**Appendix A**

In our own experiments, we have contributed to the knowledge on potential biomedical applications of nanocellulose/carbon nanotube composites, namely regarding their application in tissue engineering. In these experiments, a PurCotton® cellulose mesh (Winner Industrial Park, Shenzhen, China) was modified with an aqueous dispersion of positively-charged (i.e., cationic) wood-derived CNFs, described in our earlier review article [2], and MWCNTs. Two types of composite samples were prepared, namely the samples with "thick" and "thin" coating of the fibers in the cellulose mesh. For thick coating, square-shaped samples of the cellulose mesh were fully immersed in an aqueous suspension of CNFs+MWCNTs, and during this immersion, the samples were homogeneously impregnated with the nanoparticles. For thin coating, only one corner of the cellulose samples was submerged into the CNF+MWCNT suspension, which resulted in infiltration of the suspension throughout the cellulose mesh by capillary forces. Both types of samples were then dried for 5 h at 60 ◦C. Both types of samples displayed a grayish color, which was more intense in samples with a thick coating. A pure CNF coating was prepared similarly to the thick coatings, and the meshes without coating were used as a control.

The samples were sterilized by UV light (20 min for each side), fixed into CellCrowns™ inserts (Scaffdex Ltd., Tampere, Finland), placed into 24-well cell culture plates (TPP, Trasadingen, Switzerland) and seeded with human adipose tissue-derived stem cells (hASC) or porcine adipose tissue-derived stem cells (pASC). Human ASC were isolated from subcutaneous fat tissue, obtained by liposuction from the abdominal region of healthy female donors after their informed consent, under ethical approval issued by the Ethics Committee of Hospital "Na Bulovce" in Prague, and in compliance with the tenets of the Declaration of Helsinki on experiments involving human tissues. The isolation was described in more details in our earlier studies [173,174]. Porcine ASC were isolated by enzymatic disintegration of subcutaneous fat tissue samples obtained by excision from laboratory pigs in collaboration with the Institute of Clinical and Experimental Medicine (IKEM) in Prague, Czech Republic. Characterization of cells by flow cytometry revealed the positivity of cells for standard surface markers of ASCs, namely CD105, CD90, CD73 and CD29 in hASC, and CD105, CD90, CD29 and CD44 in pASCs. The ASCs from both species were negative or almost negative for hematopoietic markers, such as CD34 and CD45, and for CD31, an endothelial marker [174,175].

The cells were seeded on the material samples at a density of 50,000 cells per well into 1.5 mL of the culture media. Human ASCs were cultivated in Dulbecco's modified Eagle's Medium (DMEM; Life Technologies, Gibco, Carlsbad, CA, USA) with 10% of fetal bovine serum (FBS; Life Technologies, Gibco), 40 μg/mL of gentamicin (LEK, Ljubljana, Slovenia) and 10 ng/mL of recombinant human fibroblast growth factor basic (FGF2; GenScript, Piscataway, NJ, USA). Porcine ASCs were cultivated in Dulbecco's modified Eagle's Medium (Low glucose, Sigma-Aldrich Co., St. Louis, MO, USA) and Ham's Nutrient Mixture F12 medium (DMEM/F 12, Sigma-Aldrich Co.) in a ratio of 1:1 with 10% of fetal bovine serum (FBS; Life Technologies, Gibco), 1% Antibiotic Antimycotic solution (Sigma-Aldrich Co.) and 10 ng/mL of recombinant human fibroblast growth factor basic (FGF2; GenScript).

After one or seven days of cultivation, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich Co.) for 20 min, and 0.1% Triton X-100 (Sigma-Aldrich Co.) diluted in phosphate-buffered saline (PBS) was applied for 20 min at room temperature in order to permeabilize the cell membranes. Nonspecific binding sites for antibodies were then blocked by a solution of 1% bovine serum albumin and 0.1% Tween 20 in PBS (all Sigma-Aldrich Co.). Vinculin, a protein of focal adhesion plaques associated with integrin adhesion receptors, was visualized by treating the samples for 1 h at 37 ◦C with primary antibody against human vinculin (V9131, monoclonal mouse antibody, clone hVIN-1, Sigma-Aldrich Co.), diluted in the blocking solution (1% albumin and 0.1% Tween 20 in PBS) in a ratio of 1:200. After washing with PBS, the samples were incubated for 1 h at room temperature in the dark with a secondary antibody, i.e., goat anti-mouse F(ab')2 fragments of IgG (H + L), conjugated with Alexa Fluor® 488 (A11017; Molecular Probes, Eugene, OR, USA; Thermo Fisher Scientific, Waltham, MA, USA), diluted in PBS to a ratio of 1:400. Finally, cytoskeletal F-actin filaments were stained by phalloidin conjugated with tetramethylrhodamine isothiocynate (TRITC) fluorescent dye (Sigma-Aldrich Co.), diluted in PBS to a final concentration of 5 μg/mL, for 1 h at room temperature in the dark. Microscopy images were acquired using spinning disk confocal system Dragonfly 503 (Andor, Belfast, UK) with Zyla 4.2 PLUS sCMOS camera (Andor, Belfast, UK) mounted on microscope Leica DMi8 (Leica Microsystems, Wetzlar, Germany) with objective HC PL APO 20x/0.75 IMM CORR CS2; Free Working Distance = 0.66 mm or HC PL APO 40x/1.10 W CORR CS2; Free Working Distance = 0.65 mm.

