Impact of PEG sensitization on the efficacy of PEG hydrogel-mediated tissue engineering

Abstract

While poly(ethylene glycol) (PEG) hydrogels are generally regarded as biologically inert blank slates, concerns over PEG immunogenicity are growing, and the implications for tissue engineering are unknown. Here, we investigate these implications by immunizing mice against PEG to stimulate anti-PEG antibody production and evaluating bone defect regeneration after treatment with bone morphogenetic protein-2-loaded PEG hydrogels. Quantitative analysis reveals that PEG sensitization increases bone formation compared to naive controls, whereas histological analysis shows that PEG sensitization induces an abnormally porous bone morphology at the defect site, particularly in males. Furthermore, immune cell recruitment is higher in PEG-sensitized mice administered the PEG-based treatment than their naive counterparts. Interestingly, naive controls that were administered a PEG-based treatment also develop anti-PEG antibodies. Sex differences in bone formation and immune cell recruitment are also apparent. Overall, these findings indicate that anti-PEG immune responses can impact tissue engineering efficacy and highlight the need for further investigation.

Fig. 1. The impact of PEG sensitization and anti-PEG antibodies on the efficacy of a PEG hydrogel-based tissue engineering strategy was assessed via implantation of PEG-based MAP hydrogels in a calvarial defect model.

Schematic of A materials and fabrication of PEG microparticles and annealing of MAP hydrogels, and B timeline of the animal study. The calvarial defect model was chosen because it is widely used to evaluate the efficacy of biomaterials and therapeutics for bone regeneration. Tissue samples were collected at 3 and 6 weeks post-implantation. Tissue processing and data analysis were conducted immediately following the end of each time point. Created with BioRender.com.

Fig. 2. PEG sensitization contributes to increased bone in PEG MAP + BMP-2 treatment groups.

A Representative 3D reconstructions of µCT scans and axial images of PEG sensitized male mice 6 weeks post-implantation. B Bone volume measurements from µCT scans of calvarial defects at 3 and 6 weeks post-implantation (n = 4 mice per treatment group). Statistical significance was calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, an alpha threshold of 0.05, and 95% confidence interval. Data are represented as mean ± SD, and dots represent individual specimens.

Fig. 3. PEG MAP + BMP-2 treatment groups demonstrate increased matrix deposition at the defect site and morphological changes to the surrounding tissue.

Images of H&E staining of calvarial defects of naive and sensitized top male and bottom female mice treated with PEG MAP + BMP-2 at 3 and 6 weeks post-implantation. Tissue samples from n = 4 mice per treatment group were analyzed. The entire calvaria was imaged to show the defect site (left side) and healthy, uninjured bone in the contralateral tissue (scale bar = 3 mm). Defect sites are indicated in solid-line boxes and magnified at ×4 (scale bar = 1 mm), and portions of the defect site that are magnified at ×10 are indicated in dashed-line boxes (scale bar = 200 μm).

Fig. 4. PEG sensitization among PEG MAP + BMP-2 treatment groups contributed to disorganized collagen structure when compared to their naive counterparts.

Masson’s Trichrome staining of calvarial defects of naive and sensitized top male and bottom female mice treated with PEG MAP + BMP-2 at 3 and 6 weeks post-implantation. Tissue samples from n = 4 mice per treatment group were analyzed. The entire calvaria was imaged to show the defect site and healthy, uninjured bone in the contralateral tissue (scale bar = 3 mm). Defect sites are indicated in solid-line boxes and magnified at ×4 (scale bar = 1 mm), and portions of the defect site that are magnified at ×10 are indicated in dashed-line boxes (scale bar = 200 μm).

Fig. 5. Quantitative analysis of the matrix at defect sites supports that PEG sensitization contributes to increased collagen deposition.

Collagen deposition was determined from tissue samples subjected to Masson’s Trichrome staining (n = 4 mice per treatment group). Statistical significance was calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, an alpha threshold of 0.05, and 95% confidence interval. Data are represented as mean ± SD, and dots represent individual specimens.

Fig. 6. Immune cell recruitment at defect sites administered PEG MAP + BMP-2 treatment was influenced by PEG sensitization at the 3- and 6-week timepoints, as well as by biological sex at the 6-week timepoint.

Quantification of A CD68+ cells and B CD3+ cells at defect sites 3 and 6 weeks post-implantation. Statistical significance was calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test with a single pooled variance, an alpha threshold of 0.05, and 95% confidence interval. Center line = median; box limits = upper and lower quartiles; whiskers = 1.5× interquartile range (n = 4 mice per treatment group). Staining is representative of n = 4 sections per sample. Scale bar = 60 µm.

Fig. 7. Antibody formation persisted in PEG-sensitized mice and was detected in naive mice after PEG hydrogel implantation.

A PEG sensitization contributed to IgM and IgG antibody formation post-implantation of treatment groups in sensitized mice. B Naive mice also formed IgM antibodies post-implantation of PEG treatment groups. Dots represent individual specimens, and the black bars are the median of the sample group (n = 4 mice per sensitized treatment group; n = 3 mice per naïve treatment group).