Light-Responsive and Antibacterial Graphenic Materials as a Holistic Approach to Tissue Engineering

While the continuous development of advanced bioprinting technologies is under fervent study, enhancing the regenerative potential of hydrogel-based constructs using external stimuli for wound dressing has yet to be tackled. Fibroblasts play a significant role in wound healing and tissue implants at different stages, including extracellular matrix production, collagen synthesis, and wound and tissue remodeling. This study explores the synergistic interplay between photothermal activity and nanomaterial-mediated cell proliferation. The use of different graphene-based materials (GBM) in the development of photoactive bioinks is investigated. In particular, we report the creation of a skin-inspired dressing for wound healing and regenerative medicine. Three distinct GBM, namely, graphene oxide (GO), reduced graphene oxide (rGO), and graphene platelets (GP), were rigorously characterized, and their photothermal capabilities were elucidated. Our investigations revealed that rGO exhibited the highest photothermal efficiency and antibacterial properties when irradiated, even at a concentration as low as 0.05 mg/mL, without compromising human fibroblast viability. Alginate-based bioinks alongside human fibroblasts were employed for the bioprinting with rGO. The scaffold did not affect the survival of fibroblasts for 3 days after bioprinting, as cell viability was not affected. Remarkably, the inclusion of rGO did not compromise the printability of the hydrogel, ensuring the successful fabrication of complex constructs. Furthermore, the presence of rGO in the final scaffold continued to provide the benefits of photothermal antimicrobial therapy without detrimentally affecting fibroblast growth. This outcome underscores the potential of rGO-enhanced hydrogels in tissue engineering and regenerative medicine applications. Our findings hold promise for developing game-changer strategies in 4D bioprinting to create smart and functional tissue constructs with high fibroblast proliferation and promising therapeutic capabilities in drug delivery and bactericidal skin-inspired dressings.


Determination of photothermal efficiency values
The efficiency values were calculated by following the protocol described by Feng et al. 28  = [ℎ( − )]− / [(1−10−)] (1) Being  the photothermal conversion efficiency, ℎ the heat transfer coefficient,  the surface area of the sample cuvette,  the steady-state temperature,  the temperature of the surrounding,  the heat associated with the light absorbance of the solution,  the incident laser power, and  the absorbance of the nanomaterials at a wavelength of 808 nm. is defined through the following equation ( 2): Where  is the mass of the water solution,  the water heat capacity,  the increase in water temperature, and  the duration of the irradiation.ℎ, which can be named as , is defined as follows (3): To solve for , a sample time constant  is defined (4): Also, as reported in the literature 28 , the following relation can be established (5): Therefore, the time constant is obtained from the equation of the graph when plotting time data vs .ℎ can be defined according to the obtained , also considering the mass of the solution and the heat capacity of water.

Semi-quantification of printability
The printability values were calculated by following the protocol described by L Ouyang et al. 29 When the bioink gels ideally, the extruded filament shows a smooth, consistently sized morphology, forming regular grids and square holes in the constructs.In contrast, undergelation leads to a more liquid-like state, causing the upper layer to merge with the lower layer and creating roughly circular holes in the process.It is known that circularity (C) of an enclosed area is defined as: where, L means perimeter and A means area.Circles have the highest circularity (C = 1) If the C value approaches 1, the shape is more circular.Circularity for a square shape is π/4.We establish bioink printability (Pr) for a square shape using the following function: Under ideal gelation or perfect printability, the interconnected channels in constructs exhibit a square shape, with a Pr value of 1.A higher Pr value indicates a greater bioink gelation degree, while a lower Pr value suggests a smaller gelation degree.The Pr value for each bioprinted scaffold was determined by analyzing optical images in ImageJ software to calculate the perimeter and area of interconnected channels (n = 3).Table S1.Elemental analyses of rGO and GP.

Figure S1 .
Figure S1.Representative TEM images of a) GO, b) rGO and c) GP nanomaterials.

Figure S5 .
Figure S5.Representative images of bioprinted scaffolds Alg (left panel) and Alg_rGO (right panel) before irradiation (top panel) and after irradiation with 808 nm light for 10 min at a power density of 0.5 W/cm 2 (bottom panel).

Figure S6 .
Figure S6.Representative pictures from the Live/Dead experiments of hFBs embedded into non-irradiated Alg and Alg_rGO hydrogels, at incubation time points of 0 h, 24 h and 72 h.

Figure S7 .
Figure S7.Representative pictures from the Live/Dead experiments of hFBs embedded into irradiated Alg and Alg_rGO hydrogels, at incubation time points of 0 h, 24 h and 72 h.