We found that the initial adhesion and subsequent growth of cells, evaluated by the cell number and spreading on days 1 and 7, were similar on all coated samples. There was no apparent difference between samples coated with thick and thin layers of CNFs + MWCNTs and samples coated only with CNFs. However, all types of coatings markedly improved the adhesion and growth of cells in comparison with a pure uncoated cellulose mesh (Figure A1). In general, the cell growth was relatively slow in all tested samples. On day 7, the cell number in all tested samples was only slightly higher than on day 1. In addition, hASCs grew slightly better than pASCs, particularly on samples coated with CNFs+MWCNTs. Therefore, we decided to cultivate pASCs on the tested samples under dynamic

conditions, which are known to improve the growth of cells by their mechanical stimulation, by a better supply of nutrients and oxygen and by quicker waste removal.

**Figure A1.** Human adipose tissue-derived stem cells (hASC) and porcine adipose tissue-derived stem cells (pASC) on days 1 and 7 after seeding on a cellulose mesh with thick or thin CNF+MWCNT coating (column (**a**) and (**b**), respectively), with CNF coating (column (**c**)), and without any coating (column (**d**)). Cells were stained by immunofluorescence for vinculin (green), with TRITC-conjugated phalloidin for F-actin (red) and with Hoechst #33258 for the nuclei (blue). Cellulose mesh had autofluorescence in the blue channel. Dragonfly 503 spinning disk confocal microscope with a Zyla 4.2 PLUS sCMOS camera, objective HC PL APO 20x/0.75 IMM CORR CS2. Scale bar: 50 μm.

The dynamic cultivation was held in a unique lab-made cultivation chamber (Figure A2). This chamber allows for fixing a standard well plate with tested substrates and its hermetical sealing to maintain the desired pressure. This chamber was connected to a custom pressure stimulator. This stimulator consists of a servo-controlled linear stage with piston pump and special controlling software. This software allows for setting stimulation parameters that include high/low pressure, motor speed, pulsatile frequency and the shape of the pressure wave.

Porcine ASCs were seeded on CellCrown-fixed substrates in 24-well plates at the same number and in the same cultivation medium as mentioned above. Afterwards, the well plate for dynamic cultivation was fixed into the cultivation chamber, and this chamber was sealed and connected to the pressure stimulator. In the initial phase, the cells were left for 24 h without any pressure stimulation in order to allow their adhesion to the materials. The system was opened through a 220-nm filter to atmosphere forced with slow motion of pump piston to equilibrate CO2 level and pH of medium. After this resting phase, the pressure stimulation was set to 15.9/10.6 kPa (120/80 mmHg) high/low pressure with frequency of 1 Hz (60 pulses per minute) with triangular pulse shape with 1:1 ratio. This dynamic cultivation lasted for 72 h (96 h of cultivation in total including the 24-h rest phase). Static control samples were cultivated for 96 h in a well plate with standard lid in the same CO2 incubator as the dynamic samples.

**Figure A2.** Lab-made dynamic cultivation system for pressure stimulation of cells on the tested material samples. The whole system in a cell incubator (**a**); detail of a cultivation chamber (**b**).

We found that dynamic cell cultivation markedly improved the adhesion and subsequent growth of pASCs. The cells were better spread and their number after three days of dynamic cultivation (day 4 after seeding) was markedly higher than in the corresponding samples incubated under static conditions for four days (Figure A3), and also for seven days (Figure A1). The improvement in cell colonization by dynamic cultivation was observed particularly on samples with thin CNF + MWCNT coating. On both thick and thin CNF + MWCNT coatings, the cells under dynamic conditions were distributed almost homogeneously, while on the pure CNF coating, the cells tended to form clusters. A similar picture was observed in our earlier study performed on human dermal fibroblasts in four-day-old static cultures on the same cationic CNFs, where the cells were less widespread and distributed less homogeneously than on anionic CNFs [2]. Therefore, it can be concluded that the addition of MWCNTs to cationic CNFs improved the colonization of the material with pASCs under dynamic cell culture conditions.

**Figure A3.** Porcine adipose tissue-derived stem cells (pASC) cultivated in a conventional static cell culture system for four days or in a pressure-generating dynamic cell culture system for three days (after one day of static cultivation). The cells were grown on a cellulose mesh with thick or thin CNF + MWCNT coating (column (**a**) and (**b**), respectively), with CNF coating (column (**c**)), and without any coating (column (**d**)). Cells were stained by immunofluorescence for vinculin (green), with TRITC-conjugated phalloidin for F-actin (red) and with DAPI for the nuclei (blue). Cellulose mesh had autofluorescence in the blue channel. Dragonfly 503 spinning disk confocal microscope with a Zyla 4.2 PLUS sCMOS camera, objective HC PL APO 20x/0.75 IMM CORR CS2. Scale bar: 50 μm.
