3D-printed aerogels as theranostic implants monitored by fluorescence bioimaging

Abstract

Aerogel scaffolds are nanostructured materials with beneficial properties for tissue engineering applications. The tracing of the state of the aerogels after their implantation is challenging due to their variable biodegradation rate and the lack of suitable strategies capable of in vivo monitoring the scaffolds. Upconversion nanoparticles (UCNPs) have emerged as advanced tools for in vitro bioimaging because of their fluorescence properties. In this work, highly fluorescent UCNPs were loaded into aerogels to obtain theranostic implants for tissue engineering and bioimaging applications. 3D-printed alginate-hydroxyapatite aerogels labeled with UCNPs were manufactured by 3D-printing and supercritical CO₂ drying to generate personalize-to-patient aerogels. The physicochemical performance of the resulting structures was evaluated by printing fidelity measurements, nitrogen adsorption-desorption analysis, and different microscopies (confocal, transmission and scanning electron microscopies). Stability of the aerogels in terms of physicochemical properties was also tested after 3 years of storage. Biocompatibility was evaluated in vitro by different cell and hemocompatibility assays, in ovo and in vivo by safety and bioimaging studies using different murine models. Cytokines profile, tissue index and histological evaluations of the main organs unveiled an in vivo downregulation of the inflammation after implantation of the scaffolds. UCNPs-decorated aerogels were first-time manufactured and long-term traceable by fluorescence-based bioimaging until 3 weeks post-implantation, thereby endorsing their suitability as tissue engineering and theranostic nanodevices (i.e. bifunctional implants).

Keywords: Aerogels; In vivo fluorescence; Theranostic implants; Upconversion nanoparticles.

Graphical abstract

Fig. 1

Physicochemical evaluation of UCNPs-decorated aerogels. (a) Filament drop test, general pore shape geometry and visual appearance of aerogels obtained from different ink formulations: Alg UCNPs 0.8 (left) and AlgHA UCNPs 0.4 (right). Rheological evaluation of inks by (b,c) overall viscosity with respect to the shear rate; (d) shear stress with respect to the shear rate, and (e) overall viscosity with respect to the shear stress. (f) SFF and (g) volume shrinkage (in percentage) of different UCNPs-decorated aerogels. No statistically significant differences among groups were obtained after post hoc Tukey HSD multiple comparison test (p < 0.05). (h) ATR/FT-IR spectra of UCNPs and aerogel formulations.

Fig. 2

Microscopical analysis of UCNPs-decorated aerogels. SEM images of different aerogel formulations observed at two different magnifications: (a,b) Alg UCNPs 0.4; (c,d) Alg UCNPs 0.8; (e,f) AlgHA UCNPs 0.4. (g) EDX spectrum of Alg UCNPs 0.8 aerogels. (h) Particle size distribution of the UCNPs loaded in Alg UCNPs 0.8 aerogels. (i, j) TEM images of UCNPs. (i) UCNPs obtained just after synthesis; (j) UCNPs obtained 3 years after synthesis. (k) Particle size distribution of UCNPs. (l,m) Confocal images of different formulations of UCNPs-decorated aerogels (scale bar: 100 μm). (n–s) SEM images of different aerogel formulations 3 years after synthesis observed at two different magnifications: (n,o) Alg; (p,q) Alg UCNPs 0.8; (r,s) AlgHA UCNPs 0.4.

Fig. 3

Cellular studies of different aerogel formulations. (a) Cell viability test on BALB/c 3T3 cells. Viability (expressed in %) of BALB/c 3T3 cells after 24 and 48 h in contact with aerogels measured by WST-1 test. Positive controls: BALB/c 3T3 cells without contact with UCNPs or aerogels. * Data obtained from Ref. [10]. (b) Scratch test on BALB/c 3T3 cells. Quantitative analysis (expressed in %) of the BALB/c 3T3 cells migration area after 24 h in cell culture and microscopy images after 0 and 24 h. Positive controls: BALB/c 3T3 cells without contact with UCNPs or aerogels. (c) Cell attachment test on MSCs. Quantitative analysis (expressed in nuclei number per mm² of scaffold) after 13 days in cell culture (left) and confocal images of DAPI-stained MSCs seeded on aerogels (middle) and of aerogels after cell attachment tests (right). No statistically significant differences among groups were obtained after post hoc Tukey HSD multiple comparison test (p < 0.05).

Fig. 4

Hemocompatibility evaluation of different aerogel formulations. (a) Hemolysis percentage of UCNPs and UCNPs-labeled aerogels. Positive control: 4 % v/v Triton X-100 and negative control 0.9 % w/v NaCl. * Data obtained from Ref. [10]. Statistically significant differences among groups were denoted as *** (post hoc Tukey HSD multiple comparison test, p < 0.001). (b) HET-CAM test of aerogels. Negative control: 0.9 % w/v NaCl, positive control: 0.1 N NaOH.

Fig. 5

Plasmatic cytokines (TNF-α and IL-10) concentration (expressed in pg/mL) and tissue index (of spleen, liver, and kidney) values (expressed in %) of (a) mice and (b) rats 1 week after subcutaneous implantation of different aerogel formulations (Alg, Alg UCNPs 0.8 or AlgHA UCNPs 0.4, respectively). Control: animals with a surgical incision without scaffold implantation. Plasmatic concentration of IL-6 was not detected. No statistically significant differences among groups were obtained after post hoc Tukey HSD multiple comparison test (p < 0.01). (c) Images of the connective tissue capsule developed 1 week after subcutaneous implantation of UCNPs-labeled aerogels (AlgHA UCNPs 0.4).

Fig. 6

Histological images obtained by H&E staining of different tissues (skin, spleen, liver, and kidney) of (a) mice and (b) rats 1 week after subcutaneous implantation of different aerogel formulations (Alg, Alg UCNPs 0.8 or AlgHA UCNPs 0.4, respectively). Control: animals with a surgical incision without scaffold implantation.

Fig. 7

Fluorescence intensity maps (expressed in normalized counts) obtained at the excitation wavelength of 745 nm and the emission wavelength of 840 nm. (a) Alg (left) and Alg UCNPs 0.8 aerogel implants (right) without (top) and with (bottom) fluorescence excitation. (b)in vivo fluorescence image of Alg implant (control of fluorescence emission). (c–h)in vivo fluorescence images recorded at different timepoints post-implantation of mice bearing Alg UCNPs 0.8 aerogel implants. (i) Fluorescence image of excised brain, liver, heart, lungs, kidneys, spleen, and skin surrounding the implant (rows from top to bottom) of a control animal (without scaffold implantation) and of the 3 animals with Alg UCNPs 0.8 theranostic implant (columns from left to right).