**2.5. Antibacterial assays**

*E. coli* and *S. aureus* were activated and diluted to the desired concentration of 108 CFU/mL (CFU is an abbreviation of colony‐forming unit). The inoculum of 10 μL *E. coli* and 10 μL *S. aureus* was separately added to the 1‐L growth broth consisting of 30‐g tryptic soy broth (BD 211825) and 1‐L DI water. Two broth media were shaken at 37°C overnight. The bottom‐ layer media were taken and rinsed by sterile PBS via the centrifugation at 4000 revolutions per minute (rpm) for 3 min, and the rinsing process was repeated three times. The bacterial pellets obtained for two bacteria were re‐dispersed, respectively, in the fresh growth broth and then adjusted to achieve 0.1 of optical density (OD) value for bacterial media (equal to 108 CFU/mL). The composite materials prepared were cut into identical round size with a diameter (D) of 10.5 mm. One piece of sample was placed into 12‐well culture plate and UV sterilized for 1 h. Each bacterial medium of 16 μL was dripped onto the sample and another piece of sample was immediately covered on the bacterial medium. The culture plate with samples and bacterial media was cultivated at 37°C for 2 h. Next, the samples and the culture plate were rinsed by sterile PBS at least three times. The collected bacterial solution was centrifuged at 4000 rpm for 3 min and the bacterial pellet was re‐dispersed in 250‐μL fresh broth. The as‐prepared bacterial medium of 100 μL was taken and spread out on the agar medium consisting of 40 g of tryptic soy agar (BD 236950) in 1‐L DI water. The bacteria with the agar medium were cultivated at 37°C for 15 h. The number of bacteria growing on the agar was counted. The antibacterial ability was calculated according to Eq. (1).

$$\text{Amitbackward ability} \left( \% \right) = \frac{\text{CFU}\_{\text{blank}} - \text{CFU}\_{\text{composite}}}{\text{CFU}\_{\text{blank}}} \times 100 \tag{1}$$

where CFUblank is the number of bacterial colonies growing on the agar medium without the presence of composite samples and CFUcomposite is the number of bacterial colonies growing on the agar medium in the presence of composite samples.

#### **2.6. Biocompatibility**

9213) were purchased from the American‐Type Culture Collection (Manassas, VA, USA). *Escherichia coli* (*E. coli*, Trans5α) was purchased from Transgen Biotech (Beijing, China).

An amount of PVA, AE, and QCS was dissolved in deionized (DI) water with a final concen‐ tration of 5% (m:v). The mass ratios of PVA, AE, and QCS in the PVA/AE/QCS (PAQ) mixture were selected at three levels which were 6:3:1 (PAQ1), 7:2:1 (PAQ2), and 8:1:1 (PAQ3). The mixture was poured into a mold with a depth of 0.5 mm and kept at 4°C for 4 h. The cold mixture was then frozen at ‐20°C for 4 h following a defrozen operation at room temperature for 4 h. This freezing‐defreezing operation was repeated three times until a homogeneous gel was achieved. Next, the gel was lyophilized and cut into an identical size for next studies.

PAQ composite samples prepared in the last section were coated with gold and then were imaged by the scanning electron microscopy (SEM, FEI Nova NanoSEM450, USA) operating at 5 kV. Both surface and cross section of samples were examined by SEM. Examination of Fourier transform infrared spectroscopy (FTIR, Bruker Vertex 70, USA) spectra for all compo‐ site samples was performed under conditions that FTIR data were taken from 500 to 4000 cm‐1. OMNIC software (Thermo Electron Corporation) was used to correct and normalize the baseline of FTIR spectra. Thermogravimetric analyses (TGA) of all composite samples were performed by the TGA Q600 (TA Instrument, USA). Thermograms of samples were recorded between 36 and 600°C at a heating rate of 10°C/min and a nitrogen flow of 100 mL/min. TA Universal Analysis 2000 (TA Instrument, USA) was used to calculate the percentage of weight loss, the first derivatives of the thermograms (DTG), and the decomposition temperatures.

The lyophilized PAQ composite samples were soaked in phosphate‐buffered saline (PBS, pH 7.4) and acetic acid/sodium acetate buffer (HAc‐NaAc, pH 5.0). At different assigned times, the sample was taken out and the excessive solution on the surface of the sample was removed by Kimwipes. The water absorbability was the ratio of weight difference of lyophilized sample before and after the soaking versus the weight of lyophilized sample (the amount of adsorbed

*E. coli* and *S. aureus* were activated and diluted to the desired concentration of 108 CFU/mL (CFU is an abbreviation of colony‐forming unit). The inoculum of 10 μL *E. coli* and 10 μL *S. aureus* was separately added to the 1‐L growth broth consisting of 30‐g tryptic soy broth (BD 211825) and 1‐L DI water. Two broth media were shaken at 37°C overnight. The bottom‐ layer media were taken and rinsed by sterile PBS via the centrifugation at 4000 revolutions per minute (rpm) for 3 min, and the rinsing process was repeated three times. The bacterial pellets obtained for two bacteria were re‐dispersed, respectively, in the fresh growth broth

**2.2. Preparation of QCS/AE/PVA composites**

258 Composites from Renewable and Sustainable Materials

**2.3. Material characterization by SEM, FTIR, TGA**

**2.4. Water absorbability**

**2.5. Antibacterial assays**

water vs. dry weight of lyophilized sample).

The effect of the composite samples on the proliferation of L929 mouse fibroblast was first examined. The composite samples of 10.5‐mm D round size were sterilized under UV light for 1 h and then activated in the α‐minimum essential media (α‐MEM, ThermoFisher 11095072) at 37°C and 5% CO2 for 24 h. The activated samples were placed in a 12‐well culture plate and 100 μL of activated L929 cells (20,000 cells/mL/well) was then added to the culture plate. The growth media were exchanged to α‐MEM plus 10% of fetal bovine serum (FBS, ATCC 30‐2021, USA) and added to the culture plate. The cultivation was conducted at 37°C and 5% CO2 for 5 days. At days 1, 3, and 5, the culture medium in the culture plate was taken out and rinsed by sterile PBS via centrifugation at 120× g relative centrifugal force (RCF), respectively. The collected cell pellet was added to the cell‐counting kit‐8 (CCK‐8, Sigma 96992, USA) consisting of 10% CCK‐8 plus 90% α‐MEM. The cell mixture was cultivated at 37°C and 5% CO2 for 2 h and then OD value of cell mixture was examined by ultraviolet‐visible (UV‐VIS) spectrometry (Shimadzu UV‐3600, Japan).

For the cell attachment and morphology of HFCs on the composite samples, HFCs were first activated according to the protocol described in our previous study [18]. The complete growth medium of HFCs was composed by Eagle's minimum essential medium (EMEM) (ATCC 30‐ 2003, USA) plus 10% of FBS. The composite samples were sterilized under UV light for 1 h and then activated by growth medium of HFCs in a 16‐well culture plate. Next, the initial cell suspension (2000 cells/mL/well) was added directly onto the samples following the addition of adequate growth medium to each well, and the cultivation was conducted at 37°C and 5% CO2. At the assigned day, the culture medium was removed and the attached cells on the samples were rinsed by PBS. The samples were subsequently fixed with 2.5% glutaraldehyde PBS solution at room temperature for 2 h and stained by a small drop of fluorescent isothio‐ cyanate dye (FITC) at 4°C for 1 h. Laser scanning confocal microscope (LSCM, Leica SD AF) with an excitation wavelength of 488 nm was used to examine the cell attachment and morphology.
