Natural and Synthetic Polymer Scaffolds Comprising Upconversion Nanoparticles as a Bioimaging Platform for Tissue Engineering

Abstract

Modern biocompatible materials of both natural and synthetic origin, in combination with advanced techniques for their processing and functionalization, provide the basis for tissue engineering constructs (TECs) for the effective replacement of specific body defects and guided tissue regeneration. Here we describe TECs fabricated using electrospinning and 3D printing techniques on a base of synthetic (polylactic-co-glycolic acids, PLGA) and natural (collagen, COL, and hyaluronic acid, HA) polymers impregnated with core/shell β-NaYF₄:Yb³⁺,Er³⁺/NaYF₄ upconversion nanoparticles (UCNPs) for in vitro control of the tissue/scaffold interaction. Polymeric structures impregnated with core/shell β-NaYF₄:Yb³⁺,Er³⁺/NaYF₄ nanoparticles were visualized with high optical contrast using laser irradiation at 976 nm. We found that the photoluminescence spectra of impregnated scaffolds differ from the spectrum of free UCNPs that could be used to control the scaffold microenvironment, polymer biodegradation, and cargo release. We proved the absence of UCNP-impregnated scaffold cytotoxicity and demonstrated their high efficiency for cell attachment, proliferation, and colonization. We also modified the COL-based scaffold fabrication technology to increase their tensile strength and structural stability within the living body. The proposed approach is a technological platform for "smart scaffold" development and fabrication based on bioresorbable polymer structures impregnated with UCNPs, providing the desired photoluminescent, biochemical, and mechanical properties for intravital visualization and monitoring of their behavior and tissue/scaffold interaction in real time.

Keywords: 3D printing; PLGA; bioimaging; collagen; electrospinning; hyaluronic acid; tissue engineering; upconversion nanoparticles.

Figure 1

Characterization of upconversion core/shell β-NaYF₄:Yb³⁺:Er³⁺/NaYF₄ nanoparticles: (a) schematic design of core/shell β-NaYF₄:Yb³⁺:Er³⁺/NaYF₄ UCNPs; (b) energy level diagram of the UCNPs; (c) photoluminescence spectrum of the UCNPs; (d) photograph of the UCNPs under excitation at 976 nm; (e) dependence of the ratio of the intensity of the red luminescence band at a wavelength of 658 nm to that of the green one at a wavelength of 544 nm on the power density of exciting laser radiation for UCNPs; (f) dependences of the photoluminescence intensity on the power density of exciting radiation on a double logarithmic scale at wavelengths of 544 and 658 nm for the UCNPs.

Figure 2

Tensile test results for ELS COL samples immersed in isopropanol with 15 wt.% BDDGE for 6 days and in PBS for 1 day: (a) Young’s modulus, (b) tensile strength, (c) maximum elongation. * p < 0.05, **p < 0.01 in Student’s t-test; data are the mean ± SD of at least 3 replicates.

Figure 3

Tensile test results for dry ELS COL samples immersed in isopropanol with 15 wt.% BDDGE for 6 days, dried and immersed in PBS for 1 day: (a) Young’s modulus, (b) tensile strength, (c) maximum elongation. * p < 0.05 in Student’s t-test; data are the mean ± SD of at least 3 replicates.

Figure 4

Tensile test results for ELS PLGA samples dried and immersed in PBS for 1 day: (a) Young’s modulus, (b) tensile strength, (c) maximum elongation; data are the mean ± SD of at least 3 replicates.

Figure 5

Schematic illustration of custom-build experimental setup for (a) electrospinning of polymer solutions; (b) anti-solvent extrusion 3D printing; (c) extrusion 3D printing with simultaneous photocuring and corresponding photoluminescent imaging of scaffolds.

Figure 6

Electrospun collagen scaffold characterization: SEM image (a); normalized photoluminescence spectra of the UCNPs and the UCNPs included in ELS COL scaffolds (b); photographs of ELS COL scaffolds with 0 (control) and 1 mg of UCNPs per 100 mg of polymer at 976 nm (c). Ratio of the intensity of the red peak at 658 nm to the intensity of the green one at a wavelength of 544 nm of UCNPs and ELS COL scaffolds (0.1 and 1% UCNPs) * p < 0.05 in Student’s t-test (d).

Figure 7

Electrospun polylactic-co-glycolic acid scaffolds: SEM image (a); normalized photoluminescence spectra of the UCNPs and the UCNPs included in ELS PLGA scaffolds (b); photographs of ELS PLGA scaffolds with 0 (control) and 1 mg of UCNPs per 100 mg of polymer at 976 nm (c). Ratio of the intensity of the red peak at 658 nm to the intensity of the green at a wavelength of 544 nm of UCNPs and ELS PLGA scaffolds (0.1 and 1% UCNPs) ** p < 0.01 in Student’s t-test (d).

Figure 8

SEM image of 3D-printed polylactic-co-glycolic acids scaffolds (a); normalized photoluminescence spectra of the UCNPs and the UCNPs included in 3D PLGA scaffolds (b); photograph of 3D PLGA scaffolds with 0 (control) and 1 mg of UCNPs per 100 mg of polymer at 976 nm (c). Ratio of the intensity of the red peak at 658 nm to the intensity of the green at a wavelength of 544 nm of UCNPs and 3D PLGA (0.1 and 1% UCNPs) ** p < 0.01 in Student’s t-test (d).

Figure 9

SEM image of 3D-printed HAGM scaffolds (a); normalized photoluminescence spectra of the UCNPs and the UCNPs included in 3D HAGM scaffolds (b); photograph of 3D HAGM scaffolds with 0 and 1 mg of UCNPs per 100 mg of polymer at 976 nm (c). Ratio of the intensity of the red peak at 658 nm to the intensity of the green at a wavelength of 544 nm of UCNPs and 3D HAGM scaffolds (0.1 and 1% UCNPs) * p < 0.05, ** p < 0.01 in Student’s t-test (d).

Figure 10

The viability of Bj-5ta fibroblasts in extract assay, 24 h incubation. Data are the mean ± SD; the viability of non-treated (intact) cells was taken as 100%.

Figure 11

The growth of Bj-5ta fibroblasts on the UCNPs-loaded scaffolds, 4 and 8 days of incubation. Data are the mean ± SD, * p < 0.05 in Mann–Whitney U test.

Figure 12

Confocal images of ELS COL (a), ELS COL 0.1% (b), and ELS COL 1% (c) scaffolds cultured with Bj-5ta fibroblasts, 8 days of incubation. Green is for calcein AM staining (live cells), blue is for Hoechst 33342 staining (cell nuclei).