Needle-injectable microcomposite cryogel scaffolds with antimicrobial properties

Abstract

Porous three-dimensional hydrogel scaffolds have an exquisite ability to promote tissue repair. However, because of their high water content and invasive nature during surgical implantation, hydrogels are at an increased risk of bacterial infection. Recently, we have developed elastic biomimetic cryogels, an advanced type of polymeric hydrogel, that are syringe-deliverable through hypodermic needles. These needle-injectable cryogels have unique properties, including large and interconnected pores, mechanical robustness, and shape-memory. Like hydrogels, cryogels are also susceptible to colonization by microbial pathogens. To that end, our minimally invasive cryogels have been engineered to address this challenge. Specifically, we hybridized the cryogels with calcium peroxide microparticles to controllably produce bactericidal hydrogen peroxide. Our novel microcomposite cryogels exhibit antimicrobial properties and inhibit antibiotic-resistant bacteria (MRSA and Pseudomonas aeruginosa), the most common cause of biomaterial implant failure in modern medicine. Moreover, the cryogels showed negligible cytotoxicity toward murine fibroblasts and prevented activation of primary bone marrow-derived dendritic cells ex vivo. Finally, in vivo data suggested tissue integration, biodegradation, and minimal host inflammatory responses when the antimicrobial cryogels, even when purposely contaminated with bacteria, were subcutaneously injected in mice. Collectively, these needle-injectable microcomposite cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

Figure 1

Engineering antimicrobial and injectable microcomposite cryogels. (a) Overview of the fabrication process of antimicrobial CP-containing injectable cryogels: (1) cryogels were fabricated using 4% HAGM with different amounts of CP (0–0.2% CaO₂); an initiator system (APS/TEMED) is added to an aqueous HAGM solution prior to cryopolymerization at − 20 °C. (2) Cryotreatment involves phase separation with ice crystal formation, cross-linking and gelation. Thawing of ice crystals (porogens) results in an interconnected macroporous cryogel network. (b) Cryogel partially dehydrated over Kimwipe regains its original shape and size after hydration. HAGM cryogels were stained with rhodamine for visualization. (c) Following injection through a 16G hypodermic needle, cryogels regain their original shape and dimensions. (d) Cryogels retain their encapsulated CP after needle injection as indicated by the Alizarin Red S staining (n = 5).

Figure 2

Antimicrobial cryogels have advantageous mechanical properties and microarchitectural features. A total of three types of cryogels were fabricated using 4% HAGM and different amounts of CP (0–0.2% CaO₂). (a) swelling ratio, (b) Young’s moduli, and (c) evaluation of pore connectivity. Cross-sectional SEM images showing interconnected macroporous network of (d) CP-free cryogels (0% CaO₂), (e) CP-containing (0.1% CaO₂) cryogels, and (f) CP-containing (0.2% CaO₂) cryogels. SEM samples (n = 3) were scanned and representative images have been presented. Cross-sectional SEM images for visualization of CP (pseudocolored in pink): (g) CP-free cryogel (0% CaO₂), (h) CP-containing (0.1% CaO₂) cryogel, and (i) CP-containing (0.2% CaO₂) cryogel. Pore size distribution histograms of (j) CP-free cryogels (0% CaO₂), (k) CP-containing (0.1% CaO₂) cryogels, and (l) CP-containing (0.2% CaO₂) cryogels. Scale bars = 100 µm (d, e, and f) and 50 µm (g, h, and i).

Figure 3

CP-containing cryogels generate antimicrobial hydrogen peroxide (H₂O₂). (a) Release of H₂O₂ from the cryogels following injection through a 16G needle, n = 3. (b) Growth monitoring absorbance values of MRSA after 24 h of incubation with CP alone, CP with catalase and Ca(OH)₂ along with a control (CP-free medium), average values of three experiments have been plotted. (c) Schematic representing the biocidal action of CP-containing cryogels against bacteria.

Figure 4

Antibacterial activity of CP-containing microcomposite cryogels. (a) Residual CFU i.e., colony forming units per ml of MRSA after 6 h of contact period within different cryogels; namely CP-free cryogels (0% CaO₂), CP-containing (0.1% CaO₂) cryogels, and CP-containing (0.2% CaO₂) cryogels. (b) Cross-sectional SEM of CP-free cryogels (0% CaO₂), and (c) CP-containing (0.1% CaO₂) cryogels. (d) Residual CFU/ml P. aeruginosa after 6 h of contact period across different cryogels. (e) cross-sectional SEM of CP-free cryogel (0% CaO₂), and (f) CP-containing (0.2% CaO₂) cryogels. Data are representative of three independent experiments and are presented as the mean ± standard error of the mean (n = 4). For visualization, MRSA and P. aeruginosa were pseudocolored in yellow and green, respectively. Scale bars = 10 µm.

Figure 5

Antimicrobial CP-containing cryogels are cytocompatible towards murine fibroblasts. Confocal images showing mouse embryonic fibroblast NIH/3T3 cells cultured for 24 h in three CP-containing HAGM cryogels: (a) 0%, (b) 0.1%, and (c) 0.2% CaO₂. Red color depicts dead cells, blue color represents cell nuclei, green color highlights cell cytoskeleton and yellow color shows the polymer network of cryogels. (d) Evaluation of NIH/3T3 cell viability (%). Scale bars = 200 µm (large confocal images) and 100 µm (inset confocal images). Data are presented as the mean ± standard error of the mean (n = 4).

Figure 6

Antimicrobial CP-containing cryogels do not activate BMDCs. BMDCs were cultured for 24 h in the following conditions: in the presence of CP-free cryogels (0% CaO₂), CP-containing (0.1% CaO₂) cryogels, CP-containing (0.2% CaO₂) cryogels, cryogel-free medium (untreated, negative control) and LPS-containing medium (100 ng/mL, positive control). Fractions of activated (a) CD11c⁺ CD86⁺ BMDCs and (b) CD11c⁺ MHCII⁺ BMDCs after 24 h of incubation in the different conditions. (c) Expression profile of CD86 and MHCII in CD11c⁺ DCs. The results shown are representative of 4 independent experiments. Concentrations of DC-secreted (d) TNF-α, (e) IL-6, and (f) IL-12 cytokines from cell culture supernatants after 24 h of incubation in the different conditions. Data are presented as the mean ± standard error of the mean (n = 5) and were analyzed using one-way analysis of variance (ANOVA) and the Dunnett′s post-hoc test (*p < 0.05, **p < 0.01, compared to the untreated condition).

Figure 7

Antimicrobial microcomposite cryogels are biodegradable and elicit minimal host inflammatory responses. H&E staining of HAGM cryogel scaffold sections explanted 4 days following subcutaneous injections in the dorsal flanks of C57BL/6 mice: (a) CP-free cryogel (0% CaO₂), (b) CP-containing (0.1% CaO₂) cryogel, and (c) CP-containing (0.1% CaO₂) cryogel contaminated with P. aeruginosa. H&E staining highlights the macroporous polymeric network of cryogels (interconnected dark blue fibers), infiltrated leukocytes (dark blue dots), fibrin formation (purple), and surrounding tissues (cryogel-free). Masson's trichrome (MT) (d) and H&E (e) staining of square-shaped implants explanted 2 months following subcutaneous injections in the dorsal flanks of C57BL/6 mice: CP-free (0% CaO₂) and CP-containing (0.2% CaO₂) cryogels (dimensions: 4 mm × 4 mm × 1 mm). The yellow doted lines indicate the boundary between the cryogel and the host tissue. Images are representative of n = 5 samples per condition.