confirmed the BMP-2-loaded MAP hydrogels stimulated the largest volume of bone when compared to the other treatment groups, regardless of sex or PEG sensitization (Figure 1). This was particularly significant for PEG-sensitized mice at 3 and 6 weeks post-implantation. Interestingly, significant differences as a result of PEG sensitization were only observed between naïve and sensitized males treated with MAP + BMP-2, as the latter had a 1.22-fold increase in bone formation. Additionally, sex appeared to impact bone formation at 6 weeks, where sensitized males had a 1.49-fold increase in bone volume compared to their female counterparts. Histology images corroborated μCT results and provided further insight on bone morphology at the calvarial defect sites. Mice implanted with BMP-2-loaded MAP hydrogels demonstrated noticeably thicker matrix deposition at the defect site and the immediately neighboring parietal bone when compared to the contralateral side (Figure 2). Furthermore, the newly formed bone appeared to have the greatest overall thickness among PEG-sensitized mice. Masson’s trichrome staining further elucidated that collagen deposition was less dense among PEG-sensitized mice (Figure 3). PEG-sensitized males in particular demonstrated multiple sites of bone marrow cavities, which could indicate differences in the structural integrity of the generated bone.
PEG immunogenicity was found to impact the efficacy of PEG-based tissue engineering therapies using a bone defect model and implanting a PEG hydrogel platform. Specifically, we found that PEG sensitization contributed to differences in bone morphology when compared to naïve controls, and this pattern was accompanied with observations of ectopic bone growth when BMP-2 was loaded in the hydrogel platform. These observations were prevalent among females and males, but were more pronounced in the male group. Overall, our findings suggest that PEG sensitization must be considered when implementing PEG-based platforms, as the anti-PEG antibodies can contribute to unfavorable clinical outcomes. Future studies will evaluate systemic anti-PEG antibody titer formation, characterize the cell type recruited at sites of critical-sized defects over time, and further investigate how biological sex contributes to PEG-based immunological responses.


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