Fast, Easy, and Reproducible Fingerprint Methods for Endotoxin Characterization in Nanocellulose and Alginate-Based Hydrogel Scaffolds

Abstract

Nanocellulose- and alginate-based hydrogels have been suggested as potential wound-healing materials, but their utilization is limited by the Food and Drug Administration (FDA) requirements regarding endotoxin levels. Cytotoxicity and the presence of endotoxin were assessed after gel sterilization using an autoclave and UV treatment. A new fingerprinting method was developed to characterize the compounds detected in cellulose nanocrystal (CNC)- and cellulose-nanofiber (CNF)-based hydrogels using both positive- and negative-ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectroscopy (ESI FT-ICR MS). These biobased hydrogels were used as scaffolds for the cultivation and growth of human dermal fibroblasts to test their biocompatibility. A resazurin-based assay was preferred over all other biocompatibility methodologies since it allowed for the evaluation of viability over time in the same sample without causing cell lysis. The CNF dispersion of 6 EU mL^-1 was slightly above the limits, and it did not affect the cell viability, whereas CNC hydrogels induced a reduction of metabolic activity by the fibroblasts. The chemical structure of the detected endotoxins did not contain phosphates, but it encompassed hydrophobic sulfonate groups, requiring the development of new high-pressure sterilization methods for the use of cellulose hydrogels in medicine.

Figure 1

Preparation of hydrogels by the osmotic dehydration process with the following compounds: (A) 0.25 wt % methylcelluloses (MC156S, SGA150) and CNC/CNF; (B) pure methylcellulose SGA150; and (C) methylcellulose (MC156S; SGA150) and alginate with CaCl₂ as a cross-linker.

Figure 2

Effects of cellulose hydrogels on human dermal fibroblast viability. (a) Indirect assay, in which fibroblasts are treated in a conditioned medium and incubated for 24 h in the presence of the hydrogels. (b, c) Direct assay, in which fibroblasts are cultured directly on the hydrogels: (b) with testing of all hydrogels at 24 and/or 48 h. (c) Visualization of cell viability of each hydrogel at 24 and 48 h. The site letters mean (CTR) control medium; (A) 0.25 wt % MC156S–0.25 wt % alginate; (B) 0.25 wt % MC156S–1 wt % CNF; (C) 0.25 wt % SGA150 solution; (D) 0.25 wt % SGA150–0.25 wt % alginate; and (E) 0.25 wt % MC156S–1 wt % CNC.

Figure 3

Morphology and organization of fibroblasts cultured in different cellulose hydrogels and in control conditions on culture plastic after 48 h. Black arrows point to the cells. The letters mean (CTR) control medium; (A) 0.25 wt % MC156S–0.25 wt % alginate; (B) 0.25 wt % MC156S–1 wt % CNF; (C) 0.25 wt % SGA150 solution; (D) 0.25 wt % SGA150–0.25 wt % alginate; and (E) 0.25 wt % MC156S–1 wt % CNC.

Figure 4

Endotoxins measured in pure solutions of H2SO₄-hydrolyzed CNCs, TEMPO-oxidized CNC, TEMPO-oxidized CNF, methylcelluloses (MC156S, SGA150), and alginic acid dissolved in DI water.

Figure 5

Van Krevelen plots were generated from the ESIFT-ICR-MS data of the CNC and CNF samples. The compound classes were assigned according to previous studies,^, and displayed color-coded, according to the description at the bottom. The observed intensity is presented logarithmically and color-coded (blue: low intensity, yellow: medium intensity, and red: high intensity; see the color bar).

Figure 6

n_C–DBE plots for compound classes O₁–O₄ of the ESI FT-ICR MS data for all three analyzed CNC and CNF samples. The observed intensity is presented logarithmically and color-coded (blue: low intensity, yellow: medium intensity, and red: high intensity).