Dual roles of photosynthetic hydrogel with sustained oxygen generation in promoting cell survival and eradicating anaerobic infection

Abstract

Tissue engineering offers a promising alternative for oral and maxillofacial tissue defect rehabilitation; however, cells within a sizeable engineered tissue construct after transplantation inevitably face prolonged and severe hypoxic conditions, which may compromise the survivability of the transplanted cells and arouse the concern of anaerobic infection. Microalgae, which can convert carbon dioxide and water into oxygen and glucose through photosynthesis, have been studied as a source of oxygen supply for several biomedical applications, but their promise in orofacial tissue regeneration remains unexplored. Here, we demonstrated that through photosynthetic oxygenation, Chlamydomonas reinhardtii (C. reinhardtii) supported dental pulp stem cell (DPSC) energy production and survival under hypoxia. We developed a multifunctional photosynthetic hydrogel by embedding DPSCs and C. reinhardtii encapsulated alginate microspheres (CAMs) within gelatin methacryloyl hydrogel (GelMA) (CAMs@GelMA). This CAMs@GelMA hydrogel can generate a sustainable and sufficient oxygen supply, reverse intracellular hypoxic status, and enhance the metabolic activity and viability of DPSCs. Furthermore, the CAMs@GelMA hydrogel exhibited selective antibacterial activity against oral anaerobes and remarkable antibiofilm effects on multispecies biofilms by disrupting the hypoxic microenvironment and increasing reactive oxygen species generation. Our work presents an innovative photosynthetic strategy for oral tissue engineering and opens new avenues for addressing other hypoxia-related challenges.

Keywords: Anaerobic bacteria; Antibacterial activity; Chlamydomonas reinhardtii; Oxygen; Photosynthesis; Tissue engineering.

Graphical abstract

Scheme 1

The photosynthetic hydrogel could provide sustained and sufficient oxygen supply through photosynthesis by encapsulating C. reinhardtii alginate microspheres to promote DPSC survival and eradicate anaerobic infection.

Fig. 1

Characterization of C. reinhardtii. (a) Bright-field and (b) fluorescence images of C. reinhardtii. Inset: large-scale culture of microalgae. (c) Dissolved oxygen produced by C. reinhardtii at different concentrations under light conditions at 25 °C and 37 °C. (d) Dissolved oxygen released by C. reinhardtii under light (ON, yellow) and dark (OFF, gray) conditions at 25 °C and 37 °C. (e) Comparison of the photosynthetic activity of C. reinhardtii at 25 °C and 37 °C. Proliferation of DPSCs cocultured with different concentrations of C. reinhardtii under (f) normoxic and (g) hypoxic conditions. Scale bars: 25 μm (a, b). *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Photosynthetic oxygen produced by C. reinhardtii improves DPSC survival under hypoxia. (a) Schematic illustration of the indirect coculture of DPSCs and C. reinhardtii. (b) Dissolved oxygen concentrations in the coculture medium of DPSCs incubated under different conditions. (c) Representative fluorescence images of DPSCs stained with Hypoxyprobe Image-iT (green) and Hoechst (blue) under different conditions. (d) Western blotting images and (e) corresponding quantification of HIF-1α expression in DPSCs after 24 h of different treatments. (f) Metabolic activity of DPSCs cultured with or without C. reinhardtii under hypoxia for 7 days. (g) Representative fluorescence images of live/dead staining and (h) corresponding quantification of the survival fraction of DPSCs cultured with or without C. reinhardtii under hypoxia for 4 days. (i) Intracellular ATP content in DPSCs cultured with or without C. reinhardtii under hypoxia for 24 h. Scale bars: 50 μm (c), 100 μm (g). *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Characterization of C. reinhardtii alginate microspheres (CAMs). (a) Schematic illustration of the immobilization of C. reinhardtii in alginate microspheres. (b) Bright-field images and (c) diameter distribution of alginate microspheres. (d) Representative bright-field and (e) fluorescence images of CAMs. (f, g) Representative SEM images of the external surface and (h, i) internal structure of alginate microspheres. White arrows indicate C. reinhardtii. (j) Macroscopic images of CAMs incubated for 14 days. (k) Cell density of C. reinhardtii in alginate microspheres. (l) Representative fluorescence images of live/dead staining for C. reinhardtii immobilized in alginate microspheres. (m) Comparison of the oxygen-producing capability of immobilized and free C. reinhardtii. Scale bars: 5 μm (g), 10 μm (i), 50 μm (f, h), 100 μm (d, e), 200 μm (b, l). ***p < 0.001.

Fig. 4

Characterization of photosynthetic GelMA hydrogel. (a) 3D fluorescent images showing the distribution of DPSCs (green) and C. reinhardtii (red) within different GelMA hydrogels. (b, c) Representative SEM images of the inner pore structure of different GelMA hydrogels. Asterisks denote GelMA hydrogels, white arrows point to alginate microspheres, and green arrows indicate immobilized C. reinhardtii. (d) Comparison of oxygen concentration in different GelMA hydrogels incubated under hypoxia. (e) Western blotting images and (f) corresponding quantification of HIF-1α expression in DPSCs encapsulated in different GelMA hydrogels for 24 h. (g) Representative fluorescence images of DPSCs stained with Hypoxyprobe Image-iT (green) and Hoechst (blue) within different GelMA hydrogels. Scale bars: 50 μm (c, g), 200 μm (a), 250 μm (b). **p < 0.01. ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

High survivability of DPSCs within photosynthetic hydrogel under hypoxia. (a) Metabolic activity of DPSCs encapsulated in different GelMA hydrogels under hypoxia for 7 days. (b) Representative fluorescence images of live/dead staining and (c) corresponding quantification of the survival fraction of DPSCs encapsulated in different GelMA hydrogels. Scale bars: 100 μm (b). **p < 0.01. ***p < 0.001.

Fig. 6

Antibacterial activity of photosynthetic hydrogel. (a) Antibacterial effects of different GelMA hydrogels on planktonic bacteria after 24 h. (b) Representative CFU images and (c) corresponding colony counts of bacteria treated with different GelMA hydrogels for 24 h. (d) Representative fluorescence images of ROS stained by DCFH-DA (green) in multi-planktonic bacteria (P. gingivalis, F. nucleatum, and S. gordonii) treated with different GelMA hydrogels for 24 h. Scale bars: 10 μm (d). ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Antibiofilm effects of photosynthetic hydrogel on multispecies biofilms. (a, c) Representative 3D fluorescent images of live/dead staining for multispecies biofilms treated with different GelMA hydrogels. (b, d) Average thickness of biofilms. (e, f) Representative SEM images of multispecies biofilms treated with different GelMA hydrogels. Scale bars: 5 μm (e, f), 20 μm (a, c). ***p < 0.001.