Immobilization of Heparin on Bacterial Nanocellulose Hydrogels Induces Tubulogenesis of Human Endothelial Cells

Materials

Reagents and chemicals were purchased from Sigma- Aldrich do Brasil Ltd. (São Paulo, SP). Culture media and supplements were purchased from Thermo Fisher Scientific do Brasil Ltd. (São Paulo, SP). Heparin (HEPAMAX-S® sodium heparin - 5000 IU/mL) was supplied by Blau Farmacêutica S.A. (Cotia, SP).

BNC-HEP Hydrogels Synthesis

Gluconacetobacter hansenii (strain ATCC 23769) was cultured in a sterile medium composed of mannitol (25 g), yeast extract (5.0 g), and bactopeptone (3.0 g), diluted in 1 L of water and pH adjusted to 6.5. Mixture was stirred and transferred to 24 wells plates (1 mL/well). After four days, on static culture conditions at 26°C, hydrogels were removed and purified in 0.1 M NaOH solution at 50°C for 24 h and then rinsed with distilled water to pH 6.5. Finally, the hydrogels were sterilized by autoclaving (121°C for 20 min).

BNC hydrogels were oxidized, with HNO3/H3PO4- NaNO2, to convert free hydroxyl groups of BNC in carboxyl groups [6]. Those carboxyl groups were activated with EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) allowing the covalent immobilization of heparin (Figure 1a). Briefly, oxidized BNC were immersed in 0.1 M citrate buffer solution (pH 4.8) containing 2 mM of EDC. Those hydrogels were shaking during 24 h at 4°C and after washed with deionized water. Samples were placed in citrate buffer (pH 4.8) containing 1000 IU heparin (v/v) for 24 h at 4°C with shaking. Then, BNC- HEP hydrogels were washed with alkaline phosphate buffered saline and rinsed with distilled water. Hydrogels were lyophilized at -50°C for 24h to perform the chemical and microstructural characterization and they were sterilized by UV before to perform in vitro tests.

Characterization of BNC-HEP

The functional groups on BNC and BNC-HEP were analyzed by Fourier Transformed Infrared Spectroscopy (FTIR). Infrared spectra were recorded on an Agilent spectrophotometer (model Carry 600), with a resolution of 4 cm-1, in a range of 4000-600 cm-1, and using attenuated total reflectance. Scanning electron microscope (SEM) was performed to analyze the microstructure of top and bottom surfaces of BNC and BNC-HEP, using a JEOL JSM-6390LV microscope at 10 kV. Toluidine blue colorimetric assay was used to determine the amount of heparin immobilized on BNC-HEP. The amount of heparin immobilized on BNC-HEP hydrogels was quantitated in comparison with the calibration curve prepared [7].

Citotoxic potential of BNC-HEP hydrogels was evaluated following the direct contact assay. For that, immortalized murine fibroblast cells (L929) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% of FBS and 1% penicillin/streptomycin. L929 cells were seeded in each well of 24 wells culture plate at 9.0 × 103 cells/cm2 for 24 h in a humidified atmosphere (5% CO2) at 37°C. BNC hydrogels, non-cytotoxic material, were used as control group [8]. After that, BNC and BNC-HEP hydrogels were added into each well. After 1, 3 and 7 days of in vitro culture, the metabolic activity of L929 cells were determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5- (3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium] colorimetric assay from Promega Biotecnologia do Brasil, Ltda. (São Paulo, SP), performed according to the manufacturer’s instructions.

Adhesion, Proliferation and Tubulogenesis Induction of Huvecs on Hydrogels

HUVECs were seeded at a density of 105 cells/cm2 on the bottom surface of BNC-HEP hydrogels to evaluate tubulogenesis induction. After 24 h, in a humidified atmosphere containing 5% of CO2 at 37°C, samples were fixed with 4% of formaldehyde for 30 min at room temperature. Phase contrast images were taken using an Olympus BX4 microscope (Olympus America Inc.).

Statistical Analysis

Data were analyzed statistically by one-way analysis of variance (ANOVA) with Tukey test. Values represent the mean ± standard medium error, with p< 0.05. All experiments were performed in triplicate at two separated experimental times.

Functionalization of BNC Hydrogels

Characteristic bands of BNC appeared at 3345 cm-1 and 2898 cm-1, where the absorption band assigned to hydroxyl groups and hydrogen bonds, respectively [9] (Figure 1b). BNC-HEP showed a broad absorption at 3100–3500 cm-1 which was assigned to N–H stretching absorption and hydrogen bonded hydroxyl groups; Other heparin characteristic bands were observed at 1701 cm−1 (C=O) and 1248 cm-1 (S=O) [9, 10].

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SEM micrographs related to the top and bottom surfaces of BNC and BNC‒HEP hydrogels were shown in Figure 2a. BNC hydrogels were characterized by an entangle arrangement of BNC fibers on top surface and with a porous arrangement of fibers on the bottom surface. The top surface of BNC-HEP kept the same microstructure observed on the top surface of BNC. Moreover, the bottom surface of BNC-HEP showed a similar microstructure observed on the bottom surface of BNC, however within more pores. In addition, results showed that BNC and BNC-HEP hydrogels did not induce any citotoxic effect on L929 cells during 7 days of in vitro culture Figure 2b. The amount of heparin immobilized on BNC hydrogels was quantified by toluidine blue dye assay which revealed a density of HEP immobilization corresponding to 4.7 ± 0.2 μg⋅cm-2 approximately 1% of BNC dry weight.

Tubulogenesis Induction

After 24 hours of culture, HUVECs were aligned and organized as tubulogenic networks on the bottom surface of BNC-HEP. In detail, Figure 3a showed the formation of endothelial tubulogenic networks containing loops and branch points. Figure 3b showed the presence of endothelial tip cells, characterized by cell protrusions called filopodia followed by endothelial stalk cells.

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bars represent 100 µm.